Miles, Dale R; Mesfin, Mimi; Mody, Tarak D; Stiles, Mark; Lee, Jean; Fiene, John; Denis, Bernie; Boswell, Garry W
2006-05-01
Liquid chromatography-fluorescence (LC-FLS), liquid chromatography-tandem mass spectrometry (LC-MS/MS) and inductively coupled plasma-mass spectrometry (ICP-MS) methods were developed and validated for the evaluation of motexafin gadolinium (MGd, Xcytrin) pharmacokinetics and biodistribution in plasma and tissues. The LC-FLS method exhibited the greatest sensitivity (0.0057 microg mL(-1)), and was used for pharmacokinetic, biodistribution, and protein binding studies with small sample sizes or low MGd concentrations. The LC-MS/MS method, which exhibited a short run time and excellent selectivity, was used for routine clinical plasma sample analysis. The ICP-MS method, which measured total Gd, was used in conjunction with LC methods to assess MGd stability in plasma. All three methods were validated using human plasma. The LC-FLS method was also validated using plasma, liver and kidneys from mice and rats. All three methods were shown to be accurate, precise and robust for each matrix validated. For three mice, the mean (standard deviation) concentration of MGd in plasma/tissues taken 5 hr after dosing with 23 mg kg(-1) MGd was determined by LC-FLS as follows: plasma (0.025+/-0.002 microg mL(-1)), liver (2.89+/-0.45 microg g(-1)), and kidney (6.09+/-1.05 microg g(-1)). Plasma samples from a subset of patients with brain metastases from extracranial tumors were analyzed using both LC-MS/MS and ICP-MS methods. For a representative patient, > or = 90% of the total Gd in plasma was accounted for as MGd over the first hour post dosing. By 24 hr post dosing, 63% of total Gd was accounted for as MGd, indicating some metabolism of MGd.
DBS-LC-MS/MS assay for caffeine: validation and neonatal application.
Bruschettini, Matteo; Barco, Sebastiano; Romantsik, Olga; Risso, Francesco; Gennai, Iulian; Chinea, Benito; Ramenghi, Luca A; Tripodi, Gino; Cangemi, Giuliana
2016-09-01
DBS might be an appropriate microsampling technique for therapeutic drug monitoring of caffeine in infants. Nevertheless, its application presents several issues that still limit its use. This paper describes a validated DBS-LC-MS/MS method for caffeine. The results of the method validation showed an hematocrit dependence. In the analysis of 96 paired plasma and DBS clinical samples, caffeine levels measured in DBS were statistically significantly lower than in plasma but the observed differences were independent from hematocrit. These results clearly showed the need for extensive validation with real-life samples for DBS-based methods. DBS-LC-MS/MS can be considered to be a good alternative to traditional methods for therapeutic drug monitoring or PK studies in preterm infants.
Dubreil, Estelle; Gautier, Sophie; Fourmond, Marie-Pierre; Bessiral, Mélaine; Gaugain, Murielle; Verdon, Eric; Pessel, Dominique
2017-04-01
An approach is described to validate a fast and simple targeted screening method for antibiotic analysis in meat and aquaculture products by LC-MS/MS. The strategy of validation was applied for a panel of 75 antibiotics belonging to different families, i.e., penicillins, cephalosporins, sulfonamides, macrolides, quinolones and phenicols. The samples were extracted once with acetonitrile, concentrated by evaporation and injected into the LC-MS/MS system. The approach chosen for the validation was based on the Community Reference Laboratory (CRL) guidelines for the validation of screening qualitative methods. The aim of the validation was to prove sufficient sensitivity of the method to detect all the targeted antibiotics at the level of interest, generally the maximum residue limit (MRL). A robustness study was also performed to test the influence of different factors. The validation showed that the method is valid to detect and identify 73 antibiotics of the 75 antibiotics studied in meat and aquaculture products at the validation levels.
USDA-ARS?s Scientific Manuscript database
A liquid chromatography-mass spectrometry (LC/MS) method for simultaneous quantitation of seven ergot alkaloids (lysergic acid, ergonovine, ergovaline, ergocornine, ergotamine, ergocryptine and ergocrystine) in vascular tissue was developed and validated. Reverse-phase chromatography, coupled to an...
Determination of aliskiren in tablet dosage forms by a validated stability-indicating RP-LC method.
Wrasse-Sangoi, M; Sangoi, M S; Oliveira, P R; Secretti, L T; Rolim, C M B
2011-02-01
A reversed-phase liquid chromatography (RP-LC) method is validated for the determination of aliskiren in tablet dosage form. The LC method is carried out on a Waters XBridge C(18) column (150 × 4.6 mm i.d.), maintained at 25°C. The mobile phase consisted of acetonitrile:water (95:5, v/v)/phosphoric acid (25 mM, pH 3.0) (40:60, v/v), run at a flow rate of 1.0 mL/min, with photodiode array detector set at 229 nm. The chromatographic separation is obtained with aliskiren retention time of 3.68 min, and it is linear in the range of 10-300 μg/mL (r = 0.9999). The limits of detection and quantitation are 2.38 and 7.93 μg/mL, respectively. The specificity and stability-indicating capability of the method are proven through degradation studies, which also showed that there is no interference of the formulation excipients, showing that peak is free from any coeluting peak. The method showed adequate precision, with a relative standard deviation (RSD) values lower than 0.92%. Good values of accuracy were also obtained, with a mean value of 99.55%. Experimental design is used during validation to calculate method robustness. The proposed method is applied for the analysis of the tablet dosage forms, contributing to improve the quality control and to assure the therapeutic efficacy.
Yang, He S; Wu, Alan H B; Lynch, Kara L
2016-06-01
With the rise in the use and misuse of prescription opioids, there is an increasing need for the confirmed identification of opioid analgesics in toxicology laboratories. The goals of this study were to (i) systematically evaluate the hydrolysis efficiency of four β-glucuronidase enzymes under optimized condition; (ii) evaluate compound recovery, matrix effects and precision of three protein precipitation plates and (iii) develop and validate a qualitative liquid-chromatography mass spectrometry (LC-MS/MS) assay to identify 13 opioids in urine. A recombinant β-glucuronidase exhibited the best overall hydrolysis efficiency for seven opioid glucuronide conjugates compared with β-glucuronidase from red abalone, Escherichia coli and Patella vulgata One of the protein precipitation plates tested exhibited overall better recovery of the opioids and lower ion suppression compared with the other two plates. An ESI positive mode LC-MS/MS assay for qualitative opioid analysis was developed and validated. Linearity, LOD, precision, matrix effect, recovery, carryover and interference of the method were evaluated. Sixty-two patient samples were analyzed by both a legacy GC-MS opioid method and the LC-MS/MS method, and 22 samples were analyzed by the LC-MS/MS and an LC-MS/MS reference method. The results of the comparisons showed good concordance. Overall, we described an efficient sample preparation procedure for a sensitive qualitative opioid confirmation assay in urine. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.
Wang, Zhenyu; Li, Shiming; Ferguson, Stephen; Goodnow, Robert; Ho, Chi-Tang
2008-01-01
Polymethoxyflavones (PMFs), which exist exclusively in the citrus genus, have biological activities including anti-inflammatory, anticarcinogenic, and antiatherogenic properties. A validated RPLC method was developed for quantitative analysis of six major PMFs, namely nobiletin, tangeretin, sinensetin, 5,6,7,4'-tetramethoxyflavone, 3,5,6,7,3',4'-hexamethoxyflavone, and 3,5,6,7,8,3',4'-heptamethoxyflavone. The polar embedded LC stationary phase was able to fully resolve the six analogues. The developed method was fully validated in terms of linearity, accuracy, precision, sensitivity, and system suitability. The LOD of the method was calculated as 0.15 microg/mL and the recovery rate was between 97.0 and 105.1%. This analytical method was successfully applied to quantify the individual PMFs in four commercially available citrus peel extracts (CPEs). Each extract shows significant difference in the PMF composition and concentration. This method may provide a simple, rapid, and reliable tool to help reveal the correlation between the bioactivity of the PMF extracts and the individual PMF content.
Analytical Stability-Indicating Methods for Alogliptin in Tablets by LC-CAD and LC-UV.
Bertol, Charise Dallazem; Friedrich, Maria Tereza; Carlos, Graciela; Froehlich, Pedro Eduardo
2017-03-01
Stability-indicating LC methods using a UV detector and a charged aerosol detector (CAD) simultaneously were validated for the assessment of alogliptin (ALG) in tablets. The analysis was performed on a C8 column (250 × 4.6 mm, 5 μm) at a flow of 0.8 mL/min, using acetonitrile-10 mM ammonium acetate buffer (pH 3.5; 90 + 10, v/v) as mobile phase and UV detection at 275 nm. Validation followed the International Conference on Harmonization guidelines. The method was linear over the range of 25-200 μg/mL. Normality of the residuals showed a normal distribution, no autocorrelation, and homoscedasticity. LODs were 6.25 and 2.65 µg/mL and LOQs were 20.85 and 8.84 µg/mL for the CAD and the UV detector, respectively. The methods were precise and accurate. Excipients and degradation products did not interfere in the methods in studies of specificity. None of the factors studied in the analysis of robustness had a significant effect on the quantification of the ALG by the Pareto chart. The results of the assay obtained with LC-CAD and LC-UV were similar. The methods could be considered interchangeable and stability-indicating, and can be applied as an appropriate QC tool for analysis of ALG in tablets.
Zhang, Jie; Wei, Shimin; Ayres, David W; Smith, Harold T; Tse, Francis L S
2011-09-01
Although it is well known that automation can provide significant improvement in the efficiency of biological sample preparation in quantitative LC-MS/MS analysis, it has not been widely implemented in bioanalytical laboratories throughout the industry. This can be attributed to the lack of a sound strategy and practical procedures in working with robotic liquid-handling systems. Several comprehensive automation assisted procedures for biological sample preparation and method validation were developed and qualified using two types of Hamilton Microlab liquid-handling robots. The procedures developed were generic, user-friendly and covered the majority of steps involved in routine sample preparation and method validation. Generic automation procedures were established as a practical approach to widely implement automation into the routine bioanalysis of samples in support of drug-development programs.
Kahsay, Getu; Song, Huiying; Eerdekens, Fran; Tie, Yaxin; Hendriks, Danny; Van Schepdael, Ann; Cabooter, Deirdre; Adams, Erwin
2015-01-01
Misoprostol is a synthetic prostaglandin E1 analogue which is mainly used for prevention and treatment of gastric ulcers, but also for abortion due to its labour inducing effect. Misoprostol exists as a mixture of diastereoisomers (1:1) and has several related impurities owing to its instability at higher temperatures and moisture. A simple and robust reversed phase liquid chromatographic (RPLC) method is described for the separation of the related substances and a normal phase (NP) LC method for the separation of misoprostol diastereoisomers. The RPLC method was performed using an Ascentis Express C18 (150 mm × 4.6 mm, 5 μm) column kept at 35 °C. The mobile phase was a gradient mixture of mobile phase A (ACN-H2O-MeOH, 28:69:3 v/v/v) and mobile phase B (ACN-H2O-MeOH, 47:50:3 v/v/v) eluted at a flow rate of 1.5 mL/min. UV detection was performed at 200 nm. The NPLC method was undertaken by using an XBridge bare silica (150 mm × 2.1 mm, 3.5 μm) column at 35 °C. The mobile phase contained 1-propanol-heptane-TFA (4:96:0.1%, v/v/v), pumped at a flow rate of 0.5 mL/min. UV detection was performed at 205 nm. This LC method can properly separate the two diastereoisomers (Rs > 2) within an analysis time of less than 20 min. Both methods were validated according to the ICH guidelines. Furthermore, these new LC methods have been successfully applied for purity control and diastereoisomers ratio determination of misoprostol bulk drug, tablets and dispersion. Copyright © 2015 Elsevier B.V. All rights reserved.
This document is a standardized single laboratory validated liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for the detection and quantification of cyanotoxins (combined intracellular and extracellular) in ambient freshwaters.
Zhang, Yanhua; Dufield, Dawn; Klover, Jon; Li, Wenlin; Szekely-Klepser, Gabriella; Lepsy, Christopher; Sadagopan, Nalini
2009-02-15
An LC-MS/MS method was developed and validated to quantify endogenous cyclic guanosine 3',5'-monophosphate (cGMP) in human plasma. The LC-MS/MS and competitive enzyme immunoassay (EIA) assays were compared. cGMP concentrations of 20 human plasma samples were measured by both methods. For the MS-based assay, plasma samples were subjected to a simple protein precipitation procedure by acetonitrile prior to analysis by electrospray ionization LC-MS/MS. De-protonated analytes generated in negative ionization mode were monitored through multiple reaction monitoring (MRM). A stable isotope-labeled internal standard, (13)C(10),(15)N(5)-cGMP, which was biosynthesized in-house, was used in the LC-MS/MS method. The competitive EIA was validated using a commercially available cGMP fluorescence assay kit. The intra-assay accuracy and precision for MS-based assay for cGMP were 6-10.1% CV and -3.6% to 7.3% relative error (RE), respectively, while inter-assay precision and accuracy were 5.6-8.1% CV and -2.1% to 6.3% RE, respectively. The intra-assay accuracy and precision for EIA were 17.9-27.1% CV and -4.9% to 24.5% RE, respectively, while inter-assay precision and accuracy were 15.1-39.5% CV and -30.8% to 4.37% RE, respectively. Near the lower limits of detection, there was little correlation between the cGMP concentration values in human plasma generated by these two methods (R(2)=0.197, P=0.05). Overall, the MS-based assay offered better selectivity, recovery, precision and accuracy over a linear range of 0.5-20ng/mL. The LC-MS/MS method provides an effective tool for the quantitation of cGMP to support clinical mechanistic studies of curative pharmaceuticals.
Emory, Joshua F.; Seserko, Lauren A.; Marzinke, Mark A.
2014-01-01
Background Maraviroc is a CCR5 antagonist that has been utilized as a viral entry inhibitor in the management of HIV-1. Current clinical trials are pursuing maraviroc drug efficacy in both oral and topical formulations. Therefore, in order to fully understand drug pharmacokinetics, a sensitive method is required to quantify plasma drug concentrations. Methods Maraviroc-spiked plasma was combined with acetonitrile containing an isotopically-labeled internal standard, and following protein precipitation, samples were evaporated to dryness and reconstituted for liquid chromatographic-tandem mass spectrometric (LC-MS/MS) analysis. Chromatographic separation was achieved on a Waters BEH C8, 50 × 2.1 mm UPLC column, with a 1.7 μm particle size and the eluent was analyzed using an API 4000 mass analyzer in selected reaction monitoring mode. The method was validated as per FDA Bioanalytical Method Validation guidelines. Results The analytical measuring range of the LC-MS/MS method is 0.5-1000 ng/ml. Calibration curves were generated using weighted 1/x2 quadratic regression. Inter-and intra-assay precision was ≤ 5.38% and ≤ 5.98%, respectively; inter-and intra-assay accuracy (%DEV) was ≤ 10.2% and ≤ 8.44%, respectively. Additional studies illustrated similar matrix effects between maraviroc and its internal standard, and that maraviroc is stable under a variety of conditions. Method comparison studies with a reference LC-MS/MS method show a slope of 0.948 with a Spearman coefficient of 0.98. Conclusions Based on the validation metrics, we have generated a sensitive and automated LC-MS/MS method for maraviroc quantification in human plasma. PMID:24561264
This product is an LC/MS/MS single laboratory validated method for the determination of cylindrospermopsin and anatoxin-a in ambient waters. The product contains step-by-step instructions for sample preparation, analyses, preservation, sample holding time and QC protocols to ensu...
Hummert, Pamela; Parsons, Teresa L; Ensign, Laura M; Hoang, Thuy; Marzinke, Mark A
2018-04-15
The nucleotide reverse transcriptase inhibitor tenofovir (TFV) is widely administered in a disoproxil prodrug form (tenofovir disoproxil fumarate, TDF) for HIV management and prevention. Recently, novel prodrugs tenofovir alafenamide fumarate (TAF) and hexadecyloxypropyl tenofovir (CMX157) have been pursued for HIV treatment while minimizing adverse effects associated with systemic TFV exposure. Dynamic and sensitive bioanalytical tools are required to characterize the pharmacokinetics of these prodrugs in systemic circulation. Two parallel methods have been developed, one to combinatorially quantify TAF and TFV, and a second method for CMX157 quantification, in plasma. K 2 EDTA plasma was spiked with TAF and TFV, or CMX157. Following the addition of isotopically labeled internal standards and sample extraction via solid phase extraction (TAF and TFV) or protein precipitation (CMX157), samples were subjected to liquid chromatographic-tandem mass spectrometric (LC-MS/MS) analysis. For TAF and TFV, separation occurred using a Zorbax Eclipse Plus C18 Narrow Bore RR, 2.1 × 50 mm, 3.5 μm column and analytes were detected on an API5000 mass analyzer; CMX157 was separated using a Kinetex C8, 2.1 × 50 mm, 2.6 μm column and quantified using an API4500 mass spectrometer. Methods were validated according to FDA Bioanalytical Method Validation guidelines. Analytical methods: were optimized for the multiplexed monitoring of TAF and TFV, and CMX157 in plasma. The lower limits of quantification (LLOQs) for TAF, TFV, and CMX157 were 0.03, 1.0, and 0.25 ng/mL, respectively. Calibration curves were generated via weighted linear regression of standards. Intra- and inter-assay precision and accuracy studies demonstrated %CVs ≤ 14.4% and %DEVs ≤ ± 7.95%, respectively. Stability and matrix effects studies were also performed. All results were acceptable and in accordance with the recommended guidelines for bioanalytical methods. Assays were also
This document is a standardized, single laboratory validated liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for the detection of cyanotoxins—microsystins and nodularin (combined intracellular and extracellular)—in ambient freshwaters.
da Luz, Suzane Rickes; Pazdiora, Paulo Cesar; Dallagnol, Leandro José; Dors, Giniani Carla; Chaves, Fábio Clasen
2017-04-01
Wheat (Triticum aestivum) is an annual crop, cultivated in the winter and spring and susceptible to several pathogens, especially fungi, which are managed with fungicides. It is also one of the most consumed cereals, and can be contaminated by mycotoxins and fungicides. The objective of this study was to validate an analytical method by LC-MS for simultaneous determination of mycotoxins and fungicide residues in wheat grains susceptible to fusarium head blight treated with fungicides, and to evaluate the relationship between fungicide application and mycotoxin production. All parameters of the validated analytical method were within AOAC and ANVISA limits. Deoxynivalenol was the prevalent mycotoxin in wheat grain and epoxiconazole was the fungicide residue found in the highest concentration. All fungicidal treatments induced an increase in AFB2 production when compared to the control (without application). AFB1 and deoxynivalenol, on the contrary, were reduced in all fungicide treatments compared to the control. Copyright © 2016 Elsevier Ltd. All rights reserved.
Sato, Tamaki; Miyamoto, Iori; Uemura, Masako; Nakatani, Tadashi; Kakutani, Naoya; Yamano, Tetsuo
2016-01-01
A validation study was carried out on a rapid method for the simultaneous determination of pesticide residues in vegetables and fruits by LC-MS/MS. Preparation of the test solution was performed by a solid-phase extraction technique with QuEChERS (STQ method). Pesticide residues were extracted with acetonitrile using a homogenizer, followed by salting-out and dehydration at the same time. The acetonitrile layer was purified with C18 and PSA mini-columns. The method was assessed for 130 pesticide residues in 14 kinds of vegetables and fruits at the concentration level of 0.01 μg/g according to the method validation guideline of the Ministry of Health, Labour and Welfare of Japan. As a result 75 to 120 pesticide residues were determined satisfactorily in the tested samples. Thus, this method could be useful for a rapid and simultaneous determination of multi-class pesticide residues in various vegetables and fruits.
Ananthula, Suryatheja; Janagam, Dileep R; Jamalapuram, Seshulatha; Johnson, James R; Mandrell, Timothy D; Lowe, Tao L
2015-10-15
Rapid, sensitive, selective and accurate LC/MS/MS method was developed for quantitative determination of levonorgestrel (LNG) in rat plasma and further validated for specificity, linearity, accuracy, precision, sensitivity, matrix effect, recovery efficiency and stability. Liquid-liquid extraction procedure using hexane:ethyl acetate mixture at 80:20 v:v ratio was employed to efficiently extract LNG from rat plasma. Reversed phase Luna column C18(2) (50×2.0mm i.d., 3μM) installed on a AB SCIEX Triple Quad™ 4500 LC/MS/MS system was used to perform chromatographic separation. LNG was identified within 2min with high specificity. Linear calibration curve was drawn within 0.5-50ng·mL(-1) concentration range. The developed method was validated for intra-day and inter-day accuracy and precision whose values fell in the acceptable limits. Matrix effect was found to be minimal. Recovery efficiency at three quality control (QC) concentrations 0.5 (low), 5 (medium) and 50 (high) ng·mL(-1) was found to be >90%. Stability of LNG at various stages of experiment including storage, extraction and analysis was evaluated using QC samples, and the results showed that LNG was stable at all the conditions. This validated method was successfully used to study the pharmacokinetics of LNG in rats after SubQ injection, providing its applicability in relevant preclinical studies. Copyright © 2015 Elsevier B.V. All rights reserved.
Goswami, Dipanjan; Kumar, Ajay; Khuroo, Arshad H; Monif, Tausif; Rab, Shamsur
2009-11-01
A LC-MS/MS method for plasma topiramate analysis is delineated involving least number of healthy volunteers. Topiramate and amlodipine internal standard (IS) were extracted by simple centrifuge-coupled solid-phase extraction and reverse-phase chromatographic separation was performed on an Ascentis C(18) column. Turbo-spray negative-ion mode multiple-reaction monitoring was selected for mass pair detection at m/z 338.3 --> 78.0 and m/z 407.3 --> 295.5 for analyte and IS respectively. The method showed a dynamic linearity range from 10.4 to 2045.0 ng/mL, lower limit of quantitation achieved at 10.4 ng/mL and finally a mass spectrometric total run time of within 2.5 min for human sample analysis. Bioequivalence was assessed successfully using this fully validated method on 16 fasted Indian male subjects with 25 mg topiramate tablet administration. An appropriate study design describes plasma samples collection up to 216 h post dose in two periods, separated by a 28 day washout period. The challenge of half-life matching for test and reference drug was achieved with 73.43 +/- 9.68 and 73.06 +/- 14.03 h, respectively, and intra-subject coefficient of variation achieved within 11% for AUCs and C(max) evaluated by non-compartmental pharmacokinetic analysis. The results of LCMS topiramate complete method validation supported by pharmacokinetic study have not been published before, and are presented and discussed for the first time in this article. Copyright (c) 2009 John Wiley & Sons, Ltd.
Zimmermann, Estevan Sonego; da Silva, Lucélia Magalhães; Calegari, Guilherme Zanini; Stamm, Fernanda Pavani; Souto, Ricardo Bizogne; Dalmora, Sérgio Luiz
2013-01-01
A size-exclusion LC method was validated for the determination of interferon-a2a (rhlFN-alpha2a) in pharmaceutical formulations without interference from human serum albumin. Chromatographic separation was performed on a BioSep-SEC-S 2000 column (300 x 7.8 mm id). The mobile phase consisted of 0.001 M monobasic potassium phosphate, 0.008 M sodium phosphate dibasic; 0.2 M sodium chloride buffer, pH 7.4, run at a gradient flow rate and using photodiode array detection at 214 nm, was used. Chromatographic separation was achieved with a retention time of 17.2 min, and the analysis was linear over the concentration range of 1.98 to 198 microg/mL (r2 = 0.9996). The accuracy was 101.39%, with bias lower than 1.67%. The LOD and LOQ were 0.87 and 1.98 microg/mL, respectively. Moreover, method validation demonstrated acceptable results for precision and robustness. The method was applied to the assessment of rhlFN-alpha2a and related proteins in biopharmaceutical dosage forms, and the content/potencies were correlated to those given by a validated RP-LC method and an in vitro bioassay. It was concluded that use of the methods in conjunction allows a great improvement in monitoring stability and QC, thereby ensuring the therapeutic efficacy of the biotechnology-derived medicine.
Noegrohati, Sri; Hernadi, Elan; Asviastuti, Syanti
2018-06-01
Production of red flesh dragon fruit (Hylocereus polyrhizus) was hampered by Colletotrichum sp. Pre-harvest application of azoxystrobin and difenoconazole mixture is recommended, therefore, a selective and sensitive multi residues analytical method is required in monitoring and evaluating the commodity's safety. LC-MS/MS is a well-established analytical technique for qualitative and quantitative determination in complex matrices. However, this method is hurdled by co-eluted coextractives interferences. This work evaluated the pH effect of acetate buffered and citrate buffered QuEChERS sample preparation in their effectiveness of matrix effect reduction. Citrate buffered QuEChERS proved to produce clean final extract with relative matrix effect 0.4%-0.7%. Method validation of the selected sample preparation followed by LC-MS/MS for whole dragon fruit, flesh and peel matrices fortified at 0.005, 0.01, 0.1 and 1 g/g showed recoveries 75%-119%, intermediate repeatability 2%-14%. The expanded uncertainties were 7%-48%. Based on the international acceptance criteria, this method is valid.
Bansal, Gulshan; Singh, Ranjit; Saini, Balraj; Bansal, Yogita
2013-01-01
The present study reports the characterization of forced degradation products of bosentan and a validated stability-indicating HPLC method for the stability testing of bosentan tablets. The forced degradation was carried out under the conditions of hydrolysis, oxidation, dry heat and photolysis. The drug was found unstable in acid, alkali and oxidative media whereas stable to the hydrolysis in water, to dry heat and to photolysis. In total, six degradation products were formed in all conditions which were resolved in a single run on a C-18 column with gradient elution using ammonium acetate buffer (pH 4.5, 5.0mM), methanol and acetonitrile. Structures of all the degradation products were characterized through +ESI-MS(n) and LC-ESI-MS spectral data of bosentan as well as LC-ESI-MS spectral data of the products. The products II-VI were characterized as 6-amino-[2,2']bipyrimidinyl-4,5-diol, 6-amino-5-(2-methoxyphenoxy)-[2,2']-bipyrimidinyl-4-ol, 2-[6-amino-5-(2-methoxyphenoxy)-[2,2']-bipyrimidinyl-4-yloxy]-ethanol, 4-tert-butyl-N-[6-(1-methoxyethoxy)-5-(2-methoxyphenoxy)-[2,2']-bipyrimidinyl-4-yl]-benzenesulfonamide and 4-tert-butyl-N-[6-hydroxy-5-(2-methoxyphenoxy)-[2,2']bipyrimidinyl-4-yl]-benzenesulfonamide, respectively. The peak of the product I was found to be due to two secondary degradation products which co-eluted and were characterized as β-hydroxyethyl p-tert-butylphenylsulfonate (Ia) and 2-[2-(2-hydroxyethoxy)-phenoxy]-ethanol (Ib). These products were formed due to hydrolysis of sulfonamide and alkylaryl ether and the diaryl ether linkages as well as dehydration of the primary alcohol group. The most probable degradation mechanisms were proposed. The HPLC method was found to be stability-indicating, linear (2-100 μg ml(-1)), accurate, precise, sensitive, specific, rugged and robust for quantitation of the drug. The method was applied to the stability testing of the commercially available bosentan tablets successfully. Copyright © 2012 Elsevier B.V. All
Rower, Joseph E.; Bushman, Lane R.; Hammond, Kyle P.; Kadam, Rajendra S.; Aquilante, Christina L.
2011-01-01
Gemfibrozil, a fibric acid hypolipidemic agent, is increasingly being used in clinical drug-drug interaction studies as an inhibitor of drug metabolizing enzymes and drug transporters. The validation of a fast, accurate, and precise LC/MS method is described for the quantitative determination of gemfibrozil in an EDTA-anticoagulated human plasma matrix. Briefly, gemfibrozil was extracted from human plasma by an acetonitrile protein precipitation method. The assay was reproducible with intra-assay precision between 1.6% and 10.7%, and inter-assay precision ranging from 4.4% to 7.8%. The assay also showed good accuracy, with intra-assay concentrations within 85.6% and 108.7% of the expected value, and inter-assay concentrations within 89.4 to 104.0% of the expected value. The linear concentration range was between 0.5 and 50 μg/mL with a lower limit of quantitation of 0.5 μg/mL when 125 μL of plasma were extracted. This LC/MS method yielded a quick, simple, and reliable protocol for determining gemfibrozil concentrations in plasma and is applicable to clinical pharmacokinetic studies. PMID:21077249
Rower, Joseph E; Bushman, Lane R; Hammond, Kyle P; Kadam, Rajendra S; Aquilante, Christina L
2010-12-01
Gemfibrozil, a fibric acid hypolipidemic agent, is increasingly being used in clinical drug-drug interaction studies as an inhibitor of drug metabolizing enzymes and drug transporters. The validation of a fast, accurate and precise LC/MS method is described for the quantitative determination of gemfibrozil in an EDTA-anticoagulated human plasma matrix. Briefly, gemfibrozil was extracted from human plasma by an acetonitrile protein precipitation method. The assay was reproducible with intra-assay precision between 1.6 and 10.7%, and inter-assay precision ranging from 4.4 to 7.8%. The assay also showed good accuracy, with intra-assay concentrations within 85.6-108.7% of the expected value, and inter-assay concentrations within 89.4-104.0% of the expected value. The linear concentration range was between 0.5 and 50 µg/mL with a lower limit of quantitation of 0.5 µg/mL when 125 µL of plasma were extracted. This LC/MS method yielded a quick, simple and reliable protocol for determining gemfibrozil concentrations in plasma and is applicable to clinical pharmacokinetic studies. Copyright © 2010 John Wiley & Sons, Ltd.
Xiong, Shan; Deng, Zhipeng; Sun, Peilu; Mu, Yanling; Xue, Mingxing
2017-11-01
Osimertinib is a new-generation epidermal growth factor inhibitor for the treatment of non-small cell lung cancer. In the present study, a rapid and sensitive LC with tandem MS method was developed and validated for the determination of osimertinib in rat plasma. Chromatographic separation was carried out on a C18 column using acetonitrile and water containing 0.1% formic acid. The assay was validated over a concentration range of 1.0-1000 ng/mL for osimertinib, with a lower LOQ of 1.0 ng/mL. The intra- and interday accuracy values for osimertinib ranged from 92.66 to 101.50% and from 97.08 to 99.15%, respectively, and the intra- and interday precision values for osimertinib ranged from 6.25 to 10.34% and from 3.43 to 10.44%, respectively. The method was successfully applied in a pharmacokinetic study of osimertinib after oral administration of osimertinib (4.5 mg/kg) to rats.
Jiang, Hongliang; Wang, Yurong; Shet, Manjunath S; Zhang, Yang; Zenke, Duane; Fast, Douglas M
2011-09-01
A rapid, specific, and reliable LC-MS/MS based bioanalytical method was developed and validated for the simultaneous determination of naloxone (NLX) and its two metabolites, 6β-naloxol (NLL) and naloxone-3β-D-glucuronide (NLG) in mouse plasma. The optimal chromatographic behavior of these analytes was achieved on an Aquasil C18 column (50 mm × 2.1 mm, 5 μm) using reversed phase chromatography. The total LC analysis time per injection was 2.5 min with a flow rate of 1.0 mL/min with gradient elution. Sample preparation via protein precipitation with acetonitrile in a 96-well format was applied for analyses of these analytes. The analytes were monitored by electrospray ionization in positive ion multiple reaction monitoring (MRM) mode. Modification of collision energy besides chromatographic separation was applied to further eliminate interference peaks for NLL and NLG. The method validation was conducted over the curve range of 0.200/0.400/0.500 to 100/200/250 ng/mL for NLX/NLL/NLG, respectively, using 0.0250 mL of plasma sample. The intra- and inter-day precision and accuracy of the quality control samples at low, medium, and high concentration levels showed ≤ 6.5% relative standard deviation (RSD) and -8.3 to -2.5% relative error (RE). The method was successfully applied to determine the concentrations of NLX, NLL, and NLG in incurred mouse plasma samples. Copyright © 2011 Elsevier B.V. All rights reserved.
Kinyua, Juliet; Covaci, Adrian; Maho, Walid; McCall, Ann-Kathrin; Neels, Hugo; van Nuijs, Alexander L N
2015-09-01
Sewage-based epidemiology (SBE) employs the analysis of sewage to detect and quantify drug use within a community. While SBE has been applied repeatedly for the estimation of classical illicit drugs, only few studies investigated new psychoactive substances (NPS). These compounds mimic effects of illicit drugs by introducing slight modifications to chemical structures of controlled illicit drugs. We describe the optimization, validation, and application of an analytical method using liquid chromatography coupled to positive electrospray tandem mass spectrometry (LC-ESI-MS/MS) for the determination of seven NPS in sewage: methoxetamine (MXE), butylone, ethylone, methylone, methiopropamine (MPA), 4-methoxymethamphetamine (PMMA), and 4-methoxyamphetamine (PMA). Sample preparation was performed using solid-phase extraction (SPE) with Oasis MCX cartridges. The LC separation was done with a HILIC (150 x 3 mm, 5 µm) column which ensured good resolution of the analytes with a total run time of 19 min. The lower limit of quantification (LLOQ) was between 0.5 and 5 ng/L for all compounds. The method was validated by evaluating the following parameters: sensitivity, selectivity, linearity, accuracy, precision, recoveries and matrix effects. The method was applied on sewage samples collected from sewage treatment plants in Belgium and Switzerland in which all investigated compounds were detected, except MPA and PMA. Furthermore, a consistent presence of MXE has been observed in most of the sewage samples at levels higher than LLOQ. Copyright © 2015 John Wiley & Sons, Ltd.
Determination of lipophilic toxins by LC/MS/MS: single-laboratory validation.
Villar-González, Adriano; Rodríguez-Velasco, María Luisa; Gago-Martínez, Ana
2011-01-01
An LC/MS/MS method has been developed, assessed, and intralaboratory-validated for the analysis of the lipophilic toxins currently regulated by European Union legislation: okadaic acid (OA) and dinophysistoxins 1 and 2, including their ester forms; azaspiracids 1, 2, and 3; pectenotoxins 1 and 2; yessotoxin (YTX), and the analogs 45 OH-YTX, Homo YTX, and 45 OH-Homo YTX; as well as for the analysis of 13-desmetil-spirolide C. The method consists of duplicate sample extraction with methanol and direct analysis of the crude extract without further cleanup or concentration. Ester forms of OA and dinophysistoxins are detected as the parent ions after alkaline hydrolysis of the extract. The validation process of this method was performed using both fortified and naturally contaminated samples, and experiments were designed according to International Organization for Standardization, International Union of Pure and Applied Chemistry, and AOAC guidelines. With the exception of YTX in fortified samples, RSDr below 15% and RSDR were below 25%. Recovery values were between 77 and 95%, and LOQs were below 60 microg/kg. These data together with validation experiments for recovery, selectivity, robustness, traceability, and linearity, as well as uncertainty calculations, are presented in this paper.
Jiang, Hui; Sidhu, Rohini; Fujiwara, Hideji; De Meulder, Marc; de Vries, Ronald; Gong, Yong; Kao, Mark; Porter, Forbes D.; Yanjanin, Nicole M.; Carillo-Carasco, Nuria; Xu, Xin; Ottinger, Elizabeth; Woolery, Myra; Ory, Daniel S.; Jiang, Xuntian
2014-01-01
2-Hydroxypropyl-β-cyclodextrin (HP-β-CD), a widely used excipient for drug formulation, has emerged as an investigational new drug for the treatment of Niemann-Pick type C1 (NPC1) disease, a neurodegenerative cholesterol storage disorder. Development of a sensitive quantitative LC-MS/MS assay to monitor the pharmacokinetics (PKs) of HP-β-CD required for clinical trials has been challenging owing to the dispersity of the HP-β-CD. To support a phase 1 clinical trial for ICV delivery of HP-β-CD in NPC1 patients, novel methods for quantification of HP-β-CD in human plasma and cerebrospinal fluid (CSF) using LC-MS/MS were developed and validated: a 2D-LC-in-source fragmentation-MS/MS (2D-LC-IF-MS/MS) assay and a reversed phase ultra performance LC-MS/MS (RP-UPLC-MS/MS) assay. In both assays, protein precipitation and “dilute and shoot” procedures were used to process plasma and CSF, respectively. The assays were fully validated and in close agreement, and allowed determination of PK parameters for HP-β-CD. The LC-MS/MS methods are ∼100-fold more sensitive than the current HPLC assay, and were successfully employed to analyze HP-β-CD in human plasma and CSF samples to support the phase 1 clinical trial of HP-β-CD in NPC1 patients. PMID:24868096
Xiong, Yeping; Zhao, Yuan-Yuan; Goruk, Sue; Oilund, Kirsten; Field, Catherine J; Jacobs, René L; Curtis, Jonathan M
2012-12-12
A hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC LC-MS/MS) method was developed and validated to simultaneously quantify six aqueous choline-related compounds and eight major phospholipids classes in a single run. HILIC chromatography was coupled to positive ion electrospray mass spectrometry. A combination of multiple scan modes including precursor ion scan, neutral loss scan and multiple reaction monitoring was optimized for the determination of each compound or class in a single LC/MS run. This work developed a simplified extraction scheme in which both free choline and related compounds along with phospholipids were extracted into a homogenized phase using chloroform/methanol/water (1:2:0.8) and diluted into methanol for the analysis of target compounds in a variety of sample matrices. The analyte recoveries were evaluated by spiking tissues and food samples with two isotope-labeled internal standards, PC-d(3) and Cho-d(3). Recoveries of between 90% and 115% were obtained by spiking a range of sample matrices with authentic standards containing all 14 of the target analytes. The precision of the analysis ranged from 1.6% to 13%. Accuracy and precision was comparable to that obtained by quantification of selected phospholipid classes using (31)P NMR. A variety of sample matrices including egg yolks, human diets and animal tissues were analyzed using the validated method. The measurements of total choline in selected foods were found to be in good agreement with values obtained from the USDA choline database. Copyright © 2012 Elsevier B.V. All rights reserved.
Jiang, Hui; Sidhu, Rohini; Fujiwara, Hideji; De Meulder, Marc; de Vries, Ronald; Gong, Yong; Kao, Mark; Porter, Forbes D; Yanjanin, Nicole M; Carillo-Carasco, Nuria; Xu, Xin; Ottinger, Elizabeth; Woolery, Myra; Ory, Daniel S; Jiang, Xuntian
2014-07-01
2-Hydroxypropyl-β-cyclodextrin (HP-β-CD), a widely used excipient for drug formulation, has emerged as an investigational new drug for the treatment of Niemann-Pick type C1 (NPC1) disease, a neurodegenerative cholesterol storage disorder. Development of a sensitive quantitative LC-MS/MS assay to monitor the pharmacokinetics (PKs) of HP-β-CD required for clinical trials has been challenging owing to the dispersity of the HP-β-CD. To support a phase 1 clinical trial for ICV delivery of HP-β-CD in NPC1 patients, novel methods for quantification of HP-β-CD in human plasma and cerebrospinal fluid (CSF) using LC-MS/MS were developed and validated: a 2D-LC-in-source fragmentation-MS/MS (2D-LC-IF-MS/MS) assay and a reversed phase ultra performance LC-MS/MS (RP-UPLC-MS/MS) assay. In both assays, protein precipitation and "dilute and shoot" procedures were used to process plasma and CSF, respectively. The assays were fully validated and in close agreement, and allowed determination of PK parameters for HP-β-CD. The LC-MS/MS methods are ∼100-fold more sensitive than the current HPLC assay, and were successfully employed to analyze HP-β-CD in human plasma and CSF samples to support the phase 1 clinical trial of HP-β-CD in NPC1 patients. Copyright © 2014 by the American Society for Biochemistry and Molecular Biology, Inc.
Sun, Hanwen; Wang, Fengchi; Ai, Lianfeng; Guo, Chunhai; Chen, Ruichun
2009-01-01
A sensitive method based on solid-phase extraction-liquid chromatography-tandem mass spectrometry interfaced with electrospray ionization (SPE-LC-MS/MS-ESI) was developed for the simultaneous determination of 8 banned nitroimidazole (NOZ) drugs including metronidazole (MNZ), ronidazole (RNZ), dimetridazole (DMZ), tinidazole, ornidazole, secnidazole, metronidazole-OH (MNZOH, the metabolite of MNZ), and 2-hydroxymethyl-1-methyl-5-nitroimidazole (HMMNI, the metabolite of RNZ and DMZ) in natural casings. After extraction with ethyl acetate and evaporation, the NOZs were reconstituted in ethyl acetate and purified on a strong cation-exchange SPE column, and then LC/MS/MS analysis was performed by positive ESI applying multiple reaction monitoring of 2 transition reactions for each compound. The method was validated according to the European Union requirements (Commission Decision 2002/657/EC). Specificity, linearity, decision limit (CCalpha), detection capability (CCbeta), accuracy, and precision were determined. Average recoveries of the 8 NOZs from natural animal casing fortified at 3 levels (0.1, 0.5, and 1 microg/kg) ranged from 87.3 to 116.5%. The calculated CCalpha for NOZs ranged from 0.029 to 0.049 microg/kg, and CCbeta ranged from 0.049 to 0.083 microg/kg. Repeatability was in the range of 3.35-10.1%, and within-laboratory reproducibility was <10.3%.
Ramadan, Gouda; Al Jabir, Muna; Alabdulmalik, Najat; Mohammed, Ali
2016-05-01
Most countries have clearly defined regulations governing the use of pesticides in agricultural activity. The application of pesticides in agriculture usually leads to a residual amount of these pesticides on food products such as fruit and vegetables. The presence of pesticide residues on these foods destined for human consumption may pose food safety risks to consumers. To protect consumers, national authorities have established maximum limits for pesticide residues in foods. These limits can only be enforced if there are methods available to detect and monitor their concentrations in the applicable food products. To support the enforcement of this legislation, we have developed a multi-residue liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the analysis of 120 pesticide residues in apples and cucumbers which has been validated and implemented in the routine monitoring and surveillance programme for these pesticides. In this method, apple and cucumber samples are extracted using the QuEChERS method (quick, easy, cheap, effective, rugged, and safe) and the extracts were analyzed directly by LC-MS/MS. The mean recoveries at three different concentrations of 0.01 µg/g , 0.05 µg/g, and 0.1 µg/g over the analytical range varied between 70 and 120%. The repeatability of the method expressed as %RSD was less than 20%. The limit of detection (LOD) of the method ranged between 0.0014 and 0.0110 µg/g for apples and between 0.0012 and 0.0075 µg/g for cucumbers. The limit of quantification (LOQ) of the method was 0.01 µg/g for apples and cucumbers. The method has been used for the analysis of over 600 apple and 550 cucumber samples over the past two years. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.
Peixoto, Maria Paula Garofo; Kaiser, Samuel; Verza, Simone Gasparin; de Resende, Pedro Ernesto; Treter, Janine; Pavei, Cabral; Borré, Gustavo Luís; Ortega, George González
2012-01-01
Ilex paraguariensis A. St. Hil. (mate) is known in several South American countries because of the use of its leaves in stimulant herbal beverages. High saponin contents were reported in mate leaves and unripe fruits that possess a dissimilar composition. Two LC-UV methods previously reported for mate saponins assay focused on mate leaves and the quantification of the less polar saponin fraction in mate fruits. To develop and validate a LC-UV method to assay the total content of saponins in unripe mate fruits and characterise the chemical structure of triterpenic saponins by UPLC/Q-TOF-MS. From unripe fruits of mate a crude ethanolic extract was prepared (EX40) and the mate saponin fraction (MSF) purified by solid phase extraction. The LC-UV method was validated using ilexoside II as external standard. UPLC/Q-TOF-MS was adjusted from the LC-UV method to obtain the fragmentation patterns of the main saponins present in unripe fruits. Both LC-UV and UPLC/Q-TOF-MS methods indicate a wide range of Ilex saponins polarity. The ilexoside II and total saponin content of EX40 were 8.20% (w/w) and 47.60% (w/w), respectively. The total saponin content in unripe fruits was 7.28% (w/w). The saponins present in MSF characterised by UPLC/Q-TOF-MS are derived mainly from ursolic/oleanolic, acetyl ursolic or pomolic acid. The validated LC-UV method was shown to be linear, precise, accurate and to cover several saponins previously isolated from Ilex species and could be applied for the quality control of unripe fruit saponins. Copyright © 2011 John Wiley & Sons, Ltd.
Yang, Samuel H; Wang, Jenny; Zhang, Kelly
2017-04-07
Despite the advantages of 2D-LC, there is currently little to no work in demonstrating the suitability of these 2D-LC methods for use in a quality control (QC) environment for good manufacturing practice (GMP) tests. This lack of information becomes more critical as the availability of commercial 2D-LC instrumentation has significantly increased, and more testing facilities begin to acquire these 2D-LC capabilities. It is increasingly important that the transferability of developed 2D-LC methods be assessed in terms of reproducibility, robustness and performance across different laboratories worldwide. The work presented here focuses on the evaluation of a heart-cutting 2D-LC method used for the analysis of a pharmaceutical material, where a key, co-eluting impurity in the first dimension ( 1 D) is resolved from the main peak and analyzed in the second dimension ( 2 D). A design-of-experiments (DOE) approach was taken in the collection of the data, and the results were then modeled in order to evaluate method robustness using statistical modeling software. This quality by design (QBD) approach gives a deeper understanding of the impact of these 2D-LC critical method attributes (CMAs) and how they affect overall method performance. Although there are multiple parameters that may be critical from method development point of view, a special focus of this work is devoted towards evaluation of unique 2D-LC critical method attributes from method validation perspective that transcend conventional method development and validation. The 2D-LC method attributes are evaluated for their recovery, peak shape, and resolution of the two co-eluting compounds in question on the 2 D. In the method, linearity, accuracy, precision, repeatability, and sensitivity are assessed along with day-to-day, analyst-to-analyst, and lab-to-lab (instrument-to-instrument) assessments. The results of this validation study demonstrate that the 2D-LC method is accurate, sensitive, and robust and is
A computer method of finding valuations forcing validity of LC formulae
NASA Astrophysics Data System (ADS)
Godlewski, Łukasz; Świetorzecka, Kordula; Mulawka, Jan
2014-11-01
The purpose of this paper is to present the computer implementation of a system known as LC temporal logic [1]. Firstly, to become familiar with some theoretical issues, a short introduction to this logic is discussed. The algorithms allowing a deep analysis of the formulae of LC logic are considered. In particular we discuss how to determine if a formula is a tautology, contrtautology or it is satisfable. Next, we show how to find all valuations to satisfy the formula. Finally, we consider finding histories generated by the formula and transforming these histories into the state machine. Moreover, a description of the experiments that verify the implementation are briefly presented.
Athar Masood, M; Veenstra, Timothy D
2017-08-26
Urine Drug Testing (UDT) is an important analytical/bio-analytical technique that has inevitably become an integral and vital part of a testing program for diagnostic purposes. This manuscript presents a tailor-made LC-MS/MS quantitative assay method development and validation for a custom group of 33 pain panel drugs and their metabolites belonging to different classes (opiates, opioids, benzodiazepines, illicit, amphetamines, etc.) that are prescribed in pain management and depressant therapies. The LC-MS/MS method incorporates two experiments to enhance the sensitivity of the assay and has a run time of about 7 min. with no prior purification of the samples required and a flow rate of 0.7 mL/min. The method also includes the second stage metabolites for some drugs that belong to different classes but have first stage similar metabolic pathways that will enable to correctly identify the right drug or to flag the drug that might be due to specimen tampering. Some real case examples and difficulties in peak picking were provided with some of the analytes in subject samples. Finally, the method was deliberated with some randomly selected de-identified clinical subject samples, and the data evaluated from "direct dilute and shoot analysis" and after "glucuronide hydrolysis" were compared. This method is now used to run routinely more than 100 clinical subjects samples on a daily basis. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.
Kostić, Nađa; Dotsikas, Yannis; Jović, Nebojša; Stevanović, Galina; Malenović, Anđelija; Medenica, Mirjana
2015-05-10
In this paper, novel LC-MS/MS methods for the determination of antiepileptic drug pregabalin in dried matrix spots (DMS) are presented. This attractive technique of sample collection in micro amount was utilized in the form of dried blood spots (DBS) and dried plasma spots (DPS). Following a pre-column derivatization procedure, using n-propyl chloroformate in the presence of n-propanol, and consecutive liquid-liquid extraction, derivatized pregabalin and its internal standard, 4-aminocyclohexanecarboxylic acid, were detected in positive ion mode by applying two SRM transitions per analyte. A YMC-Pack Octyl column (50mm×4.0mm, 3μm particle size) maintained at 30°C, was utilized with running mobile phase composed of acetonitrile: 0.15% formic acid (85:15, v/v). Flow rate was 550μL/min and total run time 2min. Established methods were fully validated over the concentration range of 0.200-20.0μg/mL for DBS and 0.400-40.0μg/mL for DPS, respectively, while specificity, accuracy, precision, recovery, matrix-effect, stability, dilution integrity and spot homogeneity were found within acceptance criteria. Validated methods were applied for the determination of pregabalin levels in dried blood and plasma samples obtained from patients with epilepsy, after per os administration of commercial capsules. Comparison of drug level in blood and plasma, as well as correction steps undertaken in order to overcome hematocrit issue, when analyzing DBS, are also given. Copyright © 2015 Elsevier B.V. All rights reserved.
Grigori, Katerina; Loukas, Yannis L; Malenović, Anđelija; Samara, Vicky; Kalaskani, Anastasia; Dimovasili, Efi; Kalovidouri, Magda; Dotsikas, Yannis
2017-10-25
A sensitive Liquid Chromatography tandem mass spectrometry (LC-MS/MS) method was developed and validated for the quantitative analysis of three potential genotoxic impurities (318BP, M9, S5) in meropenem Active Pharmaceutical Ingredient (API). Due to the requirement for LOD values in ppb range, a high concentration of meropenem API (30mg/mL) had to be injected. Therefore, efficient determination of meropenem from its impurities became a critical aim of this study, in order to divert meropenem to waste, via a switching valve. After the selection of the important factors affecting analytes' elution, a Box-Behnken design was utilized to set the plan of experiments conducted with UV detector. As responses, the separation factor s between the last eluting impurity and meropenem, as well as meropenem retention factor k were used. Grid point search methodology was implemented aiming to obtain the optimal conditions that simultaneously comply to the conflicted criteria. Optimal mobile phase consisted of ACN, methanol and 0.09% HCOOH at a ratio 71/3.5/15.5v/v. All impurities and internal standard omeprazole were eluted before 7.5min and at 8.0min the eluents were directed to waste. The protocol was transferred to LC-MS/MS and validated according to ICH guidelines. Copyright © 2017 Elsevier B.V. All rights reserved.
Gil, Jeovanis; Cabrales, Ania; Reyes, Osvaldo; Morera, Vivian; Betancourt, Lázaro; Sánchez, Aniel; García, Gerardo; Moya, Galina; Padrón, Gabriel; Besada, Vladimir; González, Luis Javier
2012-02-23
Growth hormone-releasing peptide 6 (GHRP-6, His-(DTrp)-Ala-Trp-(DPhe)-Lys-NH₂, MW=872.44 Da) is a potent growth hormone secretagogue that exhibits a cytoprotective effect, maintaining tissue viability during acute ischemia/reperfusion episodes in different organs like small bowel, liver and kidneys. In the present work a quantitative method to analyze GHRP-6 in human plasma was developed and fully validated following FDA guidelines. The method uses an internal standard (IS) of GHRP-6 with ¹³C-labeled Alanine for quantification. Sample processing includes a precipitation step with cold acetone to remove the most abundant plasma proteins, recovering the GHRP-6 peptide with a high yield. Quantification was achieved by LC-MS in positive full scan mode in a Q-Tof mass spectrometer. The sensitivity of the method was evaluated, establishing the lower limit of quantification at 5 ng/mL and a range for the calibration curve from 5 ng/mL to 50 ng/mL. A dilution integrity test was performed to analyze samples at higher concentration of GHRP-6. The validation process involved five calibration curves and the analysis of quality control samples to determine accuracy and precision. The calibration curves showed R² higher than 0.988. The stability of the analyte and its internal standard (IS) was demonstrated in all conditions the samples would experience in a real time analyses. This method was applied to the quantification of GHRP-6 in plasma from nine healthy volunteers participating in a phase I clinical trial. Copyright © 2011 Elsevier B.V. All rights reserved.
De Kesel, Pieter M M; Lambert, Willy E; Stove, Christophe P
2015-11-01
Caffeine is the probe drug of choice to assess the phenotype of the drug metabolizing enzyme CYP1A2. Typically, molar concentration ratios of paraxanthine, caffeine's major metabolite, to its precursor are determined in plasma following administration of a caffeine test dose. The aim of this study was to develop and validate an LC-MS/MS method for the determination of caffeine and paraxanthine in hair. The different steps of a hair extraction procedure were thoroughly optimized. Following a three-step decontamination procedure, caffeine and paraxanthine were extracted from 20 mg of ground hair using a solution of protease type VIII in Tris buffer (pH 7.5). Resulting hair extracts were cleaned up on Strata-X™ SPE cartridges. All samples were analyzed on a Waters Acquity UPLC® system coupled to an AB SCIEX API 4000™ triple quadrupole mass spectrometer. The final method was fully validated based on international guidelines. Linear calibration lines for caffeine and paraxanthine ranged from 20 to 500 pg/mg. Precision (%RSD) and accuracy (%bias) were below 12% and 7%, respectively. The isotopically labeled internal standards compensated for the ion suppression observed for both compounds. Relative matrix effects were below 15%RSD. The recovery of the sample preparation procedure was high (>85%) and reproducible. Caffeine and paraxanthine were stable in hair for at least 644 days. The effect of the hair decontamination procedure was evaluated as well. Finally, the applicability of the developed procedure was demonstrated by determining caffeine and paraxanthine concentrations in hair samples of ten healthy volunteers. The optimized and validated method for determination of caffeine and paraxanthine in hair proved to be reliable and may serve to evaluate the potential of hair analysis for CYP1A2 phenotyping. Copyright © 2015 Elsevier B.V. All rights reserved.
Dillon, Gerald Patrick; Keegan, Jason D; Wallace, Geoff; Yiannikouris, Alexandros; Moran, Colm Anthony
2018-06-01
Docosahexaenoic acid (DHA), is an omega 3 fatty acid (n-3 FA) that has been shown to play a role in canine growth and physiological integrity and improvements in skin and coat condition. However, potential adverse effects of n-3 FA specifically, impaired cellular immunity has been observed in dogs fed diets with elevated levels of n-3 FA. As such, a safe upper limit (SUL) for total n-3 FAs (DHA and EPA) in dogs has been established. Considering this SUL, sensitive methods detecting DHA in blood serum as a biomarker when conducting n-3 FA supplementation trials involving dogs are required. In this study, an LC-ESI-MS/MS method of DHA detection in dog serum was validated and verified. Recovery of DHA was optimized and parallelism tests were conducted with spiked samples demonstrating that the serum matrix did not interfere with quantitation. The stability of DHA in serum was also investigated, with -80 °C considered suitable when storing samples for up to six months. The method was linear over a calibration range of 1-500 μg/mL and precision and accuracy were found to meet the requirements for validation. This method was verified in an alternative laboratory using a different analytical system and operator, with the results meeting the criteria for verification. Copyright © 2018. Published by Elsevier Inc.
Tan, Zhirong; Ouyang, Dongsheng; Chen, Yao; Zhou, Gan; Cao, Shan; Wang, Yicheng; Peng, Xiujuan; Zhou, Honghao
2010-08-01
A sensitive and specific liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS/MS) method has been developed and validated for the identification and quantification of clebopride in human plasma using itopride as an internal standard. The method involves a simple liquid-liquid extraction. The analytes were separated by isocratic gradient elution on a CAPCELL MG-III C(18) (5 microm, 150 mm x 2.1 mm i.d.) column and analyzed in multiple reaction monitoring (MRM) mode with positive electrospray ionization (ESI) interface using the respective [M+H](+) ions, m/z 373.9-->m/z184.0 for clebopride, m/z 359.9-->m/z71.5 for itopride. The method was validated over the concentration range of 69.530-4450.0 pg/ml for clebopride. Within- and between-batch precision (RSD%) was all within 6.83% and accuracy ranged from -8.16 to 1.88%. The LLOQ was 69.530 pg/ml. The extraction recovery was on an average 77% for clebopride. The validated method was used to study the pharmacokinetics profile of clebopride in human plasma after oral administration of clebopride. Copyright 2010. Published by Elsevier B.V.
Wabuyele, Simuli L; Wald, David; Xu, Yan
2014-06-01
(-)-Securinine (SE) is a major alkaloid found in plant Securinega suffruticosa, which has a wide range of pharmacological activities including anticancer, anti-parasitic and central nervous system stimulating effects, etc. To aid the pharmacological study of SE, we developed an LC-MS/MS method for quantitative determination of SE in mouse plasma. In this method, plasma samples were first prepared with salting-out assisted liquid-liquid extraction using cold acetonitrile (-20°C) and 2.00 M ammonium acetate. Separation of SE and the internal standard (IS) from sample matrix was achieved on a Gemini Nx C18 column using 40% acetonitrile and 60% 10.0mM ammonium acetate at a flow rate of 0.200 mL min(-1). Quantification of SE was accomplished with positive electrospray ionization tandem mass spectrometry using mass transitions m/z 218.1→84.1 for SE, and m/z 204.1→70.2 for the IS. This method has a lower limit of quantitation (LLOQ) of 0.600 ng mL(-1) and a linear calibration range up to 600 ng mL(-1) in mouse plasma. The intra- and inter-run accuracy (%RE) and precision (%CV) were ≤ ± 6% and 6%, respectively. The IS normalized matrix factors from six lots of plasma matrices ranged 0.92-1.07, and the recoveries of plasma SE were 99-109%. The validated method has been applied to the measurement of SE in plasma samples of a mouse study. Copyright © 2014 Elsevier B.V. All rights reserved.
Ramisetti, Nageswara Rao; Kuntamukkala, Ramakrishna; Lakshetti, Sridhar; Sripadi, Prabhakar
2014-07-01
The current study dealt with the degradation behavior of lacosamide (LAC) under ICH prescribed stress conditions. LAC was found to be labile under acid and base hydrolytic stress conditions, while it was stable to neutral hydrolytic, oxidative, photolytic and thermal stress. In total, seven degradation products (DPs) were formed, which were separated on a C18 column using a stability-indicating method. LC-MS analyses indicated that one of the DPs had the same molecular mass as that of the drug. Structural characterization of DPs was carried out using ESI-Q-TOF-MS/MS technique. The degradation pathways and mechanisms of degradation of the drug were delineated by carrying out the degradation in different co-solvents viz. methanol, deuterated methanol, ethanol, 1-propanol and acetonitrile. The developed LC method was validated for the determination of related substances and assay of LAC as per ICH guidelines. This study demonstrates a comprehensive approach of LAC degradation studies during its development phase. Copyright © 2014. Published by Elsevier B.V.
A simple, fast and sensitive screening LC-ESI-MS/MS method for antibiotics in fish.
Guidi, Letícia Rocha; Santos, Flávio Alves; Ribeiro, Ana Cláudia S R; Fernandes, Christian; Silva, Luiza H M; Gloria, Maria Beatriz A
2017-01-15
The objective of this study was to develop and validate a fast, sensitive and simple liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) method for the screening of six classes of antibiotics (aminoglycosides, beta-lactams, macrolides, quinolones, sulfonamides and tetracyclines) in fish. Samples were extracted with trichloroacetic acid. LC separation was achieved on a Zorbax Eclipse XDB C18 column and gradient elution using 0.1% heptafluorobutyric acid in water and acetonitrile as mobile phase. Analysis was carried out in multiple reaction monitoring mode via electrospray interface operated in the positive ionization mode, with sulfaphenazole as internal standard. The method was suitable for routine screening purposes of 40 antibiotics, according to EC Guidelines for the Validation of Screening Methods for Residues of Veterinary Medicines, taking into consideration threshold value, cut-off factor, detection capability, limit of detection, sensitivity and specificity. Real fish samples (n=193) from aquaculture were analyzed and 15% were positive for enrofloxacin (quinolone), one of them at a higher concentration than the level of interest (50µgkg -1 ), suggesting possible contamination or illegal use of that antibiotic. Copyright © 2016 Elsevier B.V. All rights reserved.
LC-PROM: Validation of a patient reported outcomes measure for liver cirrhosis patients.
Zhang, Ying; Yang, Yuanyuan; Lv, Jing; Zhang, Yanbo
2016-05-10
The aim of the study is to develop a specific patient-reported scale of liver cirrhosis according to the Patient Reported Outcome guidelines of the Food and Drug Administration (FDA), and to examine its capacity to fill gaps in this field. A conceptual framework was developed and a preliminary item pool developed through literature review and interviews of 10 patients with liver cirrhosis. With the preliminary items, we performed a pilot survey that included a cognitive test with patients and interviews with experts; the focus was on content and language of the scale. In the item selection stage, seven statistical methods including discrete trends method, discrimination analysis, exploratory factor analysis, Cronbach's α coefficient, correlation coefficient, test-retest reliability, Item-Response Theory were applied to survey data from 200 subjects (150 liver cirrhosis patients and 50 controls). This produced the preliminary Liver Cirrhosis Patient-reported Outcome Measure (LC-PROM). In the next stage, we conducted the survey with 620 subjects (500 patients and 120 controls) to validate reliability, validity and acceptability of this scale. The 55 items and 13 dimensions addressed four domains: physical, psychological, social, and therapeutic. Cronbach's α coefficients were 0.921 for the total scale; the confirmatory factor analysis, t-tests and ANOVA supported scale validity; the model fit index as Root Mean Square Error of Approximation (RMSEA), Root Mean Square Residual (RMR), Normed Fit Index (NFI), Non-Normed Fit Index (NNFI), Comparative Fit Index (CFI) and Incremental Fit Index (IFI) met the criterion generally. The acceptance ratio and response rate indicated good feasibility. This study developed an accurate and stable patient-reported outcome scale of liver cirrhosis, which is able to evaluate clinical effects effectively, is helpful to patients in recognizing their health condition, and contributes to clinical decision making both for patients and
Liu, Huaming; Zhang, Dandan; Tang, Zhidan; Sun, Mengjie; Azietaku, John Teye; Ouyang, Huizi; Chang, Yanxu; Wang, Meng; He, Jun; Gao, Xiumei
2018-05-29
Periplocin is a cardiac glycoside and has been used widely in the clinic for its cardiotonic, anti-inflammatory and anti-tumor effects. Though it was taken frequently by oral administration in clinic, there were no reports demonstrated that periplocin could be detected in vivo after an oral administration of periplocin, so it is badly need of searching the characteristic of periplocin in vivo after an oral administration. In this study, a sensitive and reliable liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated to identify and quantify periplocin and its two metabolites in rat tissue after a single dosage of perplocin at 50 mg/kg. The results demonstrated that periplocin and its two metabolites were detected in all of the selected tissues, periplocin could be reach peak concentration quickly after administration, while periplocymarin and periplogenin reached maximum concentration more than 4.83 h after administration. The tissue distribution of analytes tended to be mostly in the liver, and higher analytes concentrations were found in the heart, liver, spleen, lung, kidney, but a small amount of chemical constituents were distributed into the brain. The consequences obtained using this method might provide a meaningful insight for the clinical investigations and applications. This article is protected by copyright. All rights reserved.
Andersen, Wendy C; Casey, Christine R; Schneider, Marilyn J; Turnipseed, Sherri B
2015-01-01
Prior to conducting a collaborative study of AOAC First Action 2012.25 LC-MS/MS analytical method for the determination of residues of three triphenylmethane dyes (malachite green, crystal violet, and brilliant green) and their metabolites (leucomalachite green and leucocrystal violet) in seafood, a single-laboratory validation of method 2012.25 was performed to expand the scope of the method to other seafood matrixes including salmon, catfish, tilapia, and shrimp. The validation included the analysis of fortified and incurred residues over multiple weeks to assess analyte stability in matrix at -80°C, a comparison of calibration methods over the range 0.25 to 4 μg/kg, study of matrix effects for analyte quantification, and qualitative identification of targeted analytes. Method accuracy ranged from 88 to 112% with 13% RSD or less for samples fortified at 0.5, 1.0, and 2.0 μg/kg. Analyte identification and determination limits were determined by procedures recommended both by the U. S. Food and Drug Administration and the European Commission. Method detection limits and decision limits ranged from 0.05 to 0.24 μg/kg and 0.08 to 0.54 μg/kg, respectively. AOAC First Action Method 2012.25 with an extracted matrix calibration curve and internal standard correction is suitable for the determination of triphenylmethane dyes and leuco metabolites in salmon, catfish, tilapia, and shrimp by LC-MS/MS at a residue determination level of 0.5 μg/kg or below.
Ring, P R; Bostick, J M
2000-04-01
A sensitive and selective high-performance liquid chromatography (HPLC) method was developed for the determination of zolpidem in human plasma. Zolpidem and the internal standard (trazodone) were extracted from human plasma that had been made basic. The basic sample was loaded onto a conditioned Bond Elut C18 cartridge, rinsed with water and eluted with methanol. Forty microliters were then injected onto the LC system. Separation was achieved on a C18 column (150 x 4.6 mm, 5 microm) with a mobile phase composed of acetonitrile:50 mM potassium phosphate monobasic at pH 6.0 (4:6, v/v). Detection was by fluorescence, with excitation at 254 nm and emission at 400 nm. The retention times of zolpidem and internal standard were approximately 4.7 and 5.3 min, respectively. The LC run time was 8 min. The assay was linear in concentration range 1-400 ng/ml for zolpidem in human plasma. The analysis of quality control samples for zolpidem (3, 30, and 300 ng/ml) demonstrated excellent precision with relative standard deviations (RSD) of 3.7, 4.6, and 3.0%, respectively (n = 18). The method was accurate with all intraday (n = 6) and overall (n = 18) mean concentrations within 5.8% from nominal at all quality control sample concentrations. This method was also performed using a Gilson Aspec XL automated sample processor and autoinjector. The samples were manually fortified with internal standard and made basic. The aspec then performed the solid phase extraction and made injections of the samples onto the LC system. Using the automated procedure for analysis, quality control samples for zolpidem (3, 30, and 300 ng/ml) demonstrated acceptable precision with RSD values of 9.0, 4.9, and 5.1%, respectively (n = 12). The method was accurate with all intracurve (n = 4) and overall (n = 12) mean values being less than 10.8% from nominal at all quality control sample concentrations.
2014-01-01
Background Yakuchinone A has a plethora of beneficial biological effects. However, the pharmacokinetic (PK) data of yakuchinone A still remain unknown so far. Furthermore, the quantification of yakuchinone A in biological samples has not been reported in the literature. Therefore, in the present study we aimed to develop a new method for the fast, efficient and accurate assessment of yakuchinone A concentration in plasma, as a means for facilitating the PK evaluation of yakuchinone A. Results A liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) method was developed and validated for the determination of yakuchinone A in rat plasma. Mass spectrometric and chromatographic conditions were optimized. Plasma samples were pretreated by protein precipitation with methanol. LC separation was performed on a Phenomenex Luna C18 column with gradient elution using a mobile phase consisting of methanol–water containing 0.5 mM formic acid (HCOOH) at a flow rate of 0.28 mL/min. ESI-MS spectra were acquired in positive ion multiple reaction monitoring mode (MRM). The precursor-to-product ion pairs used for MRM of yakuchinone A and yakuchinone B were m/z 313.1 → 137.0 and 311.2 → 117.1, respectively. Low concentration of HCOOH reduced the ion suppression caused by matrix components and clearly improved the analytical sensitivity. Yakuchinone A showed good linearity over a wide concentration range (r > 0.99). The accuracy, precision, stability and linearity were found to be within the acceptable criteria. This new method was successfully applied to analyze the rat plasma concentration of parent yakuchinone A after a single oral administration of SuoQuan capsules. Low systemic exposure to parent yakuchinone A was observed. Conclusion The proposed method is sensitive and reliable. It is hoped that this new method will prove useful for the future PK studies. PMID:24422995
Maas, Alexandra; Maier, Christoph; Iwersen-Bergmann, Stefanie; Madea, Burkhard; Hess, Cornelius
2017-11-30
Besides its clinical application, the anaesthetic agent propofol is being increasingly misused, mostly by healthcare professionals, and its abuse potential gained worldwide attention after the tragic death of Michael Jackson in 2009. Due to the short duration of its narcotic effects, propofol abuse is especially easy to hide compared with the use of other recreational drugs. However, propofol possesses a very narrow therapeutic window between the desired effect and potentially fatal toxicity, making abuse of the drug extremely dangerous even in experienced physicians. Consequently, it is important that forensic laboratories possess a sensitive and specific method for the detection of chronic propofol abuse. We present a simple, fast and reliable method to simultaneously extract propofol and its main metabolite propofol glucuronide from hair, followed by sensitive LC-MS/MS analyses, allowing to determine a chronic propofol abuse. Difficulties regarding the detection of propofol using LC-MS/MS were solved by using a derivatization reaction with 2-fluoro-1-methylpyridinium-p-toluene-sulfonate and triethylamine. Reliability of extraction method and subsequent LC-MS/MS analyses was confirmed under consideration of the validation parameters selectivity, linearity, accuracy and precision, analytical limits, processed sample stability, matrix effects and recovery. Appropriate quantification (LLOQ=10pg/mg hair) and detection limits (3.6pg/mg hair for propofol and 7.8 pg/mg hair for propofol glucuronide) could be achieved, enabling to detect even small amounts of both analytes. Applicability of the method was confirmed by analysis of three human hair samples from deceased with suspicion of chronic propofol abuse. Copyright © 2017 Elsevier B.V. All rights reserved.
Neiens, Patrick; De Simone, Angela; Ramershoven, Anna; Höfner, Georg; Allmendinger, Lars; Wanner, Klaus T
2018-03-03
MS Binding Assays represent a label-free alternative to radioligand binding assays. In this study, we present an LC-ESI-MS/MS method for the quantification of (R,R)-4-(2-benzhydryloxyethyl)-1-(4-fluorobenzyl)piperidin-3-ol [(R,R)-D-84, (R,R)-1], (S,S)-reboxetine [(S,S)-2], and (S)-citalopram [(S)-3] employed as highly selective nonlabeled reporter ligands in MS Binding Assays addressing the dopamine [DAT, (R,R)-D-84], norepinephrine [NET, (S,S)-reboxetine] and serotonin transporter [SERT, (S)-citalopram], respectively. The developed LC-ESI-MS/MS method uses a pentafluorphenyl stationary phase in combination with a mobile phase composed of acetonitrile and ammonium formate buffer for chromatography and a triple quadrupole mass spectrometer in the multiple reaction monitoring mode for mass spectrometric detection. Quantification is based on deuterated derivatives of all three analytes serving as internal standards. The established LC-ESI-MS/MS method enables fast, robust, selective and highly sensitive quantification of all three reporter ligands in a single chromatographic run. The method was validated according to the Center for Drug Evaluation and Research (CDER) guideline for bioanalytical method validation regarding selectivity, accuracy, precision, calibration curve and sensitivity. Finally, filtration-based MS Binding Assays were performed for all three monoamine transporters based on this LC-ESI-MS/MS quantification method as read out. The affinities determined in saturation experiments for (R,R)-D-84 toward hDAT, for (S,S)-reboxetine toward hNET, and for (S)-citalopram toward hSERT, respectively, were in good accordance with results from literature, clearly demonstrating that the established MS Binding Assays have the potential to be an efficient alternative to radioligand binding assays widely used for this purpose so far. Copyright © 2018 John Wiley & Sons, Ltd.
Validated enantiospecific LC method for determination of (R)-enantiomer impurity in (S)-efavirenz.
Seshachalam, U; Narasimha Rao, D V L; Chandrasekhar, K B
2008-02-01
A high-performance liquid chromatographic method was developed for separation of the enantiomers of efavirenz. The developed method was applied for the determination of (R)-enantiomer in (S)-efavirenz and satisfactory results were achieved. The base line separation with a resolution of more than 4.0 was achieved on Chiralcel OD (250 mm x 4.6 mm, 10 microm) column containing tris-(3,5-dimethylphenylcarbomate) as stationary phase. The mobile phase consists of n-hexane: isopropyl alcohol (80:20 v/v) with 0.1% (v/v) of formic acid as additive. The flow rate was kept at 1.0 ml/min and the UV detection was monitored at 254 nm. The (R)-enantiomer was found linear over the range of 0.1 microg/ml--6 microg/ml. The limit of detection (LOD) was 0.03 microg/ml and the limit of quantification (LOQ) was 0.1 microg/ml (n=3. The precision of (R)-enantiomer at LOQ level was evaluated through six replicate injections and the RSD of the peak response was achieved as 1.34%. The results demonstrated that the developed LC method was simple, precise, robust and applicable for the purity determination of efavirenz.
Kumar, Thangarathinam; Ramya, Mohandass; Srinivasan, Viswanathan; Xavier, N
2017-08-01
Hydroxylamine is a known genotoxic impurity compound that needs to be controlled down to ppm level in pharmaceutical processes. It is difficult to detect using conventional analytical techniques due to its physio-chemical properties like lack of chromophore, low molecular weight, absence of carbon atom and high polarity. In addition to that, analysis of the pharmaceutical samples encounters considerable obstruction from matrix components that greatly overshadow the response of hydroxylamine. This study describes a simple, sensitive and direct Liquid Chromatographic-Mass Spectrometric method (LC-MS) for detection of hydroxylamine in pharmaceutical compounds. The LC-MS method was detected up to 0.008 ppm of hydroxylamine with S/N > 3.0 and quantified up to 0.025 ppm of hydroxylamine with S/N ratio >10.0. This validated method can be applied as a generic method to detect the hydroxylamine for pharmaceutical process control and drug substance release. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.
Identification of Forced Degradation Products of Itopride by LC-PDA and LC-MS.
Joshi, Payal; Bhoir, Suvarna; Bhagwat, A M; Vishwanath, K; Jadhav, R K
2011-05-01
Degradation products of itopride formed under different forced conditions have been identified using LC-PDA and LC-MS techniques. Itopride was subjected to forced degradation under the conditions of hydrolysis, photolysis, oxidation, dry and wet heat, in accordance with the International Conference on Harmonization. The stress solutions were chromatographed on reversed phase C18 (250×4.6 mm, 5 μm) column with a mobile phase methanol:water (55:45, v/v) at a detection wavelength of 215 nm. Itopride degraded in acid, alkali and oxidative stress conditions. The stability indicating method was developed and validated. The degradation pathway of the drug to products II-VIII is proposed.
Identification of Forced Degradation Products of Itopride by LC-PDA and LC-MS
Joshi, Payal; Bhoir, Suvarna; Bhagwat, A. M.; Vishwanath, K.; Jadhav, R. K.
2011-01-01
Degradation products of itopride formed under different forced conditions have been identified using LC-PDA and LC-MS techniques. Itopride was subjected to forced degradation under the conditions of hydrolysis, photolysis, oxidation, dry and wet heat, in accordance with the International Conference on Harmonization. The stress solutions were chromatographed on reversed phase C18 (250×4.6 mm, 5 μm) column with a mobile phase methanol:water (55:45, v/v) at a detection wavelength of 215 nm. Itopride degraded in acid, alkali and oxidative stress conditions. The stability indicating method was developed and validated. The degradation pathway of the drug to products II-VIII is proposed. PMID:22457552
EL-Houssini, Ola M.; Zawilla, Nagwan H.; Mohammad, Mohammad A.
2013-01-01
Specific stability indicating reverse-phase liquid chromatography (RP-LC) assay method (SIAM) was developed for the determination of cinnarizine (Cinn)/piracetam (Pira) and cinnarizine (Cinn)/heptaminol acefyllinate (Hept) in the presence of the reported degradation products of Cinn. A C18 column and gradient mobile phase was applied for good resolution of all peaks. The detection was achieved at 210 nm and 254 nm for Cinn/Pira and Cinn/Hept, respectively. The responses were linear over concentration ranges of 20–200, 20–1000 and 25–1000 μgmL−1 for Cinn, Pira, and Hept respectively. The proposed method was validated for linearity, accuracy, repeatability, intermediate precision, and robustness via statistical analysis of the data. The method was shown to be precise, accurate, reproducible, sensitive, and selective for the analysis of Cinn/Pira and Cinn/Hept in laboratory prepared mixtures and in pharmaceutical formulations. PMID:24137049
Comparison of ESI- and APCI-LC-MS/MS methods: A case study of levonorgestrel in human plasma.
Wang, Rulin; Zhang, Lin; Zhang, Zunjian; Tian, Yuan
2016-12-01
Electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) techniques for liquid chromatography-tandem mass spectrometry (LC-MS/MS) determination of levonorgestrel were evaluated. In consideration of difference in ionization mechanism, the two ionization sources were compared in terms of LC conditions, MS parameters and performance of method. The sensitivity for detection of levonorgestrel with ESI was 0.25 ng/mL which was lower than 1 ng/mL with APCI. Matrix effects were evaluated for levonorgestrel and canrenone (internal standard, IS) in human plasma, and the results showed that APCI source appeared to be slightly less liable to matrix effect than ESI source. With an overall consideration, ESI was chosen as a better ionization technique for rapid and sensitive quantification of levonorgestrel. The optimized LC-ESI-MS/MS method was validated for a linear range of 0.25-50 ng/mL with a correlation coefficient ≥0.99. The intra- and inter-batch precision and accuracy were within 11.72% and 6.58%, respectively. The application of this method was demonstrated by a bioequivalence study following a single oral administration of 1.5 mg levonorgestrel tablets in 21 Chinese healthy female volunteers.
Satyanarayana Raju, T; Vishweshwari Kutty, O; Ganesh, V; Yadagiri Swamy, P
2012-08-01
Although a number of methods are available for evaluating Linezolid and its possible impurities, a common method for separation if its potential impurities, degradants and enantiomer in a single method with good efficiency remain unavailable. With the objective of developing an advanced method with shorter runtimes, a simple, precise, accurate stability-indicating LC method was developed for the determination of purity of Linezolid drug substance and drug products in bulk samples and pharmaceutical dosage forms in the presence of its impurities and degradation products. This method is capable of separating all the related substances of Linezolid along with the chiral impurity. This method can also be used for the estimation of assay of Linezolid in drug substance as well as in drug product. The method was developed using Chiralpak IA (250 mm×4.6 mm, 5 μm) column. A mixture of acetonitrile, ethanol, n-butyl amine and trifluoro acetic acid in 96:4:0.10:0.16 (v/v/v/v) ratio was used as a mobile phase. The eluted compounds were monitored at 254 nm. Linezolid was subjected to the stress conditions of oxidative, acid, base, hydrolytic, thermal and photolytic degradation. The degradation products were well resolved from main peak and its impurities, proving the stability-indicating power of the method. The developed method was validated as per International Conference on Harmonization (ICH) guidelines with respect to specificity, limit of detection, limit of quantification, precision, linearity, accuracy, robustness and system suitability.
Seraglio, Siluana Katia Tischer; Valese, Andressa Camargo; Daguer, Heitor; Bergamo, Greici; Azevedo, Mônia Stremel; Gonzaga, Luciano Valdemiro; Fett, Roseane; Costa, Ana Carolina Oliveira
2016-09-01
A simple, reproducible and sensitive method has been optimized and validated for simultaneous determination of 32 phenolic compounds in bracatinga (Mimosa scabrella Benth.) with the diluted-and-shoot approach, without the need of any additional clean-up steps. It has been based on high performance liquid chromatography coupled with triple quadrupole tandem mass spectrometry and electrospray ionization (HPLC-ESI-MS/MS). The chromatography conditions were optimized, and due to the selectivity provided by MRM monitoring, LC separation required only 9min. The developed method was validated on the basis of Eurachem and European Commission Decision 2002/657/EC guidelines. Mean recoveries ranged from 70.4 to 110%. Intra-day and inter-day precision with RSD (relative standard deviations) from 0.14 to 18.9% and 0.34 to 20.0%, respectively were achieved. Limits of detection (LOD) and quantification (LOQ) ranged from 0.03 to 3.20μgL -1 and 0.20-12.8μgL -1 . Finally, the method was applied to samples and 20 phenolic compounds were quantified in all the samples analyzed, representing a contribution to the characterization and quantification of phenolic compounds from bracatinga (M. scabrella Bentham) honeydew honey. Copyright © 2016 Elsevier Ltd. All rights reserved.
Carter, Melissa D; Calcutt, M Wade; Malow, Beth A; Rose, Kristie L; Hachey, David L
2012-03-01
Melatonin (MEL) and its chemical precursor N-acetylserotonin (NAS) are believed to be potential biomarkers for sleep-related disorders. Measurement of these compounds, however, has proven to be difficult due to their low circulating levels, especially that of NAS. Few methods offer the sensitivity, specificity and dynamic range needed to monitor MEL and its precursors and metabolites in small blood samples, such as those obtained from pediatric patients. In support of our ongoing study to determine the safety, tolerability and PK dosing strategies for MEL in treating insomnia in children with autism spectrum disorder, two highly sensitive LC-MS/MS assays were developed for the quantitation of MEL and precursor NAS at pg/mL levels in small volumes of human plasma. A validated electrospray ionization (ESI) method was used to quantitate high levels of MEL in PK studies, and a validated nanospray (nESI) method was developed for quantitation of MEL and NAS at endogenous levels. In both assays, plasma samples were processed by centrifugal membrane dialysis after addition of stable isotopic internal standards, and the components were separated by either conventional LC using a Waters SymmetryShield RP18 column (2.1 × 100 mm, 3.5 µm) or on a polyimide-coated, fused-silica capillary self-packed with 17 cm AquaC18 (3 µm, 125 Å). Quantitation was done using the SRM transitions m/z 233 → 174 and m/z 219 → 160 for MEL and NAS, respectively. The analytical response ratio versus concentration curves were linear for MEL (nanoflow LC: 11.7-1165 pg/mL, LC: 1165-116,500 pg/mL) and for NAS (nanoflow LC: 11.0-1095 pg/mL). Copyright © 2012 John Wiley & Sons, Ltd.
Carter, Melissa D.; Calcutt, M. Wade; Malow, Beth A.; Rose, Kristie L.; Hachey, David L.
2012-01-01
Melatonin (MEL) and its chemical precursor N-acetylserotonin (NAS) are believed to be potential biomarkers for sleep-related disorders. Measurement of these compounds, however, has proven to be difficult due to their low circulating levels, especially that of NAS. Few methods offer the sensitivity, specificity and dynamic range needed to monitor MEL and its precursors and metabolites in small blood samples, such as those obtained from pediatric patients. In support of our ongoing study to determine the safety, tolerability, and PK dosing strategies for MEL in treating insomnia in children with autism spectrum disorder, two highly sensitive LC-MS/MS assays were developed for the quantitation of MEL and precursor NAS at pg/mL levels in small volumes of human plasma. A validated electrospray ionization (ESI) method was used to quantitate high levels of MEL in PK studies and a validated nanospray (nESI) method was developed for quantitation of MEL and NAS at endogenous levels. In both assays plasma samples were processed by centrifugal membrane dialysis after addition of stable isotopic internal standards, and the components were separated by either conventional LC using a Waters SymmetryShield RP18 column (2.1×100 mm, 3.5 μm) or on a polyimide-coated, fused-silica capillary self-packed with 17 cm AquaC18 (3 μm, 125 Å). Quantitation was done using the SRM transitions m/z 233→174 and m/z 219→160 for MEL and NAS, respectively. The analytical response ratio vs. concentration curves were linear for MEL (nanoflow LC: 11.7–1165 pg/mL, LC: 1165–116500 pg/mL) and for NAS (nanoflow LC: 11.0–1095 pg/mL). PMID:22431453
Sanghvi, M; Ramamoorthy, A; Strait, J; Wainer, I W; Moaddel, R
2013-08-15
Due to the lack of sensitivity in current methods for the determination of fenoterol (Fen), a rapid LC-MS/MS method was developed for the determination of (R,R')-Fen and (R,R';S,S')-Fen in plasma and urine. The method was fully validated and was linear from 50pg/ml to 2000pg/ml for plasma and from 2.500ng/ml to 160ng/ml for urine with a lower limit of quantitation of 52.8pg/ml in plasma. The coefficient of variation was <15% for the high QC standards and <10% for the low QC standards in plasma and was <15% for the high and low QC standards in urine. The relative concentrations of (R,R')-Fen and (S,S')-Fen were determined using a chirobiotic T chiral stationary phase. The method was used to determine the concentration of (R,R')-Fen in plasma and urine samples obtained in an oral cross-over study of (R,R')-Fen and (R,R';S,S')-Fen formulations. The results demonstrated a potential pre-systemic enantioselective interaction in which the (S,S')-Fen reduces the sulfation of the active (R,R')-Fen. The data suggest that a non-racemic mixture of the Fen enantiomers may provide better bioavailability of the active (R,R')-Fen for use in the treatment of cardiovascular disease. Published by Elsevier B.V.
Sanghvi, M.; Ramamoorthy, A.; Strait, J.; Wainer, I. W.; Moaddel, R.
2013-01-01
Due to the lack of sensitivity in current methods for the determination of fenoterol (Fen). A rapid, LC-MS/MS method was developed for the determination of (R,R′)-Fen and (R,R′;S,S′)-Fen in plasma and urine. The method was fully validated and was linear from 50 pg/ml to 2000 pg/ml for plasma and from 2.500 ng/ml to 160 ng/ml for urine with a lower limit of quantitation of 52.8 pg/ml in plasma. The coefficient of variation was <15% for the high QC standards and <10% for the low QC standards in plasma and was <15% for the high and low QC standards in urine. The relative concentrations of (R,R′)-Fen and (S,S′)-Fen were determined using a chirobiotic T chiral stationary phase. The method was used to determine the concentration of (R,R′)-Fen in plasma and urine samples obtained in an oral cross-over study of (R,R′)-Fen and (R,R′;S,S′)-Fen formulations. The results demonstrated a potential pre-systemic enantioselective interaction in which the (S,S′)-Fen reduces the sulfation of the active (R,R′)-Fen. The data suggests that a non-racemic mixture of the Fen enantiomers may provide better bioavailability of the active (R,R′)-Fen for use in the treatment of cardiovascular disease PMID:23872161
Magotra, Asmita; Sharma, Anjna; Gupta, Ajai Prakash; Wazir, Priya; Sharma, Shweta; Singh, Parvinder Pal; Tikoo, Manoj Kumar; Vishwakarma, Ram A; Singh, Gurdarshan; Nandi, Utpal
2017-08-15
In the present study, a simple, sensitive, specific and rapid liquid chromatography (LC) tandem mass spectrometry (MS/MS) method was developed and validated according to the Food and Drug Administration (FDA) guidelines for estimation of IIIM-MCD-211 (a potent oral candidate with promising action against tuberculosis) in mice plasma using carbamazepine as internal standard (IS). Bioanalytical method consisted of one step protein precipitation for sample preparation followed by quantitation in LC-MS/MS using positive electrospray ionization technique (ESI) operating in multiple reaction monitoring (MRM) mode. Elution was achieved in gradient mode on High Resolution Chromolith RP-18e column with mobile phase comprised of acetonitrile and 0.1% (v/v) formic acid in water at the flow rate of 0.4mL/min. Precursor to product ion transitions (m/z 344.5/218.4 and m/z 237.3/194.2) were used to measure analyte and IS, respectively. All validation parameters were well within the limit of acceptance criteria. The method was successfully applied to assess the pharmacokinetics of the candidate in mice following oral (10mg/kg) and intravenous (IV; 2.5mg/kg) administration. It was also effectively used to quantitate metabolic stability of the compound in mouse liver microsomes (MLM) and human liver microsomes (HLM) followed by its in-vitro-in-vivo extrapolation. Copyright © 2017 Elsevier B.V. All rights reserved.
Vacondio, Federica; Silva, Claudia; Fioni, Alessandro; Mor, Marco; Rivara, Mirko; Bordi, Fabrizio; Flammini, Lisa; Ballabeni, Vigilio; Barocelli, Elisabetta
2008-01-07
A rapid, simple and sensitive liquid chromatography-mass spectrometry (LC-MS) method was developed and validated for the determination of the imidazole H(3) antagonist ROS203 in rat plasma, using the superior homologue ROS287 as internal standard. Analyses were performed on an Agilent 1100 Series HPLC system employing a Supelco Ascentis C(18) column and isocratic elution with acetonitrile-10mM ammonium acetate buffer pH 4.0 (30:70, v/v) at a flow rate of 0.25 mL/min. An Applied Biosystems/MDS Sciex 150-EX single quadrupole mass spectrometer, equipped with an electrospray ionization interface was employed, operating in the positive ion mode. Plasma samples were deproteinized with acetonitrile (1:2), evaporated under nitrogen stream, reconstituted in the mobile phase and 5 microL were injected into the system. The retention times of ROS203 and IS were 2.20 and 2.90 min, respectively. Calibration curves in spiked plasma were linear over the concentration range of 2610-2.61 ng/mL with determination coefficients >0.99. The lower limit of quantification (LLOQ) was 2.61 ng/mL. The accuracy of the method was within 15%. Intra- and inter-day relative standard deviations were less or equal to 9.50% or 7.19%, respectively. The applicability of the LC-MS method was tested employing plasma samples obtained after i.p. administration of ROS203 to female Wistar rats to support a behavioral in vivo study. The specificity of the method was confirmed by the absence of interferences from endogenous substances. The reported method can provide the necessary sensitivity, linearity, precision, accuracy and specificity to allow the determination of ROS203 in rat plasma samples to support further pharmacokinetic assays.
A new LC-MS/MS bioanalytical method for atenolol in human plasma and milk.
Phyo Lwin, Ei Mon; Gerber, Cobus; Song, Yunmei; Leggett, Catherine; Ritchie, Usha; Turner, Sean; Garg, Sanjay
2017-04-01
A new sensitive LC-MS/MS method for the quantification of atenolol in human plasma and milk has been developed for clinical lactation studies. Atenolol and the internal standard, phenazone, were extracted from biological matrices by protein precipitation. A Phenomenex ® C-18 column and gradient chromatographic conditions were used for separation of the analyte, followed by detection with MS. Stability of samples was confirmed for atenolol in human plasma and milk for up to 3 months. Linearity range of 1-800 ng/ml (r 2 = 0.9995), the precision within 15% CV and the recovery of the analyte (80-100% range) were achieved. A new validated analytical method for atenolol in plasma and milk was developed.
Ediage, Emmanuel Njumbe; Di Mavungu, José Diana; Monbaliu, Sofie; Van Peteghem, Carlos; De Saeger, Sarah
2011-05-25
This study was designed to develop a sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the simultaneous detection and quantification of 25 mycotoxins in cassava flour, peanut cake and maize samples with particular focus on the optimization of the sample preparation protocol and method validation. All 25 mycotoxins were extracted in a single step with a mixture of methanol/ethyl acetate/water (70:20:10, v/v/v). The method limits of quantification (LOQ) varied from 0.3 μg/kg to 106 μg/kg. Good precision and linearity were observed for most of the mycotoxins. The method was applied for the analysis of naturally contaminated peanut cake, cassava flour and maize samples from the Republic of Benin. All samples analyzed (fifteen peanut cakes, four maize flour and four cassava flour samples) tested positive for one or more mycotoxins. Aflatoxins (total aflatoxins; 10-346 μg/kg) and ochratoxin A (
Kakiyama, Genta; Muto, Akina; Takei, Hajime; Nittono, Hiroshi; Murai, Tsuyoshi; Kurosawa, Takao; Hofmann, Alan F.; Pandak, William M.; Bajaj, Jasmohan S.
2014-01-01
We have developed a simple and accurate HPLC method for measurement of fecal bile acids using phenacyl derivatives of unconjugated bile acids, and applied it to the measurement of fecal bile acids in cirrhotic patients. The HPLC method has the following steps: 1) lyophilization of the stool sample; 2) reconstitution in buffer and enzymatic deconjugation using cholylglycine hydrolase/sulfatase; 3) incubation with 0.1 N NaOH in 50% isopropanol at 60°C to hydrolyze esterified bile acids; 4) extraction of bile acids from particulate material using 0.1 N NaOH; 5) isolation of deconjugated bile acids by solid phase extraction; 6) formation of phenacyl esters by derivatization using phenacyl bromide; and 7) HPLC separation measuring eluted peaks at 254 nm. The method was validated by showing that results obtained by HPLC agreed with those obtained by LC-MS/MS and GC-MS. We then applied the method to measuring total fecal bile acid (concentration) and bile acid profile in samples from 38 patients with cirrhosis (17 early, 21 advanced) and 10 healthy subjects. Bile acid concentrations were significantly lower in patients with advanced cirrhosis, suggesting impaired bile acid synthesis. PMID:24627129
Park, Ah Yeon; Park, So-Young; Lee, Jaehyun; Jung, Mihye; Kim, Jinwoong; Kang, Sam Sik; Youm, Jeong-Rok; Han, Sang Beom
2009-10-01
Rapid, simple and reliable HPLC/UV and LC-ESI-MS/MS methods for the simultaneous determination of five active coumarins of Angelicae dahuricae Radix, byakangelicol (1), oxypeucedanin (2), imperatorin (3), phellopterin (4) and isoimperatorin (5) were developed and validated. The separation condition for HPLC/UV was optimized using a Develosil RPAQUEOUS C(30) column using 70% acetonitrile in water as the mobile phase. This HPLC/UV method was successful for providing the baseline separation of the five coumarins with no interfering peaks detected in the 70% ethanol extract of Angelicae dahuricae Radix. The specific determination of the five coumarins was also accomplished by a triple quadrupole tandem mass spectrometer equipped with an electrospray ionization source (LC-ESI-MS/MS). Multiple reaction monitoring (MRM) in the positive mode was used to enhance the selectivity of detection. The LC-ESI-MS/MS methods were successfully applied for the determination of the five major coumarins in Angelicae dahuricae Radix. These HPLC/UV and LC-ESI-MS/MS methods were validated in terms of recovery, linearity, accuracy and precision (intra- and inter-day validation). Taken together, the shorter analysis time involved makes these HPLC/UV and LC-ESI-MS/MS methods valuable for the commercial quality control of Angelicae dahuricae Radix extracts and its pharmaceutical preparations. Copyright (c) 2009 John Wiley & Sons, Ltd.
Gören, Ahmet C; Bilsel, Gökhan; Şimşek, Adnan; Bilsel, Mine; Akçadağ, Fatma; Topal, Kevser; Ozgen, Hasan
2015-05-15
High Performance Liquid Chromatography LC-UV and LC-MS/MS methods were developed and validated for quantitative analyses of sodium benzoate and potassium sorbate in foods and beverages. HPLC-UV and LC-MS/MS methods were compared for quantitative analyses of sodium benzoate and potassium sorbate in a representative ketchup sample. Optimisation of the methods enabled the chromatographic separation of the analytes in less than 4 min. A correlation coefficient of 0.999 was achieved over the measured calibration range for both compounds and methods (HPLC and LC-MS/MS). The uncertainty values of sodium benzoate and potassium sorbate were found as 0.199 and 0.150 mg/L by HPLC and 0.072 and 0.044 mg/L by LC-MS/MS, respectively. Proficiency testing performance of Turkish accredited laboratories between the years 2005 and 2013 was evaluated and reported herein. The aim of the proficiency testing scheme was to evaluate the performance of the laboratories, analysing benzoate and sorbate in tomato ketchup. Copyright © 2014 Elsevier Ltd. All rights reserved.
Maier, Barbara; Vogeser, Michael
2013-04-01
Isotope dilution LC-MS/MS methods used in the clinical laboratory typically involve multi-point external calibration in each analytical series. Our aim was to test the hypothesis that determination of target analyte concentrations directly derived from the relation of the target analyte peak area to the peak area of a corresponding stable isotope labelled internal standard compound [direct isotope dilution analysis (DIDA)] may be not inferior to conventional external calibration with respect to accuracy and reproducibility. Quality control samples and human serum pools were analysed in a comparative validation protocol for cortisol as an exemplary analyte by LC-MS/MS. Accuracy and reproducibility were compared between quantification either involving a six-point external calibration function, or a result calculation merely based on peak area ratios of unlabelled and labelled analyte. Both quantification approaches resulted in similar accuracy and reproducibility. For specified analytes, reliable analyte quantification directly derived from the ratio of peak areas of labelled and unlabelled analyte without the need for a time consuming multi-point calibration series is possible. This DIDA approach is of considerable practical importance for the application of LC-MS/MS in the clinical laboratory where short turnaround times often have high priority.
Assessment of data pre-processing methods for LC-MS/MS-based metabolomics of uterine cervix cancer.
Chen, Yanhua; Xu, Jing; Zhang, Ruiping; Shen, Guoqing; Song, Yongmei; Sun, Jianghao; He, Jiuming; Zhan, Qimin; Abliz, Zeper
2013-05-07
A metabolomics strategy based on rapid resolution liquid chromatography/tandem mass spectrometry (RRLC-MS/MS) and multivariate statistics has been implemented to identify potential biomarkers in uterine cervix cancer. Due to the importance of the data pre-processing method, three popular software packages have been compared. Then they have been used to acquire respective data matrices from the same LC-MS/MS data. Multivariate statistics was subsequently used to identify significantly changed biomarkers for uterine cervix cancer from the resulting data matrices. The reliabilities of the identified discriminated metabolites have been further validated on the basis of manually extracted data and ROC curves. Nine potential biomarkers have been identified as having a close relationship with uterine cervix cancer. Considering these in combination as a biomarker group, the AUC amounted to 0.997, with a sensitivity of 92.9% and a specificity of 95.6%. The prediction accuracy was 96.6%. Among these potential biomarkers, the amounts of four purine derivatives were greatly decreased, which might be related to a P2 receptor that might lead to a decrease in cell number through apoptosis. Moreover, only two of them were identified simultaneously by all of the pre-processing tools. The results have demonstrated that the data pre-processing method could seriously bias the metabolomics results. Therefore, application of two or more data pre-processing methods would reveal a more comprehensive set of potential biomarkers in non-targeted metabolomics, before a further validation with LC-MS/MS based targeted metabolomics in MRM mode could be conducted.
NASA Astrophysics Data System (ADS)
Al-Qaim, Fouad Fadhil; Abdullah, Md Pauzi; Othman, Mohamed Rozali
2013-11-01
In this work, a developed method using solid - phase extraction (SPE) followed by liquid chromatography - time of flight mass spectrometry (LC-ESI-TOF/MS) was developed and validated for quantification and confirmation of eleven pharmaceuticals with different therapeutic classes in water samples, Malaysia. These compounds are caffeine (CAF), prazosin (PRZ), enalapril (ENL), carbamazepine (CBZ), nifedipine (NFD), levonorgestrel (LNG), simvastatin (SMV), hydrochlorothiazide (HYD), gliclazide (GLIC), diclofenac-Na (DIC-Na) and mefenamic acid (MEF). LC was performed on a Dionex Ultimate 3000/LC 09115047 (USA) system. Chromatography was performed on a Thermo Scientific C18 (250 mm × 2.1 mm, i.d.: 5μm) column. Several parameters were optimised such as; mobile phase, gradient elution, collision energy and solvent elution for extraction of compounds from water. The recoveries obtained ranged from 30-148 % in river water. Five pharmaceutical compounds were detected in the surface water samples: caffeine, prazosin, enalpril, diclofenac-Na and mefenamic acid. The developed method is precise and accepted recoveries were got. In addition, this method is suitable to identify and quantify trace concentrations of pharmaceuticals in surface water.
Simultaneous analysis of 17 diuretics in dietary supplements by HPLC and LC-MS/MS.
Woo, H; Kim, J W; Han, K M; Lee, J H; Hwang, I S; Lee, J H; Kim, J; Kweon, S J; Cho, S; Chae, K R; Han, S Y; Kim, J
2013-01-01
In order to test health foods for illegally added diuretics for weight loss, we developed simple, rapid, selective, and sensitive methods using HPLC and LC-MS/MS for the simultaneous analysis of 17 diuretics in dietary supplements. HPLC conditions were set with a Capcell-pak C18, using a mobile phase consisting of gradient conditions, UV detection at 254 nm and validated for linearity (r(2)> 0.999), precision (CV ≤ 3%), recoveries (90.4-102.8%) and reproducibility. Identification and quantification of 17 diuretics were accomplished by ion-spray LC-MS/MS using multiple reaction monitoring (MRM). The chromatographic separation was carried out under the reversed-phase mechanism on an HSS-T3 column. The LC-MS/MS method was validated for linearity (r(2)> 0.99) and precision (CV < 13%). Sixteen dietary supplements were tested with the developed methods. Diuretics were not detected in all samples. Extraction recovery was also investigated and the extraction recoveries in different formulations were from 88% to 110% and from 81% to 116% using HPLC and LC-MS/MS, respectively. There was no significant difference in recoveries in the type of dietary supplements. Based on this result, the developed methods to monitor illegal drug adulterations in dietary supplements using HPLC and LC-MS/MS are simple, fast and reliable. Therefore, it is applicable to routine drug-adulteration screening.
Spanjer, Martien C; Rensen, Peter M; Scholten, Jos M
2008-04-01
Mycotoxin analysis is usually carried out by high performance liquid chromatography after immunoaffinity column cleanup or in enzyme-linked immunosorbent assay tests. These methods normally involve determination of single compounds only. EU legislation already exists for the aflatoxins, ochratoxin A and patulin in food, and legislation will come into force for deoxynivalenol, zearalenone and the fumonisins in 2007. To enforce the various legal limits, it would be preferable to determine all mycotoxins by routine analysis in different types of matrices in one single extract. This would also be advantageous for HACCP control purposes. For this reason, a multi-method was developed with which 33 mycotoxins in various products could be analysed simultaneously. The mycotoxins were extracted with an acetonitrile/water mixture, diluted with water and then directly injected into a LC-MS/MS system. The mycotoxins were separated by reversed-phase HPLC and detected using an electrospray ionisation interface (ESI) and tandem MS, using MRM in the positive ion mode, to increase specificity for quality control. The following mycotoxins could be analysed in a single 30-min run: Aflatoxins B1, B2, G1 and G2, ochratoxin A, deoxynivalenol, zearalenone, T-2 toxin, HT-2 toxin, alpha-zearalenol, alpha-zearalanol, beta-zearalanol, sterigmatocystin, cyclopiazonic acid, penicillic acid, fumonisins B1, B2 and B3, diacetoxyscirpenol, 3- and 15-acetyl-deoxynivalenol, zearalanone, ergotamin, ergocornin, ergocristin, alpha-ergocryptin, citrinin, roquefortin C, fusarenone X, nivalenol, mycophenolic acid, alternariol and alternariol monomethyl ether. The limit of quantification for the aflatoxins and ochratoxin A was 1.0 microg kg(-1) and for deoxynivalenol 50 microg kg(-1). The quantification limits for the other mycotoxins were in the range 10-200 microg kg(-1). The matrix effect and validation data are presented for between 13 and 24 mycotoxins in peanuts, pistachios, wheat, maize, cornflakes
Gielecińska, Iwona; Mojska, Hanna
2013-01-01
Numerous studies have demonstrated acrylamide to be both neurotoxic and carcinogenic. At present it is widely recognised that acrylamide is mainly formed through the Maillard reaction from free asparagine and reducing sugars. The major sources of dietary acrylamide are potato products, processed cereals and coffee. To optimise and validate an analytical method for determining acrylamide in coffee by liquid chromatography and tandem mass spectrometry analysis (LC/MS/MS) using SPE clean-up. Analytical separation of acrylamide from roasted coffee was performed by liquid chromatography using a Hypercarb column followed by LC/MS/MS analysis, with 2,3,3-d3 acrylamide as an internal standard. The method was based on two purification steps: the first with hexane and Carrez solutions in order to remove of fat and to precipitate proteins, respectively; and the second with a solid-phase extraction (SPE) column which proved to be efficient in the elimination of the main chromatographic interferences. Limit of quantification (LOQ) for measuring acrylamide in coffee was 50 microg/kg. The described method demonstrates satisfactory precision (RSD = 2.5%), repeatability (RSD = 9.2%) and accuracy (mean recovery - 97.4%). Our results confirm that LC-MS/MS with SPE clean-up is selective and suitable for determination of acrylamide in coffee. Indeed, this method meets the criteria of EU Commission Recommendations (No. 2007/331/EC and No. 2010/307/EU), on the monitoring of acrylamide levels in food.
Koželj, Gordana; Perharič, Lucija; Stanovnik, Lovro; Prosen, Helena
2014-08-05
A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the determination of atropine and scopolamine in 100μL human plasma was developed and validated. Sample pretreatment consisted of protein precipitation with acetonitrile followed by a concentration step. Analytes and levobupivacaine (internal standard) were separated on a Zorbax XDB-CN column (75mm×4.6mm i.d., 3.5μm) with gradient elution (purified water, acetonitrile, formic acid). The triple quadrupole MS was operated in ESI positive mode. Matrix effect was estimated for deproteinised plasma samples. Selected reaction monitoring (SRM) was used for quantification in the range of 0.10-50.00ng/mL. Interday precision for both tropanes and intraday precision for atropine was <10%, intraday precision for scopolamine was <14% and <18% at lower limit of quantification (LLOQ). Mean interday and intraday accuracies for atropine were within ±7% and for scopolamine within ±11%. The method can be used for determination of therapeutic and toxic levels of both compounds and has been successfully applied to a study of pharmacodynamic and pharmacokinetic properties of tropanes, where plasma samples of volunteers were collected at fixed time intervals after ingestion of a buckwheat meal, spiked with five low doses of tropanes. Copyright © 2014 Elsevier B.V. All rights reserved.
Famiglini, Giorgio; Termopoli, Veronica; Palma, Pierangela; Capriotti, Fabiana; Cappiello, Achille
2014-05-01
An LC-MS method for the analysis of personal care and household products without sample preparation is presented. The method takes advantage of the Direct-electron ionization (EI) LC-MS interface for the quantitation of principal components, as well as for the identification of unknown or undeclared ingredients. The technique has proven its inertness toward matrix effects and the electron ionization allows quantitation and library identification. Commercially available products (shower gel, perfume, and hand cream) were diluted with methanol and injected directly into a nano-LC column. Limonene, linalool, and citral were selected as target compounds because of their use as fragrances in toiletry and detergent products. These and all other fragrances are commonly determined with GC-MS analysis, prior to sample cleanup, a procedure that can lead to analytes loss. The selected compounds are not detected with ESI because of their poor or very low response. Figures of merit and validation studies were executed and special attention was devoted to matrix-effects evaluation, because a sample preparation procedure is not involved. No matrix effects were observed, and the repeatability was excellent even after several weeks of operation. Products composition was investigated in full scan mode to determine the presence of unknown or not listed ingredients. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
L-C Measurement Acquisition Method for Aerospace Systems
NASA Technical Reports Server (NTRS)
Woodard, Stanley E.; Taylor, B. Douglas; Shams, Qamar A.; Fox, Robert L.
2003-01-01
This paper describes a measurement acquisition method for aerospace systems that eliminates the need for sensors to have physical connection to a power source (i.e., no lead wires) or to data acquisition equipment. Furthermore, the method does not require the sensors to be in proximity to any form of acquisition hardware. Multiple sensors can be interrogated using this method. The sensors consist of a capacitor, C(p), whose capacitance changes with changes to a physical property, p, electrically connected to an inductor, L. The method uses an antenna to broadcast electromagnetic energy that electrically excites one or more inductive-capacitive sensors via Faraday induction. This method facilitates measurements that were not previously possible because there was no practical means of providing power and data acquisition electrical connections to a sensor. Unlike traditional sensors, which measure only a single physical property, the manner in which the sensing element is interrogated simultaneously allows measurement of at least two unrelated physical properties (e.g., displacement rate and fluid level) by using each constituent of the L-C element. The key to using the method for aerospace applications is to increase the distance between the L-C elements and interrogating antenna; develop all key components to be non-obtrusive and to develop sensing elements that can easily be implemented. Techniques that have resulted in increased distance between antenna and sensor will be presented. Fluid-level measurements and pressure measurements using the acquisition method are demonstrated in the paper.
Tokumura, Masahiro; Miyake, Yuichi; Wang, Qi; Nakayama, Hayato; Amagai, Takashi; Ogo, Sayaka; Kume, Kazunari; Kobayashi, Takeshi; Takasu, Shinji; Ogawa, Kumiko
2018-04-16
Organophosphorus flame retardants (PFRs) are extensively used as alternatives to banned polybrominated diphenyl ethers (PBDEs) and hexabromocyclododecane (HBCD). In this study, we analyzed 14 PFRs by means of four mass-spectrometry-based methods: gas chromatography combined with electron-impact mass spectrometry (GC-EI-MS) or negative-chemical-ionization mass spectrometry (GC-NCI-MS) and liquid chromatography combined with tandem mass spectrometry using electrospray ionization (LC-ESI-MS/MS) or atmospheric pressure chemical ionization (LC-APCI-MS/MS). The limits of quantification (LOQs) for LC-ESI-MS/MS and LC-APCI-MS/MS (0.81-970 pg) were 1-2 orders of magnitude lower than the LOQs for GC-EI-MS and GC-NCI-MS (2.3-3900 pg). LC-APCI-MS/MS showed the lowest LOQs (mean = 41 pg; median = 3.4 pg) for all but two of the PFRs targeted in this study. For LC-APCI-MS/MS, the lowest LOQ was observed for tributyl phosphate (TBP) (0.81 pg), and the highest was observed for tris(butoxyethyl) phosphate (TBOEP) (36 pg). The results of this study indicate that LC-APCI-MS/MS is the optimum analytical method for the target PFRs, at least in terms of LOQ.
Brêtas, Juliana Machado; César, Isabela Costa; Brêtas, Camila Machado; Teixeira, Leonardo de Souza; Bellorio, Karini Bruno; Mundim, Iram Moreira; Pianetti, Gerson Antônio
2016-06-01
A sensitive and fast liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) method was developed and validated for the simultaneous quantification of naproxen and sumatriptan in human plasma. A simple liquid-liquid extraction procedure, with a mixture of ethyl acetate, methyl tert-butyl ether, and dichloromethane (4:3:3, v/v), was used for the cleanup of plasma. Naratriptan and aceclofenac were employed as internal standards. The analyses were carried out using an ACE C18 column (50 × 4.6 mm i.d.; particle size 5 μm) and a mobile phase consisting of 2 mM aqueous ammonium acetate with 0.025 % formic acid and methanol (38:62, v/v). A triple-quadrupole mass spectrometer equipped with an electrospray source in the positive mode was set up in the selective reaction monitoring mode to detect the ion transitions m/z 231.67 → m/z 185.07, m/z 296.70 → m/z 157.30, m/z 354.80 → m/z 215.00, and m/z 336.80 → m/z 97.94 for naproxen, sumatriptan, aceclofenac, and naratriptan, respectively. The method was validated and proved to be linear, accurate, precise, and selective over the ranges of 2.5-130 μg mL(-1) for naproxen and 1-50 ng mL(-1) for sumatriptan. The validated method was successfully applied to a pharmacokinetic study with simultaneous administration of naproxen sodium and sumatriptan succinate tablet formulations in healthy volunteers.
Ma, Jianshe; Cai, Jinzhang; Lin, Guanyang; Chen, Huilin; Wang, Xianqin; Wang, Xianchuan; Hu, Lufeng
2014-05-15
Corynoxeine(CX), isolated from the extract of Uncaria rhynchophylla, is a useful and prospective compound in the prevention and treatment for vascular diseases. A simple and selective liquid chromatography mass spectrometry (LC-MS) method was developed to determine the concentration of CX in rat plasma. The chromatographic separation was achieved on a Zorbax SB-C18 (2.1 mm × 150 mm, 5 μm) column with acetonitrile-0.1% formic acid in water as mobile phase. Selective ion monitoring (SIM) mode was used for quantification using target ions m/z 383 for CX and m/z 237 for the carbamazepine (IS). After the LC-MS method was validated, it was applied to a back-propagation artificial neural network (BP-ANN) pharmacokinetic model study of CX in rats. The results showed that after intravenous administration of CX, it was mainly distributed in blood and eliminated quickly, t1/2 was less than 1h. The predicted concentrations generated by BP-ANN model had a high correlation coefficient (R>0.99) with experimental values. The developed BP-ANN pharmacokinetic model can be used to predict the concentration of CX in rats. Copyright © 2014 Elsevier B.V. All rights reserved.
Bureš, Jan; Jansová, Hana; Stariat, Ján; Filipský, Tomáš; Mladěnka, Přemysl; Šimůnek, Tomáš; Kučera, Radim; Klimeš, Jiří; Wang, Qin; Franz, Katherine J.; Kovaříková, Petra
2015-01-01
Salicylaldehyde isonicotinoyl hydrazone (SIH) is an intracellular iron chelator with well documented potential to protect against oxidative injury both in vitro and in vivo. However, it suffers from short biological half-life caused by fast hydrolysis of the hydrazone bond. Recently, a concept of boronate prochelators has been introduced as a strategy that might overcome these limitations. This study presents two complementary analytical methods for detecting the prochelator BSIH (boronyl salicylaldehyde isonicotinoyl hydrazone) along with its active metal-binding chelator SIH in different solution matrices and concentration ranges. An LC-UV method for determination of BSIH and SIH in buffer and cell culture medium was validated over concentrations of 7 – 115 and 4 – 115 μM, respectively, and applied to BSIH activation experiments in vitro. An LC-MS assay was validated for quantification of BSIH and SIH in plasma over the concentration range of 0.06 – 23 and 0.24 – 23 μM, respectively, and applied to stability studies in plasma in vitro as well as analysis of plasma taken after i.v. administration of BSIH to rats. A Zorbax-RP bonus column and mobile phases containing either phosphate buffer with EDTA or ammonium formate and methanol/acetonitrile mixture provided suitable conditions for the LC-UV and LC-MS analysis, respectively. Samples were diluted or precipitated with methanol prior to analysis. These separative analytical techniques establish the first validated protocols to investigate BSIH activation by hydrogen peroxide in multiple matrices, directly compare the stabilities of the prochelator and chelator in plasma, and provide the first basic pharmacokinetic data of this prochelator. Experiments reveal that BSIH is stable in all media tested and is partially converted to SIH by H2O2. The observed integrity of BSIH in plasma samples from the in vivo study suggest that the concept of prochelation might be a promising strategy for further development
Boison, Joe; Lee, Stephen; Gedir, Ron
2009-01-01
A liquid chromatographic-mass spectrometric (LC-MS) method was developed and validated for the determination and confirmation of virginiamycin (VMY) M1 residues in porcine liver, kidney, and muscle tissues at concentrations > or =2 ng/g. Porcine liver, kidney, or muscle tissue is homogenized with methanol-acetonitrile. After centrifugation, the supernatant is diluted with phosphate buffer and cleaned up on a C18 solid-phase extraction cartridge. VMY in the eluate is partitioned into chloroform and the aqueous upper layer is removed by aspiration. After evaporating the chloroform in the residual mixture to dryness, the dried extract is reconstituted in mobile phase and VMY is quantified by LC-MS. Any samples eliciting quantifiable levels of VMY M1 (i.e., at concentrations > or =2 ng/g) are subjected to confirmatory analysis by LC-MSIMS. VMY S1, a minor component of the VMY complex, is monitored but not quantified or confirmed.
Ge, Shufan; Gao, Song; Yin, Taijun; Hu, Ming
2015-03-25
Chrysin, a flavone found in many plants, is also available as a dietary supplement because of its reported anticancer activities. However, its bioavailability is very poor due to extensive phase II metabolism. The purpose of this study was to develop an UPLC-MS/MS method to simultaneously quantify chrysin and its phase II metabolites, and to determine its pharmacokinetics in FVB wild-type and Bcrp knockout (Bcrp1 -/-) mice. In addition, the role of BCRP in chrysin phase II disposition was further investigated in Caco-2 cells. The results showed that our sensitive and reproducible UPLC-MS/MS method was successfully applied to the pharmacokinetic study of chrysin in wild-type and Bcrp1 (-/-) FVB mice after oral administration (20 mg/kg). Although there was no significant change in systemic exposure of chrysin and its metabolites, it was found that the Tmax for chrysin glucuronide was significantly shorter (p < 0.01) in Bcrp1-deficient mice. Furthermore, it was shown that inhibition of BCRP by Ko143 significantly reduced the efflux of chrysin sulfate in Caco-2 cells. In conclusion, BCRP had significant but less than expected impact on pharmacokinetics of chrysin and its conjugates, which were determined using a newly developed and validated LC-MS/MS method.
Jenkinson, Carl; Taylor, Angela E; Hassan-Smith, Zaki K; Adams, John S; Stewart, Paul M; Hewison, Martin; Keevil, Brian G
2016-03-01
Recent studies suggest that vitamin D-deficiency is linked to increased risk of common human health problems. To define vitamin D 'status' most routine analytical methods quantify one particular vitamin D metabolite, 25-hydroxyvitamin D3 (25OHD3). However, vitamin D is characterized by complex metabolic pathways, and simultaneous measurement of multiple vitamin D metabolites may provide a more accurate interpretation of vitamin D status. To address this we developed a high-throughput liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to analyse multiple vitamin D analytes, with particular emphasis on the separation of epimer metabolites. A supportive liquid-liquid extraction (SLE) and LC-MS/MS method was developed to quantify 10 vitamin D metabolites as well as separation of an interfering 7α-hydroxy-4-cholesten-3-one (7αC4) isobar (precursor of bile acid), and validated by analysis of human serum samples. In a cohort of 116 healthy subjects, circulating concentrations of 25-hydroxyvitamin D3 (25OHD3), 3-epi-25-hydroxyvitamin D3 (3-epi-25OHD3), 24,25-dihydroxyvitamin D3 (24R,25(OH)2D3), 1,25-dihydroxyvitamin D3 (1α,25(OH)2D3), and 25-hydroxyvitamin D2 (25OHD2) were quantifiable using 220μL of serum, with 25OHD3 and 24R,25(OH)2D3 showing significant seasonal variations. This high-throughput LC-MS/MS method provides a novel strategy for assessing the impact of vitamin D on human health and disease. Copyright © 2016 Elsevier B.V. All rights reserved.
Zou, Quanfei; Gu, Yuan; Lu, Rong; Zhang, Tiejun; Zhao, Guang-Rong; Liu, Changxiao; Si, Duanyun
2013-09-01
In this study, a simple and sensitive LC/MS/MS method was developed and validated for the determination of arctigenin in rat plasma. The MS detection was performed using multiple reaction monitoring at the transitions of m/z 373.2 → 137.3 for arctigenin and m/z 187.1 → 131.0 for psoralen (internal standard) with a Turbo IonSpray electrospray in positive mode. The calibration curves fitted a good linear relationship over the concentration range of 0.2-500 ng/mL. It was found that arctigenin is not stable enough at both room temperature and -80 °C unless mixed with methanol before storage. The validated LC/MS/MS method was successfully applied for the pharmacokinetic study of arctigenin in rats. After intravenous injection of 0.3 mg/kg arctigenin injection to rats, the maximum concentration, half-life and area under the concentration-time curve were 323 ± 65.2 ng/mL, 0.830 ± 0.166 and 81.0 ± 22.1 h ng/mL, respectively. Copyright © 2013 John Wiley & Sons, Ltd.
Method of preparing a tunable-focus liquid-crystal (LC) lens
NASA Astrophysics Data System (ADS)
Li, Xiaolong; Zhou, Zuowei; Ren, Hongwen
2018-02-01
A liquid crystal (LC) lens is prepared by controlling the alignment of a LC using a homogeneous polyimide (PI) layer and a homeotropic PI layer. The rubbed homogeneous PI layer has a concave surface and the homeotropic PI layer is flat. The LC sandwiched between the two PI layers obtains a hybrid alignment which has the largest gradient of refractive index (GRIN) distribution. The LC layer exhibits a lens character because of its convex shape. Since the effective refractive index of the LC is larger than that of the homogeneous PI, the LC lens can focus a light with the shortest focal length in the voltage-off state. By applying an external voltage, the LC molecules can be reoriented along the electric field. As a result, the focal length of the LC lens is reduced. The focal length of the LC lens can be tuned from 30 to 120 μm when the voltage is changed from 0 to 7 Vrms. This LC lens has the advantages of no threshold, low operating voltage, and simple fabrication.
Jardim, Andréia Nunes Oliveira; Mello, Denise Carvalho; Goes, Fernanda Caroline Silva; Frota Junior, Elcio Ferreira; Caldas, Eloisa Dutra
2014-12-01
A multiresidue method for the determination of 46 pesticides in fruits was validated. Samples were extracted with acidified ethyl acetate, MgSO4 and CH3COONa and cleaned up by dispersive SPE with PSA. The compounds were analysed by GC-FPD, GC-μECD or LC-MS/MS, with LOQs from 1 to 8 μg/kg. The method was used to analyse 238 kaki, cashew apple, guava, and peach fruit and pulp samples, which were also analysed for dithiocarbamates (DTCs) using a spectrophotometric method. Over 70% of the samples were positive, with DTC present in 46.5%, λ-cyhalothrin in 37.1%, and omethoate in 21.8% of the positive samples. GC-MS/MS confirmed the identities of the compounds detected by GC. None of the pesticides found in kaki, cashew apple and guava was authorised for these crops in Brazil. The risk assessment has shown that the cumulative acute intake of organophosphorus or pyrethroid compounds from the consumption of these fruits is unlikely to pose a health risk to consumers. Copyright © 2014 Elsevier Ltd. All rights reserved.
Broekaert, N; Devreese, M; De Mil, T; Fraeyman, S; De Baere, S; De Saeger, S; De Backer, P; Croubels, S
2014-11-15
This study aims to develop an LC-MS/MS method allowing the determination of 3-acetyl-deoxynivalenol, 15-acetyl-deoxynivalenol, deoxynivalenol and its main in vivo metabolite, deepoxy-deoxynivalenol, in broiler chickens and pigs. These species have a high exposure to these toxins, given their mainly cereal based diet. Several sample cleanup strategies were tested and further optimized by means of fractional factorial designs. A simple and straightforward sample preparation method was developed consisting out of a deproteinisation step with acetonitrile, followed by evaporation of the supernatant and reconstitution in water. The method was single laboratory validated according to European guidelines and found to be applicable for the intended purpose, with a linear response up to 200ngml(-1) and limits of quantification of 0.1-2ngml(-1). As a proof of concept, biological samples from a broiler chicken that received either deoxynivalenol, 3- or 15-acetyl-deoxynivalenol were analyzed. Preliminary results indicate nearly complete hydrolysis of 3-acetyl-deoxynivalenol to deoxynivalenol; and to a lesser extent of 15-acetyl-deoxynivalenol to deoxynivalenol. No deepoxy-deoxynivalenol was detected in any of the plasma samples. The method will be applied to study full toxicokinetic properties of deoxynivalenol, 3-acetyl-deoxynivalenol and 15-acetyl-deoxynivalenol in broiler chickens and pigs. Copyright © 2014 Elsevier B.V. All rights reserved.
Development and validation of an LC-UV method for the determination of sulfonamides in animal feeds.
Kumar, P; Companyó, R
2012-05-01
A simple LC-UV method was developed for the determination of residues of eight sulfonamides (sulfachloropyridazine, sulfadiazine, sulfadimidine, sulfadoxine, sulfamethoxypyridazine, sulfaquinoxaline, sulfamethoxazole, and sulfadimethoxine) in six types of animal feed. C18, Oasis HLB, Plexa and Plexa PCX stationary phases were assessed for the clean-up step and the latter was chosen as it showed greater efficiency in the clean-up of interferences. Feed samples spiked with sulfonamides at 2 mg/kg were used to assess the trueness (recovery %) and precision of the method. Mean recovery values ranged from 47% to 66%, intra-day precision (RSD %) from 4% to 15% and inter-day precision (RSD %) from 7% to 18% in pig feed. Recoveries and intra-day precisions were also evaluated in rabbit, hen, cow, chicken and piglet feed matrices. Calibration curves with standards prepared in mobile phase and matrix-matched calibration curves were compared and the matrix effects were ascertained. The limits of detection and quantification in the feeds ranged from 74 to 265 µg/kg and from 265 to 868 µg/kg, respectively. Copyright © 2011 John Wiley & Sons, Ltd.
Processing methods for differential analysis of LC/MS profile data
Katajamaa, Mikko; Orešič, Matej
2005-01-01
Background Liquid chromatography coupled to mass spectrometry (LC/MS) has been widely used in proteomics and metabolomics research. In this context, the technology has been increasingly used for differential profiling, i.e. broad screening of biomolecular components across multiple samples in order to elucidate the observed phenotypes and discover biomarkers. One of the major challenges in this domain remains development of better solutions for processing of LC/MS data. Results We present a software package MZmine that enables differential LC/MS analysis of metabolomics data. This software is a toolbox containing methods for all data processing stages preceding differential analysis: spectral filtering, peak detection, alignment and normalization. Specifically, we developed and implemented a new recursive peak search algorithm and a secondary peak picking method for improving already aligned results, as well as a normalization tool that uses multiple internal standards. Visualization tools enable comparative viewing of data across multiple samples. Peak lists can be exported into other data analysis programs. The toolbox has already been utilized in a wide range of applications. We demonstrate its utility on an example of metabolic profiling of Catharanthus roseus cell cultures. Conclusion The software is freely available under the GNU General Public License and it can be obtained from the project web page at: . PMID:16026613
Processing methods for differential analysis of LC/MS profile data.
Katajamaa, Mikko; Oresic, Matej
2005-07-18
Liquid chromatography coupled to mass spectrometry (LC/MS) has been widely used in proteomics and metabolomics research. In this context, the technology has been increasingly used for differential profiling, i.e. broad screening of biomolecular components across multiple samples in order to elucidate the observed phenotypes and discover biomarkers. One of the major challenges in this domain remains development of better solutions for processing of LC/MS data. We present a software package MZmine that enables differential LC/MS analysis of metabolomics data. This software is a toolbox containing methods for all data processing stages preceding differential analysis: spectral filtering, peak detection, alignment and normalization. Specifically, we developed and implemented a new recursive peak search algorithm and a secondary peak picking method for improving already aligned results, as well as a normalization tool that uses multiple internal standards. Visualization tools enable comparative viewing of data across multiple samples. Peak lists can be exported into other data analysis programs. The toolbox has already been utilized in a wide range of applications. We demonstrate its utility on an example of metabolic profiling of Catharanthus roseus cell cultures. The software is freely available under the GNU General Public License and it can be obtained from the project web page at: http://mzmine.sourceforge.net/.
Tsuchiya, Megumi; Karim, M Rezaul; Matsumoto, Taro; Ogawa, Hidesato; Taniguchi, Hiroaki
2017-01-24
Transcriptional coregulators are vital to the efficient transcriptional regulation of nuclear chromatin structure. Coregulators play a variety of roles in regulating transcription. These include the direct interaction with transcription factors, the covalent modification of histones and other proteins, and the occasional chromatin conformation alteration. Accordingly, establishing relatively quick methods for identifying proteins that interact within this network is crucial to enhancing our understanding of the underlying regulatory mechanisms. LC-MS/MS-mediated protein binding partner identification is a validated technique used to analyze protein-protein interactions. By immunoprecipitating a previously-identified member of a protein complex with an antibody (occasionally with an antibody for a tagged protein), it is possible to identify its unknown protein interactions via mass spectrometry analysis. Here, we present a method of protein preparation for the LC-MS/MS-mediated high-throughput identification of protein interactions involving nuclear cofactors and their binding partners. This method allows for a better understanding of the transcriptional regulatory mechanisms of the targeted nuclear factors.
Hessling, Bernd; Büttner, Knut; Hecker, Michael; Becher, Dörte
2013-10-01
Quantitative LC-MALDI is an underrepresented method, especially in large-scale experiments. The additional fractionation step that is needed for most MALDI-TOF-TOF instruments, the comparatively long analysis time, and the very limited number of established software tools for the data analysis render LC-MALDI a niche application for large quantitative analyses beside the widespread LC-electrospray ionization workflows. Here, we used LC-MALDI in a relative quantification analysis of Staphylococcus aureus for the first time on a proteome-wide scale. Samples were analyzed in parallel with an LTQ-Orbitrap, which allowed cross-validation with a well-established workflow. With nearly 850 proteins identified in the cytosolic fraction and quantitative data for more than 550 proteins obtained with the MASCOT Distiller software, we were able to prove that LC-MALDI is able to process highly complex samples. The good correlation of quantities determined via this method and the LTQ-Orbitrap workflow confirmed the high reliability of our LC-MALDI approach for global quantification analysis. Because the existing literature reports differences for MALDI and electrospray ionization preferences and the respective experimental work was limited by technical or methodological constraints, we systematically compared biochemical attributes of peptides identified with either instrument. This genome-wide, comprehensive study revealed biases toward certain peptide properties for both MALDI-TOF-TOF- and LTQ-Orbitrap-based approaches. These biases are based on almost 13,000 peptides and result in a general complementarity of the two approaches that should be exploited in future experiments.
Castilhos, Tamara S; Barreto, Fabiano; Meneghini, Leonardo; Bergold, Ana Maria
2016-07-01
A reliable and simple method for the detection and quantification of residues of 14 non-steroidal anti-inflammatory drugs and a metamizole metabolite in swine muscle was developed using liquid chromatography-electrospray ionisation-tandem mass spectrometry (LC-ESI-MS/MS). The samples were extracted with acetonitrile (ACN) in solid-liquid extraction followed by a low-temperature partitioning (LLE-LTP) process at -20 ± 2°C. After evaporation to dryness, the residue was reconstituted with hexane and a mixture of water:acetonitrile (1:1). LC separation was achieved on a reversed-phase (RP18) column with gradient elution using water (phase A) and ACN (phase B) both containing 1 mmol l(-)(1) ammonium acetate (NH4COO) with 0.025% acetic acid. Analysis was carried out on a triple-quadrupole tandem mass spectrometer (LC-MS/MS) in multiple reaction monitoring mode using an electrospray interface in negative and positive mode in a single run. Method validation was performed according to the criteria of Commission Decision No. 2002/657/EC. The matrix effect and linearity were evaluated. Decision limit (CCα), detection capability (CCβ), accuracy and repeatability of the method are also reported. The proposed method proved to be simple, easy and adequate for high-throughput analysis and was applied to routine analysis by the Brazilian Ministry of Agriculture, Livestock and Food Supply.
Glauser, Gaétan; Grund, Baptiste; Gassner, Anne-Laure; Menin, Laure; Henry, Hugues; Bromirski, Maciej; Schütz, Frédéric; McMullen, Justin; Rochat, Bertrand
2016-03-15
A paradigm shift is underway in the field of quantitative liquid chromatography-mass spectrometry (LC-MS) analysis thanks to the arrival of recent high-resolution mass spectrometers (HRMS). The capability of HRMS to perform sensitive and reliable quantifications of a large variety of analytes in HR-full scan mode is showing that it is now realistic to perform quantitative and qualitative analysis with the same instrument. Moreover, HR-full scan acquisition offers a global view of sample extracts and allows retrospective investigations as virtually all ionized compounds are detected with a high sensitivity. In time, the versatility of HRMS together with the increasing need for relative quantification of hundreds of endogenous metabolites should promote a shift from triple-quadrupole MS to HRMS. However, a current "pitfall" in quantitative LC-HRMS analysis is the lack of HRMS-specific guidance for validated quantitative analyses. Indeed, false positive and false negative HRMS detections are rare, albeit possible, if inadequate parameters are used. Here, we investigated two key parameters for the validation of LC-HRMS quantitative analyses: the mass accuracy (MA) and the mass-extraction-window (MEW) that is used to construct the extracted-ion-chromatograms. We propose MA-parameters, graphs, and equations to calculate rational MEW width for the validation of quantitative LC-HRMS methods. MA measurements were performed on four different LC-HRMS platforms. Experimentally determined MEW values ranged between 5.6 and 16.5 ppm and depended on the HRMS platform, its working environment, the calibration procedure, and the analyte considered. The proposed procedure provides a fit-for-purpose MEW determination and prevents false detections.
Sandra, Koen; Mortier, Kjell; Jorge, Lucie; Perez, Luis C; Sandra, Pat; Priem, Sofie; Poelmans, Sofie; Bouche, Marie-Paule
2014-05-01
Nanobodies(®) are therapeutic proteins derived from the smallest functional fragments of heavy chain-only antibodies. The development and validation of an LC-MS/MS-based method for the quantification of an IgE binding Nanobody in cynomolgus monkey plasma is presented. Nanobody quantification was performed making use of a proteotypic tryptic peptide chromatographically enriched prior to LC-MS/MS analysis. The validated LLOQ at 36 ng/ml was measured with an intra- and inter-assay precision and accuracy <20%. The required sensitivity could be obtained based on the selectivity of 2D LC combined with MS/MS. No analyte specific tools for affinity purification were used. Plasma samples originating from a PK/PD study were analyzed and compared with the results obtained with a traditional ligand-binding assay. Excellent correlations between the two techniques were obtained, and similar PK parameters were estimated. A 2D LC-MS/MS method was successfully developed and validated for the quantification of a next generation biotherapeutic.
Machado, J C; Lange, A D; Todeschini, V; Volpato, N M
2014-02-01
A dissolution method to analyze atorvastatin tablets using in vivo data for RP and test pilot (PB) was developed and validated. The appropriate conditions were determined after solubility tests using different media, and sink conditions were established. The conditions used were equipment paddle at 50 rpm and 900 mL of potassium phosphate buffer pH 6.0 as dissolution medium. In vivo release profiles were obtained from the bioequivalence study of RP and the generic candidate PB. The fraction of dose absorbed was calculated using the Loo-Riegelman method. It was necessary to use a scale factor of time similar to 6.0, to associate the values of absorbed fraction and dissolved fraction, obtaining an in vivo-in vitro correlation level A. The dissolution method to quantify the amount of drug dissolved was validated using high-performance liquid chromatography and ultraviolet spectrophotometry, and validated according to the USP protocol. The discriminative power of dissolution conditions was assessed using two different pilot batches of atorvastatin tablets (PA and PB) and RP. The dissolution test was validated and may be used as a discriminating method in quality control and in the development of the new formulations.
Valkenborg, Dirk; Baggerman, Geert; Vanaerschot, Manu; Witters, Erwin; Dujardin, Jean-Claude; Burzykowski, Tomasz; Berg, Maya
2013-01-01
Abstract Combining liquid chromatography-mass spectrometry (LC-MS)-based metabolomics experiments that were collected over a long period of time remains problematic due to systematic variability between LC-MS measurements. Until now, most normalization methods for LC-MS data are model-driven, based on internal standards or intermediate quality control runs, where an external model is extrapolated to the dataset of interest. In the first part of this article, we evaluate several existing data-driven normalization approaches on LC-MS metabolomics experiments, which do not require the use of internal standards. According to variability measures, each normalization method performs relatively well, showing that the use of any normalization method will greatly improve data-analysis originating from multiple experimental runs. In the second part, we apply cyclic-Loess normalization to a Leishmania sample. This normalization method allows the removal of systematic variability between two measurement blocks over time and maintains the differential metabolites. In conclusion, normalization allows for pooling datasets from different measurement blocks over time and increases the statistical power of the analysis, hence paving the way to increase the scale of LC-MS metabolomics experiments. From our investigation, we recommend data-driven normalization methods over model-driven normalization methods, if only a few internal standards were used. Moreover, data-driven normalization methods are the best option to normalize datasets from untargeted LC-MS experiments. PMID:23808607
MacMahon, Shaun; Ridge, Clark D; Begley, Timothy H
2014-12-03
A new analytical method has been developed and validated for the detection and quantification of 2-monochloropropanediol (2-MCPD) esters in edible oils. The target compounds are potentially carcinogenic contaminants formed during the processing of edible oils. As the 2-MCPD esters that occur most frequently in refined edible oils were not commercially available, standards were synthesized with identity and purity (95+%) confirmed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and (1)H NMR. Target analytes are separated from edible oil matrices using a two-step solid-phase extraction (SPE) procedure. The extracts are then analyzed using LC-MS/MS with electrospray ionization (ESI). The method has been validated for 11 2-MCPD diesters and 3 2-MCPD monoesters in soybean oil, olive oil, and palm oil using an external calibration curve. The ranges of average recoveries and relative standard deviations (RSD) across the three oil matrices at three spiking concentrations are 79-106% (3-13% RSD) for the 2-MCPD diesters and 72-108% (4-17% RSD) for the 2-MCPD monoesters, with limits of quantitation at or below 30 ng/g for the diesters and 90 ng/g for the monoesters.
Comprehensive Optimization of LC-MS Metabolomics Methods Using Design of Experiments (COLMeD)
Rhoades, Seth D.
2017-01-01
Introduction Both reverse-phase and HILIC chemistries are deployed for liquid-chromatography mass spectrometry (LC-MS) metabolomics analyses, however HILIC methods lag behind reverse-phase methods in reproducibility and versatility. Comprehensive metabolomics analysis is additionally complicated by the physiochemical diversity of metabolites and array of tunable analytical parameters. Objective Our aim was to rationally and efficiently design complementary HILIC-based polar metabolomics methods on multiple instruments using Design of Experiments (DoE). Methods We iteratively tuned LC and MS conditions on ion-switching triple quadrupole (QqQ) and quadrupole-time-of-flight (qTOF) mass spectrometers through multiple rounds of a workflow we term COLMeD (Comprehensive optimization of LC-MS metabolomics methods using design of experiments). Multivariate statistical analysis guided our decision process in the method optimizations. Results LC-MS/MS tuning for the QqQ method on serum metabolites yielded a median response increase of 161.5% (p<0.0001) over initial conditions with a 13.3% increase in metabolite coverage. The COLMeD output was benchmarked against two widely used polar metabolomics methods, demonstrating total ion current increases of 105.8% and 57.3%, with median metabolite response increases of 106.1% and 10.3% (p<0.0001 and p<0.05 respectively). For our optimized qTOF method, 22 solvent systems were compared on a standard mix of physiochemically diverse metabolites, followed by COLMeD optimization, yielding a median 29.8% response increase (p<0.0001) over initial conditions. Conclusions The COLMeD process elucidated response tradeoffs, facilitating improved chromatography and MS response without compromising separation of isobars. COLMeD is efficient, requiring no more than 20 injections in a given DoE round, and flexible, capable of class-specific optimization as demonstrated through acylcarnitine optimization within the QqQ method. PMID:28348510
Dhanure, Shivanand; Savalia, Atulkumar; More, Pravinkumar; Shirode, Prashant; Kapse, Kailas; Shah, Virag
2014-01-01
A simple, sensitive, and selective LC-MS/MS method was developed and validated for the quantification of carbocisteine in human plasma. Rosiglitazone was used as the internal standard and heparin was used as the anticoagulant. The chromatographic separation was performed by using the Waters Symmetry Shield RP 8, 150 × 3.9 mm, 5 μ column at 40°C with a mobile phase consisting of a mixture of methanol and 0.5% formic acid solution in a 40:60 proportion. The flow rate was 500 μl/min along with a 5 μl injection volume. Protein precipitation was used as the extraction method. Mass spectrometric data were detected in positive ion mode. The MRM mode of the ions for carbocisteine was 180.0 > 89.0 and for rosiglitazone it was 238.1 > 135.1. The method was validated in the concentration curve range of 50.000 ng/mL to 6000.000 ng/mL. The retention times of carbocisteine and the internal standard rosiglitazone were approximately 2.20 and 3.01 min, respectively. The overall run time was 4.50 min. This method was found suitable to analyze human plasma samples for the application in pharmacokinetic and BA/BE studies.
Satoh, Mamoru; Ishige, Takayuki; Ogawa, Shoujiro; Nishimura, Motoi; Matsushita, Kazuyuki; Higashi, Tatsuya; Nomura, Fumio
2016-11-01
The quantification of serum 25-hydroxyvitamin D [25(OH)D] as an indicator of vitamin D status is currently primarily conducted by immunoassays, yet LC-MS/MS would allow more accurate determination. Furthermore, LC-MS/MS would allow simultaneous measurement of multiple analytes. The aim of this study was to develop and validate an LC-MS/MS method to simultaneously measure four vitamin D metabolites (25(OH)D 3 , 3-epi-25(OH)D 3 , 25(OH)D 2 , and 24,25(OH) 2 D 3 ) in serum for clinical laboratory applications. Serum samples were first prepared in a 96-well supported liquid extraction plate and the eluate was derivatized using the Cookson-type reagent 4-(4'-dimethylaminophenyl)-1,2,4-triazoline-3,5-dione (DAPTAD), which rapidly and quantitatively reacts with the s-cis-diene structure of vitamin D metabolites. The derivatized samples were subjected to LC-MS/MS, ionized by electrospray ionization (positive-ion mode), and detected by selected reaction monitoring. The lower limits of quantification for 25(OH)D 3 , 3-epi-25(OH)D 3 , 25(OH)D 2 , and 24,25(OH) 2 D 3 were 0.091, 0.020, 0.013, and 0.024 ng/mL, respectively. The accuracy values and the extraction recoveries for these four metabolites were satisfactory. Serum 25(OH)D levels determined by our LC-MS/MS were compared with those obtained by conventional radioimmunoassay (RIA) that cannot distinguish 25(OH)D 3 and 25(OH)D 2 . The values obtained by the RIA method exhibited a mean bias of about 8.35 ng/mL, most likely as a result of cross reaction of the antibody with low-abundance metabolites, including 24,25(OH) 2 D 3 . Various preanalytical factors, such as long sample sitting prior to serum separation, repeated freeze-thaw cycles, and the presence of anticoagulants, had no significant effects on these determinations. This high-throughput LC-MS/MS simultaneous assay of the four vitamin D metabolites 25(OH)D 3 , 3-epi-25(OH)D 3 , 25(OH)D 2 , and 24,25(OH) 2 D 3 required as little as 20 μL serum. This method
Jank, Louise; Martins, Magda Targa; Arsand, Juliana Bazzan; Hoff, Rodrigo Barcellos; Barreto, Fabiano; Pizzolato, Tânia Mara
2015-01-01
This study describes the development and validation procedures for scope extension of a method for the determination of β-lactam antibiotic residues (ampicillin, amoxicillin, penicillin G, penicillin V, oxacillin, cloxacillin, dicloxacillin, nafcillin, ceftiofur, cefquinome, cefoperazone, cephapirine, cefalexin and cephalonium) in bovine milk. Sample preparation was performed by liquid-liquid extraction (LLE) followed by two clean-up steps, including low temperature purification (LTP) and a solid phase dispersion clean-up. Extracts were analysed using a liquid chromatography-electrospray-tandem mass spectrometry system (LC-ESI-MS/MS). Chromatographic separation was performed in a C18 column, using methanol and water (both with 0.1% of formic acid) as mobile phase. Method validation was performed according to the criteria of Commission Decision 2002/657/EC. Main validation parameters such as linearity, limit of detection, decision limit (CCα), detection capability (CCβ), accuracy, and repeatability were determined and were shown to be adequate. The method was applied to real samples (more than 250) and two milk samples had levels above maximum residues limits (MRLs) for cloxacillin - CLX and cefapirin - CFAP.
Evaluation of tamoxifen and metabolites by LC-MS/MS and HPLC methods.
Heath, D D; Flat, S W; Wu, A H B; Pruitt, M A; Rock, C L
2014-01-01
Epidemiological and laboratory evidence suggests that quantification of serum or plasma levels of tamoxifen and its metabolites, 4-hydroxy-N-desmethyl-tamoxifen (endoxifen), Z-4-hydroxytamoxifen (4HT), N-desmethyl-tamoxifen (ND-tam), is a clinically useful tool in the assessment and monitoring of breast cancer status in patients taking adjuvant tamoxifen. A liquid chromatographic mass spectrometric method (LC-MS/MS) was used to measure the blood levels of tamoxifen and its metabolites. This fully automated analytical method is specific, accurate and sensitive. The LC-MS/MS automated technique has now become a widely accepted reference method. This study analysed a randomly selected batch of blood samples from participants enrolled in a breast cancer study to compare results from this reference method in 40 samples with those obtained from a recently developed high-performance liquid chromatography (HPLC) method with fluorescence detection. The mean (SD) concentrations for the LC-MS/MS method (endoxifen 12.6 [7.5] ng/mL, tamoxifen 105 [44] ng/mL, 4-HT 1.9 [1.0] ng/mL, ND-tam 181 [69] ng/mL) and the HPLC method (endoxifen 13.1 [7.8] ng/mL, tamoxifen 108 [55] ng/mL, 4-HT 1.8 [0.8] ng/mL, ND-tam 184 [81] ng/mL) did not show any significant differences. The results confirm that the HPLC method offers an accurate and comparable alternative for the quantification of tamoxifen and tamoxifen metabolites.
Comprehensive Optimization of LC-MS Metabolomics Methods Using Design of Experiments (COLMeD).
Rhoades, Seth D; Weljie, Aalim M
2016-12-01
Both reverse-phase and HILIC chemistries are deployed for liquid-chromatography mass spectrometry (LC-MS) metabolomics analyses, however HILIC methods lag behind reverse-phase methods in reproducibility and versatility. Comprehensive metabolomics analysis is additionally complicated by the physiochemical diversity of metabolites and array of tunable analytical parameters. Our aim was to rationally and efficiently design complementary HILIC-based polar metabolomics methods on multiple instruments using Design of Experiments (DoE). We iteratively tuned LC and MS conditions on ion-switching triple quadrupole (QqQ) and quadrupole-time-of-flight (qTOF) mass spectrometers through multiple rounds of a workflow we term COLMeD (Comprehensive optimization of LC-MS metabolomics methods using design of experiments). Multivariate statistical analysis guided our decision process in the method optimizations. LC-MS/MS tuning for the QqQ method on serum metabolites yielded a median response increase of 161.5% (p<0.0001) over initial conditions with a 13.3% increase in metabolite coverage. The COLMeD output was benchmarked against two widely used polar metabolomics methods, demonstrating total ion current increases of 105.8% and 57.3%, with median metabolite response increases of 106.1% and 10.3% (p<0.0001 and p<0.05 respectively). For our optimized qTOF method, 22 solvent systems were compared on a standard mix of physiochemically diverse metabolites, followed by COLMeD optimization, yielding a median 29.8% response increase (p<0.0001) over initial conditions. The COLMeD process elucidated response tradeoffs, facilitating improved chromatography and MS response without compromising separation of isobars. COLMeD is efficient, requiring no more than 20 injections in a given DoE round, and flexible, capable of class-specific optimization as demonstrated through acylcarnitine optimization within the QqQ method.
Rama Raju, Kanumuri Siva; Taneja, Isha; Singh, Sheelendra Pratap; Tripathi, Amit; Mishra, Durga Prasad; Hussain, K Mahaboob; Gayen, Jiaur Rahman; Singh, Shio Kumar; Wahajuddin, Muhammad
2015-01-01
Tamoxifen and centchroman are two non-steroidal, selective estrogen receptors modulators, intended for long term therapy in the woman. Because of their wide spread use, there is a possibility of co-prescription of these agents. We studied the probable pharmacokinetic interaction between these agents in breast cancer model rats. A simple, sensitive and rapid LC-ESI-MS/MS method was developed and validated for the simultaneous determination of tamoxifen, centchroman and their active metabolites. The method was linear over a range of 0.2-200 ng/ml. All validation parameters met the acceptance criteria according to regulatory guidelines. LC-MS/MS method for determination of tamoxifen, centchroman and their metabolites was developed and validated. Results show the potential of drug-drug interaction upon co-administration these two marketed drugs.
Shaik, Rihana Parveen; Puttagunta, Srinivasa Babu; Kothapalli, Chandrasekhar Bannoth; Awen, Bahlul Zayed Sh; Challa, B R
2013-12-01
Liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was used for simultaneous quantification of tolterodine and its metabolite 5-hydroxy methyl tolterodine in rat plasma. Tolterodine-d6 and 5-hydroxy methyl tolterodine-d14 were used as internal standards (IS). Chromatographic separation was performed on Ascentis Express RP amide (50 mm×4.6 mm, 2.7 μm) column with an isocratic mobile phase composed of 10 mM ammonium acetate and acetonitrile in the ratio of 20:80 (v/v), at a flow-rate of 0.5 mL/min. Tolterodine, tolterodine-d6, 5-hydroxy methyl tolterodine and 5-hydroxy methyl tolterodine-d14 were detected with proton adducts at m / z 326.1→147.1, 332.3→153.1, 342.2→223.1 and 356.2→223.1 in multiple reaction monitoring (MRM) positive mode respectively. The drug, metabolite and internal standards were extracted by liquid-liquid extraction method. The method was validated over a linear concentration range of 20.00-5000.00 pg/mL for tolterodine and 20.00-5000.00 pg/mL for 5-hydroxy methyl tolterodine. This method demonstrated intra- and inter-day precision of 0.62-6.36% and 1.73-4.84% for tolterodine, 1.38-4.22% and 1.62-4.25% for 5-hydroxy methyl tolterodine respectively. This method also demonstrated intra- and inter-day accuracy of 98.75-103.56% and 99.20-104.40% for tolterodine, 98.08-104.67% and 98.73-103.06% for 5-hydroxy methyl tolterodine respectively. Both analytes were found to be stable throughout freeze-thaw cycles, bench top and postoperative stability studies. This method was successfully applied for the pharmacokinetic analysis of rat plasma samples following i.v. administration.
Kumar, Ajay; Monif, Tausif; Khuroo, Arshad; Sasmal, Dinakar; Goswami, Dipanjan; Lahkar, Vijay Kumar
2011-06-01
LC- ESI- MS/MS simultaneous bioanalytical method was developed to determine acitretin and its metabolite isoacitretin in human plasma using acitretin-d3 used as the internal standard for both analytes. The compounds were extracted using protein precipitation coupled with liquid-liquid extraction with flash freezing technique. Negative mass transitions (m/z) of acitretin, isoacitretin and acitretin-d3 were detected in multiple reactions monitoring (MRM) mode at 325.4 → 266.3, 325.2 → 266.1 and 328.3 → 266.3, respectively, with a turbo ion spray interface. The chromatographic separation was achieved on an Ascentis-RP amide column (4.6 × 150 mm, 5 µm) with mobile phase delivered in isocratic mode. The method was validated over a concentration range of 1.025-753.217 ng/mL for acitretin and 0.394-289.234 ng/mL for isoacitretin with a limit of quantification of 1.025 and 0.394 ng/mL. The intra-day and inter-day precisions were below 8.1% for acitretin and below 13.8% for isoacitretin, while accuracy was within ±7.0 and ±10.6% respectively. For the first time, the best possible conditions for plasma stability of acitretin and isoacitretin are presented and discussed with application to clinical samples. Copyright © 2010 John Wiley & Sons, Ltd.
Urine Multi-drug Screening with GC-MS or LC-MS-MS Using SALLE-hybrid PPT/SPE.
Lee, Junhui; Park, Jiwon; Go, Ahra; Moon, Heesung; Kim, Sujin; Jung, Sohee; Jeong, Wonjoon; Chung, Heesun
2018-05-14
To intoxicated patients in the emergency room, toxicological analysis can be considerably helpful for identifying the involved toxicants. In order to develop a urine multi-drug screening (UmDS) method, gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-tandem mass spectrometry (LC-MS-MS) were used to determine targeted and unknown toxicants in urine. A GC-MS method in scan mode was validated for selectivity, limit of detection (LOD) and recovery. An LC-MS-MS multiple reaction monitoring (MRM) method was validated for lower LOD, recovery and matrix effect. The results of the screening analysis were compared with patient medical records to check the reliability of the screen. Urine samples collected from an emergency room were extracted through a combination of salting-out assisted liquid-liquid extraction (SALLE) and hybrid protein precipitation/solid phase extraction (hybrid PPT/SPE) plates and examined by GC-MS and LC-MS-MS. GC-MS analysis was performed as unknown drug screen and LC-MS-MS analysis was conducted as targeted drug screen. After analysis by GC-MS, a library search was conducted using an in-house library established with the automated mass spectral deconvolution and identification system (AMDISTM). LC-MS-MS used Cliquid®2.0 software for data processing and acquisition in MRM mode. An UmDS method by GC-MS and LC-MS-MS was developed by using a SALLE-hybrid PPT/SPE and in-house library. The results of UmDS by GC-MS and LC-MS-MS showed that toxicants could be identified from 185 emergency room patient samples containing unknown toxicants. Zolpidem, acetaminophen and citalopram were the most frequently encountered drugs in emergency room patients. The UmDS analysis developed in this study can be used effectively to detect toxic substances in a short time. Hence, it could be utilized in clinical and forensic toxicology practices.
Kotnala, A; Senthilkumari, S; Halder, N; Kumar, A; Velpandian, T
2018-01-15
To develop a microwave assisted method for the rapid synthesis of A2E and also to develop a method to quantify N-retinylidene-N-retinylethanolamine(A2E), all-trans retinal dimer (ATRD), A2-glycerophospho ethanolamine (A2GPE), dihydropyridine phosphatidyl ethanolamine (A2DHPE) and monofuran A2E (MFA2E) in age matched retina. The development of microwave assisted synthesis of A2E, its purification and characterization for its utility in quantification in human retina. The semi-quantitative method development using LC-ESI-MS, LC-ESI-MS/MS and LC-APCI-MS/MS from pooled macula and peripheral retina for the bisretinoid analysis has been done. Maximum A2E conversion using microwave assisted process took place at 80°C for 45min with a yield of 55.01%. Highly sensitive and specific mass spectrometric method was developed using reverse phase C-18 separation with positive electrospray ionization and positive atmospheric phase chemical ionization of tandom mass spectrometry. A gradient mobile phase separation was achieved using water and methanol with 0.1% TFA. Multiple reaction monitoring acquisition for ESI and APCI was performed at ATRD m/z 551.2/522.2, A2GPE m/z 746.4/729.5, A2DHPEm/z 594.4/576.5, MFA2E m/z 608.2/591.2, A2E m/z 592.4/418.2. Method was validated using LC-ESI-SIM mode to determine selectivity, linearity, sensitivity, precision and accuracy. An attempt towards optimization of the synthetic procedure of A2E was made so as to reduce the lengthy reaction time without compromising the yield. Developed method was capable enough for the detection of low level of bisretinids in retina. Copyright © 2017 Elsevier B.V. All rights reserved.
LC-MSMS assays of urinary cortisol, a comparison between four in-house assays.
Brossaud, Julie; Leban, Monique; Corcuff, Jean-Benoit; Boux de Casson, Florence; Leloupp, Anne-Gaëlle; Masson, Damien; Moal, Valérie; Bach-Ngohou, Kalyane
2018-06-27
Twenty-four hour urinary free cortisol (UFC) determination can be used for screening and follow-up of Cushing syndrome (CS). As immunoassay methods lack specificity for UFC measurement, the use of high-performance liquid chromatography coupled to mass spectrometer (LC-MSMS) is recommended. The aim of our study was to compare UFC results using four LC-MSMS methods performed in four independent laboratories in order to evaluate interlaboratory agreement. Frozen aliquots of 24-h urine samples (78 healthy volunteers and 20 patients with CS) were sent to four different laboratories for analysis. Following liquid-liquid or solid-liquid extraction, UFC were determined using four different LC-MSMS assay. UFC intra- and interassays variation coefficients were lower than 10% for each centre. External quality control results were not significantly different. UFC normal ranges (established from healthy volunteers) were 17-126, 15-134, 12-118 and 27-157 nmol/day, respectively. Classification of UFC from healthy volunteers and patients with CS using a 95th percentile threshold was similar. However, for extreme UFC values (<50 or >270 nmol/day), negative or positive bias was noted. Even for highly specific methods such as LC-MSMS, variations of results can be found depending on analytical process. Validation of LC-MSMS methods including determination of the reference range is essential.
Paoloni, Angela; Solfrizzo, Michele; Bibi, Rita; Pecorelli, Ivan
2018-05-04
The objective of this study was to develop a liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the determination of Ochratoxin A (OTA) and Ochratoxin α (OTα) in poultry tissues and eggs. The two toxins were extracted by a mixture of acetonitrile/water, purified with a reversed phase C18 solid phase extraction column (SPE) and determined by LC-MS/MS. The LC-MS/MS method performances were evaluated in terms of linearity in solvent and in matrix (ranged from 0.5 to 15.10 µg L -1 for OTA and from 0.60 to 17.85 µg L -1 for OTα), limit of detection (LOD), limit of quantitation (LOQ), specificity, accuracy and precision in repeatability conditions. Recovery experiments were performed by spiking poultry liver, kidney, muscle and eggs around 1 µg kg -1 and 10 µg kg -1 . LODs were 0.27 and 0.26 µg kg -1 while LOQs were fixed at 1.0 and 1.2 µg kg -1 for OTA and OTα, respectively. Main recoveries for OTA ranged from 82 to 109% and for OTα ranged from 55 to 89%. The values of within-laboratory relative standard deviation (RSD r ) were equal to or below 20%. Considering the results obtained and that all analytical performance criteria were fulfilled, the new extraction and purification method developed for OTA and OTα determination in animal tissues and eggs was found appropriate for control laboratories and research activities designed to ensure food safety.
Evaluation of Tamoxifen and metabolites by LC-MS/MS and HPLC Methods
Heath, D.D.; Flatt, S.W.; Wu, A.H.B.; Pruitt, M.A.; Rock, C.L.
2015-01-01
Epidemiological and laboratory evidence suggests that quantification of serum or plasma levels of tamoxifen and the metabolites of tamoxifen, 4-hydroxy-N-desmethyl-tamoxifen (endoxifen), Z-4-hydroxy-tamoxifen (4HT), N-desmethyl-tamoxifen (ND-tam) is a clinically useful tool in the assessment and monitoring of breast cancer status in patients taking adjuvant tamoxifen. A liquid chromatographic mass spectrometric method (LC-MS/MS) was used to measure the blood levels of tamoxifen and the metabolites of tamoxifen. This fully automated analytical method is specific, accurate and sensitive. The LC-MS/MS automated technique has now become a widely accepted reference method. We analyzed a randomly selected batch of blood samples from participants enrolled in a breast cancer study to compare results from this reference method in 40 samples with those obtained from a recently developed high performance liquid chromatography (HPLC) method with fluorescence detection. The mean (SD) concentration for the LC-MS/MS (endoxifen 12.6 [7.5] ng/mL, tamoxifen 105 [44] ng/mL, 4-HT 1.9 [1.0] ng/mL, ND-tam 181 [69] ng/mL) and the HPLC (endoxifen 13.1 [7.8] ng/mL, tamoxifen 108[55]ng/mL, 4-HT 1.8 [0.8] ng/mL, ND-tam 184 [81] ng/mL), the methods did not show any significant differences. Our results confirm that the HPLC method offers an accurate and comparable alternative for the quantification of tamoxifen and tamoxifen metabolites. PMID:24693573
Lawson, Graham; Cocks, Elizabeth; Tanna, Sangeeta
2012-05-15
The use of blood spot collection cards was investigated as a means of obtaining small volume samples for the quantification of therapeutic drugs for assessing medication adherence. A liquid chromatography-high resolution TOF mass spectrometry (LC-HRMS) method, based on the measurement at the accurate mass to charge ratio of the target analyte, was used to ensure specificity for atenolol in the dried blood spot (DBS) samples. A working method was developed and validated. For the preparation of DBS samples whole blood spiked with analyte was used to produce 30 μl blood spots on specimen collection cards. A 5mm disc was cut from the dried blood spot and extracted using methanol:water (60:40, v/v) containing the internal standard, atenolol-d(7). Extracts were vortexed, sonicated and then centrifuged. Gradient chromatographic elution was achieved using an Ascentis Express C18 100mm×2.1mm column and a mobile phase flow rate of 0.2 ml/min and the column oven temperature at 30 °C. MS detection was carried out in electrospray positive ion mode for target ions at accurate mass m/z 267.1703 for atenolol and 274.2143 for the IS. Drug extraction efficiency from spiked blood spots was demonstrated to be 96±5% and the drug was stable in DBS for at least 10 weeks. The developed LC-HRMS method was linear within the tested calibration range of 25-1500 ng/ml and validation showed the accuracy (relative error) and precision (coefficient of variation) values were within the pre-defined limits of ≤ 5% at all concentrations with a limit of quantification of 25 ng/ml. Factors with potential to affect drug quantification measurements such as the matrix effects, volume of blood applied onto the collection card and effect of different sampling cards were investigated. The developed LC-HRMS method was applied to blood spots on sampling card taken from adult healthy volunteers previously administered a 50mg atenolol tablet and a DBS concentration-time profile was obtained for atenolol
Bharathi, D Vijaya; Hotha, Kishore Kumar; Sagar, P V Vidya; Kumar, S Sirish; Naidu, A; Mullangi, Ramesh
2009-02-01
A highly sensitive and specific LC-MS/MS method has been developed and validated for the estimation of pramipexole (PPX) with 500 microL human plasma using memantine as an internal standard (IS). The API-4000 was operated under multiple-reaction monitoring mode (MRM) using the electrospray ionization technique. Solid-phase extraction was used to extract PPX and IS from human plasma. The resolution of peaks was achieved with 0.01 m ammonium acetate buffer (pH 4.4):acetonitrile (30:70, v/v) on a Discovery CN column. The total chromatographic run time was 3.0 min and the elution of PPX and IS occurred at approximately 2.32 and 2.52, respectively. The MS/MS ion transitions monitored were 212.10 --> 153.10 for PPX and 180.20 --> 107.30 for IS. The method was proved to be accurate and precise at linearity range of 20-3540 pg/mL with a correlation coefficient (r) of > or =0.999. The intra- and inter-day precision and accuracy values found to be within the assay variability limits as per the FDA guidelines. The developed assay method was applied to a pharmacokinetic study in human volunteers following oral administration of 0.25 mg PPX tablet. Copyright (c) 2008 John Wiley & Sons, Ltd.
Quantitation of brinzolamide in dried blood spots by a novel LC-QTOF-MS/MS method.
Foivas, Anargyros; Malenović, Anđelija; Kostić, Nađa; Božić, Marija; Knežević, Miroslav; Loukas, Yannis L; Dotsikas, Yannis
2016-02-05
In the current study, a rapid and sensitive LC-QTOF-MS/MS method for the determination of brinzolamide in dried blood spots (DBS) was developed and validated. This novel sample collection, storage and transfer technique was suitable for analyzing a drug with high distribution into red blood cells and negligible plasma levels. The method included an isocratic mobile phase consisting of methanol and 10mM ammonium formate (90:10, v/v) and detection in positive electrospray mode (ESI+). The flow rate was adjusted to 0.350mL/min yielding retention times of 1.7min for both brinzolamide and internal standard (IS) rabeprazole on a Cyano analytical column, respectively. The validation of the proposed method over the concentration range 0.500-20.0μg/mL was performed in compliance with EMEA and FDA guidelines, assessing all major performance characteristics. Inter- and intra- assay precisions were less than 14%, while inter- and intra- assay accuracies varied from 92.2 to 111%. No matrix effect was observed and the mean brinzolamide extraction recovery was 93.5%. The method was successfully applied to real DBS samples from patients in steady state condition, receiving brinzolamide ophthalmic suspension 1% (w/v) for several months. Initial concentrations were corrected due to hematocrit effect, using image processing algorithm written in Matlab. Copyright © 2015 Elsevier B.V. All rights reserved.
Methods for Studying Interactions Between Atg8/LC3/GABARAP and LIR-Containing Proteins.
Johansen, T; Birgisdottir, Å B; Huber, J; Kniss, A; Dötsch, V; Kirkin, V; Rogov, V V
2017-01-01
LC3/GABARAP proteins (LC3/GABARAPs) are mammalian orthologues of yeast Atg8, small ubiquitin (Ub)-like proteins (UBLs) whose covalent attachment to lipid membranes is crucial for the growth and closure of the double membrane vesicle called the autophagosome. In the past decade, it was demonstrated that Atg8/LC3/GABARAPs are also required for autophagic degradation of cargos in a selective fashion. Cargo selectivity is ensured by receptor proteins, such as p62/SQSTM1, NBR1, Cue5, Atg19, NIX, Atg32, NCOA4, and FAM134B, which simultaneously bind Atg8/LC3/GABARAPs and the cargo together, thereby linking the core autophagic machinery to the target structure: a protein, an organelle, or a pathogen. LC3-interacting regions (LIRs) are short linear motifs within selective autophagy receptors and some other structural and signaling proteins (e.g., ULK1, ATG13, FIP200, and Dvl2), which mediate binding to Atg8/LC3/GABARAPs. Identification and characterization of LIR-containing proteins have provided important insights into the biology of the autophagy pathway, and studying their interactions with the core autophagy machinery represents a growing area of autophagy research. Here, we present protocols for the identification of LIR-containing proteins, i.e., by yeast-two-hybrid screening, glutathione S-transferase (GST) pulldown experiments, and peptide arrays. The use of two-dimensional peptide arrays also represents a powerful method to identify the residues of the LIR motif that are critical for binding. We also describe a biophysical method for studying interactions between Atg8/LC3/GABARAP and LIR-containing proteins and a protocol for preparation and purification of LIR peptides. © 2017 Elsevier Inc. All rights reserved.
Narayana, M B V; Chandrasekhar, K B; Rao, B M
2014-09-01
A validated specific stability-indicating reverse-phase liquid chromatographic method was developed for the quantitative determination of Ambrisentan as well as its related substances in bulk samples, pharmaceutical dosage forms in the presence of degradation products and its related impurities. Forced degradation studies were performed on bulk samples of Ambrisentan as per the ICH-prescribed stress conditions using acid, base, oxidative, thermal stress and photolytic degradation to show the stability-indicating power of the LC method. Significant degradation in acidic, basic stress conditions was observed and no degradation was observed in other stress conditions. The chromatographic method was optimized using the samples generated from the forced degradation studies and the impurity-spiked solution. Good resolution between the peaks corresponds to Ambrisentan-related impurities and degradation products from the analyte were achieved on a SunFire C18 column using a mobile phase consisting of a mixture of potassium dihydrogen orthophosphate at a pH adjusted to 2.5 with ortho-phosphoric acid in water and a mixture of acetonitrile:methanol using a simple linear gradient. The detection was carried out at 225 nm. The limit of detection and the limit of quantification for the Ambrisentan and its related impurities were established. The stressed test solutions were assayed against the qualified working standard of Ambrisentan and the mass balance in each case was between 98.9 and 100.3%, indicating that the developed LC method was stability indicating. Validation of the developed LC method was carried out as per the ICH requirements. The developed method was found to be suitable to check the quality of bulk samples of Ambrisentan at the time of batch release and also during its storage (long-term and accelerated stability). © The Author [2013]. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.
Misra, A; Kushwaha, H N; Gautam, N; Singh, B; Verma, P C; Pratap, R; Singh, S K
2014-08-01
A sensitive and selective LC-MS/MS method has been developed and validated for CDRI antidiabetic candidate S007-1261 in rat plasma using 16-dehydropregnenolone as an internal standard. The API 4000 triple quadrupole LC-MS/MS system was operated under multiple reaction monitoring mode using electrospray ionization technique in positive mode. The sample processing method involves 2-step liquid-liquid extraction using n-hexane as an extracting solvent. The analyte was chromatographed on RP 18, waters column (3.5 µm, 2.1 mm i.d. × 30 mm) with guard using acetonitrile and ammonium acetate buffer (pH 5.0, 10 mM) in 90:10 (v/v) composition at a flow rate of 0.40 mL min(-1). The chromatographic run time was 5.30 min. Calibration curve shows linearity over concentration range 1.56-200 ng mL(-1). The lower limit of detection was 0.39 ng mL(-1) and lower limit of quantitation was 1.56 ng mL(-1). The inter- and intra-day accuracy and precision were found to be within the assay variability limits as per US FDA guidelines. The absolute recovery of S007-1261 was found to be >90%. S007-1261 does not show any stability problems as it was stable at room temperature for 8 h. S007-1261 was also stable up to 3 freeze-thaw cycles and can be stored up to 30 days at -60 °C. The assay was successfully applied to both oral (40 mg kg(-1)) and intravenous (10 mg kg(-1)) pharmacokinetic studies in male Sprague-Dawley rats. The oral bioavailability of S007-1261 was found to be 33.61%. © Georg Thieme Verlag KG Stuttgart · New York.
Multi-mycotoxin stable isotope dilution LC-MS/MS method for Fusarium toxins in beer.
Habler, Katharina; Gotthardt, Marina; Schüler, Jan; Rychlik, Michael
2017-03-01
A stable isotope dilution LC-MS/MS multi-mycotoxin method was developed for 12 different Fusarium toxins including modified mycotoxins in beer (deoxynivalenol-3-glucoside, deoxynivalenol, 3-acetyldeoxynivalenol, 15-acetyl-deoxynivalenol, HT2-toxin, T2-toxin, enniatin B, B1, A1, A, beauvericin and zearalenone). As sample preparation and purification of beer a combined solid phase extraction for trichothecenes, enniatins, beauvericin and zearalenone was firstly developed. The validation of the new method gave satisfying results: intra-day and inter-day precision and recoveries were 1-5%, 2-8% and 72-117%, respectively. In total, 61 different organic and conventional beer samples from Germany and all over the world were analyzed by using the newly developed multi-mycotoxin method. In summary, deoxynivalenol, deoxynivalenol-3-glucoside, 3-acetyldeoxynivaleneol and enniatin B were quantified in rather low contents in the investigated beer samples. None of the other monitored Fusarium toxins like 15-acetyldeoxynivalenol, HT2- and T2-toxin, zearalenone, enniatin B1, A1, A or beauvericin were detectable. Copyright © 2016 Elsevier Ltd. All rights reserved.
NASA Astrophysics Data System (ADS)
Stefanović, S.; Đorđevic, V.; Jelušić, V.
2017-09-01
The aim of this paper is to verify the performance characteristics and fitness for purpose of rapid and simple QuEChERS-based LC-MS/MS method for determination of acrylamide in potato chips and coffee. LC-MS/MS is by far the most suitable analytical technique for acrylamide measurements given its inherent sensitivity and selectivity, as well as capability of analyzing underivatized molecule. Acrylamide in roasted coffee and potato chips wasextracted with water:acetonitrile mixture using NaCl and MgSO4. Cleanup was carried out with MgSO4 and PSA. Obtained results were satisfactory. Recoveries were in the range of 85-112%, interlaboratory reproducibility (Cv) was 5.8-7.6% and linearity (R2) was in the range of 0.995-0.999. LoQ was 35 μg kg-1 for coffee and 20 μg kg-1 for potato chips. Performance characteristic of the method are compliant with criteria for analytical methods validation. Presented method for quantitative determination of acrylamide in roasted coffee and potato chips is fit for purposes of self-control in food industry as well as regulatory controls carried out by the governmental agencies.
Reference Proteome Extracts for Mass Spec Instrument Performance Validation and Method Development
Rosenblatt, Mike; Urh, Marjeta; Saveliev, Sergei
2014-01-01
Biological samples of high complexity are required to test protein mass spec sample preparation procedures and validate mass spec instrument performance. Total cell protein extracts provide the needed sample complexity. However, to be compatible with mass spec applications, such extracts should meet a number of design requirements: compatibility with LC/MS (free of detergents, etc.)high protein integrity (minimal level of protein degradation and non-biological PTMs)compatibility with common sample preparation methods such as proteolysis, PTM enrichment and mass-tag labelingLot-to-lot reproducibility Here we describe total protein extracts from yeast and human cells that meet the above criteria. Two extract formats have been developed: Intact protein extracts with primary use for sample preparation method development and optimizationPre-digested extracts (peptides) with primary use for instrument validation and performance monitoring
Oliveira, Fabiano Aurélio da Silva; Pereira, Elba Nathália Corrêa; Gobbi, Jennifer Mattedi; Soto-Blanco, Benito; Melo, Marília Martins
2018-01-01
Beef meat is an important food that can be contaminated by pesticides. This study aimed to optimize a multiresidue method for identification and quantification of pesticides in beef meat by liquid chromatography coupled to mass spectrometry detection (LC-MS). The extraction and clean-up procedures were adapted from the QuECHERS method. From the 188 analytes tested, the method was validated as qualitative method for 19 compounds and as quantitative method for 152 compounds. The results were satisfactory, yielding coefficients of variation of less than 20% and recoveries ranging from 70% to 120% and expanded uncertainty of less than 50%. The quantification limit was typically 10 µg kg -1 (but 25 µg kg -1 for 12 of the compounds) and the detection limit was 5.0 µg kg -1 . Thirty-two real samples of commercialized beef meat were analyzed without any residual pesticide being found. Thus, the results showed that the multiresidue method for detecting 171 pesticides, using adapted QuECHERS for extraction and LC-MS for detection, is suitable for analyzing beef meat.
Guale, Fessessework; Shahreza, Shahriar; Walterscheid, Jeffrey P; Chen, Hsin-Hung; Arndt, Crystal; Kelly, Anna T; Mozayani, Ashraf
2013-01-01
Liquid chromatography time-of-flight mass spectrometry (LC-TOF-MS) analysis provides an expansive technique for identifying many known and unknown analytes. This study developed a screening method that utilizes automated solid-phase extraction to purify a wide array of analytes involving stimulants, benzodiazepines, opiates, muscle relaxants, hypnotics, antihistamines, antidepressants and newer synthetic "Spice/K2" cannabinoids and cathinone "bath salt" designer drugs. The extract was applied to LC-TOF-MS analysis, implementing a 13 min chromatography gradient with mobile phases of ammonium formate and methanol using positive mode electrospray. Several common drugs and metabolites can share the same mass and chemical formula among unrelated compounds, but they are structurally different. In this method, the LC-TOF-MS was able to resolve many isobaric compounds by accurate mass correlation within 15 ppm mass units and a narrow retention time interval of less than 10 s of separation. Drug recovery yields varied among spiked compounds, but resulted in overall robust area counts to deliver an average match score of 86 when compared to the retention time and mass of authentic standards. In summary, this method represents a rapid, enhanced screen for blood and urine specimens in postmortem, driving under the influence, and drug facilitated sexual assault forensic toxicology casework.
van Dooren, Ines; Foubert, Kenn; Theunis, Mart; Naessens, Tania; Pieters, Luc; Apers, Sandra
2018-01-30
The berries of Vaccinium macrocarpon, cranberry, are widely used for the prevention of urinary tract infections. This species contains A-type proanthocyanidins (PACs), which intervene in the initial phase of the development of urinary tract infections by preventing the adherence of Escherichia coli by their P-type fimbriae to uroepithelial cells. Unfortunately, the existing clinical studies used different cranberry preparations, which were poorly standardized. Because of this, the results were hard to compare, which led sometimes to conflicting results. Currently, PACs are quantified using the rather non-specific spectrophotometric 4-dimethylaminocinnamaldehyde (DMAC) method. In addition, a normal phase HPTLC-densitometric method, a HPLC-UV method and three LC-MS/MS methods for quantification of procyanidin A2 were recently published. All these methods contain some shortcomings and errors. Hence, the development and validation of a fast and sensitive standard addition LC-MS/MS method for the simultaneous quantification of A-type dimers and trimers in a cranberry dry extract was carried out. A linear calibration model could be adopted for dimers and, after logaritmic transformation, for trimers. The maximal interday and interconcentration precision was found to be 4.86% and 4.28% for procyanidin A2, and 5.61% and 7.65% for trimeric PACs, which are all acceptable values for an analytical method using LC-MS/MS. In addition, twelve different cranberry extracts were analyzed by means of the newly validated method and other widely used methods. There appeared to be an enormous variation in dimeric and trimeric PAC content. Comparison of these results with LC-MS/MS analysis without standard addition showed the presence of matrix effects for some of the extracts and proved the necessity of standard addition. A comparison of the well-known and widely used DMAC method, the butanol-HCl assay and this newly developed LC-MS/MS method clearly indicated the need for a reliable
Ozkan Cansel, Kose; Ozgur, Esim; Sevinc, Kurbanoglu; Ayhan, Savaser; Ozkan, Sibel A; Yalcin, Ozkan
2016-01-01
Pharmaceutical preparations of ezetimibe and atorvastatin are generally used to regulate the lipid level in blood. It decreases the secondary events for patients with high cholesterol and clinical cardiovascular disease such as non-fatal or fatal heart attack. There is no any pharmacopoeia method available for the dissolution testing recommended by the FDA. Development of dissolution tests method is very critical parameter especially for the pharmaceutical preparations that contain Class II drugs (slightly soluble, good permeable). In the proposed method, the effects of pH and surfactant on the dissolution of poorly water soluble combined drug therapy with a different pKa values in an in vitro environment is investigated. The content of our study was designed to answer these open-ended questions. The optimized test conditions achieved under sink conditions with USP apparatus 2 at a paddle rotation speed of 75 rpm and 900 ml in 0.01 M Acetate buffer (pH= 6.8) containing 0.45% SDS as a dissolution medium. Quantification of dissolution samples were analyzed with a new fully validated RP-LC method with UV detection at 242 nm.
Krishnaiah, Ch; Sri, Khagga Bhavya
2012-05-01
A simple, selective and sensitive gradient reversed-phase liquid chromatography method has been developed for the separation and determination of 2,3-dichlorobenzoic acid, which is an intermediate of the lamotrizine drug substance, and its regio isomers. The separation was achieved on a reversed-phase United States Pharmacopeia L1 (C-18) column using 0.01 M ammonium acetate buffer at pH 2.5 and methanol (50:50 v/v) mixture as mobile phase A and a methanol and water mixture (80:20 v/v) as mobile phase B in a gradient elution at flow rate 1.2 mL/min with ultraviolet detection at 210 nm. The method is found to be selective, precise, linear, accurate and robust. It was used for quality assurance and monitoring the synthetic reactions involved in the process development of lamotrizine. The method is found to be simple, rapid, specific and reliable for the determination of unreacted levels of raw materials and isomers in reaction mixtures and finished product lamotrizine. The method was fully validated as per International Conference of Harmonization guidelines and results from validation confirm that the method is highly suitable for its intended purpose. © The Author [2012]. Published by Oxford University Press. All rights reserved.
DOE Office of Scientific and Technical Information (OSTI.GOV)
Zhou, Jianying; Dann, Geoffrey P.; Shi, Tujin
2012-03-10
Sodium dodecyl sulfate (SDS) is one of the most popular laboratory reagents used for highly efficient biological sample extraction; however, SDS presents a significant challenge to LC-MS-based proteomic analyses due to its severe interference with reversed-phase LC separations and electrospray ionization interfaces. This study reports a simple SDS-assisted proteomic sample preparation method facilitated by a novel peptide-level SDS removal protocol. After SDS-assisted protein extraction and digestion, SDS was effectively (>99.9%) removed from peptides through ion substitution-mediated DS- precipitation with potassium chloride (KCl) followed by {approx}10 min centrifugation. Excellent peptide recovery (>95%) was observed for less than 20 {mu}g of peptides.more » Further experiments demonstrated the compatibility of this protocol with LC-MS/MS analyses. The resulting proteome coverage from this SDS-assisted protocol was comparable to or better than those obtained from other standard proteomic preparation methods in both mammalian tissues and bacterial samples. These results suggest that this SDS-assisted protocol is a practical, simple, and broadly applicable proteomic sample processing method, which can be particularly useful when dealing with samples difficult to solubilize by other methods.« less
In 't Veld, P H; van der Laak, L F J; van Zon, M; Biesta-Peters, E G
2018-04-12
A method for the quantification of the Bacillus cereus emetic toxin (cereulide) was developed and validated. The method principle is based on LC-MS as this is the most sensitive and specific method for cereulide. Therefore the study design is different from the microbiological methods validated under this mandate. As the method had to be developed a two stage validation study approach was used. The first stage (pre-study) focussed on the method applicability and the experience of the laboratories with the method. Based on the outcome of the pre-study and comments received during voting at CEN and ISO level a final method was agreed to be used for the second stage the (final) validation of the method. In the final (validation) study samples of cooked rice (both artificially contaminated with cereulide or contaminated with B. cereus for production of cereulide in the rice) and 6 other food matrices (fried rice dish, cream pastry with chocolate, hotdog sausage, mini pancakes, vanilla custard and infant formula) were used. All these samples were spiked by the participating laboratories using standard solutions of cereulide supplied by the organising laboratory. The results of the study indicate that the method is fit for purpose. Repeatability values were obtained of 0.6 μg/kg at low level spike (ca. 5 μg/kg) and 7 to 9.6 μg/kg at high level spike (ca. 75 μg/kg). Reproducibility at low spike level ranged from 0.6 to 0.9 μg/kg and from 8.7 to 14.5 μg/kg at high spike level. Recovery from the spiked samples ranged between 96.5% for mini-pancakes to 99.3% for fries rice dish. Copyright © 2018. Published by Elsevier B.V.
Barco, Sebastiano; Castagnola, Elio; Moscatelli, Andrea; Rudge, James; Tripodi, Gino; Cangemi, Giuliana
2017-10-25
In this paper we show the development and validation of a volumetric absorptive microsampling (VAMS™)-LC-MS/MS method for the simultaneous quantification of four antibiotics: piperacillin-tazobactam, meropenem, linezolid and ceftazidime in 10μL human blood. The novel VAMS-LC-MS/MS method has been compared with a dried blood spot (DBS)-based method in terms of impact of hematocrit (HCT) on accuracy, reproducibility, recovery and matrix effect. Antibiotics were extracted from VAMS and DBS by protein precipitation with methanol after a re-hydration step at 37°C for 10min. LC-MS/MS was carried out on a Thermo Scientific™ TSQ Quantum™ Access MAX triple quadrupole coupled to an Accela ™UHPLC system. The VAMS-LC-MS/MS method is selective, precise and reproducible. In contrast to DBS, it allows an accurate quantification without any HCT influence. It has been applied to samples derived from pediatric patients under therapy. VAMS is a valid alternative sampling strategy for the quantification of antibiotics and is valuable in support of clinical PK/PD studies and consequently therapeutic drug monitoring (TDM) in pediatrics. Copyright © 2017 Elsevier B.V. All rights reserved.
Improved LC-MS/MS method for the quantification of hepcidin-25 in clinical samples.
Abbas, Ioana M; Hoffmann, Holger; Montes-Bayón, María; Weller, Michael G
2018-06-01
Mass spectrometry-based methods play a crucial role in the quantification of the main iron metabolism regulator hepcidin by singling out the bioactive 25-residue peptide from the other naturally occurring N-truncated isoforms (hepcidin-20, -22, -24), which seem to be inactive in iron homeostasis. However, several difficulties arise in the MS analysis of hepcidin due to the "sticky" character of the peptide and the lack of suitable standards. Here, we propose the use of amino- and fluoro-silanized autosampler vials to reduce hepcidin interaction to laboratory glassware surfaces after testing several types of vials for the preparation of stock solutions and serum samples for isotope dilution liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS). Furthermore, we have investigated two sample preparation strategies and two chromatographic separation conditions with the aim of developing a LC-MS/MS method for the sensitive and reliable quantification of hepcidin-25 in serum samples. A chromatographic separation based on usual acidic mobile phases was compared with a novel approach involving the separation of hepcidin-25 with solvents at high pH containing 0.1% of ammonia. Both methods were applied to clinical samples in an intra-laboratory comparison of two LC-MS/MS methods using the same hepcidin-25 calibrators with good correlation of the results. Finally, we recommend a LC-MS/MS-based quantification method with a dynamic range of 0.5-40 μg/L for the assessment of hepcidin-25 in human serum that uses TFA-based mobile phases and silanized glass vials. Graphical abstract Structure of hepcidin-25 (Protein Data Bank, PDB ID 2KEF).
Stypulkowska, Karolina; Fijalek, Zbigniew; Sarna, Katarzyna
2010-01-01
A new, simple and repeatable liquid chromatography method with charged aerosol detection (LC-CAD) for the determination of gentamicin sulphate composition and related substances has been developed. Gentamicin lacks of chromophores, therefore its determination is quite problematic. Using a universal CAD enables to achieve good separation without sample derivatization. Mass spectrometry was employed to confirm the LC-CAD peak profile. The proposed method was validated and applied for the determination of gentamicin sulphate composition and related substances in pharmaceutical preparations. PMID:21212825
Mezzullo, Marco; Fanelli, Flaminia; Fazzini, Alessia; Gambineri, Alessandra; Vicennati, Valentina; Di Dalmazi, Guido; Pelusi, Carlotta; Mazza, Roberta; Pagotto, Uberto; Pasquali, Renato
2016-10-01
Salivary steroid testing represents a valuable source of biological information; however, the proper measurement of low salivary levels is challenging for direct immunoassays, lacking adequate sensitivity and specificity and causing poor inter-laboratory reproducibility. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) has overcome previous analytical limits, often providing results deviating from previous knowledge. Nowadays, LC-MS/MS is being introduced in clinical laboratories for salivary cortisol testing; however, so far only a few studies have reported thorough biological validation based on LC-MS/MS data. In this study, we provide a thorough analytical, pre-analytical and biological validation of an LC-MS/MS method for the measurement of salivary cortisol (F) and of its inactive metabolite cortisone (E). Analytes were extracted from 50μl of saliva, were then separated in 7.5min LC-gradient and detected by negative electrospray ionization-multiple reaction monitoring. The reliability of a widely diffused collection device, Salivette(®), was assessed and the overall procedure was validated. The diurnal cortisol and cortisone fluctuation in saliva and serum was described by a four paired collection protocol (8 am, 12 am, 4 pm and 8 pm) in 19 healthy subjects. The assay allowed the quantitation of F and E down to 39.1 and 78.1pg/ml, with an imprecision range of 5.5-9.5%, 3.9-14.1% and 2.6-14.4%, and an accuracy range of 105.5-113.1%, 88.5-98.7% and 90.7-96.7% for both analytes at low, medium and high levels, respectively. Salivette(®) provided comparable results and better precision (CV<1.0%) as referred to direct spitting (CV<13.0%). A parallel diurnal rhythm in saliva and serum was observed for cortisol and cortisone, with values lowering from the morning to the evening time points (P<0.0001). While salivary E linearly correlated to total serum F (R(2)=0.854, P<0.001), salivary F showed an exponential relationship (R(2)=0.903, P<0.001) with serum F
Downes, Katherine; Terry, Leon A
2010-06-30
Onion soluble non-structural carbohydrates consist of fructose, glucose and sucrose plus fructooligosaccharides (FOS) with degrees of polymerisation (DP) in the range of 3-19. In onion, sugars and FOS are typically separated using liquid chromatography (LC) with acetonitrile (ACN) as a mobile phase. In recent times, however, the production of ACN has diminished due, in part, to the current worldwide economic recession. A study was therefore undertaken, to find an alternative LC method to quantify sugars and FOS from onion without the need for ACN. Two mobile phases were compared; the first taken from a paper by Vågen and Slimestad (2008) using ACN mobile phase, the second, a newly reported method using ethanol (EtOH). The EtOH mobile phase eluted similar concentrations of all FOS compared to the ACN mobile phase. In addition, limit of detection, limit of quantification and relative standard deviation values were sufficiently and consistently lower for all FOS using the EtOH mobile phase. The drawback of the EtOH mobile phase was mainly the inability to separate all individual sugar peaks, yet FOS could be successfully separated. However, using the same onion extract, a previously established LC method based on an isocratic water mobile phase could be used in a second run to separate sugars. Although the ACN mobile phase method is more convenient, in the current economic climate a method based on inexpensive and plentiful ethanol is a valid alternative and could potentially be applied to other fresh produce types. In addition to the mobile phase solvent, the effect of extraction solvents on sugar and FOS concentration was also investigated. EtOH is still widely used to extract sugars from onion although previous literature has concluded that MeOH is a superior solvent. For this reason, an EtOH-based extraction method was compared with a MeOH-based method to extract both sugars and FOS. The MeOH-based extraction method was more efficacious at extracting sugars and
Bohm, Detlef A; Stachel, Carolin S; Gowik, Petra
2012-07-01
The presented multi-method was developed for the confirmation of 37 antibiotic substances from the six antibiotic groups: macrolides, lincosamides, quinolones, tetracyclines, pleuromutilines and diamino-pyrimidine derivatives. All substances were analysed simultaneously in a single analytical run with the same procedure, including an extraction with buffer, a clean-up by solid-phase extraction, and the measurement by liquid chromatography tandem mass spectrometry in ESI+ mode. The method was validated on the basis of an in-house validation concept with factorial design by combination of seven factors to check the robustness in a concentration range of 5-50 μg kg(-1). The honeys used were of different types with regard to colour and origin. The values calculated for the validation parameters-decision limit CCα (range, 7.5-12.9 μg kg(-1)), detection capability CCβ (range, 9.4-19.9 μg kg(-1)), within-laboratory reproducibility RSD(wR) (<20% except for tulathromycin with 23.5% and tylvalosin with 21.4 %), repeatability RSD(r) (<20% except for tylvalosin with 21.1%), and recovery (range, 92-106%)-were acceptable and in agreement with the criteria of Commission Decision 2002/657/EC. The validation results showed that the method was applicable for the residue analysis of antibiotics in honey to substances with and without recommended concentrations, although some changes had been tested during validation to determine the robustness of the method.
LC-MS/MS method for the determination of clodronate in human plasma.
Hasan, Mahmoud; Schumacher, Gitta; Seekamp, Anne; Taedken, Tobias; Siegmund, Werner; Oswald, Stefan
2014-11-01
Clodronate belongs to the class of bisphosphonates which are used for the treatment of bone disorders. Due to its high polarity it has a low and highly variable oral bioavailability which results in low plasma concentrations and requires sensitive bioanalytical methods to characterize its pharmacokinetics in human. Here, we describe for the first time the development and validation of a LC-MS/MS assay for the quantification of clodronate in human plasma. The bisphosphonate was isolated from the biological matrix by protein precipitation using perchloric acid (10%), and derivatized with trimethylorthoacetate prior sample clean-up with liquid-liquid extraction using methyl tert-butyl ether. The chromatography was performed using an isocratic elution with ammonium acetate 5mM (85% v/v, pH 3.8) and acetonitrile (15% v/v) as mobile phase with a flow rate of 300μl/min on a reversed-phase column (Supelco Ascentis(®), C18) temporized at 50°C. The mass spectrometric detection was done using the API4000 triple quadruple mass spectrometer monitoring the mass/charge transitions 301.0/145 for clodronate and 305.2/137.1 for the internal standard etidronate. The analytical range was set to 5-800ng/ml, allowing an evaluation of the plasma concentration-time profiles of clodronate for approximately 7-8 half-life (∼24h). The method was validated according to current FDA/EMA guidelines on bioanalytical method validation with respect to specificity, linearity, intra- and inter-day accuracy and precision, matrix effect, recovery as well as stability. The precision of the assay was 0.6-6.9% and 0.6-8.1% for the intra-day and inter-day variability, respectively. The intra-day and inter-day accuracy (error) was 0.6-8.8% and 2.2-4.5%. The recovery of the analyte was low (2-3%) but reproducible over the entire validation range and sufficient to monitor the target concentrations in human plasma. The drug was shown to be stable in plasma at room temperature for at least 3h (96.0±6%) and
Gandhi, Adarsh; Matta, Murali; Garimella, Narayana; Zere, Tesfalem; Weaver, James
2018-06-01
Extensive use and misuse of antibiotics over the past 50 years has contributed to the emergence and spread of antibiotic-resistant bacterial strains, rendering them as a global health concern. To address this issue, a dynamic in vitro hollow-fiber system, which mimics the in vivo environment more closely than the static model, was used to study the emergence of bacterial resistance of Escherichia coli against fosfomycin (FOS). To aid in this endeavor we developed and validated a liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for quantitative analysis of FOS in lysogeny broth. FOS was resolved on a Kinetex HILIC (2.1 × 50 mm, 2.6 μm) column with 2 mm ammonium acetate (pH 4.76) and acetonitrile as mobile phase within 3 min. Multiple reaction monitoring was used to acquire data on a triple quadrupole mass spectrometer. The assay was linear from 1 to 1000 μg/mL. Inter- and intra-assay precision and accuracy were <15% and between ±85 and 115% respectively. No significant matrix effect was observed when corrected with the internal standard. FOS was stable for up to 24 h at room temperature, up to three freeze-thaw cycles and up to 24 h when stored at 4°C in the autosampler. In vitro experimental data were similar to the simulated plasma pharmacokinetic data, further confirming the appropriateness of the experimental design to quantitate antibiotics and study occurrence of antimicrobial resistance in real time. The validated LC-MS/MS assays for quantitative determination of FOS in lysogeny broth will help antimicrobial drug resistance studies. Published 2018. This article is a U.S. Government work and is in the public domain in the USA.
Hessling, Bernd; Büttner, Knut; Hecker, Michael; Becher, Dörte
2013-01-01
Quantitative LC-MALDI is an underrepresented method, especially in large-scale experiments. The additional fractionation step that is needed for most MALDI-TOF-TOF instruments, the comparatively long analysis time, and the very limited number of established software tools for the data analysis render LC-MALDI a niche application for large quantitative analyses beside the widespread LC–electrospray ionization workflows. Here, we used LC-MALDI in a relative quantification analysis of Staphylococcus aureus for the first time on a proteome-wide scale. Samples were analyzed in parallel with an LTQ-Orbitrap, which allowed cross-validation with a well-established workflow. With nearly 850 proteins identified in the cytosolic fraction and quantitative data for more than 550 proteins obtained with the MASCOT Distiller software, we were able to prove that LC-MALDI is able to process highly complex samples. The good correlation of quantities determined via this method and the LTQ-Orbitrap workflow confirmed the high reliability of our LC-MALDI approach for global quantification analysis. Because the existing literature reports differences for MALDI and electrospray ionization preferences and the respective experimental work was limited by technical or methodological constraints, we systematically compared biochemical attributes of peptides identified with either instrument. This genome-wide, comprehensive study revealed biases toward certain peptide properties for both MALDI-TOF-TOF- and LTQ-Orbitrap-based approaches. These biases are based on almost 13,000 peptides and result in a general complementarity of the two approaches that should be exploited in future experiments. PMID:23788530
Noyes, Pamela D.; Lema, Sean C.; Roberts, Simon C.; Cooper, Ellen M.
2014-01-01
Thyroid hormones are critical regulators of normal development and physiological functioning in all vertebrates. Radioimmunoassay (RIA) approaches have been the method of choice for measuring circulating levels of thyroid hormones in vertebrates. While sensitive, RIA-based approaches only allow for a single analyte measurement per assay, can lack concordance across platforms and laboratories, and can be prone to analytical interferences especially when used with fish plasma. Ongoing advances in liquid chromatography tandem mass spectrometry (LC/MS/MS) have led to substantial decreases in detection limits for thyroid hormones and other biomolecules in complex matrices, including human plasma. Despite these advances, current analytical approaches do not allow for the measurement of native thyroid hormone in teleost fish plasma by mass spectrometry and continue to rely on immunoassay. In this study, we developed a new method that allows for the rapid extraction and simultaneous measurement of total T4 (TT4) and total T3 (TT3) in low volumes (50 μL) of fish plasma by LC/MS/MS. Methods were optimized initially in plasma from rainbow trout (Oncorhynchus mykiss) and applied to plasma from other teleost fishes, including fathead minnows (Pimephales promelas), mummichogs (Fundulus heteroclitus), sockeye salmon (Oncorhynchus nerka), and coho salmon (Oncorhynchus kisutch). Validation of method performance with T4- and T3-spiked rainbow trout plasma at 2 and 4 ng/mL produced mean recoveries ranging from 82 to 95 % and 97 to 105 %, respectively. Recovery of 13C12-T4 internal standard in plasma extractions was: 99±1.8 % in rainbow trout, 85±11 % in fathead minnow, 73±5.0 % in mummichog, 73±1.7 % in sockeye salmon, and 80±8.4 % in coho salmon. While absolute levels of thyroid hormones measured in identical plasma samples by LC/MS/MS and RIA varied depending on the assay used, T4/T3 ratios were generally consistent across both techniques. Less variability was measured among
Cirrincione, Lauren R; Penchala, Sujan Dilly; Scarsi, Kimberly K; Podany, Anthony T; Winchester, Lee C; Back, David J; Khoo, Saye H; Fletcher, Courtney V; Siccardi, Marco; Else, Laura J
2018-05-01
Levonorgestrel (LNG) is a synthetic progestin that is available in oral contraceptive tablets, a subdermal implant, and an intrauterine system for contraception. LNG pharmacokinetics are a pivotal determinant of contraceptive efficacy and essential in assessing drug-drug interactions influencing LNG exposure following different routes of LNG administration. A highly sensitive LC-MS/MS method was developed and validated to quantify levonorgestrel in human plasma. Liquid-liquid extraction was utilized with a sample volume of 500 μL to extract levonorgestrel from plasma. Chromatographic separation of LNG was achieved with a Fortis™ C18 (3 μm: 100 mm × 2.1 mm) reverse phase analytical column. The mobile phases consisted of de-ionized water plus 0.1% NH 4 OH (100:0.1%, v/v) (A), and methanol plus 0.1% NH 4 OH (100:0.1%, v/v) (B) delivered as a gradient at a flow rate of 400 μL/min. Detection of LNG and internal standard (D-(-)-norgestrel-d7) was achieved using positive polarity mode monitoring at 313.2-245.2 amu and 320.1-251.2 amu, respectively. The assay was linear over the calibration range of 49.6 to 1500 pg/mL. This method was used to quantify plasma LNG released by subdermal implant in support of a drug interaction study among women with HIV receiving efavirenz- or nevirapine-based antiretroviral therapy. Copyright © 2018 Elsevier B.V. All rights reserved.
Frank, Nancy; Bessaire, Thomas; Tarres, Adrienne; Goyon, Alexandre; Delatour, Thierry
2017-11-01
The increasing number of food frauds using exogenous nitrogen-rich adulterants to artificially raise the protein content for economically motivated adulteration has demonstrated the need for a robust analytical methodology. This method should be applicable for quality control in operations covering a wide range of analyte concentrations to be able to analyse high levels as usually found in adulteration, as well as low levels due to contamination. The paper describes a LC-MS/MS method covering 14 nitrogen-rich adulterants using a simple and fast sample preparation based on dilution and clean-up by dispersive SPE. Quantification is carried out by isotopic dilution reaching LOQs of 0.05-0.20 mg/kg in a broad range of food matrices (infant formula, liquid milk, dairy ingredient, high protein meal, cereal, infant cereal, and meat/fish powder). Validation of seven commodity groups was performed according to SANCO 12571/2013, giving satisfactory results demonstrating the method's fitness for purpose at the validated range at contamination level. Method ruggedness was further assessed by transferring the developed method into another laboratory devoted to routine testing for quality control. Next to the method description, emphasis is placed on challenges and problems appearing during method development as well as validation. They are discussed in detail and solutions are provided.
Validation of a mass spectrometry-based method for milk traces detection in baked food.
Lamberti, Cristina; Cristina, Lamberti; Acquadro, Elena; Elena, Acquadro; Corpillo, Davide; Davide, Corpillo; Giribaldi, Marzia; Marzia, Giribaldi; Decastelli, Lucia; Lucia, Decastelli; Garino, Cristiano; Cristiano, Garino; Arlorio, Marco; Marco, Arlorio; Ricciardi, Carlo; Carlo, Ricciardi; Cavallarin, Laura; Laura, Cavallarin; Giuffrida, Maria Gabriella; Gabriella, Giuffrida Maria
2016-05-15
A simple validated LC-MS/MS-based method was set up to detect milk contamination in bakery products, taking the effects of food processing into account for the evaluation of allergen recovery and quantification. Incurred cookies were prepared at eight levels of milk contamination and were cooked to expose all milk components, including allergenic proteins, to food processing conditions. Remarkable results were obtained in term of sufficiently low LOD and LOQ (1.3 and 4 mg/kg cookies, respectively). Precision was calculated as intra-day repeatability (RSD in the 5-20% range) and inter-day repeatability (4 days; RSD never exceeded 12%). The extraction recovery values ranged from 20% to 26%. Method applicability was evaluated by analysing commercial cookies labelled either as "milk-free" or "may contain milk". Although the ELISA methodology is considered the gold standard for detecting allergens in foods, this robust LC-MS/MS approach should be a useful confirmatory method for assessing and certifying "milk-free" food products. Copyright © 2015 Elsevier Ltd. All rights reserved.
Kovarikova, Petra; Pasakova-Vrbatova, Ivana; Vavrova, Anna; Stariat, Jan; Klimes, Jiri; Simunek, Tomas
2013-03-25
Dexrazoxane (DEX) is the only clinically used drug effective against anthracycline-induced cardiotoxicity and extravasation injury. However, the mechanism of its cardioprotective action still remains elusive. This paucity of comprehensive data is at least partially caused by the analytical difficulties associated with selective and sensitive simultaneous determination of the parent drug and its putative active metabolite ADR-925 in the relevant biological material. The aim of this study was to develop and validate the first LC-MS/MS method for simultaneous determination of DEX and ADR-925 in the isolated rat neonatal ventricular cardiomyocytes (NVCMs) and the cell culture medium. The analysis was performed on a Synergi Polar-RP column using the gradient profile of the mobile phase composed of 2mM ammonium formate and methanol. Electrospray ionization and ion trap mass analyzer were used as ionization and detection techniques, respectively. NVCMs were precipitated with methanol and the cell culture medium samples were diluted with the same solvent prior the LC-MS/MS analysis. The method was validated within the range of 4-80pmol/10(6) NVCMs and 7-70pmol/10(6) NVCMs for DEX and ADR-925, respectively, and at the concentrations of 8-100μM for both compounds in the culture cell medium. The practical applicability of this method was confirmed by the pilot analysis of NVCMs and the corresponding cell medium samples from relevant in vitro experiment. Hence, the LC-MS/MS method developed in this study represents a modern analytical tool suitable for investigation of DEX bioactivation inside the cardiomyocytes. In addition, the basic utility of the method for the analysis of DEX and ADR-925 in plasma samples was proved in a pilot experiment. Copyright © 2013 Elsevier B.V. All rights reserved.
Yuan, Yin; Zhou, Xuan; Li, Jian; Ye, Suofu; Ji, Xiwei; Li, Liang; Zhou, Tianyan; Lu, Wei
2015-04-01
In the current study, a simple, sensitive and rapid analytical method for the determination of dexamethasone was developed and applied to a pharmacokinetic study in nude mice. Using testosterone as an internal standard, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) approach after one-step precipitation with acetonitrile was validated and used to determine the concentrations of dexamethasone in nude mice plasma. The method utilized a simple isocratic reverse phase separation over a Dionex C18 column with a mobile phase composed of acetonitrile-water (40:60, v/v). The analyte was detected by a triple quadrupole tandem mass spectrometer via electrospray and multiple reaction monitoring was employed to select both dexamethasone at m/z 393.0/147.1 and testosterone at m/z 289.5/97.3 in the positive ion mode. The calibration curves were linear (r >0.99) ranging from 2.5 to 500 ng/mL with a lower limit of quantitation of 2.5 ng/mL. The relative standard deviation ranged from 1.69 to 9.22% while the relative error ranged from -1.92 to -8.46%. This method was successfully applied to a preclinical pharmacokinetic study of dexamethasone and its pharmacokinetics was characterized by a two-compartment model with first-order absorption in female nude mice. Copyright © 2014 John Wiley & Sons, Ltd.
Simithy, Johayra; Gill, Gobind; Wang, Yu; Goodwin, Douglas C; Calderón, Angela I
2015-02-17
A simple and reliable liquid chromatography-mass spectrometry (LC-MS) assay has been developed and validated for the kinetic characterization and evaluation of inhibitors of shikimate kinase from Mycobacterium tuberculosis (MtSK), a potential target for the development of novel antitubercular drugs. This assay is based on the direct determination of the reaction product shikimate-3-phosphate (S3P) using electrospray ionization (ESI) and a quadrupole time-of-flight (Q-TOF) detector. A comparative analysis of the kinetic parameters of MtSK obtained by the LC-MS assay with those obtained by a conventional UV-assay was performed. Kinetic parameters determined by LC-MS were in excellent agreement with those obtained from the UV assay, demonstrating the accuracy, and reliability of this method. The validated assay was successfully applied to the kinetic characterization of a known inhibitor of shikimate kinase; inhibition constants and mode of inhibition were accurately delineated with LC-MS.
Yang, Guangjie; Zhang, Fangrong; Deng, Linfang; Chen, Chang; Cheng, Zhongzhe; Huang, Jiangeng; Liu, Jiangyun; Jiang, Hongliang
2016-09-01
Mucuna pruriens, an ancient Indian herbal medicine containing levodopa, is widely used for Parkinson's disease. In order to simultaneously determine levodopa and 1,1-dimethyl-3-carboxy-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline (MD01) in rat plasma, an improved LC-MS/MS method was developed and validated for a pharmacokinetic study in rats orally administered levodopa or Mucuna pruriens extract (MPE). Elimination of matrix effect and improvement of extraction recovery were achieved through systematic optimization of reversed-phase and hydrophilic interaction chromatographic conditions together with sample clean-up procedures. A satisfactory chromatographic performance was obtained with a Thermo Aquasil C18 column (50 × 2.1 mm, 3 µm) using acetonitrile and water containing 0.2% formic acid as mobile phases. Futhermore, sodium metabisulfite and formic acid were used as stabilizers in neat solutions as well as rat plasma. The method was validated in a dynamic range of 20.0-10,000 ng/mL for levodopa and MD01; the intra- and inter-day precision and accuracy were acceptable. The method was successfully utilized to determine the levodopa level in plasma samples of rats administered levodopa or MPE. Pharmacokinetic results showed that an increase in the AUC of levodopa was observed in rats following oral administration of multiple doses of MPE. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.
Cordero, Chiara; Canale, Francesca; Del Rio, Daniele; Bicchi, Carlo
2009-11-01
The present study is focused on flavan-3-ols characterizing the antioxidant properties of fermented tea (Camellia sinensis). These bioactive compounds, object of nutritional claims in commercial products, should be quantified with rigorous analytical procedures whose accuracy and precision have been stated with a certain level of confidence. An HPLC-UV/DAD method, able to detect and quantify flavan-3-ols in infusions and ready-to-drink teas, has been developed for routine analysis and validated by characterizing several performance parameters. The accuracy assessment has been run through a series of LC-MS/MS analyses. Epigallocatechin, (+)-catechin, (-)-epigallocatechingallate, (-)-epicatechin, (-)-gallocatechingallate, (-)-epicatechingallate, and (-)-catechingallate were chosen as markers of the polyphenolic fraction. Quantitative results showed that samples obtained from tea leaves infusion were richer in polyphenolic antioxidants than those obtained through other industrial processes. The influence of shelf-life and packaging material on the flavan-3-ols content was also considered; markers decreased, with an exponential trend, as a function of time within the shelf life while packaging materials demonstrated to influence differently the flavan-3-ol fraction composition over time. The method presented here provides quantitative results with a certain level of confidence and is suitable for a routine quality control of iced teas whose antioxidant properties are object of nutritional claim.
Gajbhiye, Narendra A; Makasana, Jayanti; Kumar, Satyanshu
2015-01-01
A comprehensive experiment was conducted to study the accumulation pattern and determination of three important bioactive compounds namely withaferin-A (WA), 12-deoxywithastramonolide (WO) and withanolide-A (WD) and its determination by the liquid chromatography/electrospray ionization tandem mass spectrometry (LC-ESI-MS-MS) method in root, stem, fruits and leaves of Withania somnifera. A rapid and sensitive LC-ESI-MS-MS method was developed and validated for the determination of these three important bioactive compounds, having same molecular weight. The multiple reaction monitoring method was established by two transitions for each analyte and intense transition used for quantification. Separation of the three analytes was achieved within a run time of 5 min on an RP-18 column using a mobile phase consisting of acetonitrile and 0.1% acetic acid in water in an isocratic condition. The developed method was validated as per the ICH guidelines. The developed method was found to be suitable for identification and quantification of WA, WO and WD in different plant parts such as roots, stems, fruits and leaves of W. somnifera. The accumulation of WA was highest in leaves samples (8.84 ± 0.37 mg/g) and it was 2.23, 5.85 and 27.26 times higher than its concentration in fruits, stems and roots, respectively. WO and WD contents were highest (0.44 ± 0.016 and 0.72 ± 0.016 mg/g, respectively) in root. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.
Li, Xiaofang; Parks, Elizabeth J; McLaren, David G; Lambert, Jennifer E; Cardasis, Helene L; Chappell, Derek L; McAvoy, Thomas; Salituro, Gino; Alon, Achilles; Dennie, Justin; Chakravarthy, Manu; Shankar, Sudha S; Laterza, Omar F; Lassman, Michael E
2016-06-01
A traditional oral fatty acid challenge assesses absorption of triacylglycerol (TG) into the periphery through the intestines, but cannot distinguish the composition or source of fatty acid in the TG. Stable isotope-labeled tracers combined with LC-MRM can be used to identify and distinguish TG synthesized with dietary and stored fatty acids. Concentrations of three abundant TGs (52:2, 54:3 and 54:4) were monitored for incorporation of one or two (2)H11-oleate molecules per TG. This method was subjected to routine assay validation and meets typical requirements for an assay to be used to support clinical studies. Calculations for the fractional appearance rate of TG in plasma are presented along with the intracellular enterocyte precursor pool for 12 study participants.
van Nuland, M; Hillebrand, M J X; Rosing, H; Burgers, J A; Schellens, J H M; Beijnen, J H
2018-03-20
In microdose clinical trials a maximum of 100 μg of drug substance is administered to participants, in order to determine the pharmacokinetic properties of the agents. Measuring low plasma concentrations after administration of a microdose is challenging and requires the use of ulta-sensitive equipment. Novel liquid chromatography-mass spectrometry (LC-MS/MS) platforms can be used for quantification of low drug plasma levels. Here we describe the development and validation of an LC-MS/MS method for quantification of gemcitabine and its metabolite 2',2'-difluorodeoxyuridine (dFdU) in the low picogram per milliliter range to support a microdose trial. The validated assay ranges from 2.5-500 pg/mL for gemcitabine and 250-50,000 pg/mL for dFdU were linear, with a correlation coefficient (r 2 ) of 0.996 or better. Sample preparation with solid phase extraction provided a good and reproducible recovery. All results were within the acceptance criteria of the latest US FDA guidance and EMA guidelines. In addition, the method was successfully applied to measure plasma concentrations of gemcitabine in a patient after administration of a microdose of gemcitabine. Copyright © 2017 Elsevier B.V. All rights reserved.
Napolitano, José G.; Gödecke, Tanja; Lankin, David C.; Jaki, Birgit U.; McAlpine, James B.; Chen, Shao-Nong; Pauli, Guido F.
2013-01-01
The development of analytical methods for parallel characterization of multiple phytoconstituents is essential to advance the quality control of herbal products. While chemical standardization is commonly carried out by targeted analysis using gas or liquid chromatography-based methods, more universal approaches based on quantitative 1H NMR (qHNMR) measurements are being used increasingly in the multi-targeted assessment of these complex mixtures. The present study describes the development of a 1D qHNMR-based method for simultaneous identification and quantification of green tea constituents. This approach utilizes computer-assisted 1H iterative Full Spin Analysis (HiFSA) and enables rapid profiling of seven catechins in commercial green tea extracts. The qHNMR results were cross-validated against quantitative profiles obtained with an orthogonal LC-MS/MS method. The relative strengths and weaknesses of both approaches are discussed, with special emphasis on the role of identical reference standards in qualitative and quantitative analyses. PMID:23870106
Analysis of catecholamines in urine by unique LC/MS suitable ion-pairing chromatography.
Bergmann, Marianne L; Sadjadi, Seyed; Schmedes, Anne
2017-07-01
The catecholamines, epinephrine (E) and norepinephrine (NE) are small polar, hydrophilic molecules, posing significant challenges to liquid chromatography - tandem mass spectrometry (LC-MS/MS) method development. Specifically, these compounds show little retention on conventional reversed-phase liquid chromatography columns. This work presents development and validation of an LC-MS/MS method for determining catecholamines in urine, based on a new approach to ion-pairing chromatography (IPC), in which the ion-pairing reagent (IPR), 1-Heptane Sulfonic Acid (HSA), is added to the extracted samples instead of the mobile phases. A Hamilton STARlet workstation carried out the solid phase extraction of urine samples. The extracted samples were diluted with 60mmol/L HSA and injected on a Kinetex core-shell biphenyl column with conventional LC-MS/MS suitable mobile phases. Chromatographic separation of E and NE was achieved successfully with very stable retention times (RT). In 484 injections, the RTs were steady with a CV of less than ±4%. Furthermore, HSA was separated from E and NE, allowing HSA to be diverted to waste instead of entering the mass spectrometer ion chamber. The method was validated with good analytical performance, and even though the analysis for urinary catecholamines is increasingly being replaced by plasma free metanephrines in diagnosing pheochromocytomas, this work represents the application of a new analytical technique that can be transferred to other small polar molecules, that are difficult to chromatograph on traditional reversed phase columns. Copyright © 2017 Elsevier B.V. All rights reserved.
Simões, Rodrigo Almeida; Bonato, Pierina Sueli; Mirnaghi, Fatemeh S; Bojko, Barbara; Pawliszyn, Janusz
2015-01-01
A high-throughput bioanalytical method using 96-blade thin film microextraction (TFME) and LC-MS/MS for the analysis of repaglinide (RPG) and two of its main metabolites was developed and used for an in vitro metabolism study. The target analytes were extracted from human microsomal medium by a 96-blade-TFME system employing the low-cost prototype 'SPME multi-sampler' using C18 coating. Method validation showed recoveries around 90% for all analytes and was linear over the concentration range of 2-1000 ng ml(-1) for RPG and of 2-500 ng ml(-1) for each RPG metabolite. The method was applied to an in vitro metabolism study of RPG employing human liver microsomes and proved to be very useful for this purpose.
Camino-Sánchez, F J; Zafra-Gómez, A; Oliver-Rodríguez, B; Ballesteros, O; Navalón, A; Crovetto, G; Vílchez, J L
2010-11-01
A rapid, simple and sensitive multi-residue method was developed and validated for the simultaneous quantification and confirmation of 69 pesticides in fruit and vegetables using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The samples were extracted following the quick, easy, cheap, effective, rugged and safe method known as QuEChERS. Mass spectrometric conditions were individually optimised for each analyte in order to achieve maximum sensitivity in multiple reaction monitoring (MRM) mode. Using the developed chromatographic conditions, 69 pesticides can be separated in less than 17 min. Two selected reaction monitoring (SRM) assays were used for each pesticide to obtain simultaneous quantification and identification in one run. With this method in SRM mode, more than 150 pesticides can be analysed and quantified, but their confirmation is not possible in all cases according to the European regulations on pesticide residues. Nine common representative matrices (zucchini, melon, cucumber, watermelon, tomato, garlic, eggplant, lettuce and pepper) were selected to investigate the effect of different matrices on recovery and precision. Mean recoveries ranged from 70% to 120%, with relative standard deviations (RSDs) lower than 20% for all the pesticides. The proposed method was applied to the analysis of more than 2000 vegetable samples from the extensive greenhouse cultivation in the province of Almeria, Spain, during one year. The methodology combines the advantages of both QuEChERS and LC-MS/MS producing a very rapid, sensitive, accurate and reliable procedure that can be applied in routine analytical laboratories. The method was validated and accredited according to UNE-EN-ISO/IEC 17025:2005 international standard (accreditation number 278/LE1027).
Reagen, William K; Lindstrom, Kent R; Thompson, Kathy L; Flaherty, John M
2004-09-01
The widespread use of semi- and nonvolatile organofluorochemicals in industrial facilities, concern about their persistence, and relatively recent advancements in liquid chromatography/mass spectrometry (LC/MS) technology have led to the development of new analytical methods to assess potential worker exposure to airborne organofluorochemicals. Techniques were evaluated for the determination of 19 organofluorochemicals and for total fluorine in ambient air samples. Due to the potential biphasic nature of most of these fluorochemicals when airborne, Occupational Safety and Health Administration (OSHA) versatile sampler (OVS) tubes were used to simultaneously trap fluorochemical particulates and vapors from workplace air. Analytical methods were developed for OVS air samples to quantitatively analyze for total fluorine using oxygen bomb combustion/ion selective electrode and for 17 organofluorochemicals using LC/MS and gas chromatography/mass spectrometry (GC/MS). The experimental design for this validation was based on the National Institute of Occupational Safety and Health (NIOSH) Guidelines for Air Sampling and Analytical Method Development and Evaluation, with some revisions of the experimental design. The study design incorporated experiments to determine analytical recovery and stability, sampler capacity, the effect of some environmental parameters on recoveries, storage stability, limits of detection, precision, and accuracy. Fluorochemical mixtures were spiked onto each OVS tube over a range of 0.06-6 microg for each of 12 compounds analyzed by LC/MS and 0.3-30 microg for 5 compounds analyzed by GC/MS. These ranges allowed reliable quantitation at 0.001-0.1 mg/m3 in general for LC/MS analytes and 0.005-0.5 mg/m3 for GC/MS analytes when 60 L of air are sampled. The organofluorochemical exposure guideline (EG) is currently 0.1 mg/m3 for many analytes, with one exception being ammonium perfluorooctanoate (EG is 0.01 mg/m3). Total fluorine results may be used
An improved method for fast and selective separation of carotenoids by LC-MS.
Abate-Pella, Daniel; Freund, Dana M; Slovin, Janet P; Hegeman, Adrian D; Cohen, Jerry D
2017-11-01
Carotenoids are a large class of compounds that are biosynthesized by condensation of isoprene units in plants, fungi, bacteria, and some animals. They are characteristically highly conjugated through double bonds, which lead to many isomers as well susceptibility to oxidation and other chemical modifications. Carotenoids are important because of their potent antioxidant activity and are the pigments responsible for color in a wide variety of foods. Human consumption is correlated to many health benefits including prevention of cancer, cardiovascular disease, and age-related disease. Extreme hydrophobicity, poor stability, and low concentration in biological samples make these compounds difficult to analyze and difficult to develop analytical methods for aimed towards identification and quantification. Examples in the literature frequently report the use of exotic stationary phases, solvents, and additives, such as ethyl acetate, dichloromethane, and methyl tert-butyl ether that are incompatible with liquid chromatography mass spectrometry (LC-MS). In order to address these issues, we implemented the use of LC-MS friendly conditions using a low-hydrophobicity cyano-propyl column (Agilent Zorbax SB-CN). We successfully differentiated between isomeric carotenoids by optimizing two gradient methods and using a mixture of 11 standards and LC-MS in positive ionization mode. Three complex biological samples from strawberry leaf, chicken feed supplement, and the photosynthetic bacterium Chloroflexus aurantiacus were analyzed and several carotenoids were resolved in these diverse backgrounds. Our results show this methodology is a significant improvement over other alternatives for analyzing carotenoids because of its ease of use, rapid analysis time, high selectivity, and, most importantly, its compatibility with typical LC-MS conditions. Copyright © 2017 Elsevier B.V. All rights reserved.
Automated solid-phase extraction workstations combined with quantitative bioanalytical LC/MS.
Huang, N H; Kagel, J R; Rossi, D T
1999-03-01
An automated solid-phase extraction workstation was used to develop, characterize and validate an LC/MS/MS method for quantifying a novel lipid-regulating drug in dog plasma. Method development was facilitated by workstation functions that allowed wash solvents of varying organic composition to be mixed and tested automatically. Precision estimates for this approach were within 9.8% relative standard deviation (RSD) across the calibration range. Accuracy for replicate determinations of quality controls was between -7.2 and +6.2% relative error (RE) over 5-1,000 ng/ml(-1). Recoveries were evaluated for a wide variety of wash solvents, elution solvents and sorbents. Optimized recoveries were generally > 95%. A sample throughput benchmark for the method was approximately equal 8 min per sample. Because of parallel sample processing, 100 samples were extracted in less than 120 min. The approach has proven useful for use with LC/MS/MS, using a multiple reaction monitoring (MRM) approach.
Analysis of benzodiazepines and their metabolites using DBS cards and LC-MS/MS.
Lee, Heesang; Park, Yujin; Jo, Jiyeong; In, Sangwhan; Park, Yonghoon; Kim, Eunmi; Pyo, Jaesung; Choe, Sanggil
2015-10-01
Dried Blood Spot (DBS) has been used a blood extraction method for inherited metabolic disorder screening since 1960s. With introduction of LC-MS/MS, not only DBS could be used to analysis drugs in small blood volume, but in various fields, such as toxicology, drug therapeutic monitoring, drug diagnostic screening, and illicit drugs. In toxicology field, many drugs (e.g. benzodiazepines, acetaminophen, small molecule drugs) have been tested with DBS. Compared with earlier blood extraction methods (SPE and LLE), DBS has lots of advantages; lower blood volume (less than 50μL), shorter analysis time caused by a more concise analysis procedure and lower cost. We optimized the DBS procedure and LC-MS/MS conditions for 18 benzodiazepines, seven benzodiazepine metabolites, and one z-drug (zolpidem) analysis in blood. 30μL of whole blood was spotted on FTA DMPK card C and dried for 2h in a desiccator. A 6-mm disk was punched and vortexed for 1min in a centrifuge tube with 300μL methanol/acetonitrile mixture (1:1, v/v). After evaporation, redissolved in 100μL mobile phase of LC-MS/MS and 5μL was injected. In the analysis for 26 target compounds in blood, all of the method validation parameters - LLOD, LLOQ, accuracy (intra- and inter-assay), and precision (intra- and inter-assay) - were satisfied with method validation criteria, within 15%. The results of matrix effect, recovery, and process efficiency were good. We developed a fast and reliable sample preparation method using DBS for 26 benzodiazepines, benzodiazepine metabolites, and z-drug (zolpidem). Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.
Determination of chlormequat in pig serum and sow milk by LC-MS/MS.
Poulsen, M E; Christensen, H B; Sørensen, M T; Leffers, H; Andersen, J H
2007-11-01
Chlormequat is a plant growth regulator widely used on cereals, and there is general concern that it may impair human fertility. A LC-MS/MS method for the analysis of chlormequat in milk and serum was developed and validated in connection with an investigation on the effect of chlormequat on pig reproduction. Validation of the method was based on recovery tests at three spiking levels, determined as double determinations and repeated at least four times. Samples were extracted with methanol-water-acetic acid, centrifuged, filtrated and determined by LC-MS/MS. The mean recoveries were in the range 80-110%, and the LOD was 0.2 ng/g for serum and 0.3 ng/g for milk. The values for repeatability and reproducibility were within 2/3 of the limits given by the Horwitz equation. Samples of pig serum (59) and sow milk (27) were analyzed using the method. Chlormequat was determined in four milk samples in the range of 0.4 ng/g to 1.2 ng/g and in all serum samples in the range of 0.2 ng/g-4.0 ng/g.
Lei, Q Paula; Lamb, David H; Shannon, Anthony G; Cai, Xinxing; Heller, Ronald K; Huang, Michael; Zablackis, Earl; Ryall, Robert; Cash, Patricia
2004-12-25
An LC-MS/MS method for determination of the break down product of N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide (EDC) urea derivative, EDU, has been developed and validated for monitoring the residual coupling reagents. Results indicate that the method exhibits suitable specificity, sensitivity, precision, linearity and accuracy for quantification of residual EDU in the presence of meningococcal polysaccharide-diphtheria toxoid conjugate vaccine and other vaccine matrix compounds. The assay has been validated for a detection range of 10-100 ng/mL and then successfully transferred to quality control (QC) lab. This same method has also been applied to the determination of residual diaminohexane (DAH) in the presence of EDU. LC-MS/MS has proven to be useful as a quick and sensitive approach for simultaneous determination of multiple residual compounds in glycoconjugate vaccine samples.
Li, Tengfei; Cui, Zhimin; Wang, Yan; Yang, Wen; Li, Duo; Song, QinXin; Sun, Luning; Ding, Li
2018-03-20
As an orally active iron chelator, deferasirox forms its ion complexes in the prepared plasma samples and LC-MS mobile phase where ferric ion exists, and then comparing with the nominal concentration level, a lower detected concentration level of deferasirox would be obtained after LC-MS analysis, if no proper treatment was adopted. Meanwhile, the phenomenon would be observed that multiple repeat injections of the same deferasirox plasma sample in the same tube would show the lower and lower detected concentration levels of deferasirox, which caused by more and more ferric ions from the injection needle dissolved in the sample solution as multiple repeated injections. The addition of a proper concentration of EDTA in the mobile phase and the sample will competitively inhibit deferasirox from complexing with ferric ion, and prevent the decrease of deferasirox concentration. In this paper, an LC-MS/MS method was developed and validated for the determination of deferasirox in human plasma. To achieve the protein precipitation, the analytes were extracted from aliquots of 200 μL human plasma with acetonitrile. Chromatographic separation was performed on an ODS-C18 column with the mobile phase consisted of methanol and 0.1% formic acid containing 0.04 mM ethylenediamine tetraacetate dihydrate (EDTA) (80:20, v/v) at a flow rate of 0.5 mL/min. Deferasirox and the internal standard (IS, mifepristone) were detected using electrospray ionization in positive ion multiple reaction monitoring mode by monitoring the precursor-to-product ion transitions m/z 374.2 → 108.1 for deferasirox and m/z 430.1 → 372.2 for the IS. The method exhibited good linearity over the concentration range of 0.04-40 μg/mL for deferasirox. The method was successfully applied to a pharmacokinetic study in 10 Chinese healthy volunteers after oral administration of deferasirox. Copyright © 2018 Elsevier B.V. All rights reserved.
Mochizuki, Ayumi; Ieki, Katsunori; Kamimori, Hiroshi; Nagao, Akemi; Nakai, Keiko; Nakayama, Akira; Nanba, Eitaro
2018-04-01
The guidance and several guidelines on bioanalytical method validation, which were issued by the US FDA, EMA and Ministry of Health, Labour and Welfare, list the 'full' validation parameters; however, none of these provide any details for 'partial' validation. Japan Bioanalysis Forum approved a total of three annual discussion groups from 2012 to 2014. In the discussion groups, members from pharmaceutical companies and contract research organizations discussed the details of partial validation from a risk assessment viewpoint based on surveys focusing on bioanalysis of small molecules using LC-MS/MS in Japan. This manuscript presents perspectives and recommendations for most conceivable changes that can be made to full and partial validations by members of the discussion groups based on their experiences and discussions at the Japan Bioanalysis Forum Symposium.
Xu, Mingzhen; Ni, Yang; Li, Shihong; Du, Juan; Li, Huqun; Zhou, Ying; Li, Weiyong; Chen, Hui
2016-08-01
A simple liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was firstly developed and validated for simultaneous determination of netupitant and palonosetron in human plasma using ibrutinib as the internal standard (IS). Following liquid-liquid extraction, the compounds were eluted isocratically on a Phenomenex C18 column (50mm×2.0mm, 3μm) with the mobile phase consisting of acetonitrile and 10mM ammonium acetate buffer (pH 9.0) (89:11, v/v) at the flow rate of 0.3mL/min. The monitored ion transitions were m/z 579.5→522.4 for netupitant, m/z 297.3→110.2 for palonosetron and m/z 441.2→138.1 for IS. Chromatographic run time was 2.5min per injection, which made it possible to analyze more than 300 of samples per day. The assay exhibited a linear dynamic range of 5-1000ng/mL for netupitant and 0.02-10ng/mL for palonosetron in plasma. The values for both within- and between-day precision and accuracy were well within the generally accepted criteria for analytical methods (<15%). Selectivity, linearity, lower limit of quantification (LLOQ), accuracy, precision, stability, matrix effect, recovery and carry-over effect were evaluated for all analytes. The method is simple, rapid, and has been applied successfully to a pharmacokinetic study of netupitant and palonosetron in healthy volunteers. Copyright © 2016 Elsevier B.V. All rights reserved.
Kallem, Raja Reddy; Jillela, Bhupathi; Ravula, Arun Reddy; Samala, Ramakrishna; Andy, Adinarayana; Ramesh, Mullangi; Rao, Jvln Seshagiri
2016-06-01
A selective, sensitive and rapid LC-MS/MS method has been developed and validated for quantification of the phenelzine (PZ) in 200μL of human plasma using hydroxyzine (HZ) as an internal standard (IS) as per regulatory guidelines. The sample preparation involved the derivatization of PZ using pentaflurobenzaldehyde followed by solid phase extraction process to extract PZ and HZ from human plasma. LC-MS/MS was operated under the multiple reaction-monitoring mode (MRM) using the electro spray ionization technique in positive ion mode and the transitions of m/z 305.1→105.1 and m/z 375.3→201.1 were used to measure the derivative of PZ and IS, respectively. The total run time was 3.5min and the elution of PZ and HZ occurred at 2.53, and 1.92min, respectively; this was achieved with a mobile phase consisting of 10mM ammonium acetate: acetonitrile (20:80, v/v) at a flow rate of 1.0mL/min on an Ace C18 column with a split ratio of 70:30. The developed method was validated in human plasma with a lower limit of quantitation 0.51ng/mL. A linear response function was established for the range of concentrations 0.51-25.2ng/mL (r>0.995) for PZ. The intra- and inter-day precision values met the acceptance criteria. PZ was stable in the battery of stability studies viz., stock solution, bench-top, auto-sampler, long-term and freeze/thaw cycles. The developed assay method was applied to an oral bioequivalence study in humans. Copyright © 2016 Elsevier B.V. All rights reserved.
Glycidyl fatty acid esters in food by LC-MS/MS: method development.
Becalski, A; Feng, S Y; Lau, B P-Y; Zhao, T
2012-07-01
An improved method based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the analysis of glycidyl fatty acid esters in oils was developed. The method incorporates stable isotope dilution analysis (SIDA) for quantifying the five target analytes: glycidyl esters of palmitic (C16:0), stearic (C18:0), oleic (C18:1), linoleic (C18:2) and linolenic acid (C18:3). For the analysis, 10 mg sample of edible oil or fat is dissolved in acetone, spiked with deuterium labelled analogs of glycidyl esters and purified by a two-step chromatography on C18 and normal silica solid phase extraction (SPE) cartridges using methanol and 5% ethyl acetate in hexane, respectively. If the concentration of analytes is expected to be below 0.5 mg/kg, 0.5 g sample of oil is pre-concentrated first using a silica column. The dried final extract is re-dissolved in 250 μL of a mixture of methanol/isopropanol (1:1, v/v), 15 μL is injected on the analytical C18 LC column and analytes are eluted with 100% methanol. Detection of target glycidyl fatty acid esters is accomplished by LC-MS/MS using positive ion atmospheric pressure chemical ionization operating in Multiple Reaction Monitoring mode monitoring 2 ion transitions for each analyte. The method was tested on replicates of a virgin olive oil which was free of glycidyl esters. The method detection limit was calculated to be in the range of 70-150 μg/kg for each analyte using 10 mg sample and 1-3 μg/kg using 0.5 g sample of oil. Average recoveries of 5 glycidyl esters spiked at 10, 1 and 0.1 mg/kg were in the range 84% to 108%. The major advantage of our method is use of SIDA for all analytes using commercially available internal standards and detection limits that are lower by a factor of 5-10 from published methods when 0.5 g sample of oil is used. Additionally, MS/MS mass chromatograms offer greater specificity than liquid chromatography-mass spectrometry operated in selected ion monitoring mode. The method will be applied to
A new LC-MS/MS bioanalytical method for perindopril and perindoprilat in human plasma and milk.
Lwin, Ei Mon Phyo; Gerber, Cobus; Song, Yunmei; Leggett, Catherine; Ritchie, Usha; Turner, Sean; Garg, Sanjay
2017-10-01
A first of its kind, simple, rapid, and sensitive liquid chromatography mass spectrometry (LC-MS/MS) method was developed and validated for quantification of perindopril and perindoprilat in both human plasma and breast milk. The analytes and internal standards (phenazone and acetyl salicylic acid) were extracted from biological matrices by protein precipitation. A Phenomenex® C-18 column was used to provide an appropriate chromatographic separation of the analytes, followed by detection with tandem mass spectrometry. Gradient chromatographic and mass spectrometric detection conditions with mobile phases (A: 5% methanol + 0.1% formic acid in water v/v, and B: 95% methanol + 0.1% formic acid in water v/v) were developed to achieve a LOQ of 0.5 ng/mL in both human plasma and milk. The method was suitable of evaluating clinical samples. The mass transition was followed as m/z 369.10/172.00 for perindopril, m/z 339.00/168.10 for perindoprilat, m/z 188.90/55.95 for phenazone, and m/z 179.04/137.02 for acetyl salicylic acid. The developed method was optimized and validated with a linear range of 0.1-200 ng/mL (r 2 = better than 0.99 for both perindopril and perindoprilat). The precision and accuracy values were within 15% CV. The overall recovery of the analytes was 80-110%. The method has good specificity and repeatability. Stability studies were conducted in both human plasma and bovine milk for up to 3 months, at the storage conditions of 25, 4, and -80 °C.
Reassessment of the Access Testosterone chemiluminescence assay and comparison with LC-MS method.
Dittadi, Ruggero; Matteucci, Mara; Meneghetti, Elisa; Ndreu, Rudina
2018-03-01
To reassess the imprecision and Limit of Quantitation, to evaluate the cross-reaction with dehydroepiandrosterone-sulfate (DHEAS), the accuracy toward liquid chromatography-mass spectrometry (LC-MS) and the reference interval of the Access Testosterone method, performed by DxI immunoassay platform (Beckman Coulter). Imprecision was evaluated testing six pool samples assayed in 20 different run using two reagents lots. The cross-reaction with DHEAS was studied both by a displacement curve and by spiking DHEAS standard in two serum samples with known amount of testosterone. The comparison with LC-MS was evaluated by Passing-Bablock analysis in 21 routine serum samples and 19 control samples from an External Quality Assurance (EQA) scheme. The reference interval was verified by an indirect estimation on 2445 male and 2838 female outpatients. The imprecision study showed a coefficient of variation (CV) between 2.7% and 34.7% for serum pools from 16.3 and 0.27 nmol/L. The value of Limit of Quantitation at 20% CV was 0.53 nmol/L. The DHEAS showed a cross-reaction of 0.0074%. A comparison with LC-MS showed a trend toward a slight underestimation of immunoassay vs LC-MS (Passing-Bablock equations: DxI=-0.24+0.906 LCMS in serum samples and DxI=-0.299+0.981 LCMS in EQA samples). The verification of reference interval showed a 2.5th-97.5th percentile distribution of 6.6-24.3 nmol/L for male over 14 years and <0.5-2.78 nmol/L for female subjects, in accord with the reference intervals reported by the manufacturer. The Access Testosterone method could be considered an adequately reliable tool for the testosterone measurement. © 2017 Wiley Periodicals, Inc.
Quantifying MMA by SLE LC-MS/MS: Unexpected challenges in assay development.
Lo, Sheng-Ying; Gordon, Cindy; Sadilkova, Katerina; Jack, Rhona M; Dickerson, Jane A
2016-09-01
Analysis of serum/plasma methylmalonic acid (MMA) is important for the diagnosis and management of methylmalonic acidemia in pediatric populations. This work focuses on developing and validating a liquid chromatography tandem mass spectrometry (LC-MS/MS) method to monitor methylmalonic acidemia using a simple method preparation. MMA and stable isotope labeled d3-MMA was extracted using supported liquid extraction (SLE). Assay imprecision, bias, linearity, recovery and carryover were determined. The relationship between MMA and propionyl acylcarnitine (C3-acylcarnitine) was also evaluated using historical paired results from 51 unique individuals. Baseline separation between MMA and succinic acid was completed in 7min. The assay was linear from 0.1 to 500μM. The intra-day and inter-day imprecision CV ranged from 4.1 to 13.2% (0.3 to 526μM) and 5.0 to 15.7% (0.3 to 233μM), respectively. Recovery ranged from 93 to 125%. The correlation with a national reference laboratory LC-MS/MS assay showed a Deming regression of 1.026 and intercept of -1.335. Carryover was determined to be <0.04%. Patient-specific correlation was observed between MMA and C3-acylcarnitine. This report describes the first LC-MS/MS method using SLE for MMA extraction. In addition, we illustrate the challenges encountered during this method development that should be assessed and resolved by any laboratory implementing a SLE LC-MS/MS assay designed to quantify analytes across several orders of magnitude. Copyright © 2016 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
Analysis of Ethanolamines: Validation of Semi-Volatile Analysis by HPLC-MS/MS by EPA Method MS888
DOE Office of Scientific and Technical Information (OSTI.GOV)
Owens, J; Vu, A; Koester, C
The Environmental Protection Agency's (EPA) Region 5 Chicago Regional Laboratory (CRL) developed a method titled 'Analysis of Diethanolamine, Triethanolamine, n-Methyldiethanolamine, and n-Ethyldiethanolamine in Water by Single Reaction Monitoring Liquid Chromatography/Tandem Mass Spectrometry (LC/MS/MS): EPA Method MS888'. This draft standard operating procedure (SOP) was distributed to multiple EPA laboratories and to Lawrence Livermore National Laboratory, which was tasked to serve as a reference laboratory for EPA's Environmental Reference Laboratory Network (ERLN) and to develop and validate analytical procedures. The primary objective of this study was to validate and verify the analytical procedures described in 'EPA Method MS888' for analysis of themore » listed ethanolamines in aqueous samples. The gathered data from this validation study will be used to: (1) demonstrate analytical method performance; (2) generate quality control acceptance criteria; and (3) revise the SOP to provide a validated method that would be available for use during a homeland security event. The data contained in this report will be compiled, by EPA CRL, with data generated by other EPA Regional laboratories so that performance metrics of 'EPA Method MS888' can be determined.« less
A multiclass multiresidue LC-MS/MS method for analysis of veterinary drugs in bovine kidney
USDA-ARS?s Scientific Manuscript database
The increased efficiency permitted by multiclass, multiresidue methods has made such approaches very attractive to laboratories involved in monitoring veterinary drug residues in animal tissues. In this current work, evaluation of a multiclass multiresidue LC-MS/MS method in bovine kidney is describ...
Balaji, Narayanan; Sulochana, Suresh P; Saini, Neeraj Kumar; A, Siva Kumar; Mullangi, Ramesh
2018-05-01
A simple, selective and reliable LC-MS/MS method was validated for simultaneous quantitation of darolutamide diastereomers in 50 μL mouse plasma using warfarin as an internal standard (IS) as per regulatory guidelines. Plasma samples were extracted by liquid-liquid extraction and the chromatographic separation was achieved on a Chiralpak IA column with an isocratic mobile phase 5 mm ammonium acetate-absolute alcohol (20:80, v/v) at a flow rate of 1.0 mL/min. Detection and quantitation was done in multiple reaction monitoring mode following the transitions m/z 397 → 202 and 307 → 250 for darolutamide diastereomers and the IS, respectively, in the negative ionization mode. The linearity range was 100-2400 ng/mL for each diastereomer. The intra- and inter-day precisions were in the ranges of 1.78-4.20 and 4.34-14.6, and 3.63-4.74 and 4.78-5.15 for diastereomer-1 and diastereomer-2, respectively. Both diastereomers were found to be stable under different stability conditions. The validated method was applied to a pharmacokinetic study in mice. Following oral administration of darolutamide at 10 mg/kg, maximum concentration in plasma was 4189 and 726 ng/mL for diastereomer-1 and diastereomer-2, respectively. The terminal half-life was found to be ~0.50 h for both the diastereomers. The AUC (0-t) was found to be 18,961 ng*h/mL for diastereomer-1 and 1340 ng*h/mL diastereomer-2. Copyright © 2018 John Wiley & Sons, Ltd.
Gesquiere, Laurence R.; Ziegler, Toni E.; Chen, Patricia A.; Epstein, Katherine A.; Alberts, Susan C.; Altmann, Jeanne
2014-01-01
The development of non-invasive methods, particularly fecal determination, has made possible the assessment of hormone concentrations in wild animal populations. However, measuring fecal metabolites needs careful validation for each species and for each sex. We investigated whether radioimmunoassays (RIAs) previously used to measure fecal testosterone (fT) in male baboons and fecal estrogens (fE) in female baboons were well suited to measure these hormones in the opposite sex. We compared fE and fT concentrations determined by RIA to those measured by liquid chromatography combined with triple quadropole mass spectrometry (LC/MS/MS), a highly specific method. Additionally, we conducted a biological validation to assure that the measurements of fecal concentrations reflected physiological levels of the hormone of interest. Several tests produced expected results that led us to conclude that our RIAs can reliably measure fT and fE in both sexes, and that within-sex comparisons of these measures are valid: (i) fTRIA were significantly correlated to fTLC/MS/MS for both sexes; (ii) fTRIA were higher in adult than in immature males; (iii) fTRIA were higher in pregnant than non-pregnant females; (iv) fERIA were correlated with 17β-estradiol (fE2) and with estrone (fE1) determined by LC/MS/MS in pregnant females; (v) fERIA were significantly correlated with fE2 in non-pregnant females and nearly significantly correlated in males; (vi) fERIA were higher in adult males than in immature males. fERIA were higher in females than in males, as predicted, but unexpectedly, fTRIA were higher in females than in males, suggesting a difference in steroid metabolism in the two sexes; consequently, we conclude that while within-sex comparisons are valid, fTRIA should not be used for intersexual comparisons. Our results should open the field to important additional studies, as to date the roles of testosterone in females and estrogens in males have been little investigated. PMID
Milosheska, Daniela; Roškar, Robert
2017-05-10
The aim of the present report was to develop and validate simple, sensitive and reliable LC-MS/MS method for quantification of topiramate (TPM) and its main metabolites: 2,3-desisopropylidene TPM, 4,5-desisopropylidene TPM, 10-OH TPM and 9-OH TPM in human plasma samples. The most abundant metabolite 2,3-desisopropylidene TPM was isolated from patients urine, characterized and afterwards used as an authentic standard for method development and validation. Sample preparation method employs 100μL of plasma sample and liquid-liquid extraction with a mixture of ethyl acetate and diethyl ether as extraction solvent. Chromatographic separation was achieved on a 1290 Infinity UHPLC coupled to 6460 Triple Quad Mass Spectrometer operated in negative MRM mode using Kinetex C18 column (50×2.1mm, 2.6μm) by gradient elution using water and methanol as a mobile phase and stable isotope labeled TPM as internal standard. The method showed to be selective, accurate, precise and linear over the concentration ranges of 0.10-20μg/mL for TPM, 0.01-2.0μg/mL for 2,3-desisopropylidene TPM, and 0.001-0.200μg/mL for 4,5-desisopropylidene TPM, 10-OH TPM and 9-OH TPM. The described method is the first fully validated method capable of simultaneous determination of TPM and its main metabolites in plasma over the selected analytical range. The suitability of the method was successfully demonstrated by the quantification of all analytes in plasma samples of patients with epilepsy and can be considered as reliable analytical tool for future investigations of the TPM metabolism. Copyright © 2017 Elsevier B.V. All rights reserved.
Dash, Alekha K; Sawhney, Angeli
2002-07-31
The objective of this study was to develop a simple and sensitive LC method for the determination of creatine and creatinine in various creatine supplement formulations. The chromatographic system comprised of a LC-600 pump, SCL-6B system controller, and SPD-6AV detector (Shimadzu, Japan). The mobile phase consisted of 0.045 M ammonium sulfate in water. The chromatographic separation was achieved at ambient temperature on a Betabasic C-18 column (250 x 4.6 mm, Keystone Sci.). The flow rate was maintained at 0.75 ml/min and effluents are monitored at 205 nm. 4-(2-Aminoethyl)benzene sulfonamide was used as an internal standard (IS). This method required less than 7 min of chromatographic time. The standard curves were linear over the concentration range of 1-100 microg/ml for creatine and 2-100 microg/ml for creatinine, respectively. The relative standard deviations (RSD) for the within-day and day-to-day precision for creatine were within 1.0-4.6 and 2.2-4.7%, respectively. The RSD for the accuracy of creatine assay was in the range of 2.4-4.7%. The RSD values for the within-day precision, day-to-day precision and accuracy for creatinine validation were 1.7-4.4, 2.3-5.4 and 2.4-4.8%, respectively. This method was used to determine: (i) the creatine concentration in various marketed products; (ii) saturated solubility of various creatine salts; and (iii) stability of creatine in aqueous solution. In conclusion, a simple and sensitive LC method with UV detection was developed for the simultaneous determination of creatine and creatinine in formulations. Di-creatine citrate salt showed a higher aqueous solubility (at 25 degrees C) as compared to creatine and creatine monohydrate. Some of the over-the-counter (OTC) products tested contained a very low level of creatine in contrast to their label claim. Substantial conversion of creatine into creatinine was noticed in liquid formulation.
Workshop Report: Crystal City VI-Bioanalytical Method Validation for Biomarkers.
Arnold, Mark E; Booth, Brian; King, Lindsay; Ray, Chad
2016-11-01
With the growing focus on translational research and the use of biomarkers to drive drug development and approvals, biomarkers have become a significant area of research within the pharmaceutical industry. However, until the US Food and Drug Administration's (FDA) 2013 draft guidance on bioanalytical method validation included consideration of biomarker assays using LC-MS and LBA, those assays were created, validated, and used without standards of performance. This lack of expectations resulted in the FDA receiving data from assays of varying quality in support of efficacy and safety claims. The AAPS Crystal City VI (CC VI) Workshop in 2015 was held as the first forum for industry-FDA discussion around the general issues of biomarker measurements (e.g., endogenous levels) and specific technology strengths and weaknesses. The 2-day workshop served to develop a common understanding among the industrial scientific community of the issues around biomarkers, informed the FDA of the current state of the science, and will serve as a basis for further dialogue as experience with biomarkers expands with both groups.
Vialaret, Jérôme; Picas, Alexia; Delaby, Constance; Bros, Pauline; Lehmann, Sylvain; Hirtz, Christophe
2018-06-01
Hepcidin-25 peptide is a biomarker which is known to have considerable clinical potential for diagnosing iron-related diseases. Developing analytical methods for the absolute quantification of hepcidin is still a real challenge, however, due to the sensitivity, specificity and reproducibility issues involved. In this study, we compare and discuss two MS-based assays for quantifying hepcidin, which differ only in terms of the type of liquid chromatography (nano LC/MS versus standard LC/MS) involved. The same sample preparation, the same internal standards and the same MS analyzer were used with both approaches. In the field of proteomics, nano LC chromatography is generally known to be more sensitive and less robust than standard LC methods. In this study, we established that the performances of the standard LC method are equivalent to those of our previously developed nano LC method. Although the analytical performances were very similar in both cases. The standard-flow platform therefore provides the more suitable alternative for accurately determining hepcidin in clinical settings. Copyright © 2018 Elsevier B.V. All rights reserved.
Born, Christoph; Amann, Benedikt L; Grunze, Heinz; Post, Robert M; Schärer, Lars
2014-05-07
Careful observation of the longitudinal course of bipolar disorders is pivotal to finding optimal treatments and improving outcome. A useful tool is the daily prospective Life-Chart Method, developed by the National Institute of Mental Health. However, it remains unclear whether the patient version is as valid as the clinician version. We compared the patient-rated version of the Lifechart (LC-self) with the Young-Mania-Rating Scale (YMRS), Inventory of Depressive Symptoms-Clinician version (IDS-C), and Clinical Global Impression-Bipolar version (CGI-BP) in 108 bipolar I and II patients who participated in the Naturalistic Follow-up Study (NFS) of the German centres of the Bipolar Collaborative Network (BCN; formerly Stanley Foundation Bipolar Network). For statistical evaluation, levels of severity of mood states on the Lifechart were transformed numerically and comparison with affective scales was performed using chi-square and t tests. For testing correlations Pearson´s coefficient was calculated. Ratings for depression of LC-self and total scores of IDS-C were found to be highly correlated (Pearson coefficient r = -.718; p < .001), whilst the correlation of ratings for mania with YMRS compared to LC-self were slightly less robust (Pearson coefficient r = .491; p = .001). These results were confirmed by good correlations between the CGI-BP IA (mania), IB (depression) and IC (overall mood state) and the LC-self ratings (Pearson coefficient r = .488, r = .721 and r = .65, respectively; all p < .001). The LC-self shows a significant correlation and good concordance with standard cross sectional affective rating scales, suggesting that the LC-self is a valid and time and money saving alternative to the clinician-rated version which should be incorporated in future clinical research in bipolar disorder. Generalizability of the results is limited by the selection of highly motivated patients in specialized bipolar centres and by the
Li, Bo; Tang, Jing; Yang, Qingxia; Cui, Xuejiao; Li, Shuang; Chen, Sijie; Cao, Quanxing; Xue, Weiwei; Chen, Na; Zhu, Feng
2016-12-13
In untargeted metabolomics analysis, several factors (e.g., unwanted experimental &biological variations and technical errors) may hamper the identification of differential metabolic features, which requires the data-driven normalization approaches before feature selection. So far, ≥16 normalization methods have been widely applied for processing the LC/MS based metabolomics data. However, the performance and the sample size dependence of those methods have not yet been exhaustively compared and no online tool for comparatively and comprehensively evaluating the performance of all 16 normalization methods has been provided. In this study, a comprehensive comparison on these methods was conducted. As a result, 16 methods were categorized into three groups based on their normalization performances across various sample sizes. The VSN, the Log Transformation and the PQN were identified as methods of the best normalization performance, while the Contrast consistently underperformed across all sub-datasets of different benchmark data. Moreover, an interactive web tool comprehensively evaluating the performance of 16 methods specifically for normalizing LC/MS based metabolomics data was constructed and hosted at http://server.idrb.cqu.edu.cn/MetaPre/. In summary, this study could serve as a useful guidance to the selection of suitable normalization methods in analyzing the LC/MS based metabolomics data.
Li, Bo; Tang, Jing; Yang, Qingxia; Cui, Xuejiao; Li, Shuang; Chen, Sijie; Cao, Quanxing; Xue, Weiwei; Chen, Na; Zhu, Feng
2016-01-01
In untargeted metabolomics analysis, several factors (e.g., unwanted experimental & biological variations and technical errors) may hamper the identification of differential metabolic features, which requires the data-driven normalization approaches before feature selection. So far, ≥16 normalization methods have been widely applied for processing the LC/MS based metabolomics data. However, the performance and the sample size dependence of those methods have not yet been exhaustively compared and no online tool for comparatively and comprehensively evaluating the performance of all 16 normalization methods has been provided. In this study, a comprehensive comparison on these methods was conducted. As a result, 16 methods were categorized into three groups based on their normalization performances across various sample sizes. The VSN, the Log Transformation and the PQN were identified as methods of the best normalization performance, while the Contrast consistently underperformed across all sub-datasets of different benchmark data. Moreover, an interactive web tool comprehensively evaluating the performance of 16 methods specifically for normalizing LC/MS based metabolomics data was constructed and hosted at http://server.idrb.cqu.edu.cn/MetaPre/. In summary, this study could serve as a useful guidance to the selection of suitable normalization methods in analyzing the LC/MS based metabolomics data. PMID:27958387
Determination of benzoylurea insecticides in food by pressurized liquid extraction and LC-MS.
Brutti, Monia; Blasco, Cristina; Picó, Yolanda
2010-01-01
A method based on pressurized liquid extraction and LC-MS/MS has been developed for determining nine benzoylureas (BUs) in fruit, vegetable, cereals, and animal products. Samples (5 g) were homogenized with diatomaceous earth and extracted in a 22 mL cell with 22 mL of ethyl acetate at 80 degrees C and 1500 psi. After solvent concentration and exchange to methanol, BUs were analyzed by LC-MS/MS using an IT mass analyzer, which achieved several transitions of precursor ions that increase selectivity providing identification. LOQs were between 0.002 and 0.01 mg/kg, which are equal or lower than maximum residue limits established by the Codex Alimentarius. Excellent linearity was achieved over a range of concentrations from 0.01 to 1 mg/kg with correlation coefficients 0.995-0.999 (n=7). Validation of the total method was performed by analyzing in quintuplicate seven different commodities (milk, eggs, meat, rice, lettuce, avocado, and lemon) at three concentration levels (0.01, 0.1, and 1 mg/kg). The recoveries ranged from 58 to 97% and the RSDs from 5 to 19% depending on the compound and the commodity. The combination of pressurized liquid extraction with LC-MS/MS provides a sensitive and selective method for the determination of BUs in food.
Dolder, Patrick C; Liechti, Matthias E; Rentsch, Katharina M
2015-02-01
Lysergic acid diethylamide (LSD) is a widely used recreational drug. The aim of the present study is to develop a quantitative turboflow LC-MS/MS method that can be used for rapid quantification of LSD and its main metabolite 2-oxo-3-hydroxy LSD (O-H-LSD) in serum and urine in emergency toxicological cases without time-consuming extraction steps. The method was developed on an ion-trap LC-MS/MS instrument coupled to a turbulent-flow extraction system. The validation data showed no significant matrix effects and no ion suppression has been observed in serum and urine. Mean intraday accuracy and precision for LSD were 101 and 6.84%, in urine samples and 97.40 and 5.89% in serum, respectively. For O-H-LSD, the respective values were 97.50 and 4.99% in urine and 107 and 4.70% in serum. Mean interday accuracy and precision for LSD were 100 and 8.26% in urine and 101 and 6.56% in serum, respectively. For O-H-LSD, the respective values were 101 and 8.11% in urine and 99.8 and 8.35% in serum, respectively. The lower limit of quantification for LSD was determined to be 0.1 ng/ml. LSD concentrations in serum were expected to be up to 8 ng/ml. 2-Oxo-3-hydroxy LSD concentrations in urine up to 250 ng/ml. The new method was accurate and precise in the range of expected serum and urine concentrations in patients with a suspected LSD intoxication. Until now, the method has been applied in five cases with suspected LSD intoxication where the intake of the drug has been verified four times with LSD concentrations in serum in the range of 1.80-14.70 ng/ml and once with a LSD concentration of 1.25 ng/ml in urine. In serum of two patients, the O-H-LSD concentration was determined to be 0.99 and 0.45 ng/ml. In the urine of a third patient, the O-H-LSD concentration was 9.70 ng/ml.
Teitelbaum, Aaron M.; Flaker, Alicia M.; Kharasch, Evan D.
2016-01-01
A stereoselective analytical method was developed and validated for the quantification of bupropion, and principle metabolites hydroxybupropion, erythrohydrobupropion and threohydrobupropion in human plasma. Separation of individual enantiomers (R)-bupropion, (S)-bupropion, (R,R)-hydroxybupropion, (S,S-hydroxybupropion), (1S,2S)-threohydrobupropion, (1R,2R)-threohydrobupropion, (1R,2S)-erythrohydrobupropion, and (1S,2R)-erythrohydrobupropion was achieved utilizing an α1-acid glycoprotein column within a 12-minute run time. Chromatograph separation was significantly influenced by mobile phase pH and variability between columns. Analytes were quantified by positive ion electrospray tandem mass spectrometry following plasma protein precipitation with 20% trichloroacetic acid. Identification of erythrohydrobupropion enantiomer peaks and threohydrobupropion enantiomer peaks was achieved by sodium borohydride reduction of enantiopure (R)- and (S)-bupropion. Initial assay validation and sensitivity determination was on AB Sciex 3200, 4000 QTRAP, and 6500 mass spectrometers. Accuracy and precision were within 15% for each analyte. The assay was fully validated over analyte-specific concentrations using an AB Sciex 3200 mass spectrometer. Intra- and inter-assay precision and accuracy were within 12% for each analyte. The limits of quantification for bupropion (R and S), hydroxybupropion (R,R and S,S), threohydrobupropion (1S,2S and 1R,2R), and erythrohydrobupropion (1R,2S and 1S,2R) were 0.5, 2, 1, and 1 ng/mL, respectively. All analytes were stable following freeze thaw cycles at −80°C and while stored at 4°C in the instrument autosampler. This method was applicable to clinical pharmacokinetic investigations of bupropion in patients. This is the first chromatographic method to resolve erythrohydrobupropion and threohydrobupropion enantiomers, and the first stereoselective LC-MS/MS assay to quantify bupropion, and principle metabolites hydroxybupropion
den Brok, Monique W J; Nuijen, Bastiaan; Hillebrand, Michel J X; Grieshaber, Charles K; Harvey, Michael D; Beijnen, Jos H
2005-09-01
C1311 (5-[[2-(diethylamino)ethyl]amino]-8-hydroxyimidazo [4,5,1-de]-acridin-6-one-dihydrochloride trihydrate) is the lead compound from the group of imidazoacridinones, a novel group of rationally designed anticancer agents. The pharmaceutical development of C1311 necessitated the availability of an assay for the quantification and purity determination of C1311 active pharmaceutical ingredient (API) and its pharmaceutical dosage form. A reversed-phase liquid chromatographic method (RP-LC) with ultraviolet (UV) detection was developed, consisting of separation on a C18 column with phosphate buffer (60 mM; pH 3 with 1 M citric acid)-acetonitrile-triethylamine (83:17:0.05, v/v/v) as the mobile phase and UV-detection at 280 nm. The method was found to be linear over a concentration range of 2.50-100 microg/mL, precise and accurate. Accelerated stress testing showed degradation products, which were well separated from the parent compound, confirming its stability-indicating capacity. Moreover, the use of LC-MS and on-line photo diode array detection enabled us to propose structures for four degradation products. Two of these products were also found as impurities in the API and more abundantly in an impure lot of API.
McGuffey, James E.; Wei, Binnian; Bernert, John T.; Morrow, John C.; Xia, Baoyun; Wang, Lanqing; Blount, Benjamin C.
2014-01-01
Tobacco use is a major contributor to premature morbidity and mortality. The measurement of nicotine and its metabolites in urine is a valuable tool for evaluating nicotine exposure and for nicotine metabolic profiling—i.e., metabolite ratios. In addition, the minor tobacco alkaloids—anabasine and anatabine—can be useful for monitoring compliance in smoking cessation programs that use nicotine replacement therapy. Because of an increasing demand for the measurement of urinary nicotine metabolites, we developed a rapid, low-cost method that uses isotope dilution liquid chromatography-tandem mass spectrometry (LC-MS/MS) for simultaneously quantifying nicotine, six nicotine metabolites, and two minor tobacco alkaloids in smokers' urine. This method enzymatically hydrolyzes conjugated nicotine (primarily glucuronides) and its metabolites. We then use acetone pretreatment to precipitate matrix components (endogenous proteins, salts, phospholipids, and exogenous enzyme) that may interfere with LC-MS/MS analysis. Subsequently, analytes (nicotine, cotinine, hydroxycotinine, norcotinine, nornicotine, cotinine N-oxide, nicotine 1′-N-oxide, anatabine, and anabasine) are chromatographically resolved within a cycle time of 13.5 minutes. The optimized assay produces linear responses across the analyte concentrations typically found in urine collected from daily smokers. Because matrix ion suppression may influence accuracy, we include a discussion of conventions employed in this procedure to minimize matrix interferences. Simplicity, low cost, low maintenance combined with high mean metabolite recovery (76–99%), specificity, accuracy (0–10% bias) and reproducibility (2–9% C.V.) make this method ideal for large high through-put studies. PMID:25013964
Ens, Waldemar; Senner, Frank; Gygax, Benjamin; Schlotterbeck, Götz
2014-05-01
A new method for the simultaneous determination of iodated X-ray contrast media (ICM) and artificial sweeteners (AS) by liquid chromatography-tandem mass spectrometry (LC-MS/MS) operated in positive and negative ionization switching mode was developed. The method was validated for surface, ground, and drinking water samples. In order to gain higher sensitivities, a 10-fold sample enrichment step using a Genevac EZ-2 plus centrifugal vacuum evaporator that provided excellent recoveries (90 ± 6 %) was selected for sample preparation. Limits of quantification below 10 ng/L were obtained for all compounds. Furthermore, sample preparation recoveries and matrix effects were investigated thoroughly for all matrix types. Considerable matrix effects were observed in surface water and could be compensated by the use of four stable isotope-labeled internal standards. Due to their persistence, fractions of diatrizoic acid, iopamidol, and acesulfame could pass the whole drinking water production process and were observed also in drinking water. To monitor the fate and occurrence of these compounds, the validated method was applied to samples from different stages of the drinking water production process of the Industrial Works of Basel (IWB). Diatrizoic acid was found as the most persistent compound which was eliminated by just 40 % during the whole drinking water treatment process, followed by iopamidol (80 % elimination) and acesulfame (85 % elimination). All other compounds were completely restrained and/or degraded by the soil and thus were not detected in groundwater. Additionally, a direct injection method without sample preparation achieving 3-20 ng/L limits of quantification was compared to the developed method.
Validation of an ultra-fast UPLC-UV method for the separation of antituberculosis tablets.
Nguyen, Dao T-T; Guillarme, Davy; Rudaz, Serge; Veuthey, Jean-Luc
2008-04-01
A simple method using ultra performance LC (UPLC) coupled with UV detection was developed and validated for the determination of antituberculosis drugs in combined dosage form, i. e. isoniazid (ISN), pyrazinamide (PYR) and rifampicin (RIF). Drugs were separated on a short column (2.1 mm x 50 mm) packed with 1.7 mum particles, using an elution gradient procedure. At 30 degrees C, less than 2 min was necessary for the complete separation of the three antituberculosis drugs, while the original USP method was performed in 15 min. Further improvements were obtained with the combination of UPLC and high temperature (up to 90 degrees C), namely HT-UPLC, which allows the application of higher mobile phase flow rates. Therefore, the separation of ISN, PYR and RIF was performed in less than 1 min. After validation (selectivity, trueness, precision and accuracy), both methods (UPLC and HT-UPLC) have proven suitable for the routine quality control analysis of antituberculosis drugs in combined dosage form. Additionally, a large number of samples per day can be analysed due to the short analysis times.
Granada, Andréa; Murakami, Fabio S; Sartori, Tatiane; Lemos-Senna, Elenara; Silva, Marcos A S
2008-01-01
A simple, rapid, and sensitive reversed-phase column high-performance liquid chromatographic method was developed and validated to quantify camptothecin (CPT) in polymeric nanocapsule suspensions. The chromatographic separation was performed on a Supelcosil LC-18 column (15 cm x 4.6 mm id, 5 microm) using a mobile phase consisting of methanol-10 mM KH2PO4 (60 + 40, v/v; pH 2.8) at a flow rate of 1.0 mL/min and ultraviolet detection at 254 nm. The calibration graph was linear from 0.5 to 3.0 microg/mL with a correlation coefficient of 0.9979, and the limit of quantitation was 0.35 microg/mL. The assay recovery ranged from 97.3 to 105.0%. The intraday and interday relative standard deviation values were < 5.0%. The validation results confirmed that the developed method is specific, linear, accurate, and precise for its intended use. The current method was successfully applied to the evaluation of CPT entrapment efficiency and drug content in polymeric nanocapsule suspensions during the early stage of formulation development.
Dalmora, Sergio Luiz; Nogueira, Daniele Rubert; D'Avila, Felipe Bianchini; Souto, Ricardo Bizogne; Leal, Diogo Paim
2011-01-01
A stability-indicating capillary zone electrophoresis (CZE) method was validated for the analysis of entecavir in pharmaceutical formulations, using nimesulide as an internal standard. A fused-silica capillary (50 µm i.d.; effective length, 40 cm) was used while being maintained at 25°C; the applied voltage was 25 kV. A background electrolyte solution consisted of a 20 mM sodium tetraborate solution at pH 10. Injections were performed using a pressure mode at 50 mbar for 5 s, with detection at 216 nm. The specificity and stability-indicating capability were proven through forced degradation studies, evaluating also the in vitro cytotoxicity test of the degraded products. The method was linear over the concentration range of 1-200 µg mL(-1) (r(2) = 0.9999), and was applied for the analysis of entecavir in tablet dosage forms. The results were correlated to those of validated conventional and fast LC methods, showing non-significant differences (p > 0.05).
Khaksari, Maryam; Mazzoleni, Lynn R; Ruan, Chunhai; Kennedy, Robert T; Minerick, Adrienne R
2017-04-01
Two separate liquid chromatography (LC)-mass spectrometry (MS) methods were developed for determination and quantification of water-soluble and fat-soluble vitamins in human tear and blood serum samples. The water-soluble vitamin method was originally developed to detect vitamins B 1 , B 2 , B 3 (nicotinamide), B 5 , B 6 (pyridoxine), B 7 , B 9 and B 12 while the fat-soluble vitamin method detected vitamins A, D 3 , 25(OH)D 3, E and K 1 . These methods were then validated with tear and blood serum samples. In this data in brief article, we provide details on the two LC-MS methods development, methods sensitivity, as well as precision and accuracy for determination of vitamins in human tears and blood serum. These methods were then used to determine the vitamin concentrations in infant and parent samples under a clinical study which were reported in "Determination of Water-Soluble and Fat-Soluble Vitamins in Tears and Blood Serum of Infants and Parents by Liquid Chromatography/Mass Spectrometry DOI:10.1016/j.exer.2016.12.007 [1]". This article provides more details on comparison of vitamin concentrations in the samples with the ranges reported in the literature along with the medically accepted normal ranges. The details on concentrations below the limits of detection (LOD) and limits of quantification (LOQ) are also discussed. Vitamin concentrations were also compared and cross-correlated with clinical data and nutritional information. Significant differences and strongly correlated data were reported in [1]. This article provides comprehensive details on the data with slight differences or slight correlations.
Burrai, Lucia; Nieddu, Maria; Carta, Antonio; Trignano, Claudia; Sanna, Raimonda; Boatto, Gianpiero
2016-04-01
A multi-residue analytical method was developed for the determination in amniotic fluid (AF) of 13 illicit phenethylamines, including 12 compounds never investigated in this matrix before. Samples were subject to solid-phase extraction using; hydrophilic-lipophilic balance cartridges which gave good recoveries and low matrix effects on analysis of the extracts. The quantification was performed by liquid chromatography electrospray tandem mass spectrometry. The water-acetonitrile mobile phase containing 0.1% formic acid, used with a C18 reversed phase column, provided adequate separation, resolution and signal-to-noise ratio for the analytes and the internal standard. The final optimized method was validated according to international guidelines. A monitoring campaign to assess fetal exposure to these 13 substances of abuse has been performed on AF test samples obtained from pregnant women. All mothers (n = 194) reported no use of drugs of abuse during pregnancy, and this was confirmed by the analytical data. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.
Analysis of anabolic steroids in human hair using LC-MS/MS.
Deshmukh, Nawed; Hussain, Iltaf; Barker, James; Petroczi, Andrea; Naughton, Declan P
2010-10-01
New highly sensitive, specific, reliable, reproducible and robust LC-MS/MS methods were developed to detect the anabolic steroids, nandrolone and stanozolol, in human hair for the first time. Hair samples from 180 participants (108 males, 72 females, 62% athletes) were screened using ELISA which revealed 16 athletes as positive for stanozolol and 3 for nandrolone. Positive samples were confirmed on LC-MS/MS in selective reaction monitoring (SRM) mode. The assays for stanozolol and nandrolone showed good linearity in the range 1-400pg/mg and 5-400pg/mg, respectively. The methods were validated for LLOD, interday precision, intraday precision, specificity, extraction recovery and accuracy. The assays were capable of detecting 0.5pg stanozolol and 3.0pg nandrolone per mg of hair, when approximately 20mg of hair were processed. Analysis using LC-MS/MS confirmed 11 athletes' positive for stanozolol (5.0pg/mg to 86.3pg/mg) and 1 for nandrolone (14.0pg/mg) thus avoiding false results from ELISA screening. The results obtained demonstrate the application of these hair analysis methods to detect both steroids at low concentrations, hence reducing the amount of hair required significantly. The new methods complement urinalysis or blood testing and facilitate improved doping testing regimes. Hair analysis benefits from non-invasiveness, negligible risk of infection and facile sample storage and collection, whilst reducing risks of tampering and cross-contamination. Owing to the wide detection window, this approach may also offer an alternative approach for out-of-competition testing.
Hroch, Miloš; Mičuda, Stanislav; Havelek, Radim; Cermanová, Jolana; Cahlíková, Lucie; Hošťálková, Anna; Hulcová, Daniela; Řezáčová, Martina
2016-07-01
Evidence gathered in various studies points to the fact that haemanthamine, an isoquinoline alkaloid, has multiple medicinally interesting characteristics, including antitumor, antileukemic, antioxidant, antiviral, anticonvulsant and antimalarial activity. This work presents, for the first time, a universal LC-MS/MS method for analysis of haemanthamine in plasma, bile and urine which has been verified in a pilot pharmacokinetic experiment on rats. Chromatographic separation was performed on a pentafluorophenyl core-shell column in gradient elution mode with a mobile phase consisting of acetonitrile-methanol-ammonium formate buffer. A sample preparation based on liquid-liquid extraction with methyl tert-butyl ether was employed with ambelline used as an internal standard. Quantification was performed using LC-MS-ESI(+) in Selected Reaction Monitoring mode. The method was validated according to the European Medicines Agency guideline in a concentration range of 0.1-10 μmol/L in plasma, bile and urine. The concentration-time profiles of haemanthamine in plasma, bile and urine after a single i.v. bolus of 10 mg/kg have been described for the first time. The presented study addresses the lack of information on haemanthamine pharmacokinetics and also introduces a new universal method of haemanthamine analysis in complex biological matrices. Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.
Dolder, Patrick C; Liechti, Matthias E; Rentsch, Katharina M
2018-02-01
Lysergic acid diethylamide (LSD) is a widely used recreational drug. The aim of this study was to develop and validate a liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the quantification of LSD, iso-LSD, 2-oxo-3-hydroxy LSD (O-H-LSD), and nor-LSD in plasma samples from 24 healthy subjects after controlled administration of 100 μg LSD in a clinical trial. In addition, metabolites that have been recently described in in vitro studies, including lysergic acid monoethylamide (LAE), lysergic acid ethyl-2-hydroxyethylamide (LEO), 2-oxo-LSD, trioxylated-LSD, and 13/14-hydroxy-LSD, should be identified. Separation of LSD and its metabolites was achieved on a reversed phase chromatography column after turbulent-flow online extraction. For the identification and quantification, a triple-stage quadrupole LC-MS/MS instrument was used. The validation data showed slight matrix effects for LSD, iso-LSD, O-H-LSD, or nor-LSD. Mean intraday and interday accuracy and precision were 105%/4.81% and 105%/4.35% for LSD, 98.7%/5.75% and 99.4%/7.21% for iso-LSD, 106%/4.54% and 99.4%/7.21% for O-H-LSD, and 107%/5.82% and 102%/5.88% for nor-LSD, respectively. The limit of quantification was 0.05 ng/mL for LSD, iso-LSD, and nor-LSD and 0.1 ng/mL for O-H-LSD. The limit of detection was 0.01 ng/mL for all compounds. The method described herein was accurate, precise, and the calibration range within the range of expected plasma concentrations. LSD was quantified in the plasma samples of the 24 subjects of the clinical trial, whereas iso-LSD, O-H-LSD, nor-LSD, LAE, LEO, 13/14-hydroxy-LSD, and 2-oxo-LSD could only sporadically be detected but were too low for quantification. © 2017 Wiley Periodicals, Inc.
ANALYTICAL METHODS AND QUALITY ASSURANCE CRITERIA FOR LC/ES/MS DETERMINATION OF PFOS IN FISH
PFOS, perfluorooctanesulfonate, has recently received much attention from environmental researchers. Previous analytical methods were based upon complexing with a strong ion-pairing reagent and extraction into MTBE. Detection was done on a concentrate using negative ion LC/ES/MS/...
Zhang, Donglu; Raghavan, Nirmala; Chando, Theodore; Gambardella, Janice; Fu, Yunlin; Zhang, Duxi; Unger, Steve E; Humphreys, W Griffith
2007-12-01
An LC-MS/MS-based approach that employs authentic radioactive metabolites as reference standards was developed to estimate metabolite exposures in early drug development studies. This method is useful to estimate metabolite levels in studies done with non-radiolabeled compounds where metabolite standards are not available to allow standard LC-MS/MS assay development. A metabolite mixture obtained from an in vivo source treated with a radiolabeled compound was partially purified, quantified, and spiked into human plasma to provide metabolite standard curves. Metabolites were analyzed by LC-MS/MS using the specific mass transitions and an internal standard. The metabolite concentrations determined by this approach were found to be comparable to those determined by valid LC-MS/MS assays. This approach does not requires synthesis of authentic metabolites or the knowledge of exact structures of metabolites, and therefore should provide a useful method to obtain early estimates of circulating metabolites in early clinical or toxicological studies.
Cho, Doo-Yeoun; Bae, Soo Hyeon; Shon, Ji-Hong; Bae, Soo Kyung
2013-03-01
A high-sensitivity LC/MS/MS method was developed and validated for the simultaneous determination of mirodenafil and its major metabolite, SK-3541, in human plasma. Mirodenafil, SK-3541, and udenafil as an internal standard were extracted from plasma samples with methyl tert-butyl ether. Chromatographic separation was performed on a Luna phenyl-hexyl column (100 × 2.0 mm) with an isocratic mobile phase consisting of 5 mM ammonium formate and ACN (23:77, v/v) at a flow rate of 0.35 mL/min. Detection and quantification were performed using a mass spectrometer in selected reaction monitoring mode with positive ESI at m/z 532.3 → 296.1 for mirodenafil, m/z 488.1 → 296.1 for SK-3541, and m/z 517.3 → 283.2 for udenafil. The calibration curves were linear over a concentration range of 2-500 pg/mL using 0.5 mL plasma for the microdose of mirodenafil (100 μg). Analytical method validation of the clinical dose (100 mg), with a calibration curve range of 2-500 ng/mL using 0.025-mL plasma, was also conducted. The other LC-MS/MS conditions were similar to those used for the microdosing. Each method was applied successfully to pharmacokinetic studies after a microdose or clinical dose of mirodenafil to six healthy Korean male volunteers. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
ERIC Educational Resources Information Center
Parker, Patrick D.; Beers, Brandon; Vergne, Matthew J.
2017-01-01
Laboratory experiments were developed to introduce students to the quantitation of drugs of abuse by high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS). Undergraduate students were introduced to internal standard quantitation and the LC-MS/MS method optimization for cocaine. Cocaine extracted from paper currency was…
Dufield, Dawn R; Radabaugh, Melissa R
2012-02-01
There is an increased emphasis on hyphenated techniques such as immunoaffinity LC/MS/MS (IA-LC/MS/MS) or IA-LC/MRM. These techniques offer competitive advantages with respect to sensitivity and selectivity over traditional LC/MS and are complementary to ligand binding assays (LBA) or ELISA's. However, these techniques are not entirely straightforward and there are several tips and tricks to routine sample analysis. We describe here our methods and procedures for how to perform online IA-LC/MS/MS including a detailed protocol for the preparation of antibody (Ab) enrichment columns. We have included sample trapping and Ab methods. Furthermore, we highlight tips, tricks, minimal and optimal approaches. This technology has been shown to be viable for several applications, species and fluids from small molecules to proteins and biomarkers to PK assays. Copyright © 2011 Elsevier Inc. All rights reserved.
Ellingson, David J; Shippar, Jeffrey J; Gilmore, Justin M
2016-01-01
Analytical methods for the analysis of both L-carnitine and choline are needed for reliable and accurate determination in infant formula and adult/pediatric nutritional formula. These compounds are different in how they are utilized by the human body, but are structurally similar. L-carnitine and choline are quaternary ammonium compounds, enabling both to be retained under acidic conditions with strong cation exchange (SCX) chromatography. This method analyzes both compounds simultaneously as either the free forms or as a total amount that includes bound sources such as phosphatidylcholine or acetylcarnitine. The free analysis consists of water extraction and analysis by LC/MS/MS, while the total analysis consists of extraction by acid assisted microwave hydrolysis and analysis by LC/MS/MS. Calibration standards used for calculations are extracted with all samples in the batch. A single laboratory validation (SLV) was performed following the guidelines of the AOAC Stakeholder Panel on Infant Formula and Adult Nutritionals (SPIFAN) utilizing the kit of materials provided. The results achieved meet the requirements of SMPR 2012.010 and 2012.013 for L-carnitine and total choline, respectively.
ERIC Educational Resources Information Center
Ember, Lois R.
1977-01-01
The procedures utilized by the Association of Official Analytical Chemists (AOAC) to develop, evaluate, and validate analytical methods for the analysis of chemical pollutants are detailed. Methods validated by AOAC are used by the EPA and FDA in their enforcement programs and are granted preferential treatment by the courts. (BT)
Roth, Nadine; Moosmann, Bjoern; Auwärter, Volker
2013-02-01
For analysis of hair samples derived from a pilot study ('in vivo' contamination of hair by sidestream marijuana smoke), an LC-MS/MS method was developed and validated for the simultaneous quantification of Δ9-tetrahydrocannabinolic acid A (THCA-A), Δ9-tetrahydrocannabinol (THC), cannabinol (CBN) and cannabidiol (CBD). Hair samples were extracted in methanol for 4 h under occasional shaking at room temperature, after adding THC-D(3), CBN-D(3), CBD-D(3) and THCA-A-D(3) as an in-house synthesized internal standard. The analytes were separated by gradient elution on a Luna C18 column using 0.1% HCOOH and ACN + 0.1% HCOOH. Data acquisition was performed on a QTrap 4000 in electrospray ionization-multi reaction monitoring mode. Validation was carried out according to the guidelines of the German Society of Toxicological and Forensic Chemistry (GTFCh). Limit of detection and lower limit of quantification were 2.5 pg/mg for THCA-A and 20 pg/mg for THC, CBN and CBD. A linear calibration model was applicable for all analytes over a range of 2.5 pg/mg or 20 pg/mg to 1000 pg/mg, using a weighting factor 1/x. Selectivity was shown for 12 blank hair samples from different sources. Accuracy and precision data were within the required limits for all analytes (bias between -0.2% and 6.4%, RSD between 3.7% and 11.5%). The dried hair extracts were stable over a time period of one to five days in the dark at room temperature. Processed sample stability (maximum decrease of analyte peak area below 25%) was considerably enhanced by adding 0.25% lecithin (w/v) in ACN + 0.1% HCOOH for reconstitution. Extraction efficiency for CBD was generally very low using methanol extraction. Hence, for effective extraction of CBD alkaline hydrolysis is recommended. Copyright © 2013 John Wiley & Sons, Ltd.
Harun, Norlida; Anderson, Robert A; Miller, Eleanor I
2009-01-01
An ELISA and a liquid chromatography-tandem mass spectrometry (LC-MS-MS) confirmation method were developed and validated for the identification and quantitation of ketamine and its major metabolite norketamine in urine samples. The Neogen ketamine microplate ELISA was optimized with respect to sample and enzyme conjugate volumes and the sample preincubation time before addition of the enzyme conjugate. The ELISA kit was validated to include an assessment of the dose-response curve, intra- and interday precision, limit of detection (LOD), and cross-reactivity. The sensitivity and specificity were calculated by comparison to the results from the validated LC-MS-MS confirmation method. An LC-MS-MS method was developed and validated with respect to LOD, lower limit of quantitation (LLOQ), linearity, recovery, intra- and interday precision, and matrix effects. The ELISA dose-response curve was a typical S-shaped binding curve, with a linear portion of the graph observed between 25 and 500 ng/mL for ketamine. The cross-reactivity of 200 ng/mL norketamine to ketamine was 2.1%, and no cross-reactivity was detected with 13 common drugs tested at 10,000 ng/mL. The ELISA LOD was calculated to be 5 ng/mL. Both intra- (n = 10) and interday (n = 50) precisions were below 5.0% at 25 ng/mL. The LOD for ketamine and norketamine was calculated statistically to be 0.6 ng/mL. The LLOQ values were also calculated statistically and were 1.9 ng/mL and 2.1 ng/mL for ketamine and norketamine, respectively. The test linearity was 0-1200 ng/mL with correlation coefficient (R(2)) > 0.99 for both analytes. Recoveries at 50, 500, and 1000 ng/mL range from 97.9% to 113.3%. Intra- (n = 5) and interday (n = 25) precisions between extracts for ketamine and norketamine were excellent (< 10%). Matrix effects analysis showed an average ion suppression of 5.7% for ketamine and an average ion enhancement of 13.0% for norketamine for urine samples collected from six individuals. A comparison of ELISA
Codevilla, Cristiane Franco; Lemos, Alice Machado; Delgado, Leila Schreiner; Rolim, Clarice Madalena Bueno; Adams, Andréa Inês Horn; Bergold, Ana Maria
2011-08-01
A stability-indicating liquid chromatographic method has been developed for the quantitative determination of lodenafil carbonate in tablets. The method employs a Synergi Fusion C18 column (250 × 4.6 mm, i.d., 4 μm particle size), with mobile phase consisting of a mixture of methanol-acetic acid 0.1% pH 4.0 (65:35, v/v) and UV detection at 290 nm, using a photodiode array detector. A linear response (r = 0.9999) was observed in the range of 10-80 μg/mL. The method showed good recoveries (average 100.3%) and also intra and inter-day precision (RSD < 2.0%). Validation parameters as specificity and robustness were also determined. Specificity analysis showed that no impurities or degradation products were co-eluting with the lodenafil carbonate peak. The method was found to be stability-indicating and due to its simplicity and accuracy can be applied for routine quality control analysis of lodenafil carbonate in tablets.
Bajerski, Lisiane; Rossi, Rochele Cassanta; Dias, Carolina Lupi; Bergold, Ana Maria; Fröehlich, Pedro Eduardo
2010-06-01
A dissolution test for tablets containing 40 mg of olmesartan medoxomil (OLM) was developed and validated using both LC-UV and UV methods. After evaluation of the sink condition, dissolution medium, and stability of the drug, the method was validated using USP apparatus 2, 50 rpm rotation speed, and 900 ml of deaerated H(2)O + 0.5% sodium lauryl sulfate (w/v) at pH 6.8 (adjusted with 18% phosphoric acid) as the dissolution medium. The model-independent method using difference factor (f(1)) and similarity factor (f(2)), model-dependent method, and dissolution efficiency were employed to compare dissolution profiles. The kinetic parameters of drug release were also investigated. The obtained results provided adequate dissolution profiles. The developed dissolution test was validated according to international guidelines. Since there is no monograph for this drug in tablets, the dissolution method presented here can be used as a quality control test for OLM in this dosage form, especially in a batch to batch evaluation.
Barrett, Yu Chen; Akinsanya, Billy; Chang, Shu-Ying; Vesterqvist, Ole
2005-07-25
A sensitive method for quantitation of urinary 6beta-hydroxycortisol (6beta-HC) and cortisol using on-line SPE and LC-MS/MS was developed and validated. Human urine samples were injected directly onto an on-line solid phase extraction apparatus, Prospekt-2, followed by HPLC separation and electrospray triple quadrupole LC-MS/MS detection. The inter-day precision for the 6beta-HC:cortisol ratio was 7-9%. The lower limit of quantitation was 1 and 0.2 ng/mL for 6beta-HC and cortisol, respectively. Using the method we observed a diurnal variation on the 6beta-HC:cortisol ratio in healthy volunteers with the maximal ratio observed in the 2-10 pm urine collection period.
Method 544 is an accurate and precise analytical method to determine six microcystins (including MC-LR) and nodularin in drinking water using solid phase extraction and liquid chromatography tandem mass spectrometry (SPE-LC/MS/MS). The advantage of this SPE-LC/MS/MS is its sensi...
Carvalho, J J; Jerónimo, P C A; Gonçalves, C; Alpendurada, M F
2008-11-01
European Council Directive 98/83/EC on the quality of water intended for human consumption brought a new challenge for water-quality control routine laboratories, mainly on pesticides analysis. Under the guidelines of ISO/IEC 17025:2005, a multiresidue method was developed, validated, implemented in routine, and studied with real samples during a one-year period. The proposed method enables routine laboratories to handle a large number of samples, since 28 pesticides of 14 different chemical groups can be quantitated in a single procedure. The method comprises a solid-phase extraction step and subsequent analysis by liquid chromatography-mass spectrometry (LC-MS-MS). The accuracy was established on the basis of participation in interlaboratory proficiency tests, with encouraging results (majority |z-score| <2), and the precision was consistently analysed over one year. The limits of quantitation (below 0.050 microg L(-1)) are in agreement with the enforced threshold value for pesticides of 0.10 microg L(-1). Overall method performance is suitable for routine use according to accreditation rules, taking into account the data collected over one year.
2010-01-01
Chrom LC –MS...Literature 3. DATES COVERED (From - To) 4. TITLE AND SUBTITLE A liquid chromatographic–mass spectrometric ( LC –MS) method for the analysis of the 5a...Journal of Chromatography B journa l homepage: www.e lsev ier .com/ locate /chromb A liquid chromatographic–mass spectrometric ( LC –MS) method for
Mei, Shenghui; Luo, Xuying; Li, Xingang; Li, Qian; Huo, Jiping; Yang, Li; Zhu, Leting; Feng, Weixing; Zhou, Jianxin; Shi, Guangzhi; Zhao, Zhigang
2016-12-01
Tigecycline (TGC) is an important antibiotic in treating various drug-resistant bacteria. The dosage regimen for cerebral intraventricular TGC is still unknown. The aim of the study was to develop and validate liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods for the determination of TGC in human plasma and cerebrospinal fluid (CSF) to obtain an applicable regimen. The ion transitions under ESI positive model were performed at m/z 586.3 > 513.2 and m/z 595.3 > 514.3 for TGC and d9-TGC internal standard (IS). For plasma and CSF samples, the calibration curve of TGC was linear within the ranges 25-2000 and 250-100,000 ng/mL; the IS normalized matrix effect was within the ranges 96.46-101.06% and 101.13-103.58%, respectively, for all. TGC was stable under all tested conditions. The patient received 1 mg intraventricular and 49 mg intravenous administration of TGC. The AUC 0-12 in plasma and CSF calculated according to our noncompartment model were 4713 and 23,0238 h ng/mL, respectively. Given our findings cerebral intraventricular TGC may be a choice for clinicians to treat drug-resistant Gram-negative bacterial-induced meningitis and the safety and efficacy of this administration route warrants further study. Copyright © 2016 John Wiley & Sons, Ltd.
Establishment of a method for determination of arsenic species in seafood by LC-ICP-MS.
Zmozinski, Ariane V; Llorente-Mirandes, Toni; López-Sánchez, José F; da Silva, Márcia M
2015-04-15
An analytical method for determination of arsenic species (inorganic arsenic (iAs), methylarsonic acid (MA), dimethylarsinic acid (DMA), arsenobetaine (AB), trimethylarsine oxide (TMAO) and arsenocholine (AC)) in Brazilian and Spanish seafood samples is reported. This study was focused on extraction and quantification of inorganic arsenic (iAs), the most toxic form. Arsenic speciation was carried out via LC with both anionic and cationic exchange with ICP-MS detection (LC-ICP-MS). The detection limits (LODs), quantification limits (LOQs), precision and accuracy for arsenic species were established. The proposed method was evaluated using eight reference materials (RMs). Arsenobetaine was the main species found in all samples. The total and iAs concentration in 22 seafood samples and RMs ranged between 0.27-35.2 and 0.02-0.71 mg As kg(-1), respectively. Recoveries ranging from 100% to 106% for iAs, based on spikes, were achieved. The proposed method provides reliable iAs data for future risk assessment analysis. Copyright © 2014 Elsevier Ltd. All rights reserved.
Gowda, Nagaraj; Kumar, Pradeep; Panghal, Surender; Rajshree, Mashru
2010-02-01
This study presents the development and validation of a reversed-phase liquid chromatographic method for the determination of mangiferin (MGN) in alcoholic extracts of mangifera indica. A Lichrospher 100 C(18)-ODS (250 x 4.6 mm, 5 microm size) (Merck, Whitehouse Station, NJ) prepacked column and a mobile phase of potassium dihydrogen orthophosphate (0.01M) pH 2.7 +/- 0.2-acetonitrile (15:85, v/v) with the flow rate of 1 mL/min was used. MGN detection was achieved at a wavelength monitored at 254 nm with SPD-M 10A vp PDA detector or SPD 10AD vp UV detector in combination with class LC 10A software. The proposed method was validated as prescribed by International Conference on Harmonization (ICH) with respect to linearity, specificity, accuracy, precision, stability, and quantification. The method validation was realized using alcoholic extracts and raw materials of leaves and barks. All the validation parameters were within the acceptable limits, and the developed analytical method can successfully be applied for MGN determination.
Zuben, E S Von; Oliveira, A G; Chorilli, M; Scarpa, M V
2018-03-05
A reversed-phase liquid chromatography (RP-LC) method was successfully developed and validated for the determination of methotrexate in nanostructured liquid crystalline systems composed by polyether functional siloxane and silicone polyether copolymer. The LC method was performed on RP C18-ODS column, Agilent Zorbax® (4.6 x 250 mm, 5 μm), maintained at room temperature, with a mobile phase constituted by a mixture of 50 mM ammonium acetate buffer (pH 6.0) and methanol (77:23,v/v) with a flow rate of 1.0 mL/min, using ultraviolet detection at 313 nm. The parameters used in the validation process were linearity, specificity, intra and inter-day precision, accuracy, robustness. The quantitation and detection limits yielded good results. The calibration plot assumed linear behavior from 5.0-150.0 μg. mL-1 (r2 = 0.9999). The methotrexate was subjected to oxidation, acid, base and neutral degradation, photolysis and heat as stress conditions. There were no interfering peaks at or near the retention time of methotrexate. The nanostructured liquid crystalline systems did not interfere with the analysis and the recovery was quantitative. The intra and inter-day assay relative standard deviation were less than 0.20 %. The method developed proved to be simple, sensitive, accurate, precise, reproducible and therefore adequate for routine analysis of methotrexate in nanostructured liquid crystalline systems.
Lin, Long-Ze; Harnly, James M
2008-11-12
A screening method using LC-DAD-ESI/MS was developed for the identification of common hydroxycinnamoylquinic acids based on direct comparison with standards. A complete standard set for mono-, di-, and tricaffeoylquinic isomers was assembled from commercially available standards, positively identified compounds in common plants (artichokes, asparagus, coffee bean, honeysuckle flowers, sweet potato, and Vernonia amygdalina leaves) and chemically modified standards. Four C18 reversed phase columns were tested using the standardized profiling method (based on LC-DAD-ESI/MS) for 30 phenolic compounds, and their elution order and retention times were evaluated. Using only two columns under standardized LC condition and the collected phenolic compound database, it was possible to separate all of the hydroxycinnamoylquinic acid conjugates and to identify 28 and 18 hydroxycinnamoylquinic acids in arnica flowers (Arnica montana L.) and burdock roots (Arctium lappa L.), respectively. Of these, 22 are reported for the first time.
USDA-ARS?s Scientific Manuscript database
Prior to conducting a collaborative study of AOAC First Action 2012.25 LC-MS/MS analytical method for the determination of residues of three triphenylmethane dyes (malachite green, crystal violet, and brilliant green) and their metabolites (leucomalachite green and leucocrystal violet) in seafood, a...
Lakshmanan, Rajeswari; Wolff, Jeremy J.; Alvarado, Rudy; Loo, Joseph A.
2014-01-01
A comparison of different data-independent fragmentation methods combined with liquid chromatography (LC) coupled to high resolution Fourier-transform ion cyclotron resonance (FT-ICR) tandem mass spectrometry (MS) is presented for top-down MS of protein mixtures. Proteins composing the 20S and 19S proteasome complex and their post-translational modifications were identified using a 15-Tesla FT-ICR mass spectrometer. The data-independent fragmentation modes with LC timescales allowed for higher duty cycle measurements that better suit on-line LC-FT-ICR-MS. Protein top-down dissociation was effected by funnel-skimmer collisionally activated dissociation (FS-CAD) and CASI (Continuous Accumulation of Selected Ions)-CAD. The N-terminus for 9 out of the 14 20S proteasome proteins were found to be modified, and the α3 protein was found to be phosphorylated; these results are consistent with previous reports. Mass measurement accuracy with the LC-FT-ICR system for the 20–30 kDa 20S proteasome proteins was 1 ppm. The intact mass of the 100 kDa Rpn1 subunit from the 19S proteasome complex regulatory particle was measured with a deviation of 17 ppm. The CASI-CAD technique is a complementary tool for intact protein fragmentation and is an effective addition to the growing inventory of dissociation methods which are compatible with on-line protein separation coupled to FT-ICR MS. PMID:24478249
Tiryaki, Osman
2016-10-02
This study was undertaken to validate the "quick, easy, cheap, effective, rugged and safe" (QuEChERS) method using Golden Delicious and Starking Delicious apple matrices spiked at 0.1 maximum residue limit (MRL), 1.0 MRL and 10 MRL levels of the four pesticides (chlorpyrifos, dimethoate, indoxacarb and imidacloprid). For the extraction and cleanup, original QuEChERS method was followed, then the samples were subjected to liquid chromatography-triple quadrupole mass spectrometry (LC-MS/MS) for chromatographic analyses. According to t test, matrix effect was not significant for chlorpyrifos in both sample matrices, but it was significant for dimethoate, indoxacarb and imidacloprid in both sample matrices. Thus, matrix-matched calibration (MC) was used to compensate matrix effect and quantifications were carried out by using MC. The overall recovery of the method was 90.15% with a relative standard deviation of 13.27% (n = 330). Estimated method detection limit of analytes blew the MRLs. Some other parameters of the method validation, such as recovery, precision, accuracy and linearity were found to be within the required ranges.
LC-MS/MS and GC-MS methods in propofol detection: Evaluation of the two analytical procedures.
Vaiano, Fabio; Serpelloni, Giovanni; Focardi, Martina; Fioravanti, Alessia; Mari, Francesco; Bertol, Elisabetta
2015-11-01
Propofol is a short-acting hypnotic agent that is commonly used to induce and maintain anesthesia. Propofol abuse and its involvement in suicide deaths have increased in recent years, especially among healthcare personnel. An example is the suicide of a 61-year-old nurse found with a propofol drip in his left arm. We describe the postmortem concentration of propofol in various tissues (femoral and cardiac blood, bile, urine, brain, and liver) and in the drip. The toxicological analyses were performed through two analytical methods, differing in derivatization reaction and in instrumentation: silylation for gas chromatograph-mass spectrometer (GC-MS), as routinely performed in our laboratory for this kind of analyses (lower limits of quantification-LLOQ-in urine and blood: 0.3 and 5ng/ml); for liquid chromatograph-tandem mass spectrometer (LC-MS/MS) an innovative azo-coupling derivatization (LLOQ: 0.0004 and 0.1ng/ml). This latter produces an azo-derivative (molecular composition: C18H22ON2; molecular weight: 282Da) highly ionizable in electro-spray ion source, both in negative and positive ionizations. These two methods were compared to evaluate the effectiveness of this new LC-MS/MS analysis. An acidic hydrolysis (HCl 6N, 100°C, and 1h) was performed for the biological samples (1ml or 1g) irrespective of the analytical method applied. The drip content was extracted adding phosphate buffer (pH 8) and a dichloromethane/ethylacetate 8:2 (v:v) mixture. Derivatization steps were: silylation with N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA)+tetramethylammonium hydroxide (TMAH) for GC-MS; regarding LC-MS/MS, azo-coupling reaction with the aryl-diazonium salt (0-5°C, and 30min). The analyses were achieved in selected-ion monitoring for GC-MS (m/z, 235,250,73 propofol"; m/z, 252,267,27 propofol-d17) and in multiple reaction monitoring ([M-H](-): m/z 283→241,77, azo-propofol; m/z 299→251,77, azo-propofol-d17) for LC-MS/MS. Autopsy showed no significant findings
Buckley, Mike
2016-03-24
Collagen is one of the most ubiquitous proteins in the animal kingdom and the dominant protein in extracellular tissues such as bone, skin and other connective tissues in which it acts primarily as a supporting scaffold. It has been widely investigated scientifically, not only as a biomedical material for regenerative medicine, but also for its role as a food source for both humans and livestock. Due to the long-term stability of collagen, as well as its abundance in bone, it has been proposed as a source of biomarkers for species identification not only for heat- and pressure-rendered animal feed but also in ancient archaeological and palaeontological specimens, typically carried out by peptide mass fingerprinting (PMF) as well as in-depth liquid chromatography (LC)-based tandem mass spectrometric methods. Through the analysis of the three most common domesticates species, cow, sheep, and pig, this research investigates the advantages of each approach over the other, investigating sites of sequence variation with known functional properties of the collagen molecule. Results indicate that the previously identified species biomarkers through PMF analysis are not among the most variable type 1 collagen peptides present in these tissues, the latter of which can be detected by LC-based methods. However, it is clear that the highly repetitive sequence motif of collagen throughout the molecule, combined with the variability of the sites and relative abundance levels of hydroxylation, can result in high scoring false positive peptide matches using these LC-based methods. Additionally, the greater alpha 2(I) chain sequence variation, in comparison to the alpha 1(I) chain, did not appear to be specific to any particular functional properties, implying that intra-chain functional constraints on sequence variation are not as great as inter-chain constraints. However, although some of the most variable peptides were only observed in LC-based methods, until the range of
LC-MS-based quantification method for Achyranthes root saponins.
Kawahara, Yuki; Hoshino, Tatsuro; Morimoto, Hidetaka; Shinizu, Tomofumi; Narukawa, Yuji; Fuchino, Hiroyuki; Kawahara, Nobuo; Kiuchi, Fumiyuki
2016-01-01
A liquid chromatography mass spectrometry (LC-MS) method was developed for simultaneous quantitative analysis of Achyranthes root saponins: chikusetsusaponins IVa (1) and V (2), achyranthosides B (3), C (4), D (5), E (6), and G (7), sulfachyranthosides B (8) and D (9), and betavulgarosides II (10) and IV (11). Satisfactory separation of the saponins was achieved with the use of a volatile ion-pair reagent (dihexyl ammonium acetate) on a phenyl-hexylated silica gel column, and the amounts of saponins extracted under three different conditions were determined. When Achyranthes root was extracted with water at room temperature, achyranthosides B (3) and D (5) were the major saponins, and smaller amounts of other saponins (4, 6-11) were present. However, the amounts of chikusetsusaponins (1 and 2) were negligible. Under the condition to make a standard decoction of a Kampo formula, the major saponins were achyranthosides B (3), C (4), and D (5), and small amounts of chikusetsusaponins IVa (1) and V (2) appeared, whereas prolonged heating largely increased the amounts of chikusetsusaponins. This method can be used for quality control of Achyranthes root.
Lalitha Devi, M; Chandrasekhar, K B
2009-12-05
The objective of current study was to develop a validated specific stability indicating reversed-phase liquid chromatographic method for the quantitative determination of levofloxacin as well as its related substances determination in bulk samples, pharmaceutical dosage forms in the presence of degradation products and its process related impurities. Forced degradation studies were performed on bulk sample of levofloxacin as per ICH prescribed stress conditions using acid, base, oxidative, water hydrolysis, thermal stress and photolytic degradation to show the stability indicating power of the method. Significant degradation was observed during oxidative stress and the degradation product formed was identified by LCMS/MS, slight degradation in acidic stress and no degradation was observed in other stress conditions. The chromatographic method was optimized using the samples generated from forced degradation studies and the impurity spiked solution. Good resolution between the peaks corresponds to process related impurities and degradation products from the analyte were achieved on ACE C18 column using the mobile phase consists a mixture of 0.5% (v/v) triethyl amine in sodium dihydrogen orthophosphate dihydrate (25 mM; pH 6.0) and methanol using a simple linear gradient. The detection was carried out at 294 nm. The limit of detection and the limit of quantitation for the levofloxacin and its process related impurities were established. The stressed test solutions were assayed against the qualified working standard of levofloxacin and the mass balance in each case was in between 99.4 and 99.8% indicating that the developed LC method was stability indicating. Validation of the developed LC method was carried out as per ICH requirements. The developed LC method was found to be suitable to check the quality of bulk samples of levofloxacin at the time of batch release and also during its stability studies (long term and accelerated stability).
Suhr, Anna Catharina; Vogeser, Michael; Grimm, Stefanie H
2016-05-30
For quotable quantitative analysis of endogenous analytes in complex biological samples by isotope dilution LC-MS/MS, the creation of appropriate calibrators is a challenge, since analyte-free authentic material is in general not available. Thus, surrogate matrices are often used to prepare calibrators and controls. However, currently employed validation protocols do not include specific experiments to verify the suitability of a surrogate matrix calibration for quantification of authentic matrix samples. The aim of the study was the development of a novel validation experiment to test whether surrogate matrix based calibrators enable correct quantification of authentic matrix samples. The key element of the novel validation experiment is the inversion of nonlabelled analytes and their stable isotope labelled (SIL) counterparts in respect to their functions, i.e. SIL compound is the analyte and nonlabelled substance is employed as internal standard. As a consequence, both surrogate and authentic matrix are analyte-free regarding SIL analytes, which allows a comparison of both matrices. We called this approach Isotope Inversion Experiment. As figure of merit we defined the accuracy of inverse quality controls in authentic matrix quantified by means of a surrogate matrix calibration curve. As a proof-of-concept application a LC-MS/MS assay addressing six corticosteroids (cortisol, cortisone, corticosterone, 11-deoxycortisol, 11-deoxycorticosterone, and 17-OH-progesterone) was chosen. The integration of the Isotope Inversion Experiment in the validation protocol for the steroid assay was successfully realized. The accuracy results of the inverse quality controls were all in all very satisfying. As a consequence the suitability of a surrogate matrix calibration for quantification of the targeted steroids in human serum as authentic matrix could be successfully demonstrated. The Isotope Inversion Experiment fills a gap in the validation process for LC-MS/MS assays
Lee, Ji Hyun; Kang, Gihaeng; Park, Han Na; Kim, Jihee; Kim, Nam Sook; Park, Seongsoo; Park, Sung-Kwan; Baek, Sun Young; Kang, Hoil
2018-02-01
In this study, we developed a UPLC-PDA and LC-Q-TOF/MS method to identify and measure the following prohibited substances that may be found in dietary supplements:triaminodil, minoxidil, bimatoprost, alimemazine, diphenylcyclopropenone, α-tradiol, finasteride, methyltestosterone, spironolatone, flutamide, cyproterone, dutasteride, and testosterone 17-propionate.The method was validated according to International Conference on Harmonization guidelines in terms of specificity, linearity, accuracy, precision, LOD, LOQ, recovery, and stability. The method was completely validated showing satisfactory data for all method validation parameters. The linearity was good (R 2 > 0.999) with intra- and inter-day precision values of 0.2-3.4% and 0.3-2.9%, respectively. Moreover, the intra- and inter-day accuracies were 87-102% and 86-103%, respectively, and the precision was better than 9.4% (relative standard deviation).Hence, the proposed method is precise and has high quality,and can be utilised to comprehensively and continually monitor illegal drug adulteration in various forms of dietary supplements. Furthermore, to evaluate the applicability of the proposed method, we analysed 13 hair-growth compounds in 78 samples including food and dietary supplements. Minoxidil and triaminodil were detected in capsules at concentrations of 4.69 mg/g and 6.54 mg/g. In addition, finasteride was detected in a tablet at 13.45 mg/g. In addition, the major characteristic fragment ions were confirmed once again using LC-Q-TOF/MS for higher accuracy.
Kaymak, Tugrul; Türker, Levent; Tulay, Hüseyin; Stroka, Joerg
2018-04-27
Background : Pekmez and pestil are traditional Turkish foods made from concentrated grapejuice, which can be contaminated with mycotoxins such as aflatoxins and ochratoxin A (OTA). Objective : To carry out a single-laboratory validation of a method to simultaneously determine aflatoxins B 1 , B₂, G 1 , and G₂ and ochratoxin A in pekmez and pestil. Methods : The homogenized sample is extracted with methanol-water (80 + 20) using a high-speed blender. The (sample) extract is filtered, diluted with phosphate-buffered saline solution, and applied to a multi-immunoaffinity column (AFLAOCHRA PREP®). Aflatoxins and ochratoxin A are removed with (neat) methanol and then directly analyzed by reversed-phase LC with fluorescence detection using post-column bromination (Kobra cell®). Results : Test portions of blank pekmez and pestil were spiked with a mixture of aflatoxins and ochratoxin A to give levels ranging from 2.6 to 10.4 μg/kg and 1.0-4.0 μg/kg, respectively. Recoveries for total aflatoxins and ochratoxin A ranged from 84 to 106% and 80-97%, respectively, for spiked samples. Based on results for spiked pekmez and pestil (30 replicates each at three levels), the repeatability RSD ranged from 1.6 to 12% and 2.7-11% for total aflatoxins and ochratoxin A, respectively. Conclusions : The method performance in terms of recovery, repeatability, and detection limits has been demonstrated to be suitable for use as an Official Method. Highlights : First immunoaffinity column method validated for simultaneous analysis of aflatoxins and ochratoxin A in pekmez and pestil. Suitability for use for official purposes in Turkey, demonstrated by single-laboratory validation. Co-occurrence of aflatoxins and OTA in mulberry and carob pekmez reported for the first time.
Baira, Shandilya Mahamuni; Khurana, Amit; Somagoni, Jaganmohan; Srinivas, R; Godugu, Chandraiah; Talluri, M V N Kumar
2018-06-02
Nimbolide is a novel, natural compound with promising potential as a drug candidate for anticancer activity. It is isolated from the Indian traditional medicinal plant Azadirachta indica popularly known as neem. The present study was undertaken to explore the oral bioavailability and pharmacokinetic characteristics of nimbolide in rats using the LC/QTOF/MS method. A simple protein precipitation method using acetonitrile was employed for extracting nimbolide from rat plasma. The chromatographic separation of nimbolide and the internal standard (regorafenib) was attained on an Aquity BEH C18 column (100 × 2.1 mm, 2.7 μm), using ACN and 0.1% of formic acid in water as mobile phase components in a gradient elution mode at a flow rate of 0.45 mL/min over a short run time of 4 min. A mass detection was carried out using target ions of [M + H] + at m/z 467.2074 for nimbolide and m/z 483.0847 for the internal standard. The LC/MS method was validated and all the parameters were found well within the specified limits. The calibration curve was constructed in the range of 1-1000 ng/mL. The method shows good accuracy (91.66-97.12%) and precision (intra 2.21-6.92% CV and inter-day 2.56-4.62% CV). This developed LC/MS method was effectively applied to the pharmacokinetic study of nimbolide upon oral and intravenous administration in rats. In concordance with its physicochemical properties and high lipophilicity, we found that it shows poor oral absorption at different doses (10, 30 and 50 mg/kg). As expected, higher plasma levels were observed upon intravenous (10 mg/kg) administration. This method can be extended for evaluation of drug interaction and drug metabolism in rats as well as in humans. Moreover, our rapid and sensitive method may cater the need to accelerate the preclinical formulation development and lead optimization for future drug development of this potent anticancer agent. Further, our oral bioavailability studies demonstrated that
Yi, Deliang; Wang, Zhihua; Yi, Longzhi
2015-04-01
A selective and sensitive liquid chromatography-mass spectrometry (MS) method was developed and validated for the determination of karanjin in rat plasma. The target analyte, together with the internal standard (warfarin), was extracted from rat plasma by liquid-liquid extraction with ethyl acetate. Chromatographic separation was performed on a ZORBAX SB-C18 column using a mixture of acetonitrile and 0.1% aqueous formic acid as the mobile phase with linear gradient elution. MS detection was performed on a single quadrupole MS by selected ion monitoring mode via a positive electrospray ionization source. The assay exhibited a linear dynamic range of 2.50-3,000 ng/mL for karanjin. The intra- and inter-day precision was <10.8%, and the intra- and inter-day accuracy was <9.2%. The validated method has been applied to the preclinical pharmacokinetic studies of karanjin following oral administration of 5, 10 and 20 mg/kg karanjin to rats. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.
Cronly, Mark; Behan, P; Foley, B; Malone, E; Earley, S; Gallagher, M; Shearan, P; Regan, L
2010-12-01
A confirmatory method has been developed to allow for the analysis of fourteen prohibited medicinal additives in pig and poultry compound feed. These compounds are prohibited for use as feed additives although some are still authorised for use in medicated feed. Feed samples are extracted by acetonitrile with addition of sodium sulfate. The extracts undergo a hexane wash to aid with sample purification. The extracts are then evaporated to dryness and reconstituted in initial mobile phase. The samples undergo an ultracentrifugation step prior to injection onto the LC-MS/MS system and are analysed in a run time of 26 min. The LC-MS/MS system is run in MRM mode with both positive and negative electrospray ionisation. The method was validated over three days and is capable of quantitatively analysing for metronidazole, dimetridazole, ronidazole, ipronidazole, chloramphenicol, sulfamethazine, dinitolimide, ethopabate, carbadox and clopidol. The method is also capable of qualitatively analysing for sulfadiazine, tylosin, virginiamycin and avilamycin. A level of 100 microg kg(-1) was used for validation purposes and the method is capable of analysing to this level for all the compounds. Validation criteria of trueness, precision, repeatability and reproducibility along with measurement uncertainty are calculated for all analytes. Copyright (c) 2010 Elsevier B.V. All rights reserved.
Waitt, Catriona; Diliiy Penchala, Sujan; Olagunju, Adeniyi; Amara, Alieu; Else, Laura; Lamorde, Mohammed; Khoo, Saye
2017-08-15
To present the validation and clinical application of a LC-MS/MS method for the quantification of lamivudine (3TC), emtricitabine (FTC) and tenofovir (TFV) in dried blood spots (DBS) and dried breast milk spots (DBMS). DBS and DBMS were prepared from 50 and 30μL of drug-spiked whole blood and human breast milk, respectively. Following extraction with acetonitrile and water, chromatographic separation utilised a Synergi polar column with a gradient mobile phase program consisting of 0.1% formic acid in water and 0.1% formic acid in acetonitrile. Detection and quantification was performed using a TSQ Quantum Ultra triple quadrupole mass spectrometer. The analytical method was used to evaluate NRTI drug levels in HIV-positive nursing mothers-infant pairs. The assay was validated over the concentration range of 16.6-5000ng/mL for 3TC, FTC and TFV in DBS and DBMS except for TFV in DBMS where linearity was established from 4.2-1250ng/mL. Intra and inter-day precision (%CV) ranged from 3.5-8.7 and accuracy was within 15% for all analytes in both matrices. The mean recovery in DBS was >61% and in DBMS >43% for all three analytes. Matrix effect was insignificant. Median AUC 0-8 values in maternal DBS and DBMS, respectively, were 4683 (4165-6057) and 6050 (5217-6417)ngh/mL for 3TC, 3312 (2259-4312) and 4853 (4124-6691)ngh/mL for FTC and 1559 (930-1915) and 56 (45-80)ngh/mL for TFV. 3TC and FTC were quantifiable (>16.6ng/mL) in DBS from 2/6 and 1/6 infants respectively whereas TFV was undetectable in all infants. DBS and DBMS sampling for bioanalysis of 3TC, FTC and TFV is straightforward, robust, accurate and precise, and ideal for use in low-resource settings. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.
This presentation will describe the U.S. EPA’s drinking water and ambient water method development program in relation to the process employed and the typical challenges encountered in developing standardized LC/MS/MS methods for chemicals of emerging concern. The EPA&rsquo...
Chiu, Huai-Hsuan; Liao, Hsiao-Wei; Shao, Yu-Yun; Lu, Yen-Shen; Lin, Ching-Hung; Tsai, I-Lin; Kuo, Ching-Hua
2018-08-17
Monoclonal antibody (mAb) drugs have generated much interest in recent years for treating various diseases. Immunoglobulin G (IgG) represents a high percentage of mAb drugs that have been approved by the Food and Drug Administration (FDA). To facilitate therapeutic drug monitoring and pharmacokinetic/pharmacodynamic studies, we developed a general liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to quantify the concentration of IgG-based mAbs in human plasma. Three IgG-based drugs (bevacizumab, nivolumab and pembrolizumab) were selected to demonstrate our method. Protein G beads were used for sample pretreatment due to their universal ability to trap IgG-based drugs. Surrogate peptides that were obtained after trypsin digestion were quantified by using LC-MS/MS. To calibrate sample preparation errors and matrix effects that occur during LC-MS/MS analysis, we used two internal standards (IS) method that include the IgG-based drug-IS tocilizumab and post-column infused IS. Using two internal standards was found to effectively improve quantification accuracy, which was within 15% for all mAb drugs that were tested at three different concentrations. This general method was validated in term of its precision, accuracy, linearity and sensitivity for 3 demonstration mAb drugs. The successful application of the method to clinical samples demonstrated its' applicability in clinical analysis. It is anticipated that this general method could be applied to other mAb-based drugs for use in precision medicine and clinical studies. Copyright © 2018 Elsevier B.V. All rights reserved.
Application of LC/MS/MS Techniques to Development of US ...
This presentation will describe the U.S. EPA’s drinking water and ambient water method development program in relation to the process employed and the typical challenges encountered in developing standardized LC/MS/MS methods for chemicals of emerging concern. The EPA’s Drinking Water Contaminant Candidate List and Unregulated Contaminant Monitoring Regulations, which are the driving forces behind drinking water method development, will be introduced. Three drinking water LC/MS/MS methods (Methods 537, 544 and a new method for nonylphenol) and two ambient water LC/MS/MS methods for cyanotoxins will be described that highlight some of the challenges encountered during development of these methods. This presentation will provide the audience with basic understanding of EPA's drinking water method development program and an introduction to two new ambient water EPA methods.
Bayesian Normalization Model for Label-Free Quantitative Analysis by LC-MS
Nezami Ranjbar, Mohammad R.; Tadesse, Mahlet G.; Wang, Yue; Ressom, Habtom W.
2016-01-01
We introduce a new method for normalization of data acquired by liquid chromatography coupled with mass spectrometry (LC-MS) in label-free differential expression analysis. Normalization of LC-MS data is desired prior to subsequent statistical analysis to adjust variabilities in ion intensities that are not caused by biological differences but experimental bias. There are different sources of bias including variabilities during sample collection and sample storage, poor experimental design, noise, etc. In addition, instrument variability in experiments involving a large number of LC-MS runs leads to a significant drift in intensity measurements. Although various methods have been proposed for normalization of LC-MS data, there is no universally applicable approach. In this paper, we propose a Bayesian normalization model (BNM) that utilizes scan-level information from LC-MS data. Specifically, the proposed method uses peak shapes to model the scan-level data acquired from extracted ion chromatograms (EIC) with parameters considered as a linear mixed effects model. We extended the model into BNM with drift (BNMD) to compensate for the variability in intensity measurements due to long LC-MS runs. We evaluated the performance of our method using synthetic and experimental data. In comparison with several existing methods, the proposed BNM and BNMD yielded significant improvement. PMID:26357332
Liu, Yazhong; Zhou, Fang; Sang, Hua; Ye, Hui; Chen, Qianying; Yao, Lan; Ni, Ping; Wang, Guangji; Zhang, Jingwei
2017-04-15
Lysine-MCC-DM1, MCC-DM1 and DM1 are potential catabolites of trastuzumab emtansine (T-DM1). A convenient liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated to detect these catabolites simultaneously in in vitro investigations for the first time. Protein precipitation was utilized to prepare the samples. Chromatographic separation was achieved on a Phenomenex Kinetex C18 column (100×2.1mm, 2.6μm) with mobile-phase gradient elution. The calibration curves of each analyte ranging from 1 to 100nM showed good linearity (r 2 >0.995). The method was validated successfully and applied to the intracellular catabolism and regulation of T-DM1. Copyright © 2017 Elsevier B.V. All rights reserved.
Bellur Atici, Esen; Yazar, Yücel; Ağtaş, Çağan; Ridvanoğlu, Nurten; Karlığa, Bekir
2017-03-20
Antibacterial combinations consisting of the semisynthetic antibiotic amoxicillin (amox) and the β-lactamase inhibitor potassium clavulanate (clav) are commonly used and several chromatographic methods were reported for their quantification in mixtures. In the present work, single HPLC method for related substances analyses of amoxicillin and potassium clavulanate mixtures was developed and validated according to international conference on harmonization (ICH) guidelines. Eighteen amoxicillin and six potassium clavulanate impurities were successfully separated from each other by using triple gradient elution using a Thermo Hypersil Zorbax BDS C18 (250 mm×4.6mm, 3μm) column with 50μL injection volumes at a wavelength of 215nm. Commercially unavailable impurities were formed by degradation of amoxicillin and potassium clavulanate, identified by LC-MS studies and used during analytical method development and validation studies. Also, process related amoxicillin impurity-P was synthesized and characterized by using nuclear magnetic resonance (NMR) and mass spectroscopy (MS) for the first time. As complementary of this work, an assay method for amoxicillin and potassium clavulanate mixtures was developed and validated; stress-testing and stability studies of amox/clav mixtures was carried out under specified conditions according to ICH and analyzed by using validated stability-indicating assay and related substances methods. Copyright © 2016 Elsevier B.V. All rights reserved.
Kostić, Nađa; Dotsikas, Yannis; Jović, Nebojša; Stevanović, Galina; Malenović, Anđelija; Medenica, Mirjana
2014-07-01
This paper presents a LC-MS/MS method for the determination of antiepileptic drug vigabatrin in dried plasma spots (DPS). Due to its zwitterionic chemical structure, a pre-column derivatization procedure was performed, aiming to yield enhanced ionization efficiency and improved chromatographic behaviour. Propyl chloroformate, in the presence of propanol, was selected as the best derivatization reagent, providing a strong signal along with reasonable run time. A relatively novel sample collection technique, DPS, was utilized, offering easy sample handling and analysis, using a sample in micro amount (∼5μL). Derivatized vigabatrin and its internal standard, 4-aminocyclohexanecarboxylic acid, were extracted by liquid-liquid extraction (LLE) and determined in positive ion mode by applying two SRM transitions per analyte. A Zorbax Eclipse XDB-C8 column (150×4.6mm, 5μm particle size) maintained at 30°C, was utilized with running mobile phase composed of acetonitrile: 0.15% formic acid (85:15, v/v). Flow rate was 550μL/min and total run time 4.5min. The assay exhibited excellent linearity over the concentration range of 0.500-50.0μg/mL, which is suitable for the determination of vigabatrin level after per os administration in children and youths with epilepsy, who were on vigabatrin therapy, with or without co-medication. Specificity, accuracy, precision, recovery, matrix-effect and stability were also estimated and assessed within acceptance criteria. Copyright © 2014 Elsevier B.V. All rights reserved.
Qiu, Chen; Zhu, Hongbin; Ruzicka, Connie; Keire, David; Ye, Hongping
2018-05-15
Penicillins and some non-penicillin β-lactams may cause potentially life-threatening allergic reactions. Thus, possible cross contamination of β-lactams in food or drugs can put people at risk. Therefore, when there is a reasonable possibility that a non-penicillin product could be contaminated by penicillin, the drug products are tested for penicillin contamination. Here, a sensitive and rapid method for simultaneous determination of multiple β-lactam antibiotics using high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed and validated. Mass spectral acquisition was performed on a Q-Exactive HF mass spectrometer in positive ion mode with parallel reaction monitoring (PRM). The method was validated for seven β-lactam antibiotics including one or two from each class and a synthetic intermediate. The quantification precision and accuracy at 200 ppb were in the range of ± 1.84 to ± 4.56 and - 5.20 to 3.44%, respectively. The limit of detection (LOD) was 0.2 ppb, and the limit of quantitation (LOQ) was 2 ppb with a linear dynamic range (LDR) of 2-2000 ppb for all eight β-lactams. From various drug products, the recoveries of eight β-lactams at 200 and 2 ppb ranged from 93.8 ± 3.2 to 112.1 ± 4.2% and 89.7 ± 4.6 to 110.6 ± 1.9%, respectively. The application of the method for detecting cross contamination of trace β-lactams (0.2 ppb) and for monitoring facility surface cleaning was also investigated. This sensitive and fast method was fit-for-purpose for detecting and quantifying trace amount of β-lactam contamination, monitoring cross contamination in manufacturing processes, and determining potency for regulatory purposes and for quality control.
Multiclass determination and confirmation of antibiotic residues in honey using LC-MS/MS.
Lopez, Mayda I; Pettis, Jeffery S; Smith, I Barton; Chu, Pak-Sin
2008-03-12
A multiclass method has been developed for the determination and confirmation in honey of tetracyclines (chlortetracycline, doxycycline, oxytetracycline, and tetracycline), fluoroquinolones (ciprofloxacin, danofloxacin, difloxacin, enrofloxacin, and sarafloxacin), macrolides (tylosin), lincosamides (lincomycin), aminoglycosides (streptomycin), sulfonamides (sulfathiazole), phenicols (chloramphenicol), and fumagillin residues using liquid chromatography tandem mass spectrometry (LC-MS/MS). Erythromycin (a macrolide) and monensin (an ionophore) can be detected and confirmed but not quantitated. Honey samples (approximately 2 g) are dissolved in 10 mL of water and centrifuged. An aliquot of the supernatant is used to determine streptomycin. The remaining supernatant is filtered through a fine-mesh nylon fabric and cleaned up by solid phase extraction. After solvent evaporation and sample reconstitution, 15 antibiotics are assayed by LC-MS/MS using electrospray ionization (ESI) in positive ion mode. Afterward, chloramphenicol is assayed using ESI in negative ion mode. The method has been validated at the low part per billion levels for most of the drugs with accuracies between 65 and 104% and coefficients of variation less than 17%. The evaluation of matrix effects caused by honey of different floral origin is presented.
USDA-ARS?s Scientific Manuscript database
An LC-MS/MS method was developed and validated to determine 7alpha-OH cholesterol in liver microsome. This method was convenient and fast with high specificity and sensitivity. Briefly, a gradient elution was performed on a Synergi polar-C18 column (50 x 4.6mm i.d., 3microm). The mobile phase (cons...
Boison, Joe O; Asea, Philip A; Matus, Johanna L
2012-08-01
A new and sensitive multi-residue method (MRM) with detection by LC-MS/MS was developed and validated for the screening, determination, and confirmation of residues of 7 nitroimidazoles and 3 of their metabolites in turkey muscle tissues at concentrations ≥ 0.05 ng/g. The compounds were extracted into a solvent with an alkali salt. Sample clean-up and concentration was then done by solid-phase extraction (SPE) and the compounds were quantified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The characteristic parameters including repeatability, selectivity, ruggedness, stability, level of quantification, and level of confirmation for the new method were determined. Method validation was achieved by independent verification of the parameters measured during method characterization. The seven nitroimidazoles included are metronidazole (MTZ), ronidazole (RNZ), dimetridazole (DMZ), tinidazole (TNZ), ornidazole (ONZ), ipronidazole (IPR), and carnidazole (CNZ). It was discovered during the single laboratory validation of the method that five of the seven nitroimidazoles (i.e. metronidazole, dimetridazole, tinidazole, ornidazole and ipronidazole) and the 3 metabolites (1-(2-hydroxyethyl)-2-hydroxymethyl-5-nitroimidazole (MTZ-OH), 2-hydroxymethyl-1-methyl-5-nitroimidazole (HMMNI, the common metabolite of ronidazole and dimetridazole), and 1-methyl-2-(2'-hydroxyisopropyl)-5-nitroimidazole (IPR-OH) included in this study could be detected, confirmed, and quantified accurately whereas RNZ and CNZ could only be detected and confirmed but not accurately quantified. © Her Majesty the Queen in Right of Canada as Represented by the Minister of Agriculture and Agri-food Canada 2012.
Chen, Xiabin; Zheng, Xirong; Ding, Kai; Zhou, Ziyuan; Zhan, Chang-Guo; Zheng, Fang
2016-01-01
As new metabolic pathways of cocaine were recently identified, a high performance liquid chromatography tandem mass spectrometry (LC-MS/MS) method was developed to simultaneously determine cocaine and nine cocaine-related metabolites in whole blood samples. One-step solid phase extraction was used to extract all of the ten compounds and corresponding internal standards from blood samples. All compounds and internal standards extracted were separated on an Atlantis T3 (100Å, 3 µm, 2.1 mm × 150 mm I.D) column and detected in positive ion and high sensitivity mode with multiple reaction monitoring. This method was validated for its sensitivity, linearity, specificity, accuracy, precision, recovery, and stability. All of the ten compounds were quantifiable ranging from the lower limit of quantification (LLOQs) of ~10 nM (1.9–3.2 ng/ml) to ~1000 nM (190–320 ng/ml) without any interfering substance. Accuracy and precision were determined, and both of them were within the acceptance criteria of the United States (US) Food and Drug Administration (FDA) and European Medicines Agency (EMA) guidelines. The recovery was above 66.7% for all compounds. Stability tests demonstrated the stability of compounds under different storage conditions in whole blood samples. The method was successfully applied to a pharmacokinetic study with co-administration of cocaine and alcohol in rats. PMID:27923200
Reichert, Bárbara; de Kok, André; Pizzutti, Ionara Regina; Scholten, Jos; Cardoso, Carmem Dickow; Spanjer, Martien
2018-04-03
This paper describes the optimization and validation of an acetonitrile based method for simultaneous extraction of multiple pesticides and mycotoxins from raw coffee beans followed by LC-ESI-MS/MS determination. Before extraction, the raw coffee samples were milled and then slurried with water. The slurried samples were spiked with two separate standard solutions, one containing 131 pesticides and a second with 35 mycotoxins, which were divided into 3 groups of different relative concentration levels. Optimization of the QuEChERS approach included performance tests with acetonitrile acidified with acetic acid or formic acid, with or without buffer and with or without clean-up of the extracts before LC-ESI-MS/MS analysis. For the clean-up step, seven d-SPE sorbents and their various mixtures were evaluated. After method optimization a complete validation study was carried out to ensure adequate performance of the extraction and chromatographic methods. The samples were spiked at 3 concentrations levels with both mycotoxins and pesticides (with 6 replicates at each level, n = 6) and then submitted to the extraction procedure. Before LC-ESI-MS/MS analysis, the acetonitrile extracts were diluted 2-fold with methanol, in order to improve the chromatographic performance of the early-eluting polar analytes. Calibration standard solutions were prepared in organic solvent and in blank coffee extract at 7 concentration levels and analyzed 6 times each. The method was assessed for accuracy (recovery %), precision (RSD%), selectivity, linearity (r 2 ), limit of quantification (LOQ) and matrix effects (%). Copyright © 2017 Elsevier B.V. All rights reserved.
Ichibangase, Tomoko; Sugawara, Yasuhiro; Yamabe, Akio; Koshiyama, Akiyo; Yoshimura, Akari; Enomoto, Takemi; Imai, Kazuhiro
2012-01-01
Systems biology aims to understand biological phenomena in terms of complex biological and molecular interactions, and thus proteomics plays an important role in elucidating protein networks. However, many proteomic methods have suffered from their high variability, resulting in only showing altered protein names. Here, we propose a strategy for elucidating cellular protein networks based on an FD-LC-MS/MS proteomic method. The strategy permits reproducible relative quantitation of differences in protein levels between different cell populations and allows for integration of the data with those obtained through other methods. We demonstrate the validity of the approach through a comparison of differential protein expression in normal and conditional superoxide dismutase 1 gene knockout cells and believe that beginning with an FD-LC-MS/MS proteomic approach will enable researchers to elucidate protein networks more easily and comprehensively. PMID:23029042
Naveed, Safila; Usmanghani, Khan; Sana, Aisha; Ali, Huma; Zafar, Farya; Qamar, Fatima; Sarwer, Ghulam; Abbas, Sarah; Alam, M Tanweer; Shinwari, Muhammad Ibrar
2018-01-01
Sensitive, simple, reliable and rapid HPLC technique for the estimation of simvastatin (SMV) and cetirizine has been designed in this study. The chromatographic conditions were set using Shimadzu LC-10 AT VP pump, with UV detector (SPD-10 AV-VP). System integration was performed with CBM-102 (Bus Module). Partitioning of components was attained with pre-packed C-18 column of Purospher Star (5 μm, 250 x 4.6 mm) at ambient conditions. Injected volume of sample was 10 μl. Mobile phase was composed of 50:50 v/v ratio of Acetonitrile/water (pH 3.0 adjusted with ortho-phosphoric acid) having 2 ml/minutes rate of flow. Compounds were detected in UV region at 225 nm. Percent Recovery of simvastatin was observed in the range of 98-102%. All results were found in accept table range of specification. The projected method is consistent, specific, precise, and rapid, that can be employed to quantitate the SMV along with cetirizine HCl. It was estimated by 3 successive cycles of freeze and thaw stability. Results of FT samples were found within accept table limits the method was developed and validated in raw materials, bulk formulations and final drug products.
Cheng, Keding; Sloan, Angela; McCorrister, Stuart; Peterson, Lorea; Chui, Huixia; Drebot, Mike; Nadon, Celine; Knox, J David; Wang, Gehua
2014-12-01
The need for rapid and accurate H typing is evident during Escherichia coli outbreak situations. This study explores the transition of MS-H, a method originally developed for rapid H antigen typing of E. coli using LC-MS/MS of flagella digest of reference strains and some clinical strains, to E. coli isolates in clinical scenario through quantitative analysis and method validation. Motile and nonmotile strains were examined in batches to simulate clinical sample scenario. Various LC-MS/MS batch run procedures and MS-H typing rules were compared and summarized through quantitative analysis of MS-H data output for a standard method development. Label-free quantitative data analysis of MS-H typing was proven very useful for examining the quality of MS-H result and the effects of some sample carryovers from motile E. coli isolates. Based on this, a refined procedure and protein identification rule specific for clinical MS-H typing was established and validated. With LC-MS/MS batch run procedure and database search parameter unique for E. coli MS-H typing, the standard procedure maintained high accuracy and specificity in clinical situations, and its potential to be used in a clinical setting was clearly established. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Jähne, Evelyn A; Eigenmann, Daniela E; Culot, Maxime; Cecchelli, Roméo; Walter, Fruzsina R; Deli, Mária A; Tremmel, Robin; Fricker, Gert; Smiesko, Martin; Hamburger, Matthias; Oufir, Mouhssin
2014-09-01
The compound (E,Z)-3-(4-hydroxy-3,5-dimethoxybenzylidene)indolin-2-one (indolinone) was identified from lipophilic woad extracts (Isatis tinctoria L., Brassicaceae) as a compound possessing potent histamine release inhibitory and anti-inflammatory properties [1]. To further evaluate the potential of indolinone in terms of crossing the blood-brain barrier (BBB), we screened the compound in several in vitro cell-based human and animal BBB models. Therefore, we developed a quantitative LC-MS/MS method for the compound in modified Ringer HEPES buffer (RHB) and validated it according to FDA and EMA guidelines [2,3]. The calibration curve of indolinone in the range between 30.0 and 3000ng/ml was quadratic, and the limit of quantification was 30.0ng/ml. Dilution of samples up to 100-fold did not affect precision and accuracy. The carry-over was within acceptance criteria. Indolinone proved to be stable in RHB for 3h at room temperature (RT), and for three successive freeze/thaw cycles. The processed samples could be stored in the autosampler at 10°C for at least 28h. Moreover, indolinone was stable for at least 16 days in RHB when stored below -65°C. This validation study demonstrates that our method is specific, selective, precise, accurate, and capable to produce reliable results. In the immortalized human BBB mono-culture model, the apparent permeability coefficient from apical to basolateral (PappA→B), and the Papp from basolateral to apical (PappB→A) were 19.2±0.485×10(-6)cm/s and 21.7±0.326×10(-6)cm/s, respectively. For the primary rat/bovine BBB co-culture model a PappA→B of 27.1±1.67×10(-6)cm/s was determined. In the primary rat BBB triple co-culture model, the PappA→B and the PappB→A were 56.2±3.63×10(-6)cm/s and 34.6±1.41×10(-6)cm/s, respectively. The data obtained with the different models showed good correlation and were indicative of a high BBB permeation potential of indolinone confirmed by in silico prediction calculations. P
Sharma, Kuldeep; Giri, Kalpeshkumar; Dhiman, Vinay; Dixit, Abhishek; Zainuddin, Mohd; Mullangi, Ramesh
2015-05-01
A highly sensitive, specific and rapid LC-ESI-MS/MS method has been developed and validated for simultaneous quantification of methotrexate (MTX) and tofacitinib (TFB) in rat plasma (50 μL) using phenacetin as an internal standard (IS), as per the US Food and Drug Administration guidelines. After a solid-phase extraction procedure, the separation of the analytes and IS was performed on a Chromolith RP₁₈e column using an isocratic mobile phase of 5 m m ammonium acetate (pH 5.0) and acetonitrile at a ratio of 25:75 (v/v) using flow-gradient with a total run time of 3.5 min. The detection was performed in multiple reaction monitoring mode, using the transitions of m/z 455.2 → 308.3, m/z 313.2 → 149.2 and m/z 180.3 → 110.2 for MTX, TFB and IS, respectively. The calibration curves were linear over the range of 0.49-91.0 and 0.40-74.4 ng/mL for MTX and TFB, respectively. The intra- and interday accuracy and precision values for MTX and TFB were <15% at low quality control (QC), medium QC and high QC and <20% at lower limit of quantification. The validated assay was applied to derive the pharmacokinetic parameters for MTX and TFB post-dosing of MTX and TFB orally and intravenously to rats. Copyright © 2014 John Wiley & Sons, Ltd.
Liu, Yanhong; Zhang, Weihua; Yang, Yuhui
2008-10-19
A hydrophilic interaction high performance liquid chromatography-tandem mass spectrometric method has been developed and validated for simultaneous quantification of dacarbazine (DTIC) and its terminal metabolite, 5-amino-4-imidazole-carboxamide (AIC) in human plasma. The plasma samples are first extracted by a C8+SCX mixed-mode 96-well plate to extend the extraction stability of DTIC and AIC. The extracted residues are further cleaned by a primary and secondary amine (PSA) adsorbent for minimization of matrix effect. Analyses are done on an Amide-80 HPLC column coupled to a tandem mass spectrometer fitted with an atmospheric pressure turbo ion spray ionization interface in the positive-ion mode. Both DTIC and AIC have reproducible retention times on the Amide-80 HPLC column. This type of column not only has an excellent column life (over 4000 injections), but also has zero carryover effect. The injection volume should be limited at 10 microL or less to avoid the peak splitting. The validated concentration ranges are from 0.5 to 500 ng/mL for DTIC and from 2.0 to 500 ng/mL for AIC. The validated method has been successfully applied to determine the pharmacokinetic profiles for human patients receiving DTIC infusions.
A survey of free glutamic acid in foods using a robust LC-MS/MS method.
Cebi, Nur; Dogan, Canan Ekinci; Olgun, Elmas Oktem; Sagdic, Osman
2018-05-15
An effective and simultaneous liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was used with the aim of quantifying monosodium glutamate (MSG) in foodstuffs, such as chips, taste cubes, sauces and soups. The results were linear (R 2 = 1), with very low LOD and LOQ values, 1.0 µg/kg, 5.0 µg/kg, respectively. Excellent repeatability and reproducibility were also achieved. This highly sensitive and robust LC-MS/MS technique was applied successfully for the detection and quantification of MSG in a wide variety of foodstuffs. MSG contents ranged from 0.01 g/100 g to 15.39 g/100 g in food samples. Importantly, determination of free glutamic acid in the daily diet could also prevent various side effects associated with consumption of excess free glutamic acid. Copyright © 2017 Elsevier Ltd. All rights reserved.
Song, Yuelin; Song, Qingqing; Li, Jun; Zheng, Jiao; Li, Chun; Zhang, Yuan; Zhang, Lingling; Jiang, Yong; Tu, Pengfei
2016-07-08
Direct analysis is of great importance to understand the real chemical profile of a given sample, notably biological materials, because either chemical degradation or diverse errors and uncertainties might be resulted from sophisticated protocols. In comparison with biofluids, it is still challenging for direct analysis of solid biological samples using high performance liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Herein, a new analytical platform was configured by online hyphenating pressurized liquid extraction (PLE), turbulent flow chromatography (TFC), and LC-MS/MS. A facile, but robust PLE module was constructed based on the phenomenon that noticeable back-pressure can be generated during rapid fluid passing through a narrow tube. TFC column that is advantageous at extracting low molecular analytes from rushing fluid was employed to link at the outlet of the PLE module to capture constituents-of-interest. An electronic 6-port/2-position valve was introduced between TFC column and LC-MS/MS to fragment each measurement into extraction and elution phases, whereas LC-MS/MS took the charge of analyte separation and monitoring. As a proof of concept, simultaneous determination of 24 endogenous substances including eighteen steroids, five eicosanoids, and one porphyrin in feces was carried out in this paper. Method validation assays demonstrated the analytical platform to be qualified for directly simultaneous measurement of diverse endogenous analytes in fecal matrices. Application of this integrated platform on homolog-focused profiling of feces is discussed in a companion paper. Copyright © 2016 Elsevier B.V. All rights reserved.
Goryński, Krzysztof; Kiedrowicz, Alicja; Bojko, Barbara
2016-08-05
The current work describes the development and validation of a simple, efficient, and fast method using solid phase microextraction coupled to liquid chromatography-tandem mass spectrometry (SPME-LC-MS/MS) for the concomitant measurement of eight beta-blockers and bronchodilators in plasma and urine. The presented assay enables quantitative determination of acebutolol, atenolol, fenoterol, nadolol, pindolol, procaterol, sotalol, and timolol. In this work, samples were prepared on a high-throughput platform using the 96-well plate format of the thin film solid phase microextraction (TFME) system, and a biocompatible extraction phase made of hydrophilic-lipophilic balance particles. Analytes were separated on a pentafluorophenyl column (100mm×2.1mm, 3μm) by gradient elution using an UPLC Nexera coupled with an LCMS-8060 mass spectrometer. The mobile phase consisted of water-acetonitrile (0.1% formic acid) at a flow rate of 0.4mLmin(-1). The linearity of the method was checked within therapeutic blood-plasma concentrations, and shown to adequately reflect typically expected concentrations of future study samples. Post-extraction addition experiments showed that the matrix effect ranged in plasma from 98% for procaterol to 115% for nadolol, and in urine, from 85% for nadolol and pindolol to 119% for atenolol. The method was successfully validated using Food and Drug Administration (FDA) guidelines, and met all acceptance criteria for bioanalytical assays at five concentration levels for all selected drugs. The final protocol can be successfully applied for monitoring concentrations of the selected drugs in both plasma and urine matrices obtained from patients or athletes. Copyright © 2016 Elsevier B.V. All rights reserved.
Gaudin, Valérie
2017-09-01
Screening methods are used as a first-line approach to detect the presence of antibiotic residues in food of animal origin. The validation process guarantees that the method is fit-for-purpose, suited to regulatory requirements, and provides evidence of its performance. This article is focused on intra-laboratory validation. The first step in validation is characterisation of performance, and the second step is the validation itself with regard to pre-established criteria. The validation approaches can be absolute (a single method) or relative (comparison of methods), overall (combination of several characteristics in one) or criterion-by-criterion. Various approaches to validation, in the form of regulations, guidelines or standards, are presented and discussed to draw conclusions on their potential application for different residue screening methods, and to determine whether or not they reach the same conclusions. The approach by comparison of methods is not suitable for screening methods for antibiotic residues. The overall approaches, such as probability of detection (POD) and accuracy profile, are increasingly used in other fields of application. They may be of interest for screening methods for antibiotic residues. Finally, the criterion-by-criterion approach (Decision 2002/657/EC and of European guideline for the validation of screening methods), usually applied to the screening methods for antibiotic residues, introduced a major characteristic and an improvement in the validation, i.e. the detection capability (CCβ). In conclusion, screening methods are constantly evolving, thanks to the development of new biosensors or liquid chromatography coupled to tandem-mass spectrometry (LC-MS/MS) methods. There have been clear changes in validation approaches these last 20 years. Continued progress is required and perspectives for future development of guidelines, regulations and standards for validation are presented here.
Wang, Jin; Cao, Xianshuang; Jiang, Hao; Qi, Yadong; Chin, Kit L; Yue, Yongde
2014-12-17
Hibiscus sabdariffa has gained attention for its antioxidant activity. There are many accessions of H. sabdariffa in the world. However, information on the quantification of antioxidant compounds in different accessions is rather limited. In this paper, a liquid chromatography/quadrupole-time-of-flight mass spectrometry (LC-Q-TOF-MS) method for simultaneous determination of five antioxidant compounds (neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, rutin, and isoquercitrin) in H. sabdariffa leaves was developed. The method was validated for linearity, sensitivity, precision, repeatability and accuracy. The validated method has been successfully applied for determination of the five analytes in eight accessions of H. sabdariffa. The eight accessions of H. sabdariffa were evaluated for their antioxidant activities by DPPH free radical scavenging assay. The investigated accessions of H. sabdariffa were rich in rutin and exhibited strong antioxidant activity. The two accessions showing the highest antioxidant activities were from Cuba (No. 2) and Taiwan (No. 5). The results indicated that H. sabdariffa leaves could be considered as a potential antioxidant source for the food industry. The developed LC-Q-TOF-MS method is helpful for quality control of H. sabdariffa.
Naraharisetti, Suresh Babu; Kirby, Brian J.; Hebert, Mary F.; Easterling, Thomas R.; Unadkat, Jashvant D.
2009-01-01
Glyburide (glibenclamide, INN), a second generation sulfonylurea is widely used in the treatment of gestational diabetes mellitus (GDM). None of the previously reported analytical methods provide adequate sensitivity for the expected sub-nanogram/mL maternal and umbilical cord plasma concentrations of glyburide during pregnancy. We developed and validated a sensitive and low sample volume liquid chromatographic-mass spectrometric (LC-MS) method for simultaneous determination of glyburide (GLY) and its metabolite, 4-transhydroxy glyburide (M1) in human plasma (0.5 ml) or urine (0.1 ml). The limits of quantitation (LOQ) for GLY and M1 in plasma were 0.25 and 0.40 ng/mL, respectively whereas it was 1.06 ng/mL for M1 in urine. As measured by quality control samples, precision (% coefficient of variation) of the assay was < 15% whereas the accuracy (% deviation from expected) ranged from −10.1–14.3%. We found that the GLY metabolite, M1 is excreted in the urine as the glucuronide-conjugate. PMID:17980680
Ayoub, Bassam M; Mowaka, Shereen; Elzanfaly, Eman S; Ashoush, Nermeen; Elmazar, Mohamed M; Mousa, Shaker A
2017-05-31
The present study considered the pharmacokinetic evaluation of empagliflozin after administration to Egyptian volunteers, and the results were compared with other ethnic populations. The FDA recognizes that standard methods of defining racial subgroups are necessary to compare results across pharmacokinetic studies and to assess potential subgroup differences. The design of the study was as an open labeled, randomized, one treatment, one period, single dose pharmacokinetic study. The main pharmacokinetic parameters estimated were C max , T max , t 1/2 , elimination rate constant, AUC 0-t and AUC 0-inf . The insignificant difference in pharmacokinetic parameters between Egyptians and white German subjects suggests that no dose adjustment should be considered with administration of 25 mg empagliflozin to Egyptian population. A new LC-MS/MS method was developed and validated, allowing sensitive estimation of empagliflozin (25-600 ng mL -1 ) in human plasma using dapagliflozin as an internal standard (IS). The method was applied successfully on the underlying pharmacokinetic study with enhanced sample preparation that involved liquid-liquid extraction. Multiple Reaction Monitoring (MRM) of the transition pairs of m/z 449.01 to 371.21 for empagliflozin and m/z 407.00 to 328.81 for dapagliflozin (IS) was employed utilizing negative mode Electro Spray Ionization (ESI). The validated LC-MS/MS method is suitable for further toxicodynamic and bioequivalence studies.
Determination of Dextromethorphan in Oral Fluid by LC-MS-MS.
Amaratunga, Piyadarsha; Clothier, Morgan; Lorenz Lemberg, Bridget; Lemberg, Dave
2016-06-01
Dextromethorphan (DXM) is an antitussive drug found in commonly used nonprescription cold and cough medications. At low doses, DXM is a safe drug that does not produce adverse reactions. However, abuse of DXM has been reported among adolescents and young adults using the drug at higher doses. DXM is not a scheduled drug in the USA, and the primary reason for its abuse is the ease of availability. DXM is available to purchase in the form of over-the-counter cough medications, such as Robitussin(®) and Coricidin(®), or it can be purchased over the Internet in the form of a powder. In this research work, we developed an LC-MS-MS method that can quantify DXM and dextrorphan (DXO) in oral fluid in a high-throughput toxicology laboratory setting. The developed method was validated according to the Scientific Working Group for Forensic Toxicology guidelines. The linear dynamic range was 5-100 ng/mL with a lowest limit of quantitation (LLOQ) of 5.0 ng/mL for DXM and DXO. Overall, the results of the accuracy and the precision values were within the acceptance criteria for both drugs. In addition, selectivity, matrix effect and recovery were calculated for the LC-MS-MS method. Authentic samples (n = 59) were tested to evaluate the applicability of the method. Thirty samples were found to be positive for DXM and DXO and two samples were found to be positive for DXM only. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.
Zeric Stosic, Marina Z; Jaksic, Sandra M; Stojanov, Igor M; Apic, Jelena B; Ratajac, Radomir D
2016-11-01
High-performance liquid chromatography (HPLC) method with diode array detection (DAD) were optimized and validated for separation and determination of tetramethrin in an antiparasitic human shampoo. In order to optimize separation conditions, two different columns, different column oven temperatures, as well as mobile phase composition and ratio, were tested. Best separation was achieved on the Supelcosil TM LC-18- DB column (4.6 x 250 mm), particle size 5 jim, with mobile phase methanol : water (78 : 22, v/v) at a flow rate of 0.8 mL/min and at temperature of 30⁰C. The detection wavelength of the detector was set at 220 nm. Under the optimum chromatographic conditions, standard calibration curve was measured with good linearity [r2 = 0.9997]. Accuracy of the method defined as a mean recovery of tetramethrin from shampoo matrix was 100.09%. The advantages of this method are that it can easily be used for the routine analysis of drug tetramethrin in pharmaceutical formulas and in all pharmaceutical researches involving tetramethrin.
2009-01-01
Background Aliphatic molecules containing free carboxyl groups are important intermediates in many metabolic and signalling reactions, however, they accumulate to low levels in tissues and are not efficiently ionized by electrospray ionization (ESI) compared to more polar substances. Quantification of aliphatic molecules becomes therefore difficult when small amounts of tissue are available for analysis. Traditional methods for analysis of these molecules require purification or enrichment steps, which are onerous when multiple samples need to be analyzed. In contrast to aliphatic molecules, more polar substances containing free carboxyl groups such as some phytohormones are efficiently ionized by ESI and suitable for analysis by LC-MS/MS. Thus, the development of a method with which aliphatic and polar molecules -which their unmodified forms differ dramatically in their efficiencies of ionization by ESI- can be simultaneously detected with similar sensitivities would substantially simplify the analysis of complex biological matrices. Results A simple, rapid, specific and sensitive method for the simultaneous detection and quantification of free aliphatic molecules (e.g., free fatty acids (FFA)) and small polar molecules (e.g., jasmonic acid (JA), salicylic acid (SA)) containing free carboxyl groups by direct derivatization of leaf extracts with Picolinyl reagent followed by LC-MS/MS analysis is presented. The presence of the N atom in the esterified pyridine moiety allowed the efficient ionization of 25 compounds tested irrespective of their chemical structure. The method was validated by comparing the results obtained after analysis of Nicotiana attenuata leaf material with previously described analytical methods. Conclusion The method presented was used to detect 16 compounds in leaf extracts of N. attenuata plants. Importantly, the method can be adapted based on the specific analytes of interest with the only consideration that the molecules must contain at
Székely, Gy; Henriques, B; Gil, M; Ramos, A; Alvarez, C
2012-11-01
The present study reports on a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method development strategy supported by design of experiments (DoE) for the trace analysis of 4-dimethylaminopyridine (DMAP). The conventional approaches for development of LC-MS/MS methods are usually via trial and error, varying intentionally the experimental factors which is time consuming and interactions between experimental factors are not considered. The LC factors chosen for the DoE study include flow (F), gradient (G) and injection volume (V(inj)) while cone voltage (E(con)) and collision energy (E(col)) were chosen as MS parameters. All of the five factors were studied simultaneously. The method was optimized with respect to four responses: separation of peaks (Sep), peak area (A(peak)), length of the analysis (T) and the signal to noise ratio (S/N). A quadratic model, namely central composite face (CCF) featuring 29 runs was used instead of a less powerful linear model since the increase in the number of injections was insignificant. In order to determine the robustness of the method a new set of DoE experiments was carried out applying robustness around the optimal conditions was evaluated applying a fractional factorial of resolution III with 11 runs, wherein additional factors - such as column temperature and quadrupole resolution - were considered. The method utilizes a Phenomenex Gemini NX C-18 HPLC analytical column with electrospray ionization and a triple quadrupole mass detector in multiple reaction monitoring (MRM) mode, resulting in short analyses with a 10min runtime. Drawbacks of derivatization, namely incomplete reaction and time consuming sample preparation, have been avoided and the change from SIM to MRM mode resulted in increased sensitivity and lower LOQ. The DoE method development strategy led to a method allowing the trace analysis of DMAP at 0.5 ng/ml absolute concentration which corresponds to a 0.1 ppm limit of quantification in 5mg
Chan, Sue Hay; Lee, Warren; Asmawi, Mohd Zaini; Tan, Soo Choon
2016-07-01
A sequential solid-phase extraction (SPE) method was developed and validated using liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) for the detection and quantification of salbutamol enantiomers in porcine urine. Porcine urine samples were hydrolysed with β-glucuronidase/arylsulfatase from Helix pomatia and then subjected to a double solid-phase extraction (SPE) first using the Abs-Elut Nexus SPE and then followed by the Bond Elut Phenylboronic Acid (PBA) SPE. The salbutamol enantiomers were separated using the Astec CHIROBIOTIC™ T HPLC column (3.0mm×100mm; 5μm) maintained at 15°C with a 15min isocratic run at a flow rate of 0.4mL/min. The mobile phase constituted of 5mM ammonium formate in methanol. Salbutamol and salbutamol-tert-butyl-d9 (internal standard, IS) was monitored and quantified with the multiple reaction monitoring (MRM) mode. The method showed good linearity for the range of 0.1-10ng/mL with limit of quantification at 0.3ng/mL. Analysis of the QC samples showed intra- and inter-assay precisions to be less than 5.04%, and recovery ranging from 83.82 to 102.33%. Copyright © 2016 Elsevier B.V. All rights reserved.
Vaka, Venkata Rami Reddy; Inamadugu, Jaswanth Kumar; Pilli, Nageswara Rao; Ramesh, Mullangi; Katreddi, Hussain Reddy
2013-11-01
An improved, simple and highly sensitive LC-MS/MS method has been developed and validated for quantification of febuxostat with 100 μL human plasma using febuxostat-d7 as an internal standard (IS) according to regulatory guidelines. The analyte and IS were extracted from human plasma via liquid-liquid extraction using diethyl ether. The chromatographic separation was achieved on a Zorbax C18 column using a mixture of acetonitrile and 5 mm ammonium formate (60:40, v/v) as the mobile phase at a flow rate of 0.5 mL/min. The total run time was 5.0 min and the elution of febuxostat and IS occurred at 1.0 and 1.5 min, respectively. A linear response function was established for the range of concentrations 1-6000 ng/mL (r > 0.99). The precursor to product ion transitions monitored for febuxostat and IS were m/z 317.1 → 261.1 and 324.2 → 262.1, respectively. The intra- and inter-day precisions (%RSD) were within 1.29-9.19 and 2.85-7.69%, respectively. The proposed method was successfully applied to pharmacokinetic studies in humans. Copyright © 2013 John Wiley & Sons, Ltd.
Patyra, Ewelina; Nebot, Carolina; Gavilán, Rosa Elvira; Cepeda, Alberto; Kwiatek, Krzysztof
2018-05-01
A new multi-compound method for the analysis of veterinary drugs, namely tiamulin, trimethoprim, tylosin, sulfadiazine and sulfamethazine was developed and validated in medicated feeds. After extraction, the samples were centrifuged, diluted in Milli-Q water, filtered and analysed by high performance liquid chromatography coupled to tandem mass spectrometry. The separation of the analytes was performed on a biphenyl column with a gradient of 0.1% formic acid in acetonitrile and 0.1% formic acid in Milli-Q water. Quantitative validation was done in accordance with the guidelines laid down in European Commission Decision 2002/657/EC. Method performances were evaluated by the following parameters: linearity (R 2 < 0.99), precision (repeatability <14% and within-laboratory reproducibility <24%), recovery (73.58-115.21%), sensitivity, limit of detection (LOD), limit of quantification (LOQ), selectivity and expanded measurement uncertainty (k. = 2). The validated method was successfully applied to the 2 medicated feeds obtained from the interlaboratory studies and feed manufactures from Spain in August 2017. In these samples, tiamulin, tylosin and sulfamethazine were detected at the concentration levels declared by the manufacturers. The developed method can therefore be successfully used to routinely control the content and homogeneity of these antibacterial substances in medicated feed. Abbreviations AAFCO - Association of American Feed Control Officials; TYL - tylosin; TIAM - tiamulin fumarate; TRIM - trimethoprim; SDZ - sulfadiazine; SMZ - sulfamethazine; UV - ultraviolet detector; FLD - fluorescence detector; HPLC - high performance liquid chromatography; MS/MS - tandem mass spectrometry; LOD - limit of detection; LOQ - limit of quantification; CV - coefficient of variation; SD - standard deviation; U - uncertainty.
Malinsky, Michelle Duval; Jacoby, Cliffton B; Reagen, William K
2011-01-10
We report herein a simple protein precipitation extraction-liquid chromatography tandem mass spectrometry (LC/MS/MS) method, validation, and application for the analysis of perfluorinated carboxylic acids (C7-C12), perfluorinated sulfonic acids (C4, C6, and C8), and perfluorooctane sulfonamide (FOSA) in fish fillet tissue. The method combines a rapid homogenization and protein precipitation tissue extraction procedure using stable-isotope internal standard (IS) calibration. Method validation in bluegill (Lepomis macrochirus) fillet tissue evaluated the following: (1) method accuracy and precision in both extracted matrix-matched calibration and solvent (unextracted) calibration, (2) quantitation of mixed branched and linear isomers of perfluorooctanoate (PFOA) and perfluorooctanesulfonate (PFOS) with linear isomer calibration, (3) quantitation of low level (ppb) perfluorinated compounds (PFCs) in the presence of high level (ppm) PFOS, and (4) specificity from matrix interferences. Both calibration techniques produced method accuracy of at least 100±13% with a precision (%RSD) ≤18% for all target analytes. Method accuracy and precision results for fillet samples from nine different fish species taken from the Mississippi River in 2008 and 2009 are also presented. Copyright © 2010 Elsevier B.V. All rights reserved.
Accurate LC Peak Boundary Detection for 16 O/ 18 O Labeled LC-MS Data
Cui, Jian; Petritis, Konstantinos; Tegeler, Tony; Petritis, Brianne; Ma, Xuepo; Jin, Yufang; Gao, Shou-Jiang (SJ); Zhang, Jianqiu (Michelle)
2013-01-01
In liquid chromatography-mass spectrometry (LC-MS), parts of LC peaks are often corrupted by their co-eluting peptides, which results in increased quantification variance. In this paper, we propose to apply accurate LC peak boundary detection to remove the corrupted part of LC peaks. Accurate LC peak boundary detection is achieved by checking the consistency of intensity patterns within peptide elution time ranges. In addition, we remove peptides with erroneous mass assignment through model fitness check, which compares observed intensity patterns to theoretically constructed ones. The proposed algorithm can significantly improve the accuracy and precision of peptide ratio measurements. PMID:24115998
Quantitation of Tenofovir and Emtricitabine in Dried Blood Spots (DBS) with LC-MS/MS
Zheng, Jia-Hua; Guida, Louis A; Rower, Caitlin; Castillo-Mancilla, Jose; Meditz, Amie; Klein, Brandon; Kerr, Becky Jo; Langness, Jacob; Bushman, Lane; Kiser, Jennifer; Anderson, Peter L.
2013-01-01
A reversed-phase high performance liquid chromatographic (LC), tandem mass spectrometry (MS/MS) assay for the determination of tenofovir (TFV) and emtricitabine (FTC) in dried blood spots (DBS) from human whole blood was developed and validated. Whole blood samples were spotted, dried, and a 3mm punch was extracted with methanol for analysis by LC-MS/MS utilizing stable isotope labeled internal standards. The assay was validated over the range of 2.5ng/mL to 1,000ng/mL for TFV and 2.5ng/mL to 5,000ng/mL for FTC. The method was accurate (within ± 15% of control) and precise (coefficient of variation ≤ 15%) for hematocrit concentrations ranging from 25% to 76%; using edge punches versus center punches; and spot volumes of 10µL to 50µL. Analytes were stable for five freeze/thaw cycles and up to 6 days at room temperature, whereas long-term storage required −20°C or −80°C. Comparison of TFV and FTC in DBS versus plasma yielded r2 ≥ 0.96, indicating that DBS can be used as a plasma alternative for pharmacokinetic analyses in vivo. PMID:24055850
Tang, Wai-Tong; Wong, Sui-Kay; Law, Tin-Yau; Pang, Kwok-Chu; Sin, Della; Tam, Yin-King
2008-01-01
A study of single-laboratory validation (SLV) of a reversed-phase liquid Chromatography (RP-LC) method was conducted for the determination of diester-diterpene Aconitum alkaloids, viz., aconitine, mesaconitine, and hypaconitine, in a variety of dietary supplements, including single-arid multiple-ingredient dry powder extracts, pills, capsules, and raw materials. The Aconitum alkaloids in the samples were extracted by diethyl ether in the presence of ammonia. After cleanup with solid-phase extraction to remove the matrix interferences, the alkaloids were determined by RP-LC with UV detection at 235 nm, and the results were confirmed by tandem mass Spectrometry. The linear responses for aconitine, mesaconitine, and hypaconitine based on the present LC system ranged from 0.5 to 200 μg/mL. Relative standard deviations of 2.0 to 6.9% were obtained from duplicate analysis of 6 test materials of different matrixes for the 3 Aconitum alkaloids performed by 2 analysts on 5 different days. The recoveries determined for supplements and raw materials spiked with 3 Aconitum alkaloids at levels of 2.5–10 μg/g were in the range of 86–99%. In view of the attainment of satisfactory results for accuracy, precision, and recovery in the SLV study, it is recommended that the method validation process proceed to a collaborative study. PMID:17225594
Validation of alternative methods for toxicity testing.
Bruner, L H; Carr, G J; Curren, R D; Chamberlain, M
1998-01-01
Before nonanimal toxicity tests may be officially accepted by regulatory agencies, it is generally agreed that the validity of the new methods must be demonstrated in an independent, scientifically sound validation program. Validation has been defined as the demonstration of the reliability and relevance of a test method for a particular purpose. This paper provides a brief review of the development of the theoretical aspects of the validation process and updates current thinking about objectively testing the performance of an alternative method in a validation study. Validation of alternative methods for eye irritation testing is a specific example illustrating important concepts. Although discussion focuses on the validation of alternative methods intended to replace current in vivo toxicity tests, the procedures can be used to assess the performance of alternative methods intended for other uses. Images Figure 1 PMID:9599695
ICPD-a new peak detection algorithm for LC/MS.
Zhang, Jianqiu; Haskins, William
2010-12-01
The identification and quantification of proteins using label-free Liquid Chromatography/Mass Spectrometry (LC/MS) play crucial roles in biological and biomedical research. Increasing evidence has shown that biomarkers are often low abundance proteins. However, LC/MS systems are subject to considerable noise and sample variability, whose statistical characteristics are still elusive, making computational identification of low abundance proteins extremely challenging. As a result, the inability of identifying low abundance proteins in a proteomic study is the main bottleneck in protein biomarker discovery. In this paper, we propose a new peak detection method called Information Combining Peak Detection (ICPD ) for high resolution LC/MS. In LC/MS, peptides elute during a certain time period and as a result, peptide isotope patterns are registered in multiple MS scans. The key feature of the new algorithm is that the observed isotope patterns registered in multiple scans are combined together for estimating the likelihood of the peptide existence. An isotope pattern matching score based on the likelihood probability is provided and utilized for peak detection. The performance of the new algorithm is evaluated based on protein standards with 48 known proteins. The evaluation shows better peak detection accuracy for low abundance proteins than other LC/MS peak detection methods.
ICPD-A New Peak Detection Algorithm for LC/MS
2010-01-01
Background The identification and quantification of proteins using label-free Liquid Chromatography/Mass Spectrometry (LC/MS) play crucial roles in biological and biomedical research. Increasing evidence has shown that biomarkers are often low abundance proteins. However, LC/MS systems are subject to considerable noise and sample variability, whose statistical characteristics are still elusive, making computational identification of low abundance proteins extremely challenging. As a result, the inability of identifying low abundance proteins in a proteomic study is the main bottleneck in protein biomarker discovery. Results In this paper, we propose a new peak detection method called Information Combining Peak Detection (ICPD ) for high resolution LC/MS. In LC/MS, peptides elute during a certain time period and as a result, peptide isotope patterns are registered in multiple MS scans. The key feature of the new algorithm is that the observed isotope patterns registered in multiple scans are combined together for estimating the likelihood of the peptide existence. An isotope pattern matching score based on the likelihood probability is provided and utilized for peak detection. Conclusions The performance of the new algorithm is evaluated based on protein standards with 48 known proteins. The evaluation shows better peak detection accuracy for low abundance proteins than other LC/MS peak detection methods. PMID:21143790
LC-MS/MS assay for the quantitation of the tyrosine kinase inhibitor neratinib in human plasma.
Kiesel, Brian F; Parise, Robert A; Wong, Alvin; Keyvanjah, Kiana; Jacobs, Samuel; Beumer, Jan H
2017-02-05
Neratinib is an orally available tyrosine kinase inhibitor targeting HER2 (ERBB2) and EGFR (ERBB). It is being clinically evaluated for the treatment of breast and other solid tumors types as a single agent or in combination with other chemotherapies. In support of several phase I/II clinical trials investigating neratinib combinations, we developed and validated a novel LC-MS/MS assay for the quantification of neratinib in 100μL of human plasma with a stable isotopic internal standard. Analytes were extracted from plasma using protein precipitation and evaporation of the resulting supernatant followed by resuspension. Chromatographic separation was achieved using an Acquity UPLC BEH Shield RP18 column and a gradient methanol-water mobile phase containing 10% ammonium acetate. An ABI 4000 mass spectrometer and electrospray positive mode ionization were used for detection. The assay was linear from 2 to 1,000ng/mL and proved to be accurate (98.9-106.5%) and precise (<6.2%CV), and met the FDA guidance for bioanalytical method validation. This LC-MS/MS assay will be an essential tool to further define the pharmacokinetics of neratinib. Copyright © 2016 Elsevier B.V. All rights reserved.
Shen, Hong-Wu; Jiang, Xi-Ling; Yu, Ai-Ming
2010-01-01
Introduction 5-Methoxy-N,N-dimethyltryptamine (5-MeO-DMT) is a psychoactive indolealkylamine substance that has been used for recreational purpose and may lead to fatal toxicity. While 5-MeO-DMT is mainly inactivated via deamination, it is O-demethylated to an active metabolite, bufotenine. Quantitation of 5-MeO-DMT and bufotenine is essential to understand the exposure to and the effects of drug and metabolite. This study, therefore, aimed to develop and validate a LC-MS/MS method for simultaneous analysis of 5-MeO-DMT and bufotenine in mouse serum. Methods A simple protein precipitation method coupled with an optimal gradient elution was used for sample preparation and separation. Detection of 5-MeO-DMT and bufotenine was accomplished using multiple reaction monitoring of m/z 219.2→174.2 and 205.2→160.2, respectively, in the positive ion mode. 5-Methyl-N,N-dimethyltrypamine (m/z 203.2→158.3) was used as internal standard for quantification. Accuracy and precision were determined after the analyses of quality control samples. Validated assay was then employed to determine drug and metabolite concentrations in serum samples collected from mice at different time points after intraperitoneal administration of 5-MeO-DMT (2 mg/kg). Results With a total run time of 9 min, 5-MeO-DMT and bufotenine were eluted at 2.8 and 5.6 min, respectively. The assay was linear over the range 0.90–5,890 ng/mL (1.12–7,360 pg on-column) for 5-MeO-DMT and 2.52–5,510 ng/mL (3.14–6,890 pg) for bufotenine. Intra- and inter-day precision and accuracy were within 15% for both analytes. The recovery of each analyte from 20 µL of serum containing 8.08, 72.7 and 655 ng/mL of 5-MeO-DMT and 7.56, 68.1 and 613 ng/mL of bufotenine was more than 75%. Pharmacokinetic analysis revealed that the systemic exposure (area under the curve) to metabolite bufotenine was about 1/14 of that to 5-MeO-DMT. Conclusion This LC-MS/MS method is a sensitive and reliable assay for quantitation of blood 5
Shen, Hong-Wu; Jiang, Xi-Ling; Yu, Ai-Ming
2009-04-01
INTRODUCTION: 5-Methoxy-N,N-dimethyltryptamine (5-MeO-DMT) is a psychoactive indolealkylamine substance that has been used for recreational purpose and may lead to fatal toxicity. While 5-MeO-DMT is mainly inactivated via deamination, it is O-demethylated to an active metabolite, bufotenine. Quantitation of 5-MeO-DMT and bufotenine is essential to understand the exposure to and the effects of drug and metabolite. This study, therefore, aimed to develop and validate a LC-MS/MS method for simultaneous analysis of 5-MeO-DMT and bufotenine in mouse serum. METHODS: A simple protein precipitation method coupled with an optimal gradient elution was used for sample preparation and separation. Detection of 5-MeO-DMT and bufotenine was accomplished using multiple reaction monitoring of m/z 219.2→174.2 and 205.2→160.2, respectively, in the positive ion mode. 5-Methyl-N,N-dimethyltrypamine (m/z 203.2→158.3) was used as internal standard for quantification. Accuracy and precision were determined after the analyses of quality control samples. Validated assay was then employed to determine drug and metabolite concentrations in serum samples collected from mice at different time points after intraperitoneal administration of 5-MeO-DMT (2 mg/kg). RESULTS: With a total run time of 9 min, 5-MeO-DMT and bufotenine were eluted at 2.8 and 5.6 min, respectively. The assay was linear over the range 0.90-5,890 ng/mL (1.12-7,360 pg on-column) for 5-MeO-DMT and 2.52-5,510 ng/mL (3.14-6,890 pg) for bufotenine. Intra- and inter-day precision and accuracy were within 15% for both analytes. The recovery of each analyte from 20 µL of serum containing 8.08, 72.7 and 655 ng/mL of 5-MeO-DMT and 7.56, 68.1 and 613 ng/mL of bufotenine was more than 75%. Pharmacokinetic analysis revealed that the systemic exposure (area under the curve) to metabolite bufotenine was about 1/14 of that to 5-MeO-DMT. CONCLUSION: This LC-MS/MS method is a sensitive and reliable assay for quantitation of blood 5-Me
Lozano, Ana; Pérez-Parada, Andrés; Heinzen, Horacio; Fernández-Alba, Amadeo R
2012-01-01
In spite of high plant growth regulator application rates, little has been reported in the literature on determination of their residues in fruits and vegetables. This would be useful in monitoring good manufacturing practices and overall safety through the enforcement of maximum residue levels (MRLs). The present work describes method validation for the determination of 1-naphthylacetic acid (NAA) and 2(1-naphthyl)acetamide (NAAm) in tomato and zucchini using the mini-Luke, ethyl acetate (EtOAc) and acetate-buffered quick, easy, cheap, effective, rugged, and safe (QuEChERS) methods. Samples were spiked at two different levels: 50 and 100 pg/kg for NAA and 20 and 100 pg/kg for NAAm. These compounds were analyzed within the same chromatographic run with LC coupled to triple quadrupole MS (LC/(QqQ)MS/MS) in positive and negative electrospray ionization [ESI(+) and ESI(-)] modes for NAAm and NAA, respectively. For analyte confirmation, LC/ESI(-)QTOF-MS was also investigated given that NAA has only one multiple reaction monitoring transition (185.1-*140.9 m/z). These three common methods were used to determine linearity, recoveries, precision (RSD), matrix effects, repeatability, and reproducibility (n = 5) for the selected matrixes. In terms of the Directorate-General for Health and Consumers (DG-SANCO) guidelines, only insignificant differences were found for the multiresidue methods tested, regardless of the commodity. Matrix-matched calibration was used, and LODs were below 10.1 pg/kg for NAA and 6.0 pg/kg for NAAm, which were lower than the MRLs established in current European Union legislation for these compounds. Obtained recoveries for NAA ranged from 87 to 107% with RSD values below 10% for mini-Luke, 83 to 107% with RSD <11% for EtOAc, and 76 to 85% with RSD <7% for QuEChERS. NAAm recoveries ranged from 74 to 102% with RSD 5 15% for mini-Luke, 76 to 97% with RSD <4% for EtOAc, and 76 to 93% with RSD < 5% for QuEChERS. The linearity of the response over two
Jank, Louise; Martins, Magda Targa; Arsand, Juliana Bazzan; Campos Motta, Tanara Magalhães; Hoff, Rodrigo Barcellos; Barreto, Fabiano; Pizzolato, Tânia Mara
2015-11-01
A fast and simple method for residue analysis of the antibiotics classes of macrolides (erythromycin, azithromycin, tylosin, tilmicosin and spiramycin) and lincosamides (lincomycin and clindamycin) was developed and validated for cattle, swine and chicken muscle and for bovine milk. Sample preparation consists in a liquid-liquid extraction (LLE) with acetonitrile, followed by liquid chromatography-electrospray-tandem mass spectrometry analysis (LC-ESI-MS/MS), without the need of any additional clean-up steps. Chromatographic separation was achieved using a C18 column and a mobile phase composed by acidified acetonitrile and water. The method was fully validated according the criteria of the Commission Decision 2002/657/EC. Validation parameters such as limit of detection, limit of quantification, linearity, accuracy, repeatability, specificity, reproducibility, decision limit (CCα) and detection capability (CCβ) were evaluated. All calculated values met the established criteria. Reproducibility values, expressed as coefficient of variation, were all lower than 19.1%. Recoveries range from 60% to 107%. Limits of detection were from 5 to 25 µg kg(-1).The present method is able to be applied in routine analysis, with adequate time of analysis, low cost and a simple sample preparation protocol. Copyright © 2015. Published by Elsevier B.V.
Tré-Hardy, Marie; Capron, Arnaud; Antunes, Marina Venzon; Linden, Rafael; Wallemacq, Pierre
2016-11-01
The purpose of this study was to develop and validate a new liquid chromatography-tandem mass spectrometric (LC-MSMS) assay for the simultaneous quantification of tamoxifen (TAM) and its main therapeutically active metabolites, N-desmethyltamoxifen (NDT), 4-hydroxytamoxifen (4HT) and endoxifen (END) in dried blood spots. Ultrasound assisted methanolic extraction was used for TAM and metabolites extraction from dried blood spot. After evaporation and methanol reconstitution, the extract was injected into a LC-MSMS system. Reversed phase chromatography was performed on a C18 grafted column in gradient mode. TAM, metabolites, and internal standard (diazepam-d 5 ; IS) were identified in positive electrospray ionization mode using m/z transition of 372.5>72.1 (TAM); 374.23>58.10 (END); 358.27>58.10 (NDT); 388.23>44.80 (4HT) and 290.00>198.00 (IS). Total analytical run time was 6.5min. Assay was linear from 1 to 500ng/mL for all substances and presented intra and inter-assay precision and accuracy <15%. TAM, NDT, 4HT and END limits of quantification and detection were of 1 and 0.5ng/mL; 1 and 3ng/mL; 1.7 and 3ng/mL; 0.6 and 2ng/mL, respectively. Recovery ranged from 83.8 to 96.3% with matrix effect ranged from 4.3 to 29.8% for TAM and its metabolites. Hematocrit value ≤40% appeared to negatively influence accuracy of the method. In conclusion, the method described here is somewhat accessible, relatively fast, sensitive and selective with no interference. This assay might be used to investigate the level of TAM and its metabolites in DBS for therapeutic drug monitoring purposes. Copyright © 2016 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
Wiegers, Ann L
2003-07-01
Third-party accreditation is a valuable tool to demonstrate a laboratory's competence to conduct testing. Accreditation, internationally and in the United States, has been discussed previously. However, accreditation is only I part of establishing data credibility. A validated test method is the first component of a valid measurement system. Validation is defined as confirmation by examination and the provision of objective evidence that the particular requirements for a specific intended use are fulfilled. The international and national standard ISO/IEC 17025 recognizes the importance of validated methods and requires that laboratory-developed methods or methods adopted by the laboratory be appropriate for the intended use. Validated methods are therefore required and their use agreed to by the client (i.e., end users of the test results such as veterinarians, animal health programs, and owners). ISO/IEC 17025 also requires that the introduction of methods developed by the laboratory for its own use be a planned activity conducted by qualified personnel with adequate resources. This article discusses considerations and recommendations for the conduct of veterinary diagnostic test method development, validation, evaluation, approval, and transfer to the user laboratory in the ISO/IEC 17025 environment. These recommendations are based on those of nationally and internationally accepted standards and guidelines, as well as those of reputable and experienced technical bodies. They are also based on the author's experience in the evaluation of method development and transfer projects, validation data, and the implementation of quality management systems in the area of method development.
Armstrong, Jenna L; Dills, Russell L; Yu, Jianbo; Yost, Michael G; Fenske, Richard A
2014-01-01
A rapid liquid chromatography tandem mass spectrometry (LC-MS/MS) method has been developed for determination of levels of the organophosphorus (OP) pesticides chlorpyrifos (CPF), azinphos methyl (AZM), and their oxygen analogs chlorpyrifos-oxon (CPF-O) and azinphos methyl-oxon (AZM-O) on common active air sampling matrices. XAD-2 resin and polyurethane foam (PUF) matrices were extracted with acetonitrile containing stable-isotope labeled internal standards (ISTD). Analysis was accomplished in Multiple Reaction Monitoring (MRM) mode, and analytes in unknown samples were identified by retention time (±0.1 min) and qualifier ratio (±30% absolute) as compared to the mean of calibrants. For all compounds, calibration linearity correlation coefficients were ≥0.996. Limits of detection (LOD) ranged from 0.15-1.1 ng/sample for CPF, CPF-O, AZM, and AZM-O on active sampling matrices. Spiked fortification recoveries were 78-113% from XAD-2 active air sampling tubes and 71-108% from PUF active air sampling tubes. Storage stability tests also yielded recoveries ranging from 74-94% after time periods ranging from 2-10 months. The results demonstrate that LC-MS/MS is a sensitive method for determining these compounds from two different matrices at the low concentrations that can result from spray drift and long range transport in non-target areas following agricultural applications. In an inter-laboratory comparison, the limit of quantification (LOQ) for LC-MS/MS was 100 times lower than a typical gas chromatography-mass spectrometry (GC-MS) method.
t'Kindt, Ruben; De Veylder, Lieven; Storme, Michael; Deforce, Dieter; Van Bocxlaer, Jan
2008-08-01
This study treats the optimization of methods for homogenizing Arabidopsis thaliana plant leaves as well as cell cultures, and extracting their metabolites for metabolomics analysis by conventional liquid chromatography electrospray ionization mass spectrometry (LC-ESI/MS). Absolute recovery, process efficiency and procedure repeatability have been compared between different pre-LC-MS homogenization/extraction procedures through the use of samples fortified before extraction with a range of representative metabolites. Hereby, the magnitude of the matrix effect observed in the ensuing LC-MS based metabolomics analysis was evaluated. Based on relative recovery and repeatability of key metabolites, comprehensiveness of extraction (number of m/z-retention time pairs) and clean-up potential of the approach (minimum matrix effects), the most appropriate sample pre-treatment was adopted. It combines liquid nitrogen homogenization for plant leaves with thermomixer based extraction using MeOH/H(2)O 80/20. As such, an efficient and highly reproducible LC-MS plant metabolomics set-up is achieved, as illustrated by the obtained results for both LC-MS (8.88%+/-5.16 versus 7.05%+/-4.45) and technical variability (12.53%+/-11.21 versus 9.31%+/-6.65) data in a comparative investigation of A. thaliana plant leaves and cell cultures, respectively.
Absolute quantification of histone PTM marks by MRM-based LC-MS/MS.
Gao, Jun; Liao, Rijing; Yu, Yanyan; Zhai, Huili; Wang, Yingqi; Sack, Ragna; Peters, Antoine H F M; Chen, Jiajia; Wu, Haiping; Huang, Zheng; Hu, Min; Qi, Wei; Lu, Chris; Atadja, Peter; Oyang, Counde; Li, En; Yi, Wei; Zhou, Shaolian
2014-10-07
The N-terminal tails of core histones harbor the sites of numerous post-translational modifications (PTMs) with important roles in the regulation of chromatin structure and function. Profiling histone PTM marks provides data that help understand the epigenetics events in cells and their connections with cancer and other diseases. Our previous study demonstrated that specific derivatization of histone peptides by NHS propionate significantly improved their chromatographic performance on reversed phase columns for LC/MS analysis. As a step forward, we recently developed a multiple reaction monitoring (MRM) based LC-MS/MS method to analyze 42 targeted histone peptides. By using stable isotopic labeled peptides as internal standards that are spiked into the reconstituted solutions, this method allows to measure absolute concentration of the tryptic peptides of H3 histone proteins extracted from cancer cell lines. The method was thoroughly validated for the accuracy and reproducibility through analyzing recombinant histone proteins and cellular samples. The linear dynamic range of the MRM assays was achieved in 3 orders of magnitude from 1 nM to 1 μM for all targeted peptides. Excellent intrabatch and interbatch reproducibility (<15% CV) was obtained. This method has been used to study translocated NSD2 (a histone lysine methyltransferase that catalyzes the histone lysine 36 methylation) function with its overexpression in KMS11 multiple myeloma cells. From the results we have successfully quantitated both individual and combinatorial histone marks in parental and NSD2 selective knockout KMS11 cells.
Chandra, A; Rana, J; Li, Y
2001-08-01
A method has been established and validated for identification and quantification of individual, as well as total, anthocyanins by HPLC and LC/ES-MS in botanical raw materials used in the herbal supplement industry. The anthocyanins were separated and identified on the basis of their respective M(+) (cation) using LC/ES-MS. Separated anthocyanins were individually calculated against one commercially available anthocyanin external standard (cyanidin-3-glucoside chloride) and expressed as its equivalents. Amounts of each anthocyanin calculated as external standard equivalent were then multiplied by a molecular-weight correction factor to afford their specific quantities. Experimental procedures and use of a molecular-weight correction factors are substantiated and validated using Balaton tart cherry and elderberry as templates. Cyanidin-3-glucoside chloride has been widely used in the botanical industry to calculate total anthocyanins. In our studies on tart cherry and elderberry, its use as external standard followed by use of molecular-weight correction factors should provide relatively accurate results for total anthocyanins, because of the presence of cyanidin as their major anthocyanidin backbone. The method proposed here is simple and has a direct sample preparation procedure without any solid-phase extraction. It enables selection and use of commercially available anthocyanins as external standards for quantification of specific anthocyanins in the sample matrix irrespective of their commercial availability as analytical standards. It can be used as a template and applied for similar quantification in several anthocyanin-containing raw materials for routine quality control procedures, thus providing consistency in analytical testing of botanical raw materials used for manufacturing efficacious and true-to-the-label nutritional supplements.
Ates, Ebru; Mittendorf, Klaus; Senyuva, Hamide
2013-01-01
An automated sample preparation technique involving cleanup and analytical separation in a single operation using an online coupled TurboFlow (RP-LC system) is reported. This method eliminates time-consuming sample preparation steps that can be potential sources for cross-contamination in the analysis of plasticizers. Using TurboFlow chromatography, liquid samples were injected directly into the automated system without previous extraction or cleanup. Special cleanup columns enabled specific binding of target compounds; higher MW compounds, i.e., fats and proteins, and other matrix interferences with different chemical properties were removed to waste, prior to LC/MS/MS. Systematic stepwise method development using this new technology in the food safety area is described. Selection of optimum columns and mobile phases for loading onto the cleanup column followed by transfer onto the analytical column and MS detection are critical method parameters. The method was optimized for the assay of 10 phthalates (dimethyl, diethyl, dipropyl, butyl benzyl, diisobutyl, dicyclohexyl, dihexyl, diethylhexyl, diisononyl, and diisododecyl) and one adipate (diethylhexyl) in beverages and milk.
Shibata, Kaito; Naito, Takafumi; Okamura, Jun; Hosokawa, Seiji; Mineta, Hiroyuki; Kawakami, Junichi
2017-11-30
Proteomic approaches using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) without an immunopurification technique have not been applied to the determination of serum cetuximab. This study developed a simple and rapid LC-MS/MS method for the absolute determination of cetuximab in human serum and applied it to clinical settings. Surrogate peptides derived from cetuximab digests were selected using a Fourier transform mass spectrometer. Reduced-alkylated serum cetuximab without immunopurification was digested for 20minutes using immobilized trypsin, and the digestion products were purified by solid-phase extraction. The LC-MS/MS was run in positive ion multiple reaction monitoring mode. This method was applied to the determination of serum samples in head and neck cancer patients treated with cetuximab. The chromatographic run time was 10minutes and no peaks interfering with surrogate peptides in serum digestion products were observed. The calibration curve of absolute cetuximab in serum was linear over the concentration range of 4-200μg/mL. The lower limit of quantification of cetuximab in human serum was 4μg/mL. The intra-assay and inter-assay precision and accuracy were less than 13.2% and 88.0-100.7%, respectively. The serum concentration range of cetuximab was 19-140μg/mL in patients. The serum cetuximab concentrations in LC-MS/MS were correlated with those in ELISA (r=0.899, P <0.01) and the mean bias was 1.5% in cancer patients. In conclusion, the present simple and rapid method with acceptable analytical performance can be helpful for evaluating the absolute concentration of serum cetuximab in clinical settings. Copyright © 2017 Elsevier B.V. All rights reserved.
Schofield, Ryan C.; Ramanathan, Lakshmi V.; Murata, Kazunori; Grace, Marie; Fleisher, Martin; Pessin, Melissa S.; Carlow, Dean C.
2016-01-01
A rapid and simple turbulent flow liquid chromatography (TFC–LC) method implementing positive heated electrospray ionization (HESI) for the accurate and precise determination of methotrexate (MTX), 7-hydroxy methotrexate (7-OH MTX), and 4-amino-4-deoxy-N10-methylpteroic acid (DAMPA) concentrations in serum was developed. MTX was isolated from serum samples (100 μL) after protein precipitation with methanol containing formic acid and internal standard (MTX-D3) followed by centrifugation. The supernatant was injected into the turbulent flow liquid chromatography which is followed by electrospray positive ionization tandem mass spectrometry (TFC–LC–MS/MS) and quantified using a six-point calibration curve. For MTX and DAMPA the assays were linear from 10 to 1000 nmol/L and for 7-OH MTX from 20 to 2000 nmol/L. Dilutions of 10, 100 and 1000-fold were validated giving a clinically reportable range of 10 nmol/L to 5 × 105 nmol/L. Within-day and between-day precisions at concentrations spanning the analytical measurement ranges were less than 10% for all three analytes. MTX, DAMPA and 7-OH MTX were sufficiently stable under all relevant analytical conditions. No significant matrix effect was observed during the method validation. The TFC–LC-MS/MS MTX method was also compared with three other clinically validated MTX assays: a dihydrofolate reductase (DHFR) inhibition assay, an immunoassay based on fluorescence polarization and a previously developed LC–MS/MS assay. PMID:26322588
DOE Office of Scientific and Technical Information (OSTI.GOV)
Desai, Meera Jay
The purpose of this research was to develop sensitive LC-MS methods for enantiomeric separation and detection, and then apply these methods for determination of enantiomeric composition and for the study of pharmacokinetic and pharmacodynamic properties of a chiral nutraceutical. Our first study, evaluated the use of reverse phase and polar organic mode for chiral LC-API/MS method development. Reverse phase methods containing high water were found to decrease ionization efficiency in electrospray, while polar organic methods offered good compatibility and low limits of detection with ESI. The use of lower flow rates dramatically increased the sensitivity by an order of magnitude.more » Additionally, for rapid chiral screening, the coupled Chirobiotic column afforded great applicability for LC-MS method development. Our second study, continued with chiral LC-MS method development in this case for the normal phase mode. Ethoxynonafluorobutane, a fluorocarbon with low flammability and no flashpoint, was used as a substitute solvent for hexane/heptane mobile phases for LC-APCI/MS. Comparable chromatographic resolutions and selectivities were found using ENFB substituted mobile phase systems, although, peak efficiencies were significantly diminished. Limits of detection were either comparable or better for ENFB-MS over heptane-PDA detection. The miscibility of ENFB with a variety of commonly used organic modifiers provided for flexibility in method development. For APCI, lower flow rates did not increase sensitivity as significantly as was previously found for ESI-MS detection. The chiral analysis of native amino acids was evaluated using both APCI and ESI sources. For free amino acids and small peptides, APCI was found to have better sensitivities over ESI at high flow rates. For larger peptides, however, sensitivity was greatly improved with the use of electrospray. Additionally, sensitivity was enhanced with the use of non-volatile additives, This optimized method
Lattice-patterned LC-polymer composites containing various nanoparticles as additives
2012-01-01
In this study, we show the effect of various nanoparticle additives on phase separation behavior of a lattice-patterned liquid crystal [LC]-polymer composite system and on interfacial properties between the LC and polymer. Lattice-patterned LC-polymer composites were fabricated by exposing to UV light a mixture of a prepolymer, an LC, and SiO2 nanoparticles positioned under a patterned photomask. This resulted in the formation of an LC and prepolymer region through phase separation. We found that the incorporation of SiO2 nanoparticles significantly affected the electro-optical properties of the lattice-patterned LC-polymer composites. This effect is a fundamental characteristic of flexible displays. The electro-optical properties depend on the size and surface functional groups of the SiO2 nanoparticles. Compared with untreated pristine SiO2 nanoparticles, which adversely affect the performance of LC molecules surrounded by polymer walls, SiO2 nanoparticles with surface functional groups were found to improve the electro-optical properties of the lattice-patterned LC-polymer composites by increasing the quantity of SiO2 nanoparticles. The surface functional groups of the SiO2 nanoparticles were closely related to the distribution of SiO2 nanoparticles in the LC-polymer composites, and they influenced the electro-optical properties of the LC molecules. It is clear from our work that the introduction of nanoparticles into a lattice-patterned LC-polymer composite provides a method for controlling and improving the composite's electro-optical properties. This technique can be used to produce flexible substrates for various flexible electronic devices. PMID:22222011
Bertuzzi, Terenzio; Rastelli, Silvia; Mulazzi, Annalisa; Pietri, Amedeo
2017-12-01
A survey on the occurrence of acrylamide (AA) in roasted coffee, barley, and potato crisps was carried out using an intra-lab validated liquid chromatography (LC)-MS (mass spectrometry)/MS method. Over the years 2015-2016, 66 samples of coffee, 22 of roasted barley, and 22 of potato crisps were collected from retail outlets in Italy. AA was detected in almost all samples. In roasted coffee, the level exceeded 450 µg kg -1 , the limit recommended by the European Commission (EC), in 36.4% of the samples. In roasted barley, mean contamination was slightly lower than in coffee and no sample exceeded the EC limit of 2000 µg kg -1 . The AA contamination in potato crisps was remarkable. A percentage of 36.4 (n = 8) showed a value higher than the EC limit of 1000 µg kg -1 . Considering the average consumption of coffee and potato crisps by Italian people, AA exposure is significant and should be decreased.
Mostafa, Nadia M; Elsayed, Ghada M; Hassan, Nagiba Y; El Mously, Dina A
2017-11-01
Five simple, sensitive, and eco-friendly LC and UV spectrophotometric methods have been developed for the simultaneous determination of phenylephrine hydrochloride (PHE) and prednisolone acetate (PRD) in their combined dosage form. The first method was reversed-phase (RP) LC using methanol-water-heptane-1-sulfonic acid sodium salt (75 + 25 + 0.1, v/v/w) as a mobile phase. Separation was achieved using an XSelect HSS reversed-phase C18 analytical column (250 × 4.6 mm, 5µm). The flow rate was 1.0 mL/min and UV detection was done at 230 nm. Quantification was achieved over the concentration ranges of 5-50 µg/mL for PHE and 2-90 µg/mL for PRD. Four spectrophotometric methods were proposed, namely dual wavelength, first derivative of ratio spectra, ratio difference, and mean-centering of ratio spectra. Linearity was observed in the concentration ranges of 10-120 and 5-35 µg/mL for PHE and PRD, respectively, for the spectrophotometric methods. Green solvents were used in the proposed methods because they play a vital role in the analytical methods' influence on the environment. The suggested methods were validated regarding linearity, accuracy, and precision according to the International Conference on Harmonization guidelines, with satisfactory results. These methods could be used as harmless substitutes for routine analysis of the mentioned drugs, with no interference from excipients.
Boenzi, Sara; Deodato, Federica; Taurisano, Roberta; Martinelli, Diego; Verrigni, Daniela; Carrozzo, Rosalba; Bertini, Enrico; Pastore, Anna; Dionisi-Vici, Carlo; Johnson, David W
2014-11-01
Two oxysterols, cholestan-3β,5α,6β-triol (C-triol) and 7-ketocholesterol (7-KC), have been recently proposed as diagnostic markers of Niemann-Pick type C (NP-C) disease, representing a potential alternative diagnostic tool to the more invasive and time consuming filipin test in cultured fibroblasts. Usually, the oxysterols are detected and quantified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) method using atmospheric pressure chemical ionization (APCI) or electro-spray-ionization (ESI) sources, after a variety of derivatization procedures to enhance sensitivity. We developed a sensitive LC-MS/MS method to quantify the oxysterols in plasma as dimethylaminobutyrate ester, suitable for ESI analysis. This method, with an easy liquid-phase extraction and a short derivatization procedure, has been validated to demonstrate specificity, linearity, recovery, lowest limit of quantification, accuracy and precision. The assay was linear over a concentration range of 0.5-200ng/mL for C-triol and 1.0-200ng/mL for 7-KC. Intra-day and inter-day coefficients of variation (CV%) were <15% for both metabolites. Receiver operating characteristic analysis estimates that the area under curve was 0.998 for C-triol, and 0.972 for 7-KC, implying a significant discriminatory power for the method in this patient population of both oxysterols. In summary, our method provides a simple, rapid and non-invasive diagnostic tool for the biochemical diagnosis of NP-C disease. Copyright © 2014 Elsevier B.V. All rights reserved.
NASA Astrophysics Data System (ADS)
Sitnikov, Dmitri G.; Monnin, Cian S.; Vuckovic, Dajana
2016-12-01
The comparison of extraction methods for global metabolomics is usually executed in biofluids only and focuses on metabolite coverage and method repeatability. This limits our detailed understanding of extraction parameters such as recovery and matrix effects and prevents side-by-side comparison of different sample preparation strategies. To address this gap in knowledge, seven solvent-based and solid-phase extraction methods were systematically evaluated using standard analytes spiked into both buffer and human plasma. We compared recovery, coverage, repeatability, matrix effects, selectivity and orthogonality of all methods tested for non-lipid metabolome in combination with reversed-phased and mixed-mode liquid chromatography mass spectrometry analysis (LC-MS). Our results confirmed wide selectivity and excellent precision of solvent precipitations, but revealed their high susceptibility to matrix effects. The use of all seven methods showed high overlap and redundancy which resulted in metabolite coverage increases of 34-80% depending on LC-MS method employed as compared to the best single extraction protocol (methanol/ethanol precipitation) despite 7x increase in MS analysis time and sample consumption. The most orthogonal methods to methanol-based precipitation were ion-exchange solid-phase extraction and liquid-liquid extraction using methyl-tertbutyl ether. Our results help facilitate rational design and selection of sample preparation methods and internal standards for global metabolomics.
Applications of LC-MS in PET Radioligand Development and Metabolic Elucidation
Ma, Ying; Kiesewetter, Dale O.; Lang, Lixin; Gu, Dongyu; Chen, Xiaoyuan
2013-01-01
Positron emission tomography (PET) is a very sensitive molecular imaging technique that when employed with an appropriate radioligand has the ability to quantititate physiological processes in a non-invasive manner. Since the imaging technique detects all radioactive emissions in the field of view, the presence and biological activity of radiolabeled metabolites must be determined for each radioligand in order to validate the utility of the radiotracer for measuring the desired physiological process. Thus, the identification of metabolic profiles of radiolabeled compounds is an important aspect of design, development, and validation of new radiopharmaceuticals and their applications in drug development and molecular imaging. Metabolite identification for different chemical classes of radiopharmaceuticals allows rational design to minimize the formation and accumulation of metabolites in the target tissue, either through enhanced excretion or minimized metabolism. This review will discuss methods for identifying and quantitating metabolites during the pre-clinical development of radiopharmaceuticals with special emphasis on the application of LC/MS. PMID:20540692
Geng, Chunmei; Li, Pingli; Chen, Xuwang; Yuan, Guiyan; Guo, Nan; Liu, Huanjun; Zhang, Rui; Guo, Ruichen
2017-05-23
To determine the feasibility of using a nanoparticle immunoassay for clinical therapeutic drug monitoring (TDM) of docetaxel concentrations, a sensitive and simple method of liquid chromatography-tandem mass spectrometry (LC-MS/MS) was established to measure the docetaxel concentration in human plasma and the results of LC-MS/MS and the immunoassay were compared. Docetaxel and paclitaxel (the internal standard, or IS) in human plasma were extracted through protein precipitation, separated on a Diamonsil C18 column (150 mm × 4.6 mm, 5 μm), ionized with positive ions, and detected with LC-MS/MS in multi-reaction monitoring (MRM) mode. Plasma samples from 248 cancer patients were assayed with LC-MS/MS and a nanoparticle immunoassay. Data from the samples were analyzed with the statistical software SPSS and the software MedCalc. Results indicated that the calibration curve of the validated method of LC-MS/MS was linear over the range of 10-2,000 ng/mL, with an lowest limit of quantitation (LLOQ) of 10 ng/mL, and the intra- and inter- day precision and accuracy were both < ± 15%. Comparison of the two methods indicated that results of the LC-MS/MS were closely related to those of the nanoparticle immunoassay, with a correlation coefficient (R) of 0.965 and acceptable 95% confidence intervals (CI) of ‒ 231.7-331.1 ng/mL. Overall, the established method of LC-MC/MS and the nanoparticle immunoassay were both suitable for measurement of the docetaxel concentration in human plasma, and the immunoassay was far more cost-effective and better at clinical TDM of docetaxel in clinical practice.
Imbert, L; Dulaurent, S; Mercerolle, M; Morichon, J; Lachâtre, G; Gaulier, J-M
2014-01-01
The two major challenges in hair analysis are the limited amount of samples usually available and the low targeted concentrations. To overcome these limitations, a liquid chromatography-electrospray-tandem mass spectrometry method (LC-ESI-MS/MS) allowing the simultaneous analysis of 17 amphetamines (amphetamine, BDB, m-CPP, dexfenfluramine, DOB, DOM, ephedrine, MBDB, MDA, MDEA, MDMA, methamphetamine, methylphenidate, 4-MTA, norephedrine, norfenfluramine and PMA), 5 opiates (morphine, codeine, heroin, ethylmorphine, and 6AM), cocaine and 5 metabolites [ecgonine methyl ester (EME), benzoylecgonine (BZE), anhydroecgonine methyl ester (AME), cocaethylene, and norcocaine] has been developed. The validation procedure included linearity, intra-day and inter-day variability and accuracy for 5 days (5 replicates at 3 concentration levels). Proficiency studies were used to check the accuracy of the method. As a result, all amphetamines, opiates and cocaine derivatives were satisfactory identified by 2 MRM transitions in 15 min. Calibration curves were performed by a quadratic 1/X weighted regression. The calibration model fits from 0.05 to 10 ng/mg. The limits of detection (LODs) range between 0.005 and 0.030 ng/mg. Precision has been checked by intra-day and inter-day RSD, and associated relative bias, which were lower than 25% for the limits of quantifications (LOQs) and lower than 20% for the other levels tested. This method was routinely applied to hair samples: two positive results of adult drug addicts are presented. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.
Sun, Qi-ran; Xiang, Ping; Yan, Hui; Shen, Min
2008-12-01
A sensitive LC-MS/MS method to determine cocaine and its major metabolite benzoylecgonine in guinea pig' s hair has been established. About 20 mg of decontaminated hair sample was hydrolyzed with 0. 1 mol x L(-1) HCl at 50 degrees C overnight, in the presence of cocaine-d3 and benzoylecgonine-d8 used as internal standards, and then extracted with dichlormethane. The analysis was performed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Positive electrospray ionization (ESI +) and multiple reactions monitoring (MRM) mode were used. The limit of detection (LOD) for cocaine and benzoylecgonine was 1 pg x mg(-1). The calibration curves of extracted standards were linear over the range from 5 pg x mg(-1) to 250 pg x mg(-1) (r2 > or = 0.9997). The method was validated and applied to the analysis of guinea pig's hair after a single dose administration of cocaine hydrochloride. Cocaine and benzoylecgonine were not only detected, but also quantified in guinea pigs hair.
Tache, Florentin; Farca, Alexandru; Medvedovici, Andrei; David, Victor
2002-05-15
Both derivatization of free captopril in human plasma samples using monobromobimane as fluorescent label and the corresponding HPLC-fluorescence detection (FLD) method were validated. Calibration curve for the fluorescent captopril derivative in plasma samples is linear in the ppb-ppm range with a detection limit of 4 ppb and an identification limit of 10 ppb (P%: 90; nu > or = 5). These methods were successfully applied on bioequivalence studies carried out on some marketed pharmaceutical formulations.
Honda, Akira; Yamashita, Kouwa; Ikegami, Tadashi; Hara, Takashi; Miyazaki, Teruo; Hirayama, Takeshi; Numazawa, Mitsuteru; Matsuzaki, Yasushi
2009-01-01
We describe a new sensitive and specific method for the quantification of serum malonate (malonic acid, MA), which could be a new biomarker for de novo lipogenesis (fatty acid synthesis). This method is based upon a stable isotope-dilution technique using LC-MS/MS. MA from 50 μl of serum was derivatized into di-(1-methyl-3-piperidinyl)malonate (DMP-MA) and quantified by LC-MS/MS using the positive electrospray ionization mode. The detection limit of the DMP-MA was approximately 4.8 fmol (500 fg) (signal-to-noise ratio = 10), which was more than 100 times more sensitive compared with that of MA by LC-MS/MS using the negative electrospray ionization mode. The relative standard deviations between sample preparations and measurements made using the present method were 4.4% and 3.2%, respectively, by one-way ANOVA. Recovery experiments were performed using 50 μl aliquots of normal human serum spiked with 9.6 pmol (1 ng) to 28.8 pmol (3 ng) of MA and were validated by orthogonal regression analysis. The results showed that the estimated amount within a 95% confidence limit was 14.1 ± 1.1 pmol, which was in complete agreement with the observed X¯0 = 15.0 ± 0.6 pmol, with a mean recovery of 96.0%. This method provides reliable and reproducible results for the quantification of MA in human serum. PMID:19403942
Cha, Eunju; Kim, Sohee; Kim, Ho Jun; Lee, Kang Mi; Kim, Ki Hun; Kwon, Oh-Seung; Lee, Jaeick
2015-01-01
This study compared the sensitivity of various separation and ionization methods, including gas chromatography with an electron ionization source (GC-EI), liquid chromatography with an electrospray ionization source (LC-ESI), and liquid chromatography with a silver ion coordination ion spray source (LC-Ag(+) CIS), coupled to a mass spectrometer (MS) for steroid analysis. Chromatographic conditions, mass spectrometric transitions, and ion source parameters were optimized. The majority of steroids in GC-EI/MS/MS and LC-Ag(+) CIS/MS/MS analysis showed higher sensitivities than those obtained with other analytical methods. The limits of detection (LODs) of 65 steroids by GC-EI/MS/MS, 68 steroids by LC-Ag(+) CIS/MS/MS, 56 steroids by GC-EI/MS, 54 steroids by LC-ESI/MS/MS, and 27 steroids by GC-ESI/MS/MS were below cut-off value of 2.0 ng/mL. LODs of steroids that formed protonated ions in LC-ESI/MS/MS analysis were all lower than the cut-off value. Several steroids such as unconjugated C3-hydroxyl with C17-hydroxyl structure showed higher sensitivities in GC-EI/MS/MS analysis relative to those obtained using the LC-based methods. The steroids containing 4, 9, 11-triene structures showed relatively poor sensitivities in GC-EI/MS and GC-ESI/MS/MS analysis. The results of this study provide information that may be useful for selecting suitable analytical methods for confirmatory analysis of steroids. Copyright © 2015 John Wiley & Sons, Ltd.
USDA-ARS?s Scientific Manuscript database
The purpose of this study was to develop a Single-Lab Validated Method using high-performance liquid chromatography (HPLC) with different detectors (diode array detector - DAD, fluorescence detector - FLD, and mass spectrometer - MS) for determination of seven B-complex vitamins (B1 - thiamin, B2 – ...
High-Throughput Quantification of GFP-LC3+ Dots by Automated Fluorescence Microscopy.
Bravo-San Pedro, J M; Pietrocola, F; Sica, V; Izzo, V; Sauvat, A; Kepp, O; Maiuri, M C; Kroemer, G; Galluzzi, L
2017-01-01
Macroautophagy is a specific variant of autophagy that involves a dedicated double-membraned organelle commonly known as autophagosome. Various methods have been developed to quantify the size of the autophagosomal compartment, which is an indirect indicator of macroautophagic responses, based on the peculiar ability of microtubule-associated protein 1 light chain 3 beta (MAP1LC3B; best known as LC3) to accumulate in forming autophagosomes upon maturation. One particularly convenient method to monitor the accumulation of mature LC3 within autophagosomes relies on a green fluorescent protein (GFP)-tagged variant of this protein and fluorescence microscopy. In physiological conditions, cells transfected temporarily or stably with a GFP-LC3-encoding construct exhibit a diffuse green fluorescence over the cytoplasm and nucleus. Conversely, in response to macroautophagy-promoting stimuli, the GFP-LC3 signal becomes punctate and often (but not always) predominantly cytoplasmic. The accumulation of GFP-LC3 in cytoplasmic dots, however, also ensues the blockage of any of the steps that ensure the degradation of mature autophagosomes, calling for the implementation of strategies that accurately discriminate between an increase in autophagic flux and an arrest in autophagic degradation. Various cell lines have been engineered to stably express GFP-LC3, which-combined with the appropriate controls of flux, high-throughput imaging stations, and automated image analysis-offer a relatively straightforward tool to screen large chemical or biological libraries for inducers or inhibitors of autophagy. Here, we describe a simple and robust method for the high-throughput quantification of GFP-LC3 + dots by automated fluorescence microscopy. © 2017 Elsevier Inc. All rights reserved.
Desmarchelier, Aurélien; Anizan, Sébastien; Minh Tien, Mai; Savoy, Marie-Claude; Bion, Cindy
2018-04-01
An LC-MS/MS method is presented for screening five tetracyclines and their epimers in a broad range of food products. The scope of matrices includes meat-, fish-, seafood-based products, various dairy ingredients, infant formulae and fats. The method principle is based on a liquid-liquid extraction with aqueous ethylenediaminetetraacetic acid (EDTA) and acetonitrile followed by a freezing step to promote phase separation at low temperature. After defatting with hexane, sample extracts were evaporated and reconstituted before injection onto the LC-MS/MS system. The addition of oxalic acid in the aqueous mobile phase was mandatory to maintain good peak shape and sensitivity over the run. The screening is based upon a double preparation of each sample, one 'as such' and a second one with the analytes spiked in the sample, in order to mitigate the risk of false negative response. The method was validated according to the European Community Reference Laboratories Residues Guidelines. A total of 93 samples were included in the validation by two independent laboratories giving both false-negative and false-positive rates at 0% for all compounds. Over the last two years, 2600 samples were analysed routinely and only one chicken sample was found above the regulatory limit.
Data reduction of isotope-resolved LC-MS spectra.
Du, Peicheng; Sudha, Rajagopalan; Prystowsky, Michael B; Angeletti, Ruth Hogue
2007-06-01
Data reduction of liquid chromatography-mass spectrometry (LC-MS) spectra can be a challenge due to the inherent complexity of biological samples, noise and non-flat baseline. We present a new algorithm, LCMS-2D, for reliable data reduction of LC-MS proteomics data. LCMS-2D can reliably reduce LC-MS spectra with multiple scans to a list of elution peaks, and subsequently to a list of peptide masses. It is capable of noise removal, and deconvoluting peaks that overlap in m/z, in retention time, or both, by using a novel iterative peak-picking step, a 'rescue' step, and a modified variable selection method. LCMS-2D performs well with three sets of annotated LC-MS spectra, yielding results that are better than those from PepList, msInspect and the vendor software BioAnalyst. The software LCMS-2D is available under the GNU general public license from http://www.bioc.aecom.yu.edu/labs/angellab/as a standalone C program running on LINUX.
He, Jun; Bo, Fang; Tu, Yaru; Azietaku, John Teye; Dou, Ting; Ouyang, Huizi; Chang, Yanxu; Liu, Hong; Gao, Xiumei
2015-10-10
A sensitive and reliable LC-MS/MS method was developed and validated for the simultaneous determination of periplocin and its two metabolites (periplocymarin and periplogenin) in rat plasma using psoralen as the internal standard (IS). After liquid-liquid extraction with ethyl acetate, chromatographic separation was performed on a C18 column with a 13 min gradient elution using 0.1% formic acid and acetonitrile as mobile phase at a flow rate of 0.3 mL/min. The detection was accomplished on a tandem mass spectrometer via an electrospray ionization (ESI) source by multiple reaction monitoring (MRM) in the positive ionization mode. The lower limits of quantitation (LLOQs) for periplocin, periplocymarin and periplogenin were 0.5, 1 and 0.1 ng/mL, respectively. The mean recoveries of the analytes and IS were higher than 67.7%. The proposed method was successfully applied to evaluating the pharmacokinetic studies of periplocin and its metabolites (periplocymarin and periplogenin) in rats after a single oral administration of periplocin at 50 mg/kg. Copyright © 2015 Elsevier B.V. All rights reserved.
Hess, Cornelius; Ritke, Natalie; Broecker, Sebastian; Madea, Burkhard; Musshoff, Frank
2013-05-01
The antihelminthic drug Levamisole can enhance cocaine effects by conversion into the amphetamine-like drug aminorex. We describe an LC-MS method for the determination of levamisole and its metabolite aminorex in human urine. Selectivity is given, calibration curves were linear within the calibration range 2.5-250 ng/mL; limits of the method were LoD 0.51 ng/mL, LoQ 1.02 ng/mL for levamisole and LoD 0.65 ng/mL, LoQ 0.76 ng/mL for aminorex. Precision data was in accordance with the guidelines (intraday precision for aminorex ranged between 5.75 and 11.0 % for levamisole between 8.36 and 10.9 %; interday precision for levamisole 10.9-16.9 % and for aminorex 7.64-12.7 %; accuracy data for levamisole -1.96 to -14.3 % and for aminorex-11.9 to-18.5 %). The validated method was successfully applied to study the urinary excretion of levamisole after the administration of 100 mg of levamisole orally. Levamisole and aminorex could be detected in post-administration urine samples. Levamisole could be detected up to 39 h after ingestion, while aminorex was detectable up to 54 h. Maximum aminorex concentrations were 45 ng/mL urine. Further metabolites of levamisole after oral ingestion by means of liquid chromatography hybrid quadrupole time-of-flight high-resolution mass spectrometry (LC-QTOF-HRMS) were identified. Only 0.5 % of the ingested drug was quantified as unchanged levamisole in urine. Besides aminorex, five isomers of aminorex and 4 hydroxy-metabolites of aminorex or its isomers were found. Furthermore, levamisole is also hydroxylated and eliminated free or conjugated with sulfate or glucuronide into urine.
Wang, Ling-Chi; Okitsu, Cindy Yen; Kochounian, Harold; Rodriguez, Anthony; Hsieh, Chih-Lin; Zandi, Ebrahim
2008-05-01
A modified sol-gel method for a one-step on-column frit preparation for fused-silica capillaries and its utility for peptide separation in LC-MS/MS is described. This method is inexpensive, reproducible, and does not require specialized equipments. Because the frit fabrication process does not damage polyimide coating, the frit-fabricated column can be tightly connected on-line for high pressure LC. These columns can replace any capillary liquid transfer tubing without any specialized connections up-stream of a spray tip column. Therefore multiple columns with different phases can be connected in series for one- or multiple-dimensional chromatography.
Balaji, Narayanan; Chinnapattu, Murugan; Dixit, Abhishek; Sahu, Promod; P S, Suresh; Mullangi, Ramesh
2017-04-01
A highly sensitive, specific and enantioselective assay has been validated for the quantitation of OTX015 enantiomers [(+)-OTX015 and (-)-OTX015] in mice plasma on LC-MS/MS-electrospray ionization as per regulatory guidelines. Protein precipitation was used to extract (±)-OTX015 enantiomers and internal standard (IS) from mice plasma. The active [(-)-OTX015] and inactive [(+)-OTX015] enantiomers were resolved on a Chiralpak-IA column using an isocratic mobile phase (0.2% ammonia/acetonitrile 20 : 80, v/v) at a flow rate of 1.2 mL/min. The total run time was 6.0 min. (+)-OTX015, (-)-OTX015 and IS eluted at 3.34, 4.08 and 4.77 min, respectively. The MS/MS ion transitions monitored were m/z 492 → 383 for OTX015 and m/z 457 → 401 for IS. The standard curves for OTX015 enantiomers were linear (r 2 > 0.998) in the concentration range 1.03-1030 ng/mL. The inter- and intraday precisions were in the range 2.20-13.3 and 8.03-12.1% and 3.80-14.4 and 8.97-13.6% for (+)-OTX015 and (-)-OTX015, respectively. Both the enantiomers were found to be stable in a battery of stability studies. This novel method has been applied to the study of stereoselective oral pharmacokinetics of (-)-OTX015 and unequivocally demonstrated that (-)-OTX015 does not undergo chiral inversion to its antipode in vivo in mice. Copyright © 2016 John Wiley & Sons, Ltd.
A method for the determination of acrylamide in bakery products using ion trap LC-ESI-MS/MS.
Claus, Achim; Weisz, Georg M; Kammerer, Dietmar R; Carle, Reinhold; Schieber, Andreas
2005-10-01
Acrylamide levels of bakery products, e. g., bread and bread rolls, are usually below 100 microg/kg , often even below 50 microg/kg. Therefore, usual analytical methods which have an LOQ greater, not dbl equals 25 microg/kg are not sensitive enough for detailed investigations on acrylamide formation within these commodities. An improved method for trace level determination of acrylamide in bakery products was developed using ion trap LC-ESI-MS/MS. Samples were divided into crumbs and crusts to achieve an initial concentration by removing the crumbs since these are devoid of acrylamide. After sample extraction and clean-up using multimode SPE cartridges, further analyte enrichment was accomplished by solid-phase-supported liquid-liquid extraction with ethyl acetate prior to LC-MS/MS analysis. The method was evaluated using bread, bread rolls, alkali-baked bread rolls, and toast. LOQ was calculated from the confidence interval of the calibration curve and found to be 1.7 ng/mL, corresponding to 17 microg/kg of product. When crumbs and crusts were separated, an LOQ of 10.2 microg/kg of bakery product could be obtained. As demonstrated in preliminary comparative analyses, accuracy of the method met the requirements for determination of trace level acrylamide formation in bakery products. Mean recovery was 102.4% (CV 4.5%), intermediate reproducibility revealed a CV of 2.1%, and a repeatability of CV 6.0%.
Jiang, Juanjuan; Tian, Lei; Huang, Yiling; Yan, Yan; Li, Yishi
2016-08-01
A liquid chromatography-tandem mass spectrometry (LC-MS) method to quantify tolvaptan and its two main metabolites and applied to human study was first developed and validated as a measure of compliance in clinical research. Because of the structure similarity of tolvaptan and its multiple metabolites, the method was optimized to obtain a chromatographic and MS separation of the endogenous interference and isotope ions as well as high analysis throughput. Tolvaptan, its two main metabolites and the internal standard were extracted from human serum (0.1mL) using solid-phase extraction, separated on a Waters nova-pak C18 column (150×3.9mm, 5μm) using isocratic elution with a mobile phase composed of acetonitrile, water and formic acid (65:35:0.25, v/v/v). The total run-time was shortened to 3.5min. The mass transition ranges under positive electrospray ionisation that were monitored for quantitation included m/z 449-252 for tolvaptan, m/z 479-252 for metabolite DM-4103, m/z 481-252 for metabolite DM-4107 and m/z 463-266 for the internal standard (IS). The limit of quantification in plasma for all three analytes was 1ng/mL. The method was validated over a linear range from 1 to 500ng/mL for all three analytes with acceptable inter- and intra-assay precision and accuracy. The stability of the analytes was determined to be suitable for routine laboratory practices. The method was successfully applied to samples taken from research volunteers who ingested a 15mg tolvaptan tablet. Copyright © 2016. Published by Elsevier B.V.
ARMSTRONG, JENNA L.; DILLS, RUSSELL L.; YU, JIANBO; YOST, MICHAEL G.; FENSKE, RICHARD A.
2018-01-01
A rapid liquid chromatography tandem mass spectrometry (LC-MS/MS) method has been developed for determination of levels of the organophosphorus (OP) pesticides chlorpyrifos (CPF), azinphos methyl (AZM), and their oxygen analogs chlorpyrifos-oxon (CPF-O) and azinphos methyl-oxon (AZM-O) on common active air sampling matrices. XAD-2 resin and polyurethane foam (PUF) matrices were extracted with acetonitrile containing stable-isotope labeled internal standards (ISTD). Analysis was accomplished in Multiple Reaction Monitoring (MRM) mode, and analytes in unknown samples were identified by retention time (±0.1 min) and qualifier ratio (±30% absolute) as compared to the mean of calibrants. For all compounds, calibration linearity correlation coefficients were ≥0.996. Limits of detection (LOD) ranged from 0.15–1.1 ng/sample for CPF, CPF-O, AZM, and AZM-O on active sampling matrices. Spiked fortification recoveries were 78–113% from XAD-2 active air sampling tubes and 71–108% from PUF active air sampling tubes. Storage stability tests also yielded recoveries ranging from 74–94% after time periods ranging from 2–10 months. The results demonstrate that LC-MS/MS is a sensitive method for determining these compounds from two different matrices at the low concentrations that can result from spray drift and long range transport in non-target areas following agricultural applications. In an inter-laboratory comparison, the limit of quantification (LOQ) for LC-MS/MS was 100 times lower than a typical gas chromatography-mass spectrometry (GC-MS) method. PMID:24328542
Liu, Xiaoying; Liu, Qian; Wang, Dongmei; Wang, Xueya; Zhang, Peng; Xu, Haiyan; Zhao, Hui; Zhao, Huaiqing
2010-05-01
A highly sensitive and selective LC-MS/MS method was developed and validated for the determination of dauricine in human plasma, using protopine as internal standard (IS). The analyte and IS were extracted by liquid-liquid extraction and analyzed by LC-MS/MS. Chromatographic separation was performed on Agilent TC-C(18) column with a mobile phase of methanol-water-glacial acetic acid (60:40:0.8, v/v/v) at a flow rate of 0.7 mL/min. Detection was performed on a triple quadrupole tandem mass spectrum by multiple reaction monitoring (MRM) mode using the electrospray ionization technique in positive mode. The method was linear over the concentration range of 1-200 ng/mL. The lower limit of quantification (LLOQ) was 1 ng/mL in human plasma with acceptable precision and accuracy. The intra- and inter-day precision was less than 5.9% determined from quality control (QC) samples at concentrations of 2.0, 20.0 and 160 ng/mL, and the accuracy was within +/-9.9%. This method was successfully applied for the evaluation of pharmacokinetics of dauricine after oral doses of 100, 300 and 600 mg phenolic alkaloids of menispermum dauricum tablet (PAMDT) to 12 Chinese healthy volunteers. 2010 Elsevier B.V. All rights reserved.
French, Deborah
2013-01-16
At our institution, serum testosterone in adult males is measured by immunoassay while female and pediatric specimens are sent to a reference laboratory for liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis due to low concentrations. As this is of significant cost, a testosterone LC-MS/MS assay was developed in-house. A 5500 QTRAP® using electrospray ionization and a Shimadzu Prominence with a C18 column were used. Gradient elution with formic acid, water and methanol:acetonitrile at 0.5 ml/min had a 7-min run-time. A liquid-liquid extraction with hexane:ethyl acetate was carried out on 200 μl of serum. Multiple reaction monitoring was employed. Sample preparation took ~80 min for 21 samples. Six calibrators were used (0-1263 ng/dl; concentration assigned by NIST SRM 971) with 3 quality controls (9, 168 and 532 ng/dl). The limits of detection and quantitation were 1 and 2 ng/dl respectively. Extraction recovery was ~90% and ion suppression ~5%. Within-run and total precision studies yielded <15% CV at the limit of quantitation and <7% CV through the rest of the linear range. Isobaric interferences were baseline separated from testosterone. Method comparisons between this assay, an immunoassay, and another LC-MS/MS assay were completed. An accurate and sensitive LC-MS/MS assay for total testosterone was developed. Bringing this assay in-house reduces turnaround time for clinicians and patients and saves our institution funds. Copyright © 2012 Elsevier B.V. All rights reserved.
Muñoz, Eva; Muñoz, Gloria; Pineda, Laura; Serrahima, Eulalia; Centrich, Francesc
2012-01-01
A multiresidue method based on GC or LC and MS or MS/MS for the determination of 204 pesticides in diverse food matrixes of animal and plant origin is described. The method can include different stages of cleanup according to the chemical characteristics of each sample. Samples were extracted using accelerated solvent extraction. Those with a high fat content or that contained chlorophyll required further purification by gel permeation chromatography and/or SPE (ENVI-Carb). The methodology developed here was fully validated; the LOQs for the 204 pesticides are presented. The LOQ values lie between 0.01 to 0.02 mg/kg. However, in some cases, mainly in baby food, they were as low as 0.003 mg/kg, thereby meeting European Union requirements on maximum residue levels for pesticides, as outlined in European regulation 396/2005 and the Commission Directive 2003/13/EC. The procedure has been accredited for a wide scope of pesticides and matrixes by the Spanish Accreditation Body (ENAC) following ISO/IEC 17025:2005, as outlined in ENAC technical note NT-19.
Seserko, Lauren A; Emory, Joshua F; Hendrix, Craig W; Marzinke, Mark A
2013-11-01
Dapivirine is a non-nucleoside reverse transcriptase inhibitor designed to prevent HIV-1 viral replication and subsequent propagation. A sensitive method is required to quantify plasma concentrations to assess drug efficacy. Dapivirine-spiked plasma was combined with acetonitrile containing deuterated IS and was processed for analysis. The method has an analytical measuring range from 20 to 10,000 pg/ml. For the LLOQ, low, mid and high QCs, intra- and inter-assay precision (%CV) ranged from 5.58 to 13.89% and 5.23 to 13.36%, respectively, and intra- and inter-day accuracy (% deviation) ranged from -5.61 to 0.75% and -4.30 to 6.24%, respectively. A robust and sensitive LC-MS/MS assay for the high-throughput quantification of the antiretroviral drug dapivirine in human plasma was developed and validated following bioanalytical validation guidelines. The assay meets criteria for the analysis of samples from large research trials.
MassCascade: Visual Programming for LC-MS Data Processing in Metabolomics.
Beisken, Stephan; Earll, Mark; Portwood, David; Seymour, Mark; Steinbeck, Christoph
2014-04-01
Liquid chromatography coupled to mass spectrometry (LC-MS) is commonly applied to investigate the small molecule complement of organisms. Several software tools are typically joined in custom pipelines to semi-automatically process and analyse the resulting data. General workflow environments like the Konstanz Information Miner (KNIME) offer the potential of an all-in-one solution to process LC-MS data by allowing easy integration of different tools and scripts. We describe MassCascade and its workflow plug-in for processing LC-MS data. The Java library integrates frequently used algorithms in a modular fashion, thus enabling it to serve as back-end for graphical front-ends. The functions available in MassCascade have been encapsulated in a plug-in for the workflow environment KNIME, allowing combined use with e.g. statistical workflow nodes from other providers and making the tool intuitive to use without knowledge of programming. The design of the software guarantees a high level of modularity where processing functions can be quickly replaced or concatenated. MassCascade is an open-source library for LC-MS data processing in metabolomics. It embraces the concept of visual programming through its KNIME plug-in, simplifying the process of building complex workflows. The library was validated using open data.
Becalski, Adam; Lau, Benjamin P Y; Lewis, David; Seaman, Stephen W; Sun, Wing F
2005-01-01
Recent concerns surrounding the presence of acrylamide in many types of thermally processed food have brought about the need for the development of analytical methods suitable for determination of acrylamide in diverse matrices with the goals of improving overall confidence in analytical results and better understanding of method capabilities. Consequently, the results are presented of acrylamide testing in commercially available food products--potato fries, potato chips, crispbread, instant coffee, coffee beans, cocoa, chocolate and peanut butter, obtained by using the same sample extract. The results obtained by using LC-MS/MS, GC/MS (El), GC/HRMS (El)--with or without derivatization--and the use of different analytical columns, are discussed and compared with respect to matrix borne interferences, detection limits and method complexities.
Gao, Xinliu; Lin, Hui; Krantz, Carsten; Garnier, Arlette; Flarakos, Jimmy; Tse, Francis L S; Li, Wenkui
2016-01-01
Factor P (Properdin), an endogenous glycoprotein, plays a key role in innate immune defense. Its quantification is important for understanding the pharmacodynamics (PD) of drug candidate(s). In the present work, an immunoaffinity capturing LC-MS/MS method has been developed and validated for the first time for the quantification of factor P in monkey serum with a dynamic range of 125 to 25,000 ng/ml using the calibration standards and QCs prepared in factor P depleted monkey serum. The intra- and inter-run precision was ≤7.2% (CV) and accuracy within ±16.8% (%Bias) across all QC levels evaluated. Results of other evaluations (e.g., stability) all met the acceptance criteria. The validated method was robust and implemented in support of a preclinical PK/PD study.
Sitnikov, Dmitri G.; Monnin, Cian S.; Vuckovic, Dajana
2016-01-01
The comparison of extraction methods for global metabolomics is usually executed in biofluids only and focuses on metabolite coverage and method repeatability. This limits our detailed understanding of extraction parameters such as recovery and matrix effects and prevents side-by-side comparison of different sample preparation strategies. To address this gap in knowledge, seven solvent-based and solid-phase extraction methods were systematically evaluated using standard analytes spiked into both buffer and human plasma. We compared recovery, coverage, repeatability, matrix effects, selectivity and orthogonality of all methods tested for non-lipid metabolome in combination with reversed-phased and mixed-mode liquid chromatography mass spectrometry analysis (LC-MS). Our results confirmed wide selectivity and excellent precision of solvent precipitations, but revealed their high susceptibility to matrix effects. The use of all seven methods showed high overlap and redundancy which resulted in metabolite coverage increases of 34–80% depending on LC-MS method employed as compared to the best single extraction protocol (methanol/ethanol precipitation) despite 7x increase in MS analysis time and sample consumption. The most orthogonal methods to methanol-based precipitation were ion-exchange solid-phase extraction and liquid-liquid extraction using methyl-tertbutyl ether. Our results help facilitate rational design and selection of sample preparation methods and internal standards for global metabolomics. PMID:28000704
Accurate LC peak boundary detection for ¹⁶O/¹⁸O labeled LC-MS data.
Cui, Jian; Petritis, Konstantinos; Tegeler, Tony; Petritis, Brianne; Ma, Xuepo; Jin, Yufang; Gao, Shou-Jiang S J; Zhang, Jianqiu Michelle
2013-01-01
In liquid chromatography-mass spectrometry (LC-MS), parts of LC peaks are often corrupted by their co-eluting peptides, which results in increased quantification variance. In this paper, we propose to apply accurate LC peak boundary detection to remove the corrupted part of LC peaks. Accurate LC peak boundary detection is achieved by checking the consistency of intensity patterns within peptide elution time ranges. In addition, we remove peptides with erroneous mass assignment through model fitness check, which compares observed intensity patterns to theoretically constructed ones. The proposed algorithm can significantly improve the accuracy and precision of peptide ratio measurements.
Poetzsch, Michael; Steuer, Andrea E; Roemmelt, Andreas T; Baumgartner, Markus R; Kraemer, Thomas
2014-12-02
Single hair analysis normally requires extensive sample preparation microscale protocols including time-consuming steps like segmentation and extraction. Matrix assisted laser desorption and ionization mass spectrometric imaging (MALDI-MSI) was shown to be an alternative tool in single hair analysis, but still, questions remain. Therefore, an investigation of MALDI-MSI in single hair analysis concerning the extraction process, usage of internal standard (IS), and influences on the ionization processes were systematically investigated to enable the reliable application to hair analysis. Furthermore, single dose detection, quantitative correlation to a single hair, and hair strand LC-MS/MS results were performed, and the performance was compared to LC-MS/MS single hair monitoring. The MALDI process was shown to be independent from natural hair color and not influenced by the presence of melanin. Ionization was shown to be reproducible along and in between different hair samples. MALDI image intensities in single hair and hair snippets showed good semiquantitative correlation to zolpidem hair concentrations obtained from validated routine LC-MS/MS methods. MALDI-MSI is superior to LC-MS/MS analysis when a fast, easy, and cheap sample preparation is necessary, whereas LC-MS/MS showed higher sensitivity with the ability of single dose detection for zolpidem. MALDI-MSI and LC-MS/MS segmental single hair analysis showed good correlation, and both are suitable for consumption monitoring of drugs of abuse with a high time resolution.
Askenazi, David J; Moore, John F; Fineberg, Naomi; Koralkar, Rajesh; Clevenger, Stephanie; Sharer, Jon Daniel
2014-09-01
Measurement of serum creatinine (SCr) and urine creatinine (UCr) is regularly used in clinical and research settings. For small animal experiments and for studies in which sample collection is spare (i.e. neonatal cohorts), measuring SCr and UCr using tiny amounts of sample (as low as 10 mcl) would maximize exploration and minimize iatrogenic blood loss. We performed an evaluation in six healthy adults to determine differences between SCr and UCr values in different methodologies and storage environments and time. Study was conducted using 20 mcl of sample. Analyses were done using two-way repeated measures of ANOVA. Scr values showed no significant differences between LC/MS vs. Jaffe. However, the SCr using LC/MS method was lowest when measured immediately compared to other time points (F = 7.2; P< 0.001). Similarly, Jaffe measurements showed changes in the mean differences over time; however, these were not significant. UCr values were consistently higher using LC/MS than Jaffe (F = 19; P< 0.01), and UCr changed over time (F = 8.7; P < 0.02). In addition, the interaction term for method and time was also significant (F = 5.8; P < 0.04) which reflects the stability of the Jaffe measurements over time whereas the LC/MS measurements declined; especially after being frozen for 1 year (P < 0.001). UCr measured by Jaffe is lower than samples measured by LC/MS. UCr measurements by LC/MS vary more over time, mostly due to the sample measured after 1 year; therefore, storage of urine for more than 90 days measured by LC/MS may provide altered results. © 2014 Wiley Periodicals, Inc.
Jiang, Jian; James, Christopher A; Wong, Philip
2016-09-05
A LC-MS/MS method has been developed and validated for the determination of glycine in human cerebrospinal fluid (CSF). The validated method used artificial cerebrospinal fluid as a surrogate matrix for calibration standards. The calibration curve range for the assay was 100-10,000ng/mL and (13)C2, (15)N-glycine was used as an internal standard (IS). Pre-validation experiments were performed to demonstrate parallelism with surrogate matrix and standard addition methods. The mean endogenous glycine concentration in a pooled human CSF determined on three days by using artificial CSF as a surrogate matrix and the method of standard addition was found to be 748±30.6 and 768±18.1ng/mL, respectively. A percentage difference of -2.6% indicated that artificial CSF could be used as a surrogate calibration matrix for the determination of glycine in human CSF. Quality control (QC) samples, except the lower limit of quantitation (LLOQ) QC and low QC samples, were prepared by spiking glycine into aliquots of pooled human CSF sample. The low QC sample was prepared from a separate pooled human CSF sample containing low endogenous glycine concentrations, while the LLOQ QC sample was prepared in artificial CSF. Standard addition was used extensively to evaluate matrix effects during validation. The validated method was used to determine the endogenous glycine concentrations in human CSF samples. Incurred sample reanalysis demonstrated reproducibility of the method. Copyright © 2016 Elsevier B.V. All rights reserved.
Barbosa, Marta O; Ribeiro, Ana R; Pereira, Manuel F R; Silva, Adrián M T
2016-11-01
Organic micropollutants present in drinking water (DW) may cause adverse effects for public health, and so reliable analytical methods are required to detect these pollutants at trace levels in DW. This work describes the first green analytical methodology for multi-class determination of 21 pollutants in DW: seven pesticides, an industrial compound, 12 pharmaceuticals, and a metabolite (some included in Directive 2013/39/EU or Decision 2015/495/EU). A solid-phase extraction procedure followed by ultra-high-performance liquid chromatography coupled to tandem mass spectrometry (offline SPE-UHPLC-MS/MS) method was optimized using eco-friendly solvents, achieving detection limits below 0.20 ng L -1 . The validated analytical method was successfully applied to DW samples from different sources (tap, fountain, and well waters) from different locations in the north of Portugal, as well as before and after bench-scale UV and ozonation experiments in spiked tap water samples. Thirteen compounds were detected, many of them not regulated yet, in the following order of frequency: diclofenac > norfluoxetine > atrazine > simazine > warfarin > metoprolol > alachlor > chlorfenvinphos > trimethoprim > clarithromycin ≈ carbamazepine ≈ PFOS > citalopram. Hazard quotients were also estimated for the quantified substances and suggested no adverse effects to humans. Graphical Abstract Occurrence and removal of multi-class micropollutants in drinking water, analyzed by an eco-friendly LC-MS/MS method.
Koesukwiwat, Urairat; Vaclavik, Lukas; Mastovska, Katerina
2018-05-08
According to the European Commission directive 2006/141/EC, haloxyfop residue levels should not exceed 0.003 mg/kg in ready-to-feed infant formula, and the residue definition includes sum of haloxyfop, its esters, salts, and conjugates expressed as haloxyfop. A simple method for total haloxyfop analysis in infant formula and related ingredient matrices was developed and validated using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The sample preparation consisted of an alkaline hydrolysis with methanolic sodium hydroxide to release haloxyfop (parent acid) from its bound forms prior to the extraction with acetonitrile. A mixture of magnesium sulfate (MgSO 4 ) and sodium chloride (NaCl) (4:1, w/w) was added to the extract to induce phase separation and force the analyte into the upper acetonitrile-methanol layer and then a 1-mL aliquot was subsequently cleaned up by dispersive solid phase extraction with 150 mg of MgSO 4 and 50 mg of octadecyl (C 18 ) sorbent. The analytical procedure was developed and carefully optimized to enable low-level, total haloxyfop analysis in a variety of challenging matrices, including infant formulas and their important high-carbohydrate, high-protein, high-fat, and emulsifier ingredients. The final method was validated in two different laboratories by fortifying samples with haloxyfop and haloxyfop-methyl, which was used as a model compound simulating bound forms of the analyte. Mean recoveries of haloxyfop across all fortification levels and evaluated matrices ranged between 92.2 and 114% with repeatability, within-lab reproducibility, and reproducibility RSDs ≤ 14%. Based on the validation results, this method was capable to convert the haloxyfop ester into the parent acid in a wide range of sample types and to reliably identify and quantify total haloxyfop at the target 0.003 mg/kg level in infant formulas (both powdered and ready-to-feed liquid forms). Graphical abstract LC-MS/MS-based workflow for the
Experimental validation of structural optimization methods
NASA Technical Reports Server (NTRS)
Adelman, Howard M.
1992-01-01
The topic of validating structural optimization methods by use of experimental results is addressed. The need for validating the methods as a way of effecting a greater and an accelerated acceptance of formal optimization methods by practicing engineering designers is described. The range of validation strategies is defined which includes comparison of optimization results with more traditional design approaches, establishing the accuracy of analyses used, and finally experimental validation of the optimization results. Examples of the use of experimental results to validate optimization techniques are described. The examples include experimental validation of the following: optimum design of a trussed beam; combined control-structure design of a cable-supported beam simulating an actively controlled space structure; minimum weight design of a beam with frequency constraints; minimization of the vibration response of helicopter rotor blade; minimum weight design of a turbine blade disk; aeroelastic optimization of an aircraft vertical fin; airfoil shape optimization for drag minimization; optimization of the shape of a hole in a plate for stress minimization; optimization to minimize beam dynamic response; and structural optimization of a low vibration helicopter rotor.
Liu, Jingjing; Li, Qing; Yin, Yidi; Liu, Ran; Xu, Huarong; Bi, Kaishun
2014-01-01
A fast, sensitive, and efficient ultra-fast LC-ESI-MS/MS method was developed for the simultaneous quantitation of six highly toxic Aconitum alkaloids, that is, aconitine, mesaconitine, hypaconitine, benzoylaconine, benzoylmesaconine, and benzoylhypaconine, in rat plasma after oral administration of crude ethanol extracts from Aconiti kusnezoffii radix by ultrasonic extraction, reflux extraction for 1 h, and reflux extraction for 3 h, respectively. The separation of six Aconitum alkaloids and aminopyrine (internal standard) was performed on an InertSustain® C18 column, and the quantification of the analytes was performed on a 4000Q ultra-fast LC-MS/MS system with turbo ion spray source in the positive ion and multiple-reaction monitoring mode. Absolute recoveries ranged within 65.06-85.1% for plasma samples. The intra- and interday precision and accuracy of analytes were satisfactory. The methods were validated with sensitivity reaching the lower LOQ for aconitine, mesaconitine, hypaconitine, benzoylaconine, benzoylmesaconine, and benzoylhypaconine, which were 0.025, 0.025, 0.050, 0.025, 0.025, and 0.100 ng/mL, respectively. The method was successfully applied to a pharmacokinetic study of six Aconitum alkaloids in rat plasma after oral administration of crude ethanol extracts from the raw root of Aconitum kusnezoffii Reichb. by three different extraction processes. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Cheng, Yung-Yi; Tsai, Tung-Hu
2016-06-05
Rhodamine B is an illegal and potentially carcinogenic food dye. The aim of this study was to develop a convenient, rapid, and sensitive UHPLC-MS/MS method for pharmacokinetic studies in rats. Rat plasma samples were deproteinized with acetonitrile and separated by UHPLC on a reverse-phase C18e column (100mm×2.1mm, 2μm) using a mobile phase consisting of methanol-5mM ammonium acetate (90:10, v/v). Detection was performed using a triple quadrupole tandem mass spectrometer in the selected reaction monitoring mode at [M](+) ion m/z 443.39→399.28 for rhodamine B and [M+H](+) ion m/z 253.17→238.02 for 5-methoxyflavone as the internal standard. This method was specific and produced linear results over a concentration range of 0.5-100ng/mL, with a lower limit of quantitation of 0.5ng/mL. All validation parameters, including the inter-day, intra-day, matrix effect, recovery, and stability in rat plasma, were acceptable according to the biological method validation guidelines developed by the FDA (2001). This method was successfully applied to a pharmacokinetic study in rats; oral administration of 1mg/kg of rhodamine B yielded a time to maximum concentration (Tmax) of 1.3±0.4h and an elimination half-life of 8.8±1.4h, with a clearance of 229.7±19.4mL/h/kg. These pharmacokinetic results provide a constructive contribution to our understanding of the absorption mechanism of rhodamine B and support additional food safety evaluations. Copyright © 2016 Elsevier B.V. All rights reserved.
Srinubabu, Gedela; Ratnam, Bandaru Veera Venkata; Rao, Allam Appa; Rao, Medicherla Narasimha
2008-01-01
A rapid tandem mass spectrometric (MS-MS) method for the quantification of Oxcarbazepine (OXB) in human plasma using imipramine as an internal standard (IS) has been developed and validated. Chromatographic separation was achieved isocratically on a C18 reversed-phase column within 3.0 min, using a mobile phase of acetonitrile-10 mM ammonium formate (90 : 10 v/v) at a flow rate of 0.3 ml/min. Quantitation was achieved using multiple reaction monitoring (MRM) scan at MRM transitions m/z 253>208 and m/z 281>86 for OXB and the IS respectively. Calibration curves were linear over the concentration range of 0.2-16 mug/ml (r>0.999) with a limit of quantification of 0.2 mug/ml. Analytical recoveries of OXB from spiked human plasma were in the range of 74.9 to 76.3%. Plackett-Burman design was applied for screening of chromatographic and mass spectrometric factors; factorial design was applied for optimization of essential factors for the robustness study. A linear model was postulated and a 2(3) full factorial design was employed to estimate the model coefficients for intermediate precision. More specifically, experimental design helps the researcher to verify if changes in factor values produce a statistically significant variation of the observed response. The strategy is most effective if statistical design is used in most or all stages of the screening and optimizing process for future method validation of pharmacokinetic and bioequivalence studies.
Bae, Jung-Woo; Choi, Chang-Ik; Jang, Choon-Gon; Lee, Seok-Yong
2011-11-01
A simple and sensitive liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) technique was developed and validated for the determination of sibutramine and its N-desmethyl metabolites (M1 and M2) in human plasma. After extraction with methyl t-butyl ether, chromatographic separation of analytes in human plasma was performed using a reverse-phase Luna C18 column with a mobile phase of acetonitrile-10 mm ammonium formate buffer (50:50, v/v) and quantified by ESI-MS/MS detection in positive ion mode. The flow rate of the mobile phase was 200 μL/min and the retention times of sibutramine, M1, M2 and internal standard (chlorpheniramine) were 1.5, 1.4, 1.3 and 0.9 min, respectively. The calibration curves were linear over the range 0.05-20 ng/mL, for sibutramine, M1 and M2. The lower limit of quantification was 0.05 ng/mL using 500 μL of human plasma. The mean accuracy and the precision in the intra- and inter-day validation for sibutramine, M1 and M2 were acceptable. This LC-MS/MS method showed improved sensitivity and a short run time for the quantification of sibutramine and its two active metabolites in plasma. The validated method was successfully applied to a pharmacokinetic study in human. Copyright © 2011 John Wiley & Sons, Ltd.
Bystrowska, Beata; Smaga, Irena; Tyszka-Czochara, Małgorzata; Filip, Małgorzata
2014-05-01
In recent years, a potential participation of endocannabinoids (eCBs) and related endocannabinoid-like molecules, including N-acylethanolamines (NAEs), in the physiological and pathophysiological processes has been highlighted, whereas measurement of their levels still remains difficult. The aim of this study was to develop a bioanalytical method that would enable researchers to simultaneously determine quantitatively eCBs (anandamide - AEA and 2-arachidonoylglycerol - 2-AG) and NAEs (oleoylethanolamide or oleoylethanolamine - OEA, palmitoylethanolamide or palmitoylethanolamine - PEA and linoleoylethanolamide or linoleoylethanolamine - LEA) in the rat brain. The analytical problems with analysis and possible solutions have been also shown. The methodology for quantifying eCBs/NAEs by means of a sensitive and selective liquid chromatography tandem mass spectrometry (LC-MS/MS) with electrospray positive ionization and multiple reaction monitoring (MRM) mode was developed and validated. Analytical problems with analyzed compounds were estimated. Reasonably high precision and accuracy of the method were demonstrated in the validation process. The method is linear up to 200 ng/g for AEA, OEA, PEA and LEA and up to 100 μg/g for 2-AG, while the quantification limit reaches 0.2 ng/g and 0.8 μg/g, respectively. Simplicity and rapidity of the assay allows analyzing many samples on a routine basis. This article presents the new procedure applied to the analysis of brain tissues.
Random Qualitative Validation: A Mixed-Methods Approach to Survey Validation
ERIC Educational Resources Information Center
Van Duzer, Eric
2012-01-01
The purpose of this paper is to introduce the process and value of Random Qualitative Validation (RQV) in the development and interpretation of survey data. RQV is a method of gathering clarifying qualitative data that improves the validity of the quantitative analysis. This paper is concerned with validity in relation to the participants'…
Dong, Xin; Ding, Li; Cao, Xiaomei; Jiang, Liyuan; Zhong, Shuisheng
2013-04-01
Amoxicillin (AMO) degrades in plasma at room temperature and readily undergoes hydrolysis by the plasma amidase. In this paper, a novel, rapid and sensitive LC-MS/MS method operated in segmental and multiple reaction monitoring has been developed for the simultaneous determination of amoxicillin and ambroxol in human plasma. The degradation of amoxicillin in plasma was well prevented by immediate addition of 20 μL glacial acetic acid to 200 μL aliquot of freshly collected plasma samples before storage at -80°C. The sensitivity of the method was improved with segmental monitoring of the analytes, and lower limits of quantitation of 0.5 ng/mL for ambroxol and 5 ng/mL for amoxicillin were obtained. The sensitivity of our method was five times better than those of the existing methods. Furthermore, the mass response saturation problem with amoxicillin was avoided by diluting the deproteinized plasma samples with water before injection into the LC-MS/MS system. The method was successfully employed in a pharmacokinetic study of the compound amoxicillin and ambroxol hydrochloride tablets. Copyright © 2012 John Wiley & Sons, Ltd.
Simultaneous quantitative analysis of nine vitamin D compounds in human blood using LC-MS/MS.
Abu Kassim, Nur Sofiah; Gomes, Fabio P; Shaw, Paul Nicholas; Hewavitharana, Amitha K
2016-01-01
It has been suggested that each member of the family of vitamin D compounds may have different function(s). Therefore, selective quantification of each compound is important in clinical research. Development and validation attempts of a simultaneous determination method of 12 vitamin D compounds in human blood using precolumn derivatization followed by LC-MS/MS is described. Internal standard calibration with 12 stable isotope labeled analogs was used to correct for matrix effects in MS detector. Nine vitamin D compounds were quantifiable in blood samples with detection limits within femtomole levels. Serum (compared with plasma) was found to be a more suitable sample type, and protein precipitation (compared with saponification) a more effective extraction method for vitamin D assay.
Chen, Hui; Wang, Xu; Chopra, Shruti; Adams, Erwin; Van Schepdael, Ann
2014-03-01
A new method for monitoring the enzyme inhibition of Abl1 tyrosine kinase by liquid chromatography-indirect pulsed electrochemical detection (LC-InPED) was developed. In this method, adsorption of a peptide analyte at the noble metal electrode suppresses the oxidation of polyols under alkaline condition to elicit an indirect response resulting in a negative peak of the target peptide. Among the reagents tested, D-gluconic acid sodium salt gave the best overall signal to noise (S/N) values for the indirect detection of p-Abltide, the product of Abl1 enzymatic reaction. 50 μM D-gluconic acid sodium salt dissolved in a mixture of 78% water-22% acetonitrile-0.03% trifluoroacetic acid (TFA) was used as the mobile phase. Chromatographic separation was achieved on an Alltima C18 (I.D. 5 μm; 250 mm × 4.6 mm) column with the mobile phase flow rate of 0.5 ml/min. 0.5M sodium hydroxide was added post-column to maintain alkaline conditions in the PED cell. The limit of quantification (LOQ) was 0.2 μM for p-Abltide, which was about 50-fold lower than direct PED analysis. The residual plot of the linear calibration curve indicated a good fit with a linear model within the investigated concentration range of p-Abltide. Intra- and inter-day precision was not more than 6.5% and accuracy was from -5.75% to +1.54%. The validated LC-InPED method was successfully applied for monitoring of p-Abltide in Abl1 enzyme reaction and the inhibition study of Abl1. The determined IC50 values of model inhibitors, imatinib, nilotinib and dasatinib, were 601.4 nM (R(2)=0.99), 32.3 nM (R(2)=0.99) and 1.3 nM (R(2)=0.98), respectively. These results were consistent with literature data. To the best of our knowledge this is the first time a LC-InPED method has been used to monitor an enzyme reaction. Copyright © 2013 Elsevier B.V. All rights reserved.
Vigneau, Olivier; Machuron-Mandard, Xavier
2009-03-15
The introduction of chloroform into the nebulising gas of a LC/MS electrospray interface (ESI), in a perfectly controlled way, leads to the formation of intense adducts ([M+Cl](-)) when a mobile phase containing HMX (1,3,5,7-tetranitro-1,3,5,7-tetrazacyclooctane or octogen) and RDX (1,3,5-trintro-1,3,5-triazacyclohexane or hexogen) is eluted. This LC/MS method allows the direct analysis of aqueous samples containing HMX and RDX at the pictogram level without a concentration step. The method is used to determine HMX and RDX concentrations in ground water samples from a military site.
Multiplexed LC-MS/MS analysis of horse plasma proteins to study doping in sport.
Barton, Chris; Beck, Paul; Kay, Richard; Teale, Phil; Roberts, Jane
2009-06-01
The development of protein biomarkers for the indirect detection of doping in horse is a potential solution to doping threats such as gene and protein doping. A method for biomarker candidate discovery in horse plasma is presented using targeted analysis of proteotypic peptides from horse proteins. These peptides were first identified in a novel list of the abundant proteins in horse plasma. To monitor these peptides, an LC-MS/MS method using multiple reaction monitoring was developed to study the quantity of 49 proteins in horse plasma in a single run. The method was optimised and validated, and then applied to a population of race-horses to study protein variance within a population. The method was finally applied to longitudinal time courses of horse plasma collected after administration of an anabolic steroid to demonstrate utility for hypothesis-driven discovery of doping biomarker candidates.
Simultaneous Determination of 11 Illicit Phenethylamines in Hair by LC-MS-MS: In Vivo Application.
Nieddu, Maria; Burrai, Lucia; Demontis, Maria Piera; Varoni, Maria Vittoria; Baralla, Elena; Trignano, Claudia; Boatto, Gianpiero
2015-09-01
Existing phenethylamines are a class of synthetic compounds that differ from each other only in small changes to a largely conserved chemical structure. The recreational and illicit use of phenethylamines is a widespread problem. A simple procedure for the simultaneous quantitative determination in hair of 11 phenethylamines that are officially recognized as illicit by Italian legislation (p-methoxyamphetamine; p-methoxymethamphetamine; 3,4,5-trimethoxyamphetamine; 2,5-dimethoxyamphetamine; 2,5-dimethoxy-4-methylamphetamine; 2,5-dimethoxy-4-ethylamphetamine; 2,5-dimethoxy-4-bromoamphetamine; 2,5-dimethoxy-4-bromophenethylamine; 2,5-dimethoxy-4-iodophenethylamine; 2,5-dimethoxy-4-ethylthiophenethylamine and 2,5-dimethoxy-4-n-propylthiophenethylamine) has been developed and validated. Extraction from the matrix was performed after incubation in methanolic HCl and filtered reconstituted extracts were injected into a liquid chromatography/tandem mass spectrometry system (LC-MS-MS) without any further purification steps. This validated LC-MS-MS method has been used to determine the in vivo accumulation/retention of the above target analytes in hair after repeat oral administration to rats. This experiment further permitted investigation of the effect of pigmentation on the uptake of these phenethylamines by hair and the effect of hair pigmentation. The developed method could potentially be used for forensic and toxicological purposes, in the detection and quantitation of these illicit substances in human hair in workplace drug testing; drug-facilitated crime investigation; driver re-licensing; determining drug abuse history and postmortem toxicology. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.
Konieczna, Lucyna; Kaźmierska, Katarzyna; Roszkowska, Anna; Szlagatys-Sidorkiewicz, Agnieszka; Bączek, Tomasz
2016-05-30
Cystic fibrosis (CF) is one of the most common genetic diseases in children and affects mainly respiratory and digestive system functions. Despite the prolonged supplementation of vitamins, malnutrition manifested by poor growth and weight loss in children is a major complication in CF related to pancreatic insufficiency and difficulty in absorbing fat-soluble vitamins. In the present study, we have developed and validated a sensitive and accurate high-performance liquid chromatography coupled to mass spectrometry (LC-MS) method for the simultaneous quantification of three fat-soluble vitamins (A, E and K1) and two vitamin D3 active metabolites: 25-hydroxyvitamin D3 and 1,25-dihydroxyvitamin D3 in serum samples obtained from pediatric patients with CF. In optimized conditions, the LC-MS method was highly sensitive and presented excellent linearity with a regression coefficient higher than 0.999. The accuracy was in the range of 87.55-95.58 % for all analytes. The precision of the method, expressed as% RSD, ranged from 1.36 % to 3.74 % as the intra-day variability and from 2.35 % to 7.98 % as the inter-day precision for all the studied compounds. Sample preparation included a protein precipitation step with the use of methanol followed by liquid-liquid extraction with n-hexane. The statistical analysis (t-test and principal component analysis (PCA)) of the obtained results revealed significant changes in the plasma level of the analyzed compounds, with 25-hydroxyvitamin D3, vitamin E and K1 present at extremely low concentrations in patients with cystic fibrosis in comparison to healthy controls. The elaborated method reached the expectations for the fast and reliable assessment of fat-soluble vitamin status in children with cystic fibrosis in order to diagnose the disease and monitor the treatment process. Copyright © 2016 Elsevier B.V. All rights reserved.
Kumar, Thangarathinam; Ramya, Mohandass; Arockiasamy Xavier, S J
2016-11-01
Stress degradation studies using high-performance liquid chromatography (HPLC) was performed and validated for Droxidopa (L-DOPS). Droxidopa was susceptible to acid hydrolysis (0.1 N HCl), alkaline hydrolysis (0.15 N NaOH) and thermal degradation (105°C). It was found to be resistant to white light, oxidation and UV light exposure (72 h). The thermal, acid and alkali degradation impurities were detected with the retention time (RT) of 12.7, 19.25 and 22.95 min. Our HPLC method detected process impurities (2R,3R)-2-amino-3-(3,4-dihydroxyphenyl)-3-hydroxypropionic acid (Impurity H), N-Hydroxypthalimide (Impurity N), (2R,3S)-2-amino-3-(benzo[d][1,3]dioxol-5-yl)-3-hydroxypropionic acid (Impurity L) and L-threo n-phthaloyl-3-(3, 4-dihydroxyphenyl)-serine (Intermediate) with RTs of 3.48, 15.5, 25.76 and 28.0 min. The related substances were further characterized and confirmed by liquid chromatography-mass spectroscopy (LC-MS), and nuclear magnetic resonance spectroscopy analysis. Our HPLC method detected up to 0.05 µg/mL of Droxidopa with S/N > 3.0 and quantified up to 0.10 µg /mL of Droxidopa with S/N ratio > 10.0. Droxidopa was highly stable for 12 h after its preparation for HPLC analysis. Our newly developed HPLC method was highly precise, specific, reliable and accurate for the analysis of Droxidopa and its related substances. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.
USDA-ARS?s Scientific Manuscript database
A liquid chromatographic-tandem mass spectrometric (LC-MS/MS) multi-residue method for the simultaneous quantification and identification of 38 residues of the most widely used anthelmintic veterinary drugs (including benzimidazoles, macrocyclic lactones, and flukicides) in milk and liver has been d...
Extension of the validation of AOAC Official Method 2005.06 for dc-GTX2,3: interlaboratory study.
Ben-Gigirey, Begoña; Rodríguez-Velasco, María L; Gago-Martínez, Ana
2012-01-01
AOAC Official Method(SM) 2005.06 for the determination of saxitoxin (STX)-group toxins in shellfish by LC with fluorescence detection with precolumn oxidation was previously validated and adopted First Action following a collaborative study. However, the method was not validated for all key STX-group toxins, and procedures to quantify some of them were not provided. With more STX-group toxin standards commercially available and modifications to procedures, it was possible to overcome some of these difficulties. The European Union Reference Laboratory for Marine Biotoxins conducted an interlaboratory exercise to extend AOAC Official Method 2005.06 validation for dc-GTX2,3 and to compile precision data for several STX-group toxins. This paper reports the study design and the results obtained. The performance characteristics for dc-GTX2,3 (intralaboratory and interlaboratory precision, recovery, and theoretical quantification limit) were evaluated. The mean recoveries obtained for dc-GTX2,3 were, in general, low (53.1-58.6%). The RSD for reproducibility (RSD(r)%) for dc-GTX2,3 in all samples ranged from 28.2 to 45.7%, and HorRat values ranged from 1.5 to 2.8. The article also describes a hydrolysis protocol to convert GTX6 to NEO, which has been proven to be useful for the quantification of GTX6 while the GTX6 standard is not available. The performance of the participant laboratories in the application of this method was compared with that obtained from the original collaborative study of the method. Intralaboratory and interlaboratory precision data for several STX-group toxins, including dc-NEO and GTX6, are reported here. This study can be useful for those laboratories determining STX-group toxins to fully implement AOAC Official Method 2005.06 for official paralytic shellfish poisoning control. However the overall quantitative performance obtained with the method was poor for certain toxins.
Fast targeted analysis of 132 acidic and neutral drugs and poisons in whole blood using LC-MS/MS.
Di Rago, Matthew; Saar, Eva; Rodda, Luke N; Turfus, Sophie; Kotsos, Alex; Gerostamoulos, Dimitri; Drummer, Olaf H
2014-10-01
The aim of this study was to develop an LC-MS/MS based screening technique that covers a broad range of acidic and neutral drugs and poisons by combining a small sample volume and efficient extraction technique with simple automated data processing. After protein precipitation of 100μL of whole blood, 132 common acidic and neutral drugs and poisons including non-steroidal anti-inflammatory drugs, barbiturates, anticonvulsants, antidiabetics, muscle relaxants, diuretics and superwarfarin rodenticides (47 quantitated, 85 reported as detected) were separated using a Shimadzu Prominence HPLC system with a C18 separation column (Kinetex XB-C18, 4.6mm×150mm, 5μm), using gradient elution with a mobile phase of 25mM ammonium acetate buffer (pH 7.5)/acetonitrile. The drugs were detected using an ABSciex(®) API 2000 LC-MS/MS system (ESI+ and -, MRM mode, two transitions per analyte). The method was fully validated in accordance with international guidelines. Quantification data obtained using one-point calibration compared favorably to that using multiple calibrants. The presented LC-MS/MS assay has proven to be applicable for determination of the analytes in blood. The fast and reliable extraction method combined with automated processing gives the opportunity for high throughput and fast turnaround times for forensic and clinical toxicology. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.
Wang, Xijun; Lv, Haitao; Zhang, Guangmei; Sun, Wenjun; Zhou, Dixin; Jiao, Guozheng; Yu, Yang
2008-09-01
Ultra-performance LC coupled to quadrupole TOF/MS (UPLC-QTOF/MS) in positive and negative ESI was developed and validated to analyze metabolite profiles for urine from healthy men during the day and at night. Data analysis using principal components analysis (PCA) revealed differences between metabolic phenotypes of urine in healthy men during the day and at night. Positive ions with mass-to-charge ratio (m/z) 310.24 (5.35 min), 286.24 (4.74 min) and 310.24 (5.63 min) were elevated in the urine from healthy men at night compared to that during the day. Negative ions elevated in day urine samples of healthy men included m/z 167.02 (0.66 min), 263.12 (2.55 min) and 191.03 (0.73 min), whilst ions m/z 212.01 (4.77 min) were at a lower concentration in urine of healthy men during the day compared to that at night. The ions m/z 212.01 (4.77 min), 191.03 (0.73 min) and 310.24 (5.35 min) preliminarily correspond to indoxyl sulfate, citric acid and N-acetylneuraminic acid, providing further support for an involvement of phenotypic difference in urine of healthy men in day and night samples, which may be associated with notably different activities of gut microbiota, velocity of tricarboxylic acid cycle and activity of sialic acid biosynthesis in healthy men as regulated by circadian rhythm of the mammalian bioclock.
Han, Minlu; Xie, Mengyu; Han, Jun; Yuan, Daoyi; Yang, Tian; Xie, Ying
2018-04-01
A validated liquid chromatography-tandem mass spectrometry method was developed for the simultaneous determination of D- and L-amino acids in human serum. Under the optimum conditions, except for DL-proline, L-glutamine, and D-lysine, the enantioseparation of the other 19 enantiomeric pairs of proteinogenic amino acids and nonchiral glycine was achieved with a CROWNPAK CR-I(+) chiral column within 13 min. The lower limits of quantitation for L-amino acids (including glycine) and D-amino acids were 5-56.25 μM and 0.625-500 nM, respectively, in human serum. The intraday precision and interday precision for all the analytes were less than 15%, and the accuracy ranged from -12.84% to 12.37% at three quality control levels. The proposed method, exhibiting high rapidity, enantioresolution, and sensitivity, was successfully applied to the quantification of D- and L-amino acid levels in serum from hepatocellular carcinoma patients and healthy individuals. The serum concentrations of L-arginine, L-isoleucine, L-aspartate, L-tryptophan, L-alanine, L-methionine, L-serine, glycine, L-valine, L-leucine, L-phenylalanine, L-threonine, D-isoleucine, D-alanine, D-glutamate, D-glutamine, D-methionine, and D-threonine were significantly reduced in the hepatocellular carcinoma patients compared with the healthy individuals (P < 0.01). D-Glutamate and D-glutamine were identified as the most downregulated serum markers (fold change greater than 1.5), which deserves further attention in hepatocellular carcinoma research. Graphical abstract Simultaneous determination of D- and L-amino acids in human serum from hepatocellular carcinoma patients and healthy individuals. AA amino acid, HCC hepatocellular carcinoma, LC liquid chromatography, MS/MS tandem mass spectrometry, NC normal control, TIC total ion chromatogram.
Park, Min-Ho; Lee, Yun Young; Cho, Kyung Hee; La, Sookie; Lee, Hee Joo; Yim, Dong-Seok; Ban, Sooho; Park, Moon-Young; Kim, Yong-Chul; Kim, Yoon-Gyoon; Shin, Young G
2016-03-01
A liquid chromatography-triple quadrupole mass spectrometric (LC-MS/MS) method was developed and validated for the determination of 5-nitro-5'-hydroxy-indirubin-3'-oxime (AGM-130) in human plasma to support a microdose clinical trial. The method consisted of a liquid-liquid extraction for sample preparation and LC-MS/MS analysis in the positive ion mode using TurboIonSpray(TM) for analysis. d3 -AGM-130 was used as the internal standard. A linear regression (weighted 1/concentration) was used to fit calibration curves over the concentration range of 10-2000 pg/mL for AGM-130. There were no endogenous interference components in the blank human plasma tested. The accuracy at the lower limit of quantitation was 96.6% with a precision (coefficient of variation, CV) of 4.4%. For quality control samples at 30, 160 and 1600 pg/mL, the between run CV was ≤5.0 %. Between-run accuracy ranged from 98.1 to 101.0%. AGM-130 was stable in 50% acetonitrile for 168 h at 4°C and 6 h at room temperature. AGM-130 was also stable in human plasma at room temperature for 6 h and through three freeze-thaw cycles. The variability of selected samples for the incurred sample reanalysis was ≤12.7% when compared with the original sample concentrations. This validated LC-MS/MS method for determination of AGM-130 was used to support a phase 0 microdose clinical trial. Copyright © 2015 John Wiley & Sons, Ltd.
LC Circuits for Diagnosing Embedded Piezoelectric Devices
NASA Technical Reports Server (NTRS)
Chattin, Richard L.; Fox, Robert Lee; Moses, Robert W.; Shams, Qamar A.
2005-01-01
A recently invented method of nonintrusively detecting faults in piezoelectric devices involves measurement of the resonance frequencies of inductor capacitor (LC) resonant circuits. The method is intended especially to enable diagnosis of piezoelectric sensors, actuators, and sensor/actuators that are embedded in structures and/or are components of multilayer composite material structures.
Swortwood, Madeleine J; Boland, Diane M; DeCaprio, Anthony P
2013-02-01
Recently, clandestine drug lab operators have attempted to bypass controlled substances laws and regulations with "designer" compounds chemically and pharmacologically similar to controlled substances. For example, "bath salts" have erupted onto the scene as "legal highs" containing cathinone analogs that have produced severe side effects in users worldwide. These products have sparked concern among law enforcement agencies, and emergency bans have been placed on the sale of such items. Despite the increasing number of designer drugs available, there are few comprehensive screening techniques for their detection and quantification in biological specimens. The liquid chromatography triple quadrupole tandem mass spectrometry (LC-QQQ-MS/MS) method presented here encompasses over thirty important compounds within the phenethylamine, tryptamine, and piperazine designer drug classes. Analytes were determined by LC-QQQ-MS/MS in the multiple-reaction monitoring mode after mixed-mode solid-phase extraction. The bioanalytical method was fully validated according to recommended international guidelines. The assay was selective for all analytes with acceptable accuracy and precision. Limits of quantification were in the range of 1-10 ng/mL for each compound with limits of detection near 10 pg/mL. In order to evaluate its applicability in a forensic toxicological setting, the validated method was used to analyze post-mortem specimens from two cases that were suspected of containing designer drugs. The method was able to identify and quantify seven of these compounds at concentrations as low as 11 ng/mL. The method should have wide applicability for rapid screening of important new drugs of abuse at high sensitivity in both post- and ante-mortem forensic analysis.
Breindahl, Torben; Evans-Brown, Michael; Hindersson, Peter; McVeigh, Jim; Bellis, Mark; Stensballe, Allan; Kimergård, Andreas
2015-02-01
New methods were developed and validated to determine the identity, contents, and purity of samples of melanotan II, a synthetic melanocortin receptor agonist, sold in vials as injectable skin-tanning products that were purchased from three online shops. Methods were based on liquid chromatography with ultra-violet detection (LC-UV) at wavelength 218 nm, and tandem mass spectrometric detection (MS/MS) after collision-induced fragmentation of the double charged [M+2H](2+) precursor ion (m/z 513). Identification of melanotan II was verified by correct chromatographic retention time, and relative abundance ratios of five qualifying fragment ions. LC-UV was used to quantify melanotan II as well as impurities. Method validation was performed with reference to guidelines for assessing active substances in authorized medicinal products to reach acceptable accuracy and precision. Vials from two shops contained unknown impurities ranging from 4.1 to 5.9%; impurities from one shop were below the quantification limit. The total amount of melanotan II in vials ranged between 4.32 and 8.84 mg, although each shop claimed that vials contained 10 mg melanotan II. A broad range of drugs used for enhancement purposes can be obtained from the illicit market. However, users of these drugs may be exposed to a range of potential harms, as shown in this study, given that these products are manufactured, distributed and supplied from an illicit market. Copyright © 2014 John Wiley & Sons, Ltd.
Jimmerson, Leah C.; Ray, Michelle L.; Bushman, Lane R.; Anderson, Peter L.; Klein, Brandon; Rower, Joseph E.; Zheng, Jia-Hua; Kiser, Jennifer J.
2014-01-01
Ribavirin (RBV) is a nucleoside analog used to treat a variety of DNA and RNA viruses. RBV undergoes intracellular phosphorylation to a mono- (MP), di- (DP), and triphosphate (TP). The phosphorylated forms have been associated with the mechanisms of antiviral effect observed in vitro, but the intracellular pharmacology of the drug has not been well characterized in vivo. A highly sensitive LC-MS/MS method was developed and validated for the determination of intracellular RBV MP, DP, and TP in multiple cell matrix types. For this method, the individual MP, DP, and TP fractions were isolated from lysed intracellular matrix using strong anion exchange solid phase extraction, dephosphorylated to parent RBV, desalted and concentrated and quantified using LC-MS/MS. The method utilized a stable labeled internal standard (RBV-13C5) which facilitated accuracy (% deviation within ±15%) and precision (coefficient of variation of ≤15%). The quantifiable linear range for the assay was 0.50 to 200 pmol/sample. The method was applied to the measurement of RBV MP, DP, and TP in human peripheral blood mononuclear cells (PBMC), red blood cells (RBC), and dried blood spot (DBS) samples obtained from patients taking RBV for the treatment of chronic Hepatitis C virus infection. PMID:25555148
Ok, Hyun Ee; Tian, Fei; Hong, Eun Young; Paek, Ockjin; Kim, Sheen-Hee; Kim, Dongsul; Chun, Hyang Sook
2016-01-01
An interlaboratory study was performed in eight laboratories to validate a liquid chromatography–tandem mass spectrometry (LC/MS/MS) method for the simultaneous determination of aflatoxins and sterigmatocystin (STC) in white rice and sorghum (Sorghum bicolor). Fortified samples (at three different levels) of white rice and sorghum were extracted, purified through a solid-phase extraction (SPE) column, and then analyzed by LC/MS/MS. The apparent recoveries (ARs) ranged from 78.8% to 95.0% for aflatoxins and from 85.3% to 96.7% for STC. The relative standard deviations for repeatability (RSDr) and reproducibility (RSDR) of aflatoxins were in the ranges 7.9%–33.8% and 24.4%–81.0%, respectively. For STC, the RSDr ranged from 7.1% to 40.2% and the RSDR ranged from 28.1% to 99.2%. The Horwitz ratio values for the aflatoxins and STC ranged from 0.4 to 1.2 in white rice and from 0.3 to 1.0 in sorghum, respectively. These results validated this method for the simultaneous determination of aflatoxins and STC by LC/MS/MS after SPE column cleanup. The percentages of satisfactory Z-score values (|Z| ≤ 2) were the following: for white rice, 100% for aflatoxins and STC; for sorghum, 100%, except in data from two laboratories for STC (0.3 μg/kg). This validated that the LC/MS/MS method was successfully applied for the determination of aflatoxins and STC in 20 white rice and 20 sorghum samples sourced from Korean markets. PMID:27983588
Plassmann, Merle M; Schmidt, Magdalena; Brack, Werner; Krauss, Martin
2015-09-01
Exposure to environmental pollution and consumer products may result in an uptake of chemicals into human tissues. Several studies have reported the presence of diverse environmental contaminants in human blood samples. However, previously developed multi-target methods for the analysis of human blood include a fairly limited amount of compounds stemming from one or two related compound groups. Thus, the sample preparation method QuEChERS (quick easy cheap effective rugged and safe) was tested for the extraction of 64 analytes covering a broad compound domain followed by detection using liquid and gas chromatography coupled to mass spectrometry (LC- and GC-MS). Forty-seven analytes showed absolute recoveries above 70% in the first QuEChERS step, being a simple liquid-liquid extraction (LLE) using acetonitrile and salt. The second QuEChERS step, being a dispersive solid phase extraction, did not result in an overall improvement of recoveries or removal of background signals. Using solely the LLE step, eight analytes could subsequently be detected in human blood samples from the German Environmental Specimen Bank. Using a LC-multiple reaction monitoring (MRM) method with a triple quadrupole instrument, better recoveries were achieved than with an older LC-high-resolution (HR) MS full scan orbitrap instrument, which required a higher concentration factor of the extracts. However, the application of HRMS full scan methods could be used for the detection of additional compounds retrospectively.
Li, Kefeng; Xia, Yonghong; Ma, Guolin; Zhao, Yanna; Pidatala, Venkataramana R
2017-03-29
Allura red is a widely used synthetic food dye. In this study, we developed and validated a LC-MS/MS method for the quantification of allura red in three popular takeaway Chinese dishes (braised pork, soy sauce chicken, sweet and sour pork) and human urine samples. High levels of allura red ranging from 2.85 to 8.38 mg/g wet weight were detected in the surveyed Chinese dishes. Of 113 participants who frequently consume the surveyed Chinese dishes (>once a week in the past 2 years), the median of their urinary allura red level was 22.29 nM/mM creatinine (95% CI = 19.48-25.03) . Risk assessment using Cox proportional hazard models showed that a 10-fold increase in urinary allura red was positively associated with high blood pressure (odds ratio of 1.75 (95% CI = 0.78-3.96)). Our findings provide new insights for the potential risk of hypertension for long-term allura red overconsumption.
Rugged LC-MS/MS survey analysis for acrylamide in foods.
Roach, John A G; Andrzejewski, Denis; Gay, Martha L; Nortrup, David; Musser, Steven M
2003-12-17
The described liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the detection of acrylamide in food entails aqueous room temperature extraction, SPE cleanup, and analysis by LC-MS/MS. The method is applicable to a wide variety of foods. [(13)C(3)]acrylamide is the internal standard. The limit of quantitation is 10 ppb (microg/kg). Data were obtained in duplicate from >450 products representing >35 different food types. The variability in analyte levels in certain food types suggests that it may be possible to reduce acrylamide levels in those foods.
Tang, Dao-quan; Bian, Ting-ting; Zheng, Xiao-xiao; Li, Ying; Wu, Xiao-wen; Li, Yin-jie; Du, Qian; Jiang, Shui-shi
2014-09-01
Three liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods were respectively developed and validated for the simultaneous or independent determination of taurine and edaravone in rat plasma using 3-methyl-1-p-tolyl-5-pyrazolone and sulfanilic acid as the internal standards (IS). Chromatographic separations were achieved on an Agilent Zorbax SB-Aq (100 × 2.1 mm, 3.5 µm) column. Gradient 0.03% formic acid-methanol, isocratic 0.1% formic acid-methanol (90:10) and 0.02% formic acid-methanol (40:60) were respectively selected as the mobile phase for the simultaneous determination of two analytes, taurine or edaravone alone. The MS acquisition was performed in multiple reaction monitoring mode with a positive and negative electrospray ionization source. The mass transitions monitored were m/z [M + H](+) 175.1 → 133.0 and [M + H](+) 189.2 → 147.0 for edaravone and its IS, m/z [M - H](-) 124.1 → 80.0 and [M - H](-) 172.0 → 80.0 for taurine and its IS, respectively. The validated methods were successfully applied to study the pharmacokinetic interaction of taurine and edaravone in rats after independent intravenous administration and co-administration with a single dose. Our collective results showed that there were no significant alterations on the main pharmacokinetic parameters (area under concentration-time curve, mean residence time, half-life and clearance) of taurine and edaravone, implying that the proposed combination therapy was pharmacologically feasible. Copyright © 2014 John Wiley & Sons, Ltd.
Kim, Junghyun; Suh, Joon Hyuk; Cho, Hyun-Deok; Kang, Wonjae; Choi, Yong Seok; Han, Sang Beom
2016-01-01
A multi-class, multi-residue analytical method based on LC-MS/MS detection was developed for the screening and confirmation of 28 veterinary drug and metabolite residues in flatfish, shrimp and eel. The chosen veterinary drugs are prohibited or unauthorised compounds in Korea, which were categorised into various chemical classes including nitroimidazoles, benzimidazoles, sulfones, quinolones, macrolides, phenothiazines, pyrethroids and others. To achieve fast and simultaneous extraction of various analytes, a simple and generic liquid extraction procedure using EDTA-ammonium acetate buffer and acetonitrile, without further clean-up steps, was applied to sample preparation. The final extracts were analysed by ultra-high-performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS). The method was validated for each compound in each matrix at three different concentrations (5, 10 and 20 ng g(-1)) in accordance with Codex guidelines (CAC/GL 71-2009). For most compounds, the recoveries were in the range of 60-110%, and precision, expressed as the relative standard deviation (RSD), was in the range of 5-15%. The detection capabilities (CCβs) were below or equal to 5 ng g(-1), which indicates that the developed method is sufficient to detect illegal fishery products containing the target compounds above the residue limit (10 ng g(-1)) of the new regulatory system (Positive List System - PLS).
Schläfli, A M; Berezowska, S; Adams, O; Langer, R; Tschan, M P
2015-05-05
Autophagy assures cellular homeostasis, and gains increasing importance in cancer, where it impacts on carcinogenesis, propagation of the malignant phenotype and development of resistance. To date, its tissue-based analysis by immunohistochemistry remains poorly standardized. Here we show the feasibility of specifically and reliably assessing the autophagy markers LC3B and p62 (SQSTM1) in formalin fixed and paraffin embedded human tissue by immunohistochemistry. Preceding functional experiments consisted of depleting LC3B and p62 in H1299 lung cancer cells with subsequent induction of autophagy. Western blot and immunofluorescence validated antibody specificity, knockdown efficiency and autophagy induction prior to fixation in formalin and embedding in paraffin. LC3B and p62 antibodies were validated on formalin fixed and paraffin embedded cell pellets of treated and control cells and finally applied on a tissue microarray with 80 human malignant and non-neoplastic lung and stomach formalin fixed and paraffin embedded tissue samples. Dot-like staining of various degrees was observed in cell pellets and 18/40 (LC3B) and 22/40 (p62) tumors, respectively. Seventeen tumors were double positive for LC3B and p62. P62 displayed additional significant cytoplasmic and nuclear staining of unknown significance. Interobserver-agreement for grading of staining intensities and patterns was substantial to excellent (kappa values 0.60 - 0.83). In summary, we present a specific and reliable IHC staining of LC3B and p62 on formalin fixed and paraffin embedded human tissue. Our presented protocol is designed to aid reliable investigation of dysregulated autophagy in solid tumors and may be used on large tissue collectives.
Pareja, Lucía; Cesio, Verónica; Heinzen, Horacio; Fernández-Alba, Amadeo R
2011-02-15
Four different extraction and clean-up protocols based on the QuEChERS method were compared for the development of an optimized sample preparation procedure for the multiresidue analysis of 16 commonly applied herbicides in rice crops using LC-QqQ/MS. Additionally the methods were evaluated for the analysis of 26 insecticides and fungicides currently used in rice crops. The methods comprise, in general, the hydratation of the sample with water followed by the extraction with acetonitrile, phase separation with the addition of different salts and finally a clean-up step with various sorbents. Matrix effects were evaluated for the 4 studied methods using LC-QqQ/MS. Additionally LC-TOF/MS was used to compare the co-extractants obtained with the four assayed methodologies. Thirty-six pesticides presented good performance with recoveries in the range 70-120% and relative standard deviations below 20% using 7.5 g of milled polished rice and the buffered acetate QuEChERS method without clean-up at both fortification levels: 10 and 300 μg kg(-1). The other six pesticides presented low recovery rates, nevertheless all these analytes could be analyzed with at least one of the other three studied procedures. Copyright © 2010. Published by Elsevier B.V.
Ewles, Matthew; Mannu, Ranbir; Fox, Chris; Stanta, Johannes; Evans, Graeme; Goodwin, Lee; Duffy, James; Bell, Len; Estdale, Sian; Firth, David
2016-12-01
We aimed to establish novel, high-throughput LC-MS/MS strategies for quantification of monoclonal antibodies in human serum and examine the potential impact of antidrug antibodies. We present two strategies using a thermally stable immobilized trypsin. The first strategy uses whole serum digestion and the second introduces Protein G enrichment to improve the selectivity. The impact of anti-trastuzumab antibodies on the methods was tested. Whole serum digestion has been validated for trastuzumab (LLOQ 0.25 µg/ml). Protein G enrichment has been validated for trastuzumab (LLOQ 0.1 µg/ml), bevacizumab (LLOQ 0.1 µg/ml) and adalimumab (LLOQ 0.25 µg/ml). We have shown the potential for anti-drug antibodies to impact on the quantification and we have subsequently established a strategy to overcome this impact where total quantification is desired.
NASA Technical Reports Server (NTRS)
Wang, Zuwei; Smith, Scott M.
2016-01-01
Urinary free 8-hydroxy-2'-deoxyguanosine (8OHdG), an oxidized product of DNA, and is frequently chosen as a biomarker of oxidative stress in humans, including studies of oxidative DNA damage during space flight. It is challenging to accurately and efficiently quantify urinary free 8OHdG in large scale human studies. LC-MS/MS is emerging as a preferable analytical technique owing its high sensitivity, selectivity and efficiency, compared to some traditional methods such as ELISA and HPLC. A simple and sensitive LC-MS/MS method has been developed for the determination of free 8OHdG in human urine. Sample preparation was done by solid phase extraction with a Waters Oasis HLB 96 well plate. A Waters Alliance 2795 HT Separation Module combined with a Quattro Micro tandem mass spectrometer was used as the LC-MS/MS system. The runtime of one injection can be less than 5 minutes using a reversed phase C18 column and an isocratic flow of methanol/water. ESI positive ions were quantified in the multiple reaction modes (MRM) using m/z 284 yields 168 for 8OHdG and m/z 289 yields173 for stable isotope labeled internal standard [(15)N5] 8OHdG. With this method for 8OHdG, a lower limit of quantitation of 1.0 nM (0.28 ng/mL) has been achieved using 100 microliter urine sample. The analytical range is between 1.0 and 100 nM with a correlation coefficient greater than or equal to 0.99. Good reproducibility can be obtained with intra-assay and inter-assay CVs less than or equal to 10% for 8OHdG spiked urine QC samples. This method can be used in high-throughput routine analysis of free 8OHdG in human urine.
NASA Astrophysics Data System (ADS)
Gupta, Lokesh Kumar
2012-11-01
Seven process related impurities were identified by LC-MS in the atorvastatin calcium drug substance. These impurities were identified by LC-MS. The structure of impurities was confirmed by modern spectroscopic techniques like 1H NMR and IR and physicochemical studies conducted by using synthesized authentic reference compounds. The synthesized reference samples of the impurity compounds were used for the quantitative HPLC determination. These impurities were detected by newly developed gradient, reverse phase high performance liquid chromatographic (HPLC) method. The system suitability of HPLC analysis established the validity of the separation. The analytical method was validated according to International Conference of Harmonization (ICH) with respect to specificity, precision, accuracy, linearity, robustness and stability of analytical solutions to demonstrate the power of newly developed HPLC method.
Sim, Juhyun; Kim, Eunmi; Yang, Wonkyung; Woo, Sanghee; In, Sangwhan
2017-05-01
In recent years, the inappropriate use of antipsychotics by young Korean men has become a social problem. As military service exemptions are given for mental illness, some men pose as mental health patients to avoid military service. In order to verify the authenticity of mental illnesses, we developed simultaneous analytical methods for the detection of 15 antipsychotics and 2 of their metabolites in hair using liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. The target drugs were modafinil, atomoxetine, aripiprazole, benztropine, buspirone, duloxetine, gabapentin, oxcarbazepine, topiramate, escitalopram, paliperidone, ziprasidone, lamotrigine, clonazepam, levetiracetam, and metabolites of oxcarbazepine and clonazepam. To remove possible contaminants on the hair surface, hair samples were washed twice with methanol and distilled water, and then were extracted with methanol overnight at 38°C. Desipramine-d 3 was used as an internal standard. LC-MS/MS analysis was performed on an Agilent 1290 Infinity UHPLC coupled to an AB Sciex Qtrap ® 5500 MS/MS. The total chromatographic run time was 14min. The following validation parameters were evaluated: selectivity, linearity, limit of detection (LOD), limit of quantification (LOQ), precision, accuracy, matrix effect, and recovery. The LOD and LOQ values for all analytes, except modafinil, ranged from 0.2 to 10pg/mg hair and from 0.2 to 20pg/mg hair, respectively. Good linearity was achieved for most of the analytes in the range of 20-200pg/mg hair. The method showed acceptable precision and accuracy, which were less than 15%, as well as satisfactory matrix effects and recoveries. Furthermore, this method was also applied to the analysis of rat hair samples. The study in rats showed that the concentrations of atomoxetine and aripiprazole in pigmented hair were significantly higher than those in non-pigmented hair. However, no significant difference was observed in the concentration of topiramate between
A novel approach for quantitation of glucosylceramide in human dried blood spot using LC-MS/MS.
Ji, Allena Ji; Wang, Haixing; Ziso-Qejvanaj, Enida; Zheng, Kefei; Chung, Lee Lee; Foley, Timothy; Chuang, Wei-Lien; Richards, Susan; Sung, Crystal
2015-01-01
Glucosylceramide, an efficacy biomarker for Gaucher Type 1 disease, exhibits poor solubility in polar solvents and whole blood which makes it difficult to prepare a homogenous blood standard. We developed a novel method using standard addition approach by spiking a small volume of analyte solution on the surface of prespotted dried blood spot. The whole spots were punched out for subsequent extraction and LC-MS/MS analysis. The assay performance met all validation acceptance criteria. Glucosylceramide concentrations in 50 paired plasma and dry blood spot samples obtained from Gaucher Type 1 patients were tested and the results demonstrated the feasibility of using the DBS method for clinical biomarker monitoring. The new approach greatly improves assay precision and accuracy.
Uclés, A; Ulaszewska, M M; Hernando, M D; Ramos, M J; Herrera, S; García, E; Fernández-Alba, A R
2013-07-01
This work introduces a liquid chromatography-electrospray ionization-hybrid quadrupole/time-of-flight mass spectrometry (LC-ESI-QTOF-MS)-based method for qualitative and quantitative analysis of poly(amidoamine) (PAMAM) dendrimers of generations 0 to 3 in an aqueous matrix. The multiple charging of PAMAM dendrimers generated by means of ESI has provided key advantages in dendrimer identification by assignation of charge state through high resolution of isotopic clusters. Isotopic distribution in function of abundance of isotopes (12)C and (13)C yielded valuable and complementarity data for confident characterization. A mass accuracy below 3.8 ppm for the most abundant isotopes (diagnostic ions) provided unambiguous identification of PAMAM dendrimers. Validation of the LC-ESI-QTOF-MS method and matrix effect evaluation enabled reliable and reproducible quantification. The validation parameters, limits of quantification in the range of 0.012 to 1.73 μM, depending on the generation, good linear range (R > 0.996), repeatability (RSD < 13.4%), and reproducibility (RSD < 10.9%) demonstrated the suitability of the method for the quantification of dendrimers in aqueous matrices (water and wastewater). The added selectivity, achieved by multicharge phenomena, represents a clear advantage in screening aqueous mixtures due to the fact that the matrix had no significant effect on ionization, with what is evidenced by an absence of sensitivity loss in most generations of PAMAM dendrimers. Fig Liquid chromatography-electrospray ionization-hybrid quadrupole/time of flight mass spectrometry (LC-ESI-QTOF-MS) based method for qualitative and quantitative analysis of PAMAM dendrimers in aqueous matrix.
Yang, Ya-Jun; Li, Jian-Yong; Liu, Xi-Wang; Zhang, Ji-Yu; Liu, Yu-Rong; Li, Bing
2013-01-01
To develop a non-biological method for screening active components against influenza virus from traditional Chinese medicine (TCM) extraction, a liquid chromatography (LC) column prepared with oseltamivir molecularly imprinted polymer (OSMIP) was employed with LC-mass spectrometry (LC-MS). From chloroform extracts of compound TCM liquid preparation, we observed an affinitive component m/z 249, which was identified to be matrine following analysis of phytochemical literatures, OSMIP-LC column on-line of control compounds and MS/MS off-line. The results showed that matrine had similar bioactivities with OS against avian influenza virus H9N2 in vitro for both alleviating cytopathic effect and hemagglutination inhibition and that the stereostructures of these two compounds are similar while their two-dimensional structures were different. In addition, our results suggested that the bioactivities of those affinitive compounds were correlated with their chromatographic behaviors, in which less difference of the chromatographic behaviors might have more similar bioactivities. This indicates that matrine is a potential candidate drug to prevent or cure influenza for human or animal. In conclusion, the present study showed that molecularly imprinted polymers can be used as a non-biological method for screening active components against influenza virus from TCM. PMID:24386385
Munyeza, Chiedza F; Shobo, Adeola; Baijnath, Sooraj; Bratkowska, Dominika; Naiker, Suhashni; Bester, Linda A; Singh, Sanil D; Maguire, Glenn E M; Kruger, Hendrik G; Naicker, Tricia; Govender, Thavendran
2016-06-01
Tigecycline (TIG), a derivative of minocycline, is the first in the novel class of glycylcyclines and is currently indicated for the treatment of complicated skin structure and intra-abdominal infections. A selective, accurate and reversed-phase high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method was developed for the determination of TIG in rat brain tissues. Sample preparation was based on protein precipitation and solid phase extraction using Supel-Select HLB (30 mg/1 mL) cartridges. The samples were separated on a YMC Triart C18 column (150 mm x 3.0 mm. 3.0 µm) using gradient elution. Positive electrospray ionization (ESI+) was used for the detection mechanism with the multiple reaction monitoring (MRM) mode. The method was validated over the concentration range of 150-1200 ng/mL for rat brain tissue. The precision and accuracy for all brain analyses were within the acceptable limit. The mean extraction recovery in rat brain was 83.6%. This validated method was successfully applied to a pharmacokinetic study in female Sprague Dawley rats, which were given a dose of 25 mg/kg TIG intraperitoneally at various time-points. Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.
A LC/MS METHOD FOR THE DETERMINATION OF CYANOBACTERIA TOXINS IN WATER
The cyanobacteria toxins anatoxin-a, microcystin-LR, microcystin-RR, microcystin-YR, and nodularin were separated in less than 30 minutes on several 1 mm x 15 cm reverse phase liquid chromatography (LC) columns, and their electrospray mass spectra were measured with 50 ng or less...
NASA Technical Reports Server (NTRS)
Wang, Zuwei; Vaksman, Zalman; Putcha, Lakshmi
2008-01-01
Intranasal scopolamine is a choice drug for the treatment of motion sickness during space flight because of its quick onset of action, short half-life and favorable sideeffects profile. The dose administered usually ranges between 0.1 and 0.4 mg. Such small doses make it difficult to detect concentrations of scopolamine in biological fluids using existing sensitive LC/MS/MS method, especially when the biological sample volumes are limited. To measure scopolamine in human plasma to facilitate pharmacokinetic evaluation of the drug, we developed a sensitive LC/MS/MS method using 96 well micro elution plates for solid phase extraction (SPE) of scopolamine in human plasma. Human plasma (100-250 micro L) were loaded onto Waters Oasis HLB 96 well micro elution plate and eluted with 50 L of organic solvent without evaporation and reconstitution. HPLC separation of the eluted sample was performed using an Agilent Zorbax SB-CN column (50 x 2.1 mm) at a flow rate of 0.2 mL/min for 3 minutes. The mobile phase for separation was 80:20 (v/v) methanol: ammonium acetate (30 mM) in water. Concentrations of scopolamine were determined using a Micromass Quattro Micro(TM) mass spectrometer with electrospray ionization (ESI). ESI mass spectra were acquired in positive ion mode with multiple reaction monitoring for the determination of scopolamine m/z = 304.2 right arrow 138.1 and internal standard hyoscyamine m/z = 290.2 right arrow 124.1. The method is rapid, reproducible, specific and has the following parameters: scopolamine and the IS are eluted at about 1.1 and 1.7 min respectively. The linear range is 25-10000 pg/mL for scopolamine in human plasma with correlation coefficients greater than 0.99 and CV less than 0.5%. The intra-day and inter-day CVs are less than 15% for quality control samples with concentrations of 75,300, and 750 pg/mL of scopolamine in human plasma. SPE using 96 well micro elution plates allows rapid sample preparation and enhanced sensitivity for the LC
[Validation and verfication of microbiology methods].
Camaró-Sala, María Luisa; Martínez-García, Rosana; Olmos-Martínez, Piedad; Catalá-Cuenca, Vicente; Ocete-Mochón, María Dolores; Gimeno-Cardona, Concepción
2015-01-01
Clinical microbiologists should ensure, to the maximum level allowed by the scientific and technical development, the reliability of the results. This implies that, in addition to meeting the technical criteria to ensure their validity, they must be performed with a number of conditions that allows comparable results to be obtained, regardless of the laboratory that performs the test. In this sense, the use of recognized and accepted reference methodsis the most effective tool for these guarantees. The activities related to verification and validation of analytical methods has become very important, as there is continuous development, as well as updating techniques and increasingly complex analytical equipment, and an interest of professionals to ensure quality processes and results. The definitions of validation and verification are described, along with the different types of validation/verification, and the types of methods, and the level of validation necessary depending on the degree of standardization. The situations in which validation/verification is mandatory and/or recommended is discussed, including those particularly related to validation in Microbiology. It stresses the importance of promoting the use of reference strains as controls in Microbiology and the use of standard controls, as well as the importance of participation in External Quality Assessment programs to demonstrate technical competence. The emphasis is on how to calculate some of the parameters required for validation/verification, such as the accuracy and precision. The development of these concepts can be found in the microbiological process SEIMC number 48: «Validation and verification of microbiological methods» www.seimc.org/protocols/microbiology. Copyright © 2013 Elsevier España, S.L.U. y Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica. All rights reserved.
Binz, Tina M; Braun, Ueli; Baumgartner, Markus R; Kraemer, Thomas
2016-10-15
Hair cortisol levels are increasingly applied as a measure for stress in humans and mammals. Cortisol is an endogenous compound and is always present within the hair matrix. Therefore, "cortisol-free hair matrix" is a critical point for any analytical method to accurately quantify especially low cortisol levels. The aim of this project was to modify current methods used for hair cortisol analysis to more accurately determine low endogenous cortisol concentrations in hair. For that purpose, (13)C3-labeled cortisol, which is not naturally present in hair (above 13C natural abundance levels), was used for calibration and comparative validation applying cortisol versus (13)C3-labeled cortisol. Cortisol was extracted from 20mg hair (standard sample amount) applying an optimized single step extraction protocol. An LC-MS/MS method was developed for the quantitative analysis of cortisol using either cortisol or (13)C3-cortisol as calibrators and D7-cortisone as internal standard (IS). The two methods (cortisol/(13)C3-labeled cortisol) were validated in a concentration range up to 500pg/mg and showed good linearity for both analytes (cortisol: R(2)=0.9995; (13)C3-cortisol R(2)=0.9992). Slight differences were observed for limit of detection (LOD) (0.2pg/mg/0.1pg/mg) and limit of quantification (LOQ) (1pg/mg/0.5pg/mg). Precision was good with a maximum deviation of 8.8% and 10% for cortisol and (13)C3-cortisol respectively. Accuracy and matrix effects were good for both analytes except for the quality control (QC) low cortisol. QC low (2.5pg/mg) showed matrix effects (126.5%, RSD 35.5%) and accuracy showed a deviation of 26% when using cortisol to spike. These effects are likely to be caused by the unknown amount of endogenous cortisol in the different hair samples used to determine validation parameters like matrix effect, LOQ and accuracy. No matrix effects were observed for the high QC (400pg/mg) samples. Recovery was good with 92.7%/87.3% (RSD 9.9%/6.2%) for QC low and
Dong, Ying; Yan, Kuan; Ma, Yanhua; Yang, Zhiyong; Zhao, Jun; Ding, Jinglin
2016-06-01
Glycerol and mannitol have the potential to act as plasma volume expanders and have been prohibited as masking agents by the World Anti-Doping Agency (WADA) accordingly. In this study, an improved strategy was developed and validated for the determination of urinary glycerol and mannitol levels simultaneously using a liquid chromatography/tandem mass spectrometry technique within 7min in an initial testing procedure. For confirmation, mannitol and all possible hexitols (allitol, altritol, galactitol, iditol and sorbitol) that can occur in human urine were baseline separated. This method made use of the derivatization of glycerol and mannitol by benzoyl chloride followed by analysis via LC-ESI-MS/MS with limited sample preparation. The limit of detection (LOD) for glycerol and mannitol was lower than 50ng/mL. The limit of quantitation (LOQ) for both substances was below 150ng/mL. The assay was linear from 0.15 to 1000μg/mL for glycerol and mannitol in human urine. The coefficients of variation of all inter- and intra-assay determinations at three concentration levels (0.5, 500, 900μg/mL) were better than 13% for glycerol and under 15% for mannitol. The method also afforded satisfactory results in terms of accuracy, derivatization yield, extraction recovery, matrix effect and specificity for both substances. Copyright © 2016 Elsevier B.V. All rights reserved.
[LC-MS/MS method for determination of tripterine in plasma: pharmacokinetic study in Beagles].
Zhang, Jun; Liu, Shi-Jia; Hu, Jie-Hui; Xu, Mei-Juan; Liu, Zi-Xiu; Zhou, Ling; Ju, Wen-Zheng
2016-07-01
To establish a LC-MS/MS method for determination of tripterine in Beagle plasma and study its pharmacokinetics after oral administration of tripterygium tablet. Plasma samples were extracted with dichloromethane and separated on a Phenomenex Luna C₈ (2.0 mm×50 mm, 3 μm) column with methanol-acetonitrile isopropanol(1∶1)-1‰formic acid (15∶55 ∶30) as the mobile phase. Tripterine ([M+H] ⁺, m/z 451.3/201.1) and internal standard prednisolone ([M+H] ⁺, m/z 361.1/147.1) were monitored in multiple reaction monitoring (MRM). The concentration-time curves were simulated by drug and statistic software 1.0 and the pharmacokinetic parameters were calculated. There was a good linear relationship between peak area ratio and concentration of tripterine and internal standard prednisolone within range of 0.680 0-136.0 μg•L⁻¹. The limit of quantitation was 0.680 0 μg•L⁻¹ and the intra- and inter-day precision was within 6.15%. The absolute recovery rate was between 50.42% to 51.65%. The concentration-time curves were consistent with the one-compartment model(w=1/cc). The main pharmacokinetic parameters after a single dose were as follows: Cmax (35.64±9.540) μg •L⁻¹,Tmax(2.62±0.69) h,T1/2(2.93±0.29) h, CL (0.308±0.056) L•kg⁻¹•h⁻¹, AUC0-12 (131.16±31.94) μg•L•h⁻¹, AUC0-∞ (142.83±37.57) μg•L•h⁻¹. The established LC-MS/MS method was proved to be sensitive, accurate and convenient, suitable for the pharmacokinetic study of Tripterygium tablet in Beagle dogs. Copyright© by the Chinese Pharmaceutical Association.
Du, Jiangbo; Ma, Zhiyu; Zhang, Yifan; Wang, Ting; Chen, Xiaoyan; Zhong, Dafang
2014-09-01
Ornidazole is a 5-nitroimidazole antimicrobial agent used for almost 40 years. A novel LC-MS/MS assay was developed and validated for the simultaneous determination of ornidazole and its main metabolites (M3, M6, M16-1, and M16-2) in human plasma. After extraction from 100 μl of plasma by protein precipitation with acetonitrile, all the analytes were separated on a Capcell PAK MG C18 column (100 × 4.6 mm, 5 μm) within 5.0 min and detected by ESI-MS/MS in the positive mode. The validation results met the acceptance criteria as per the US FDA and EMA guidelines. The validated method was successfully applied to a pharmacokinetic study after oral administration of 1000 mg ornidazole to six healthy Chinese volunteers.
Li, Bing; Gong, Shi-Yue; Zhou, Xu-Zheng; Yang, Ya-Jun; Li, Jian-Yong; Wei, Xiao-Juan; Cheng, Fu-Sheng; Niu, Jian-Rong; Liu, Xi-Wang; Zhang, Ji-Yu
2017-03-01
A rapid and sensitive high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated to quantify tilmicosin in pig plasma. Plasma samples were prepared by liquid-liquid extraction. Chromatographic separation was achieved on a C 18 column (2.1 × 30 mm, 3.5 μm) using acetonitrile-water (90:10, v/v; water included 0.1% formic acid) as the mobile phase. Mass detection was carried out using positive electrospray ionization in multiple reaction monitoring mode. The calibration curve was linear from 0.5 to 2000 ng/mL (r 2 = 0.9998). The intra- and inter-day accuracy and precision were within the acceptable limits of ±10% for all tilmicosin concentrations. The recoveries ranged from 95 to 99% for the three tested concentrations. The LC-MS/MS method described herein was simple, fast and less laborious than other methods, achieved high sensitivity using a small sample volume, and was successfully applied to pharmacokinetic studies of tilmicosin enteric granules after oral delivery to pigs. In comparison with tilmicosin premix, tilmicosin enteric granules slowed the elimination rate of tilmicosin, prolonged its period of action and significantly improved its bioavailability. Copyright © 2016 John Wiley & Sons, Ltd.
Quantitative Method for Simultaneous Analysis of Acetaminophen and 6 Metabolites.
Lammers, Laureen A; Achterbergh, Roos; Pistorius, Marcel C M; Romijn, Johannes A; Mathôt, Ron A A
2017-04-01
Hepatotoxicity after ingestion of high-dose acetaminophen [N-acetyl-para-aminophenol (APAP)] is caused by the metabolites of the drug. To gain more insight into factors influencing susceptibility to APAP hepatotoxicity, quantification of APAP and metabolites is important. A few methods have been developed to simultaneously quantify APAP and its most important metabolites. However, these methods require a comprehensive sample preparation and long run times. The aim of this study was to develop and validate a simplified, but sensitive method for the simultaneous quantification of acetaminophen, the main metabolites acetaminophen glucuronide and acetaminophen sulfate, and 4 Cytochrome P450-mediated metabolites by using liquid chromatography with mass spectrometric (LC-MS) detection. The method was developed and validated for the human plasma, and it entailed a single method for sample preparation, enabling quick processing of the samples followed by an LC-MS method with a chromatographic run time of 9 minutes. The method was validated for selectivity, linearity, accuracy, imprecision, dilution integrity, recovery, process efficiency, ionization efficiency, and carryover effect. The method showed good selectivity without matrix interferences. For all analytes, the mean process efficiency was >86%, and the mean ionization efficiency was >94%. Furthermore, the accuracy was between 90.3% and 112% for all analytes, and the within- and between-run imprecision were <20% for the lower limit of quantification and <14.3% for the middle level and upper limit of quantification. The method presented here enables the simultaneous quantification of APAP and 6 of its metabolites. It is less time consuming than previously reported methods because it requires only a single and simple method for the sample preparation followed by an LC-MS method with a short run time. Therefore, this analytical method provides a useful method for both clinical and research purposes.
Frey, Alexander J; Wang, Qingqing; Busch, Christine; Feldman, Daniel; Bottalico, Lisa; Mesaros, Clementina A; Blair, Ian A; Vachani, Anil; Snyder, Nathaniel W
2016-12-01
A multiplexed quantitative method for the analysis of three major unconjugated steroids in human serum by stable isotope dilution liquid chromatography-high resolution mass spectrometry (LC-HRMS) was developed and validated on a Q Exactive Plus hybrid quadrupole/Orbitrap mass spectrometer. This quantification utilized isotope dilution and Girard P derivatization on the keto-groups of testosterone (T), androstenedione (AD) and dehydroepiandrosterone (DHEA) to improve ionization efficiency using electrospray ionization. Major isomeric compounds to T and DHEA; the inactive epimer of testosterone (epiT), and the metabolite of AD, 5α-androstanedione (5α-AD) were completely resolved on a biphenyl column within an 18min method. Inter- and intra-day method validation using LC-HRMS with qualifying product ions was performed and acceptable analytical performance was achieved. The method was further validated by comparing steroid levels from 100μL of serum from young vs older subjects. Since this approach provides high-dimensional HRMS data, untargeted analysis by age group was performed. DHEA and T were detected among the top analytes most significantly different across the two groups after untargeted LC-HRMS analysis, as well as a number of other still unknown metabolites, indicating the potential for combined targeted/untargeted analysis in steroid analysis. Copyright © 2016 Elsevier Inc. All rights reserved.
Schmidt, Kathrin S; Mankertz, Joachim
2018-06-01
A sensitive and robust LC-MS/MS method allowing the rapid screening and confirmation of selective androgen receptor modulators in bovine urine was developed and successfully validated according to Commission Decision 2002/657/EC, chapter 3.1.3 'alternative validation', by applying a matrix-comprehensive in-house validation concept. The confirmation of the analytes in the validation samples was achieved both on the basis of the MRM ion ratios as laid down in Commission Decision 2002/657/EC and by comparison of their enhanced product ion (EPI) spectra with a reference mass spectral library by making use of the QTRAP technology. Here, in addition to the MRM survey scan, EPI spectra were generated in a data-dependent way according to an information-dependent acquisition criterion. Moreover, stability studies of the analytes in solution and in matrix according to an isochronous approach proved the stability of the analytes in solution and in matrix for at least the duration of the validation study. To identify factors that have a significant influence on the test method in routine analysis, a factorial effect analysis was performed. To this end, factors considered to be relevant for the method in routine analysis (e.g. operator, storage duration of the extracts before measurement, different cartridge lots and different hydrolysis conditions) were systematically varied on two levels. The examination of the extent to which these factors influence the measurement results of the individual analytes showed that none of the validation factors exerts a significant influence on the measurement results.
Zweigenbaum, J; Henion, J
2000-06-01
The high-throughput determination of small molecules in biological matrixes has become an important part of drug discovery. This work shows that increased throughput LC/MS/MS techniques can be used for the analysis of selected estrogen receptor modulators in human plasma where more than 2000 samples may be analyzed in a 24-h period. The compounds used to demonstrate the high-throughput methodology include tamoxifen, raloxifene, 4-hydroxytamoxifen, nafoxidine, and idoxifene. Tamoxifen and raloxifene are used in both breast cancer therapy and osteoporosis and have shown prophylactic potential for the reduction of the risk of breast cancer. The described strategy provides LC/MS/MS separation and quantitation for each of the five test articles in control human plasma. The method includes sample preparation employing liquid-liquid extraction in the 96-well format, an LC separation of the five compounds in less than 30 s, and selected reaction monitoring detection from low nano- to microgram per milliter levels. Precision and accuracy are determined where each 96-well plate is considered a typical "tray" having calibration standards and quality control (QC) samples dispersed through each plate. A concept is introduced where 24 96-well plates analyzed in 1 day is considered a "grand tray", and the method is cross-validated with standards placed only at the beginning of the first plate and the end of the last plate. Using idoxifene-d5 as an internal standard, the results obtained for idoxifene and tamoxifen satisfy current bioanalytical method validation criteria on two separate days where 2112 and 2304 samples were run, respectively. Method validation included 24-h autosampler stability and one freeze-thaw cycle stability for the extracts. Idoxifene showed acceptable results with accuracy ranging from 0.3% for the high quality control (QC) to 15.4% for the low QC and precision of 3.6%-13.9% relative standard deviation. Tamoxifen showed accuracy ranging from 1.6% to 13
The EIPeptiDi tool: enhancing peptide discovery in ICAT-based LC MS/MS experiments.
Cannataro, Mario; Cuda, Giovanni; Gaspari, Marco; Greco, Sergio; Tradigo, Giuseppe; Veltri, Pierangelo
2007-07-15
Isotope-coded affinity tags (ICAT) is a method for quantitative proteomics based on differential isotopic labeling, sample digestion and mass spectrometry (MS). The method allows the identification and relative quantification of proteins present in two samples and consists of the following phases. First, cysteine residues are either labeled using the ICAT Light or ICAT Heavy reagent (having identical chemical properties but different masses). Then, after whole sample digestion, the labeled peptides are captured selectively using the biotin tag contained in both ICAT reagents. Finally, the simplified peptide mixture is analyzed by nanoscale liquid chromatography-tandem mass spectrometry (LC-MS/MS). Nevertheless, the ICAT LC-MS/MS method still suffers from insufficient sample-to-sample reproducibility on peptide identification. In particular, the number and the type of peptides identified in different experiments can vary considerably and, thus, the statistical (comparative) analysis of sample sets is very challenging. Low information overlap at the peptide and, consequently, at the protein level, is very detrimental in situations where the number of samples to be analyzed is high. We designed a method for improving the data processing and peptide identification in sample sets subjected to ICAT labeling and LC-MS/MS analysis, based on cross validating MS/MS results. Such a method has been implemented in a tool, called EIPeptiDi, which boosts the ICAT data analysis software improving peptide identification throughout the input data set. Heavy/Light (H/L) pairs quantified but not identified by the MS/MS routine, are assigned to peptide sequences identified in other samples, by using similarity criteria based on chromatographic retention time and Heavy/Light mass attributes. EIPeptiDi significantly improves the number of identified peptides per sample, proving that the proposed method has a considerable impact on the protein identification process and, consequently, on
Screening Antioxidants Using LC-MS: A Case Study with Cocoa
Calderón, Angela I.; Wright, Brian J.; Hurst, W. Jeffrey; van Breemen, Richard B.
2009-01-01
Oxidative stress enhances pathological processes contributing to cancer, cardiovascular disease and neurodegenerative diseases, and dietary antioxidants may counteract these deleterious processes. Since rapid methods to evaluate and compare food products for antioxidant benefits are needed, a new assay based on liquid chromatography-mass spectrometry (LC-MS) was developed for the identification and quantitative analysis of antioxidants in complex natural product samples such as food extracts. This assay is based on the comparison of electrospray LC-MS profiles of sample extracts before and after treatment with reactive oxygen species such as hydrogen peroxide or DPPH (2,2-diphenyl-1-picrylhydrazyl radical). Using this assay, methanolic extracts of cocoa powder were analyzed, and procyanidins were found to be the most potent antioxidant species. These species were identified using LC-MS, LC-MS-MS, accurate mass measurement, and comparison with reference standards. Furthermore, LC-MS was used to determine the levels of these species in cocoa samples. Catechin and epicatechin were the most abundant antioxidants followed by their dimers and trimers. The most potent antioxidants in cocoa were trimers and dimers of catechin and epicatechin, such as procyanidin B2, followed by catechin and epicatechin. This new LC-MS assay facilitates the rapid identification and then the determination of the relative antioxidant activities of individual antioxidant species in complex natural product samples and food products such as cocoa. PMID:19489609
Pedada, Kiran K; Zhou, Xiang; Jogiraju, Harini; Carroll, Richard T; Geldenhuys, Werner J; Lin, Li; Anderson, David J
2014-01-15
Thiazolidinedione (TZD) compounds have shown promise as antidiabetic, antibiotics, antifungal and neuroprotective agents. The mitochondrial effect of a novel mitoNEET ligand, NL-1 {5-[(3,5-di-tert-butyl-4-hydroxyphenyl)methyl]-1,3-thiazolidine-2,4-dione}, and other TZD compounds, is a newly proposed mechanism for the neuroprotective action of these TZD compounds. In this work, a sensitive LC-MS/MS assay has been developed and validated for quantification of NL-1 in mouse serum. Sample preparation involved an acetonitrile protein precipitation procedure with addition of an internal standard NL-2 {5-[(4-hydroxy-3,5-dimethyl-phenyl)methyl]thiazolidine-2,4-dione}. LC-MS/MS analysis utilized a Columbus C-18 HPLC column (2mm×50mm, 5μm). Chromatography employed a multiple step gradient program that featured a steep linear gradient (25-95% in 0.5min) of 15μM ammonium acetate (additive for eliminating carry-over) in 2% methanol mixing with increasing proportions of 100% methanol. The HPLC was interfaced to a QTrap 5500 mass spectrometer (AB Sciex) equipped with an electrospray ionization source used in a negative ionization mode. Multiple reaction monitoring (MRM) of m/z 334→263 for NL-1 and m/z 250→179 for NL-2 was done. The method had a linear range of at least 1-100ng/mL in serum. The intra-assay and inter-assay percent coefficient of variation (%CV) were less than 4% and accuracies (%RE) ranged from -2.7% to 2.0%. The analytical procedure gave 96-115% absolute extraction recovery of NL-1. The relative matrix effect was measured and found to be insignificant. The analyte in serum was confirmed to be stable during storage and treatment. The method is suitable for pharmacokinetic (PK) studies of the parent drug NL-1 based on the preliminary serum results from dosed NL-1 mouse studies. Copyright © 2013 Elsevier B.V. All rights reserved.
Measurement of methionine level with the LC-ESI-MS/MS method in schizophrenic patients.
Kulaksizoglu, S; Kulaksizoglu, B; Ellidag, H Y; Eren, E; Yilmaz, N; Baykal, A
2016-01-01
The purpose of this study was to evaluate plasma methionine levels by using liquid chromatography electrospray ionization-tandem mass spectroscopy (LC-ESI-MS/MS) in schizophrenic patients. A twelve-point standard graph was drawn, and the recovery rate, the intra-day and inter-day coefficients of variation (CV), the limit of detection (LOD), and the limit of quantification (LOQ) were evaluated. The y and R2 values of the standard graph equation were determined as 0.011x + 0.0179 and 0.9989, respectively, and the graph remained linear until the 200 µmol/l level. The intra-day coefficients of variation of the samples (n = 10) containing 8, 28, and 58 µmol/l methionine were determined as 2.68, 3.10, and 3.79%, respectively; while their inter-day coefficients of variation were determined as 2.98, 3.19, and 3.84%. The LOD and LOQ values were determined as 0.04 and 0.1 µmol/l, respectively, while the mean recovery rates were determined as 101.7 and 99.3%. Plasma methionine values were measured as 21.5 (19.5-24,6) µmol/l for the patient group, 17.8 (16.3-20.1) µmol/l for the control group, and the difference between the two groups was statistically significant (p = 0.03). LC-ESI-MS/MS method represents a fairly sensitive, economic, and rapid analysis that requires very little sample and is suitable for measuring methionine levels in schizophrenic patients.
A rapid LC/MS/MS method for the analysis of nonvolatile antiinflammatory agents from Mentha spp.
Shen, Diandian; Pan, Min-Hsiung; Wu, Qing-Li; Park, Chung-Heon; Juliani, H Rodolfo; Ho, Chi-Tang; Simon, James E
2011-08-01
Mints (Mentha spp.), aromatic crops grown largely for their essential oils, also are rich sources of nonvolatile antiinflammatory agents. Identification and quantitation of the constituents responsible for their antiinflammatory activity is challenging owing to the lack of suitable chromatographic methodology. In the present research, the simultaneous quantitation of antiinflammatory constituents rosmarinic acid, oleanolic acid, and ursolic acid in mints was attained by using a unique tandem HPLC column system coupled with an electrospray ionization mass detection (MRM mode). The ion mode optimization for rosmarinic acid under negative and triterpenoid acids under positive was achieved by setting 2 time segments in a single run where the polarity mode was switched from negative (0 to 10 min) to positive (10 to 40 min). For the investigated concentration ranges of antiinflammatory agents in mints, good linearities (r² ≥ 0.998) were obtained for each calibration curve. Validation of precision and accuracy for this method showed that intra- and inter-day repeatabilities for all analytes were less than 5.51%, and the recoveries varied from 97.8% to 99.3%. The developed LC/MS/MS assay provides a suitable quality control method for the determination of antiinflammatory constituents in Mentha spp. There is a wide range of diversity in the natural product composition for these acids across the Mentha germplasm collection evaluated. The presence of these antiinflammatory acids in post-distilled mints shows that value-added nutraceutical enriched products can be developed with proper processing and recovery systems in addition to the distillation and capture of the valuable volatile essential oils. Results from this research would benefit both commercial farmers growing mint for essential oil and those in the food industry where value-added phytopharmaceutical enriched products can be developed with proper processing, quality control, and recovery systems during
Gervasoni, Jacopo; Schiattarella, Arcangelo; Giorgio, Valentina; Primiano, Aniello; Russo, Consuelo; Tesori, Valentina; Scaldaferri, Franco; Urbani, Andrea; Zuppi, Cecilia; Persichilli, Silvia
2016-01-01
Aim . Lactulose/mannitol ratio is used to assess intestinal barrier function. Aim of this work was to develop a robust and rapid method for the analysis of lactulose and mannitol in urine by liquid chromatography coupled to tandem mass spectrometry. Lactulose/mannitol ratio has been measured in pediatric patients suffering from irritable bowel syndrome. Methods . Calibration curves and raffinose, used as internal standard, were prepared in water : acetonitrile 20 : 80. Fifty μ L of urine sample was added to 450 μ L of internal standard solution. The chromatographic separation was performed using a Luna NH 2 column operating at a flow rate of 200 μ L/min and eluted with a linear gradient from 20% to 80% water in acetonitrile. Total run time is 9 minutes. The mass spectrometry operates in electrospray negative mode. Method was fully validated according to European Medicine Agency guidelines. Results and Conclusions . Linearity ranged from 10 to 1000 mg/L for mannitol and 2.5 to 1000 mg/L for lactulose. Imprecision in intra- and interassay was lower than 15% for both analytes. Accuracy was higher than 85%. Lactulose/mannitol ratio in pediatric patients is significantly higher than that measured in controls. The presented method, rapid and sensitive, is suitable in a clinical laboratory.
LC-MS based analysis of endogenous steroid hormones in human hair.
Gao, Wei; Kirschbaum, Clemens; Grass, Juliane; Stalder, Tobias
2016-09-01
The quantification of endogenous steroid hormone concentrations in hair is increasingly used as a method for obtaining retrospective information on long-term integrated hormone exposure. Several different analytical procedures have been employed for hair steroid analysis, with liquid chromatography-mass spectrometry (LC-MS) being recognized as a particularly powerful analytical tool. Several methodological aspects affect the performance of LC-MS systems for hair steroid analysis, including sample preparation and pretreatment, steroid extraction, post-incubation purification, LC methodology, ionization techniques and MS specifications. Here, we critically review the differential value of such protocol variants for hair steroid hormones analysis, focusing on both analytical quality and practical feasibility issues. Our results show that, when methodological challenges are adequately addressed, LC-MS protocols can not only yield excellent sensitivity and specificity but are also characterized by relatively simple sample processing and short run times. This makes LC-MS based hair steroid protocols particularly suitable as a high-quality option for routine application in research contexts requiring the processing of larger numbers of samples. Copyright © 2016 Elsevier Ltd. All rights reserved.
McMahon, Gillian; Wall, Rachel; Nolan, Kieran; Diamond, Dermot
2002-07-19
A series of derivatisation reactions between p-t-butyl calix[4]arene and ethyl bromoacetate were carried out in order to prepare 1,3 diester substituted calix[4]arene. Mass spectral data, obtained from direct injection of samples, indicated that the reactions were rich in the desired product. Since the ultra violet (UV) spectra of the desired product and possible impurities are very similar, liquid chromatography (LC) chromatographic data seemed to corroborate these results. However, when on-line LC-UV-MS was carried out and each LC peak subjected to MS analysis as it eluted, a very different picture emerged. It was found that many of these reactions actually contained high levels of the monoester product which, having less affinity for sodium in the MS, is therefore seriously underestimated in any direct injection assay. LC-diode array detection (DAD) methods were also used to help successfully identify and characterise the compounds being formed in these complex reactions. The overall results obtained in this paper allowed the optimal reaction conditions to be determined for this reaction. LC-MS analysis of the chromatographic peaks also identified the presence of two isomers of the diester substituted calix[4]arene (1,3 and 1,2 diesters). The combination of LC and UV/MS detection is required for accurate analysis of the products of such reactions.
Sarah, S A; Faradalila, W N; Salwani, M S; Amin, I; Karsani, S A; Sazili, A Q
2016-05-15
The purpose of this study was to identify porcine-specific peptide markers from thermally processed meat that could differentiate pork from beef, chevon and chicken meat. In the initial stage, markers from tryptic digested protein of chilled, boiled and autoclaved pork were identified using LC-QTOF-MS. An MRM method was then established for verification. A thorough investigation of LC-QTOF-MS data showed that only seven porcine-specific peptides were consistently detected. Among these peptides, two were derived from lactate dehydrogenase, one from creatine kinase, and four from serum albumin protein. However, MRM could only detect four peptides (EVTEFAK, LVVITAGAR, FVIER and TVLGNFAAFVQK) that were consistently present in pork samples. In conclusion, meat species determination through a tandem mass spectrometry platform shows high potential in providing scientifically valid and reliable results even at peptide level. Besides, the specificity and selectivity offered by the proteomics approach also provide a robust platform for Halal authentication. Copyright © 2015 Elsevier Ltd. All rights reserved.
Nirogi, Ramakrishna; Ajjala, Devender Reddy; Kandikere, Vishwottam; Aleti, Raghupathi; Pantangi, Hanumanth Rao; Srikakolapu, Surya Rao; Benade, Vijay; Bhyrapuneni, Gopinadh; Vurimindi, Himabindu
2013-01-01
A sensitive LC-MS/MS method was developed and validated for the quantification of almotriptan in rat brain and blood dialysates. Almotriptan is a 5HT1B/1D receptor agonist used for the treatment of migraine pain. Method consists of rapid gradient elution program with 10mM ammonium formate (pH 3) and acetonitrile on a Xbridge column. The MRM transitions monitored were m/z 336.2-58.1 for almotriptan and m/z 448.2-285.3 for the IS. The assay was linear in the range of 0.1-20 ng/ml, with acceptable precision and accuracy along with adequate sensitivity. The between batch accuracy was in the range of 99.0-104.3% with precision in between 0.6% and 5.8%. Microdialysis is an important sampling technique, with the capability of capturing the concentrations of various analytes in different bio fluids, at a single time point. This method was applied to quantify brain and blood dialysate samples obtained from a microdialysis study of rats treated with almotriptan (10mg/kg, p.o.). In vivo recovery experiments were performed to correct the dialysate concentrations into extracellular concentrations. Mean peak dialysate concentrations of almotriptan were found to be 152 ± 78 and 7.4 ± 1.0 ng/ml in blood and prefrontal cortex, respectively. The brain penetration of almotriptan is characterized by the AUCbrain/AUCblood found to be 0.07 ± 0.05. The results revealed the importance of measuring the unbound almotriptan concentrations in the brain over the blood for understanding its PK/PD relationship. Copyright © 2013 Elsevier B.V. All rights reserved.
Meng, Qingfang; Buchanan, Beth; Zuccolo, Jonathan; Poulin, Mathieu-Marc; Gabriele, Joseph; Baranowski, David Charles
2018-01-01
In the past 50 years, Cannabis sativa (C. sativa) has gone from a substance essentially prohibited worldwide to one that is gaining acceptance both culturally and legally in many countries for medicinal and recreational use. As additional jurisdictions legalize Cannabis products and the variety and complexity of these products surpass the classical dried plant material, appropriate methods for measuring the biologically active constituents is paramount to ensure safety and regulatory compliance. While there are numerous active compounds in C. sativa the primary cannabinoids of regulatory and safety concern are (-)-Δ⁹-tetrahydrocannabinol (THC), cannabidiol (CBD), and their respective acidic forms THCA-A and CBDA. Using the US Food and Drug Administration (FDA) bioanalytical method validation guidelines we developed a sensitive, selective, and accurate method for the simultaneous analysis CBD, CBDA, THC, and THCA-A in oils and THC & CBD in more complex matrices. This HPLC-MS/MS method was simple and reliable using standard sample dilution and homogenization, an isocratic chromatographic separation, and a triple quadrupole mass spectrometer. The lower limit of quantification (LLOQ) for analytes was 0.195 ng/mL over a 0.195-50.0 ng/mL range of quantification with a coefficient of correlation of >0.99. Average intra-day and inter-day accuracies were 94.2-112.7% and 97.2-110.9%, respectively. This method was used to quantify CBD, CBDA, THC, and THCA-A in 40 commercial hemp products representing a variety of matrices including oils, plant materials, and creams/cosmetics. All products tested met the federal regulatory restrictions on THC content in Canada (<10 μg/g) except two, with concentrations of 337 and 10.01 μg/g. With respect to CBD, the majority of analyzed products contained low CBD levels and a CBD: CBDA ratio of <1.0. In contrast, one product contained 8,410 μg/g CBD and a CBD: CBDA ratio of >1,000 (an oil-based product). Overall, the method proved
Meng, Qingfang; Buchanan, Beth; Zuccolo, Jonathan; Poulin, Mathieu-Marc; Gabriele, Joseph
2018-01-01
In the past 50 years, Cannabis sativa (C. sativa) has gone from a substance essentially prohibited worldwide to one that is gaining acceptance both culturally and legally in many countries for medicinal and recreational use. As additional jurisdictions legalize Cannabis products and the variety and complexity of these products surpass the classical dried plant material, appropriate methods for measuring the biologically active constituents is paramount to ensure safety and regulatory compliance. While there are numerous active compounds in C. sativa the primary cannabinoids of regulatory and safety concern are (-)-Δ⁹-tetrahydrocannabinol (THC), cannabidiol (CBD), and their respective acidic forms THCA-A and CBDA. Using the US Food and Drug Administration (FDA) bioanalytical method validation guidelines we developed a sensitive, selective, and accurate method for the simultaneous analysis CBD, CBDA, THC, and THCA-A in oils and THC & CBD in more complex matrices. This HPLC-MS/MS method was simple and reliable using standard sample dilution and homogenization, an isocratic chromatographic separation, and a triple quadrupole mass spectrometer. The lower limit of quantification (LLOQ) for analytes was 0.195 ng/mL over a 0.195–50.0 ng/mL range of quantification with a coefficient of correlation of >0.99. Average intra-day and inter-day accuracies were 94.2–112.7% and 97.2–110.9%, respectively. This method was used to quantify CBD, CBDA, THC, and THCA-A in 40 commercial hemp products representing a variety of matrices including oils, plant materials, and creams/cosmetics. All products tested met the federal regulatory restrictions on THC content in Canada (<10 μg/g) except two, with concentrations of 337 and 10.01 μg/g. With respect to CBD, the majority of analyzed products contained low CBD levels and a CBD: CBDA ratio of <1.0. In contrast, one product contained 8,410 μg/g CBD and a CBD: CBDA ratio of >1,000 (an oil-based product). Overall, the method proved
Tang, Dao-quan; Zheng, Xiao-xiao; Li, Yin-jie; Bian, Ting-ting; Yu, Yan-yan; Du, Qian; Yang, Dong-zhi; Jiang, Shui-shi
2014-11-01
In this study, two independent and complementary liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods were respectively developed and validated for the determination of edaravone or taurine in rat urine, feces and bile after intravenous administration, using 3-methyl-l-p-tolyl-5-pyrazolone and sulfanilic acid as the internal standards (IS). Edaravone was separated on an Agilent Eclipse Plus C18 column (100×2.1 mm, 3.5 μm) using methanol and water (containing 5 mM ammonium formate and 0.02% formic acid) as mobile phase, while taurine was performed on a Waters Atlantis HILIC Silica column (150×2.1 mm, 3 μm) using acetonitrile and water (containing 5mM ammonium formate and 0.2% formic acid) as mobile phase. The mass analysis was performed in a Triple Quadrupole mass spectrometer via multiple reaction monitoring (MRM) with negative ionization mode. The optimized mass transition ion pairs (m/z) for quantification were 173.1→92.2 and 187.2→106.0 for edaravone and its IS, 124.1→80.0 and 172.0→80.0 for taurine and its IS, respectively. The validated methods have been successfully applied to the excretion and metabolism interaction study of edaravone and taurine in rats after independent intravenous administration and co-administration with a single dose. The results demonstrated that there were no significant alternations on the metabolism and cumulative excretion rate of edaravone and taurine, implying that the proposed combination therapy was pharmacologically viable. Copyright © 2014 Elsevier B.V. All rights reserved.
An improved brine shrimp larvae lethality microwell test method.
Zhang, Yi; Mu, Jun; Han, Jinyuan; Gu, Xiaojie
2012-01-01
This article described an improved brine shrimp larvae lethality microwell test method. A simply designed connecting vessel with alternative photoperiod was used to culture and collect high yield of active Artemia parthenogenetica nauplii for brine shrimp larvae lethality microwell test. Using this method, pure A. parthenogenetica nauplii suspension was easily cultured and harvested with high density about 100-150 larvae per milliliter and the natural mortality was reduced to near zero by elimination of unnecessary artificial disturbance. And its sensitivity was validated by determination of LC(50)-24 h of different reference toxicants including five antitumor agents, two pesticides, three organic pollutants, and four heavy metals salts, most of which exhibited LC(50)-24 h between 0.07 and 58.43 mg/L except for bleomycin and mitomycin C with LC(50)-24 h over 300 mg/L.
Bernardy, Jeffry A.; Hubert, Terrance D.; Ogorek, Jacob M.; Schmidt, Larry J.
2013-01-01
An LC/MS method was developed and validated for the quantitative determination and confirmation of antimycin-A (ANT-A) in water from lakes or streams. Three different water sample volumes (25, 50, and 250 mL) were evaluated. ANT-A was stabilized in the field by immediately extracting it from water into anhydrous acetone using SPE. The stabilized concentrated samples were then transported to a laboratory and analyzed by LC/MS using negative electrospray ionization. The method was determined to have adequate accuracy (78 to 113% recovery), precision (0.77 to 7.5% RSD with samples ≥500 ng/L and 4.8 to 17% RSD with samples ≤100 ng/L), linearity, and robustness over an LOQ range from 8 to 51 600 ng/L.
Bernardy, Jeffry A; Hubert, Terrance D; Ogorek, Jacob M; Schmidt, Larry J
2013-01-01
An LC/MS method was developed and validated for the quantitative determination and confirmation of antimycin-A (ANT-A) in water from lakes or streams. Three different water sample volumes (25, 50, and 250 mL) were evaluated. ANT-A was stabilized in the field by immediately extracting it from water into anhydrous acetone using SPE. The stabilized concentrated samples were then transported to a laboratory and analyzed by LC/MS using negative electrospray ionization. The method was determined to have adequate accuracy (78 to 113% recovery), precision (0.77 to 7.5% RSD with samples > or = 500 ng/L and 4.8 to 17% RSD with samples < or = 100 ng/L), linearity, and robustness over an LOQ range from 8 to 51 600 ng/L.
Granja, Rodrigo H M M; Nino, Alfredo M M; Reche, Karine V G; Giannotti, Fabio M; de Lima, Andreia C; Wanschel, Amarylis C B A; Salerno, Alessandro G
2013-01-01
Nitroimidazoles are a class of veterinary drugs used for the treatment and prevention of certain bacterial and protozoal diseases in poultry, swine dysentery and genital trichomoniasis in cattle. Since the safety assessment of nitroimidazoles showed them to be genotoxic, carcinogenic and mutagenic, a number of nitroimidazoles have been banned for therapeutic purposes in different countries. Moreover, nitroimidazoles have also been extensively used as growth promoters in food-producing animals. Due to their efficacious improvement in meat production and feed conversion, deliberate use still exists. Therefore, the illegal use of nitroimidazoles in animal husbandry must be monitored. A sensitive method based on LC-MS/MS for the simultaneous determination and confirmation of five banned nitroimidazole drugs including metronidazole, ronidazole, dimetridazole, metronidazole-OH (metabolite of metronidazole), and 2-hydroxymethyl-1-methyl-5-nitroimidazole (metabolite of ronidazole and dimetridazole) in bovine muscle, using ronidazole-d3 as an internal standard, was developed and validated. After extraction with ethyl acetate and evaporation, the nitroimidazoles were reconstituted in petroleum ether and purified, and LC-MS/MS analysis was performed. The method was validated according to Brazilian Regulation 24/2009 (equivalent to European Union Decision 2002/657/EC). Parameters such as decision limit (CCα), detection capability (CCβ), precision, accuracy, uncertaincy and ruggedness were determined. Average accuracy of the five nitroimidazoles from bovine muscle fortified at 5 levels (0.5, 1.0, 1.5, 2.0 and 2.5 μg kg(-1)) ranged from 96% to 103%. The calculated CCα ranged from 0.0 to 0.17 μg kg(-1); CCβ ranged from 0.08 to 0.41 μg kg(-1). A complete statistical analysis was performed and the results indicate that the method is robust when subjected to day-to-day analytical variations.
Abdelhameed, Ali S; Attwa, Mohamed W; Kadi, Adnan A
2017-02-01
A reliable, high-throughput and sensitive LC-MS/MS procedure was developed and validated for the determination of five tyrosine kinase inhibitors in human plasma. Following their extraction from human plasma, samples were eluted on a RP Luna®-PFP 100 Å column using a mobile phase system composed of acetonitrile and 0.01 m ammonium formate in water (pH ~4.1) with a ratio of (50:50, v/v) flowing at 0.3 mL min -1 . The mass spectrometer was operating with electrospray ionization in the positive ion multiple reaction monitoring mode. The proposed methodology resulted in linear calibration plots with correlation coefficients values of r 2 = 0.9995-0.9999 from concentration ranges of 2.5-100 ng mL -1 for imatinib, 5.0-100 ng mL -1 for sorafenib, tofacitinib and afatinib, and 1.0-100 ng mL -1 for cabozantinib. The procedure was validated in terms of its specificity, limit of detection (0.32-1.71 ng mL -1 ), lower limit of quantification (0.97-5.07 ng mL -1 ), intra- and inter assay accuracy (-3.83 to +2.40%) and precision (<3.37%), matrix effect and recovery and stability. Our results demonstrated that the proposed method is highly reliable for routine quantification of the investigated tyrosine kinase inhibitors in human plasma and can be efficiently applied in the rapid and sensitive analysis of their clinical samples. Copyright © 2016 John Wiley & Sons, Ltd.
Stephanson, N N; Signell, P; Helander, A; Beck, O
2017-08-01
The influx of new psychoactive substances (NPS) has created a need for improved methods for drug testing in toxicology laboratories. The aim of this work was to design, validate and apply a multi-analyte liquid chromatography-high-resolution mass spectrometry (LC-HRMS) method for screening of 148 target analytes belonging to the NPS class, plant alkaloids and new psychoactive therapeutic drugs. The analytical method used a fivefold dilution of urine with nine deuterated internal standards and injection of 2 μl. The LC system involved a 2.0 μm 100 × 2.0 mm YMC-UltraHT Hydrosphere-C 18 column and gradient elution with a flow rate of 0.5 ml/min and a total analysis time of 6.0 min. Solvent A consisted of 10 mmol/l ammonium formate and 0.005% formic acid, pH 4.8, and Solvent B was methanol with 10 mmol/l ammonium formate and 0.005% formic acid. The HRMS (Q Exactive, Thermo Scientific) used a heated electrospray interface and was operated in positive mode with 70 000 resolution. The scan range was 100-650 Da, and data for extracted ion chromatograms used ± 10 ppm tolerance. Product ion monitoring was applied for confirmation analysis and for some selected analytes also for screening. Method validation demonstrated limited influence from urine matrix, linear response within the measuring range (typically 0.1-1.0 μg/ml) and acceptable imprecision in quantification (CV <15%). A few analytes were found to be unstable in urine upon storage. The method was successfully applied for routine drug testing of 17 936 unknown samples, of which 2715 (15%) contained 52 of the 148 analytes. It is concluded that the method design based on simple dilution of urine and using LC-HRMS in extracted ion chromatogram mode may offer an analytical system for urine drug testing that fulfils the requirement of a 'black box' solution and can replace immunochemical screening applied on autoanalyzers. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.
Durden, David A
2007-05-01
Avermectin endectocides are used for the treatment of cattle against a variety of nematode and arthropod parasites, and consequently may appear in milk after normal or off-label use. The compounds abamectin, doramectin, and ivermectin, contain only C, H and O and may be expected to be detected by LC-MS in negative ion mode. The others contain nitrogen in addition and would be expected to be preferentially ionized in positive mode. The use of positive ion and negative ion methods with electrospray LC-MS-MS were compared. Using negative ion the compounds abamectin, doramectin, ivermectin, emamectin, eprinomectin, and moxidectin gave a curvilinear response and were quantified in raw milk by LC-MS-MS with a triethylamine-acetonitrile buffer over the concentration range 1-60 ppb (microg/kg) using selamectin as the internal standard. The limits of detection (LOD) were between 0.19 ppb (doramectin) and 0.38 ppb (emamectin). The compounds gave maximum sensitivity with positive ionisation from a formic acid-ammonium formate-acetonitrile buffer and were detected in milk (LC-MS-MS) also with a curvilinear response over the range 0.5-60 ppb. Although the positive ion signals were larger, with somewhat lower limits of detection (LOD between 0.06 ppb (doramectin) and 0.32 ppb (moxidectin) the negative ion procedure gave a more linear response and more consistent results. Comparison of spiked samples in the range 2-50 ppb showed a high degree of correlation between the two methods.
Bateman, J; Proctor, M; Buchnev, O; Podoliak, N; D'Alessandro, G; Kaczmarek, M
2014-07-01
The voltage transfer function is a rapid and visually effective method to determine the electrical response of liquid crystal (LC) systems using optical measurements. This method relies on crosspolarized intensity measurements as a function of the frequency and amplitude of the voltage applied to the device. Coupled with a mathematical model of the device it can be used to determine the device time constants and electrical properties. We validate the method using photorefractive LC cells and determine the main time constants and the voltage dropped across the layers using a simple nonlinear filter model.
Analytical difficulties facing today's regulatory laboratories: issues in method validation.
MacNeil, James D
2012-08-01
The challenges facing analytical laboratories today are not unlike those faced in the past, although both the degree of complexity and the rate of change have increased. Challenges such as development and maintenance of expertise, maintenance and up-dating of equipment, and the introduction of new test methods have always been familiar themes for analytical laboratories, but international guidelines for laboratories involved in the import and export testing of food require management of such changes in a context which includes quality assurance, accreditation, and method validation considerations. Decisions as to when a change in a method requires re-validation of the method or on the design of a validation scheme for a complex multi-residue method require a well-considered strategy, based on a current knowledge of international guidance documents and regulatory requirements, as well the laboratory's quality system requirements. Validation demonstrates that a method is 'fit for purpose', so the requirement for validation should be assessed in terms of the intended use of a method and, in the case of change or modification of a method, whether that change or modification may affect a previously validated performance characteristic. In general, method validation involves method scope, calibration-related parameters, method precision, and recovery. Any method change which may affect method scope or any performance parameters will require re-validation. Some typical situations involving change in methods are discussed and a decision process proposed for selection of appropriate validation measures. © 2012 John Wiley & Sons, Ltd.
Chen, Xinhui; Bushman, Lane R; McAllister, Kevin J; Anderson, Peter L; Kiser, Jennifer J
2014-12-01
The purpose of this study was to validate a reversed-phase high-performance liquid chromatographic (HPLC), tandem mass spectrometry (MS/MS) assay for the determination of telaprevir and its R-diastereomer (VRT-127394) in acidified and nonacidified human plasma. The chromatographic baseline separation of telaprevir and telaprevir-R was performed on a Waters XBridge(TM) BEH Shield C18 , 2.1 × 75 mm column with a 2.5 µm particle size, under isocratic conditions consisting of a mobile phase of 50:45:5 water-acetonitrile-isopropanol with 1% ammonia at 0.2 mL/min. This method utilized a stable isotope internal standard with 11 deuterium atoms on the structure of the telaprevir molecule (telaprevir-d11). An internal standard for the telaprevir-R (telaprevir-R-d11) was also prepared by incubating telaprevir-d11 in basic solution, which facilitated isomer inter-conversion. The detection and quantitation of telaprevir, telaprevir-R, telaprevir-IS and telaprevir-R-IS was achieved by positive ion electrospray (ESI+) MS/MS detection. The assay quantifiable limit was 5.0 ng/mL when 0.100 mL of acidified human plasma was extracted. Accuracy and precision were validated over the calibration range of 5.0-5000 ng/mL. It was demonstrated using patient samples that, contrary to previous recommendations, quantitation of telaprevir does not require acidified plasma. Copyright © 2014 John Wiley & Sons, Ltd.
Miller, Eleanor I; Wylie, Fiona M; Oliver, John S
2008-09-01
A liquid chromatography-tandem mass spectrometry method was developed and validated for the simultaneous identification and quantification of amphetamines, diazepam and its metabolites, cocaine and its metabolites, and opiates from hair using a single extraction method. As part of the method development, Gemini C18, Synergi Hydro RP, and Zorbax Stablebond-Phenyl LC columns were tested with three different mobile phases. Analyte recovery and limit of detection were evaluated for two different solid-phase extraction methods that used Bond Elut Certify and Clean Screen cartridges. Phosphate buffer (pH 5.0) was chosen as the optimum hair incubation medium because of the high stability of cocaine and 6-monoacetylmorphine using this method and faster sample preparation. The optimized method was fully validated. Linearity was established over the concentration range 0.2-10 ng/mg hair, and the correlation coefficients were all greater than 0.99. Total extraction recoveries were greater than 76%, detection limits were between 0.02 and 0.09 ng/mg, and the intra- and interday imprecisions were generally less than 20% in spiked hair. The intra- and interbatch imprecision of the method for a pooled authentic hair sample ranged from 1.4 to 23.4% relative standard deviation (RSD) and 8.3 to 25.4% RSD, respectively, for representative analytes from the different drug groups. The percent matrix effect ranged from 63.5 to 135.6%, with most analytes demonstrating ion suppression. Sixteen postmortem samples collected from suspected drug-related deaths were analyzed for the 17 drugs of abuse and metabolites included in the method. The method was sufficiently sensitive and specific for the analysis of drugs and metabolites in postmortem hair samples. There is scope for the inclusion of other target drugs and metabolites in the method.
Hubert, C; Houari, S; Rozet, E; Lebrun, P; Hubert, Ph
2015-05-22
When using an analytical method, defining an analytical target profile (ATP) focused on quantitative performance represents a key input, and this will drive the method development process. In this context, two case studies were selected in order to demonstrate the potential of a quality-by-design (QbD) strategy when applied to two specific phases of the method lifecycle: the pre-validation study and the validation step. The first case study focused on the improvement of a liquid chromatography (LC) coupled to mass spectrometry (MS) stability-indicating method by the means of the QbD concept. The design of experiments (DoE) conducted during the optimization step (i.e. determination of the qualitative design space (DS)) was performed a posteriori. Additional experiments were performed in order to simultaneously conduct the pre-validation study to assist in defining the DoE to be conducted during the formal validation step. This predicted protocol was compared to the one used during the formal validation. A second case study based on the LC/MS-MS determination of glucosamine and galactosamine in human plasma was considered in order to illustrate an innovative strategy allowing the QbD methodology to be incorporated during the validation phase. An operational space, defined by the qualitative DS, was considered during the validation process rather than a specific set of working conditions as conventionally performed. Results of all the validation parameters conventionally studied were compared to those obtained with this innovative approach for glucosamine and galactosamine. Using this strategy, qualitative and quantitative information were obtained. Consequently, an analyst using this approach would be able to select with great confidence several working conditions within the operational space rather than a given condition for the routine use of the method. This innovative strategy combines both a learning process and a thorough assessment of the risk involved
Esterhuizen-Londt, Maranda; Schwartz, Katrin; Balsano, Evelyn; Kühn, Sandra; Pflugmacher, Stephan
2016-06-01
Acetaminophen is a pharmaceutical, frequently found in surface water as a contaminant. Bioremediation, in particular, mycoremediation of acetaminophen is a method to remove this compound from waters. Owing to the lack of quantitative analytical method for acetaminophen in aquatic organisms, the present study aimed to develop a method for the determination of acetaminophen using LC-MS/MS in the aquatic fungus Mucor hiemalis. The method was then applied to evaluate the uptake of acetaminophen by M. hiemalis, cultured in pellet morphology. The method was robust, sensitive and reproducible with a lower limit of quantification of 5 pg acetaminophen on column. It was found that M. hiemalis internalize the pharmaceutical, and bioaccumulate it with time. Therefore, M. hiemalis was deemed a suitable candidate for further studies to elucidate its pharmaceutical tolerance and the longevity in mycoremediation applications. Copyright © 2016 Elsevier Inc. All rights reserved.
Determination of Acid Herbicides Using Modified QuEChERS with Fast Switching ESI(+)/ESI(-) LC-MS/MS.
Sack, Chris; Vonderbrink, John; Smoker, Michael; Smith, Robert E
2015-11-04
A method for the determination of 35 acid herbicides in food matrices was developed, validated, and implemented. It utilizes a modified QuEChERS extraction procedure coupled with quantitation by liquid chromatography tandem mass spectrometry (LC-MS/MS). The acid herbicides analyzed are all organic carboxylic acids, including the older chlorophenoxy acid herbicides such as 2,4-dichlorophenoxyacetic acid (2,4-D), dicamba, 4-chlorophenoxyacetic acid (4-CPA), quinclorac, and many of the newer imidazolinone herbicides such as imazethapyr and imazaquin. In the procedure, 10 mL of water is added to 5 g of sample and then extracted with 1% formic acid in acetonitrile for 1 min. The acetonitrile phase is salted out of the extract by adding sodium chloride and magnesium sulfate, followed by centrifugation. The acetonitrile is diluted 1:1 with water to enable quantitation by LC-MS/MS using fast switching between positive and negative electrospray ionization modes. The average recoveries for all the compounds except aminocyclopyrachlor were 95% with a precision of 8%. The method detection limits for all residues were less than 10 ng/g, and the correlation coefficients for the calibration curves was greater than 0.99 for all but two compounds tested. The method was used successfully for the quantitation of acid herbicides in the FDA's total diet study. The procedure proved to be accurate, precise, linear, sensitive, and rugged.
New 3D-printed sorbent for extraction of steroids from human plasma preceding LC-MS analysis.
Konieczna, Lucyna; Belka, Mariusz; Okońska, Magdalena; Pyszka, Magdalena; Bączek, Tomasz
2018-04-13
In recent years, there has been an increasing worldwide interest in the use of alternative sample preparation methods that are proceeded by separation techniques. Fused deposition modeling (FDM) is a 3D printing technique that is based the consecutive layering of softened/melted thermoplastic materials. In this study, a group of natural steroids and sexual hormones - namely, aldosterone, cortisol, β-estradiol, testosterone, dihydrotestosterone, and synthetic methyltestosterone and betamethasone - were separated and determined using an optimized high-performance liquid chromatography coupled to mass spectrometry (LC-MS) method in positive ionization mode. 3D-printed sorbents were selected as the pre-concentration technique because they are generally low cost, fast, and simple to make and automate. Furthermore, the use of 3D-printed sorbents helps to minimize potential errors due to their repeatability and reproducibility, and their ability to eliminate carry over by using one printed sorbent for a single extraction of steroids from biological matrices. The extraction procedure was optimized and the parameters influencing 3D-printed Layfomm 60 ® based sorbent and LC-MS were studied, including the type of extraction solvent used, sorption and desorption times, temperature, and the salting-out effect. To demonstrate this method's applicability for biological sample analysis, the SPME-LC-MS method was validated for its ability to simultaneously quantify endogenous steroids. This evaluation confirmed good linearity and an R 2 that was between 0.9970 and 0.9990. The recovery rates for human plasma samples were 86.34-93.6% for the studied steroids with intra- and inter-day RSDs of 1.44-7.42% and 1.44-9.46%, respectively. To our knowledge, this study is the first time that 3D-printed sorbents have been used to extract trace amounts of endogenous low-molecular-weight compounds, such as steroids, from biological samples, such as plasma. Copyright © 2018 Elsevier B.V. All
Faassen, Elisabeth J.; Antoniou, Maria G.; Beekman-Lukassen, Wendy; Blahova, Lucie; Chernova, Ekaterina; Christophoridis, Christophoros; Combes, Audrey; Edwards, Christine; Fastner, Jutta; Harmsen, Joop; Hiskia, Anastasia; Ilag, Leopold L.; Kaloudis, Triantafyllos; Lopicic, Srdjan; Lürling, Miquel; Mazur-Marzec, Hanna; Meriluoto, Jussi; Porojan, Cristina; Viner-Mozzini, Yehudit; Zguna, Nadezda
2016-01-01
Exposure to β-N-methylamino-l-alanine (BMAA) might be linked to the incidence of amyotrophic lateral sclerosis, Alzheimer’s disease and Parkinson’s disease. Analytical chemistry plays a crucial role in determining human BMAA exposure and the associated health risk, but the performance of various analytical methods currently employed is rarely compared. A CYANOCOST initiated workshop was organized aimed at training scientists in BMAA analysis, creating mutual understanding and paving the way towards interlaboratory comparison exercises. During this workshop, we tested different methods (extraction followed by derivatization and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) analysis, or directly followed by LC-MS/MS analysis) for trueness and intermediate precision. We adapted three workup methods for the underivatized analysis of animal, brain and cyanobacterial samples. Based on recovery of the internal standard D3BMAA, the underivatized methods were accurate (mean recovery 80%) and precise (mean relative standard deviation 10%), except for the cyanobacterium Leptolyngbya. However, total BMAA concentrations in the positive controls (cycad seeds) showed higher variation (relative standard deviation 21%–32%), implying that D3BMAA was not a good indicator for the release of BMAA from bound forms. Significant losses occurred during workup for the derivatized method, resulting in low recovery (<10%). Most BMAA was found in a trichloroacetic acid soluble, bound form and we recommend including this fraction during analysis. PMID:26938542
Faassen, Elisabeth J; Antoniou, Maria G; Beekman-Lukassen, Wendy; Blahova, Lucie; Chernova, Ekaterina; Christophoridis, Christophoros; Combes, Audrey; Edwards, Christine; Fastner, Jutta; Harmsen, Joop; Hiskia, Anastasia; Ilag, Leopold L; Kaloudis, Triantafyllos; Lopicic, Srdjan; Lürling, Miquel; Mazur-Marzec, Hanna; Meriluoto, Jussi; Porojan, Cristina; Viner-Mozzini, Yehudit; Zguna, Nadezda
2016-02-29
Exposure to β-N-methylamino-l-alanine (BMAA) might be linked to the incidence of amyotrophic lateral sclerosis, Alzheimer's disease and Parkinson's disease. Analytical chemistry plays a crucial role in determining human BMAA exposure and the associated health risk, but the performance of various analytical methods currently employed is rarely compared. A CYANOCOST initiated workshop was organized aimed at training scientists in BMAA analysis, creating mutual understanding and paving the way towards interlaboratory comparison exercises. During this workshop, we tested different methods (extraction followed by derivatization and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) analysis, or directly followed by LC-MS/MS analysis) for trueness and intermediate precision. We adapted three workup methods for the underivatized analysis of animal, brain and cyanobacterial samples. Based on recovery of the internal standard D₃BMAA, the underivatized methods were accurate (mean recovery 80%) and precise (mean relative standard deviation 10%), except for the cyanobacterium Leptolyngbya. However, total BMAA concentrations in the positive controls (cycad seeds) showed higher variation (relative standard deviation 21%-32%), implying that D₃BMAA was not a good indicator for the release of BMAA from bound forms. Significant losses occurred during workup for the derivatized method, resulting in low recovery (<10%). Most BMAA was found in a trichloroacetic acid soluble, bound form and we recommend including this fraction during analysis.
Reduction of Solvent Effect in Reverse Phase Gradient Elution LC-ICP-MS
DOE Office of Scientific and Technical Information (OSTI.GOV)
Sullivan, Patrick Allen
2005-12-17
Quantification in liquid chromatography (LC) is becoming very important as more researchers are using LC, not as an analytical tool itself, but as a sample introduction system for other analytical instruments. The ability of LC instrumentation to quickly separate a wide variety of compounds makes it ideal for analysis of complex mixtures. For elemental speciation, LC is joined with inductively coupled plasma mass spectrometry (ICP-MS) to separate and detect metal-containing, organic compounds in complex mixtures, such as biological samples. Often, the solvent gradients required to perform complex separations will cause matrix effects within the plasma. This limits the sensitivity ofmore » the ICP-MS and the quantification methods available for use in such analyses. Traditionally, isotope dilution has been the method of choice for LC-ICP-MS quantification. The use of naturally abundant isotopes of a single element in quantification corrects for most of the effects that LC solvent gradients produce within the plasma. However, not all elements of interest in speciation studies have multiple naturally occurring isotopes; and polyatomic interferences for a given isotope can develop within the plasma, depending on the solvent matrix. This is the case for reverse phase LC separations, where increasing amounts of organic solvent are required. For such separations, an alternative to isotope dilution for quantification would be is needed. To this end, a new method was developed using the Apex-Q desolvation system (ESI, Omaha, NE) to couple LC instrumentation with an ICP-MS device. The desolvation power of the system allowed greater concentrations of methanol to be introduced to the plasma prior to destabilization than with direct methanol injection into the plasma. Studies were performed, using simulated and actual linear methanol gradients, to find analyte-internal standard (AIS) pairs whose ratio remains consistent (deviations {+-} 10%) over methanol concentration ranges
Evaluation of Acrylamide in Food from China by a LC/MS/MS Method
Chen, Yong-Hong; Xia, En-Qin; Xu, Xiang-Rong; Ling, Wen-Hua; Li, Sha; Wu, Shan; Deng, Gui-Fang; Zou, Zhi-Fei; Zhou, Jing; Li, Hua-Bin
2012-01-01
Acrylamide is potential carcinogenic compound that possesses neurotoxicity activity. In this study, the levels of acrylamide in 123 selected food samples from China was evaluated using a LC/MS/MS method. One hundred and fifteen (115) out of 123 samples showed positive levels of acrylamide in the range of 0.41 to 4,126.26 µg/kg. Generally, the highest acrylamide levels were found in fried products, such as potato, prawn strips and rice crust, with average values of 604.27, 341.40, and 201.51 µg/kg, respectively. Heated protein-rich food also showed some acrylamide content (ranging from 2.31 to 78.57 µg/kg). The results revealed that a potential acrylamide public health risk occurred in processed snacks, as well as the food consumed daily. This study supplied new information on acrylamide content of a variety of heat-treated foods from China. PMID:23202837
LC-MS characterization of constituents of mesquite flour
USDA-ARS?s Scientific Manuscript database
Using an LC-MS method in conjunction with two complementary types of chromatographic retention modes—namely reversed phase and aqueous normal phase (ANP)—various compounds present in mesquite flour extracts were identified. Because of the diverse types of chemical constituents found in such natural ...
LC-MS/MS assay for the quantitation of the ATR kinase inhibitor VX-970 in human plasma.
Kiesel, Brian F; Scemama, Jonas; Parise, Robert A; Villaruz, Liza; Iffland, Andre; Doyle, Austin; Ivy, Percy; Chu, Edward; Bakkenist, Christopher J; Beumer, Jan H
2017-11-30
DNA damaging chemotherapy and radiation are widely used standard-of-care modalities for the treatment of cancer. Nevertheless, the outcome for many patients remains poor and this may be attributed, at least in part, to highly effective DNA repair mechanisms. Ataxia-telangiectasia mutated and Rad3-related (ATR) is a key regulator of the DNA-damage response (DDR) that orchestrates the repair of damaged replication forks. ATR is a serine/threonine protein kinase and ATR kinase inhibitors potentiate chemotherapy and radiation. The ATR kinase inhibitor VX-970 (NSC 780162) is in clinical development in combination with primary cytotoxic agents and as a monotherapy for tumors harboring specific mutations. We have developed and validated an LC-MS/MS assay for the sensitive, accurate and precise quantitation of VX-970 in human plasma. A dilute-and-shoot method was used to precipitate proteins followed by chromatographic separation with a Phenomenex Polar-RP 80Å (4μm, 50×2mm) column and a gradient acetonitrile-water mobile phase containing 0.1% formic acid from a 50μL sample volume. Detection was achieved using an API 4000 mass spectrometer using electrospray positive ionization mode. The assay was linear from 3 to 5,000ng/mL, proved to be accurate (94.6-104.2%) and precise (<8.4% CV), and fulfilled criteria from the FDA guidance for bioanalytical method validation. This LC-MS/MS assay will be a crucial tool in defining the clinical pharmacokinetics and pharmacology of VX-970 as it progresses through clinical development. Copyright © 2017 Elsevier B.V. All rights reserved.
Dong, Xiaona; Zhang, Yiwei; Meng, Zhiyun; Zhu, Xiaoxia; Gan, Hui; Gu, Ruolan; Wu, Zhuona; Li, Jian; Zheng, Ying; Yang, Baofeng; Dou, Guifang
2018-05-29
HYD-PEP06 is a novel RGD-modified Endostar mimetic peptide with 30 amino acids that is intended to suppress the formation of neoplasm vessels. This assay was developed and validated to monitor the level of the peptide HYD-PEP06 in rat blood, using liquid chromatography tandem mass spectrometry (LC-MS/MS). HYD-PEP10, another peptide similar to the analyte, was used as an internal standard (IS). A triple quadrupole mass spectrometry in Multiple Reaction Monitoring (MRM) mode and an electrospray interface (ESI) in the positive mode were used for MS analysis. The analysis was optimized with addition of 0.3% formic acid (FA) into the mobile phase as well as with a needle washing solution to overcome the carryover effect. In addition, the carryover was reduced by optimizing the mobile phase gradient. Methanol was used as a diluent of working solutions to avoid any adsorption. Methanol:acetonitrile (1:1, v:v) containing 0.3% FA was employed to precipitate the blood samples. Unknown blood samples must be placed in ice bath immediately, and precipitating agents should be added within 30 min to ensure the stability of blood samples. The assay was established and validated. This method showed a good linear relationship for the HYD-PEP06 in the range of 10 ng·mL -1 to 2000 ng·mL -1 , with R > 0.99. HYD-PEP06 was determined with accuracy values (RE%) of -5.06%-8.54%, intra- and inter-day precisions (RSD%) of 3.13%-4.87% and 4.81%-9.42%. The method was successfully in monitoring the concentration of HYD-PEP06 in rat blood. Copyright © 2018 Elsevier B.V. All rights reserved.
Kakde, Rajendra B; Satone, Dinesh D; Gadapayale, Kamalesh K; Kakde, Megha G
2013-07-01
The objective of the current study was to develop a validated, specific stability-indicating reversed-phase liquid chromatographic (LC) method for the quantitative determination of escitalopram oxalate and clonazepam and their related substances in bulk drugs and pharmaceutical dosage forms in the presence of degradation products. Forced degradation studies were performed on the pure drugs of escitalopram oxalate and clonazepam, as per the stress conditions prescribed by the International Conference on Harmonization (ICH) using acid, base, oxidation, thermal stress and photolytic degradation to show the stability-indicating power of the method. Significant degradation was observed during acid and alkaline hydrolysis and no degradation was observed in other stress conditions. The chromatographic method was optimized using the samples generated from forced degradation studies. Good resolution between the peaks corresponded to the active pharmaceutical ingredients, escitalopram oxalate and clonazepam, and degradation products from the analyte were achieved on an ODS Hypersil C18 column (250 × 4.6 mm) using a mobile phase consisting of a mixture of acetonitrile-50 mM phosphate buffer + 10 mM triethylamine (70:30, v/v). The detection was conducted at 268 nm. The limit of detection and the limit of quantitation for escitalopram oxalate and clonazepam were established. The stress test solutions were assayed against the qualified working standards of escitalopram oxalate and clonazepam, which indicated that the developed LC method was stability-indicating. Validation of the developed LC method was conducted as per ICH requirements. The developed LC method was found to be suitable to check the quality of bulk samples of escitalopram oxalate and clonazepam.
Bock, C; Stachel, C; Gowik, P
2007-03-14
A method for the detection and determination of nitrofuran derivatives in egg by liquid chromatography-tandem mass spectrometry (LC-MS/MS) was validated with the software InterVal and can be applied for the confirmation of nitrofuran metabolites in fresh or lyophilised eggs. The validation study comprises variations in operator, storage condition, breeding, equipment and duration of sample preparation. A comprehensive overview of the robustness of the method is obtained by analysing eight samples at six concentration levels. First results of short- and medium-term investigations for stability of analytes in solution show that standard solutions of nitrofuran metabolites are stable for at least 1 year when stored at +4 degrees C in the dark. The decision limit CCalpha expressed for the underivatised metabolite is 0.05 microg kg(-1) for 3-amino-5-methyl-morpholino-2-oxazolidinone, 0.03 microg kg(-1) for 3-amino-2-oxazolidinone, 0.20 microg kg(-1) for semicarbazide and 0.22 microg kg(-1) for 1-amino-hydantoin.
Parsons, Teresa L.; Emory, Joshua F.; Seserko, Lauren A.; Aung, Wutyi S.; Marzinke, Mark A.
2014-01-01
Background Topical microbicidal agents are being actively pursued as a modality to prevent HIV viral transmission during sexual intercourse. Quantification of antiretroviral agents in specimen sources where antiviral activity is elicited is critical, and drug measurements in cervicovaginal fluid can provide key information on local drug concentrations. Two antiretroviral drugs, dapivirine and maraviroc, have gained interest as vaginal microbicidal agents, and rugged methods are required for their quantification in cervicovaginal secretions. Methods Cervicovaginal fluid spiked with dapivirine and maraviroc were applied to ophthalmic tear strips or polyester-based swabs to mimic collection procedures used in clinical studies. Following sample extraction and the addition of isotopically-labeled internal standards, samples were subjected to liquid chromatographic-tandem mass spectrometric (LC-MS/MS) analysis using a Waters BEH C8, 50 × 2.1 mm, 1.7 µm particle size column, on an API 4000 mass analyzer operated in selective reaction monitoring mode. The method was validated according to FDA Bioanalytical Method Validation guidelines. Results Due to the disparate saturation capacity of the tested collection devices, the analytical measuring ranges for dapivirine and maravirocin cervicovaginal fluid on the ophthalmic tear strip were 0.05 to 25 ng/tear strip, and 0.025 to 25 ng/tear strip, respectively. As for the polyester-based swab, the analytical measuring ranges were 0.25 to 125 ng/swab for dapivirine and 0.125 to 125 ng/swab for maraviroc. Dilutional studies were performed for both analytes to extended ranges of 25,000 ng/tear strip and 11,250 ng/swab. Standard curves were generated via weighted (1/x2) linear or quadratic regression of calibrators. Precision, accuracy, stability and matrix effects studies were all performed and deemed acceptable according to the recommendations of the FDA Bioanalytical Method Validation guidelines. Conclusions A rugged LC
Warth, Benedikt; Sulyok, Michael; Fruhmann, Philipp; Mikula, Hannes; Berthiller, Franz; Schuhmacher, Rainer; Hametner, Christian; Abia, Wilfred Angie; Adam, Gerhard; Fröhlich, Johannes; Krska, Rudolf
2012-07-15
Mycotoxins regularly occur in food worldwide and pose serious health risks to consumers. Since individuals can be exposed to a variety of these toxic secondary metabolites of fungi at the same time, there is a demand for proper analytical methods to assess human exposure by suitable biomarkers. This study reports on the development of a liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) method for the quantitative measurement of 15 mycotoxins and key metabolites in human urine using polarity switching. Deoxynivalenol (DON), DON-3-O-glucuronide, DON-15-O-glucuronide (D15GlcA), de-epoxy DON, nivalenol (NIV), T-2 toxin, HT-2 toxin, zearalenone, zearalenone-14-O-glucuronide, α- and β-zearalenol, fumonisins B(1) and B(2) (FB(1), FB(2)), ochratoxin A (OTA) and aflatoxin M(1) (AFM(1)) were determined without the need for any cleanup using a rapid and simple dilute and shoot approach. Validation was performed in the range of 0.005-40 µg L(-1) depending on the analyte and expected urinary concentration levels. Apparent recoveries between 78 and 119% and interday precisions of 2-17% relative standard deviation (RSD) were achieved. The applicability of the method was demonstrated by the analysis of urine samples obtained from Cameroon. In naturally contaminated urine samples up to six biomarkers of exposure (AFM(1), DON, D15GlcA, NIV, FB(1), and OTA) were detected simultaneously. We conclude that the developed LC/MS/MS method is well suited to quantify multiple mycotoxin biomarkers in human urine down to the sub-ppb range within 18 min and without any prior cleanup. The co-occurrence of several mycotoxins in the investigated samples clearly emphasizes the great potential and importance of this method to assess exposure of humans and animals to naturally occurring mycotoxins. Copyright © 2012 John Wiley & Sons, Ltd.
G2LC: Resources Autoscaling for Real Time Bioinformatics Applications in IaaS.
Hu, Rongdong; Liu, Guangming; Jiang, Jingfei; Wang, Lixin
2015-01-01
Cloud computing has started to change the way how bioinformatics research is being carried out. Researchers who have taken advantage of this technology can process larger amounts of data and speed up scientific discovery. The variability in data volume results in variable computing requirements. Therefore, bioinformatics researchers are pursuing more reliable and efficient methods for conducting sequencing analyses. This paper proposes an automated resource provisioning method, G2LC, for bioinformatics applications in IaaS. It enables application to output the results in a real time manner. Its main purpose is to guarantee applications performance, while improving resource utilization. Real sequence searching data of BLAST is used to evaluate the effectiveness of G2LC. Experimental results show that G2LC guarantees the application performance, while resource is saved up to 20.14%.
Profile-Based LC-MS Data Alignment—A Bayesian Approach
Tsai, Tsung-Heng; Tadesse, Mahlet G.; Wang, Yue; Ressom, Habtom W.
2014-01-01
A Bayesian alignment model (BAM) is proposed for alignment of liquid chromatography-mass spectrometry (LC-MS) data. BAM belongs to the category of profile-based approaches, which are composed of two major components: a prototype function and a set of mapping functions. Appropriate estimation of these functions is crucial for good alignment results. BAM uses Markov chain Monte Carlo (MCMC) methods to draw inference on the model parameters and improves on existing MCMC-based alignment methods through 1) the implementation of an efficient MCMC sampler and 2) an adaptive selection of knots. A block Metropolis-Hastings algorithm that mitigates the problem of the MCMC sampler getting stuck at local modes of the posterior distribution is used for the update of the mapping function coefficients. In addition, a stochastic search variable selection (SSVS) methodology is used to determine the number and positions of knots. We applied BAM to a simulated data set, an LC-MS proteomic data set, and two LC-MS metabolomic data sets, and compared its performance with the Bayesian hierarchical curve registration (BHCR) model, the dynamic time-warping (DTW) model, and the continuous profile model (CPM). The advantage of applying appropriate profile-based retention time correction prior to performing a feature-based approach is also demonstrated through the metabolomic data sets. PMID:23929872
Turner, Andrew D; Waack, Julia; Lewis, Adam; Edwards, Christine; Lawton, Linda
2018-02-01
A simple, rapid UHPLC-MS/MS method has been developed and optimised for the quantitation of microcystins and nodularin in wide variety of sample matrices. Microcystin analogues targeted were MC-LR, MC-RR, MC-LA, MC-LY, MC-LF, LC-LW, MC-YR, MC-WR, [Asp3] MC-LR, [Dha7] MC-LR, MC-HilR and MC-HtyR. Optimisation studies were conducted to develop a simple, quick and efficient extraction protocol without the need for complex pre-analysis concentration procedures, together with a rapid sub 5min chromatographic separation of toxins in shellfish and algal supplement tablet powders, as well as water and cyanobacterial bloom samples. Validation studies were undertaken on each matrix-analyte combination to the full method performance characteristics following international guidelines. The method was found to be specific and linear over the full calibration range. Method sensitivity in terms of limits of detection, quantitation and reporting were found to be significantly improved in comparison to LC-UV methods and applicable to the analysis of each of the four matrices. Overall, acceptable recoveries were determined for each of the matrices studied, with associated precision and within-laboratory reproducibility well within expected guidance limits. Results from the formalised ruggedness analysis of all available cyanotoxins, showed that the method was robust for all parameters investigated. The results presented here show that the optimised LC-MS/MS method for cyanotoxins is fit for the purpose of detection and quantitation of a range of microcystins and nodularin in shellfish, algal supplement tablet powder, water and cyanobacteria. The method provides a valuable early warning tool for the rapid, routine extraction and analysis of natural waters, cyanobacterial blooms, algal powders, food supplements and shellfish tissues, enabling monitoring labs to supplement traditional microscopy techniques and report toxicity results within a short timeframe of sample receipt. The new
Peifer, Susanne; Schneider, Konstantin; Nürenberg, Gudrun; Volmer, Dietrich A; Heinzle, Elmar
2012-11-01
Intermediates of the purine biosynthesis pathway play key roles in cellular metabolism including nucleic acid synthesis and signal mediation. In addition, they are also of major interest to the biotechnological industry as several intermediates either possess flavor-enhancing characteristics or are applied in medical therapy. In this study, we have developed an analytical method for quantitation of 12 intermediates from the purine biosynthesis pathway including important nucleotides and their corresponding nucleosides and nucleobases. The approach comprised a single-step acidic extraction/quenching procedure, followed by quantitative electrospray LC-MS/MS analysis. The assay was validated in terms of accuracy, precision, reproducibility, and applicability for complex biological matrices. The method was subsequently applied for determination of free intracellular pool sizes of purine biosynthetic pathway intermediates in the two Gram-positive bacteria Corynebacterium glutamicum and Corynebacterium ammoniagenes. Importantly, no ion pair reagents were applied in this approach as usually required for liquid chromatography analysis of large classes of diverse metabolites.
Gika, Helen G; Theodoridis, Georgios A; Earll, Mark; Wilson, Ian D
2012-09-01
An approach to the determination of day-to-day analytical robustness of LC-MS-based methods for global metabolic profiling using a pooled QC sample is presented for the evaluation of metabonomic/metabolomic data. A set of 60 urine samples were repeatedly analyzed on five different days and the day-to-day reproducibility of the data obtained was determined. Multivariate statistical analysis was performed with the aim of evaluating variability and selected peaks were assessed and validated in terms of retention time stability, mass accuracy and intensity. The methodology enables the repeatability/reproducibility of extended analytical runs in large-scale studies to be determined, allowing the elimination of analytical (as opposed to biological) variability, in order to discover true patterns and correlations within the data. The day-to-day variability of the data revealed by this process suggested that, for this particular system, 3 days continuous operation was possible without the need for maintenance and cleaning. Variation was generally based on signal intensity changes over the 7-day period of the study, and was mainly a result of source contamination.
Kim, Jihyun; Ji, Dajeong; Kang, Soyoung; Park, Meejung; Yang, Wonkyung; Kim, Eunmi; Choi, Hwakyung; Lee, Sooyeun
2014-02-01
Natural and synthetic opioids have efficient analgesic activity but can also be addictive. Thus, the determination of opioids and their metabolites in biological specimens is of interest in clinical and forensic toxicology laboratories. The analysis of drugs in hair provides valuable information on previous chronic drug use and has been successfully applied to the diagnosis of drug abuse, tolerance, compliance and gestational drug exposure. Despite the abuse of prescription opioids along with heroin and other illegal opiates, few studies have been conducted on the simultaneous determination of the broad range of opioids covering those drugs in hair. In the present study, an analytical method for the simultaneous detection in hair of 18 opioids and metabolites considered to have a high abuse risk based on the results of urine drug screening was established and validated using liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the purpose of clinical and forensic applications. The drugs and metabolites were extracted from hair using methanol and analyzed using LC-MS/MS. The validation results proved that the method was selective, accurate and precise with acceptable linearity within calibration ranges. No significant variation was observed by different sources of matrices. The limits of detection and the limits of quantification ranged from 0.05 to 0.25ng/10mg hair and from 0.05 to 0.5ng/10mg hair, respectively. The developed method was successfully applied to 15 hair samples from opioids users. This method will be very useful for monitoring the inappropriate use of opioid drugs. Copyright © 2013 Elsevier B.V. All rights reserved.
Bushman, Lane R; Kiser, Jennifer J; Rower, Joseph E; Klein, Brandon; Zheng, Jia-Hua; Ray, Michelle L; Anderson, Peter L
2011-09-10
An ultra-sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) assay was developed and validated to facilitate the assessment of clinical pharmacokinetics of nucleotide analogs from lysed intracellular matrix. The method utilized a strong anion exchange isolation of mono-(MP), di-(DP), and tri-phosphates (TP) from intracellular matrix. Each fraction was then dephosphorylated to the parent moiety yielding a molar equivalent to the original nucleotide analog intracellular concentration. The analytical portion of the methodology was optimized in specific nucleoside analog centric modes (i.e. tenofovir (TFV) centric, zidovudine (ZDV) centric), which included desalting/concentration by solid phase extraction and detection by LC-MS/MS. Nucleotide analog MP-, DP-, and TP-determined on the TFV centric mode of analysis include TFV, lamivudine (3TC), and emtricitibine (FTC). The quantifiable linear range for TFV was 2.5-2000 fmol/sample, and that for 3TC/FTC was 0.1 200 pmol/sample. Nucleoside analog MP-, DP-, and TP-determined on the ZDV centric mode of analysis included 3TC and ZDV. The quantifiable linear range for 3TC was 0.1 100 pmol/sample, and 5-2000 fmol/sample for ZDV. Stable labeled isotopic internal standards facilitated accuracy and precision in alternative cell matrices, which supported the intended use of the method for MP, DP, and TP determinations in various cell types. The method was successfully applied to clinical research samples generating novel intracellular information for TFV, FTC, ZDV, and 3TC nucleotides. This document outlines method development, validation, and application to clinical research. Copyright © 2011 Elsevier B.V. All rights reserved.
Schwaninger, Andrea E.; Meyer, Markus R.; Huestis, Marilyn A.; Maurer, Hans H.
2013-01-01
3,4-Methylenedioxymethamphetamine (MDMA) is a racemic drug of abuse and its R- and S-enantiomers are known to differ in their dose-response curve. The S-enantiomer was shown to be eliminated at a higher rate than the R-enantiomer most likely explained by stereoselective metabolism that was observed in various in vitro experiments. The aim of this work was the development and validation of methods for evaluating the stereoselective elimination of phase I and particularly phase II metabolites of MDMA in human urine. Urine samples were divided into three different methods. Method A allowed stereoselective determination of the 4-hydroxy-3-methoxymethamphetamine (HMMA) glucuronides and only achiral determination of the intact sulfate conjugates of HMMA and 3,4-dihydroxymethamphetamine (DHMA) after C18 solid-phase extraction by liquid chromatography–high-resolution mass spectrometry with electrospray ionization. Method B allowed the determination of the enantiomer ratios of DHMA and HMMA sulfate conjugates after selective enzymatic cleavage and chiral analysis of the corresponding deconjugated metabolites after chiral derivatization with S-heptafluorobutyrylprolyl chloride using gas chromatography–mass spectrometry with negativeion chemical ionization. Method C allowed the chiral determination of MDMA and its unconjugated metabolites using method B without sulfate cleavage. The validation process including specificity, recovery, matrix effects, process efficiency, accuracy and precision, stabilities and limits of quantification and detection showed that all methods were selective, sensitive, accurate and precise for all tested analytes. PMID:21656610
Nakata, Katsunori; Saitoh, Ryoichi; Ishigai, Masaki; Imai, Kazuhiro
2018-02-01
Biological functions in organisms are usually controlled by a set of interacting proteins, and identifying the proteins that interact is useful for understanding the mechanism of the functions. Immunoprecipitation is a method that utilizes the affinity of an antibody to isolate and identify the proteins that have interacted in a biological sample. In this study, the FD-LC-MS/MS method, which involves fluorogenic derivatization followed by separation and quantification by HPLC and finally identification of proteins by HPLC-tandem mass spectrometry, was used to identify proteins in immunoprecipitated samples, using heat shock protein 90 (HSP90) as a model of an interacting protein in HepaRG cells. As a result, HSC70 protein, which was known to form a complex with HSP90, was isolated, together with three different types of HSP90-beta. The results demonstrated that the proposed immunoaffinity-FD-LC-MS/MS method could be useful for simultaneously detecting and identifying the proteins that interact with a certain protein. Copyright © 2017 John Wiley & Sons, Ltd.
Míguez, J; Herrero, C; Quintás, I; Rodríguez, C; Gigosos, P G; Mariz, O C
2012-12-01
A new and simple liquid chromatography tandem mass-spectrometry method for the determination of different bisphenol A (BPA) derivatives such as bisphenol A diglycidyl ether (BADGE), bisphenol F diglycidyl ether (BFDGE) and their reaction products with water and hydrochloric acid in different fish food products was developed. The extraction procedure and the chromatographic conditions were optimised for complex food matrices such as fish products. Food samples were homogenised and extracted with a 1:1 solution of acetonitrile-hexane, the solvent was eliminated in a N(2) stream and the extract was reconstituted with 0.5mL of a 0.01M solution of ammonium formate. The sample solution obtained was directly measured by LC-MS/MS without any further purification under the developed conditions. The use of a mobile phase composed by ammonium formate-methanol in a binary gradient mode produced [M+NH(4)](+) aducts for the different BADGEs and BFDGEs. These aduct's fragmentations were employed for the LC-MS/MS quantification of BPA derivatives in canned fish samples. The results of the validation were appropriate: the method was linear for BADGE and its hydrolysed derivatives up to 1000μgkg(-1), for the remaining compounds linearity achieved up to 100μgkg(-1). Quantification limits were in the range 2-10μgkg(-1). RSD (intra and inter-day) was 6-12% and the recovery was comprised between 89% and 109%. Under the optimised conditions, the chromatographic separation was performed in 8min per sample. The method was applied to the determination of BADGE, BFDGE and their reaction products in different samples of canned fish from Spanish origin. Migration results obtained were in compliance with the EU regulations. Copyright © 2012 Elsevier Ltd. All rights reserved.
Penchala, Sujan Dilly; Tjia, John; El Sherif, Omar; Back, David J; Khoo, Saye H; Else, Laura J
2013-08-01
A sensitive high-performance reverse phase liquid chromatography-positive ion electrospray tandem mass spectrometry method was developed and validated for the quantification of telaprevir and its inactive R-diastereomer (VRT-127394) in human plasma. The analytes and the internal standard (telaprevir-d11) were extracted from plasma by liquid-liquid extraction using tert-Butyl methyl ether (TBME). Chromatographic separation was achieved on a reversed-phase Accucore C18 column with a gradient programme consisting of water:ammonia (25%), 100:0.01 (v/v) (mobile phase A) and ACN:MeOH:ammonia (25%), 15:85:0.01 (v/v/v) (mobile phase B). The MS acquisition was performed with selective reaction monitoring mode using the respective [M+H](+) ions, m/z 680.59→322.42 for telaprevir and VRT-127394, and 691.15→110.13 for telaprevir-d11. The assay exhibited a linear dynamic range of 5-5000ng/mL for telaprevir and VRT-127394. Acceptable precision (%RSD<6.5%) and accuracy (94-108%) were obtained for concentrations over the range of the standard curve. A procedure was established to stabilise the plasma to prevent ex vivo interconversion of the isomers. Copyright © 2013 Elsevier B.V. All rights reserved.
Baldo, Matías N; Angeli, Emmanuel; Gareis, Natalia C; Hunzicker, Gabriel A; Murguía, Marcelo C; Ortega, Hugo H; Hein, Gustavo J
2018-04-01
A relative bioavailability study (RBA) of two phenytoin (PHT) formulations was conducted in rabbits, in order to compare the results obtained from different matrices (plasma and blood from dried blood spot (DBS) sampling) and different experimental designs (classic and block). The method was developed by liquid chromatography tandem-mass spectrometry (LC-MS/MS) in plasma and blood samples. The different sample preparation techniques, plasma protein precipitation and DBS, were validated according to international requirements. The analytical method was validated with ranges 0.20-50.80 and 0.12-20.32 µg ml -1 , r > 0.999 for plasma and blood, respectively. Accuracy and precision were within acceptance criteria for bioanalytical assay validation (< 15 for bias and CV% and < 20 for limit of quantification (LOQ)). PHT showed long-term stability, both for plasma and blood, and under refrigerated and room temperature conditions. Haematocrit values were measured during the validation process and RBA study. Finally, the pharmacokinetic parameters (C max , T max and AUC 0-t ) obtained from the RBA study were tested. Results were highly comparable for matrices and experimental designs. A matrix correlation higher than 0.975 and a ratio of (PHT blood) = 1.158 (PHT plasma) were obtained. The results obtained herein show that the use of classic experimental design and DBS sampling for animal pharmacokinetic studies should be encouraged as they could help to prevent the use of a large number of animals and also animal euthanasia. Finally, the combination of DBS sampling with LC-MS/MS technology showed to be an excellent tool not only for therapeutic drug monitoring but also for RBA studies.
Data Dependent Peak Model Based Spectrum Deconvolution for Analysis of High Resolution LC-MS Data
2015-01-01
A data dependent peak model (DDPM) based spectrum deconvolution method was developed for analysis of high resolution LC-MS data. To construct the selected ion chromatogram (XIC), a clustering method, the density based spatial clustering of applications with noise (DBSCAN), is applied to all m/z values of an LC-MS data set to group the m/z values into each XIC. The DBSCAN constructs XICs without the need for a user defined m/z variation window. After the XIC construction, the peaks of molecular ions in each XIC are detected using both the first and the second derivative tests, followed by an optimized chromatographic peak model selection method for peak deconvolution. A total of six chromatographic peak models are considered, including Gaussian, log-normal, Poisson, gamma, exponentially modified Gaussian, and hybrid of exponential and Gaussian models. The abundant nonoverlapping peaks are chosen to find the optimal peak models that are both data- and retention-time-dependent. Analysis of 18 spiked-in LC-MS data demonstrates that the proposed DDPM spectrum deconvolution method outperforms the traditional method. On average, the DDPM approach not only detected 58 more chromatographic peaks from each of the testing LC-MS data but also improved the retention time and peak area 3% and 6%, respectively. PMID:24533635
Delocalized periodic vibrations in nonlinear LC and LCR electrical chains
NASA Astrophysics Data System (ADS)
Chechin, G. M.; Shcherbinin, S. A.
2015-05-01
We consider electrical LC- and LCR-chains consisting of N cells. In the LC-chain each cell contains a linear inductor L and a nonlinear capacitor C, while the cell in the LCR-chain include additionally a resistor R and an voltage source. It is assumed that voltage dependence of capacitors represents an even function. Such capacitors have implemented by some experimental groups studying propagation of electrical signals in the lines constructed on MOS and CMOS substrates. In these chains, we study dynamical regimes representing nonlinear normal modes (NNMs) by Rosenberg. We prove that maximum possible number of symmetry-determined NNMs which can be excited in the considered chains is equal to 5. The stability of these modes for different N is studied with the aid of the group-theoretical method [Physical Review E 73 (2006) 36216] which allows to simplify radically the variational systems appearing in the Floquet stability analysis. For NNMs in LC-chain, the scaling of the voltage stability threshold in the thermodynamic limit (N → ∞) is determined. It is shown that the above group theoretical method can be also used for studying stability of NNMs in the LCR-chains.
Xu, Wei; Chen, Deying; Wang, Nan; Zhang, Ting; Zhou, Ruokun; Huan, Tao; Lu, Yingfeng; Su, Xiaoling; Xie, Qing; Li, Liang; Li, Lanjuan
2015-01-20
Human fecal samples contain endogenous human metabolites, gut microbiota metabolites, and other compounds. Profiling the fecal metabolome can produce metabolic information that may be used not only for disease biomarker discovery, but also for providing an insight about the relationship of the gut microbiome and human health. In this work, we report a chemical isotope labeling liquid chromatography-mass spectrometry (LC-MS) method for comprehensive and quantitative analysis of the amine- and phenol-containing metabolites in fecal samples. Differential (13)C2/(12)C2-dansyl labeling of the amines and phenols was used to improve LC separation efficiency and MS detection sensitivity. Water, methanol, and acetonitrile were examined as an extraction solvent, and a sequential water-acetonitrile extraction method was found to be optimal. A step-gradient LC-UV setup and a fast LC-MS method were evaluated for measuring the total concentration of dansyl labeled metabolites that could be used for normalizing the sample amounts of individual samples for quantitative metabolomics. Knowing the total concentration was also useful for optimizing the sample injection amount into LC-MS to maximize the number of metabolites detectable while avoiding sample overloading. For the first time, dansylation isotope labeling LC-MS was performed in a simple time-of-flight mass spectrometer, instead of high-end equipment, demonstrating the feasibility of using a low-cost instrument for chemical isotope labeling metabolomics. The developed method was applied for profiling the amine/phenol submetabolome of fecal samples collected from three families. An average of 1785 peak pairs or putative metabolites were found from a 30 min LC-MS run. From 243 LC-MS runs of all the fecal samples, a total of 6200 peak pairs were detected. Among them, 67 could be positively identified based on the mass and retention time match to a dansyl standard library, while 581 and 3197 peak pairs could be putatively
Roberts, Michael S; Turner, David C; Owens, Thandranese S; Ramachandran, Abhijit; Wetmore, Cynthia; Throm, Stacy L; Stewart, Clinton F
2013-06-15
A LC-ESI-MS/MS method for the determination of crenolanib (CP-868,596) in human serum was developed and validated employing d4-CP-868,596 as an internal standard (ISTD). In addition to human serum, the method was also partially validated for crenolanib determination in human cerebrospinal fluid (CSF) samples. Sample aliquots (50μl of serum or CSF) were prepared for analysis using liquid-liquid extraction (LLE) with tert-butyl methyl ether. Chromatography was performed using a phenomenex Gemini C18 column (3μm, 100mm×4.6mm I.D.) in a column heater set at 50°C and an isocratic mobile phase (methanol/water/formic acid at a volume ratio of 25/25/0.15, v/v/v). The flow rate was 0.45mL/min, and the retention time for both analyte and ISTD was less than 3.5min. Samples were analyzed with an API-5500 LC-MS/MS system (ESI) in positive ionization mode coupled to a Shimadzu HPLC system. The ion transitions monitored were m/z 444.4→373.1 and m/z 448.2→374.2 for crenolanib and ISTD, respectively. The method was linear over the range of 5-1000ng/mL for serum and 0.5-1000ng/mL for CSF. For human serum, both intra-day and inter-day precision were <4%, while intra-day and inter-day accuracy were within 8% of nominal values. Recovery was greater than 50% for both the analyte and ISTD. For CSF samples, both intra-day and inter-day precision were <9% except at the lower limit of quantification (LLOQ) which was <17%. The intra-day and inter-day accuracy were within 11% of the nominal CSF concentrations. After validation, this method was successfully applied to the analysis of serial pharmacokinetic samples obtained from a child treated with oral crenolanib. Copyright © 2013 Elsevier B.V. All rights reserved.
Jasiecka-Mikołajczyk, A; Jaroszewski, J J
2017-03-01
Tigecycline (TIG), a novel glycylcycline antibiotic, plays an important role in the management of complicated skin and intra-abdominal infections. The available data lack any description of a method for determination of TIG in avian plasma. In our study, a selective, accurate and reversed-phase high performance liquid chromatography-tandem mass spectrometry method was developed for the determination of TIG in turkey plasma. Sample preparation was based on protein precipitation and liquid-liquid extraction using 1,2-dichloroethane. Chromatographic separation of TIG and minocycline (internal standard, IS) was achieved on an Atlantis T3 column (150 mm × 3.0 mm, 3.0 μm) using gradient elution. The selected reaction monitoring transitions were performed at 293.60 m/z → 257.10 m/z for TIG and 458.00 m/z → 441.20 m/z for IS. The developed method was validated in terms of specificity, selectivity, linearity, lowest limit of quantification, limit of detection, precision, accuracy, matrix effect, carry-over effect, extraction recovery and stability. All parameters of the method submitted to validation met the acceptance criteria. The assay was linear over the concentration range of 0.01-100 μg/ml. This validated method was successfully applied to a TIG pharmacokinetic study in turkey after intravenous and oral administration at a dose of 10 mg/kg at various time-points.
Laryea, Maurice D; Herebian, Diran; Meissner, Thomas; Mayatepek, Ertan
2010-12-01
Inborn errors of urea metabolism result in hyperammonemia. Treatment of urea cycle disorders can effectively lower plasma ammonium levels and results in survival in the majority of patients. Available medications for treating urea cycle disorders include sodium benzoate (BA), sodium phenylacetate (PAA), and sodium phenylbutyrate (PBA) and are given to provide alternate routes for disposition of waste nitrogen excretion. In this study, we develop and validate a liquid chromatography tandem mass spectrometry (LC-MS/MS) method for simultaneous determination of benzoic acid, phenylacetic acid, phenylbutyric acid, phenylacetylglutamine, and hippuric acid in plasma and urine from children with inborn errors of urea synthesis. Plasma extracts and diluted urine samples were injected on a reverse-phase column and identified and quantified by selected reaction monitoring (SRM) in negative ion mode. Deuterated analogues served as internal standards. Analysis time was 7 min. Assay precision, accuracy, and linearity and sample stability were determined using enriched samples. Quantification limits of the method were 100 ng/ml (0.3-0.8 μmol/L) for all analytes, and recoveries were >90%. Inter- and intraday relative standard deviations were <10%. Our newly developed LC-MS/MS represents a robust, sensitive, and rapid method that allows simultaneous determination of the five compounds in plasma and urine.
Peng, Can; Tian, Jixin; Lv, Mengying; Huang, Yin; Tian, Yuan; Zhang, Zunjian
2014-02-01
Artificial Calculus Bovis is a major substitute in clinical treatment for Niuhuang, a widely used, efficacious but rare traditional Chinese medicine. However, its chemical structures and the physicochemical properties of its components are complicated, which causes difficulty in establishing a set of effective and comprehensive methods for its identification and quality control. In this study, a simple, sensitive and reliable liquid chromatography-tandem mass spectrometry method was successfully developed and validated for the simultaneous determination of bilirubin, taurine and major bile acids (including six unconjugated bile acids, two glycine-conjugated bile acids and three taurine-conjugated bile acids) in artificial Calculus Bovis using a Zorbax SB-C18 column with a gradient elution of methanol and 10 mmol/L ammonium acetate in aqueous solution (adjusted to pH 3.0 with formic acid). The mass spectra were obtained in the negative ion mode using dehydrocholic acid as the internal standard. The content of each analyte in artificial Calculus Bovis was determined by monitoring specific ion pairs in the selected reaction monitoring mode. All analytes demonstrated perfect linearity (r(2) > 0.994) in a wide dynamic range, and 10 batches of samples from different sources were further analyzed. This study provided a comprehensive method for the quality control of artificial Calculus Bovis.
Parsons, Teresa L; Emory, Joshua F; Seserko, Lauren A; Aung, Wutyi S; Marzinke, Mark A
2014-09-01
Topical microbicidal agents are being actively pursued as a modality to prevent HIV viral transmission during sexual intercourse. Quantification of antiretroviral agents in specimen sources where antiviral activity is elicited is critical, and drug measurements in cervicovaginal fluid can provide key information on local drug concentrations. Two antiretroviral drugs, dapivirine and maraviroc, have gained interest as vaginal microbicidal agents, and rugged methods are required for their quantification in cervicovaginal secretions. Cervicovaginal fluid spiked with dapivirine and maraviroc were applied to ophthalmic tear strips or polyester-based swabs to mimic collection procedures used in clinical studies. Following sample extraction and the addition of isotopically labeled internal standards, samples were subjected to liquid chromatographic-tandem mass spectrometric (LC-MS/MS) analysis using a Waters BEH C8, 50mm×2.1mm, 1.7μm particle size column, on an API 4000 mass analyzer operated in selective reaction monitoring mode. The method was validated according to FDA Bioanalytical Method Validation guidelines. Due to the disparate saturation capacity of the tested collection devices, the analytical measuring ranges for dapivirine and maravirocin cervicovaginal fluid on the ophthalmic tear strip were 0.05-25ng/tear strip, and 0.025-25ng/tear strip, respectively. As for the polyester-based swab, the analytical measuring ranges were 0.25-125ng/swab for dapivirine and 0.125-125ng/swab for maraviroc. Dilutional studies were performed for both analytes to extended ranges of 25,000ng/tear strip and 11,250ng/swab. Standard curves were generated via weighted (1/x(2)) linear or quadratic regression of calibrators. Precision, accuracy, stability and matrix effects studies were all performed and deemed acceptable according to the recommendations of the FDA Bioanalytical Method Validation guidelines. A rugged LC-MS/MS method for the dual quantification of dapivirine and
Mudge, Elizabeth M; Liu, Ying; Lund, Jensen A; Brown, Paula N
2016-11-01
Suitably validated analytical methods that can be used to quantify medicinally active phytochemicals in natural health products are required by regulators, manufacturers, and consumers. Hawthorn ( Crataegus ) is a botanical ingredient in natural health products used for the treatment of cardiovascular disorders. A method for the quantitation of vitexin-2″- O - rhamnoside, vitexin, isovitexin, rutin, and hyperoside in hawthorn leaf and flower raw materials and finished products was optimized and validated according to AOAC International guidelines. A two-level partial factorial study was used to guide the optimization of the sample preparation. The optimal conditions were found to be a 60-minute extraction using 50 : 48 : 2 methanol : water : acetic acid followed by a 25-minute separation using a reversed-phased liquid chromatography column with ultraviolet absorbance detection. The single-laboratory validation study evaluated method selectivity, accuracy, repeatability, linearity, limit of quantitation, and limit of detection. Individual flavonoid content ranged from 0.05 mg/g to 17.5 mg/g in solid dosage forms and raw materials. Repeatability ranged from 0.7 to 11.7 % relative standard deviation corresponding to HorRat ranges from 0.2 to 1.6. Calibration curves for each flavonoid were linear within the analytical ranges with correlation coefficients greater than 99.9 %. Herein is the first report of a validated method that is fit for the purpose of quantifying five major phytochemical marker compounds in both raw materials and finished products made from North American ( Crataegus douglasii ) and European ( Crataegus monogyna and Crataegus laevigata) hawthorn species. The method includes optimized extraction of samples without a prolonged drying process and reduced liquid chromatography separation time. Georg Thieme Verlag KG Stuttgart · New York.
Chen, Xun; Stout, Steven; Mueller, Uwe; Boykow, George; Visconti, Richard; Siliphaivanh, Phieng; Spencer, Kerrie; Presland, Jeremy; Kavana, Michael; Basso, Andrea D; McLaren, David G; Myers, Robert W
2017-08-01
We have developed and validated label-free, liquid chromatography-mass spectrometry (LC-MS)-based equilibrium direct and competition binding assays to quantitate small-molecule antagonist binding to recombinant human and mouse BLT1 receptors expressed in HEK 293 cell membranes. Procedurally, these binding assays involve (1) equilibration of the BLT1 receptor and probe ligand, with or without a competitor; (2) vacuum filtration through cationic glass fiber filters to separate receptor-bound from free probe ligand; and (3) LC-MS analysis in selected reaction monitoring mode for bound probe ligand quantitation. Two novel, optimized probe ligands, compounds 1 and 2, were identified by screening 20 unlabeled BLT1 antagonists for direct binding. Saturation direct binding studies confirmed the high affinity, and dissociation studies established the rapid binding kinetics of probe ligands 1 and 2. Competition binding assays were established using both probe ligands, and the affinities of structurally diverse BLT1 antagonists were measured. Both binding assay formats can be executed with high specificity and sensitivity and moderate throughput (96-well plate format) using these approaches. This highly versatile, label-free method for studying ligand binding to membrane-associated receptors should find broad application as an alternative to traditional methods using labeled ligands.
G2LC: Resources Autoscaling for Real Time Bioinformatics Applications in IaaS
Hu, Rongdong; Liu, Guangming; Jiang, Jingfei; Wang, Lixin
2015-01-01
Cloud computing has started to change the way how bioinformatics research is being carried out. Researchers who have taken advantage of this technology can process larger amounts of data and speed up scientific discovery. The variability in data volume results in variable computing requirements. Therefore, bioinformatics researchers are pursuing more reliable and efficient methods for conducting sequencing analyses. This paper proposes an automated resource provisioning method, G2LC, for bioinformatics applications in IaaS. It enables application to output the results in a real time manner. Its main purpose is to guarantee applications performance, while improving resource utilization. Real sequence searching data of BLAST is used to evaluate the effectiveness of G2LC. Experimental results show that G2LC guarantees the application performance, while resource is saved up to 20.14%. PMID:26504488
Presgrave, Octavio; Moura, Wlamir; Caldeira, Cristiane; Pereira, Elisabete; Bôas, Maria H Villas; Eskes, Chantra
2016-03-01
The need for the creation of a Brazilian centre for the validation of alternative methods was recognised in 2008, and members of academia, industry and existing international validation centres immediately engaged with the idea. In 2012, co-operation between the Oswaldo Cruz Foundation (FIOCRUZ) and the Brazilian Health Surveillance Agency (ANVISA) instigated the establishment of the Brazilian Center for the Validation of Alternative Methods (BraCVAM), which was officially launched in 2013. The Brazilian validation process follows OECD Guidance Document No. 34, where BraCVAM functions as the focal point to identify and/or receive requests from parties interested in submitting tests for validation. BraCVAM then informs the Brazilian National Network on Alternative Methods (RENaMA) of promising assays, which helps with prioritisation and contributes to the validation studies of selected assays. A Validation Management Group supervises the validation study, and the results obtained are peer-reviewed by an ad hoc Scientific Review Committee, organised under the auspices of BraCVAM. Based on the peer-review outcome, BraCVAM will prepare recommendations on the validated test method, which will be sent to the National Council for the Control of Animal Experimentation (CONCEA). CONCEA is in charge of the regulatory adoption of all validated test methods in Brazil, following an open public consultation. 2016 FRAME.
Jensen, Pamela K; Wujcik, Chad E; McGuire, Michelle K; McGuire, Mark A
2016-01-01
Simple high-throughput procedures were developed for the direct analysis of glyphosate [N-(phosphonomethyl)glycine] and aminomethylphosphonic acid (AMPA) in human and bovine milk and human urine matrices. Samples were extracted with an acidified aqueous solution on a high-speed shaker. Stable isotope labeled internal standards were added with the extraction solvent to ensure accurate tracking and quantitation. An additional cleanup procedure using partitioning with methylene chloride was required for milk matrices to minimize the presence of matrix components that can impact the longevity of the analytical column. Both analytes were analyzed directly, without derivatization, by liquid chromatography tandem mass spectrometry using two separate precursor-to-product transitions that ensure and confirm the accuracy of the measured results. Method performance was evaluated during validation through a series of assessments that included linearity, accuracy, precision, selectivity, ionization effects and carryover. Limits of quantitation (LOQ) were determined to be 0.1 and 10 µg/L (ppb) for urine and milk, respectively, for both glyphosate and AMPA. Mean recoveries for all matrices were within 89-107% at three separate fortification levels including the LOQ. Precision for replicates was ≤ 7.4% relative standard deviation (RSD) for milk and ≤ 11.4% RSD for urine across all fortification levels. All human and bovine milk samples used for selectivity and ionization effects assessments were free of any detectable levels of glyphosate and AMPA. Some of the human urine samples contained trace levels of glyphosate and AMPA, which were background subtracted for accuracy assessments. Ionization effects testing showed no significant biases from the matrix. A successful independent external validation was conducted using the more complicated milk matrices to demonstrate method transferability.
Cai, Shuang; Wang, Lei
2012-05-15
A simple and rapid high-performance liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) method has been developed and validated for the determination of 4-p-anisamidobutyric acid (ABA; or N-anysoyl-γ-aminobutiryc acid, N-anisoyl-GABA), a major active metabolite of aniracetam, in human plasma. After protein precipitation of plasma sample with methanol, ABA and the internal standard lisinopril were separated on a Venusil ASB C₁₈ column at 25 °C. The mobile phase consisted of methanol-ammonium acetate (10 mmol/L) (30:70, v/v). The detection was performed on a triple quadrupole tandem mass spectrometer with an ESI source in negative ion mode. Multiple reaction monitoring (MRM) using the precursor→product ion combinations of m/z 235.8→m/z 106.6, and m/z 403.8→m/z 113.6 was used to quantify ABA and lisinopril, respectively. This is the first LC-MS/MS method for ABA with advantages of short analysis time (4.5 min per sample run) and high selectivity attributable to the MRM detection and optimized HPLC conditions. The response was linear in a concentration range of 0.0485-19.4 μg/mL in plasma. The extraction recovery of ABA was between 89.1% and 100.7%. The precision (RSD) and accuracy (RE) of the method were evaluated to be within 7.3% and from 2.5% to 6.9%. The validated method has been applied to the pharmacokinetic study after a single oral administration of aniracetam dispersible tablets to human beings. Copyright © 2012 Elsevier B.V. All rights reserved.
Identification of RIP-II toxins by affinity enrichment, enzymatic digestion and LC-MS.
Fredriksson, Sten-Åke; Artursson, Elisabet; Bergström, Tomas; Östin, Anders; Nilsson, Calle; Åstot, Crister
2015-01-20
Type 2 ribosome-inactivating protein toxins (RIP-II toxins) were enriched and purified prior to enzymatic digestion and LC-MS analysis. The enrichment of the RIP-II family of plant proteins, such as ricin, abrin, viscumin, and volkensin was based on their affinity for galactosyl moieties. A macroporous chromatographic material was modified with a galactose-terminated substituent and packed into miniaturized columns that were used in a chromatographic system to achieve up to 1000-fold toxin enrichment. The galactose affinity of the RIP-II proteins enabled their selective enrichment from water, beverages, and extracts of powder and wipe samples. The enriched fractions were digested with trypsin and RIP-II peptides were identified based on accurate mass LC-MS data. Their identities were unambiguously confirmed by LC-MS/MS product ion scans of peptides unique to each of the toxins. The LC-MS detection limit achieved for ricin target peptides was 10 amol and the corresponding detection limit for the full method was 10 fmol/mL (0.6 ng/mL). The affinity enrichment method was applied to samples from a forensic investigation into a case involving the illegal production of ricin and abrin toxins.
Remily-Wood, Elizabeth R; Benson, Kaaron; Baz, Rachid C; Chen, Y Ann; Hussein, Mohamad; Hartley-Brown, Monique A; Sprung, Robert W; Perez, Brianna; Liu, Richard Z; Yoder, Sean J; Teer, Jamie K; Eschrich, Steven A; Koomen, John M
2014-10-01
Quantitative MS assays for Igs are compared with existing clinical methods in samples from patients with plasma cell dyscrasias, for example, multiple myeloma (MM). Using LC-MS/MS data, Ig constant region peptides, and transitions were selected for LC-MRM MS. Quantitative assays were used to assess Igs in serum from 83 patients. RNA sequencing and peptide-based LC-MRM are used to define peptides for quantification of the disease-specific Ig. LC-MRM assays quantify serum levels of Igs and their isoforms (IgG1-4, IgA1-2, IgM, IgD, and IgE, as well as kappa (κ) and lambda (λ) light chains). LC-MRM quantification has been applied to single samples from a patient cohort and a longitudinal study of an IgE patient undergoing treatment, to enable comparison with existing clinical methods. Proof-of-concept data for defining and monitoring variable region peptides are provided using the H929 MM cell line and two MM patients. LC-MRM assays targeting constant region peptides determine the type and isoform of the involved Ig and quantify its expression; the LC-MRM approach has improved sensitivity compared with the current clinical method, but slightly higher inter-assay variability. Detection of variable region peptides is a promising way to improve Ig quantification, which could produce a dramatic increase in sensitivity over existing methods, and could further complement current clinical techniques. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Li, Wenkui; Doherty, John P; Kulmatycki, Kenneth; Smith, Harold T; Tse, Francis Ls
2012-06-01
In support of a pilot clinical trial using acetaminophen as the model compound to assess dried blood spot (DBS) sampling as the method for clinical pharmacokinetic sample collection, a novel sensitive LC-MS/MS method was developed and validated for the simultaneous determination of acetaminophen and its major metabolites, acetaminophen glucuronide and sulfate, in human DBS samples collected by subjects via fingerprick. The validated assay dynamic range was from 50.0 to 5000 ng/ml for each compound using a 1/8´´ (3-mm) disc punched from a DBS sample. Baseline separation of the three analytes was achieved to eliminate the possible impact of insource fragmentation of the conjugated metabolites on the analysis of the parent. The overall extraction efficiency was from 61.3 to 78.8% for the three analytes by direct extraction using methanol. The validated method was successfully implemented in the pilot clinical study with the obtained pharmacokinetic parameters in agreement with the values reported in literature.
Application of survival analysis methodology to the quantitative analysis of LC-MS proteomics data.
Tekwe, Carmen D; Carroll, Raymond J; Dabney, Alan R
2012-08-01
Protein abundance in quantitative proteomics is often based on observed spectral features derived from liquid chromatography mass spectrometry (LC-MS) or LC-MS/MS experiments. Peak intensities are largely non-normal in distribution. Furthermore, LC-MS-based proteomics data frequently have large proportions of missing peak intensities due to censoring mechanisms on low-abundance spectral features. Recognizing that the observed peak intensities detected with the LC-MS method are all positive, skewed and often left-censored, we propose using survival methodology to carry out differential expression analysis of proteins. Various standard statistical techniques including non-parametric tests such as the Kolmogorov-Smirnov and Wilcoxon-Mann-Whitney rank sum tests, and the parametric survival model and accelerated failure time-model with log-normal, log-logistic and Weibull distributions were used to detect any differentially expressed proteins. The statistical operating characteristics of each method are explored using both real and simulated datasets. Survival methods generally have greater statistical power than standard differential expression methods when the proportion of missing protein level data is 5% or more. In particular, the AFT models we consider consistently achieve greater statistical power than standard testing procedures, with the discrepancy widening with increasing missingness in the proportions. The testing procedures discussed in this article can all be performed using readily available software such as R. The R codes are provided as supplemental materials. ctekwe@stat.tamu.edu.
Coupling of Ultrafast LC with Mass Spectrometry by DESI
NASA Astrophysics Data System (ADS)
Cai, Yi; Liu, Yong; Helmy, Roy; Chen, Hao
2014-10-01
Recently we reported a desorption electrospray ionization (DESI) interface to combine liquid chromatography (LC) with mass spectrometry (MS) using a new LC eluent splitting strategy through a tiny orifice on LC capillary tube [ J. Am. Soc. Mass Spectrom. 25, 286 (2014)]. The interface introduces negligible dead volume and back pressure, thereby allowing "near real-time" MS detection, fast LC elution, and online MS-directed purification. This study further evaluates the LC/DESI-MS performance with focus of using ultra-fast LC. Using a monolithic C18 column, metabolites in urine can be separated within 1.6 min and can be online collected for subsequent structure elucidation (e.g., by NMR, UV, IR) in a recovery yield up to 99%. Using a spray solvent with alkaline pH, negative ions could be directly generated for acidic analytes (e.g., ibuprofen) in acidic LC eluent by DESI, offering a novel protocol to realize "wrong-way around" ionization for LC/MS analysis. In addition, DESI-MS is found to be compatible with ultra-performance liquid chromatography (UPLC) for the first time.
Alcántara-Concepción, Victor; Cram, Silke; Gibson, Richard; Ponce de León, Claudia; Mazari-Hiriart, Marisa
2013-01-01
The Xochimilco area in the southeastern part of Mexico City has a variety of socioeconomic activities, such as periurban agriculture, which is of great importance in the Mexico City metropolitan area. Pesticides are used extensively, some being legal, mostly chlorpyrifos and malathion, and some illegal, mostly DDT. Sediments are a common sink for pesticides in aquatic systems near agricultural areas, and Xochimilco sediments have a complex composition with high contents of organic matter and clay that are ideal adsorption sites for organochlorine (OC) and organophosphorus (OP) pesticides. Therefore, it is important to have a quick, affordable, and reliable method to determine these pesticides. Conventional methods for the determination of OC and OP pesticides are long, laborious, and costly owing to the high volume of solvents and adsorbents. The present study developed and validated a method for determining 18 OC and five OP pesticides in sediments with high organic and clay contents. In contrast with other methods described in the literature, this method allows isolation of the 23 pesticides with a 12 min microwave-assisted extraction (MAE) and one-step cleanup of pesticides. The method developed is a simpler, time-saving procedure that uses only 3.5 g of dry sediment. The use of MAE eliminates excessive handling and the possible loss of analytes. It was shown that the use of LC-Si cartridges with hexane-ethyl acetate (75+25, v/v) in the cleanup procedure recovered all pesticides with rates between 70 and 120%. The validation parameters demonstrated good performance of the method, with intermediate precision ranging from 7.3 to 17.0%, HorRat indexes all below 0.5, and tests of accuracy with the 23 pesticides at three concentration levels demonstrating recoveries ranging from 74 to 114% and RSDs from 3.3 to 12.7%.
Tölgyesi, Adám; Sharma, Virender K; Fekete, Jeno
2011-02-15
A new method was developed to determine five corticosteroids (prednisolone, methylprednisone, flumethasone, dexamethasone, and methylprednisolone) in pig fat samples by liquid chromatography-tandem mass spectrometry (LC-MS/MS) utilizing an optimized liquid-liquid extraction (LLE) and subsequent solid-phase extraction (SPE) for sample clean-up. In the sample preparation, a pig fat sample was dissolved in n-hexane and then extracted into the methanol-water (50/50, v/v) mixture that enabled extraction of only medium polar corticosteroids and not the non-polar components of matrices. This extract was cleaned-up and concentrated on polymeric Oasis HLB SPE cartridge. Separation involved isocratic solvent (methanol-acetate buffer, pH 5.4) and Ascentis Express Fused-Core type HLPC column; reduced the analysis time to 7.5 min, which is at least two times lower than time required for separation using conventional techniques. Other advantage of the developed method is the minimized ion suppression of LC-MS/MS analysis, which allowed detection of corticosteroids in sub μg/kg. Method was validated according to European Union (EU) Commission Decision 2002/657/EC. Measured parameters such as selectivity, linearity, recovery, within-laboratory reproducibility, decision limit, and detection capability satisfied the EU Directive. Ranges of mean recoveries and within-laboratory reproducibility were 81-100% and 8.0-20.5%, respectively. Decision limits were calculated in the range from 4.5 to 11.9 μg/kg for MRL compounds and varied from 0.1 to 0.2 μg/kg for banned substances. Limit of detections (LODs), calculated as three time signal-to-noise ratio, were in the range of 0.1-0.3 μg/kg. Copyright © 2010 Elsevier B.V. All rights reserved.
Matrix effects break the LC behavior rule for analytes in LC-MS/MS analysis of biological samples
USDA-ARS?s Scientific Manuscript database
High-performance liquid chromatography (HPLC) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) are generally accepted as the preferred techniques for detecting and quantitating analytes of interest in biological matrices on the basis of the rule that one chemical compound yields one LC-...
Confirmation by LC-MS of drugs in oral fluid obtained from roadside testing.
Concheiro, Marta; de Castro, Ana; Quintela, Oscar; Cruz, Angelines; López-Rivadulla, Manuel
2007-08-06
The aim of this study was to assess the effectiveness of two current on-site oral fluid (OF) drug detection devices (OraLab and Dräger), as part of the Spanish participation in the Roadside Testing Assessment Project (ROSITA Project). The study was done in collaboration with the Spanish Traffic Police, in Galicia (NW Spain), during 2004 and 2005. A total of 468 drivers selected at the police controls agreed to participate through informed consent. In addition, saliva samples were collected and sent to the laboratory to confirm the on-site results. For this purpose, two different analytical liquid chromatography-mass spectrometry (LC-MS) methods were used to detect 11 drugs or metabolites in a 300 microL sample. Simultaneous analysis of morphine, 6-acetylmorphine, amphetamine, methamphetamine, MDA, MDMA, MDEA, MBDB, cocaine and benzoylecgonine was carried out using 100 microL of oral fluid, after an automated solid phase extraction. A different LC-MS method was performed to detect Delta(9)-THC in 200 microL of oral fluid using liquid-liquid extraction with hexane at pH 6. Both methods were fully validated, including linearity (1-250 ng/mL, 2-250 ng/mL) recovery (>50%), within-day and between-day precision (CV<15%), accuracy (mean relative error<15%), limit of detection (0.5 and 1 ng/mL), quantitation (1 and 2 ng/mL) and matrix effect. All of the positive cases and a random selection of 30% of the negatives were analyzed for confirmation analysis. Good results (sensitivity, specificity, accuracy, positive predictive value and negative predictive value>90%) were obtained for cocaine and opiates by OraLab, and for cocaine by Dräger. However, the results for the other compounds could be improved for both detection devices. Differences in the ease of use and in the interpretation mode (visual or instrumental) were observed.
LC/MS Method for the Determination of Stable Isotope Labeled Promethazine in Human Plasma
NASA Technical Reports Server (NTRS)
Zuwei, Wang; Boyd, Jason; Berens, Kurt L.; Putcha, Lakshmi
2004-01-01
Promethazine (PMZ) is taken by astronauts orally (PO), intramuscularly (IM) or rectally (PR) for space motion sickness. LC/MS method was developed with off-line solid phase extraction to measure plasma concentrations of PMZ given as stable isotope-labeled (SIL) formulations by the three different routes of administration simultaneously. Samples (0.5ml) were loaded on to Waters Oasis HLB co-polymer cartridges and eluted with 1.0 mL methanol. HPLC separation of the eluted sample was performed using an Agilent Zorbax SB-CN column (50 x 2.1 mm) at a flow rate of 0.2 mL/min for 6 min. Acetonitrile/ ammonium acetate (30 mM) in water (3:2, v/v), pH 5.6 plus or minus 0.1, was used as the mobile phase for separation. Concentrations of PMZ, PMZ-d4 and PMZ-d7 and chlorpromazine (internal standard) were determined using a Micromass ZMD single quadrupole mass spectrometer with Electrospray Ionization (ESI). ESI mass spectra were acquired in positive ion mode with selected ion monitoring of [M+ H]dot plus. The method is rapid, reproducible and the assay specific parameters are listed in a table. A novel, sensitive and specific method for the measurement of PMZ and SIL PMZ in human plasma is reported.
Déglon, Julien; Versace, François; Lauer, Estelle; Widmer, Christèle; Mangin, Patrice; Thomas, Aurélien; Staub, Christian
2012-06-01
Dried blood spots (DBS) sampling has gained popularity in the bioanalytical community as an alternative to conventional plasma sampling, as it provides numerous benefits in terms of sample collection and logistics. The aim of this work was to show that these advantages can be coupled with a simple and cost-effective sample pretreatment, with subsequent rapid LC-MS/MS analysis for quantitation of 15 benzodiazepines, six metabolites and three Z-drugs. For this purpose, a simplified offline procedure was developed that consisted of letting a 5-µl DBS infuse directly into 100 µl of MeOH, in a conventional LC vial. The parameters related to the DBS pretreatment, such as extraction time or internal standard addition, were investigated and optimized, demonstrating that passive infusion in a regular LC vial was sufficient to quantitatively extract the analytes of interest. The method was validated according to international criteria in the therapeutic concentration ranges of the selected compounds. The presented strategy proved to be efficient for the rapid analysis of the selected drugs. Indeed, the offline sample preparation was reduced to a minimum, using a small amount of organic solvent and consumables, without affecting the accuracy of the method. Thus, this approach enables simple and rapid DBS analysis, even when using a non-DBS-dedicated autosampler, while lowering the costs and environmental impact.
Li, Jin; Zhang, Qiu-Hong; He, Jun; Liu, Er-wei; Gao, Xiu-mei; Chang, Yan-xu
2015-01-01
An improved LC-MS/MS method was developed for simultaneous determination of eleven bioactive constituents of Radix Angelicae Pubescentis and its related preparations. It was the first report on the quantification of bioactive constituents in different preparations of Radix Angelicae Pubescentis by LC-MS/MS analytical method. These samples were separated with an Agilent Zorbax Extend reversed-phase C18 column (1.8 μm, 4.6 × 100 mm) by linear gradient elution using aqueous ammonium acetate and acetonitrile as mobile phase. The flow rate was 0.3 mL min−1. The eleven bioactive constituents showed good regression (R > 0.990) within test ranges and the recoveries were in the range of 87.1–110%. The limit of detections and quantifications for most of the major constituents were less than 0.5 and 1.0 ng mL−1, respectively. All results indicated that the developed method could be readily utilized as a suitable quality control method for Radix Angelicae Pubescentis and related preparations. PMID:26078992
Comparative assessment of bioanalytical method validation guidelines for pharmaceutical industry.
Kadian, Naveen; Raju, Kanumuri Siva Rama; Rashid, Mamunur; Malik, Mohd Yaseen; Taneja, Isha; Wahajuddin, Muhammad
2016-07-15
The concepts, importance, and application of bioanalytical method validation have been discussed for a long time and validation of bioanalytical methods is widely accepted as pivotal before they are taken into routine use. United States Food and Drug Administration (USFDA) guidelines issued in 2001 have been referred for every guideline released ever since; may it be European Medical Agency (EMA) Europe, National Health Surveillance Agency (ANVISA) Brazil, Ministry of Health and Labour Welfare (MHLW) Japan or any other guideline in reference to bioanalytical method validation. After 12 years, USFDA released its new draft guideline for comments in 2013, which covers the latest parameters or topics encountered in bioanalytical method validation and approached towards the harmonization of bioanalytical method validation across the globe. Even though the regulatory agencies have general agreement, significant variations exist in acceptance criteria and methodology. The present review highlights the variations, similarities and comparison between bioanalytical method validation guidelines issued by major regulatory authorities worldwide. Additionally, other evaluation parameters such as matrix effect, incurred sample reanalysis including other stability aspects have been discussed to provide an ease of access for designing a bioanalytical method and its validation complying with the majority of drug authority guidelines. Copyright © 2016. Published by Elsevier B.V.
BMAA extraction of cyanobacteria samples: which method to choose?
Lage, Sandra; Burian, Alfred; Rasmussen, Ulla; Costa, Pedro Reis; Annadotter, Heléne; Godhe, Anna; Rydberg, Sara
2016-01-01
β-N-Methylamino-L-alanine (BMAA), a neurotoxin reportedly produced by cyanobacteria, diatoms and dinoflagellates, is proposed to be linked to the development of neurological diseases. BMAA has been found in aquatic and terrestrial ecosystems worldwide, both in its phytoplankton producers and in several invertebrate and vertebrate organisms that bioaccumulate it. LC-MS/MS is the most frequently used analytical technique in BMAA research due to its high selectivity, though consensus is lacking as to the best extraction method to apply. This study accordingly surveys the efficiency of three extraction methods regularly used in BMAA research to extract BMAA from cyanobacteria samples. The results obtained provide insights into possible reasons for the BMAA concentration discrepancies in previous publications. In addition and according to the method validation guidelines for analysing cyanotoxins, the TCA protein precipitation method, followed by AQC derivatization and LC-MS/MS analysis, is now validated for extracting protein-bound (after protein hydrolysis) and free BMAA from cyanobacteria matrix. BMAA biological variability was also tested through the extraction of diatom and cyanobacteria species, revealing a high variance in BMAA levels (0.0080-2.5797 μg g(-1) DW).
Wang, Qing; Zhao, Hua; Xi, Cunxian; Wang, Guomin; Chen, Dongdong; Ding, Shijia
2014-01-01
An immunoaffinity column clean-up and LC-MS/MS method was successfully developed for simultaneous determination of chloramphenicol, zearalanone, α-zearalanol, β-zearalanol, zearalenone, α-zearalenol and β-zearalenol in pig muscle. The sample was extracted with diethyl ether after enzymatic digestion by β-glucuronidase/sulfatase. The extracted solution was evaporated to dryness and the residue was then dissolved in 1 ml of 50% acetonitrile solution. After filtration and dilution with phosphate buffer solution (PBS), the reconstituted solution was cleaned-up with an IAC-CZ immunoaffinity column and then analysed by HPLC-MS/MS. The established method were validated by linearity (r ≥ 0.9990), precision (RSD ≥ 2.9%), average recovery (74.5-105.0%) and limit of detection (0.04-0.10 μg kg(-1)). The developed method is rapid, reliable, sensitive, accurate and has good applicability for real samples.
Plenis, Alina; Olędzka, Ilona; Bączek, Tomasz
2013-05-05
This paper focuses on a comparative study of the column classification system based on the quantitative structure-retention relationships (QSRR method) and column performance in real biomedical analysis. The assay was carried out for the LC separation of moclobemide and its metabolites in human plasma, using a set of 24 stationary phases. The QSRR models established for the studied stationary phases were compared with the column test performance results under two chemometric techniques - the principal component analysis (PCA) and the hierarchical clustering analysis (HCA). The study confirmed that the stationary phase classes found closely related by the QSRR approach yielded comparable separation for moclobemide and its metabolites. Therefore, the QSRR method could be considered supportive in the selection of a suitable column for the biomedical analysis offering the selection of similar or dissimilar columns with a relatively higher certainty. Copyright © 2013 Elsevier B.V. All rights reserved.
Bourgogne, Emmanuel; Culot, Benoit; Dell'Aiera, Sylvie; Chanteux, Hugues; Stockis, Armel; Nicolas, Jean-Marie
2018-06-01
Brivaracetam (BRV) is a new high affinity synaptic vesicle protein 2A ligand recently approved for adults with partial-onset seizures. As a support to in vitro metabolism assays, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method coupled to off-line solid phase extraction (SPE) was developed to quantify BRV acid metabolites, that is, BRV-AC (carboxylic derivative derived from BRV hydrolysis) and BRV-OHAC (corresponding to hydroxylated BRV-AC). The method was validated for various incubates (liver and kidney tissue homogenates and blood, all from humans) and applied to in vitro metabolism assays. The analytes were isolated from buffered samples using ISOLUTE C8 96-well SPE plates. Chromatographic separation was achieved on a Waters Atlantis T3 C18 analytical column (2.1 mm × 50 mm, 5 μm) with detection accomplished using a Waters Premier tandem mass spectrometer in positive ion electrospray and multiple reaction monitoring (MRM) mode. The standard curves, which ranged from 1.00 to 200 ng/mL for BRV-AC, BRV-OHAC, were fitted to a 1/x 2 weighted linear regression model. The intra-assay precision and inter-assay precision (expressed as coefficient of variation -%CV) were <8.5%, and the assay accuracy (deviation - %Dev) was within ±7.1% for the different matrices. This accurate, precise, and selective SPE/LC-MS/MS method has been successfully applied to in vitro assays aimed at characterizing the kinetics of BRV hydrolysis. BRV was found to be a better substrate for hydrolysis than its hydroxylated metabolite BRV-OH. BRV hydrolysis was detected in blood, liver and kidneys, demonstrating the broad distribution of the enzyme catalyzing the reaction. Copyright © 2018 Elsevier B.V. All rights reserved.
Lai, Kuan-Ming; Cheng, Yung-Yi; Tsai, Tung-Hu
2015-06-09
The herbal decoction process is generally inconvenient and unpleasant. To avoid using herbal medicine decoctions, various high-quality industrial and pharmaceutical herbal decoction products have been used in clinical applications for more than ten years in Taiwan. However, the consistency and standardization of the quality of these herbal medicines are goals that remain to be achieved. The aim of study was to develop a validated liquid chromatography-tandem electrospray ionization mass spectrometry (LC-MS/MS) method to determine the biomarkers astragaloside I, astragaloside IV, formononetin, cinnamic acid, paeoniflorin and gingerol in the herbal preparation known as Huangqi-Guizhi-Wuwu (HGW). To investigate the physical quality of HGW, methods such as scanning electron microscopy, light microscopy with Congo red and potassium iodine staining, solubility measurements, swelling power tests, and crude fiber analysis were used to identify additives in commercial pharmaceutical products. The optimal LC-MS/MS multiple reaction-monitoring system included a gradient program using 5 mM ammonium acetate buffer with 0.05% formic acid/methanol. The results demonstrate deviations in biomarker content across different brands. In addition to the herbal extract, starch and excipients in the pharmaceutical granule, and crushed crude herb powder was added to the pharmaceutical products to increase their herbal ingredient content. In conclusion, a rigorous examination should be performed to certify the quality of the herbal products.
Oliva, Alexis; Fariña, José B; Llabrés, Matías
2013-10-15
A simple and reproducible UPLC method was developed and validated for the quantitative analysis of finasteride in low-dose drug products. Method validation demonstrated the reliability and consistency of analytical results. Due to the regulatory requirements of pharmaceutical analysis in particular, evaluation of robustness is vital to predict how small variations in operating conditions affect the responses. Response surface methodology as an optimization technique was used to evaluate the robustness. For this, a central composite design was implemented around the nominal conditions. Statistical treatment of the responses (retention factor and drug concentrations expressed as percentage of label claim) showed that methanol content in mobile-phase and flow rate were the most influential factors. In the optimization process, the compromise decision support problem (cDSP) strategy was used. Construction of the robust domain from response-surfaces provided tolerance windows for the factors affecting the effectiveness of the method. The specified limits for the USP uniformity of dosage units assay (98.5-101.5%) and the purely experimental variations based on the repeatability test for center points (nominal conditions repetitions) were used as criteria to establish the tolerance windows, which allowed definition design space (DS) of analytical method. Thus, the acceptance criteria values (AV) proposed by the USP-uniformity of assay only depend on the sampling error. If the variation in the responses corresponded to approximately twice the repeatability standard deviation, individual values for percentage label claim (%LC) response may lie outside the specified limits; this implies the data are not centered between the specified limits, and that this term plus the sampling error affects the AV value. To avoid this fact, the limits specified by the Uniformity of Dosage Form assay (i.e., 98.5-101.5%) must be taken into consideration to fix the tolerance windows for each
Wang, Shunhai; Bobst, Cedric E.; Kaltashov, Igor A.
2018-01-01
Transferrin (Tf) is an 80 kDa iron-binding protein which is viewed as a promising drug carrier to target the central nervous system due to its ability to penetrate the blood-brain barrier (BBB). Among the many challenges during the development of Tf-based therapeutics, sensitive and accurate quantitation of the administered Tf in cerebrospinal fluid (CSF) remains particularly difficult due to the presence of abundant endogenous Tf. Herein, we describe the development of a new LC-MS based method for sensitive and accurate quantitation of exogenous recombinant human Tf in rat CSF. By taking advantage of a His-tag present in recombinant Tf and applying Ni affinity purification, the exogenous hTf can be greatly enriched from rat CSF, despite the presence of the abundant endogenous protein. Additionally, we applied a newly developed O18-labeling technique that can generate internal standards at the protein level, which greatly improved the accuracy and robustness of quantitation. The developed method was investigated for linearity, accuracy, precision and lower limit of quantitation, all of which met the commonly accepted criteria for bioanalytical method validation. PMID:26307718
Metz, Thomas O.; Zhang, Qibin; Page, Jason S.; Shen, Yufeng; Callister, Stephen J.; Jacobs, Jon M.; Smith, Richard D.
2008-01-01
SUMMARY The future utility of liquid chromatography-mass spectrometry (LC-MS) in metabolic profiling and metabolomic studies for biomarker discover will be discussed, beginning with a brief description of the evolution of metabolomics and the utilization of the three most popular analytical platforms in such studies: NMR, GC-MS, and LC-MS. Emphasis is placed on recent developments in high-efficiency LC separations, sensitive electrospray ionization approaches, and the benefits to incorporating both in LC-MS-based approaches. The advantages and disadvantages of various quantitative approaches are reviewed, followed by the current LC-MS-based tools available for candidate biomarker characterization and identification. Finally, a brief prediction on the future path of LC-MS-based methods in metabolic profiling and metabolomic studies is given. PMID:19177179
LC3/GABARAP family proteins: autophagy-(un)related functions.
Schaaf, Marco B E; Keulers, Tom G; Vooijs, Marc A; Rouschop, Kasper M A
2016-12-01
From yeast to mammals, autophagy is an important mechanism for sustaining cellular homeostasis through facilitating the degradation and recycling of aged and cytotoxic components. During autophagy, cargo is captured in double-membraned vesicles, the autophagosomes, and degraded through lysosomal fusion. In yeast, autophagy initiation, cargo recognition, cargo engulfment, and vesicle closure is Atg8 dependent. In higher eukaryotes, Atg8 has evolved into the LC3/GABARAP protein family, consisting of 7 family proteins [LC3A (2 splice variants), LC3B, LC3C, GABARAP, GABARAPL1, and GABARAPL2]. LC3B, the most studied family protein, is associated with autophagosome development and maturation and is used to monitor autophagic activity. Given the high homology, the other LC3/GABARAP family proteins are often presumed to fulfill similar functions. Nevertheless, substantial evidence shows that the LC3/GABARAP family proteins are unique in function and important in autophagy-independent mechanisms. In this review, we discuss the current knowledge and functions of the LC3/GABARAP family proteins. We focus on processing of the individual family proteins and their role in autophagy initiation, cargo recognition, vesicle closure, and trafficking, a complex and tightly regulated process that requires selective presentation and recruitment of these family proteins. In addition, functions unrelated to autophagy of the LC3/GABARAP protein family members are discussed.-Schaaf, M. B. E., Keulers, T. G, Vooijs, M. A., Rouschop, K. M. A. LC3/GABARAP family proteins: autophagy-(un)related functions. © FASEB.
Montoro, Paola; Maldini, Mariateresa; Russo, Mariateresa; Postorino, Santo; Piacente, Sonia; Pizza, Cosimo
2011-02-20
Liquid chromatography electrospray mass spectrometry (LC-ESI/MS) has been applied to the full characterization of saponins and phenolics in hydroalcoholic extracts of roots of liquorice (Glycyrrhiza glabra). Relative quantitative analyses of the samples with respect to the phenolic constituents and to a group of saponins related to glycyrrhizic acid were performed using LC-ESI/MS. For the saponin constituents, full scan LC-MS/MS fragmentation of the protonated (positive ion mode) or deprotonated (negative ion mode) molecular species generated diagnostic fragment ions that provided information concerning the triterpene skeleton and the number and nature of the substituents. On the basis of the specific fragmentation of glycyrrhizic acid, an LC-MS/MS method was developed in order to quantify the analyte in the liquorice root samples. Chinese G. glabra roots contained the highest levels of glycyrrhizic acid, followed by those from Italy (Calabria). Copyright © 2010 Elsevier B.V. All rights reserved.
Abu-Awwad, Ahmad; Arafat, Tawfiq; Schmitz, Oliver J
2016-09-01
Nicotine (Nic) distribution in human fluids and tissues has a deleterious effect on human health. In addition to its poisoning profile, Nic may contribute to the particular impact of smoking on human reproduction. Although present in seminal fluid, still nobody knows whether nicotine is available in sperm or not. Herein, we developed and validated a new bioanalytical method, for simultaneous determination of Nic, cotinine (Cot), and nicotine N'-oxide (Nox) in human plasma, semen, and sperm by LC-ESI-orbitrap-MS. Blood and semen samples were collected from 12 healthy smoking volunteers in this study. Sperm bodies were then separated quantitatively from 1 mL of semen samples by centrifugation. The developed method was fully validated for plasma following European and American guidelines for bioanalytical method validation, and partial validation was applied to semen analysis. Plasma, semen, and sperm samples were treated by trichloroacetic acid solution for protein direct precipitation in single extraction step. The established calibration range for Nic and Nox in plasma and semen was linear between 5 and 250 ng/mL, and for Cot between 10 and 500 ng/mL. Nic and Cot were detected in human sperm at concentrations as high as in plasma. In addition, Nox was present in semen and sperm but not in plasma. Graphical abstract Nicotine correlation between plasma and semen a; Nicotine correlation between semen and sperm c; Cotinine correlation between plasma and semen b; Cotinine correlation between semen and sperm d.
Zhang, Xing; Wang, Xiaoming; Vernikovskaya, Daria I; Fokina, Valentina M; Nanovskaya, Tatiana N; Hankins, Gary D V; Ahmed, Mahmoud S
2015-04-01
This report describes the development and validation of an LC-MS/MS method for the quantitative determination of glyburide (GLB), its five metabolites (M1, M2a, M2b, M3 and M4) and metformin (MET) in plasma and urine of pregnant patients under treatment with a combination of the two medications. The extraction recovery of the analytes from plasma samples was 87-99%, and that from urine samples was 85-95%. The differences in retention times among the analytes and the wide range of the concentrations of the medications and their metabolites in plasma and urine patient samples required the development of three LC methods. The lower limit of quantitation (LLOQ) of the analytes in plasma samples was as follows: GLB, 1.02 ng/mL; its five metabolites, 0.100-0.113 ng/mL; and MET, 4.95 ng/mL. The LLOQ in urine samples was 0.0594 ng/mL for GLB, 0.984-1.02 ng/mL for its five metabolites and 30.0 µg/mL for MET. The relative deviation of this method was <14% for intra-day and inter-day assays in plasma and urine samples, and the accuracy was 86-114% in plasma, and 94-105% in urine. The method described in this report was successfully utilized for determining the concentrations of the two medications in patient plasma and urine. Copyright © 2014 John Wiley & Sons, Ltd.
Zhang, Xing; Wang, Xiaoming; Vernikovskaya, Daria I.; Fokina, Valentina M.; Nanovskaya, Tatiana N.; Hankins, Gary D.V.; Ahmed, Mahmoud S.
2014-01-01
This report describes the development and validation of an LC-MS/MS method for the quantitative determination of glyburide (GLB), its five metabolites (M1, M2a, M2b, M3, and M4) and metformin (MET) in plasma and urine of pregnant patients under treatment with a combination of the two medications. The extraction recovery of the analytes from plasma samples ranged between 87% and 99%, and 85%–95% for urine samples. The differences in retention times among the analytes, and the wide range of the concentrations of the medications and their metabolites in plasma and urine patient samples required the development of three LC methods. The lower limit of quantitation (LLOQ) of the analytes in plasma samples was as follows: GLB, 1.02 ng/mL; its five metabolites, 0.100–0.113 ng/mL and 4.95 ng/mL for MET. LLOQ in urine samples was 0.0594 ng/mL for GLB, 0.984–1.02 ng/mL for its five metabolites and 30.0 μg/mL for MET. The relative deviation of this method was < 14% for intra-day and inter-day assays in plasma and urine samples, and the accuracy ranged between 86% and 114% in plasma, and 94% to 105% in urine. The method described in this report was successfully utilized for determining the concentrations of the two medications in patient plasma and urine. PMID:25164921
ASTM Validates Air Pollution Test Methods
ERIC Educational Resources Information Center
Chemical and Engineering News, 1973
1973-01-01
The American Society for Testing and Materials (ASTM) has validated six basic methods for measuring pollutants in ambient air as the first part of its Project Threshold. Aim of the project is to establish nationwide consistency in measuring pollutants; determining precision, accuracy and reproducibility of 35 standard measuring methods. (BL)
Chip-LC-MS for label-free profiling of human serum.
Horvatovich, Peter; Govorukhina, Natalia I; Reijmers, Theo H; van der Zee, Ate G J; Suits, Frank; Bischoff, Rainer
2007-12-01
The discovery of biomarkers in easily accessible body fluids such as serum is one of the most challenging topics in proteomics requiring highly efficient separation and detection methodologies. Here, we present the application of a microfluidics-based LC-MS system (chip-LC-MS) to the label-free profiling of immunodepleted, trypsin-digested serum in comparison to conventional capillary LC-MS (cap-LC-MS). Both systems proved to have a repeatability of approximately 20% RSD for peak area, all sample preparation steps included, while repeatability of the LC-MS part by itself was less than 10% RSD for the chip-LC-MS system. Importantly, the chip-LC-MS system had a two times higher resolution in the LC dimension and resulted in a lower average charge state of the tryptic peptide ions generated in the ESI interface when compared to cap-LC-MS while requiring approximately 30 times less (~5 pmol) sample. In order to characterize both systems for their capability to find discriminating peptides in trypsin-digested serum samples, five out of ten individually prepared, identical sera were spiked with horse heart cytochrome c. A comprehensive data processing methodology was applied including 2-D smoothing, resolution reduction, peak picking, time alignment, and matching of the individual peak lists to create an aligned peak matrix amenable for statistical analysis. Statistical analysis by supervised classification and variable selection showed that both LC-MS systems could discriminate the two sample groups. However, the chip-LC-MS system allowed to assign 55% of the overall signal to selected peaks against 32% for the cap-LC-MS system.
Quantitative method for the determination of Iso-fludelone (KOS-1803) in human plasma by LC-MS/MS
Christner, Susan M.; Parise, Robert A.; Levine, Erica D.; Rizvi, Naiyer A.; Gounder, Mrinal M.; Beumer, Jan H.
2014-01-01
Epothilones are relatively new tubulin-poison anticancer drugs. Iso-fludelone (KOS-1803) is a synthetic third generation epothilone drug discovered at Memorial Sloan Kettering Cancer Center, and currently in Phase I clinical trials. We report an LC-MS/MS assay for the sensitive, accurate and precise quantitation of Iso-fludelone in 0.2 mL of human plasma. Validation was performed according to FDA guidance. The assay comprised of KOS-1724 as the internal standard and an MTBE liquid-liquid extraction with a water wash step. Separation was achieved with an YMC-Pack ODS-AQ column and an isocratic mobile phase of 0.1% formic acid in acetonitrile and water (70:30, v/v) at 0.3 mL/min for 4 min. Chromatographic separation was followed by electrospray, positive-mode ionization tandem mass spectrometric detection in the multiple reaction monitoring (MRM) mode. The assay was linear from 0.1– 300 ng/mL and was accurate (−9.41–7.07%) and precise (1.03–13.7%) which fulfilled FDA criteria for validation. Recovery from plasma was 73.9–79.7% and ion suppression was negligible (−22.8 to −31.3%). Plasma freeze thaw stability (99.97–105.7%), stability for 11 months at −80 °C (94.93–107.9%), and stability for 6 h at room temperature (94.75–105.5%) were all acceptable. This assay is currently being applied to quantitate Iso-fludelone in clinical samples. PMID:25168219
Quantitative method for the determination of iso-fludelone (KOS-1803) in human plasma by LC-MS/MS.
Christner, Susan M; Parise, Robert A; Levine, Erica D; Rizvi, Naiyer A; Gounder, Mrinal M; Beumer, Jan H
2014-11-01
Epothilones are relatively new tubulin-poison anticancer drugs. Iso-fludelone (KOS-1803) is a synthetic third generation epothilone drug discovered at Memorial Sloan Kettering Cancer Center, and currently in phase I clinical trials. We report an LC-MS/MS assay for the sensitive, accurate and precise quantitation of iso-fludelone in 0.2mL of human plasma. Validation was performed according to FDA guidance. The assay comprised of KOS-1724 as the internal standard and an MTBE liquid-liquid extraction with a water wash step. Separation was achieved with an YMC-Pack ODS-AQ column and an isocratic mobile phase of 0.1% formic acid in acetonitrile and water (70:30, v/v) at 0.3mL/min for 4min. Chromatographic separation was followed by electrospray, positive-mode ionization tandem mass spectrometric detection in the multiple reaction monitoring (MRM) mode. The assay was linear from 0.1 to 300ng/mL and was accurate (-9.41 to -7.07%) and precise (1.03-13.7%) which fulfilled FDA criteria for validation. Recovery from plasma was 73.9-79.7% and ion suppression was negligible (-22.8 to -31.3%). Plasma freeze-thaw stability (99.97-105.7%), stability for 11 months at -80°C (94.93-107.9%), and stability for 6h at room temperature (94.75-105.5%) were all acceptable. This assay is currently being applied to quantitate iso-fludelone in clinical samples. Copyright © 2014 Elsevier B.V. All rights reserved.
Huang, Rongrong; Pomin, Vitor H.; Sharp, Joshua S.
2011-01-01
Improved methods for structural analyses of glycosaminoglycans (GAGs) are required to understand their functional roles in various biological processes. Major challenges in structural characterization of complex GAG oligosaccharides using liquid chromatography-mass spectrometry (LC-MS) include the accurate determination of the patterns of sulfation due to gas-phase losses of the sulfate groups upon collisional activation and inefficient on-line separation of positional sulfation isomers prior to MS/MS analyses. Here, a sequential chemical derivatization procedure including permethylation, desulfation, and acetylation was demonstrated to enable both on-line LC separation of isomeric mixtures of chondroitin sulfate (CS) oligosaccharides and accurate determination of sites of sulfation by MSn. The derivatized oligosaccharides have sulfate groups replaced with acetyl groups, which are sufficiently stable to survive MSn fragmentation and reflect the original sulfation patterns. A standard reversed-phase LC-MS system with a capillary C18 column was used for separation, and MSn experiments using collision-induced dissociation (CID) were performed. Our results indicate that the combination of this derivatization strategy and MSn methodology enables accurate identification of the sulfation isomers of CS hexasaccharides with either saturated or unsaturated nonreducing ends. Moreover, derivatized CS hexasaccharide isomer mixtures become separable by LC-MS method due to different positions of acetyl modifications. PMID:21953261
Yuan, Long; Ji, Qin C
2018-06-05
Metabolite interferences represent a major risk of inaccurate quantification when using LC-MS/MS bioanalytical assays. During LC-MS/MS bioanalysis of BMS-919194, a phosphate ester prodrug, in plasma samples from rat and monkey GLP toxicology studies, an unknown peak was detected in the MRM channel of the prodrug. This peak was not observed in previous discovery toxicology studies, in which a fast gradient LC-MS/MS method was used. We found out that this unknown peak would co-elute with the prodrug peak when the discovery method was used, therefore, causing significant overestimation of the exposure of the prodrug in the discovery toxicology studies. To understand the nature of this interfering peak and its impact to bioanalytical assay, we further investigated its formation and identification. The interfering compound and the prodrug were found to be isobaric and to have the same major product ions in electrospray ionization positive mode, thus, could not be differentiated using a triple quadrupole mass spectrometer. By using high-resolution mass spectrometry (HRMS), the interfering metabolite was successfully identified to be an isobaric sulfate metabolite of BMS-919194. To the best of our knowledge, this is the first report that a phosphate prodrug was metabolized in vivo to an isobaric sulfate metabolite, and this metabolite caused significant interference to the analysis of the prodrug. This work demonstrated the presence of the interference risk from isobaric sulfate metabolites to the bioanalysis of phosphate prodrugs in real samples. It is critical to evaluate and mitigate potential metabolite interferences during method development, therefore, minimize the related bioanalytical risks and ensure assay quality. Our work also showed the unique advantages of HRMS in identifying potential metabolite interference during LC-MS/MS bioanalysis. Copyright © 2018 Elsevier B.V. All rights reserved.
Gu, Haiwei; Huang, Yuan; Filgueira, Marcelo; Carr, Peter W.
2012-01-01
In this study, we examined the effect of first dimension column selectivity in reversed phase (RP) online comprehensive two dimensional liquid chromatography (LC × LC). The second dimension was always a carbon clad metal oxide reversed phase material. The hydrophobic subtraction model (HSM) and the related phase selective triangles were used to guide the selection of six different RP first dimension columns. Various kinds of samples were investigated and thus two different elution conditions were needed to cause full elution from the first dimension columns. We compared LC × LC chromatograms, contours plots, and fcoverage plots by measuring peak capacities, peak numbers, relative spatial coverage, correlation values, etc. The major finding of this study is that the carbon phase due to its rather different selectivity from other reversed phases is reasonably orthogonal to a variety of common types of bonded reversed phases. Thus quite surprisingly the six different first dimension stationary phases all showed generally similar separation patterns when paired to the second dimension carbon phase. This result greatly simplifies the task of choosing the correct pair of phases for RP × RP. PMID:21840009
Teijlingen, Raymond van; Meijer, John; Takusagawa, Shin; Gelderen, Marcel van; Beld, Cas van den; Usui, Takashi
2012-03-01
Mirabegron is being developed for the treatment of overactive bladder. To support the development of mirabegron, including pharmacokinetic studies, liquid chromatography/tandem mass spectrometry methods for mirabegron and eight metabolites (M5, M8, M11-M16) were developed and validated for heparinized human plasma containing sodium fluoride. Four separate bioanalytical methods were developed for the analysis of: (1) mirabegron; (2) M5 and M16; (3) M8; and (4) M11-M15. Either solid-phase extraction or liquid-liquid extraction was used to extract the analytes of interest from matrix constituents. For mirabegron, an Inertsil C₈-3 analytical column was used and detection was performed using a triple-quad mass spectrometer equipped with an atmospheric pressure chemical ionization interface. For the metabolite assays, chromatographic separation was performed through a Phenomenex Synergi Fusion-RP C₁₈ analytical column and detection was performed using a triple-quad mass spectrometer equipped with a Heated Electrospray Ionization interface. The validation results demonstrated that the developed liquid chromatography/tandem mass spectrometry methods were precise, accurate, and selective for the determination of mirabegron and its metabolites in human plasma. All methods were successfully applied in evaluating the pharmacokinetic parameters of mirabegron and metabolites in human plasma. Copyright © 2012 Elsevier B.V. All rights reserved.
Determination of toxins involved in ciguatera fish poisoning in the Pacific by LC/MS.
Yogi, Kentaro; Sakugawa, Satsuki; Oshiro, Naomasa; Ikehara, Tsuyoshi; Sugiyama, Kiminori; Yasumoto, Takeshi
2014-01-01
Ciguatera fish poisoning is the most extensive and difficult to control of the seafood poisonings. To facilitate monitoring of fish toxicity, toxin profiles were investigated by an LC/MS/MS method using 14 reference toxins on eight representative species of fish collected in four different areas of the Pacific. Snappers and groupers from Okinawa contained ciguatoxin-1B (CTX1B) and two deoxy congeners at variable but species-specific ratios, while red snapper, Lutjanus bohar, from Minamitorishima, and amberjack, Seriola dumerili, from Hawaii, contained both CTX1B-type and CTX3C-type toxins. Spotted knifejaw, Oplegnathus punctatus, from Okinawan waters, contained mainly CTX4A and CTX4B, but the same species caught at Miyazaki was contaminated primarily with the CTX3C-type toxins. Otherwise, the toxin profiles were consistently species-specific in fish collected from various locations around Okinawa over 20 years. The LC/MS/MS and mouse bioassay results agreed well, indicating the LC/MS/MS method is a promising alternative to the mouse bioassay. Pure CTX1B and CTX3C were prepared for use in future LC/MS/MS analysis.
Analysis of cocaine/crack biomarkers in meconium by LC-MS.
D'Avila, Felipe Bianchini; Ferreira, Pâmela C Lukasewicz; Salazar, Fernanda Rodrigues; Pereira, Andrea Garcia; Santos, Maíra Kerpel Dos; Pechansky, Flavio; Limberger, Renata Pereira; Fröehlich, Pedro Eduardo
2016-02-15
Fetal exposure to illicit drugs is a worldwide problem, since many addicted women do not stop using it during pregnancy. Cocaine consumed in powdered (snorted or injected) or smoked (crack cocaine) form are harmful for the baby and its side effects are not completely known. Meconium, the first stool of a newborn, is a precious matrix usually discarded, that may contain amounts of substances consumed in the last two trimesters of pregnancy. Analyzing this biological matrix it is possible to detect the unaltered molecule of cocaine (COC) or its metabolite benzoylecgonine (BZE) and pyrolytic products anhydroecgonine methyl ester (AEME) and anhydroecgonine (AEC). A liquid chromatography mass spectrometry (LC-MS) method was validated for meconium samples after solvent extraction, followed by direct injection of 10μL. Linearity covered a concentration range of 15 to 500ng/mg with a lower limit of quantification (LLOQ) of 15ng/mg for all analytes. Matrix effect was evaluated and showed adequate results. Detection of illicit substances usage can be crucial for the baby, since knowing that can help provide medical care as fast as possible. The method proved to be simple and fast, and was applied to 17 real meconium samples. Copyright © 2016 Elsevier B.V. All rights reserved.
Method and platform standardization in MRM-based quantitative plasma proteomics.
Percy, Andrew J; Chambers, Andrew G; Yang, Juncong; Jackson, Angela M; Domanski, Dominik; Burkhart, Julia; Sickmann, Albert; Borchers, Christoph H
2013-12-16
There exists a growing demand in the proteomics community to standardize experimental methods and liquid chromatography-mass spectrometry (LC/MS) platforms in order to enable the acquisition of more precise and accurate quantitative data. This necessity is heightened by the evolving trend of verifying and validating candidate disease biomarkers in complex biofluids, such as blood plasma, through targeted multiple reaction monitoring (MRM)-based approaches with stable isotope-labeled standards (SIS). Considering the lack of performance standards for quantitative plasma proteomics, we previously developed two reference kits to evaluate the MRM with SIS peptide approach using undepleted and non-enriched human plasma. The first kit tests the effectiveness of the LC/MRM-MS platform (kit #1), while the second evaluates the performance of an entire analytical workflow (kit #2). Here, these kits have been refined for practical use and then evaluated through intra- and inter-laboratory testing on 6 common LC/MS platforms. For an identical panel of 22 plasma proteins, similar concentrations were determined, regardless of the kit, instrument platform, and laboratory of analysis. These results demonstrate the value of the kit and reinforce the utility of standardized methods and protocols. The proteomics community needs standardized experimental protocols and quality control methods in order to improve the reproducibility of MS-based quantitative data. This need is heightened by the evolving trend for MRM-based validation of proposed disease biomarkers in complex biofluids such as blood plasma. We have developed two kits to assist in the inter- and intra-laboratory quality control of MRM experiments: the first kit tests the effectiveness of the LC/MRM-MS platform (kit #1), while the second evaluates the performance of an entire analytical workflow (kit #2). In this paper, we report the use of these kits in intra- and inter-laboratory testing on 6 common LC/MS platforms. This
Klepacki, Jacek; Brunner, Nina; Schmitz, Volker; Klawitter, Jelena; Christians, Uwe; Klawitter, Jost
2013-06-05
Although increased levels of S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) have been implicated as markers for renal and vascular dysfunction, until now there have been no studies investigating their association with clinical post-transplant events such as organ rejection and immunosuppressant nephrotoxicity. A newly developed and validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for the quantification of SAM and SAH in human EDTA plasma was used for a clinical proof-of-concept pilot study. Retrospective analysis was performed using samples from a longitudinal clinical study following de novo kidney transplant patients for the first year (n=16). The ranges of reliable response were 8 to 1024 nmol/l for SAM and 16 to 1024 nmol/l for SAH. The inter-day accuracies were 96.7-103.9% and 97.9-99.3% for SAM and SAH, respectively. Inter-day imprecisions were 8.1-9.1% and 8.4-9.8%. The total assay run time was 5 min. SAM and SAH concentrations were significantly elevated in renal transplant patients preceding documented acute rejection and nephrotoxicity events when compared to healthy controls (n=8) as well as transplant patients void of allograft dysfunction (n=8). The LC-MS/MS assay will provide the basis for further large-scale clinical studies to explore these thiol metabolites as molecular markers for the management of renal transplant patients. Copyright © 2013 Elsevier B.V. All rights reserved.
Development of a SIDA-LC-MS/MS Method for the Determination of Phomopsin A in Legumes.
Schloß, Svenja; Koch, Matthias; Rohn, Sascha; Maul, Ronald
2015-12-09
A novel method for the determination of phomopsin A (1) in lupin flour, pea flour, and bean flour as well as whole lupin plants was established based on stable isotope dilution assay (SIDA) LC-MS/MS using (15)N6-1 as an isotopically labeled internal standard. Artificially infected samples were used to develop an optimized extraction procedure and sample pretreatment. The limits of detection were 0.5-1 μg/kg for all matrices. The limits of quantitation were 2-4 μg/kg. The method was used to analyze flour samples generated from selected legume seeds and lupin plant samples that had been inoculated with Diaporthe toxica and two further fungal strains. Finally, growing lupin plants infected with D. toxica were investigated to simulate a naturally in-field mycotoxicosis. Toxin levels of up to 10.1 μg/kg of 1 were found in the pods and 7.2 μg/kg in the stems and leaves.
Screening of 23 β-lactams in foodstuffs by LC-MS/MS using an alkaline QuEChERS-like extraction.
Bessaire, Thomas; Mujahid, Claudia; Beck, Andrea; Tarres, Adrienne; Savoy, Marie-Claude; Woo, Pei-Mun; Mottier, Pascal; Desmarchelier, Aurélien
2018-04-01
A fast and robust high performance LC-MS/MS screening method was developed for the analysis of β-lactam antibiotics in foods of animal origin: eggs, raw milk, processed dairy ingredients, infant formula, and meat- and fish-based products including baby foods. QuEChERS extraction with some adaptations enabled 23 drugs to be simultaneously monitored. Screening target concentrations were set at levels adequate to ensure compliance with current European, Chinese, US and Canadian regulations. The method was fully validated according to the European Community Reference Laboratories Residues Guidelines using 93 food samples of different composition. False-negative and false-positive rates were below 5% for all analytes. The method is adequate for use in high-routine laboratories. A 1-year study was additionally conducted to assess the stability of the 23 analytes in the working standard solution.
[Simultaneous determination of pesticide residues in agricultural products by LC-MS/MS].
Watanabe, Minae; Ueno, Eiji; Inoue, Tomomi; Ohno, Haruka; Ikai, Yoshitomo; Morishita, Toshio; Oshima, Harumi; Hayashi, Rumiko
2013-01-01
A method for the simultaneous determination of multiple pesticide residues in agricultural products was developed by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). The sample was extracted with acetonitrile. Co-extractives were removed by GPC/graphitized carbon column SPE, and silica gel/PSA cartridge column SPE. Pesticides in the test solution were determined by LC-MS/MS using scheduled MRM. Recoveries of 124 pesticides from spinach, brown rice, soybean, orange and tomato were tested at the level of 0.1 µg/g, and those of 121 pesticides ranged from 70 to 120% (RSD≤15%). Pesticide residues in 239 agricultural products were investigated by this method, and residues of 49 pesticides were detected in 98 agricultural products.
Hurtaud-Pessel, D; Jagadeshwar-Reddy, T; Verdon, E
2011-10-01
A liquid chromatography-high resolution mass spectrometry (LC-HRMS) method was developed for screening meat for a wide range of antibiotics used in veterinary medicine. Full-scan mode under high resolution mass spectral conditions using an LTQ-Orbitrap mass spectrometer with resolving power 60,000 full width at half maximum (FWHM) was applied for analysis of the samples. Samples were prepared using two extraction protocols prior to LC-HRMS analysis. The scope of the method focuses on screening the following main families of antibacterial veterinary drugs: penicillins, cephalosporins, sulfonamides, macrolides, tetracyclines, aminoglucosides and quinolones. Compounds were successfully identified in spiked samples from their accurate mass and LC retention times from the acquired full-scan chromatogram. Automated data processing using ToxId software allowed rapid treatment of the data. Analyses of muscle tissues from real samples collected from antibiotic-treated animals was carried out using the above methodology and antibiotic residues were identified unambiguously. Further analysis of the data for real samples allowed the identification of the targeted antibiotic residues but also non-targeted compounds, such as some of their metabolites.
Jang, Moonhee; Kim, Jihyun; Han, Inhoi; Yang, Wonkyung
2015-11-10
Lysergic acid diethylamide (LSD) is administered in low dosages, which makes its detection in biological matrices a major challenge in forensic toxicology. In this study, two sensitive and reliable methods based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) were established and validated for the simultaneous determination of LSD and its metabolite, 2-oxo-3-hydroxy-LSD (O-H-LSD), in hair and urine. Target analytes in hair were extracted using methanol at 38°C for 15h and analyzed by LC-MS/MS. For urine sample preparation, liquid-liquid extraction was performed. Limits of detection (LODs) in hair were 0.25pg/mg for LSD and 0.5pg/mg for O-H-LSD. In urine, LODs were 0.01 and 0.025ng/ml for LSD and O-H-LSD, respectively. Method validation results showed good linearity and acceptable precision and accuracy. The developed methods were applied to authentic specimens from two legal cases of LSD ingestion, and allowed identification and quantification of LSD and O-H-LSD in the specimens. In the two cases, LSD concentrations in hair were 1.27 and 0.95pg/mg; O-H-LSD was detected in one case, but its concentration was below the limit of quantification. In urine samples collected from the two suspects 8 and 3h after ingestion, LSD concentrations were 0.48 and 2.70ng/ml, respectively, while O-H-LSD concentrations were 4.19 and 25.2ng/ml, respectively. These methods can be used for documenting LSD intake in clinical and forensic settings. Copyright © 2015 Elsevier B.V. All rights reserved.
Toward a Unified Validation Framework in Mixed Methods Research
ERIC Educational Resources Information Center
Dellinger, Amy B.; Leech, Nancy L.
2007-01-01
The primary purpose of this article is to further discussions of validity in mixed methods research by introducing a validation framework to guide thinking about validity in this area. To justify the use of this framework, the authors discuss traditional terminology and validity criteria for quantitative and qualitative research, as well as…
Sullards, M. Cameron; Liu, Ying; Chen, Yanfeng; Merrill, Alfred H.
2011-01-01
Sphingolipids are a highly diverse category of molecules that serve not only as components of biological structures but also as regulators of numerous cell functions. Because so many of the structural features of sphingolipids give rise to their biological activity, there is a need for comprehensive or “sphingolipidomic” methods for identification and quantitation of as many individual subspecies as possible. This review defines sphingolipids as a class, briefly discusses classical methods for their analysis, and focuses primarily on liquid chromatography tandem mass spectrometry (LC-MS/MS) and tissue imaging mass spectrometry (TIMS). Recently, a set of evolving and expanding methods have been developed and rigorously validated for the extraction, identification, separation, and quantitation of sphingolipids by LC-MS/MS. Quantitation of these biomolecules is made possible via the use of an internal standard cocktail. The compounds that can be readily analyzed are free long-chain (sphingoid) bases, sphingoid base 1-phosphates, and more complex species such as ceramides, ceramide 1-phosphates, sphingomyelins, mono- and di-hexosylceramides sulfatides, and novel compounds such as the 1-deoxy- and 1-(deoxymethyl)-sphingoid bases and their N-acyl-derivatives. These methods can be altered slightly to separate and quantitate isomeric species such as glucosyl/galactosylceramide. Because these techniques require the extraction of sphingolipids from their native environment, any information regarding their localization in histological slices is lost. Therefore, this review also describes methods for TIMS. This technique has been shown to be a powerful tool to determine the localization of individual molecular species of sphingolipids directly from tissue slices. PMID:21749933
DOE Office of Scientific and Technical Information (OSTI.GOV)
2013-03-07
LC-IMS-MS Feature Finder is a command line software application which searches for possible molecular ion signatures in multidimensional liquid chromatography, ion mobility spectrometry, and mass spectrometry data by clustering deisotoped peaks with similar monoisotopic mass values, charge states, elution times, and drift times. The software application includes an algorithm for detecting multiple conformations and co-eluting species in the ion mobility dimension. LC-IMS-MS Feature Finder is designed to create an output file with detected features that includes associated information about the detected features.
Nonclinical dose formulation analysis method validation and sample analysis.
Whitmire, Monica Lee; Bryan, Peter; Henry, Teresa R; Holbrook, John; Lehmann, Paul; Mollitor, Thomas; Ohorodnik, Susan; Reed, David; Wietgrefe, Holly D
2010-12-01
Nonclinical dose formulation analysis methods are used to confirm test article concentration and homogeneity in formulations and determine formulation stability in support of regulated nonclinical studies. There is currently no regulatory guidance for nonclinical dose formulation analysis method validation or sample analysis. Regulatory guidance for the validation of analytical procedures has been developed for drug product/formulation testing; however, verification of the formulation concentrations falls under the framework of GLP regulations (not GMP). The only current related regulatory guidance is the bioanalytical guidance for method validation. The fundamental parameters for bioanalysis and formulation analysis validations that overlap include: recovery, accuracy, precision, specificity, selectivity, carryover, sensitivity, and stability. Divergence in bioanalytical and drug product validations typically center around the acceptance criteria used. As the dose formulation samples are not true "unknowns", the concept of quality control samples that cover the entire range of the standard curve serving as the indication for the confidence in the data generated from the "unknown" study samples may not always be necessary. Also, the standard bioanalytical acceptance criteria may not be directly applicable, especially when the determined concentration does not match the target concentration. This paper attempts to reconcile the different practices being performed in the community and to provide recommendations of best practices and proposed acceptance criteria for nonclinical dose formulation method validation and sample analysis.
de Jager, Lowri S; Perfetti, Gracia A; Diachenko, Gregory W
2007-03-23
A LC-MS method was developed for the determination of coumarin, vanillin, and ethyl vanillin in vanilla products. Samples were analyzed using LC-electrospray ionization (ESI)-MS in the positive ionization mode. Limits of detection for the method ranged from 0.051 to 0.073 microg mL(-1). Using the optimized method, 24 vanilla products were analyzed. All samples tested negative for coumarin. Concentrations ranged from 0.38 to 8.59 mg mL(-1) (x =3.73) for vanillin and 0.33 to 2.27 mg mL(-1) (x =1.03) for ethyl vanillin. The measured concentrations are compared to values calculated using UV monitoring and to results reported in a similar survey in 1988. Analytical results, method precision, and accuracy data are presented.
Nucleocytoplasmic Distribution and Dynamics of the Autophagosome Marker EGFP-LC3
Drake, Kimberly R.; Kang, Minchul; Kenworthy, Anne K.
2010-01-01
The process of autophagy involves the formation of autophagosomes, double-membrane structures that encapsulate cytosol. Microtubule-associated protein light chain 3 (LC3) was the first protein shown to specifically label autophagosomal membranes in mammalian cells, and subsequently EGFP-LC3 has become one of the most widely utilized reporters of autophagy. Although LC3 is currently thought to function primarily in the cytosol, the site of autophagosome formation, EGFP-LC3 often appears to be enriched in the nucleoplasm relative to the cytoplasm in published fluorescence images. However, the nuclear pool of EGFP-LC3 has not been specifically studied in previous reports, and mechanisms by which LC3 shuttles between the cytoplasm and nucleoplasm are currently unknown. In this study, we therefore investigated the regulation of the nucleo-cytoplasmic distribution of EGFP-LC3 in living cells. By quantitative fluorescence microscopy analysis, we demonstrate that soluble EGFP-LC3 is indeed enriched in the nucleus relative to the cytoplasm in two commonly studied cell lines, COS-7 and HeLa. Although LC3 contains a putative nuclear export signal (NES), inhibition of active nuclear export or mutation of the NES had no effect on the nucleo-cytoplasmic distribution of EGFP-LC3. Furthermore, FRAP analysis indicates that EGFP-LC3 undergoes limited passive nucleo-cytoplasmic transport under steady state conditions, and that the diffusional mobility of EGFP-LC3 was substantially slower in the nucleus and cytoplasm than predicted for a freely diffusing monomer. Induction of autophagy led to a visible decrease in levels of soluble EGFP-LC3 relative to autophagosome-bound protein, but had only modest effects on the nucleo-cytoplasmic ratio or diffusional mobility of the remaining soluble pools of EGFP-LC3. We conclude that the enrichment of soluble EGFP-LC3 in the nucleus is maintained independently of active nuclear export or induction of autophagy. Instead, incorporation of soluble
Boix, C; Ibáñez, M; Fabregat-Safont, D; Morales, E; Pastor, L; Sancho, J V; Sánchez-Ramírez, J E; Hernández, F
2016-01-01
In this work, two analytical methodologies based on liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) were developed for quantification of emerging pollutants identified in sewage sludge after a previous wide-scope screening. The target list included 13 emerging contaminants (EC): thiabendazole, acesulfame, fenofibric acid, valsartan, irbesartan, salicylic acid, diclofenac, carbamazepine, 4-aminoantipyrine (4-AA), 4-acetyl aminoantipyrine (4-AAA), 4-formyl aminoantipyrine (4-FAA), venlafaxine and benzoylecgonine. The aqueous and solid phases of the sewage sludge were analyzed making use of Solid-Phase Extraction (SPE) and UltraSonic Extraction (USE) for sample treatment, respectively. The methods were validated at three concentration levels: 0.2, 2 and 20 μg L(-1) for the aqueous phase, and 50, 500 and 2000 μg kg(-1) for the solid phase of the sludge. In general, the method was satisfactorily validated, showing good recoveries (70-120%) and precision (RSD < 20%). Regarding the limit of quantification (LOQ), it was below 0.1 μg L(-1) in the aqueous phase and below 50 μg kg(-1) in the solid phase for the majority of the analytes. The method applicability was tested by analysis of samples from a wider study on degradation of emerging pollutants in sewage sludge under anaerobic digestion. The key benefits of these methodologies are: • SPE and USE are appropriate sample procedures to extract selected emerging contaminants from the aqueous phase of the sewage sludge and the solid residue. • LC-MS/MS is highly suitable for determining emerging contaminants in both sludge phases. • Up to our knowledge, the main metabolites of dipyrone had not been studied before in sewage sludge.
Striegel, Lisa; Chebib, Soraya; Netzel, Michael E.; Rychlik, Michael
2018-01-01
Folates play an important role in the human body and a deficiency of this vitamin can cause several diseases. Therefore, a reliable analytical method is crucial for the determination of folate vitamers in strawberries and other dietary folate sources. A stable isotope dilution LC-MS/MS method for analyzing folates in food was developed and validated. The folate vitamers Pteroylmonoglutamic acid, tetrahydrofolate, 5-methyltetrahydrofolate, and 5-formyltetrahydrofolate were quantified using 13C-labeled internal standards. Validation of the assay was accomplished by determining linearity, precision, recovery, limit of detection, and limit of quantification and revealed to be a precise, sensitive, and accurate method to determine folate vitamers. Strawberries are worldwide consumed and known to be a good dietary source of nutritive compounds. Using this method, folate concentrations in selected commercial strawberry cultivars and experimental breeding lines grown in Germany and Australia were investigated. Total folates varied from 59 to 153 μg/100 g on fresh weight basis. Furthermore, folate content after lyophilizing or freezing did not show any significant differences compared to fresh strawberries. However, significant losses of total folates in pureed strawberries could be observed after 5 days of storage with only 16% of the original concentration retained. In summary, some of the investigated strawberry cultivars/breeding lines can be considered as rich dietary sources of natural folates. PMID:29468149
NASA Astrophysics Data System (ADS)
Striegel, Lisa; Chebib, Soraya; Netzel, Michael E.; Rychlik, Michael
2018-02-01
Folates play an important role in the human body and a deficiency of this vitamin can cause several diseases. Therefore, a reliable analytical method is crucial for the determination of folate vitamers in strawberries and other dietary folate sources. A stable isotope dilution LC-MS/MS method for analyzing folates in food was developed and validated. The folate vitamers Pteroylmonoglutamic acid, tetrahydrofolate, 5-methyltetrahydrofolate and 5-formyltetrahydrofolate were quantified using 13C-labelled internal standards. Validation of the assay was accomplished by determining linearity, precision, recovery, limit of detection and limit of quantification and revealed to be a precise, sensitive and accurate method to determine folate vitamers. Strawberries are worldwide consumed and known to be a good dietary source of nutritive compounds. Using this method, folate concentrations in selected commercial strawberry cultivars and experimental breeding lines grown in Germany were investigated. Total folates varied from 59 to 153 µg/100 g on fresh weight basis. Furthermore, folate content after lyophilizing or freezing did not show any significant differences compared to fresh strawberries. However, significant losses of total folates in pureed strawberries could be observed after 5 days of storage with only 16 % of the original concentration retained. In summary, some of the investigated strawberry cultivars/breeding lines can be considered as rich dietary sources of natural folates.
Preprocessing and Analysis of LC-MS-Based Proteomic Data
Tsai, Tsung-Heng; Wang, Minkun; Ressom, Habtom W.
2016-01-01
Liquid chromatography coupled with mass spectrometry (LC-MS) has been widely used for profiling protein expression levels. This chapter is focused on LC-MS data preprocessing, which is a crucial step in the analysis of LC-MS based proteomics. We provide a high-level overview, highlight associated challenges, and present a step-by-step example for analysis of data from LC-MS based untargeted proteomic study. Furthermore, key procedures and relevant issues with the subsequent analysis by multiple reaction monitoring (MRM) are discussed. PMID:26519169
Yang, Yang; Zhang, Yongmin; Wei, Chong; Li, Jing; Sun, Wenji
2018-09-01
Silver ion chromatography, utilizing columns packed with silver ions bonded to silica gel, has proved to be an invaluable technique for the analysis of some positional isomers. In this work, silver ion chromatography by combination with online heart-cutting LC-LC technique for the preparative separation of two sesquiterpenes positional isomers from a natural product was investigated. On the basis of the evaluation that silver ion content impacts on the separation, the laboratory-made silver ion columns, utilizing silica gel impregnated with 15% silver nitrate as column packing materials, were used for peak resolution improvement of these two isomers and the preparative separation of them in heart-cutting LC-LC. The relationship among the maximal sample load, flow rate and peak resolution in the silver ion column were optimized, and the performance of the silver ion column was compared with conventional C 18 column and silica gel column. Based on the developed chromatographic conditions, online heart-cutting LC-LC chromatographic separation system in combination with a silica gel column and a silver ion column that was applied to preparative separation of these two isomers from a traditional Chinese medicine, Inula racemosa Hook.f., was established. The results showed that the online heart-cutting LC-LC technique by combination of a silica gel column and a silver ion column for the preparative separation of these two positional isomers from this natural plant was superior to the preparative separation performed on a single-column system with C 18 column or silica gel column. Copyright © 2018 Elsevier B.V. All rights reserved.
Structural features of LC8-induced self-association of swallow.
Kidane, Ariam I; Song, Yujuan; Nyarko, Afua; Hall, Justin; Hare, Michael; Löhr, Frank; Barbar, Elisar
2013-09-03
Cell functions depend on the collective activity of protein networks within which a few proteins, called hubs, participate in a large number of interactions. Dynein light chain LC8, first discovered as a subunit of the motor protein dynein, is considered to have a role broader than that of dynein, and its participation in diverse systems fits the description of a hub. Among its partners is Swallow with which LC8 is essential for proper localization of bicoid mRNA at the anterior cortex of Drosophila oocytes. Why LC8 is essential in this process is not clear, but emerging evidence suggests that LC8 functions by promoting self-association and/or structural organization of its diverse binding partners. This work addresses the energetics and structural features of LC8-induced Swallow self-association distant from LC8 binding. Mutational design based on a hypothetical helical wheel, intermonomer nuclear Overhauser effects assigned to residues expected at interface positions, and circular dichroism spectral characteristics indicate that the LC8-promoted dimer of Swallow is a coiled coil. Secondary chemical shifts and (15)N backbone relaxation identify the boundaries and distinguishing structural features of the coiled coil. Thermodynamic analysis of Swallow polypeptides designed to decouple self-association from LC8 binding reveals that the higher binding affinity of the engineered bivalent Swallow is of purely entropic origin and that the linker separating the coiled coil from the LC8 binding site remains disordered. We speculate that the LC8-promoted coiled coil is critical for bicoid mRNA localization because it favors structural organization of Swallow, which except for the central LC8-promoted coiled coil is primarily disordered.
Structural Features of LC8-Induced Self Association of Swallow†
Kidane, Ariam I.; Song, Yujuan; Nyarko, Afua; Hall, Justin; Hare, Michael; Löhr, Frank; Barbar, Elisar
2013-01-01
Cell function depends on the collective activity of protein networks within which a few proteins, called hubs, participate in a large number of interactions. Dynein light chain LC8, first discovered as a subunit of the motor protein dynein, is considered to have a role broader than dynein and its participation in diverse systems fits the description of a hub. Among its partners is Swallow with which LC8 is essential for proper localization of bicoid mRNA at the anterior cortex of Drosophila oocytes. Why LC8 is essential in this process is not clear, but emerging evidence suggests that LC8 functions by promoting self-association and/or structural organization of its diverse binding partners. This work addresses the mechanistic and structural features of LC8-induced Swallow self-association distant from LC8 binding. Mutational design based on a hypothetical helical wheel, inter-monomer NOEs assigned to residues expected at interface positions and circular dichroism spectral characteristics indicate that the LC8-promoted dimer of Swallow is a coiled-coil. Secondary chemical shifts and 15N backbone relaxation identify the boundaries and distinguishing structural features of the coiled-coil. Thermodynamic analysis of Swallow polypeptides designed to decouple self-association from LC8 binding reveals that the higher binding affinity of the engineered bivalent Swallow is of purely entropic origin and that the linker separating the coiled-coil from the LC8 binding site remains disordered. We speculate that the LC8-promoted coiled-coil is critical for bicoid mRNA localization because it could induce structural organization of Swallow, which except for the central LC8-promoted coiled-coil is primarily disordered. PMID:23914803
de-Torres, Juan P; Wilson, David O; Sanchez-Salcedo, Pablo; Weissfeld, Joel L; Berto, Juan; Campo, Arantzazu; Alcaide, Ana B; García-Granero, Marta; Celli, Bartolome R; Zulueta, Javier J
2015-02-01
Patients with chronic obstructive pulmonary disease (COPD) are at high risk for lung cancer (LC) and represent a potential target to improve the diagnostic yield of screening programs. To develop a predictive score for LC risk for patients with COPD. The Pamplona International Early Lung Cancer Detection Program (P-IELCAP) and the Pittsburgh Lung Screening Study (PLuSS) databases were analyzed. Only patients with COPD on spirometry were included. By logistic regression we determined which factors were independently associated with LC in PLuSS and developed a COPD LC screening score (COPD-LUCSS) to be validated in P-IELCAP. By regression analysis, age greater than 60, body mass index less than 25 kg/m(2), pack-years history greater than 60, and emphysema presence were independently associated with LC diagnosis and integrated into the COPD-LUCSS, which ranges from 0 to 10 points. Two COPD-LUCSS risk categories were proposed: low risk (scores 0-6) and high risk (scores 7-10). In comparison with low-risk patients, in both cohorts LC risk increased 3.5-fold in the high-risk category. The COPD-LUCSS is a good predictor of LC risk in patients with COPD participating in LC screening programs. Validation in two different populations adds strength to the findings.
Model-Based Method for Sensor Validation
NASA Technical Reports Server (NTRS)
Vatan, Farrokh
2012-01-01
Fault detection, diagnosis, and prognosis are essential tasks in the operation of autonomous spacecraft, instruments, and in situ platforms. One of NASA s key mission requirements is robust state estimation. Sensing, using a wide range of sensors and sensor fusion approaches, plays a central role in robust state estimation, and there is a need to diagnose sensor failure as well as component failure. Sensor validation can be considered to be part of the larger effort of improving reliability and safety. The standard methods for solving the sensor validation problem are based on probabilistic analysis of the system, from which the method based on Bayesian networks is most popular. Therefore, these methods can only predict the most probable faulty sensors, which are subject to the initial probabilities defined for the failures. The method developed in this work is based on a model-based approach and provides the faulty sensors (if any), which can be logically inferred from the model of the system and the sensor readings (observations). The method is also more suitable for the systems when it is hard, or even impossible, to find the probability functions of the system. The method starts by a new mathematical description of the problem and develops a very efficient and systematic algorithm for its solution. The method builds on the concepts of analytical redundant relations (ARRs).