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Sample records for ms quantification method

  1. A stable isotope dilution LC-ESI-MS/MS method for the quantification of pyridoxal-5'-phosphate in whole blood.

    PubMed

    van Zelst, Bertrand D; de Jonge, Robert

    2012-08-15

    Vitamin B6 is a cofactor in numerous biologic processes that include gluconeogenesis, neurotransmitter synthesis and amino acid metabolism. The aim of this study was to develop a method to measure the concentration of the biologically active form of vitamin B6 (pyridoxal-5'-phosphate, PLP) in whole blood with stable isotope dilution LC-ESI-MS/MS and compare this new procedure with an established HPLC method based on derivatization of pyridoxal-5'-phosphate. 50 μl of stable isotope (PLP-d3) was added to 250 μl of sample, followed by deproteinization with 10% trichloroacetic acid. After centrifugation, 20 μl of the supernatant was injected into the LC-ESI-MS/MS. Reversed phase chromatography was performed on a UPLC system, using a Waters™ Symmetry C18 column, with a gradient of 0.1% formic acid in methanol. PLP was measured on a tandem MS with a mass transition of 247.8>149.8 in the positive ion mode with a collision energy of 14 eV. The chromatographic run lasted 4 min. The method was linear from 4 to 8000 nmol/l. The intra-day and inter-day precision ranged between 1.7-2.8% and 3.0-4.1%, respectively. The mean absolute matrix-effect was 99.3% [97-102%]. The relative matrix-effect was 98.8%. The mean recovery was 98% [89-103%]. The lower limit of quantification was 4 nmol/l. The comparison of the LC-ESI-MS/MS method with our current HPLC method yielded the following equation: LC-ESI-MS/MS=1.11 [confidence interval, CI: 1.03-1.20] × HPLC+4.6 [CI: -1.3 to 11.0] (r²=0.94). This LC-ESI-MS/MS based method is characterized by simple sample processing and a short run time. The comparison with the current HPLC method is excellent although a significant proportional bias was detected. To conclude, the LC-ESI-MS/MS method is an appropriate method to determine PLP in whole blood.

  2. GC-MS/MS method for the quantification of α-cedrene in rat plasma and its pharmacokinetic application.

    PubMed

    Hong, Joo Yeon; Lee, Byung Ho; Kim, Tae Hwan; Hong, Jongki; Lee, Kyoung Mee; Yoo, Sun Dong; Lee, Hye Suk

    2013-11-01

    α-Cedrene is a pharmacologically active ingredient isolated from the essential oil of cedar. A selective and sensitive GC-MS/MS method was developed for the quantification of α-cedrene in rat plasma for the first time. α-Cedrene was extracted from rat plasma using ethyl acetate at neutral pH. The analytes were determined in selective reaction monitoring mode using MS/MS: m/z 204.3→119.0 for α-cedrene and m/z 146.0→111.0 for 1,4-dichlorobenzene (internal standard). The standard curve was linear (r(2) ≥ 0.995) over the concentration ranges of 5-800 ng/mL. The lower limit of quantification was 5 ng/mL using 50 μL of rat plasma. The coefficient of variation and relative error for intra- and interassays at four quality control levels were 3.1-13.9% and -4.0-2.6%, respectively. The stability of processing (freeze-thaw, long-term storage at -80°C, and short-term storage at room temperature) and chromatography (reinjection) was shown to be of insignificant effect. The present method was applied successfully to the pharmacokinetic study of α-cedrene after its intravenous (10 mg/kg) and oral (25 mg/kg) administration in male Sprague-Dawley rats. PMID:23996797

  3. Multiclass method for pesticides quantification in honey by means of modified QuEChERS and UHPLC-MS/MS.

    PubMed

    Tette, Patrícia Amaral Souza; da Silva Oliveira, Fabiano Aurélio; Pereira, Elba Nathália Corrêa; Silva, Gilsara; de Abreu Glória, Maria Beatriz; Fernandes, Christian

    2016-11-15

    Bee products can be produced in an environment contaminated by pesticides that can be transported by honey bees to the hive and incorporated into the honey. Therefore, rapid and modern methods to determine pesticide residues in honey samples are essential to guarantee consumers' health. In this study, a simple multiresidue method for the quantification of 116 pesticides in honey is proposed. It involves the use of a modified QuEChERS procedure followed by UHPLC-MS/MS analysis. The method was validated according to the European Union SANCO/12571/2013 guidelines. Acceptable values were obtained for the following parameters: linearity, limit of detection (0.005mg/kg) and limit of quantification (0.010 and 0.025mg/kg), trueness (for the four tested levels the recovery assays values were between 70 and 120%), intermediate precision (RSD<20.0%) and measurement uncertainty tests (<50.0%). The validated method was applied for determination of 100 honey samples from five states of Brazil. PMID:27283616

  4. Multiclass method for pesticides quantification in honey by means of modified QuEChERS and UHPLC-MS/MS.

    PubMed

    Tette, Patrícia Amaral Souza; da Silva Oliveira, Fabiano Aurélio; Pereira, Elba Nathália Corrêa; Silva, Gilsara; de Abreu Glória, Maria Beatriz; Fernandes, Christian

    2016-11-15

    Bee products can be produced in an environment contaminated by pesticides that can be transported by honey bees to the hive and incorporated into the honey. Therefore, rapid and modern methods to determine pesticide residues in honey samples are essential to guarantee consumers' health. In this study, a simple multiresidue method for the quantification of 116 pesticides in honey is proposed. It involves the use of a modified QuEChERS procedure followed by UHPLC-MS/MS analysis. The method was validated according to the European Union SANCO/12571/2013 guidelines. Acceptable values were obtained for the following parameters: linearity, limit of detection (0.005mg/kg) and limit of quantification (0.010 and 0.025mg/kg), trueness (for the four tested levels the recovery assays values were between 70 and 120%), intermediate precision (RSD<20.0%) and measurement uncertainty tests (<50.0%). The validated method was applied for determination of 100 honey samples from five states of Brazil.

  5. Rapid method for quantification of seven synthetic pigments in colored Chinese steamed buns using UFLC-MS/MS without SPE.

    PubMed

    Gao, He-Gang; Gong, Wen-Jie; Zhao, Yong-Gang

    2015-01-01

    Synthetic pigments are still used instead of natural pigments in many foods and their residues in food could be an important risk to human health. A simple and rapid analytical method combining the low-cost extraction protocol with ultra-fast liquid chromatography-tandem quadrupole mass spectrometry (UFLC-MS/MS) was developed for the simultaneous determination of seven synthetic pigments used in colored Chinese steamed buns. For the first time, ethanol/ammonia solution/water (7:2:1, v/v/v) was used as extraction solution for the synthetic pigments in colored Chinese steamed buns. The results showed that the property of the extraction solution used in this method was more effective than critic acid solution, which is used in the polyamide adsorption method. The limits of quantification for the seven synthetic pigments ranged from 0.15 to 0.50 μg/kg. The present method was successfully applied to samples of colored Chinese steamed buns for food-safety risk monitoring in Zhejiang Province, China. The results found sunset yellow pigment in six out of 300 colored Chinese steamed buns (from 0.50 to 32.6 μg/kg).

  6. A sensitive LC-MS/MS method for the quantification of febuxostat in human plasma and its pharmacokinetic application.

    PubMed

    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.

  7. Rapid and simple UPLC-MS/MS method for precise phytochelatin quantification in alga extracts.

    PubMed

    Bräutigam, Anja; Wesenberg, Dirk; Preud'homme, Hugues; Schaumlöffel, Dirk

    2010-09-01

    Quantitative phytochelatin (PC) analysis is, due to oxidation sensitivity of the PCs, matrix effects, and time consuming sample preparation, still a challenging analytical task. In this study, a rapid, simple, and sensitive method for precise determination of native PCs in crude extracts of the green alga Chlamydomonas reinhardtii was developed. Algae were exposed 48 h to 70 μM Cd. Coupling of ultra performance liquid chromatography and electrospray ionization tandem mass spectrometry with multi-reaction mode transitions for detection permitted the required short-time, high-resolution separation and detection specificity. Thus, under optimized chromatographic conditions, 10 thiol peptides were baseline-separated within 7 min. Relative detection limits in the nanomolar range in microliter sample volumes were achieved (corresponding to absolute detection limits at femtomole level). Next to glutathione (GSH), the most abundant cadmium-induced PCs in C. reinhardtii, namely CysGSH, PC(2), PC(3), CysPC(2), and CysPC(3), were quantified with high reproducibility at concentrations between 15 and 198 nmol g(-1) fresh weight. The biological variation of PC synthesis of nine independently grown alga cultures was determined to be on average 13.7%. PMID:20632163

  8. Development and validation of UHPLC-MS/MS methods for the quantification of colistin in plasma and dried plasma spots.

    PubMed

    Cangemi, Giuliana; Barco, Sebastiano; Castagnola, Elio; Tripodi, Gino; Favata, Fabio; D'Avolio, Antonio

    2016-09-10

    Quantification of colistin in plasma samples may be very useful in optimizing therapy especially in special patients' population. Nevertheless, therapeutic drug monitoring of colistin is still limited probably for the low number of laboratories which perform this analysis and for high shipment costs. We developed and validated new UHPLC-MS/MS methods to quantify colistin in plasma and in dried plasma spots (DPS) collected on dried sample spots devices (DSSD). Colistin A, Colistin B and polimixin B, used as internal standard, were detected using multiple reaction monitoring (MRM) of the following specific transitions: 585.5→534.9; 576, 578.5→527.9; 568.9 and 602.5→100.9, 551.9, 592.8, respectively. Colistin A and B were extracted from plasma using protein precipitation and from DSSD using an extraction basic solution. Both methods were validated, and the mean intra and inter-day accuracies and precisions were in accordance with FDA and EMA guidelines. Colistin in DPS was found to be stable for at least one week at room temperature (20-25°C). A statistically significant linear correlation was found between colistin extracted from plasma and from DPS [r(2) 0.9864 (P<0.0001, 95% CI 0.9699-0.9939) for colistin A and 0.9695 (P<0.0001, 95% CI 0.9310-0.9866) for colistin B, respectively]. DPS on DSSD represents a safe and cheap strategy to store and ship at room temperature plasma samples. Thus, it is suited for pharmacokinetic studies and therapeutic drug monitoring of colistin. PMID:27505127

  9. Development and validation of a sensitive UPLC-ESI-MS/MS method for the simultaneous quantification of 15 endocannabinoids and related compounds in milk and other biofluids.

    PubMed

    Gouveia-Figueira, Sandra; Nording, Malin L

    2014-01-21

    The endocannabinoid (eCB) system has gained an increasing interest over the past decades since the discovery of anandamide and 2-arachidonoyl glycerol (2-AG). These, and structurally related compounds, are associated with a wide variety of physiological processes. For instance, eCB levels in milk have been associated with infants' feeding and sleeping behavior. A method based on ultraperformance liquid chromatography coupled with electrospray ionization tandem mass spectrometry (UPLC-ESI-MS/MS) was developed and validated for the simultaneous quantification of 15 eCBs and related compounds, including both fatty acid amides and glycerols. Linearity (0.9845 < R(2) < 1), limit of detection and quantification (0.52-293 pg on column), inter- and intraday accuracy (>70%) and precision (CV < 15%), stability, and recovery (in milk and plasma) were established in accordance to the U.S. Food and Drug Administration guidelines. The method was successfully applied to bovine and elk milk revealing species-specific eCB profiles, with significant different levels of 2-AG, 2-linoleoyl glycerol, docosahexaenoyl ethanolamide, palmitoyl ethanolamide, and oleoyl ethanolamide. Furthermore, stearoyl ethanolamide and docosatetraenoyl ethanolamide were only detected in elk milk. In summary, our UPLC-ESI-MS/MS method may be used for quantification of eCBs and related compounds in different biofluids and applied to investigations of the role of these emerging compounds in various physiological processes. PMID:24377270

  10. A validated SPME-GC-MS method for simultaneous quantification of club drugs in human urine.

    PubMed

    Brown, Stacy D; Rhodes, Daniel J; Pritchard, Boyd J

    2007-09-13

    A solid-phase microextraction-gas chromatographic-mass spectrometric (SPME-GC-MS) method has been developed and validated for measuring four club drugs in human urine. These drugs include gamma-hydroxybutyrate (GHB), ketamine (KET), methamphetamine (MAMP), and methylenedioxymethamphetamine (MDMA). These drugs are referred to as 'club drugs' because of their prevalence at parties and raves. Deuterium labeled internal standards for each of the four drugs was included in the assay to aid in quantitation. The drugs were spiked into human urine and derivatized using pyridine and hexylchloroformate to make them suitable for GC-MS analysis. The SPME conditions of extraction time/temperature and desorption time/temperature were optimized to yield the highest peak area for each of the four drugs. The final SPME parameters included a 90 degrees C extraction for 20min with a 1min desorption in the GC injector at 225 degrees C using a splitless injection. All SPME work was done using a 100microm PDMS fiber by Supelco. The ratio of pyridine to hexylchloroformate for derivatization was also optimized. The GC separation was carried out on a VF-5ht column by Varian (30m, 0.25mm i.d., 0.10microm film thickness) using a temperature program of 150-270 degrees C at 10 degrees C/min. The instrument used was a ThermoFinnigan Trace GC-Polaris Q interfaced with a LEAP CombiPal autosampler. The data was collected by using extracted ion chromatograms of marker m/z values for each drug from the total ion chromatograms (TIC) (full scan mode). Calibration curves with R(2)>0.99 were generated each day using the peak area ratios (peak area drug/peak area internal standard) versus concentration. The validated method resulted in intra-day and inter-day precision (% R.S.D.) of less than 15% and a % error of less than 15% for four concentrations in the range of 0.05-20microg/mL (MAMP) and 0.10-20microg/mL (GHB, KET, and MDMA). This method has the advantage of an easy sample preparation with

  11. Development and Validation of an LC-MS/MS Method for the Quantification of Agaritine in Mushrooms.

    PubMed

    Merdivan, Simon; Willke, Christoph; Lindequist, Ulrike

    2016-01-01

    Agaritine, an aromatic hydrazine, is found as a secondary metabolite in mushroom species. It is among others suspected to exhibit genotoxic activity. This publication describes the validation of a method for the quantification of agaritine in mushrooms (i.e., extraction and purification by solid phase extraction) and measurement by liquid chromatography with tandem mass spectrometry detection in positive ionization mode. The results show this method to be selective, accurate, and precise. This method could be used for the quality control of pharmaceutical preparations containing mushrooms. PMID:27279441

  12. Validated LC-MS/MS Method for the Quantification of Ponatinib in Plasma: Application to Metabolic Stability

    PubMed Central

    Kadi, Adnan A.; Darwish, Hany W.; Attwa, Mohamed W.; Amer, Sawsan M.

    2016-01-01

    In the current work, a rapid, specific, sensitive and validated liquid chromatography tandem mass-spectrometric method was developed for the quantification of ponatinib (PNT) in human plasma and rat liver microsomes (RLMs) with its application to metabolic stability. Chromatographic separation of PNT and vandetanib (IS) were accomplished on Agilent eclipse plus C18 analytical column (50 mm × 2.1 mm, 1.8 μm particle size) maintained at 21±2°C. Flow rate was 0.25 mLmin-1 with run time of 4 min. Mobile phase consisted of solvent A (10 mM ammonium formate, pH adjusted to 4.1 with formic acid) and solvent B (acetonitrile). Ions were generated by electrospray (ESI) and multiple reaction monitoring (MRM) was used as basis for quantification. The results revealed a linear calibration curve in the range of 5–400 ngmL-1 (r2 ≥ 0.9998) with lower limit of quantification (LOQ) and lower limit of detection (LOD) of 4.66 and 1.53 ngmL-1 in plasma, 4.19 and 1.38 ngmL-1 in RLMs. The intra- and inter-day precision and accuracy in plasma ranged from1.06 to 2.54% and -1.48 to -0.17, respectively. Whereas in RLMs ranged from 0.97 to 2.31% and -1.65 to -0.3%. The developed procedure was applied for quantification of PNT in human plasma and RLMs for study metabolic stability of PNT. PNT disappeared rapidly in the 1st 10 minutes of RLM incubation and the disappearance plateaued out for the rest of the incubation. In vitro half-life (t1/2) was 6.26 min and intrinsic clearance (CLin) was 15.182± 0.477. PMID:27764191

  13. A highly sensitive quantification method for the accumulation of alarmone ppGpp in Arabidopsis thaliana using UPLC-ESI-qMS/MS.

    PubMed

    Ihara, Yuta; Ohta, Hiroyuki; Masuda, Shinji

    2015-05-01

    Recently, a bacterial second messenger, guanosine 5'-diphosphate 3'-diphosphate (ppGpp), has been detected in chloroplasts. However, because ppGpp concentration in plants is much lower than that in bacteria, detailed analysis of ppGpp in plants has not been performed. A highly sensitive quantification method is required for further characterization of ppGpp function in chloroplasts. Here, we report a new method that allows for the highly sensitive and selective high-throughput quantification of ppGpp by ultra-performance liquid chromatography (UPLC) coupled with a tandem quadrupole mass spectrometer (qMS/MS) equipped with an electrospray interface (ESI). This method requires only ~100 mg of plant tissue for ppGpp quantification. We used this method to measure ppGpp levels in Arabidopsis thaliana under different light conditions. A. thaliana accumulated ppGpp during dark periods. This method will be helpful to further characterize the stringent response in higher plants.

  14. Rapid method for quantification of nine sulfonamides in bovine milk using HPLC/MS/MS and without using SPE.

    PubMed

    Nebot, Carolina; Regal, Patricia; Miranda, Jose Manuel; Fente, Cristina; Cepeda, Alberto

    2013-12-01

    Sulfonamides are antimicrobial agents widely employed in animal production and their residues in food could be an important risk to human health. In the dairy industry, large quantities of milk are monitored daily for the presence of sulfonamides. A simple and low-cost extraction protocol followed by a liquid chromatographic-tandem mass spectrometry method was developed for the simultaneous detection of nine sulfonamides in whole milk. The method was validated at the maximum residue limits established by European legislation. The limits of quantification obtained for most sulfonamides were between 12.5 and 25 μg kg(-1), detection capabilities ranged from 116 to 145 μg kg(-1), and recoveries, at 100 μg kg(-1), were greater than 89±12.5%. The method was employed to analyse 100 raw whole bovine milk samples collected from dairy farms in the northwest region of Spain. All of the samples were found to be compliant, but two were positive; one for sulfadiazine and the other for sulfamethoxipyridazine.

  15. Quantification of sofosbuvir and ledipasvir in human plasma by UPLC-MS/MS method: Application to fasting and fed bioequivalence studies.

    PubMed

    Rezk, Mamdouh R; Bendas, Ehab R; Basalious, Emad B; Karim, Iman A

    2016-08-15

    A rapid and sensitive LC-MS/MS method was developed, optimized and validated for quantification of sofosbuvir (SF) and ledipasvir (LD) in human plasma using eplerenone as an internal standard (IS). Analytes and IS were extracted from plasma by simple liquid-liquid extraction technique using methyl tertiary butyl ether. The prepared samples were chromatographed on Acquity UPLC BEH C18 column. Separation was done using a mobile phase formed of 0.1% formic acid and acetonitrile (50:50, v/v) in an isocratic mode at a flow rate of 0.4ml/min. The Xevo TQD LC-MS/MS was operated under the multiple-reaction monitoring mode using electrospray ionization. A full validation of the method was performed according to the FDA guidelines. Linearity was found to be in the range of 0.25-3500ng/ml for SF and 5-2000ng/ml for LD. The intra-day and inter-day precision and accuracy results were within the acceptable limits. A short run time of 2min allows analysis of more than 400 plasma samples per day. The developed method was successfully applied to both fasting and fed bioequivalence studies in healthy human volunteers. PMID:27322631

  16. Bioanalytical LC-MS Method for the Quantification of Plasma Androgens and Androgen Glucuronides in Breast Cancer.

    PubMed

    Kalogera, Eleni; Pistos, Constantinos; Provatopoulou, Xeni; Christophi, Costas A; Zografos, George C; Stefanidou, Maria; Spiliopoulou, Chara; Athanaselis, Sotirios; Gounaris, Antonia

    2016-04-01

    The physiological and pathological development of the breast is strongly affected by the hormonal milieu consisting of steroid hormones. Mass spectrometry (MS) technologies of high sensitivity and specificity enable the quantification of androgens and consequently the characterization of the hormonal status. The aim of this study is the assessment of plasma androgens and androgen glucuronides, in the par excellence hormone-sensitive tissue of the breast, through the application of liquid chromatography-mass spectrometry (LC-MS). A simple and efficient fit-for-purpose method for the simultaneous identification and quantification of dehydroepiandrosterone sulfate (DHEAS), androstenedione (A4), androsterone glucuronide (ADTG) and androstane-3α, 17β-diol-17-glucuronide (3α-diol-17G) in human plasma was developed and validated. The presented method permits omission of derivatization, requires a single solid-phase extraction procedure and the chromatographic separation can be achieved on a single C18 analytical column, for all four analytes. The validated method was successfully applied for the analysis of 191 human plasma samples from postmenopausal women with benign breast disease (BBD), lobular neoplasia (LN), ductal carcinoma in situ and invasive ductal carcinoma (IDC). DHEAS plasma levels exhibited significant differences between LN, IDC and BBD patients (P < 0.05). Additionally, ADTG levels were significantly higher in patients with LN compared with those with BBD (P < 0.05). PMID:26762957

  17. Development and validation of an UPLC-MS/MS method for the quantification of tamoxifen and its main metabolites in human scalp hair.

    PubMed

    Drooger, Jan C; Jager, Agnes; Lam, Mei-Ho; den Boer, Mathilda D; Sleijfer, Stefan; Mathijssen, Ron H J; de Bruijn, Peter

    2015-10-10

    The aim of this study was to validate an earlier developed high-performance highly sensitive ultra performance liquid chromatography/tandem mass spectrometry (UPLC-MS/MS) method for quantification of tamoxifen and its three main metabolites (N-desmethyl-tamoxifen, 4-hydroxy-tamoxifen and 4-hydroxy-N-desmethyl-tamoxifen) in scalp hair. This non-invasive method might, by segmental analysis of hair, be useful in the determination of the concentration of drugs and its metabolites over time, which can be used to study a wide variety of clinical relevant questions. Hair samples (150-300 hair strands, cut as close to the scalp as possible from the posterior vertex region of the head) were collected from female patients taking tamoxifen 20mg daily (n=19). The analytes were extracted using a liquid-liquid extraction procedure with carbonate buffer at pH 8.8 and a mixture of n-hexane/isopropranol method, followed by UPLC-MS/MS chromatography, based on an earlier validated method. The calibration curves were linear in the range of 1.00-200 pmol for tamoxifen and N-desmethyl-tamoxifen, with lower limit of quantitation of 1.00 pmol and 0.100-20.0 pmol with lower limit of quantitation of 0.100 pmol for endoxifen and 4-hydroxy-tamoxifen. Assay performance was fair with a within-run and between-run variability less than 9.24 at the three quality control samples and less than 15.7 for the lower limit of quantitation. Importantly, a steep linear decline was observed from distal to proximal hair segments. Probably, this is due to UV exposure as we showed degradation of tamoxifen and its metabolites after exposure to UV-light. Furthermore, higher concentrations of tamoxifen were found in black hair samples compared to blond and brown hair samples. We conclude that measurement of the concentration of tamoxifen and its main metabolites in hair is possible, with the selective, sensitive, accurate and precise UPLC-MS/MS method. However, for tamoxifen, it seems not possible to determine

  18. Method development for quantification of the environmental neurotoxin annonacin in Rat plasma by UPLC-MS/MS and application to a pharmacokinetic study.

    PubMed

    Bonneau, Natacha; Schmitz-Afonso, Isabelle; Brunelle, Alain; Touboul, David; Champy, Pierre

    2015-11-01

    Annonacin is an environmental neurotoxin identified in the pulp of several fruits of the Annonaceae family (for example in Annona muricata, Asimina triloba), whose consumption was linked with the occurrence of sporadic atypical Parkinsonism with dementia. Pharmacokinetic parameters of this molecule are unknown. A method for its quantification in Rat plasma was developed, using its analogue annonacinone as an internal standard. Extraction from plasma was performed using ethylacetate with a good recovery. Quantification was performed by UPLC-MS/MS in SRM mode, based on the loss of the γ-methyl-γ-lactone (-112amu) from the sodium-cationized species [M+Na](+) of both annonacin and internal standard. The limit of quantification was 0.25ng/mL. Despite strong matrix effects, a good linearity was obtained over two distinct ranges 0.25-10ng/mL and 10-100ng/mL. The intra- and inter-day precisions (RSD) were lower than 10%, while accuracy was within ±10%. This method was applied to a pharmacokinetic study in the Rat. After oral administration of 10mg/kg annonacin, a Cmax of 7.9±1.5ng/mL was reached at Tmax 0.25h; T1/2 was 4.8±0.7h and apparent distribution volume was 387.9±64.6L. The bioavailability of annonacin was estimated to be 3.2±0.3% of the ingested dose.

  19. Rapid, sensitive and selective HPLC-MS/MS method for the quantification of topically applied besifloxacin in rabbit plasma and ocular tissues: Application to a pharmacokinetic study.

    PubMed

    Gu, Xiao-Fei; Mao, Bai-Yang; Xia, Min; Yang, Yang; Zhang, Jia-Li; Yang, Da-Song; Wu, Wei-Xin; Du, Ying-Xiang; Di, Bin; Su, Meng-Xiang

    2016-01-01

    Besifloxacin is a fourth-generation broad-spectrum fluoroquinolone registered for the topical treatment of bacterial conjunctivitis. In this study, a rapid, sensitive and selective liquid chromatography tandem mass spectrometry (LC-MS/MS) method was developed for quantification of besifloxacin in rabbit plasma and ocular tissues using nateglinide as the internal standard (IS). The analyte and IS were separated on a Sepax GP-Phenyl column by isocratic elution with methanol-acetonitrile-5 mM ammonium formate-formic acid (29:55:16:0.1, v/v/v/v) as the mobile phase at a flow rate of 1.2 mL/min, and the total run time was 3.0 min. An electrospray ionization (ESI) source was applied and operated in the positive ion mode; multiple reaction monitoring (MRM) mode was used for quantification, and the monitored transitions were 394.2→377.1 for besifloxacin and m/z 318.3→166.1 for the IS. The calibration curve was linear over the range of 0.103-206 ng/mL for plasma and 2.06-2060 ng/mL for tears, aqueous humor, conjunctiva and cornea with correlation coefficient (r) greater than 0.99. The lower limit of quantification (LLOQ) for besifloxacin was 0.103 ng/mL for plasma and 2.06 ng/mL for other ocular tissues with good accuracy and precision. Intra- and inter-batch precision were both lower than 15% and accuracy ranged from 85% to 115% at all QC levels. The method was successfully applied to the pharmacokinetic study of besifloxacin in rabbit plasma and ocular tissues after single and multiple topical administrations. PMID:26340560

  20. LC-MS/MS method for the simultaneous quantification of 11 compounds of Ginkgo biloba extract in lysates of mesangial cell cultured by high glucose.

    PubMed

    Qiu, Jing-ying; Chen, Xu; Li, Zheng; Wang, Shi-rui; Wu, Xiao-wen; Li, Yin-jie; Yang, Dong-zhi; Yu, Yan-yan; Yin, Xiao-xing; Tang, Dao-quan

    2015-08-01

    The mesangial cell (MC) cultured with high glucose has been used to observe the protective effect of Ginkgo biloba extract (GBE) against diabetic nephropathy (DN), but the compounds interacting with cell are still unknown, which may be potential bioactive components. Based on this, the determination of GBE in MC lysates was proposed by high performance liquid chromatography tandem mass spectrometry (LC-MS/MS) in this study. The MC was cultured with normal or high glucose with GBE for 4, 8, 12, 16, 24 and 48h. The harvested cell was extracted with 40% acetic acid in water and further analyzed by LC-MS/MS. All the validation data including linearity, intra-day and inter-day precision, limit of detection and quantification, matrix effect, and stability were within the required limits. The validated method was successfully applied to quantify 11 compounds of GBE in cell lysates. The results showed that high glucose prolonged the peak time of all observed 11 compounds and peak concentrations of bilobalide, ginkgolide C, ginkgolide B, quercetin, luteolin, kaempferol, isorhamnetin and genkwanin in cell lysates, which hinted that these compounds may be the potential bioactive components of GBE with preventive effect against DN.

  1. Stable isotope dilution HILIC-MS/MS method for accurate quantification of glutamic acid, glutamine, pyroglutamic acid, GABA and theanine in mouse brain tissues.

    PubMed

    Inoue, Koichi; Miyazaki, Yasuto; Unno, Keiko; Min, Jun Zhe; Todoroki, Kenichiro; Toyo'oka, Toshimasa

    2016-01-01

    In this study, we developed the stable isotope dilution hydrophilic interaction liquid chromatography with tandem mass spectrometry (HILIC-MS/MS) technique for the accurate, reasonable and simultaneous quantification of glutamic acid (Glu), glutamine (Gln), pyroglutamic acid (pGlu), γ-aminobutyric acid (GABA) and theanine in mouse brain tissues. The quantification of these analytes was accomplished using stable isotope internal standards and the HILIC separating mode to fully correct the intramolecular cyclization during the electrospray ionization. It was shown that linear calibrations were available with high coefficients of correlation (r(2)  > 0.999, range from 10 pmol/mL to 50 mol/mL). For application of the theanine intake, the determination of Glu, Gln, pGlu, GABA and theanine in the hippocampus and central cortex tissues was performed based on our developed method. In the region of the hippocampus, the concentration levels of Glu and pGlu were significantly reduced during reality-based theanine intake. Conversely, the concentration level of GABA increased. This result showed that transited theanine has an effect on the metabolic balance of Glu analogs in the hippocampus.

  2. Separation, identification, quantification, and method validation of anthocyanins in botanical supplement raw materials by HPLC and HPLC-MS.

    PubMed

    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.

  3. Robust MS quantification method for phospho-peptides using 18O/16O labeling

    PubMed Central

    Andersen, Claus A; Gotta, Stefano; Magnoni, Letizia; Raggiaschi, Roberto; Kremer, Andreas; Terstappen, Georg C

    2009-01-01

    Background Quantitative measurements of specific protein phosphorylation sites, as presented here, can be used to investigate signal transduction pathways, which is an important aspect of cell dynamics. The presented method quantitatively compares peptide abundances from experiments using 18O/16O labeling starting from elaborated MS spectra. It was originally developed to study signaling cascades activated by amyloid-β treatment of neurons used as a cellular model system with relevance to Alzheimer's disease, but is generally applicable. Results The presented method assesses, in complete cell lysates, the degree of phosphorylation of specific peptide residues from MS spectra using 18O/16O labeling. The abundance of each observed phospho-peptide from two cell states was estimated from three overlapping isotope contours. The influence of peptide-specific labeling efficiency was removed by performing a label swapped experiment and assuming that the labeling efficiency was unchanged upon label swapping. Different degrees of phosphorylation were reported using the fold change measure which was extended with a confidence interval found to reflect the quality of the underlying spectra. Furthermore a new way of method assessment using simulated data is presented. Using simulated data generated in a manner mimicking real data it was possible to show the method's robustness both with increasing noise levels and with decreasing labeling efficiency. Conclusion The fold change error assessable on simulated data was on average 0.16 (median 0.10) with an error-to-signal ratio and labeling efficiency distributions similar to the ones found in the experimentally observed spectra. Applied to experimentally observed spectra a very good match was found to the model (<10% error for 85% of spectra) with a high degree of robustness, as assessed by data removal. This new method can thus be used for quantitative signal cascade analysis of total cell extracts in a high throughput mode

  4. Development and validation of a HILIC-MS/MS method for quantification of decitabine in human plasma by using lithium adduct detection.

    PubMed

    Hua, Wenyi; Ierardi, Thomas; Lesslie, Michael; Hoffman, Brian T; Mulvana, Daniel

    2014-10-15

    A highly sensitive, selective, and rugged quantification method was developed and validated for decitabine (5-aza-2'-deoxycytidine) in human plasma treated with 100μg/mL of tetrahydrouridine (THU). Chromatographic separation was accomplished using hydrophilic interaction liquid chromatography (HILIC) and detection used electrospray ionization (ESI) tandem mass spectrometry (MS/MS) by monitoring lithiated adducts of the analytes as precursor ions. The method involves simple acetonitrile precipitation steps (in an ice bath) followed by injection of the supernatant onto a Thermo Betasil Silica-100, 100×3.0mm, 5μm LC column. Protonated ([M+H](+)), sodiated ([M+Na](+)), and lithiated ([M+Li](+)) adducts as precursor ions for MS/MS detection were evaluated for best sensitivity and assay performance. During initial method development abundant sodium [M+Na](+) and potassium [M+K](+) adducts were observed while the protonated species [M+H](+) was present at a relative abundance of less than 5% in Q1. The alkali adducts were not be able to be minimized by the usual approach of increasing acid content in mobile phases. Significant analyte/internal standard (IS) co-suppression and inter-lot response differences were observed when using the sodium adduct as the precursor ion for quantification. By adding 2mM lithium acetate in aqueous mobile phase component, the lithium adduct effectively replaced other cationic species and was successfully used as the precursor ion for selected reaction monitoring (SRM) detection. The method demonstrated the separation of anomers and from other endogenous interferences using a 3-min gradient elution. Decitabine stock, working solution stabilities were investigated during method development. Three different peaks, including one from anomerization, were observed in the SRM transition of the analyte when it was in neutral aqueous solution. The assay was validated over a concentration range of 0.5-500ng/mL (or 0.44-440pg injected on column) in

  5. Fully automated standard addition method for the quantification of 29 polar pesticide metabolites in different water bodies using LC-MS/MS.

    PubMed

    Kowal, Sebastian; Balsaa, Peter; Werres, Friedrich; Schmidt, Torsten C

    2013-07-01

    A reliable quantification by LC-ESI-MS/MS as the most suitable analytical method for polar substances in the aquatic environment is usually hampered by matrix effects from co-eluting compounds, which are unavoidably present in environmental samples. The standard addition method (SAM) is the most appropriate method to compensate matrix effects. However, when performed manually, this method is too labour- and time-intensive for routine analysis. In the present work, a fully automated SAM using a multi-purpose sample manager "Open Architecture UPLC®-MS/MS" (ultra-performance liquid chromatography tandem mass spectrometry) was developed for the sensitive and reliable determination of 29 polar pesticide metabolites in environmental samples. A four-point SAM was conducted parallel to direct-injection UPLC-ESI-MS/MS determination that was followed by a work flow to calculate the analyte concentrations including monitoring of required quality criteria. Several parameters regarding the SAM, chromatography and mass spectrometry conditions were optimised in order to obtain a fast as well as reliable analytical method. The matrix effects were examined by comparison of the SAM with an external calibration method. The accuracy of the SAM was investigated by recovery tests in samples of different catchment areas. The method detection limit was estimated to be between 1 and 10 ng/L for all metabolites by direct injection of a 10-μL sample. The relative standard deviation values were between 2 and 10% at the end of calibration range (30 ng/L). About 200 samples from different water bodies were examined with this method in the Rhine and Ruhr region of North Rhine-Westphalia (Germany). Approximately 94% of the analysed samples contained measurable amounts of metabolites. For most metabolites, low concentrations ≤0.10 μg/L were determined. Only for three metabolites were the concentrations in ground water significantly higher (up to 20 μg/L). In none of the examined drinking

  6. Quantification of protodioscin and rutin in asparagus shoots by LC/MS and HPLC methods.

    PubMed

    Wang, Mingfu; Tadmor, Yaakov; Wu, Qing-Li; Chin, Chee-Kok; Garrison, Stephen A; Simon, James E

    2003-10-01

    A liquid chromatography/mass spectrometry (LC/MS) method with selected ion monitoring was developed and validated to analyze the contents of protodioscin and rutin in asparagus. The distribution of rutin and protodioscin within the shoots was found to vary by location, with the tissue closest to the rhizome found to be a rich source of protodioscin, at an average level of 0.025% tissue fresh weight in the three tested lines, while the upper youngest shoot tissue contained the highest amount of rutin at levels of 0.03-0.06% tissue fresh weight. The lower portions of the asparagus shoots that are discarded during grading and processing should instead be considered a promising source of a new value-added nutraceutical product.

  7. Validated LC-MS/MS method for simultaneous quantification of resveratrol levels in mouse plasma and brain and its application to pharmacokinetic and brain distribution studies.

    PubMed

    Ramalingam, Prakash; Ko, Young Tag

    2016-02-01

    A rapid, selective, and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated to simultaneously determine resveratrol levels in plasma and brain tissue in mice for supporting pharmacokinetic and brain distribution studies. Analytes were separated using a Sepax BR-C18 analytical column (5μm, 120Å, 1.0×100mm) and eluted using an isocratic elution mobile phase acetonitrile and 0.01% formic acid [60:40, v/v] at a flow rate of 0.1mL/min. Precursor and product ion transitions for analyte resveratrol m/z 226.9>184.8 and curcumin m/z 367.1>148.9 were monitored using triple quadrupole mass spectrometer with multiple reaction monitoring (MRM) in negative ionization mode. The method was validated with respect to accuracy, within- and between-day precision, linearity, limit of quantification, recovery, and matrix effects of analyte. The inter- and intra-day accuracy and precision were within the range of the US Food and Drug Administration (FDA) acceptance criteria, for both matrices. The method was also successfully applied to pharmacokinetic and brain distribution studies of resveratrol after intravenous administration of free resveratrol and resveratrol-loaded solid lipid nanoparticles to mice. The combined use of serial blood sampling, small sample volume, simple extraction, and capillary depletion method drastically improved resveratrol analysis from biological matrices.

  8. A rapid and highly sensitive UPLC-MS-MS method for the quantification of zolpidem tartrate in human EDTA plasma and its application to pharmacokinetic study.

    PubMed

    Reddy, Dendhi Chandrapal; Bapuji, Akula Tukaram; Rao, Vepakomma Suryanarayana; Himabindu, Vurimindi; Ravinder, Sreedasyam

    2012-07-01

    A rapid and high sensitive liquid chromatography-tandem mass spectrometry (LC-MS-MS) method was developed and validated for the quantification of zolpidem in human EDTA plasma using ondansetron (IS) as an internal standard. The analyte and IS were extracted from human plasma using ethyl acetate and separated on a C18 column (Inertsil-ODS, 5 µm, 4.6 × 50 mm) interfaced with a triple quadrupole tandem mass spectrometer. The mobile phase, which consisted of a mixture of methanol and 20 mM ammonium formate (pH 5.00 ± 0.05; 75:25 v/v), was injected at a flow rate of 0.40 mL/min. The retention times of zolpidem and IS were approximately 1.76 and 1.22. The LC run time was 3 min. The electrospray ionization source was operated in positive ion mode. Multiple reaction monitoring used the [M + H](+) ions m/z 308.13 → 235.21 for zolpidem and m/z 294.02 → 170.09 for the ondansetron, respectively. Five freeze-thaw cycles was established at -20 and -70°C.The linearity of the response/concentration curve was established in human EDTA plasma over the concentration range 0.10-149.83 ng/mL. The lower detection limit [(signal-to-noise (S/N) > 3] was 0.04 ng/mL and the lower limit of quantification (S/N > 10) was 0.10 ng/mL. This LC-MS-MS method was validated with intra-batch and inter-batch precision of 0.52-8.66.The intra-batch and inter-batch accuracy was 96.66-106.11. Recovery of zolpidem in human plasma was 87.00% and IS recovery was 81.60%. The primary pharmacokinetic parameters were T(max) (h) = (1.25 ± 0.725), C(max) (ng/mL) (127.80 ± 34.081), AUC(0→t), = (665.37 ± 320.982) and AUC(0→∞), 686.03 ± 342.952, respectively. PMID:22689921

  9. Application of an LC-MS/MS method for the simultaneous quantification of human intestinal transporter proteins absolute abundance using a QconCAT technique.

    PubMed

    Harwood, M D; Achour, B; Russell, M R; Carlson, G L; Warhurst, G; Rostami-Hodjegan, A

    2015-06-10

    Transporter proteins expressed in the gastrointestinal tract play a major role in the oral absorption of some drugs, and their involvement may lead to drug-drug interaction (DDI) susceptibility when given in combination with drugs known to inhibit gut wall transporters. Anticipating such liabilities and predicting the magnitude of the impact of transporter proteins on oral drug absorption and DDIs requires quantification of their expression in human intestine, and linking these to data obtained through in vitro experiments. A quantitative targeted absolute proteomic method employing liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) together with a quantitative concatenation (QconCAT) strategy to provide proteotypic peptide standards has been applied to quantify ATP1A1 (sodium/potassium-ATPase; Na/K-ATPase), CDH17 (human peptide transporter 1; HPT1), ABCB1 (P-glycoprotein; P-gp), ABCG2 (breast cancer resistance protein; BCRP), ABCC2 (multidrug resistance-associated protein 2; MRP2) and SLC51A (Organic Solute Transporter subunit alpha; OST-α), in human distal jejunum (n=3) and distal ileum (n=1) enterocyte membranes. Previously developed selected reaction monitoring (SRM) schedules were optimised to enable quantification of the proteotypic peptides for each transporter. After harvesting enterocytes by calcium chelation elution and generating a total membrane fraction, the proteins were subjected to proteolytic digestion. To account for losses of peptides during the digestion procedure, a gravimetric method is also presented. The linearity of quantifying the QconCAT from an internal standard (correlation coefficient, R(2)=0.998) and quantification of all target peptides in a pooled intestinal quality control sample (R(2)≥ 0.980) was established. The assay was also assessed for within and between-day precision, demonstrating a <15% coefficient of variation for all peptides across 3 separate analytical runs, over 2 days. The methods were applied to

  10. Development and validation of an LC-MS/MS method for quantification of Δ9-tetrahydrocannabinolic acid A (THCA-A), THC, CBN and CBD in hair.

    PubMed

    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.

  11. A sensitive LC-ESI-MS/MS method for the quantification of avobenzone in rat plasma and skin layers: Application to a topical administration study.

    PubMed

    Kim, Min Gi; Kim, Tae Hwan; Shin, Beom Soo; Kim, Min Gyu; Seok, Su Hyun; Kim, Kyu-Bong; Lee, Jong Bong; Choi, Hyeon Gwan; Lee, Young Sung; Yoo, Sun Dong

    2015-10-15

    This study describes the development of a sensitive high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) method for the quantification of avobenzone in rat plasma and skin layers. Separations were performed on a Zorbax SB C8 column using a binary gradient mobile phase composed of acetonitrile and 0.1% formic acid in water. The assay achieved LLOQ of 0.5ng/ml for plasma, 5ng/ml for stratum corneum, and 10ng/ml for epidermis and dermis. This method was applied to a percutaneous absorption study of avobenzone in rats. At 12h following topical application of emulsion and lotion (applied amount of avobenzone 11.7mg/kg), avobenzone was found primarily in the stratum corneum (16.3-17.8%) followed by epidermis (2.0-3.4%) and dermis (0.11-0.15%). Avobenzone was not quantifiable in the plasma samples collected over a 12h sampling period. Given the excellent plasma assay sensitivity, this study provides evidence that the systemic absorption of avobenzone is insignificant, if any, after topical application.

  12. A sensitive LC-ESI-MS/MS method for the quantification of avobenzone in rat plasma and skin layers: Application to a topical administration study.

    PubMed

    Kim, Min Gi; Kim, Tae Hwan; Shin, Beom Soo; Kim, Min Gyu; Seok, Su Hyun; Kim, Kyu-Bong; Lee, Jong Bong; Choi, Hyeon Gwan; Lee, Young Sung; Yoo, Sun Dong

    2015-10-15

    This study describes the development of a sensitive high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) method for the quantification of avobenzone in rat plasma and skin layers. Separations were performed on a Zorbax SB C8 column using a binary gradient mobile phase composed of acetonitrile and 0.1% formic acid in water. The assay achieved LLOQ of 0.5ng/ml for plasma, 5ng/ml for stratum corneum, and 10ng/ml for epidermis and dermis. This method was applied to a percutaneous absorption study of avobenzone in rats. At 12h following topical application of emulsion and lotion (applied amount of avobenzone 11.7mg/kg), avobenzone was found primarily in the stratum corneum (16.3-17.8%) followed by epidermis (2.0-3.4%) and dermis (0.11-0.15%). Avobenzone was not quantifiable in the plasma samples collected over a 12h sampling period. Given the excellent plasma assay sensitivity, this study provides evidence that the systemic absorption of avobenzone is insignificant, if any, after topical application. PMID:26409261

  13. GC-MS quantification of suspected volatile allergens in fragrances. 2. Data treatment strategies and method performances.

    PubMed

    Bassereau, Maud; Chaintreau, Alain; Duperrex, Stéphanie; Joulain, Daniel; Leijs, Hans; Loesing, Gerd; Owen, Neil; Sherlock, Alan; Schippa, Christine; Thorel, Pierre-Jean; Vey, Matthias

    2007-01-10

    The performances of the GC-MS determination of suspected allergens in fragrance concentrates have been investigated. The limit of quantification was experimentally determined (10 mg/L), and the variability was investigated for three different data treatment strategies: (1) two columns and three quantification ions; (2) two columns and one quantification ion; and (3) one column and three quantification ions. The first strategy best minimizes the risk of determination bias due to coelutions. This risk was evaluated by calculating the probability of coeluting a suspected allergen with perfume constituents exhibiting ions in common. For hydroxycitronellal, when using a two-column strategy, this may statistically occur more than once every 36 analyses for one ion or once every 144 analyses for three ions in common.

  14. Isotope Inversion Experiment evaluating the suitability of calibration in surrogate matrix for quantification via LC-MS/MS-Exemplary application for a steroid multi-method.

    PubMed

    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

  15. Quantification and interpretation of total petroleum hydrocarbons in sediment samples by a GC/MS method and comparison with EPA 418.1 and a rapid field method.

    PubMed

    Xie, G; Barcelona, M J; Fang, J

    1999-05-01

    Total petroleum hydrocarbons (TPH) as a lumped parameter can be easily and rapidly measured or monitored. Despite interpretational problems, it has become an accepted regulatory benchmark used widely to evaluate the extent of petroleum product contamination. Three currently used methods (GC/MS, conventional EPA 418.1, and a rapid field method PetroFLAG) were performed to quantify the TPH content in samples collected from a site contaminated by transformer oil. To standardize the method and improve the comparability of TPH data, crucial GC-based quantification issues were examined, e.g., quantification based on internal standards (ISTD) vs external standards (ESTD), single vs multiple ISTD, and various area integration approaches. The interpretation of hydrocarbon chromatographic results was examined in the context of field samples. The performance of the GC/MS method was compared with those of EPA 418.1 and PetroFLAG. As a result, it was observed that the ISTD quantification method was preferred to the ESTD method, multiple ISTD might be better than single ISTD, and three different area integration approaches did not have a significant effect on TPH results. Evaluation of the chromatograms between a reference sample and three unknown samples showed that the extent of contamination varied appreciably with sample depth. It was also found that there existed a good positive correlation between GC/MS and both EPA 418.1 and PetroFLAG, and that EPA 418.1 produced the higher overall estimate while GC/MS and PetroFLAG resulted in lower, more statistically comparable TPH values.

  16. Development and validation of LC-MS methods for peptaibol quantification in fungal extracts according to their lengths.

    PubMed

    Van Bohemen, Anne-Isaline; Zalouk-Vergnoux, Aurore; Poirier, Laurence; Phuong, Nam Ngoc; Inguimbert, Nicolas; Ben Haj Salah, Khoubaib; Ruiz, Nicolas; Pouchus, Yves François

    2016-01-15

    Some terrestrial Trichoderma sp. strains are already used as biological control agents (BCAs). They all produce peptaibols, small antimicrobial peptides which are supposed to play a role in the anti-phytopathogenic activity of Trichoderma sp. Trichoderma strains producing high amounts of peptaibols could represent new potential BCAs. In this context, marine-derived Trichoderma strains from the marine fungal strain collection of the "Mer, Molécules, Santé" (MMS) laboratory were investigated for their peptaibol production. Previously, the quantification of peptaibols was performed using alamethicin, as standard (20-amino acid residues peptaibol). In this study, the development and validation of quantification LC/ESI-TI-MS methods using different standards of peptaibols (11-, 14- and 20-amino acid residues) was performed in order to quantify all of them, in a single analysis, in Trichoderma crude extracts according to their chain length. The developed and validated methods were used to study the peptaibol production kinetic of a marine-derived Trichoderma strain, i.e., Trichoderma longibrachiatum (MMS 151). The results showed the optimal culture time at the 9th day with concentrations reaching 1.4±0.2% and 2.3±0.4% of the fungal biomass respectively for 11- and 20-residue peptaibols. Then, the different peptaibol subgroups produced by 13 Trichoderma strains were quantified. According to their 18-, 19- and 20-residue peptaibol production, three strains referenced as MMS 1541, MMS 639 and MMS 151 seemed to be good candidates as potential new biological control agents with respective production of 0.4, 0.4 and 2.1%.

  17. Development and validation of LC-MS methods for peptaibol quantification in fungal extracts according to their lengths.

    PubMed

    Van Bohemen, Anne-Isaline; Zalouk-Vergnoux, Aurore; Poirier, Laurence; Phuong, Nam Ngoc; Inguimbert, Nicolas; Ben Haj Salah, Khoubaib; Ruiz, Nicolas; Pouchus, Yves François

    2016-01-15

    Some terrestrial Trichoderma sp. strains are already used as biological control agents (BCAs). They all produce peptaibols, small antimicrobial peptides which are supposed to play a role in the anti-phytopathogenic activity of Trichoderma sp. Trichoderma strains producing high amounts of peptaibols could represent new potential BCAs. In this context, marine-derived Trichoderma strains from the marine fungal strain collection of the "Mer, Molécules, Santé" (MMS) laboratory were investigated for their peptaibol production. Previously, the quantification of peptaibols was performed using alamethicin, as standard (20-amino acid residues peptaibol). In this study, the development and validation of quantification LC/ESI-TI-MS methods using different standards of peptaibols (11-, 14- and 20-amino acid residues) was performed in order to quantify all of them, in a single analysis, in Trichoderma crude extracts according to their chain length. The developed and validated methods were used to study the peptaibol production kinetic of a marine-derived Trichoderma strain, i.e., Trichoderma longibrachiatum (MMS 151). The results showed the optimal culture time at the 9th day with concentrations reaching 1.4±0.2% and 2.3±0.4% of the fungal biomass respectively for 11- and 20-residue peptaibols. Then, the different peptaibol subgroups produced by 13 Trichoderma strains were quantified. According to their 18-, 19- and 20-residue peptaibol production, three strains referenced as MMS 1541, MMS 639 and MMS 151 seemed to be good candidates as potential new biological control agents with respective production of 0.4, 0.4 and 2.1%. PMID:26688345

  18. A selective and sensitive method based on UPLC-MS/MS for quantification of momordin Ic in rat plasma: application to a pharmacokinetic study.

    PubMed

    Yan, Huiyu; Song, Yanqing; Zhou, Wei; Zhang, Sixi

    2015-11-10

    A selective and sensitive method was developed and validated based on ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). This method was applied to quantify momordin Ic in rat plasma. Chromatographic separation was performed on a Hypersil GOLD HPLC C18 column (150mm×4.6mm, 5μm) using an isocratic mobile phase of acetonitrile/water (80:20, v/v) at a flow rate of 0.6mL/min. An electrospray ionization source was applied and operated in negative ion mode; selected reaction monitoring (SRM) mode was used for quantification by monitoring the precursor-to-product ion transitions of m/z 763.4→m/z 455.3 for momordin Ic, and m/z 649.4→m/z 487.3 for IS. Calibration curves showed good linearity over the range of 22.0-2200ng/mL for momordin Ic in rat plasma. The developed method was applied to a pharmacokinetic study of momordin Ic in rats after single intravenous doses at 0.52, 1.56, and 4.67mg/kg. The elimination half-life (t1/2) values were 1.22±0.39, 1.14±0.10, and 1.83±0.39h, respectively. The plasma concentration at 2min (C2min) and area under the curve (AUC) for the intravenous doses of momordin Ic were approximately dose proportional. PMID:26218506

  19. Development and application of an analytical method for curdione quantification in pregnant Sprague-Dawley rats by LC-MS/MS.

    PubMed

    Meng, Xiang; Zhang, Ting; Li, Ying; Pan, Qi; Jiang, Juan; Luo, Yongwei; Chong, Liming; Yang, Yang; Xu, Sichong; Zhou, Li; Sun, Zuyue

    2015-10-01

    The vaginal administration route suffers from relatively low absorption efficiency, which may hinder the identification of the toxicokinetics of curdione in pregnant women. A sensitive analytical method for determining the plasma concentration of curdione was developed and applied in the determination of curdione in pregnant Sprague-Dawley rats as a simulated model. Glimepiride was used as an internal standard and chromatographic separation was achieved on a Capcell Pak C18 MGIII column. A gradient elution profile with 0.5% formic acid (A)-0.5% formic acid-acetonitrile (B) was selected as mobile phase. The selected reaction monitoring mode was used for quantification based on the target fragment ions m/z 237.2 to m/z 135.1 for curdione and m/z 491.3 to m/z 352.1 for the glimepiride. The standard curve was linear over the range of 0.5-500 ng/mL for curdione in rat plasma and yielded a consistent peak pattern, even at the lower limit of quantitation of 0.5 ng/mL. The retention times of curdione and IS were 6.55 and 6.59 min, respectively. The mean recovery of curdione in rat plasma was 95.5-101.1%. The intra-day and inter-day precisions were between 2.35 and 9.08%. This LC-MS/MS method provides a simple and sensitive means for determining the plasma concentration. PMID:25736727

  20. Development and validation of a LC-MS/MS method for the quantification of the regioisomers of dihydroxybutylether in human plasma.

    PubMed

    Yuan, Bo; Li, Li; Fu, Yao; Jin, Yi; Guo, Lixin; Xu, Haiyan

    2012-12-12

    Dihydroxybutylether (DHBE), a strong choleretic drug, is a mixture of three regioisomers: 4-(3-hydroxybutoxy)-2-butanol (I), 3-(4-hydroxy-2-butoxy)-1-butanol (II) and 3-(3-hydroxylbutoxy)-1-butanol (III). A liquid chromatography-tandem mass spectrometry method was developed and validated for the quantification of dihydroxybutylether (DHBE) regioisomers in human plasma. After plasma samples were deproteinized with 10% perchloric acid, the post-treatment samples were analyzed on a Capcell Pak C(18) MGII column interfaced with a triple quadrupole tandem mass spectrometer in positive electrospray ionization mode. Methanol and water was used as the mobile phase with a gradient elution at a flow rate of 1mL/min. Acetaminophen was used as an internal standard (IS). Multiple selected reaction monitoring was performed using the transitions m/z 163→55 and m/z 152→110 to quantify DHBE regioisomers and IS, respectively. Five DHBE isomers (a, b, c, d and e) were separated under the present chromatographic condition. The assay was linear over the concentration range of 5.0-200ng/mL for DHBE isomers a, b and c, and 10.0-400ng/mL for DHBE isomers d and e. The intra- and inter-day precision was within 13.6% in terms of relative standard deviation (RSD%) and the accuracy within 7.3% in terms of relative error. This simple and sensitive and easily reproducible LC-MS/MS method was successfully applied to the pharmacokinetic study of DHBE regioisomers in healthy male Chinese volunteers after an oral dose of 1.0g DHBE.

  1. Validation and application of an UPLC-MS/MS method for the quantification of synthetic cannabinoids in urine samples and analysis of seized materials from the Portuguese market.

    PubMed

    Simões, Susana Sadler; Silva, Inês; Ajenjo, Antonio Castañera; Dias, Mário João

    2014-10-01

    An UPLC-MS/MS method using ESI+ionization and MRM was developed and fully validated according to international guidelines for the qualitative and quantitative analysis of nine synthetic cannabinoids and/or their metabolites in urine samples (1mL). Prior to extraction the samples were subjected to an enzymatic hydrolysis using β-glucuronidase followed by a SPE procedure using Oasis(®) HLB 3cc (60mg) columns. The chromatographic separation was performed with an Acquity UPLC(®) HSS T3 (50mm×2.1mm i.d., 1.8μm) reversed-phase column using a gradient with methanol-ammonium formate 2mM (0.1% formic acid) and with a run time of 9.5min. The method was validated in terms of selectivity, capacity of identification, limits of detection (0.01-0.5ng/mL) and quantification (0.05-0.5ng/mL), recovery (58-105%), carryover, matrix effect, linearity (0.05-50ng/mL), intra-assay precision, inter-assay accuracy and precision (CV<20%). The method was applied to 80 authentic samples, five of them (6.2%) were confirmed or suspected to be positive for the metabolites JWH-018 N-hydroxypentyl and JWH-018 N-pentanoic acid of JWH-018 and for the metabolite JWH-122 N-(5-hydroxypentyl) of JWH-122, and three of them in association with THC and/or THCCOOH (substances included in the method, together with the 11-OH-THC). Additionally, 17 spice products were analyzed, for which were confirmed the presence of the following substances: AM-2201, JWH-018, JWH-022 JWH-073, JWH-122, JWH-203, JWH-210, JWH-250, HU-210 and RCS-4, according to the comparison with authentic reference material and published data. The analytical method developed allowed the analysis of synthetic cannabinoids and the notification of the first cases in Portugal. PMID:25127518

  2. Validation and application of an UPLC-MS/MS method for the quantification of synthetic cannabinoids in urine samples and analysis of seized materials from the Portuguese market.

    PubMed

    Simões, Susana Sadler; Silva, Inês; Ajenjo, Antonio Castañera; Dias, Mário João

    2014-10-01

    An UPLC-MS/MS method using ESI+ionization and MRM was developed and fully validated according to international guidelines for the qualitative and quantitative analysis of nine synthetic cannabinoids and/or their metabolites in urine samples (1mL). Prior to extraction the samples were subjected to an enzymatic hydrolysis using β-glucuronidase followed by a SPE procedure using Oasis(®) HLB 3cc (60mg) columns. The chromatographic separation was performed with an Acquity UPLC(®) HSS T3 (50mm×2.1mm i.d., 1.8μm) reversed-phase column using a gradient with methanol-ammonium formate 2mM (0.1% formic acid) and with a run time of 9.5min. The method was validated in terms of selectivity, capacity of identification, limits of detection (0.01-0.5ng/mL) and quantification (0.05-0.5ng/mL), recovery (58-105%), carryover, matrix effect, linearity (0.05-50ng/mL), intra-assay precision, inter-assay accuracy and precision (CV<20%). The method was applied to 80 authentic samples, five of them (6.2%) were confirmed or suspected to be positive for the metabolites JWH-018 N-hydroxypentyl and JWH-018 N-pentanoic acid of JWH-018 and for the metabolite JWH-122 N-(5-hydroxypentyl) of JWH-122, and three of them in association with THC and/or THCCOOH (substances included in the method, together with the 11-OH-THC). Additionally, 17 spice products were analyzed, for which were confirmed the presence of the following substances: AM-2201, JWH-018, JWH-022 JWH-073, JWH-122, JWH-203, JWH-210, JWH-250, HU-210 and RCS-4, according to the comparison with authentic reference material and published data. The analytical method developed allowed the analysis of synthetic cannabinoids and the notification of the first cases in Portugal.

  3. Development and Validation of a GC-MS Method for the Detection and Quantification of Clotiapine in Blood and Urine Specimens and Application to a Postmortem Case

    PubMed Central

    Mannocchi, Giulio; Pantano, Flaminia; Tittarelli, Roberta; Catanese, Miriam; Umani Ronchi, Federica; Busardò, Francesco Paolo

    2015-01-01

    Introduction. Clotiapine is an atypical antipsychotic of the dibenzothiazepine class introduced in a few European countries since 1970, efficient in treatment-resistant schizophrenic patients. There is little published data on the therapeutic and toxic concentrations of this drug. Aims. The aim of the present study is the development and validation of a method that allows the detection and quantification of clotiapine in blood and urine specimens by gas chromatography-mass spectrometry (GC-MS). Methods. Validation was performed working on spiked postmortem blood and urine samples. Samples were extracted with liquid-liquid extraction (LLE) technique at pH 8.5 with n-hexane/dichloromethane (85/15 v/v) and analysis was followed by GC-MS. Methadone-d9 was used as internal standard. Results. The limit of detection (LOD) was 1.2 and 1.3 ng/mL for urine and blood, respectively, while the lower limit of quantification (LLOQ) was 3.9 and 4.3 ng/mL, respectively. Linearity, precision, selectivity, accuracy, and recovery were also determined. The method was applied to a postmortem case. The blood and urine clotiapine concentrations were 1.32 and 0.49 μg/mL, respectively. Conclusions. A reliable GC-MS method for the detection and quantification of clotiapine in blood and urine samples has been developed and fully validated and then applied to a postmortem case. PMID:26236337

  4. Development and Validation of a GC-MS Method for the Detection and Quantification of Clotiapine in Blood and Urine Specimens and Application to a Postmortem Case.

    PubMed

    Mannocchi, Giulio; Pantano, Flaminia; Tittarelli, Roberta; Catanese, Miriam; Umani Ronchi, Federica; Busardò, Francesco Paolo

    2015-01-01

    Introduction. Clotiapine is an atypical antipsychotic of the dibenzothiazepine class introduced in a few European countries since 1970, efficient in treatment-resistant schizophrenic patients. There is little published data on the therapeutic and toxic concentrations of this drug. Aims. The aim of the present study is the development and validation of a method that allows the detection and quantification of clotiapine in blood and urine specimens by gas chromatography-mass spectrometry (GC-MS). Methods. Validation was performed working on spiked postmortem blood and urine samples. Samples were extracted with liquid-liquid extraction (LLE) technique at pH 8.5 with n-hexane/dichloromethane (85/15 v/v) and analysis was followed by GC-MS. Methadone-d9 was used as internal standard. Results. The limit of detection (LOD) was 1.2 and 1.3 ng/mL for urine and blood, respectively, while the lower limit of quantification (LLOQ) was 3.9 and 4.3 ng/mL, respectively. Linearity, precision, selectivity, accuracy, and recovery were also determined. The method was applied to a postmortem case. The blood and urine clotiapine concentrations were 1.32 and 0.49 μg/mL, respectively. Conclusions. A reliable GC-MS method for the detection and quantification of clotiapine in blood and urine samples has been developed and fully validated and then applied to a postmortem case. PMID:26236337

  5. Comparison of sample preparation methods, validation of an UPLC-MS/MS procedure for the quantification of tetrodotoxin present in marine gastropods and analysis of pufferfish.

    PubMed

    Nzoughet, Judith Kouassi; Campbell, Katrina; Barnes, Paul; Cooper, Kevin M; Chevallier, Olivier P; Elliott, Christopher T

    2013-02-15

    Tetrodotoxin (TTX) is one of the most potent marine neurotoxins reported. The global distribution of this toxin is spreading with the European Atlantic coastline now being affected. Climate change and increasing pollution have been suggested as underlying causes for this. In the present study, two different sample preparation techniques were used to extract TTX from Trumpet shells and pufferfish samples. Both extraction procedures (accelerated solvent extraction (ASE) and a simple solvent extraction) were shown to provide good recoveries (80-92%). A UPLC-MS/MS method was developed for the analysis of TTX and validated following the guidelines contained in the Commission Decision 2002/657/EC for chemical contaminant analysis. The performance of this procedure was demonstrated to be fit for purpose. This study is the first report on the use of ASE as a mean for TTX extraction, the use of UPLC-MS/MS for TTX analysis, and the validation of this method for TTX in gastropods.

  6. Development and validation of a highly sensitive LC-MS/MS-ESI method for quantification of IIIM-019-A novel nitroimidazole derivative with promising action against Tuberculosis: Application to drug development.

    PubMed

    Kour, Gurleen; Chandan, Bal Krishan; Khullar, Mowkshi; Munagala, Gurunadham; Singh, Parvinder Pal; Bhagat, Asha; Gupta, Ajai Prakash; Vishwakarma, Ram A; Ahmed, Zabeer

    2016-05-30

    The study aims to illustrate an analytical validation of a rapid and sensitive liquid chromatography (LC) coupled to tandem mass spectrometry (MS-MS) and electrospray ionization (ESI) method for quantification of IIIM-019 (a novel nitroimidazole derivative with potential activity against Tuberculosis) in mice plasma. The extraction of the analyte and the internal standard (Tolbutamide) from the plasma samples involves protein precipitation using acetonitrile. The chromatographic separation was accomplished using a gradient mode and the mobile phase comprised of acetonitrile and 0.1% formic acid in water. The flow rate used was 0.7 ml/min on a C18e high performance Chromolith column. IIIM-019 and Tolbutamide (IS) were analyzed by combined reversed-phase LC/MS-MS with positive ion electrospray ionization. The MS-MS ion transitions used were 533>170.1, 533>198 for IIIM-019 and 271>74, 271>155 for internal standard (IS) respectively. The method was linear over a concentration range of 0.5-1000 ng/ml and the lower limit of quantification was 0.50 ng/ml. The entire study was validated for accuracy, precision, linearity, range, selectivity, lower limit of quantification (LLOQ), recovery, and matrix effect in accordance with the FDA guidelines of method validation. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. The intra and inter-day precisions were in the range of 0.51-11.18% and 0.51-7.55%. The pharmacokinetics was performed on male Balb/c mice by oral (2.5mg/kg), intraperitoneal (2.5mg/kg) and intravenous (1mg/kg) routes. The oral bioavailability of IIIM-019 was 51.6%. The method was also applied successfully in determining microsomal stability wherein the compound was found to be very slightly metabolized by rat liver microsomes.

  7. Direct injection LC-MS/MS method for identification and quantification of amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine and 3,4-methylenedioxymethamphetamine in urine drug testing.

    PubMed

    Andersson, M; Gustavsson, E; Stephanson, N; Beck, O

    2008-01-01

    A method based on direct injection of diluted urine for the identification and quantification of amphetamine, methamphetamine, 3,4-methylenedioxymetamphetamine and 3,4-methylenedioxyamphetamine in human urine by electrospray ionisation liquid chromatography-tandem mass spectrometry was validated for use as a confirmation procedure in urine drug testing. Two deuterium labelled analogues, amphetamine-D5 and 3,4-methylenedioxymetamphetamine-D5, were used as internal standards. Twenty microliter aliquots of urine were mixed with 80 microL internal standard solution in autosampler vials and 10 microL was injected. The chromatographic system consisted of a 2.0 mmx100 mm C18 column and the gradient elution buffers used acetonitrile and 25 mmol/L formic acid. Two product ions produced from the protonated molecules were monitored in the selected reaction monitoring mode. The intra- and inter-assay variability (coefficient of variation) was between 5 and 16% for all analytes at 200 and 6000 ng/mL levels. Ion suppression occurred early after injection but did not affect the identification and quantification of the analytes in authentic urine samples. The method was further validated by comparison with a reference gas chromatographic-mass spectrometric method using 479 authentic urine samples. The two methods agreed almost completely (99.8%) regarding identified analytes when applying a 150 ng/mL reporting limit. Four deviating results were observed for 3,4-methylenedioxymethamphetamine and this was due to uncertainty in quantification around the reporting limit. For the quantitative results the slope of the regression lines were between 0.9769 and 1.0146, with correlation coefficients>0.9339. We conclude that the presented liquid chromatographic-tandem mass spectrometric method is robust and reliable, and suitable for use as a confirmation method in urine drug testing for amphetamines.

  8. Validation and application of HPLC-ESI-MS/MS method for the quantification of RBBR decolorization, a model for highly toxic molecules, using several fungi strains.

    PubMed

    Perlatti, Bruno; da Silva, Maria Fátima das Graças Fernandes; Fernandes, João Batista; Forim, Moacir Rossi

    2012-11-01

    A novel analytical method using HPLC-MS/MS operating in selected reaction monitoring (SRM) for evaluation of fungi efficacy to decolorize Remazol Brilliant Blue R (RBBR) dye solution was developed, validated and applied. The method shows high sensibility allowing the detection of 4.6 pM of RBBR. Four fungal strains were tested in liquid medium, three strains of Aspergillus (Aspergillus aculeatus, Aspergillus flavus and Aspergillus fumigatus) and Phanerochaete chrysosporium. All fungi were able to degrade the dye, with efficiencies ranging from 40% for P. chrysosporium up to 99% for A. flavus during a 30-day incubation period. During the experiment, increased accumulation of degradation products was observed in A. flavus cultures containing RBBR. Through the use of full scan HPLC-MS technique it was possible to propose the biogenesis of the microbial metabolic degradation pathway. Screening using microorganisms and RBBR may be hereafter used to investigate microbial biodegradation of high toxicity molecules such as dioxins.

  9. Development and validation of a simple and sensitive method for quantification of levodopa and carbidopa in rat and monkey plasma using derivatization and UPLC-MS/MS.

    PubMed

    Junnotula, Venkatraman; Licea-Perez, Hermes

    2013-05-01

    A simple, selective, and sensitive quantitative method has been developed for the simultaneous determination of levodopa and carbidopa in rat and monkey plasma by protein precipitation using acetonitrile containing the derivatizing reagent, flourescamine. Derivatized products of levodopa and carbidopa were separated on a BEH C18 column (2.1 mm × 50 mm; 1.7 μm particle size) using ultra high performance liquid chromatography (UHPLC) without any further purification. Tandem mass spectrometry (MS/MS) was used for detection. The method was validated over the concentration range of 5-5000 ng/mL and 3-3000 ng/mL for levodopa and carbidopa, respectively in rat and monkey plasma. Due to the poor stability of the investigated analytes in biological matrices, a mixture of sodium metabisulfite and hydrazine dihydrochloride was used as a stabilizer. This method was successfully utilized to support pharmacokinetic studies in both species. The results from assay validations and incurred samples re-analysis show that the method is selective, sensitive and robust. To our knowledge, this is the first UHPLC-MS/MS based method that utilizes derivatization with fluorescamine and provides adequate sensitivity for both levodopa and carbidopa with 50 μL of sample and a run time of 3.5 min.

  10. Development and validation of an ESI-LC-MS/MS method for simultaneous identification and quantification of 24 analytes of forensic relevance in vitreous humour, whole blood and plasma.

    PubMed

    Arora, Beauty; Velpandian, Thirumurthy; Saxena, Rohit; Lalwani, Sanjeev; Dogra, T D; Ghose, Supriyo

    2016-01-01

    Detection and quantification of drugs from various biological matrices are of immense importance in forensic toxicological analysis. Despite the various reported methods, development of a new method for the detection and quantification of drugs is still an active area of research. However, every method and biological matrix has its own limitation, which further encourage forensic toxicologists to develop new methods and to explore new matrices for the analysis of drugs. In this study, an electrospray ionization-liquid chromatograph-tandem mass spectrometry (ESI-LC-MS/MS) method is developed and validated for simultaneous identification and quantification of 24 drugs of forensic relevance in various body fluids, namely, whole blood, plasma and vitreous humour. The newly developed method has been validated for intra-day and inter-day accuracy, precision, selectivity and sensitivity. Absolute recovery shows a mean of 84.5, 86.2, and 103% in the vitreous humour, whole blood and plasma respectively, which is suitable for the screening procedure. Further, the absolute matrix effect (AME) shows a mean of 105, 96.5, and 109% in the vitreous humour, whole blood and plasma, respectively. In addition, to examine the practical utility of this method, it has been applied for screening of drugs in post-mortem samples of the vitreous humour, whole blood and plasma collected at autopsy from ten cadavers. Experimental results show that the newly developed method is well applicable for screening of analytes in all the three matrices.

  11. Development of a sensitive UPLC-ESI-MS/MS method for quantification of sofosbuvir and its metabolite, GS-331007, in human plasma: Application to a bioequivalence study.

    PubMed

    Rezk, Mamdouh R; Basalious, Emad B; Karim, Iman A

    2015-10-10

    A rapid and simple LC-MS/MS method was developed and validated for the simultaneous estimation of sofosbuvir (SF) and its metabolite GS-331007 (GS) using famotidine as an internal standard (IS). The Xevo TQD LC-MS/MS was operated under the multiple-reaction monitoring mode using electrospray ionization. Extraction with ethyl acetate was used in sample preparation. The prepared samples were chromatographed on Acquity UPLC HSS C₁₈ (50 mm × 2.1 mm, 1.8 μm) column by pumping 0.1% formic acid and acetonitrile (50:50, v/v) in an isocratic mode at a flow rate of 0.3 ml/min. Method validation was performed as per the FDA guidelines and the standard curves were found to be linear in the range of 10-2500 ng/ml for both SF and its metabolite. The intra-day and inter-day precision and accuracy results were within the acceptable limits. A very short run time of 1.2 min made it possible to analyze more than 300 human plasma samples per day. The developed assay method was successfully applied to a bioequivalence study in human volunteers.

  12. Quantification of intact covalently metal labeled proteins using ESI-MS/MS.

    PubMed

    Benda, David; Schwarz, Gunnar; Beck, Sebastian; Linscheid, Michael W

    2014-01-01

    Mass spectrometric methods matured from the successful qualitative characterization of proteins in complex mixtures into methods for quantitative proteomics often based on chemical tags with stable isotope labeling. In the study presented here, we extended the application of lanthanide-ion-based tags from the quantification using inductively coupled plasma-MS into the quantification of labeled intact proteins using electrospray ionization (ESI)-MS and ESI-MS/MS. We applied the metal chelate tag MeCAT-iodoacetamide (IA) (1,4,7,10-tetraazacyclododecane N,N',N″,N″ '-tetra acetic acid with a IA reactive site). Labeled proteins were separated using C3-reversed phase-high-performance liquid chromatography interfaced to ESI-MS. We could prove that even large proteins were completely labeled at all available cysteine residues using MeCAT-IA with only a small excess of reagent. Fragmentation of labeled proteins either using infrared multiphoton dissociation in Fourier transform ion cyclotron resonance-MS or higher-energy collision dissociation with an Orbitrap gave characteristic fragments. We used these fragments to quantify several intact proteins avoiding digestion. To demonstrate the applicability, human serum albumin was quantified in blood serum. The high-performance liquid chromatography/ESI-MS/MS quantification data were validated using inductively coupled plasma-MS. Because the metal within the tag may be any of the lanthanides, multiplexing capabilities are inherent.

  13. Determination of heterocyclic aromatic amines in food products: automation of the sample preparation method prior to HPLC and HPLC-MS quantification.

    PubMed

    Fay, L B; Ali, S; Gross, G A

    1997-05-12

    Heat-processing protein-rich foods may cause the formation of heterocyclic aromatic amines (HAAs), all of which have mutagenic and some also carcinogenic potential. Accurately measuring HAA levels in food products is therefore a necessary to realistically assess this risk factor. A solid-phase extraction method for quantitative HAA analysis has been developed by us over the last few years. This method has recently been automated using a robotic workstation and now allows almost unattended sample preparation, a process which saves a human operator about five hours of benchwork. Cleaned-up samples were analyzed by high performance liquid chromatography (HPLC) and ultraviolet (UV) or mass spectrometric (MS) detection. While HPLC-UV remains the daily tool to quantify HAAs, we found HPLC-electrospray-MS to be an alternative detection method with unique advantages, suited for both HAA identification and quantification.

  14. A rapid and sensitive UPLC-MS/MS method for the simultaneous quantification of serum androsterone glucuronide, etiocholanolone glucuronide, and androstan-3α, 17β diol 17-glucuronide in postmenopausal women.

    PubMed

    Ke, Yuyong; Gonthier, Renaud; Isabelle, Maxim; Bertin, Jonathan; Simard, Jean-Nicolas; Dury, Alain Y; Labrie, Fernand

    2015-05-01

    Quantification of steroidal glucuronide conjugates by the indirect methods of immunoassay and GC-MS/MS may underestimate some conjugates since hydrolysis is needed in sample processing. In the present work, a sensitive and rapid liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed and validated for the simultaneous direct quantification of androsterone glucuronide, etiocholanolone glucuronide, and androstan-3α, 17β diol 17-glucuronide in postmenopausal women's serum. The quantification limits are 0.1ng/mL for 3α-diol-17G and 4ng/mL for both ADT-G and Etio-G, respectively, with an extraction from 200μL serum while the total run time is less than 6min for all three glucuronides. In this method, solid phase extraction is used for sample preparation. The assay has been validated in compliance with EndoCeutics SOPs and FDA guidelines for bioanalytical method development and validation. The recovery of glucuronides in stripped serum is consistent with that in unstripped serum, where the average difference in stripped and unstripped is less than 10%. A linear regression model fits well the standard curves of all three compounds with R≥0.99 where the weighting factor is 1/X. Interday accuracy and CV for all levels of QCs are within the range of 15% in both stripped and unstripped serum while all calibration curves are within the range of 6% except for LLOQs, which are within the range of 9%. Other parameters have also been assessed such as selectivity, matrix, lipemic and hemolysis effects as well as stabilities in solution and matrix. Incurred sample reanalysis has been performed with a result of over 93% within 20% of the original values. This reliable, sensitive and fast method is ready for large-scale clinical sample assays. PMID:25701608

  15. Development and Bioanalytical Validation of a Liquid Chromatographic-Tandem Mass Spectrometric (LC-MS/MS) Method for the Quantification of the CCR5 Antagonist Maraviroc in Human Plasma

    PubMed Central

    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

  16. Liquid chromatography-tandem mass spectrometry (LC/APCI-MS/MS) methods for the quantification of captan and folpet phthalimide metabolites in human plasma and urine.

    PubMed

    Berthet, Aurélie; Bouchard, Michèle; Schüpfer, Patrick; Vernez, David; Danuser, Brigitta; Huynh, Cong Khanh

    2011-02-01

    Captan and folpet are fungicides largely used in agriculture. They have similar chemical structures, except that folpet has an aromatic ring unlike captan. Their half-lives in blood are very short, given that they are readily broken down to tetrahydrophthalimide (THPI) and phthalimide (PI), respectively. Few authors measured these biomarkers in plasma or urine, and analysis was conducted either by gas chromatography coupled to mass spectrometry or liquid chromatography with UV detection. The objective of this study was thus to develop simple, sensitive and specific liquid chromatography-atmospheric pressure chemical ionization-tandem mass spectrometry (LC/APCI-MS/MS) methods to quantify both THPI and PI in human plasma and urine. Briefly, deuterated THPI was added as an internal standard and purification was performed by solid-phase extraction followed by LC/APCI-MS/MS analysis in negative ion mode for both compounds. Validation of the methods was conducted using spiked blank plasma and urine samples at concentrations ranging from 1 to 250 μg/L and 1 to 50 μg/L, respectively, along with samples of volunteers and workers exposed to captan or folpet. The methods showed a good linearity (R (2) > 0.99), recovery (on average 90% for THPI and 75% for PI), intra- and inter-day precision (RSD, <15%) and accuracy (<20%), and stability. The limit of detection was 0.58 μg/L in urine and 1.47 μg/L in plasma for THPI and 1.14 and 2.17 μg/L, respectively, for PI. The described methods proved to be accurate and suitable to determine the toxicokinetics of both metabolites in human plasma and urine.

  17. Two simple cleanup methods combined with LC-MS/MS for quantification of steroid hormones in in vivo and in vitro assays.

    PubMed

    Weisser, Johan Juhl; Hansen, Cecilie Hurup; Poulsen, Rikke; Larsen, Lizette Weber; Cornett, Claus; Styrishave, Bjarne

    2016-07-01

    Measuring both progestagens, androgens, corticosteroids as well as estrogens with a single method makes it possible to investigate the effects of endocrine-disrupting chemicals (EDCs) on the main pathways in the mammalian steroidogenesis. This paper presents two simple methods for the determination of the major steroid hormones in biological matrixes using liquid chromatography tandem mass spectrometry (LC-MS(2)). A novel method was developed for the determination of 14 steroids in the H295R in vitro assay without the need for solid phase extraction (SPE) purification prior to LC-MS(2) analysis. The in vitro assay was validated by exposing H295R cells to prochloraz for inhibiting steroid hormone secretion and by exposing cells to forskolin for inducing steroid hormone secretion. The developed method fulfills the recommendations for the H295R assay suggested by the OECD. Furthermore, a simple off-line SPE methodology was developed for the necessary clean-up of in vivo assays. Samples, such as gonad tissue, plasma and serum, are complex biological matrixes, and the SPE methodology was optimized to remove salts and proteins prior to elution of target analytes. At the same time, lipophilic compounds were retained on the SPE cartridge during elution. This, combined with the multi-steroid LC-MS(2) method, made it possible to determine 10 steroids in male Sprague-Dawley rat gonad tissue. Furthermore, it was possible to quantify 6 steroids in the plasma. In general, the observed concentration of steroid hormones in plasma, testes, and H295R cell medium corresponded well with previous studies. The off-line SPE method was validated using spiked charcoal-stripped serum. Method recovery, accuracy, precision and robustness were all good. Instrument sensitivity was in the range of 55-530 pg/mL (LLOQ). PMID:27150205

  18. Development and validation of a specific and sensitive HPLC-ESI-MS method for quantification of lysophosphatidylinositols and evaluation of their levels in mice tissues.

    PubMed

    Masquelier, Julien; Muccioli, Giulio G

    2016-07-15

    Increasing evidence suggests that lysophosphatidylinositols (LPIs), a subspecies of lysophospholipids, are important endogenous mediators. Although LPIs long remained among the less studied lysophospholipids, the identification of GPR55 as their molecular target sparked a renewed interest in the study of these bioactive lipids. Furthermore, increasing evidence points towards a role for LPIs in cancer development. However, a better understanding of the role and functions of LPIs in physiology and disease requires methods that allow for the quantification of LPI levels in cells and tissues. Because dedicated efficient methods for quantifying LPIs were missing, we decided to develop and validate an HPLC-ESI-MS method for the quantification of LPI species from tissues. LPIs are extracted from tissues by liquid/liquid extraction, pre-purified by solid-phase extraction, and finally analyzed by HPLC-ESI-MS. We determined the method's specificity and selectivity, we established calibration curves, determined the carry over (< 2%), LOD and LLOQ (between 0.116-7.82 and 4.62-92.5pmol on column, respectively), linearity (0.988 80%), intermediate precision (CV<20%) as well as the recovery from tissues. We then applied the method to determine the relative abundance of the LPI species in 15 different mouse tissues. Finally, we quantified the absolute LPI levels in six different mouse tissues. We found that while 18:0 LPI represents more than 60% of all the LPI species in the periphery (e.g. liver, gastrointestinal tract, lungs, spleen) it is much less abundant in the central nervous system where the levels of 20:4 LPI are significantly higher. Thus this validated HPLC-ESI-MS method for quantifying LPIs represents a powerful tool that will facilitate the comprehension of the pathophysiological roles of LPIs.

  19. A simple, rapid and sensitive UFLC-MS/MS method for the quantification of oral contraceptive norgestrel in human plasma and its pharmacokinetic applications.

    PubMed

    Batta, N; Pigili, R K; Pallapothu, L M K; Yejella, R P

    2014-09-01

    A simple, rapid and sensitive ultra flow liquid chromatography-tandem mass spectrometry (UFLC-MS/MS) assay for the determination of norgestrel in human plasma was developed using levonorgestrel D6 as an internal standard (IS). Norgestrel and IS were extracted from human plasma via liquid-liquid extraction. Chromatographic separation was achieved on a Zorbax XDB-Phenyl column under isocratic conditions. Detection was done by tandem mass spectrometry, operating in positive ion mode. The protonated precursor to product ion transitions monitored for norgestrel and IS were at m/z 313.30→245.40 and 319.00→251.30 respectively. The method was fully validated as per current regulatory guidelines. Anticoagulant counter ion effect was also assessed with K2EDTA and K3EDTA. The method was validated with a linearity range of 304.356-50 807.337 pg/mL having run time of 2.0 min per sample. The method has shown tremendous reproducibility with intra- and inter-day precision (%CV) less than 11.0% and intra- and inter-day accuracy less than 9.0% of nominal values. The validated method was applied to a pharmacokinetic study in human plasma samples generated after administrating a single oral dose of 0.3 mg norgestrel tablets to healthy female volunteers and has proved to be highly reliable for the analysis of clinical samples. PMID:24310360

  20. A highly sensitive HPLC-MS/MS method for quantification of 20(S)-protopanaxadiol in human plasma and its application in phase IIa clinical trial of a novel antidepressant agent.

    PubMed

    Ling, Jin; Yu, Yingjia; Zhu, Jiajun; Li, Yan; Ling, Li; Wang, Liping; Xu, Changjiang; Duan, Gengli

    2016-09-15

    A highly sensitive HPLC-MS/MS assay method was established to quantify 20(S)-protopanaxadiol (PPD) in human plasma with dexamethasone as an internal standard. The electrospray ion mass spectrometry (ESI-MS) was operated under the multiple reactions monitoring mode (MRM) using positive ion mode. PPD was extracted from 500μL plasma samples by liquid-liquid extraction then separated by a C18 analytical column with gradient elution. The concentration of PPD could be determined by this HPLC-MS/MS method over the range of 0.05-20ng/mL with the lower limit of quantification (LLOQ) of 0.05ng/mL. The method was successfully applied to phase IIa clinical trial of Yuxintine (PPD capsule) in which plasma samples of 87 subjects were analyzed following 6 weeks of oral administration of placebo or PPD capsules in 5 different doses. In this study, the measured concentration was linearly related to the oral dosage with R=0.9901. The minimum and maximum values of measured concentration were 0.06 and 11.60ng/mL, respectively. In addition, plasma concentrations of PPD in depression patients were reported for the first time in our study. PMID:27507666

  1. Impact of the grinding process on the quantification of ethyl glucuronide in hair using a validated UPLC-ESI-MS-MS method.

    PubMed

    Kummer, Natalie; Wille, Sarah M R; Di Fazio, Vincent; Ramírez Fernández, Maria Del Mar; Yegles, Michel; Lambert, Willy E E; Samyn, Nele

    2015-01-01

    The Society of Hair Testing (SoHT) has provided cutoffs for the quantification of ethyl glucuronide (EtG) in hair to indicate occasional or chronic/excessive alcohol consumption. Although several sensitive methods have been reported, past proficiency test results show a lack of reproducibility. An ultra-performance liquid chromatographic mass spectrometric method (LLOQ of 10 pg EtG/mg hair) has been validated according to the international guidelines, including the successful participation in five proficiency tests. This method was subsequently used to evaluate the impact of different grinding conditions (cut, weakly or extensively pulverized hair samples) on the final measured EtG concentration. Hair from alcohol consumers (n = 2) and commercially available quality control samples (QCs) (n = 2) was used. For the QCs, extensive pulverization led to a significantly higher amount of measured EtG. In the hair samples obtained from volunteers, cut or weakly pulverized hair resulted in EtG concentrations below the LLOQ, while the mean concentrations of 14 and 40 pg EtG/mg hair were determined after extensive pulverization. Differences in sample preparation could partially explain inter-laboratory variability. As the differences in results can lead to a different interpretation even when applying the SoHT cutoffs, it is of interest to standardize sample preparation techniques in the field of EtG hair testing. PMID:25274495

  2. The Development of a Specific and Sensitive LC-MS-Based Method for the Detection and Quantification of Hydroperoxy- and Hydroxydocosahexaenoic Acids as a Tool for Lipidomic Analysis

    PubMed Central

    Derogis, Priscilla B. M. C.; Freitas, Florêncio P.; Marques, Anna S. F.; Cunha, Daniela; Appolinário, Patricia P.; de Paula, Fernando; Lourenço, Tiago C.; Murgu, Michael; Di Mascio, Paolo; Medeiros, Marisa H. G.; Miyamoto, Sayuri

    2013-01-01

    Docosahexaenoic acid (DHA) is an n-3 polyunsaturated fatty acid that is highly enriched in the brain, and the oxidation products of DHA are present or increased during neurodegenerative disease progression. The characterization of the oxidation products of DHA is critical to understanding the roles that these products play in the development of such diseases. In this study, we developed a sensitive and specific analytical tool for the detection and quantification of twelve major DHA hydroperoxide (HpDoHE) and hydroxide (HDoHE) isomers (isomers at positions 4, 5, 7, 8, 10, 11, 13, 14, 16, 17, 19 and 20) in biological systems. In this study, HpDoHE were synthesized by photooxidation, and the corresponding hydroxides were obtained by reduction with NaBH4. The isolated isomers were characterized by LC-MS/MS, and unique and specific fragment ions were chosen to construct a selected reaction monitoring (SRM) method for the targeted quantitative analysis of each HpDoHE and HDoHE isomer. The detection limits for the LC-MS/MS-SRM assay were 1−670 pg for HpDoHE and 0.5−8.5 pg for HDoHE injected onto a column. Using this method, it was possible to detect the basal levels of HDoHE isomers in both rat plasma and brain samples. Therefore, the developed LC-MS/MS-SRM can be used as an important tool to identify and quantify the hydro(pero)xy derivatives of DHA in biological system and may be helpful for the oxidative lipidomic studies. PMID:24204871

  3. UHPLC-MS/MS method with protein precipitation extraction for the simultaneous quantification of ten antihypertensive drugs in human plasma from resistant hypertensive patients.

    PubMed

    De Nicolò, Amedeo; Avataneo, Valeria; Rabbia, Franco; Bonifacio, Gabriele; Cusato, Jessica; Tomasello, Cristina; Perlo, Elisa; Mulatero, Paolo; Veglio, Franco; Di Perri, Giovanni; D'Avolio, Antonio

    2016-09-10

    Today the management of resistant hypertension is a critical health problem: the main difficulty on this field is the discrimination of cases of poor therapeutic adherence from cases of real resistance. This gives rise to the need of high throughput and reliable quantification methods for the Therapeutic Drug Monitoring (TDM) of antihypertensive drugs. The aim of this work was the development and validation of a UHPLC-Tandem mass spectrometry assay for this application and its use in plasma from patients with resistant hypertension. The novelty of this method resides in the ability to simultaneously quantify a wide panel of antihypertensive drugs: amlodipine, atenolol, clonidine, chlortalidone, doxazosin, hydrochlorothiazide, nifedipine, olmesartan, ramipril and telmisartan. Moreover, this method stands out for its simplicity and cheapness, resulting feasible for clinical routine. Both standards and quality controls were prepared in human plasma. After the addition of internal standard, each sample underwent protein precipitation with acetonitrile and was then dried. Extracts were resuspended in water:acetonitrile 90:10 (0.05% formic acid) and then injected into the chromatographic system. Chromatographic separation was performed on an Acquity(®) UPLC HSS T3 1.8μm 2.1×150mm column, with a gradient of water and acetonitrile, both added with 0.05% formic acid. Accuracy, intra-day and inter-day precision fitted FDA guidelines for all analytes, while matrix effects and recoveries resulted stable between samples for each analyte. Finally, we tested this method by monitoring plasma concentrations in 22 hypertensive patients with good results. This simple analytical method could represent a useful tool for the management of antihypertensive therapy. PMID:27497654

  4. Development and validation of an UPLC-MS/MS method for the quantification of ethoxzolamide in plasma and bioequivalent buffers: Applications to absorption, brain distribution, and pharmacokinetic studies

    PubMed Central

    Gao, Song; Zhao, Jing; Yin, Taijun; Ma, Yong; Xu, Beibei; Moore, Anthony N.; Dash, Pramod K.; Hu, Ming

    2015-01-01

    The purpose of this study is to develop and validate an UPLC-MS/MS method to quantify ethoxzolamide in plasma (EZ) and apply the method to absorption, brain distribution, as well as pharmacokinetic studies. A C18 column was used with 0.1% of formic acid in acetonitrile and 0.1% of formic acid in water as the mobile phases to resolve EZ. The mass analysis was performed in a triple quadrupole mass spectrometer using multiple reaction monitoring (MRM) with positive scan mode. The results show that the linear range of EZ is 4.88–10,000.00 nM. The intra-day variance is less than 12.43 % and the accuracy is between 88.88–08.00 %. The inter-day variance is less than 12.87 % and accuracy is between 89.27–115.89 %. Protein precipitation was performed using methanol to extract EZ from plasma and brain tissues. Only 40 µL of plasma is needed for analysis due to the high sensitivity of this method, which could be completed in less than three minutes. This method was used to study the pharmacokinetics of EZ in SD rats, and the transport of EZ in Caco-2 and MDCK-MDR1 overexpressing cell culture models. Our data show that EZ is not a substrate for p-glycoprotein (P-gp) and its entry into the brain may not limited by the blood-brain barrier. PMID:25706567

  5. Evaluation of a rapid method for the simultaneous quantification of ribavirin, sofosbuvir and its metabolite in rat plasma by UPLC-MS/MS.

    PubMed

    Shi, Xiaojun; Zhu, Dedong; Lou, Jie; Zhu, Bo; Hu, Ai-rong; Gan, Dongmei

    2015-10-01

    A rapid and sensitive ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method for the determination of ribavirin, sofosbuvir and its metabolite GS-331007 in rat plasma was established. The analytes and the internal standard (midazolam) were separated on an Acquity UPLC BEH C18 chromatography column (2.1mm×50mm, 1.7μm) using gradient elution with a mobile phase of acetonitrile and 0.1% formic acid in water at a flow rate of 0.4mL/min. The detection was performed on a triple quadrupole tandem mass spectrometer by multiple reaction monitoring (MRM) mode to monitor the precursor-to-product ion transitions of m/z 245.1→113.1 for ribavirin, m/z 530.3→243.1 for sofosbuvir, m/z 261.5→113.1 for GS-331007 and m/z 326.2→291.1 for midazolam (IS) using a positive electrospray ionization interface. The method was validated over a concentration range of 5-1000ng/mL for ribavirin, 10-2000ng/mL for sofosbuvir and 10-2000ng/mL for GS-331007. Total time for each chromatograph was 3.0min. The intra- and inter-day precision and accuracy of the quality control samples at low, medium, and high concentration levels exhibited relative standard deviations (RSD) <10.0% and the accuracy values ranged from -10.6% to 11.6%. The method was successfully applied to a pharmacokinetic study of ribavirin, sofosbuvir and GS-331007 in rats.

  6. Development of a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantification of glucocorticoid residues in edible tissues of swine, cattle, sheep, and chicken.

    PubMed

    Chen, Dongmei; Tao, Yanfei; Liu, Zhaoying; Liu, Zhenli; Wang, Yulian; Huang, Lingli; Yuan, Zonghui

    2010-10-01

    A confirmatory and quantitative method using liquid chromatography-tandem mass spectrometry (LC-MS/MS) to determine the presence of eight glucocorticoids (prednisone, prednisolone, hydrocortisone, methylprednisolone, dexamethasone, betamethasone, beclomethasone, and fludrocortisone) in the muscles and livers of swine, cattle, and sheep and the muscle of chicken is described. After deconjugation in alkali media, samples were extracted with ethyl acetate for glucocorticoids followed by solid-phase extraction clean-up and reconstitution in the LC mobile phase. The hydrolysis procedure with sodium hydroxide was used to reduce handling time. A single-step solid-phase extraction method was optimized which is suitable for the clean-up of the compounds of interest in many diverse tissue matrices. LC separations were performed on a C(18) column with gradient elution using acetonitrile and water (containing 0.2% formic acid) and the two epimers betamethasone and dexamethasone were successfully separated. LC-electrospray ionization (ESI)-MS/MS in negative mode with selected reaction monitoring (SRM) mode was performed to improve method sensitivity and reduce matrix interference. Two SRM transitions were used for each compound. The recovery of glucocorticoids spiked at levels of 0.5-16 microg kg(-1) ranged from 55% to 107%; the between-day relative standard deviations were no more than 15%. The limits of quantification were 0.5-2.0 microg kg(-1) in muscle and 1-4 microg kg(-1) in liver. The optimized procedure was successfully applied to monitor the food at the 2008 Summer Olympics Games in Beijing, China, demonstrating the method to be simple, fast, robust, and suitable for identification and quantification of glucocorticoids residues in foods of animal origin.

  7. QbD-Driven Development and Validation of a Bioanalytical LC-MS Method for Quantification of Fluoxetine in Human Plasma.

    PubMed

    Hasnain, Mohammad Saquib; Siddiqui, Salman; Rao, Shireen; Mohanty, Priyadarsan; Ara, Tahseen Jahan; Beg, Sarwar

    2016-01-01

    The current studies describe the Quality by Design (QbD)-based development and validation of a LC-MS-MS method for quantification of fluoxetine in human plasma using fluoxetine-D5 as an internal standard (IS). Solid-phase extraction was employed for sample preparation, and linearity was observed for drug concentrations ranging between 2 and 30 ng/mL. Systematic optimization of the method was carried out by employing Box-Behnken design with mobile phase flow rate (X1), pH (X2) and mobile phase composition (X3) as the method variables, followed by evaluating retention time (Rt) (Y1) and peak area (Y2) as the responses. The optimization studies revealed reduction in the variability associated with the method variables for improving the method robustness. Validation studies of the developed method revealed good linearity, accuracy, precision, selectivity and sensitivity of fluoxetine in human plasma. Stability studies performed in human plasma through freeze-thaw, bench-top, short-term and long-term cycles, and autosampler stability revealed lack of any change in the percent recovery of the drug. In a nutshell, the developed method demonstrated satisfactory results for analysis of fluoxetine in human plasma with plausible utility in pharmacokinetic and bioequivalence studies.

  8. A rapid and sensitive LC-MS/MS method for quantification of quercetin-3-O-β-d-glucopyranosyl-7-O-β-d-gentiobioside in plasma and its application to a pharmacokinetic study.

    PubMed

    He, Xin; Tao, Guizhou; Gao, Hang; Li, Keyan; Zhang, Yazhuo; Sun, Limin; Zhang, Yingjie

    2016-09-01

    A rapid and sensitive LC-MS/MS method with good accuracy and precision was developed and validated for the pharmacokinetic study of quercetin-3-O-β-d-glucopyranosyl-7-O-β-d-gentiobioside (QGG) in Sprague-Dawley rats. Plasma samples were simply precipitated by methanol and then analyzed by LC-MS/MS. A Venusil® ASB C18 column (2.1 × 50 mm, i.d. 5 μm) was used for separation, with methanol-water (50:50, v/v) as the mobile phase at a flow rate of 300 μL/min. The optimized mass transition ion-pairs (m/z) for quantitation were 787.3/301.3 for QGG, and 725.3/293.3 for internal standard. The linear range was 7.32-1830 ng/mL with an average correlation coefficient of 0.9992, and the limit of quantification was 7.32 ng/mL. The intra- and inter-day precision and accuracy were less than ±15%. At low, medium and high quality control concentrations, the recovery and matrix effect of the analyte and IS were in the range of 89.06-92.43 and 88.58-97.62%, respectively. The method was applied for the pharmacokinetic study of QGG in Sprague-Dawley rats. Copyright © 2016 John Wiley & Sons, Ltd. PMID:26848536

  9. Quantification of sphingosine 1-phosphate by validated LC-MS/MS method revealing strong correlation with apolipoprotein M in plasma but not in serum due to platelet activation during blood coagulation.

    PubMed

    Frej, Cecilia; Andersson, Anders; Larsson, Benny; Guo, Li Jun; Norström, Eva; Happonen, Kaisa E; Dahlbäck, Björn

    2015-11-01

    Sphingosine 1-phosphate (S1P) is a signalling sphingolipid affecting multiple cellular functions of vascular and immune systems. It circulates at submicromolar levels bound to HDL-associated apolipoprotein M (apoM) or to albumin. S1P in blood is mainly produced by platelets and erythrocytes, making blood sampling for S1P quantification delicate. Standardisation of sampling is thereby of great importance to obtain robust data. By optimising and characterising the extraction procedure and the LC-MS/MS analysis, we have developed and validated a highly specific and sensitive method for S1P quantification. Blood was collected from healthy individuals (n = 15) to evaluate the effects of differential blood sampling on S1P levels. To evaluate correlation between S1P and apoM in different types of plasma and serum, apoM was measured by ELISA. The method showed good accuracy and precision in the range of 0.011 to 0.9 μM with less than 0.07 % carryover. We found that the methanol precipitation used to extract S1P co-extracted apoM and several other HDL-proteins from plasma. The platelet-associated S1P was released during coagulation, thus increasing the S1P concentration to double in serum as compared to that in plasma. Gel filtration chromatography revealed that the platelet-released S1P was mainly bound to albumin. This explains why the strong correlation between S1P and apoM levels in plasma is lost upon the clotting process and hence not observed in serum. We have developed, characterised and validated an efficient, highly sensitive and specific method for the quantification of S1P in biological material.

  10. Development, validation, and application of a novel LC-MS/MS trace analysis method for the simultaneous quantification of seven iodinated X-ray contrast media and three artificial sweeteners in surface, ground, and drinking water.

    PubMed

    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.

  11. Development and validation of a rapid turboflow LC-MS/MS method for the quantification of LSD and 2-oxo-3-hydroxy LSD in serum and urine samples of emergency toxicological cases.

    PubMed

    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. PMID:25542574

  12. Development and validation of a rapid turboflow LC-MS/MS method for the quantification of LSD and 2-oxo-3-hydroxy LSD in serum and urine samples of emergency toxicological cases.

    PubMed

    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.

  13. Antidepressants detection and quantification in whole blood samples by GC-MS/MS, for forensic purposes.

    PubMed

    Truta, Liliana; Castro, André L; Tarelho, Sónia; Costa, Pedro; Sales, M Goreti F; Teixeira, Helena M

    2016-09-01

    Depression is among the most prevalent psychiatric disorders of our society, leading to an increase in antidepressant drug consumption that needs to be accurately determined in whole blood samples in Forensic Toxicology Laboratories. For this purpose, this work presents a new gas chromatography tandem mass spectrometry (GC-MS/MS) method targeting the simultaneous and rapid determination of 14 common Antidepressants in whole blood: 13 Antidepressants (amitriptyline, citalopram, clomipramine, dothiepin, fluoxetine, imipramine, mianserin, mirtazapine, nortryptiline, paroxetine, sertraline, trimipramine and venlafaxine) and 1 Metabolite (N-desmethylclomipramine). Solid-phase extraction was used prior to chromatographic separation. Chromatographic and MS/MS parameters were selected to improve sensitivity, peak resolution and unequivocal identification of the eluted analyte. The detection was performed on a triple quadrupole tandem MS in selected ion monitoring (SIM) mode in tandem, using electronic impact ionization. Clomipramine-D3 and trimipramine-D3 were used as deutered internal standards. The validation parameters included linearity, limits of detection, lower limit of quantification, selectivity/specificity, extraction efficiency, carry-over, precision and robustness, and followed internationally accepted guidelines. Limits of quantification and detection were lower than therapeutic and sub-therapeutic concentration ranges. Overall, the method offered good selectivity, robustness and quick response (<16min) for typical concentration ranges, both for therapeutic and lethal levels. PMID:27376459

  14. A Confirmatory Method Based on HPLC-MS/MS for the Detection and Quantification of Residue of Tetracyclines in Nonmedicated Feed

    PubMed Central

    Gavilán, Rosa E.; Veiga-Gómez, Maria; Roca-Saavedra, Paula; Vazquez Belda, Beatriz; Franco, Carlos M.; Cepeda, Alberto

    2016-01-01

    The Commission Regulation 574/2011/EC set up maximum levels of coccidiostats and histomonostats in nonmedicated feed as a consequence of carry-over during manufacturing. Carry-over takes place from medicated to nonmedicated feed during feed production. Similar contamination could also occur for other pharmaceuticals such as tetracyclines, a group of antibiotics commonly employed in food production animal. The objective of this work is to present a simple and fast method for the simultaneous detection of four tetracyclines (chlortetracycline, doxycycline, oxytetracycline, and tetracycline) in nontarget feed at a μg/kg level. Validation of the method was performed according to the guideline included in the Commission Decision 2002/657/EC for official method. The validated method was successfully applied to 50 feed samples collected from different milk farms and 25 samples obtained from feed manufacturers. While oxytetracycline was the tetracycline most frequently detected, chlortetracycline was the analyte measured at the highest concentration 15.14 mg/Kg. From 75 nonmedicated feed analysed 15% resulted to be positive for the presence of one tetracycline. PMID:27595038

  15. A Confirmatory Method Based on HPLC-MS/MS for the Detection and Quantification of Residue of Tetracyclines in Nonmedicated Feed.

    PubMed

    Gavilán, Rosa E; Nebot, Carolina; Veiga-Gómez, Maria; Roca-Saavedra, Paula; Vazquez Belda, Beatriz; Franco, Carlos M; Cepeda, Alberto

    2016-01-01

    The Commission Regulation 574/2011/EC set up maximum levels of coccidiostats and histomonostats in nonmedicated feed as a consequence of carry-over during manufacturing. Carry-over takes place from medicated to nonmedicated feed during feed production. Similar contamination could also occur for other pharmaceuticals such as tetracyclines, a group of antibiotics commonly employed in food production animal. The objective of this work is to present a simple and fast method for the simultaneous detection of four tetracyclines (chlortetracycline, doxycycline, oxytetracycline, and tetracycline) in nontarget feed at a μg/kg level. Validation of the method was performed according to the guideline included in the Commission Decision 2002/657/EC for official method. The validated method was successfully applied to 50 feed samples collected from different milk farms and 25 samples obtained from feed manufacturers. While oxytetracycline was the tetracycline most frequently detected, chlortetracycline was the analyte measured at the highest concentration 15.14 mg/Kg. From 75 nonmedicated feed analysed 15% resulted to be positive for the presence of one tetracycline. PMID:27595038

  16. Simple and sensitive GC/MS-method for the quantification of urinary phenol, o- and m-cresol and ethylphenols as biomarkers of exposure to industrial solvents.

    PubMed

    Schettgen, T; Alt, A; Dewes, P; Kraus, T

    2015-07-15

    We have developed and validated a simple and sensitive method for the determination of urinary phenol as well as the urinary metabolites of toluene and ethylbenzene in one analytical run. After enzymatic hydrolysis for the cleavage of conjugates overnight, the analytes are extracted from the matrix with a liquid-liquid extraction procedure using toluene as solvent under acidic conditions. The analytes are then derivatised to volatile ethers using N,O-bis(trimethylsilyl) trifluoroacetamid (BSTFA) for cresols and ethylphenols as well as N-tert-butyldimethylsilyl-N-methyltrifluoroacetamid (MTBSTFA) for the determination of phenol. Separation and detection was carried out using capillary gas chromatography with mass-selective detection (GC-MS). Deuterium-labeled o-cresol served as internal standard for the quantification of all metabolites and guaranteed good accuracy of the results. No matrix effects were observed in the quantification of the analytes. The limit of detection for o- and m-cresol and 2- and 4-ethylphenol was 10 and 20μg/l urine and linearity ranged up to 3 and 12mg/L urine, respectively. The limit of detection for urinary phenol was 0.5mg/L with a linear range up to 200mg/L. The relative standard deviation of the within-series imprecision ranged between 3.0 and 7.2% at two spiked concentrations of 60 and 400μg/l and the relative recovery was between 84 and 104%, depending on the analyte. The method was successfully applied in proficiency testing for urinary o-cresol and phenol. This method was used for the analysis of urine samples of 17 non-smoking and 13 smoking persons from the general population without known exposure to solvents. Smokers showed a significantly higher excretion of o-cresol (median: 23 vs. 33μg/l), m-cresol (median: 43 vs. 129μg/l) as well as 4-ethylphenol (median: 25 vs. 124μg/l). Especially excretion of 4-ethylphenol was significantly correlated to smoking habits. The method seems to be suitable for biological monitoring of

  17. Highly sensitive simultaneous quantification of estrogenic tamoxifen metabolites and steroid hormones by LC-MS/MS.

    PubMed

    Johänning, Janina; Heinkele, Georg; Precht, Jana C; Brauch, Hiltrud; Eichelbaum, Michel; Schwab, Matthias; Schroth, Werner; Mürdter, Thomas E

    2015-09-01

    Tamoxifen is a mainstay in the treatment of estrogen receptor-positive breast cancer and is metabolized to more than 30 different compounds. Little is known about in vivo concentrations of estrogenic metabolites E-metabolite E, Z-metabolite E, and bisphenol and their relevance for tamoxifen efficacy. Therefore, we developed a highly sensitive HPLC-ESI-MS/MS quantification method for tamoxifen metabolites bisphenol, E-metabolite E, and Z-metabolite E as well as for the sex steroid hormones estradiol, estrone, testosterone, androstenedione, and progesterone. Plasma samples were subjected to protein precipitation followed by solid phase extraction. Upon derivatization with 3-[(N-succinimide-1-yl)oxycarbonyl]-1-methylpyridinium iodide, all analytes were separated on a sub-2-μm column with a gradient of acetonitrile in water with 0.1 % of formic acid. Analytes were detected on a triple-quadrupole mass spectrometer with positive electrospray ionization in the multiple reaction monitoring mode. Our method demonstrated high sensitivity, accuracy, and precision. The lower limits of quantification were 12, 8, and 25 pM for bisphenol, E-metabolite E, and Z-metabolite E, respectively, and 4 pM for estradiol and estrogen, 50 pM for testosterone and androstenedione, and 25 pM for progesterone. The method was applied to plasma samples of postmenopausal patients taken at baseline and under tamoxifen therapy. Graphical Abstract Sample preparation and derivatization for highly sensitive quantification of estrogenic tamoxifen metabolites and steroid hormones by HPLC-MS/MS.

  18. Highly sensitive simultaneous quantification of estrogenic tamoxifen metabolites and steroid hormones by LC-MS/MS.

    PubMed

    Johänning, Janina; Heinkele, Georg; Precht, Jana C; Brauch, Hiltrud; Eichelbaum, Michel; Schwab, Matthias; Schroth, Werner; Mürdter, Thomas E

    2015-09-01

    Tamoxifen is a mainstay in the treatment of estrogen receptor-positive breast cancer and is metabolized to more than 30 different compounds. Little is known about in vivo concentrations of estrogenic metabolites E-metabolite E, Z-metabolite E, and bisphenol and their relevance for tamoxifen efficacy. Therefore, we developed a highly sensitive HPLC-ESI-MS/MS quantification method for tamoxifen metabolites bisphenol, E-metabolite E, and Z-metabolite E as well as for the sex steroid hormones estradiol, estrone, testosterone, androstenedione, and progesterone. Plasma samples were subjected to protein precipitation followed by solid phase extraction. Upon derivatization with 3-[(N-succinimide-1-yl)oxycarbonyl]-1-methylpyridinium iodide, all analytes were separated on a sub-2-μm column with a gradient of acetonitrile in water with 0.1 % of formic acid. Analytes were detected on a triple-quadrupole mass spectrometer with positive electrospray ionization in the multiple reaction monitoring mode. Our method demonstrated high sensitivity, accuracy, and precision. The lower limits of quantification were 12, 8, and 25 pM for bisphenol, E-metabolite E, and Z-metabolite E, respectively, and 4 pM for estradiol and estrogen, 50 pM for testosterone and androstenedione, and 25 pM for progesterone. The method was applied to plasma samples of postmenopausal patients taken at baseline and under tamoxifen therapy. Graphical Abstract Sample preparation and derivatization for highly sensitive quantification of estrogenic tamoxifen metabolites and steroid hormones by HPLC-MS/MS. PMID:26206706

  19. A validated HPLC-ESI-MS/MS method for quantification of 2-hydroxy-4-methoxy benzoic acid from rat plasma and its application to pharmacokinetic study using sparse sampling methodology.

    PubMed

    Nair, Sreenath Nandakumar; Mhatre, Mandar; Menon, Sasikumar; Shailajan, Sunita

    2014-11-01

    The phenolic compound, 2-hydroxy-4-methoxy benzoic acid (HMBA), is one of the major phytoconstituents of Decalepis arayalpathra (Joseph & Chandra.) Venter, a rare and endemic medicinal plant found in the Western Ghats of India. HMBA has been attributed to possess several biological effects including anti-inflammatory, anti-pyretic, anti-oxidant and anti-diabetic. The present article describes a rapid and sensitive liquid chromatography-tandem mass spectrometric method (HPLC-MS/MS) for the determination of HMBA in rat plasma. In brief, the developed assay involves pre-treatment of the plasma samples by an optimized solid phase extraction method (recoveries for HMBA greater than 90%) followed by chromatographic separation on a Cosmosil C18 (150mm×4.6mm i.d.; 5μm particle size) analytical column with mobile phase of methanol and 10mM ammonium formate (95:5 v/v; 0.2% formic acid) delivered at a constant flow rate of 1.0mL/min. The detection and quantification was performed using an Applied Biosystems Hybrid Q-Trap API 2000 mass spectrometer equipped with electrospray ionization source (ESI) functioning in negative mode. The developed assay was validated as per the US FDA bioanalytical guidelines with the calibration curve linear over the concentration range of 5.05-2019.60ng/mL (r(2)≥0.9936) for HMBA from rat plasma. Further, the validated HPLC-MS/MS method was successfully applied to pharmacokinetic study of HMBA after oral administration of D. arayalpathra tuber extracts to female albino Wistar rats using sparse sampling methodology. Following oral administration, the maximum mean concentration in rat plasma (Cmax -1301.57±128.22ng/mL) was achieved at 1.5h (Tmax) and the area under the curve (AUC0-48h) was 8985.02±229.54ngh/mL. The elimination half-life (t1/2) and terminal elimination rate constant (Kel) were 2.48h and 0.28 L/h, respectively.

  20. Rapid screening and quantification of sulfonate derivatives in white peony root by UHPLC-MS-MS.

    PubMed

    Yan, Zhixiang; Chen, Chen; Xie, Xiabing; Fu, Bo; Yang, Xinghao

    2012-02-01

    A rapid ultra-high-performance liquid chromatographic-tandem mass spectrometric (UHPLC-MS-MS) method has been developed for rapid screening and quantitative analysis of sulfonate derivatives (SDs) in commercial white peony root. Separation was performed on an Agilent Zorbax Eclipse Plus-C18 column by gradient elution with acetonitrile-0.1% (v/v) formic acid as the mobile phase. In-source fragmentation was used to generate the characteristic fragment ion at m/z 259 and to screen for nine SDs. Detection of these SDs was further performed in multiple reaction monitoring (MRM) mode to improve sensitivity and to quantify the two SDs paeoniflorin sulfonate and benzoylpaeoniflorin sulfonate. The method was validated for specificity, linearity, limits of detection and quantification, precision, accuracy, and matrix effects. Nine commercial white peony root samples were examined by use of this method, which revealed great variety in the paeoniflorin sulfonate and benzoylpaeoniflorin sulfonate content.

  1. Retinoid quantification by HPLC/MS(n)

    NASA Technical Reports Server (NTRS)

    McCaffery, Peter; Evans, James; Koul, Omanand; Volpert, Amy; Reid, Kevin; Ullman, M. David

    2002-01-01

    Retinoic acid (RA) mediates most of the biological effects of vitamin A that are essential for vertebrate survival. It acts through binding to receptors that belong to the nuclear receptor transcription factor superfamily (Mangelsdorf et al. 1994). It is also a highly potent vertebrate teratogen. To determine the function and effects of endogenous and exogenous RA, it is important to have a highly specific, sensitive, accurate, and precise analytical procedure. Current analyses of RA and other retinoids are labor intensive, of poor sensitivity, have limited specificity, or require compatibility with RA reporter cell lines (Chen et al. 1995. BIOCHEM: Pharmacol. 50: 1257-1264; Creech Kraft et al. 1994. BIOCHEM: J. 301: 111-119; Lanvers et al. 1996. J. Chromatogr. B Biomed. Appl. 685: 233-240; Maden et al. 1998. DEVELOPMENT: 125: 4133-4144; Wagner et al. 1992. DEVELOPMENT: 116: 55-66). This paper describes an HPLC/mass spectrometry/mass spectrometry product ion scan (HPLC/MS(n)) procedure for the analysis of retinoids that employs atmospheric pressure chemical ionization MS. The retinoids are separated by normal-phase column chromatography with a linear hexane-isopropanol-dioxane gradient. Each retinoid is detected by a unique series of MS(n) functions set at optimal collision-induced dissociation energy (30% to 32%) for all MS(n) steps. The scan events are divided into three segments, based on HPLC elution order, to maximize the mass spectrometer duty cycle. The all-trans, 9-cis, and 13-cis RA isomers are separated, if desired, by an isocratic hexane-dioxane-isopropanol mobile phase. This paper describes an HPLC/MS(n) procedure possessing high sensitivity and specificity for retinoids.

  2. The Effect of Peptide Identification Search Algorithms on MS2-Based Label-Free Protein Quantification

    PubMed Central

    Degroeve, Sven; Staes, An; De Bock, Pieter-Jan

    2012-01-01

    Abstract Several approaches exist for the quantification of proteins in complex samples processed by liquid chromatography-mass spectrometry followed by fragmentation analysis (MS2). One of these approaches is label-free MS2-based quantification, which takes advantage of the information computed from MS2 spectrum observations to estimate the abundance of a protein in a sample. As a first step in this approach, fragmentation spectra are typically matched to the peptides that generated them by a search algorithm. Because different search algorithms identify overlapping but non-identical sets of peptides, here we investigate whether these differences in peptide identification have an impact on the quantification of the proteins in the sample. We therefore evaluated the effect of using different search algorithms by examining the reproducibility of protein quantification in technical repeat measurements of the same sample. From our results, it is clear that a search engine effect does exist for MS2-based label-free protein quantification methods. As a general conclusion, it is recommended to address the overall possibility of search engine-induced bias in the protein quantification results of label-free MS2-based methods by performing the analysis with two or more distinct search engines. PMID:22804230

  3. UHPLC-MS/MS quantification of buprenorphine, norbuprenorphine, methadone, and glucuronide conjugates in umbilical cord plasma.

    PubMed

    Kyle, Amy Redmond; Carmical, Jennifer; Shah, Darshan; Pryor, Jason; Brown, Stacy

    2015-10-01

    Opioid use during pregnancy can result in the newborn being physically dependent on the substance, thus experiencing drug withdrawal, termed neonatal abstinence syndrome (NAS). Buprenorphine and methadone are two drugs used to treat opioid withdrawal and are approved for use in pregnancy. Quantification of these compounds in umbilical cord plasma would help assess in utero exposure of neonates in cases of buprenorphine or methadone use during pregnancy. An LC-MS/MS method using solid-phase extraction sample preparation was developed and validated for the simultaneous quantification of methadone, buprenorphine, norbuprenorphine, and glucuronide metabolites in umbilical cord plasma. The average accuracy (percentage error) and precision (relative standard deviation) were <15% for each validated concentration. Our data establishes a 2 week maximum freezer storage window in order to achieve the most accurate cord plasma concentrations of these analytes. Additionally, we found that the umbilical cord tissue analysis was less sensitive compared with analysis with umbilical cord blood plasma, indicating that this may be a more appropriate matrix for determination of buprenorphine and metabolite concentrations. This method was successfully applied to the analysis of cord blood from women with known buprenorphine or methadone use during pregnancy. PMID:25808363

  4. Identification and Quantification of Loline-Type Alkaloids in Endophyte-Infected Grasses by LC-MS/MS.

    PubMed

    Adhikari, Khem B; Boelt, Birte; Fomsgaard, Inge S

    2016-08-10

    Lolines, fungal metabolites of the grass-endophyte association, were identified and quantified using newly developed LC-MS/MS methods in endophyte-infected grasses belonging to the Lolium and Festuca genera after extraction with three different solvents using two extraction methods. The shaking extraction method with isopropanol/water was superior to the other methods due to its high sensitivity, high accuracy (recovery within or close to the range of 80-120%), and high precision (coefficient of variation of <10%). Seven loline alkaloids were identified and quantified using our newly established LC-MS/MS methods, and N-formylloline was the most abundant (5 mg/g dry matter), followed by N-acetylloline. These LC-MS/MS methods used the shortest sample handling time and the fewest sample preparation steps and proved to be good alternatives to existing GC and GC-MS analytical methods without compromising analytical efficiency. In conclusion, we developed for the first time a highly sensitive quantitative LC-MS/MS analytical method for the accurate and reproducible quantification and a LightSight-assisted LC-QTRAP/MS qualitative method for the tentative identification of loline-type alkaloids in endophyte-infected grasses. PMID:27434508

  5. Quantification of 4-hydroxy-2-nonenal-protein adducts in the in vivo gastric digesta of mini-pigs using a GC-MS/MS method with accuracy profile validation.

    PubMed

    Delosière, Mylène; Santé-Lhoutellier, Véronique; Chantelauze, Céline; Durand, Denys; Thomas, Agnès; Joly, Charlotte; Pujos-Guillot, Estelle; Rémond, Didier; Comte, Blandine; Gladine, Cécile; Guy, Alexandre; Durand, Thierry; Laurentie, Michel; Dufour, Claire

    2016-08-10

    Hydroxyalkenals are lipid oxidation end-products resulting from the oxidation of polyunsaturated fatty acids (PUFA). This study aimed at quantifying the production of 4-hydroxy-2-nonenal-protein adducts (HNE-P) via Michael addition from n-6 PUFA oxidation in the gastric digesta of mini-pigs after the consumption of meat-based meals with different plant antioxidant contents. Using the accuracy profile procedure, we validated an extraction protocol for the quantification of HNE-P by GC-MS/MS in gastric contents. The formation of HNE-P in the gastric compartment was observed for the first time, with concentrations ranging from less than 0.52 to 1.33 nmol HNE-P per 500 mg digesta. Nevertheless, most gastric HNE-P levels were below the limit of quantification of 0.52 nmol HNE-P per 500 mg digesta. In this animal study, the protective effect of plant antioxidant sources on HNE-P formation was not evidenced contrasting with the results using TBARS as markers. PMID:27418316

  6. Chemical derivatization for enhancing sensitivity during LC/ESI-MS/MS quantification of steroids in biological samples: a review.

    PubMed

    Higashi, Tatsuya; Ogawa, Shoujiro

    2016-09-01

    Sensitive and specific methods for the detection, characterization and quantification of endogenous steroids in body fluids or tissues are necessary for the diagnosis, pathological analysis and treatment of many diseases. Recently, liquid chromatography/electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS) has been widely used for these purposes due to its specificity and versatility. However, the ESI efficiency and fragmentation behavior of some steroids are poor, which lead to a low sensitivity. Chemical derivatization is one of the most effective methods to improve the detection characteristics of steroids in ESI-MS/MS. Based on this background, this article reviews the recent advances in chemical derivatization for the trace quantification of steroids in biological samples by LC/ESI-MS/MS. The derivatization in ESI-MS/MS is based on tagging a proton-affinitive or permanently charged moiety on the target steroid. Introduction/formation of a fragmentable moiety suitable for the selected reaction monitoring by the derivatization also enhances the sensitivity. The stable isotope-coded derivatization procedures for the steroid analysis are also described.

  7. Quantification of flavonol glycosides in Camellia sinensis by MRM mode of UPLC-QQQ-MS/MS.

    PubMed

    Wu, Yahui; Jiang, Xiaolan; Zhang, Shuxiang; Dai, Xinlong; Liu, Yajun; Tan, Huarong; Gao, Liping; Xia, Tao

    2016-04-01

    Phenolic compounds are major components of tea flavour, in which catechins and flavonol glycosides play important roles in the astringent taste of tea infusion. However, the flavonol glycosides are difficult to quantify because of the large variety, as well as the inefficient seperation on chromatography. In this paper, a total of 15 flavonol glycosides in the tea plant (Camellia sinensis) were identified by the high performance liquid chromatography (HPLC) coupled to a time-of-flight mass spectrometer (TOF-MS), and a quantitative method was established based on multiple reaction monitoring (MRM) mode of ultra-high performance liquid chromatography (UPLC) coupled to a triple quadrupole mass spectrometer (QQQ-MS/MS). It provided the limit of detection and quantification to the order of picogram, which was more sensitive than the HPLC detection of the order of nanogram. The relative standard deviations of the intra- and inter-day variations in retention time and signal intensity (peak area) of six analytes were less than 0.26% and 4%, respectively. The flavonol glycosides of four tea cultivars were relatively quantified using the signal intensity (peak area) of product ion, in which six flavonol glycosides were quantified by the authentic standards. The results showed that the flavonol mono-, di- and tri-glycoside mostly accumulated in young leaves of the four tea cultivars. Notably, the myricetin 3-O-galactoside was the major component among the six flavonol glycosides detected.

  8. Identification and quantification of 30 antipsychotics in blood using LC-MS/MS.

    PubMed

    Saar, Eva; Gerostamoulos, Dimitri; Drummer, Olaf H; Beyer, Jochen

    2010-08-01

    Over the last decade, the prescription rates of antipsychotic (AP) drugs have increased worldwide. Studies have shown that the risk of sudden cardiac death is threefold higher among patients treated with APs. To investigate the presence of APs in postmortem cases, a liquid chromatography (LC)-MS/MS method was developed using only 0.1 ml of blood sample with 10 microl of internal standard (IS) (haloperidol-d(4), 1 microg/ml). After the addition of 0.2 ml of Trizma buffer, the blood sample was extracted using liquid-liquid extraction (LLE) with 1 ml of 1-chlorobutane for 5 min on a shaker at 1500 rpm. After centrifugation at 12,000 rpm for 1 min, the separated solvent layer was transferred to an autosampler vial and evaporated to dryness under N(2). The residue was reconstituted in 0.05 ml acetonitrile containing 0.1% formic acid, vortexed for 30 s and an additional 0.45 ml of 50 mmol/l ammonium formate pH 3.5 was added and the sample vortexed; 0.1 ml of the final extract was injected into a Shimadzu Prominence HPLC system, with detection of drugs achieved using an Applied Biosystems 3200 Q-TRAP LC-MS/MS system equipped with a Turbo V ion source [electron spray ionization (ESI), multiple reaction monitoring (MRM) mode]. The method has been validated according to international guidelines and was found to be selective for all tested compounds. Calibration was satisfactory for all drugs, except olanzapine, from subtherapeutic to toxic concentrations. The lower limits of quantifications (LLOQs) corresponded to the lowest concentrations used for the calibration curves. With the exception of the lowest concentrations of bromperidol, buspirone and perphenazine, accuracy data were within the acceptance interval of +/- 15% (+/- 20% at LLOQ) of the nominal values for all drugs. The method has been proven to be useful for the routine analysis of APs in postmortem blood samples.

  9. Fast MS/MS acquisition without dynamic exclusion enables precise and accurate quantification of proteome by MS/MS fragment intensity

    PubMed Central

    Zhang, Shen; Wu, Qi; Shan, Yichu; Zhao, Qun; Zhao, Baofeng; Weng, Yejing; Sui, Zhigang; Zhang, Lihua; Zhang, Yukui

    2016-01-01

    Most currently proteomic studies use data-dependent acquisition with dynamic exclusion to identify and quantify the peptides generated by the digestion of biological sample. Although dynamic exclusion permits more identifications and higher possibility to find low abundant proteins, stochastic and irreproducible precursor ion selection caused by dynamic exclusion limit the quantification capabilities, especially for MS/MS based quantification. This is because a peptide is usually triggered for fragmentation only once due to dynamic exclusion. Therefore the fragment ions used for quantification only reflect the peptide abundances at that given time point. Here, we propose a strategy of fast MS/MS acquisition without dynamic exclusion to enable precise and accurate quantification of proteome by MS/MS fragment intensity. The results showed comparable proteome identification efficiency compared to the traditional data-dependent acquisition with dynamic exclusion, better quantitative accuracy and reproducibility regardless of label-free based quantification or isobaric labeling based quantification. It provides us with new insights to fully explore the potential of modern mass spectrometers. This strategy was applied to the relative quantification of two human disease cell lines, showing great promises for quantitative proteomic applications. PMID:27198003

  10. Development and validation of two LC-MS/MS methods for the detection and quantification of amphetamines, designer amphetamines, benzoylecgonine, benzodiazepines, opiates, and opioids in urine using turbulent flow chromatography.

    PubMed

    Schaefer, Nadine; Peters, Benjamin; Schmidt, Peter; Ewald, Andreas H

    2013-01-01

    In the context of driving ability diagnostics in Germany, administrative cutoffs for various drugs and pharmaceuticals in urine have been established. Two liquid chromatography-tandem mass spectrometry methods for simultaneous detection and quantification of amphetamines, designer amphetamines, benzoylecgonine, benzodiazepines, opiates, and opioids in urine were developed and validated. A 500-μL aliquot of urine was diluted and fortified with an internal standard solution. After enzymatic cleavage, online extraction was performed by an ion-exchange/reversed-phase turbulent flow column. Separation was achieved by using a reversed-phase column and gradient elution. For detection, a Thermo Fisher TSQ Quantum Ultra Accurate Mass tandem mass spectrometer with positive electrospray ionization was used, and the analytes were measured in multiple-reaction monitoring mode detecting two transitions per precursor ion. The total run time for both methods was about 15 min. Validation was performed according to the guidelines of the Society of Toxicological and Forensic Chemistry. The results of matrix effect determination were between 78% and 116%. The limits of detection and quantification for all drugs, except zopiclone, were less than 10 ng/mL and less than 25 ng/mL, respectively. Calibration curves ranged from 25 to 200 ng/mL for amphetamines, designer amphetamines, and benzoylecgonine, from 25 to 250 ng/mL for benzodiazepines, from 12.5 to 100 ng/mL for morphine, codeine, and dihydrocodeine, and from 5 to 50 ng/mL for buprenorphine and norbuprenorphine. Intraday and interday precision values were lower than 15%, and bias values within ± 15% were achieved. Turbulent flow chromatography needs no laborious sample preparation, so the workup is less time-consuming compared with gas chromatography-mass spectrometry methods. The methods are suitable for quantification of multiple analytes at the cutoff concentrations required for driving ability diagnostics in Germany.

  11. Development and validation of two LC-MS/MS methods for the detection and quantification of amphetamines, designer amphetamines, benzoylecgonine, benzodiazepines, opiates, and opioids in urine using turbulent flow chromatography.

    PubMed

    Schaefer, Nadine; Peters, Benjamin; Schmidt, Peter; Ewald, Andreas H

    2013-01-01

    In the context of driving ability diagnostics in Germany, administrative cutoffs for various drugs and pharmaceuticals in urine have been established. Two liquid chromatography-tandem mass spectrometry methods for simultaneous detection and quantification of amphetamines, designer amphetamines, benzoylecgonine, benzodiazepines, opiates, and opioids in urine were developed and validated. A 500-μL aliquot of urine was diluted and fortified with an internal standard solution. After enzymatic cleavage, online extraction was performed by an ion-exchange/reversed-phase turbulent flow column. Separation was achieved by using a reversed-phase column and gradient elution. For detection, a Thermo Fisher TSQ Quantum Ultra Accurate Mass tandem mass spectrometer with positive electrospray ionization was used, and the analytes were measured in multiple-reaction monitoring mode detecting two transitions per precursor ion. The total run time for both methods was about 15 min. Validation was performed according to the guidelines of the Society of Toxicological and Forensic Chemistry. The results of matrix effect determination were between 78% and 116%. The limits of detection and quantification for all drugs, except zopiclone, were less than 10 ng/mL and less than 25 ng/mL, respectively. Calibration curves ranged from 25 to 200 ng/mL for amphetamines, designer amphetamines, and benzoylecgonine, from 25 to 250 ng/mL for benzodiazepines, from 12.5 to 100 ng/mL for morphine, codeine, and dihydrocodeine, and from 5 to 50 ng/mL for buprenorphine and norbuprenorphine. Intraday and interday precision values were lower than 15%, and bias values within ± 15% were achieved. Turbulent flow chromatography needs no laborious sample preparation, so the workup is less time-consuming compared with gas chromatography-mass spectrometry methods. The methods are suitable for quantification of multiple analytes at the cutoff concentrations required for driving ability diagnostics in Germany. PMID

  12. Branched perfluorooctane sulfonate isomer quantification and characterization in blood serum samples by HPLC/ESI-MS(/MS).

    PubMed

    Riddell, Nicole; Arsenault, Gilles; Benskin, Jonathan P; Chittim, Brock; Martin, Jonathan W; McAlees, Alan; McCrindle, Robert

    2009-10-15

    Perfluorooctane sulfonate (PFOS) is a global contaminant and is currently among the most prominent contaminants in human blood and wildlife samples. Although "total PFOS" (SigmaPFOS) analytical methods continue to be the most commonly used for quantification, recent analytical method developments have made it possible to resolve the various isomers of PFOS by HPLC-MS/MS. Characterized technical PFOS standards (i.e., containing a mixture of PFOS isomers) are now available that enable isomer specific quantification of PFOS, however the advantages of such an analysis have notyet been examined systematically. Herein, PFOS isomers have been individually quantified for the first time in real samples and the results are compared to a traditional SigmaPFOS method; the influence of analytical standards and isomer specific electrospray and MS/ MS behavior were also investigated. The two human serum standard reference materials chosen for analysis contained dramaticallydifferent PFOS isomer profiles (approximately 30-50% total branched isomers) emphasizing that isomer patterns should not be ignored and may provide useful information on exposure sources (i.e., direct exposure to PFOS vs indirect exposure from PFOS-precursors). Depending on the sample and the particular MS/MS transition chosen for SigmaPFOS analysis (i.e., 499-->80 or 499-->99), SigmaPFOS concentrations may be over- or underestimated compared to the isomer specific analysis. Differences in the extent of in-source fragmentation and MS/MS dissociation contributed to the systematic analytical bias. It was also shown that SigmaPFOS data are prone to interlaboratory variation due to various choices of PFOS standards and instrumental conditions used. In the future, for either SigmaPFOS or isomer specific PFOS analyses, we suggest that accuracy can be maximized and interlaboratory discrepancies minimized by using a common chemically pure technical PFOS standard characterized by 19F NMR.

  13. A new sensitive method for the quantification of glyoxal and methylglyoxal in snow and ice by stir bar sorptive extraction and liquid desorption-HPLC-ESI-MS.

    PubMed

    Müller-Tautges, Christina; Eichler, Anja; Schwikowski, Margit; Hoffmann, Thorsten

    2014-04-01

    In this study, the development of a new sensitive method for the analysis of alpha-dicarbonyls glyoxal (G) and methylglyoxal (MG) in environmental ice and snow is presented. Stir bar sorptive extraction with in situ derivatization and liquid desorption (SBSE-LD) was used for sample extraction, enrichment, and derivatization. Measurements were carried out using high-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS). As part of the method development, SBSE-LD parameters such as extraction time, derivatization reagent, desorption time and solvent, and the effect of NaCl addition on the SBSE efficiency as well as measurement parameters of HPLC-ESI-MS/MS were evaluated. Calibration was performed in the range of 1-60 ng/mL using spiked ultrapure water samples, thus incorporating the complete SBSE and derivatization process. 4-Fluorobenzaldehyde was applied as internal standard. Inter-batch precision was <12 % RSD. Recoveries were determined by means of spiked snow samples and were 78.9 ± 5.6 % for G and 82.7 ± 7.5 % for MG, respectively. Instrumental detection limits of 0.242 and 0.213 ng/mL for G and MG were achieved using the multiple reaction monitoring mode. Relative detection limits referred to a sample volume of 15 mL were 0.016 ng/mL for G and 0.014 ng/mL for MG. The optimized method was applied for the analysis of snow samples from Mount Hohenpeissenberg (close to the Meteorological Observatory Hohenpeissenberg, Germany) and samples from an ice core from Upper Grenzgletscher (Monte Rosa massif, Switzerland). Resulting concentrations were 0.085-16.3 ng/mL for G and 0.126-3.6 ng/mL for MG. Concentrations of G and MG in snow were 1-2 orders of magnitude higher than in ice core samples. The described method represents a simple, green, and sensitive analytical approach to measure G and MG in aqueous environmental samples.

  14. Analysis of Glyoxal and Methylglyoxal in atmospheric particulate matter - Qualification and Quantification using a derivatisation method for HPLC-ESI-MS

    NASA Astrophysics Data System (ADS)

    Kampf, Christopher; Hoffmann, Thorsten

    2010-05-01

    In recent years much effort has been put into the analysis of so called secondary organic aerosols (SOA). SOA is produced through gas phase oxidation of volatile organic compounds (VOC's) by atmospheric oxidants like OH- or NO3-radicals or ozone with subsequent gas-particle partitioning of the low volatility products. VOC's are emitted by both biogenic and anthropogenic sources in large amounts into the atmosphere. However, it is found that gas to particle partitioning alone cannot explain the complete amount of SOA produced in the atmosphere. It is therefore proposed that heterogeneous reactions on the particle surface or in the particles themselves could lead to the formation of additional SOA mass from semi-volatile compounds such as the reactive dialdehydes glyoxal and methylglyoxal[1]. Global glyoxal and methylglyoxal emissions are estimated to be 45 Tg a-1 and 140 Tg a-1, respectively. The oxidation of biogenic isoprene contributes to about 47% of the total glyoxal mass formed and even to about 79% for methylglyoxal[2]. Due to their high solubility in water (hydration of aldehyde functions), glyoxal and methylglyoxal have a high potential to form SOA via heterogeneous reactions in the particle phase although their volatility is relatively high. Several studies propose oligomerisation or formation of imidazole derivatives as potential reaction pathways to reduce their volatility[1,3,4,5]. Here we present a method for the qualification and quantification of both glyoxal and methylglyoxal in atmospheric PM2.5 filter samples via derivatisation with phenylhydrazine. Reproducibility, recovery and limits of detection and quantification are given. The method is found to be easily suitable for measurements at atmospheric concentration levels for both substances. First results of a measurement campaign in Mainz, Germany in August 2009 are shown for a proof of principle. Initial problems of the method development due to the chemical nature of the analytes und future

  15. Quantification of Al2O3 nanoparticles in human cell lines applying inductively coupled plasma mass spectrometry (neb-ICP-MS, LA-ICP-MS) and flow cytometry-based methods.

    PubMed

    Böhme, Steffi; Stärk, Hans-Joachim; Meißner, Tobias; Springer, Armin; Reemtsma, Thorsten; Kühnel, Dana; Busch, Wibke

    2014-01-01

    In order to quantify and compare the uptake of aluminum oxide nanoparticles of three different sizes into two human cell lines (skin keratinocytes (HaCaT) and lung epithelial cells (A549)), three analytical methods were applied: digestion followed by nebulization inductively coupled plasma mass spectrometry (neb-ICP-MS), direct laser ablation ICP-MS (LA-ICP-MS), and flow cytometry. Light and electron microscopy revealed an accumulation and agglomeration of all particle types within the cell cytoplasm, whereas no particles were detected in the cell nuclei. The internalized Al2O3 particles exerted no toxicity in the two cell lines after 24 h of exposure. The smallest particles with a primary particle size (x BET) of 14 nm (Alu1) showed the lowest sedimentation velocity within the cell culture media, but were calculated to have settled completely after 20 h. Alu2 (x BET = 111 nm) and Alu3 (x BET = 750 nm) were calculated to reach the cell surface after 7 h and 3 min, respectively. The internal concentrations determined with the different methods lay in a comparable range of 2-8 µg Al2O3/cm(2) cell layer, indicating the suitability of all methods to quantify the nanoparticle uptake. Nevertheless, particle size limitations of analytical methods using optical devices were demonstrated for LA-ICP-MS and flow cytometry. Furthermore, the consideration and comparison of particle properties as parameters for particle internalization revealed the particle size and the exposure concentration as determining factors for particle uptake. PMID:25285033

  16. Simultaneous quantification of cholesterol sulfate, androgen sulfates, and progestagen sulfates in human serum by LC-MS/MS[S

    PubMed Central

    Sánchez-Guijo, Alberto; Oji, Vinzenz; Hartmann, Michaela F.; Traupe, Heiko; Wudy, Stefan A.

    2015-01-01

    Steroids are primarily present in human fluids in their sulfated forms. Profiling of these compounds is important from both diagnostic and physiological points of view. Here, we present a novel method for the quantification of 11 intact steroid sulfates in human serum by LC-MS/MS. The compounds analyzed in our method, some of which are quantified for the first time in blood, include cholesterol sulfate, pregnenolone sulfate, 17-hydroxy-pregnenolone sulfate, 16-α-hydroxy-dehydroepiandrosterone sulfate, dehydroepiandrosterone sulfate, androstenediol sulfate, androsterone sulfate, epiandrosterone sulfate, testosterone sulfate, epitestosterone sulfate, and dihydrotestosterone sulfate. The assay was conceived to quantify sulfated steroids in a broad range of concentrations, requiring only 300 μl of serum. The method has been validated and its performance was studied at three quality controls, selected for each compound according to its physiological concentration. The assay showed good linearity (R2 > 0.99) and recovery for all the compounds, with limits of quantification ranging between 1 and 80 ng/ml. Averaged intra-day and between-day precisions (coefficient of variation) and accuracies (relative errors) were below 10%. The method has been successfully applied to study the sulfated steroidome in diseases such as steroid sulfatase deficiency, proving its diagnostic value. This is, to our best knowledge, the most comprehensive method available for the quantification of sulfated steroids in human blood. PMID:26239050

  17. LC-MS/MS quantification of salvinorin A from biological fluids.

    PubMed

    Caspers, Michael J; Williams, Todd D; Lovell, Kimberly M; Lozama, Anthony; Butelman, Eduardo R; Kreek, Mary Jeanne; Johnson, Matthew; Griffiths, Roland; Maclean, Katherine; Prisinzano, Thomas E

    2013-12-21

    A facile method for quantifying the concentration of the powerful and widely available hallucinogen salvinorin A (a selective kappa opioid agonist) from non-human primate cerebrospinal fluid (CSF) and human plasma has been developed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) in positive electrospray ionization (ESI) mode. With CSF solid phase extraction can be avoided completely by simply diluting each sample to 10 % (v/v) acetonitrile, 1 % (v/v) formic acid and injecting under high aqueous conditions for analyte focusing. Extensive plasma sample preparation was investigated including protein precipitation, SPE column selection, and plasma particulate removal. Human plasma samples were centrifuged at 21,000 × gravity for 4 minutes to obtain clear particulate-free plasma, from which 300 μl was spiked with internal standard and loaded onto a C18 SPE column with a 100 mg mL(-1) loading capacity. Guard columns (C18, hand packed 1 mm × 20 mm) were exchanged after backpressure increased above 4600psi, about 250 injections. A shallow acetonitrile/water gradient was used, 29 to 33% CH3CN over 8 minutes to elute salvinorin A. Reduction of chemical noise was achieved using tandem mass spectrometry with multiple reaction monitoring while sensitivity increases were observed using a 50 μL injection volume onto a small bore analytical column (C18, 1 mm ID × 50 mm) thus increasing peak concentration. Limits of quantification were found to be 0.0125 ng mL(-1) (CSF) and 0.05 ng mL(-1) (plasma) with interday precision and accuracy below 1.7 % and 9.42 % (CSF) and 3.47 % and 12.37 % (plasma) respectively. This method was used to determine the concentration of salvinorin A from an in vivo Rhesus monkey study and a trial of healthy human research participants, using behaviorally active doses. PMID:24416081

  18. Biodiesel production from microalgal isolates of southern Pakistan and quantification of FAMEs by GC-MS/MS analysis

    PubMed Central

    2012-01-01

    Background Microalgae have attracted major interest as a sustainable source for biodiesel production on commercial scale. This paper describes the screening of six microalgal species, Scenedesmus quadricauda, Scenedesmus acuminatus, Nannochloropsis sp., Anabaena sp., Chlorella sp. and Oscillatoria sp., isolated from fresh and marine water resources of southern Pakistan for biodiesel production and the GC-MS/MS analysis of their fatty acid methyl esters (FAMEs). Results Growth rate, biomass productivity and oil content of each algal species have been investigated under autotrophic condition. Biodiesel was produced from algal oil by acid catalyzed transesterification reaction and resulting fatty acid methyl esters (FAMEs) content was analyzed by GC/MS. Fatty acid profiling of the biodiesel, obtained from various microalgal oils showed high content of C-16:0, C-18:0, cis-Δ9C-18:1, cis-Δ11C-18:1 (except Scenedesmus quadricauda) and 10-hydroxyoctadecanoic (except Scenedesmus acuminatus). Absolute amount of C-14:0, C-16:0 and C-18:0 by a validated GC-MS/MS method were found to be 1.5-1.7, 15.0-42.5 and 4.2-18.4 mg/g, respectively, in biodiesel obtained from various microalgal oils. Biodiesel was also characterized in terms of cetane number, kinematic viscosity, density and higher heating value and compared with the standard values. Conclusion Six microalgae of local origin were screened for biodiesel production. A method for absolute quantification of three important saturated fatty acid methyl esters (C-14, C-16 and C-18) by gas chromatography-tandem mass spectrometry (GC-MS/MS), using multiple reactions monitoring (MRM) mode, was employed for the identification and quantification of biodiesels obtained from various microalgal oils. The results suggested that locally found microalgae can be sustainably harvested for the production of biodiesel. This offers the tremendous economic opportunity for an energy-deficient nation. PMID:23216896

  19. UPLC-MS/MS method with automated on-line SPE for the isomer-specific quantification of the first-generation anti-HCV protease inhibitors in peripheral blood mononuclear cells.

    PubMed

    De Nicolò, Amedeo; Abdi, Adnan Mohamed; Boglione, Lucio; Baiett, Lorena; Allegra, Sarah; Di Perri, Giovanni; D'Avolio, Antonio

    2015-11-10

    HCV infection affects over 170 million people worldwide. The current standard for treatment of genotype 1 infection is the association of the first generation protease inhibitors boceprevir or telaprevir to ribavirin and peginterferon α. Although the response rate has been improved with these new drugs, some pharmacokinetic/pharmacodinamic issues emerged in the past years. To date, some analytical methods are available for the quantification of these drugs in plasma; however, the real active concentrations of the two drugs are those in hepatocytes. Being the withdrawal of hepatocytes too invasive, in this work we aimed to develop and validate a chromatographic method coupled with tandem mass spectrometry capable of quantifying boceprevir and telaprevir isomers in peripheral blood mononuclear cells, used as an "in-vivo" cellular model of compartmentalization. The method used an on-line solid phase extraction protocol based on the new OSM(®) platform and was fully validated following FDA guidelines. This method showed mean intra- and inter-day inaccuracy and imprecision both lower than 15%, high and stable recovery and contained matrix effect, with a run time of 6min, comprehensive of SPE extraction. The method was then applied on 35 real samples from patients treated with boceprevir or telaprevir, with good analytical performances, thus assessing its eligibility for a possible future routine use. Peculiar pharmacokinetic data have been observed, suggesting the usefulness of investigating intracellular pharmacokinetics of these drugs. Further studies will be required to test the correlation of intracellular concentrations with effectiveness and toxicity of triple therapy.

  20. Development and validation of an UPLC-MS/MS method for the quantification of irinotecan, SN-38 and SN-38 glucuronide in plasma, urine, feces, liver and kidney: Application to a pharmacokinetic study of irinotecan in rats.

    PubMed

    Basu, Sumit; Zeng, Min; Yin, Taijun; Gao, Song; Hu, Ming

    2016-03-15

    The objective of this research is to develop and validate a sensitive and reproducible UPLC-MS/MS method to quantify irinotecan, its active metabolite SN-38 and SN-38 glucuronide (phase II metabolite of SN-38) simultaneously in different bio-matrices (plasma, urine, feces), tissues (liver and kidney) and to use the method to investigate its pharmacokinetic behavior in rats. Irinotecan, SN-38 and SN-38 glucuronide has been resolved and separated by C18 column using acetonitrile and 0.1% formic acid in water used as the mobile phases. Triple quadruple mass spectrometer using multiple reaction monitoring (MRM) with positive scan mode were employed to perform mass analysis. The results showed that the linear response range of irinotecan and SN-38 in plasma, feces, liver and kidney is 4.88-10000 nM, 39-5000 nM, 48.8-6250 nM and 48.8-6250 nM, respectively (R(2)>0.99). In case of SN-38 glucuronide, the standard curves were linear in the concentration range of 6.25-2000 nM, 4.88-1250 nM, 9.8-1250 nM and 9.8-1250 nM in plasma, feces, liver and kidney homogenates, respectively. The lower limit of detection (LLOD) of irinotecan, SN-38 and SN-38 glucuronide was determined to be less than 25 nM in all bio-matrices as well as tissue homogenates. Recoveries of irinotecan, SN-38 and SN-38 glucuronide at three different concentrations (low, medium and high) were not less than 85% at three different concentrations in plasma and feces. The percentage matrix factors in different bio-matrices and tissues were within 20%. The UPLC-MS/MS method was validated with intra-day and inter-day precision of less than 15% in plasma, feces, liver and kidney. Owing to the high sensitivity of this method, only 20 μl of plasma, urine and homogenates of liver, kidney and feces is needed. The validated method has been successfully employed for pharmacokinetic evaluation of irinotecan in male wistar rats to quantify irinotecan, SN-38 and SN-38 glucuronide in plasma, feces, and urine samples. PMID

  1. Quantification of 33 antidepressants by LC-MS/MS--comparative validation in whole blood, plasma, and serum.

    PubMed

    Montenarh, Deborah; Wernet, Mathias P; Hopf, Markus; Maurer, Hans H; Schmidt, Peter H; Ewald, Andreas H

    2014-09-01

    In the present study, a liquid chromatography-mass spectrometry (LC-MS/MS) multi-analyte approach based on a simple liquid-liquid extraction was developed for fast target screening and quantification of 33 antidepressants in whole blood, plasma, and serum. The method was validated with respect to selectivity, matrix effects, recovery, process efficiency, accuracy and precision, stabilities, and limits. In addition, cross-calibration between the three biosamples was done to assess the impact of the different matrices on the calibration. Whole blood, plasma, and serum (500 μL each) were extracted twice at pH 7.4 and at pH 10 with ether-ethyl acetate (1:1). Separation, detection, and quantification were performed using LC-MS/MS with electrospray ionization in positive mode. For accuracy and precision, full calibration was performed with ranges from subtherapeutic to toxic concentrations. The approach was sensitive and selective for 33 analytes in whole blood and 31 analytes in plasma and serum and accurate and precise for 30 of the 33 tested drugs in whole blood, 31 in plasma, and 28 in serum. Cross-calibration was successful only for 13 analytes in whole blood and 16 analytes in serum calculated over a calibration curve made in plasma, 12 analytes in whole blood and 15 analytes in plasma calculated over a calibration curve made in serum, and 10 analytes in plasma and 15 analytes in serum calculated over a calibration curve made in whole blood.

  2. The importance of peptide detectability for protein identification, quantification, and experiment design in MS/MS proteomics

    PubMed Central

    Li, Yong Fuga; Arnold, Randy J.; Tang, Haixu

    2010-01-01

    Peptide detectability is defined as the probability that a peptide is identified in an LC-MS/MS experiment and has been useful in providing solutions to protein inference and label-free quantification. Previously, predictors for peptide detectability trained on standard or complex samples were proposed. Although the models trained on complex samples may benefit from the large training data sets, it is unclear to what extent they are affected by the unequal abundances of identified proteins. To address this challenge and improve detectability prediction, we present a new algorithm for the iterative learning of peptide detectability from complex mixtures. We provide evidence that the new method approximates detectability with useful accuracy and, based on its design, can be used to interpret the outcome of other learning strategies. We studied the properties of peptides from the bacterium Deinococcus radiodurans and found that at standard quantities, its tryptic peptides can be roughly classified as either detectable or undetectable, with a relatively small fraction having medium detectability. We extend the concept of detectability from peptides to proteins and apply the model to predict the behavior of a replicate LC-MS/MS experiment from a single analysis. Finally, our study summarizes a theoretical framework for peptide/protein identification and label-free quantification. PMID:21067214

  3. SPE-LC/ESI/MS: a simple and reproducible method for detection and quantification of 17beta-estradiol in aqueous samples

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Steroid estrogens contained in wastewater discharge from sewage treatment plants and agricultural run-off can alter endocrine function in exposed wildlife at part per trillion (ng/L) levels. Detection and quantification of estrogens in the environment at these levels pose numerous analytical challen...

  4. Development and validation of an LC-MS/MS method for simultaneous quantification of levodopa and MD01 in rat plasma and its application to a pharmacokinetic study of mucuna pruriens extract.

    PubMed

    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. PMID:26928470

  5. Free amino acid quantification by LC-MS/MS using derivatization generated isotope-labelled standards.

    PubMed

    Johnson, David W

    2011-05-15

    The further development of derivatizing reagents for plasma amino acid quantification by tandem mass spectrometry is described. The succinimide ester of 4-methylpiperazineacetic acid (MPAS), the iTRAQ reagent, was systematically modified to improve tandem mass spectrometer (MS/MS) product ion intensity. 4-Methylpiperazinebutyryl succinimide (MPBS) and dimethylaminobutyryl succinimide (DMABS) afforded one to two orders of magnitude greater MS/MS product ion signal intensity than the MPAS derivative for simple amino acids. CD(3) analogues of the modified derivatizing reagents were evaluated for preparation of amino acid isotope-labelled quantifying standards. Acceptable accuracy and precision was obtained with d(3)-DMABS as the amino acid standards derivatizing reagent. The product ion spectra of the DMABS amino acid derivatives are diagnostic for structural isomers including valine/norvaline, alanine/sarcosine and leucine/isoleucine. Improved analytical sensitivity and specificity afforded by these derivatives may help to establish liquid chromatography tandem mass spectrometry (LC-MS/MS) with derivatization generated isotope-labelled standards a viable alternative to amino acids analysers.

  6. UPLC-MS/MS method with automated on-line SPE for the isomer-specific quantification of the first-generation anti-HCV protease inhibitors in peripheral blood mononuclear cells.

    PubMed

    De Nicolò, Amedeo; Abdi, Adnan Mohamed; Boglione, Lucio; Baiett, Lorena; Allegra, Sarah; Di Perri, Giovanni; D'Avolio, Antonio

    2015-11-10

    HCV infection affects over 170 million people worldwide. The current standard for treatment of genotype 1 infection is the association of the first generation protease inhibitors boceprevir or telaprevir to ribavirin and peginterferon α. Although the response rate has been improved with these new drugs, some pharmacokinetic/pharmacodinamic issues emerged in the past years. To date, some analytical methods are available for the quantification of these drugs in plasma; however, the real active concentrations of the two drugs are those in hepatocytes. Being the withdrawal of hepatocytes too invasive, in this work we aimed to develop and validate a chromatographic method coupled with tandem mass spectrometry capable of quantifying boceprevir and telaprevir isomers in peripheral blood mononuclear cells, used as an "in-vivo" cellular model of compartmentalization. The method used an on-line solid phase extraction protocol based on the new OSM(®) platform and was fully validated following FDA guidelines. This method showed mean intra- and inter-day inaccuracy and imprecision both lower than 15%, high and stable recovery and contained matrix effect, with a run time of 6min, comprehensive of SPE extraction. The method was then applied on 35 real samples from patients treated with boceprevir or telaprevir, with good analytical performances, thus assessing its eligibility for a possible future routine use. Peculiar pharmacokinetic data have been observed, suggesting the usefulness of investigating intracellular pharmacokinetics of these drugs. Further studies will be required to test the correlation of intracellular concentrations with effectiveness and toxicity of triple therapy. PMID:26291788

  7. PTMScan direct: identification and quantification of peptides from critical signaling proteins by immunoaffinity enrichment coupled with LC-MS/MS.

    PubMed

    Stokes, Matthew P; Farnsworth, Charles L; Moritz, Albrecht; Silva, Jeffrey C; Jia, Xiaoying; Lee, Kimberly A; Guo, Ailan; Polakiewicz, Roberto D; Comb, Michael J

    2012-05-01

    Proteomic studies of post-translational modifications by metal affinity or antibody-based methods often employ data-dependent analysis, providing rich data sets that consist of randomly sampled identified peptides because of the dynamic response of the mass spectrometer. This can complicate the primary goal of programs for drug development, mutational analysis, and kinase profiling studies, which is to monitor how multiple nodes of known, critical signaling pathways are affected by a variety of treatment conditions. Cell Signaling Technology has developed an immunoaffinity-based LC-MS/MS method called PTMScan Direct for multiplexed analysis of these important signaling proteins. PTMScan Direct enables the identification and quantification of hundreds of peptides derived from specific proteins in signaling pathways or specific protein types. Cell lines, tissues, or xenografts can be used as starting material. PTMScan Direct is compatible with both SILAC and label-free quantification. Current PTMScan Direct reagents target key nodes of many signaling pathways (PTMScan Direct: Multipathway), serine/threonine kinases, tyrosine kinases, and the Akt/PI3K pathway. Validation of each reagent includes score filtering of MS/MS assignments, filtering by identification of peptides derived from expected targets, identification of peptides homologous to expected targets, minimum signal intensity of peptide ions, and dependence upon the presence of the reagent itself compared with a negative control. The Multipathway reagent was used to study sensitivity of human cancer cell lines to receptor tyrosine kinase inhibitors and showed consistent results with previously published studies. The Ser/Thr kinase reagent was used to compare relative levels of kinase-derived phosphopeptides in mouse liver, brain, and embryo, showing tissue-specific activity of many kinases including Akt and PKC family members. PTMScan Direct will be a powerful quantitative method for elucidation of changes in

  8. Detection and quantification of MS lesions using fuzzy topological principles

    NASA Astrophysics Data System (ADS)

    Udupa, Jayaram K.; Wei, Luogang; Samarasekera, Supun; Miki, Yukio; van Buchem, M. A.; Grossman, Robert I.

    1996-04-01

    Quantification of the severity of the multiple sclerosis (MS) disease through estimation of lesion volume via MR imaging is vital for understanding and monitoring the disease and its treatment. This paper presents a novel methodology and a system that can be routinely used for segmenting and estimating the volume of MS lesions via dual-echo spin-echo MR imagery. An operator indicates a few points in the images by pointing to the white matter, the gray matter, and the CSF. Each of these objects is then detected as a fuzzy connected set. The holes in the union of these objects correspond to potential lesion sites which are utilized to detect each potential lesion as a fuzzy connected object. These 3D objects are presented to the operator who indicates acceptance/rejection through the click of a mouse button. The volume of accepted lesions is then computed and output. Based on several evaluation studies and over 300 3D data sets that were processed, we conclude that the methodology is highly reliable and consistent, with a coefficient of variation (due to subjective operator actions) of less than 1.0% for volume.

  9. Validation of a novel method to identify in utero ethanol exposure: simultaneous meconium extraction of fatty acid ethyl esters, ethyl glucuronide, and ethyl sulfate followed by LC-MS/MS quantification

    PubMed Central

    Himes, Sarah K.; Concheiro, Marta; Scheidweiler, Karl B.

    2015-01-01

    Presence of fatty acid ethyl esters (FAEE), ethyl glucuronide (EtG), and ethyl sulfate (EtS) in meconium, the first neonatal feces, identifies maternal alcohol consumption during pregnancy. Current meconium alcohol marker assays require separate analyses for FAEE and EtG/EtS. We describe development and validation of the first quantitative liquid chromatography tandem mass spectrometry assay for 9 FAEEs, EtG, and EtS in 100 mg meconium. For the first time, these alcohol markers are analyzed in the same meconium aliquot, enabling comparison of the efficiency of gestational ethanol exposure detection. 100 mg meconium was homogenized in methanol and centrifuged. The supernatant was divided, and applied to two different solid phase extraction columns for optimized analyte recovery. Limits of quantification for ethyl laurate, myristate, linolenate, palmitoleate, arachidonate, linoleate, palmitate, oleate, and stearate ranged from 25–50 ng/g, with calibration curves to 2,500–5,000 ng/g. EtG and EtS linear dynamic ranges were 5–1,000 and 2.5–500 ng/g, respectively. Mean bias and between-day imprecision were <15 %. Extraction efficiencies were 51.2–96.5 %. Matrix effects ranged from −84.7 to 16.0 %, but were compensated for by matched deuterated internal standards when available. All analytes were stable (within ±20 % change from baseline) in 3 authentic positive specimens, analyzed in triplicate, after 3 freeze/thaw cycles (−20 °C). Authentic EtG and EtS also were stable after 12 h at room temperature and 72 h at 4 °C; some FAEE showed instability under these conditions, although there was large inter-subject variability. This novel method accurately detects multiple alcohol meconium markers and enables comparison of markers for maternal alcohol consumption. PMID:24408304

  10. Development and validation of sensitive LC-MS/MS assays for quantification of HP-β-CD in human plasma and CSF

    PubMed Central

    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

  11. Improved Quantification of Free and Ester-Bound Gallic Acid in Foods and Beverages by UHPLC-MS/MS.

    PubMed

    Newsome, Andrew G; Li, Yongchao; van Breemen, Richard B

    2016-02-17

    Hydrolyzable tannins are measured routinely during the characterization of food and beverage samples. Most methods for the determination of hydrolyzable tannins use hydrolysis or methanolysis to convert complex tannins to small molecules (gallic acid, methyl gallate, and ellagic acid) for quantification by HPLC-UV. Often unrecognized, analytical limitations and variability inherent in these approaches for the measurement of hydrolyzable tannins include the variable mass fraction (0-0.90) that is released as analyte, contributions of sources other than tannins to hydrolyzable gallate (can exceed >10 wt %/wt), the measurement of both free and total analyte, and lack of controls to account for degradation. An accurate, specific, sensitive, and higher-throughput approach for the determination of hydrolyzable gallate based on ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) that overcomes these limitations was developed. PMID:26804199

  12. Improved Quantification of Free and Ester-Bound Gallic Acid in Foods and Beverages by UHPLC-MS/MS.

    PubMed

    Newsome, Andrew G; Li, Yongchao; van Breemen, Richard B

    2016-02-17

    Hydrolyzable tannins are measured routinely during the characterization of food and beverage samples. Most methods for the determination of hydrolyzable tannins use hydrolysis or methanolysis to convert complex tannins to small molecules (gallic acid, methyl gallate, and ellagic acid) for quantification by HPLC-UV. Often unrecognized, analytical limitations and variability inherent in these approaches for the measurement of hydrolyzable tannins include the variable mass fraction (0-0.90) that is released as analyte, contributions of sources other than tannins to hydrolyzable gallate (can exceed >10 wt %/wt), the measurement of both free and total analyte, and lack of controls to account for degradation. An accurate, specific, sensitive, and higher-throughput approach for the determination of hydrolyzable gallate based on ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) that overcomes these limitations was developed.

  13. Quantification of proteins on gold nanoparticles by combining MALDI-TOF MS and proteolysis

    NASA Astrophysics Data System (ADS)

    Ju, Soomi; Yeo, Woon-Seok

    2012-04-01

    Protein-coated nanoparticles have been used in many studies, including those related to drug delivery, disease diagnosis, therapeutics, and bioassays. The number and density of proteins on the particles’ surface are important parameters that need to be calculable in most applications. While quantification methods for two-dimensional surface-bound proteins are commonly found, only a few methods for the quantification of proteins on three-dimensional surfaces such as nanoparticles have been reported. In this paper, we report on a new method of quantifying proteins on nanoparticles using matrix assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry (MS). In this method, the nanoparticle-bound proteins are digested by trypsin and the resulting peptide fragments are analyzed by MALDI-TOF MS after the addition of an isotope-labeled internal standard (IS) which has the same sequence as a reference peptide of the surface-bound protein. Comparing the mass intensities between the reference peptide and the IS allows the absolute quantification of proteins on nanoparticles, because they have the same molecular milieu. As a model system, gold nanoparticles were examined using bovine serum albumin (BSA) as a coating protein. We believe that our strategy will be a useful tool that can provide researchers with quantitative information about the proteins on surfaces of three-dimensional materials.

  14. Quantification of proteins on gold nanoparticles by combining MALDI-TOF MS and proteolysis.

    PubMed

    Ju, Soomi; Yeo, Woon-Seok

    2012-04-01

    Protein-coated nanoparticles have been used in many studies, including those related to drug delivery, disease diagnosis, therapeutics, and bioassays. The number and density of proteins on the particles' surface are important parameters that need to be calculable in most applications. While quantification methods for two-dimensional surface-bound proteins are commonly found, only a few methods for the quantification of proteins on three-dimensional surfaces such as nanoparticles have been reported. In this paper, we report on a new method of quantifying proteins on nanoparticles using matrix assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry (MS). In this method, the nanoparticle-bound proteins are digested by trypsin and the resulting peptide fragments are analyzed by MALDI-TOF MS after the addition of an isotope-labeled internal standard (IS) which has the same sequence as a reference peptide of the surface-bound protein. Comparing the mass intensities between the reference peptide and the IS allows the absolute quantification of proteins on nanoparticles, because they have the same molecular milieu. As a model system, gold nanoparticles were examined using bovine serum albumin (BSA) as a coating protein. We believe that our strategy will be a useful tool that can provide researchers with quantitative information about the proteins on surfaces of three-dimensional materials. PMID:22417878

  15. Quantification of the Triazole Antifungal Compounds Voriconazole and Posaconazole in Human Serum or Plasma Using Liquid Chromatography Electrospray Tandem Mass Spectrometry (HPLC-ESI-MS/MS).

    PubMed

    Molinelli, Alejandro R; Rose, Charles H

    2016-01-01

    Voriconazole and posaconazole are triazole antifungal compounds used in the treatment of fungal infections. Therapeutic drug monitoring of both compounds is recommended in order to guide drug dosing to achieve optimal blood concentrations. In this chapter we describe an HPLC-ESI-MS/MS method for the quantification of both compounds in human plasma or serum following a simple specimen preparation procedure. Specimen preparation consists of protein precipitation using methanol and acetonitrile followed by a cleanup step that involves filtration through a cellulose acetate membrane. The specimen is then injected into an HPLC-ESI-MS/MS equipped with a C18 column and separated over an acetonitrile gradient. Quantification of the drugs in the specimen is achieved by comparing the response of the unknown specimen to that of the calibrators in the standard curve using multiple reaction monitoring.

  16. Quantification of phytochelatins in plants by reversed-phase HPLC-ESI-MS-MS.

    PubMed

    El-Zohri, M H A; Cabala, R; Frank, H

    2005-08-01

    An on-line HPLC-ESI-MS-MS method has been developed for determination of glutathione and phytochelatins (PC) in plant tissues. For sample pretreatment, dithiothreitol (DTT) must be added at the very beginning, as an anti-oxidant. Optimization of instrumental conditions i.e. composition of HPLC mobile phase, ionization efficiency of the electrospray interface, and MS-MS detection in the multiple ion-monitoring mode, are the central aspects of this work. A polystyrene-packed column was found to be superior to a standard silica-packed reversed-phase column. A concave quadratic gradient of ammonium formate buffer and acetonitrile was found to be optimum. The limits of quantitation were 0.2 micromol kg(-1) plant tissue for glutathione and PC. The method has been applied to analysis of tissue samples from Vicia faba grown in Cd-containing nutrient solutions. PMID:16001238

  17. Quantification of phytochelatins in plants by reversed-phase HPLC-ESI-MS-MS.

    PubMed

    El-Zohri, M H A; Cabala, R; Frank, H

    2005-08-01

    An on-line HPLC-ESI-MS-MS method has been developed for determination of glutathione and phytochelatins (PC) in plant tissues. For sample pretreatment, dithiothreitol (DTT) must be added at the very beginning, as an anti-oxidant. Optimization of instrumental conditions i.e. composition of HPLC mobile phase, ionization efficiency of the electrospray interface, and MS-MS detection in the multiple ion-monitoring mode, are the central aspects of this work. A polystyrene-packed column was found to be superior to a standard silica-packed reversed-phase column. A concave quadratic gradient of ammonium formate buffer and acetonitrile was found to be optimum. The limits of quantitation were 0.2 micromol kg(-1) plant tissue for glutathione and PC. The method has been applied to analysis of tissue samples from Vicia faba grown in Cd-containing nutrient solutions.

  18. Analysis of sterigmatocystin in cereals, animal feed, seeds, beer and cheese by immunoaffinity column clean-up and HPLC and LC-MS/MS quantification.

    PubMed

    Marley, Elaine; Brown, Phyllis; Mackie, Jennifer; Donnelly, Carol; Wilcox, Joyce; Pietri, Amedeo; Macdonald, Susan

    2015-01-01

    A method is reported for the analysis of sterigmatocystin in various food and feed matrices using a commercial sterigmatocystin immunoaffinity column (IAC) for sample clean-up prior to HPLC analysis by UV with mass spectrometric detection (LC-MS/MS). Cereals (wheat, oats, rye, maize and rice), sunflower seeds and animal feed were spiked with sterigmatocystin at levels from 0.75 to 50 µg kg(-1) to establish method performance. Using acetonitrile/water extraction followed by IAC clean-up, and analysis by HPLC with detection at 325 nm, recoveries ranged from 68% to 106%, with repeatability from 4.2% to 17.5%. The limit of quantification with UV detection in these matrices was 1.5 µg kg(-1). For the analysis of beer and cheese the sample preparation prior to IAC clean-up was changed to accommodate the different properties of the matrix, prior to analysis by LC-MS/MS. For beer and cheese spiked at 5.0 µg kg(-1) the recoveries were 94% and 104%, and precision (RSDs) were 1.9% and 2.9% respectively. The limits of quantification by LC-MS/MS in beer and cheese were 0.02 and 0.6 µg kg(-1) respectively. The sterigmatocystin IAC was demonstrated to provide an efficient clean-up of various matrices to enable this mycotoxin to be determined by either HPLC with UV detection or LC-MS/MS.

  19. Analysis of sterigmatocystin in cereals, animal feed, seeds, beer and cheese by immunoaffinity column clean-up and HPLC and LC-MS/MS quantification.

    PubMed

    Marley, Elaine; Brown, Phyllis; Mackie, Jennifer; Donnelly, Carol; Wilcox, Joyce; Pietri, Amedeo; Macdonald, Susan

    2015-01-01

    A method is reported for the analysis of sterigmatocystin in various food and feed matrices using a commercial sterigmatocystin immunoaffinity column (IAC) for sample clean-up prior to HPLC analysis by UV with mass spectrometric detection (LC-MS/MS). Cereals (wheat, oats, rye, maize and rice), sunflower seeds and animal feed were spiked with sterigmatocystin at levels from 0.75 to 50 µg kg(-1) to establish method performance. Using acetonitrile/water extraction followed by IAC clean-up, and analysis by HPLC with detection at 325 nm, recoveries ranged from 68% to 106%, with repeatability from 4.2% to 17.5%. The limit of quantification with UV detection in these matrices was 1.5 µg kg(-1). For the analysis of beer and cheese the sample preparation prior to IAC clean-up was changed to accommodate the different properties of the matrix, prior to analysis by LC-MS/MS. For beer and cheese spiked at 5.0 µg kg(-1) the recoveries were 94% and 104%, and precision (RSDs) were 1.9% and 2.9% respectively. The limits of quantification by LC-MS/MS in beer and cheese were 0.02 and 0.6 µg kg(-1) respectively. The sterigmatocystin IAC was demonstrated to provide an efficient clean-up of various matrices to enable this mycotoxin to be determined by either HPLC with UV detection or LC-MS/MS. PMID:26515281

  20. Development, validation and application of a stable isotope dilution liquid chromatography electrospray ionization/selected reaction monitoring/mass spectrometry (SID-LC/ESI/SRM/MS) method for quantification of keto-androgens in human serum.

    PubMed

    Tamae, Daniel; Byrns, Michael; Marck, Brett; Mostaghel, Elahe A; Nelson, Peter S; Lange, Paul; Lin, Daniel; Taplin, Mary-Ellen; Balk, Steven; Ellis, William; True, Larry; Vessella, Robert; Montgomery, Bruce; Blair, Ian A; Penning, Trevor M

    2013-11-01

    Prostate cancer is the most frequently diagnosed form of cancer in males in the United States. The disease is androgen driven and the use of orchiectomy or chemical castration, known as androgen deprivation therapy (ADT) has been employed for the treatment of advanced prostate cancer for over 70 years. Agents such as GnRH agonists and non-steroidal androgen receptor antagonists are routinely used in the clinic, but eventually relapse occurs due to the emergence of castration-resistant prostate cancer. With the appreciation that androgen signaling still persists in these patients and the development of new therapies such as abiraterone and enzalutamide that further suppresses androgen synthesis or signaling, there is a renewed need for sensitive and specific methods to quantify androgen precursor and metabolite levels to assess drug efficacy. We describe the development, validation and application of a stable isotope dilution liquid chromatography electrospray ionization selected reaction monitoring mass spectrometry (SID-LC/ESI/SRM/MS) method for quantification of serum keto-androgens and their sulfate and glucuronide conjugates using Girard-T oxime derivatives. The method is robust down to 0.2-4pg on column, depending on the androgen metabolite quantified, and can also quantify dehydroepiandrosterone sulfate (DHEA-S) in only 1μL of serum. The clinical utility of this method was demonstrated by analyzing serum androgens from patients enrolled in a clinical trial assessing combinations of pharmacological agents to maximally suppress gonadal and adrenal androgens (Targeted Androgen Pathway Suppression, TAPS clinical trial). The method was validated by correlating the results obtained with a hydroxylamine derivatization procedure coupled with tandem mass spectrometry using selected reaction monitoring that was conducted in an independent laboratory.

  1. A Validated HPLC/MS Limit Test Method for a Potential Genotoxic Impurity in Cilostazol and its Quantification in the API and in the Commercially Available Drug Product.

    PubMed

    Bray, Luigi; Monzani, Luca; Brunoldi, Enrico; Allegrini, Pietro

    2015-01-01

    Cilostazol is a selective inhibitor of type 3 phosphodiesterase. 5-(3-Chloropropyl)-1-cyclohexyl-1H-tetrazole, used as an intermediate in the synthesis of cilostazol, has a primary alkyl chloride group, a well-known alerting function for genotoxic activity. Upon request from a regulatory agency, a limit test in accordance with ICH Q2(R1) added with the accuracy of a recovery test of 5-(4-chlorobutyl)-1-cyclohexyl-1H-tetrazole in cilostazol was developed and validated. The application of the method highlighted the need to optimize the purification process to ensure levels of this potential genotoxic impurity in the final active pharmaceutical ingredient below the established limit. Also, the analytical method was suitable to determine the amount of the impurity in samples of the commercially available drug product, which showed the levels to be above the established threshold of toxicological concern (TTC). PMID:26839820

  2. A Validated HPLC/MS Limit Test Method for a Potential Genotoxic Impurity in Cilostazol and its Quantification in the API and in the Commercially Available Drug Product.

    PubMed

    Bray, Luigi; Monzani, Luca; Brunoldi, Enrico; Allegrini, Pietro

    2015-01-01

    Cilostazol is a selective inhibitor of type 3 phosphodiesterase. 5-(3-Chloropropyl)-1-cyclohexyl-1H-tetrazole, used as an intermediate in the synthesis of cilostazol, has a primary alkyl chloride group, a well-known alerting function for genotoxic activity. Upon request from a regulatory agency, a limit test in accordance with ICH Q2(R1) added with the accuracy of a recovery test of 5-(4-chlorobutyl)-1-cyclohexyl-1H-tetrazole in cilostazol was developed and validated. The application of the method highlighted the need to optimize the purification process to ensure levels of this potential genotoxic impurity in the final active pharmaceutical ingredient below the established limit. Also, the analytical method was suitable to determine the amount of the impurity in samples of the commercially available drug product, which showed the levels to be above the established threshold of toxicological concern (TTC).

  3. A Validated HPLC/MS Limit Test Method for a Potential Genotoxic Impurity in Cilostazol and its Quantification in the API and in the Commercially Available Drug Product

    PubMed Central

    Bray, Luigi; Monzani, Luca; Brunoldi, Enrico; Allegrini, Pietro

    2015-01-01

    Cilostazol is a selective inhibitor of type 3 phosphodiesterase. 5-(3-Chloropropyl)-1-cyclohexyl-1H-tetrazole, used as an intermediate in the synthesis of cilostazol, has a primary alkyl chloride group, a well-known alerting function for genotoxic activity. Upon request from a regulatory agency, a limit test in accordance with ICH Q2(R1) added with the accuracy of a recovery test of 5-(4-chlorobutyl)-1-cyclohexyl-1H-tetrazole in cilostazol was developed and validated. The application of the method highlighted the need to optimize the purification process to ensure levels of this potential genotoxic impurity in the final active pharmaceutical ingredient below the established limit. Also, the analytical method was suitable to determine the amount of the impurity in samples of the commercially available drug product, which showed the levels to be above the established threshold of toxicological concern (TTC). PMID:26839820

  4. Simple and accurate quantification of BTEX in ambient air by SPME and GC-MS.

    PubMed

    Baimatova, Nassiba; Kenessov, Bulat; Koziel, Jacek A; Carlsen, Lars; Bektassov, Marat; Demyanenko, Olga P

    2016-07-01

    Benzene, toluene, ethylbenzene and xylenes (BTEX) comprise one of the most ubiquitous and hazardous groups of ambient air pollutants of concern. Application of standard analytical methods for quantification of BTEX is limited by the complexity of sampling and sample preparation equipment, and budget requirements. Methods based on SPME represent simpler alternative, but still require complex calibration procedures. The objective of this research was to develop a simpler, low-budget, and accurate method for quantification of BTEX in ambient air based on SPME and GC-MS. Standard 20-mL headspace vials were used for field air sampling and calibration. To avoid challenges with obtaining and working with 'zero' air, slope factors of external standard calibration were determined using standard addition and inherently polluted lab air. For polydimethylsiloxane (PDMS) fiber, differences between the slope factors of calibration plots obtained using lab and outdoor air were below 14%. PDMS fiber provided higher precision during calibration while the use of Carboxen/PDMS fiber resulted in lower detection limits for benzene and toluene. To provide sufficient accuracy, the use of 20mL vials requires triplicate sampling and analysis. The method was successfully applied for analysis of 108 ambient air samples from Almaty, Kazakhstan. Average concentrations of benzene, toluene, ethylbenzene and o-xylene were 53, 57, 11 and 14µgm(-3), respectively. The developed method can be modified for further quantification of a wider range of volatile organic compounds in air. In addition, the new method is amenable to automation. PMID:27154647

  5. Relative quantification of biomarkers using mixed-isotope labeling coupled with MS

    PubMed Central

    Chapman, Heidi M; Schutt, Katherine L; Dieter, Emily M; Lamos, Shane M

    2013-01-01

    The identification and quantification of important biomarkers is a critical first step in the elucidation of biological systems. Biomarkers take many forms as cellular responses to stimuli and can be manifested during transcription, translation, and/or metabolic processing. Increasingly, researchers have relied upon mixed-isotope labeling (MIL) coupled with MS to perform relative quantification of biomarkers between two or more biological samples. MIL effectively tags biomarkers of interest for ease of identification and quantification within the mass spectrometer by using isotopic labels that introduce a heavy and light form of the tag. In addition to MIL coupled with MS, a number of other approaches have been used to quantify biomarkers including protein gel staining, enzymatic labeling, metabolic labeling, and several label-free approaches that generate quantitative data from the MS signal response. This review focuses on MIL techniques coupled with MS for the quantification of protein and small-molecule biomarkers. PMID:23157360

  6. Towards absolute quantification of therapeutic monoclonal antibody in serum by LC-MS/MS using isotope-labeled antibody standard and protein cleavage isotope dilution mass spectrometry.

    PubMed

    Heudi, Olivier; Barteau, Samuel; Zimmer, Dieter; Schmidt, Joerg; Bill, Kurt; Lehmann, Natalie; Bauer, Christian; Kretz, Olivier

    2008-06-01

    Although LC-MS methods are increasingly used for the absolute quantification of proteins, the lack of appropriate internal standard (IS) hinders the development of rapid and standardized analytical methods for both in vitro and in vivo studies. Here, we have developed a novel method for the absolute quantification of a therapeutic protein, which is monoclonal antibody (mAb). The method combines liquid chromatography tandem mass spectrometry (LC-MS/MS) and protein cleavage isotope dilution mass spectrometry with the isotope-labeled mAb as IS. The latter was identical to the analyzed mAb with the exception that each threonine contains four (13)C atoms and one (15)N atom. Serum samples were spiked with IS prior to the overnight trypsin digestion and subsequent sample cleanup. Sample extracts were analyzed on a C18 ACE column (150 mm x 4.6 mm) using an LC gradient time of 11 min. Endogenous mAb concentrations were determined by calculating the peak height ratio of its signature peptide to the corresponding isotope-labeled peptide. The linear dynamic range was established between 5.00 and 1000 microg/mL mAb with accuracy and precision within +/-15% at all concentrations and below +/-20% at the LLOQ (lower limit of quantification). The overall method recovery in terms of mAb was 14%. The losses due to sample preparation (digestion and purification) were 72% from which about 32% was due to the first step of the method, the sample digestion. This huge loss during sample preparation strongly emphasizes the necessity to employ an IS right from the beginning. Our method was successfully applied to the mAb quantification in marmoset serum study samples, and the precision obtained on duplicate samples was, in most cases, below 20%. The comparison with enzyme-linked immunosorbent assay (ELISA) showed higher exposure in terms of AUC and Cmax with the LC-MS/MS method. Possible reasons for this discrepancy are discussed in this study. The results of this study indicate that our LC-MS/MS

  7. Quantification of Terpenes by 1DGC-MS and 2DGC-TOF-MS

    NASA Astrophysics Data System (ADS)

    Flores, R. M.; Perlinger, J. A.; Doskey, P. V.

    2009-12-01

    Biogenic emissions are the primary source of volatile organic compounds in the global troposphere. Deciduous and coniferous forests are the principal emitters of a complex mixture of isoprene (C5H8), monoterpenes (C10H16), and sesquiterpenes (C15H24). Sesquiterpenes are readily oxidized in the atmosphere producing secondary organic aerosols (SOA) with 100% yields. The SOA are hydrophilic and scatter light, and thus, increase albedo and lead to a cooling effect. In addition, both monoterpene and sesquiterpene generated SOA are effective cloud condensation nuclei leading to an increase in the particle number concentration and to the formation of clouds that also increase albedo. To quantify the complex mixture of terpenes and their oxidation products requires development of on-line extraction and comprehensive two-dimensional gas chromatographic techniques. One objective of this work was to compare one-dimensional gas chromatography-mass spectrometry (1DGC-MS) and two-dimensional gas chromatography time-of-flight mass spectrometry (2DGC-TOFMS) for quantifying eight monoterpenes (alpha- and beta-pinene, limonene, 3-carene, linalool, terpinolene, myrcene and ocimene) and eight sesquiterpenes (beta-caryophyllene, humulene, alpha-cedrene, cis-nerolidol, trans-nerolidol, cedrol, camphene and farnesene) in air samples collected in Northern Michigan. Future research involves coupling thermal desorption and supercritical fluid extraction devices to a GC×2GC for routine quantification of the complex mixture of terpenes and their oxidation products in rural and urban air.

  8. LC-ESI MS/MS quantification of atropine and six other antimuscarinic tropane alkaloids in plasma.

    PubMed

    John, Harald; Binder, Tobias; Höchstetter, Hans; Thiermann, Horst

    2010-01-01

    We have developed and validated a quantitative liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI MS/MS) procedure for the simultaneous determination of seven natural and semisynthetic tropane alkaloids in plasma: atropine (d-hyoscyamine/l-hyoscyamine), cocaine, homatropine, ipratropium, littorine, N-butylscopolamine, and scopolamine. Plasma and serum samples were precipitated for deproteinization (recovery 88-94%), followed by reversed-phase-based liquid chromatography prior to positive electrospray ionization for detection by multiple reaction monitoring using a linear ion trap quadrupole mass spectrometer. All analytes were quantified using cocaine-d3 as an internal standard suitable and reliable for robust, precise (coefficient of variation 2-13%), and accurate (87-122%) measurement within a linear range of 3 orders of magnitude (0.05-50 ng/ml plasma). The method was exemplarily applied to stability studies in phosphate-buffered saline, human serum, and rabbit serum. Each alkaloid was incubated separately and samples were taken at distinct incubation time points. Supernatants of diverse alkaloids at corresponding time points were pooled and subjected to simultaneous LC-ESI MS/MS quantification. This combinatorial analysis design allowed us to analyze the stability of samples with a drastically reduced number of chromatographic runs. In the presence of rabbit serum, all tropane alkaloids tested were degraded significantly within minutes to hours, with the exception of the stable semisynthetic compounds ipratropium and N-butylscopolamine. In contrast, in the presence of equal concentrations of human serum, no degradation was observed for any of the compounds, with the exception of cocaine. Relevant enzymes involved in enzymatic degradation are discussed.

  9. Ultrapressure liquid chromatography-tandem mass spectrometry assay using atmospheric pressure photoionization (UPLC-APPI-MS/MS) for quantification of 4-methoxydiphenylmethane in pharmacokinetic evaluation.

    PubMed

    Farhan, Nashid; Fitzpatrick, Sean; Shim, Yun M; Paige, Mikell; Chow, Diana Shu-Lian

    2016-09-01

    4-Methoxydiphenylmethane (4-MDM), a selective augmenter of Leukotriene A4 Hydrolase (LTA4H), is a new anti-inflammatory compound for potential treatment of chronic obstructive pulmonary disease (COPD). Currently, there is no liquid chromatography tandem mass spectrometric (LC-MS/MS) method for the quantification of 4-MDM. A major barrier for developing the LC-MS/MS method is the inability of electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) to ionize 4-MDM due to its hydrophobicity and lack of any functional group for ionization. With the advent of atmospheric pressure photoionization (APPI) technique, many hydrophobic compounds have been demonstrated to ionize by charge transfer reactions. In this study, a highly sensitive ultrapressure liquid chromatography tandem mass spectrometry assay using atmospheric pressure photoionization (UPLC-APPI-MS/MS) for the quantifications of 4-MDM in rat plasma has been developed and validated. 4-MDM was extracted from the plasma by solid phase extraction (SPE) and separated chromatographically using a reverse phase C8 column. The photoionization (PI) was achieved by introducing anisole as a dopant to promote the reaction of charge transfer. The assay with a linear range of 5 (LLOQ)-400ngmL(-1) met the regulatory requirements for accuracy, precision and stability. The validated assay was employed to quantify the plasma concentrations of 4-MDM after an oral dosing in Sprague Dawley (SD) rats.

  10. Detection and quantification of bacterial spoilage in milk and pork meat using MALDI-TOF-MS and multivariate analysis.

    PubMed

    Nicolaou, Nicoletta; Xu, Yun; Goodacre, Royston

    2012-07-17

    Microbiological safety is one of the cornerstones of quality control in the food industry. Identification and quantification of spoilage bacteria in pasteurized milk and meat in the food industry currently relies on accurate and sensitive yet time-consuming techniques which give retrospective values for microbial contamination. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), a proven technique in the field of protein and peptide identification and quantification, may be a valuable alternative approach for the rapid assessment of microbial spoilage. In this work we therefore developed MALDI-TOF-MS as a novel analytical approach for the assessment of food that when combined with chemometrics allows for the detection and quantification of milk and pork meat spoilage bacteria. To develop this approach, natural spoilage of pasteurized milk and raw pork meat samples incubated at 15 °C and at room temperature, respectively, was conducted. Samples were collected for MALDI-TOF-MS analysis (which took 4 min per sample) at regular time intervals throughout the spoilage process, with concurrent calculation and documentation of reference total viable counts using traditional microbiological methods (these took 2 days). Multivariate statistical techniques such as principal component discriminant function analysis, canonical correlation analysis, partial least-squares (PLS) regression, and kernel PLS (KPLS) were used to analyze the data. The results from MALDI-TOF-MS combined with PLS or KPLS gave excellent bacterial quantification results for both milk and meat spoilage, and typical root mean squared errors for prediction in test spectra were between 0.53 and 0.79 log unit. Overall these novel findings strongly indicate that MALDI-TOF-MS when combined with chemometric approaches would be a useful adjunct for routine use in the milk and meat industry as a fast and accurate viable bacterial detection and quantification method.

  11. High temperature liquid chromatography hyphenated with ESI-MS and ICP-MS detection for the structural characterization and quantification of halogen containing drug metabolites.

    PubMed

    de Vlieger, Jon S B; Giezen, Mark J N; Falck, David; Tump, Cornelis; van Heuveln, Fred; Giera, Martin; Kool, Jeroen; Lingeman, Henk; Wieling, Jaap; Honing, Maarten; Irth, Hubertus; Niessen, Wilfried M A

    2011-07-18

    In this paper we describe the hyphenation of high temperature liquid chromatography with ICP-MS and ESI-MS for the characterization of halogen containing drug metabolites. The use of temperature gradients up to 200°C enabled the separation of metabolites with low organic modifier content. This specific property allowed the use of detection methods that suffer from (significant) changes in analyte response factors as a function of the organic modifier content such as ICP-MS. Metabolites of two kinase inhibitors (SB-203580-Iodo and MAPK inhibitor VIII) produced by bacterial cytochrome P450 BM3 mutants and human liver microsomes were identified based on high resolution MS(n) data. Quantification was done using their normalized and elemental specific response in the ICP-MS. The importance of these kinds of quantification strategies is stressed by the observation that the difference of the position of one oxygen atom in a structure can greatly affect its response in ESI-MS and UV detection.

  12. Evaluation of tamoxifen and metabolites by LC-MS/MS and HPLC methods.

    PubMed

    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. PMID:24693573

  13. Pseudo isobaric peptide termini labelling for relative proteome quantification by SWATH MS acquisition.

    PubMed

    Zhang, Shen; Chen, Lingfan; Shan, Yichu; Sui, Zhigang; Wu, Qi; Zhang, Lihua; Liang, Zhen; Zhang, Yukui

    2016-08-01

    The pseudo isobaric peptide termini labeling (IPTL) method is a remarkable strategy in quantitative proteomics, and has been efficiently applied in biological studies due to its high quantitative accuracy. However, irreproducible precursor ion selection caused by data dependent acquisition and the chromatographic shift caused by isotope effects limit the wide application of this method. Herein, we expand the use of pseudo IPTL to SWATH MS application and develop a novel quantitative strategy, termed SWATH-pseudo-IPTL, by which the relative quantification could be achieved by comparing the "complete" extracted ion chromatogram (XIC) intensity of MS/MS scan instead of a single intensity measurement in DDA-pseudo-IPTL which only reflected the peptide abundances at that given time. The quantitative analysis of various proportions of mixed HeLa samples revealed the strong accuracy and precision of our SWATH-pseudo-IPTL method, both of which were better than that of the DDA-pseudo-IPTL strategy. SWATH-pseudo-IPTL was also applied to the quantitative profiling of the proteome from human hepatocellular carcinoma cell lines with high and low metastatic potential, and most of the differentially expressed proteins were related to tumorigenesis and tumor metastasis, demonstrating the feasibility of this methodology for biological applications.

  14. Simultaneous quantification of cardiolipin, bis(monoacylglycero)phosphate and their precursors by hydrophilic interaction LC-MS/MS including correction of isotopic overlap.

    PubMed

    Scherer, Max; Schmitz, Gerd; Liebisch, Gerhard

    2010-11-01

    Cardiolipin (CL) and bis(monoacylglycero)phosphate (BMP) are unique lipid structures with important biological roles for mitochondrial integrity and endolysosomal degradation, respectively. They are synthesized from common precursors, phosphatidylglycerol (PG) and phosphatidic acid (PA). Here we present a rapid method for the simultaneous quantification of BMP, CL, PG, and PA using hydrophilic interaction chromatography coupled with electrospray ionization tandem mass spectrometry (HILIC-MS/MS). HILIC provides coelution of lipid species and their internal standards required for accurate quantification. Coelution leads to isotope overlap of lipid species which was successfully corrected. This assay was validated in mouse heart tissue and primary human skin fibroblasts. It shows reproducibility and limits of detection sufficient for biomarker studies contributing to basic research on BMP and CL metabolism. PMID:20945919

  15. Simultaneous quantification of niacin and its three main metabolites in human plasma by LC-MS/MS.

    PubMed

    Liu, Man; Zhang, Dan; Wang, Xiaolin; Zhang, Lina; Han, Jing; Yang, Man; Xiao, Xue; Zhang, Yanan; Liu, Huichen

    2012-09-01

    A sensitive and specific LC-MS/MS method for the simultaneous quantification of niacin (NA) and its three main metabolites nicotinamide (NAM), nicotinuric acid (NUA) and N-methyl-2-pyridone-5-carboxamide (2-Pyr) in human plasma has been developed and validated. Plasma samples (200 μL) were prepared by deproteinization with acetonitrile (500 μL), then the supernatant after centrifugation was evaporated and reconstituted. Chromatography was performed on a phenomenex synergi hydro-RP column with an isocratic elution of methanol-0.1% formic acid (5:95, v/v). The full separation of all analytes was achieved within 9 min. Multiple-reaction monitoring (MRM) using the fragmentation transitions of m/z 124.1 → 80.1, 123.1 → 80.0, 181.0 → 79.0 and 153.1 → 110.2 in positive electrospray ionization (ESI) mode was performed to quantify NA, NAM, NUA and 2-Pyr, respectively. The calibration curves were linear over the concentration range of 2.0-3000 ng/mL for NA and NUA, 10.0-1600 ng/mL for NAM and 50.0-5000 ng/mL for 2-Pyr. This method has been validated in accordance with the US FDA guidelines for bioanalytical method development and applied to the determination of NA and its three main metabolites in Chinese subjects following a single oral dose of niacin extended-release and simvastatin 1000 mg/20mg. In particular, because of the endogenous NAM and 2-Pyr in human plasma, the concentrations of NAM and 2-Pyr in human plasma after dosing were determined by subtracting blank values of them.

  16. Absolute quantification of transferrin in blood samples of harbour seals using HPLC-ICP-MS.

    PubMed

    Grebe, Mechthild; Pröfrock, Daniel; Kakuschke, Antje; Broekaert, Jose A C; Prange, Andreas

    2011-02-01

    Harbour seals (Phoca vitulina) are bio-indicators for the assessment of their habitat and environmental changes. Besides population parameters and trends (survival, age structure, sex ratio), the individual health status represents a further important parameter for this assessment. The health status of seals is a complex and vague term, determined by a wide range of diagnostic parameters. Quantities of important blood proteins such as transferrin (Tf), as well as altered distribution patterns of its glycoforms, are frequently used as biomarkers in clinical diagnosis. Within this context Tf quantities and a varying pattern of its glycoforms are used as indicator for e.g. certain liver diseases, which also represents one of the most frequently observed pathological indication in harbour seals of the North Sea. Currently, most assay based quantification methods for Tf are limited since they often provide only information regarding the total Tf concentration rather than information of its different glycoforms. Due to a lack of suitable seal Tf antibodies also the application of more specific antibody based approaches is not possible. Within this background a new approach for the absolute quantification of the iron-transport protein Tf in the blood of harbour seals using its characteristic iron content and HPLC-ICP-MS detection is described. Method validation was performed using a certified human serum reference material (ERM-DA470K/IFCC). A Tf concentration of 2.33 ± 0.03 g L(-1) (sum of all quantified glycoforms) has been calculated, which is in good agreement with the certified total Tf concentration of 2.35 ± 0.08 g L(-1), confirming the accuracy of the proposed analytical method. Finally, different seal samples were analysed to demonstrate the suitability of the procedure for the quantification of Tf in real samples as well as to observe modified glycoform patterns. Compared to our previous studies for the first time it was possible to quantify the serum Tf

  17. Quantification of Hydrazine in Human Urine by HPLC-MS-MS.

    PubMed

    Isenberg, Samantha L; Carter, Melissa D; Crow, Brian S; Graham, Leigh Ann; Johnson, Darryl; Beninato, Nick; Steele, Kandace; Thomas, Jerry D; Johnson, Rudolph C

    2016-05-01

    Currently used on F-16 fighter jets and some space shuttles, hydrazine could be released at toxic levels to humans as a result of an accidental leakage or spill. Lower-level exposures occur in industrial workers or as a result of the use of some pharmaceuticals. A method was developed for the quantitation of hydrazine in human urine and can be extended by dilution with water to cover at least six orders of magnitude, allowing measurement at all clinically significant levels of potential exposure. Urine samples were processed by isotope dilution, filtered, derivatized and then quantified by HPLC-MS-MS. The analytical response ratio was linearly proportional to the urine concentration of hydrazine from 0.0493 to 12.3 ng/mL, with an average correlation coefficientRof 0.9985. Inter-run accuracy for 21 runs, expressed as percent relative error (% RE), was ≤14%, and the corresponding precision, expressed as percent relative standard deviation (% RSD), was ≤15%. Because this method can provide a quantitative measurement of clinical samples over six orders of magnitude, it can be used to monitor trace amounts of hydrazine exposure as well as industrial and environmental exposure levels.

  18. Quantification of Hydrazine in Human Urine by HPLC-MS-MS.

    PubMed

    Isenberg, Samantha L; Carter, Melissa D; Crow, Brian S; Graham, Leigh Ann; Johnson, Darryl; Beninato, Nick; Steele, Kandace; Thomas, Jerry D; Johnson, Rudolph C

    2016-05-01

    Currently used on F-16 fighter jets and some space shuttles, hydrazine could be released at toxic levels to humans as a result of an accidental leakage or spill. Lower-level exposures occur in industrial workers or as a result of the use of some pharmaceuticals. A method was developed for the quantitation of hydrazine in human urine and can be extended by dilution with water to cover at least six orders of magnitude, allowing measurement at all clinically significant levels of potential exposure. Urine samples were processed by isotope dilution, filtered, derivatized and then quantified by HPLC-MS-MS. The analytical response ratio was linearly proportional to the urine concentration of hydrazine from 0.0493 to 12.3 ng/mL, with an average correlation coefficientRof 0.9985. Inter-run accuracy for 21 runs, expressed as percent relative error (% RE), was ≤14%, and the corresponding precision, expressed as percent relative standard deviation (% RSD), was ≤15%. Because this method can provide a quantitative measurement of clinical samples over six orders of magnitude, it can be used to monitor trace amounts of hydrazine exposure as well as industrial and environmental exposure levels. PMID:26977107

  19. Trace quantification of 1-triacontanol in beagle plasma by GC-MS/MS and its application to a pharmacokinetic study.

    PubMed

    Wang, Chunfeng; Fan, Ali; Zhu, Xiaojie; Lu, Yang; Deng, Shuhua; Gao, Wenchao; Zhang, Wei; Liu, Qi; Chen, Xijing

    2015-05-01

    1-Triacontanol (TA), a member of long chain fatty alcohol, has recently been received great attention owing to its antitumor activity. In this study, an accurate, sensitive and selective gas chromatography-tandem mass spectrometry method was developed and validated for the quantification of TA in beagle plasma using 1-octacosanal as the internal standard (IS) for the first time. With temperature programming, chromatographic separation was carried out on an HP-5MS column, using helium as carrier gas and argon as collision gas, both at a flow rate of 1 mL/min. TA was analyzed using positive ion electrospray ionization in multiple-reaction monitoring mode, with the precursor to product ion transitions of m/z 495.6 → 97.0 and m/z 467.5 → 97.0 for TA and the IS, respectively. The lower limit of quantitation, linearity, intra- and interday precision, accuracy, stability, extraction recovery and matrix effect of TA were within the acceptable limits. The validated method was successfully applied to a pharmacokinetic study of TA in beagles.

  20. Quantification of taraxasterol in rat plasma by LC/MS/MS: application to a pharmacokinetic study.

    PubMed

    Zhang, Nan; Pang, Li; Dong, Ning; Xu, Dahai; Xu, Hong

    2015-11-01

    Taraxasterol, a pentacyclic triterpene from Taraxacum officinale, is one of the main active constituents of the herb. This study developed and validated a highly selective and sensitive liquid chromatography/tandem mass spectrometry for the determination of taraxasterol in rat plasma over the range of 9.0-5000 ng/mL. Chromatographic separation was achieved on a C18 (4.6 × 50 mm, 5.0 µm) column with methanol-isopropanol-water-formic acid (80:10:10:0.1, v/v/v/v) as mobile phase with an isocratic elution. The flow rate was 0.7 mL/min. After adding cucurbitacin IIa as an internal standard (IS), liquid-liquid extraction was used for sample preparation using ethyl acetate. The atmospheric pressure chemical ionization source was applied and operated in positive ion mode. Selected reaction monitoring mode was used for the quantification of transition ions m/z 409.4 → 137.1 for taraxasterol and m/z 503.4 → 113.1 for IS. The mean recoveries of taraxasterol in rat plasma ranged from 85.3 to 87.2%. The matrix effects for taraxasterol were between 98.5 and 104.0%. Intra- and inter-day precision were both <11.8%, and the accuracy of the method ranged from -7.0 to 12.9%. The method was successfully applied to a pharmacokinetic study of taraxasterol after oral administration of 7.75, 15.5 and 31.0 mg/kg in rats. PMID:25873241

  1. Simultaneous HPLC-APCI-MS/MS quantification of endogenous cannabinoids and glucocorticoids in hair.

    PubMed

    Mwanza, Christopher; Chen, Zheng; Zhang, Quan; Chen, Shenghuo; Wang, Weiwen; Deng, Huihua

    2016-08-15

    Hair matrix could retrospectively record association of endogenous cannabinoids (e.g. 2-arachidonoyl glycerol, 2-AG and N-arachidonoyl-ethanolamine, AEA) and glucocorticoids (e.g. cortisol and cortisone) in a myriad of physiological functions. However, depending on the extraction conditions, the spontaneous isomerization of 2-AG to 1-arachidonoylglycerol (1-AG) and the possible rearrangement of O-arachidonoyl ethanolamine (OAEA) to AEA in various sample matrices could be major obstacles encountered in the detection of both 2-AG and AEA. This study aimed to develop a novel method for simultaneous quantification of 2-AG, AEA, cortisol and cortisone in hair. Methanol was used as the incubation solution and an acidic mixture of deionized water and methanol were utilized as mobile phase in order to avert possible rearrangements of both OAEA and 2-AG. The analyses were performed on a high-performance liquid chromatography tandem mass spectrometer with atmosphere pressure chemical ionization in positive mode. The method showed good linearity in the range of 3.0-250pg/mg for AEA, 15.0-1250pg/mg for 2-AG and 1-250pg/mg for cortisol and cortisone. Limit of detection was 1.5pg/mg for AEA, 6.0pg/mg for 2-AG and 0.5pg/mg for cortisol and cortisone. For all four analytes, intra and inter-day coefficients of variation were less than 20% and recovery above 90%. Population analyses in 473 hair samples established that 2-AG was significantly correlated with AEA. 2-AG was significantly and positively correlated with cortisol and cortisone. There was a significant positive correlation of AEA with cortisol, but not with cortisone. Obese participants showed a significantly higher concentration of cortisone and 2-AG. Males showed significantly higher 2-AG and cortisone levels but significantly lower AEA levels than females. PMID:27318292

  2. LC-MS/MS and GC-MS methods in propofol detection: Evaluation of the two analytical procedures.

    PubMed

    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

  3. Detection and quantification of gas-phase oxidized mercury compounds by GC/MS

    NASA Astrophysics Data System (ADS)

    Jones, Colleen P.; Lyman, Seth N.; Jaffe, Daniel A.; Allen, Tanner; O'Neil, Trevor L.

    2016-05-01

    Most mercury pollution is emitted to the atmosphere, and the location and bioavailability of deposited mercury largely depends on poorly understood atmospheric chemical reactions that convert elemental mercury into oxidized mercury compounds. Current measurement methods do not speciate oxidized mercury, leading to uncertainty about which mercury compounds exist in the atmosphere and how oxidized mercury is formed. We have developed a gas chromatography/mass spectrometry (GC-MS)-based system for identification and quantification of atmospheric oxidized mercury compounds. The system consists of an ambient air collection device, a thermal desorption module, a cryofocusing system, a gas chromatograph, and an ultra-sensitive mass spectrometer. It was able to separate and identify mercury halides with detection limits low enough for ambient air collection (90 pg), but an improved ambient air collection device is needed. The GC/MS system was unable to quantify HgO or Hg(NO3)2, and data collected cast doubt upon the existence of HgO in the gas phase.

  4. Antibody-free workflows for protein quantification by LC-MS/MS.

    PubMed

    Wilffert, Daniel; Bischoff, Rainer; van de Merbel, Nico C

    2015-01-01

    Antibody-free approaches for quantitative LC-MS/MS-based protein bioanalysis are reviewed and critically evaluated, and compared with the more widely used immunoaffinity-based approaches. Antibody-free workflows will be divided into four groups and discussed in the following order: direct analysis of signature peptides after proteolytic digestion; enrichment of target proteins and signature peptides by fractionated protein precipitation; enrichment of target proteins and signature peptides by reversed-phase and ion-exchange solid-phase extraction; and enrichment of target proteins and signature peptides by (antibody-free) affinity-solid-phase extraction. PMID:25871591

  5. Development and clinical application of an LC-MS-MS method for mescaline in urine.

    PubMed

    Björnstad, Kristian; Helander, Anders; Beck, Olof

    2008-04-01

    Mescaline (3,4,5-trimethoxyphenylethylamine) is an hallucinogenic psychoactive substance present in several species of cacti. Mescaline has a documented use dating back 5700 years. In more recent years, the interest in hallucinogenic designer drugs such as ecstasy has also triggered interest in the naturally occurring mescaline. This study was undertaken to develop a liquid chromatography-tandem mass spectrometry (LC-MS-MS) method for the screening and confirmation of mescaline in human urine samples and to apply this method to routine testing in patient samples. For the screening procedure, chromatographic separation was achieved on a 5-microm HyPURITY C(18) column, using a methanol gradient in ammonium acetate buffer. The MS-MS analysis was performed using selected reaction monitoring; the transitions monitored were m/z 212.3 --> m/z 180.3 for mescaline and m/z 221.3 --> m/z 186.3 for the deuterated internal standard (mescaline-d(9)). The detection limit for mescaline in urine matrix was 3-5 microg/L, the upper limit of quantification was 10,000 microg/L, and the total coefficient of variation for spiked samples containing 10 to 1025 microg/L was < 8.5%. The confirmation procedure included a sample clean-up by solid-phase extraction on a C(18) cartridge, and one extra transition for mescaline (m/z 212.3 --> m/z 195.2) was monitored. The LC-MS-MS method was found to be sensitive and specific for the routine detection of mescaline in urine. Among 462 urine samples collected from young people with alcohol or drug problems, 32% were positive for illicit drugs, but none for mescaline.

  6. An Efficient Approach to Evaluate Reporter Ion Behavior from MALDI-MS/MS Data for Quantification Studies Using Isobaric Tags.

    PubMed

    Cologna, Stephanie M; Crutchfield, Christopher A; Searle, Brian C; Blank, Paul S; Toth, Cynthia L; Ely, Alexa M; Picache, Jaqueline A; Backlund, Peter S; Wassif, Christopher A; Porter, Forbes D; Yergey, Alfred L

    2015-10-01

    Protein quantification, identification, and abundance determination are important aspects of proteome characterization and are crucial in understanding biological mechanisms and human diseases. Different strategies are available to quantify proteins using mass spectrometric detection, and most are performed at the peptide level and include both targeted and untargeted methodologies. Discovery-based or untargeted approaches oftentimes use covalent tagging strategies (i.e., iTRAQ, TMT), where reporter ion signals collected in the tandem MS experiment are used for quantification. Herein we investigate the behavior of the iTRAQ 8-plex chemistry using MALDI-TOF/TOF instrumentation. The experimental design and data analysis approach described is simple and straightforward, which allows researchers to optimize data collection and proper analysis within a laboratory. iTRAQ reporter ion signals were normalized within each spectrum to remove peptide biases. An advantage of this approach is that missing reporter ion values can be accepted for purposes of protein identification and quantification without the need for ANOVA analysis. We investigate the distribution of reporter ion peak areas in an equimolar system and a mock biological system and provide recommendations for establishing fold-change cutoff values at the peptide level for iTRAQ data sets. These data provide a unique data set available to the community for informatics training and analysis. PMID:26288259

  7. New Method for the Analysis of Flukicide and Other Anthelmintic Residues in Bovine Milk and Liver using LC-MS/MS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  8. Matrine and oxymatrine in corroborant plant extracts and fertilizers: HPLC/MS-MS method development and single-laboratory validation.

    PubMed

    Sabatino, Leonardo; Scarangella, Michele; Lazzaro, Francesco; Scordino, Monica; Picariello, Giavanna; Leotta, Claudia; Traulo, Pasqualino; Gagliano, Giacomo

    2015-01-01

    A reversed phase high-performance liquid chromatographic method (HPLC/MS-MS) has been developed and validated for detection of alkaloids matrine and oxymatrine in fertilizer with labeled enhancer plant defense activities. The analytical method was validated statistically. The results show a strong matrix effect, requiring quantification by standard addition method. The regression lines showed r(2) > 0.994. Recoveries ranging from 97 to 104% were obtained for the fortification level of 0.01% wt wt(-1) and the relative standard deviations ranged from 3 to 4% (n = 10). The limits of detection were below 0.0001% wt wt(-1), while the limits of quantification did not exceed 0.0004% wt wt(-1). The method is currently applied in ICQRF Laboratory of Catania on fertilized and corroborant plant extract collected in the Italian market in the frame of MIPAAF institutional quality control activity, with the aim to dectect these unpermitted active substances. PMID:26252197

  9. Matrine and oxymatrine in corroborant plant extracts and fertilizers: HPLC/MS-MS method development and single-laboratory validation.

    PubMed

    Sabatino, Leonardo; Scarangella, Michele; Lazzaro, Francesco; Scordino, Monica; Picariello, Giavanna; Leotta, Claudia; Traulo, Pasqualino; Gagliano, Giacomo

    2015-01-01

    A reversed phase high-performance liquid chromatographic method (HPLC/MS-MS) has been developed and validated for detection of alkaloids matrine and oxymatrine in fertilizer with labeled enhancer plant defense activities. The analytical method was validated statistically. The results show a strong matrix effect, requiring quantification by standard addition method. The regression lines showed r(2) > 0.994. Recoveries ranging from 97 to 104% were obtained for the fortification level of 0.01% wt wt(-1) and the relative standard deviations ranged from 3 to 4% (n = 10). The limits of detection were below 0.0001% wt wt(-1), while the limits of quantification did not exceed 0.0004% wt wt(-1). The method is currently applied in ICQRF Laboratory of Catania on fertilized and corroborant plant extract collected in the Italian market in the frame of MIPAAF institutional quality control activity, with the aim to dectect these unpermitted active substances.

  10. Quantification of isoflavones in coffee by using solid phase extraction (SPE) and high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS).

    PubMed

    Caprioli, Giovanni; Navarini, Luciano; Cortese, Manuela; Ricciutelli, Massimo; Torregiani, Elisabetta; Vittori, Sauro; Sagratini, Gianni

    2016-09-01

    A new method for extracting isoflavones from espresso coffee (EC) was coupled with high-performance liquid chromatography-tandem mass spectrometry (MS/MS) for the first time to analyse five isoflavones, which included both a glycosilated form, genistin and the aglycons daidzein, genistein, formononetin and biochanin A. Isoflavones were extracted from coffee samples using methanol, stored in a freezer overnight to precipitate proteic or lipidic residues and purified on SPE C18 cartridges before high-performance liquid chromatography-MS/MS analysis. The recovery percentages obtained by spiking the matrix at concentrations of 10 and 100 µg l(-1) with a standard mixture of isoflavones were in the range of 70 to 104%. The limits of detection and limits of quantification were in the range of 0.015-0.3 µg l(-1) and 0.05-1 µg l(-1) , respectively. Once validated, the method was used to analyze the concentrations of isoflavones in six ECs and ten ground coffee samples. Only formononetin and biochanin A were found, and their respective concentrations ranged from 0.36 to 0.41 µg l(-1) and from 0.58 to 3.26 µg l(-1) in ECs and from 0.36 to 4.27 µg kg(-1) and from 0.71 to 3.95 µg kg(-1) in ground coffees. This method confirms the high specificity and selectivity of MS/MS systems for detecting bioactives in complex matrices such as coffee.Copyright © 2016 John Wiley & Sons, Ltd. PMID:27628757

  11. Identification and Quantification Analysis on the Chemical Constituents from Traditional Mongolian Medicine Flos Scabiosae Using UHPLC-DAD-Q-TOF-MS Combined with UHPLC-QqQ-MS.

    PubMed

    Ouyang, Hui; Li, Tianer; He, Mingzhen; Li, Zhifeng; Tan, Ting; Zhang, Wugang; Li, Yan; Feng, Yulin; Yang, Shilin

    2016-07-01

    In this study, a systematic method was established for the qualitative and quantitative analysis of the major constituents in Flos Scabiosae (FS). First, Ultra-high performance liquid chromatography tandem quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS) was developed for the identification of the multi-constituents in FS. A total of 48 compounds (9 phenolic acids, 24 flavonoids, 8 iridoids and 7 others) were unambiguously or tentatively identified, including 25 compounds (flavonoids, phenolic acids) identified in FS for the first time. Second, ultra-high performance liquid chromatography combined with triple quadrupole mass spectrometry (UHPLC-MS-MS) was developed for the quantitative analysis of 10 phenolic compounds. Ten compounds, either with high contents or strong bioactivities, were chosen as markers. This analytical method was validated through intra- and inter-day precision, repeatability and stability, with respective relative standard deviations <4.43, <8.64, <4.60 and <3.65%, respectively. The limits of detection and quantification were <1.09 and <16.96 ng/mL, respectively. The overall recoveries ranged from 96.47 to 103.94% .Then the validated method was applied to determine 10 batches of FS. The results indicated that the new method can be applied for the qualitative and quantitative analysis of FS.

  12. Multidimensional Separation Using HILIC and SCX Pre-fractionation for RP LC-MS/MS Platform with Automated Exclusion List-based MS Data Acquisition with Increased Protein Quantification

    PubMed Central

    Zhou, Yu; Meng, Zhen; Edman-Woolcott, Maria; Hamm-Alvarez, Sarah F; Zandi, Ebrahim

    2015-01-01

    Liquid chromatography–mass spectrometry (LC-MS) based proteomics is one of the most widely used analytical platforms for global protein discovery and quantification. One of the challenges is the difficulty of identifying low abundance biomarker proteins from limited biological samples. Extensive fractionation could expand proteomics dynamic range, however, at the cost of high sample and time consumption. Extensive fractionation would increase the sample need and the labeling cost. Also quantitative proteomics depending on high resolution MS have the limitation of spectral acquisition speed. Those practical problems hinder the in-depth quantitative proteomics analysis such as tandem mass tag (TMT) experiments. We found the joint use of hydrophilic interaction liquid chromatography (HILIC) and strong cation exchange Chromatography (SCX) prefractionation at medium level could improve MS/MS efficiency, increase proteome coverage, shorten analysis time and save valuable samples. In addition, we scripted a program, Exclusion List Convertor (ELC), which automates and streamlines data acquisition workflow using the precursor ion exclusion (PIE) method. PIE reduces redundancy of high abundance MS/MS analyses by running replicates of the sample. The precursor ions detected in the initial run(s) are excluded for MS/MS in the subsequent run. We compared PIE methods with standard data dependent acquisition (DDA) methods running replicates without PIE for their effectiveness in quantifying TMT-tagged peptides and proteins in mouse tears. We quantified a total of 845 proteins and 1401 peptides using the PIE workflow, while the DDA method only resulted in 347 proteins and 731 peptides. This represents a 144% increase of protein identifications as a result of PIE analysis. PMID:26807013

  13. High-throughput and rapid quantification of lipids by nanoflow UPLC-ESI-MS/MS: application to the hepatic lipids of rabbits with nonalcoholic fatty liver disease.

    PubMed

    Byeon, Seul Kee; Lee, Jong Cheol; Chung, Bong Chul; Seo, Hong Seog; Moon, Myeong Hee

    2016-07-01

    A rapid and high-throughput quantification method (approximately 300 lipids within 20 min) was established using nanoflow ultrahigh-pressure liquid chromatography-tandem mass spectrometry (nUPLC-ESI-MS/MS) with selective reaction monitoring (SRM) and applied to the quantitative profiling of the hepatic lipids of rabbits with different metabolic conditions that stimulate the development of nonalcoholic fatty liver disease (NAFLD). Among the metabolic conditions of rabbits in this study [inflammation (I), high-cholesterol diet (HC), and high-cholesterol diet combined with inflammation (HCI)], significant perturbation in hepatic lipidome (>3-fold and p < 0.01) was observed in the HC and HCI groups, while no single lipid showed a significant change in group I. In addition, this study revealed a dramatic increase (>2-fold) in relatively high-abundant monohexosylceramides (MHCs), sphingomyelins (SMs), and triacylglycerols (TGs) in both the HC and HCI groups, especially in MHCs as all 11 MHCs increased by larger than 3- to 12-fold. As the levels of the relatively high-abundant lipids in the above classes increased, the total lipidome level of each class increased significantly by approximately 2-fold to 5-fold. Other classes of lipids also generally increased, which was likely induced by the increase in mitogenic and nonapoptotic MHCs and SMs, as they promote cell proliferation. On the other hand, a slight decrease in the level of apoptotic ceramides (Cers) was observed, which agreed with the general increase in total lipid level. As distinct changes in hepatic lipidome were observed from HC groups, this suggests that HC or HCI is highly associated with NAFLD but not inflammation alone itself. Graphical Abstract Schematic of lipidomic analysis from hepatic tissue using nanoflow LC-ESI-MS/MS and nUPLC-ESI-MS/MS.

  14. Tutorial examples for uncertainty quantification methods.

    SciTech Connect

    De Bord, Sarah

    2015-08-01

    This report details the work accomplished during my 2015 SULI summer internship at Sandia National Laboratories in Livermore, CA. During this internship, I worked on multiple tasks with the common goal of making uncertainty quantification (UQ) methods more accessible to the general scientific community. As part of my work, I created a comprehensive numerical integration example to incorporate into the user manual of a UQ software package. Further, I developed examples involving heat transfer through a window to incorporate into tutorial lectures that serve as an introduction to UQ methods.

  15. Quantification of reactive carbonyl compounds in icodextrin-based peritoneal dialysis fluids by combined UHPLC-DAD and -MS/MS detection.

    PubMed

    Gensberger-Reigl, Sabrina; Huppert, Jochen; Pischetsrieder, Monika

    2016-01-25

    During heat sterilization of peritoneal dialysis (PD) fluids, the glucose component is partially degraded. The formed glucose degradation products impair biocompatibility and limit the long-term application of PD fluids. As an alternative to glucose, icodextrin, a polyglucose, is used as osmotic agent in PD fluids. After targeted screening for reactive carbonyl compounds, NMR- and MS-analyses very recently revealed 4-deoxyglucosone (4-DG), 3-deoxyglucosone (3-DG), 3-deoxygalactosone (3-DGal), 3,4-dideoxypentosone (3,4-DDPS), and 5-hydroxymethylfurfural (5-HMF) as main polyglucose degradation products (pGDPs) in icodextrin-based PD fluids. Now, the present study established and validated a UHPLC method with DAD as well as a UHPLC-MS/MS method for the first-time quantification of those five major pGDPs in commercial icodextrin PD fluids after derivatization with o-phenylenediamine. Thus, 4-DG was identified to be the main degradation product (in concentrations up to 20 μM). In contrast to the values measured in glucose-based products, the concentration of 3-DGal (≤ 16 μM) was higher than the concentration of 3-DG (≤ 7 μM) indicating different reaction pathways starting from polyglucose compared to glucose. The compounds 3,4-DDPS and 5-HMF were present in minor quantities (≤ 0.3 μM each).

  16. Sensitive analytical methods for 22 relevant insecticides of 3 chemical families in honey by GC-MS/MS and LC-MS/MS.

    PubMed

    Paradis, Delphine; Bérail, Géraldine; Bonmatin, Jean-Marc; Belzunces, Luc P

    2014-01-01

    Several methods for analyzing pesticides in honey have been developed. However, they do not always reach the sufficiently low limits of quantification (LOQ) needed to quantify pesticides toxic to honey bees at low doses. To properly evaluate the toxicity of pesticides, LOQ have to reach at least 1 ng/g. In this context, we developed extraction and analytical methods for the simultaneous detection of 22 relevant insecticides belonging to three chemical families (neonicotinoids, pyrethroids, and pyrazoles) in honey. The insecticides were extracted with the QuEChERS method that consists in an extraction and a purification with mixtures of salts adapted to the matrix and the substances to be extracted. Analyses were performed by gas chromatography coupled with tandem mass spectrometry (GC-MS/MS) for the pyrazoles and the pyrethroids and by high-performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS) for the neonicotinoids and ethiprole. Calibration curves were built from various honey types fortified at different concentrations. Linear responses were obtained between 0.2 and 5 ng/g. Limits of detection (LOD) ranged between 0.07 and 0.2 ng/g, and LOQ ranged between 0.2 and 0.5 ng/g. The mean extraction yields ranged between 63 % and 139 % with RSD <25 %. A complete validation of the methods also examined recovery rates and specificity. These methods were applied to 90 honey samples collected during a 2009-2010 field study in two apiaries placed in different anthropic contexts.

  17. Multi-residues determination of antimicrobials in fish tissues by HPLC-ESI-MS/MS method.

    PubMed

    Rezk, Mamdouh R; Riad, Safa'a M; Khattab, Fatma I; Marzouk, Hoda M

    2015-01-26

    A rapid, simple, sensitive and specific LC-MS/MS method was developed and validated for the simultaneous quantification of four antimicrobials commonly used in aquaculture, namely ciprofloxacin (CPX), trimethoprim (TMP), sulphadimethoxine (SDM) and florphenicol (FLOR) in fish tissues. The LC-MS/MS was operated under the multiple-reaction monitoring mode using electrospray ionization. Sample preparation involves simple liquid extraction step followed by post-extraction clean-up step with n-hexane. The purified extracts were chromatographed on Agilent Poroshell 120 EC, C18 (50 mm × 3 mm, 2.7 μm) column by pumping an isocratic mobile phase consisting of 0.1% formic acid in water:0.1% formic acid in methanol (20:80, by volume) at a flow rate of 0.4 mL/min. A detailed validation of the method was performed as per FDA guidelines and the standard curves were found to be linear in the range of 1-100 ng/g for both CPX and TMP, 0.5-100 ng/g for SDM and 1-50 ng/g for FLOR. The intra-day and inter-day precision and accuracy of the results were within the acceptable limits. A run time of 1.5 min for each sample made it possible to analyze multiple fish tissue samples per day. The developed assay method was successfully applied for the detection of antimicrobials in real fish tissue samples obtained from different fish farms. PMID:25531877

  18. Development of a LC-MS/MS method to monitor palmitoyl peptides content in anti-wrinkle cosmetics.

    PubMed

    Chirita, Raluca-Ioana; Chaimbault, Patrick; Archambault, Jean-Christophe; Robert, Isabelle; Elfakir, Claire

    2009-05-01

    Palmitoyl peptides are anti-aging agents widely used in cosmetics. This article describes the development of a LC-MS/MS analytical procedure that allows, after a liquid-liquid extraction procedure, their unambiguous detection in cosmetic formulation. MS/MS detection is shown to be specific regarding placebo formulations. Limits of quantification, linearity, accuracy and precision of the method were estimated. The results presented show that palmitoyl peptides can be thus reliably assayed. The palmitoylated pentapeptide palmitoyl-lysyl-threonyl-threonyl-lysyl-serine (pal-KTTKS) was assayed in anti-wrinkle creams using palmitoyl-glycyl-histidyl-lysine (pal-GHK) as internal standard. From the results obtained, the influence of the formulation on pal-KTTKS availability is evidenced. PMID:19393372

  19. Novel LC- ESI-MS/MS method for desvenlafaxine estimation human plasma: application to pharmacokinetic study.

    PubMed

    Kancharla, Pushpa Kumari; Kondru, Venu Gopal Raju; Dannana, Gowri Sankar

    2016-02-01

    A simple, sensitive and specific liquid chromatography tandem mass spectrometry (LC-ESI-MS/MS) method was developed for the quantification of desvenlafaxine in human plasma using desvenlafaxine d6 as an internal standard (IS). Chromatographic separation was performed using a Thermo-BDS hypersil C8 column (50 × 4.6 mm, 3 µm) with an isocratic mobile phase composed of 5 mM ammonium acetate buffer: methanol (20:80, v/v), at a flow rate of 0.80 mL/min. Desvenlafaxine and desvenlafaxine d6 were detected with proton adducts at m/z 264.2/58.1 and 270.2/ 64.1 in multiple reaction monitoring positive mode, respectively. Liquid-liquid extraction was used to extract the drug and the IS. The method was linear over the concentration range 1.001-400.352 ng/mL with a correlation coefficient of ≥0.9994. This method demonstrated intra and inter-day precision within 0.7-5.5 and 1.9-6.8%, and accuracy within 95.3-107.4 and 93.4-99.5%. Desvenlafaxine was found to be stable throughout the freeze-thaw cycles, bench-top and long-term matrix stability studies. The developed and validated method can be successfully applied for the bioequivalence/pharmacokinetic studies of desvenlafaxine in pharmaceutical dosage forms.

  20. Evaluation of an automatic multiple sclerosis lesion quantification tool in an informatics-based MS e-folder system

    NASA Astrophysics Data System (ADS)

    Ma, Kevin; Fernandez, James; Amezcua, Lilyana; Lerner, Alex; Liu, Brent

    2011-03-01

    Multiple sclerosis (MS) is a demyelinating disease of the central nervous system. The chronic nature of MS necessitates multiple MRI studies to track disease progression. We have presented an imaging informatics decision-support system, called MS eFolder, designed to integrate patient clinical data with MR images and a computer-aided detection (CAD) component for automatic white matter lesion quantification. The purpose of the MS eFolder is to comprehensively present MS patient data for clinicians and radiologists, while providing a lesion quantification tool that can be objective and consistent for MS tracking in longitudinal studies. The MS CAD algorithm is based on the K-nearest neighbor (KNN) principles and has been integrated within the eFolder system. Currently, the system has been completed and the CAD algorithm for quantifying MS lesions has undergone the expert evaluation in order to validate system performance and accuracy. The evaluation methodology has been developed and the data has been collected, including over 100 MS MRI cases with various age and ethnic backgrounds. The preliminary results of the evaluation are expected to include sensitivity and specificity of lesion and non-lesion voxels in the white matter, the effectiveness of different probability thresholds for each voxel, and comparison between CAD quantification results and radiologists' manual readings. The results aim to show the effectiveness of a MS lesion CAD system to be used in a clinical setting, as well as a step closer to full clinical implementation of the eFolder system.

  1. Development and validation of a LC-MS/MS method to determine sulforaphane in honey.

    PubMed

    Ares, Ana M; Valverde, Silvia; Bernal, José L; Nozal, María J; Bernal, José

    2015-08-15

    A new method was developed to determine sulforaphane (SFN) in honey using liquid chromatography tandem mass spectrometry (LC-MS/MS) with electrospray ionization (ESI). An efficient extraction procedure was proposed (average analyte recoveries were between 92% and 99%); this involved a solid phase extraction (SPE) with a polymeric sorbent. Chromatography was performed on a Synergi™ Hydro analytical column with a mobile phase of 0.02 M ammonium formate in water and acetonitrile, at a flow rate of 0.5 mL/min. The method was fully validated in terms of selectivity, limits of detection (LOD) and quantification (LOQ), linearity, carry-over effect, reinjection reproducibility, precision and accuracy. The LOD and LOQ values were below 0.8 μg/kg and 2.6 μg/kg, respectively. The proposed method was applied to analyze SFN in honey from different botanical origins (rosemary, multifloral, orange blossom and heather), and SFN was detected at trace levels in some of the honey samples examined.

  2. Quantification of Iohexol in Serum by High-Performance Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS).

    PubMed

    Vicente, Faye B; Vespa, Gina; Miller, Alan; Haymond, Shannon

    2016-01-01

    Iohexol is a nonradioactive contrast medium, and its clearance from serum or urine is used to measure glomerular filtration rate (GFR). GFR is the most useful indicator of kidney function and progression of kidney disease. GFR determination using iohexol clearance is increasingly being applied in clinical practice, given its advantages over and correlation with inulin. We describe a high-performance liquid chromatography tandem mass spectrometry (LC-MS/MS) method for iohexol clearance, requiring only 50 μL of serum. The sample preparation involves protein precipitation with LC/MS-grade methanol, containing ioversol as the internal standard. Samples are centrifuged and supernatant is dried under nitrogen gas at room temperature. Samples are reconstituted with mobile phase (ammonium acetate-formic acid-water). Iohexol is separated using an HPLC gradient method on a C-8 analytical column. MS/MS detection is in the multiple-reaction monitoring (MRM) mode and the transitions monitored are m/z 822.0 to m/z 804.0 and m/z 807.0 to m/z 588.0 for iohexol and ioversol, respectively.

  3. LC-APCI-MS/MS Quantification and Topical Bioavailability of Chloroacetamide in Rats.

    PubMed

    Kim, Tae Hwan; Seok, Su Hyun; Kim, Kyu-Bong; Shin, Beom Soo; Lee, Jeong Pyo; Choi, Yong-Kyu; Kim, Min Gi; Kim, Min Kyu; Kim, Sang Min; Yoo, Sun Dong

    2015-08-01

    Chloroacetamide (CAA) is a preservative used in various cosmetic, personal care and household products. Due to the hazard potential for allergic reaction and reproductive toxicity, CAA is being considered a high priority for screening assessment and toxicological re-evaluation. This study describes the development of a highly specific and sensitive atmospheric pressure chemical ionization tandem mass spectrometry method for the determination of CAA in rat plasma and its application to a topical bioavailability study. Chromatographic separations were achieved on a C8 column using a highly aqueous mobile phase with a binary gradient elution. The assay was linear in the concentration range of 5-2,500 ng/mL (r ≥ 0.995) using a small sample volume (100 µL). Applicability of the assay was demonstrated in a bioavailability study in rats after i.v. injection (0.5 or 2 mg/kg) and topical application (7.02 mg/kg). Average elimination half-life and clearance ranged from 26.6 to 30.5 min and 53.9 to 57.3 mL/min/kg, respectively. Upon topical application, CAA was slowly but steadily absorbed for a prolonged time period (12 h). The topical bioavailability was 53.5 and 48.3% for emulsion and lotion, respectively. The developed assay may be useful to examine the relationship between exposure and toxic potential of CAA in risk assessment.

  4. Identification and simultaneous quantification of five alkaloids in Piper longum L. by HPLC-ESI-MS(n) and UFLC-ESI-MS/MS and their application to Piper nigrum L.

    PubMed

    Liu, Hao-Long; Luo, Rong; Chen, Xiao-Qing; Ba, Yin-Ying; Zheng, Li; Guo, Wei-Wei; Wu, Xia

    2015-06-15

    A simple, effective and suitable UFLC-ESI-MS/MS method was firstly developed to simultaneously determine five characteristic constituents (piperine, piperlonguminine, Δα,β-dihydropiperlonguminine, pellitorine and piperanine) of Piper longum L. The total alkaloids of P. longum L. was prepared. The alkaloid contents of Piper nigrum L. and P. longum L. were compared. The analysis was carried out in multiple reaction monitoring scan mode. The method showed a good specificity, linearity (R(2)>0.995), stability (RSD<2.53%), repeatability (RSD<2.58%), and recovery (90.0-103.5%). The limits of detection and limits of quantification of five alkaloids were in the range of 0.02-0.03 and 0.05-0.10 ng/mL, respectively. The intra- and inter-day precision was less than 9.30% and 9.55%, respectively. The validation results confirmed that the method could simultaneously determine the target alkaloids in the sample. Furthermore, the identities of the alkaloids were verified by HPLC-ESI-MS/MS. Compared with P. nigrum, P. longum had lower piperine content but was enriched in the other four alkaloids. PMID:25660876

  5. Identification and simultaneous quantification of five alkaloids in Piper longum L. by HPLC-ESI-MS(n) and UFLC-ESI-MS/MS and their application to Piper nigrum L.

    PubMed

    Liu, Hao-Long; Luo, Rong; Chen, Xiao-Qing; Ba, Yin-Ying; Zheng, Li; Guo, Wei-Wei; Wu, Xia

    2015-06-15

    A simple, effective and suitable UFLC-ESI-MS/MS method was firstly developed to simultaneously determine five characteristic constituents (piperine, piperlonguminine, Δα,β-dihydropiperlonguminine, pellitorine and piperanine) of Piper longum L. The total alkaloids of P. longum L. was prepared. The alkaloid contents of Piper nigrum L. and P. longum L. were compared. The analysis was carried out in multiple reaction monitoring scan mode. The method showed a good specificity, linearity (R(2)>0.995), stability (RSD<2.53%), repeatability (RSD<2.58%), and recovery (90.0-103.5%). The limits of detection and limits of quantification of five alkaloids were in the range of 0.02-0.03 and 0.05-0.10 ng/mL, respectively. The intra- and inter-day precision was less than 9.30% and 9.55%, respectively. The validation results confirmed that the method could simultaneously determine the target alkaloids in the sample. Furthermore, the identities of the alkaloids were verified by HPLC-ESI-MS/MS. Compared with P. nigrum, P. longum had lower piperine content but was enriched in the other four alkaloids.

  6. A validated LC-MS-MS method for simultaneous identification and quantitation of rodenticides in blood.

    PubMed

    Bidny, Sergei; Gago, Kim; David, Mark; Duong, Thanh; Albertyn, Desdemona; Gunja, Naren

    2015-04-01

    A rapid, highly sensitive and specific analytical method for the extraction, identification and quantification of nine rodenticides from whole blood has been developed and validated. Commercially available rodenticides in Australia include coumatetralyl, warfarin, brodifacoum, bromadiolone, difenacoum, flocoumafen, difethialone, diphacinone and chlorophacinone. A Waters ACQUITY UPLC TQD system operating in multiple reaction monitoring mode was used to conduct the analysis. Two different ionization techniques, ES+ and ES-, were examined to achieve optimal sensitivity and selectivity resulting in detection by MS-MS using electrospray ionization in positive mode for difenacoum and brodifacoum and in negative mode for all other analytes. All analytes were extracted from 200 µL of whole blood with ethylacetate and separated on a Waters ACQUITY UPLC BEH-C18 column using gradient elution. Ammonium acetate (10 mM, pH 7.5) and methanol were used as mobile phases with a total run time of 8 min. Recoveries were between 70 and 105% with limits of detection ranging from 0.5 to 1 ng/mL. The limit of quantitation was 2 ng/mL for all analytes. Calibration curves were linear within the range 2-200 ng/mL for all analytes with the coefficient of determination ≥0.98. The application of the proposed method using liquid-liquid extraction in a series of clinical investigations and forensic toxicological analyses was successful. PMID:25595137

  7. Simultaneous extraction, identification and quantification of phenolic compounds in Eclipta prostrata using microwave-assisted extraction combined with HPLC-DAD-ESI-MS/MS.

    PubMed

    Fang, Xinsheng; Wang, Jianhua; Hao, Jifu; Li, Xueke; Guo, Ning

    2015-12-01

    A simple and rapid method was developed using microwave-assisted extraction (MAE) combined with HPLC-DAD-ESI-MS/MS for the simultaneous extraction, identification, and quantification of phenolic compounds in Eclipta prostrata, a common herb and vegetable in China. The optimized parameters of MAE were: employing 50% ethanol as solvent, microwave power 400 W, temperature 70 °C, ratio of liquid/solid 30 mL/g and extraction time 2 min. Compared to conventional extraction methods, the optimized MAE can avoid the degradation of the phenolic compounds and simultaneously obtained the highest yields of all components faster with less consumption of solvent and energy. Six phenolic acids, six flavonoid glycosides and one coumarin were firstly identified. The phenolic compounds were quantified by HPLC-DAD with good linearity, precision, and accuracy. The extract obtained by MAE showed significant antioxidant activity. The proposed method provides a valuable and green analytical methodology for the investigation of phenolic components in natural plants.

  8. Simultaneous identification and quantification of canrenone and 11-α-hydroxy-canrenone by LC-MS and HPLC-UVD.

    PubMed

    Huang, Da-Ming; Zhang, Tian-Zhen; Cui, Feng-Jie; Sun, Wen-Jing; Zhao, Li-Ming; Yang, Meng-Yi; Wang, Ya-Juan

    2011-01-01

    A procedure for simultaneous identification and quantification of canrenone and its biotransformed product 11-α-hydroxy-canrenone by high-performance liquid chromatography with ultraviolet detector (HPLC-UVD) and mass spectrometry (LC-MS) methods was proposed. The optimal determination variables on the HPLC-UVD or LC-MS coupled with a ZORBAX Eclipse XDB-C18 column (150 mm × 4.6 mm, 5 μm) were set as follows: detection wavelength of 280 nm, mobile phase of water and methanol gradient elution, temperature for the chromatographic column of 30°C, flow rate of mobile phase of 0.8 mL/min, sample injection volume of 5 μL, and elution time of 40 min. The MS conditions were set as follows: the flow rate of sheath gas, aux gas, and sweep gas were kept at 35 arb, 5 arb, and 0 arb, respectively. The temperature of capillary was held at 300°C, and capillary voltage was set at 30.00 V. Tube lens were performed at 100.00 V. The proposed method was validated by linearity (r² ≥ 0.9910), average recovery (94.93%, RSD1.21%), precision (RSD ≤ 1.31%), limit of detection, and limit of quantification (LOD 0.1~0.12 mg/L, LOQ 0.5~0.67 mg/L), which proved to be affordable for simultaneously determining canrenone and its bio-transformed product 11-α-hydroxy-canrenone. PMID:22203787

  9. Simultaneous Identification and Quantification of Canrenone and 11-α-Hydroxy-Canrenone by LC-MS and HPLC-UVD

    PubMed Central

    Huang, Da-Ming; Zhang, Tian-Zhen; Cui, Feng-Jie; Sun, Wen-Jing; Zhao, Li-Ming; Yang, Meng-Yi; Wang, Ya-Juan

    2011-01-01

    A procedure for simultaneous identification and quantification of canrenone and its biotransformed product 11-α-hydroxy-canrenone by high-performance liquid chromatography with ultraviolet detector (HPLC-UVD) and mass spectrometry (LC-MS) methods was proposed. The optimal determination variables on the HPLC-UVD or LC-MS coupled with a ZORBAX Eclipse XDB-C18 column (150 mm × 4.6 mm, 5 μm) were set as follows: detection wavelength of 280 nm, mobile phase of water and methanol gradient elution, temperature for the chromatographic column of 30°C, flow rate of mobile phase of 0.8 mL/min, sample injection volume of 5 μL, and elution time of 40 min. The MS conditions were set as follows: the flow rate of sheath gas, aux gas, and sweep gas were kept at 35 arb, 5 arb, and 0 arb, respectively. The temperature of capillary was held at 300°C, and capillary voltage was set at 30.00 V. Tube lens were performed at 100.00 V. The proposed method was validated by linearity (r2 ≥ 0.9910), average recovery (94.93%, RSD1.21%), precision (RSD ≤ 1.31%), limit of detection, and limit of quantification (LOD 0.1~0.12 mg/L, LOQ 0.5~0.67 mg/L), which proved to be affordable for simultaneously determining canrenone and its bio-transformed product 11-α-hydroxy-canrenone. PMID:22203787

  10. Identification and quantification of 14 phthalates and 5 non-phthalate plasticizers in PVC medical devices by GC-MS.

    PubMed

    Gimeno, Pascal; Thomas, Sébastien; Bousquet, Claudine; Maggio, Annie-Françoise; Civade, Corinne; Brenier, Charlotte; Bonnet, Pierre-Antoine

    2014-02-15

    A GC/MS method was developed for the identification and quantification of 14 phthalates: 8 phthalates classified H360 (DBP, DEHP, BBP, DMEP, DnPP, DiPP, DPP and DiBP), 3 phthalates proposed to be forbidden in medical devices (DnOP, DiNP and DiDP) and 3 other phthalates none regulated (DMP, DCHP and DEP) which may interfere with hormone function. In order to identify and quantify other plasticizers that are commonly used in PVC medical devices such as DEHP substitute, 5 non-phthalate plasticizers (ATBC, DEHA, DEHT, TOTM, and DINCH) were included in this study. Analyses are carried out on a GC/MS system with electron impact ionization mode (EI). The separation of plasticizers is obtained on a cross-linked 5%-phenyl/95%-dimethylpolysiloxane capillary column 30m×0.25mm (i.d.)×0.25μm film thickness using a gradient temperature. Compounds quantification is performed by external calibration using an internal standard. Validation elements on standard solutions were determined using the ISO 12787 standard approach. Plasticizers are extracted from PVC medical devices using THF for dissolving the PVC part of the sample followed by precipitation of the PVC by addition of ethanol. The supernatant is injected into a GC/MS system after dilution in ethanol. Different validation elements, including extraction recoveries for all compounds or for DEHP a cross-validation of the extraction process using the European pharmacopoeia monograph 3.1.14 as reference method, are discussed. Results obtained on 61 medical devices in PVC and 12 raw materials used as plasticizers are given.

  11. Detection and quantification of traces of bisphenol A and bisphenol S in paper samples using analytical pyrolysis-GC/MS.

    PubMed

    Becerra, Valentina; Odermatt, Jürgen

    2012-05-01

    In this paper a simplified method based on analytical pyrolysis gas chromatography mass spectrometry (Py-GC/MS) for the detection and quantification of bisphenol A and bisphenol S in paper samples is presented. The method enables a direct analysis of the samples without tedious sample preparation. As the analytes are thermally desorbed, a solvent extraction is not needed. The method is applicable to small samples of ~120 μg. The limits of detection are below 1 mg kg(-1) for bisphenol A and for bisphenol S. The limits of quantification are about 1.3 mg kg(-1). Several validation characteristics of the method developed like standard error of calibration, limit of determination, linearity, and accuracy are given. To prove the accuracy of the method, interferences and matrix dependencies were also investigated. The influence of the pyrolysis crucibles, a special effect of analytical pyrolysis, was additionally investigated. It was found that the impact of the crucibles on the results is significant and one cause for matrix effects.

  12. Simultaneous Quantification of Six Major Flavonoids From Fructus sophorae by LC-ESI-MS/MS and Statistical Analysis

    PubMed Central

    Chang, L.; Zhang, X. X.; Ren, Y. P.; Cao, L.; Zhi, X. R.; Zhang, L. T.

    2013-01-01

    A new, sensitive and selective high-performance liquid chromatography–tandem mass spectrometric method has been developed for the determination of six major flavonoids including sophoricoside, genistin, genistein, rutin, quercetin, kaempferol in Fructus sophorae. Principal component analysis and hierarchical clustering analysis were used to classify and differentiate these samples. Chromatographic separation was performed on a C18 column with linear gradient elution of methanol and 0.05% acetic acid (v/v) at a flow rate of 0.8 ml/min. The detection was accomplished in the negative mode using multiple-reaction monitoring. The total run time was 8.0 min. Full validation of the assay was carried out including linearity, precision, accuracy, recovery, limit of detection and limit of quantification. The validated method was successfully applied to the simultaneous determination of these active components in Fructus sophorae. The results demonstrated that the quantitative difference in content of six active compounds was useful for chemotaxonomy of many samples from different sources and the standardization and differentiation of many similar samples. Simultaneous quantification of bioactive components by high-performance liquid chromatography–tandem mass spectrometric method coupled with chemometric techniques would be a well-acceptable strategy to comprehensively control the quality of Fructus sophorae. PMID:24082349

  13. Identification and quantification of components in extracts of Uncaria tomentosa by HPLC-ES/MS.

    PubMed

    Montoro, P; Carbone, V; Quiroz, J de Dioz Zuniga; De Simone, F; Pizza, C

    2004-01-01

    The two main classes of secondary metabolites, alkaloids and quinovic acid glycosides, of Uncaria tomentosa (Willd.) DC. (Rubiaceae), a Peruvian plant commonly known as 'uña de gato', have been analysed. Separation of the alkaloidal fraction was achieved using a solid phase extraction method based on cationic exchange, and an analytical method employing HPLC-ES/MS has been developed. Quantitative data for commercial wild bark, cultivated bark and leaves are reported. The analysis of quinovic acid glycosides was performed directly on the crude extract using both a fast analytical method based on flow injection ES/MS, and a more complete analytical technique using HPLC-MS.

  14. Identification and quantification of components in extracts of Uncaria tomentosa by HPLC-ES/MS.

    PubMed

    Montoro, P; Carbone, V; Quiroz, J de Dioz Zuniga; De Simone, F; Pizza, C

    2004-01-01

    The two main classes of secondary metabolites, alkaloids and quinovic acid glycosides, of Uncaria tomentosa (Willd.) DC. (Rubiaceae), a Peruvian plant commonly known as 'uña de gato', have been analysed. Separation of the alkaloidal fraction was achieved using a solid phase extraction method based on cationic exchange, and an analytical method employing HPLC-ES/MS has been developed. Quantitative data for commercial wild bark, cultivated bark and leaves are reported. The analysis of quinovic acid glycosides was performed directly on the crude extract using both a fast analytical method based on flow injection ES/MS, and a more complete analytical technique using HPLC-MS. PMID:14979528

  15. Development of analytical strategies using U-HPLC-MS/MS and LC-ToF-MS for the quantification of micropollutants in marine organisms.

    PubMed

    Wille, Klaas; Kiebooms, Julie A L; Claessens, Michiel; Rappé, Karen; Vanden Bussche, Julie; Noppe, Herlinde; Van Praet, Nander; De Wulf, Eric; Van Caeter, Peter; Janssen, Colin R; De Brabander, Hubert F; Vanhaecke, Lynn

    2011-05-01

    Organic micropollutants such as pharmaceuticals, perfluorinated compounds (PFCs), and pesticides, are important environmental contaminants. To obtain more information regarding their presence in marine organisms, an increasing demand exists for reliable analytical methods for quantification of these micropollutants in biotic matrices. Therefore, we developed extraction procedures and new analytical methods for the quantification of 14 pesticides, 10 PFCs, and 11 pharmaceuticals in tissue of marine organisms, namely blue mussels (Mytilus edulis). This paper presents these optimized analytical procedures and their application to M. edulis, deployed at five stations in the Belgian coastal zone. The methods consisted of a pressurized liquid extraction and solid-phase extraction (SPE) followed by ultra high-performance liquid chromatography coupled to triple quadrupole mass spectrometry for pharmaceuticals and pesticides, and of a liquid extraction using acetonitrile and SPE, followed by liquid chromatography coupled to time-of-flight mass spectrometry for PFCs. The limits of quantification of the three newly optimized analytical procedures in M. edulis tissue varied between 0.1 and 10 ng g(-1), and satisfactory linearities (≥0.98) and recoveries (90-106%) were obtained. Application of these methods to M. edulis revealed the presence of five pharmaceuticals, two PFCs, and seven pesticides at levels up to 490, 5, and 60 ng g(-1), respectively. The most prevalent micropollutants were salicylic acid, paracetamol, perfluorooctane sulfonate, chloridazon, and dichlorvos. PMID:21442366

  16. Bioanalytical validation for simultaneous quantification of non-aromatic steroids in follicular fluid from cattle via ESI-LC-MS/MS.

    PubMed

    Kunze, Martin; Wirthgen, Elisa; Walz, Christina; Spitschak, Marion; Brenmoehl, Julia; Vanselow, Jens; Schwerin, Manfred; Wimmers, Klaus; Hoeflich, Andreas

    2015-12-15

    The family of steroid hormones is quite attractive for the approach of phenotype monitoring in farm animals. Therefore, we developed a new protocol for the quantitative analysis of natural steroids in follicular fluid from dairy cows. The corresponding steroid profile, which consists of progesterone, corticosterone, hydrocortisone, testosterone, and androstenedione covering three distinct steroid classes, was determined by LC/MS. Quantification is achieved by use of steroid standards diluted in steroid-free follicular fluid as calibrators. Thus, the new protocol does not require deuterated standards. In order to correct for conditional performance of the analytical system we have used dexamethasone as an internal standard. The method was validated according to EMA guidelines. Within- and between-day variations were below 20% for most parameters assessed. All steroids assessed had lower limits of quantification in the range of 2.1 to 4.4ng/ml. We have established a simple and sensitive analytical system in order to step towards a broader and cost-efficient phenotyping analysis in follicular fluid from dairy cows.

  17. Simultaneous quantification of five steroid saponins from Dioscorea zingiberensis C.H. Wright in rat plasma by HPLC-MS/MS and its application to the pharmacokinetic studies.

    PubMed

    Zhang, Xinxin; Li, Jing; Ito, Yoichiro; Sun, Wenji

    2015-01-01

    A simple, reliable and sensitive high-performance liquid chromatography tandem mass spectrometry method (HPLC-MS/MS) was established for simultaneous analyses of the following 5 steroid saponins in rat plasma after the single dose administration of total steroid saponins extracted from the rhizome of Dioscorea zingiberensis C.H. Wright for the first time. Protodioscin, huangjiangsu A, zingiberensis new saponin, dioscin, and gracillin were quantified using ginsenoside Rb1 as the internal standard (IS). The plasma samples were pretreated by a single step acetonitrile-mediated protein precipitation. The chromatographic separation was performed on an Inersil ODS-3 C18 column (250mm×4.6mm, 5μm) with the mobile phase composed of acetonitrile and water containing 0.1% formic acid under a gradient elution mode at 0.2mLmin(-1) using a microsplit after the eluent from the HPLC apparatus. The quantification was accomplished on a triple quadrupole tandem mass spectrometer using the multiple reaction monitoring (MRM) in the positive ionization mode. The above five analytes were stable under sample storage and preparation conditions applied in the present study. The linearity, precision, accuracy, and recoveries of the analysis confirmed the requirements for quality-control purposes. After validation, this proposed method was successfully adopted to investigate the pharmacokinetic parameters of these five analytes.

  18. Bromadiolone poisoning: LC-MS method and pharmacokinetic data.

    PubMed

    Vindenes, Vigdis; Karinen, Ritva; Hasvold, Inger; Bernard, Jean-Paul; Mørland, Jørg G; Christophersen, Asbjørg S

    2008-07-01

    Poisoning with superwarfarins, like bromadiolone, is a growing public health problem, and the mortality is high. Pharmacokinetic data on bromadiolone in humans are however scarce, and there are no reports following repeated exposures to bromadiolone. We have developed a method for quantification of bromadiolone in whole blood, using liquid chromatography-mass spectrometry (LC-MS). The analytical method is reported. Limit of detection was 0.005 mg/L and limit of quantification was 0.01 mg/L. The concentrations of bromadiolone in whole blood and plasma in serial samples from a 62-year-old woman were measured. The half-life of bromadiolone in blood was estimated to be about 6 days in the initial phase of elimination and about 10-13 days in the terminal phase. The mean plasma/blood ratio of bromadiolone was 1.7 +/- 0.6. Stability testing of bromadiolone in whole blood samples after two cycles of freeze and thaw revealed that bromadiolone concentrations decreased. PMID:18482378

  19. Comparison of analysis methods for airway quantification

    NASA Astrophysics Data System (ADS)

    Odry, Benjamin L.; Kiraly, Atilla P.; Novak, Carol L.; Naidich, David P.

    2012-03-01

    Diseased airways have been known for several years as a possible contributing factor to airflow limitation in Chronic Obstructive Pulmonary Diseases (COPD). Quantification of disease severity through the evaluation of airway dimensions - wall thickness and lumen diameter - has gained increased attention, thanks to the availability of multi-slice computed tomography (CT). Novel approaches have focused on automated methods of measurement as a faster and more objective means that the visual assessment routinely employed in the clinic. Since the Full-Width Half-Maximum (FWHM) method of airway measurement was introduced two decades ago [1], several new techniques for quantifying airways have been detailed in the literature, but no approach has truly become a standard for such analysis. Our own research group has presented two alternative approaches for determining airway dimensions, one involving a minimum path and the other active contours [2, 3]. With an increasing number of techniques dedicated to the same goal, we decided to take a step back and analyze the differences of these methods. We consequently put to the test our two methods of analysis and the FWHM approach. We first measured a set of 5 airways from a phantom of known dimensions. Then we compared measurements from the three methods to those of two independent readers, performed on 35 airways in 5 patients. We elaborate on the differences of each approach and suggest conclusions on which could be defined as the best one.

  20. Simple and rapid quantification of brominated vegetable oil in commercial soft drinks by LC-MS.

    PubMed

    Chitranshi, Priyanka; Gamboa da Costa, Gonçalo

    2016-12-15

    We report here a simple and rapid method for the quantification of brominated vegetable oil (BVO) in soft drinks based upon liquid chromatography-electrospray ionization mass spectrometry. Unlike previously reported methods, this novel method does not require hydrolysis, extraction or derivatization steps, but rather a simple "dilute and shoot" sample preparation. The quantification is conducted by mass spectrometry in selected ion recording mode and a single point standard addition procedure. The method was validated in the range of 5-25μg/mL BVO, encompassing the legal limit of 15μg/mL established by the US FDA for fruit-flavored beverages in the US market. The method was characterized by excellent intra- and inter-assay accuracy (97.3-103.4%) and very low imprecision [0.5-3.6% (RSD)]. The direct nature of the quantification, simplicity, and excellent statistical performance of this methodology constitute clear advantages in relation to previously published methods for the analysis of BVO in soft drinks.

  1. Simple and rapid quantification of brominated vegetable oil in commercial soft drinks by LC-MS.

    PubMed

    Chitranshi, Priyanka; Gamboa da Costa, Gonçalo

    2016-12-15

    We report here a simple and rapid method for the quantification of brominated vegetable oil (BVO) in soft drinks based upon liquid chromatography-electrospray ionization mass spectrometry. Unlike previously reported methods, this novel method does not require hydrolysis, extraction or derivatization steps, but rather a simple "dilute and shoot" sample preparation. The quantification is conducted by mass spectrometry in selected ion recording mode and a single point standard addition procedure. The method was validated in the range of 5-25μg/mL BVO, encompassing the legal limit of 15μg/mL established by the US FDA for fruit-flavored beverages in the US market. The method was characterized by excellent intra- and inter-assay accuracy (97.3-103.4%) and very low imprecision [0.5-3.6% (RSD)]. The direct nature of the quantification, simplicity, and excellent statistical performance of this methodology constitute clear advantages in relation to previously published methods for the analysis of BVO in soft drinks. PMID:27451219

  2. Development and validation of UHPLC-MS/MS method for determination of eight naturally occurring catechin derivatives in various tea samples and the role of matrix effects.

    PubMed

    Svoboda, Pavel; Vlčková, Hana; Nováková, Lucie

    2015-10-10

    A complete analytical procedure combining optimized tea infusion preparation and validated UHPLC-MS/MS method was developed for routine quantification of eight naturally occurring catechin derivatives in various tea samples. The preparation of tea infusions was optimized in terms of temperature, time and water-to-tea ratio in green, white and black teas. The catechins were analyzed using ultra-high performance liquid chromatography coupled with triple quadrupole mass spectrometry in a run of only 4 min including equilibration of the system. The UHPLC-MS/MS method was fully validated in terms of inter/intra-day precision, accuracy, linearity (r(2)>0.9991), range (50-5000 ng/ml), LOD (1.5-7.5 ng/ml) and LOQ (5-25 ng/ml). Validation of the method included also the determination of the matrix effects that were evaluated in both flavored and unflavored green, white and black teas. Dilution of the resulting tea infusions appeared to be crucial for the matrix effects and also for subsequent catechin quantification in real tea samples in order to fit into the linear range of the UHPLC-MS/MS method. This complete procedure for catechin quantification was finally applied to real sample analysis represented by 70 commercial tea samples.

  3. Development and validation of UHPLC-MS/MS method for determination of eight naturally occurring catechin derivatives in various tea samples and the role of matrix effects.

    PubMed

    Svoboda, Pavel; Vlčková, Hana; Nováková, Lucie

    2015-10-10

    A complete analytical procedure combining optimized tea infusion preparation and validated UHPLC-MS/MS method was developed for routine quantification of eight naturally occurring catechin derivatives in various tea samples. The preparation of tea infusions was optimized in terms of temperature, time and water-to-tea ratio in green, white and black teas. The catechins were analyzed using ultra-high performance liquid chromatography coupled with triple quadrupole mass spectrometry in a run of only 4 min including equilibration of the system. The UHPLC-MS/MS method was fully validated in terms of inter/intra-day precision, accuracy, linearity (r(2)>0.9991), range (50-5000 ng/ml), LOD (1.5-7.5 ng/ml) and LOQ (5-25 ng/ml). Validation of the method included also the determination of the matrix effects that were evaluated in both flavored and unflavored green, white and black teas. Dilution of the resulting tea infusions appeared to be crucial for the matrix effects and also for subsequent catechin quantification in real tea samples in order to fit into the linear range of the UHPLC-MS/MS method. This complete procedure for catechin quantification was finally applied to real sample analysis represented by 70 commercial tea samples. PMID:26025813

  4. An improved method for analysis of glucosylceramide species having cis-8 and trans-8 isomers of sphingoid bases by LC-MS/MS.

    PubMed

    Imai, Hiroyuki; Hattori, Hideyasu; Watanabe, Masayuki

    2012-12-01

    High-performance liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) approaches have enabled high selectivity and sensitivity for the identification and quantification of glucosylceramide molecular species. Here we demonstrate that HPLC-ESI-MS/MS is an efficient method for characterizing plant glucosylceramide species having the cis-8 and trans-8 isomers of sphingoid bases. Complete baseline separation was achieved using a high-carbon-content octadecylsilyl column and a simple binary gradient comprising methanol and water. The result of 2-hydroxy fatty acid composition achieved by HPLC-ESI-MS/MS was compared with that achieved by gas chromatography with flame ionization detection (GC-FID), indicating that the two methods yield similar molar compositions. The current method should be applicable to seeking the active components of glucosylceramide species from plant materials in response to biological challenges. PMID:23108960

  5. Quantification of Complex Polycyclic Aromatic Hydrocarbon Mixtures in Standard Reference Materials Using GC×GC/ToF-MS

    PubMed Central

    Manzano, Carlos; Hoh, Eunha; Massey Simonich, Staci L.

    2014-01-01

    This research is the first to quantify complex PAH mixtures in NIST SRMs using comprehensive two-dimensional gas chromatography coupled to time-of-flight mass spectrometry (GC×GC/ToF-MS), with and without extract cleanup, and reports previously unidentified PAH isomers in the NIST SRMs. We tested a novel, high orthogonality GC column combination (LC-50×NSP-35), as well as with a commonly used column combination (Rtx-5ms×Rxi-17) for the quantification of a complex mixture of 85 different PAHs, including parent (PAHs), alkyl- (MPAHs), nitro- (NPAHs), oxy- (OPAHs), thio- (SPAHs), bromo- (BrPAHs), and chloro-PAHs (ClPAHs) in extracts from two standard reference materials: NIST SRM1650b (diesel particulate matter), with cleanup and NIST SRM1975 (diesel particulate extract), with and without extract cleanup. The LC-50×NSP-35 column combination resulted in an average absolute percent difference of 33.8%, 62.2% and 30.8% compared to the NIST certified PAH concentrations for NIST SRM1650b, NIST SRM1975 with cleanup and NIST SRM1975 without cleanup, while the Rtx-5ms×Rxi-17 resulted in an absolute percent difference of 38.6%, 67.2% and 79.6% for NIST SRM1650b, NIST SRM1975 with cleanup and NIST SRM1975 without cleanup, respectively. This GC×GC/ToF-MS method increases the number of PAHs detected and quantified in complex environmental extracts using a single chromatographic run. Without clean-up, 7 additional compounds were detected and quantified in NIST SRM1975 using the LC-50×NSP-35 column combination. These results suggest that the use of the LC-50×NSP-35 column combination in GC×GC/ToF-MS not only results in better chromatographic resolution and greater orthogonality for the separation of complex PAH mixtures, but can also be used for the accurate quantification of complex PAH mixtures in environmental extracts without cleanup. PMID:23932031

  6. Validation and uncertainties evaluation of an isotope dilution-SPE-LC-MS/MS for the quantification of drug residues in surface waters.

    PubMed

    Brieudes, V; Lardy-Fontan, S; Lalere, B; Vaslin-Reimann, S; Budzinski, H

    2016-01-01

    The present work describes the development and validation of a reference method conducted at the French National Institute of Metrology (LNE) for the quantitative determination of psychoactive compounds in the dissolved fraction of surface waters. More specifically an isotope dilution-SPE-LC-MS/MS based method has been implemented for the characterization of a broad range of analytes belonging to different classes of psychotropic drugs such as benzodiazepines, antidepressants, stimulants, opiates and opioids, anticonvulsants, anti-dementia drugs, analgesics as well as the anti-inflammatory drug diclofenac in the low ng L(-1) range of concentration. Full validation of the method was performed following procedures described by the French standard NF T90-210. Limits of quantification between 0.14 and 3.54 ng L(-1) were obtained. Method recoveries from 71 to 123% were observed with standard deviation below 10% in intermediate precision conditions. Accuracy was determined for every compound: measurement errors were between -4 and +1% and standard deviations in intermediate precision conditions were included within a 1-9% interval. Finally, measurement uncertainties were evaluated following the Guide to the expression of uncertainty in measurement (GUM). Expanded uncertainties (k=2) ranged from 2% for carbamazepine, EDDP (2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine) and venlafaxine to 17% for diazepam. The validated method was implemented to Seine river surface waters demonstrating its fitness for purpose. All compounds were detected and 22 out of 25 analytes were quantified. More specifically, measured concentration ranged from 0.39 ng L(-1) for MDMA (3,4-methylene-dioxy-N-methylamphetamine) to 182 ng L(-1) for gabapentine.

  7. Validation and uncertainties evaluation of an isotope dilution-SPE-LC-MS/MS for the quantification of drug residues in surface waters.

    PubMed

    Brieudes, V; Lardy-Fontan, S; Lalere, B; Vaslin-Reimann, S; Budzinski, H

    2016-01-01

    The present work describes the development and validation of a reference method conducted at the French National Institute of Metrology (LNE) for the quantitative determination of psychoactive compounds in the dissolved fraction of surface waters. More specifically an isotope dilution-SPE-LC-MS/MS based method has been implemented for the characterization of a broad range of analytes belonging to different classes of psychotropic drugs such as benzodiazepines, antidepressants, stimulants, opiates and opioids, anticonvulsants, anti-dementia drugs, analgesics as well as the anti-inflammatory drug diclofenac in the low ng L(-1) range of concentration. Full validation of the method was performed following procedures described by the French standard NF T90-210. Limits of quantification between 0.14 and 3.54 ng L(-1) were obtained. Method recoveries from 71 to 123% were observed with standard deviation below 10% in intermediate precision conditions. Accuracy was determined for every compound: measurement errors were between -4 and +1% and standard deviations in intermediate precision conditions were included within a 1-9% interval. Finally, measurement uncertainties were evaluated following the Guide to the expression of uncertainty in measurement (GUM). Expanded uncertainties (k=2) ranged from 2% for carbamazepine, EDDP (2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine) and venlafaxine to 17% for diazepam. The validated method was implemented to Seine river surface waters demonstrating its fitness for purpose. All compounds were detected and 22 out of 25 analytes were quantified. More specifically, measured concentration ranged from 0.39 ng L(-1) for MDMA (3,4-methylene-dioxy-N-methylamphetamine) to 182 ng L(-1) for gabapentine. PMID:26695245

  8. Identification and quantification of constituents of Gardenia jasminoides Ellis (Zhizi) by HPLC-DAD-ESI-MS.

    PubMed

    Bergonzi, M C; Righeschi, C; Isacchi, B; Bilia, A R

    2012-09-15

    A simple, rapid and specific HPLC method was carried out for the analysis of characteristic constituents in Gardenia jasminoides Ellis (Zhizi), namely iridoids, caffeoyl quinic acid derivatives and crocins. The separation was successfully obtained using a C(18) column by gradient elution with mixtures of methanol and water as mobile phases; detection wavelength was set at 240 nm for iridoid glycosides, 315 nm for quinic acid derivatives and 438 nm for crocins. The analytical method was validated and the quantification of active compounds, namely iridoids, was performed. Linearity, precision, repeatability, stability, accuracy, limit of detection (LOD) and limit of quantification (LOQ) were also reported. This assay was successfully applied for qualitative and quantitative analysis of five commercial samples of G. jasminoides Ellis. PMID:23107748

  9. Development and validation of LC-MS/MS method for the determination of cyproheptadine in several pharmaceutical syrup formulations.

    PubMed

    Feás, Xesús; Ye, Lei; Hosseini, Seyed V; Fente, Cristina A; Cepeda, Alberto

    2009-12-01

    A rapid and sensitive liquid chromatographic-tandem mass spectrometric (LC-MS/MS) method was developed and validated for the qualitative and quantitative assay of cyproheptadine (CP) in pharmaceutical samples. Diphenylpyraline hydrochloride (DPP) was used as an internal standard (IS). Two multiple reaction-monitoring (MRM) transitions for each analyte were observed: 288.1/96.1 and 288.1/191.2 for CP and 282.1/167.2 and 282.1/116.3 for DPP. The retention time of the drug was 7.29 min. The analytical method was successfully validated for linearity (1-100 ng/ml), intra-day precision, inter-day precision, and accuracy. The limit of detection (LOD) and limit of quantification (LOQ) were 0.86 and 0.98 ng/ml, respectively. The proposed method was applied to analyse the cyproheptadine content from seven different syrup formulations.

  10. QuEChERS-based method for the determination of carbamate residues in aromatic herbs by UHPLC-MS/MS.

    PubMed

    Nantia, Edouard Akono; Moreno-González, David; Manfo, Faustin P T; Gámiz-Gracia, Laura; García-Campaña, Ana M

    2017-02-01

    A new reliable, fast and highly sensitive method based on ultra-high performance liquid chromatography tandem mass spectrometry has been developed and validated for the determination of 28 carbamates in aromatic herbs. A modified QuEChERS-based method was optimized for the extraction of carbamate residues from a wide variety of fresh herbal products. The proposed method allowed recoveries higher than 72%, achieving quantification limits of 2μgkg(-1), therefore below maximum residue limits established for this type of samples. The combination of QuEChERS with UHPLC-MS/MS introduces a high-throughput methodology for the monitoring of these residues in this type of matrices scarcely explored. The analysis of the real samples revealed that several samples sold in the European Union and in the North West region of Cameroon contain pesticides in concentrations below the maximum residue limits.

  11. QuEChERS-based method for the determination of carbamate residues in aromatic herbs by UHPLC-MS/MS.

    PubMed

    Nantia, Edouard Akono; Moreno-González, David; Manfo, Faustin P T; Gámiz-Gracia, Laura; García-Campaña, Ana M

    2017-02-01

    A new reliable, fast and highly sensitive method based on ultra-high performance liquid chromatography tandem mass spectrometry has been developed and validated for the determination of 28 carbamates in aromatic herbs. A modified QuEChERS-based method was optimized for the extraction of carbamate residues from a wide variety of fresh herbal products. The proposed method allowed recoveries higher than 72%, achieving quantification limits of 2μgkg(-1), therefore below maximum residue limits established for this type of samples. The combination of QuEChERS with UHPLC-MS/MS introduces a high-throughput methodology for the monitoring of these residues in this type of matrices scarcely explored. The analysis of the real samples revealed that several samples sold in the European Union and in the North West region of Cameroon contain pesticides in concentrations below the maximum residue limits. PMID:27596428

  12. An automatic quantification system for MS lesions with integrated DICOM structured reporting (DICOM-SR) for implementation within a clinical environment

    NASA Astrophysics Data System (ADS)

    Jacobs, Colin; Ma, Kevin; Moin, Paymann; Liu, Brent

    2010-03-01

    Multiple Sclerosis (MS) is a common neurological disease affecting the central nervous system characterized by pathologic changes including demyelination and axonal injury. MR imaging has become the most important tool to evaluate the disease progression of MS which is characterized by the occurrence of white matter lesions. Currently, radiologists evaluate and assess the multiple sclerosis lesions manually by estimating the lesion volume and amount of lesions. This process is extremely time-consuming and sensitive to intra- and inter-observer variability. Therefore, there is a need for automatic segmentation of the MS lesions followed by lesion quantification. We have developed a fully automatic segmentation algorithm to identify the MS lesions. The segmentation algorithm is accelerated by parallel computing using Graphics Processing Units (GPU) for practical implementation into a clinical environment. Subsequently, characterized quantification of the lesions is performed. The quantification results, which include lesion volume and amount of lesions, are stored in a structured report together with the lesion location in the brain to establish a standardized representation of the disease progression of the patient. The development of this structured report in collaboration with radiologists aims to facilitate outcome analysis and treatment assessment of the disease and will be standardized based on DICOM-SR. The results can be distributed to other DICOM-compliant clinical systems that support DICOM-SR such as PACS. In addition, the implementation of a fully automatic segmentation and quantification system together with a method for storing, distributing, and visualizing key imaging and informatics data in DICOM-SR for MS lesions improves the clinical workflow of radiologists and visualizations of the lesion segmentations and will provide 3-D insight into the distribution of lesions in the brain.

  13. How Many proteins are Missed in Quantitative proteomics Based on Ms/Ms sequencing Methods?

    PubMed Central

    Mulvey, Claire; Thur, Bettina; Crawford, Mark; Godovac-Zimmermann, Jasminka

    2014-01-01

    Current bottom-up quantitative proteomics methods based on MS/MS sequencing of peptides are shown to be strongly dependent on sample preparation. Using cytosolic proteins from MCF-7 breast cancer cells, it is shown that protein pre-fractionation based on pI and MW is more effective than pre-fractionation using only MW in increasing the number of observed proteins (947 vs. 704 proteins) and the number of spectral counts per protein. Combination of MS data from the different pre-fractionation methods results in further improvements (1238 proteins). We discuss that at present the main limitation on quantitation by MS/MS sequencing is not MS sensitivity and protein abundance, but rather extensive peptide overlap and limited MS/MS sequencing throughput, and that this favors internally calibrated methods such as SILAC, ICAT or ITRAQ over spectral counting methods in attempts to drastically improve proteome coverage of biological samples. PMID:25729266

  14. Quantification of Tricyclic Antidepressants in Serum Using Liquid Chromatography Electrospray Tandem Mass Spectrometry (HPLC-ESI-MS/MS).

    PubMed

    Crutchfield, Christopher A; Breaud, Autumn R; Clarke, William A

    2016-01-01

    Tricyclic antidepressants (TCA) are used to treat major depressive disorder and other psychological conditions. The efficacy of these drugs is tied to a narrow therapeutic window. Inappropriately high drug concentrations can result in serious side effects such as hypotension, tachycardia, or coma. As a result, concentrations of tricyclic antidepressants are routinely monitored to ensure compliance and to prevent adverse side effects by dose adjustments. We describe a method for the determination of concentrations of amitriptyline, desipramine, imipramine, and nortriptyline in human serum using high-performance liquid chromatography coupled to a tandem mass spectrometer with electrospray ionization (HPLC-ESI-MS/MS). The method is rapid, requiring only 3.5 min per analysis. The method requires 100 μL of serum. Concentrations of each TCA were quantified by a calibration curve relating the peak area ratio of each TCA analyte to a deuterated internal standard (amitriptyline-D3, desipramine-D3, imipramine-D3, and nortriptyline-D3). The method was linear from ~70 ng/mL to ~1000 ng/mL for all TCAs, with imprecision ≤ 12%.

  15. Quantification of Docetaxel in Serum Using Turbulent Flow Liquid Chromatography Electrospray Tandem Mass Spectrometry (TFC-HPLC-ESI-MS/MS).

    PubMed

    Crutchfield, Christopher A; Marzinke, Mark A; Clarke, William A

    2016-01-01

    Docetaxel is a second-generation taxane and is used clinically as an anti-neoplastic agent in cancer chemotherapy via an anti-mitotic mechanism. Its efficacy is limited to a narrow therapeutic window. Inappropriately high concentrations may cause erythema, fluid retention, nausea, diarrhea, and neutropenia. As a result, dosing recommendations have changed from high dosage loading every 3 weeks to lower dosage loading weekly. We describe a method that can be used for therapeutic drug monitoring of docetaxel levels using turbulent flow liquid chromatography electrospray tandem mass spectrometry (TFC-HPLC-ESI-MS/MS). The method is rapid, requiring only 6.3 min per analytical run following a simple protein crash. The method requires only 100 μL of serum. Concentrations of docetaxel were quantified by a calibration curve relating the peak-area ratio of docetaxel to a deuterated internal standard (docetaxel-D9). The method was linear from 7.8 to 1000 ng/mL, with imprecision ≤6.2 %.

  16. A LC-MS-MS method for determination of low doxazosin concentrations in plasma after oral administration to dogs.

    PubMed

    Erceg, Marijana; Cindric, Mario; Pozaic Frketic, Lidija; Vertzoni, Maria; Cetina-Cizmek, Biserka; Reppas, Christos

    2010-02-01

    A rapid and sensitive reversed phase liquid chromatography- tandem mass spectrometry (LC-MS-MS) method is developed for the determination of doxazosin in canine plasma. The samples are prepared by precipitation of proteins using a mixture of methanol and acetonitrile, followed by freezing and evaporation of the organic solvent. The remaining dry residue is redissolved in mobile phase and analyzed by LC-MS-MS with positive electrospray ionization using the selected reactions monitoring mode. An XTerra MS C(18) column, a mobile phase composed of acetonitrile and 2mM ammonium acetate with gradient elution, and a flow rate of 400 microL/min are employed. The elution times for prazosin (internal standard) and doxazosin are approximately 8 and 10 min, respectively. Calibration curves are linear in the 1-20 ng/mL concentration range. Limits of detection and quantification are 0.4 ng/mL and 1.2 ng/mL, respectively. Recovery is higher than 94%. Intra- and inter-day relative standard deviations are below 7% and 8%, respectively. The method is applied for the determination of doxazosin plasma levels following a single administration of doxazosin base and doxazosin mesylate tablets (2 mg dose) to dogs in the fed state. The results indicate possible superiority of the mesylate salt on the plasma input rates of doxazosin.

  17. Simultaneous quantification of picfeltarraenins IA and IB in rat plasma by UPLC-MS/MS: Application to a pharmacokinetic study.

    PubMed

    He, Xin; Zhang, Yingjie; Gao, Hang; Li, Keyan; Zhang, Yazhuo; Sun, Limin; Tao, Guizhou

    2016-02-20

    A simple and rapid quantitative UPLC-MS/MS method for simultaneous determination of picfeltarraenins IA and IB in rat plasma was developed and validated in accordance with the US FDA Bioanalytical Guidance (2001). Analytes were extracted from rat plasma by using methanol and separated on Agilent ZORBAX SB-C18 (50mm×2.1mm, 1.8μm) column by using a mobile phase composed of methanol and water (70:30, v/v). Eluents were monitored by ESI tandem mass spectrometry detection with SRM mode using ion transitions m/z 785.4→639.5, m/z 815.5→669.5, and m/z 763.5→455.3 for picfeltarraenin IA, picfeltarraenin IB, and internal standard, respectively. The method was validated over the linear range of 11.5-1150ng/mL and 13.0-1300ng/mL. The developed analytical method was applied to support a pharmacokinetic study on simultaneous estimation of picfeltarraenins IA and IB in rats.

  18. Analysis of 136 pesticides in avocado using a modified QuEChERS method with LC-MS/MS and GC-MS/MS.

    PubMed

    Chamkasem, Narong; Ollis, Lisa W; Harmon, Tiffany; Lee, Sookwang; Mercer, Greg

    2013-03-13

    A simple and high-throughput screening method for the analysis of 136 pesticides in avocado ( Persea americana ) by LC-(+)-ESI-MS/MS and GC-MS/MS is presented. A modified QuEChERS sample preparation method was developed to improve the extraction recovery of highly lipophilic pesticides. Extracts from minced avocados after acetonitrile (MeCN) extraction were directly injected to LC-MS/MS, whereas other GC-amenable compounds were treated with the modified QuEChERS procedure for GC-MS/MS analysis. The average recoveries for 79 pesticides quantified by LC-MS/MS at 10, 50, and 200 ng/g fortifying levels were 86.1% or better (with maximum RSD at 9.2%), whereas GC-MS/MS analysis demonstrated 70.2% or better (RSD < 18%) for average recovery from 57 compounds at the same spike levels. The application of LC- and GC-MS/MS combined with the improved extraction procedures led to the current method, which can quantitate these pesticides even if they are present in avocados below the targeted action level by FDA. This method demonstrated the improved recovery of several challenging lipophilic pesticides in highly fat-rich avocados. PMID:23362971

  19. Development of an Advanced HPLC–MS/MS Method for the Determination of Carotenoids and Fat-Soluble Vitamins in Human Plasma

    PubMed Central

    Hrvolová, Barbora; Martínez-Huélamo, Miriam; Colmán-Martínez, Mariel; Hurtado-Barroso, Sara; Lamuela-Raventós, Rosa Maria; Kalina, Jiří

    2016-01-01

    The concentration of carotenoids and fat-soluble vitamins in human plasma may play a significant role in numerous chronic diseases such as age-related macular degeneration and some types of cancer. Although these compounds are of utmost interest for human health, methods for their simultaneous determination are scarce. A new high pressure liquid chromatography (HPLC)-tandem mass spectrometry (MS/MS) method for the quantification of selected carotenoids and fat-soluble vitamins in human plasma was developed, validated, and then applied in a pilot dietary intervention study with healthy volunteers. In 50 min, 16 analytes were separated with an excellent resolution and suitable MS signal intensity. The proposed HPLC–MS/MS method led to improvements in the limits of detection (LOD) and quantification (LOQ) for all analyzed compounds compared to the most often used HPLC–DAD methods, in some cases being more than 100-fold lower. LOD values were between 0.001 and 0.422 µg/mL and LOQ values ranged from 0.003 to 1.406 µg/mL, according to the analyte. The accuracy, precision, and stability met with the acceptance criteria of the AOAC (Association of Official Analytical Chemists) International. According to these results, the described HPLC-MS/MS method is adequately sensitive, repeatable and suitable for the large-scale analysis of compounds in biological fluids. PMID:27754400

  20. Multi-residue method for detecting coccidiostats at carry-over level in feed by HPLC-MS/MS.

    PubMed

    Delahaut, Ph; Pierret, G; Ralet, N; Dubois, M; Gillard, N

    2010-06-01

    A multi-residue HPLC-ESI-MS/MS method has been developed for the simultaneous extraction, detection and confirmation of the 11 coccidiostats referenced by Regulation 2009/8/EC (lasalocid sodium, narasin, salinomycin sodium, monensin sodium, semduramicin sodium, maduramicin ammonium alpha, robenidine hydrochloride, decoquinate, halofuginone hydrobromide, nicarbazin, and diclazuril) in feedstuffs at carry-over level. The sensitivity of the method allows quantification and confirmation for all coccidiostats below target concentration. The method was in-house validated and meets all criteria of European legislation (2002/657/EC). The precision of the method was determined under repeatability and within-laboratory reproducibility conditions; RSD(r) and RSD(R) were below the maximum permitted values for every tested concentration. The specificity was checked by analysing representative blank samples and blank samples fortified with potentially interfering substances (benzimidazoles, corticosteroides, triphenylmethane dyes, quinolones, nitrofurans, nitroimidazoles, phenicols); no interference were found. Concerning quantification, a quadratic regression model was fitted to every calibration curve with a regression coefficient r2 above 0.99 on each data set. Finally, the expanded uncertainty U was calculated with data obtained within the laboratory while applying the method during validation and in routine tests.

  1. A novel ultrasensitive LC-MS/MS assay for quantification of intracellular raltegravir in human cell extracts

    PubMed Central

    Robbins, Brian L.; Nelson, Sarah R.; Fletcher, Courtney V.

    2012-01-01

    An assay using ultrahigh pressure liquid chromatography and mass spectrometry detection was developed and validated for measurement of the HIV integrase inhibitor raltegravir (MK-0518) in human cell extracts. The assay is designed to utilize 200 µl of 70% MeOH cell extract derived from human peripheral blood mononuclear cells or human tissue samples. The assay is linear over a range from 0.0023 to 9.2 ng ml−1. The average % CV (SD/Mean)*100 and % Deviation ((observed – target)/target)*100 were less than 20% at the lower limit of quantification and less than 15% over the range of the curve. This assay is an accurate and highly sensitive method for determining raltegravir concentrations in cellular extracts with a lower limit 40 to over 100-fold lower than other methods in the literature. We also present a new processing method where a rapid spin through oil produced a significant increase in apparent intracellular raltegravir concentration compared with conventional processing. PMID:22727807

  2. A fast and reliable method for GHB quantitation in whole blood by GC-MS/MS (TQD) for forensic purposes.

    PubMed

    Castro, André L; Tarelho, Sónia; Dias, Mário; Reis, Flávio; Teixeira, Helena M

    2016-02-01

    Gamma-hydroxybutyric acid (GHB) is an endogenous compound with a story of clinical use since the 1960s. However, due to its secondary effects, it has become a controlled substance, entering the illicit market. A fully validated, sensitive and reproducible method for the quantification of GHB by methanolic precipitation and GC-MS/MS (TQD) in whole blood is presented. Using 100μL of whole blood, obtained results included a LOD and LLOQ of 0.1mg/L and a recovery of 86% in a working range between 0.1 and 100mg/L. This method is sensitive and specific to detect the presence of GHB in small amounts of whole blood (both ante-mortem or post-mortem), and is, to the authors' knowledge, the first GC-MS-MS TQD method that uses different precursor ions and product ions for the identification of GHB and GHB-D6 (internal standard). Hence, this method may be especially useful for the study of endogenous values in this biological sample.

  3. HPLC-MS/MS method validation for the detection of carbadox and olaquindox in poultry and swine feedingstuffs.

    PubMed

    Souza Dibai, Wagner Lutero; de Alkimin Filho, Juarez Fabiano; da Silva Oliveira, Fabiano Aurélio; Sampaio de Assis, Débora Cristina; Camargos Lara, Leonardo José; de Figueiredo, Tadeu Chaves; de Vasconcelos Cançado, Silvana

    2015-11-01

    Carbadox (CBX) and olaquindox (OLA) were used in poultry and swine feed for growth promotion, to improve feed efficiency and increase the rate of weight gain. However, the use of these agents in feedingstuffs was prohibited because of concerns about their toxicity. Regulatory laboratories are required to have suitably validated analytical methods to ensure compliance with the ban. A quantitative and confirmatory method for determining the presence of CBX and OLA in poultry and swine feed by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) was developed, optimized, and validated. The analytes extraction was performed with a mixture of water and acetonitrile (1:1v/v) and cleanup with hexane and C18 (dispersive phase). The method was evaluated by the following parameters: specificity, linearity, matrix effect, decision limits (CCα), detection capability (CCβ), accuracy, precision, limits of detection (LoD), limits of quantification (LoQ) and measurement uncertainty. The validated method presented a broad linear study range and no significant matrix effect. The limit of detection (LoD) was defined at 9 μg kg(-1) for CBX and 80 μg kg(-1) for OLA, and the limit of quantification (LoQ) was defined at 12 μg kg(-1) and 110 μg kg(-1) for CBX and OLA, respectively. The accuracy of the method was adequate for CBX and OLA. The recovery values found in the repeatability conditions were 99.41% for CBX and 104.62% for OLA. Under intralaboratory reproducibility conditions, the values were 98.63% for CBX and 95.07% for OLA. It was concluded that the performance parameters demonstrated total method adequacy for the detection and quantification of CBX and OLA in poultry and swine feedingstuffs.

  4. Development and application of a LC-MS/MS assay for the simultaneous quantification of edaravone and taurine in beagle plasma.

    PubMed

    Yu, Yan-yan; Zheng, Xiao-xiao; Bian, Ting-ting; Li, Yin-jie; Wu, Xiao-wen; Yang, Dong-zhi; Jiang, Shui-shi; Tang, Dao-quan

    2013-12-01

    An LC-MS/MS method was developed and validated for the simultaneous quantification of edaravone and taurine in beagle plasma. The plasma sample was deproteinized using acetonitrile containing formic acid. Chromatographic separations were achieved on an Agilent Zorbax SB-Aq (100 × 2.1 mm, 3.5 μm) column, with a gradient of water (containing 0.03% formic acid) and methanol as the mobile phase at a flow rate of 0.3 mL/min. The analyte detection was carried out in multiple reaction monitoring mode and the optimized precursor-to-product transitions of m/z [M+H](+) 175.1 → 133.0 (edaravone), m/z [M+H](+) 189.1 → 147.0 (3-methyl-1-p-tolyl-5-pyrazolone, internal standard, IS), m/z [M-H](-) 124.1→80.0 (taurine), and m/z [M-H](-) 172.0 → 80.0 (sulfanilic acid, IS) were employed to quantify edaravone, taurine, and their corresponding ISs, respectively. The LOD and the lower LOQ were 0.01 and 0.05 μg/mL for edaravone and 0.66 and 2 μg/mL for taurine, respectively. The calibration curves of these two analytes demonstrated good linearity (r > 0.99). All the validation data including the specificity, precision, recovery, and stability conformed to the acceptable requirements. This validated method has successfully been applied in the pharmacokinetic study of edaravone and taurine mixture in beagle dogs.

  5. Extraction and quantification of phytoestrogens in foods using automated solid-phase extraction and LC/MS/MS.

    PubMed

    Kuhnle, Gunter G C; Dell'aquila, Caterina; Low, Yen-Ling; Kussmaul, Michaela; Bingham, Sheila A

    2007-12-01

    Phytoestrogens are a group of polyphenolic plant metabolites that can induce biological responses. Their bioactivity is based on their similarity to 17beta-estradiol and their ability to bind to the beta-estrogen receptor. Although epidemiological data are inconclusive, phytoestrogens are considered to be beneficial for a variety of conditions, for example, hormone-related cancers like breast and prostate cancer. To investigate the biological effects of these compounds and to assess the exposure of larger cohorts or the general public, reliable data on the phytoestrogen content of food is necessary. Previously, food analysis for phytoestrogens was performed using either HPLC-UV or GC/MS. Here, we describe the development of the first generic method for the analysis of phytoestrogens in food, using automated solid-phase extraction and liquid chromatography-tandem mass spectrometry. The presented method shows a good reproducibility and can be easily adapted to other phytoestrogens if required.

  6. Simultaneous Quantification of Baricitinib and Methotrexate in Rat Plasma by LC-MS/MS: Application to a Pharmacokinetic Study

    PubMed Central

    Veeraraghavan, Sridhar; Thappali, Satheeshmanikandan R. S.; Viswanadha, Srikant; Vakkalanka, Swaroop; Rangaswamy, Manivannan

    2016-01-01

    Efficacy assessments using a combination of baricitinib and methotrexate necessitate the development of an analytical method for the determination of both drugs in plasma with precision. A high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the simultaneous determination of baricitinib and methotrexate in rat plasma. Extraction of baricitinib, methotrexate, and tolbutamide (internal standard; IS) from 50 µL of rat plasma was carried out by protein precipitation with methanol. Chromatographic separation of the analytes was performed on the YMC pack ODS AM (150 mm × 4.6 mm, 5 µm) column under gradient conditions with methanol: 2.0 mM ammonium acetate buffer as the mobile phases at a flow rate of 1 mL/min. The precursor ion and product ion transition for both analytes and IS were monitored on a triple quadrupole mass spectrometer, operated with selective reaction monitoring in positive ionization mode. The method was validated over a concentration range of 0.5–250.00 ng/mL for baricitinib and methotrexate. Mean extraction recoveries for baricitinib, methotrexate, and IS of 86.8%, 89.4%, and 91.8% were consistent across low, medium, and high QC levels, respectively. Precision and accuracy at low, medium, and high quality control levels were less than 15% across the analytes. Benchtop, wet, freeze-thaw, and long-term stability were evaluated for both of the analytes. The analytical method was applied to support the pharmacokinetic study of simultaneous estimation of baricitinib and methotrexate in Wistar rats. Assay reproducibility was demonstrated by reanalysis of 18 incurred samples PMID:27222609

  7. Simultaneous Quantification of Baricitinib and Methotrexate in Rat Plasma by LC-MS/MS: Application to a Pharmacokinetic Study.

    PubMed

    Veeraraghavan, Sridhar; Thappali, Satheeshmanikandan R S; Viswanadha, Srikant; Vakkalanka, Swaroop; Rangaswamy, Manivannan

    2016-01-01

    Efficacy assessments using a combination of baricitinib and methotrexate necessitate the development of an analytical method for the determination of both drugs in plasma with precision. A high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the simultaneous determination of baricitinib and methotrexate in rat plasma. Extraction of baricitinib, methotrexate, and tolbutamide (internal standard; IS) from 50 µL of rat plasma was carried out by protein precipitation with methanol. Chromatographic separation of the analytes was performed on the YMC pack ODS AM (150 mm × 4.6 mm, 5 µm) column under gradient conditions with methanol: 2.0 mM ammonium acetate buffer as the mobile phases at a flow rate of 1 mL/min. The precursor ion and product ion transition for both analytes and IS were monitored on a triple quadrupole mass spectrometer, operated with selective reaction monitoring in positive ionization mode. The method was validated over a concentration range of 0.5-250.00 ng/mL for baricitinib and methotrexate. Mean extraction recoveries for baricitinib, methotrexate, and IS of 86.8%, 89.4%, and 91.8% were consistent across low, medium, and high QC levels, respectively. Precision and accuracy at low, medium, and high quality control levels were less than 15% across the analytes. Benchtop, wet, freeze-thaw, and long-term stability were evaluated for both of the analytes. The analytical method was applied to support the pharmacokinetic study of simultaneous estimation of baricitinib and methotrexate in Wistar rats. Assay reproducibility was demonstrated by reanalysis of 18 incurred samples.

  8. Simultaneous quantification of ruxolitinib and nilotinib in rat plasma by LC-MS/MS: application to a pharmacokinetic study.

    PubMed

    Veeraraghavan, Sridhar; Thappali, Satheeshmanikandan; Viswanadha, Srikant; Chennupati, Sandhyarani; Nalla, Santhoshkumar; Golla, Manikantakumar; Vakkalanka, Swaroopkumar; Rangasamy, Manivannan

    2014-06-01

    Efficacy assessments using a combination of ruxolitinib and nilotinib necessitate the development of a high precision analytical method for determination of both drugs in plasma. A high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the simultaneous determination of ruxolitinib and nilotinib in rat plasma. Extraction of ruxolitinib, nilotinib and dasatinib (internal standard; IS) from 50μl rat plasma was carried out by protein precipitation with methanol. Chromatographic separation of analytes was performed on YMC pack ODS AM (150mm×4.6mm, 5μm) column under gradient conditions with acetonitrile:2.0mM ammonium acetate buffer as the mobile phase at a flow rate of 1ml/min. Precursor ion and product ion transition for both analytes and IS were monitored on a triple quadrupole mass spectrometer, operated in the selective reaction monitoring with positive ionization mode. Method was validated over a concentration range of 0.16-247ng/ml for ruxolitinib and 0.86-219ng/ml for nilotinib. Mean extraction recovery for ruxolitinib, nilotinib, and IS of 99.6%, 97.6% and 90.3% were consistent across low, medium, and high QC levels. Precision and accuracy at low, medium and high quality control levels were less than 15% across analytes. Bench top, wet, freeze-thaw and long term stability were evaluated for both analytes. The analytical method was applied to support a pharmacokinetic study of simultaneous estimation of ruxolitinib and nilotinib in Wistar rat. Assay reproducibility was demonstrated by re-analysis of 18 incurred samples.

  9. Rapid speciation and quantification of selenium compounds by HPLC-ICP MS using multiple standards labelled with different isotopes.

    PubMed

    Ohta, Yuki; Suzuki, Noriyuki; Kobayashi, Yayoi; Hirano, Seishiro

    2011-09-01

    Speciation analysis using high-performance liquid chromatography-inductively coupled plasma mass spectrometry (HPLC-ICP MS) is now commonly used to investigate metabolic and toxicological aspects of some metals and metalloids. We have developed a rapid method for simultaneous identification and quantification of metabolites of selenium (Se) compounds using multiple standards labelled with different isotopes. A mixture of the labelled standards was spiked in a selenised garlic extract and the sample was subjected to speciation analysis by HPLC-ICP MS. The selenised garlic contains γ-glutamyl-methylselenocysteine, methylselenocysteine, and selenomethionine and the concentrations of those Se compounds were 723.8, 414.8, and 310.7 ng Se ml(-1), respectively. The isotopically labelled standards were also applied to the speciation of Se in rat urine. Selenate, methylselenonic acid, selenosugar, and trimethyselenium ions were found to be excreted by the present speciation procedure. Multiple standards labelled with different stable isotopes enable high-throughput identification and quantitative measurements of Se metabolites.

  10. Development and validation of an UHPLC-MS/MS method for the determination of mycotoxins in grass silages.

    PubMed

    McElhinney, Cormac; O'Kiely, Pádraig; Elliott, Chris; Danaher, Martin

    2015-01-01

    An ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) multi-mycotoxin analytical method was developed to simultaneously identify and quantify 20 mycotoxins in grass silages, inclusive of mycotoxins that are currently regulated in European Union feeds. Extraction of mycotoxins from dried grass silages was performed using of a modified QuEChERS extraction employing an acidified aqueous extraction (0.1 N HCl) with no further clean-up. Following chromatographic separation, analytes were detected using a fast polarity-switching MS/MS method that allowed both positive and negative ions to be analysed from a single injection, thus the reducing time and cost of analysis. The limits of detection and quantification ranged between 3 µg kg(-1) DM (aflatoxin B1, beauvericin and enniatin A and A1) and 200 µg kg(-1) DM (deoxynivalenol), and between 10 µg kg(-1) DM (aflatoxin B1, beauvericin and enniatin A1) and 500 µg kg(-1) DM (deoxynivalenol), respectively. Inter-assay accuracy and precision ranged between 90% and 107% and between 3.9% and 15.0% CV, respectively. The accuracy of the method was assessed through the application to a range of incurred samples in an inter-laboratory study.

  11. Niacin and its metabolites: role of LC-MS/MS bioanalytical methods and update on clinical pharmacology. An overview.

    PubMed

    Mullangi, Ramesh; Srinivas, Nuggehally R

    2011-01-01

    Niacin (nicotinic acid), although an old drug, has seen a sudden surge in popularity for treatment of lipid disorders and other associated clinical conditions for the prevention of cardiovascular risk. Also, there has been considerable interest in clarifying the role of metabolic pathways of niacin in explaining the tolerability/adverse affect profile of the agent. Hence, it has become very important to quantify/monitor the levels of niacin and its metabolites in various clinical studies. This review describes the recent trends in the bioanalysis of niacin and its metabolites, where HPLC and LC-MS/MS assays have been successfully employed to measure the drug levels in various biological matrices arising from preclinical and clinical studies. In addition, this review encompass various considerations such as internal standard selection, extraction schemes, matrix effect, selectivity evaluation and optimization of mass spectral conditions to enable the development of sound bioanalytical methods for niacin alone or niacin along with its metabolites. Recent updates pertaining to the clinical pharmacology of niacin and ongoing debate for the clarification of adverse effects are also provided. Overall LC-MS/MS methods have proven to be choice of bioanalytical method for the quantification of niacin alone or with its metabolites in both preclinical and clinical studies.

  12. Acoustic processing method for MS/MS experiments

    NASA Technical Reports Server (NTRS)

    Whymark, R. R.

    1973-01-01

    Acoustical methods in which intense sound beams can be used to control the position of objects are considered. The position control arises from the radiation force experienced when a body is placed in a sound field. A description of the special properties of intense sound fields useful for position control is followed by a discussion of the more obvious methods of position, namely the use of multiple sound beams. A new type of acoustic position control device is reported that has advantages of simplicity and reliability and utilizes only a single sound beam. Finally a description is given of an experimental single beam levitator, and the results obtained in a number of key levitation experiments.

  13. LC-MS/MS bioanalytical method development for AMG 900: resolution of an isobaric interference in rodent in vivo studies.

    PubMed

    Be, Xuhai; Moore, Earl S; Zhao, Zhiyang; Wells, Mary C

    2013-02-23

    AMG 900 is an orally available small molecule that is a highly potent and selective pan-aurora kinase inhibitor currently in development for the treatment of advanced human cancers. A co-eluting, isobaric interference was discovered in preliminary LC-MS/MS analyses of rodent in vivo pharmacokinetic samples during preclinical evaluation of AMG 900. The interference was identified as a major circulating N-oxide metabolite which partially converted to an [M+H-O](+) ion under the conditions of atmospheric pressure chemical ionization. A selective liquid chromatography-tandem mass spectrometry method for the simultaneous quantification of AMG 900 and its N-oxide metabolite in plasma was developed and successfully applied for the bioanalysis of discovery stage preclinical rodent pharmacokinetic studies.

  14. Prospective Comparison of Liver Stiffness Measurements between Two Point Shear Wave Elastography Methods: Virtual Touch Quantification and Elastography Point Quantification

    PubMed Central

    Yoo, Hyunsuk; Yoon, Jeong Hee; Lee, Dong Ho; Chang, Won; Han, Joon Koo

    2016-01-01

    Objective To prospectively compare technical success rate and reliable measurements of virtual touch quantification (VTQ) elastography and elastography point quantification (ElastPQ), and to correlate liver stiffness (LS) measurements obtained by the two elastography techniques. Materials and Methods Our study included 85 patients, 80 of whom were previously diagnosed with chronic liver disease. The technical success rate and reliable measurements of the two kinds of point shear wave elastography (pSWE) techniques were compared by χ2 analysis. LS values measured using the two techniques were compared and correlated via Wilcoxon signed-rank test, Spearman correlation coefficient, and 95% Bland-Altman limit of agreement. The intraobserver reproducibility of ElastPQ was determined by 95% Bland-Altman limit of agreement and intraclass correlation coefficient (ICC). Results The two pSWE techniques showed similar technical success rate (98.8% for VTQ vs. 95.3% for ElastPQ, p = 0.823) and reliable LS measurements (95.3% for VTQ vs. 90.6% for ElastPQ, p = 0.509). The mean LS measurements obtained by VTQ (1.71 ± 0.47 m/s) and ElastPQ (1.66 ± 0.41 m/s) were not significantly different (p = 0.209). The LS measurements obtained by the two techniques showed strong correlation (r = 0.820); in addition, the 95% limit of agreement of the two methods was 27.5% of the mean. Finally, the ICC of repeat ElastPQ measurements was 0.991. Conclusion Virtual touch quantification and ElastPQ showed similar technical success rate and reliable measurements, with strongly correlated LS measurements. However, the two methods are not interchangeable due to the large limit of agreement. PMID:27587964

  15. Quantification of neonicotinoid insecticide residues in soils from cocoa plantations using a QuEChERS extraction procedure and LC-MS/MS.

    PubMed

    Dankyi, Enock; Gordon, Christopher; Carboo, Derick; Fomsgaard, Inge S

    2014-11-15

    The use of neonicotinoids as an insecticide group in Ghana has been quite significant particularly in cocoa production. The high usage has been mainly as a result of a government policy of free insecticide spraying on cocoa farms, in an effort to curb declining yields caused by pests and diseases and to prevent the use of unapproved or banned insecticides on cocoa farms. However the scale of cocoa farming, the frequency and intensity of usage coupled with the mode of application may result in large physical volumes of insecticides in the environment. This makes the knowledge of the concentration and fate of neonicotinoids in the environment extremely important. The present study was aimed at assessing the levels of five major neonicotinoids in soils from cocoa farmlands in Ghana. Extraction and cleanup of analytes were performed by use of a method based on the original QuEChERS procedure after optimizing salts, sorbents and instrumental conditions. Analyte extraction with NaCl and MgSO4 in acidified acetonitrile followed by cleanup with primary secondary amine (PSA) presented the optimum conditions for extraction. Quantification was performed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) with electrospray ionization (ESI). Validation of the procedure showed average recoveries ranging from 72.0 to 104.8% for all analytes at all fortification levels with relative standard deviation (RSD) ≤ 15.0. Limits of quantitation were <10 μg kg(-1) for all neonicotinoids studied. The results obtained from the analysis of 52 samples from cocoa farms revealed imidacloprid as the predominant neonicotinoid with concentrations ranging from 4.3 to 251.4 μg kg(-1) in >50% of samples analyzed.

  16. A novel quantification strategy of transferrin and albumin in human serum by species-unspecific isotope dilution laser ablation inductively coupled plasma mass spectrometry (ICP-MS).

    PubMed

    Feng, Liuxing; Zhang, Dan; Wang, Jun; Shen, Dairui; Li, Hongmei

    2015-07-16

    Species-specific (SS) isotope dilution analysis with gel electrophoresis (GE)-laser ablation (LA)-ICP-MS is a promising technique for the quantification of particular metal-binding proteins in biological samples. However, unavailable isotopically enriched spike and metal losses in GE separation are main limitations for SS-isotope dilution PAGE-LA-ICP-MS. In this study, we report for the first time the absolute quantification of transferrin (Tf) and albumin (Alb) in human serum by non-denaturing (native) GE combined with species-unspecific isotope dilution mass spectrometry (IDMS). In order to achieve a homogeneous distribution of both protein and isotope-enriched spike (simulated isotope equilibration), immersing the protein strips with (34)S spike solution after gel electrophoresis was demonstrated to be an effective way of spike addition. Furthermore, effects of immersion time and (34)S spike concentration were investigated to obtain optimal conditions of the post-electrophoresis isotope dilution method. The relative mass of spike and ablated sample (m(sp)/m(sam)) in IDMS equation was calculated by standard Tf and Alb proteins, which could be applied to the quantification of Tf and Alb in ERM-DA470k/IFCC for method confirmation. The results were in agreement with the certified value with good precision and small uncertainty (1.5-3%). In this method, species-specific spike protein is not necessary and the integrity of the heteroatom-protein could be maintained in sample preparation process. Moreover, the application of species-unspecific isotope dilution GE-LA-ICP-MS has the potential to offer reliable, direct and simultaneous quantification of proteins after conventional 1D and 2D gel electrophoretic separations.

  17. Improved 6-Plex Tandem Mass Tags Quantification Throughput Using a Linear Ion Trap-High-Energy Collision Induced Dissociation MS(3) Scan.

    PubMed

    Liu, Jane M; Sweredoski, Michael J; Hess, Sonja

    2016-08-01

    The use of tandem mass tags (TMT) as an isobaric labeling strategy is a powerful method for quantitative proteomics, yet its accuracy has traditionally suffered from interference. This interference can be largely overcome by selecting MS(2) fragment precursor ions for high-energy collision induced dissociation (HCD) MS(3) analysis in an Orbitrap scan. While this approach minimizes the interference effect, sensitivity suffers due to the high AGC targets and long acquisition times associated with MS(3) Orbitrap detection. We investigated whether acquiring the MS(3) scan in a linear ion trap with its lower AGC target would increase overall quantification levels with a minimal effect on precision and accuracy. Trypsin-digested proteins from Saccharomyces cerevisiae were tagged with 6-plex TMT reagents. The sample was subjected to replicate analyses using either the Orbitrap or the linear ion trap for the HCD MS(3) scan. HCD MS(3) detection in the linear ion trap vs Orbitrap increased protein identification by 66% with minor loss in precision and accuracy. Thus, the use of a linear ion trap-HCD MS(3) scan during a 6-plex TMT experiment can improve overall identification levels while maintaining the power of multiplexed quantitative analysis. PMID:27377715

  18. High precision quantification of human plasma proteins using the automated SISCAPA Immuno-MS workflow.

    PubMed

    Razavi, Morteza; Leigh Anderson, N; Pope, Matthew E; Yip, Richard; Pearson, Terry W

    2016-09-25

    Efficient robotic workflows for trypsin digestion of human plasma and subsequent antibody-mediated peptide enrichment (the SISCAPA method) were developed with the goal of improving assay precision and throughput for multiplexed protein biomarker quantification. First, an 'addition only' tryptic digestion protocol was simplified from classical methods, eliminating the need for sample cleanup, while improving reproducibility, scalability and cost. Second, methods were developed to allow multiplexed enrichment and quantification of peptide surrogates of protein biomarkers representing a very broad range of concentrations and widely different molecular masses in human plasma. The total workflow coefficients of variation (including the 3 sequential steps of digestion, SISCAPA peptide enrichment and mass spectrometric analysis) for 5 proteotypic peptides measured in 6 replicates of each of 6 different samples repeated over 6 days averaged 3.4% within-run and 4.3% across all runs. An experiment to identify sources of variation in the workflow demonstrated that MRM measurement and tryptic digestion steps each had average CVs of ∼2.7%. Because of the high purity of the peptide analytes enriched by antibody capture, the liquid chromatography step is minimized and in some cases eliminated altogether, enabling throughput levels consistent with requirements of large biomarker and clinical studies.

  19. Chiral liquid chromatography-mass spectrometry (LC-MS/MS) method development for the detection of salbutamol in urine samples.

    PubMed

    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%. PMID:27232053

  20. Ultra-trace quantification method for chlordecone in human fluids and tissues.

    PubMed

    Bichon, Emmanuelle; Guiffard, Ingrid; Vénisseau, Anaïs; Marchand, Philippe; Antignac, Jean-Philippe; Le Bizec, Bruno

    2015-08-21

    Chlordecone is an organochlorine pesticide (OCP) considered as a Persistent Organic Pollutant (POP) as it persists in the environment, bio-accumulates through the food web, causes adverse effects to human health and the environment and transports across international boundaries far from its sources. The atypical physico-chemical properties of chlordecone make its inclusion in classical analytical approaches non applicable. The aim of our work was to include chlordecone in a multi organochlorine residue method preventing any degradation during the analytical process and thus allowing quantification at ppt (ngkg(-1) or ngL(-1)) levels for a wide range of OCPs in breast milk, human serum and adipose tissue. After GC-HRMS vs. MS/MS and EI vs. APCI comparisons, the major improvement in terms of sensitivity was found in decreasing the length and film thickness of the gas chromatography column. Thanks to a linear correlation between relative response and quantity of chlordecone injected, LC-(ESI-)-MS/MS was finally preferred. An acetonitrile based gradient optimized on a C30 coreshell HPLC column has led to reaching limits of quantification as low as 8ngL(-1), 25pgmL(-1) and 0.2ngg(-1) fat for breast milk, serum and adipose tissue, respectively, allowing multiresidue OCP quantification at concentration levels compatible with biomonitoring purposes and pre-requisites. PMID:26184709

  1. Recently developed GC/MS and LC/MS methods for determining NSAIDs in water samples.

    PubMed

    Farré, M; Petrovic, M; Barceló, D

    2007-02-01

    Pharmaceuticals have become major targets in environmental chemistry due to their presence in aquatic environments (following incomplete removal in wastewater treatment or point-source contaminations), threat to drinking water sources and concern about their possible effects to wildlife and humans. Recently several methods have been developed for the determination of drugs and their metabolites in the lower nanogram per litre range, most of them using solid-phase extraction (SPE) or solid-phase microextraction (SPME), derivatisation and finally gas chromatography mass spectrometry (GC-MS), gas chromatography tandem mass spectrometry (GC-MS/MS) and liquid chromatography electrospray tandem mass spectrometry (LC-ES/MS/MS). Due to the elevated polarity of non-steroidal anti-inflamatory drugs (NSAIDs), analytical techniques based on either liquid chromatography coupled to mass spectrometry (LC-MS) and gas chromatography coupled to mass spectrometry (GC-MS) after a previous derivatisation step are essential. The most advanced aspects of current GC-MS, GC-MS/MS and LC-MS/MS methodologies for NSAID analysis are presented.

  2. Quantification of amygdalin in nonbitter, semibitter, and bitter almonds (Prunus dulcis) by UHPLC-(ESI)QqQ MS/MS.

    PubMed

    Lee, Jihyun; Zhang, Gong; Wood, Elizabeth; Rogel Castillo, Cristian; Mitchell, Alyson E

    2013-08-14

    Amygdalin is a cynaogenic diglucoside responsible for the bitterness of almonds. Almonds display three flavor phenotypes, nonbitter, semibitter, and bitter. Herein, the amygdalin content of 20 varieties of nonbitter, semibitter, and bitter almonds from four primary growing regions of California was determined using solid-phase extraction and ultrahigh-pressure liquid chromatography electrospray triple-quadrupole mass spectrometry (UHPLC-(ESI)QqQ MS/MS). The detection limit for this method is ≤ 0.1 ng/mL (3 times the signal-to-noise ratio) and the LOQ is 0.33 ng/mL (10 times the signal-to-noise ratio), allowing for the reliable quantitation of trace levels of amygdalin in nonbitter almonds (0.13 mg/kg almond). Results indicate that amygdalin concentrations for the three flavor phenotypes were significantly different (p < 0.001). The mean concentrations of amygdalin in nonbitter, semibitter, and bitter almonds are 63.13 ± 57.54, 992.24 ± 513.04, and 40060.34 ± 7855.26 mg/kg, respectively. Levels of amygdalin ranged from 2.16 to 157.44 mg/kg in nonbitter, from 523.50 to 1772.75 mg/kg in semibitter, and from 33006.60 to 53998.30 mg/kg in bitter almonds. These results suggest that phenotype classification may be achieved on the basis of amygdalin levels. Growing region had a statistically significant effect on the amygdalin concentration in commercial varieties (p < 0.05).

  3. An LC-MS/MS method to quantify acylcarnitine species including isomeric and odd-numbered forms in plasma and tissues.

    PubMed

    Giesbertz, Pieter; Ecker, Josef; Haag, Alexander; Spanier, Britta; Daniel, Hannelore

    2015-10-01

    Acylcarnitines are intermediates of fatty acid and amino acid oxidation found in tissues and body fluids. They are important diagnostic markers for inherited diseases of peroxisomal and mitochondrial oxidation processes and were recently described as biomarkers of complex diseases like the metabolic syndrome. Quantification of acylcarnitine species can become challenging because various species occur as isomers and/or have very low concentrations. Here we describe a new LC-MS/MS method for quantification of 56 acylcarnitine species with acyl-chain lengths from C2 to C18. Our method includes amino acid-derived positional isomers, like methacrylyl-carnitine (2-M-C3:1-CN) and crotonyl-carnitine (C4:1-CN), and odd-numbered carbon species, like pentadecanoyl-carnitine (C15:0-CN) and heptadecanoyl-carnitine (C17:0-CN), occurring at very low concentrations in plasma and tissues. Method validation in plasma and liver samples showed high sensitivity and excellent accuracy and precision. In an application to samples from streptozotocin-treated diabetic mice, we identified significantly increased concentrations of acylcarnitines derived from branched-chain amino acid degradation and of odd-numbered straight-chain species, recently proposed as potential biomarkers for the metabolic syndrome. In conclusion, the LC-MS/MS method presented here allows robust quantification of isomeric acylcarnitine species and extends the palette of acylcarnitines with diagnostic potential derived from fatty acid and amino acid metabolism. PMID:26239049

  4. An LC-MS/MS method to quantify acylcarnitine species including isomeric and odd-numbered forms in plasma and tissues[S

    PubMed Central

    Giesbertz, Pieter; Ecker, Josef; Haag, Alexander; Spanier, Britta; Daniel, Hannelore

    2015-01-01

    Acylcarnitines are intermediates of fatty acid and amino acid oxidation found in tissues and body fluids. They are important diagnostic markers for inherited diseases of peroxisomal and mitochondrial oxidation processes and were recently described as biomarkers of complex diseases like the metabolic syndrome. Quantification of acylcarnitine species can become challenging because various species occur as isomers and/or have very low concentrations. Here we describe a new LC-MS/MS method for quantification of 56 acylcarnitine species with acyl-chain lengths from C2 to C18. Our method includes amino acid-derived positional isomers, like methacrylyl-carnitine (2-M-C3:1-CN) and crotonyl-carnitine (C4:1-CN), and odd-numbered carbon species, like pentadecanoyl-carnitine (C15:0-CN) and heptadecanoyl-carnitine (C17:0-CN), occurring at very low concentrations in plasma and tissues. Method validation in plasma and liver samples showed high sensitivity and excellent accuracy and precision. In an application to samples from streptozotocin-treated diabetic mice, we identified significantly increased concentrations of acylcarnitines derived from branched-chain amino acid degradation and of odd-numbered straight-chain species, recently proposed as potential biomarkers for the metabolic syndrome. In conclusion, the LC-MS/MS method presented here allows robust quantification of isomeric acylcarnitine species and extends the palette of acylcarnitines with diagnostic potential derived from fatty acid and amino acid metabolism. PMID:26239049

  5. QUANTIFICATION OF 2,4-D ON SOLID-PHASE EXPOSURE SAMPLING MEDIA BY LC/MS/MS

    EPA Science Inventory

    Three types of solid phase chemical exposure sampling media: cellulose, polyurethane foam (PUF) and XAD-2, were analyzed for 2,4-D and the amine salts of 2,4-D. Individual samples were extracted into acidified methanol and the extracts were analyzed via LC/MS/MS using electrospra...

  6. Validation of a method for the determination of 120 pesticide residues in apples and cucumbers by LC-MS/MS.

    PubMed

    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. PMID:27443204

  7. Accurate quantification of the mercapturic acids of acrylonitrile and its genotoxic metabolite cyanoethylene-epoxide in human urine by isotope-dilution LC-ESI/MS/MS.

    PubMed

    Schettgen, T; Bertram, J; Kraus, T

    2012-08-30

    Acrylonitrile is a highly important industrial chemical with a high production volume worldwide, especially in the plastics industry. It is classified as a possible human carcinogen by the International Agency for Research on Cancer (IARC group 2B). During metabolism of acrylonitrile, the genotoxic metabolite cyanoethylene-epoxide is formed. The urinary mercapturic acids of acrylonitrile, namely N-acetyl-S-(2-cyanoethyl)-L-cysteine (CEMA) and cyanoethylene-epoxide, namely N-acetyl-S-(1-cyano-2-hydroxyethyl)-L-cysteine (CHEMA) are specific biomarkers for the determination of individual internal exposure to acrylonitrile and its highly reactive metabolite. We have developed and validated a sensitive method for the accurate determination of CEMA and CHEMA in human urine with a multidimensional LC/MS/MS-method using deuterium-labelled analogues for both analytes as internal standards. Analytes were stripped from urinary matrix by online extraction on a restricted access material, transferred to the analytical column and determined by tandem mass spectrometry. The limit of quantification (LOQ) for CEMA and CHEMA was 1 μg/L urine and allowed to quantify the background exposure of the (smoking) general population. Precision within and between series for CHEMA ranged from 2.6 to 8.0% at four concentrations ranging from 8.3 to 86 μg/L urine, mean accuracy was between 94 and 100%. For CEMA, precision within and between series ranged from 2.4 to 14.5% at four concentrations ranging from 15.1 to 196 μg/L urine, mean accuracy was between 91 and 104%. We applied the method to spot urine samples of 83 subjects of the general population with no known occupational exposure to acrylonitrile. Median levels (range) for CEMA and CHEMA in urine samples of non-smokers (n=47) were 1.9 μg/L (<1-16.4 μg/L) and<1 μg/L (<1-3 μg/L), while in urine samples of smokers (n=36), median levels were 184 μg/L (2-907 μg/L) and 29.3 μg/L (<1-147 μg/L), respectively. Smokers showed a

  8. Identification of phenolic compounds in Equisetum giganteum by LC-ESI-MS/MS and a new approach to total flavonoid quantification.

    PubMed

    Francescato, Leandro N; Debenedetti, Silvia L; Schwanz, Thiago G; Bassani, Valquiria L; Henriques, Amélia T

    2013-02-15

    Equisetum giganteum L., commonly called "giant horsetail", is an endemic species of Latin America. Its aerial parts have been widely used in ethnomedicine as a diuretic and in herbal medicine and food supplements as a raw material. The phenolic composition of E. giganteum stems was studied by liquid chromatography coupled to diode array detection (LC-DAD) and liquid chromatography coupled to electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS), which identified caffeic acid derivatives, flavonoids and styrylpyrones. The most abundant glycosilated flavonoids in this sample were kaempferol derivatives. Other rare phenolic components, namely, quercetin-3-O-(caffeoyl)-glucoside and 3-hydroxyhispidin-3,4'-di-O-glucoside, were reported for first time in the Equisetum genus. An LC-UV method for the simultaneous quantification of flavonoid aglycones in E. giganteum obtained after hydrolysis was developed and validated. The method exhibited excellent linearity for all analytes, with regression coefficients above 0.998, LOD ≥ 0.043μg mL(-1), LOQ ≥ 0.158 μg mL(-1) and recovery rates of 96.89-103.33% and 98.22-102.49% for quercetin and kaempferol, respectively. The relative standard deviation for the intra- and inter-day precision was ≤ 3.75%. The hydrolysis process was optimized by central composite rotational design and response surface analysis. The second-order response models for the aglycones contents were as follows: quercetin (μg g(-1))=24.8102+55.2823 × HCl+0.776997 × Time-7.23852 × HCl(2)-7.46528E-04 × Time(2)-0.229167 × HCl × Time; kaempferol (μg g(-1))=-9.66755+974.822 × HCl+11.8059 × Time-130.612 × HCl(2)-0.0125694×Time(2) -3.22917 × HCl × Time, with estimated optimal conditions of 1.18 M HCl and 205 min of hydrolysis. The results obtained with these new methods were compared to those from a spectrophotometric assay used to determine the total flavonoids in the Equisetum arvense monograph (Horsetail, British Pharmacopoeia 2011

  9. A quick colorimetric method for total lipid quantification in microalgae.

    PubMed

    Byreddy, Avinesh R; Gupta, Adarsha; Barrow, Colin J; Puri, Munish

    2016-06-01

    Discovering microalgae with high lipid productivity are among the key milestones for achieving sustainable biodiesel production. Current methods of lipid quantification are time intensive and costly. A rapid colorimetric method based on sulfo-phospho-vanillin (SPV) reaction was developed for the quantification of microbial lipids to facilitate screening for lipid producing microalgae. This method was successfully tested on marine thraustochytrid strains and vegetable oils. The colorimetric method results correlated well with gravimetric method estimates. The new method was less time consuming than gravimetric analysis and is quantitative for lipid determination, even in the presence of carbohydrates, proteins and glycerol. PMID:27050419

  10. Analysis of 18 urinary mercapturic acids by two high-throughput multiplex-LC-MS/MS methods.

    PubMed

    Pluym, Nikola; Gilch, Gerhard; Scherer, Gerhard; Scherer, Max

    2015-07-01

    Mercapturic acids (MAs) are metabolic end products formed from conjugates between glutathione and electrophilic compounds. MAs are, therefore, suitable biomarkers of exposure to toxicants, which are either electrophiles by themselves or metabolized to electrophilic intermediates. We developed and validated two LC-MS/MS methods which allow the complementary, rapid, and sensitive determination of MAs derived from acrolein, acrylamide, acrylonitrile, benzene, 1,3-butadiene, crotonaldehyde, N,N-dimethylformamide, ethylene, ethylene oxide, vinyl chloride, propylene oxide, styrene, toluene as well as methylating and ethylating agents. Since separate determinations of single or small groups of MAs are time-consuming and expensive, we multiplexed several individual methods into two LC-MS/MS methods covering 18 individual mercapturic acids. Method validation according to FDA guidelines showed excellent results in terms of robustness, accuracy, and sensitivity of the methods. Moreover, the use of a minimal, simple, and straightforward sample cleanup procedure further accelerated the analytical workflow, which allows a time- and cost-efficient analysis of up to 18 MAs derived from various toxicants in environmental levels. The methods were applied to urine samples derived from a strictly diet-controlled clinical study, including 25 smoking and 25 non-smoking subjects. Significant increase in the urine concentrations in smokers as compared to non-smokers (p < 0.01; Student t test) was observed for 13 individual MAs. Moreover, a dose dependence was obtained for the majority of the analytes. In conclusion, the newly developed assays represent a powerful tool for the fast and reliable quantification of 18 MAs in clinical studies. A first method application suggests several suitable biomarkers for nine relevant toxicants in tobacco smoke. PMID:25935678

  11. A Probabilistic Framework for Peptide and Protein Quantification from Data-Dependent and Data-Independent LC-MS Proteomics Experiments

    PubMed Central

    Richardson, Keith; Denny, Richard; Hughes, Chris; Skilling, John; Sikora, Jacek; Dadlez, Michał; Manteca, Angel; Jung, Hye Ryung; Jensen, Ole Nørregaard; Redeker, Virginie; Melki, Ronald; Langridge, James I.; Vissers, Johannes P.C.

    2013-01-01

    A probability-based quantification framework is presented for the calculation of relative peptide and protein abundance in label-free and label-dependent LC-MS proteomics data. The results are accompanied by credible intervals and regulation probabilities. The algorithm takes into account data uncertainties via Poisson statistics modified by a noise contribution that is determined automatically during an initial normalization stage. Protein quantification relies on assignments of component peptides to the acquired data. These assignments are generally of variable reliability and may not be present across all of the experiments comprising an analysis. It is also possible for a peptide to be identified to more than one protein in a given mixture. For these reasons the algorithm accepts a prior probability of peptide assignment for each intensity measurement. The model is constructed in such a way that outliers of any type can be automatically reweighted. Two discrete normalization methods can be employed. The first method is based on a user-defined subset of peptides, while the second method relies on the presence of a dominant background of endogenous peptides for which the concentration is assumed to be unaffected. Normalization is performed using the same computational and statistical procedures employed by the main quantification algorithm. The performance of the algorithm will be illustrated on example data sets, and its utility demonstrated for typical proteomics applications. The quantification algorithm supports relative protein quantification based on precursor and product ion intensities acquired by means of data-dependent methods, originating from all common isotopically-labeled approaches, as well as label-free ion intensity-based data-independent methods. PMID:22871168

  12. Analysis of Thiodiglycol: Validation of Semi-Volatile Analysis by HPLC-MS/MS by EPA Method MS777

    SciTech Connect

    Owens, J; Koester, C

    2008-07-24

    The Environmental Protection Agency's (EPA) Region 5 Chicago Regional Laboratory (CRL) developed a method for the analysis of thiodiglycol, the breakdown product of the sulfur mustard HD, in water by high performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS), titled Method EPA MS777 (hereafter referred to as EPA CRL SOP MS777). 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 verify the analytical procedures described in MS777 for analysis of thiodiglycol in aqueous samples. The gathered data from this 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 Method EPA MS777 can be determined.

  13. Simple and sensitive analysis of histamine and tyramine in Japanese soy sauces and their intermediates using the stable isotope dilution HILIC-MS/MS method.

    PubMed

    Todoroki, Kenichiro; Ishii, Yasuhiro; Miyauchi, Chiemi; Kitagawa, Sachiyo; Min, Jun Zhe; Inoue, Koichi; Yamanaka, Tomoyuki; Suzuki, Kuniaki; Yoshikawa, Yuko; Ohashi, Norio; Toyo'oka, Toshimasa

    2014-07-01

    We established a simple, sensitive, and reproducible method to analyze the histamine and tyramine levels in Japanese soy sauce and its mash (called moromi) using hydrophilic interaction liquid chromatography with tandem mass spectrometry (HILIC-MS/MS). Histamine and tyramine quantification was performed using their stable isotopes for electrospray ionization-tandem mass spectrometry in the selected reaction monitoring mode. The sample pretreatment process was a simple, one-step liquid-liquid extraction. HILIC separation was performed with a gradient elution of aqueous ammonium formate and acetonitrile. Because of validation tests, the linearity, the accuracies, and precisions were sufficient. The limit of detection and the limit of quantification were 0.09 and 0.29 ppm for histamine and 0.13 and 0.42 ppm for tyramine, respectively. We successfully applied this method to histamine and tyramine determination in four kinds of commercial Japanese soy sauces and also in moromi samples during soy sauce production.

  14. Development and Validation of a UPLC-MS/MS Method to Monitor Cephapirin Excretion in Dairy Cows following Intramammary Infusion

    PubMed Central

    Ray, Partha; Knowlton, Katharine F.; Shang, Chao; Xia, Kang

    2014-01-01

    Cephapirin, a cephalosporin antibiotic, is used by the majority of dairy farms in the US. Fecal and urinary excretion of cephapirin could introduce this compound into the environment when manure is land applied as fertilizer, and may cause development of bacterial resistance to antibiotics critical for human health. The environmental loading of cephapirin by the livestock industry remains un-assessed, largely due to a lack of appropriate analytical methods. Therefore, this study aimed to develop and validate a cephapirin quantification method to capture the temporal pattern of cephapirin excretion in dairy cows following intramammary infusion. The method includes an extraction with phosphate buffer and methanol, solid-phase extraction (SPE) clean-up, and quantification using ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The LOQ values of the developed method were 4.02 µg kg−1 and 0.96 µg L−1 for feces and urine, respectively. This robust method recovered >60% and >80% cephapirin from spiked blank fecal and urine samples, respectively, with acceptable intra- and inter-day variation (<10%). Using this method, we detected trace amounts (µg kg−1) of cephapirin in dairy cow feces, and cephapirin in urine was detected at very high concentrations (133 to 480 µg L−1). Cephapirin was primarily excreted via urine and its urinary excretion was influenced by day (P = 0.03). Peak excretion (2.69 mg) was on day 1 following intramammary infusion and decreased sharply thereafter (0.19, 0.19, 0.08, and 0.17 mg on day 2, 3, 4, and 5, respectively) reflecting a quadratic pattern of excretion (Quadratic: P = 0.03). The described method for quantification of cephapirin in bovine feces and urine is sensitive, accurate, and robust and allowed to monitor the pattern of cephapirin excretion in dairy cows. This data will help develop manure segregation and treatment methods to minimize the risk of antibiotic loading to the environment

  15. Analysis of Ethanolamines: Validation of Semi-Volatile Analysis by HPLC-MS/MS by EPA Method MS888

    SciTech Connect

    Owens, J; Vu, A; Koester, C

    2008-10-08

    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 the 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.

  16. LC-MS-MS Method for Stimulants in Wastewater During Football Games.

    PubMed

    Gul, Waseem; Stamper, Brandon J; Godfrey, Murrell; ElSohly, Mahmoud A

    2016-03-01

    A method was developed for the analysis of amphetamines and cocaine (Coc) in wastewater samples using liquid chromatography coupled with tandem mass spectrometry (LC-MS-MS). Seven stimulant-type drugs and metabolites were analyzed. These drugs included amphetamine (Amp), methamphetamine (Meth), methylenedioxyamphetamine (MDA), methylenedioxymethamphetamine (MDMA), methylenedioxyethylamphetamine (MDEA), Coc and benzoylecgonine (BE, the major metabolite of Coc). These drugs were chosen because of their widespread use. Wastewater samples were collected at both the Oxford Waste Water Treatment Plant in Oxford, Mississippi (MS) and the University Wastewater Treatment Plant in University, MS. Samples were collected on weekends in which the Ole Miss Rebel football team held home games (Vaught-Hemingway Stadium, University, MS 38677). The collected samples were analyzed using a validated method and found to contain Amp, Meth, MDMA, Coc and BE. The concentrations of Amp and BE significantly rose in the university wastewater during football games. PMID:26538543

  17. LC-MS-MS Method for Stimulants in Wastewater During Football Games.

    PubMed

    Gul, Waseem; Stamper, Brandon J; Godfrey, Murrell; ElSohly, Mahmoud A

    2016-03-01

    A method was developed for the analysis of amphetamines and cocaine (Coc) in wastewater samples using liquid chromatography coupled with tandem mass spectrometry (LC-MS-MS). Seven stimulant-type drugs and metabolites were analyzed. These drugs included amphetamine (Amp), methamphetamine (Meth), methylenedioxyamphetamine (MDA), methylenedioxymethamphetamine (MDMA), methylenedioxyethylamphetamine (MDEA), Coc and benzoylecgonine (BE, the major metabolite of Coc). These drugs were chosen because of their widespread use. Wastewater samples were collected at both the Oxford Waste Water Treatment Plant in Oxford, Mississippi (MS) and the University Wastewater Treatment Plant in University, MS. Samples were collected on weekends in which the Ole Miss Rebel football team held home games (Vaught-Hemingway Stadium, University, MS 38677). The collected samples were analyzed using a validated method and found to contain Amp, Meth, MDMA, Coc and BE. The concentrations of Amp and BE significantly rose in the university wastewater during football games.

  18. LC-MS-MS Method for Analysis of Opiates in Wastewater During Football Games II.

    PubMed

    Gul, Waseem; Stamper, Brandon; Godfrey, Murrell; Gul, Shahbaz W; ElSohly, Mahmoud A

    2016-06-01

    Continuing our previous studies analyzing drugs of abuse in municipal wastewater, a method was developed for the analysis of opiates in wastewater samples using liquid chromatography coupled with tandem mass spectrometry (LC-MS-MS). Eight opiate drugs and metabolites were analyzed including codeine, hydrocodone, hydromorphone, 6-monoacetylmorphine (6-MAM, the primary urinary metabolite of heroin), morphine, norhydrocodone (the primary urinary metabolite of hydrocodone), oxycodone and oxymorphone. These drugs were chosen because of their widespread abuse. Wastewater samples were collected at both the Oxford Waste Water Treatment Plant in Oxford, Mississippi (MS) and the University Wastewater Treatment Plant in University, MS. These wastewater samples were collected on weekends in which the Ole Miss Rebel football team held home games (Vaught-Hemingway Stadium, University, MS 38677). The collected samples were analyzed using a validated method and found to contain codeine, hydrocodone, hydromorphone, morphine, norhydrocodone, oxycodone and oxymorphone. None of the samples contained 6-MAM. PMID:27052850

  19. Quantitative analysis of unconjugated and total bisphenol A in human urine using solid-phase extraction and UPLC-MS/MS: method implementation, method qualification and troubleshooting.

    PubMed

    Buscher, Brigitte; van de Lagemaat, Dick; Gries, Wolfgang; Beyer, Dieter; Markham, Dan A; Budinsky, Robert A; Dimond, Stephen S; Nath, Rajesh V; Snyder, Stephanie A; Hentges, Steven G

    2015-11-15

    The aim of the presented investigation was to document challenges encountered during implementation and qualification of a method for bisphenol A (BPA) analysis and to develop and discuss precautions taken to avoid and to monitor contamination with BPA during sample handling and analysis. Previously developed and published HPLC-MS/MS methods for the determination of unconjugated BPA (Markham et al. Journal of Analytical Toxicology, 34 (2010) 293-303) [17] and total BPA (Markham et al. Journal of Analytical Toxicology, 38 (2014) 194-203) [20] in human urine were combined and transferred into another laboratory. The initial method for unconjugated BPA was developed and evaluated in two independent laboratories simultaneously. The second method for total BPA was developed and evaluated in one of these laboratories to conserve resources. Accurate analysis of BPA at sub-ppb levels is a challenging task as BPA is a widely used material and is ubiquitous in the environment at trace concentrations. Propensity for contamination of biological samples with BPA is reported in the literature during sample collection, storage, and/or analysis. Contamination by trace levels of BPA is so pervasive that even with extraordinary care, it is difficult to completely exclude the introduction of BPA into biological samples and, consequently, contamination might have an impact on BPA biomonitoring data. The applied UPLC-MS/MS method was calibrated from 0.05 to 25ng/ml. The limit of quantification was 0.1ng/ml for unconjugated BPA and 0.2ng/ml for total BPA, respectively, in human urine. Finally, the method was applied to urine samples derived from 20 volunteers. Overall, BPA can be analyzed in human urine with acceptable recovery and repeatability if sufficient measures are taken to avoid contamination throughout the procedure from sample collection until UPLC-MS/MS analysis. PMID:26465088

  20. Analysis of Carbamate Pesticides: Validation of Semi-Volatile Analysis by HPLC-MS/MS by EPA Method MS666

    SciTech Connect

    Owens, J; Koester, C

    2008-05-14

    The Environmental Protection Agency's (EPA) Region 5 Chicago Regional Laboratory (CRL) developed a method for analysis of aldicarb, bromadiolone, carbofuran, oxamyl, and methomyl in water by high performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS), titled Method EPA MS666. 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 MS666 for analysis of carbamate pesticides 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 Method EPA MS666 can be determined.

  1. Rapid and sensitive quantification of levoglucosan in aerosols by high-performance anion-exchange chromatography with positive electrospray ionization mass spectrometry (HPAEC-positive ESI-MS)

    NASA Astrophysics Data System (ADS)

    Asakawa, Daichi; Furuichi, Yuko; Yamamoto, Atsushi; Oku, Yuichiro; Funasaka, Kunihiro

    2015-12-01

    A convenient quantification method for underivatized levoglucosan, which is a tracer for biomass burning influenced particulate matter (PM), has been established using high-performance anion-exchange chromatography (HPAEC) coupled to positive electrospray ionization mass spectrometry ((+)ESI-MS). Levoglucosan was chromatographically separated from its isomers (mannosan and galactosan) and detected selectively with positive ESI-MS. Limits of detection and quantification for this method were 0.40 and 1.3 ng mL-1, respectively. A comparison of simultaneous measurements by this method and conventional derivatization gas chromatography/mass spectrometry showed a good linearity with a slope of 1.008 and a determination coefficient of 0.9932. The developed method was applied to ambient suspended particulate matter hourly collected by continuous particulate monitors at 10 stations. The hourly concentration of levoglucosan during August 9-11, 2011, was 1.7-918 ng m-3 and its distribution indicated the transportation of biomass burning aerosols of a forest fire. This is the first report of horizontal distribution of the hourly levoglucosan concentration in Japan.

  2. Development and UFLC-MS/MS Characterization of a Product-Specific Standard for Phenolic Quantification of Maple-Derived Foods.

    PubMed

    Liu, Yongqiang; Ma, Hang; Seeram, Navindra P

    2016-05-01

    The phenolic contents of plant foods are commonly quantified by the Folin-Ciocalteu assay based on gallic acid equivalents (GAEs). However, this may lead to inaccuracies because gallic acid is not always representative of the structural heterogeneity of plant phenolics. Therefore, product-specific standards have been developed for the phenolic quantification of several foods. Currently, maple-derived foods (syrup, sugar, sap/water, and extracts) are quantified for phenolic contents based on GAEs. Because lignans are the predominant phenolics present in maple, herein, a maple phenolic lignan-enriched standard (MaPLES) was purified (by chromatography) and characterized (by UFLC-MS/MS with lignans previously isolated from maple syrup). Using MaPLES and secoisolariciresinol (a commercially available lignan), the phenolic contents of the maple-derived foods increased 3-fold compared to GAEs. Therefore, lignan-based standards are more appropriate for phenolic quantification of maple-derived foods versus GAEs. Also, MaPLES can be utilized for the authentication and detection of fake label claims on maple products.

  3. Development and UFLC-MS/MS Characterization of a Product-Specific Standard for Phenolic Quantification of Maple-Derived Foods.

    PubMed

    Liu, Yongqiang; Ma, Hang; Seeram, Navindra P

    2016-05-01

    The phenolic contents of plant foods are commonly quantified by the Folin-Ciocalteu assay based on gallic acid equivalents (GAEs). However, this may lead to inaccuracies because gallic acid is not always representative of the structural heterogeneity of plant phenolics. Therefore, product-specific standards have been developed for the phenolic quantification of several foods. Currently, maple-derived foods (syrup, sugar, sap/water, and extracts) are quantified for phenolic contents based on GAEs. Because lignans are the predominant phenolics present in maple, herein, a maple phenolic lignan-enriched standard (MaPLES) was purified (by chromatography) and characterized (by UFLC-MS/MS with lignans previously isolated from maple syrup). Using MaPLES and secoisolariciresinol (a commercially available lignan), the phenolic contents of the maple-derived foods increased 3-fold compared to GAEs. Therefore, lignan-based standards are more appropriate for phenolic quantification of maple-derived foods versus GAEs. Also, MaPLES can be utilized for the authentication and detection of fake label claims on maple products. PMID:27101225

  4. High-throughput ultra-performance LC-MS-MS method for analysis of diclofenac sodium in rabbit plasma.

    PubMed

    Alam, Mohd Aftab; Al-Jenoobi, Fahad I; Al-Mohizea, Abdullah M

    2015-01-01

    A new UPLC-MS-MS method was developed and validated for quantification of diclofenac sodium in rabbit plasma. Acetonitrile-based protein precipitation method was used to extract the drug from plasma samples. Chromatographic separation was carried out on Acquity UPLC(®)BEH phenyl C18 1.7 µm, 2.1 × 50 mm column. Drug elution was facilitated by using mobile phase containing acetonitrile (0.1% glacial acetic) and water (pH 3.5), in a ratio of 75 : 25, flowing at 0.2 mL/min. Molecular ions were generated by using the positive electrospray ionization mode (ESI(+)) and analyzed on a triple-quadrupole mass spectrometer. The ionic transitions of diclofenac (m/z 296 > 214 and 249.9) and flufenamic acid (internal standard) (m/z 282.1 > 166.9 and 244) were measured in multiple reaction modes. Developed method is simple, quick, precise and accurate over a linearity range of 80-4,000 ng/mL. The lower limit of quantification (LLOQ) for diclofenac was 80 ng/mL. The percentage recoveries of diclofenac at three quality control samples were 54 ± 6.1, 67.1 ± 5.4 and 62.3 ± 1.4%. Precision and accuracy of the assay at LLOQ, middle limit of quantitation and higher limit of quantitation were 100 ± 7.0, 100 ± 1.0 and 100 ± 2.0% and 81.8 ± 4.6, 106.6 ± 3.1 and 103.3 ± 4.0%, respectively.

  5. Accumulation of three important bioactive compounds in different plant parts of Withania somnifera and its determination by the LC-ESI-MS-MS (MRM) method.

    PubMed

    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.

  6. Validation and Application of an LC-MS-MS Method for the Determination of Ceftizoxime in Human Serum and Urine.

    PubMed

    Wang, Lu; Zheng, Xin; Zhong, Wen; Chen, Jia; Jiang, Ji; Hu, Pei

    2016-01-01

    Ceftizoxime sodium is a third-generation cephalosporin available for parenteral administration, which is mainly excreted through urine. A rapid and sensitive ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS-MS) method was developed and validated for the determination of ceftizoxime in human serum and urine. The samples were purified by protein precipitation and separated on an XTerra Phenyl column (4.6 × 50 mm, 5 µm). Electrospray ionization in the positive ion mode and multiple reaction monitoring were used to monitor the ion transitions at m/z 383.9/227.0. The results revealed that the method had excellent selectivity. The linear range covered from 2.50 to 10,000 ng/mL in serum and from 0.500 to 50.0 µg/mL in urine, respectively. Intra-batch and inter-batch precisions (in terms of relative standard deviation) were all <15% and the accuracies (in terms of relative error) were within the range of ± 15%. The lower limit of quantification, stability and extraction recovery were also validated and satisfied the criteria of validation. Finally, the method was successfully applied to a pharmacokinetic study of Chinese elderly healthy subjects after intravenous administration. The Cmax values in serum were 34,721.3 ± 5,697.3 ng/mL. Serum concentrations declined with t1/2 of 2.57 ± 0.22 h. PMID:26896348

  7. Accurate LC-ESI-MS/MS quantification of 2'-deoxymugineic acid in soil and root related samples employing porous graphitic carbon as stationary phase and a ¹³C₄-labeled internal standard.

    PubMed

    Schindlegger, Yvonne; Oburger, Eva; Gruber, Barbara; Schenkeveld, Walter D C; Kraemer, Stephan M; Puschenreiter, Markus; Koellensperger, Gunda; Hann, Stephan

    2014-05-01

    For the first time the phytosiderophore 2'-deoxymugineic acid (DMA) could be accurately quantified by LC-MS/MS in plant and soil related samples. For this purpose a novel chromatographic method employing porous graphitic carbon as stationary phase combined with ESI-MS/MS detection in selected reaction monitoring was developed. Isotope dilution was implemented by using in-house synthesized DMA as external calibrant and ¹³C₄-labeled DMA as internal standard (concentration levels of standards 0.1-80 μM, determination coefficient of linear regression R² > 0.9995). Sample preparation involved acidification of the samples in order to obtain complete dissociation of metal-DMA complexes. Excellent matrix related LOD and LOQ depending on different experimental setups were obtained in the range of 3-34 nM and 11-113 nM, respectively. Standard addition experiments and the implementation of the internal ¹³C₄-DMA standard proved the accuracy of the quantification strategy even in complex matrices such as soil solution. The repeatability of the method, including sample preparation, expressed as short- and long term precision was below 4 and 5% RSD, respectively. Finally, application in the context of plant and soil research to samples from rhizosphere sampling via micro suction cups, from soil solutions and soil adsorption/extraction studies revealed a DMA concentration range from 0.1 to 235 μM.

  8. Quantification of serum apolipoproteins A-I and B-100 in clinical samples using an automated SISCAPA-MALDI-TOF-MS workflow.

    PubMed

    van den Broek, Irene; Nouta, Jan; Razavi, Morteza; Yip, Richard; Bladergroen, Marco R; Romijn, Fred P H T M; Smit, Nico P M; Drews, Oliver; Paape, Rainer; Suckau, Detlev; Deelder, André M; van der Burgt, Yuri E M; Pearson, Terry W; Anderson, N Leigh; Cobbaert, Christa M

    2015-06-15

    A fully automated workflow was developed and validated for simultaneous quantification of the cardiovascular disease risk markers apolipoproteins A-I (apoA-I) and B-100 (apoB-100) in clinical sera. By coupling of stable-isotope standards and capture by anti-peptide antibodies (SISCAPA) for enrichment of proteotypic peptides from serum digests to matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS detection, the standardized platform enabled rapid, liquid chromatography-free quantification at a relatively high throughput of 96 samples in 12h. The average imprecision in normo- and triglyceridemic serum pools was 3.8% for apoA-I and 4.2% for apoB-100 (4 replicates over 5 days). If stored properly, the MALDI target containing enriched apoA-1 and apoB-100 peptides could be re-analyzed without any effect on bias or imprecision for at least 7 days after initial analysis. Validation of the workflow revealed excellent linearity for daily calibration with external, serum-based calibrators (R(2) of 0.984 for apoA-I and 0.976 for apoB-100 as average over five days), and absence of matrix effects or interference from triglycerides, protein content, hemolysates, or bilirubins. Quantification of apoA-I in 93 normo- and hypertriglyceridemic clinical sera showed good agreement with immunoturbidimetric analysis (slope = 1.01, R(2) = 0.95, mean bias = 4.0%). Measurement of apoB-100 in the same clinical sera using both methods, however, revealed several outliers in SISCAPA-MALDI-TOF-MS measurements, possibly as a result of the lower MALDI-TOF-MS signal intensity (slope = 1.09, R(2) = 0.91, mean bias = 2.0%). The combination of analytical performance, rapid cycle time and automation potential validate the SISCAPA-MALDI-TOF-MS platform as a valuable approach for standardized and high-throughput quantification of apoA-I and apoB-100 in large sample cohorts.

  9. Comparison of Two Methods for Anthocyanin Quantification

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The pH differential method (AOAC method 2005.02) by spectrophotometer, and high performance liquid chromatography (HPLC) are methods commonly used by researchers and the food industry for quantifying anthocyanins of samples or products. This study was carried out to establish a relationship between ...

  10. Comparison of Two Methods for Anthocyanin Quantification

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The pH differential method (AOAC method 2005.02) by spectrophotometer and high performance liquid chromatography (HPLC) are methods commonly used by researchers and the food industry for quantifying anthocyanins of samples or products. This study was carried out to establish a relationship between t...

  11. Comparison of Two Methods for Anthocyanin Quantification

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The pH differential method (AOAC method 2005.02) by spectrophotometer, and high performance liquid chromatography (HPLC) are methods commonly used by researchers and the food industry for quantifying anthocyanins of samples or products. This study was carried out to establish a relationship between...

  12. Development and validation of LC/MS/MS method for the simultaneous determination of montelukast, gliclazide, and nifedipine and its application to a pharmacokinetic study

    PubMed Central

    2014-01-01

    Background Montelukast is a leukotriene receptor antagonist for treatment of asthma, gliclazide is an oral hypoglycemic antidiabetic agent, and nifedipine is a calcium channel blocker used for treatment of angina pectoris and hypertension. These drugs may be prescribed to patients suffering from these chronic diseases. A survey of the literature reveals that there is no reported method for the simultaneous determination of montelukast, gliclazide, and nifedipine in pharmaceutical preparations or biological fluids. Results A simple, sensitive, and rapid method for the simultaneous quantification of montelukast, gliclazide, and nifedipine in human plasma was developed and validated. Montelukast, gliclazide, and nifedipine were resolved using rapid resolution LC/MS/MS Agilent system and SB-C18 (50 × 4.6 mm) 1.8 μm particle size column. The mobile phase consisted of acetonitrile: 0.1% formic acid (84:16). The three drugs were simultaneously extracted from plasma by protein precipitation with acetonitrile using zaferolukast as an internal standard. The method was validated according to FDA guidelines with good reproducibility and linearity of 0.999 and the limits of quantification were 0.11, 0.04, and 0.07 ng/mL for montelukast, gliclazide, and nifedipine, respectively. The accuracies of the three QCs for the three drugs were 99.48% (montelukast), 106.53% (gliclazide), and 108.03% (nifedipine) in human plasma. The validated method was applied to a pharmacokinetic study in human volunteers after oral administration of the three drugs. The applied LC/MS/MS method was shown to be sufficiently sensitive and suitable for pharmacokinetic studies. Conclusion The LC/MS/MS method was validated and successfully applied for the determination of montelukast, gliclazide, and nifedipine concentrations in human plasma. PMID:24618480

  13. Simultaneous quantification of the glucagon-like peptide-1 (GLP-1) and cholecystokinin (CCK) receptor agonists in rodent plasma by on-line solid phase extraction and LC-MS/MS.

    PubMed

    Wang, Yan; Roth, Jonathan D; Taylor, Steven W

    2014-04-15

    Peptide agonists of the glucagon-like peptide-1 receptor (GLP-1R) and the cholecystokinin-1 receptor (CCK1-R) have therapeutic potential because of their marked anorexigenic and weight lowering effects. Furthermore, recent studies in rodents have shown that co-administration of these agents may prove more effective than treatment either of the peptide classes alone. To correlate the pharmacodynamic effects to the pharmacokinetics of these peptide drugs in vivo, a sensitive and robust bioanalytical method is essential. Furthermore, the simultaneous determination of both analytes in plasma samples by a single method offers obvious advantages. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is well suited to this goal through its ability to simultaneously monitor multiple analytes through selected reaction monitoring (SRM). However, it is a challenge to find appropriate conditions that allow two peptides with widely disparate physiochemical properties to be simultaneously analyzed while maintaining the necessary sensitivity for their accurate plasma concentrations. Herein, we report an on-line solid phase extraction (SPE) LC-MS/MS method for simultaneous quantification of the CCK1-R agonist AC170222 and the GLP-1R agonist AC3174 in rodent plasma. The assay has a linear range from 0.0975 to 100ng/mL, with lower limits of quantification of 0.0975ng/mL and 0.195ng/mL for AC3174 and AC170222, respectively. The intra- and inter-day precisions were below 15%. The developed LC-MS/MS method was used to simultaneously quantify AC3174 and AC170222, the results showed that the terminal plasma concentrations of AC3174 or AC170222 were comparable between groups of animals that were administered with the peptides alone (247±15pg/mL of AC3174 and 1306±48pg/mL of AC170222), or in combination (222±32pg/mL and 1136±47pg/mL of AC3174 and AC170222, respectively). These data provide information on the drug exposure to aid in assessing the combination effects of AC3174 and AC

  14. The detection and quantification of lorazepam and its 3-O-glucuronide in fingerprint deposits by LC-MS/MS.

    PubMed

    Goucher, Edward; Kicman, Andrew; Smith, Norman; Jickells, Sue

    2009-07-01

    The use of fingerprints as an alternative biological matrix to test for the presence of drugs and/or their metabolites is a novel area of research in analytical toxicology. This investigation describes quantitative analysis for the benzodiazepine lorazepam and its 3-O-glucuronide conjugate in fingerprints following the oral administration of a single 2 mg dose of lorazepam to five volunteers. Creatinine was also measured to investigate whether the amount of drug relative to that of creatinine would help to account for the variable amount of secretory material deposited. Fingerprints were deposited on glass cover slips and extracted by dissolving them in a solution of dichloromethane/methanol, containing tetradeuterated lorazepam as an internal standard. The samples were evaporated, reconstituted with mobile phase and analysed by LC-MS/MS. Chromatography was achieved using an RP (C18) column for the analysis of lorazapem and its glucuronide, and a hydrophilic interaction column (HILIC) for the analysis of creatinine. Lorazepam and its glucuronide were only detected where ten prints had been combined, up to 12 h following drug administration. In every case, the amount of lorazepam glucuronide exceeded that of lorazepam, the peak amounts being 210 and 11 pg, respectively. Adjusting for creatinine smoothed the elimination profile. To our knowledge, this represents the first time a drug glucuronide has been detected in deposited fingerprints.

  15. Development of LC-MS/MS method for analysis of polyphenolic compounds in juice, tea and coffee samples

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A simple and fast method for the analysis of a wide range of polyphenolic compounds in juice, tea, and coffee samples was developed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The method was based on a simple sample preparation “dilute and shoot” approach, and LC-MS/MS triple qu...

  16. Comparison of DNA Quantification Methods for Next Generation Sequencing

    PubMed Central

    Robin, Jérôme D.; Ludlow, Andrew T.; LaRanger, Ryan; Wright, Woodring E.; Shay, Jerry W.

    2016-01-01

    Next Generation Sequencing (NGS) is a powerful tool that depends on loading a precise amount of DNA onto a flowcell. NGS strategies have expanded our ability to investigate genomic phenomena by referencing mutations in cancer and diseases through large-scale genotyping, developing methods to map rare chromatin interactions (4C; 5C and Hi-C) and identifying chromatin features associated with regulatory elements (ChIP-seq, Bis-Seq, ChiA-PET). While many methods are available for DNA library quantification, there is no unambiguous gold standard. Most techniques use PCR to amplify DNA libraries to obtain sufficient quantities for optical density measurement. However, increased PCR cycles can distort the library’s heterogeneity and prevent the detection of rare variants. In this analysis, we compared new digital PCR technologies (droplet digital PCR; ddPCR, ddPCR-Tail) with standard methods for the titration of NGS libraries. DdPCR-Tail is comparable to qPCR and fluorometry (QuBit) and allows sensitive quantification by analysis of barcode repartition after sequencing of multiplexed samples. This study provides a direct comparison between quantification methods throughout a complete sequencing experiment and provides the impetus to use ddPCR-based quantification for improvement of NGS quality. PMID:27048884

  17. UPLC-MS-MS Method for the Determination of Vilazodone in Human Plasma: Application to a Pharmacokinetic Study.

    PubMed

    El-Bagary, Ramzia; Hashem, Hanaa; Fouad, Marwa; Tarek, Sally

    2016-09-01

    A sensitive, rapid and simple liquid chromatographic-electrospray ionization tandem mass spectrometric (LC-ESI-MS-MS) method was developed for the quantitative determination of vilazodone in human plasma and for the study of the pharmacokinetic behavior of vilazodone in healthy Egyptian volunteers. With escitalopram as internal standard (IS), liquid-liquid extraction was used for the purification and preconcentration of analytes from human plasma matrix using diethyl ether. The separation was performed on an Acquity UPLC BEH shield RP C18 column (1.7 µm, 2.1 × 150 mm). Isocratic elution was applied using methanol-0.2% formic acid (90:10, v/v). Detection was performed on a triple-quadrupole tandem mass spectrometer with multiple reaction monitoring mode via an electrospray ionization source at m/z 442.21 → 155.23 for vilazodone and m/z 325.14 → 109.2 for escitalopram. Linear calibration curves were obtained over the range of 1-200 ng/mL with the lower limit of quantification at 1 ng/mL. The intra- and inter-day precision showed relative standard deviation ≤3.3%. The total run time was 1.5 min. This method was successfully applied for clinical pharmacokinetic investigation, and a preliminary metabolic study was also carried out. PMID:27209054

  18. Development of an UPLC-MS/MS method for the determination of antibiotic ertapenem on dried blood spots.

    PubMed

    la Marca, Giancarlo; Giocaliere, Elisa; Villanelli, Fabio; Malvagia, Sabrina; Funghini, Silvia; Ombrone, Daniela; Filippi, Luca; De Gaudio, Marina; De Martino, Maurizio; Galli, Luisa

    2012-03-01

    Ertapenem (Invanz) is a newly developed carbapenem β-lactam antimicrobial agent. The drug usage in pediatric age needs an accurate drug monitoring for effective patient management. The aim of this study was to evaluate the use of dried blood spot (DBS) specimens to measure ertapenem concentration during treatment. The analysis was performed by UPLC-MS/MS operating in multiple reaction monitoring (MRM) mode. The calibration curve in matrix was linear in the concentration range of 0.5-100 mg/L with correlation coefficient value higher than 0.997. Performance parameters of this method like lower limit of detection (LLOD, 0.2 mg/L), lower limit of quantification (LLOQ, 0.5 mg/L), matrix effect (20%), intra- and inter-day imprecision (CV within than 15%) and accuracy (between 94 and 155%) of drug concentrations have been evaluated. The drug stability at different temperatures was tested for one month, to evaluate the risks of sample delivery at different climatic conditions. The reported method allows now ertapenem analysis and offers many advantages for patients including the possibility of collecting samples at home. This new assay is both precise and accurate and is especially suitable for therapeutic drug monitoring and pharmacokinetic studies in neonates in whom obtaining larger blood samples is not convenient or possible.

  19. Direct injection method for HPLC/MS/MS analysis of acrylamide in aqueous solutions: application to adsorption experiments.

    PubMed

    Mnif, Ines; Hurel, Charlotte; Marmier, Nicolas

    2015-05-01

    Polyacrylamides are polymers used in many fields and represent the main source of release of the highly toxic acrylamide in the environment. In this work, a simple, rapid, and sensitive analytical method was developed with HPLC/MS/MS and direct injection for acrylamide analysis in water and adsorption samples. AFNOR standards NF T90-210 and NF T90-220 were used for the analytical method validation and uncertainty estimation. Limit of quantification (LOQ) for acrylamide was 1 μg/L, and accuracy was checked at three acrylamide levels (1, 6, and 10 μg/L). Uncertainties were estimated at 34.2, 22, and 12.4 % for acrylamide concentrations at LOQ, 6 μg/L, and 10 μg/L, respectively. Acrylamide adsorption on clays (kaolinite, illite) and sludge was then studied as a function of pH, time, and acrylamide concentrations. Acrylamide adsorption on kaolinite, illite, and sludge was found to be very weak since adsorption percentages were inferior to 10 %, whatever the pH value and the initial acrylamide concentration. The low affinity of acrylamide for clays and sludge is likely due to its hydrophilic property, small size, and charge neutrality.

  20. Event-specific qualitative and quantitative PCR detection methods for transgenic rapeseed hybrids MS1xRF1 and MS1xRF2.

    PubMed

    Wu, Yuhua; Wu, Gang; Xiao, Ling; Lu, Changming

    2007-10-17

    Except for the events RT73, MS8, RF3, and T45, event-specific detection methods for most commercialized genetically modified (GM) rapeseed varieties have not been established, and as a result, the enforcement of genetically modified organism labeling policies has been hindered. The genetically modified rapeseeds, MS1xRF1 and MS1xRF2, are 2 of 11 approved GM-rapeseed varieties for commercialization. In this study, the right border junction fragments between the gene construct and the rapeseed genome of events RF1, RF2, and MS1 were isolated using the commercially available GenomeWalker technology. Homology analysis indicated that the gene construct of RF1 integrated upstream of the nuclease gene, and that of the RF2 and MS1 inserted into the exon region of a gene encoding for an unknown protein. The event-specific primer pairs and corresponding probes were designed on the basis of the revealed right border junction fragments. Then, we successfully developed the identification and quantification methods for the gene-stacked hybrids MS1xRF1 and MS1xRF2 using those primers and probes. The relative limit of detection in the qualitative polymerase chain reaction (PCR) was 0.013% for the RF2 and MS1 assays using 100 ng of rapeseed DNA per reaction and 0.13% for the RF1 assay. The absolute limit of detection in the quantitative PCR was approximately one to two initial copies for each of the three event-specific assays. The evaluation of the real-time PCR assays revealed that the qualitative and quantitative methods developed by focusing on the gene-stacked hybrids MS1xRF1 and MS1xRF2 were highly specific, sensitive, and suitable for samples with a low quantity of DNA.

  1. Sensitive quantification of clozapine and its main metabolites norclozapine and clozapine-N-oxide in serum and urine using LC-MS/MS after simple liquid-liquid extraction work-up.

    PubMed

    Wohlfarth, Ariane; Toepfner, Nicole; Hermanns-Clausen, Maren; Auwärter, Volker

    2011-05-01

    An LC-MS/MS method for the determination of the atypic neuroleptic clozapine and its two main metabolites norclozapine and clozapine-N-oxide has been developed and validated for serum and urine. After addition of d4-clozapine as deuterated internal standard a fast single-step liquid-liquid extraction under alkaline conditions and with ethyl acetate as organic solvent followed. The analytes were chromatographically separated on a Synergi Polar RP column using gradient elution with 1 mM ammonium formate and methanol. Data acquisition was performed on a QTrap 2000 tandem mass spectrometer in multiple reaction monitoring mode with positive electrospray ionization. Two transitions were monitored for each analyte in order to fulfill the established identification criteria. The validation included the determination of the limits of quantification (1.0 ng/mL for all analytes in serum and 2.0 ng/mL for all analytes in urine), assessment of matrix effects (77% to 92% in serum, 21 to 78% in urine) and the determination of extraction efficiencies (52% to 85% for serum, 59% to 88% for urine) and accuracy data. Imprecision was <10%, only the quantification of norclozapine in urine yielded higher relative standard deviations (11.2% and 15.7%). Bias values were below ±10%. Dilution of samples had no impact on the correctness for clozapine and norclozapine in both matrices and for clozapine-N-oxide in serum. For quantification of clozapine-N-oxide in urine a calibration with diluted calibrators has to be used. Calibration curves were measured from the LOQ up to 2,000 ng/mL and proved to be linear over the whole range with regression coefficients higher than 0.98. The method was finally applied to several clinical serum and urine samples and a cerebro-spinal fluid sample of an intoxicated 13-month-old girl.

  2. Determination and quantification of 2'-O-fucosyllactose and 3-O-fucosyllactose in human milk by GC-MS as O-trimethylsilyl-oxime derivatives.

    PubMed

    Balogh, Réka; Szarka, Szabolcs; Béni, Szabolcs

    2015-11-10

    Human milk oligosaccharides possess various biological functions by protecting the infant from several bacterial and viral infections, modulating the immune system, serving as prebiotics and also contributing to the brain development. Hence, huge effort is underway by manufacturers to produce infant formulas enriched with human milk oligosaccharides which could mimic its diverse biological role the most. For this purpose, quantification of the natural oligosaccharide composition of the human milk is a key task. This study reports a fit for purpose GC-MS method for the quantification of the TMS ether oxime derivatives of 2'-O-fucosyllactose and 3-O-fucosyllactose, the two most abundant trisaccharides in human milk. The EI fragmentation pattern of the linkage isomers is discussed in details, focusing also on specific fragment ions. The GC-MS method with external standard calibration was applied for the monitoring of concentration changes of the trisaccharides throughout the first week of lactation in human milks samples collected from two volunteers. The results showed high concentration of both 2'-FL (4525-6266μg/mL in donor A and 2694-3551μg/mL in donor B) and 3-FL (271-441μg/mL in donor A and 99-208μg/mL in donor B), while no significant change has been observed throughout the one-week lactation period. The presented GC-MS method can serve as a quality control technique for the infant formulas and also offers an alternative to existing chromatographic methods to investigate HMOs in milk samples.

  3. Determination and quantification of 2'-O-fucosyllactose and 3-O-fucosyllactose in human milk by GC-MS as O-trimethylsilyl-oxime derivatives.

    PubMed

    Balogh, Réka; Szarka, Szabolcs; Béni, Szabolcs

    2015-11-10

    Human milk oligosaccharides possess various biological functions by protecting the infant from several bacterial and viral infections, modulating the immune system, serving as prebiotics and also contributing to the brain development. Hence, huge effort is underway by manufacturers to produce infant formulas enriched with human milk oligosaccharides which could mimic its diverse biological role the most. For this purpose, quantification of the natural oligosaccharide composition of the human milk is a key task. This study reports a fit for purpose GC-MS method for the quantification of the TMS ether oxime derivatives of 2'-O-fucosyllactose and 3-O-fucosyllactose, the two most abundant trisaccharides in human milk. The EI fragmentation pattern of the linkage isomers is discussed in details, focusing also on specific fragment ions. The GC-MS method with external standard calibration was applied for the monitoring of concentration changes of the trisaccharides throughout the first week of lactation in human milks samples collected from two volunteers. The results showed high concentration of both 2'-FL (4525-6266μg/mL in donor A and 2694-3551μg/mL in donor B) and 3-FL (271-441μg/mL in donor A and 99-208μg/mL in donor B), while no significant change has been observed throughout the one-week lactation period. The presented GC-MS method can serve as a quality control technique for the infant formulas and also offers an alternative to existing chromatographic methods to investigate HMOs in milk samples. PMID:26291789

  4. Development and validation of an LC-ESI/MS/MS method with precolumn derivatization for the determination of betulin in rat plasma.

    PubMed

    Hu, Zhiwei; Guo, Na; Wang, Ziming; Liu, Yong; Wang, Yu; Ding, Weimin; Zhang, Dehui; Wang, Yang; Yan, Xiufeng

    2013-11-15

    Neutral pentacyclic triterpenes with only one or two hydroxyl groups, such as betulin, are not easily ionized by electrospray ionization (ESI). However, because betulin is reactive and neutral, derivatization may improve ionization efficiency. In the present study, the potency of different derivatization reagents was evaluated and p-toluenesulfonyl isocyanate (PTSI) was proven to be the optimal. The derivative generated by the reaction of betulin with PTSI was ionizable and fragmentable in the negative mode by liquid chromatography-electrospray ionization/tandem mass spectrometry (LC-ESI/MS/MS). Based on this chemical derivatization, an LC-ESI/MS/MS method was developed and validated for the quantification of betulin in rat plasma. The sample was extracted with ethyl acetate, derivatized with PTSI, separated on an ACQ UPLC BEH phenyl column, and analyzed in negative multiple reaction monitoring (MRM) mode. The calibration curve was linear over the betulin concentration range 2.5-200ng/mL. The lower limit of quantification was 2.5ng/mL. The inter- and intra-day accuracy and precision were within ±15%. Betulin recoveries were 86.7% or higher at three quality control levels (5, 50, and 160ng/mL). This validated method was subsequently applied to a pharmacokinetic study of betulin in rat plasma after oral administration.

  5. Quantification of Bufadienolides in Bryophyllum pinnatum Leaves and Manufactured Products by UHPLC-ESIMS/MS.

    PubMed

    Oufir, Mouhssin; Seiler, Christina; Gerodetti, Manon; Gerber, Julia; Fürer, Karin; Mennet-von Eiff, Monica; Elsas, Siegward-M; Brenneisen, Rudolf; von Mandach, Ursula; Hamburger, Matthias; Potterat, Olivier

    2015-08-01

    A quantitative assay for determination of the main bufadienolides bersaldegenin-1-acetate (1), bersaldegenin-3-acetate (2), bryophyllin A (3), and bersaldegenin-1,3,5-orthoacetate (4) in Bryophyllum pinnatum leaves and manufactured products was developed and validated. The assay involved extraction by pressurised liquid extraction, followed by quantification by ultrahigh performance liquid chromatography-tandem mass spectroscopy. The ultrahigh performance liquid chromatography-tandem mass spectroscopy method was applied to various batches of leaves harvested on several dates from plants grown at two locations (Brazil and Germany). In addition, press juices prepared from plants cultivated in Germany and Brazil were analysed. The total bufadienolide content ranged from 16.28 to 40.50 mg/100 g dry weight in leaves from plants grown in Brazil. The total content of these four bufadienolides was significantly lower in plants cultivated in Germany (3.78-12.49 mg/100 g dry weight, resp.). The total amounts of bufadienolides were 0.091-0.163 mg/100 mL and 0.89-1.16 mg/100 mL in press juices obtained from plants cultivated in Germany and Brazil, respectively. When analysing single leaves from individual plants, the content of bufadienolides was markedly higher in young leaves. For comparative purposes, the content of these bufadienolides was also determined in Bryophyllum daigremontianum and Bryophyllum tubiflorum. Bersaldegenin-1,3,5-orthoacetate (4) was predominant in the leaves of B. daigremontianum and in the stems of B. tubiflorum, while the leaves of B. tubiflorum contained very low amounts of 1-4. PMID:26132852

  6. Tandem DART™ MS Methods for Methadone Analysis in Unprocessed Urine.

    PubMed

    Beck, Rachel; Carter, Patrick; Shonsey, Erin; Graves, David

    2016-03-01

    Current methods of methadone analysis in untreated urine are traditionally limited to enzyme immunoassays (EIA) while confirmation techniques require specimen processing (i.e., sample clean-up) before analyzing by gas or liquid chromatography coupled with mass spectrometry (GC-MS or LC-MS-MS). EIA and traditional confirmation techniques can be costly and, at times inefficient. As an alternative approach, we present Direct Analysis in Real Time (DART™) coupled with both time-of-flight and triple quadrupole linear ion trap (Q-TRAP™) mass spectrometers for screening and confirming methadone in untreated urine specimens. These approaches require neither expensive kits nor sample clean-up for analysis. More importantly, the total combined analysis time for both screening and confirmation methods was <5 min per sample; in contrast to the 3-5 day process required by traditional EIA, GC-MS and LC-MS-MS techniques. To examine the fundamental protocol and its applicability for routine drug screening, studies were performed that included limits of detection, precision, selectivity and specificity, sample recovery and stability and method robustness. The methods described in this report were determined to be highly specific and selective; allowing for detection of methadone at 250 ng/mL, consistent with cutoffs for current EIA techniques (300 ng/mL). The results reported here demonstrate the DART™ MS platform provides rapid and selective methadone analysis and the potential for providing savings of both time and resources compared with current analysis procedures. PMID:26590378

  7. Analysis of Phosphonic Acids: Validation of Semi-Volatile Analysis by HPLC-MS/MS by EPA Method MS999

    SciTech Connect

    Owens, J; Vu, A; Koester, C

    2008-10-31

    The Environmental Protection Agency's (EPA) Region 5 Chicago Regional Laboratory (CRL) developed a method titled Analysis of Diisopropyl Methylphosphonate, Ethyl Hydrogen Dimethylamidophosphate, Isopropyl Methylphosphonic Acid, Methylphosphonic Acid, and Pinacolyl Methylphosphonic Acid in Water by Multiple Reaction Monitoring Liquid Chromatography/Tandem Mass Spectrometry: EPA Version MS999. 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 MS999 for analysis of the listed phosphonic acids and surrogates 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 MS999 can be determined.

  8. Comparative study of antioxidant properties and total phenolic content of the extracts of Humulus lupulus L. and quantification of bioactive components by LC-MS/MS and GC-MS.

    PubMed

    Önder, Ferah Cömert; Ay, Mehmet; Sarker, Satyajit D

    2013-11-01

    In this research, antioxidant activities of various extracts obtained from Humulus lupulus L. were compared by DPPH, ABTS, FRAP, and CUPRAC assays. The amount of total phenolic components determined by the Folin-Ciocalteu reagent was found to be highest for 25% aqueous ethanol (9079 ± 187.83 mg Ferulic acid equivalent/100 g extract) and methanol-1 (directly) (8343 ± 158.39 mg Ferulic acid equivalent/100 g extract) extracts. The n-hexane extract of H. lupulus exhibited the greatest with DPPH (14.95 ± 0.03 μg Trolox equivalent/g sample). The highest phenolic content in the ethanolic extract could be the major contributor to its highest CUPRAC activity (3.15 ± 0.44 mmol Trolox equivalent/g sample). Methanol-2 (n-hexane, acetone, and methanol) and methanol-3 (n-hexane, dichloromethane, ethylacetate, and methanol) extracts, respectively, exhibited the most potent ABTS (7.35 ± 0.03 mM Trolox equivalent) and FRAP (1.56 ± 0.35 mmol Fe(2+)/g sample) activities. Some of the components from the crude extracts were determined by LC-MS/MS and GC-MS analyses. Comparative screening of antioxidant activities of H. lupulus extracts and quantification of some major components by LC-MS/MS, qualitatively analysis of the reported ones which were optimal under negative ion SIM mode and coinjection, are going to be valuable for food and health applications. PMID:24079371

  9. Optimization of a QuEChERS based method by means of central composite design for pesticide multiresidue determination in orange juice by UHPLC-MS/MS.

    PubMed

    Rizzetti, Tiele M; Kemmerich, Magali; Martins, Manoel L; Prestes, Osmar D; Adaime, Martha B; Zanella, Renato

    2016-04-01

    In this study, different extraction procedures based on the QuEChERS method were compared for the multiresidue determination of pesticides in orange juice by ultra high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS). After choosing preliminary conditions, an experimental design was carried out with the variables C18, PSA, NaOH and CH3COONa to optimize the sample preparation step. The validation results of the validation were satisfactory, since the method presented recoveries between 70% and 118%, with RSD lower than 19% for spike levels between 10 and 100 μg L(-1). The method limit of detection (LOD) and limit of quantification (LOQ) ranged from 3.0 to 7.6 μg L(-1) and from 4.9 to 26 μg L(-1), respectively. The method developed was adequate for the determination of 74 pesticide residues in orange juice. PMID:26593461

  10. Optimization of a QuEChERS based method by means of central composite design for pesticide multiresidue determination in orange juice by UHPLC-MS/MS.

    PubMed

    Rizzetti, Tiele M; Kemmerich, Magali; Martins, Manoel L; Prestes, Osmar D; Adaime, Martha B; Zanella, Renato

    2016-04-01

    In this study, different extraction procedures based on the QuEChERS method were compared for the multiresidue determination of pesticides in orange juice by ultra high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS). After choosing preliminary conditions, an experimental design was carried out with the variables C18, PSA, NaOH and CH3COONa to optimize the sample preparation step. The validation results of the validation were satisfactory, since the method presented recoveries between 70% and 118%, with RSD lower than 19% for spike levels between 10 and 100 μg L(-1). The method limit of detection (LOD) and limit of quantification (LOQ) ranged from 3.0 to 7.6 μg L(-1) and from 4.9 to 26 μg L(-1), respectively. The method developed was adequate for the determination of 74 pesticide residues in orange juice.

  11. A novel method for the quantification of quinic acid in food using stable isotope dilution analysis.

    PubMed

    Erk, Thomas; Bergmann, Hannah; Richling, Elke

    2009-01-01

    Organic acids play an important role in the flavor and taste of plant-derived foods. Quinic acid (QA) is one of the major acids. In the past, several methods like HPLC/UV, GC, and capillary electrophoresis were used for identification and quantification of QA. For the first time, a novel, sensitive, and selective method for the quantification of QA in food using stable isotope dilution analysis with HPLC/MS/MS has been established. Uniformly labeled 13C-QA was used as a standard to reduce sample preparations and to overcome matrix and ionization effects. The method was used to determine the QA content of red wines, instant coffees, and cloudy apple juices. QA contents of instant coffees were 64.4 and 63.6 g/kg powder. The concentrations in red wines were 24.0 and 25.1 mg/L, and 1493.3 and 1705.2 mg/L in cloudy apple juices.

  12. Quantification of andrographolide sodium bisulphite in urine after intravenous injection to rats by LC-MS/MS.

    PubMed

    Zhang, Shuang-Qing; Chen, Xiang-Hong; Yu, Min; Sun, Xu; Li, Zuo-Gang

    2012-01-01

    A liquid chromatography-tandem mass spectrometry method for the determination of andrographolide sodium bisulphite (ASB) in rat urine was established and validated. To our knowledge, the analytical method is the first developed assay for the determination of ASB in urine samples. Dehydroandrographolide (DAG) was used as an internal standard. ASB and DAG were separated on a C(18) column and detected at negative ion mode using the mass transitions of m/z 413.2→287.2 and m/z 331.2→303.3, respectively. Good linearity was obtained over the range of 50-5000 ng/mL and the correlation coefficient was better than 0.99. The intra- and inter-day accuracy at all levels fell in the ranges of 85.8-101.4% and 87.9-97.5%, and the intra- and inter-day precision (RSD) were in the ranges of 4.3-11.2% and 8.4-13.3%, respectively. The recovery ranged from 96.1% to 98.3% and the matrix effects from 96.2% to 98.1%. Good stability was found under tested conditions. The method was successfully applied to a urinary excretion study of ASB in rats following intravenous administration of 80 mg/kg ASB.

  13. LA-ICP-MS of magnetite: Methods and reference materials

    USGS Publications Warehouse

    Nadoll, P.; Koenig, A.E.

    2011-01-01

    Magnetite (Fe3O4) is a common accessory mineral in many geologic settings. Its variable geochemistry makes it a powerful petrogenetic indicator. Electron microprobe (EMPA) analyses are commonly used to examine major and minor element contents in magnetite. Laser ablation ICP-MS (LA-ICP-MS) is applicable to trace element analyses of magnetite but has not been widely employed to examine compositional variations. We tested the applicability of the NIST SRM 610, the USGS GSE-1G, and the NIST SRM 2782 reference materials (RMs) as external standards and developed a reliable method for LA-ICP-MS analysis of magnetite. LA-ICP-MS analyses were carried out on well characterized magnetite samples with a 193 nm, Excimer, ArF LA system. Although matrix-matched RMs are sometimes important for calibration and normalization of LA-ICP-MS data, we demonstrate that glass RMs can produce accurate results for LA-ICP-MS analyses of magnetite. Cross-comparison between the NIST SRM 610 and USGS GSE-1G indicates good agreement for magnetite minor and trace element data calibrated with either of these RMs. Many elements show a sufficiently good match between the LA-ICP-MS and the EMPA data; for example, Ti and V show a close to linear relationship with correlation coefficients, R2 of 0.79 and 0.85 respectively. ?? 2011 The Royal Society of Chemistry.

  14. A High-Throughput UHPLC-QqQ-MS Method for Polyphenol Profiling in Rosé Wines.

    PubMed

    Lambert, Marine; Meudec, Emmanuelle; Verbaere, Arnaud; Mazerolles, Gérard; Wirth, Jérémie; Masson, Gilles; Cheynier, Véronique; Sommerer, Nicolas

    2015-01-01

    A rapid, sensitive and selective analysis method using Ultra High Performance Liquid Chromatography coupled to triple-quadrupole Mass Spectrometry (UHPLC-QqQ-MS) has been developed for the quantification of polyphenols in rosé wines. The compound detection being based on specific MS transitions in Multiple Reaction Monitoring (MRM) mode, the present method allows the selective quantification of up to 152 phenolic and two additional non-phenolic wine compounds in 30 min without sample purification or pre-concentration, even at low concentration levels. This method was repeatably applied to a set of 12 rosé wines and thus proved to be suitable for high-throughput and large-scale metabolomics studies.

  15. A rapid and sensitive method for the simultaneous analysis of aliphatic and polar molecules containing free carboxyl groups in plant extracts by LC-MS/MS

    PubMed Central

    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

  16. Accurate Quantification of Selenoprotein P (SEPP1) in Plasma Using Isotopically Enriched Seleno-peptides and Species-Specific Isotope Dilution with HPLC Coupled to ICP-MS/MS.

    PubMed

    Deitrich, Christian L; Cuello-Nuñez, Susana; Kmiotek, Diana; Torma, Frank Attila; Del Castillo Busto, Maria Estela; Fisicaro, Paola; Goenaga-Infante, Heidi

    2016-06-21

    A novel strategy for the absolute quantification of selenium (Se) included in selenoprotein P (SEPP1), an important biomarker for human nutrition and disease, including diabetes and cancer, is presented here for the first time. It is based on the use of species-specific double isotope dilution mass spectrometry (SSIDA) in combination with HPLC-ICP-MS/MS for the determination of protein bound Se down to the peptide level in a complex plasma matrix with a total content of Se of 105.5 μg kg(-1). The method enabled the selective Se speciation analysis of human plasma samples without the need of extensive cleanup or preconcentration steps as required for traditional protein mass spectrometric approaches. To assess the method accuracy, two plasma reference materials, namely, BCR-637 and SRM1950, for which literature data and a reference value for SEPP1 have been reported, were analyzed using complementary hyphenated methods and the species-specific approach developed in this work. The Se mass fractions obtained via the isotopic ratios (78)Se/(76)Se and (82)Se/(76)Se for each of the Se-peptides, namely, ENLPSLCSUQGLR (ENL) and AEENITESCQUR (AEE) (where U is SeCys), were found to agree within 2.4%. A relative expanded combined uncertainty (k = 2) of 5.4% was achieved for a Se (as SEPP1) mass fraction of approximately 60 μg kg(-1). This work represents a systematic approach to the accurate quantitation of plasma SEPP1 at clinical levels using SSIDA quantification. Such methodology will be invaluable for the certification of reference materials and the provision of reference values to clinical measurements and clinical trials. PMID:27108743

  17. Simple validated LC-MS/MS method for the determination of atropine and scopolamine in plasma for clinical and forensic toxicological purposes.

    PubMed

    Koželj, Gordana; Perharič, Lucija; Stanovnik, Lovro; Prosen, Helena

    2014-08-01

    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.

  18. Assessment methods for angiogenesis and current approaches for its quantification

    PubMed Central

    AlMalki, Waleed Hassan; Shahid, Imran; Mehdi, Abeer Yousaf; Hafeez, Muhammad Hassan

    2014-01-01

    Angiogenesis is a physiological process which describes the development of new blood vessels from the existing vessels. It is a common and the most important process in the formation and development of blood vessels, so it is supportive in the healing of wounds and granulation of tissues. The different assays for the evaluation of angiogenesis have been described with distinct advantages and some limitations. In order to develop angiogenic and antiangiogenic techniques, continuous efforts have been resulted to give animal models for more quantitative analysis of angiogenesis. Most of the studies on angiogenic inducers and inhibitors rely on various models, both in vitro, in vivo and in ova, as indicators of efficacy. The angiogenesis assays are very much helpful to test efficacy of both pro- and anti- angiogenic agents. The development of non-invasive procedures for quantification of angiogenesis will facilitate this process significantly. The main objective of this review article is to focus on the novel and existing methods of angiogenesis and their quantification techniques. These findings will be helpful to establish the most convenient methods for the detection, quantification of angiogenesis and to develop a novel, well tolerated and cost effective anti-angiogenic treatment in the near future. PMID:24987169

  19. Thallium lung-to-heart quantification: three methods of evaluation

    SciTech Connect

    Harler, M.B.; Mahoney, M.; Bartlett, B.; Patel, K.; Turbiner, E.

    1986-12-01

    Lung-to-heart quantification, when used in conjunction with visual assessment of /sup 201/Tl stress test images, has been found useful in diagnosing cardiac dysfunction. The authors evaluated three methods of quantification in terms of inter- and intraobserver variability and reproducibility. Fifty anterior /sup 201/Tl stress images were quantified by each of the following methods: Method A (sum region), which involved one region of interest (ROI) in the measurement of pulmonary activity relative to that of the myocardium; Method B (count density), which required two ROIs, the lung-to-heart ratio being dependent on count density; and Method C (maximum pixel), which used the gray scale of the computer to determine the most intense pixels in the lung field and myocardium. Statistical evaluation has shown that the three methods assess clinical data equally well. Method C was found to be most reproducible in terms of inter- and intraobserver variability followed by Methods A and B. Although nearly equivalent in terms of statistics, the three methods possess inherent differences and therefore should not be used interchangeably without conversion factors.

  20. A novel HPLC-MS/MS method for the simultaneous determination of astemizole and its major metabolite in dog or monkey plasma and application to pharmacokinetics.

    PubMed

    Back, Hyun-moon; Lee, Jong-Hwa; Chae, Jung-woo; Song, Byungjeong; Seo, Joung-Wook; Yun, Hwi-yeol; Kwon, Kwang-il

    2015-10-10

    Astemizole (AST), a second-generation antihistamine, is metabolized to desmethyl astemizole (DEA), and although it has been removed from the market for inducing QT interval prolongation, it has reemerged as a potential anticancer and antimalarial agent. This report describes a novel high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method for simultaneously determining the concentrations of AST and DEA in beagle dog and cynomolgus monkey plasma with simple preparation method and short retention time. Prior to HPLC analyses, the plasma samples were extracted with simple liquid-liquid extraction method. The isocratic mobile phase was 0.025% trifluoroacetic acid (TFA dissolved in acetonitrile) and 20 mM ammonium acetate (94:6) at a flow rate of 0.25 mL/min and diphenhydramine used as internal standard. In MS/MS analyses, precursor ions of the analytes were optimized as protonated molecular ions: [M+H](+). The lower limit of quantification of astemizole was 2.5 ng/mL in both species and desmethyl astemizole were 7.5 ng/mL and 10 ng/mL in dog and monkey plasma, respectively. The accuracy, precision, and stability of the method were in accordance with FDA guidelines for the validation of bioanalytical methods. Finally this validated method was successfully applied to a pharmacokinetic study in dogs and monkeys after oral administration of 10 mg/kg AST. PMID:26037160

  1. A novel HPLC-MS/MS method for the simultaneous determination of astemizole and its major metabolite in dog or monkey plasma and application to pharmacokinetics.

    PubMed

    Back, Hyun-moon; Lee, Jong-Hwa; Chae, Jung-woo; Song, Byungjeong; Seo, Joung-Wook; Yun, Hwi-yeol; Kwon, Kwang-il

    2015-10-10

    Astemizole (AST), a second-generation antihistamine, is metabolized to desmethyl astemizole (DEA), and although it has been removed from the market for inducing QT interval prolongation, it has reemerged as a potential anticancer and antimalarial agent. This report describes a novel high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method for simultaneously determining the concentrations of AST and DEA in beagle dog and cynomolgus monkey plasma with simple preparation method and short retention time. Prior to HPLC analyses, the plasma samples were extracted with simple liquid-liquid extraction method. The isocratic mobile phase was 0.025% trifluoroacetic acid (TFA dissolved in acetonitrile) and 20 mM ammonium acetate (94:6) at a flow rate of 0.25 mL/min and diphenhydramine used as internal standard. In MS/MS analyses, precursor ions of the analytes were optimized as protonated molecular ions: [M+H](+). The lower limit of quantification of astemizole was 2.5 ng/mL in both species and desmethyl astemizole were 7.5 ng/mL and 10 ng/mL in dog and monkey plasma, respectively. The accuracy, precision, and stability of the method were in accordance with FDA guidelines for the validation of bioanalytical methods. Finally this validated method was successfully applied to a pharmacokinetic study in dogs and monkeys after oral administration of 10 mg/kg AST.

  2. Glycidyl fatty acid esters in food by LC-MS/MS: method development.

    PubMed

    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

  3. Comparison of MS/MS Methods for Characterization of DNA/cisplatin Adducts

    PubMed Central

    Xu, Zhe; Shaw, Jared B.; Brodbelt, Jennifer S.

    2012-01-01

    The development of activation/dissociation techniques such as ultraviolet photodissociation (UVPD), infrared multiphoton dissociation (IRMPD) and electron transfer dissociation (ETD) as alternatives to collision induced dissociation (CID) has extended the range of strategies for characterizing biologically relevant molecules. Here, we describe a comprehensive comparison of CID, IRMPD, UVPD, ETD and hybrid processes termed ETcaD and ET-IRMPD (and analogous hybrid methods in the negative mode NETcaD and NET-IRMPD) for generating sequence specific-fragment ions and allowing adduction sites to be pinpointed for DNA/cisplatin adducts. Among the six MS/MS methods, the numerous products generated by the IRMPD and UVPD techniques resulted in the most specific and extensive backbone cleavages. We conclude that IRMPD and UVPD methods generally offer the best characteristics for pinpointing the cisplatin adduction sites in the fragment-rich spectra. PMID:23264150

  4. Development of a LC-MS/MS method for the simultaneous screening of seven water-soluble vitamins in processing semi-coarse wheat flour products.

    PubMed

    Nurit, Eric; Lyan, Bernard; Piquet, Agnès; Branlard, Gérard; Pujos-Guillot, Estelle

    2015-05-01

    Wheat is the second largest crop cultivated around the world and constitutes a major part of the daily diet in Europe. It is therefore important to determine the content of micronutrient in wheat and wheat-based food products to define the contribution of wheat-based foods to the nutrition of the consumers. The aim of the present work was to develop a simple and rapid method based on liquid chromatography tandem mass spectrometry (LC-MS/MS) for the simultaneous determination of seven water-soluble vitamins in various wheat-based food materials. The vitamins present in the test material were separated in less than 15 min by using a reverse-phase C18 column, and analyzed by positive ion electrospray selected reaction monitoring MS/MS. The MS response for all the vitamins was linear over the working range (0.05 to 9 μg/mL) with correlation coefficients ranging between 0.991 and 1. Limits of quantification in the different food materials ranged from 0.09 to 3.5 μg/g. Intra-day and inter-day precision were found satisfactory. The developed method was applied for the simultaneous analysis of the water-soluble vitamin natural content of different semi-coarse wheat flours and in their corresponding baking products.

  5. UNE-EN ISO/IEC 17025:2005-accredited method for the determination of pesticide residues in fruit and vegetable samples by LC-MS/MS.

    PubMed

    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). PMID:20730643

  6. Why do Ladybugs Smell Bad? In-vivo Quantification of Odorous Insect Kairomones with SPME and Multidimensional GC-MS-Olfactometry

    NASA Astrophysics Data System (ADS)

    Cai, Lingshuang; Koziel, Jacek A.; O'Neal, Matthew E.

    2009-05-01

    Winemakers, small fruit growers, and homeowners are concerned with noxious compounds released by multicolored Asian ladybird beetles (Harmonia axyridis, Coleoptera: Coccinellidae). New method based on headspace solid phase microextraction (HS-SPME) coupled with multidimensional gas chromatography mass spectrometry—olfactometry (MDGC-MS-O) system was developed for extraction, isolation and simultaneous identification of compounds responsible for the characteristic odor of live H. axyridis. Four methoxypyrazines (MPs) were identified in headspace volatiles of live H. axyridis as those responsible for the characteristic odor: 2, 5-dimethy1-3-methoxypyrazine (DMMP), 2-isopropy1-3-methoxypyrazine (IPMP), 2-sec-buty1-3-methoxypyrazine (SBMP), and 2-isobuty1-3-methoxypyrazine (IBMP). To the best of our knowledge this is the first report of H. axyridis releasing DMMP and the first report of this compound being a component of the H. axyridis characteristic odor. Quantification of three MPs (IPMP, SBMP and IBMP) emitted from live H. axyridis were performed using external calibration with HS-SPME and direct injections. A linear relationship (R2>0.9958 for all 3 MPs) between MS response and concentration of standard was observed over a concentration range from 0.1 ng L-1 to 0.05 μg L-1 for HS-SPME-GC-MS. The method detection limits (MDL) based on multidimensional GC-MS approach for three MPs were estimated to be between 0.020 ng L-1. to 0.022 ng L-1. This methodology is applicable for in vivo determination of odor-causing chemicals associated with emissions of volatiles from insects.

  7. Why do Ladybugs Smell Bad? In-vivo Quantification of Odorous Insect Kairomones with SPME and Multidimensional GC-MS-Olfactometry

    SciTech Connect

    Cai Lingshuang; Koziel, Jacek A.; O'Neal, Matthew E.

    2009-05-23

    Winemakers, small fruit growers, and homeowners are concerned with noxious compounds released by multicolored Asian ladybird beetles (Harmonia axyridis, Coleoptera: Coccinellidae). New method based on headspace solid phase microextraction (HS-SPME) coupled with multidimensional gas chromatography mass spectrometry--olfactometry (MDGC-MS-O) system was developed for extraction, isolation and simultaneous identification of compounds responsible for the characteristic odor of live H. axyridis. Four methoxypyrazines (MPs) were identified in headspace volatiles of live H. axyridis as those responsible for the characteristic odor: 2, 5-dimethy1-3-methoxypyrazine (DMMP), 2-isopropy1-3-methoxypyrazine (IPMP), 2-sec-buty1-3-methoxypyrazine (SBMP), and 2-isobuty1-3-methoxypyrazine (IBMP). To the best of our knowledge this is the first report of H. axyridis releasing DMMP and the first report of this compound being a component of the H. axyridis characteristic odor. Quantification of three MPs (IPMP, SBMP and IBMP) emitted from live H. axyridis were performed using external calibration with HS-SPME and direct injections. A linear relationship (R{sup 2}>0.9958 for all 3 MPs) between MS response and concentration of standard was observed over a concentration range from 0.1 ng L{sup -1} to 0.05 {mu}g L{sup -1} for HS-SPME-GC-MS. The method detection limits (MDL) based on multidimensional GC-MS approach for three MPs were estimated to be between 0.020 ng L{sup -1}. to 0.022 ng L{sup -1}. This methodology is applicable for in vivo determination of odor-causing chemicals associated with emissions of volatiles from insects.

  8. Development of a validated RP-LC/ESI-MS-MS method for separation, identification and determination of related substances of tamsulosin in bulk drugs and formulations.

    PubMed

    Nageswara Rao, R; Kumar Talluri, M V N; Narasa Raju, A; Shinde, Dhananjay D; Ramanjaneyulu, G S

    2008-01-01

    A reversed-phase high performance liquid chromatographic (RP-HPLC) method for evaluation of purity of tamsulosin in bulk drugs and pharmaceuticals was developed. The separation was accomplished on an Inertsil C(18) column using 10 mM ammonium acetate: acetonitrile as a mobile phase in a gradient elution mode. A photodiode array detector set at 280 nm was used for detection. The impurities were identified by ESI-MS-MS. The detection limits were 0.06-0.11 microg/ml. The method was validated with respect to accuracy, precision, linearity, ruggedness and limits of detection and quantification. It finds application not only for monitoring the reactions during the process development but also on quality assurance of tamsulosin.

  9. Method validation and analysis of nine dithiocarbamates in fruits and vegetables by LC-MS/MS.

    PubMed

    Schmidt, B; Christensen, H B; Petersen, A; Sloth, J J; Poulsen, M E

    2013-01-01

    An analytical method for separation and quantitative determination of nine dithiocarbamates (DTCs) in fruits and vegetables by using LC-MS/MS was developed, validated and applied to samples purchased in local supermarkets. The nine DTCs were ziram, ferbam, thiram, maneb, zineb, nabam, metiram, mancozeb and propineb. Validation parameters of mean recovery for two matrices at two concentration levels, relative repeatability (RSDr), relative within-laboratory reproducibility (RSDR) and LOD were obtained for the nine DTCs. The results from the analysis of fruits and vegetables served as the basis for an exposure assessment within the given commodities and a risk assessment by comparing the calculated exposure to the acceptable daily intake and acute reference dose for various exposure groups. The analysis indicated positive findings of DTCs in apples, pears, plums, table grapes, papaya and broccoli at concentrations ranging from 0.03 mg/kg to 2.69 mg/kg expressed as the equivalent amount of CS2. None of the values exceeded the Maximum residue level (MRL) set by the European Union, and furthermore, it was not possible to state whether illegal use had taken place or not, because a clear differentiation between the various DTCs in the LC-MS/MS analysis was lacking. The exposure and risk assessment showed that only for maneb in the case of apples and apple juice, the acute reference dose was exceeded for infants in the United Kingdom and for children in Germany, respectively. PMID:23799268

  10. Ultra-performance liquid chromatography mass spectrometry and sensitive bioassay methods for quantification of posaconazole plasma concentrations after oral dosing.

    PubMed

    Rochat, Bertrand; Pascual, Andres; Pesse, Benoît; Lamoth, Frédéric; Sanglard, Dominique; Decosterd, Laurent A; Bille, Jacques; Marchetti, Oscar

    2010-12-01

    Posaconazole (POS) is a new antifungal agent for prevention and therapy of mycoses in immunocompromised patients. Variable POS pharmacokinetics after oral dosing may influence efficacy: a trough threshold of 0.5 μg/ml has been recently proposed. Measurement of POS plasma concentrations by complex chromatographic techniques may thus contribute to optimize prevention and management of life-threatening infections. No microbiological analytical method is available. The objective of this study was to develop and validate a new simplified ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method and a sensitive bioassay for quantification of POS over the clinical plasma concentration range. The UPLC-MS/MS equipment consisted of a triple quadrupole mass spectrometer, an electrospray ionization (ESI) source, and a C(18) analytical column. The Candida albicans POS-hypersusceptible mutant (MIC of 0.002 μg/ml) Δcdr1 Δcdr2 Δflu Δmdr1 Δcan constructed by targeted deletion of multidrug efflux transporters and calcineurin genes was used for the bioassay. POS was extracted from plasma by protein precipitation with acetonitrile-methanol (75%/25%, vol/vol). Reproducible standard curves were obtained over the range 0.014 to 12 (UPLC-MS/MS) and 0.028 to 12 μg/ml (bioassay). Intra- and interrun accuracy levels were 106% ± 2% and 103% ± 4% for UPLC-MS/MS and 102% ± 8% and 104% ± 1% for bioassay, respectively. The intra- and interrun coefficients of variation were 7% ± 4% and 7% ± 3% for UPLC-MS/MS and 5% ± 3% and 4% ± 2% for bioassay, respectively. An excellent correlation between POS plasma concentrations measured by UPLC-MS/MS and bioassay was found (concordance, 0.96). In 26 hemato-oncological patients receiving oral POS, 27/69 (39%) trough plasma concentrations were lower than 0.5 μg/ml. The UPLC-MS/MS method and sensitive bioassay offer alternative tools for accurate and precise quantification of the plasma concentrations in patients

  11. UPLC-UV-MS(E) analysis for quantification and identification of major carotenoid and chlorophyll species in algae.

    PubMed

    Fu, Weiqi; Magnúsdóttir, Manuela; Brynjólfson, Sigurður; Palsson, Bernhard Ø; Paglia, Giuseppe

    2012-12-01

    A fast method for quantification and identification of carotenoid and chlorophyll species utilizing liquid chromatography coupled with UV detection and mass spectrometry has been demonstrated and validated for the analysis of algae samples. This method allows quantification of targeted pigments and identification of unexpected compounds, providing isomers separation, UV detection, accurate mass measurements, and study of fragment ions for structural elucidation in a single run. This is possible using parallel alternating low- and high-energy collision spectral acquisition modes, which provide accurate mass full scan chromatograms and accurate mass high-energy chromatograms. Here, it is shown how this approach can be used to confirm carotenoid and chlorophyll species by identification of key diagnostic fragmentations during high-energy mode. The developed method was successfully applied for the analysis of Dunaliella salina samples during defined red LED lighting growth conditions, identifying 37 pigments including 19 carotenoid species and 18 chlorophyll species, and providing quantification of 7 targeted compounds. Limit of detections for targeted pigments ranged from 0.01 ng/mL for lutein to 0.24 ng/mL for chlorophyll a. Inter-run precision ranged for of 3 to 24 (RSD%) while inter-run inaccuracy ranged from -17 to 11. PMID:23052878

  12. A confirmatory HPLC-MS/MS method for ten synthetic corticosteroids in bovine urines.

    PubMed

    Savu, S R; Silvestro, L; Haag, A; Sörgel, F

    1996-12-01

    In the present study, an HPLC-MS/MS method to confirm, in bovine urine, the most common synthetic corticosteroids illegally used as growth promoters in livestock breeding will be presented. An API III-Plus (PE-Sciex) triple quadrupole mass spectrometer, interfaced by means of an atmospheric pressure chemical ionization source to the HPLC system, was used. Urine samples were treated with a sulfatase-glucuronidase mixture to cleave the drug-conjugates and then extracted on C18 disposable columns. LC separations were performed on a reversed-phase C18 column with ammonium acetate 0.1 M/acetonitrile (60/40, v/v) as mobile phase. Detection was performed in multiple reaction monitoring mode, negative ions, selecting fragmentations characteristic of 10 corticosteroids used more frequently. Good results, in terms of sensitivity and specificity have been obtained for nine corticosteroids that can be analyzed in the same HPLC run; the limits of sensitivity achieved were 0.05-1.0 ng/ml in urine. Only a more polar corticosteroid, required a different HPLC separation. Practical applications of this technique to real samples proved that it is an effective method to confirm the illegal use of corticosteroids as growth promoter in animal. In comparison with the chemical GC-MS methods the simpler sample preparation and the faster time of analysis permit a considerable increase of sample testing per day without compromising on analytical sensitivity and specificity.

  13. Development of high-throughput multi-residue method for non-steroidal anti-inflammatory drugs monitoring in swine muscle by LC-MS/MS.

    PubMed

    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. PMID:27268755

  14. Simultaneous, rapid, and sensitive quantification of 8-hydroxy-2'-deoxyguanosine and cotinine in human urine by on-line solid-phase extraction LC-MS/MS: correlation with tobacco exposure biomarkers NNAL.

    PubMed

    Chen, Chao-Yu; Jhou, Yu-Ting; Lee, Hui-Ling; Lin, Yu-Wen

    2016-09-01

    Cigarette smoke can increase oxidative DNA damage. The main component in cigarette smoke is nicotine. Nicotine is metabolized to cotinine, which can be regarded as a biomarker for measuring exposure to tobacco smoke. A sensitive, simple, and robust method based on on-line solid-phase extraction liquid chromatography with tandem mass spectrometry (on-line SPE LC-MS/MS) has been developed and validated for the simultaneous determination of 8-OHdG and cotinine. The matrix effects of 8-OHdG and cotinine were measured at 97.1 and 91.7 %, with values for CV at 4.4 and 4.2 %, respectively. The limits of detection of 8-OHdG and cotinine were 10.0 and 5.5 pg mL(-1), and the limits of quantification were 40.0 and 20.0 pg mL(-1), respectively. The total run time was 12 min. We quantified 8-OHdG and cotinine in the urine of 80 male subjects. The results showed the levels of 8-OHdG and cotinine in smokers were significantly higher than that in non-smokers. Furthermore, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol and its glucuronide conjugate (defined as total NNAL) are the nitrosation metabolites of nicotine. In this study, urinary levels of 8-OHdG and cotinine were well correlated with urinary levels of total NNAL. This is also the first study to focus on the future risk of oxidative stress from exposure to cigarette smoke based on the relationship between 8-OHdG levels, cotinine levels, and total NNAL concentrations in the urine of humans. Graphical Abstract On-line SPE LC-MS/MS for the simultaneous determination of 8-OHdG and cotinine in human urine. PMID:27422644

  15. Quantification of brain endocannabinoid levels: methods, interpretations and pitfalls

    PubMed Central

    Buczynski, Matthew W; Parsons, Loren H

    2010-01-01

    Endocannabinoids play an important role in a diverse range of neurophysiological processes including neural development, neuroimmune function, synaptic plasticity, pain, reward and affective state. This breadth of influence and evidence for altered endocannabinoid signalling in a variety of neuropathologies has fuelled interest in the accurate quantification of these lipids in brain tissue. Established methods for endocannabinoid quantification primarily employ solvent-based lipid extraction with further sample purification by solid phase extraction. In recent years in vivo microdialysis methods have also been developed for endocannabinoid sampling from the brain interstitial space. However, considerable variability in estimates of endocannabinoid content has led to debate regarding the physiological range of concentrations present in various brain regions. This paper provides a critical review of factors that influence the quantification of brain endocannabinoid content as determined by lipid extraction from bulk tissue and by in vivo microdialysis. A variety of methodological issues are discussed including analytical approaches, endocannabinoid extraction and purification, post-mortem changes in brain endocannabinoid content, cellular reactions to microdialysis probe implantation and caveats related to lipid sampling from the extracellular space. The application of these methods for estimating brain endocannabinoid content and the effects of endocannabinoid clearance inhibition are discussed. The benefits, limitations and pitfalls associated with each approach are emphasized, with an eye toward the appropriate interpretation of data gathered by each method. This article is part of a themed issue on Cannabinoids. To view the editorial for this themed issue visit http://dx.doi.org/10.1111/j.1476-5381.2010.00831.x PMID:20590555

  16. Volatile organic silicon compounds in biogases: development of sampling and analytical methods for total silicon quantification by ICP-OES.

    PubMed

    Chottier, Claire; Chatain, Vincent; Julien, Jennifer; Dumont, Nathalie; Lebouil, David; Germain, Patrick

    2014-01-01

    Current waste management policies favor biogases (digester gases (DGs) and landfill gases (LFGs)) valorization as it becomes a way for energy politics. However, volatile organic silicon compounds (VOSiCs) contained into DGs/LFGs severely damage combustion engines and endanger the conversion into electricity by power plants, resulting in a high purification level requirement. Assessing treatment efficiency is still difficult. No consensus has been reached to provide a standardized sampling and quantification of VOSiCs into gases because of their diversity, their physicochemical properties, and the omnipresence of silicon in analytical chains. Usually, samplings are done by adsorption or absorption and quantification made by gas chromatography-mass spectrometry (GC-MS) or inductively coupled plasma-optical emission spectrometry (ICP-OES). In this objective, this paper presents and discusses the optimization of a patented method consisting in VOSiCs sampling by absorption of 100% ethanol and quantification of total Si by ICP-OES.

  17. Volatile Organic Silicon Compounds in Biogases: Development of Sampling and Analytical Methods for Total Silicon Quantification by ICP-OES

    PubMed Central

    Julien, Jennifer; Dumont, Nathalie; Lebouil, David; Germain, Patrick

    2014-01-01

    Current waste management policies favor biogases (digester gases (DGs) and landfill gases (LFGs)) valorization as it becomes a way for energy politics. However, volatile organic silicon compounds (VOSiCs) contained into DGs/LFGs severely damage combustion engines and endanger the conversion into electricity by power plants, resulting in a high purification level requirement. Assessing treatment efficiency is still difficult. No consensus has been reached to provide a standardized sampling and quantification of VOSiCs into gases because of their diversity, their physicochemical properties, and the omnipresence of silicon in analytical chains. Usually, samplings are done by adsorption or absorption and quantification made by gas chromatography-mass spectrometry (GC-MS) or inductively coupled plasma-optical emission spectrometry (ICP-OES). In this objective, this paper presents and discusses the optimization of a patented method consisting in VOSiCs sampling by absorption of 100% ethanol and quantification of total Si by ICP-OES. PMID:25379538

  18. Dissipation kinetics and degradation mechanism of amicarbazone in soil revealed by a reliable LC-MS/MS method.

    PubMed

    Dong, Maofeng; Han, Wei; Ediage, Emmanuel Njumbe; Fan, Liangxiu; Tang, Hongxia; Wang, Weimin; Han, Lijun; Zhao, Zhihui; Song, Weiguo; Han, Zheng

    2015-11-01

    A sensitive and reliable analytical method was developed for simultaneous determination of amicarbazone (AMZ) and its two major metabolites including desamino amicarbazone (DA) and isopropyl-2-hydroxy-DA-amicarbazone (Ipr-2-OH-DA-AMZ) in soil for the first time. Targeted analytes were extracted and purified using a modified quick, easy, cheap, effective, rugged, and safe (QuEChERS) procedure, and then analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) with a total run time of 9 min. The established approach was extensively validated by determining the linearity (R (2) ≥ 0.99), recovery (84-96 ), sensitivity (limits of quantification at 5-10 μg kg(-1)), and precision (RSDs ≤12 %). Based on the methodological advances, the subsequent dissipation kinetics and degradation mechanism of amicarbazone in soil were thoroughly investigated in an illumination incubator. As revealed, AMZ was easily degraded with the half-lives of 13.9-19.7 days in soil. Field trial results of AMZ (40 g a.i. ha(-1)) in Shanghai showed that the residues of AMZ and its metabolite Ipr-2-OH-DA-AMZ decreased from 0.505 mg kg(-1) (day 50) to 0.038 mg kg(-1) (day 365) and from 0.099 mg kg(-1) (day 50) to 0.028 mg kg(-1) (day 365), respectively, while the content of DA increased from 0.097 mg kg(-1) (day 50) to 0.245 mg kg(-1) (day 365). This study provided valuable data to understand the toxicity of AMZ and substantially promote its safe application to protect environment and human health. PMID:26139399

  19. Development and validation of a selective, sensitive and stability indicating UPLC-MS/MS method for rapid, simultaneous determination of six process related impurities in darunavir drug substance.

    PubMed

    A, Vijaya Bhaskar Reddy; Yusop, Zulkifli; Jaafar, Jafariah; Aris, Azmi B; Majid, Zaiton A; Umar, Khalid; Talib, Juhaizah

    2016-09-01

    In this study a sensitive and selective gradient reverse phase UPLC-MS/MS method was developed for the simultaneous determination of six process related impurities viz., Imp-I, Imp-II, Imp-III, Imp-IV, Imp-V and Imp-VI in darunavir. The chromatographic separation was performed on Acquity UPLC BEH C18 (50 mm×2.1mm, 1.7μm) column using gradient elution of acetonitrile-methanol (80:20, v/v) and 5.0mM ammonium acetate containing 0.01% formic acid at a flow rate of 0.4mL/min. Both negative and positive electrospray ionization (ESI) modes were operated simultaneously using multiple reaction monitoring (MRM) for the quantification of all six impurities in darunavir. The developed method was fully validated following ICH guidelines with respect to specificity, linearity, limit of detection (LOD), limit of quantification (LOQ), accuracy, precision, robustness and sample solution stability. The method was able to quantitate Imp-I, Imp-IV, Imp-V at 0.3ppm and Imp-II, Imp-III, and Imp-VI at 0.2ppm with respect to 5.0mg/mL of darunavir. The calibration curves showed good linearity over the concentration range of LOQ to 250% for all six impurities. The correlation coefficient obtained was >0.9989 in all the cases. The accuracy of the method lies between 89.90% and 104.60% for all six impurities. Finally, the method has been successfully applied for three formulation batches of darunavir to determine the above mentioned impurities, however no impurity was found beyond the LOQ. This method is a good quality control tool for the trace level quantification of six process related impurities in darunavir during its synthesis.

  20. An HPLC-MS/MS method for simultaneous determination of the active metabolites of febuxostat (67M-1, 67M-2 and 67M-4) in human plasma.

    PubMed

    Xie, Huiru; Wang, Zhijun; Deng, Ke; Jiang, Xuehua; Wang, Ling; Lv, Guoyu

    2014-11-01

    An HPLC-MS/MS method for simultaneously determination of the active metabolites (67M-1, 67M-2 and 67M-4) in human plasma using clopidogrel as the internal standard was developed and validated. The compounds were extracted by protein precipitation using acetonitrile and separated using a C8 column by a gradient elution with the mobile phase consisting of acetonitrile (containing 0.1% formic acid) and 0.1% formic acid. Quantification was performed using multiple reaction monitoring in positive mode with m/z transitions of 333.1-261.0, 333.1-261.0, 347.0-261.0 and 322.2-184.1 for 67M-1, 67M-2, 67M-4 and clopidogrel (Internal Standard), respectively. This method was validated in terms of specificity, linearity, precision, accuracy, and stability. The lower limit of quantification of this method was 0.5 ng/mL and the calibration curve was linear over the concentration range of 0.5-150 ng/mL. The intra- and inter-run precision was less than 11.67% and 8.64%, respectively, with the accuracy between 98.33% and 108.38%. The samples were stable under all the tested conditions. This method has been successfully applied to the pharmacokinetic study of febuxostat in healthy Chinese volunteers following oral administration of 40 mg and 80 mg febuxostat.

  1. A fast and effective routine method based on HS-SPME-GC-MS/MS for the analysis of organotin compounds in biota samples.

    PubMed

    Noventa, Seta; Barbaro, Jvan; Formalewicz, Malgorzata; Gion, Claudia; Rampazzo, Federico; Brusà, Rossella Boscolo; Gabellini, Massimo; Berto, Daniela

    2015-02-01

    This work validated an automated, fast, and low solvent- consuming methodology suited for routine analysis of tributyltin (TBT) and degradation products (dibutyltin, DBT; monobutyltin, MBT) in biota samples. The method was based on the headspace solid-phase microextraction methodology (HS-SPME), coupled with gas chromatographic separation and tandem mass-spectrometry (GC-MS/MS). The effectiveness of the matrix-matched signal ratio external calibration was tested for quantification purposes. The exclusion of matrix influences in the calibration curves proved the suitability of this versatile quantification method. The method detection limits obtained were of 3 ng Sn g(-1) dw for all the analytes. The analysis of references materials showed satisfying accuracy under optimum calibration conditions (% recovery between 87-111%; |Z-scores|<2). The repeatability RSD% and intra-laboratory reproducibility RSD% were lower than 9.6% and 12.6%, respectively. The work proved the remarkable analytical performances of the method and its high potential for routine application in monitoring organotin compounds (OTC). PMID:25597804

  2. The parallel reaction monitoring method contributes to a highly sensitive polyubiquitin chain quantification

    SciTech Connect

    Tsuchiya, Hikaru; Tanaka, Keiji Saeki, Yasushi

    2013-06-28

    Highlights: •The parallel reaction monitoring method was applied to ubiquitin quantification. •The ubiquitin PRM method is highly sensitive even in biological samples. •Using the method, we revealed that Ufd4 assembles the K29-linked ubiquitin chain. -- Abstract: Ubiquitylation is an essential posttranslational protein modification that is implicated in a diverse array of cellular functions. Although cells contain eight structurally distinct types of polyubiquitin chains, detailed function of several chain types including K29-linked chains has remained largely unclear. Current mass spectrometry (MS)-based quantification methods are highly inefficient for low abundant atypical chains, such as K29- and M1-linked chains, in complex mixtures that typically contain highly abundant proteins. In this study, we applied parallel reaction monitoring (PRM), a quantitative, high-resolution MS method, to quantify ubiquitin chains. The ubiquitin PRM method allows us to quantify 100 attomole amounts of all possible ubiquitin chains in cell extracts. Furthermore, we quantified ubiquitylation levels of ubiquitin-proline-β-galactosidase (Ub-P-βgal), a historically known model substrate of the ubiquitin fusion degradation (UFD) pathway. In wild-type cells, Ub-P-βgal is modified with ubiquitin chains consisting of 21% K29- and 78% K48-linked chains. In contrast, K29-linked chains are not detected in UFD4 knockout cells, suggesting that Ufd4 assembles the K29-linked ubiquitin chain(s) on Ub-P-βgal in vivo. Thus, the ubiquitin PRM is a novel, useful, quantitative method for analyzing the highly complicated ubiquitin system.

  3. A novel screening method for 64 new psychoactive substances and 5 amphetamines in blood by LC-MS/MS and application to real cases.

    PubMed

    Vaiano, Fabio; Busardò, Francesco P; Palumbo, Diego; Kyriakou, Chrystalla; Fioravanti, Alessia; Catalani, Valeria; Mari, Francesco; Bertol, Elisabetta

    2016-09-10

    Identification and quantification of new psychoactive substances (NPS), both in biological and non-biological samples, represent a hard challenge for forensic toxicologists. NPS are increasingly emerging on illegal drug market. Many cases of co-consumption of NPS and other substances have also been reported. Hence, the development of analytical methods aiming at the detection of a broad-spectrum of compounds (NPS and "traditional" drugs) could be helpful. In this paper, a fully validated screening method in blood for the simultaneous detection of 69 substances, including 64 NPS (28 synthetic cannabinoids, 19 synthetic cathinones, 5 phenethylamines, 3 indanes, 2 piperazines, 2 tryptamines, 2 phencyclidine, methoxetamine, ketamine and its metabolite) and 5 amphetamines (amphetamine, methamphetamine, MDMA, MDA, 3,4-methylenedioxy-N-ethylamphetamine - MDEA-) by a dynamic multiple reaction monitoring analysis through liquid chromatography - tandem mass spectrometry (LC-MS/MS) is described. This method is very fast, easy to perform and cheap as it only requires the deproteinization of 200μL of blood sample with acetonitrile. The chromatographic separation is achieved with a C18 column. The analysis is very sensitive, with limits of quantification ranging from 0.1 to 0.5ng/mL. The method is linear from 1 to 100ng/mL and the coefficient of determination (R(2)) was always above 0.9900. Precision and accuracy were acceptable at any quality control level and recovery efficiency range was 72-110%. Matrix effects did not negatively affect the analytical sensitivity. This method was successfully applied to three real cases, allowing identification and quantification of: mephedrone and methamphetamine (post-mortem); ketamine, MDMA and MDA (post-mortem); AB-FUBINACA (ante-mortem). PMID:27490334

  4. A novel screening method for 64 new psychoactive substances and 5 amphetamines in blood by LC-MS/MS and application to real cases.

    PubMed

    Vaiano, Fabio; Busardò, Francesco P; Palumbo, Diego; Kyriakou, Chrystalla; Fioravanti, Alessia; Catalani, Valeria; Mari, Francesco; Bertol, Elisabetta

    2016-09-10

    Identification and quantification of new psychoactive substances (NPS), both in biological and non-biological samples, represent a hard challenge for forensic toxicologists. NPS are increasingly emerging on illegal drug market. Many cases of co-consumption of NPS and other substances have also been reported. Hence, the development of analytical methods aiming at the detection of a broad-spectrum of compounds (NPS and "traditional" drugs) could be helpful. In this paper, a fully validated screening method in blood for the simultaneous detection of 69 substances, including 64 NPS (28 synthetic cannabinoids, 19 synthetic cathinones, 5 phenethylamines, 3 indanes, 2 piperazines, 2 tryptamines, 2 phencyclidine, methoxetamine, ketamine and its metabolite) and 5 amphetamines (amphetamine, methamphetamine, MDMA, MDA, 3,4-methylenedioxy-N-ethylamphetamine - MDEA-) by a dynamic multiple reaction monitoring analysis through liquid chromatography - tandem mass spectrometry (LC-MS/MS) is described. This method is very fast, easy to perform and cheap as it only requires the deproteinization of 200μL of blood sample with acetonitrile. The chromatographic separation is achieved with a C18 column. The analysis is very sensitive, with limits of quantification ranging from 0.1 to 0.5ng/mL. The method is linear from 1 to 100ng/mL and the coefficient of determination (R(2)) was always above 0.9900. Precision and accuracy were acceptable at any quality control level and recovery efficiency range was 72-110%. Matrix effects did not negatively affect the analytical sensitivity. This method was successfully applied to three real cases, allowing identification and quantification of: mephedrone and methamphetamine (post-mortem); ketamine, MDMA and MDA (post-mortem); AB-FUBINACA (ante-mortem).

  5. β-lactam antibiotics residues analysis in bovine milk by LC-ESI-MS/MS: a simple and fast liquid-liquid extraction method.

    PubMed

    Jank, L; Hoff, R B; Tarouco, P C; Barreto, F; Pizzolato, T M

    2012-01-01

    This study presents the development and validation of a simple method for the detection and quantification of six β-lactam antibiotics residues (ceftiofur, penicillin G, penicillin V, oxacillin, cloxacillin and dicloxacillin) in bovine milk using a fast liquid-liquid extraction (LLE) for sample preparation, followed by liquid chromatography-electrospray-tandem mass spectrometry (LC-MS/MS). LLE consisted of the addition of acetonitrile to the sample, followed by addition of sodium chloride, centrifugation and direct injection of an aliquot into the LC-MS/MS system. Separation was performed in a C(18) column, using acetonitrile 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. Limits of detection ranged from 0.4 (penicillin G and penicillin V) to 10.0 ng ml(-1) (ceftiofur), and linearity was achieved. The decision limit (CCα), detection capability (CCβ), accuracy, inter- and intra-day repeatability of the method are reported.

  6. Development and validation of sensitive and rapid UPLC-MS/MS method for quantitative determination of daclatasvir in human plasma: Application to a bioequivalence study.

    PubMed

    Rezk, Mamdouh R; Bendas, Ehab R; Basalious, Emad B; Karim, Iman A

    2016-09-01

    A rapid and sensitive UPLC-MS/MS method was developed and validated for determination of daclatasvir (DAC) in human plasma using sofosbuvir (SOF) as an internal standard (IS). The Xevo TQD LC-MS/MS was operated under the multiple-reaction monitoring mode using electrospray ionization. Precipitation with acetonitrile was used in sample preparation. The prepared samples were chromatographed on Acquity UPLC HSS C18 (50×2.1mm, 1.8μm) column by pumping 10mM ammonium formate (pH 3.5) and acetonitrile in an isocratic mode at a flow rate of 0.30ml/min. Method validation was performed as per the FDA guidelines and the standard curves were found to be linear in the range of 5-4000ng/ml for DAC. The intra-day and inter-day precision and accuracy results were within the acceptable limits. A very short run time of 1.2min made it possible to analyze more than 500 human plasma samples per day. The wider range of quantification of DAC allowed the applicability of the developed method for its determination in a bioequivalence study in human volunteers. PMID:27232152

  7. Validation of a SPE-LC-MS/MS method for the determination of ketamine and norketamine in micropulverized hair after a single IV dose.

    PubMed

    Barreto, Adriana S; Brant, Vinnicius F; Spinelli, Eliani; Rodrigues, Silvana V

    2016-10-15

    A SPE-LC-MS/MS method for the determination of ketamine (KET) and norketamine (NKET) was developed and validated. Extensive pulverization (25min at 25Hz) of previously cooled samples (5min in liquid nitrogen) allowed for extraction in a phosphate buffer (pH 6) solution after 10min vortex agitation at room temperature, simplifying the coupling of the extraction to an effective mixed-mode SPE (solid phase extraction) clean-up procedure. The extraction optimization was performed with samples fortified by drug incorporation according to a published procedure involving incubation of blank matrices for 16days. The method was validated for selectivity, matrix effect, linearity, LLQ (lower limit of quantification), precision, accuracy, recovery, carryover and stability after preparation and has proven to be accurate and reliable within a range of 0.02-10ng/mg for KET and 0.04-4ng/mg for NKET, meeting proposed KET cutoffs for discrimination from chronic use. In addition, the method was sensitive enough to detect the drugs after unique small (1mg/kg) intravenous doses received by patients submitted to general anesthesia before surgical procedures. Ketamine levels varied from 0.060 to 0.111ng/mg and norketamine was positive (MS/MS detection is probably due to the simple but very effective proposed procedure.

  8. Development and validation of a highly sensitive LC-MS/MS method for quantitation of bivalirudin in human plasma: application to a human pharmacokinetic study.

    PubMed

    Chai, Dong; Wang, Rui; Bai, Nan; Cai, Yun; Liang, Beibei

    2013-12-01

    A sensitive, specific and simple LC-MS/MS method was developed for the identification and quantification of bivalirudin in human plasma using diazepam as an internal standard (IS). The API-4000 LC-MS/MS was operated under multiple-reaction monitoring mode using electrospray ionization. The sample preparation consisted of an easy protein precipitation sample pretreatment with methanol. Chromatographic separation was achieved on a Zorbax Eclipse plus C18 100 × 2.1 mm column with a mobile phase of water-methanol-0.1% formic acid. The analytes were detected with a triple quadrupole Quantum Access with positive ionization. Ions monitored in the multiple-reaction monitoring mode were m/z 1091 → 650 for bivalirudin (at 2.70 min) and m/z 285 → 193 for diazepam (at 3.85 min). The developed method was validated in human plasma with a lower limit of quantitation of 20 µg/L for bivalirudin. A linear response function was established for the range of concentrations 20-10,000 µg/L (r > 0.998) for bivalirudin. The intra- and inter-day precision values for bivalirudin met the acceptance criteria as per US Food and Drug Administration guidelines. Bivalirudin was stable in the battery of stability studies, viz. bench-top, freeze-thaw cycles and long-term stability. The developed assay method was applied to an intravenous administration study in humans. PMID:23893840

  9. 'Dilute-and-shoot' RSLC-MS-MS method for fast detection of nerve and vesicant chemical warfare agent metabolites in urine.

    PubMed

    Rodin, Igor; Braun, Arcady; Stavrianidi, Andrey; Baygildiev, Timur; Shpigun, Oleg; Oreshkin, Dmitry; Rybalchenko, Igor

    2015-01-01

    A sensitive screening method based on fast liquid chromatography tandem mass-spectrometry (RSLC-MS-MS) has shown the feasibility of separation and detection of low concentration β-lyase metabolites of sulfur mustard and of nerve agent phosphonic acids in urine. The analysis of these compounds is of interest because they are specific metabolites of the chemical warfare agents (CWAs), sulfur mustard (HD), sarin (GB), soman (GD), VX and Russian VX (RVX). The 'dilute-and-shoot' RSLC-MS-MS method provides a sensitive and direct approach for determining CWA exposure in non-extracted non-derivatized samples from urine. Chromatographic separation of the metabolites was achieved using a reverse phase column with gradient mobile phases consisting of 0.5% formic acid in water and acetonitrile. Identification and quantification of species were achieved using electrospray ionization-tandem mass-spectrometry monitoring two precursor-to-product ion transitions for each compound. The method demonstrates linearity over at least two orders of magnitude and had detection limits of 0.5 ng/mL in urine.

  10. 'Dilute-and-shoot' RSLC-MS-MS method for fast detection of nerve and vesicant chemical warfare agent metabolites in urine.

    PubMed

    Rodin, Igor; Braun, Arcady; Stavrianidi, Andrey; Baygildiev, Timur; Shpigun, Oleg; Oreshkin, Dmitry; Rybalchenko, Igor

    2015-01-01

    A sensitive screening method based on fast liquid chromatography tandem mass-spectrometry (RSLC-MS-MS) has shown the feasibility of separation and detection of low concentration β-lyase metabolites of sulfur mustard and of nerve agent phosphonic acids in urine. The analysis of these compounds is of interest because they are specific metabolites of the chemical warfare agents (CWAs), sulfur mustard (HD), sarin (GB), soman (GD), VX and Russian VX (RVX). The 'dilute-and-shoot' RSLC-MS-MS method provides a sensitive and direct approach for determining CWA exposure in non-extracted non-derivatized samples from urine. Chromatographic separation of the metabolites was achieved using a reverse phase column with gradient mobile phases consisting of 0.5% formic acid in water and acetonitrile. Identification and quantification of species were achieved using electrospray ionization-tandem mass-spectrometry monitoring two precursor-to-product ion transitions for each compound. The method demonstrates linearity over at least two orders of magnitude and had detection limits of 0.5 ng/mL in urine. PMID:25326204

  11. A multi-analyte LC-MS/MS method for the analysis of 23 mycotoxins in different sorghum varieties: the forgotten sample matrix.

    PubMed

    Njumbe Ediage, Emmanuel; Van Poucke, Christof; De Saeger, Sarah

    2015-06-15

    An LC-MS/MS method was developed and validated for the detection and quantification of 23 mycotoxins in different varieties of sorghum. The method performance characteristics were as follows: suitable linearity ranges for all 23 mycotoxins with p-value >0.05; limits of detection (1.2-50 μg/kg), limits of quantification (2.5-100 μg/kg), repeatability (RSDr, 7-22%), intermediate precision (RSDR, 14-44%) and apparent recovery (0.2-11%, expressed as bias). The method was applied to analyze 10 samples obtained from retail shops in Belgium (n=8) and Germany (n=2). Nine of the 10 samples (90%) were positive for the following mycotoxins: aflatoxin B1 (50 μg/kg), alternariol monomethyl ether (

  12. [Determination method of ultra-high-intensity sweetener, advantame, in processed foods by HPLC and LC-MS/MS].

    PubMed

    Kobayashi, Miki; Terada, Hisaya; Nakajima, Masahiro

    2015-01-01

    A simple method using HPLC and LC-MS/MS was developed for the determination of ultra-high-intensity sweetener, advantame, in processed foods. Advantame was extracted by dialysis, and cleaned up on a Sep-Pak Plus C18 cartridge, then determined by HPLC and LC-MS/MS. The recoveries from 5 kinds of processed foods fortified at the levels of 0.001 g/kg and 0.01 g/kg were 64.1-89.9% (RSD 0.9-6.9%) by HPLC and 68.8-99.9% (RSD 0.8-4.9%) by LC-MS/MS. The quantitation limit was 0.0004 g/kg by HPLC and 0.00004 g/kg by LC-MS/MS.

  13. A validated GC/MS method for the determination of amisulpride in whole blood.

    PubMed

    Papoutsis, Ioannis; Rizopoulou, Anna; Nikolaou, Panagiota; Pistos, Constantinos; Spiliopoulou, Chara; Athanaselis, Sotiris

    2014-02-01

    A sensitive GC/MS method for the determination of amisulpride in whole blood was developed, optimized and validated. Sample preparation included solid-phase extraction using HF Bond Elut C18 cartridges and further derivatization with heptafluorobutyric anhydride (HFBA). The limits of detection and quantification were 3.00 and 10.0 μg/L, respectively. The calibration curves were linear up to 1000 μg/L (R(2)≥0.991). Absolute recovery ranged from 94.2 to 101%. Accuracy was found to be between -8.7 and 1.9% and imprecision was less than 10.0%. The developed method covers the generally accepted therapeutic range but it can also cover levels above them. This makes our method suitable for the determination of amisulpride not only for clinical purposes on psychiatric patients, but also during the investigation of forensic cases where amisulpride is involved.

  14. Simultaneous detection of antibiotics and other drug residues in the dissolved and particulate phases of water by an off-line SPE combined with on-line SPE-LC-MS/MS: Method development and application.

    PubMed

    Tlili, Ines; Caria, Giovanni; Ouddane, Baghdad; Ghorbel-Abid, Ibtissem; Ternane, Riadh; Trabelsi-Ayadi, Malika; Net, Sopheak

    2016-09-01

    Due to their widespread use in human and animal healthcare, antibiotics and other drug residues are ubiquitous in the aquatic environment. Given their potential impacts on ecosystem functioning and public health, the quantification of environmental drug residues has become a necessity. Various analysis techniques have been found to be suitable for reliable detection of such compounds. However, quantification can be difficult because these compounds are present at trace or ultra-trace levels. Consequently, the accuracy of environmental analyses depends on both the efficiency and the robustness of the extraction and quantification method. In this work, an off-line solid-phase extraction (SPE) combined with on-line SPE-LC-MS/MS was applied to the simultaneous extraction and quantification of 26 pharmaceutical products, including 18 antibiotics, dissolved in a water phase. Optimal conditions were determined and then applied to assess the contamination level of the targeted drug residues in water collected from four sites in Northern France: a river, the input and output of an aerated lagoon, and a wastewater treatment plant. Drug residues associated with suspended solid matter (SSM) were also quantified in this work using pressurized liquid extraction (PLE) combined with an on-line SPE-LC-MS/MS system in order to complete an assessment of the degree of total background pollution.

  15. Simultaneous detection of antibiotics and other drug residues in the dissolved and particulate phases of water by an off-line SPE combined with on-line SPE-LC-MS/MS: Method development and application.

    PubMed

    Tlili, Ines; Caria, Giovanni; Ouddane, Baghdad; Ghorbel-Abid, Ibtissem; Ternane, Riadh; Trabelsi-Ayadi, Malika; Net, Sopheak

    2016-09-01

    Due to their widespread use in human and animal healthcare, antibiotics and other drug residues are ubiquitous in the aquatic environment. Given their potential impacts on ecosystem functioning and public health, the quantification of environmental drug residues has become a necessity. Various analysis techniques have been found to be suitable for reliable detection of such compounds. However, quantification can be difficult because these compounds are present at trace or ultra-trace levels. Consequently, the accuracy of environmental analyses depends on both the efficiency and the robustness of the extraction and quantification method. In this work, an off-line solid-phase extraction (SPE) combined with on-line SPE-LC-MS/MS was applied to the simultaneous extraction and quantification of 26 pharmaceutical products, including 18 antibiotics, dissolved in a water phase. Optimal conditions were determined and then applied to assess the contamination level of the targeted drug residues in water collected from four sites in Northern France: a river, the input and output of an aerated lagoon, and a wastewater treatment plant. Drug residues associated with suspended solid matter (SSM) were also quantified in this work using pressurized liquid extraction (PLE) combined with an on-line SPE-LC-MS/MS system in order to complete an assessment of the degree of total background pollution. PMID:27151499

  16. [Analytical method of hydramethylnon in agricultural products by LC-MS/MS].

    PubMed

    Takahashi, Kunihiko; Matsumoto, Ryuji; Nemoto, Satoru; Matsuda, Rieko

    2011-01-01

    A simple determination method of hydramethylnon in agricultural products by liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed. The sample was homogenized with phosphoric acid and extracted with acetone. An aliquot of crude extract was re-extracted with hexane and sat. NaCl solution. In the case of tea leaf, solidification processing using ammonium chloride and phosphoric acid was performed before re-extraction with hexane. Clean-up was performed using a silica-gel mini column (500 mg). The LC separation was performed on a C18 column using methanol-water (8 : 2) containing 10 mM ammonium acetate as the mobile phase and MS detection with positive ion electrospray ionization. The calibration curve was linear between 0.002-0.2 µg/mL of hydramethylnon. Recoveries (n=5) of hydromethylnon from 10 kinds of agricultural products fortified at 0.01 µg/g (0.05 µg/g for pineapple) were 82-110%, and their relative standard deviations were 2-12%.

  17. LC-MS/MS screening method for designer amphetamines, tryptamines, and piperazines in serum.

    PubMed

    Wohlfarth, Ariane; Weinmann, Wolfgang; Dresen, Sebastian

    2010-04-01

    Since the late 1990s and early 2000s, derivatives of well-known designer drugs as well as new psychoactive compounds have been sold on the illicit drug market and have led to intoxications and fatalities. The LC-MS/MS screening method presented covers 31 new designer drugs as well as cathinone, methcathinone, phencyclidine, and ketamine which were included to complete the screening spectrum. All but the last two are modified molecular structures of amphetamine, tryptamine, or piperazine. Among the amphetamine derivatives are cathinone, methcathinone, 3,4-DMA, 2,5-DMA, DOB, DOET, DOM, ethylamphetamine, MDDMA, 4-MTA, PMA, PMMA, 3,4,5-TMA, TMA-6 and members of the 2C group: 2C-B, 2C-D, 2C-H, 2C-I, 2C-P, 2C-T-2, 2C-T-4, and 2C-T-7. AMT, DPT, DiPT, MiPT, DMT, and 5MeO-DMT are contained in the tryptamine group, BZP, MDBP, TFMPP, mCPP, and MeOPP in the piperazine group. Using an Applied Biosystems LC-MS/MS API 365 TurboIonSpray it is possible to identify all 35 substances. After addition of internal standards and mixed-mode solid-phase extraction the analytes are separated using a Synergi Polar RP column and gradient elution with 1 mM ammonium formate and methanol/0.1% formic acid as mobile phases A and B. Data acquisition is performed in MRM mode with positive electro spray ionization. The assay is selective for all tested substances. Limits of detection were determined by analyzing S/N-ratios and are between 1.0 and 5.0 ng/mL. Matrix effects lie between 65% and 118%, extraction efficiencies range from 72% to 90%.

  18. Development and validation of an LC-MS/MS method for determination of p-phenylenediamine and its metabolites in blood samples.

    PubMed

    Mohamed, Khaled M; Cromarty, Duncan; Steenkamp, Vanessa

    2015-08-01

    In some developing countries, p-phenylenediamine (PPD) is used in combination with Henna as hair dye or skin decoration. A sensitive LC-MS/MS method was developed and validated for the simultaneous determination of p-phenylenediamine (PPD) and its metabolites N-acetyl-p-phenylenediamine (MAPPD) and N,N-diacetyl-p-phenylenediamine (DAPPD) in human blood. Acetanilide was used as an internal standard (IS). The LC-MS/MS was operated under multiple reaction-monitoring mode using the electrospray positive ionization technique. The transition ions m/z 109→92, m/z 151→92, m/z 193→92, and m/z 136→77 were selected for the quantification of PPD, MAPPD, DAPPD, and IS, respectively. The linear range was 10-2000ng/mL for all the compounds. The absolute recoveries were 51.94, 56.20 and 54.88% for PPD, MAPPD and DAPPD, respectively. Intra- and inter-assay imprecision were lower than 14% (RSD), and the bias of the assay was lower than 15% for all the compounds. The stability studies demonstrated that critical degradation for PPD in blood samples and autosampler occurred after 6h, while MAPPD and DAPPD were stable in blood samples and the autosampler up to 48h and 24h, respectively. This newly developed method allows for the detection of PPD and its metabolites in blood samples in the clinical and forensic setting. PMID:26073911

  19. Method for factor analysis of GC/MS data

    DOEpatents

    Van Benthem, Mark H; Kotula, Paul G; Keenan, Michael R

    2012-09-11

    The method of the present invention provides a fast, robust, and automated multivariate statistical analysis of gas chromatography/mass spectroscopy (GC/MS) data sets. The method can involve systematic elimination of undesired, saturated peak masses to yield data that follow a linear, additive model. The cleaned data can then be subjected to a combination of PCA and orthogonal factor rotation followed by refinement with MCR-ALS to yield highly interpretable results.

  20. Quantitation of brinzolamide in dried blood spots by a novel LC-QTOF-MS/MS method.

    PubMed

    Foivas, Anargyros; Malenović, Anđelija; Kostić, Nađa; Božić, Marija; Knežević, Miroslav; Loukas, Yannis L; Dotsikas, Yannis

    2016-02-01

    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.

  1. A surrogate accelerated multicanonical Monte Carlo method for uncertainty quantification

    NASA Astrophysics Data System (ADS)

    Wu, Keyi; Li, Jinglai

    2016-09-01

    In this work we consider a class of uncertainty quantification problems where the system performance or reliability is characterized by a scalar parameter y. The performance parameter y is random due to the presence of various sources of uncertainty in the system, and our goal is to estimate the probability density function (PDF) of y. We propose to use the multicanonical Monte Carlo (MMC) method, a special type of adaptive importance sampling algorithms, to compute the PDF of interest. Moreover, we develop an adaptive algorithm to construct local Gaussian process surrogates to further accelerate the MMC iterations. With numerical examples we demonstrate that the proposed method can achieve several orders of magnitudes of speedup over the standard Monte Carlo methods.

  2. [Comparison of methods for quantification of MVOC in indoor environments].

    PubMed

    Fischer, G; Möller, M; Gabrio, T; Palmgren, U; Keller, R; Richter, H; Dott, W; Paul, R

    2005-01-01

    For several years now, MVOC have been regarded as indicators for microbial growth in indoor environments. Until now, a direct correlation between the occurrence of microfungi and MVOC could not be shown in scientific investigations. One reason may be that different analytical methods were applied, and moreover they were not validated sufficiently. The present investigation aimed to test the comparability of both methods (Tenax adsorption/thermal desorption; charcoal adsorption/elution). It turned out that with both methods comparable results can be achieved if the technical handling of the calibration is standardized to a wide extent. Thus, highest demands have to be made on quality assurance. Therefore, it is necessary to formulate technical regulations for the quantification of MVOC.

  3. Development and application of a QuEChERS-based extraction method for the analysis of 55 pesticides in the bivalve Scrobicularia plana by GC-MS/MS.

    PubMed

    Cruzeiro, Catarina; Rodrigues-Oliveira, Nádia; Velhote, Susana; Pardal, Miguel Ângelo; Rocha, Eduardo; Rocha, Maria João

    2016-05-01

    A method for quantitative determination of 55 pesticides in a bivalve matrix was established, based on QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) extraction and using gas chromatography (GC)-ion trap (IT) mass spectrometry (MS/MS). Accomplishing the European SANCO guidelines, this method was validated using 5 g of homogenized soft tissue, allowing the quantification of pesticides at ng/g of wet weight (ww). Quantification limits and recovery rates ranged from 0.33 to 10.3 μg/L and from 78 to 119 %, respectively. As an important mollusc, not only from an ecological perspective but also for food consumption, the peppery furrow shell (Scrobicularia plana) was sampled at three strategical sites (Ria Formosa Lagoon, in the south of Portugal) during 2012-2013, over six campaigns. A total of 2160 animals were pooled by place and sex. No statistical differences were found among sites or between sexes. Forty percent of the sampled pools were above quantification limits, reaching total annual average concentrations of ∑800 ng/g ww. Additionally, 83 % of the selected compounds showed concentrations above the legal limits set by the European Directive 2013/39/EU. In conclusion, the applied method was successful and proved that bivalves were contaminated by the selected pesticides. In future work, this methodology can be used to monitor body burdens and obtain data for predicting impacts in shellfish consumers. Graphical Abstract Resume of pesticides extraction and analyses process from S. plana. PMID:27032408

  4. Rapid quantification method for Legionella pneumophila in surface water.

    PubMed

    Wunderlich, Anika; Torggler, Carmen; Elsässer, Dennis; Lück, Christian; Niessner, Reinhard; Seidel, Michael

    2016-03-01

    World-wide legionellosis outbreaks caused by evaporative cooling systems have shown that there is a need for rapid screening methods for Legionella pneumophila in water. Antibody-based methods for the quantification of L. pneumophila are rapid, non-laborious, and relatively cheap but not sensitive enough for establishment as a screening method for surface and drinking water. Therefore, preconcentration methods have to be applied in advance to reach the needed sensitivity. In a basic test, monolithic adsorption filtration (MAF) was used as primary preconcentration method that adsorbs L. pneumophila with high efficiency. Ten-liter water samples were concentrated in 10 min and further reduced to 1 mL by centrifugal ultrafiltration (CeUF). The quantification of L. pneumophila strains belonging to the monoclonal subtype Bellingham was performed via flow-based chemiluminescence sandwich microarray immunoassays (CL-SMIA) in 36 min. The whole analysis process takes 90 min. A polyclonal antibody (pAb) against L. pneumophila serogroup 1-12 and a monoclonal antibody (mAb) against L. pneumophila SG 1 strain Bellingham were immobilized on a microarray chip. Without preconcentration, the detection limit was 4.0 × 10(3) and 2.8 × 10(3) CFU/mL determined by pAb and mAb 10/6, respectively. For samples processed by MAF-CeUF prior to SMIA detection, the limit of detection (LOD) could be decreased to 8.7 CFU/mL and 0.39 CFU/mL, respectively. A recovery of 99.8 ± 15.9% was achieved for concentrations between 1-1000 CFU/mL. The established combined analytical method is sensitive for rapid screening of surface and drinking water to allow fast hygiene control of L. pneumophila. PMID:26873217

  5. Stochastic methods for uncertainty quantification in radiation transport

    SciTech Connect

    Fichtl, Erin D; Prinja, Anil K; Warsa, James S

    2009-01-01

    The use of generalized polynomial chaos (gPC) expansions is investigated for uncertainty quantification in radiation transport. The gPC represents second-order random processes in terms of an expansion of orthogonal polynomials of random variables and is used to represent the uncertain input(s) and unknown(s). We assume a single uncertain input-the total macroscopic cross section-although this does not represent a limitation of the approaches considered here. Two solution methods are examined: The Stochastic Finite Element Method (SFEM) and the Stochastic Collocation Method (SCM). The SFEM entails taking Galerkin projections onto the orthogonal basis, which, for fixed source problems, yields a linear system of fully -coupled equations for the PC coefficients of the unknown. For k-eigenvalue calculations, the SFEM system is non-linear and a Newton-Krylov method is employed to solve it. The SCM utilizes a suitable quadrature rule to compute the moments or PC coefficients of the unknown(s), thus the SCM solution involves a series of independent deterministic transport solutions. The accuracy and efficiency of the two methods are compared and contrasted. The PC coefficients are used to compute the moments and probability density functions of the unknown(s), which are shown to be accurate by comparing with Monte Carlo results. Our work demonstrates that stochastic spectral expansions are a viable alternative to sampling-based uncertainty quantification techniques since both provide a complete characterization of the distribution of the flux and the k-eigenvalue. Furthermore, it is demonstrated that, unlike perturbation methods, SFEM and SCM can handle large parameter uncertainty.

  6. Rapid quantification method for Legionella pneumophila in surface water.

    PubMed

    Wunderlich, Anika; Torggler, Carmen; Elsässer, Dennis; Lück, Christian; Niessner, Reinhard; Seidel, Michael

    2016-03-01

    World-wide legionellosis outbreaks caused by evaporative cooling systems have shown that there is a need for rapid screening methods for Legionella pneumophila in water. Antibody-based methods for the quantification of L. pneumophila are rapid, non-laborious, and relatively cheap but not sensitive enough for establishment as a screening method for surface and drinking water. Therefore, preconcentration methods have to be applied in advance to reach the needed sensitivity. In a basic test, monolithic adsorption filtration (MAF) was used as primary preconcentration method that adsorbs L. pneumophila with high efficiency. Ten-liter water samples were concentrated in 10 min and further reduced to 1 mL by centrifugal ultrafiltration (CeUF). The quantification of L. pneumophila strains belonging to the monoclonal subtype Bellingham was performed via flow-based chemiluminescence sandwich microarray immunoassays (CL-SMIA) in 36 min. The whole analysis process takes 90 min. A polyclonal antibody (pAb) against L. pneumophila serogroup 1-12 and a monoclonal antibody (mAb) against L. pneumophila SG 1 strain Bellingham were immobilized on a microarray chip. Without preconcentration, the detection limit was 4.0 × 10(3) and 2.8 × 10(3) CFU/mL determined by pAb and mAb 10/6, respectively. For samples processed by MAF-CeUF prior to SMIA detection, the limit of detection (LOD) could be decreased to 8.7 CFU/mL and 0.39 CFU/mL, respectively. A recovery of 99.8 ± 15.9% was achieved for concentrations between 1-1000 CFU/mL. The established combined analytical method is sensitive for rapid screening of surface and drinking water to allow fast hygiene control of L. pneumophila.

  7. Isotope dilution quantification of ultratrace gamma-glutamyl-Se-methylselenocysteine species using HPLC with enhanced ICP-MS detection by ultrasonic nebulisation or carbon-loaded plasma.

    PubMed

    Goenaga Infante, Heidi; Ovejero Bendito, María del Carmen; Cámara, Carmen; Evans, Linda; Hearn, Ruth; Moesgaard, Sven

    2008-04-01

    A method for the accurate determination of ultratrace selenium species of relevance to cancer research, such as gamma-glutamyl-Se-methylselenocysteine (gamma-glutamyl-SeMC), using species-specific double isotope dilution analysis (IDA) with HPLC-ICP-MS is reported for the first time. The (77)Se-enriched gamma-glutamyl-SeMC spike was produced in-house by collecting the fraction at the retention time of the gamma-glutamyl-SeMC peak from a chromatographed aqueous extract of (77)Se-enriched yeast, pooling the collected fractions and freeze-drying the homogenate. The Se content of this spike was characterised using reverse isotope dilution mass spectrometry (IDMS) and the isotopic composition of this spike was checked prior to quantification of the natural abundance dipeptide species in garlic using speciated IDA. The extraction of the gamma-glutamyl-SeMC species in water was performed in a sonication bath for 2 h after adding an appropriate quantity of (77)Se-enriched gamma-glutamyl-SeMC to 50 mg of garlic to give optimal (78)Se/(77)Se and (82)Se/(77)Se ratios of 1.5 and 0.6, respectively. The effect of ultrasonic nebulisation, in comparison with the loading of the ICP with carbon (through the addition of methane gas on-line), on the detection of Se associated with gamma-glutamyl-SeMC using collision/reaction cell ICP-MS with hydrogen as collision gas was investigated. Sensitivity enhancements of approximately fourfold and twofold were achieved using USN and methane mixed plasma, respectively, in comparison with conventional nebulisation and conventional Ar ICP-MS. However, an approximately twofold improvement in the detection limit was achieved using both approaches (42 ng kg(-1) for (78)Se using peak height measurements). The use of species-specific IDMS enabled quantification of the dipeptide species at ng g(-1) levels (603 ng g(-1) Se) in the complex food matrix with a relative standard deviation (RSD, n = 4) of 4.5%, which was approximately half that obtained

  8. A simplified diphenylamine colorimetric method for growth quantification.

    PubMed

    Zhao, Youbao; Xiang, Sihai; Dai, Xida; Yang, Keqian

    2013-06-01

    Cell growth needs to be monitored in biological studies and bioprocess optimization. In special circumstances, such as microbial fermentations in media containing insoluble particles, accurate cell growth quantification is a challenge with current methods. Only the Burton method is applicable in such circumstances. The original Burton method was previously simplified by adopting a two-step sample pretreatment in perchloric acid procedure to eliminate the need for DNA extraction. Here, we further simplified the Burton method by replacing the previous two-step perchloric acid pretreatment with a new and one-step diphenylamine reagent pretreatment. The reliability and accuracy of this simplified method were assessed by measuring the biomass of four model microorganisms: Escherichia coli, Streptomyces clavuligerus, Saccharomyces cerevisiae, and Trichoderma reesei grown in normal media or those containing solid particles. The results demonstrate that this new simplified method performs comparably to the conventional methods, such as OD600 or the previously modified Burton method, and is much more sensitive than the dry weight method. Overall, the new method is simple, reliable, easy to perform, and generally applicable in most circumstances, and it reduces the operation time from more than 12 h (for the previously simplified Burton method) to about 2 h.

  9. Powerful GC-TOF-MS Techniques for Screening, Identification and Quantification of Halogenated Natural Products

    PubMed Central

    S. Haglund, Peter; Löfstrand, Karin; Siek, Kevin; Asplund, Lillemor

    2013-01-01

    Comprehensive two-dimensional gas chromatography/time-of-flight mass spectrometry (GC×GC TOFMS) and gas chromatography/high-resolution time-of-flight mass spectrometry (GC-HRT) were used to detect and identify halogenated natural products (HNPs) in tissue homogenate, in this case brominated analytes present in a marine snail. Two classes of brominated anthropogenic compounds, polybrominated diphenyl ethers (PBDEs) and brominated dibenzofurans, were analyzed for comparison. Following conventional preparation, the sample was analyzed using GC×GC-TOF-MS. Isotope ratio scripts were used to compile a list of putatively brominated analytes from amongst the thousands of features resolved in the two-dimensional chromatogram. The structured nature of the chromatogram was exploited to propose identifications for several classes of brominated compounds, and include additional candidates that fell marginally outside the script tolerances. The sample was subsequently analyzed by GC-HRT. The high-resolution mass spectral data confirmed many formula assignments, facilitated confident assignment of an alternate formula when an original proposal did not hold, and enabled unknown identification. Identified HNPs include hydroxylated and methoxylated PBDE analogs, polybrominated dibenzo-p-dioxins (PBDDs) and hydroxyl-PBDDs, permitting the environmental occurrence and fate of such compounds to be studied. PMID:24349937

  10. Uncertainty Quantification in State Estimation using the Probabilistic Collocation Method

    SciTech Connect

    Lin, Guang; Zhou, Ning; Ferryman, Thomas A.; Tuffner, Francis K.

    2011-03-23

    In this study, a new efficient uncertainty quantification technique, probabilistic collocation method (PCM) on sparse grid points is employed to enable the evaluation of uncertainty in state estimation. The PCM allows us to use just a small number of ensembles to quantify the uncertainty in estimating the state variables of power systems. By sparse grid points, the PCM approach can handle large number of uncertain parameters in power systems with relatively lower computational cost, when comparing with classic Monte Carlo (MC) simulations. The algorithm and procedure is outlined and we demonstrate the capability and illustrate the application of PCM on sparse grid points approach on uncertainty quantification in state estimation of the IEEE 14 bus model as an example. MC simulations have also been conducted to verify accuracy of the PCM approach. By comparing the results obtained from MC simulations with PCM results for mean and standard deviation of uncertain parameters, it is evident that the PCM approach is computationally more efficient than MC simulations.

  11. Importance of MS selectivity and chromatographic separation in LC-MS/MS-based methods when investigating pharmaceutical metabolites in water. Dipyrone as a case of study.

    PubMed

    Ibáñez, M; Gracia-Lor, E; Sancho, J V; Hernández, F

    2012-08-01

    Pharmaceuticals are emerging contaminants of increasing concern because of their presence in the aquatic environment and potential to reach drinking-water sources. After human and/or veterinary consumption, pharmaceuticals can be excreted in unchanged form, as the parent compound, and/or as free or conjugated metabolites. Determination of most pharmaceuticals and metabolites in the environment is commonly made by liquid chromatography (LC) coupled to mass spectrometry (MS). LC coupled to tandem MS is the technique of choice nowadays in this field. The acquisition of two selected reaction monitoring (SRM) transitions together with the retention time is the most widely accepted criterion for a safe quantification and confirmation assay. However, scarce attention is normally paid to the selectivity of the selected transitions as well as to the chromatographic separation. In this work, the importance of full spectrum acquisition high-resolution MS data using a hybrid quadrupole time-of-flight analyser and/or a suitable chromatographic separation (to reduce the possibility of co-eluting interferences) is highlighted when investigating pharmaceutical metabolites that share common fragment ions. For this purpose, the analytical challenge associated to the determination of metabolites of the widely used analgesic dipyrone (also known as metamizol) in urban wastewater is discussed. Examples are given on the possibilities of reporting false positives of dypirone metabolites by LC-MS/MS under SRM mode due to a wrong assignment of identity of the compounds detected. PMID:22899513

  12. DESI MS based screening method for phthalates in consumer goods.

    PubMed

    Schulz, Sabine; Wagner, Sebastian; Gerbig, Stefanie; Wächter, Herbert; Sielaff, Detlef; Bohn, Dieter; Spengler, Bernhard

    2015-05-21

    Phthalates are used as plasticizes in many everyday items, but some of them are known as hormone disruptors, being especially harmful during childhood. The European Union therefore restricted their application in children's toys and certain food packaging to 0.1%w. Due to the ever increasing number of plastic-containing consumer goods, rapid screening methods are needed to ensure and improve consumer safety in the future. In this study we evaluated the performance of desorption electrospray ionization (DESI) mass spectrometry (MS) for rapid quantitative screening of phthalates in toys. DESI allowed for direct surface sampling of the toys under atmospheric conditions with minimal sample preparation, while the high performance mass spectrometer used provided a high sensitivity and reliable identification via accurate mass measurements, high mass resolving power and MS/MS capabilities. External calibration curves for six banned phthalates (DBP, BBP, DEHP, DNOP, DINP and DIDP) were obtained from matrix-matched reference materials. Coefficients of determination were greater than 0.985, LOQs ranged from 0.02%w (DIDP) to 2.26%w (DINP) and the relative standard deviation of the calibration curve slope was less than 7.8% for intraday and 11.4% for interday comparison. The phthalate contents of eleven authentic samples were determined in a proof-of-concept approach using DESI MS and results were compared to those from confirmatory methods. The phthalate content was correctly assigned with relative deviations ranging from -20% to +10% for the majority of samples. Given further optimization and automation, DESI MS is likely to become a useful tool for rapid and accurate phthalate screening in the future. PMID:25827613

  13. DESI MS based screening method for phthalates in consumer goods.

    PubMed

    Schulz, Sabine; Wagner, Sebastian; Gerbig, Stefanie; Wächter, Herbert; Sielaff, Detlef; Bohn, Dieter; Spengler, Bernhard

    2015-05-21

    Phthalates are used as plasticizes in many everyday items, but some of them are known as hormone disruptors, being especially harmful during childhood. The European Union therefore restricted their application in children's toys and certain food packaging to 0.1%w. Due to the ever increasing number of plastic-containing consumer goods, rapid screening methods are needed to ensure and improve consumer safety in the future. In this study we evaluated the performance of desorption electrospray ionization (DESI) mass spectrometry (MS) for rapid quantitative screening of phthalates in toys. DESI allowed for direct surface sampling of the toys under atmospheric conditions with minimal sample preparation, while the high performance mass spectrometer used provided a high sensitivity and reliable identification via accurate mass measurements, high mass resolving power and MS/MS capabilities. External calibration curves for six banned phthalates (DBP, BBP, DEHP, DNOP, DINP and DIDP) were obtained from matrix-matched reference materials. Coefficients of determination were greater than 0.985, LOQs ranged from 0.02%w (DIDP) to 2.26%w (DINP) and the relative standard deviation of the calibration curve slope was less than 7.8% for intraday and 11.4% for interday comparison. The phthalate contents of eleven authentic samples were determined in a proof-of-concept approach using DESI MS and results were compared to those from confirmatory methods. The phthalate content was correctly assigned with relative deviations ranging from -20% to +10% for the majority of samples. Given further optimization and automation, DESI MS is likely to become a useful tool for rapid and accurate phthalate screening in the future.

  14. High-sensitivity MALDI-TOF MS quantification of anthrax lethal toxin for diagnostics and evaluation of medical countermeasures.

    PubMed

    Boyer, Anne E; Gallegos-Candela, Maribel; Quinn, Conrad P; Woolfitt, Adrian R; Brumlow, Judith O; Isbell, Katherine; Hoffmaster, Alex R; Lins, Renato C; Barr, John R

    2015-04-01

    Inhalation anthrax has a rapid progression and high fatality rate. Pathology and death from inhalation of Bacillus anthracis spores are attributed to the actions of secreted protein toxins. Protective antigen (PA) binds and imports the catalytic component lethal factor (LF), a zinc endoprotease, and edema factor (EF), an adenylyl cyclase, into susceptible cells. PA-LF is termed lethal toxin (LTx) and PA-EF, edema toxin. As the universal transporter for both toxins, PA is an important target for vaccination and immunotherapeutic intervention. However, its quantification has been limited to methods of relatively low analytic sensitivity. Quantification of LTx may be more clinically relevant than LF or PA alone because LTx is the toxic form that acts on cells. A method was developed for LTx-specific quantification in plasma using anti-PA IgG magnetic immunoprecipitation of PA and quantification of LF activity that co-purified with PA. The method was fast (<4 h total time to detection), sensitive at 0.033 ng/mL LTx in plasma for the fast analysis (0.0075 ng/mL LTx in plasma for an 18 h reaction), precise (6.3-9.9% coefficient of variation), and accurate (0.1-12.7%error; n ≥ 25). Diagnostic sensitivity was 100% (n = 27 animal/clinical cases). Diagnostic specificity was 100% (n = 141). LTx was detected post-antibiotic treatment in 6/6 treated rhesus macaques and 3/3 clinical cases of inhalation anthrax and as long as 8 days post-treatment. Over the course of infection in two rhesus macaques, LTx was first detected at 0.101 and 0.237 ng/mL at 36 h post-exposure and increased to 1147 and 12,107 ng/mL in late-stage anthrax. This demonstrated the importance of LTx as a diagnostic and therapeutic target. This method provides a sensitive, accurate tool for anthrax toxin detection and evaluation of PA-directed therapeutics.

  15. Simultaneous quantification of cryptotanshinone and its active metabolite tanshinone IIA in plasma by liquid chromatography/tandem mass spectrometry (LC-MS/MS).

    PubMed

    Hao, Haiping; Wang, Guangji; Li, Peng; Li, Jing; Ding, Zuoqi

    2006-02-13

    A rapid and sensitive method for the simultaneous determination of cryptotanshinone and its active metabolite tanshinone IIA in rat plasma was developed and well validated, using high-performance liquid chromatographic separation with tandem mass spectrometric detection. This method entailed a single step of liquid-liquid extraction with ethyl acetate from a small volume of plasmas. The analytes and internal standard diazepam were baseline separated on a Shim-pack VP-ODS analytical column. Detection was performed on a triple quadrupole tandem mass spectrometer equipped with electrospray ionization source operated under selected reaction monitoring (SRM) mode. The method was linear in the concentration range of 1-100 ng/ml for both tanshinone IIA and cryptotanshinone. The intra- and inter-day precisions (R.S.D.%) were within 10.2% for both analytes. Deviation of the assay accuracies was within +/-12.0% for both analytes. Both analytes were proved to be stable during all sample storing, preparation and analytic procedures. The method was successfully applied to a pharmacokinetic study after an oral administration of cryptotanshinone to rats with a dose of 20 mg/kg. With the lower limits of quantification at 1.0 ng/ml for tanshinone IIA and 0.2 ng/ml for cryptotanshinone, this method was proved to be sensitive enough and reproducible for the pharmacokinetics study of both tanshinones. PMID:16169701

  16. Simultaneous quantification of tenofovir, emtricitabine, rilpivirine, elvitegravir and dolutegravir in mouse biological matrices by LC-MS/MS and its application to a pharmacokinetic study.

    PubMed

    Prathipati, Pavan Kumar; Mandal, Subhra; Destache, Christopher J

    2016-09-10

    Combination antiretroviral (cARV) treatment is more common in human immunodeficiency virus (HIV) infection. In many instances, treatment regimen includes two or more combination of drugs from six different classes. Some of the antiretroviral combination medications are under study at preclinical and clinical stages. A precise method is required to quantify the drug concentration in biological matrices to study pharmacokinetic behavior and tissue distribution profile in animals and/or humans. We have developed and validated a sensitive and precise liquid chromatography-tandem mass spectrometry method for simultaneous quantification of selected antiretroviral drugs, tenofovir (TNF), emtricitabine (FTC), rilpivirine (RPV), dolutegravir (DTG) and elvitegravir (EVG) in mouse biological matrices. This method involves a solid phase extraction, simple isocratic chromatographic separation using Restek Pinnacle DB BiPh column (50mm×2.1mm, 5μm) and mass spectrometric detection by an API 3200 Q Trap instrument. The total run time for each sample was 6min. The method was validated in the concentration range of 5-2000ng/mL for FTC, RPV, DTG, EVG and 10-4000ng/mL for TNF respectively with correlation coefficients (r(2)) higher than 0.9976. The results of intra and inter-run assay precision and accuracy were within acceptance limits for all the five analytes. This method was used to support the study of pharmacokinetics and tissue distribution profile of nanoformulated antiretroviral drugs in mice. PMID:27497648

  17. Detection and quantification of chlordecone in contaminated soils from the French West Indies by GC-MS using the 13C10-chlordecone stable isotope as a tracer.

    PubMed

    Martin-Laurent, Fabrice; Sahnoun, Mehdi M; Merlin, Chloé; Vollmer, Guy; Lübke, Markus

    2014-04-01

    Chlordecone is an organochlorine insecticide that has been widely used to control banana weevil in the French West Indies. As a result of this intense use, up to 20,000 ha are contaminated by this insecticide in the French West Indies, and this causes environmental damage and health problems. A scenario of exposure was drawn by French authorities, based on land usage records. Many efforts have been made to monitor the occurrence of chlordecone and its main metabolites using different analytical methods, including GC, GC/MS, LC/MS, and NIRS. Although these different methods allow for the detection and quantification of chlordecone from soils, none of them estimate the bottleneck caused by extraction of this organochlorine from soils with high adsorption ability. In this study, we used (13)C10-chlordecone as a tracer to estimate chlordecone extraction yield and to quantify chlordecone in soil extracts based on the (13)C/(12)C isotope dilution. We report the optimization of (13)C10-chlordecone extraction from an Andosol. The method was found to be linear from 0.118 to 43 mg kg(-1) in the Andosol, with an instrumental detection limit estimated at 8.84 μg kg(-1). This method showed that chlordecone ranged from 35.4 down to 0.18 mg kg(-1) in Andosol, Nitisol, Ferralsol, and Fluvisol soil types. Traces of the metabolite β-monohydrochlordecone were detected in the Andosol, Nitisol, and Ferralsol soil samples. This last result indicates that this method could be useful to monitor the fate of chlordecone in soils of the French West Indies. PMID:23733305

  18. An isotope-dilution standard GC/MS/MS method for steroid hormones in water

    USGS Publications Warehouse

    Foreman, William T.; Gray, James L.; ReVello, Rhiannon C.; Lindley, Chris E.; Losche, Scott A.

    2013-01-01

    An isotope-dilution quantification method was developed for 20 natural and synthetic steroid hormones and additional compounds in filtered and unfiltered water. Deuterium- or carbon-13-labeled isotope-dilution standards (IDSs) are added to the water sample, which is passed through an octadecylsilyl solid-phase extraction (SPE) disk. Following extract cleanup using Florisil SPE, method compounds are converted to trimethylsilyl derivatives and analyzed by gas chromatography with tandem mass spectrometry. Validation matrices included reagent water, wastewater-affected surface water, and primary (no biological treatment) and secondary wastewater effluent. Overall method recovery for all analytes in these matrices averaged 100%; with overall relative standard deviation of 28%. Mean recoveries of the 20 individual analytes for spiked reagent-water samples prepared along with field samples analyzed in 2009–2010 ranged from 84–104%, with relative standard deviations of 6–36%. Detection levels estimated using ASTM International’s D6091–07 procedure range from 0.4 to 4 ng/L for 17 analytes. Higher censoring levels of 100 ng/L for bisphenol A and 200 ng/L for cholesterol and 3-beta-coprostanol are used to prevent bias and false positives associated with the presence of these analytes in blanks. Absolute method recoveries of the IDSs provide sample-specific performance information and guide data reporting. Careful selection of labeled compounds for use as IDSs is important because both inexact IDS-analyte matches and deuterium label loss affect an IDS’s ability to emulate analyte performance. Six IDS compounds initially tested and applied in this method exhibited deuterium loss and are not used in the final method.

  19. [Determination of ambroxol and clenbuterol in human plasma by LC-MS/MS method].

    PubMed

    Lin, Nan; Chen, Xiao-Yan; Song, Bo; Zhong, Da-Fang

    2007-03-01

    Ambroxol and clenbuterol were extracted from human plasma samples by liquid-liquid extraction, ambroxol was separated on a Zorbax XDB-C18 column and detected by tandem mass spectrometry with an atmospheric pressure chemical ionization interface after oral administration of a compound preparation. Clenbuterol was separated on a Zorbax XDB-C8 column and detected by tandem mass spectrometry with an electrospray ionization interface. Diphenhydramine is used as the internal standard. The linear concentration ranges of the calibration curves for ambroxol and clenbuterol were 0.080 - 400 microg x L(-1) and 5.0 - 5 000 ng x L(-1), respectively. The lower limits of quantification were 0.080 microg x L(-1) for ambroxol and 5.0 ng x L(-1) for clenbuterol, individually. The inter-day and intra-day precision (RSD) across three validation run over the entire concentration range was below 7.5%, and the accuracy (RE) was within +/- 2.5% for both ambroxol and clenbuterol. The methods were used to determine the pharmacokinetic parameters of ambroxol and clenbuterol in human plasma after oral administration of a compound preparation containing 60 mg ambroxol hydrochloride and 40 microg clenbuterol hydrochloride. The method was proved to be highly sensitive, selective and suitable for the pharmacokinetic study of different compound preparations containing ambroxol and clenbuterol.

  20. Identification and quantification of glutathione and phytochelatins from Chlorella vulgaris by RP-HPLC ESI-MS/MS and oxygen-free extraction.

    PubMed

    Simmons, Denina B D; Hayward, Allison R; Hutchinson, Thomas C; Emery, R J Neil

    2009-10-01

    Phytochelatins are short, cysteine-containing, detoxification peptides produced by plants, algae, and fungi in response to heavy metal exposure. These peptides auto-oxidize easily. Current extraction protocols do not adequately address losses of phytochelatins because of their oxidation and the use of indirect methods for quantification. Method enhancements include the use of an argon environment during extraction to reduce auto-oxidation, the use of glycine-(13)C2-labeled glutathione as an internal standard, and an electrospray ionization source with a triple quadrupole mass spectrometer as a detector. The method-detection limits were 0.081 microM for glutathione, 0.440 microM for phytochelatin 2, and 0.120 microM for phytochelatin 3. These detection limits were comparable to similar studies and were not compromised incorporating these adjustments. The use of a labeled internal standard and an inert gaseous environment during sample preparation greatly improved calibration linearity and sensitivity. Furthermore, phytochelatin degradation was significantly reduced and more accurately tracked. Previous studies involving phytochelatin analyses have likely been subject to higher variability caused by this propensity for phytochelatins to degrade rapidly in air. The method adjustments were simple and cost-effective and allowed phytochelatin analyses to be performed for hours at a time with minimal auto-oxidation. PMID:19688341

  1. Identification and quantification of glutathione and phytochelatins from Chlorella vulgaris by RP-HPLC ESI-MS/MS and oxygen-free extraction.

    PubMed

    Simmons, Denina B D; Hayward, Allison R; Hutchinson, Thomas C; Emery, R J Neil

    2009-10-01

    Phytochelatins are short, cysteine-containing, detoxification peptides produced by plants, algae, and fungi in response to heavy metal exposure. These peptides auto-oxidize easily. Current extraction protocols do not adequately address losses of phytochelatins because of their oxidation and the use of indirect methods for quantification. Method enhancements include the use of an argon environment during extraction to reduce auto-oxidation, the use of glycine-(13)C2-labeled glutathione as an internal standard, and an electrospray ionization source with a triple quadrupole mass spectrometer as a detector. The method-detection limits were 0.081 microM for glutathione, 0.440 microM for phytochelatin 2, and 0.120 microM for phytochelatin 3. These detection limits were comparable to similar studies and were not compromised incorporating these adjustments. The use of a labeled internal standard and an inert gaseous environment during sample preparation greatly improved calibration linearity and sensitivity. Furthermore, phytochelatin degradation was significantly reduced and more accurately tracked. Previous studies involving phytochelatin analyses have likely been subject to higher variability caused by this propensity for phytochelatins to degrade rapidly in air. The method adjustments were simple and cost-effective and allowed phytochelatin analyses to be performed for hours at a time with minimal auto-oxidation.

  2. 21 CFR 530.24 - Procedure for announcing analytical methods for drug residue quantification.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Procedure for announcing analytical methods for...-Producing Animals § 530.24 Procedure for announcing analytical methods for drug residue quantification. (a) FDA may issue an order announcing a specific analytical method or methods for the quantification...

  3. High resolution LC-ESI-TOF-mass spectrometry method for fast separation, identification, and quantification of 12 isoflavones in soybeans and soybean products.

    PubMed

    Lee, Mi Jin; Chung, Ill-Min; Kim, Hunjung; Jung, Mun Yhung

    2015-06-01

    A high resolution LC-ESI-TOF-MS analytical method was established for the rapid isolation, identification, and quantification of 12 isoflavones in soybean and soybean products. Individual isoflavones were identified on the basis of the accurate mass data of their respective protonated mass ions, Na or K adduct ions, fragment ions, and isotope ion patterns. The protonated mass ions of isoflavones were extracted for their quantification in soybean products. Twelve different isoflavones in the soybean products were fully separated, identified and quantified within 12min separation time. The established LC-TOF/MS was an effective analytical method for the simultaneous characterization and quantification of isoflavones with exceptionally short analytical time, high selectivity, a high linearity (r(2)>0.992) in a wide range, low LOD and LOQ, high precision, inter-and intra-day repeatability, and no significant matrix effect. Furthermore, it requires simple sample preparation procedure (solvent extraction, dilution, and syringe filtration).

  4. Extraction, thermal cleanup, and GC/MS quantification with disposable solid extractants: application to hydrophobic analytes in aqueous surfactant solutions.

    PubMed

    Begnaud, Frédéric; Debonneville, Christian; Chaintreau, Alain

    2011-02-01

    Fragranced consumer products are generally formulated together with surfactants. In application, these products are often highly diluted with water. Analyzing trace amounts of fragrance ingredients in such mixtures is challenging and usually requires either time-consuming sample cleanup or extensive cleaning of the trapping device to avoid memory effect and cross-contamination between samples. To overcome these limitations, a new disposable extraction device has been developed to be used in combination with a thermodesorption-GC-MS unit. Made of PDMS foam cylinders, it efficiently extracts trace amounts of hydrophobic compounds from complex aqueous solutions and provides an online sample cleanup, thanks to the controlled desorption temperature, which allows retaining the low volatile constituents of the matrix within the absorptive foam. Combined with a stable isotope dilution assay, accurate quantifications of Cetalox(®), Muscenone™, Helvetolide(®), Polysantol(®), Dartanol, and Myrrhone(®) from aqueous solution containing surfactant and from water from the aeration tank of a sewage plant were successfully conducted. LOQ varied between 1 and 25 ppb (20% confidence interval, α = 0.1).

  5. A novel automated image analysis method for accurate adipocyte quantification

    PubMed Central

    Osman, Osman S; Selway, Joanne L; Kępczyńska, Małgorzata A; Stocker, Claire J; O’Dowd, Jacqueline F; Cawthorne, Michael A; Arch, Jonathan RS; Jassim, Sabah; Langlands, Kenneth

    2013-01-01

    Increased adipocyte size and number are associated with many of the adverse effects observed in metabolic disease states. While methods to quantify such changes in the adipocyte are of scientific and clinical interest, manual methods to determine adipocyte size are both laborious and intractable to large scale investigations. Moreover, existing computational methods are not fully automated. We, therefore, developed a novel automatic method to provide accurate measurements of the cross-sectional area of adipocytes in histological sections, allowing rapid high-throughput quantification of fat cell size and number. Photomicrographs of H&E-stained paraffin sections of murine gonadal adipose were transformed using standard image processing/analysis algorithms to reduce background and enhance edge-detection. This allowed the isolation of individual adipocytes from which their area could be calculated. Performance was compared with manual measurements made from the same images, in which adipocyte area was calculated from estimates of the major and minor axes of individual adipocytes. Both methods identified an increase in mean adipocyte size in a murine model of obesity, with good concordance, although the calculation used to identify cell area from manual measurements was found to consistently over-estimate cell size. Here we report an accurate method to determine adipocyte area in histological sections that provides a considerable time saving over manual methods. PMID:23991362

  6. Simple and sensitive LC-MS/MS-based assay for the quantification of dimemorfan in human plasma for use in a pharmacokinetic study.

    PubMed

    Tan, Hongyi; Peng, Jinfu; Pei, Qi; Yang, Liu; Chen, Jun; Guo, Chengxian; Hua, Ye; Yuan, Hong; Yang, Guoping

    2015-05-01

    Dimemorfan phosphate has been widely used for 40 years throughout the world for the treatment of coughs. This is the first report on the use of an LC-MS/MS-based assay for the determination of dimemorfan in human plasma using estazolam as an internal standard after one-step protein precipitation with acetonitrile. Chromatographic separation was achieved on a Phenomenex Luna C18 column (3 µm, 50 × 2.0 mm) using a fast gradient method, which involves water and methanol as the mobile phase (both containing 0.1% formic acid). Dimemorfan and estazolam were detected with proton adducts at m/z values of 255.8 → 155.1 and 295.0 → 267.0, respectively, in the selected reaction monitoring positive mode. The linear dynamic range of the assay was 0.04-5.00 ng/mL. The chromatographic run time for each plasma sample was <5 min. The method was proven to be accurate, precise, and repeatable. Finally, the developed method was successfully applied for the determination of dimemorfan in a pharmacokinetic study using healthy Chinese subjects.

  7. An UPLC-MS/MS method for the analysis of glimepiride and fluoxetine in human plasma.

    PubMed

    Qiu, Xiangjun; Wang, Hong-wei; Yuan, Ye; Wang, Ying-fei; Sun, Ming; Huang, Xue-sun

    2015-02-01

    A sensitive and rapid ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method was developed to determine glimepiride (GPD) and fluoxetine (FLU) in human plasma using diazepam as the internal standard (IS) simultaneously. The presented method used an Acquity UPLC BEH C18 column for chromatographic separation with tandem mass spectrometric detection on a QTrap5500 mass spectrometer operated in positive ESI mode. The mobile phase is a mixture of acetonitrile and 1% formic acid in water with gradient elution at a flow rate of 0.40mL/min. The GPD, FLU and IS were eluted at 1.46, 1.27 and 1.39min, respectively. The MRM transitions of m/z 491.3→126.3 and m/z 310.5→148.1 were used to quantify for GPD and FLU, respectively. The linearity of this method was found to be within the concentration range of 2.5-300ng/mL and 0.1-20ng/mL for GPD and FLU in human plasma, respectively. The intra- and inter-day precision (RSD%) were less than 10.3% and accuracy (RE%) was within ±7.3%. The matrix effect were 95.3-100.7% for GPD and FLU. GPD and FLU were sufficiently stable under all relevant analytical conditions. The method was also successfully applied to the clinical samples after a single oral dose of 2mg GLP and 40mg FLU in patients. PMID:25589252

  8. Natural chlorate in the environment: Application of a new IC-ESI/MS/MS method with a Cl18O3- internal standard

    USGS Publications Warehouse

    Rao, Balaji; Hatzinger, Paul B.; Böhlke, John Karl; Sturchio, Neil C.; Andraski, Brian J.; Eckardt, Frank D.; Jackson, W. Andrew

    2010-01-01

    A new ion chromatography electrospray tandem mass spectrometry (IC-ESI/MS/MS) method has been developed for quantification and confirmation of chlorate (ClO3-) in environmental samples. The method involves the electro-chemical generation of isotopically labeled chlorate internal standard (Cl18O3-) using 18O water (H218O). The standard was added to all samples prior to analysis thereby minimizing the matrix effects that are associated with common ions without the need for expensive sample pretreatments. The method detection limit (MDL) for ClO3- was 2 ng L-1 for a 1 mL volume sample injection. The proposed method was successfully applied to analyze ClO3- in difficult environmental samples including soil and plant leachates. The IC-ESI/MS/MS method described here was also compared to established EPA method 317.0 for ClO3- analysis. Samples collected from a variety of environments previously shown to contain natural perchlorate (ClO 4-) occurrence were analyzed using the proposed method and ClO3- was found to co-occur with ClO4- at concentrations ranging from <2 ng L-1 in precipitation from Texas and Puerto Rico to >500 mg kg-1 in caliche salt deposits from the Atacama Desert in Chile. Relatively low concentrations of ClO3- in some natural groundwater samples (-1) analyzed in this work may indicate lower stability when compared to ClO4- in the subsurface. The high concentrations of ClO3- in caliches and soils (3-6 orders of magnitude greater) as compared to precipitation samples indicate that ClO3-, like ClO4-, may be atmospherically produced and deposited, then concentrated in dry soils, and is possibly a minor component in the biogeochemical cycle of chlorine.

  9. Natural chlorate in the environment: application of a new IC-ESI/MS/MS method with a Cl¹⁸O₃-internal standard.

    PubMed

    Balaji Rao, Balaji Rao; Hatzinger, Paul B; Böhlke, John Karl; Sturchio, Neil C; Andraski, Brian J; Eckardt, Frank D; Jackson, W Andrew

    2010-11-15

    A new ion chromatography electrospray tandem mass spectrometry (IC-ESI/MS/MS) method has been developed for quantification and confirmation of chlorate (ClO₃⁻) in environmental samples. The method involves the electrochemical generation of isotopically labeled chlorate internal standard (Cl¹⁸O₃⁻) using ¹⁸O water (H₂¹⁸O) he standard was added to all samples prior to analysis thereby minimizing the matrix effects that are associated with common ions without the need for expensive sample pretreatments. The method detection limit (MDL) for ClO₃⁻ was 2 ng L⁻¹ for a 1 mL volume sample injection. The proposed method was successfully applied to analyze ClO₃⁻ in difficult environmental samples including soil and plant leachates. The IC-ESI/MS/MS method described here was also compared to established EPA method 317.0 for ClO₃⁻ analysis. Samples collected from a variety of environments previously shown to contain natural perchlorate (ClO₄⁻) occurrence were analyzed using the proposed method and ClO₃⁻ was found to co-occur with ClO₄⁻ at concentrations ranging from < 2 ng L⁻¹ in precipitation from Texas and Puerto Rico to >500 mg kg⁻¹ in caliche salt deposits from the Atacama Desert in Chile. Relatively low concentrations of ClO₃⁻ in some natural groundwater samples (0.1 µg L⁻¹) analyzed in this work may indicate lower stability when compared to ClO₄⁻ in the subsurface. The high concentrations ClO₃⁻ in caliches and soils (3-6 orders of magnitude greater) as compared to precipitation samples indicate that ClO₃⁻, like ClO₄⁻, may be atmospherically produced and deposited, then concentrated in dry soils, and is possibly a minor component in the biogeochemical cycle of chlorine.

  10. Natural chlorate in the environment: application of a new IC-ESI/MS/MS method with a Cl¹⁸O₃-internal standard.

    PubMed

    Balaji Rao, Balaji Rao; Hatzinger, Paul B; Böhlke, John Karl; Sturchio, Neil C; Andraski, Brian J; Eckardt, Frank D; Jackson, W Andrew

    2010-11-15

    A new ion chromatography electrospray tandem mass spectrometry (IC-ESI/MS/MS) method has been developed for quantification and confirmation of chlorate (ClO₃⁻) in environmental samples. The method involves the electrochemical generation of isotopically labeled chlorate internal standard (Cl¹⁸O₃⁻) using ¹⁸O water (H₂¹⁸O) he standard was added to all samples prior to analysis thereby minimizing the matrix effects that are associated with common ions without the need for expensive sample pretreatments. The method detection limit (MDL) for ClO₃⁻ was 2 ng L⁻¹ for a 1 mL volume sample injection. The proposed method was successfully applied to analyze ClO₃⁻ in difficult environmental samples including soil and plant leachates. The IC-ESI/MS/MS method described here was also compared to established EPA method 317.0 for ClO₃⁻ analysis. Samples collected from a variety of environments previously shown to contain natural perchlorate (ClO₄⁻) occurrence were analyzed using the proposed method and ClO₃⁻ was found to co-occur with ClO₄⁻ at concentrations ranging from < 2 ng L⁻¹ in precipitation from Texas and Puerto Rico to >500 mg kg⁻¹ in caliche salt deposits from the Atacama Desert in Chile. Relatively low concentrations of ClO₃⁻ in some natural groundwater samples (0.1 µg L⁻¹) analyzed in this work may indicate lower stability when compared to ClO₄⁻ in the subsurface. The high concentrations ClO₃⁻ in caliches and soils (3-6 orders of magnitude greater) as compared to precipitation samples indicate that ClO₃⁻, like ClO₄⁻, may be atmospherically produced and deposited, then concentrated in dry soils, and is possibly a minor component in the biogeochemical cycle of chlorine. PMID:20968289

  11. Simultaneous quantification of fosinopril and its active metabolite fosinoprilat in rat plasma by UFLC-MS/MS: Application of formic acid in the stabilization of an ester-containing drug.

    PubMed

    Xue, Kewen; Li, Gao; Sun, Xiao; Hu, Yang; Hu, Lei; Huang, Jiangeng; Si, Luqin

    2015-05-15

    Fosinopril is an angiotensin-converting enzyme inhibitor containing a phosphate ester group which undergoes esterase hydrolysis to its active metabolite, fosinoprilat. EDTA was utilized as an anticoagulant to inhibit the hydrolysis of fosinopril in whole blood during blood collection and processing. To prevent the ex vivo conversion to fosinoprilat, formic acid was added to rat plasma to effectively stabilize fosinopril. A sensitive, rapid and robust ultra-fast liquid chromatography-tandem mass spectrometry (UFLC-MS/MS) method was developed and validated for simultaneous determination of fosinopril and fosinoprilat in rat plasma. Protein precipitation was employed for plasma sample clean-up. Chromatographic separation was achieved on a Welch Ultimate XB-C18 column using gradient elution with a total run time of 5min. Analytes and their stable isotope labeled internal standards were detected by positive ion electrospray tandem mass spectrometric assay. The assay involves quantitation of both analytes in small-volume (50μL) plasma, with the lower limit of quantification of 0.1 and 1ng/mL for fosinopril and fosinoprilat, respectively. The method was fully validated in linear calibration ranges of 0.1-150ng/mL for fosinopril and 1-1500ng/mL for fosinoprilat with acceptable accuracy and precision. Assay recoveries were high (>95% for fosinopril and >91% for fosinoprilat) and matrix effect was negligible. Both analytes were found to be stable in stabilized rat plasma for 6h at room temperature, 30 days at -80°C, and following three freeze-thaw cycles and were also stable in processed samples for 36h at 4°C. The validated method was successfully applied to sample analyses for pharmacokinetic study of fosinopril and can be extended to the measurement of fosinopril in other biological samples. PMID:25875586

  12. Trace Level Quantification of the (−)2-(2-amino-5-chlorophenyl)-4-cyclopropyl-1,1,1-trifluoro-3-butyn-2-ol Genotoxic Impurity in Efavirenz Drug Substance and Drug Product Using LC–MS/MS

    PubMed Central

    Jaishetty, Nagadeep; Palanisamy, Kamaraj; Maruthapillai, Arthanareeswari; Jaishetty, Rajamanohar

    2015-01-01

    Efavirenz is a non-nucleoside reverse transcriptase inhibitor used in the treatment of human immunodeficiency virus type-1 (HIV). (2S)-(2-Amino-5-chlorophenyl)-4-cyclopropyl-1,1,1-trifluoro-3-butyn-2-ol (AMCOL), used as an intermediate in the synthesis of efavirenz and a degradation impurity, has an aminoaryl derivative which is a well-known alerting function for genotoxic activity. Upon request from a regulatory agency, a selective and sensitive liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed for trace level quantitative determination of AMCOL related compound of efavirenz, for a risk assessment and comparison of impurity levels with the commercially available innovator product (brand name: Sustiva). The method provided excellent sensitivity at a typical target analyte level of <2.5 ppm, an established threshold of toxicological concern (TTC), when the drug substance and drug product samples were prepared at 15.0 mg/mL. The AMCOL sample was analyzed on a Luna C18 (2) (100 mm × 4.6 mm, 3 µm) column interfaced with a triple quadrupole tandem mass spectrometer operated in a multiple reaction monitoring (MRM) mode. Positive electrospray ionization (ESI) was employed as the ionization source and the mobile phase used was 5.0 mM ammonium acetate-methanol (35:65, v/v). The calibration curve showed good linearity over the concentration range of 0.2–5.0 ppm with a correlation coefficient of >0.999. The limit of detection (LOD) and limit of quantification (LOQ) were found to be 0.07 and 0.2 ppm, respectively. The developed method was validated as per international council on harmonization (ICH) guidelines in terms of LOD, LOQ, linearity, precision, accuracy, specificity, and robustness.

  13. Current analytical methods for plant auxin quantification--A review.

    PubMed

    Porfírio, Sara; Gomes da Silva, Marco D R; Peixe, Augusto; Cabrita, Maria J; Azadi, Parastoo

    2016-01-01

    Plant hormones, and especially auxins, are low molecular weight compounds highly involved in the control of plant growth and development. Auxins are also broadly used in horticulture, as part of vegetative plant propagation protocols, allowing the cloning of genotypes of interest. Over the years, large efforts have been put in the development of more sensitive and precise methods of analysis and quantification of plant hormone levels in plant tissues. Although analytical techniques have evolved, and new methods have been implemented, sample preparation is still the limiting step of auxin analysis. In this review, the current methods of auxin analysis are discussed. Sample preparation procedures, including extraction, purification and derivatization, are reviewed and compared. The different analytical techniques, ranging from chromatographic and mass spectrometry methods to immunoassays and electrokinetic methods, as well as other types of detection are also discussed. Considering that auxin analysis mirrors the evolution in analytical chemistry, the number of publications describing new and/or improved methods is always increasing and we considered appropriate to update the available information. For that reason, this article aims to review the current advances in auxin analysis, and thus only reports from the past 15 years will be covered.

  14. Numerical Continuation Methods for Intrusive Uncertainty Quantification Studies

    SciTech Connect

    Safta, Cosmin; Najm, Habib N.; Phipps, Eric Todd

    2014-09-01

    Rigorous modeling of engineering systems relies on efficient propagation of uncertainty from input parameters to model outputs. In recent years, there has been substantial development of probabilistic polynomial chaos (PC) Uncertainty Quantification (UQ) methods, enabling studies in expensive computational models. One approach, termed ”intrusive”, involving reformulation of the governing equations, has been found to have superior computational performance compared to non-intrusive sampling-based methods in relevant large-scale problems, particularly in the context of emerging architectures. However, the utility of intrusive methods has been severely limited due to detrimental numerical instabilities associated with strong nonlinear physics. Previous methods for stabilizing these constructions tend to add unacceptably high computational costs, particularly in problems with many uncertain parameters. In order to address these challenges, we propose to adapt and improve numerical continuation methods for the robust time integration of intrusive PC system dynamics. We propose adaptive methods, starting with a small uncertainty for which the model has stable behavior and gradually moving to larger uncertainty where the instabilities are rampant, in a manner that provides a suitable solution.

  15. Validated method for the quantification of free and total carnitine, butyrobetaine, and acylcarnitines in biological samples.

    PubMed

    Minkler, Paul E; Stoll, Maria S K; Ingalls, Stephen T; Kerner, Janos; Hoppel, Charles L

    2015-09-01

    A validated quantitative method for the determination of free and total carnitine, butyrobetaine, and acylcarnitines is presented. The versatile method has four components: (1) isolation using strong cation-exchange solid-phase extraction, (2) derivatization with pentafluorophenacyl trifluoromethanesulfonate, (3) sequential ion-exchange/reversed-phase (ultra) high-performance liquid chromatography [(U)HPLC] using a strong cation-exchange trap in series with a fused-core HPLC column, and (4) detection with electrospray ionization multiple reaction monitoring (MRM) mass spectrometry (MS). Standardized carnitine along with 65 synthesized, standardized acylcarnitines (including short-chain, medium-chain, long-chain, dicarboxylic, hydroxylated, and unsaturated acyl moieties) were used to construct multiple-point calibration curves, resulting in accurate and precise quantification. Separation of the 65 acylcarnitines was accomplished in a single chromatogram in as little as 14 min. Validation studies were performed showing a high level of accuracy, precision, and reproducibility. The method provides capabilities unavailable by tandem MS procedures, making it an ideal approach for confirmation of newborn screening results and for clinical and basic research projects, including treatment protocol studies, acylcarnitine biomarker studies, and metabolite studies using plasma, urine, tissue, or other sample matrixes. PMID:26270397

  16. The development and assessment of high-throughput mass spectrometry-based methods for the quantification of a nanoparticle drug delivery agent in cellular lysate.

    PubMed

    Buse, Joshua; Purves, Randy W; Verrall, Ronald E; Badea, Ildiko; Zhang, Haixia; Mulligan, Christopher C; Peru, Kerry M; Bailey, Jonathan; Headley, John V; El-Aneed, Anas

    2014-11-01

    The safe use of lipid-based drug delivery agents requires fast and sensitive qualitative and quantitative assessment of their cellular interactions. Many mass spectrometry (MS) based analytical platforms can achieve such task with varying capabilities. Therefore, four novel high-throughput MS-based quantitative methods were evaluated for the analysis of a small organic gene delivery agent: N,N-bis(dimethylhexadecyl)-1,3-propane-diammonium dibromide (G16-3). Analysis utilized MS instruments that detect analytes using low-resolution tandem MS (MS/MS) analysis (i.e. QTRAP or linear ion trap in this work) or high-resolution MS analysis (i.e. time of flight (ToF) or Orbitrap). Our results indicate that the validated fast chromatography (FC)-QTRAP-MS/MS, FC- LTQ-Orbitrap-MS, desorption electrospray ionization-collision-induced dissociation (CID)-MS/MS and matrix assisted laser desorption ionization-ToF/ToF-MS MS methods were superior in the area of method development and sample analysis time to a previously developed liquid chromatography (LC)-CID-MS/MS. To our knowledge, this is the first evaluation of the abilities of five MS-based quantitative methods that target a single pharmaceutical analyte. Our findings indicate that, in comparison to conventional LC-CID-MS/MS, the new MS-based methods resulted in a (1) substantial reduction in the analysis time, (2) reduction in the time required for method development and (3) production of either superior or comparable quantitative data. The four new high-throughput MS methods, therefore, were faster, more efficient and less expensive than a conventional LC-CID-MS/MS for the quantification of the G16-3 analyte within tissue culture. When applied to cellular lysate, no significant change in the concentration of G16-3 gemini surfactant within PAM212 cells was observed between 5 and 53 h, suggesting the absence of any metabolism/excretion from PAM212 cells.

  17. Development and validation of a method for the quantification of milk proteins in food products based on liquid chromatography with mass spectrometric detection.

    PubMed

    Lutter, Petra; Parisod, Véronique; Weymuth, Hans

    2011-01-01

    The protection of allergic consumers is crucial to the food industry. Therefore, accurate methods for the detection of food allergens are required. Targeted detection of selected molecules by MS combines high selectivity with accurate quantification. A confirmatory method based on LC/selected reaction monitoring (SRM)-MS/MS was established and validated for the quantification of milk traces in food. Tryptic peptides of the major milk proteins beta-lactoglobulin, beta-casein, alphaS2-casein, and K-casein were selected as quantitative markers. Precise quantification was achieved using internal standard peptides containing isotopically labeled amino acids. For each peptide, qualifier and quantifier fragments were selected according to Commission Decision 2002/657/EC. A simple sample preparation method was established without immunoaffinity or SPE enrichment steps for food matrixes containing different amounts of protein, such as baby food, breakfast cereals, infant formula, and cereals. Intermediate reproducibility, repeatability, accuracy, and measurement uncertainty were determined for each matrix. LOD values of 0.2-0.5 mg/kg, e.g., for beta-lactoglobulin, were comparable to those obtained with ELISA kits. An LOQ of approximately 5 mg/kg, expressed as mass fraction skim milk powder, was validated in protein-rich infant cereals. The obtained validation data show that the described LC/SRM-MS/MS approach can serve as a confirmatory method for the determination of milk traces in selected food matrixes. PMID:21919337

  18. Direct field method for root biomass quantification in agroecosystems.

    PubMed

    Frasier, Ileana; Noellemeyer, Elke; Fernández, Romina; Quiroga, Alberto

    2016-01-01

    The present article describes a field auger sampling method for row-crop root measurements. In agroecosystems where crops are planted in a specific design (row crops), sampling procedures for root biomass quantification need to consider the spatial variability of the root system. This article explains in detail how to sample and calculate root biomass considering the sampling position in the field and the differential weight of the root biomass in the inter-row compared to the crop row when expressing data per area unit. This method is highly reproducible in the field and requires no expensive equipment and/or special skills. It proposes to use a narrow auger thus reducing field labor with less destructive sampling, and decreases laboratory time because samples are smaller. The small sample size also facilitates the washing and root separation with tweezers. This method is suitable for either winter- or summer crop roots. •Description of a direct field method for row-crop root measurements.•Description of data calculation for total root-biomass estimation per unit area.•The proposed method is simple, less labor- and less time consuming. PMID:27630821

  19. Using Visualized Matrix Effects to Develop and Improve LC-MS/MS Bioanalytical Methods, Taking TRAM-34 as an Example

    PubMed Central

    Ye, Jia-Hung; Pao, Li-Heng

    2015-01-01

    Matrix effects (MEs) continue to be an obstacle in the development of the LC-MS/MS method, with phospholipids being the major cause of MEs. Changing the mobile phase has been a common strategy to reduce MEs; however, the underlying mechanism is unclear. "In-source multiple-reaction monitoring" (IS-MRM) for glycerophosphocholines (PCs) has been commonly applied in many bioanalytical methods. "Visualized MEs" is a suitable term to describe the application of IS-MRM to visualize the elution pattern of phospholipids. We selected a real case to discuss the relationship of MEs and phospholipids in different mobile phases by quantitative, qualitative, and visualized MEs in LC-MS/MS bioanalysis. The application of visualized MEs not only predicts the ion-suppression zone but also helps in selecting an appropriate (1) mobile phase, (2) column, (3) needle wash solvent for the residue of analyte and phospholipids, and (4) evaluates the clean-up efficiency of sample preparation. The TRAM-34 LC-MS/MS method, improved by using visualized MEs, was shown to be a precise and accurate analytical method. All data indicated that the use of visualized MEs indeed provided useful information about the LC-MS/MS method development and improvement. In this study, an integrative approach for the qualitative, quantitative, and visualized MEs was used to decipher the complexity of MEs. PMID:25909956

  20. An improved competitive inhibition enzymatic immunoassay method for tetrodotoxin quantification.

    PubMed

    Stokes, Amber N; Williams, Becky L; French, Susannah S

    2012-01-01

    Quantifying tetrodotoxin (TTX) has been a challenge in both ecological and medical research due to the cost, time and training required of most quantification techniques. Here we present a modified Competitive Inhibition Enzymatic Immunoassay for the quantification of TTX, and to aid researchers in the optimization of this technique for widespread use with a high degree of accuracy and repeatability.

  1. Simultaneous quantification of catechin, epicatechin, liquiritin, isoliquiritin, liquiritigenin, isoliquiritigenin, piperine and glycyrrhetinic acid in rat plasma by HPLC-MS/MS: application to a pharmacokinetic study of Longhu Rendan pills.

    PubMed

    Wang, Tianming; Ding, Liqing; Jin, Huajia; Shi, Rong; Li, Yuanyuan; Wu, Jiasheng; Li, Yifei; Zhu, Li; Ma, Yueming

    2016-08-01

    A sensitive, specific, accurate HPLC-MS/MS method was developed and validated for the simultaneous quantification of catechin, epicatechin, liquiritin, isoliquiritin, liquiritigenin, isoliquiritigenin, piperine and glycyrrhetinic acid from Longhu Rendan pills in rat plasma. Chromatographic separation was performed with a Hypersil Gold C18 column using a gradient of methanol and 0.01% acetic acid containing 0.2 mm ammonium acetate as mobile phase. The analytes were quantified on a triple quadrupole mass spectrometer, operating in selected reaction monitoring mode and switching the electrospray ion source polarity between positive and negative modes in a single run. The calibration curves of catechin, epicatechin, liquiritin, isoliquiritin, liquiritigenin, isoliquiritigenin, piperine and glycyrrhetinic acid were linear over the concentration ranges of 5-2000, 5-2000, 0.5-200, 0.5-200, 0.25-100, 0.25-100, 0.025-10 and 0.50-200 ng mL(-1) , respectively. The intra- and inter-assay precisions and accuracies were <11.6 and 91.9-108.2%, respectively, for all analytes. Matrix effects for all analytes were between 88.2 and 114.2%. Stability testing showed that all analytes were stable in plasma at 24 °C for 3 h, at 4 °C for 24 h, after three freeze-thaw cycles, and at -80 °C for 15 days. The method was successfully applied to an in vivo study evaluating the pharmacokinetics of multiple nonvolatile compounds following intragastric administration of Longhu Rendan pills to rats. Copyright © 2016 John Wiley & Sons, Ltd.

  2. Automated sample preparation and LC-MS for high-throughput ADME quantification.

    PubMed

    O'Connor, Desmond

    2002-01-01

    Bioanalytical groups in the pharmaceutical industry provide quantitative data to support all stages of drug discovery. The increased use of 96-well plates and robotic liquid handling systems, the availability of robust triple quadruple mass spectrometers, and developments in chromatographic and samples preparation techniques, have all increased the rate at which this data can be generated. This review describes currently used methods and emerging technologies for automation of high-throughput quantitative bioanalysis. The focus is on recent applications of sample preparation and chromatography techniques compatible with detection by triple quadruple mass spectrometers.

  3. Development of a validated LC-MS/MS method for determination of doxofylline on rat dried blood spots and urine: application to pharmacokinetics.

    PubMed

    Rao, R Nageswara; Prasad, K Guru; Naidu, Ch Gangu; Saida, Shaik; Agwane, Sachin B

    2013-05-01

    A rapid and highly sensitive liquid chromatography-tandem mass spectrometric (LC-MS/MS) method for determination of doxofylline on rat dried blood spots and urine was developed and validated. The chromatographic separation was achieved on a reverse phase C18 column (250 mm × 4.6 mm, 5 μm), using 20 mM ammonium acetate (pH adjusted to 3.5 with trifluoroacetic acid) and acetonitrile (75:25 v/v) as a mobile phase at 25 °C. LC-MS detection was performed with selective ion monitoring using target ions at m/z 267 and m/z 195 for doxofylline and caffeine used as internal standard respectively. The calibration curve showed a good linearity in the concentration range of 1-5000 ng/mL. The effect of hematocrit on extraction of doxofylline from DBS was evaluated. The mean recoveries of doxofylline from DBS and urine were 93.46 and 89.86% respectively. The intra and inter-day precisions were less than 4.28% in DBS as well as urine. The limit of detection and quantification were 0.24 and 0.84 ng/mL in DBS and 0.28 and 1.00 ng/mL in urine samples respectively. The method was validated as per ICH guidelines and successfully applied to a pharmacokinetic study of doxofylline in rats.

  4. A new analytical method for the quantification of glycidol fatty acid esters in edible oils.

    PubMed

    Masukawa, Yoshinori; Shiro, Hiroki; Nakamura, Shun; Kondo, Naoki; Jin, Norikazu; Suzuki, Nobuyoshi; Ooi, Naoki; Kudo, Naoto

    2010-01-01

    A novel method to quantify glycidol fatty acid esters (GEs), supposed to present as food processing contaminants in edible oils, has been developed in combination with double solid-phase extractions (SPEs) and LC-MS measurements. The analytes were five species of synthetic GEs: glycidol palmitic, stearic, oleic, linoleic and linolenic acid esters. The use of selected ion monitoring in a positive ion mode of atmospheric chemical ionization-MS with a reversed-phase gradient LC provided a limit of quantification of 0.0045-0.012 microg/mL for the standard GEs, which enables the detection of GEs in microg ranges per gram of edible oil. Using the double SPE procedure first in reversed-phase and then in normal-phase second, allowed large amounts of co-existing acylglycerols in the oils to be removed, which improved the robustness and stability of the method in sequential runs of LC-MS measurements. When the method was used to quantify GEs in three commercial sources of edible oils, the recovery% ranged from 71.3 to 94.6% (average 79.4%) with a relative standard deviation of 2.9-12.1% for the two oils containing triacylglycerols as major components, and ranged from 90.8 to 105.1% (average 97.2%) with a relative standard deviation of 2.1-12.0% for the other, diacylglycerol-rich oil. Although the accuracy and precision of the method may not be yet sufficient, it is useful for determining trace levels of GEs and will be helpful for the quality control of edible oils.

  5. Rapid method for the quantification of hydroquinone concentration: chemiluminescent analysis.

    PubMed

    Chen, Tung-Sheng; Liou, Show-Yih; Kuo, Wei-Wen; Wu, Hsi-Chin; Jong, Gwo-Ping; Wang, Hsueh-Fang; Shen, Chia-Yao; Padma, V Vijaya; Huang, Chih-Yang; Chang, Yen-Lin

    2015-11-01

    Topical hydroquinone serves as a skin whitener and is usually available in cosmetics or on prescription based on the hydroquinone concentration. Quantification of hydroquinone content therefore becomes an important issue in topical agents. High-performance liquid chromatography (HPLC) is the commonest method for determining hydroquinone content in topical agents, but this method is time-consuming and uses many solvents that can become an environmental issue. We report a rapid method for quantifying hydroquinone content by chemiluminescent analysis. Hydroquinone induces the production of hydrogen peroxide in the presence of basic compounds. Hydrogen peroxide induced by hydroquinone oxidized light-emitting materials such as lucigenin, resulted in the production of ultra-weak chemiluminescence that was detected by a chemiluminescence analyzer. The intensity of the chemiluminescence was found to be proportional to the hydroquinone concentration. We suggest that the rapid (measurement time, 60 s) and virtually solvent-free (solvent volume, <2 mL) chemiluminescent method described here for quantifying hydroquinone content may be an alternative to HPLC analysis. PMID:25693839

  6. An improved method for the quantification of SOA bound peroxides

    NASA Astrophysics Data System (ADS)

    Mutzel, Anke; Rodigast, Maria; Iinuma, Yoshiteru; Böge, Olaf; Herrmann, Hartmut

    2013-03-01

    An improvement is made to a method for the quantification of SOA-bound peroxides. The procedure is based on an iodometric-spectrophotometric method that has been commonly used for the determination of peroxides in a wide range of biological and environmental samples. The improved method was applied to determine the peroxide content of laboratory-generated SOA from α-pinene ozonolysis. Besides main improvements for the detection conditions, the use of more environmentally sound solvents is considered instead of carcinogenic solvents. In addition to the improved method for peroxide determination, the present study provides evidence for artefact formation caused by ultrasonic agitation for the extraction of organic compounds in SOA filter samples. The concentration of SOA-bound peroxides in the extracts from ultrasonic agitation were up to three times higher than those from a laboratory orbital shaker under the same extraction conditions, indicating peroxide formation caused by acoustic cavitation during extraction. In contrast, pinic acid, terebic acid and terpenylic acid showed significantly lower concentrations in the sample extract prepared using ultrasonic agitation, indicating that these compounds react with OH radicals that are formed from acoustic cavitation. Great care should be taken when extracting SOA samples and the use of ultrasound should be avoided.

  7. Development and validation of an on-line multidimensional SPE-LC-MS/MS method for the quantitation of Tetrandrine in blood samples.

    PubMed

    Caglar, Sena; Morello, Rosa; Boos, Karl-Siegfried

    2015-04-15

    On-line solid-phase extraction (SPE) is becoming an increasingly widespread technique in the clean-up of complex matrices such as body fluids, prior to chromatographic analysis. The use of small SPE columns instead of disposable SPE cartridges allows multiple injections and complete automation. In addition, it decreases the cost of consumables and improves the quality of the overall analysis. Coupling of SPE with HPLC combines sample preparation and separation in one system. In this paper a validated on-line multidimensional (MD) SPE-LC-MS/MS method is described for the determination of Tetrandrine (model drug) in human blood samples. The developed method showed the applicability of direct injection of plasma samples to an on-line MD-SPE-LC-MS/MS system to determine small molecules i.e. drugs. The experimental design is unique. Quantification was through tandem mass spectrometry with positive electrospray ionization (ESI) and multiple reactions monitoring (MRM). The limit of detection was calculated as 31.98 ng/mL. The linear range of the method was between 40.0 and 800.0 ng/mL. Pharmacokinetic parameters are usually determined by analysis of drug concentrations in plasma rather than whole blood. Parameters determined using plasma data may be misleading if concentrations of drug differ between plasma and red blood cells. We successfully applied the developed method for the determination of the distribution coefficient of the model drug Tetrandrine between human red blood cells and blood plasma proteins. The determination of distribution coefficient study results demonstrated that the developed method can provide direct and accurate measurement of RBC partitioning in a model drug and could be applied for screening of other compounds for potential high RBC partition, predicting potential drug toxicity and investigating mechanisms associated with RBC partitions. PMID:25746132

  8. AN IMPROVED HPLC-MS/MS METHOD FOR DETERMINATION OF ISOXAFLUTOLE (BALANCE) AND ITS METABOLITES IN SOILS AND FORAGE PLANTS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    An analytical method using turbo-spray and heat-nebulizer high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) was developed for the analysis of isoxaflutole (IXF) and its two metabolites, diketonitrile (DKN) and the benzoic acid metabolite (BA), at sub 'g/kg levels in soil a...

  9. Validated methods for determination of neurotransmitters and metabolites in rodent brain tissue and extracellular fluid by reversed phase UHPLC-MS/MS.

    PubMed

    Bergh, Marianne Skov-Skov; Bogen, Inger Lise; Lundanes, Elsa; Øiestad, Åse Marit Leere

    2016-08-15

    Fast and sensitive methods for simultaneous determination of dopamine (DA), the two DA-metabolites homovanillic acid (HVA) and 3-methoxytyramine (3-MT), serotonin (5-HT) and the 5-HT-metabolite 5-hydroxyindoleacetic acid (5-HIAA), norepinephrine (NE), acetylcholine (ACh), glutamic acid (Glu) and γ-aminobutyric acid (GABA) in rodent brain tissue (1.0-4000nM) and extracellular fluid (ECF) (0.5-2000nM) based on ultra high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) have been developed. Of the three different sample preparation methods for brain tissue samples tested, a simple and rapid protein precipitation procedure with formic acid was found to give the best results. The neurotransmitters (NTs) and NT metabolites were separated using UHPLC with an Acquity UPLC HSS T3 C18 column (2.1×100mm, 1.8μm particle size) with acidic mobile phase. Gradient elution with methanol was used and quantification was performed using multiple reaction monitoring (MRM). The total run time was 5.2min including equilibration time. The methods were validated by determining calibration model, intra- and inter-day precision and accuracy, limit of detection (LOD), lower limit of quantification (LLOQ), matrix effects (ME), carry-over and stability. Surrogate analytes were used to enable determination of the recovery and ME of the endogenous analytes in brain tissue. The methods were applied for determination of NTs at basal levels in rodent brain ECF and brain tissue homogenate. The developed methods are valuable tools in the studies of mechanisms of drugs of abuse, and neurologic and psychiatric disease. PMID:27336704

  10. Chemical quantification and antioxidant assay of four active components in Ficus hirta root using UPLC-PAD-MS fingerprinting combined with cluster analysis

    PubMed Central

    2013-01-01

    Background Root of Ficus hirta (RFH) is widely consumed in China as a plant-derived popular food. However, contents of the active constituents of RFH are unknown, and the chemical as well as bioactive properties of RFH may be affected by growing area. In order to ensure the standard efficacy of health products made with RFH, its active constituents should firstly be determined and, secondly, a means of assessing samples for their contents of these constituents is needed. Results Four active components, including two coumarins, namely psoralen and bergapten, and two flavonoids, namely luteolin and apigenin, in twenty RFH samples were quantified using a new ultra performance liquid chromatography coupled with photodiode array detector and mass spectrometry (UPLC-PAD-MS) method, and the content level in descending order was psoralen > bergapten > luteolin > apigenin. Chromatographic fingerprint similarity evaluation and cluster analysis were used to assess geographical origin of RFH, and the results revealed a high level of similarity for the tested RFH samples obtained from Hainan, Guangdong, Guangxi provinces and Hong Kong. 2, 2-Diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay was conducted to evaluate the antioxidant potencies of the four components, and the results clearly demonstrated that luteolin was most effective; apigenin exhibited a moderate potency, whereas psoralen and bergapten possessed little effect against free radical reactions. Structure-activity relationship of the components was elucidated, and the 3′-hydroxyl group of luteolin was found to be directly responsible for its antioxidant activity. Conclusion The present UPLC-PAD-MS method and DPPH radical scavenging assay performed well for the purpose of constituent quantification and antioxidant assay. Global profiles were highly similar for RFH samples from different origins. Both the coumarins and flavonoids were involved in the health benefit of RFH. PMID:23835498

  11. Dynamic of the ageing of ballpoint pen inks: quantification of phenoxyethanol by GC-MS.

    PubMed

    Lociciro, S; Dujourdy, L; Mazzella, W; Margot, P; Lock, E

    2004-01-01

    The ageing process of some inks were studied to evaluate whether it is possible to date them. We used gas chromatography coupled with mass spectrometry to measure the evaporation of volatile components. The selected approach thus follows the disappearance of one ink volatile component (phenoxyethanol) as a function of time. The ink ageing curve represents the ratio of an evaporating compound to a stable compound of ink according to time. The results obtained are thus independent of the quantity of ink sampled for analysis. We obtained for two pens, containing two different inks, a curve highlighting an exponential decrease of the evaporating compound. By fitting these curves we determined the limitations of dating a ballpoint pen ink. Two distinct behaviours were observed in two distinct modes, the first called 'fast mode' and the second called 'slow mode'. In order to try to explain the phenomenon, the studies were based on solvent diffusion theory in complex matrix (such as polymer on varnish). Calculations from certain parameters showed an extremely fast evaporation of ink solvents, as well as varying behaviour depending on the paper used. The results showed that it is not possible to date ballpoint pen inks with this method in document examination casework.

  12. Simultaneous quantification of three iridoids in rat plasma after oral administration of Zhi-zi-chi decoction using LC-MS.

    PubMed

    Long, Zhi-Min; Bi, Kai-Shun; Huo, Yan-Shuang; Yan, Xiao-Yu; Zhao, Xu; Chen, Xiao-Hui

    2011-10-01

    A sensitive and efficient liquid chromatography-mass spectrometry (LC-MS) method was developed and validated for the simultaneous determination of geniposide, 6α-hydroxygeniposide, and genipin gentiobioside in rat plasma. After the addition of internal standard (I.S.) salidroside and acidification (formic acid, 0.1%), plasma samples were carried out by protein precipitation with acetonitrile and separated on a Kromasil C(18) column (200 mm × 4.6 mm, 5 μm) within a runtime of 15.0 min. The linear ranges were 2-250 ng/mL for both 6α-hydroxygeniposide and genipin gentiobioside and 2-2000 ng/mL for geniposide, respectively. The lower limit of quantification (LLOQ) was 2 ng/mL for all the analytes. The validated method was successfully applied to the pharmacokinetics study of geniposide, 6α-hydroxygeniposide, and genipin gentiobioside in rats after oral administration of Zhi-zi-chi decoction.

  13. External calibration strategy for trace element quantification in botanical samples by LA-ICP-MS using filter paper.

    PubMed

    Nunes, Matheus A G; Voss, Mônica; Corazza, Gabriela; Flores, Erico M M; Dressler, Valderi L

    2016-01-28

    The use of reference solutions dispersed on filter paper discs is proposed for the first time as an external calibration strategy for matrix matching and determination of As, Cd, Co, Cr, Cu, Mn, Ni, Pb, Sr, V and Zn in plants by laser ablation-inductively coupled plasma mass spectrometry (LA-ICP-MS). The procedure is based on the use of filter paper discs as support for aqueous reference solutions, which are further evaporated, resulting in solid standards with concentrations up to 250 μg g(-1) of each element. The use of filter paper for calibration is proposed as matrix matched standards due to the similarities of this material with botanical samples, regarding to carbon concentration and its distribution through both matrices. These characteristics allowed the use of (13)C as internal standard (IS) during the analysis by LA-ICP-MS. In this way, parameters as analyte signal normalization with (13)C, carrier gas flow rate, laser energy, spot size, and calibration range were monitored. The calibration procedure using solution deposition on filter paper discs resulted in precision improvement when (13)C was used as IS. The method precision was calculated by the analysis of a certified reference material (CRM) of botanical matrix, considering the RSD obtained for 5 line scans and was lower than 20%. Accuracy of LA-ICP-MS determinations were evaluated by analysis of four CRM pellets of botanical composition, as well as by comparison with results obtained by ICP-MS using solution nebulization after microwave assisted digestion. Plant samples of unknown elemental composition were analyzed by the proposed LA method and good agreement were obtained with results of solution analysis. Limits of detection (LOD) established for LA-ICP-MS were obtained by the ablation of 10 lines on the filter paper disc containing 40 μL of 5% HNO3 (v v(-1)) as calibration blank. Values ranged from 0.05 to 0.81  μg g(-1). Overall, the use of filter paper as support for dried aqueous

  14. External calibration strategy for trace element quantification in botanical samples by LA-ICP-MS using filter paper.

    PubMed

    Nunes, Matheus A G; Voss, Mônica; Corazza, Gabriela; Flores, Erico M M; Dressler, Valderi L

    2016-01-28

    The use of reference solutions dispersed on filter paper discs is proposed for the first time as an external calibration strategy for matrix matching and determination of As, Cd, Co, Cr, Cu, Mn, Ni, Pb, Sr, V and Zn in plants by laser ablation-inductively coupled plasma mass spectrometry (LA-ICP-MS). The procedure is based on the use of filter paper discs as support for aqueous reference solutions, which are further evaporated, resulting in solid standards with concentrations up to 250 μg g(-1) of each element. The use of filter paper for calibration is proposed as matrix matched standards due to the similarities of this material with botanical samples, regarding to carbon concentration and its distribution through both matrices. These characteristics allowed the use of (13)C as internal standard (IS) during the analysis by LA-ICP-MS. In this way, parameters as analyte signal normalization with (13)C, carrier gas flow rate, laser energy, spot size, and calibration range were monitored. The calibration procedure using solution deposition on filter paper discs resulted in precision improvement when (13)C was used as IS. The method precision was calculated by the analysis of a certified reference material (CRM) of botanical matrix, considering the RSD obtained for 5 line scans and was lower than 20%. Accuracy of LA-ICP-MS determinations were evaluated by analysis of four CRM pellets of botanical composition, as well as by comparison with results obtained by ICP-MS using solution nebulization after microwave assisted digestion. Plant samples of unknown elemental composition were analyzed by the proposed LA method and good agreement were obtained with results of solution analysis. Limits of detection (LOD) established for LA-ICP-MS were obtained by the ablation of 10 lines on the filter paper disc containing 40 μL of 5% HNO3 (v v(-1)) as calibration blank. Values ranged from 0.05 to 0.81  μg g(-1). Overall, the use of filter paper as support for dried aqueous

  15. Simultaneous quantification of vortioxetine, carvedilol and its active metabolite 4-hydroxyphenyl carvedilol in rat plasma by UPLC-MS/MS: Application to their pharmacokinetic interaction study.

    PubMed

    Huang, Yi; Zheng, Shuangli; Pan, Yongyang; Li, Tao; Xu, Zhi-Sheng; Shao, Meng-Meng

    2016-09-01

    To establish a rapid and sensitive ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method for the determination of vortioxetine, carvedilol and its metabolite 4-hydroxyphenyl carvedilol in rat plasma. The analytes and the internal standard (diazepam) were separated on an Acquity UPLC BEH C18 chromatography column (2.1mm×50mm, 1.7μm) using gradient elution with a mobile phase of acetonitrile and 0.1% formic acid in water at a flow rate of 0.4mL/min. The detection was performed on a triple quadrupole tandem mass spectrometer by multiple reaction monitoring (MRM) mode to monitor the precursor-to-product ion transitions of m/z 299.2→150.1 for vortioxetine, m/z 407.2→100.3 for carvedilol, m/z 423.2→100.1 for 4-hydroxyphenyl carvedilol and m/z 285.2→193.1 for diazepam (IS) using a positive electrospray ionization interface. The method was validated over a concentration range of 0.5-100ng/mL for vortioxetine, 0.5-1000ng/mL for carvedilol and 0.1-50ng/mL for 4-hydroxyphenyl carvedilol. Total time for each chromatograph was 3.0min. The intra- and inter-day precision and accuracy of the quality control samples at low, medium, and high concentration levels exhibited relative standard deviations (RSD)<11.6% and the accuracy values ranged from -12.2% to 11.3%. The analytical method was successfully applied to a pharmacokinetic interaction study of vortioxetine and carvedilol after oral administration vortioxetine and carvedilol in rats. Results suggested that the co-administration of vortioxetine and carvedilol results in a significant drug interaction in rats. PMID:27262994

  16. Determination of sotolon content in South African white wines by two novel HPLC-UV and UPLC-MS methods.

    PubMed

    Gabrielli, Mario; Buica, Astrid; Fracassetti, Daniela; Stander, Marietjie; Tirelli, Antonio; du Toit, Wessel J

    2015-02-15

    Sotolon has been reported to play an important role in the atypical ageing and aroma character of many wines. A number of analytical techniques for sotolon analysis in wine have been reported, but these often require extensive sample preparation. In this work we report a HPLC-UV method and a novel UPLC-MS method to determine sotolon concentrations in white wines with little sample preparation applied for the first time for the evaluation of sotolon levels in South African wines. The validation showed that the instrumental methods had good accuracy, repeatability and linearity, but the UPLC-MS method proved more sensitive. For both methods, quantification limits were lower than the sotolon odour threshold in wine (10μg/L), 0.86μg/L and 0.013μg/L, for HPLC-UV and UPLC-MS methods, respectively. Sotolon levels in 65 South African white wines were often found to be lower than the reported odour threshold, with the highest concentration being 9.11μg/L. However, for low levels (<1μg/L), unknown interferences in certain wines led to sotolon not being quantified with the HPLC-UV method, which made the UPLC-MS method more suitable.

  17. A Comparative Analysis of Computational Approaches to Relative Protein Quantification Using Peptide Peak Intensities in Label-free LC-MS Proteomics Experiments

    SciTech Connect

    Matzke, Melissa M.; Brown, Joseph N.; Gritsenko, Marina A.; Metz, Thomas O.; Pounds, Joel G.; Rodland, Karin D.; Shukla, Anil K.; Smith, Richard D.; Waters, Katrina M.; McDermott, Jason E.; Webb-Robertson, Bobbie-Jo M.

    2013-02-01

    Liquid chromatography coupled with mass spectrometry (LC-MS) is widely used to identify and quantify peptides in complex biological samples. In particular, label-free shotgun proteomics is highly effective for the identification of peptides and subsequently obtaining a global protein profile of a sample. As a result, this approach is widely used for discovery studies. Typically, the objective of these discovery studies is to identify proteins that are affected by some condition of interest (e.g. disease, exposure). However, for complex biological samples, label-free LC-MS proteomics experiments measure peptides and do not directly yield protein quantities. Thus, protein quantification must be inferred from one or more measured peptides. In recent years, many computational approaches to relative protein quantification of label-free LC-MS data have been published. In this review, we examine the most commonly employed quantification approaches to relative protein abundance from peak intensity values, evaluate their individual merits, and discuss challenges in the use of the various computational approaches.

  18. A Method for Simultaneous Determination of 25-Hydroxyvitamin D3 and Its 3-Sulfate in Newborn Plasma by LC/ESI-MS/MS after Derivatization with a Proton-Affinitive Cookson-Type Reagent.

    PubMed

    Higashi, Tatsuya; Yokota, Mai; Goto, Ayaka; Komatsu, Kenji; Sugiura, Takahiro; Ogawa, Shoujiro; Satoh, Mamoru; Nomura, Fumio

    2016-01-01

    A method for the simultaneous determination of 25-hydroxyvitamin D3 [25(OH)D3] and its 3-sulfate [25(OH)D3S] in newborn plasma, which is expected to be helpful in the assessment of the vitamin D status, using stable isotope-dilution liquid chromatography/electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS) has been developed and validated. The plasma was pretreated based on the deproteinization and solid-phase extraction, then subjected to derivatization with 4-(4-dimethylaminophenyl)-1,2,4-triazoline-3,5-dione (DAPTAD). The derivatization enabled the accurate quantification of 25(OH)D3 without interference from 3-epi-25(OH)D3 and also facilitated the simultaneous determination of the two metabolites by LC/positive ESI-MS/MS. Quantification was based on the selected reaction monitoring with the characteristic fragmentation of the DAPTAD-derivatives during MS/MS. This method was reproducible (intra- and inter-assay relative standard deviations of 7.8% or lower for both metabolites) and accurate (analytical recovery, 95.4-105.6%). The limits of quantification were 1.0 ng/mL and 2.5 ng/mL for 25(OH)D3 and 25(OH)D3S, respectively, when using a 20-μL sample. The developed method was applied to the simultaneous determination of plasma 25(OH)D3 and 25(OH)D3S in newborns; it was recognized that the plasma concentration of 25(OH)D3S is significantly higher than that of 25(OH)D3, and preterm newborns have lower plasma 25(OH)D3S concentrations than full-term newborns. PMID:27656337

  19. A Method for Simultaneous Determination of 25-Hydroxyvitamin D3 and Its 3-Sulfate in Newborn Plasma by LC/ESI-MS/MS after Derivatization with a Proton-Affinitive Cookson-Type Reagent

    PubMed Central

    Higashi, Tatsuya; Yokota, Mai; Goto, Ayaka; Komatsu, Kenji; Sugiura, Takahiro; Ogawa, Shoujiro; Satoh, Mamoru; Nomura, Fumio

    2016-01-01

    A method for the simultaneous determination of 25-hydroxyvitamin D3 [25(OH)D3] and its 3-sulfate [25(OH)D3S] in newborn plasma, which is expected to be helpful in the assessment of the vitamin D status, using stable isotope-dilution liquid chromatography/electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS) has been developed and validated. The plasma was pretreated based on the deproteinization and solid-phase extraction, then subjected to derivatization with 4-(4-dimethylaminophenyl)-1,2,4-triazoline-3,5-dione (DAPTAD). The derivatization enabled the accurate quantification of 25(OH)D3 without interference from 3-epi-25(OH)D3 and also facilitated the simultaneous determination of the two metabolites by LC/positive ESI-MS/MS. Quantification was based on the selected reaction monitoring with the characteristic fragmentation of the DAPTAD-derivatives during MS/MS. This method was reproducible (intra- and inter-assay relative standard deviations of 7.8% or lower for both metabolites) and accurate (analytical recovery, 95.4–105.6%). The limits of quantification were 1.0 ng/mL and 2.5 ng/mL for 25(OH)D3 and 25(OH)D3S, respectively, when using a 20-μL sample. The developed method was applied to the simultaneous determination of plasma 25(OH)D3 and 25(OH)D3S in newborns; it was recognized that the plasma concentration of 25(OH)D3S is significantly higher than that of 25(OH)D3, and preterm newborns have lower plasma 25(OH)D3S concentrations than full-term newborns. PMID:27656337

  20. A Method for Simultaneous Determination of 25-Hydroxyvitamin D3 and Its 3-Sulfate in Newborn Plasma by LC/ESI-MS/MS after Derivatization with a Proton-Affinitive Cookson-Type Reagent

    PubMed Central

    Higashi, Tatsuya; Yokota, Mai; Goto, Ayaka; Komatsu, Kenji; Sugiura, Takahiro; Ogawa, Shoujiro; Satoh, Mamoru; Nomura, Fumio

    2016-01-01

    A method for the simultaneous determination of 25-hydroxyvitamin D3 [25(OH)D3] and its 3-sulfate [25(OH)D3S] in newborn plasma, which is expected to be helpful in the assessment of the vitamin D status, using stable isotope-dilution liquid chromatography/electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS) has been developed and validated. The plasma was pretreated based on the deproteinization and solid-phase extraction, then subjected to derivatization with 4-(4-dimethylaminophenyl)-1,2,4-triazoline-3,5-dione (DAPTAD). The derivatization enabled the accurate quantification of 25(OH)D3 without interference from 3-epi-25(OH)D3 and also facilitated the simultaneous determination of the two metabolites by LC/positive ESI-MS/MS. Quantification was based on the selected reaction monitoring with the characteristic fragmentation of the DAPTAD-derivatives during MS/MS. This method was reproducible (intra- and inter-assay relative standard deviations of 7.8% or lower for both metabolites) and accurate (analytical recovery, 95.4–105.6%). The limits of quantification were 1.0 ng/mL and 2.5 ng/mL for 25(OH)D3 and 25(OH)D3S, respectively, when using a 20-μL sample. The developed method was applied to the simultaneous determination of plasma 25(OH)D3 and 25(OH)D3S in newborns; it was recognized that the plasma concentration of 25(OH)D3S is significantly higher than that of 25(OH)D3, and preterm newborns have lower plasma 25(OH)D3S concentrations than full-term newborns.

  1. A Method for Simultaneous Determination of 25-Hydroxyvitamin D3 and Its 3-Sulfate in Newborn Plasma by LC/ESI-MS/MS after Derivatization with a Proton-Affinitive Cookson-Type Reagent.

    PubMed

    Higashi, Tatsuya; Yokota, Mai; Goto, Ayaka; Komatsu, Kenji; Sugiura, Takahiro; Ogawa, Shoujiro; Satoh, Mamoru; Nomura, Fumio

    2016-01-01

    A method for the simultaneous determination of 25-hydroxyvitamin D3 [25(OH)D3] and its 3-sulfate [25(OH)D3S] in newborn plasma, which is expected to be helpful in the assessment of the vitamin D status, using stable isotope-dilution liquid chromatography/electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS) has been developed and validated. The plasma was pretreated based on the deproteinization and solid-phase extraction, then subjected to derivatization with 4-(4-dimethylaminophenyl)-1,2,4-triazoline-3,5-dione (DAPTAD). The derivatization enabled the accurate quantification of 25(OH)D3 without interference from 3-epi-25(OH)D3 and also facilitated the simultaneous determination of the two metabolites by LC/positive ESI-MS/MS. Quantification was based on the selected reaction monitoring with the characteristic fragmentation of the DAPTAD-derivatives during MS/MS. This method was reproducible (intra- and inter-assay relative standard deviations of 7.8% or lower for both metabolites) and accurate (analytical recovery, 95.4-105.6%). The limits of quantification were 1.0 ng/mL and 2.5 ng/mL for 25(OH)D3 and 25(OH)D3S, respectively, when using a 20-μL sample. The developed method was applied to the simultaneous determination of plasma 25(OH)D3 and 25(OH)D3S in newborns; it was recognized that the plasma concentration of 25(OH)D3S is significantly higher than that of 25(OH)D3, and preterm newborns have lower plasma 25(OH)D3S concentrations than full-term newborns.

  2. Application of a validated LC-MS/MS method for JWH-073 and its metabolites in blood and urine in real forensic cases.

    PubMed

    Ozturk, Serkan; Ozturk, Yeter Erol; Yeter, Oya; Alpertunga, Buket

    2015-12-01

    Synthetic cannabinoids, which were synthesized to improve the therapeutic effects of cannabis, have become a major issue when they are abused. They have different chemical structures from tetrahydrocannabinol (THC) but similar effects on endocannabinoid receptors. "Spice" named products have more serious side effects than cannabis and can even cause death. These mixtures are prepared by spraying chemicals onto small pieces of herbs and are being dishonestly sold as "natural" and "legal" products over the internet. Their popularity is continuously increasing. Studies on detecting synthetic cannabinoids in biological samples as well as pharmacology and toxicology studies of these chemicals are very limited. A fast, specific and robust method for the detection and quantification of JWH-073, JWH-073 N-butanoic acid, and JWH-073 N-(4-hydroxybutyl) in blood and urine has been developed that uses solid-phase extraction (SPE) followed by UPLC-MS/MS analysis. This method has been validated in terms of its linearity (0.1-50 ng/mL), selectivity, intra-assay and inter-assay accuracy and precision (CV<10%), recovery (75-95%), limits of detection (LODs) (0.08-0.13 ng/mL), and limits of quantification (LOQs) (0.11-0.17 ng/mL). Matrix effects, stability, and process efficiency parameters of this method have also been assessed. This method was applied to 2596 authentic samples received by the Department of Toxicology (Istanbul) in the Presidency of Council of Forensic Medicine (Turkey) between September 1, 2012, and February 28, 2015. PMID:26360591

  3. Asymmetric Flow-Field Flow Fractionation Hyphenated ICP-MS as an Alternative to Cloud Point Extraction for Quantification of Silver Nanoparticles and Silver Speciation: Application for Nanoparticles with a Protein Corona.

    PubMed

    Mudalige, Thilak K; Qu, Haiou; Linder, Sean W

    2015-07-21

    Production and application of nanoparticles in consumer products is at an all-time high due to the emerging field of nanotechnology. Direct detection and quantification of trace levels of nanoparticles within consumer products is very challenging and problematic. Although multiple methodologies are available for this purpose, each method has its own set of limitations. Herein, we developed an analytical platform consisting of asymmetric flow-field flow fractionation (AF4) coupled with inductively coupled plasma mass spectroscopy (ICP-MS) for the speciation and quantification of silver ions and silver nanoparticles at the ng/kg level (ppt). AF4 is utilized to concentrate the nanoparticles, and ICP-MS acts as the detector. The protein corona that forms upon exposure of nanoparticles to bovine serum albumin was utilized as a nanoparticle stabilization and AF4 recovery enhancement mechanism. Speciation of silver ions and nanoparticles was achieved with the assistance of penicillamine as a complexation ligand. The effect of nanoparticle size, surface coating, and ionization state toward the detection and quantification of the developed methodology was evaluated. The detection limit was found to be 4 ng/kg with the application of a 5 mL sample loop. Further application of this developed methodology on environmentally relevant samples was demonstrated by the analysis of Arkansas River water spiked with silver nanoparticles and nanoparticle spiked into humic acid solution (50 mg/L) at an environmentally relevant level.

  4. An experimental database for evaluating PIV uncertainty quantification methods

    NASA Astrophysics Data System (ADS)

    Warner, Scott; Neal, Douglas; Sciacchitano, Andrea

    2014-11-01

    Uncertainty quantification for particle image velocimetry (PIV) data has recently become a topic of great interest as shown by the publishing of several different methods within the past few years. A unique experiment has been designed to test the efficacy of PIV uncertainty methods, using a rectangular jet as the flow field. The novel aspect of the experimental setup consists of simultaneous measurements by means of two different time-resolved PIV systems and a hot-wire anemometer (HWA). The first PIV system, called the ``PIV-Measurement'' system, collects the data for which uncertainty is to be evaluated. It is based on a single camera and features a dynamic velocity range (DVR) representative of many PIV experiments. The second PIV system, called the ``PIV-HDR'' (high dynamic range) system, has a significantly higher DVR obtained with a higher digital imaging resolution. The hot-wire was placed in close proximity to the PIV measurement domain. All three of the measurement systems were carefully set to simultaneously collect time-resolved data on a point-by-point basis. The HWA validates the PIV-HDR system as the reference velocity so that it can be used to evaluate the instantaneous error in the PIV-measurement system.

  5. LC-MS/MS method for the simultaneous determination of PA-824, moxifloxacin and pyrazinamide in rat plasma and its application to pharmacokinetic study.

    PubMed

    Wang, Libin; Xu, Yue; Liang, Li; Diao, Chunyan; Liu, Xueying; Zhang, Jianchun; Zhang, Shengyong

    2014-08-01

    A simple, sensitive and rapid LC-MS/MS method has been developed and validated for simultaneous determination of PA-824, moxifloxacin, and pyrazinamide in rat plasma using metronidazole as internal standard. Sample preparation involved a simple one-step protein precipitation with methanol, followed by centrifugation and evaporation of the organic solvent. The residue was redissolved in mobile phase and analyzed by LC-MS/MS. An Inertsil(®) ODS3 C18 column (150mm×4.6mm, 5μm), a mobile phase composed of methanol-0.03% TEA (triethylamine) in water (85:15, v/v), and a flow rate of 0.5mL/min were employed, and the total run time was 6.0min. The mass spectrometer was run in positive ion ESI-APCI combined mode using multiple reaction monitoring (MRM) to monitor the mass transitions. The method was validated for accuracy, precision, linearity, range, selectivity, lower limit of quantification (LLOQ), recovery, and matrix effect. All validation parameters met the acceptance criteria according to regulatory guidelines. The LLOQ was 1.0μg/mL for pyrazinamide and 0.1μg/mL for PA-824 and moxifloxacin. The recoveries obtained for PA-824, moxifloxacin and pyrazinamide were ≥85%. Intra-day and inter-day coefficients of variation were less than 10%. The method had been successfully applied to a pharmacokinetic study of fixed dose administration of PA-824, moxifloxacin, pyrazinamide and their combination in SD rat. Significant differences of Tmax, Cmax, AUC(0-t) and CLz/F were observed between the single and combined groups after equal dose of PA-824 and moxifloxacin administration, which revealed the possibility of drug-drug interaction (DDI) between the PaMZ combination. PMID:24798753

  6. A method of analysis for T-2 toxin and neosolaniol by UPLC-MS/MS in apple fruit inoculated with Trichothecium roseum.

    PubMed

    Tang, Yamei; Xue, Huali; Bi, Yang; Li, Yongcai; Wang, Yi; Zhao, Ying; Shen, Keping

    2015-01-01

    Trichothecenes are one of the most important groups of mycotoxins produced by Trichothecium roseum, which causes core rot of apple. A reliable and sensitive method was developed and successfully applied for the rapid detection of trichothecenes including T-2 toxin and neosolaniol in harvested apple using UPLC-MS/MS. After the extraction of the two mycotoxins from the apple matrix with methanol/water (80/20, v/v), the concentrated extracts were cleaned-up by PriboFast M270 columns and then analysed by UPLC-MS/MS. T-2 toxin and neosolaniol were effectively separated as unique peaks. The validity of this method was established by its linearity (R(2) ≥ 0.9995), precision (relative standard deviation ≤ 3.6%), accuracy, selectivity, limit of detection of 2-5 μg kg(-1), limit of quantification of 5-10 μg kg(-1) and average recovery of 73-96%. Levels of T-2 toxin were found in the range 7.1-128.4 µg kg(-1) in the core rot lesion of three cultivars apple (cvs. Red Delicious, Fuji and Ralls). T-2 was detected not only in the lesion, but also in the tissue without any disease symptoms. However, neosolaniol was only detected in the lesion on 'Red Delicious' apples. In addition, the concentration of T-2 toxin in the susceptible cultivar (cv. Fuji) was significantly higher than that in the resistant one (cv. Ralls). This method proved to be suitable at detecting T-2 and neosolaniol simultaneously in apples infected with T. roseum.

  7. Highly sensitive LC-MS/MS-ESI method for determination of phenelzine in human plasma and its application to a human pharmacokinetic study.

    PubMed

    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. PMID:27085800

  8. Data-independent liquid chromatography/mass spectrometry (LC/MS(E)) detection and quantification of the secreted Apium graveolens pathogen defense protein mannitol dehydrogenase.

    PubMed

    Blackburn, Kevin; Cheng, Fang-Yi; Williamson, John D; Goshe, Michael B

    2010-04-15

    Plant cells secrete a wide variety of defense-related proteins into the extracellular space or apoplast in response to pathogen attack. One of these, mannitol dehydrogenase (MTD), is normally a cytoplasmic enzyme whose primary role is the regulation of intracellular levels of the sugar alcohol mannitol in plants. Recent immunological and biochemical evidence, however, suggests that MTD is also secreted into the apoplast in response to pathogen attack, despite lacking a known peptide signal sequence for Golgi-mediated secretion. Because many plant pathogenic fungi secrete mannitol to overcome pathogen-induced generation of reactive oxygen species (ROS) by the plant, extracellular localization of MTD is hypothesized to have a defensive role of catabolizing pathogen-secreted mannitol. In the current study, LC/MS(E) was used to analyze proteins in the secretome of Apium graveolens (celery) following treatment with salicylic acid (SA), an endogenous elicitor of defense responses in plants. Levels of MTD in the secretome of SA-treated celery cell cultures were found to be induced at least 18-fold over secretome samples from cell cultures not exposed to SA. This value is in close agreement with published immunological and biochemical observations. Overall, this report provides the first mass spectrometry identification and quantification measurements supporting the hypothesis that MTD is secreted in response to simulated pathogen attack via a non-classical secretion mechanism. As demonstrated with MTD secretion, LC/MS(E) can be implemented as a discovery-driven MRM-based quantitative approach which can be used to reveal potential post-translational modifications, thus providing a new method in the area of gel-free and label-free proteomic analysis.

  9. Quantification of camalexin, a phytoalexin from Arabidopsis thaliana: a comparison of five analytical methods.

    PubMed

    Beets, Caryn; Dubery, Ian

    2011-12-15

    Camalexin is a phytoalexin of Arabidopsis thaliana and an important component of inducible defenses. Accurate quantification of low concentrations suffers from interference by structurally related metabolites. A. thaliana plants were induced with silver nitrate and camalexin was extracted using methanol and identified and quantified by (i) TLC as a blue fluorescent band, (ii) microtiter plate-based fluorescence spectroscopy, (iii) GC on a midpolar column coupled to flame ionization detection, (iv) C(18) HPLC coupled to a photodiode detector, and (v) UPLC coupled to a mass spectrometer detector. Standard curves over the range of 0.1-15 μg ml(-1) gave R(2) values from 0.996 to 0.999. The different methods were compared and evaluated for their ability to detect and quantify increasing concentrations (<0.4-8 μgg(-1) FW) of camalexin. Each of the techniques presented advantages and disadvantages with regard to accuracy, precision, interference, analytical sensitivity, and limits of detection. TLC is a good qualitative technique for the identification of camalexin and fluorescence spectroscopy is subject to quenching when performed on crude extracts. Comparable results were obtained with GC-FID, HPLC-PDA, and UPLC-MS, with UPLC-MS having the added advantage of short analysis times and detection based on accurate mass. PMID:21910963

  10. Development and validation of an LC-MS/MS method for the toxicokinetic study of deoxynivalenol and its acetylated derivatives in chicken and pig plasma.

    PubMed

    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.

  11. Nonpolar organic compounds in fine particles: quantification by thermal desorption-GC/MS and evidence for their significant oxidation in ambient aerosols in Hong Kong.

    PubMed

    Yu, Jian Zhen; Huang, X H Hilda; Ho, Steven S H; Bian, Qijing

    2011-12-01

    Nonpolar organic compounds (NPOCs) in ambient particulate matter (PM) commonly include n-alkanes, branched alkanes, hopanes and steranes, and polycyclic aromatic hydrocarbons (PAHs). The recent development of thermal desorption-gas chromatography/mass spectrometry (TD-GC/MS) has greatly reduced time and labor in their quantification by eliminating the laborious solvent extraction and sample concentration steps in the traditional approach that relies on solvent extraction. The simplicity of the TD-GCMS methods has afforded us concentration data of NPOCs in more than 90 aerosol samples in two aerosol field studies and 20 vehicular emissions-dominated source samples in Hong Kong over the past few years. In this work, we examine the interspecies relationships between select NPOCs and their concentration ratios to elemental carbon (EC) among the ambient samples and among the source samples. Our analysis indicates that hopanes were mainly from vehicular emissions and they were significantly oxidized in ambient PM. The hopane/EC ratio in ambient samples was on average less than half of the ratio in vehicular emissions-dominated source samples. This highlights the necessity in considering oxidation loss in applying organic tracer data in source apportionment studies. Select PAH/EC ratio-ratio plots reveal that PAHs had diverse sources and vehicular emissions were unlikely a dominant source for PAHs in Hong Kong. Biomass burning and other regional sources likely dominated ambient PAHs in Hong Kong. PMID:21983947

  12. Determination of paraquat and diquat: LC-MS method optimization and validation.

    PubMed

    Pizzutti, Ionara R; Vela, Giovana M E; de Kok, André; Scholten, Jos M; Dias, Jonatan V; Cardoso, Carmem D; Concenço, Germani; Vivian, Rafael

    2016-10-15

    This study describes the optimization and single-laboratory validation of a single residue method for determination of two bipyridylium herbicides, paraquat and diquat, in cowpeas by UPLC-MS/MS in a total run time of 9.3min. The method is based on extraction with an acidified methanol-water mixture. Different extraction parameters (extraction solvent composition, temperature, sample extract filtration, and pre-treatment of the laboratory sample) were evaluated in order to optimize the extraction method efficiency. Isotopically labeled internal standards, Paraquat-D6 and Diquat-D4, were used and added to the test portions prior to extraction. The method validation was performed by analyzing spiked samples at three concentrations (10, 20 and 50μgkg(-1)), with seven replicates (n=7) for each concentration. Linearity (r(2)) of analytical curves, accuracy (trueness as recovery % and precision as RSD%), instrument and method limits of detection and quantification (LOD and LOQ) and matrix effects were determined. Average recoveries obtained for diquat were between 77 and 85% with RSD values ⩽20%, for all spike levels studied. On the other hand, paraquat showed average recoveries between 68 and 103% with RSDs in the range 14.4-25.4%. The method LOQ was 10 and 20μgkg(-1) for diquat and paraquat, respectively. The matrix effect was significant for both pesticides. Consequently, matrix-matched calibration standards and using isotopically labeled (IL) analogues as internal standards for the target analytes are required for application in routine analysis. The validated method was successfully applied for cowpea samples obtained from various field studies. PMID:27173559

  13. In vivo cell tracking and quantification method in adult zebrafish

    NASA Astrophysics Data System (ADS)

    Zhang, Li; Alt, Clemens; Li, Pulin; White, Richard M.; Zon, Leonard I.; Wei, Xunbin; Lin, Charles P.

    2012-03-01

    Zebrafish have become a powerful vertebrate model organism for drug discovery, cancer and stem cell research. A recently developed transparent adult zebrafish using double pigmentation mutant, called casper, provide unparalleled imaging power in in vivo longitudinal analysis of biological processes at an anatomic resolution not readily achievable in murine or other systems. In this paper we introduce an optical method for simultaneous visualization and cell quantification, which combines the laser scanning confocal microscopy (LSCM) and the in vivo flow cytometry (IVFC). The system is designed specifically for non-invasive tracking of both stationary and circulating cells in adult zebrafish casper, under physiological conditions in the same fish over time. The confocal imaging part in this system serves the dual purposes of imaging fish tissue microstructure and a 3D navigation tool to locate a suitable vessel for circulating cell counting. The multi-color, multi-channel instrument allows the detection of multiple cell populations or different tissues or organs simultaneously. We demonstrate initial testing of this novel instrument by imaging vasculature and tracking circulating cells in CD41: GFP/Gata1: DsRed transgenic casper fish whose thrombocytes/erythrocytes express the green and red fluorescent proteins. Circulating fluorescent cell incidents were recorded and counted repeatedly over time and in different types of vessels. Great application opportunities in cancer and stem cell researches are discussed.

  14. Direct Quantification of Rare Earth Elements Concentrations in Urine of Workers Manufacturing Cerium, Lanthanum Oxide Ultrafine and Nanoparticles by a Developed and Validated ICP-MS.

    PubMed

    Li, Yan; Yu, Hua; Zheng, Siqian; Miao, Yang; Yin, Shi; Li, Peng; Bian, Ying

    2016-03-22

    Rare earth elements (REEs) have undergone a steady spread in several industrial, agriculture and medical applications. With the aim of exploring a sensitive and reliable indicator of estimating exposure level to REEs, a simple, accurate and specific ICP-MS method for simultaneous direct quantification of 15 REEs ((89)Y, (139)La, (140)Ce, (141)Pr, (146)Nd, (147)Sm, (153)Eu, (157)Gd, (159)Tb, (163)Dy, (165)Ho, (166)Er, (169)Tm, (172)Yb and (175)Lu) in human urine has been developed and validated. The method showed good linearity for all REEs in human urine in the concentrations ranging from 0.001-1.000 μg ∙ L(-1) with r² > 0.997. The limits of detection and quantification for this method were in the range of 0.009-0.010 μg ∙ L(-1) and 0.029-0.037 μg ∙ L(-1), the recoveries on spiked samples of the 15 REEs ranged from 93.3% to 103.0% and the relative percentage differences were less than 6.2% in duplicate samples, and the intra- and inter-day variations of the analysis were less than 1.28% and less than 0.85% for all REEs, respectively. The developed method was successfully applied to the determination of 15 REEs in 31 urine samples obtained from the control subjects and the workers engaged in work with manufacturing of ultrafine and nanoparticles containing cerium and lanthanum oxide. The results suggested that only the urinary levels of La (1.234 ± 0.626 μg ∙ L(-1)), Ce (1.492 ± 0.995 μg ∙ L(-1)), Nd (0.014 ± 0.009 μg ∙ L(-1)) and Gd (0.023 ± 0.010 μg ∙ L(-1)) among the exposed workers were significantly higher (p < 0.05) than the levels measured in the control subjects. From these, La and Ce were the primary components, and accounted for 88% of the total REEs. Lanthanum comprised 27% of the total REEs while Ce made up the majority of REE content at 61%. The remaining elements only made up 1% each, with the exception of Dy which was not detected. Comparison with the previously published data, the levels of urinary La and Ce in workers and

  15. Direct Quantification of Rare Earth Elements Concentrations in Urine of Workers Manufacturing Cerium, Lanthanum Oxide Ultrafine and Nanoparticles by a Developed and Validated ICP-MS

    PubMed Central

    Li, Yan; Yu, Hua; Zheng, Siqian; Miao, Yang; Yin, Shi; Li, Peng; Bian, Ying

    2016-01-01

    Rare earth elements (REEs) have undergone a steady spread in several industrial, agriculture and medical applications. With the aim of exploring a sensitive and reliable indicator of estimating exposure level to REEs, a simple, accurate and specific ICP-MS method for simultaneous direct quantification of 15 REEs (89Y, 139La, 140Ce, 141Pr, 146Nd, 147Sm, 153Eu, 157Gd, 159Tb, 163Dy, 165Ho, 166Er, 169Tm, 172Yb and 175Lu) in human urine has been developed and validated. The method showed good linearity for all REEs in human urine in the concentrations ranging from 0.001–1.000 μg∙L−1 with r2 > 0.997. The limits of detection and quantification for this method were in the range of 0.009–0.010 μg∙L−1 and 0.029–0.037 μg∙L−1, the recoveries on spiked samples of the 15 REEs ranged from 93.3% to 103.0% and the relative percentage differences were less than 6.2% in duplicate samples, and the intra- and inter-day variations of the analysis were less than 1.28% and less than 0.85% for all REEs, respectively. The developed method was successfully applied to the determination of 15 REEs in 31 urine samples obtained from the control subjects and the workers engaged in work with manufacturing of ultrafine and nanoparticles containing cerium and lanthanum oxide. The results suggested that only the urinary levels of La (1.234 ± 0.626 μg∙L−1), Ce (1.492 ± 0.995 μg∙L−1), Nd (0.014 ± 0.009 μg∙L−1) and Gd (0.023 ± 0.010 μg∙L−1) among the exposed workers were significantly higher (p < 0.05) than the levels measured in the control subjects. From these, La and Ce were the primary components, and accounted for 88% of the total REEs. Lanthanum comprised 27% of the total REEs while Ce made up the majority of REE content at 61%. The remaining elements only made up 1% each, with the exception of Dy which was not detected. Comparison with the previously published data, the levels of urinary La and Ce in workers and the control subjects show a higher trend

  16. Development and validation of a LC-MS/MS method for determination of pinoresinol diglucoside in rat plasma: Application to pharmacokinetic study.

    PubMed

    Song, Yanqing; Yan, Huiyu; Sun, Zhihui; Teng, Shiyong; Sun, Lirui; Zhang, Sixi

    2015-11-01

    Pinoresinol diglucoside (PD), a typical marker compound in Ecommia ulmoides Oliv., is an important and natural antihypertensive drug. A selective, sensitive, and rapid liquid chromatography tandem mass spectrometric (LC-MS/MS) analytical method was developed for the determination of PD in rats. After simple protein precipitation with acetonitrile, chromatographic separation of PD was conducted using a reversed-phase ZORBAX SB C18 analytical column (4.6mm × 150 mm, 5 μm particles) with a mobile phase of 10mM ammonium acetate-methanol-acetic acid (50:50:0.15, v/v/v) and quantified by selected reaction monitoring mode under positive electrospray ionization condition. The chromatographic run time was 3.4 min for each sample, in which the retention times of PD and the internal standard were 2.87 and 2.65 min, respectively. The calibration curves were linear over the range of 1.00~3000 ng/mL and the lower limit of quantification was 1.00 ng/mL in rat plasma. The precision expressed by relative standard deviations were <8.9% for intra-batch precision and <2.0% for inter-batch precision, and the intra- and inter-batch accuracy by relative error was within the range of -3.9% ~7.3%, which met acceptable criteria. The LC-MS/MS method was successfully applied to investigate the pharmacokinetics and oral bioavailability of PD in rats, with the bioavailability being only 2.5%.

  17. A rapid method for the simultaneous quantification of the major tocopherols, carotenoids, free and esterified sterols in canola (Brassica napus) oil using normal phase liquid chromatography.

    PubMed

    Flakelar, Clare L; Prenzler, Paul D; Luckett, David J; Howitt, Julia A; Doran, Gregory

    2017-01-01

    A normal phase high performance liquid chromatography (HPLC) method was developed to simultaneously quantify several prominent bioactive compounds in canola oil vis. α-tocopherol, γ-tocopherol, δ-tocopherol, β-carotene, lutein, β-sitosterol, campesterol and brassicasterol. The use of sequential diode array detection (DAD) and tandem mass spectrometry (MS/MS) allowed direct injection of oils, diluted in hexane without derivatisation or saponification, greatly reducing sample preparation time, and permitting the quantification of both free sterols and intact sterol esters. Further advantages over existing methods included increased analytical selectivity, and a chromatographic run time substantially less than other reported normal phase methods. The HPLC-DAD-MS/MS method was applied to freshly extracted canola oil samples as well as commercially available canola, palm fruit, sunflower and olive oils. PMID:27507459

  18. Functionalized quantum dots with dopamine dithiocarbamate as the matrix for the quantification of efavirenz in human plasma and as affinity probes for rapid identification of microwave tryptic digested proteins in MALDI-TOF-MS.

    PubMed

    Kailasa, Suresh Kumar; Wu, Hui-Fen

    2012-06-01

    Functionalized quantum dots with dopamine dithiocarbamate (QDs-DDTC) were utilized for the first time as an efficient material for the quantification of efavirenz in human plasma of HIV infected patients and rapid identification of microwave tryptic digest proteins (cytochrome c, lysozyme and BSA) by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The synthesized QDs-DDTC was characterized by using spectroscopic (UV-visible, FT-IR and (1)H NMR) and microscopic (SEM and TEM) techniques. Functionalized QDs-DDTC exhibited a high desorption/ionization efficiency for the rapid quantification of small molecules (efavirenz, tobramycin and aspartame) at low-mass region. QDs-DDTC has well ability to trap target species, and capable to transfer laser energy for efficient desorption/ionization of analytes with background-free detection. The use of QDs-DDTC as a matrix provided good linearity for the quantification of small molecules (R(2)=~0.9983), with good reproducibility (RSD<10%), in the analysis of efavirenz in the plasma of HIV infected patients by the standard addition method. We also demonstrated that the use of functionalized QDs-DDTC as affinity probes for the rapid identification of microwave tryptic digested proteins (cytochrome c, lysozyme and BSA) by MALDI-TOF-MS. QDs-DDTC-based MALDI-TOF-MS approach provides simplicity, rapidity, accuracy, and precision for the determination of efavirenz in human plasma of HIV infected patients and rapid identification of microwave tryptic digested proteins. This new material presents a marked advance in the development of matrix-free mass spectrometric methods for the rapid and precise quantitative determination of a variety of molecules. This article is part of a Special Issue entitled: Proteomics: The clinical link.

  19. Interplay between misfit strain relief and Stranski-Krastanov growth in fcc (111)/bcc (110) ultrathin film epitaxy. Part 2. Quantification using embedded atom method potentials

    NASA Astrophysics Data System (ADS)

    van der Merwe, J. H.; Tönsing, D. L.

    2000-03-01

    The main objective of this investigation has been to study the influence of misfit strain (MS) relief on the transition from monolayer-by-monolayer (ML-by-ML) Frank-van der Merwe (FM) to Stranski-Krastanov (SK) growth. The investigation involves (a) selection of appropriate materials [Cu, Pd, Pt on Fe(110)] to hopefully meet the appropriate stability criteria for misfit accommodation and bond strength, and of atomic interaction potentials [embedded atom method (EAM)] needed for quantification, and (b) development of appropriate analytical procedures to facilitate the description and quantification of the relevant parameters. The computations revealed that MS relief by misfit dislocations (MDs), for the selected materials, falls outside the SK transition thickness regime. The investigation has thus been limited to MS relief by a misfit vernier. The main objective of assessing the influence of MS relief on the transition to SK growth has been accomplished by showing that the growth mode discriminants, needed to predict quasi-equilibrium growth mode development, change with MS relief. The computation that culminated in the quantification of growth mode discriminants also yielded as by-product several interesting results of wider application in the field of epitaxy — the dependence on substrate proximity and bond strengths of: interlayer and intralayer relaxation, equilibrium misfit, critical misfit and thickness, substrate coverage change, optimum Fourier coefficients of periodic epilayer-substrate interaction potentials and stability of misfit accommodation and growth modes.

  20. Method validation for determination of heavy metals in wine and slightly alcoholic beverages by ICP-MS

    NASA Astrophysics Data System (ADS)

    Voica, Cezara; Dehelean, Adriana; Pamula, A.

    2009-08-01

    The Organisation International de la Vigne et du Vin (OIV) fixed an uppermost level for some heavy metals in wine. Consequently, the need to determine very low concentration of elements that may be present in wine in trace and ultra trace levels occurred. Inductively coupled plasma mass spectrometry ICP-MS is considered an excellent tool for detailed characterization of the elementary composition of many samples, including samples of drinks. In this study a method of quantitative analysis for the determination of toxic metals (Cr, As, Cd, Ni, Hg, Pb) in wines and slightly alcoholic beverages by ICP-MS was validated. Several parameters have been taken into account and evaluated for the validation of method, namely: linearity, the minimum detection limit, the limit of quantification, accuracy and uncertainty.

  1. A rapid and sensitive LC-MS/MS method for determination of lercanidipine in human plasma and its application in a bioequivalence study in Chinese healthy volunteers.

    PubMed

    Li, Xiaobing; Shi, Fuguo; He, Xiaojing; Jian, Lingyan; Ding, Li

    2016-09-01

    A rapid and highly sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed and validated for the determination of lercanidipine (LER) in human plasma. The plasma sample was deproteinized with methanol after addition of diazepam (internal standard, IS) and separated on a 38°C Hedera ODS-2 analytical column with a mobile phase of methanol and 5mM ammonium acetate buffer solution containing 0.1% formic acid at an isocratic flow rate of 400μL/min. The detection was performed on an API 4000 tandem mass spectrometer coupled with electrospray ionization (ESI) source in positive ESI mode. Quantification was conducted by multiple reaction monitoring (MRM) of the transitions of m/z 612.2→280.2 for LER and m/z 285.1→193.1 for IS, respectively. The method exhibited high sensitivity (LLOQ of 0.015ng/mL) and good linearity over the concentration range of 0.015-8.0ng/mL. No matrix effect and carry-over effect were observed. The values on both the occasions (intra- and inter-day) were all within 15% at three concentration levels. This robust method was successfully applied in a bioequivalence study to evaluate the pharmacokinetics of LER in 59 healthy male Chinese volunteers after a single oral administration of 10mg LER. PMID:27232153

  2. Development and validation of a RP-UHPLC-ESI-MS/MS method for the chiral separation and determination of flavanone, naringenin and hesperetin enantiomers.

    PubMed

    Baranowska, Irena; Hejniak, Judyta; Magiera, Sylwia

    2016-10-01

    A quick and sensitive RP-UHPLC-ESI-MS/MS method for the separation of flavanone, naringenin and hesperetin enantiomers was developed. The separation of analytes was performed using a Chiralpak AD-3R column, and methanol was used as the mobile phase. Detection was carried out using a triple quadrupole tandem mass spectrometer with an electrospray ionisation source. Positive ionisation and multiple reaction monitoring (MRM) were used. The developed method showed satisfactory linearity with determination coefficients greater than 0.996 in the concentration ranges of 2.5-100.0ngmL(-1) for naringenin and flavanone enantiomers and 0.5-100.0ngmL(-1) for hesperetin enantiomers. The limits of quantification varied from 0.1 to 2.0ngmL(-1). The intra-day and inter-day precisions were below 15%, and the accuracy varied from -13.6% to 13.5%. The described method was successfully applied for the chiral separation and determination of flavonoid enantiomers in real samples of spices and herbal root. Due to the occurrence of the natural compounds in the forms of free aglycones and their glycosides, these samples were subjected to hydrolysis in order to obtain free aglycones from the glycosylated forms. Acid and enzymatic hydrolysis techniques were also compared. In the course of this study, the enzymatic hydrolysis technique was selected.

  3. UHPLC/MS-MS Analysis of Six Neonicotinoids in Honey by Modified QuEChERS: Method Development, Validation, and Uncertainty Measurement.

    PubMed

    Proietto Galeano, Michele; Scordino, Monica; Sabatino, Leonardo; Pantò, Valentina; Morabito, Giovanni; Chiappara, Elena; Traulo, Pasqualino; Gagliano, Giacomo

    2013-01-01

    Rapid and reliable multiresidue analytical methods were developed and validated for the determination of 6 neonicotinoids pesticides (acetamiprid, clothianidin, imidacloprid, nitenpyram, thiacloprid, and thiamethoxam) in honey. A modified QuEChERS method has allowed a very rapid and efficient single-step extraction, while the detection was performed by UHPLC/MS-MS. The recovery studies were carried out by spiking the samples at two concentration levels (10 and 40 μg/kg). The methods were subjected to a thorough validation procedure. The mean recovery was in the range of 75 to 114% with repeatability below 20%. The limits of detection were below 2.5 μg/kg, while the limits of quantification did not exceed 4.0 μg/kg. The total uncertainty was evaluated taking the main independent uncertainty sources under consideration. The expanded uncertainty did not exceed 49% for the 10 μg/kg concentration level and was in the range of 16-19% for the 40 μg/kg fortification level. PMID:26904611

  4. A simple LC-MS/MS method for quantitative analysis of underivatized neurotransmitters in rats urine: assay development, validation and application in the CUMS rat model.

    PubMed

    Zhai, Xue-jia; Chen, Fen; Zhu, Chao-ran; Lu, Yong-ning

    2015-11-01

    Many amino acid neurotransmitters in urine are associated with chronic stress as well as major depressive disorders. To better understand depression, an analytical LC-MS/MS method for the simultaneous determination of 11 underivatized neurotransmitters (4-aminohippurate, 5-HIAA, glutamate, glutamine, hippurate, pimelate, proline, tryptophan, tyramine, tyrosine and valine) in a single analytical run was developed. The advantage of this method is the simple preparation in that there is no need to deconjugate the urine samples. The quantification range was 25-12,800 ng mL(-1) with >85.8% recovery for all analytes. The nocturnal urine concentrations of the 11 neurotransmitters in chronic unpredictable mild stress (CUMS) model rats and control group (n = 12) were analyzed. A series of significant changes in urinary excretion of neurotransmitters could be detected: the urinary glutamate, glutamine, hippurate and tyramine concentrations were significantly lower in the CUMS group. In addition, the urinary concentrations of tryptophan as well as tyrosine were significantly higher in chronically stressed rats. This method allows the assessment of the neurotransmitters associated with CUMS in rat urine in a single analytical run, making it suitable for implementation as a routine technique in depression research.

  5. Sewage-based epidemiology in monitoring the use of new psychoactive substances: Validation and application of an analytical method using LC-MS/MS.

    PubMed

    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.

  6. UHPLC/MS-MS Analysis of Six Neonicotinoids in Honey by Modified QuEChERS: Method Development, Validation, and Uncertainty Measurement

    PubMed Central

    Proietto Galeano, Michele; Scordino, Monica; Sabatino, Leonardo; Pantò, Valentina; Morabito, Giovanni; Chiappara, Elena; Traulo, Pasqualino; Gagliano, Giacomo

    2013-01-01

    Rapid and reliable multiresidue analytical methods were developed and validated for the determination of 6 neonicotinoids pesticides (acetamiprid, clothianidin, imidacloprid, nitenpyram, thiacloprid, and thiamethoxam) in honey. A modified QuEChERS method has allowed a very rapid and efficient single-step extraction, while the detection was performed by UHPLC/MS-MS. The recovery studies were carried out by spiking the samples at two concentration levels (10 and 40 μg/kg). The methods were subjected to a thorough validation procedure. The mean recovery was in the range of 75 to 114% with repeatability below 20%. The limits of detection were below 2.5 μg/kg, while the limits of quantification did not exceed 4.0 μg/kg. The total uncertainty was evaluated taking the main independent uncertainty sources under consideration. The expanded uncertainty did not exceed 49% for the 10 μg/kg concentration level and was in the range of 16–19% for the 40 μg/kg fortification level. PMID:26904611

  7. Development and validation of a RP-UHPLC-ESI-MS/MS method for the chiral separation and determination of flavanone, naringenin and hesperetin enantiomers.

    PubMed

    Baranowska, Irena; Hejniak, Judyta; Magiera, Sylwia

    2016-10-01

    A quick and sensitive RP-UHPLC-ESI-MS/MS method for the separation of flavanone, naringenin and hesperetin enantiomers was developed. The separation of analytes was performed using a Chiralpak AD-3R column, and methanol was used as the mobile phase. Detection was carried out using a triple quadrupole tandem mass spectrometer with an electrospray ionisation source. Positive ionisation and multiple reaction monitoring (MRM) were used. The developed method showed satisfactory linearity with determination coefficients greater than 0.996 in the concentration ranges of 2.5-100.0ngmL(-1) for naringenin and flavanone enantiomers and 0.5-100.0ngmL(-1) for hesperetin enantiomers. The limits of quantification varied from 0.1 to 2.0ngmL(-1). The intra-day and inter-day precisions were below 15%, and the accuracy varied from -13.6% to 13.5%. The described method was successfully applied for the chiral separation and determination of flavonoid enantiomers in real samples of spices and herbal root. Due to the occurrence of the natural compounds in the forms of free aglycones and their glycosides, these samples were subjected to hydrolysis in order to obtain free aglycones from the glycosylated forms. Acid and enzymatic hydrolysis techniques were also compared. In the course of this study, the enzymatic hydrolysis technique was selected. PMID:27474296

  8. UHPLC-MS/MS method for the determination of bisphenol A and its chlorinated derivatives, bisphenol S, parabens, and benzophenones in human urine samples.

    PubMed

    Vela-Soria, F; Ballesteros, O; Zafra-Gómez, A; Ballesteros, L; Navalón, A

    2014-06-01

    In the present work, a new method based on a sample treatment by dispersive liquid-liquid microextraction (DLLME) for the extraction of six bisphenols (bisphenol A, bisphenol S, and monochloro-, dichloro-, trichloro-, and tetrachlorobisphenol A), four parabens (methyl-, ethyl-, propyl-, and butylparaben), and six benzophenones (benzophenone-1, benzophenone-2, benzophenone-3, benzophenone-6, benzophenone-8, and 4-hydroxybenzophenone) in human urine samples, followed by ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) analysis, is validated. An enzymatic treatment allows determining the total content of the target EDCs. The extraction parameters were accurately optimized using multivariate optimization strategies. Ethylparaben ring-(13)C6, benzophenone-d10, and bisphenol A-d16 were used as surrogates. Limits of quantification ranging from 0.1 to 0.6 ng mL(-1) and interday variabilities (evaluated as relative standard deviations) from 2.0 to 13.8% were obtained. The method was validated using matrix-matched standard calibration followed by a recovery assay with spiked samples. Recovery rates ranged from 94 to 106%. A good linearity, for concentrations up to 300 ng mL(-1) for parabens and 40 ng mL(-1) for benzophenones and bisphenols, was also obtained. The method was satisfactorily applied for the determination of target compounds in human urine samples from 20 randomly selected individuals.

  9. Analysis of thyroid hormones in serum of Baikal seals and humans by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and immunoassay methods: application of the LC-MS/MS method to wildlife tissues.

    PubMed

    Kunisue, Tatsuya; Eguchi, Akifumi; Iwata, Hisato; Tanabe, Shinsuke; Kannan, Kurunthachalam

    2011-12-01

    Thyroid hormones (THs) are essential for the regulation of growth and development in both humans and wildlife. Until recently, TH concentrations in the tissues of animals have been examined by immunoassay (IA) methods. IA methods are sensitive, but for TH analysis, they are compromised by a lack of adequate specificity. In this study, we determined the concentrations of six THs, L-thyroxine (T(4)), 3,3',5-triiodo-L-thyronine (T(3)), 3,3',5'-triiodo-L-thyronine (rT(3)), 3,5-diiodo-L-thyronine (3,5-T(2)), 3,3'-diiodo-L-thyronine (3,3'-T(2)), and 3-iodo-L-thyronine (3-T(1)), in the serum of humans (n = 79) and wild Baikal seals (n = 37), by isotope ([(13)C(6)]-T(4))-dilution liquid chromatography (LC)-tandem mass spectrometry (MS/MS), and compared the TH levels with those measured by an electrochemiluminescent immunoassay (ECLIA) method. T(3) and T(4) were detected in all serum samples of both humans and Baikal seals, whereas T(1), 3,3'-T(2), and 3,5-T(2) were below the limit of detection (LOD). rT(3) was detected in Baikal seal sera at concentrations higher than T(3) in 28 seal samples, indicating an anomaly in deiodinase activity in Baikal seals. In humans, regression analyses of TH concentrations, measured by ECLIA and LC-MS/MS methods, showed significant correlations for T(4) (r = 0.852) and T(3) (r = 0.676; after removal of a serum sample with abnormal T(3) levels). In Baikal seals, a low correlation coefficient (r = 0.466) for T(4) levels and no correlation for T(3) levels (p = 0.093) were found between ECLIA and LC-MS/MS methods. These results suggest that interference by a nonspecific reaction against anti-T(3) and anti-T(4) antibodies used in the ECLIA can contribute to inaccuracies in TH measurement in Baikal seals. When the relationship between concentrations of THs in sera and dioxin-like toxic equivalents in blubber samples of Baikal seals (n = 19) was examined, a significantly negative correlation was found for serum T(4) levels measured by the LC-MS/MS

  10. Validated method to measure yakuchinone A in plasma by LC-MS/MS and its application to a pharmacokinetic study in rats

    PubMed Central

    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

  11. Simultaneous quantification of amphetamine and methamphetamine in meconium using ISOLUTE HM-N-supported liquid extraction columns and GC-MS.

    PubMed

    Gunn, Joshua A; Sweeney, Brenda; Dahn, Timothy; Bell, Suzanne; Newhouse, Rebecca; Terrell, Andrea R

    2008-09-01

    A procedure is described for the rapid extraction and quantification of amphetamine and methamphetamine from meconium using ISOLUTE HM-N-supported liquid extraction columns and gas chromatography-mass spectrometry (GC-MS). Because of the matrix complexity of meconium samples, extraction and sample preparation prior to instrumental analysis can prove difficult and time-consuming. The present study introduces a novel sample preparation technique for the simultaneous quantification of amphetamine and methamphetamine in meconium using GC-MS. Extraction of both analytes was achieved using ISOLUTE HM-N-supported liquid extraction columns containing a modified form of diatomaceous earth. Limits of detection for both analytes were 30 ng/g and the lower limit of quantitation was 75 ng/g. Linearity was achieved over the range 75-3000 ng/g. The methodology showed excellent intrarun precision with %CV values ranging from 2 to 8% for both analytes. Interrun precision experiments produced %CV values between 7 and 10% for both analytes. The reported methodology proved suitable for the accurate quantification of amphetamine and methamphetamine in meconium samples and greatly reduced the sample preparation time normally required for traditional solid-phase extraction. The development and validation of such analytical methodologies will prove beneficial for the identification of prenatal substance abuse, which is an ongoing concern across socioeconomic lines.

  12. Collaborative validation of the quantification method for suspected allergens and test of an automated data treatment.

    PubMed

    Chaintreau, Alain; Cicchetti, Esmeralda; David, Nathalie; Earls, Andy; Gimeno, Pascal; Grimaud, Béatrice; Joulain, Daniel; Kupfermann, Nikolai; Kuropka, Gryta; Saltron, Frédéric; Schippa, Christine

    2011-10-28

    Previous publications investigated different data treatment strategies for quantification of volatile suspected allergens by GC/MS. This publication presents the validation results obtained on "ready to inject" samples under reproducibility conditions following inter-laboratory ring-testing. The approach is based on the monitoring of three selected ions per analyte using two different GC capillary columns. To aid the analysts a decisional tree is used for guidance during the interpretation of the analytical results. The method is evaluated using a fragrance oil concentrate spiked with all suspected allergens to mimic the difficulty of a real sample extract or perfume oil. At the concentrations of 10 and 100mg/kg, imposed by Directive 76/768/EEC for labeling of leave-on and rinse-off cosmetics, the mean bias is +14% and -4%, respectively. The method is linear for all analytes, and the prediction intervals for each analyte have been determined. To speed up the analyst's task, an automated data treatment is also proposed. The method mean bias is slightly shifted towards negative values, but the method prediction intervals are close to that resulting from the decisional tree.

  13. A sensitive method for the detection and quantification of ginkgo flavonols from plasma.

    PubMed

    Zhao, Yang; Wang, Li; Bao, Yuanwu; Li, Chuan

    2007-01-01

    Extracts from Ginkgo biloba leaves (family Ginkgoaceae) have antioxidant and free radical scavenging effects, largely attributed to the flavonols, which are a major class of functional components in ginkgo extracts. In order to facilitate analysis of systemic exposure to ginkgo-derived products in animals and/or humans, we developed a liquid chromatography/tandem mass spectrometry (LC/MS/MS)-based method that is capable of routinely monitoring plasma levels of ginkgo flavonols. We used an initial acidic hydrolysis step to convert the plasma ginkgo flavonol conjugates into their aglycone forms [quercetin (QCT), kaempferol (KMF) and isorhamnetin (ISR)] prior to EtOAc-based extraction and subsequent LC/MS/MS-based analysis. Comparative studies showed that the use of a mobile phase containing an extremely low concentration of HCOOH (0.01 per thousand) dramatically improved the electrospray ionization efficiency of the analytes in the negative ion mode; the efficiencies were approximately 4-, approximately 8- and approximately 20-fold higher for QCT, KMF and ISR, respectively, versus the results obtained using an electrolyte-free mobile phase, or approximately 2-, approximately 3- and approximately 4-fold higher, respectively, versus the results obtained using a mobile phase containing the more commonly utilized concentration of HCOOH (1 per thousand). In addition, use of the low concentration of HCOOH also decreased undesired matrix effects. These favorable effects have been referred to as 'LC-electrolyte effects'. Due to structural differences in the B-ring substituent, different types of precursor-to-product ion pairs (m/z 301 --> 151 for QCT, 285 --> 187 for KMF, and 315 --> 300 for ISR) were used for the selected reaction monitoring of the analytes. In addition, the chromatographic conditions were optimized on the basis of an initial scouting of matrix effects on analyte ionization. Despite the absence of an internal standard, the validation results consistently

  14. METHOD 544. DETERMINATION OF MICROCYSTINS AND NODULARIN IN DRINKING WATER BY SOLID PHASE EXTRACTION AND LIQUID CHROMATOGRAPHY/TANDEM MASS SPECTROMETRY (LC/MS/MS)

    EPA Science Inventory

    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...

  15. A HPLC-MS/MS method for determination of 6'''-feruloylspinosin in rat plasma and tissues: Pharmacokinetics and tissue distribution study.

    PubMed

    Qiao, Longdong; Liu, Yan; Chen, Xiaoyan; Xie, Junbo; Zhang, Yanqing; Yang, Ke; Zhou, Hongjian; Duan, Yayun; Zheng, Wei; Xie, Wenlin

    2016-03-20

    A sensitive, reliable and accurate HPLC-MS/MS method was developed and validated for the quantification of 6'''-feruloylspinosin in rat plasma and tissues with puerarin as the internal standard. The separation was performed on a Proshell 120 EC-C18 column (4.6×150 mm, 2.7 μm) with a mobile phase consisting of acetonitrile and 0.1% formic acid (20:80, v/v) at 0.3 mL/min. The quantification was performed by MRM with m/z [M-H](-) 783.3→427.2 for 6'''-feruloylspinosin and m/z [M-H](-) 415.4→295.4 for the internal standard, respectively. The calibration curves covered over a concentration range of 20-2000 ng/mL in plasma and various tissues samples (heart, liver, spleen, lung, kidney, stomach, intestine, muscle, cerebrum and cerebellum) with good linearity (r(2)≥0.9914). Both the intra- and inter-day precisions were less than 14.70%, and the accuracy (RE%) ranged from -5.80% to 4.93%. The extraction recoveries were within 75.21-92.96%, and the matrix effect ranged from 87.21% to 113.44%. Compared with spinosin, 6'''-feruloylspinosin was distributed in rats faster whereas more slowly eliminated from the plasma. 6'''-Feruloylspinosin could be distributed rapidly and widely in various tissues, and transfer across the blood-brain barrier. In addition, both 6'''-feruloylspinosin and spinosin could enhance the expression of GABAAα1, GABAAα5, GABABR1 mRNA in rat hippocampal neurons significantly, indicating the bioactivity mechanism of 6'''-feruloylspinosin was involved in the GABA receptors. PMID:26780157

  16. Evaluation of Normalization Methods on GeLC-MS/MS Label-Free Spectral Counting Data to Correct for Variation during Proteomic Workflows

    NASA Astrophysics Data System (ADS)

    Gokce, Emine; Shuford, Christopher M.; Franck, William L.; Dean, Ralph A.; Muddiman, David C.

    2011-12-01

    Normalization of spectral counts (SpCs) in label-free shotgun proteomic approaches is important to achieve reliable relative quantification. Three different SpC normalization methods, total spectral count (TSpC) normalization, normalized spectral abundance factor (NSAF) normalization, and normalization to selected proteins (NSP) were evaluated based on their ability to correct for day-to-day variation between gel-based sample preparation and chromatographic performance. Three spectral counting data sets obtained from the same biological conidia sample of the rice blast fungus Magnaporthe oryzae were analyzed by 1D gel and liquid chromatography-tandem mass spectrometry (GeLC-MS/MS). Equine myoglobin and chicken ovalbumin were spiked into the protein extracts prior to 1D-SDS- PAGE as internal protein standards for NSP. The correlation between SpCs of the same proteins across the different data sets was investigated. We report that TSpC normalization and NSAF normalization yielded almost ideal slopes of unity for normalized SpC versus average normalized SpC plots, while NSP did not afford effective corrections of the unnormalized data. Furthermore, when utilizing TSpC normalization prior to relative protein quantification, t-testing and fold-change revealed the cutoff limits for determining real biological change to be a function of the absolute number of SpCs. For instance, we observed the variance decreased as the number of SpCs increased, which resulted in a higher propensity for detecting statistically significant, yet artificial, change for highly abundant proteins. Thus, we suggest applying higher confidence level and lower fold-change cutoffs for proteins with higher SpCs, rather than using a single criterion for the entire data set. By choosing appropriate cutoff values to maintain a constant false positive rate across different protein levels (i.e., SpC levels), it is expected this will reduce the overall false negative rate, particularly for proteins with

  17. A HPLC-MS/MS method for determination of 6'''-feruloylspinosin in rat plasma and tissues: Pharmacokinetics and tissue distribution study.

    PubMed

    Qiao, Longdong; Liu, Yan; Chen, Xiaoyan; Xie, Junbo; Zhang, Yanqing; Yang, Ke; Zhou, Hongjian; Duan, Yayun; Zheng, Wei; Xie, Wenlin

    2016-03-20

    A sensitive, reliable and accurate HPLC-MS/MS method was developed and validated for the quantification of 6'''-feruloylspinosin in rat plasma and tissues with puerarin as the internal standard. The separation was performed on a Proshell 120 EC-C18 column (4.6×150 mm, 2.7 μm) with a mobile phase consisting of acetonitrile and 0.1% formic acid (20:80, v/v) at 0.3 mL/min. The quantification was performed by MRM with m/z [M-H](-) 783.3→427.2 for 6'''-feruloylspinosin and m/z [M-H](-) 415.4→295.4 for the internal standard, respectively. The calibration curves covered over a concentration range of 20-2000 ng/mL in plasma and various tissues samples (heart, liver, spleen, lung, kidney, stomach, intestine, muscle, cerebrum and cerebellum) with good linearity (r(2)≥0.9914). Both the intra- and inter-day precisions were less than 14.70%, and the accuracy (RE%) ranged from -5.80% to 4.93%. The extraction recoveries were within 75.21-92.96%, and the matrix effect ranged from 87.21% to 113.44%. Compared with spinosin, 6'''-feruloylspinosin was distributed in rats faster whereas more slowly eliminated from the plasma. 6'''-Feruloylspinosin could be distributed rapidly and widely in various tissues, and transfer across the blood-brain barrier. In addition, both 6'''-feruloylspinosin and spinosin could enhance the expression of GABAAα1, GABAAα5, GABABR1 mRNA in rat hippocampal neurons significantly, indicating the bioactivity mechanism of 6'''-feruloylspinosin was involved in the GABA receptors.

  18. Quantification of pyrrolizidine alkaloids in North American plants and honey by LC-MS: single laboratory validation.

    PubMed

    Mudge, Elizabeth M; Jones, A Maxwell P; Brown, Paula N

    2015-01-01

    Pyrrolizidine alkaloids (PAs) are a class of naturally occurring compounds produced by many flowering plants around the World. Their presence as contaminants in food systems has become a significant concern in recent years. For example, PAs are often found as contaminants in honey through pollen transfer. A validated method was developed for the quantification of four pyrrolizidine alkaloids and one pyrrolizidine alkaloid N-oxide in plants and honey grown and produced in British Columbia. The method was optimised for extraction efficiency from the plant materials and then subjected to a single-laboratory validation to assess repeatability, accuracy, selectivity, LOD, LOQ and method linearity. The PA content in plants ranged from1.0 to 307.8 µg/g with repeatability precision between 3.8 and 20.8% RSD. HorRat values were within acceptable limits and ranged from 0.62 to 1.63 for plant material and 0.56-1.82 for honey samples. Method accuracy was determined through spike studies with recoveries ranging from 84.6 to 108.2% from the raw material negative control and from 82.1-106.0 % for the pyrrolizidine alkaloids in corn syrup. Based on the findings in this single-laboratory validation, this method is suitable for the quantitation of lycopsamine, senecionine, senecionine N-oxide, heliosupine and echimidine in common comfrey (Symphytum officinale), tansy ragwort (Senecio jacobaea), blueweed (Echium vulgare) and hound's tongue (Cynoglossum officinale) and for PA quantitation in honey and found that PA contaminants were present at low levels in BC honey. PMID:26482059

  19. Quantification of pyrrolizidine alkaloids in North American plants and honey by LC-MS: single laboratory validation.

    PubMed

    Mudge, Elizabeth M; Jones, A Maxwell P; Brown, Paula N

    2015-01-01

    Pyrrolizidine alkaloids (PAs) are a class of naturally occurring compounds produced by many flowering plants around the World. Their presence as contaminants in food systems has become a significant concern in recent years. For example, PAs are often found as contaminants in honey through pollen transfer. A validated method was developed for the quantification of four pyrrolizidine alkaloids and one pyrrolizidine alkaloid N-oxide in plants and honey grown and produced in British Columbia. The method was optimised for extraction efficiency from the plant materials and then subjected to a single-laboratory validation to assess repeatability, accuracy, selectivity, LOD, LOQ and method linearity. The PA content in plants ranged from1.0 to 307.8 µg/g with repeatability precision between 3.8 and 20.8% RSD. HorRat values were within acceptable limits and ranged from 0.62 to 1.63 for plant material and 0.56-1.82 for honey samples. Method accuracy was determined through spike studies with recoveries ranging from 84.6 to 108.2% from the raw material negative control and from 82.1-106.0 % for the pyrrolizidine alkaloids in corn syrup. Based on the findings in this single-laboratory validation, this method is suitable for the quantitation of lycopsamine, senecionine, senecionine N-oxide, heliosupine and echimidine in common comfrey (Symphytum officinale), tansy ragwort (Senecio jacobaea), blueweed (Echium vulgare) and hound's tongue (Cynoglossum officinale) and for PA quantitation in honey and found that PA contaminants were present at low levels in BC honey.

  20. Validated LC-MS/MS method for the simultaneous determination of hyperoside and 2''-O-galloylhyperin in rat plasma: application to a pharmacokinetic study in rats.

    PubMed

    Zhou, Donghui; Jin, Yuanzhe; Yao, Fengchen; Duan, Zhiying; Wang, Qi; Liu, Jing

    2014-08-01

    An LC-MS/MS method was developed for the first time to simultaneously determine hyperoside and 2''-O-galloylhyperin, two major components in Pyrola calliantha extract, in rat plasma. Following extraction by one-step protein precipitation with methanol, the analytes were separated on a Venusil MP-C18 column within 2 min, using methanol-water-formic acid (50:50:0.1, v/v/v) as the mobile phase at a flow rate of 0.4 mL/min. Detection was performed on electrospray negative ionization mass spectrometry by multiple-reaction monitoring of the transitions of 2''-O-galloylhyperin at m/z 615.1 → 301.0, of hyperoside at m/z 463.1 → 300.1, and of internal standard at m/z 415.1 → 295.1. The limits of quantification were 2 ng/mL for both hyperoside and 2''-O-galloylhyperin. The precisions were <13.1%, and the accuracies were between -9.1 and 5.5% for both compounds. The method was successfully applied in pharmacokinetic studies following intravenous administration of the total flavonoids of P. calliantha extract in rats.

  1. Development of an LC-MS/MS method to quantify sex hormones in bovine milk and influence of pregnancy in their levels.

    PubMed

    Regal, P; Cepeda, A; Fente, C

    2012-01-01

    Hormones work in harmony in the body, and this status must be maintained to avoid metabolic disequilibrium and the subsequent illness. Besides, it has been reported that exogenous steroids (presence in the environment and food products) influence the development of several important illnesses in humans. Endogenous steroid hormones in food of animal origin are unavoidable as they occur naturally in these products. The presence of hormones in food has been connected with several human health problems. Bovine milk contains considerable quantities of hormones and it is of particular concern. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method, based on hydroxylamine derivatisation, has been developed and validated for the quantification of six sex hormones in milk [pregnenolone (P₅), progesterone (P₄), estrone (E₁), testosterone (T), androstenedione (A) and dehydroepiandrosterone (DHEA)]. This method has been applied to real raw milk samples and the existence of differences between milk from pregnant and non-pregnant cows has been statistically confirmed. Basing on a revision of existing published data, it could be concluded that maximum daily intakes for hormones are not reached through milk ingestion. Although dairy products are an important source of hormones, other products of animal origin must be considered as well for intake calculations.

  2. Development of an LC-MS/MS analytical method for the simultaneous measurement of aldehydes from polyunsaturated fatty acids degradation in animal feed.

    PubMed

    Douny, Caroline; Bayram, Pinar; Brose, François; Degand, Guy; Scippo, Marie-Louise

    2016-05-01

    Knowing that polyunsaturated fatty acids can lead to the formation of potentially toxic aldehydes as secondary oxidation products, an analytical method using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) detection was developed to measure the concentration of eight aldehydes in animal feed: malondialdehyde (MDA), 4-hydroxy-2-nonenal (4-HNE), 4-hydroxy-2-hexenal (4-HHE), crotonaldehyde (CRT), benzaldehyde (BNZ), hexanal (HXL), 2,4-nonadienal, and 2,4-decadienal. The developed method was validated according to the criteria and procedure described in international standards. The evaluated parameters were specificity/selectivity, recovery, precision, accuracy, uncertainty, limits of detection and quantification, using the concept of accuracy profiles. These parameters were determined during experiments conducted over three different days with ground Kellogg's® Corn Flakes® cereals as model matrix for animal feed and spiked at different levels of concentration. Malondialdehyde, 4-HHE, 4-HNE, crotonaldehyde, benzaldehyde, and hexanal can be analyzed in the same run in animal feed with a very good accuracy, with recovery rates ranging from 86 to 109% for a working range going from 0.16 to 12.50 mg/kg. The analysis of 2,4-nonadienal and 2,4-decadienal can also be performed but in a limited range of concentration and with a limited degree of accuracy. Their recovery rates ranged between 54 and 114% and coefficient of variation for the intermediate precision between 11 and 25% for these two compounds. Copyright © 2016 John Wiley & Sons, Ltd.

  3. Online solid phase extraction LC-MS/MS method for the analysis of succinate dehydrogenase inhibitor fungicides and its applicability to surface water samples.

    PubMed

    Gulkowska, Anna; Buerge, Ignaz J; Poiger, Thomas

    2014-10-01

    A sensitive and selective analytical method, based on online solid phase extraction coupled to LC-MS/MS, was developed and validated to determine traces of several recently introduced fungicides in surface water and wastewater. The list of target analytes included eight succinate dehydrogenase inhibitors (bixafen, boscalid, fluopyram, flutolanil, fluxapyroxad, isopyrazam, penflufen, and penthiopyrad), and two other fungicides with different modes of action, fenpyrazamine and fluopicolide. Detection and quantification limits in various matrices were in the range of 0.1 to 2 and 0.5 to 10 ng/L, respectively. Moderate signal suppression was observed in surface water (≤15%) and wastewater (≤25%) and was well compensated by the selected internal standard. The intra- and inter-day precisions were generally <10 and <20%, respectively. The applicability of the method was demonstrated in a study on the occurrence of fungicides in the river Glatt, Switzerland, that drains a catchment area of 419 km(2) with a substantial proportion of agricultural land. Of the studied compounds, only boscalid and fluopicolide were detected in flow-proportional weekly composite samples, generally at low concentrations up to 15 and 5 ng/L, respectively. While fluopicolide was detected in only 30% of the samples above the LOD of 0.5 ng/L, boscalid was detected in all samples analyzed between March and October 2012.

  4. A sensitive LC-MS/MS method for quantifying capsaicin and dihydrocapsaicin in rabbit plasma and tissue: application to a pharmacokinetic study.

    PubMed

    Wang, Dimin; Meng, Fanhua; Yu, Lin; Sun, Lu; Sun, Lili; Guo, Jifen

    2015-04-01

    Prescription and nonprescription products for topical management of pain, including cream, lotion and patch forms, contain capsaicin (CAP) and dihydrocapsaicin (DHC). There are few in vivo studies on absorption, bioavailability and disposition of CAP and DHC. We established a sensitive and rapid LC-MS/MS assay to determine CAP and DHC levels in rabbit plasma and tissue. Bio-samples prepared by liquid-liquid extraction using n-hexane-dichloromethane-isopropanol (100: 50: 5, v/v/v) mixture were separated by isocratic chromatography with an Extend C18 column. The mobile phase was acetonitrile-water-formic acid (70: 30: 0.1, v/v/v). The method was linear from 0.125 to 50 ng/mL for a 100 μL bio-sample, and the lower quantification limit was 0.125 ng/mL. Total run time to analyze each sample was 3.5 min. We used this validated method to study pharmacokinetics and tissue distribution of CAP gel administered topically to rabbits. A very small amount of CAP and DHC was absorbed into the systemic circulation. The highest plasma concentration was 2.39 ng/mL, and the mean peak plasma concentration value after 12 h of CAP gel application was 1.68 ng/mL. Drug concentration in treated skin was relatively high, with low concentration in other tissues. Thus, topical CAP gel had strong local effects and weaker systemic effects.

  5. Determination of steroidal oestrogens in tap water samples using solid-phase extraction on a molecularly imprinted polymer sorbent and quantification with gas chromatography-mass spectrometry (GC-MS).

    PubMed

    Zacs, D; Perkons, I; Bartkevics, V

    2016-07-01

    An analytical method was established and validated for the analysis of steroidal oestrogens in tap water samples. Gas chromatography coupled to high-resolution mass spectrometry (GC-HRMS) and gas chromatography coupled to tandem mass spectrometry (GC-MS/MS) were used for the identification/quantification of selected compounds and the analytical performance of these techniques was evaluated. Liquid-liquid extraction (LLE) and solid-phase extraction (SPE) with a molecularly imprinted polymer (MIP) stationary phase that was highly selective for oestrogens were used for the extraction of 100-mL aliquots of water samples. The recoveries of analytes with the described methods ranged from 92 to 114 %, while the repeatability in terms of relative standard deviations (RSDs) was in the range from 2.1 to 15.2 % (n = 5). It was concluded that SPE with MIP that was highly selective for oestrogens in combination with GC-HRMS detection is more preferable for the analysis of oestrogens in tap water samples. The typical oestrogen, 17β-estradiol (17β-E2), was detected above the method limit of quantification (m-LOQ) in 5 of 14 analysed tap water samples at concentrations from 0.09 to 0.15 ng L(-1). Despite that 17α-ethynylestradiol (17α-EE2) was not quantified in this study above m-LOQ, the presence of this chemical was qualitatively confirmed in some of the analysed samples.

  6. Quantification of Cell-Penetrating Peptide Associated with Polymeric Nanoparticles Using Isobaric-Tagging and MALDI-TOF MS/MS

    NASA Astrophysics Data System (ADS)

    Chiu, Jasper Z. S.; Tucker, Ian G.; McDowell, Arlene

    2016-09-01

    High sensitivity quantification of the putative cell-penetrating peptide di-arginine-histidine (RRH) associated with poly (ethyl-cyanoacrylate) (PECA) nanoparticles was achieved without analyte separation, using a novel application of isobaric-tagging and high matrix-assisted laser desorption/ionization coupled to time-of-flight (MALDI-TOF) mass spectrometry. Isobaric-tagging reaction equilibrium was reached after 5 min, with 90% or greater RRH peptide successfully isobaric-tagged after 60 min. The accuracy was greater than 90%, which indicates good reliability of using isobaric-tagged RRH as an internal standard for RRH quantification. The sample intra- and inter-spot coefficients of variations were less than 11%, which indicate good repeatability. The majority of RRH peptides in the nanoparticle formulation were physically associated with the nanoparticles (46.6%), whereas only a small fraction remained unassociated (13.7%). The unrecovered RRH peptide (~40%) was assumed to be covalently associated with PECA nanoparticles.

  7. Quantification of Cell-Penetrating Peptide Associated with Polymeric Nanoparticles Using Isobaric-Tagging and MALDI-TOF MS/MS

    NASA Astrophysics Data System (ADS)

    Chiu, Jasper Z. S.; Tucker, Ian G.; McDowell, Arlene

    2016-11-01

    High sensitivity quantification of the putative cell-penetrating peptide di-arginine-histidine (RRH) associated with poly (ethyl-cyanoacrylate) (PECA) nanoparticles was achieved without analyte separation, using a novel application of isobaric-tagging and high matrix-assisted laser desorption/ionization coupled to time-of-flight (MALDI-TOF) mass spectrometry. Isobaric-tagging reaction equilibrium was reached after 5 min, with 90% or greater RRH peptide successfully isobaric-tagged after 60 min. The accuracy was greater than 90%, which indicates good reliability of using isobaric-tagged RRH as an internal standard for RRH quantification. The sample intra- and inter-spot coefficients of variations were less than 11%, which indicate good repeatability. The majority of RRH peptides in the nanoparticle formulation were physically associated with the nanoparticles (46.6%), whereas only a small fraction remained unassociated (13.7%). The unrecovered RRH peptide (~40%) was assumed to be covalently associated with PECA nanoparticles.

  8. A novel GC-MS method in urinary estrogen analysis from postmenopausal women with osteoporosis[S

    PubMed Central

    Moon, Ju-Yeon; Kim, Kwang Joon; Moon, Myeong Hee; Chung, Bong Chul; Choi, Man Ho

    2011-01-01

    Estrogen metabolites play important roles in the development of female-related disorders and homeostasis of the bone. To improve detectability, a validated gas chromatography-mass spectrometry (GC-MS) method was conducted with two-phase extractive ethoxycarbonlyation (EOC) and subsequent pentafluoropropionyl (PFP) derivatization was introduced. The resulting samples were separated through a high-temperature MXT-1 column within an 8 min run and were detected in the selected ion monitoring (SIM) mode. The optimized analytical conditions led to good separation with a symmetric peak shape for 19 estrogens as their EOC-PFP derivatives. The limit of quantification (LOQ) was from 0.02 to ∼0.1 ng/ml for most estrogens analyzed, except for 2-hydroxyestriol (0.5 ng/ml). The devised method was found to be linear (r2 > 0.995) in the range from the LOQ to 40 ng/ml, whereas the precision (% CV) and accuracy (% bias) ranged from 1.4 to 10.5% and from 91.4 to 108.5%, respectively. The good sensitivity and selectivity of this method even allowed quantification of the estrogen metabolites in urine samples obtained from the postmenopausal female patients with osteoporosis. The present technique can be useful for clinical diagnosis as well as to better understand the pathogenesis of estrogen-related disorders in low-level quantification. PMID:21602563

  9. Analytical method for the accurate determination of tricothecenes in grains using LC-MS/MS: a comparison between MRM transition and MS3 quantitation.

    PubMed

    Lim, Chee Wei; Tai, Siew Hoon; Lee, Lin Min; Chan, Sheot Harn

    2012-07-01

    The current food crisis demands unambiguous determination of mycotoxin contamination in staple foods to achieve safer food for consumption. This paper describes the first accurate LC-MS/MS method developed to analyze tricothecenes in grains by applying multiple reaction monitoring (MRM) transition and MS(3) quantitation strategies in tandem. The tricothecenes are nivalenol, deoxynivalenol, deoxynivalenol-3-glucoside, fusarenon X, 3-acetyl-deoxynivalenol, 15-acetyldeoxynivalenol, diacetoxyscirpenol, and HT-2 and T-2 toxins. Acetic acid and ammonium acetate were used to convert the analytes into their respective acetate adducts and ammonium adducts under negative and positive MS polarity conditions, respectively. The mycotoxins were separated by reversed-phase LC in a 13.5-min run, ionized using electrospray ionization, and detected by tandem mass spectrometry. Analyte-specific mass-to-charge (m/z) ratios were used to perform quantitation under MRM transition and MS(3) (linear ion trap) modes. Three experiments were made for each quantitation mode and matrix in batches over 6 days for recovery studies. The matrix effect was investigated at concentration levels of 20, 40, 80, 120, 160, and 200 μg kg(-1) (n = 3) in 5 g corn flour and rice flour. Extraction with acetonitrile provided a good overall recovery range of 90-108% (n = 3) at three levels of spiking concentration of 40, 80, and 120 μg kg(-1). A quantitation limit of 2-6 μg kg(-1) was achieved by applying an MRM transition quantitation strategy. Under MS(3) mode, a quantitation limit of 4-10 μg kg(-1) was achieved. Relative standard deviations of 2-10% and 2-11% were reported for MRM transition and MS(3) quantitation, respectively. The successful utilization of MS(3) enabled accurate analyte fragmentation pattern matching and its quantitation, leading to the development of analytical methods in fields that demand both analyte specificity and fragmentation fingerprint-matching capabilities that are

  10. Quantification of strontium in human serum by ICP-MS using alternate analyte-free matrix and its application to a pilot bioequivalence study of two strontium ranelate oral formulations in healthy Chinese subjects.

    PubMed

    Zhang, Dan; Wang, Xiaolin; Liu, Man; Zhang, Lina; Deng, Ming; Liu, Huichen

    2015-01-01

    A rapid, sensitive and accurate ICP-MS method using alternate analyte-free matrix for calibration standards preparation and a rapid direct dilution procedure for sample preparation was developed and validated for the quantification of exogenous strontium (Sr) from the drug in human serum. Serum was prepared by direct dilution (1:29, v/v) in an acidic solution consisting of nitric acid (0.1%) and germanium (Ge) added as internal standard (IS), to obtain simple and high-throughput preparation procedure with minimized matrix effect, and good repeatability. ICP-MS analysis was performed using collision cell technology (CCT) mode. Alternate matrix method by using distilled water as an alternate analyte-free matrix for the preparation of calibration standards (CS) was used to avoid the influence of endogenous Sr in serum on the quantification. The method was validated in terms of selectivity, carry-over, matrix effects, lower limit of quantification (LLOQ), linearity, precision and accuracy, and stability. Instrumental linearity was verified in the range of 1.00-500ng/mL, corresponding to a concentration range of 0.0300-15.0μg/mL in 50μL sample of serum matrix and alternate matrix. Intra- and inter-day precision as relative standard deviation (RSD) were less than 8.0% and accuracy as relative error (RE) was within ±3.0%. The method allowed a high sample throughput, and was sensitive and accurate enough for a pilot bioequivalence study in healthy male Chinese subjects following single oral administration of two strontium ranelate formulations containing 2g strontium ranelate. PMID:25023847

  11. Quantification of strontium in human serum by ICP-MS using alternate analyte-free matrix and its application to a pilot bioequivalence study of two strontium ranelate oral formulations in healthy Chinese subjects.

    PubMed

    Zhang, Dan; Wang, Xiaolin; Liu, Man; Zhang, Lina; Deng, Ming; Liu, Huichen

    2015-01-01

    A rapid, sensitive and accurate ICP-MS method using alternate analyte-free matrix for calibration standards preparation and a rapid direct dilution procedure for sample preparation was developed and validated for the quantification of exogenous strontium (Sr) from the drug in human serum. Serum was prepared by direct dilution (1:29, v/v) in an acidic solution consisting of nitric acid (0.1%) and germanium (Ge) added as internal standard (IS), to obtain simple and high-throughput preparation procedure with minimized matrix effect, and good repeatability. ICP-MS analysis was performed using collision cell technology (CCT) mode. Alternate matrix method by using distilled water as an alternate analyte-free matrix for the preparation of calibration standards (CS) was used to avoid the influence of endogenous Sr in serum on the quantification. The method was validated in terms of selectivity, carry-over, matrix effects, lower limit of quantification (LLOQ), linearity, precision and accuracy, and stability. Instrumental linearity was verified in the range of 1.00-500ng/mL, corresponding to a concentration range of 0.0300-15.0μg/mL in 50μL sample of serum matrix and alternate matrix. Intra- and inter-day precision as relative standard deviation (RSD) were less than 8.0% and accuracy as relative error (RE) was within ±3.0%. The method allowed a high sample throughput, and was sensitive and accurate enough for a pilot bioequivalence study in healthy male Chinese subjects following single oral administration of two strontium ranelate formulations containing 2g strontium ranelate.

  12. Stability-indicating simultaneous determination of paracetamol and three of its related substances using a direct GC/MS method.

    PubMed

    Belal, Tarek; Awad, Tamer; Clark, C Randall

    2009-01-01

    A simple, direct, and selective stability-indicating GC/MS procedure was developed for the simultaneous determination of paracetamol (PR) and three of its related substances: 4-aminophenol (4-AP), acetanilide (AD), and 4'-chloroacetanilide (4-CA). The method involved resolution of the underivatized compounds using a 100% dimethylpolysiloxane (Rtx-1) column, and MS detection was carried out in the electron-impact mode. The four compounds were completely resolved in less than 11 min. The fragmentation pathways for the four compounds were described, and the structures of the major fragment ions peaks were proposed. Quantification of the analytes was based on measuring their peak areas. The reliability and analytical performance of the proposed method including linearity, range, precision, accuracy, and detection and quantification limits were statistically validated. Calibration curves were linear over the ranges 75-500, 25-350, 25-350, and 25-350 microg/mL for PR, 4-AP, AD, and 4-CA, respectively. The proposed method was successfully applied for the determination of PR and its related substances in laboratory-prepared mixtures of different proportions. Also, it was applied for the assay of PR in several commercially available pharmaceutical formulations with recoveries of 98.95-100.76%. PMID:20166578

  13. [Metabolites and metabolic pathways of mesaconitine in rat liver microsomal investigated by using UPLC-MS/MS method in vitro].

    PubMed

    Bi, Yun-Feng; Liu, Shu; Zhang, Rui-Xing; Song, Feng-Rui; Liu, Zhi-Qiang

    2013-12-01

    Mesaconitine was incubated with rat liver microsomes in vitro. The metabolites of mesaconitine in rat liver microsomes were identified by ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method with high resolution power. A typical reaction mixture of 100 mol L-1 Tris-HCI buffer (pH 7.4) containing 0.5 gL-1 microsomal protein and 50 micro molL-1 mesaconitine was prepared. The above reaction mixture was divided into six groups, and the volume of each group was 200 micro L. The incubation mixture was pre-incubated at 37 degrees C for 2 min and the reactions were initiated by adding NADPH generating system. After 90 min incubation at 37 degrees C, 200 micro L of acetonitrile was added to each group to stop the reaction. The metabolites of mesaconitine were investigated by UPLC-MS/MS method. Mesaconitine and 6 metabolites M1-M6 were found in the incubation system. The structures were characterized according to the data from MS/MS spectra and literatures. The metabolic reactions of mesaconitine in rat liver microsomes included the demethylation, deacetylation, dehydrogenation and hydroxylation. The major metabolic pathways of mesaconitine in rat liver microsomes were determined by UPLC-MS/MS on multiple reaction monitoring (MRM) mode combined with specific inhibitors of cytochrome P450 (CYP) isoforms, including alpha-naphthoflavone (CYP1A2), quinine (CYP2D), diethyldithiocarbamate (CYP2E1), ketoconazole (CYP3A) and sulfaphenazole (CYP2C), separately. Mesaconitine was mainly metabolized by CYP3A. CYP2C and CYP2D were also more important CYP isoforms for the metabolism reactions of mesaconitine, but CYP1A2 and CYP2E1 haven't any contribution to MA metabolism in rat liver microsomes. PMID:24689241

  14. Method development by GC-ECD and HS-SPME-GC-MS for beer volatile analysis.

    PubMed

    da Silva, Gisele C; da Silva, Abner A S; da Silva, Larissa S N; Godoy, Ronoel Luiz de O; Nogueira, Luciana C; Quitério, Simone L; Raices, Renata S L

    2015-01-15

    Two methods for the extraction, identification and quantification of the highest occurrence and lowest perception threshold off-flavours in fifteen different samples of Brazilian Pilsner beers were developed. For this purpose, headspace solid phase microextraction in combination with a gas chromatography coupled to a mass spectrometric detection (HS-SPME-GC-MS) as well as headspace extraction in combination with a gas chromatography coupled to electron capture detection (HS-GC-ECD) were evaluated. The first and the second methods were used for esters and vicinal diketones analysis, respectively. All data were comprehended below the taster's threshold detection limit: ethyl acetate 39.48 ng mL(-1) (RSD mean value 4.2%), isoamyl acetate 3.88 ng mL(-1) (RSD mean value 3.4%), ethyl hexanoate 0.61 ng mL(-1) (RSD mean value 3.1%) and 2,3-butanedione 0.10 ng mL(-1) (RSD mean value 2.9%). The validated method demonstrated to be useful for the analysis of highest incidence beer off-flavours. PMID:25148961

  15. Evaluation of the Coat-A-Count sup 125 I fentanyl RIA: Comparison of sup 12 5I RIA and GC/MS-SIM for quantification of fentanyl in case urine specimens

    SciTech Connect

    Watts, V.W.; Caplan, Y.H. )

    1990-09-01

    The Coat-A-Count solid phase {sup 125}I Fentanyl Radioimmunoassay was evaluated with respect to linearity and precision using equine urine fortified with fentanyl and then compared with a gas chromatographic/mass spectrometric method for quantification of fentanyl in urine. The RIA assay was found to be linear over the urine fentanyl concentration range of 0.25 to 7.5 ng/mL and precise with coefficients of variation (CV) ranging from 9.6 to 19.3%. The RIA calibrators, ranging in fentanyl concentrations from 0.25 to 7.5 ng/mL, and controls, at mean fentanyl concentrations of 0.46 and 1.32 ng/mL, were compared by both the RIA and GC/MS methods. The cross-reactivity with the {sup 125}I RIA test was determined for the fentanyl metabolites, norfentanyl and hydroxyfentanyl, and found to be 5% and 35%, respectively. The illicit fentanyl analogs were found to show significant cross-reactivity, ranging from 20 to 100%. The {sup 125}I RIA was compared to GC/MS quantifications of fentanyl in 35 positive and 20 negative case urine specimens.

  16. Optical methods for the quantification of the fibrillation degree of bleached MFC materials.

    PubMed

    Chinga-Carrasco, Gary

    2013-05-01

    In this study, the suitability of optical devices for quantification of the fibrillation degree of bleached microfibrillated cellulose (MFC) materials has been assessed. The techniques for optical assessment include optical scanner, UV-vis spectrophotometry, turbidity, quantification of the fiber fraction and a camera system for dynamic measurements. The results show that the assessed optical devices are most adequate for quantification of the light transmittance of bleached MFC materials. Such quantification yields an estimation of the fibrillation degree. Films made of poorly fibrillated materials are opaque, while films made of highly fibrillated materials containing a major fraction of nanofibrils are translucent, with light transmittance larger than 90%. Finally, the concept of using images acquired with a CCD camera system, for estimating the fibrillation degree in dynamic conditions was exemplified. Such systems are most interesting as this will widen the applicability of optical methods for quantification of fibrillation degree online in production lines, which is expected to appear in the years to come.

  17. Detection and quantification of glycosylated flavonoid malonates in celery, Chinese celery, and celery seed by LC-DAD-ESI/MS.

    PubMed

    Lin, Long-Ze; Lu, Shengmin; Harnly, James M

    2007-02-21

    A screening method using LC-DAD-ESI/MS was applied to the analysis of flavonoids in celery, Chinese celery, and celery seeds (Apium graveolens L. and varieties). Fifteen flavonoid glycosides were detected in the three celery materials. They were identified as luteolin 7-O-apiosylglucoside, luteolin 7-O-glucoside, apigenin 7-O-apiosylglucoside, chrysoeriol 7-O-apiosylglucoside, chrysoeriol 7-O-glucoside, and more than 10 malonyl derivatives of these glycosides. The identification of the malonyl derivatives was confirmed by their conversion into glycosides upon heating and by comparison of some of the malonates with malonates that had previously been identified in red bell pepper and parsley. The concentrations of the glycosides and the malonyl glycosides in the three materials were estimated by comparison to aglycone standards. This is the first report of the presence of these glycosylated flavonoid malonates in celery.

  18. Development and application of a method for the analysis of two trichothecenes: deoxynivalenol and T-2 toxin in meat in China by HPLC-MS/MS.

    PubMed

    Zou, Zhongyi; He, Zhifei; Li, Hongjun; Han, Pengfei; Tang, Jun; Xi, Cunxian; Li, Yingguo; Zhang, Lei; Li, Xianliang

    2012-03-01

    A reliable and sensitive method was developed and successfully applied for the determination of deoxynivalenol and T-2 toxin simultaneously in pig dorsal muscle, pig back fat and chicken muscle by high performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS) analysis. Limit of detection of deoxynivalenol and T-2 was 0.02μg/kg and 0.007μg/kg, and limit of quantification of deoxynivalenol and T-2 was 0.07μg/kg and 0.02μg/kg, respectively. Sixty-six meat samples were analyzed and deoxynivalenol was detected in the samples of pig back fat, with concentrations lower than 0.5μg/kg, and T-2 toxin was detected in the samples of pig dorsal muscle, pig back fat and chicken muscle, with concentrations lower than 0.5μg/kg. The results of sample analysis show that only trace residues of deoxynivalenol and T-2 toxin were detected in the samples analyzed.

  19. Review: DNA oxidation, its consequences and efficacy of GC-MS and SPME-GC-MS for In Vitro quantification of DNA oxidative products

    NASA Astrophysics Data System (ADS)

    Singh, Himansha; Udawat, Abhishek; Franklin, Tony; Sarathi, Sai Partha

    2012-10-01

    DNA oxidation could be one of the main factors contributing to DNA damage, eventually leading to carcinogenesis, mutations or non-carcinogenic diseases such as Parkinsonís and Alzheimerís. Only recently has the focus turned towards identifying oxidative products of DNA and their consequences. Metabolism activities in vitro produce reactive radicals, which can break DNA strands to cause lesions. These lesions could also act as biomarkers for diagnostic purposes. This review provides an insight of the DNA oxidation mechanism, its harmful consequences and the advantages/disadvantages of available techniques to quantify such DNA oxidative products, focussing mainly on the use GC-MS along with derivatization reaction. In addition, the review also discusses the use of Solid Phase Micro Extraction (SPME) before conducting GC-MS as a potential assay to overcome the discrepancies involved in using GC-MS alone for the identification of DNA oxidative products.

  20. Application of on-line electrochemistry/electrospray/tandem mass spectrometry to a quantification method for the antipsychotic drug zotepine in human serum.

    PubMed

    Nozaki, Kazuyoshi; Osaka, Issey; Kawasaki, Hideya; Arakawa, Ryuichi

    2009-10-01

    A simple, rapid, and sensitive on-line liquid chromatographic electrochemistry/electrospray/tandem mass spectrometry (LC-EC/ESI-MS/MS) method for the determination of zotepine in human serum was developed using a new generated-electrochemically fragment ion, and was validated. A recent novel technique of LC-EC/ESI-MS/MS that combines LC-MS/MS and the on-line EC reaction is potentially applicable to developing a quantification method for drugs in biological samples. Newly formed products generated by the on-line EC cell are expected to provide appropriate precursor and product ions for the MS/MS determination method. This technique was successfully applied to a drug assay in a biological matrix. After adding imipramine (IS) to a 30-microL aliquot of human serum, the resulting sample was simply deproteinated with acetonitrile for a measurement. The analytical run time was 5 min. The calibration curve was linear in the concentration range of 10-2000 ng/mL. The intra-assay precision and accuracy were in the range of 1.8-8.9 and 98.4-113%, respectively.

  1. Combination of PAGE and LA-ICP-MS as an analytical workflow in metallomics: state of the art, new quantification strategies, advantages and limitations.

    PubMed

    Sussulini, Alessandra; Becker, Johanna Sabine

    2011-12-01

    Metallomics (more specifically, metalloproteomics) is an emerging field that encompasses the role, uptake, transport and storage of trace metals, which are essential to preserve the functions of proteins within a biological system. The current strategies for metal-binding and metalloprotein analysis based on the combination of polyacrylamide gel electrophoresis (PAGE) and laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) are discussed in this review. The advantages, limitations and the most recently developed and applied quantification approaches for this methodology are also described.

  2. Methods for external event screening quantification: Risk Methods Integration and Evaluation Program (RMIEP) methods development

    SciTech Connect

    Ravindra, M.K.; Banon, H. )

    1992-07-01

    In this report, the scoping quantification procedures for external events in probabilistic risk assessments of nuclear power plants are described. External event analysis in a PRA has three important goals; (1) the analysis should be complete in that all events are considered; (2) by following some selected screening criteria, the more significant events are identified for detailed analysis; (3) the selected events are analyzed in depth by taking into account the unique features of the events: hazard, fragility of structures and equipment, external-event initiated accident sequences, etc. Based on the above goals, external event analysis may be considered as a three-stage process: Stage I: Identification and Initial Screening of External Events; Stage II: Bounding Analysis; Stage III: Detailed Risk Analysis. In the present report, first, a review of published PRAs is given to focus on the significance and treatment of external events in full-scope PRAs. Except for seismic, flooding, fire, and extreme wind events, the contributions of other external events to plant risk have been found to be negligible. Second, scoping methods for external events not covered in detail in the NRC's PRA Procedures Guide are provided. For this purpose, bounding analyses for transportation accidents, extreme winds and tornadoes, aircraft impacts, turbine missiles, and chemical release are described.

  3. Methods for external event screening quantification: Risk Methods Integration and Evaluation Program (RMIEP) methods development

    SciTech Connect

    Ravindra, M.K.; Banon, H.

    1992-07-01

    In this report, the scoping quantification procedures for external events in probabilistic risk assessments of nuclear power plants are described. External event analysis in a PRA has three important goals; (1) the analysis should be complete in that all events are considered; (2) by following some selected screening criteria, the more significant events are identified for detailed analysis; (3) the selected events are analyzed in depth by taking into account the unique features of the events: hazard, fragility of structures and equipment, external-event initiated accident sequences, etc. Based on the above goals, external event analysis may be considered as a three-stage process: Stage I: Identification and Initial Screening of External Events; Stage II: Bounding Analysis; Stage III: Detailed Risk Analysis. In the present report, first, a review of published PRAs is given to focus on the significance and treatment of external events in full-scope PRAs. Except for seismic, flooding, fire, and extreme wind events, the contributions of other external events to plant risk have been found to be negligible. Second, scoping methods for external events not covered in detail in the NRC`s PRA Procedures Guide are provided. For this purpose, bounding analyses for transportation accidents, extreme winds and tornadoes, aircraft impacts, turbine missiles, and chemical release are described.

  4. Event-specific qualitative and quantitative polymerase chain reaction methods for detection of genetically modified rapeseed Ms8xRf3 based on the right border junctions.

    PubMed

    Wu, Gang; Wu, Yuhua; Xiao, Ling; Lu, Changming

    2008-01-01

    Ms8xRf3 is a genetically modified rapeseed hybrid which is widely cultivated in Canada and exported to some other countries for production of foodstuffs or fodder. In this study, the genomic sequences flanking the right borders of the integrated transgenic sequences in the Ms8xRf3 genome were characterized and showed high similarities with the bacterial artificial chromosome clone of Chinese cabbage. Event-specific qualitative polymerase chain reaction (PCR) methods were established with the primers and probes targeting the junction regions to produce a 123 base pair (bp) product for the Ms8 event and 92 bp for the Rf3 event. The absolute detection limit of qualitative PCR was 2.5 initial template copies for the Ms8 event and 50 copies for the Rf3 event. Quantitiative detection methods were established, with the absolute quantification limit being approximately 25 initial template copies.

  5. A validated enantioselective LC-MS/MS assay for quantification of a major chiral metabolite of an achiral 11-β-hydroxysteroid-dehydrogenase 1 inhibitor in human plasma: Application to a clinical pharmacokinetic study.

    PubMed

    Furlong, Michael T; Ji, Qin C; Iacono, Lisa; Dang, Oanh; Noren, Marzena; Bruce, John; Aubry, Anne-Françoise; Arnold, Mark E

    2016-06-01

    BMS-823778 is a potent 11-β-hydroxysteroid-dehydrogenase 1 (11βHSD-1) inhibitor and a potential therapeutic agent for type 2 diabetes mellitus (T2DM). A high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay was developed and validated to enable reliable separation and quantification of both enantiomers of a chiral hydroxy metabolite (BMT-094817) in human plasma. Following liquid-liquid extraction in a 96-well plate format, chromatographic separation of the metabolite enantiomers was achieved by isocratic elution on a Chiralpak IA-3 column. Chromatographic conditions were optimized to ensure separation of both metabolite enantiomers. Metabolite enantiomers and stable isotope-labeled (SIL) internal standards were detected by positive ion electrospray tandem mass spectrometry. The LC-MS/MS assay was validated over a concentration range of 0.200-200ng/mL. Intra- and inter-assay precision values for replicate quality control samples were less than 9.9% for both enantiomers during the assay validation. Mean quality control accuracy values were within ±7.3%. Assay recoveries were high (>75%) and consistent across the assay range. The metabolite enantiomers were stable in human blood for 2h on ice. The analytes were also stable in human plasma for 25h at room temperature, 34days at -20°C and -70°C, and following five freeze-thaw cycles. No interconversion of the metabolite enantiomers was detected under any bioanalytical stress conditions, from blood collection/processing through extracted sample storage. The validated assay was successfully applied to the quantification of both metabolite enantiomers in human plasma in support of a human pharmacokinetic study.

  6. Method for Indirect Quantification of CH4 Production via H2O Production Using Hydrogenotrophic Methanogens.

    PubMed

    Taubner, Ruth-Sophie; Rittmann, Simon K-M R

    2016-01-01

    Hydrogenotrophic methanogens are an intriguing group of microorganisms from the domain Archaea. Methanogens exhibit extraordinary ecological, biochemical, and physiological characteristics and possess a huge biotechnological potential. Yet, the only possibility to assess the methane (CH4) production potential of hydrogenotrophic methanogens is to apply gas chromatographic quantification of CH4. In order to be able to effectively screen pure cultures of hydrogenotrophic methanogens regarding their CH4 production potential we developed a novel method for indirect quantification of the volumetric CH4 production rate by measuring the volumetric water production rate. This method was established in serum bottles for cultivation of methanogens in closed batch cultivation mode. Water production was estimated by determining the difference in mass increase in a quasi-isobaric setting. This novel CH4 quantification method is an accurate and precise analytical technique, which can be used to rapidly screen pure cultures of methanogens regarding their volumetric CH4 evolution rate. It is a cost effective alternative determining CH4 production of methanogens over CH4 quantification by using gas chromatography, especially if applied as a high throughput quantification method. Eventually, the method can be universally applied for quantification of CH4 production from psychrophilic, thermophilic and hyperthermophilic hydrogenotrophic methanogens. PMID:27199898

  7. Method for Indirect Quantification of CH4 Production via H2O Production Using Hydrogenotrophic Methanogens

    PubMed Central

    Taubner, Ruth-Sophie; Rittmann, Simon K.-M. R.

    2016-01-01

    Hydrogenotrophic methanogens are an intriguing group of microorganisms from the domain Archaea. Methanogens exhibit extraordinary ecological, biochemical, and physiological characteristics and possess a huge biotechnological potential. Yet, the only possibility to assess the methane (CH4) production potential of hydrogenotrophic methanogens is to apply gas chromatographic quantification of CH4. In order to be able to effectively screen pure cultures of hydrogenotrophic methanogens regarding their CH4 production potential we developed a novel method for indirect quantification of the volumetric CH4 production rate by measuring the volumetric water production rate. This method was established in serum bottles for cultivation of methanogens in closed batch cultivation mode. Water production was estimated by determining the difference in mass increase in a quasi-isobaric setting. This novel CH4 quantification method is an accurate and precise analytical technique, which can be used to rapidly screen pure cultures of methanogens regarding their volumetric CH4 evolution rate. It is a cost effective alternative determining CH4 production of methanogens over CH4 quantification by using gas chromatography, especially if applied as a high throughput quantification method. Eventually, the method can be universally applied for quantification of CH4 production from psychrophilic, thermophilic and hyperthermophilic hydrogenotrophic methanogens. PMID:27199898

  8. Enrichment and purification of six Aconitum alkaloids from Aconiti kusnezoffii radix by macroporous resins and quantification by HPLC-MS.

    PubMed

    Liu, Jingjing; Li, Qing; Liu, Ran; Yin, Yidi; Chen, Xiaohui; Bi, Kaishun

    2014-06-01

    Aconitine, mesaconitine, hypaconitine, benzoylaconine, benzoylmesaconine and benzoylhypaconine are six main Aconitum alkaloids from traditional Chinese medicine, Aconiti kusnezoffii radix, which possess highly bioactive as well as highly toxic character for medicinal use. In the present study, for the purpose of better utilizing the toxic herbal material, the performance characteristics of NKA-II, D101, X-5, AB-8, S-8, HPD722 and HPD750 macroporous resins for the enrichment and purification of these six Aconitum alkaloids were critically evaluated. Results showed that NKA-II offered the best adsorption and desorption capacities for six Aconitum alkaloids among the seven macroporous resins tested, which were affected significantly by the pH value. Subsequently, dynamic adsorption and desorption experiments had been carried out with the column packed by NKA-II resin to optimize the separation process of six Aconitum alkaloids. After one run treatment with NKA-II resin, the content of total six Aconitum alkaloids were increased from 5.87% to 60.3%, the recovery was 75.8%. Meanwhile, a validated HPLC-MS method had been developed to qualitative and quantitative these six Aconitum alkaloids. This method would provide scientific references to the large-scale production of six Aconitum alkaloids from Aconiti kusnezoffii radix or other plants and might also expand the secure application of these highly toxic components for pharmacy. PMID:24814003

  9. Development and validation of a liquid chromatography isotope dilution mass spectrometry method for the reliable quantification of alkylphenols in environmental water samples by isotope pattern deconvolution.

    PubMed

    Fabregat-Cabello, Neus; Sancho, Juan V; Vidal, Andreu; González, Florenci V; Roig-Navarro, Antoni Francesc

    2014-02-01

    We present here a new measurement method for the rapid extraction and accurate quantification of technical nonylphenol (NP) and 4-t-octylphenol (OP) in complex matrix water samples by UHPLC-ESI-MS/MS. The extraction of both compounds is achieved in 30min by means of hollow fiber liquid phase microextraction (HF-LPME) using 1-octanol as acceptor phase, which provides an enrichment (preconcentration) factor of 800. On the other hand we have developed a quantification method based on isotope dilution mass spectrometry (IDMS) and singly (13)C1-labeled compounds. To this end the minimal labeled (13)C1-4-(3,6-dimethyl-3-heptyl)-phenol and (13)C1-t-octylphenol isomers were synthesized, which coelute with the natural compounds and allows the compensation of the matrix effect. The quantification was carried out by using isotope pattern deconvolution (IPD), which permits to obtain the concentration of both compounds without the need to build any calibration graph, reducing the total analysis time. The combination of both extraction and determination techniques have allowed to validate for the first time a HF-LPME methodology at the required levels by legislation achieving limits of quantification of 0.1ngmL(-1) and recoveries within 97-109%. Due to the low cost of HF-LPME and total time consumption, this methodology is ready for implementation in routine analytical laboratories. PMID:24423386

  10. Measurement of "total" microcystins using the MMPB/LC/MS/MS method, and application to HAB impacted surface waters-presentation

    EPA Science Inventory

    The detection and quantification of microcystins, a family of toxins associated with harmful algal blooms, is complicated by their structural diversity and a lack of commercially available analytical standards for method development. As a result, most detection methods have focus...

  11. Determination of selected biogenic amines in Acacia rigidula plant materials and dietary supplements using LC-MS/MS methods.

    PubMed

    Pawar, Rahul S; Grundel, Erich; Fardin-Kia, Ali Reza; Rader, Jeanne I

    2014-01-01

    Extracts of Acacia rigidula leaves are used in weight-loss products sold in vitamin shops and over the internet with little or no published data about their potential biological effects. In our chemical investigations on authenticated A. rigidula plant material, we established a rapid and sensitive LC-MS/MS method for the quantitative determination of several phenethylamine, tyramine and tryptamine derivatives. Stable isotopically labeled compounds were used as internal standards for quantitative analysis. We found total calculated contents of 6 biogenic amines in A. rigidula leaf of 18.6 and 32.9μg/g. The content of selected amines in 21 dietary supplements labeled as containing A. rigidula was determined by a second LC-MS/MS method. Our study revealed significant differences in the amine profiles of authenticated plant materials and dietary supplements. β-Methylphenethylamine, a non-natural compound, was found in 9 of the 21 dietary supplement products. β-Methylphenethylamine was found at levels of 960-60,500μg/g while phenethylamine was found at levels of 710-171,620μg/g. β-Methylphenethylamine is a positional isomer of amphetamine and our results showed that it can be misidentified as amphetamine during LC-MS analysis. An independent GC-MS analysis was used to confirm the presence of β-methylphenethylamine and the absence of amphetamine in dietary supplements labeled as containing A. rigidula. This study demonstrates that confirmations by independent analytical methods are essential to verify findings of unusual or unexpected compounds in dietary supplements.

  12. A new objective method for acquisition and quantification of reflex receptive fields.

    PubMed

    Jensen, Michael Brun; Manresa, José Biurrun; Andersen, Ole Kæseler

    2015-03-01

    The nociceptive withdrawal reflex (NWR) is a polysynaptic spinal reflex correlated with pain perception. Assessment of this objective physiological measure constitutes the core of existing methods for quantification of reflex receptive fields (RRFs), which however still suffer from a certain degree of subjective involvement. This article proposes a strictly objective methodology for RRF quantification based on automated identification of NWR thresholds (NWR-Ts). Nociceptive withdrawal reflex thresholds were determined for 10 individual stimulation sites using an interleaved up-down staircase method. Reflexes were detected from electromyography by evaluation of interval peak z scores and application of conduction velocity analysis. Reflex receptive field areas were quantified from interpolated mappings of NWR-Ts and compared with existing RRF quantifications. A total of 3 repeated measures were performed in 2 different sessions to evaluate the test-retest reliability of the various quantifications, using coefficients of repeatability (CRs) and hypothetical sample sizes. The novel quantifications based on identification of NWR-Ts showed a similar level of reliability within and between sessions, whereas existing quantifications all demonstrated worse between-session than within-session reliability. The NWR-T-based quantifications required a smaller sample size than any of the existing RRF measures to detect a clinically relevant effect in a crossover study design involving more than 1 session. Of all measures, quantification from mapping of inversed NWR-Ts demonstrated superior reliability both within (CR, 0.25) and between sessions (CR, 0.28). The study presents a more reliable and robust quantification of the RRF to be used as biomarker of pain hypersensitivity in clinical and experimental research.

  13. Development of a UHPLC-MS/MS method for the determination of plasma histamine in various mammalian species.

    PubMed

    Liu, Jia; Wang, Lei; Hu, Wenjuan; Chen, Xiaoyan; Zhong, Dafang

    2014-11-15

    Histamine is an important mediator of anaphylactic reactions. Although several methods have been developed to measure histamine levels, each has its limitations. In this study, we developed and validated a convenient bioanalytical method for the qualitative and quantitative determination of histamine in plasma samples from humans, beagle dogs, Sprague-Dawl