Science.gov

Sample records for amino derivatized bicyclonucleosides

  1. Comparison of amino acid derivatization reagents for LC-ESI-MS analysis. Introducing a novel phosphazene-based derivatization reagent.

    PubMed

    Rebane, Riin; Oldekop, Maarja-Liisa; Herodes, Koit

    2012-09-01

    Amino acid analysis with high performance liquid chromatography with electrospray ionization mass spectrometry (LC-ESI-MS) is an emerging method. For more sensitive analysis, derivatization is used and next to commercially available derivatization reagents such as dansyl chloride (DNS), 9-fluorenylmethyl chloroformate (FMOC-Cl) and diethyl ethoxymethylenemalonate (DEEMM), new derivatization reagents are designed specially for LC-ESI-MS, like p-N,N,N-trimethylammonioanilyl N'-hydroxysuccinimidyl carbamate iodide (TAHS) which provides very low limits of detection. In this work, a novel phosphazene based derivatization reagent (FOSF) that provides comparable limits of quantitation (LoQ) to TAHS is introduced. Moreover, a thorough comparison between FOSF, TAHS, DNS, FMOC-Cl and DEEMM is carried out for 7 different amino acids - Arg, Asp, Gly, β-Ala, Pro, Trp and Phe. This is a first time that thorough comparison is carried out on the same instrument for amino acid derivatization reagents. Results on the same instrument for five amino acid derivatization reagents show that novel reagents are sensitive with LoQ values around 80 fmol but have disadvantages such as problematic chromatographic separation. Next to novel reagents, DEEMM offers very good LoQ-s (average of 150 fmol) and wide dynamic linear range.

  2. Comparison of three buffer solutions for amino acid derivatization and following analysis by liquid chromatography electrospray mass spectrometry.

    PubMed

    Rebane, Riin; Herodes, Koit

    2012-07-06

    For reversed phase separation amino acids are usually derivatized. Several derivatization reactions are carried out at basic pH. In the present work, influence of three basic buffer solutions on liquid chromatography electrospray ionization mass-spectrometric (LC-ESI-MS) analysis of amino acid derivatives was studied. Borate buffer--the most common derivatization buffer--was found to influence ESI ionization up to 23 min retention time. For 9-fluorenylmethylmethoxycarbonyl chloride (Fmoc-Cl derivatization) carbonate buffer should be preferred as it provides higher responses. Hexafluoroisopropanol (HFIP) buffer improves chromatographic peak shapes and responses for diethyl ethoxymethylenemalonate (DEEMM) derivatives.

  3. Development of amino acid derivatization reagents for liquid chromatography electrospray ionization mass spectrometric analysis and ionization efficiency measurements.

    PubMed

    Rebane, Riin; Rodima, Toomas; Kütt, Agnes; Herodes, Koit

    2015-04-17

    Derivatization is one of the most common ways for improving chromatographic separation and sensitivity for LC-ESI-MS analysis. The aim of this work was to design new derivatization reagents for LC-ESI-MS analysis of amino acids which would (1) provide good reversed phase chromatographic separation, (2) most importantly, provide low detection limits, (3) be easily synthesized, (4) produce derivatives which are less susceptible to matrix influences and (5) have convenient derivatization procedure with stable derivatives suitable for automatization. In the current work two new LC-ESI-MS compatible derivatization reagents have been designed and synthesized, dibenzyl ethoxymethylene malonate (DBEMM) and benzyl ethyl ethoxymethylene malonate (EBEMM). The DBEMM meets all the goals set with instrumental detection limits as low as 1 femtomole for amino acids and 40 attomole for selenoamino acids.

  4. Chiral derivatizations applied for the separation of unusual amino acid enantiomers by liquid chromatography and related techniques.

    PubMed

    Ilisz, István; Aranyi, Anita; Péter, Antal

    2013-06-28

    Amino acids are essential for life, and have many functions in metabolism. One particularly important function is to serve as the building blocks of peptides and proteins, giving rise complex three dimensional structures through disulfide bonds or crosslinked amino acids. Peptides are frequently cyclic and contain proteinogenic as well as nonproteinogenic amino acids in many instances. Since most of the proteinogenic α-amino acids contain at least one stereogenic center (with the exception of glycine), the stereoisomers of all these amino acids and the peptides in which they are to be found may possess differences in biological activity in living systems. The impetus for advances in chiral separation has been highest in the past 25 years and this still continues to be an area of high focus. The important analytical task of the separation of isomers is achieved mainly by chromatographic and electrophoretic methods. This paper reviews indirect separation approaches, i.e. derivatization reactions aimed at creating the basis for the chromatographic resolution of biologically and pharmaceutically important enantiomers of unusual amino acids and related compounds, with emphasis on the literature published from 1980s. The main aspects of the chiral derivatization of amino acids are discussed, i.e. derivatization on the amino group, transforming the molecules into covalently bonded diastereomeric derivatives through the use of homochiral derivatizing agents. The diastereomers formed (amides, urethanes, urea and thiourea derivatives, etc.) can be separated on achiral stationary phases. The applications are considered, and in some cases different derivatizing agents for the resolution of complex mixtures of proteinogenic d,l-amino acids, non-proteinogenic amino acids and peptides/amino acids from peptide syntheses or microorganisms are compared.

  5. Application of chiral derivatizing agents in the high-performance liquid chromatographic separation of amino acid enantiomers: a review.

    PubMed

    Ilisz, István; Berkecz, Robert; Péter, Antal

    2008-05-12

    The past 20 years has seen an explosive growth in the field of chirality, as illustrated by the rapid progress in the various facets of this intriguing field. The impetus for advances in chiral separation has been highest in the past decade and this still continues to be an area of high focus. This paper reviews indirect separation approaches, i.e. derivatization reactions aimed at creating the basis for the chromatographic resolution of biologically and pharmaceutically important enantiomers, with emphasis on the literature published in the last 12 years. The main aspects of the chiral derivatization of amino acids are discussed, i.e. derivatization on the amino group, transforming the molecules into covalently bonded diastereomeric derivatives through the use of homochiral derivatizing agents. The diastereomers formed (amides, urethanes, urea, thiourea derivatives, etc.) can be separated on achiral stationary phases. The applications are considered, and in some cases different derivatizing agents for the resolution of complex mixtures of proteinogenic d,l-amino acids, non-proteinogenic amino acids and peptides/amino acids from peptide syntheses or microorganisms are compared.

  6. Protein Quantification by Derivatization-Free High-Performance Liquid Chromatography of Aromatic Amino Acids

    PubMed Central

    Hesse, Almut

    2016-01-01

    Amino acid analysis is considered to be the gold standard for quantitative peptide and protein analysis. Here, we would like to propose a simple HPLC/UV method based on a reversed-phase separation of the aromatic amino acids tyrosine (Tyr), phenylalanine (Phe), and optionally tryptophan (Trp) without any derivatization. The hydrolysis of the proteins and peptides was performed by an accelerated microwave technique, which needs only 30 minutes. Two internal standard compounds, homotyrosine (HTyr) and 4-fluorophenylalanine (FPhe) were used for calibration. The limit of detection (LOD) was estimated to be 0.05 µM (~10 µg/L) for tyrosine and phenylalanine at 215 nm. The LOD for a protein determination was calculated to be below 16 mg/L (~300 ng BSA absolute). Aromatic amino acid analysis (AAAA) offers excellent accuracy and a precision of about 5% relative standard deviation, including the hydrolysis step. The method was validated with certified reference materials (CRM) of amino acids and of a pure protein (bovine serum albumin, BSA). AAAA can be used for the quantification of aromatic amino acids, isolated peptides or proteins, complex peptide or protein samples, such as serum or milk powder, and peptides or proteins immobilized on solid supports. PMID:27559481

  7. Quantitation of amino acids in plasma by high performance liquid chromatography: simultaneous deproteinization and derivatization with 9-fluorenylmethyloxycarbonyl chloride.

    PubMed

    Jámbor, A; Molnár-Perl, I

    2009-08-21

    This paper, as a novelty to this field, presents the deproteinization and derivatization of plasma's free amino acids (PFAAs), simultaneously, in a single step, with the acetonitrile (ACN) containing 9-fluorenylmethyloxycarbonyl chloride (FMOC) reagent. Deproteinization and derivatization, were studied with 22 amino acids, applying photodiode array (DAD) and fluorescence (FL) detection, simultaneously. Model investigations have been carried out as a function of the FMOC concentration, reaction time and reaction conditions: with standard solutions, with human plasma samples in its initial condition and fortified with standard amino acids (excluding tryptophan because it co-elutes with the hydrolyzed FMOC). Reproducibilities of 22 amino acids, including both histidine and tyrosine derivatives, obtained under optimum derivatization conditions are presented (at 3.0 mM FMOC concentration, at pH 9; derivatization time - 20 min), and characterized with the relative standard deviation percentages of their responses (amino acid FMOC derivatives proved to be 2.5 pmol, except for cystine, ornithine (5 pmol) and for the total of tyrosines (N-FMOC-tyrosine and N,O-FMOC-tyrosine 10 pmol).

  8. Assessment of the efficacy of anticancer drugs by amino acid metabolomics using fluorescence derivatization-HPLC.

    PubMed

    Tomita, Ryoko; Todoroki, Kenichiro; Machida, Kazuyuki; Nishida, Sho; Maruoka, Hiroshi; Yoshida, Hideyuki; Fujioka, Toshihiro; Nakashima, Manabu; Yamaguchi, Masatoshi; Nohta, Hitoshi

    2014-01-01

    Metabolomic studies conducted for evaluating cancer pathogenesis and progression by monitoring the amino acids metabolic balance hold great promise for assessing current and future anticancer treatments. We performed a comprehensive quantification of 21 amino acids concentrations in cultured human colorectal adenocarcinoma cells treated with the anticancer drugs 5-fluorouracil, irinotecan, and cisplatin. A precolumn fluorescence derivatization-HPLC method involving 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate was used. Amino acid concentration data were analyzed by principal-component analysis and partial least-squares multivariate statistical methods to represent samples on two-dimensional graphs. The hierarchical cluster analysis and linear discriminant analysis were used to classify the samples on the score plots. Unlike the cluster analysis approach, the linear discrimination analysis classification successfully distinguished anticancer drug-treated samples from the untreated controls. Moreover, three candidate amino acids (serine, aspartic acid, and methionine) were identified from the loading plots as potential biomarkers. Our proposed method might be able to evaluate the effectiveness of anticancer therapy even in small laboratories or medical institutions.

  9. Simultaneous extraction and derivatization of amino acids and free fatty acids in meat products.

    PubMed

    Leggio, Antonella; Belsito, Emilia Lucia; De Marco, Rosaria; Liguori, Angelo; Siciliano, Carlo; Spinella, Mariagiovanna

    2012-06-08

    In meat products the contents of free amino acids and free fatty acids are two important parameters used to establish their quality. These compounds play a very important role in defining the sensorial characteristics and acceptability of meat products. An innovative procedure for the measurement of free amino acid and fatty acid contents in meat and meat derivatives was developed. A single experiment can be performed in order to determine simultaneously the free amino acid and free fatty acid profiles. The analytes of interest are rapidly extracted from the meat matrix and derivatized by using methyl chloroformate. This reagent allows the transformation of the two groups of analytes into the corresponding N-methyloxycarbonyl amino acid methyl esters and fatty acid methyl esters that can easily be extracted and sampled for their further identification and quantitation. The measurement of the obtained amino acid and fatty acid derivatives is performed by GC/MS analysis and their concentrations are calculated by using two appropriate internal standards. The main advantage of the proposed protocol is the determination at the same time of two important classes of analytes that are of great importance in food analysis and characterization. Moreover, minimal sample manipulation and preparation, and reduced total extraction times are required to obtain the response with respect to conventional procedures, in which instead the analysis of both the two classes of compounds must be performed separately. The helpfulness of the protocol was tested in the analysis of a cured meat product that is typical of the South of Italy. The optimized protocol successfully allowed the determination of thirteen free amino acids and six free fatty acids, namely those most abundant in the lipid content of the cured meat product under evaluation. Copyright © 2012 Elsevier B.V. All rights reserved.

  10. Preparative scale isolation, purification and derivatization of mimosine, a non-proteinogenic amino acid.

    PubMed

    Nokihara, Kiyoshi; Hirata, Akiyoshi; Sogon, Tetsuya; Ohyama, Takafumi

    2012-07-01

    Focusing on drug discovery non-proteinogenic amino acids have often been used as important building blocks for construction of compound libraries in the filed of combinatorial chemistry and chemical biology. Highly homogeneous L: -mimosine, α-amino-β-(3-hydoxy-4-oxo-1,4-dihydropyridin-1-yl)-propanoic acid, a non-proteinogenic amino acid, has been successfully isolated and purified on an industrial scale from wild leaves of Leucaena (Leucaena leucocephala de Wit) which is a widely distributed legume in Okinawa, a sub-tropical island in Japan. Optical purity determinations used for quality control have been established through diastereomer formation. Physico-chemical properties and biological properties of purified mimosine have been clarified. Mimosine is sparingly soluble in water and organic solvents but can be dissolved in aqueous alkaline solution. The tyrosinase pathway is of particular interest in the cosmetic field, since mimosine is an analog of tyrosine. Thus the present purified mimosine have been tested in tyrosinase inhibitory assays. The IC50 for tyrosinase inhibitory activity of purified Mim was compared with kojic acid. Mimosine shows significant inhibition of melanin production in murine melanoma cells. The derivatization of mimosine has been investigated with a focus on its use in conventional peptide syntheses to generate mimosyl peptides. N-(9-Fluorenylmethoxycarbonyloxy)-mimosine and resin-bound mimosine for solid-phase syntheses have also been performed. Highly homogeneous Mim is a useful material for the development of functional cosmetics or active pharmaceutical ingredients.

  11. Separation and determination of B vitamins and essential amino acids in health drinks by CE-LIF with simultaneous derivatization.

    PubMed

    Zhao, Danyue; Lu, Minghua; Cai, Zongwei

    2012-08-01

    An efficient and sensitive method for the separation and determination of three essential amino acids and three B vitamins by CE-LIF with a simultaneous derivatization procedure was developed. The conditions for derivatization and separation of these micronutrients were investigated. FITC was used as the reagent for fluorescence tagging of arginine (Arg), valine (Val), tryptophan (Trp), folic acid (FA), and niacinamide (NA). Riboflavin (RF) was detected without derivatization. Derivatization of analytes dissolved in borate solution was performed by successive introduction of fluorescence reagent and analytes followed by water bathing at 43°C. The molar ratio of sample/reagent (S/R), derivatization temperature, and incubation time significantly influenced the efficiency of derivatization. To maximize the fluorescence yield, a high S/R (≥20) was required. The nonderivatized RF and five derivatized analytes were separated in the optimized CE-LIF system with the application of 22 kV voltage and 25 mM borate buffer at pH 9.85. Validation of the method showed good linearity for the corrected peak areas versus standard concentrations for the six analytes. The RSDs (n = 3) of the migration time and the peak area obtained for the analytes ranged from 0.4 to 1.1% and from 1.9 to 4.4%, respectively. The developed method, with the lowest LOD of 0.5 nM, was successfully applied for the efficient derivatization and determination of B vitamins in four health drink samples. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. Characterization of N-methylated amino acids by GC-MS after ethyl chloroformate derivatization.

    PubMed

    Reddy, B Sudarshana; Chary, V Naresh; Pavankumar, P; Prabhakar, S

    2016-08-01

    Methylation is an essential metabolic process in the biological systems, and it is significant for several biological reactions in living organisms. Methylated compounds are known to be involved in most of the bodily functions, and some of them serve as biomarkers. Theoretically, all α-amino acids can be methylated, and it is possible to encounter them in most animal/plant samples. But the analytical data, especially the mass spectral data, are available only for a few of the methylated amino acids. Thus, it is essential to generate mass spectral data and to develop mass spectrometry methods for the identification of all possible methylated amino acids for future metabolomic studies. In this study, all N-methyl and N,N-dimethyl amino acids were synthesized by the methylation of α-amino acids and characterized by a GC-MS method. The methylated amino acids were derivatized with ethyl chloroformate and analyzed by GC-MS under EI and methane/CI conditions. The EI mass spectra of ethyl chloroformate derivatives of N-methyl (1-18) and N,N-dimethyl amino acids (19-35) showed abundant [M-COOC2 H5 ](+) ions. The fragment ions due to loss of C2 H4 , CO2 , (CO2  + C2 H4 ) from [M-COOC2 H5 ](+) were of structure indicative for 1-18. The EI spectra of 19-35 showed less number of fragment ions when compared with those of 1-18. The side chain group (R) caused specific fragment ions characteristic to its structure. The methane/CI spectra of the studied compounds showed [M + H](+) ions to substantiate their molecular weights. The detected EI fragment ions were characteristic of the structure that made easy identification of the studied compounds, including isomeric/isobaric compounds. Fragmentation patterns of the studied compounds (1-35) were confirmed by high-resolution mass spectra data and further substantiated by the data obtained from (13) C2 -labeled glycines and N-ethoxycarbonyl methoxy esters. The method was applied to human plasma samples for the identification

  13. Amino acid analysis by high-performance liquid chromatography after derivatization with 9-fluorenylmethyloxycarbonyl chloride Literature overview and further study.

    PubMed

    Jámbor, A; Molnár-Perl, I

    2009-04-10

    A literature overview is given of HPLC methods currently in use to determine amino acids as their 9-fluorenylmethyloxycarbonyl (FMOC) derivatives. On the basis of the detailed literature overview an exhaustive derivatization study was performed with 22 amino acids, applying photodiode array (DAD) and fluorescence (FL) detection simultaneously, in order to clear up the controversial points of FMOC derivatization. Model investigations have been carried out as a function of the reaction time and reaction conditions, such as the molar concentration of the reagent, the molar ratios of the reactants, the pH and the solvent composition of the reaction medium. Special emphasis was put (i) on the evaluation of the blank values of the reagents, as a function of the composition and that of the pH of the reaction medium, (ii) on the unambiguous quantitation of all amino acids, including the less reactive aspartic and glutamic acids, as well as on the formation and transformation of histidine and tyrosine, existing partly, as single (N-FMOC-histidine, N-FMOC-tyrosine), partly as double labeled species (N,NH-FMOC-histidine, N,O-FMOC-tyrosine). Reproducibilities of 22 amino acids, including both histidine and tyrosine derivatives, obtained under optimum derivatization conditions are presented (at 0.5mM FMOC concentration corresponding to the molar ratios of [FMOC]/[amino acids](T)=5.5/1 (note: the superscript 'T' means the total of amino acids), with acetonitrile containing reagents, at pH 9, derivatization time=20 min), and characterized with the relative standard deviation percentages of their responses (amino acid FMOC derivatives proved to be 1 pmol, except for serine, glycine, valine (2.5 pmol), for cystine, N,NH-FMOC-histidine and for the total of N-FMOC-tyrosine and N,O-FMOC-tyrosine (5 pmol) and for tryptophan (10 pmol).

  14. An alternative derivatization method for the analysis of amino acids in cerebrospinal fluid by gas chromatography-mass spectrometry.

    PubMed

    de Paiva, Maria José Nunes; Menezes, Helvécio Costa; Christo, Paulo Pereira; Resende, Rodrigo Ribeiro; Cardeal, Zenilda de Lourdes

    2013-07-15

    The determination of the concentrations of l-amino acids in cerebrospinal fluid (CSF) has been used to gain biochemical insight into central nervous system disorders. This paper describes a microwave-assisted derivatization (MAD) method using N,O-bis-(trimethylsilyl)trifluoroacetamide (BSTFA) as a derivatizing agent for determining the concentrations of l-amino acids in human CSF by gas chromatography with mass spectrometry (GC/MS). The experimental design used to optimize the conditions showed that the optimal derivatization time was 3min with a microwave power of 210W. The method showed good performance for the validation parameters. The sensitivity was very good, with limits of detection (LODs) ranging from 0.01μmolL(-1) to 4.24μmolL(-1) and limits of quantification (LOQs) ranging from 0.02 to 7.07μmolL(-1). The precision, measured using the relative standard deviation (RSD), ranged from 4.12 to 15.59% for intra-day analyses and from 6.36 to 18.71% for inter-day analyses. The coefficients of determination (R(2)) were above 0.990 for all amino acids. The optimized and validated method was applied to the determination of amino acid concentrations in human CSF.

  15. Quantification of glycated N-terminal peptide of hemoglobin using derivatization for multiple functional groups of amino acids followed by liquid chromatography/tandem mass spectrometry.

    PubMed

    Sakaguchi, Yohei; Kinumi, Tomoya; Yamazaki, Taichi; Takatsu, Akiko

    2016-02-01

    A novel method of amino acid analysis using derivatization of multiple functional groups (amino, carboxyl, and phenolic hydroxyl groups) was applied to measure glycated amino acids in order to quantify glycated peptides and evaluate the degree of glycation of peptide. Amino and carboxyl groups of amino acids were derivatized with 1-bromobutane so that the hydrophobicities and basicities of the amino acids, including glycated amino acids, were improved. These derivatized amino acids could be detected with high sensitivity using LC-MS/MS. In this study, 1-deoxyfructosyl-VHLTPE and VHLTPE, which are N-terminal peptides of the β-chains of hemoglobin, were selected as target compounds. After reducing the peptide sample solution with sodium borohydride, the obtained peptides were hydrolyzed with hydrochloric acid. The released amino acids were then derivatized with 1-bromobutane and analyzed with LC-MS/MS. The derivatized amino acids, including glycated amino acids, could be separated using an octadecyl silylated silica column and good sharp peaks were detected. We show a confirmatory experiment that the proposed method can be applied to evaluate the degree of glycation of peptides, using mixtures of glycated and non-glycated peptide.

  16. Derivatization and fluorescence detection of amino acids and peptides with 9-fluorenylmethyl chloroformate on the surface of a solid adsorbent.

    PubMed

    Shangguan, D; Zhao, Y; Han, H; Zhao, R; Liu, G

    2001-05-01

    An approach that exploits the surface of a solid adsorbent is proposed for precolumn FMOC derivatization of amino acids and peptides. Amino acids (Ser, Glu, GABA, Val, Phe, Lys) and two neuropeptides (substance P and Leuenkephalin) were adsorbed on alkaline silica gel cartridges. After drying, they were reacted with 9-fluorenyl-methyl chloroformate (FMOC-Cl) in toluene. After washing off the excess FMOC-Cl with ethyl acetate, the derivatives were eluted with aqueous eluant. The eluates were separated and detected by means of HPLC with fluorescence detection. Compared with the traditional derivatization in the liquid phase, the extent of formation of byproducts of FMOC-Cl with water was greatly decreased, and the excess FMOC-Cl was eliminated completely.

  17. Evaluation of diazotized 4-amino-3,5-dinitrobenzoic acid (ADBA) as a new derivatizing reagent.

    PubMed

    Idowu, S O; Olaniyi, A A

    2001-09-01

    A preliminary evaluation of the reactivity of diazotized 4-amino-3,5-dinitrobenzoic acid (ADBA) towards selected aromatic compounds has been described. Successful diazo coupling of pharmaceuticals possessing aromatic rings of varying reactivities was achieved with the arenediazonium ion of ADBA at room (30 degrees C) or elevated temperature (80 degrees C). The adducts formed in spot-test reactions were coloured for some compounds (e.g, cloxacillin and chloroxylenol), others showed colour at elevated temperature of reaction (e.g., salicylic acid and aspirin), while others showed no detectable change in colour of reaction mixture, even at elevated temperature (e.g., imidazole and tinidazole). The coloured product formed at room temperature decomposed and the colour discharged at elevated temperature in some cases (e.g., beta-naphthol). However, thin layer chromatographic analysis suggested that a more lipophilic derivative, relative to the original compound was formed for some of the compounds studied which did not show any detectable colour change in the spot test reactions. The diazotized ADBA is thus shown to be a reactive coupling reagent with which a suitable derivatization methodology could be developed for a wide range of pharmaceuticals in ultraviolet/visible spectrophotometry and high performance liquid chromatographic (HPLC) analysis.

  18. Direct tandem mass spectrometric analysis of amino acids in plasma using fluorous derivatization and monolithic solid-phase purification.

    PubMed

    Tamashima, Erina; Hayama, Tadashi; Yoshida, Hideyuki; Imakyure, Osamu; Yamaguchi, Masatoshi; Nohta, Hitoshi

    2015-11-10

    In this study, we developed a novel direct tandem mass spectrometric method for rapid and accurate analysis of amino acids utilizing a fluorous derivatization and purification technique. Amino acids were perfluoroalkylated with 2H,2H,3H,3H-perfluoroundecan-1-al in the presence of 2-picoline borane via reductive amination. The derivatives were purified by perfluoroalkyl-modified silica-based monolithic solid-phase extraction (monolithic F-SPE), and directly analyzed by tandem mass spectrometry using electrospray ionization without liquid chromatographic separation. The perfluoroalkyl derivatives could be sufficiently distinguished from non-fluorous compounds, i.e. the biological matrix, due to their fluorous interaction. Thus, rapid and accurate determination of amino acids was accomplished. The method was validated with human plasma samples and applied to the analysis of amino acids in the plasma of mice with maple syrup urine disease or phenylketonuria.

  19. GC separation of amino acid enantiomers via derivatization with heptafluorobutyl chloroformate and Chirasil-L-Val column.

    PubMed

    Zahradnícková, Helena; Husek, Petr; Simek, Petr

    2009-11-01

    Heptafluorobutyl chloroformate (HFBCF), a recently introduced derivatization reagent, was examined in enantioseparation of amino acids (AAs) by GC. Twenty proteinogenic AAs, plus ornithine, cystine and 4-fluorophenylalanine (internal standard) were treated with the reagent and separation properties of the derivatives were assessed on a Chirasil-Val capillary column. Nineteen AA enantiomers were efficiently separated in 43 min except proline, arginine and cystine. The HFBCF derivatives of the studied DL-AAs show improved separation over other chloroformate-based derivatives hitherto reported. A combination of the improved and faster separation with a simple derivatization protocol, involving an immediate one-step reaction-extraction in two-phase aqueous-organic medium, and low elution temperatures extend application of HFBCF to chiral AA analysis.

  20. Single-step enantioselective amino acid flux analysis by capillary electrophoresis using on-line sample preconcentration with chemical derivatization.

    PubMed

    Ptolemy, Adam S; Tran, Lara; Britz-McKibbin, Philip

    2006-07-15

    Capillary electrophoresis (CE) represents a versatile platform for integrating sample pretreatment with chemical analysis because of its ability to tune analyte electromigration and band dispersion properties in discontinuous electrolyte systems. In this article, a single-step method that combines on-line sample preconcentration with in-capillary chemical derivatization is developed for rapid, sensitive, and enantioselective analysis of micromolar levels of amino acids that lack intrinsic chromophores by CE with UV detection. Time-resolved electrophoretic studies revealed two distinct stages of amino acid band narrowing within the original long sample injection plug occurring both prior to and after in-capillary labeling via zone passing by ortho-phthalaldehyde/N-acetyl l-cysteine (OPA/NAC). This technique enabled direct analysis of d-amino acids in a 95% enantiomeric excess mixture with sub-micromolar detection limits and minimal sample handling, where the capillary functions as a preconcentrator, microreactor, and chiral selector. On-line sample preconcentration with chemical derivatization CE (SPCD-CE) was applied to study the enantioselective amino acid flux in Escherichia coli bacteria cultures, which demonstrated a unique l-Ala efflux into the extracellular medium. New strategies for high-throughput analyses of low-abundance metabolites are important for understanding fundamental physiological processes in bacteria required for screening the efficacy of new classes of antibiotics as well as altered metabolism in genetically modified mutant strains.

  1. Gas chromatography-mass spectrometry of hexafluoroacetone derivatives: First time utilization of a gaseous phase derivatizing agent for analysis of extraterrestrial amino acids.

    PubMed

    Geffroy-Rodier, C; Buch, A; Sternberg, R; Papot, S

    2012-07-06

    Within the perspective of the current and next space missions to Mars (MSL 2011 and Exomars 2016-2018), the detection and enantioselective separation of building blocks such as the amino acids are important subjects which are becoming fundamental for the search for traces of life on the surface and subsurface of Mars. In this work, we have developed and optimized a method adapted to space experimentation to derivatize and analyze amino acids, using hexafluoroacetone as the derivatizing agent. The temperature, duration of the derivative transfer to the analyser, and chromatographic separation parameters have been optimized to meet the instrument design constraints imposed on devices for extraterrestrial experiments. The work presented in this rationale has established that hexafluoroacetone, in addition to its intrinsic qualities, such as the production of light-weight derivatives (no racemization) and great resistance to the drastic operating conditions, has indeed facilitated simple and fast derivatization that appears to be suitable for in situ analysis in space. By using hexafluoroacetone as the derivatizing agent, we successfully identified, 21 amino acids including 12 of the 20 proteinic amino acids without stirring or extraction steps. Ten of these derivatized amino acids were enantioselectively separated. The precision and accuracy measurements for the D/L ratio showed that the proposed method was also suitable for the determination of both enantioselective forms of most of the tested amino acids. The limits of detection obtained were lower than the ppb level of organic molecules detected in Martian meteorites.

  2. Pulsed electromembrane extraction for analysis of derivatized amino acids: A powerful technique for determination of animal source of gelatin samples.

    PubMed

    Rezazadeh, Maryam; Yamini, Yadollah; Seidi, Shahram; Aghaei, Ali

    2015-05-01

    Differentiation of animal sources of gelatin is required for many reasons such as some anxieties about bovine spongiform encephalopathy or a ban on consuming porcine gelatin in some religions. In the present work, an efficient method is introduced for determination of animal origin of gelatin samples. The basis of this procedure is the application of pulsed electric field for extraction, preconcentration, and analysis of derivatized amino acids in gelatin. To this end, after derivatization of amino acids of interest by means of o-phthalaldehyde (OPA) for enhancing their ultraviolet (UV) absorbance as well as increasing their lipophilicities, a 137V electric field was applied for 20min with 10min(-1) frequency to make the analytes migrate through a 200µm organic liquid membrane into an aqueous acceptor phase. Finally, the acceptor phase was analyzed by HPLC-UV. The proposed technique offered a high efficiency for analysis of amino acids, regarding 43% and 79% as extraction recoveries and 25ng mL(-1) and 50ng mL(-1) as limits of detection (LODs) for asparagine and glutamine, respectively. Therefore, due to sample cleanup ability of the proposed method and obtained preconcentration factors (29 and 53 for asparagine and glutamine, respectively), it could be carried out for differentiation of animal origins of gelatin samples, even if only small amounts of samples are available or in complicated media of foodstuffs and medicament. Copyright © 2015 Elsevier B.V. All rights reserved.

  3. Derivatization of (+/-)-5-[(2-methylphenoxy)methyl]-2-amino-2-oxazoline, an imidazoline binding sites ligand, with (+)-(R)-alpha-methylbenzyl isocyanate for drug monitoring purposes.

    PubMed

    Matoga, Myriam; Forfar, Isabelle; Chaimbault, Corinne; Guillon, Jean; Péhourcq, Fabienne; Bosc, Jean-Jacques; Rettori, Marie-Claire; Jarry, Christian

    2002-12-01

    The derivatization of racemic 5-[(2-methylphenoxy)methyl]-2-amino-2-oxazoline, developed as an imidazoline binding sites ligand, with (+)-(R)-alpha-methylbenzyl isocyanate was performed in chloroform. The reaction led to two pairs of diastereomers, which were separated by RP-HPLC. A kinetic study of the derivatization reaction was achieved in order to establish conditions suitable for experimental drug monitoring.

  4. Hollow fiber-stir bar sorptive extraction and microwave assisted derivatization of amino acids in biological matrices.

    PubMed

    Li, Jia; Qi, Huan-Yang; Wang, Yan-Bin; Su, Qiong; Wu, Shang; Wu, Lan

    2016-11-25

    A kind of solid phase microextraction configuration combining the principles of hollow fiber solid phase microextraction (HF-SPME) and stir bar sorptive extraction (SBSE) is presented. The main feature of HF-SBSE is the use of microporous hollow fiber acting as the carrier and filter, while a thin stainless steel wire and silica microspheres in the lumen of hollow fiber respectively acting as the magnetic stirrer and the dispersed sorbents for the collection and extraction of the target analytes, thus affording extraction process like SBSE. Moreover, the prepared hollow fiber stir bar was applied to direct microextraction and microwave assisted derivatization with N,O-Bis(trimethylsilyl)trifluroacetamide (BSTFA) of four amino acids in rats' urine and cerebrospinal fluid followed by gas chromatography mass spectrometric analysis. The limits of detection for four amino acids were found to be in the range of 0.0003-0.017μgmL(-1), and all the analytes did not exhibit any lack of fit. The extraction recoveries using HF-SBSE techniques ranged from 71.8% to 102.3%. The results indicated that hollow fiber stir bar sorptive extraction was a promising technique for the enrichment and direct derivatization of analytes extracted from biological matrices without sample clean-up.

  5. Analysis of amino acids by HPLC/electrospray negative ion tandem mass spectrometry using 9-fluorenylmethoxycarbonyl chloride (Fmoc-Cl) derivatization.

    PubMed

    Ziegler, Jörg; Abel, Steffen

    2014-12-01

    A new method for the determination of amino acids is presented. It combines established methods for the derivatization of primary and secondary amino groups with 9-fluorenylmethoxycarbonyl chloride (Fmoc-Cl) with the subsequent amino acid specific detection of the derivatives by LC-ESI-MS/MS using multiple reaction monitoring (MRM). The derivatization proceeds within 5 min, and the resulting amino acid derivatives can be rapidly purified from matrix by solid-phase extraction (SPE) on HR-X resin and separated by reversed-phase HPLC. The Fmoc derivatives yield several amino acid specific fragment ions which opened the possibility to select amino acid specific MRM transitions. The method was applied to all 20 proteinogenic amino acids, and the quantification was performed using L-norvaline as standard. A limit of detection as low as 1 fmol/µl with a linear range of up to 125 pmol/µl could be obtained. Intraday and interday precisions were lower than 10 % relative standard deviations for most of the amino acids. Quantification using L-norvaline as internal standard gave very similar results compared to the quantification using deuterated amino acid as internal standards. Using this protocol, it was possible to record the amino acid profiles of only a single root from Arabidopsis thaliana seedlings and to compare it with the amino acid profiles of 20 dissected root meristems (200 μm).

  6. Determination of derivatized amino acids in human embryo culture media by gas chromatography.

    PubMed

    Krizman, Mitja; Virant-Klun, Irma; Prosek, Mirko

    2007-10-15

    An adequate analytical method for determination of amino acids can provide a better insight in the metabolism of in vitro human embryo cultures, increasing the success rate of embryo implantation. Since individual amino acid amounts per embryo occur in the nanogram range, GC was the technique of choice, due to its inherent sensitivity and high sample throughput. Amino acids were analyzed as alkyl formate derivatives. The limits of detection (LOD) of all amino acids involved were in the sub-nmol range. The high risk of sample contamination proved to be the major analytical issue, but it could be overcome. For an extended method sensitivity, a simple preconcentration step could also be used.

  7. Separation of amino acids and antibiotics by narrow-bore and normal-bore high-performance liquid chromatography with pre-column derivatization.

    PubMed

    Fiedler, H P; Plaga, A

    1987-01-16

    The selectivity, efficiency and lifetime of normal- and narrow-bore columns for high-performance liquid chromatography were investigated for the separation and quantification of amino acids and the amino acid-like antibiotics phosphinothricin and phosphinothricylalanylalanine in biological samples. These compounds were determined by an automated pre-column derivatization with o-phthalaldehyde-2-mercaptoethanol reagent and UV detection at 338 nm.

  8. Enantioseparation of N-derivatized amino acids by micro-liquid chromatography/laser induced fluorescence detection using quinidine-based monolithic columns.

    PubMed

    Wu, Huihui; Wang, Qiqin; Ruan, Meng; Peng, Kun; Zhu, Peijie; Crommen, Jacques; Han, Hai; Jiang, Zhengjin

    2016-03-20

    A novel carbamoylated quinidine based monolith, namely poly(O-9-[2-(methacryloyloxy)-ethylcarbamoyl]-10,11-dihydroquinidine-co-ethylene dimethacrylate (poly(MQD-co-EDMA)), was prepared for the micro-LC enantioseparation of N-derivatized amino acids. The influence of the mobile phase composition, including the organic modifier proportion, the apparent pH and the buffer concentration, on the enantioresolution of N-derivatized amino acids was systematically investigated. Satisfactory column performance in terms of permeability, efficiency and reproducibility was obtained in most cases. The majority of the enantiomers of the tested N-protected amino acids, including 3,5-DNB, 3,5-DClB, FMOC, 3,5-DMB, p-NB, m-ClB, p-ClB and B derivatives, could be baseline separated on the poly(MQD-co-EDMA) monolithic column within 25min. A self-assembled laser induced fluorescence (LIF) detector was employed to improve sensitivity when analyzing 7-nitro-2,1,3-benzoxadiazole (NBD) derivatives of amino acids. Ten NBD-derivatized amino acids, including arginine and histidine whose enantioseparation on quinidine carbamate based CSPs has not been reported so far, were enantioresolved on the poly(MQD-co-EDMA) monolith column. It is worth noting that the d-enantiomers of NBD-derivatized amino acids eluted first, except in the case of glutamic acid. The LOD values obtained with the LIF detector were comparable to those reported using conventional LC-FL methods. The prepared poly(MQD-co-EDMA) monolithic column coupled with the LIF detector opens up interesting perspectives to the determination of trace D-amino acids in biological samples. Copyright © 2015 Elsevier B.V. All rights reserved.

  9. Amino acid analysis by reverse-phase high-performance liquid chromatography: improved derivatization and detection conditions with 9-fluorenylmethyl chloroformate.

    PubMed

    Bank, R A; Jansen, E J; Beekman, B; te Koppele, J M

    1996-09-05

    An improved method for the quantitative derivatization of amino acids with fluorenylmethyl chloroformate (FMOC-Cl) is described. Amino acids are derivatized in borate buffer at pH 11.4 for 40 min at ambient temperature. All amino acids resulted in stable derivatives. In particular, improved derivatization was obtained with the troublesome amino acids His and Tyr: exclusively monosubstituted His and disubstituted Tyr were formed, eluting as free peaks in the chromatogram. These derivatives show a higher fluorescence response than their disubstituted and monosubstituted counterparts, respectively, resulting from other protocols. Under the new conditions, considerable less of the hydrolysis product of FMOC-Cl is seen in the chromatograms. Baseline noise was substantially reduced at a higher emission wavelength (630 nm instead of 313 or 340 nm). With simple precautions, extensive adsorption of the disubstituted derivatives (Lys, Hyl, and Tyr) on plastic or glass surfaces could be prevented. Calibration curves were linear over a 10 to 300 molar ratio of FMOC-Cl to total amino acid. The detection limits are in the femtomole range and the derivatives are stable for more than 48 h, thus permitting automated analysis of multiple samples.

  10. An UPLC-ESI-MS/MS Assay Using 6-Aminoquinolyl-N-Hydroxysuccinimidyl Carbamate Derivatization for Targeted Amino Acid Analysis: Application to Screening of Arabidopsis thaliana Mutants

    PubMed Central

    Salazar, Carolina; Armenta, Jenny M.; Shulaev, Vladimir

    2012-01-01

    In spite of the large arsenal of methodologies developed for amino acid assessment in complex matrices, their implementation in metabolomics studies involving wide-ranging mutant screening is hampered by their lack of high-throughput, sensitivity, reproducibility, and/or wide dynamic range. In response to the challenge of developing amino acid analysis methods that satisfy the criteria required for metabolomic studies, improved reverse-phase high-performance liquid chromatography-mass spectrometry (RPHPLC-MS) methods have been recently reported for large-scale screening of metabolic phenotypes. However, these methods focus on the direct analysis of underivatized amino acids and, therefore, problems associated with insufficient retention and resolution are observed due to the hydrophilic nature of amino acids. It is well known that derivatization methods render amino acids more amenable for reverse phase chromatographic analysis by introducing highly-hydrophobic tags in their carboxylic acid or amino functional group. Therefore, an analytical platform that combines the 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) pre-column derivatization method with ultra performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UPLC-ESI-MS/MS) is presented in this article. For numerous reasons typical amino acid derivatization methods would be inadequate for large scale metabolic projects. However, AQC derivatization is a simple, rapid and reproducible way of obtaining stable amino acid adducts amenable for UPLC-ESI-MS/MS and the applicability of the method for high-throughput metabolomic analysis in Arabidopsis thaliana is demonstrated in this study. Overall, the major advantages offered by this amino acid analysis method include high-throughput, enhanced sensitivity and selectivity; characteristics that showcase its utility for the rapid screening of the preselected plant metabolites without compromising the quality of the metabolic data. The

  11. Determination of plasma dibasic amino acids following trimethylsilyl-trifluoroacyl derivatization using gas chromatography-mass spectrometry.

    PubMed

    Yoon, Hye-Ran

    2013-03-01

    A rapid analytical method was developed to quantify dibasic amino acids (ornithine, lysine and arginine) after two-step derivatization procedure with good sensitivity and specificity on human plasma. If early diagnosis has not been made, patients with inborn metabolic disorders such as HHH syndrome, Hyperornithinemia and dibasic aminoaciduria rapidly progress to sudden death, physical defect or mental retardation resulting in storage of the toxic material into the brain. Therefore, it is necessary to develop the analytical method for rapid screening and/or correct confirmation diagnosis. The formation of trimethylsilyl derivative of the carboxylic (COO-) functional group was performed by adding MSTFA. Five μL of methyl orange was added to the residue until the color changed into yellow. Consecutively, the trifluoroacyl derivative of the amino (-NH2) functional group was produced by adding MBTFA. Specific ions was chosen for quantification with following ions; m/z 166 and m/z 212 for ornithine, m/z 180 and m/z 395 for lysine, and m/z 292 and, m/z 519 for arginine. A calibration curve showed a linear relationship for the dibasic amino acids spiked to pooled normal plasma showing R(2) of 0.9955-0.9979 in the range of 0.1-600 ng investigated. The utility of the method for screening and diagnosis was demonstrated by recovery 80-125 % and reproducibility with RSD (9-17 %) at low, medium and high concentration fortified to pooled plasma. Collectively, the present study suggest that this method could be useful for diagnosis, screening, therapeutic monitoring of metabolic disorders on dietary therapy with excellent sensitivity and rapidity.

  12. Simultaneous determination of amino acid and monoamine neurotransmitters in PC12 cells and rats models of Parkinson's disease using a sensitizing derivatization reagent by UHPLC-MS/MS.

    PubMed

    Zhao, Xian-En; Zhu, Shuyun; Yang, Hongmei; You, Jinmao; Song, Fengrui; Liu, Zhiqiang; Liu, Shuying

    2015-07-15

    Multi-analytes simultaneous monitoring of amino acid and monoamine neurotransmitters (NTs) has important scientific significance for their related pathology, physiology and drug screening. In this work, in virtue of a mass spectrometry sensitizing reagent 10-ethyl-acridone-3-sulfonyl chloride (EASC) as derivatization reagent, an Ultra High Performance Liquid Chromatography-Tandem Mass Spectrometry (UHPLC-MS/MS) method was developed and validated for simultaneous determination of six amino acid NTs, two monoamine ones and its one metabolite. The simple and rapid derivatization reaction was innovatively combined with plasma preparation by using EASC acetonitrile solution as protein precipitant. This interesting combination brought the advantages of speediness, simpleness and high-throughput in a cost-effective way. Under the optimized conditions, LODs (0.004-3.80nM) and LOQs (0.014-13.3nM) of EASC derivatized-NTs were calculated and found to be significantly lower than those of direct UHPLC-MS/MS detection about 11.5-275.0 and 14.4-371.4 times, respectively. Moreover, EASC derivatization significantly improved chromatographic resolution and matrix effect when compared with direct UPLC-MS/MS detection method without derivatization. Meanwhile, it also brought acceptable precision (3.0-13.0%, peak area CVs%), accuracy (86.4-112.9%), recovery (88.3-107.8%) and stability (3.8-8.5%, peak area CVs%) results. This method was successfully applied for the antiparkinsonian effect evaluation of levodopa and Ginsenoside Rg1 using PC12 cells and rats models by measuring multiple NTs. This provided a new method for the NTs related studies in the future.

  13. Improvement of derivatized amino acid detection sensitivity in micellar electrokinetic capillary chromatography by means of acid-induced pH-mediated stacking technique.

    PubMed

    Dziomba, Szymon; Bekasiewicz, Adrian; Prahl, Adam; Bączek, Tomasz; Kowalski, Piotr

    2014-10-01

    Derivatization is a frequently used sample preparation procedure applicable to the enhancement of analyte detection sensitivity. Amino acids mostly require derivatization prior to electrophoretic or chromatographic analysis, especially if spectrophotometric detection is used. This study presents an on-line preconcentration technique for derivatized amino acids. The sensitivity of the method was improved by the utilization of the proposed acid-induced pH-mediated stacking mechanism. The method is demonstrated by preconcentration of amino acids labeled with 2,4-dinitrofluorobenzene. Use of optimized conditions for a large sample volume injection (40 s, 13.8 kPa) followed by electrokinetic injection of 0.1 M HCl (20 s, 10 kV) gave a 20- to 30-fold enhancement of sensitivity. The significance of the sweeping mechanism and pseudo-isotachophoresis for the on-line sample focusing and the influence of parameters on the preconcentration process were discussed. The applicability of the elaborated method was demonstrated using human urine samples.

  14. Enantioselective capillary electrophoresis-mass spectrometry of amino acids in cerebrospinal fluid using a chiral derivatizing agent and volatile surfactant.

    PubMed

    Prior, A; Moldovan, R C; Crommen, J; Servais, A C; Fillet, M; de Jong, G J; Somsen, G W

    2016-10-12

    The sensitivity of coupled enantioselective capillary electrophoresis-mass spectrometry (CE-MS) of amino acids (AAs) is often hampered by the chiral selectors in the background electrolyte (BGE). A new method is presented in which the use of a chiral selector is circumvented by employing (+)-1-(9-fluorenyl)ethyl chloroformate (FLEC) as chiral AA derivatizing agent and ammonium perfluorooctanoate (APFO) as a volatile pseudostationary phase for separation of the formed diastereomers. Efficient AA derivatization with FLEC was completed within 10 min. Infusion experiments showed that the APFO concentration hardly affects the MS response of FLEC-AAs and presents significantly less ion suppression than equal concentrations of ammonium acetate. The effect of the pH and APFO concentration of the BGE and the capillary temperature were studied in order to achieve optimized enantioseparation. Optimization of CE-MS parameters, such as sheath-liquid composition and flow rate, ESI and MS settings was performed in order to prevent analyte fragmentation and achieve sensitive detection. Selective detection and quantification of 14 chiral proteinogenic AAs was achieved with chiral resolution between 1.2 and 8.6, and limits of detection ranging from 130 to 630 nM injected concentration. Aspartic acid and glutamic acid were detected, but not enantioseparated. The optimized method was applied to the analysis of chiral AAs in cerebrospinal fluid (CSF). Good linearity (R(2) > 0.99) and acceptable peak area and electrophoretic mobility repeatability (RSDs below 21% and 2.4%, respectively) were achieved for the chiral proteinogenic AAs, with sensitivity and chiral resolution mostly similar to obtained for standard solutions. Next to l-AAs, endogenous levels of d-serine and d-glutamine could be measured in CSF revealing enantiomeric ratios of 4.8%-8.0% and 0.34%-0.74%, respectively, and indicating the method's potential for the analysis of low concentrations of d-AAs in presence of

  15. Determination of amino acids in human plasma by liquid chromatography with postcolumn ninhydrin derivatization using a hydroxyapatite cartridge for precolumn deproteination.

    PubMed

    Iwase, H; Ozawa, S; Ikuta, M; Ono, I

    1995-01-06

    Amino acids in human plasma were determined by liquid chromatography with postcolumn ninhydrin derivatization using a hydroxyapatite cartridge for precolumn deproteination. S-Carboxymethyl-L-cysteine, D-phenylglycine and S-aminoethyl-L-cysteine were found to be suitable internal standards. The proposed method is simple, rapid (deproteination time less than 1 min) and reproducible [relative standard deviation below 3% except for low-level aspartic acid (n = 3)]. The average recovery of 25 amino acids was above 90%. The elution time of amino acids in human plasma was approximately 2 h. Protein binding of tryptophan was also determined by the proposed method. The analytical data for amino acids in human plasma deproteinated using the proposed and published methods (5-sulphosalicylic acid and ethanol) were compared.

  16. Measurement of (15)N enrichment of glutamine and urea cycle amino acids derivatized with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate using liquid chromatography-tandem quadrupole mass spectrometry.

    PubMed

    Nakamura, Hidehiro; Karakawa, Sachise; Watanabe, Akiko; Kawamata, Yasuko; Kuwahara, Tomomi; Shimbo, Kazutaka; Sakai, Ryosei

    2015-05-01

    6-Aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) is an amino acid-specific derivatizing reagent that has been used for sensitive amino acid quantification by liquid chromatography-tandem quadrupole mass spectrometry (LC-MS/MS). In this study, we aimed to evaluate the ability of this method to measure the isotopic enrichment of amino acids and to determine the positional (15)N enrichment of urea cycle amino acids (i.e., arginine, ornithine, and citrulline) and glutamine. The distribution of the M and M+1 isotopomers of each natural AQC-amino acid was nearly identical to the theoretical distribution. The standard deviation of the (M+1)/M ratio for each amino acid in repeated measurements was approximately 0.1%, and the ratios were stable regardless of the injected amounts. Linearity in the measurements of (15)N enrichment was confirmed by measuring a series of (15)N-labeled arginine standards. The positional (15)N enrichment of urea cycle amino acids and glutamine was estimated from the isotopic distribution of unique fragment ions generated at different collision energies. This method was able to identify their positional (15)N enrichment in the plasma of rats fed (15)N-labeled glutamine. These results suggest the utility of LC-MS/MS detection of AQC-amino acids for the measurement of isotopic enrichment in (15)N-labeled amino acids and indicate that this method is useful for the study of nitrogen metabolism in living organisms.

  17. A rapid hydrolysis method and DABS-Cl derivatization for complete amino acid analysis of octreotide acetate by reversed phase HPLC.

    PubMed

    Akhlaghi, Yousef; Ghaffari, Solmaz; Attar, Hossein; Alamir Hoor, Amir

    2015-11-01

    Octreotide as a synthetic cyclic octapeptide is a somatostatin analog with longer half-life and more selectivity for inhibition of the growth hormone. The acetate salt of octreotide is currently used for medical treatment of somatostatin-related disorders such as endocrine and carcinoid tumors, acromegaly, and gigantism. Octreotide contains both cysteine and tryptophan residues which make the hydrolysis part of its amino acid analysis procedure very challenging. The current paper introduces a fast and additive-free method which preserves tryptophan and cysteine residues during the hydrolysis. Using only 6 M HCl, this hydrolysis process is completed in 30 min at 150 °C. This fast hydrolysis method followed by pre-column derivatization of the released amino acids with 4-N,N-dimethylaminoazobenzene-4'-sulfonyl chloride (DABS-Cl) which takes only 20 min, makes it possible to do the complete amino acid analysis of an octreotide sample in a few hours. The highly stable-colored DABS-Cl derivatives can be detected in 436 nm in a reversed phase chromatographic system, which eliminates spectral interferences to a great extent. The amino acid analysis of octreotide acetate including hydrolysis, derivatization, and reversed phase HPLC determination was validated according to International Conference of Harmonization (ICH) guidelines.

  18. [Determination of twenty free amino acids in flue-cured tobacco leaves using ultra performance liquid chromatography-single quadruple mass spectrometry and pre-column derivatization].

    PubMed

    Li, Haoli; Zhao, Chunxia; Zhang, Junjie; Fu, Jiajun; Wang, Ying; Lu, Xin; Xu, Guowang

    2013-12-01

    Free amino acids in flue-cured tobacco leaves were investigated using the ultra performance liquid chromatography-single quadruple mass spectrometry detection and pre-column derivatization method. The validation results showed that the method could meet the analytical requirements. A total of 138 tobacco leaf samples were collected from 14 provinces in China in 2011 in which the free amino acids were determined. The relative standard deviations (RSDs) of the contents of free amino acids in different growing regions ranged from 28.50%-94.20%, and those of asparagine and glutamine were over 80%. The RSDs of the contents of free amino acids in full aroma tobacco leaves were larger than those in fresh aroma and medium aroma tobacco leaves. The principal component analysis (PCA) and non-parameter Mann-Whitney U test were used for data analysis. The free amino acids of the same aroma type grown in different regions or different aroma types in the same province showed great variation. The contents of free amino acids of full aroma tobacco grown in Southeast region were much lower than those in Huanghuai region. The contents of free amino acids in Hunan province were much lower than the average contents. The results showed that free amino acids in flue-cured tobacco leaves were affected by the growing region.

  19. Determination of trace amino acids in human serum by a selective and sensitive pre-column derivatization method using HPLC-FLD-MS/MS and derivatization optimization by response surface methodology.

    PubMed

    Li, Guoliang; Cui, Yanyan; You, Jinmao; Zhao, Xianen; Sun, Zhiwei; Xia, Lian; Suo, Yourui; Wang, Xiao

    2011-04-01

    Analysis of trace amino acids (AA) in physiological fluids has received more attention, because the analysis of these compounds could provide fundamental and important information for medical, biological, and clinical researches. More accurate method for the determination of those compounds is highly desirable and valuable. In the present study, we developed a selective and sensitive method for trace AA determination in biological samples using 2-[2-(7H-dibenzo [a,g]carbazol-7-yl)-ethoxy] ethyl chloroformate (DBCEC) as labeling reagent by HPLC-FLD-MS/MS. Response surface methodology (RSM) was first employed to optimize the derivatization reaction between DBCEC and AA. Compared with traditional single-factor design, RSM was capable of lessening laborious, time and reagents consumption. The complete derivatization can be achieved within 6.3 min at room temperature. In conjunction with a gradient elution, a baseline resolution of 20 AA containing acidic, neutral, and basic AA was achieved on a reversed-phase Hypersil BDS C(18) column. This method showed excellent reproducibility and correlation coefficient, and offered the exciting detection limits of 0.19-1.17 fmol/μL. The developed method was successfully applied to determinate AA in human serum. The sensitive and prognostic index of serum AA for liver diseases has also been discussed.

  20. Micellular Electrokinetic Capillary Chromatography in the Undergraduate Curriculum: Separation and Identification of the Amino Acid Residues in an Unknown Dipeptide Using FMOC Derivatization

    NASA Astrophysics Data System (ADS)

    Strein, Timothy G.; Poechmann, James L.; Prudenti, Mark

    1999-06-01

    This manuscript describes our efforts to introduce biochemistry students to micellar electrokinetic capillary chromatography (MEKC), a mode of capillary electrophoresis that employs micelles in the operating buffer. Unlike free solution capillary electrophoresis, MEKC is capable of resolving both charged and uncharged analytes because the micellar pseudo stationary phase allows for the separation of uncharged species. The experiment described herein includes a comparison of MEKC, employing sodium dodecyl sulfate (SDS) as the micelle-forming species, with reverse-phase HPLC. Both methods are used to determine the amino acid residues in an unknown dipeptide. Advanced undergraduate chemistry, biochemistry, and biology majors perform this experiment in the Biochemical Methods course at Bucknell University. The students cleave the peptide bond, derivatize the resultant amino acids with 9-fluorenylmethyl chloroformate (FMOC), and separate the FMOC-amino acid derivatives using HPLC and MEKC. This manuscript details the analytical procedures for the MEKC separation and presents typical student data obtained using this relatively new method.

  1. Enantiomeric separation of ornithine in complex mixtures of amino acids by EKC with off-line derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate.

    PubMed

    Martínez-Girón, Ana Belén; Domínguez-Vega, Elena; García-Ruiz, Carmen; Crego, Antonio L; Marina, Maria Luisa

    2008-11-01

    A new analytical methodology was developed by EKC enabling the fast enantiomeric separation of Ornithine in complex mixtures of amino acids. A previous derivatization step with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) was achieved to enable the sensitive UV detection of amino acids as well as to make possible their interaction with the CDs employed as chiral selectors. A dual CD system containing an anionic and a neutral CD in phosphate buffer at acid pH showed a high resolving power allowing the enantiomeric separation of 18 protein amino acids and Orn. The method was applied to the analysis of fermented foods to investigate the extent of the presence of Orn enantiomers.

  2. A new strategy for the selective determination of D-amino acids: enzymatic and chemical modifications for pre-column derivatization.

    PubMed

    Oguri, Shigeyuki; Nomura, Michiko; Fujita, Youko

    2005-06-17

    A new strategy for the selective determination of D-amino acids (DAAs) employing a pre-column derivatization was designed with concepts based on both enzymatic and chemical modifications. Selective determination of DAAs was accomplished by following: DAA was enzymatically modified with D-amino acid oxidase (DAAO: EC 1.4.3.3) to form an alpha-keto acid. Subsequently, resulting alpha-keto acid was detected by high-performance liquid chromatography (HPLC) after chemical modification with o-phenylenediamine (PDA) in the presence of 2-mercaptoethanol (2ME) to give the corresponding quinoxalinol derivative (PDA-alpha-keto acid derivative). After optimizing the pre-column derivatization and HPLC separation, five peaks corresponding to DAAs (D-alanine, D-leucine, D-methionine, D-phenylalanine, D-valine (as the standard mixture of DAAs in this paper) were separately eluted and monitored by means of a conventional HPLC system with a gradient elution on octadecyl silica gel (ODS) column and a fluorescence detector (Ex.: 341 nm, Em.: 413 nm), respectively. It was confirmed that the present method was incapable of detecting L-amino acids (LAA) when a sample solution consisting of both LAAs and DAAs was examined. The linearity of the peak-area responses to their concentration range of DAAs from 10 to 500 microM is 0.994-1.000, and their detection limits were 0.2-1 microM (signal/noise = 3). When this method was applied to a methanolic extract of short-necked clams, Ruditapes philippinarum (in Japanese, Asari), a big peak, corresponding to D-alanine was detected, corresponding to 2.9 mg/g D-alanine. In this paper, we present an example of pre-column derivatization method that was newly configured to take into account both the biological and chemical properties of the substances in question.

  3. Two step derivatization for the analyses of organic, amino acids and glycines on filter paper plasma by GC-MS/SIM.

    PubMed

    Yoon, Hye-Ran

    2007-03-01

    A rapid dried-filter paper plasma-spot analytical method was developed to quantify organic acids, amino acids, and glycines simultaneously in a two-step derivatization procedure with good sensitivity and specificity. The new method involves a two-step trimethylsilyl (TMS) - trifluoroacyl (TFA) derivatization procedure using GC-MS/ selective ion monitoring (GC-MS/SIM). The dried-filter paper plasma was fortified with an internal standard (tropate) as well as a standard mixture of distilled water and methanol. Methyl orange was added to the residue as an indicator. N-methyl-N-(trimethylsilyl-trifluoroacetamide) and N-methyl-bis-trifluoroacetamide were then added and heated to 60 degrees C for 10 and 15 min to produce the TMS and TFA derivatives, respectively. Using this method, the silylation of carboxylic functional groups was carried out, which was followed by the trifluoroacyl derivatization of the amino functional group. The derivatives were analyzed by GC-MS/SIM. A calibration cure showed a linear relationship for the target compounds between concentrations of 10-500 ng/mL. The limit of detection and quantification on a plasma spot were 10-90 ng/mL (S/N=9) and 80-500 ng/ mL, respectively. The correlation coefficient ranged from 0.938 and 0.999. When applied to the samples from positive patients, the method clearly differentiated normal subjects from the patients with various metabolic disorders such as PKU, MSUD, OTC and a Propionic Aciduria. The new developed method might be useful for making a rapid, sensitive and simultaneous diagnosis of inherited organic and amino acid disorders. In addition, this method is expected to be an alternative method for screening newborns for metabolic disorders in laboratories where expensive MS/MS is unavailable.

  4. A micellar electrokinetic chromatography-mass spectrometry approach using in-capillary diastereomeric derivatization for fully automatized chiral analysis of amino acids.

    PubMed

    Moldovan, Radu-Cristian; Bodoki, Ede; Kacsó, Timea; Servais, Anne-Catherine; Crommen, Jacques; Oprean, Radu; Fillet, Marianne

    2016-10-07

    In the context of bioanalytical method development, process automatization is nowadays a necessity in order to save time, improve method reliability and reduce costs. For the first time, a fully automatized micellar electrokinetic chromatography-mass spectrometry (MEKC-MS) method with in-capillary derivatization was developed for the chiral analysis of d- and l-amino acids using (-)-1-(9-fluorenyl) ethyl chloroformate (FLEC) as labeling reagent. The derivatization procedure was optimized using an experimental design approach leading to the following conditions: sample and FLEC plugs in a 2:1 ratio (15s, 30mbar: 7.5s, 30mbar) followed by 15min of mixing using a voltage of 0.1kV. The formed diastereomers were then separated using a background electrolyte (BGE) consisting of 150mM ammonium perfluorooctanoate (APFO) (pH=9.5) and detected by mass spectrometry (MS). Complete chiral resolution was obtained for 8 amino acids, while partial separation was achieved for 6 other amino acid pairs. The method showed good reproducibility and linearity in the low micromolar concentration range. The applicability of the method to biological samples was tested by analyzing artificial cerebrospinal fluid (aCSF) samples. Copyright © 2016 Elsevier B.V. All rights reserved.

  5. Optimization of a gas chromatography-mass spectrometry method with methyl chloroformate derivatization for quantification of amino acids in plant tissue.

    PubMed

    Vancompernolle, Bram; Croes, Kim; Angenon, Geert

    2016-04-01

    Rapid, easy and reliable quantification of amino acids is crucial in research on plant amino acid metabolism and nutritional improvement of crops via enrichment of essential amino acids. A recently reported analysis method, based on solid phase extraction (SPE), derivatization with methyl chloroformate and gas chromatography-mass spectrometry was optimized and tested on three-week-old Arabidopsis thaliana leaf tissues. Optimization of the SPE cleanup yielded recovery rates of minimum 95% for all amino acids (except arginine). Variations in accuracy and precision did not exceed 12.5%, except for cysteine, histidine and tryptophane, which were excluded from analysis. Quantification of overlapping peaks for isoleucine/threonine and proline/asparagine was possible by selection of two specific fragment ions for each amino acid. Of the 16 selected amino acids, 14 were quantified successfully in at least 75% of the samples, while methionine and tyrosine were only quantifiable in 6% and 42%, respectively. A case study on the aspartate super pathway confirmed the applicability of the optimized method on wild type and genetically modified plants: external supplementation of methionine or lysine yielded a 146-fold or 27-fold increase in the respective absolute amino acid levels compared with the control treatment. Induced expression of dhdps-r1 (a mutated lysine biosynthesis gene encoding a feedback insensitive enzyme) caused an 83-fold increase in absolute lysine levels.

  6. Enantiomeric resolution of biomarkers in space analysis: Chemical derivatization and signal processing for gas chromatography-mass spectrometry analysis of chiral amino acids.

    PubMed

    Pietrogrande, M C; Basaglia, G

    2010-02-12

    The work compares two GC-MS methods for enantioselective separation of amino acids as suitable candidate for stereochemical analysis of chiral amino acids on board spacecrafts in space exploration missions of solar system body environments. Different derivatization reagents are used: a mixture of alkyl chloroformate-alcohol-pyridine to obtain the alkyl alkoxy carbonyl esters and a mixture of perfluorinated alcohols and anhydrides to form perfluoroacyl perfluoroalkyl esters. 20 proteinogenic amino acids were derivatized with the two procedures and submitted to GC-MS analysis on a Chirasil-l-Val stationary phase. The results were then compared in terms of the enantiomeric separation achieved and intensity of MS response. The combination of methyl chloroformate (MCF) and heptafluoro-1-butanol (HFB) allows separation of 14 enantiomeric pairs, five of which display a resolution (R(s)>or=1.2) supposed to be sufficient to quantify the enantiomeric excess. Three mixtures of trifluoroacetic (TFAA) and heptafluorobutyric (HFBA) anhydrides were combined with the corresponding perfluorinated alcohols - TFE (2,2,2-trifluoro-1-ethanol) and HFB (2,2,3,3,4,4,4-heptafluoro-1-butanol) - to give three different reagents (TFAA-TFE, TFAA-HFB, HFBA-HFB): the derivatives obtained show separation of the same number of proteinogenic amino acids (14 of 20) at a temperature lower than column bleeding limit (200 degrees C) and 8 of them give a separation with R(s)>or=1.2. Linearity study and limit of detection (X(LOD)) computation show that both methods are suitable for quantitative determination of several amino acid diastereomers at trace level (X(LOD) approximately 0.5nmol as derivatized quantity). Both the procedures were coupled with automatic data handling to increase their suitability for space analysis: the simplified data treatment is especially helpful to handle the low quality data recovered from space experiments and labor and time are saved, as imposed by the space experiments

  7. Characterization of Jamaican agro-industrial wastes. Part I: characterization of amino acids using HPLC: pre-column derivatization with phenylisothiocyanate.

    PubMed

    Bailey-Shaw, Y A; Golden, K D; Pearson, A G M; Porter, R B R

    2009-09-01

    Jamaican agro-industries generate large quantities of wastes, which are either discarded or under-utilized. In order to evaluate the possible utilization of these wastes, it is necessary that the profiles of the major biochemical groups be developed. This paper describes the determination of the amino acid composition of coffee, citrus, and rum distillery wastes using a reversed-phase high-performance liquid chromatography method. Acid hydrolysates of the wastes are derivatized with phenylisothiocyanate. They are analyzed as their phenylthiocarbamyl derivatives and determined quantitatively using norleucine as the internal standard. The presence of all the 17 amino acids investigated, nine of which include those essential for animal nutrition, are observed in the samples investigated, suggesting a high quality of protein with implications in the formulation of animal feeds.

  8. Determination of efficacy of fingermark enhancement reagents; the use of propyl chloroformate for the derivatization of fingerprint amino acids extracted from paper.

    PubMed

    Mink, Tineke; Voorhaar, Annelies; Stoel, Reinoud; de Puit, Marcel

    2013-09-01

    The analysis of the constituents of fingerprints has been described numerous times, mainly with the purpose of determining the aging effect on fingerprints or showing the differences between donors or groups of donors. In this paper we describe the use of derivatized amino acids to determine the efficacy of the visualization reagents 1,8-diazafluoren-9-one (DFO) and ninhydrin. At present certain conditions are used for the application of these reagents, as determined by trial-and-error investigations, to the effect on fingerprints. The recovery of amino acids from a porous surface can be used as a measure for the efficacy of a visualization agent. In this paper we describe a method for the determination of the amount of amino acid left after reaction with well known fingerprint visualization reagents. This will allow a more scientific approach to method development for fingermark enhancement techniques. Furthermore, investigations on the influence of the concentration of fingermark amino acids, the order of application of and exposure time to reagents and the influence of age of the amino acids were carried out. These studies have resulted in a broader understanding of the mechanism involved in visualization of fingermarks using DFO and ninhydrin.

  9. Appraisal of four pre-column derivatization methods for the high-performance liquid chromatographic determination of free amino acids in biological materials.

    PubMed

    Fürst, P; Pollack, L; Graser, T A; Godel, H; Stehle, P

    1990-01-19

    Reversed-phase high-performance liquid chromatography (RP-HPLC) is a powerful method for assaying physiological amino acid concentrations in biological fluids. Four pre-column derivatization methods, with o-phthaldialdehyde (OPA), 9-fluorenylmethyl chloroformate (FMOC-Cl), phenyl isothiocyanate (PITC) and 1-dimethylaminonaphthalene-5-sulphonyl chloride (dansyl-Cl), were assessed with respect to their applicability in biological research. The methods permit the measurement of 21-26 major amino acids in 13-40 min. The superior sensitivity favours the use of OPA, FMOC-Cl and dansyl-Cl techniques. Because of instability of the OPA adducts, automated on-line derivatization is required when using this method in general practice. Application of the PITC method, although less sensitive, is useful in clinical chemistry, where sample availability is rarely a problem. Cystine determination is not feasible when using OPA or FMOC-Cl and with PITC the reproducibility and linearity are poor, whereas the dansyl-Cl method allows reliable quantitation. The four methods are currently used to perform ca. 8000 OPA and 5000-6000 FMOC-Cl, PITC and dansyl-Cl analyses of biological samples per year. The results obtained with the RP-HPLC methods compare favourably with those derived from conventional ion-exchange amino acid analyses. When the guard column is regularly changed after 120 analyses, the separation remains satisfactory for at least 700 OPA, 800 FMOC-Cl, 150 PITC and 500 dansyl-Cl analyses. Careful control of factors and limitations inherent in the various methodologies is a prerequesite for proper identification and appropriate quantitation.

  10. Quantitative Metabolite Profiling of an Amino Group Containing Pharmaceutical in Human Plasma via Precolumn Derivatization and High-Performance Liquid Chromatography-Inductively Coupled Plasma Mass Spectrometry.

    PubMed

    Li, Sanwang; Klencsár, Balázs; Balcaen, Lieve; Cuyckens, Filip; Lynen, Frederic; Vanhaecke, Frank

    2017-02-07

    Quantitative determination of the candidate drug molecule and its metabolites in biofluids and tissues is an inevitable step in the development of new pharmaceuticals. Because of the time-consuming and expensive nature of the current standard technique for quantitative metabolite profiling, i.e., radiolabeling followed by high-performance liquid chromatography (HPLC) with radiodetection, the development of alternative methodologies is of great interest. In this work, a simple, fast, sensitive, and accurate method for the quantitative metabolite profiling of an amino group containing drug (levothyroxine) and its metabolites in human plasma, based on precolumn derivatization followed by HPLC-inductively coupled plasma mass spectrometry (ICPMS), was developed and validated. To introduce a suitable "heteroelement" (defined here as an element that is detectable with ICPMS), an inexpensive and commercially available reagent, tetrabromophthalic anhydride (TBPA) was used for the derivatization of free NH2-groups. The presence of a known number of I atoms in both the drug molecule and its metabolites enabled a cross-validation of the newly developed derivatization procedure and quantification based on monitoring of the introduced Br. The formation of the derivatives was quantitative, providing a 4:1 stoichiometric Br/NH2 ratio. The derivatives were separated via reversed-phase HPLC with gradient elution. Bromine was determined via ICPMS at a mass-to-charge ratio of 79 using H2 as a reaction gas to ensure interference-free detection, and iodine was determined at a mass-to-charge ratio of 127 for cross-validation purposes. The method developed shows a fit-for-purpose accuracy (recovery between 85% and 115%) and precision (repeatability <15% RSD). The limit of quantification (LoQ) for Br was approximately 100 μg/L.

  11. High-performance liquid chromatography evaluation of the enantiomeric purity of amino acids by means of automated precolumn derivatization with ortho-phthalaldehyde and chiral thiols.

    PubMed

    Kühnreich, Raphael; Holzgrabe, Ulrike

    2016-12-01

    The use of ortho-phthalaldehyde (OPA) for the derivatization of amino acids (AA) is well known. It enables the separation of the derivatives on common reversed phase columns and improves the sensitivity with fluorescence detection. With the use of a chiral thiol an indirect enantioseparation of chiral amines and AAs is feasible. The major drawback of the OPA-derivatization is the poor stability of the products. Here, a method with an in-needle derivatization procedure is optimized to facilitate a quantitative conversion of the AA with OPA and the chiral thiols N-acetyl-L-cysteine or N-isobutyryl-L-cysteine, followed by a subsequent analysis, eluding the stability issue. Both enantiomers of a single AA were separated as OPA-derivatives with a pentafluorophenyl column and a gradient program consisting of 50 mM sodium acetate buffer pH = 5.0 and acetonitrile. Fluorescence detection is commonly used to achieve sufficient sensitivity. In this study, the enantiomeric impurity of an AA can be detected indirectly with common UV spectrophotometric detection with a limit of quantitation of 0.04%. Seventeen different L-AAs were tested and the amount of D-AA for each individual AA was calculated by means of area normalization, which ranged from not detectable up to 4.29%. The recovery of the minor enantiomer of L- and D-AA was demonstrated for three AAs at a 0.04% level and ranged between 92.3 and 113.3%, with the relative standard deviation between 1.7 and 8.2%.

  12. Quantification of structurally related aliphatic amino alcohols in l-valinol by hydrophilic interaction liquid chromatography separation combined with postcolumn derivatization and fluorescence detection.

    PubMed

    Douša, Michal; Stach, Jan; Gibala, Petr; Lemr, Karel

    2016-03-01

    The amino alcohols in l-valinol were effectively separated and quantified using hydrophilic interaction chromatography with fluorescence detection. The influence of the mobile phase (salt type, buffer concentration, and pH) on retention was studied. A column TSKgel amide and mobile phase consisting of 10 mM acetate buffer pH 4.0 and acetonitrile (20:80, v/v) provided well-separated symmetric peaks of analytes. Fluorescence detection was performed using postcolumn derivatization with o-phtaldialdehyde/2-mercaptoethanol at an excitation and emission wavelength of 345 and 450 nm, respectively. Simple sample pretreatment and very high sensitivity represent the main advantages of the developed method. After validation, the method was successfully applied to the analysis of commercial samples of l-valinol.

  13. Study of a new derivatizing reagent that improves the analysis of amino acids by HPLC with fluorescence detection: application to hydrolyzed rape bee pollen.

    PubMed

    You, Jinmao; Liu, Lingjun; Zhao, Wenchen; Zhao, Xianen; Suo, Yourui; Wang, Honglun; Li, Yulin

    2007-04-01

    A simple and sensitive method for evaluating the chemical compositions of protein amino acids, including cystine (Cys)(2) and tryptophane (Try) has been developed, based on the use of a sensitive labeling reagent 2-(11H-benzo[alpha]-carbazol-11-yl) ethyl chloroformate (BCEC-Cl) along with fluorescence detection. The chromophore of the 1,2-benzo-3,4-dihydrocarbazole-ethyl chloroformate (BCEOC-Cl) molecule was replaced with the 2-(11H-benzo[alpha]-carbazol-11-yl) ethyl functional group, yielding the sensitive fluorescence molecule BCEC-Cl. The new reagent BCEC-Cl could then be substituted for labeling reagents commonly used in amino acid derivatization. The BCEC-amino acid derivatives exhibited very high detection sensitivities, particularly in the cases of (Cys)(2) and Try, which cannot be determined using traditional labeling reagents such as 9-fluorenyl methylchloroformate (FMOC-Cl) and ortho-phthaldialdehyde (OPA). The fluorescence detection intensities for the BCEC derivatives were compared to those obtained when using FMOC-Cl and BCEOC-Cl as labeling reagents. The ratios I (BCEC)/I (BCEOC) = 1.17-3.57, I (BCEC)/I (FMOC) = 1.13-8.21, and UV(BCEC)/UV(BCEOC) = 1.67-4.90 (where I is the fluorescence intensity and UV is the ultraviolet absorbance). Derivative separation was optimized on a Hypersil BDS C(18) column. The detection limits calculated from 1.0 pmol injections, at a signal-to-noise ratio of 3, ranged from 7.2 fmol for Try to 8.4 fmol for (Cys)(2). Excellent linear responses were observed, with coefficients of >0.9994. When coupled with high-performance liquid chromatography, the method established here allowed the development of a highly sensitive and specific method for the quantitative analysis of trace levels of amino acids including (Cys)(2) and Try from bee-collected pollen (bee pollen) samples.

  14. Optimal excitation and emission wavelengths to analyze amino acids and optimize neurotransmitters quantification using precolumn OPA-derivatization by HPLC.

    PubMed

    Perucho, J; Gonzalo-Gobernado, R; Bazan, E; Casarejos, M J; Jiménez-Escrig, A; Asensio, M J; Herranz, A S

    2015-05-01

    We describe an analytical methodology to obtain high sensitivity and better resolution through the study of fluorometric excitation (λex) and emission (λem) spectrum wavelengths of OPA-amino acids. The spectrum emission study revealed a maximum signal peak at 450 nm for aspartate and glutamine. For glycine, taurine, and GABA, the maximum signal peak was at 448 and for glutamate at 452 nm. The remaining amino acids analyzed showed a maximum emission around 450 nm. The best signal obtained within the spectrum excitation experiments was using 229- to 450-nm λex-λem. The drawbacks observed at these wavelengths were a baseline drift and negative peaks occurrence. Thus, the excitation wavelength of 240 nm was chosen (240- to 450-nm λex-λem) as a compromise between a very good signal response and a baseline stability to resolve the 18 amino acids studied. Furthermore, this protocol was properly validated. On the other hand, the elution gradient program used for neuroactive amino acids (aspartate, glutamate, glycine, taurine and GABA) showed separation to the baseline, in a 15-min run in all of them. Other amino acids, up to 18, also exhibited a very good separation in a 25-min run. In conclusion, we propose the use of 240- to 450-nm λex-λem wavelengths, in OPA-amino acids analysis, as the most suitable protocol to obtain the best signal response, maintaining an optimum chromatographic resolution.

  15. Use of a Novel Sub-2 µm Silica Hydride Vancomycin Stationary Phase in Nano-Liquid Chromatography. II. Separation of Derivatized Amino Acid Enantiomers.

    PubMed

    Rocchi, Silvia; Fanali, Chiara; Fanali, Salvatore

    2015-11-01

    A novel vancomycin silica hydride stationary phase was synthesized and the particles of 1.8 µm were packed into fused silica capillaries of 75 µm internal diameter (I.D.). The chiral stationary phase (CSP) was tested for the separation of some derivatized amino acid enantiomers by using nano-liquid chromatography (nano-LC). Some experimental parameters such as the type and the content of organic modifier, the pH, and the concentration of the buffer added to the mobile phase were modified and the effect on enantioselectivity, retention time, and enantioresolution factor was studied. The separation of selected dansyl amino acids (Dns-AAs), e.g., Asp, Glu, Leu, and Phe in their enantiomers was initially achieved utilizing a mobile phase containing 85% (v/v) methanol (MeOH) and formate buffer measuring the enantioresolution factor and enantioselectivity in the range 1.74-4.17 and 1.39-1.59, respectively. Better results were obtained employing a more polar organic solvent as acetonitrile (ACN) in the mobile phase. Optimum results (Rs 1.41-6.09 and α 1.28-2.36) were obtained using a mobile phase containing formate buffer pH 2.5/water/MeOH/ACN 6:19:12.5:62.5 (v/v/v/v) in isocratic elution mode at flow rate of 130 nL/min. © 2015 Wiley Periodicals, Inc.

  16. Determination of L-canavanine and other free amino acids in Vicia disperma (Fabaceae) seeds by precolumn derivatization using diethyl ethoxymethylenemalonate and reversed-phase high-performance liquid chromatography.

    PubMed

    Megías, Cristina; Cortés-Giraldo, Isabel; Girón-Calle, Julio; Vioque, Javier; Alaiz, Manuel

    2015-01-01

    A method for determination of the non-protein amino acid l-α-amino-γ-(guanidinooxy)-n-butyric acid (L-canavanine) and other free amino acids in Vicia disperma is presented. Seed extracts were derivatized by reaction with diethyl ethoxymethylenemalonate and analyzed by reverse-phase high-performance liquid chromatography. Calibration curves showed very good linearity of the response. The limit of detection and quantification were 0.15 and 0.50 μM, respectively. The method has a high intra- (RSD=0.35%) and inter-repeatability (RSD=2.86%), and a remarkable accuracy with a 99% recovery in spiked samples. The method is very easy to carry out and allows for ready analysis of large number of samples using very basic HPLC equipment because the derivatized samples are very stable and have very good chromatographic properties.

  17. Chloroformate derivatization for tracing the fate of Amino acids in cells and tissues by multiple stable isotope resolved metabolomics (mSIRM).

    PubMed

    Yang, Ye; Fan, Teresa W-M; Lane, Andrew N; Higashi, Richard M

    2017-07-11

    Amino acids have crucial roles in central metabolism, both anabolic and catabolic. To elucidate these roles, steady-state concentrations of amino acids alone are insufficient, as each amino acid participates in multiple pathways and functions in a complex network, which can also be compartmentalized. Stable Isotope-Resolved Metabolomics (SIRM) is an approach that uses atom-resolved tracking of metabolites through biochemical transformations in cells, tissues, or whole organisms. Using different elemental stable isotopes to label multiple metabolite precursors makes it possible to resolve simultaneously the utilization of these precursors in a single experiment. Conversely, a single precursor labeled with two (or more) different elemental isotopes can trace the allocation of e.g. C and N atoms through the network. Such dual-label experiments however challenge the resolution of conventional mass spectrometers, which must distinguish the neutron mass differences among different elemental isotopes. This requires ultrahigh resolution Fourier transform mass spectrometry (UHR-FTMS). When combined with direct infusion nano-electrospray ion source (nano-ESI), UHR-FTMS can provide rapid, global, and quantitative analysis of all possible mass isotopologues of metabolites. Unfortunately, very low mass polar metabolites such as amino acids can be difficult to analyze by current models of UHR-FTMS, plus the high salt content present in typical cell or tissue polar extracts may cause unacceptable ion suppression for sources such as nano-ESI. Here we describe a modified method of ethyl chloroformate (ECF) derivatization of amino acids to enable rapid quantitative analysis of stable isotope labeled amino acids using nano-ESI UHR-FTMS. This method showed excellent linearity with quantifiable limits in the low nanomolar range represented in microgram quantities of biological specimens, which results in extracts with total analyte abundances in the low to sub-femtomole range. We have

  18. Selective elimination of the free fatty acid fraction from esterified fatty acids in rat plasma through chemical derivatization and immobilization on amino functionalized silica nano-particles.

    PubMed

    Chen, Jun; Lyu, Qiang; Yang, Mingqing; Chen, Zhi; He, Junhui

    2016-01-29

    A high throughput and low cost approach to separate free fatty acids (FFAs) from phospholipid and acylglycerols (esterified fatty acids, EFAs) has been demonstrated, which may be widely used as a sample preparation method in the metabolomics and lipid research. The optimal conditions for FFAs reacting with N-hydroxysuccinimide (NHS) only need 10min at room temperature to obtain a 93.5% yield of FFAs-NHS ester. The rest 6% FFA transformed into N-cyclohexyl-fatty acid-amide which is stable to methyl esterification adopted for fatty acids analysis. 10min are taken for FFAs-NHS ester to react with amino functionalized silica nanoparticles to immobilize the FFAs. The separation of FFAs from EFAs could be carried out readily by centrifugation. The whole process including derivatization, immobilization, and centrifugation takes less than 40min. Much more accurate fatty acids composition of rat plasma EFAs could be obtained by this approach than the previous reported methods. Copyright © 2015 Elsevier B.V. All rights reserved.

  19. Ethyl-bridged hybrid column as an efficient alternative for HPLC analysis of plasma amino acids by pre-column derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate.

    PubMed

    Castellanos, Mar; Van Eendenburg, Cecile Van; Gubern, Carme; Sanchez, Juan M

    2016-09-01

    Conventional C18 silica columns have proven to be useful for the analysis of amino acids (AA) from protein hydrolysates but undesirable peak overlapping is usually found when analyzing body fluids given that a large number of AAs are present in the samples. As an alternative to silica packings, an ethyl-bridged packing for reversed-phase liquid chromatography of derivatized AAs with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) has been evaluated. The new packing material improves the separation efficiency allowing better separations when analyzing biological fluids. Moreover, this packing has advantages for routine AA analysis, such as a decrease in the total running time and an increase in the life-time of the columns. The pH of the mobile phase has a significant effect on the elution behavior of the AQC hydrolysis product (AMQ) and on the AA derivatives. It is not possible to elute AMQ before detecting the first AA derivative, which requires an accurate adjustment of the pH in the range of 5.30-5.35 to obtain good separation and resolution for the most polar compounds. Under the conditions proposed, it is possible to separate all AAs except the Gly-Gln pair, which is not a problem when hydrolyzed samples are analyzed. The AMQ-Ser pair requires either the use of a different mobile phase pH for its baseline separation or the use of fluorescence detection. Two different procedures for protein removal from plasma samples have been evaluated, solvent precipitation and ultrafiltration (UF) and it has been found that UF gives better results as no significant losses of AAs were observed. The validation of the proposed method with UV detection gives method detection limits in the range of 8-12μM, with repeatability values<8% (n=6) and inter-day precision in plasma samples ranging from 4 to 13% (n=4).

  20. Simultaneous Determination of Food-Related Biogenic Amines and Precursor Amino Acids Using in Situ Derivatization Ultrasound-Assisted Dispersive Liquid-Liquid Microextraction by Ultra-High-Performance Liquid Chromatography Tandem Mass Spectrometry.

    PubMed

    He, Yongrui; Zhao, Xian-En; Wang, Renjun; Wei, Na; Sun, Jing; Dang, Jun; Chen, Guang; Liu, Zhiqiang; Zhu, Shuyun; You, Jinmao

    2016-11-02

    A simple, rapid, sensitive, selective, and environmentally friendly method, based on in situ derivatization ultrasound-assisted dispersive liquid-liquid microextraction (in situ DUADLLME) coupled with ultra-high-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) using multiple reaction monitoring (MRM) mode has been developed for the simultaneous determination of food-related biogenic amines and amino acids. A new mass-spectrometry-sensitive derivatization reagent 4'-carbonyl chloride rosamine (CCR) was designed, synthesized, and first reported. Parameters and conditions of in situ DUADLLME and UHPLC-MS/MS were optimized in detail. Under the optimized conditions, the in situ DUADLLME was completed speedily (within 1 min) with high derivatization efficiencies (≥98.5%). With the cleanup and concentration of microextraction step, good analytical performance was obtained for the analytes. The results showed that this method was accurate and practical for quantification of biogenic amines and amino acids in common food samples (red wine, beer, wine, cheese, sausage, and fish).

  1. Rapid and simultaneous determination of twenty amino acids in complex biological and food samples by solid-phase microextraction and gas chromatography-mass spectrometry with the aid of experimental design after ethyl chloroformate derivatization.

    PubMed

    Mudiam, Mohana Krishna Reddy; Ratnasekhar, Ch; Jain, Rajeev; Saxena, Prem Narain; Chauhan, Abhishek; Murthy, R C

    2012-10-15

    Amino acids play a vital role as intermediates in many important metabolic pathways such as the biosynthesis of nucleotides, vitamins and secondary metabolites. A sensitive and rapid analytical method has been proposed for the first time for the simultaneous determination of twenty amino acids using solid-phase microextraction (SPME). The protein samples were hydrolyzed by 6M HCl under microwave radiation for 120 min. Then the amino acids were derivatized by ethyl chloroformate (ECF) and the ethoxy carbonyl ethyl esters of amino acids formed were extracted using SPME by direct immersion. Finally the extracted analytes on the SPME fiber were desorbed at 260°C and analyzed by gas chromatography-mass spectrometer (GC-MS) in electron ionization mode. Factors which affect the SPME efficiency were screened by Plackett-Burmann design; most significant factors were optimized with response surface methodology. The optimum conditions for SPME are as follows: pH of 1.7, ionic strength of 733 mg, extraction time of 30 min and fiber of divinyl benzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS). The recovery of all the amino acids was found to be in the range of 89.17-100.98%. The limit of detection (LOD) of all derivatized amino acids in urine, hair and soybean was found to be in the range of 0.20-7.52 μg L(-1), 0.21-8.40 μg L(-1) and 0.18-5.62 μg L(-1), respectively. Finally, the proposed technique was successfully applied for the determination of amino acids in complex biological (hair, urine) and food samples (soybean). The method can find wide applications in the routine analysis of amino acids in any biological as well as food samples.

  2. Liquid chromatography coupled to quadrupole-time of flight tandem mass spectrometry based quantitative structure-retention relationships of amino acid analogues derivatized via n-propyl chloroformate mediated reaction.

    PubMed

    Kritikos, Nikolaos; Tsantili-Kakoulidou, Anna; Loukas, Yannis L; Dotsikas, Yannis

    2015-07-17

    In the current study, quantitative structure-retention relationships (QSRR) were constructed based on data obtained by a LC-(ESI)-QTOF-MS/MS method for the determination of amino acid analogues, following their derivatization via chloroformate esters. Molecules were derivatized via n-propyl chloroformate/n-propanol mediated reaction. Derivatives were acquired through a liquid-liquid extraction procedure. Chromatographic separation is based on gradient elution using methanol/water mixtures from a 70/30% composition to an 85/15% final one, maintaining a constant rate of change. The group of examined molecules was diverse, including mainly α-amino acids, yet also β- and γ-amino acids, γ-amino acid analogues, decarboxylated and phosphorylated analogues and dipeptides. Projection to latent structures (PLS) method was selected for the formation of QSRRs, resulting in a total of three PLS models with high cross-validated coefficients of determination Q(2)Y. For this reason, molecular structures were previously described through the use of descriptors. Through stratified random sampling procedures, 57 compounds were split to a training set and a test set. Model creation was based on multiple criteria including principal component significance and eigenvalue, variable importance, form of residuals, etc. Validation was based on statistical metrics Rpred(2),QextF2(2),QextF3(2) for the test set and Roy's metrics rm(Av)(2) and rm(δ)(2), assessing both predictive stability and internal validity. Based on aforementioned models, simplified equivalent were then created using a multi-linear regression (MLR) method. MLR models were also validated with the same metrics. The suggested models are considered useful for the estimation of retention times of amino acid analogues for a series of applications.

  3. Analysis of amino acid and monoamine neurotransmitters and their metabolites in rat urine of Alzheimer's disease using in situ ultrasound-assisted derivatization dispersive liquid-liquid microextraction with UHPLC-MS/MS.

    PubMed

    Zhao, Xian-En; He, Yongrui; Li, Meng; Chen, Guang; Wei, Na; Wang, Xiao; Sun, Jing; Zhu, Shuyun; You, Jinmao

    2017-02-20

    Neurotransmitters (NTs) may play an important role in neurodegenerative disorders such as Alzheimer's disease (AD). In order to investigate the potential links, a new simple, fast, accurate and sensitive analytical method, based on in situ ultrasound-assisted derivatization dispersive liquid-liquid microextraction (in situ UA-DDLLME) coupled with ultra high-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS), has been developed and validated. The quantitation of amino acid neurotransmitters (AANTs) and monoamine neurotransmitters (MANTs) in urine of AD rats were performed in this work. The in situ UA-DDLLME procedure involved the rapid injection of the mixture of low toxic 4-bromoanisole (extractant) and acetonitrile (dispersant), which containing the new designed and synthesized 4'-carbonyl chloride rosamine (CCR) as derivatization reagent, into the aqueous phase of real sample and buffer. Under the selected conditions, the derivatization and microextraction of analytes were simultaneously completed within 1min. Good linearity for each analyte (R>0.992) was observed with low limit of detections (LODs, S/N>3). Moreover, the proposed method was compared with direct detection or other reported methods, and the results showed that low matrix effects and good recoveries results were obtained in this work. Taken together, in situ UA-DDLLME coupled with UHPLC-MS/MS analysis was demonstrated to be a good method for sensitive, accurate and simultaneous monitoring of AANTs and MANTs. This method would be expected to be highly useful in AD diseases' clinical diagnostics and may have potential value in monitoring the efficacy of treatment. Copyright © 2016 Elsevier B.V. All rights reserved.

  4. Qualitative and quantitative analysis of hyaluronan oligosaccharides with high performance thin layer chromatography using reagent-free derivatization on amino-modified silica and electrospray ionization-quadrupole time-of-flight mass spectrometry coupling on normal phase.

    PubMed

    Rothenhöfer, Martin; Scherübl, Rosmarie; Bernhardt, Günther; Heilmann, Jörg; Buschauer, Armin

    2012-07-27

    Purified oligomers of hyalobiuronic acid are indispensable tools to elucidate the physiological and pathophysiological role of hyaluronan degradation by various hyaluronidase isoenzymes. Therefore, we established and validated a novel sensitive, convenient, rapid, and cost-effective high performance thin layer chromatography (HPTLC) method for the qualitative and quantitative analysis of small saturated hyaluronan oligosaccharides consisting of 2-4 hyalobiuronic acid moieties. The use of amino-modified silica as stationary phase allows a simple reagent-free in situ derivatization by heating, resulting in a very low limit of detection (7-19 pmol per band, depending on the analyzed saturated oligosaccharide). By this derivatization procedure for the first time densitometric quantification of the analytes could be performed by HPTLC. The validated method showed a quantification limit of 37-71 pmol per band and was proven to be superior in comparison to conventional detection of hyaluronan oligosaccharides. The analytes were identified by hyphenation of normal phase planar chromatography to mass spectrometry (TLC-MS) using electrospray ionization. As an alternative to sequential techniques such as high performance liquid chromatography (HPLC) and capillary electrophoresis (CE), the validated HPTLC quantification method can easily be automated and is applicable to the analysis of multiple samples in parallel. Copyright © 2012 Elsevier B.V. All rights reserved.

  5. Reversed-phase liquid chromatographic resolution of diastereomers of protein and non-protein amino acids prepared with newly synthesized chiral derivatizing reagents based on cyanuric chloride.

    PubMed

    Bhushan, Ravi; Agarwal, Charu

    2011-02-01

    Two new chiral monochloro-s-triazines (MCT) were synthesized [viz N-(4-chloro-6-piperidinyl-[1,3,5]-triazine-2-yl)-L-leucine amide and N-(4-chloro-6-piperidinyl-[1,3,5]-triazine-2-yl)-L-leucine) (CDR 1 and 2, respectively)] by the nucleophilic displacement of chlorine atoms in s-triazine moiety. One of the Cl atoms was replaced with piperidine, and the second Cl atom in the 6-piperidinyl derivative was replaced with amino acid amide (viz L-Leu-NH(2)) and amino acid (L-Leu). These reagents were characterized and used as CDRs for chiral separation of protein and non-protein amino acids, and were separated on a reversed-phase C(18) column. The reaction conditions were optimized for the synthesis of diastereomers using one MCT reagent. The separation method was validated for limit of detection, linearity, accuracy, precision, and recovery.

  6. Simultaneous separation and quantitation of amino acids and polyamines of forest tree tissues and cell cultures within a single high-performance liquid chromatography run using dansyl derivatization

    Treesearch

    Rakesh Minocha; Stephanie Long

    2004-01-01

    The objective of the present study was to develop a rapid HPLC method for simultaneous separation and quantitation of dansylated amino acids and common polyamines in the same matrix for analyzing forest tree tissues and cell cultures. The major modifications incorporated into this method as compared to previously published HPLC methods for separation of only dansyl...

  7. Preparation of a novel ionic hybrid stationary phase by non-covalent functionalization of single-walled carbon nanotubes with amino-derivatized silica gel for fast HPLC separation of aromatic compounds.

    PubMed

    Aral, Hayriye; Çelik, K Serdar; Aral, Tarık; Topal, Giray

    2016-03-01

    Single-walled carbon nanotubes (SWCNTs) were immobilized on spherical silica gel with a 4-μm average particle size and a 60-Å average pore size. The amino-derivatized silica gel was non-covalently coated with carboxylated SWCNTs to preserve the structure of the nanotubes and their physico-chemical properties. The novel ionic hybrid stationary phase was characterized by scanning electron microscopy (SEM), infra-red (IR) spectroscopy and elemental analysis, and then, it was used to fill an empty 150×4.6mm(2) high-performance liquid chromatography (HPLC) column. Chromatographic parameters, such as the theoretical plate number, retention factor and peak asymmetry factor, and analytical parameters, such as the limit of detection (LOD), limit of quantification (LOQ), linear range, calibration equation, and R(2) value, and quantitative analysis parameters were calculated for all of the analytes. Using different mobile phases, five different classes of aromatic hydrocarbons were separated in a very short analysis time of 4-8min. Furthermore, a high theoretical plate number (up to 25000) and an excellent peak asymmetry factor (1.0) were obtained. The results showed that the surface of the SWNTs had very strong interactions with aromatic groups, therefore providing high selectivity for the separation of different classes of aromatic compounds. This study indicates that SWCNTs enable the extension of the application range of the newly prepared stationary phases for the fast separation of aromatic compounds by HPLC. Copyright © 2015 Elsevier B.V. All rights reserved.

  8. Derivatization in Capillary Electrophoresis.

    PubMed

    Marina, M Luisa; Castro-Puyana, María

    2016-01-01

    Capillary electrophoresis is a well-established separation technique in analytical research laboratories worldwide. Its interesting advantages make CE an efficient and potent alternative to other chromatographic techniques. However, it is also recognized that its main drawback is the relatively poor sensitivity when using optical detection. One way to overcome this limitation is to perform a derivatization reaction which is intended to provide the analyte more suitable analytical characteristics enabling a high sensitive detection. Based on the analytical step where the CE derivatization takes place, it can be classified as precapillary (before separation), in-capillary (during separation), or postcapillary (after separation). This chapter describes the application of four different derivatization protocols (in-capillary and precapillary modes) to carry out the achiral and chiral analysis of different compounds in food and biological samples with three different detection modes (UV, LIF, and MS).

  9. Reversed-phase high-performance liquid chromatographic separation of diastereomers of (R,S)-mexiletine prepared by microwave irradiation with four new chiral derivatizing reagents based on trichloro-s-triazine having amino acids as chiral auxiliaries and 10 others having amino acid amides.

    PubMed

    Bhushan, Ravi; Dixit, Shuchi

    2010-12-03

    A new series of chiral derivatizing reagents (CDRs) consisting of four dichloro-s-triazine reagents was synthesized by nucleophilic substitution of one chlorine atom in trichloro-s-triazine with amino acids, namely L-Leu, D-Phg, L-Val and L-Ala as chiral auxiliaries. Two other sets of CDRs consisting of four dichloro-s-triazine (DCT) and six monochloro-s-triazine (MCT) reagents were also prepared by nucleophilic substitution of chlorine atom(s) with different amino acid amides as chiral auxiliaries in trichloro-s-triazine and its 6-methoxy derivative, respectively. These 14 CDRs were used for the synthesis of diastereomers of (R,S)-mexiletine under microwave irradiation (i.e. 60s and 90 s at 85% power (of 800 W) using DCT and MCT reagents, respectively), which were resolved by reversed-phase high-performance liquid chromatography using C18 column and gradient eluting mixtures of methanol with aqueous trifluoroacetic acid (TFA) with UV detection at 230 nm. The resolution (R(s)), difference between retention times of resolved diastereomers (Δt) and retention factors (k) obtained for the three sets of diastereomers were compared among themselves and among the three groups. Explanations have been offered for longer retention times and better resolution of diastereomers prepared with DCT reagents in comparison of their MCT counterparts and, for the influence of hydrophobicity of the side chain R of the amino acid in the CDRs on retention times and resolution. The newly synthesized CDRs were observed to be superior as compared to their amide counterparts in terms of providing better resolution and cost effectiveness. The method was validated for limit of detection, linearity, accuracy and precision.

  10. Metabolomics relative quantitation with mass spectrometry using chemical derivatization and isotope labeling

    DOE PAGES

    O'Maille, Grace; Go, Eden P.; Hoang, Linh; ...

    2008-01-01

    Comprehensive detection and quantitation of metabolites from a biological source constitute the major challenges of current metabolomics research. Two chemical derivatization methodologies, butylation and amination, were applied to human serum for ionization enhancement of a broad spectrum of metabolite classes, including steroids and amino acids. LC-ESI-MS analysis of the derivatized serum samples provided a significant signal elevation across the total ion chromatogram to over a 100-fold increase in ionization efficiency. It was also demonstrated that derivatization combined with isotopically labeled reagents facilitated the relative quantitation of derivatized metabolites from individual as well as pooled samples.

  11. Chemically Derivatized Semiconductor Photoelectrodes.

    DTIC Science & Technology

    1982-01-04

    as Si, Ge, and GaAs derivatized with reagents based on ferrocene such as those represented by I and II. Work with p-type semiconductor photoelectrode...Concerning n-type Si it was found that EtOH/0.1 M En-Bu4N)C104 solutions containing A = ferrocene and A+ = ferri-- cinium result in a constant output of...electrical energy from an illuminated photoelectrochemical device configured as in Scheme II.(20) The ferrocene captures the photogenerated h+ at a rate -4

  12. Heavy-atom derivatization.

    PubMed

    Garman, Elspeth; Murray, James W

    2003-11-01

    Most of the standard methods of solving macromolecular structures involve producing a protein crystal that is derivatized by an anomalous scatterer or heavy atom (MIR, SIRAS, MAD, SAD etc.). The theoretical methodology which underpins the extraction of phase information from such derivatives is widely available in the literature. In addition, there are comprehensive sources of information on the chemistry of heavy-atom compounds and the ligands with which they are known to interact, as well as the Heavy Atom Databank accessible on the World Wide Web. This contribution therefore aims to provide some information on the less well documented practical problems of firstly deciding on an overall strategy for derivatization and secondly performing the physical manipulations involved in producing heavy-atom derivatives from native protein crystals and then cryocooling them. Ways to optimize the chances of isomorphous unit cells are suggested. Methods of determining whether or not the heavy atom is bound are outlined, including the powerful technique of PIXE (particle-induced X-ray emission).

  13. Resolution and isolation of enantiomers of (±)-isoxsuprine using thin silica gel layers impregnated with L-glutamic acid, comparison of separation of its diastereomers prepared with chiral derivatizing reagents having L-amino acids as chiral auxiliaries.

    PubMed

    Bhushan, Ravi; Nagar, Hariom

    2015-03-01

    Thin silica gel layers impregnated with optically pure l-glutamic acid were used for direct resolution of enantiomers of (±)-isoxsuprine in their native form. Three chiral derivatizing reagents, based on DFDNB moiety, were synthesized having l-alanine, l-valine and S-benzyl-l-cysteine as chiral auxiliaries. These were used to prepare diastereomers under microwave irradiation and conventional heating. The diastereomers were separated by reversed-phase high-performance liquid chromatography on a C18 column with detection at 340 nm using gradient elution with mobile phase containing aqueous trifluoroacetic acid and acetonitrile in different compositions and by thin-layer chromatography (TLC) on reversed phase (RP) C18 plates. Diastereomers prepared with enantiomerically pure (+)-isoxsuprine were used as standards for the determination of the elution order of diastereomers of (±)-isoxsuprine. The elution order in the experimental study of RP-TLC and RP-HPLC supported the developed optimized structures of diastereomers based on density functional theory. The limit of detection was 0.1-0.09 µg/mL in TLC while it was in the range of 22-23 pg/mL in HPLC and 11-13 ng/mL in RP-TLC for each enantiomer. The conditions of derivatization and chromatographic separation were optimized. The method was validated for accuracy, precision, limit of detection and limit of quantification.

  14. Immersed sol-gel based amino-functionalized SPME fiber and HPLC combined with post-column photochemically induced fluorimetry derivatization and fluorescence detection of pyrethroid insecticides from water samples.

    PubMed

    Bagheri, Habib; Ghanbarnejad, Hadi; Khalilian, Faezeh

    2009-09-01

    A method based on direct immersion solid-phase microextraction (DI-SPME) coupled with high performance liquid chromatography combined with post-column photochemically induced fluorimetry derivatization and fluorescence detection (HPLC-PIF-FD) was developed to extract three pyrethroid insecticides, i.e. cyfluthrin, cypermethrin, and flumethrin from water samples. A sol-gel based coating fiber using 3-(trimethoxysilyl propyl) amine as precursor was prepared and used for the extraction of the pyrethroids from groundwater samples. A post-column photochemical reactor was designed and constructed for the derivatization of these environmentally important pollutants to increase their fluorescence sensitivity and determination in HPLC. The parameters affecting extraction process (extraction time and temperature, pH, salt addition, and co-solvent) and desorption step (solvent, desorption time, and temperature) of the analytes from the sol-gel-based fiber, along with photochemical reaction conditions were investigated. The developed method proved to be relatively rapid, simple, and easy and offers high sensitivity and reproducibility. Linear dynamic ranges (LDR) for these insecticides were ranged between 0.25 to 50 microg/L. The regression coefficients were satisfactory (R(2) > 0.984) for these pyrethroids. The limits of detection and limits of quantification varied between 0.09 and 0.35 microg/L and 0.25 and 1.00 microg/L, respectively. Relative standard deviation RSDs values varied between 4.41% and 6.20%. Relative recoveries obtained from analysis of Jajroud river water sample ranged between 94% and 104%.

  15. [Development of novel fluorescence-derivatization-HPLC methods enabling highly sensitive and selective analysis of biological compounds].

    PubMed

    Todoroki, Kenichiro

    2011-01-01

    Fluorescence-derivatization-HPLC methods are powerful tools for performing the analysis of bioactive compounds with high sensitivity and selectivity. In this paper, the author reviews the development of the following four types of novel fluorescence-derivatization-HPLC analytical systems: (1) simultaneous HPLC analysis of melatonin and its related compounds through post-column electrochemical demethylation and fluorescence derivatization, (2) HPLC analysis of 5-hydroxyindoles based on fluorescence derivatization by online pre-column photocatalytic oxidation with benzylamine, (3) reagent peak-free HPLC analysis for aliphatic amines and amino acids using F-trap pyrene as a fluorous tag-bound fluorescence derivatization reagent, and (4) reagent peak-free HPLC analysis for carboxylic acids using a fluorous scavenging-derivatization method. The authors have also successfully applied these systems to biological and pharmaceutical analyses.

  16. Determination of neurotoxin 3-N-oxalyl-2,3-diaminopropionic acid and non-protein amino acids in Lathyrus sativus by precolumn derivatization with 1-fluoro-2,4-dinitrobenzene.

    PubMed

    Wang, F; Chen, X; Chen, Q; Qin, X; Li, Z

    2000-06-23

    A rapid and simple method is presented for determining neuro-excitatory nonprotein amino acid 3-N-oxalyl-2,3-diaminopropionic acid (beta-ODAP) and non-protein amino acids in Lathyrus sativus. Seed and foliage extracts of Lathyrus sativus were treated with 1-fluoro-2,4-dinitrobenzene (FDNB) and a reversed-phase high-performance liquid chromatography method (RP HPLC) for the separation of the derivatives in the pmol range is reported. The RP HPLC method and a colorimetric method were compared for measuring ODAP.

  17. Isotopic variants of light and heavy L-pyroglutamic acid succinimidyl esters as the derivatization reagents for DL-amino acid chiral metabolomics identification by liquid chromatography and electrospray ionization mass spectrometry.

    PubMed

    Mochizuki, Toshiki; Todoroki, Kenichiro; Inoue, Koichi; Min, Jun Zhe; Toyo'oka, Toshimasa

    2014-02-06

    L-Pyroglutamic acid succinimidyl ester (L-PGA-OSu) and its isotopic variant (L-PGA[d5]-OSu) were newly synthesized and evaluated as the chiral labeling reagents for the enantioseparation of amino acids, in terms of separation efficiency by reversed-phase chromatography and detection sensitivity by ESI-MS/MS. The enantiomers of amino acids were easily labeled with the reagents at 60°C within 10 min in an alkaline medium containing triethylamine. Although all the diastereomers derived from 18 proteolytic amino acids could not be satisfactorily separated, the pairs of 9 amino acids were completely separated by reversed-phase chromatography using the small particle (1.7 μm) ODS column (Rs=1.95-8.05). The characteristic daughter ions, i.e., m/z 84.04 and m/z 89.04, were detected from all the derivatives by the collision induced dissociation of the protonated molecular ions. A highly sensitive detection at a low-fmol level (0.5-3.2 fmol) was also obtained from the selected reaction monitoring (SRM) chromatograms. An isotope labeling strategy using light and heavy L-PGA-OSu for the differential analysis of the DL-amino acids in different sample groups is also presented in this paper. The differential analysis of biological sample (i.e., human serum) and food product (i.e., yogurt) were tried to demonstrate the efficiency of the proposed method. The ratios of the DL-amino acids in human serum samples, spiked with the different concentrations of D-amino acids, were determined by the procedures using L-PGA-OSu and L-PGA[d5]-OSu. The D/L ratios in the two sample groups at different concentrations of amino acids were similar to the theoretical values. Furthermore, the ratios of D/L-alanine values in different yogurt products were comparable to the ratios obtained from the d/l values using only light reagent (i.e., L-PGA-OSu). Consequently, the proposed strategy is useful for the differential analysis not only in biological samples but also in food products.

  18. GC-MS/MS survey of collision-induced dissociation of tert-butyldimethylsilyl-derivatized amino acids and its application to (13)C-metabolic flux analysis of Escherichia coli central metabolism.

    PubMed

    Okahashi, Nobuyuki; Kawana, Shuichi; Iida, Junko; Shimizu, Hiroshi; Matsuda, Fumio

    2016-09-01

    Stable isotope labeling experiments using mass spectrometry have been employed to investigate carbon flow levels (metabolic flux) in mammalian, plant, and microbial cells. To achieve a more precise (13)C-metabolic flux analysis ((13)C-MFA), novel fragmentations of tert-butyldimethylsilyl (TBDMS)-amino acids were investigated by gas chromatography-tandem mass spectrometry (GC-MS/MS). The product ion scan analyses of 15 TBDMS-amino acids revealed 24 novel fragment ions. The amino acid-derived carbons included in the five fragment ions were identified by the analyses of (13)C-labeled authentic standards. The identification of the fragment ion at m/z 170 indicated that the isotopic abundance of S-methyl carbon in methionine could be determined from the cleavage of C5 in the precursor of [M-159](+) (m/z 218). It was also confirmed that the precision of (13)C-MFA in Escherichia coli central carbon metabolism could be improved by introducing (13)C-labeling data derived from novel fragmentations. Graphical Abstract Novel collision-induced dissociation fragmentations of tert-butyldimethylsilyl amino acids were investigated and identified by GC-MS/MS.

  19. Selenium Derivatization of Nucleic Acids for Crystallography

    SciTech Connect

    Jiang,J.; Sheng, J.; Carrasco, N.; Huang, Z.

    2007-01-01

    The high-resolution structure of the DNA (5'-GTGTACA-C-3') with the selenium derivatization at the 2'-position of T2 was determined via MAD and SAD phasing. The selenium-derivatized structure (1.28 {angstrom} resolution) with the 2'-Se modification in the minor groove is isomorphorous to the native structure (2.0 {angstrom}). To directly compare with the conventional bromine derivatization, we incorporated bromine into the 5-postion of T4, determined the bromine-derivatized DNA structure at 1.5 {angstrom} resolution, and found that the local backbone torsion angles and solvent hydration patterns were altered in the structure with the Br incorporation in the major groove. Furthermore, while the native and Br-derivatized DNAs needed over a week to form reasonable-size crystals, we observed that the Se-derivatized DNAs grew crystals overnight with high-diffraction quality, suggesting that the Se derivatization facilitated the crystal formation. In addition, the Se-derivatized DNA sequences crystallized under a broader range of buffer conditions, and generally had a faster crystal growth rate. Our experimental results indicate that the selenium derivatization of DNAs may facilitate the determination of nucleic acid X-ray crystal structures in phasing and high-quality crystal growth. In addition, our results suggest that the Se derivatization can be an alternative to the conventional Br derivatization.

  20. Chemical derivatization of compact disc polycarbonate surfaces for SNPs detection.

    PubMed

    Bañuls, María-José; García-Piñón, Francisco; Puchades, Rosa; Maquieira, Angel

    2008-03-01

    Compact discs have been proposed as an efficient analytical platform, with potential to develop high-throughput affinity assays for genomics, proteomics, clinics, and health monitoring. Chemical derivatization of CD surfaces is one of the keys to developing highly efficient microarraying-based assays on discs. Approaches for mild chemical modification of polycarbonate (PC) disc surface based on nitration, reduction, and chloromethylation reactions have been developed. Derivatized surfaces as amino and thiol are obtained for PC, maintaining unchanged the mechanical and optical properties of the discs. Studies of covalent attachment of oligonucleotide probes (5' Cy5-labeled, 3' NH 2-ended) on the modified surfaces have been performed to develop microarraying assays based on hybridization of cDNA strands and single nucleotide polymorphism discrimination (SNPs). A demonstration of the applicability to the compact disc audio/video technology for its use as analytical system is performed, including the employment of a commercial CD player to read the results on disc.

  1. Detecting refractory organic matter on Mars: how derivatization will help

    NASA Astrophysics Data System (ADS)

    Freissinet, C.; Kashyap, S.; Glavin, D. P.; Buch, A.; Brault, A.; Mahaffy, P. R.

    2012-12-01

    The search for organic molecules on Mars can provide important first clues of extinct or extant biota on the planet. Gas Chromatography Mass Spectrometry (GC-MS) is currently the most relevant space-compatible analytical tool for the detection of organics. Nevertheless, GC separation is intrinsically restricted to volatile molecules, and a lot of the molecules of exobiological interest are refractory or polar. To analyze these organics such as amino acids, nucleobases and carboxylic acids, an additional derivatization step is required to transform them into volatile derivatives that are amenable to GC analysis. As part of the Sample Analysis at Mars (SAM) experiment onboard Mars Science Laboratory (MSL) Curiosity rover which successfully landed on Mars on August 5, 2012, a single-step protocol of extraction and chemical derivatization with the silylating reagent N-methyl-N-(tert-butyldimethylsilyl)-trifluoroacetamide MTBSTFA has been developed to reach a wide range of astrobiology-relevant refractory organic molecules. Seven cups on SAM are devoted to MTBSTFA derivatization. However, this chemical reaction adds a protective silyl group in place of each labile hydrogen, which make the molecule non-identifiable in common mass spectra libraries. We thus created an extended library of mass spectra of derivatized compounds of interest, considering their potential occurrence in Mars soils. We then looked specifically at these compounds using the existing and the newly created library, in various Mars analog soils. To enable a more accurate interpretation of the in situ derivatization GC-MS results that will be obtained by SAM, the lab experiments are performed in the restrictive conditions of the SAM flight instrument. First experiments display promising results, the system permitting an extraction and detection of several proteinic amino and carboxylic acids from Martian representative matrices. Preliminary results show a lack of derivatized organic molecules in

  2. A novel approach for HPLC determination of 2-cynaoacetamide using derivatization procedure with 2-hydroxyacetophenone as a new useful derivatization reagent.

    PubMed

    Douša, Michal; Břicháč, Jiří; Tkadlecová, Marcela; Man, Stanislav; Zezula, Josef; Hájíček, Josef; Pekárek, Tomáš

    2016-09-05

    A novel and sensitive derivatization procedure for the determination of 2-cynaoacetamide in pharmaceutical samples using liquid chromatography with the fluorescence detection was discovered. The method is based on derivatization of 2-cynaoacetamide using 2-hydroxyacetophenone as a new derivatization reagent. The product of derivatization reaction was isolated and characterized using spectroscopic techniques namely LC-MS, NMR and IR. The structure of 2-cyanoacetamide derivative was unambiguously assigned as a 2-amino-4-phenylfuran-3-carboxamide. Two derivatization systems were optimized in terms of reaction temperature, reaction time, pH and concentration of 2-hydroxyacetophenone, and a new pre- and post-derivatization HPLC methods were developed. The separations on HPLC with pre-column derivatization were accomplished using stationary phase based on a XBridge C18 column (100×4.6, 3.5μm) and isocratic elution using the mobile phase acetonitrile - 0.1% formic acid (30:70, v/v). The separations on the HPLC with post-column derivatization were performed on stationary phase on a TSKgel Amide-80 column (150×4.6mm, 3μm). The mobile phase was a mixture of acetonitrile, methanol and 10mM sodium formate buffer at pH=4.5 in ratio 3:2:95 (v/v). Both HPLC methods were fully validated in terms of linearity, sensitivity (limit of detection and limit of quantification), accuracy and precision according to ICH guidelines. The pre-column derivatization method was linear in the range 1.1-2000μg/l with method accuracy≥98.2% and method precision RSD≤4.8%. The post-column derivatization method was linear in the range 12-2000μg/l. Method accuracy≥96.3% and method precision RSD≤3.5%. Proposed new methods were proved to be highly sensitive, simple and rapid, and were successfully applied to the determinations of 2-cynaoacetamide in pregabalin.

  3. Interaction of psoralen-derivatized oligodeoxyribonucleoside methylphosphonates with single-stranded DNA

    SciTech Connect

    Lee, B.L.; Murakami, A.; Blake, K.R.; Lin, S.B.; Miller, P.S.

    1988-05-03

    Oligodeoxyribonucleoside methylphosphonates derivatized at the 5' end with 4'-(amino-alkyl)-4,5',8-trimethylpsoralen were prepared. The interaction of these psoralen-derivatized methyl-phosphonate oligomers with synthetic single-stranded DNAs 35 nucleotides in length was studied. Irradiation of a solution containing the 35-mer and its complementary methylphosphonate oligomer at 365 nm gave a cross-linked duplex produced by cycloaddition between the psoralen pyrone ring of the derivatized methylphosphonate oligomer and a thymine base of the DNA. Photoadduct formation could be reversed by irradiation at 254 nm. The rate and extent of cross-linking were dependent upon the length of the aminoalkyl linker between the trimethylpsoralen group and the 5' end of the methylphosphonate oligomer. Methylphosphonate oligomers derivatized with 4'-((N-(2-aminoethyl)amino)methyl)-4,5',8-trimethylpsoralen gave between 70% and 85% cross-linked product when irradiated for 20 min at 4 /sup 0/C. These results suggest that the methylphosphonate oligomers undergo both cross-linking and deactivation reactions when irradiated at 365 nm. The cross-linking reaction was dependent upon the fidelity of base-pairing interactions between the methylphosphonate oligomers and the single-stranded DNA. The extent and sequence specificity of photoinduced cross-linking, combined with the known ability of methylphosphonate oligomers to be taken up by living cells, suggest that psoralen-derivatized oligonucleoside methylphosphonates may be useful probes of cellular gene expression.

  4. In situ Analysis of Organic Compounds on Mars using Chemical Derivatization and Gas Chromatography Mass Spectrometry

    NASA Technical Reports Server (NTRS)

    Glavin, D. P.; Buch, A.; Cabane, M.; Coll, P.; Navarro-Gonzalez, R.; Mahaffy, P. R.

    2005-01-01

    One of the core science objectives of NASA's 2009 Mars Science Laboratory (MSL) mission is to determine the past or present habitability of Mars. The search for key organic compounds relevant to terrestrial life will be an important part of that assessment. We have developed a protocol for the analysis of amino acids and carboxylic acids in Mars analogue materials using gas chromatography mass spectrometry (GCMS). As shown, a variety of carboxylic acids were readily identified in soil collected from the Atacama Desert in Chile at part-per-billion levels by GCMS after extraction and chemical derivatization using the reagent N,N-tert.-butyl (dimethylsilyl) trifluoroacetamide (MTBSTFA). Several derivatized amino acids including glycine and alanine were also detected by GCMS in the Atacama soil at lower concentrations (chromatogram not shown). Lacking derivatization capability, the Viking pyrolysis GCMS instruments could not have detected amino acids and carboxylic acids, since these non-volatile compounds require chemical transformation into volatile species that are stable in a GC column. We are currently optimizing the chemical extraction and derivatization technique for in situ GCMS analysis on Mars. Laboratory results of analyses of Atacama Desert samples and other Mars analogue materials using this protocol will be presented.

  5. Amino acidis derived from Titan tholins

    NASA Technical Reports Server (NTRS)

    Khare, Bishun N.; Sagan, Carl; Ogino, Hiroshi; Nagy, Bartholomew; Er, Cevat

    1986-01-01

    The production of amino acids by acid treatment of Titan tholin is experimentally investigated. The synthesis of Titan tholin and the derivatization of amino acids to N-trifluoroacetyl isopropyl esters are described. The gas chromatography/mass spectroscopy analysis of the Titan tholins reveals the presence of glycine, alpha and beta alainine, and aspartic acid, and the total yield of amino acids is about 0.01.

  6. Amino acidis derived from Titan tholins

    NASA Technical Reports Server (NTRS)

    Khare, Bishun N.; Sagan, Carl; Ogino, Hiroshi; Nagy, Bartholomew; Er, Cevat

    1986-01-01

    The production of amino acids by acid treatment of Titan tholin is experimentally investigated. The synthesis of Titan tholin and the derivatization of amino acids to N-trifluoroacetyl isopropyl esters are described. The gas chromatography/mass spectroscopy analysis of the Titan tholins reveals the presence of glycine, alpha and beta alainine, and aspartic acid, and the total yield of amino acids is about 0.01.

  7. Derivatization and photolysis of a photoaffinity reagent for probing protein and cell surface interactions

    SciTech Connect

    Murphy, H.; Harris, H.W. Jr.

    1986-05-01

    The synthesis of the novel, heterobifunctional, cleavable, photoactivable crosslinking reagent, N-(4-(p-azido-m-(/sup 125/I) iodophenylazo)benzoyl)-3-aminopropyl-N'-oxysulfosuccinimide has been described by Denny and Blobel. This reagent is desirable because after photolysis and azo bond cleavage the /sup 125/I is transferred from the reagent to the crosslinked molecule. The authors demonstrate that using the reported synthesis 99% of the desired reagent is destroyed during the chloramine-T iodination step. They report a synthesis revision which produces high yields of the uniodinated (U) reagent. The derivatized reagent may be used in its iodinated (I) or U forms. To study the U reagent, a horseradish peroxidase (HRP) molecule is derivatized with nine reagent molecules. The derivatized HRP has 70% of its original enzymatic activity. After photolysis, 14% of this activity is retained and SDS-PAGE electrophoresis shows a crosslinked complex of HRP molecules. After endocytosis by cells, photolysis attaches the soluble derivatized HRP to membranes allowing them to be traced in the electron microscope. To study the I reagent, an amino-dextran (MW 73-400) molecule is derivatized with three U reagent molecules. The U reagent molecules are then iodinated by the chloramine-T method. With photolysis and cleavage, the /sup 125/I labeled reagent on dextran transfers its label to bovine serum albumin or ovalbumin. The authors conclude this reagent is a versatile probe for study of protein or cell surface topography.

  8. High-performance liquid chromatography determination of pipecolic acid after precolumn ninhydrin derivatization using domestic microwave.

    PubMed

    Moulin, Michaël; Deleu, Carole; Larher, François Robert; Bouchereau, Alain

    2002-09-15

    A novel procedure to specifically quantify low amounts of pipecolic acid and structurally related compounds in several types of biological materials has been characterized. From crude extracts of various types of biological material, the first step was to clear all low-molecular-weight compounds containing primary amino groups by a treatment of nitrous acid. Using a microwave-assisted reaction, the remaining substances containing secondary amino groups were then derivatized with ninhydrin and made soluble in glacial acetic acid. The derivatives produced were resolved by reverse-phase HPLC and detected by spectrophotometry at 570nm. This procedure allowed more rapid determination of pipecolic acid since microwave heating shortened the time needed for derivatization compared with heating at 95 degrees C in a water bath. The complete analysis of the chromogens for pipecolic acid and related substances was achieved in 20min. Under such conditions, the detection threshold for pipecolic acid was about 20pmol. The suitability of the technique was assessed in various biological matrices known to contain significant amounts of this amino acid. The data obtained are in accordance with those available in the literature. To our knowledge, this is the first method using the ninhydrin reaction in a precolumn, microwave-assisted derivatization procedure for detection and determination of heterocyclic alpha-amino acids.

  9. Qualitative and quantitative evaluation of derivatization reagents for different types of protein-bound carbonyl groups.

    PubMed

    Bollineni, Ravi Chand; Fedorova, Maria; Hoffmann, Ralf

    2013-09-07

    Mass spectrometry (MS) of 'carbonylated proteins' often involves derivatization of reactive carbonyl groups to facilitate their enrichment, identification and quantification. Among the many reported reagents, 2,4-dinitrophenylhydrazine (DNPH), biotin hydrazide (BHZ) and O-(biotinylcarbazoylmethyl) hydroxylamine (ARP) are the most frequently used. Despite their common use in carbonylation research, their reactivity towards protein-bound carbonyls has not been quantitatively evaluated in detail, to the best of our knowledge. Thus we studied the reactivity and specificity of these reagents towards different classes of reactive carbonyl groups (e.g. aldehydes, ketones and lactams), each being represented by a synthetic peptide carrying an accordingly modified residue. All three tagging reagents were selective for aliphatic aldehydes and ketones. Lactams and carbonyl-containing tryptophan oxidation products, however, were labelled only at low levels or not at all. Whereas DNPH derivatization was efficient under the published standard conditions, the derivatization conditions for BHZ and ARP had to be altered. Acidic conditions provided quantitative labelling yields for ARP. Peptides derivatized with DNPH, BHZ and ARP fragmented efficiently in tandem mass spectrometry, when the experimental conditions were chosen carefully for each reagent. Importantly, the tested carbonylated peptides did not cross-react with amino groups in other proteins present during sample preparations or enzymatic digestion. Thus, it appears favourable to digest proteins first and then derivatise the reactive carbonyl groups more efficiently at the peptide level under acidic conditions. The carbonylated model peptides used in this study might be valid internal standards for carbonylation proteomics.

  10. Laccase-catalyzed carbon-nitrogen bond formation: coupling and derivatization of unprotected L-phenylalanine with different para-hydroquinones.

    PubMed

    Hahn, V; Mikolasch, A; Manda, K; Gördes, D; Thurow, K; Schauer, F

    2009-07-01

    Unprotected L-phenylalanine was derivatized by an innovative enzymatic method by means of laccases from Pycnoporus cinnabarinus and Myceliophthora thermophila. During the incubation of L-phenylalanine with para-hydroquinones using laccase as biocatalyst, one or two main products were formed. Dependent on the substitution grade of the hydroquinones mono- and diaminated products were detected. Differences of the used laccases are discussed. The described reactions are of interest for the derivatization of amino acids and a synthesis of pharmacological-active amino acid structures in the field of white biotechnology.

  11. Derivatization reactions of carbamate pesticides in supercritical carbon dioxide.

    PubMed

    King, Jerry W; Zhang, Zhouyao

    2002-09-01

    Supercritical fluid carbon dioxide (SC-CO(2)) has been used to dissolve derivatizing agents (e.g. heptafluorobutyric anhydride, HFBA, and pyridine), which also act as a modifier in the fluid phase, for simultaneous extraction and derivatization of carbamates from the sample matrix. The derivatized carbamate pesticides (carbaryl, 3-hydroxycarbofuran, carbofuran, aldicarb, methiocarb) were then analyzed by GC-ECD or GC-MS with excellent sensitivity. Extraction and conversion of the carbamates was complete, as indicated by HPLC with post-column hydrolysis and o-phthalaldehyde derivatization then fluorescence detection. GC-MS (ion trap) was also used to confirm the formation of the carbamate derivatives. Compared with the same HFBA reaction in an organic solvent the derivatization reaction time was considerably shorter in SC-CO(2.) The described approach, combining both extraction and derivatization, simplifies the analysis of carbamate pesticides and eliminates the use of organic solvents associated with the derivatization step.

  12. Fluoroacetylation/fluoroethylesterification as a derivatization approach for gas chromatography-mass spectrometry in metabolomics: preliminary study of lymphohyperplastic diseases.

    PubMed

    Karamani, Anna A; Fiamegos, Yiannis Ch; Vartholomatos, George; Stalikas, Constantine D

    2013-08-09

    Metabolic fingerprinting in combination with gas chromatography and multivariate analysis is being extensively employed for the improved understanding of biological changes induced by endogenous or exogenous factors. Chemical derivatization increases the sensitivity and specificity of gas chromatography-mass spectrometry (GC-MS) for polar or thermally labile biological compounds, which bear derivatizable groups. Thus, there is a constant demand for simple methods of derivatization and separation that satisfy the need for metabolite analysis, identifying as many chemical classes of compounds as possible. In this study, an optimized protocol of extraction and derivatization is established as a generally applicable method for the analysis of a wide range of classes of metabolites in urine, whole blood and saliva. Compounds of biological relevance bearing hydroxyl- carboxyl- and amino-groups are derivatized using single-step fluoroacetylation/fluoroethylesterification after proper optimization of the protocol. Subsequently, the developed derivatization procedure is engaged in finding blood metabolic biomarkers, induced by lymphohyperplastic disease, through the metabolomic fingerprinting approach, the multivariate modeling (hierarchical cluster analysis) and GC-MS. Our preliminary, GC-MS-based metabolomic fingerprinting study underlines the contribution of certain metabolites to the discrimination of patients with lymphohyperplastic diseases.

  13. HPLC method for amino acids profile in biological fluids and inborn metabolic disorders of aminoacidopathies.

    PubMed

    Babu, S V Suresh; Shareef, M M; Shetty, A Pavan Kumar; Shetty, K Taranath

    2002-07-01

    Quantification of total and individual amino acids in biological fluids such as plasma, urine and cerebrospinal fluid has an important diagnostic implication in laboratory medicine. The present paper describes protocols for the assay of total amino acids by modified method based on dinitrophenyl and HPLC profile involving pre-column derivatization with o-pthalaldehyde (OPA) derivatization, respectively. The method, based on the alkylation of-SH groups prior to OPA derivatization of amino acids followed by reverse phase high performance liquid chromatography, provide a comprehensive profile of more than twenty amino acids (including-SH group containing) in a single run lasting about 45 minutes. The present study, apart from establishing the normal profile of amino acids in plasma of Indian sub population, also presents HPLC profile for some of the rare amino acidopathies.

  14. Analysis of Organic Molecules Extracted from Mars Analogues and Influence of Their Mineralogy Using N-Methyl-N-(tert-butyldimethylsilyl)Trifluoroacetamide Derivatization Coupled with Gas Chromatography Mass Spectrometry in Preparation for the Sample Analysis at Mars Derivatization Experiment on the Mars Science Laboratory Mission

    NASA Technical Reports Server (NTRS)

    Stalport, F.; Glavin, D. P.; Eigenbrode, J. L.; Bish, D.; Blake, D.; Coll, P.; Szopa, C.; Buch, A.; McAdam, A.; Dworkin, J. P.; hide

    2012-01-01

    The search for complex organic molecules on Mars, including important biomolecules such as amino acids and carboxylic acids will require a chemical extraction and derivatization step to transform these organic compounds into species that are sufficiently volatile to be detected by gas chromatography mass spectrometry (GCMS). We have developed, a one-pot extraction and chemical derivatization protocol using N-methyl-N-(tert-butyldimethylsilyl)trifluoroacetamide (MTBSTFA) and dimethylformamide (DMF) for the Sample Analysis at Mars (SAM) experiment on the Mars Science Laboratory (MSL). The temperature and duration the derivatization reaction, pre-concentration of chemical derivatives, and gas chromatographic separation parameters have been optimized under SAM instrument design constraints. MTBSTFA/DMF extraction and derivatization at 300 C for several minutes of a variety of terrestrial Mars analogue materials facilitated the detection of amino acids and carboxylic acids in a surface soil sample collected from the Atacama Desert and a carbonate-rich stromatolite sample from Svalbard. However, the rapid reaction of MTBSTFA with water in several analogue materials that contained high abundances of hydrated minerals and the possible deactivation of derivatized compounds by iron oxides, as detected by XRD/XRF using the CheMin field unit Terra, proved to be highly problematic for the direct extraction of organics using MTBSTFA, The combination of pyrolysis and two different chemical derivatization methods employed by SAM should enable a wide range of organic compounds to be detected by GCMS if present on Mars,

  15. Amino acid contents of infant foods.

    PubMed

    Bosch, Lourdes; Alegría, Amparo; Farré, Rosaura

    2006-01-01

    The protein quality of three milk-cereal-based infant foods (paps) was evaluated by determining their amino acid contents and calculating the amino acid score. Proteins were subjected to acid hydrolysis, prior to which cysteine and methionine were oxidized with performic acid. Amino acids were determined by reverse-phase high-performance liquid chromatography with fluorescence detection with a prior derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate. Tryptophan was determined by reverse-phase high-performance liquid chromatography with ultraviolet detection after basic hydrolysis. Glutamic acid, proline and leucine were the most abundant amino acids, whereas tryptophan and cysteine had the lowest contents. Tryptophan was the limiting amino acid in the analyzed infant foods. A pap serving (250 ml) contributes significantly to fulfillment of the recommended dietary allowances of essential and semi-essential amino acids for infants (7-12 months old) and young children (1-3 years old).

  16. Derivatization of carbohydrates for analysis by chromatography; electrophoresis and mass spectrometry.

    PubMed

    Harvey, David J

    2011-05-15

    Carbohydrates display a large diversity of structures and their analysis presents many obstacles as the result of properties such as isomeric diversity, existence of branched structures and the lack of chromophores or fluorophores. Consequently, many analytical approaches depend on the application of chemical modifications such as hydrolysis or derivative formation. This review covers various aspects of derivatization that are used for such approaches as improving thermal stability and volatility for gas-phase analyses, introduction of fluorophores for optical detectors, introduction of charge for mass spectral analyses and attachment of bioaffinity tags for bioactivity studies. Reducing carbohydrates contain, in addition to multiple hydroxyl groups, several other sites for derivatization such as the single anomeric site that has been used in numerous methods for attaching various property-enhancing tags. Other sites are restricted to specific carbohydrates but include carboxy groups in sialic acids and amino groups in glycosylamines. All of these groups have been the targets of derivatization and this review attempts to summarise the main methods used for these various functional groups.

  17. Catalytic wateroxidation on derivatized nanoITO

    SciTech Connect

    Chen, Zuofeng; Concepcion, Javier J; Hull, Jonathan F; Hoertz, Paul G.; Meyer, Thomas J.

    2010-06-22

    Electrocatalytic water oxidation occurs on high surface area, nanocrystalline ITO (nanoITO) surface-derivatized by phosphonate-binding of the catalyst [Ru(Mebimpy)(4,4'-((HO)2OPCH2)2bpy)(OH2)]2+ (Mebimpy is 2,6-bis(1-methylbenzimidazol-2-yl)pyridine; bpy is 2,2'-bipyridine). With nanoITO, spectral data can be acquired on electrochemically generated intermediates and voltammograms monitored spectrophotometrically.

  18. Research for amino acids in lunar samples.

    NASA Technical Reports Server (NTRS)

    Gehrke, C. W.; Zumwalt, R. W.; Kuo, K.; Rash, J. J.; Aue , W. A.; Stalling, D. L.; Kvenvolden, K. A.; Ponnamperuma, C.

    1972-01-01

    The study was primarily directed toward the examination of Apollo 14 lunar fines for indigenous amino acids or materials which could be converted to amino acids on hydrolysis with 6 N hydrochloric acid. Initial experiments were conducted to confirm the integrity of the derivatization reactions and reagents, and to optimize the gas-liquid chromatographic (GLC) instrumental and chromatographic system for the separation and flame ionization detection of the amino acid derivatives. In studies on the recovery of amino acids added to lunar fines, low recoveries were obtained when 10 ng of each amino acid were added to 50 mg of virgin fines, but the subsequent addition of 50 ng of each to the previously extracted sample resulted in much higher recoveries.

  19. Research for amino acids in lunar samples.

    NASA Technical Reports Server (NTRS)

    Gehrke, C. W.; Zumwalt, R. W.; Kuo, K.; Rash, J. J.; Aue , W. A.; Stalling, D. L.; Kvenvolden, K. A.; Ponnamperuma, C.

    1972-01-01

    The study was primarily directed toward the examination of Apollo 14 lunar fines for indigenous amino acids or materials which could be converted to amino acids on hydrolysis with 6 N hydrochloric acid. Initial experiments were conducted to confirm the integrity of the derivatization reactions and reagents, and to optimize the gas-liquid chromatographic (GLC) instrumental and chromatographic system for the separation and flame ionization detection of the amino acid derivatives. In studies on the recovery of amino acids added to lunar fines, low recoveries were obtained when 10 ng of each amino acid were added to 50 mg of virgin fines, but the subsequent addition of 50 ng of each to the previously extracted sample resulted in much higher recoveries.

  20. XPS depth profiling of derivatized amine and anhydride plasma polymers: Evidence of limitations of the derivatization approach

    NASA Astrophysics Data System (ADS)

    Manakhov, Anton; Michlíček, Miroslav; Felten, Alexandre; Pireaux, Jean-Jacques; Nečas, David; Zajíčková, Lenka

    2017-02-01

    The quantitative analysis of the chemistry at the surface of functional plasma polymers is highly important for the optimization of their deposition conditions and, therefore, for their subsequent applications. The chemical derivatization of amine and carboxyl-anhydride layers is a well-known technique already applied by many researchers, notwithstanding the known drawback of the derivatization procedures like side or uncomplete reactions that could lead to "unreliable" results. In this work, X-ray photoelectron spectroscopy (XPS) combined with depth profiling with argon clusters is applied for the first time to study derivatized amine and carboxyl-anhydride plasma polymer layers. It revealed an additional important parameter affecting the derivatization reliability, namely the permeation of the derivatizing molecule through the target analysed layer, i.e. the composite effect of the probe molecule size and the layer porosity. Amine-rich films prepared by RF low pressure plasma polymerization of cyclopropylamine were derivatized with trifluoromethyl benzaldehide (TFBA) and it was observed by that the XPS-determined NH2 concentration depth profile is rapidly decreasing over top ten nanometers of the layer. The anhydride-rich films prepared by atmospheric plasma co-polymerization of maleic anhydride and C2H2 have been reacted with, parafluoroaniline and trifluoroethyl amine. The decrease of the F signal in top surface layer of the anhydride films derivatized by the "large" parafluoroaniline was observed similarly as for the amine films but the derivatization with the smaller trifluoroethylamine (TFEA) led to a more homogenous depth profile. The data analysis suggests that the size of the derivatizing molecule is the main factor, showing that the very limited permeation of the TFBA molecule can lead to underestimated densities of primary amines if the XPS analysis is solely carried out at a low take-off angle. In contrast, TFEA is found to be an efficient

  1. The Influence of Mineralogy on Recovering Organic Acids from Mars Analogue Materials Using the One-Pot Derivatization Experiment on the Sample Analysis at Mars(SAM) Instrument Suite

    NASA Technical Reports Server (NTRS)

    Stalport, Fabien; Glavin, Daniel P.; Eigenbrode, J. L.; Bish, D.; Blake, D.; Coll, P.; Szopa, C.; Buch, A.; McAdam, A.; Dworkin, J. P.; hide

    2012-01-01

    The search for complex organic molecules on Mars, including important biomolecules such as amino acids and carboxylic acids, will require a chemical extraction and a derivatization step to transform these organic compounds into species that are sufficiently volatile to be detected by gas chromatography mass spectrometry (GCMS). We have developed a ''one-pot'' extraction and chemical derivatization protocol using N-methyl-N-(tert-butyldimethylsilyl) trifluoroacetamide (MTBSTFA) and dimethylformamide (DMF) for the Sample Analysis at Mars (SAM) experiment instrument suite on NASA's the Mars Science Laboratory (MSL) mission. The temperature and duration of the derivatization reaction, pre-concentration of chemical derivatives, and gas chromatographic separation parameters have been optimized under SAM instrument design constraints. MTBSTFA/DMF extraction and derivatization at 300 1C for several minutes of a variety of terrestrial Mars analog materials facilitated the detection of amino acids and carboxylic acids in a surface soil sample collected from the Atacama Desert and a carbonate-rich stromatolite sample from Svalbard. However, the rapid reaction of MTBSTFA with water in several analog materials that contained high abundances of hydrated minerals, and the possible deactivation of derivatized compounds by iron oxides, as detected by XRD/XRF using the CheMin field unit Terra, proved to be highly problematic for the direct extraction of organics using MTBSTFA. The combination of pyrolysis and two different wet-chemical derivatization methods employed by SAM should enable a wide range of organic compounds to be detected by GCMS if present on Mars.

  2. The influence of mineralogy on recovering organic acids from Mars analogue materials using the “one-pot” derivatization experiment on the Sample Analysis at Mars (SAM) instrument suite

    NASA Astrophysics Data System (ADS)

    Stalport, F.; Glavin, D. P.; Eigenbrode, J. L.; Bish, D.; Blake, D.; Coll, P.; Szopa, C.; Buch, A.; McAdam, A.; Dworkin, J. P.; Mahaffy, P. R.

    2012-07-01

    The search for complex organic molecules on Mars, including important biomolecules such as amino acids and carboxylic acids, will require a chemical extraction and a derivatization step to transform these organic compounds into species that are sufficiently volatile to be detected by gas chromatography mass spectrometry (GCMS). We have developed a "one-pot" extraction and chemical derivatization protocol using N-methyl-N-(tert-butyldimethylsilyl) trifluoroacetamide (MTBSTFA) and dimethylformamide (DMF) for the Sample Analysis at Mars (SAM) experiment instrument suite on NASA's the Mars Science Laboratory (MSL) mission. The temperature and duration of the derivatization reaction, pre-concentration of chemical derivatives, and gas chromatographic separation parameters have been optimized under SAM instrument design constraints. MTBSTFA/DMF extraction and derivatization at 300 °C for several minutes of a variety of terrestrial Mars analog materials facilitated the detection of amino acids and carboxylic acids in a surface soil sample collected from the Atacama Desert and a carbonate-rich stromatolite sample from Svalbard. However, the rapid reaction of MTBSTFA with water in several analog materials that contained high abundances of hydrated minerals, and the possible deactivation of derivatized compounds by iron oxides, as detected by XRD/XRF using the CheMin field unit Terra, proved to be highly problematic for the direct extraction of organics using MTBSTFA. The combination of pyrolysis and two different wet-chemical derivatization methods employed by SAM should enable a wide range of organic compounds to be detected by GCMS if present on Mars.

  3. Detection of in-situ derivatized peptides in microbial biofilms by laser desorption 7.87 eV postionizaton mass spectrometry.

    SciTech Connect

    Edirisinghe, P. D.; Moore, J. F.; Skinner-Nemec, K. A.; Lindberg, C.; Giometti, C. S.; Veryovkin, I. V.; Hunt, J. E.; Pellin, M. J.; Hanley, L.; Biosciences Division; Univ. of Illinois at Chicago; MassThink

    2007-01-01

    A novel analytical method based on laser desorption postionization mass spectrometry (LDPI-MS) was developed to investigate the competence and sporulation factor-a pentapeptide of amino acid sequence ERGMT-within intact Bacillus subtilis biofilms. Derivatization of the neat ERGMT peptide with quinoline- and anthracene-based tags was separately used to lower the peptide ionization potential and permit direct ionization by 7.87-eV vacuum ultraviolet radiation. The techniques of mass shifting and selective ionization of the derivatized peptide were combined here to permit detection of ERGMT peptide within intact biofilms by LDPI-MS, without any prior extraction or chromatographic separation. Finally, imaging MS specific to the derivatized peptide was demonstrated on an intact biofilm using LDPI-MS. The presence of ERGMT in the biofilms was verified by bulk extraction/LC-MS. However, MALDI imaging MS analyses were unable to detect ERGMT within intact biofilms.

  4. A reexamination of amino acids in lunar soil

    NASA Astrophysics Data System (ADS)

    Brinton, K. L. F.; Bada, J. L.; Arnold, J. R.

    1993-03-01

    Amino acids in lunar soils provide an important indicator of the level of prebiotic organic compounds on the moon. The results provide insight into the chemistry of amino acid precursors, and furthermore, given the flux of carbonaceous material to the moon, we can evaluate the survival of organics upon impact. The amino acid contents of both hydrolyzed and unhydrolyzed hot-water extracts of Apollo 17 lunar soil were determined using ophthaldialdehyde/N-acetyl cysteine (OPA/NAC) derivatization followed by HPLC analysis. Previous studies of lunar amino acids were inconclusive, as the technique used (derivatization with ninhydrin followed by HPLC analysis) was unable to discriminate between cosmogenic amino acids and terrestrial contaminants. Cosmogenic amino acids are racemic, and many of the amino acids found in carbonaceous meteorites such as Murchison, i.e., alpha-amino-i-butyric acid (aib), are extremely rare on Earth. The ninhydrin method does not distinguish amino acid enantiomers, nor does it detect alpha-alkyl amino acids such as aib, whereas the OPA/NAC technique does both.

  5. A reexamination of amino acids in lunar soil

    NASA Technical Reports Server (NTRS)

    Brinton, K. L. F.; Bada, J. L.; Arnold, J. R.

    1993-01-01

    Amino acids in lunar soils provide an important indicator of the level of prebiotic organic compounds on the moon. The results provide insight into the chemistry of amino acid precursors, and furthermore, given the flux of carbonaceous material to the moon, we can evaluate the survival of organics upon impact. The amino acid contents of both hydrolyzed and unhydrolyzed hot-water extracts of Apollo 17 lunar soil were determined using ophthaldialdehyde/N-acetyl cysteine (OPA/NAC) derivatization followed by HPLC analysis. Previous studies of lunar amino acids were inconclusive, as the technique used (derivatization with ninhydrin followed by HPLC analysis) was unable to discriminate between cosmogenic amino acids and terrestrial contaminants. Cosmogenic amino acids are racemic, and many of the amino acids found in carbonaceous meteorites such as Murchison, i.e., alpha-amino-i-butyric acid (aib), are extremely rare on Earth. The ninhydrin method does not distinguish amino acid enantiomers, nor does it detect alpha-alkyl amino acids such as aib, whereas the OPA/NAC technique does both.

  6. A reexamination of amino acids in lunar soil

    NASA Technical Reports Server (NTRS)

    Brinton, K. L. F.; Bada, J. L.; Arnold, J. R.

    1993-01-01

    Amino acids in lunar soils provide an important indicator of the level of prebiotic organic compounds on the moon. The results provide insight into the chemistry of amino acid precursors, and furthermore, given the flux of carbonaceous material to the moon, we can evaluate the survival of organics upon impact. The amino acid contents of both hydrolyzed and unhydrolyzed hot-water extracts of Apollo 17 lunar soil were determined using ophthaldialdehyde/N-acetyl cysteine (OPA/NAC) derivatization followed by HPLC analysis. Previous studies of lunar amino acids were inconclusive, as the technique used (derivatization with ninhydrin followed by HPLC analysis) was unable to discriminate between cosmogenic amino acids and terrestrial contaminants. Cosmogenic amino acids are racemic, and many of the amino acids found in carbonaceous meteorites such as Murchison, i.e., alpha-amino-i-butyric acid (aib), are extremely rare on Earth. The ninhydrin method does not distinguish amino acid enantiomers, nor does it detect alpha-alkyl amino acids such as aib, whereas the OPA/NAC technique does both.

  7. A highly sensitive quantification of phytosterols through an inexpensive derivatization.

    PubMed

    Liu, Songbai; Ruan, Huina

    2013-01-01

    A highly sensitive method for quantification of phytosterols based on HPLC has been developed by derivatization with the benzoyl chromophore. Introduction of the chromophore, benzoyl group, to phytosterols via simple and inexpensive derivatization greatly improved the UV response at 254 nm. Quantification of phytosterols was effectively performed by HPLC analysis with methyl benzoate as the internal standard after derivatization. This new method demonstrated outstanding yield of recovery (> 95%) and excellent sensitivity (ng level) and was applicable for sterols from either plant or animal sources. This method is generally useful in phytosterol studies.

  8. Highly efficient solid-phase derivatization of sugar phosphates with titanium-immobilized hydrophilic polydopamine-coated silica.

    PubMed

    Qin, Qian; Wang, Bohong; Chang, Mengmeng; Zhou, Zhihui; Shi, Xianzhe; Xu, Guowang

    2016-07-29

    Sugar phosphates are a type of key metabolic intermediates of glycolysis, gluconeogenesis and pentose phosphate pathway, which can regulate tumor energetic metabolism. Due to their low endogenous concentrations, poor chromatographic retention properties as well as ionization suppression from complex matrix interference, the determination of sugar phosphates in biological samples is very difficult. In this study, titanium-immobilized hydrophilic polydopamine-coated silica microspheres (SiO2@PD-Ti(4+)) were synthesized for highly efficient solid-phase derivatization of sugar phosphates. Sugar phosphates were selectively captured onto the surface of the SiO2@PD-Ti(4+) microspheres by chelating with phosphate groups, and then reacted with 3-amino-9-ethylcarbazole via reductive amination based on solid-phase derivatization, which could not only increase the retention and resolution of sugar phosphates on reversed-phase liquid chromatography (RPLC), but also improve the mass spectrometry (MS) sensitivity of sugar phosphates. The adsorption capacity of SiO2@PD-Ti(4+) microspheres towards glucose-6-phosphate is 0.76mg/g, which is much larger than that of commercial TiO2. Compared with the traditional liquid-phase derivatization, the solid-phase derivatization based on the SiO2@PD-Ti(4+) microspheres displayed several superiorities including shorter derivatization time (within 10min), higher product purity and much lower limit of detection (up to 38pmol/L). In addition, good linearity (R(2)≥0.99), excellent recovery (80.6-118%) and high precision (RSDs with 2.8-7.8%) were obtained when the developed method was used for quantitative analysis of sugar phosphates. Finally, the SiO2@PD-Ti(4+) microspheres combined with RPLC-MS were successfully applied to the determination of sugar phosphates from hepatocarcinoma cell lines and could even detect the trace sugar phosphates in thousands of cells. Copyright © 2016 Elsevier B.V. All rights reserved.

  9. Amino acids

    MedlinePlus

    ... this page: //medlineplus.gov/ency/article/002222.htm Amino acids To use the sharing features on this page, please enable JavaScript. Amino acids are organic compounds that combine to form proteins . ...

  10. Mass spectrometer having a derivatized sample presentation apparatus

    DOEpatents

    Nelson, Randall W.

    2000-07-25

    A mass spectrometer having a derivatized sample presentation apparatus is provided. The sample presentation apparatus has a complex bound to the surface of the sample presentation apparatus. This complex includes a molecule which may chemically modify a biomolecule.

  11. On-line derivatization for continuous and automatic monitoring of brain extracellular glutamate levels in anesthetized rats: a microdialysis study.

    PubMed

    Yang, C S; Tsai, P J; Chen, W Y; Tsai, W J; Kuo, J S

    1999-10-29

    Glutamate is an important excitatory amino acid in central nervous system. We developed a method for in vivo, continuous and automatic monitoring of brain extracellular glutamate, as well as other amino acids in anesthetized rat. This method involves the use of microdialysis perfusion technique and a high-performance liquid chromatography system equipped with a fluorescence detector. The microdialysate (perfused at a flow-rate of 1 microl/min) was on-line derivatized with o-phthaldehyde (perfused at 2 microl/min) through a mixing tee prior to the injection onto the HPLC column. The efficiency of this on-line derivatization was equivalent to that performed with an off-line manner. The effect of cerebral ischemia (2 h) and reperfusion (2 h) in brain cortex of anesthetized rats was monitored using this method. In addition to glutamate, extracellular concentrations of other amino acids, such as aspartate, glutamine, glycine, taurine and gamma-aminobutyric acid, were also simultaneously monitored with this on-line method. Since monitoring of extracellular amino acids by microdialysis perfusion is intensively used in neuroscience investigations, this simple and convenient method would be useful in the future applications.

  12. Utilization of a deuterated derivatization agent to synthesize internal standards for gas chromatography-tandem mass spectrometry quantification of silylated metabolites.

    PubMed

    Lien, Stina K; Kvitvang, Hans Fredrik Nyvold; Bruheim, Per

    2012-07-20

    GC-MS analysis of silylated metabolites is a sensitive method that covers important metabolite groups such as sugars, amino acids and non-amino organic acids, and it has become one of the most important analytical methods for exploring the metabolome. Absolute quantitative GC-MS analysis of silylated metabolites poses a challenge as different metabolites have different derivatization kinetics and as their silyl-derivates have varying stability. This report describes the development of a targeted GC-MS/MS method for quantification of metabolites. Internal standards for each individual metabolite were obtained by derivatization of a mixture of standards with deuterated N-methyl-N-trimethylsilyltrifluoroacetamide (d9-MSTFA), and spiking this solution into MSTFA derivatized samples prior to GC-MS/MS analysis. The derivatization and spiking protocol needed optimization to ensure that the behaviour of labelled compound responses in the spiked sample correctly reflected the behaviour of unlabelled compound responses. Using labelled and unlabelled MSTFA in this way enabled normalization of metabolite responses by the response of their deuterated counterpart (i.e. individual correction). Such individual correction of metabolite responses reproducibly resulted in significantly higher precision than traditional data correction strategies when tested on samples both with and without serum and urine matrices. The developed method is thus a valuable contribution to the field of absolute quantitative metabolomics.

  13. Comparison of automated pre-column and post-column analysis of amino acid oligomers

    NASA Technical Reports Server (NTRS)

    Chow, J.; Orenberg, J. B.; Nugent, K. D.

    1987-01-01

    It has been shown that various amino acids will polymerize under plausible prebiotic conditions on mineral surfaces, such as clays and soluble salts, to form varying amounts of oligomers (n = 2-6). The investigations of these surface reactions required a quantitative method for the separation and detection of these amino acid oligomers at the picomole level in the presence of nanomole levels of the parent amino acid. In initial high-performance liquid chromatography (HPLC) studies using a classical postcolumn o-phthalaldehyde (OPA) derivatization ion-exchange HPLC procedure with fluorescence detection, problems encountered included lengthy analysis time, inadequate separation and large relative differences in sensitivity for the separated species, expressed as a variable fluorescent yield, which contributed to poor quantitation. We have compared a simple, automated, pre-column OPA derivatization and reversed-phase HPLC method with the classical post-column OPA derivatization and ion-exchange HPLC procedure. A comparison of UV and fluorescent detection of the amino acid oligomers is also presented. The conclusion reached is that the pre-column OPA derivatization, reversed-phase HPLC and UV detection produces enhanced separation, improved sensitivity and faster analysis than post-column OPA derivatization, ion-exchange HPLC and fluorescence detection.

  14. High-performance liquid chromatographic determination of memantine hydrochloride in rat plasma using sensitive fluorometric derivatization.

    PubMed

    Xie, Mei-Fen; Zhou, Wei; Tong, Xin-Yi; Chen, Yi-Le; Cai, Yi; Li, Yan; Duan, Geng-Li

    2011-02-01

    In this study, we investigated a simple, sensitive and reliable liquid chromatography-fluorescence detection method for the determination of memantine hydrochloride in rat plasma which was based on derivatization with 9-fluorenylmethyl chloroformate (FMOC-Cl). For the first time, FMOC-Cl was introduced into derivatization of memantine hydrochloride in rat plasma. The amino groups of memantine hydrochloride and amantadine hydrochloride (internal standard) were trapped with FMOC-Cl to form memantine hydrochloride-FMOC-Cl and amantadine hydrochloride-FMOC-Cl compositions, which can be very compatible for LC-FLD. Precipitation of plasma proteins by acetonitrile was followed by vortex mixing and centrifugation. Chromatographic separation was performed on a C(18) column (DIAMONSIL 150 × 4.6 mm, id 5 μm) with a mobile phase consisting of acetonitrile and water at a flow rate of 1.0 mL/min. The retention times of memantine hydrochloride-FMOC-Cl and amantadine hydrochloride-FMOC-Cl compositions were 23.69 and 40.27 min, respectively. Optimal conditions for the derivatization of memantine hydrochloride were also described. The limit of quantification (LOQ) was 25 ng/mL for memantine hydrochloride in plasma, the linear range was 0.025-5.0 μg/mL in plasma with a correlation coefficient (r) of 0.9999. The relative standard deviations (RSDs) of intra-day and inter-day assays were 4.46-12.19 and 5.23-11.50%, respectively. The validated method was successfully applied to the determination of memantine hydrochloride in rat plasma samples.

  15. A non-aqueous procedure to synthesize amino group bearing nanostructured organic-inorganic hybrid materials.

    PubMed

    Göring, M; Seifert, A; Schreiter, K; Müller, P; Spange, S

    2014-09-04

    Amino-functionalized organic-inorganic hybrid materials with a narrow distributed nanostructure of 2-4 nm in size were obtained by means of a template-free and non-aqueous procedure. Simultaneous twin polymerization of novel amino group containing twin monomers with 2,2'-spirobi[4H-1,3,2-benzodioxasiline] has been applied for this purpose. The amino groups of the organic-inorganic hybrid material are useful for post derivatization.

  16. Tandem MS Analysis of Selenamide-Derivatized Peptide Ions

    PubMed Central

    Zhang, Yun; Zhang, Hao; Cui, Weidong; Chen, Hao

    2013-01-01

    Our previous study showed that selenamide reagents such as ebselen and N-(phenylseleno) phthalimide (NPSP) can be used for selective and rapid derivatization of protein/peptide thiols in high conversion yield. This paper reports the systematic investigation of MS/MS dissociation behaviors of selenamide-derivatized peptide ions upon collision induced dissociation (CID) and electron transfer dissociation (ETD). In the positive ion mode, derivatized peptide ions exhibit tag-dependent CID dissociation pathways. For instance, ebselen-derivatized peptide ions preferentially undergo Se–S bond cleavage upon CID to produce a characteristic fragment ion, the protonated ebselen (m/z 276), which allows selective identification of thiol peptides from protein digest as well as selective detection of thiol proteins from protein mixture using precursor ion scan (PIS). In contrast, NPSP-derivatized peptide ions retain their phenylselenenyl tags during CID, which is useful in sequencing peptides and locating cysteine residues. In the negative ion CID mode, both types of tags are preferentially lost via the Se–S cleavage, analogous to the S–S bond cleavage during CID of disulfide-containing peptide anions. In consideration of the convenience in preparing selenamide-derivatized peptides and the similarity of Se–S of the tag to the S–S bond, we also examined ETD of the derivatized peptide ions to probe the mechanism for electron-based ion dissociation. Interestingly, facile cleavage of Se–S bond occurs to the peptide ions carrying either protons or alkali metal ions, while backbone cleavage to form c/z ions is severely inhibited. These results are in agreement with the Utah-Washington mechanism proposed for depicting electron-based ion dissociation processes. PMID:21953264

  17. Resin-bound chiral derivatizing agents for assignment of configuration by NMR spectroscopy.

    PubMed

    Porto, Silvia; Seco, José Manuel; Espinosa, Juan Félix; Quiñoá, Emilio; Riguera, Ricardo

    2008-08-01

    A general methodology for assigning the configuration of chiral mono- and polyfunctional compounds by NMR is presented. The approach is based on the use of polystyrene-bound chiral derivatizing agents (CDA-resins) specifically designed to achieve the high-yield formation of the covalent linkages (amide or ester bonds) between the substrate and the chiral auxiliary within the NMR tube, without the need for other manipulations, on a microscale level and in a short time. The deuterated NMR solvents (CDCl3, CD3CN, CS2/CD2Cl2) are also the reaction solvents and separations, purifications or workups of any kind are not necessary prior to recording the spectra. The CDA-resins prepared included MPA, 9-AMA, BPG, MTPA, and 2-NTBA as auxiliary agents incorporated either as single enantiomers or as mixed combinations of the (R)- and the (S)-enantiomers at unequal and known ratios. The high versatility of these systems was successfully demonstrated in a variety of ways based on double and single derivatization, low temperature experiments, or the formation of metal complexes. The approach allowed the absolute configurations of chiral primary amines, primary and secondary alcohols, cyanohydrins, thiols, diols, triols, and amino alcohols to be determined. Extensive high-resolution magic angle spinning (HR-MAS) NMR experiments allowed the characterization of the new CDA-resins and enabled the study of their stability and regioselectivity.

  18. Analytical platform for metabolome analysis of microbial cells using methyl chloroformate derivatization followed by gas chromatography-mass spectrometry.

    PubMed

    Smart, Kathleen F; Aggio, Raphael B M; Van Houtte, Jeremy R; Villas-Bôas, Silas G

    2010-09-01

    This protocol describes an analytical platform for the analysis of intra- and extracellular metabolites of microbial cells (yeast, filamentous fungi and bacteria) using gas chromatography-mass spectrometry (GC-MS). The protocol is subdivided into sampling, sample preparation, chemical derivatization of metabolites, GC-MS analysis and data processing and analysis. This protocol uses two robust quenching methods for microbial cultures, the first of which, cold glycerol-saline quenching, causes reduced leakage of intracellular metabolites, thus allowing a more reliable separation of intra- and extracellular metabolites with simultaneous stopping of cell metabolism. The second, fast filtration, is specifically designed for quenching filamentous micro-organisms. These sampling techniques are combined with an easy sample-preparation procedure and a fast chemical derivatization reaction using methyl chloroformate. This reaction takes place at room temperature, in aqueous medium, and is less prone to matrix effect compared with other derivatizations. This protocol takes an average of 10 d to complete and enables the simultaneous analysis of hundreds of metabolites from the central carbon metabolism (amino and nonamino organic acids, phosphorylated organic acids and fatty acid intermediates) using an in-house MS library and a data analysis pipeline consisting of two free software programs (Automated Mass Deconvolution and Identification System (AMDIS) and R).

  19. A sensitive and selective determination method of histamine by HPLC with intramolecular excimer-forming derivatization and fluorescence detection.

    PubMed

    Yoshitake, Takashi; Ichinose, Fumio; Yoshida, Hideyuki; Todoroki, Ken-ichiro; Kehr, Jan; Inoue, Osamu; Nohta, Hitoshi; Yamaguchi, Masatoshi

    2003-12-01

    A highly sensitive, selective and simple method is described for the determination of histamine by high-performance liquid chromatography (HPLC) with fluorescence detection. The method is based on an intramolecular excimer-forming fluorescence derivatization of histamine with 4-(1-pyrene)butyric acid N-hydroxysuccinimide ester (PSE), followed by reversed-phase HPLC. Histamine, having two amino moieties in a molecule, was converted to the dipyrene-labeled derivative by reaction with PSE. The derivative afforded intramolecular excimer fluorescence (450-540 nm), which can clearly be discriminated from the monomer fluorescence (370-420 nm) emitted from PSE. Typically, a 10 micro L sample solution was mixed with 100 micro L of derivatization reagent solution, which was a mixture of 0.5 mm PSE in acetonitrile and 0.5 mm potassium carbonate in water (8:2, v/v). The derivatization was carried out at 100 degrees C for 90 min. The PSE derivative of histamine could be separated by reversed-phase ODS column with isocratic elution using acetonitrile:water (82:18, v/v) containing 0.03% triethylamine. The detection limit (singnal-to-noise ratio = 3) of histamine was 0.5 fmol for a 30 micro L injection. The method was successfully applied to the determination of histamine in human urine, and had enough selectivity and sensitivity for urinary histamine quantification.

  20. An Efficient and Rapid Method to Monitor the Oxidative Degradation of Protein Pharmaceuticals: Probing Tyrosine Oxidation with Fluorogenic Derivatization.

    PubMed

    Bommana, Rupesh; Mozziconacci, Olivier; John Wang, Y; Schöneich, Christian

    2017-07-01

    The loss of potency of protein therapeutics can be linked to the oxidation of specific amino acid residues leading to a great variety of oxidative modifications. The comprehensive identification of these oxidative modifications requires high-resolution mass spectrometry analysis, which requires time and expensive resources. Here, we propose a fluorogenic derivatization method of oxidized Tyr and Phe yielding benzoxazole derivatives, as an orthogonal technique for the rapid screening of protein oxidation. Four model proteins, IgG1, human growth hormone (hGH), insulin and bovine serum albumin (BSA) were exposed to oxidation via peroxyl radicals and metal-catalyzed reactions and efficiently screened by fluorogenic derivatization of Tyr and Phe oxidation products. Complementary LC-MS analysis was done to identify the extent of methionine oxidation in oxidized proteins. The Fluorogenic derivatization technique can easily be adapted to a 96-well plate, in which several protein formulations can be screened in short time. Representatively for hGH, we show that the formation of benzoxazole parallels the oxidation of Met to methionine sulfoxide which enables estimation of Met oxidation by just recording the fluorescence. Our rapid fluorescence based screening allows for the fast comparison of the stability of multiple formulations.

  1. Silyl-based alkyne-modifying linker for the preparation of C-terminal acetylene-derivatized protected peptides.

    PubMed

    Strack, Martin; Langklotz, Sina; Bandow, Julia E; Metzler-Nolte, Nils; Albada, H Bauke

    2012-11-16

    A novel linker for the synthesis of C-terminal acetylene-functionalized protected peptides is described. This SAM1 linker is applied in the manual Fmoc-based solid-phase peptide synthesis of Leu-enkephalin and in microwave-assisted automated synthesis of Maculatin 2.1, an antibacterial peptide that contains 18 amino acid residues. For the cleavage, treatment with tetramethylammonium fluoride results in protected acetylene-derivatized peptides. Alternatively, a one-pot cleavage-click procedure affords the protected 1,2,3-triazole conjugate in high yields after purification.

  2. Synthesis and evaluation of two coumarin-type derivatization reagents for fluorescence detection of chiral amines and chiral carboxylic acids.

    PubMed

    Mertens, Matthias D; Gütschow, Michael

    2013-12-01

    The synthesis of two fluorescent coumarin-type chiral derivatization agents (4 and 11) is reported. A chiral side chain was introduced at position 7 of the coumarin via Mitsunobu reaction. The two coumarins bear in this side chain either a free amino group or a carboxyl group, making them useful for further transformations. Conjugates of chiral prototype drugs with 4 or 11 were prepared by amide coupling of the analyte's carboxyl group to the reagent's amine group, or vice versa. The separation of seven diastereomeric conjugates through achiral high-performance liquid chromatography (HPLC) on a common C18 column is demonstrated. © 2013 Wiley Periodicals, Inc.

  3. Alkylation or Silylation for Analysis of Amino and Non-Amino Organic Acids by GC-MS?

    PubMed Central

    Villas-Bôas, Silas G.; Smart, Kathleen F.; Sivakumaran, Subathira; Lane, Geoffrey A.

    2011-01-01

    Gas chromatography–mass spectrometry (GC-MS) is a widely used analytical technique in metabolomics. GC provides the highest resolution of any standard chromatographic separation method, and with modern instrumentation, retention times are very consistent between analyses. Electron impact ionization and fragmentation is generally reproducible between instruments and extensive libraries of spectra are available that enhance the identification of analytes. The major limitation is the restriction to volatile analytes, and hence the requirement to convert many metabolites to volatile derivatives through chemical derivatization. Here we compared the analytical performance of two derivatization techniques, silylation (TMS) and alkylation (MCF), used for the analysis of amino and non-amino organic acids as well as nucleotides in microbial-derived samples. The widely used TMS derivatization method showed poorer reproducibility and instability during chromatographic runs while the MCF derivatives presented better analytical performance. Therefore, alkylation (MCF) derivatization seems to be preferable for the analysis of polyfunctional amines, nucleotides and organic acids in microbial metabolomics studies. PMID:24957242

  4. Alkylation or Silylation for Analysis of Amino and Non-Amino Organic Acids by GC-MS?

    PubMed

    Villas-Bôas, Silas G; Smart, Kathleen F; Sivakumaran, Subathira; Lane, Geoffrey A

    2011-01-17

    Gas chromatography-mass spectrometry (GC-MS) is a widely used analytical technique in metabolomics. GC provides the highest resolution of any standard chromatographic separation method, and with modern instrumentation, retention times are very consistent between analyses. Electron impact ionization and fragmentation is generally reproducible between instruments and extensive libraries of spectra are available that enhance the identification of analytes. The major limitation is the restriction to volatile analytes, and hence the requirement to convert many metabolites to volatile derivatives through chemical derivatization. Here we compared the analytical performance of two derivatization techniques, silylation (TMS) and alkylation (MCF), used for the analysis of amino and non-amino organic acids as well as nucleotides in microbial-derived samples. The widely used TMS derivatization method showed poorer reproducibility and instability during chromatographic runs while the MCF derivatives presented better analytical performance. Therefore, alkylation (MCF) derivatization seems to be preferable for the analysis of polyfunctional amines, nucleotides and organic acids in microbial metabolomics studies.

  5. Catalytically-Promoted Analyte Derivatization Inside a Gas Chromatographic Inlet

    PubMed Central

    Fowler, William K.; Gamble, Kelly J.; Wright, Amber R.

    2010-01-01

    Reported here is a preliminary assessment of the feasibility of catalyzing on-line derivatization reactions inside the inlet (i.e., the injection port) of a gas chromatograph (GC) with solid heterogeneous catalysts. The experiments described here entail the installation of candidate catalysts inside the GC inlet liner and the subsequent injection of analyte/reagent mixtures onto the catalyst beds. Two catalysts are identified, each of which clearly catalyzes one of the chosen model derivatization reactions in the inlet of a GC. This result supports our hypothesis that on-line derivatizations can, in principle, be reproducibly catalyzed inside the GC inlet by solid heterogeneous catalysts and that the presence of such catalysts in the inlet do not necessarily cause a serious loss of instrument performance or chromatographic efficiency. PMID:20822662

  6. Derivatized vancomycin stationary phases for LC chiral separations.

    PubMed

    Berthod, A; Nair, U B; Bagwill, C; Armstrong, D W

    1996-10-01

    In this work, synthetic and natural chiral selectors were combined to form two different chiral stationary phases (CSPs). These were made by bonding R- or S-(1-naphthylethyl) carbamate (R-NEC or S-NEC)-derivatized vancomycin molecules to a silica gel support. The two CSPs were evaluated using a set of 60 enantiomeric pairs. The results were compared to the ones obtained with the commercial underivatized vancomycin CSP. Three Chromatographic modes were used: (i) the normal-phase mode using a nonpolar mobile phase with different ratios of hexane and ethanol; (ii) the reversed-phase mode with hydro-organic mobile phases; and (iii) the polar aprotic organic mode with nonaqueous acetonitrile plus small amounts of methanol and an acid and/or base to control retention and selectivity. It is shown that the polarity of the underivatized vancomycin phase is higher than that of the two R- and S-NEC-derivatized CSPs. In the pH range 4-7, there is no ionization change of the chiral selector for the three CSPs. 43% of the studied compounds were resolved by the NEC-derivatized phases when they could not be resolved by the vancomycin CSP. However, the enantiorecognition for 12% of the compounds on the native vancomycin CSP was lost upon NEC derivatization. 45% of the studied compounds were resolved by the NEC-derivatized and native CSPs. The NEC derivatization procedure may block some useful active sites on the vancomycin molecule. Also, the R- and S-NEC moieties are chiral themselves and can contribute additional interaction sites not available on the native vancomycin molecule.

  7. A Derivatization and Validation Strategy for Determining the Spatial Localization of Endogenous Amine Metabolites in Tissues using MALDI Imaging Mass Spectrometry

    PubMed Central

    Manier, M. Lisa; Spraggins, Jeffrey M.; Reyzer, Michelle L.; Norris, Jeremy L.; Caprioli, Richard M.

    2014-01-01

    Imaging mass spectrometry (IMS) studies increasingly focus on endogenous small molecular weight metabolites and consequently bring special analytical challenges. Since analytical tissue blanks do not exist for endogenous metabolites, careful consideration must be given to confirm molecular identity. Here we present approaches for the improvement in detection of endogenous amine metabolites such as amino acids and neurotransmitters in tissues through chemical derivatization and matrix-assisted laser desorption/ionization (MALDI) IMS. Chemical derivatization with 4-hydroxy-3-methoxycinnamaldehyde (CA) was used to improve sensitivity and specificity. CA was applied to the tissue via MALDI sample targets precoated with a mixture of derivatization reagent and ferulic acid (FA) as a MALDI matrix. Spatial distributions of chemically derivatized endogenous metabolites in tissue were determined by high-mass resolution and MSn imaging mass spectrometry. We highlight an analytical strategy for metabolite validation whereby tissue extracts are analyzed by high-performance liquid chromatography (HPLC)-MS/MS to unambiguously identify metabolites and distinguish them from isobaric compounds. PMID:25044893

  8. GC-MS analysis of multiply derivatized opioids in urine.

    PubMed

    Chen, Bud-Gen; Wang, Sheng-Meng; Liu, Ray H

    2007-08-01

    Opiates such as hydrocodone, hydromorphone, oxycodone, noroxycodone, and oxymorphone reportedly may interfere with the analysis of morphine and codeine. The analysis of these compounds themselves also is an important issue. Thus, double derivatization approaches utilizing methoxyamine and hydroxylamine to first form oxime products with keto-opiates, followed by the derivatization with trimethylsilyl (TMS) or propionyl groups, have been developed for the simultaneous analysis of these compounds. However, these studies have not included all compounds of interest and resulted in inadequate chromatographic resolution or significant intensity cross-contribution between the ions designating the analyte and its deuterated internal standard for certain compounds. By exploring three-step derivatization approaches with the combination of various derivatization groups and orders, this study concluded that application of methoxyimino/propionyl/TMS groups, in the order listed, facilitated the simultaneous analysis of eight opiates (morphine, 6-acetylmorphine, hydromorphone, oxymorphone, codeine, hydrocodone, oxycodone and noroxycodone) in urine samples, achieving satisfactory limits of quantitation and detection. In addition, the adapted approach resulted in two usable products for morphine and codeine providing alternatives, should interferences render any of these products non-usable.

  9. Process for derivatizing carbon nanotubes with diazonium species

    NASA Technical Reports Server (NTRS)

    Tour, James M. (Inventor); Bahr, Jeffrey L. (Inventor); Yang, Jiping (Inventor)

    2007-01-01

    The invention incorporates new processes for the chemical modification of carbon nanotubes. Such processes involve the derivatization of multi- and single-wall carbon nanotubes, including small diameter (ca. 0.7 nm) single-wall carbon nanotubes, with diazonium species. The method allows the chemical attachment of a variety of organic compounds to the side and ends of carbon nanotubes. These chemically modified nanotubes have applications in polymer composite materials, molecular electronic applications and sensor devices. The methods of derivatization include electrochemical induced reactions thermally induced reactions (via in-situ generation of diazonium compounds or pre-formed diazonium compounds), and photochemically induced reactions. The derivatization causes significant changes in the spectroscopic properties of the nanotubes. The estimated degree of functionality is ca. 1 out of every 20 to 30 carbons in a nanotube bearing a functionality moiety. Such electrochemical reduction processes can be adapted to apply site-selective chemical functionalization of nanotubes. Moreover, when modified with suitable chemical groups, the derivatized nanotubes are chemically compatible with a polymer matrix, allowing transfer of the properties of the nanotubes (such as, mechanical strength or electrical conductivity) to the properties of the composite material as a whole. Furthermore, when modified with suitable chemical groups, the groups can be polymerized to form a polymer that includes carbon nanotubes ##STR00001##.

  10. Amino acid profile of milk-based infant formulas.

    PubMed

    Viadel, B; Alegriá, A; Farré, R; Abellán, P; Romero, F

    2000-09-01

    The protein content and amino acid profile of three milk-based infant formulas, two of which were powdered (adapted and follow-on) and the third liquid, were determined to check their compliance with the EU directive and to evaluate whether or not they fulfil an infant's nutritional needs. To obtain the amino acid profile proteins were subjected to acid hydrolysis, prior to which the sulfur-containing amino acids were oxidized with performic acid. The amino acids were derivatized with phenylisothiocyanate (PITC) and then determined by ion-pair reverse phase high performance liquid chromatography (HPLC) In the case of tryptophan a basic hydrolysis was applied and there was no need of derivatization. The protein contents of the analysed formulas were in the ranges established by the EU directive for these products and the amino acid contents were in the ranges reported by other authors for these types of formulas. In all cases the tryptophan content determined the value of the chemical score, which was always lower than 80% of the reference protein but in the ranges reported by other authors. The analysed adapted infant formula provides amino acids in amounts higher than the established nutritional requirements.

  11. Vortex-assisted liquid-liquid microextraction coupled with derivatization for the fluorometric determination of aliphatic amines.

    PubMed

    Chang, Wei-Yao; Wang, Chin-Yi; Jan, Jeng-Lyan; Lo, Yu-Shiu; Wu, Chien-Hou

    2012-07-27

    A new one-step derivatization and microextraction technique was developed for the fluorometric determination of C(1)-C(8) linear aliphatic primary amines in complex sample solutions containing high levels of amino acids. In this method, amines were derivatized with o-phthalaldehyde (OPA) and 2-mercaptoethanol (2-ME) in aqueous solution and extracted simultaneously by vortex-assisted liquid-liquid microextraction (VALLME). Parameters affecting the extraction efficiency were investigated in detail. The optimum conditions were as follows: 50 μL of isooctane as the extractant phase; 2.0 mL aqueous donor samples with 12 mM OPA, 24 mM 2-ME, and 0.1 M borate buffer at pH 10; 1 min vortex extraction time; centrifugation for 4 min at 6000 rpm. After centrifugation, the enriched analytes in the floated extractant phase were determined by HPLC-FL in less than 14 min. Under the optimum conditions, the limits of detection were of the order of 0.09-0.31 nM. The calibration curves showed good linearity over the investigated concentration range between 0.4 and 40 nM. The proposed method has been applied to the determination of aliphatic amines in acidophilus milk, beer, and Cu(II)/amino acid solution.

  12. Analysis of twelve amino acids in biological fluids by mass fragmentography

    NASA Technical Reports Server (NTRS)

    Summons, R. E.; Pereira, W. E.; Reynolds, W. E.; Rindfleisch, T. C.; Duffield, A. M.

    1974-01-01

    Description of a computerized method for the simultaneous quantitation of 12 amino acids in biological fluids using the technique of quadrupole mass in fragmentography. The time taken for one complete analysis, exclusive of derivatization, is 40 minutes for data collection with an additional 10 minutes before the computer presents the final analytical result.

  13. On-line sample preconcentration with chemical derivatization of bacterial biomarkers by capillary electrophoresis: a dual strategy for integrating sample pretreatment with chemical analysis.

    PubMed

    Ptolemy, Adam S; Le Bihan, Marianne; Britz-McKibbin, Philip

    2005-11-01

    Simple, selective yet sensitive methods to quantify low-abundance bacterial biomarkers derived from complex samples are required in clinical, biological, and environmental applications. In this report, a new strategy to integrate sample pretreatment with chemical analysis is investigated using on-line preconcentration with chemical derivatization by CE and UV detection. Single-step enantioselective analysis of muramic acid (MA) and diaminopimelic acid (DAP) was achieved by CE via sample enrichment by dynamic pH junction with ortho-phthalaldehyde/N-acetyl-L-cysteine labeling directly in-capillary. The optimized method resulted in up to a 100-fold enhancement in concentration sensitivity compared to conventional off-line derivatization procedures. The method was also applied toward the detection of micromolar levels of MA and DAP excreted in the extracellular medium of Escherichia coli bacterial cell cultures. On-line preconcentration with chemical derivatization by CE represents a unique approach for conducting rapid, sensitive, and high-throughput analyses of other classes of amino acid and amino sugar metabolites with reduced sample handling, where the capillary functions simultaneously as a concentrator, microreactor, and chiral selector.

  14. Determination of free amino compounds in betalainic fruits and vegetables by gas chromatography with flame ionization and mass spectrometric detection.

    PubMed

    Kugler, Florian; Graneis, Stephan; Schreiter, Pat P-Y; Stintzing, Florian C; Carle, Reinhold

    2006-06-14

    Amino acids and amines are the precursors of betalains. Therefore, the profiles of free amino compounds in juices obtained from cactus pears [Opuntia ficus-indica (L.) Mill. cv. Bianca, cv. Gialla, and cv. Rossa], pitaya fruits [Selenicereus megalanthus (K. Schumann ex Vaupel) Moran, Hylocereus polyrhizus (Weber) Britton & Rose, and Hylocereus undatus (Haworth) Britton & Rose], and in extracts from differently colored Swiss chard [Beta vulgaris L. ssp. cicla (L.) Alef. cv. Bright Lights] petioles and red and yellow beets (B. vulgaris L. ssp. vulgaris var. conditiva Alef. cv. Burpee's Golden) were investigated for the first time. Amino compounds were derivatized with propyl chloroformate. While gas chromatography (GC) with mass spectrometry was used for peak assignment, GC flame ionization detection was applied for quantification of individual compounds. Whereas proline was the major free amino compound of cactus pear and pitaya fruit juices, glutamine dominated in Swiss chard stems and beets, respectively. Interestingly, extremely high concentrations of dopamine were detected in Swiss chard stems and beets. Furthermore, the cleavage of betaxanthins caused by derivatization in alkaline reaction solutions is demonstrated for the first time. Amino acids and amines thus released might increase the actual free amino compound contents of the respective sample. To evaluate the contribution of betaxanthin cleavage to total amino acid and amine concentration, isolated betaxanthins were derivatized according to the "EZ:faast" method prior to quantification of the respective amino compounds released. On a molar basis, betaxanthin contribution to overall amino compound contents was always below 6.4%.

  15. Improving derivatization efficiency of BMAA utilizing AccQ-Tag in a complex cyanobacterial matrix.

    PubMed

    Eriksson, Johan; Jonasson, Sara; Papaefthimiou, Dimitra; Rasmussen, Ulla; Bergman, Birgitta

    2009-01-01

    Two different assays have been developed and used in order to investigate the optimal conditions for derivatization and detection of acid beta-N-methyl-amino-L-alanine (BMAA) in a cyanobacterial sample. BMAA was extracted from cyanobacterial cultures both from the cytosolic ("free") fraction and in the precipitated ("protein") fraction using a newly developed extraction scheme and the sample matrix was standardized according to protein concentration to ensure the highest possible derivative yield. A rapid and sensitive HPLC method for fluorescence detection of the non-protein amino acid BMAA in cyanobacteria, utilizing the Waters AccQ-Tag chemistry and Chromolith Performance RP-18e columns was developed. Using this new method and utilizing a different buffer system and column than that recommended by Waters, we decreased the time between injections by 75%. The limit of quantification was determined to be 12 nmol and limit of detection as 120 fmol. The linear range was in the range of 8.5 nmol-84 pmol. Accuracy and precision were well within FDA guidelines for bioanalysis.

  16. Study of Highly Selective and Efficient Thiol Derivatization using Selenium Reagents by Mass Spectrometry

    SciTech Connect

    Xu, Kehua; Zhang, Yun W.; Tang, Bo; Laskin, Julia; Roach, Patrick J.; Chen, Hao

    2010-08-15

    Biological thiols are critical physiological components and their detection often involves derivatization. This paper reports a systemic mass spectrometry (MS) investigation of the cleavage of Se-N bond by thiol to form a new Se-S bond, the new selenium chemistry for thiol labeling. Our data shows that the reaction is highly selective, rapid, reversible and efficient. For instance, among twenty amino acids, only cysteine was found to be reactive with Se-N containing reagents and the reaction takes place in seconds. By adding dithiothreitol (DTT), the newly formed Se-S bond of peptides/proteins can be reduced back to free thiol. The high selectivity and excellent reversibility of the reaction provide potential of using this chemistry for selective identification of thiol compounds or enriching and purifying thiol peptides/proteins. In addition, the derivatized thiol peptides have interesting dissociation behavior, which is tunable using different selenium reagents. For example, by introducing an adjacent nucleophilic group into the selenium reagent in the case of using ebselen, the reaction product of ebselen with glutathione (GSH) is easy to lose the selenium tag upon collision-induced dissociation (CID), which is useful to "fish out" those peptides containing free cysteine residues by precursor ion scan. By contrast, the selenium tag of N-(phenylseleno) phthalimide reagent can be stable and survive in CID process, which would be of value in pinpointing thiol location using a top-down proteomic approach. Also, the high conversion yield of the reaction allows the counting of total number of thiol in proteins. We believe that ebselen or N-(phenylseleno) phthalimide as tagging thiol-protein reagents will have important applications in both qualitative and quantitative analysis of different thiol-proteins derived from living cells by MS method.

  17. Analysis of cysteine-containing proteins using precolumn derivatization with N-(2-ferroceneethyl)maleimide and liquid chromatography/electrochemistry/mass spectrometry.

    PubMed

    Seiwert, Bettina; Karst, Uwe

    2007-08-01

    N-(2-ferroceneethyl)maleimide (FEM) is introduced as an electroactive derivatizing agent for thiol functionalities in proteins. Using appropriate reaction conditions, the derivatization is completed within five minutes and no unspecific labeling of free amino functions is observed. Liquid chromatography/electrochemistry/mass spectrometry was used to detect the reaction products. The reagent is a useful tool for determining the number of free thiol groups or the total number of free and disulfide-bound thiol groups in proteins. The electrochemical cell provides additional information, because the increase in mass spectrometric response upon electrochemical oxidation of the neutral ferrocene to the charged ferrocinium groups is monitored. The method was successfully applied to the analysis of native proteins and their tryptic digests.

  18. Modification of a micellar system for amino acid separation by MEKC--application for amino acid profiling in formulations for parenteral use.

    PubMed

    Jaworska, Małgorzata; Szulińska, Zofia; Wilk, Małgorzata; Anuszewska, Elżbieta

    2010-12-15

    The paper proposes a new method for amino acid determination which can be applied for amino acid profiling in solutions for parenteral nutrition. The MEKC method based on a mixed micellar system was developed for the separation of 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) derivatized amino acids. Background electrolyte was based on tris-borate buffer with high alkaline pH. Sodium dodecyl sulfate micelles were modified using 1,2-hexanediol as a co-surfactant. The effect of the modifier on amino acid migration was studied with respect to hydrophobicity of the analytes. The modifier appeared to be suitable to improve the separation of AQC-tagged amino acids without an adverse effect on buffer ionic strength or EOF velocity. The method was successfully validated and applied for amino acid profiling in medicinal preparations for parenteral nutrition. The results obtained were compared with a reference chromatographic method (amino acid analyser).

  19. Amino Acids Profiles in Biological Media

    SciTech Connect

    Iordache, A.; Horj, E.; Morar, S.; Cozar, O.; Culea, M.; Ani, A. R.; Mesaros, C.

    2010-08-04

    An accurate analytical method was developed to determine amino acids in some biological specimens by GC/MS technique. Stable isotopes provide useful tools for a variety of studies, offering ideal internal standards in quantitative information. Isotopic dilution gas chromatography--mass spectrometry (ID-GC/MS) is the techniques used for quantitative analysis of compounds labeled with stable isotopes. A Trace DSQ Thermo Finnigan quadrupole mass spectrometer coupled with a Trace GC was used. Amino acids were separated on a Rtx-5 MS capillary column, 30 mx0.25 mm, 0.25 {mu}m film thickness, using a temperature program from 50 deg. C, 1 min, 6 deg. C/min at 100 deg. C, 4 deg. C/min at 200 deg. C, 20 deg. C/min at 300 deg. C, (3 min). The transfer line temperature was 250 deg. C, the injector temperature 200 deg. C and ion source temperature 250 deg. C; splitter: 10:1. Electron energy was 70 eV and emission current, 100 {mu}A. The amino acids were purified on a Dowex 50W-W8 exchange resin and were derivatized in a procedure following two steps to obtain trifluoroacetyl butyl esters. The identification of amino acids was obtained by using NIST library but also by using amino acid standards.

  20. Amino acids derived from Titan tholins

    NASA Technical Reports Server (NTRS)

    Khare, B. N.; Sagan, C.; Ogino, H.; Nagy, B.; Er, C.; Schram, K. H.; Arakawa, E. T.

    1986-01-01

    An organic heteropolymer (Titan tholin) was produced by continuous dc discharge through a 0.9 N2/0.1 CH4 gas mixture at 0.2 mbar pressure, roughly simulating the cloudtop atmosphere of Titan. Treatment of this tholin with 6N HCl yielded 16 amino acids by gas chromatography after derivatization of N-trifluroacetyl isopropyl esters on two different capillary columns. Identifications were confirmed by GC/MS. Glycine, aspartic acid, and alpha- and beta-alanine were produced in greatest abundance; the total yield of amino acids was approximately 10(-2), approximately equal to the yield of urea. The presence of "nonbiological" amino acids, the absence of serine, and the fact that the amino acids are racemic within experimental error together indicate that these molecules are not due to microbial or other contamination, but are derived from the tholin. In addition to the HCN, HC2CN, and (CN)2 found by Voyager, nitriles and aminonitriles should be sought in the Titanian atmosphere and, eventually, amino acids on the surface. These results suggest that episodes of liquid water in the past or future of Titan might lead to major further steps in prebiological organic chemistry on that body.

  1. An overview of heavy-atom derivatization of protein crystals

    PubMed Central

    Pike, Ashley C. W.; Garman, Elspeth F.; Krojer, Tobias; von Delft, Frank; Carpenter, Elisabeth P.

    2016-01-01

    Heavy-atom derivatization is one of the oldest techniques for obtaining phase information for protein crystals and, although it is no longer the first choice, it remains a useful technique for obtaining phases for unknown structures and for low-resolution data sets. It is also valuable for confirming the chain trace in low-resolution electron-density maps. This overview provides a summary of the technique and is aimed at first-time users of the method. It includes guidelines on when to use it, which heavy atoms are most likely to work, how to prepare heavy-atom solutions, how to derivatize crystals and how to determine whether a crystal is in fact a derivative. PMID:26960118

  2. An overview of heavy-atom derivatization of protein crystals.

    PubMed

    Pike, Ashley C W; Garman, Elspeth F; Krojer, Tobias; von Delft, Frank; Carpenter, Elisabeth P

    2016-03-01

    Heavy-atom derivatization is one of the oldest techniques for obtaining phase information for protein crystals and, although it is no longer the first choice, it remains a useful technique for obtaining phases for unknown structures and for low-resolution data sets. It is also valuable for confirming the chain trace in low-resolution electron-density maps. This overview provides a summary of the technique and is aimed at first-time users of the method. It includes guidelines on when to use it, which heavy atoms are most likely to work, how to prepare heavy-atom solutions, how to derivatize crystals and how to determine whether a crystal is in fact a derivative.

  3. Extraction, derivatization, and analysis of vegetable oils from fire debris.

    PubMed

    Gambrel, Abby K; Reardon, Michelle R

    2008-11-01

    Vegetable oils have the ability to spontaneously heat under certain conditions, which may lead to spontaneous ignition. While the oils are not often encountered in forensic casework, they may be suspected in some fire cases. As these oils are not effectively analyzed using traditional fire debris analysis methods, a protocol must be established for extracting vegetable oils from fire debris. In this study, a protocol was developed for the extraction, derivatization, and analysis of vegetable oils from fire debris. Three derivatization methods were compared to establish an optimal derivatization procedure to convert the fatty acids found in vegetable oils to the fatty acid methyl esters (FAMEs) used in analysis. Three different gas chromatograph columns and programs were examined to determine which was best suited for the separation and analysis of FAMEs. The procedure was tested and refined using a variety of neat and burned vegetable oils, in addition to extractions from oils burned on commonly encountered fire debris materials. The findings of this research will serve as a starting point for further understanding and research of vegetable oils in fire debris.

  4. Spectroscopic and potentiometric studies on derivatized natural humic acid.

    PubMed

    Andjelkovic, Tatjana; Perovic, Jelica; Purenovic, Milovan; Blagojevic, Srdjan; Nikolic, Ruzica; Andjelkovic, Darko; Bojic, Aleksandar

    2006-12-01

    Isolated soil humic acid (HA) and commercial Aldrich HA were derivatized by esterification with methanol-thionyl and acetylation with acetic anhidride, in order to obtain derivatives with selectively blocked carboxyl and phenol groups, respectively. Results obtained by FT-IR spectroscopy and potentiometry show that the methanol-thionyl procedure is a selective, specific and efficient route for blocking carboxyl groups. The good correlation between results obtained by direct potentiometry after HA esterification and by classical calcium-acetate and baryta exchange methods suggests that esterification followed by direct acid-base potentiometric titration can be used as a method for the estimation of carboxyl and phenol group contents. Phenol groups can not be specifically identified by the acetylation method, due to the low selectivity of the acetylation method. The average values of apparent and intrinsic pK of underivatized and derivatized HAs confirm decrease in ionizable groups content due to derivatization and their values are related to the different chemical structures of the acids.

  5. Solventless mechanosynthesis of N-protected amino esters.

    PubMed

    Konnert, Laure; Lamaty, Frédéric; Martinez, Jean; Colacino, Evelina

    2014-05-02

    Mechanochemical derivatizations of N- or C-protected amino acids were performed in a ball mill under solvent-free conditions. A vibrational ball mill was used for the preparation of N-protected α- and β-amino esters starting from the corresponding N-unmasked precursors via a carbamoylation reaction in the presence of di-tert-butyl dicarbonate (Boc2O), benzyl chloroformate (Z-Cl) or 9-fluorenylmethoxycarbonyl chloroformate (Fmoc-Cl). A planetary ball mill proved to be more suitable for the synthesis of amino esters from N-protected amino acids via a one-pot activation/esterification reaction in the presence of various dialkyl dicarbonates or chloroformates. The spot-to-spot reactions were straightforward, leading to the final products in reduced reaction times with improved yields and simplified work-up procedures.

  6. Phosphopeptide Enrichment by Covalent Chromatography after Derivatization of Protein Digests Immobilized on Reversed-Phase Supports

    PubMed Central

    Nika, Heinz; Nieves, Edward; Hawke, David H.; Angeletti, Ruth Hogue

    2013-01-01

    A rugged sample-preparation method for comprehensive affinity enrichment of phosphopeptides from protein digests has been developed. The method uses a series of chemical reactions to incorporate efficiently and specifically a thiol-functionalized affinity tag into the analyte by barium hydroxide catalyzed β-elimination with Michael addition using 2-aminoethanethiol as nucleophile and subsequent thiolation of the resulting amino group with sulfosuccinimidyl-2-(biotinamido) ethyl-1,3-dithiopropionate. Gentle oxidation of cysteine residues, followed by acetylation of α- and ε-amino groups before these reactions, ensured selectivity of reversible capture of the modified phosphopeptides by covalent chromatography on activated thiol sepharose. The use of C18 reversed-phase supports as a miniaturized reaction bed facilitated optimization of the individual modification steps for throughput and completeness of derivatization. Reagents were exchanged directly on the supports, eliminating sample transfer between the reaction steps and thus, allowing the immobilized analyte to be carried through the multistep reaction scheme with minimal sample loss. The use of this sample-preparation method for phosphopeptide enrichment was demonstrated with low-level amounts of in-gel-digested protein. As applied to tryptic digests of α-S1- and β-casein, the method enabled the enrichment and detection of the phosphorylated peptides contained in the mixture, including the tetraphosphorylated species of β-casein, which has escaped chemical procedures reported previously. The isolates proved highly suitable for mapping the sites of phosphorylation by collisionally induced dissociation. β-Elimination, with consecutive Michael addition, expanded the use of the solid-phase-based enrichment strategy to phosphothreonyl peptides and to phosphoseryl/phosphothreonyl peptides derived from proline-directed kinase substrates and to their O-sulfono- and O-linked β-N-acetylglucosamine (O

  7. Ion-beam and microwave-stimulated functionalization and derivatization of carbon nanotubes

    NASA Astrophysics Data System (ADS)

    Makala, Raghuveer S.

    Derivatizing carbon nanotubes (CNTs) with other low-dimensional nanostructures is of widespread interest for creating CNT-based nanocomposites and devices. Conventional routes based on wet-chemical oxidation or hydrophobic adsorption do not allow premeditated control over the location or spatial extent of functionalization. Moreover, aggressive oxidative treatments and agitation in corrosive environments lead to CNT shortening, damage, and incorporation of excess impurity concentrations. Thus, it is imperative to explore and develop alternative functionalization methods to overcome these shortcomings. The work presented in this thesis outlines two such methodologies: one based on focused ion irradiation for siteselective functionalization and the other that employs microwave-stimulation for mild, yet rapid and homogenous CNT functionalization. The utility of 10 and 30 kcV Ga+ focused ion beams (FIB) to thin, slice, weld, and alter the structure and composition at precise locations along the CNT axis is presented. This strategy of harnessing ion-beam-induced defect generation and doping is attractive for modulating chemical and electrical properties along the CNT length, and fabricate CNT-based heterostructures and networks. A novel approach that utilizes focused ion irradiation to site-selectively derivatize preselected segments of CNTs with controlled micro-/nano-scale lateral spatial resolution is demonstrated. Irradiation followed by air-exposure results in functionalized CNT segments ranging from the nanoscopic to the macroscopic scale. The functional moieties are utilized to site-selectively anchor Au nanoparticles, fluorescent nanospheres, an amino acid---lysine, a charge-transfer metalloprotein---azurin, and a photoactive protein---bacteriorhodopsin by means of electrostatic or covalent interactions. This approach is versatile and can be extended to obtaining other molecular moieties and derivatives opening up possibilities for building new types of nano

  8. Amino acid, fatty acid, and carbohydrate content of Artocarpus altilis (breadfruit).

    PubMed

    Golden, K D; Williams, O J

    2001-06-01

    A study is conducted to determine the amino acid, fatty acid, and carbohydrate content of breadfruit using high-performance liquid chromatography (HPLC) and gas chromatography (GC). An HPLC method is used for the determination of amino acids and fatty acids in breadfruit. Representative amino acid samples are derivatized with phenylisothiocianate and the resulting phenylthiocarbamyl derivatives are separated on a reversed-phase column by gradient elution with a 0.05M ammonium acetate buffer and 0.01M ammonium acetate in acetonitrile-methanol-water (44:10:46, v/v). Representative fatty acid samples are derivatized with phenacyl bromide and the resulting fatty acid phenacyl esters are separated on a reversed-phase column by gradient elution with acetonitrile and water. Amino acid and fatty acid derivatives are detected by ultraviolet detection at 254 nm. The analysis of the carbohydrates in breadfruit employs a GC method. Carbohydrates are derivatized using trimethylchlorosilane and hexamethyldisilazane to form trimethylsilyl ethers. Compounds in the samples are separated by the temperature programming of a GC using nitrogen as the carrier gas. Percent recoveries of amino acids, fatty acids, and carbohydrates are 72.5%, 68.2%, and 81.4%, respectively. The starch content of the breadfruit is 15.52 g/100 g fresh weight.

  9. HPLC Determination of Bioactive Sulfur Compounds, Amino Acids and Biogenic Amines in Biological Specimens.

    PubMed

    Francioso, Antonio; Fanelli, Sergio; Vigli, Daniele; Ricceri, Laura; Cavallaro, Rosaria A; Conrado, Alessia Baseggio; Fontana, Mario; D'Erme, Maria; Mosca, Luciana

    2017-01-01

    There is an increasing interest for analytical methods aimed to detect biological sulfur-containing amines, because of their involvement in human diseases and metabolic disorders. This work describes an improved HPLC method for the determination of sulfur containing amino acids and amines from different biological matrices. We optimized a pre-column derivatization procedure using dabsyl chloride, in which dabsylated products can be monitored spectrophotometrically at 460 nm. This method allows the simultaneous analysis of biogenic amines, amino acids and sulfo-amino compounds including carnosine, dopamine, epinephrine, glutathione, cysteine, taurine, lanthionine, and cystathionine in brain specimens, urines, plasma, and cell lysates. Moreover, the method is suitable for the study of physiological and non-physiological derivatives of taurine and glutathione such as hypotaurine, homotaurine, homocysteic acid and S-acetylglutathione. The present method displays good efficiency of derivatization, having the advantage to give rise to stable products compared to other derivatizing agents such as o-phthalaldehyde and dansyl chloride.With this method, we provide a tool to study sulfur cycle from a metabolic point of view in relation to the pattern of biological amino-compounds, allowing researchers to get a complete scenario of organic sulfur and amino metabolism in tissues and cells.

  10. Recent advances in development and application of derivatization reagents having a benzofurazan structure: a brief overview.

    PubMed

    Santa, Tomofumi

    2014-06-01

    Chemical derivatization is often used to improve the separation efficiency and to enhance the detectability of the target compounds in high-performance liquid chromatography and capillary electrophoresis. The derivatization reagents having a benzofurazan (2,1,3-benzoxadiazole) structure are one of the most often used reagent for this purpose. In this paper, the recent advances in the development and the application of benzofurazan derivatization reagents are reviewed.

  11. Characterization of dicarboxylic naphthenic acid fraction compounds utilizing amide derivatization: proof of concept.

    PubMed

    Kovalchik, Kevin A; MacLennan, Matthew S; Peru, Kerry M; Ajaero, Chukwuemeka; McMartin, Dena W; Headley, John V; Chen, David D Y

    2017-09-25

    The characterization of naphthenic acid fraction compounds (NAFCs) in oil sands process affected water (OSPW) is of interest for both toxicology studies and regulatory reasons. Previous studies utilizing authentic standards have identified dicarboxylic naphthenic acids using two-dimensional gas chromatography hyphenated to time-of-flight mass spectrometry (GCxGC/TOFMS). The selective derivatization of hydroxyl groups has also recently aided in the characterization of oxy-NAFCs, and indirectly the characterization of dicarboxylic NAFCs. However, there has been no previous report of derivatization being used to directly aid in the standard-free characterization of NAFCs with multiple carboxylic acid functional groups. Herein we present proof-of-concept for the characterization of dicarboxylic NAFCs utilizing amide derivatization. Carboxylic acid groups in OSPW extract and in a dicarboxylic acid standard were derivatized to amides using a previously described method. The derivatized extract and derivatized standard were analyzed by direct-injection positive-mode electrospray ionization ((+)ESI) high resolution mass spectrometry (MS), and the underivatized extract was analyzed by (-)ESI MS. Tandem-MS was carried out on selected ions of the derivatized standard and derivatized OSPW. Data analysis was carried out using the Python programming language. The distribution of monocarboxylic NAFCs observed in the amide-derivatized OSPW sample by (+)ESI-MS was generally similar to that seen in underivatized OSPW by (-)ESI-MS. The dicarboxylic acid standard shows evidence of being doubly-derivatized, although the second derivatization appears to be inefficient. Furthermore, a spectrum of potential diacid NAFCs is presented, identified by both charge state and derivatization mass. Interference due to the presence of multiple derivatization products is noted, but can be eliminated using on-line separation or an isotopically labelled derivatization reagent. Proof of concept for the

  12. Quantification of candidate prostate cancer metabolite biomarkers in urine using dispersive derivatization liquid-liquid microextraction followed by gas and liquid chromatography-mass spectrometry.

    PubMed

    Shamsipur, Mojtaba; Naseri, Mohammad Taghi; Babri, Mehran

    2013-01-01

    A simple, rapid and sensitive method based on dispersive derivatization liquid-liquid microextraction (DDLLME) combined with gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS) was developed and validated for the determination of prostate cancer metabolite biomarkers, including sarcosine, alanine, leucine and proline, in human urine samples. Dispersive derivatization using isobutyl chloroformate has been successfully employed to identify the amino acids of interest in ng mL(-1) concentrations. Under the optimum experimental conditions, the detection limits of the amino acids were in the range of 0.05-0.1 ng mL(-1). The enrichment factor and relative recovery for the target amino acids were in the range of 140-155 and 93.8-106%, respectively. The proposed method showed good linearity (correlation coefficients >0.997), and good intra-day (below 7%) and inter-day precision (below 10%). This protocol provides a rapid, simple, selective and sensitive tool to quantify sarcosine and endogenous urinary metabolite for prostate cancer diagnosis and for a screening test. Copyright © 2013 Elsevier B.V. All rights reserved.

  13. Simultaneous analysis of apolar phytohormones and 1-aminocyclopropan-1-carboxylic acid by high performance liquid chromatography/electrospray negative ion tandem mass spectrometry via 9-fluorenylmethoxycarbonyl chloride derivatization.

    PubMed

    Ziegler, Jörg; Qwegwer, Jakob; Schubert, Melvin; Erickson, Jessica L; Schattat, Martin; Bürstenbinder, Katharina; Grubb, C Douglas; Abel, Steffen

    2014-10-03

    A strategy to detect and quantify the polar ethylene precursor 1-aminocyclopropan-1-carboxylic acid (ACC) along with the more apolar phytohormones abscisic acid (ABA), indole-3-acetic acid (IAA), jasmonic acid (JA), jasmonic acid-isoleucine conjugate (JA-Ile), 12-oxo-phytodienoic acid (OPDA), trans-zeatin, and trans-zeatin 9-riboside using a single extraction is presented. Solid phase resins commonly employed for extraction of phytohormones do not allow the recovery of ACC. We circumvent this problem by attaching an apolar group to ACC via derivatization with the amino group specific reagent 9-fluorenylmethoxycarbonyl chloride (Fmoc-Cl). Derivatization in the methanolic crude extract does not modify other phytohormones. The derivatized ACC could be purified and detected together with the more apolar phytohormones using common solid phase extraction resins and reverse phase HPLC/electrospray negative ion tandem mass spectrometry. The limit of detection was in the low nanomolar range for all phytohormones, a sensitivity sufficient to accurately determine the phytohormone levels from less than 50mg (fresh weight) of Arabidopsis thaliana and Nicotiana benthamiana tissues. Comparison with previously published phytohormone levels and the reported changes in phytohormone levels after stress treatments confirmed the accuracy of the method.

  14. Ion-pairing high-performance liquid chromatography determination of amitrole in apple after solid-phase extraction and precolumn derivatization.

    PubMed

    Sun, Yan; Luo, Laixin; Wang, Fang; Li, Jianqiang; Cao, Yongsong

    2009-09-01

    A novel method based on solid-phase extraction was studied for the extraction of amitrole (3-amino-1,2,4-triazole), and its residue determination in apples has been developed. The samples were derivatized with 4-chloro-3,5-dinitrobenzotrifluoride (CNBF). The derivatization conditions and the influence of elution composition on the separation were investigated. In pH 9.5 H(3)BO(3)-Na(2)B(4)O(7) media, the reaction of amitrole with CNBF was complete at 60 degrees C after 30 min. The separation of derivatized amitrole was achieved at room temperature within 13 min by gradient elution mode with cetyltrimethylammonium bromide in mobile phase as ion-pair reagent. The method correlation coefficient was 0.9996, in concentrations ranging from 1.66 to 415 mg L(-1). The calculated recoveries of the proposed method were from 94.17% to 105.67%, and relative standard deviations were 1.57% to 6.44% in the application to the quantitative determination of amitrole in apples. The detection limit of amitrole was 0.10 mg L(-1) with a signal-to-noise ratio of 3.

  15. Determination of linear aliphatic aldehydes in heavy metal containing waters by high-performance liquid chromatography using 2,4-dinitrophenylhydrazine derivatization.

    PubMed

    Lin, Yi-Liang; Wang, Po-Yen; Hsieh, Ling-Ling; Ku, Kuan-Hsuan; Yeh, Yun-Tai; Wu, Chien-Hou

    2009-09-04

    A simple and sensitive method is described for the determination of picomolar amounts of C(1)-C(9) linear aliphatic aldehydes in waters containing heavy metal ions. In this method, aldehydes were first derivatized with 2,4-dinitrophenylhydrazine (DNPH) at optimized pH 1.8 for 30 min and analyzed by HPLC with UV detector at 365 nm. Factors affecting the derivatization reaction of aldehydes and DNPH were investigated. Cupric ion, an example of heavy metals, is a common oxidative reagent, which may oxidize DNPH and greatly interfere with the determination of aldehydes. EDTA was used to effectively mask the interferences by heavy metal ions. The method detection limits for direct injection of derivatized most aldehydes except formaldehyde were of the order of 7-28 nM. The detection limit can be further lowered by using off-line C(18) adsorption cartridge enrichment. The recoveries of C(1)-C(9) aldehydes were 93-115% with a relative standard deviation of 3.6-8.1% at the 0.1 microM level for aldehydes. The HPLC-DNPH method has been applied for determining aldehyde photoproducts from Cu(II)-amino acid complex systems.

  16. SYNTHESIS OF HIGHLY FLUORINATED CHLOROFORMATES AND THEIR USE AS DERIVATIZING AGENTS FOR HYDROPHILIC COMPOUNDS AND DRINKING WATER DISINFECTION BY-PRODUCTS

    EPA Science Inventory

    A rapid, safe and efficient procedure was developed to synthesize perfluorinated chloroformates in the small scale generally required to perform analytical derivatizations. This new family of derivatizing agents allows straightforward derivatization of highly polar compounds, co...

  17. LCMS analysis of fingerprints, the amino acid profile of 20 donors.

    PubMed

    de Puit, Marcel; Ismail, Mahado; Xu, Xiaoma

    2014-03-01

    The analysis of amino acids present in fingerprints has been studied several times. In this paper, we report a method for the analysis of amino acids using an fluorenylmethyloxycarbonyl chloride-derivatization for LC separation and MS detection. We have obtained good results with regard to the calibration curves and the limit of detection and LOQ for the target compounds. The extraction of the amino acids from the substrates used proved to be very efficient. Analysis of the derivatized amino acids enabled us to obtain full amino acid profiles for 20 donors. The intervariability is as expected rather large, with serine as the most abundant constituent, and when examining the total profile of the amino acids per donor, a characteristic pattern can be observed. Some amino acids were not detected in some donors, or fell out of the range of the calibration curve, where others showed a surprisingly high amount of material in the deposition analyses. Further investigations will have to address the intravariability of the amino acid profiles of the fingerprints from donors. By the development of the analytical method and the application to the analysis of fingerprints, we were able to gain insight in the variability of the constituents of fingerprints between the donors.

  18. Free amino acids in botanicals and botanical preparations.

    PubMed

    Carratù, B; Boniglia, C; Giammarioli, S; Mosca, M; Sanzini, E

    2008-06-01

    Numerous studies were carried out about aminoacidic composition of vegetable proteins, but information about the free amino acid pool and the role of these substances is very incomplete. The aim of this paper was to contribute to the scarce knowledge concerning the composition of free amino acids in botanicals and botanical preparations widely used as food, in dietary supplements, and in pharmaceutical products. This work studied the composition of free amino acids, identified the major components of 19 species of plants, and evaluated the influence of different types of extraction on the amino acid profile. Amino acids were determined using an automatic precolumn derivatization with fluorenylmethyl-chloroformate and reversed-phase liquid chromatography with fluorescence and ultraviolet detection. The amounts of total free amino acids varied widely between plants, from approximately 12 g in 100 g of Echinacea pallida extract to less than 60 mg in the same amount of Coleus forskohlii, Garcinia cambogia, and Glycine max. In 13 plants arginine, asparagine, glutamine, proline, and gamma-aminobutyric acid were the free amino acids found in preponderant quantities. The levels of free amino acids above the quantification limit in 36 assayed samples of botanicals, extracts, and supplements are shown.

  19. [Separation and quantification of theanine enantiomers using high performance liquid chromatography coupled with chiral derivatization].

    PubMed

    Li, Yinhua; Liu, Zhonghua; Huang, Jian'an

    2007-09-01

    L-Theanine is a unique non-protein amino acid in tea, and shows many physiological functions. To directly extract L-theanine from tea is a lengthy and very costly process. Although the chemical synthesis of theanines is simple, the product is unfortunately a mixture of DL-enantiomers. A chiral derivatization method was developed for the separation and quantification of theanine enantiomers by reversed-phase high performance liquid chromatography (RP-HPLC). 1-Fluoro-2,4-dinitrophenyl-5-L-alanine amide (FDAA)was used as the chiral reagent. This method showed good linearity for both L-theanine (ranging from 1.732 x 10(-3) to 2.077 microg) and D-theanine (ranging from 1.696 x 10(-3) to 2.044 microg). The recoveries were in the range of 97.3% - 102.0% for L-theanine and 97.2% - 103.2% for D-theanine. This method also showed excellent limit of detection (approximately 5 x 10(-4) microg) and limit of quantification (approximately 1 x 10(-3) microg) for both L-theanine and D-theanine. The results demonstrated that this method is precise, accurate and can be used for the determination of theanine enantiomers.

  20. Atom transfer radical polymerization preparation and photophysical properties of polypyridylruthenium derivatized polystyrenes.

    PubMed

    Fang, Zhen; Ito, Akitaka; Keinan, Shahar; Chen, Zuofeng; Watson, Zoe; Rochette, Jason; Kanai, Yosuke; Taylor, Darlene; Schanze, Kirk S; Meyer, Thomas J

    2013-08-05

    A ruthenium containing polymer featuring a short carbonyl-amino-methylene linker has been prepared by atom transfer radical polymerization (ATRP). The polymer was derived from ATRP of the N-hydroxysuccinimide (NHS) derivative of p-vinylbenzoic acid, followed by an amide coupling reaction of the NHS-polystyrene with Ru(II) complexes derivatized with aminomethyl groups (i.e., [Ru(bpy)2(CH3-bpy-CH2NH2)](2+) where bpy is 2,2'-bipyridine, and CH3-bpy-CH2NH2 is 4-methyl-4'-aminomethyl-2,2'-bipyridine). The Ru-functionalized polymer structure was confirmed by using nuclear magnetic resonance and infrared spectroscopy, and the results suggest that a high loading ratio of polypyridylruthenium chromophores on the polystyrene backbone was achieved. The photophysical properties of the polymer were characterized in solution and in rigid ethylene glycol glasses. In solution, emission quantum yield and lifetime studies reveal that the polymer's metal-to-ligand charge transfer (MLCT) excited states are quenched relative to a model Ru complex chromophore. In rigid media, the MLCT-ground state band gap and lifetime are both increased relative to solution with time-resolved emission measurements revealing fast energy transfer hopping within the polymer. Molecular dynamics studies of the polymer synthesized here as well as similar model systems with various spatial arrangements of the pendant Ru complex chromophores suggest that the carbonyl-amino-methylene linker probed in our target polymer provides shorter Ru-Ru nearest-neighbor distances leading to an increased Ru*-Ru energy hopping rate, compared to those with longer linkers in counterpart polymers.

  1. Enhanced analysis of steroids by gas chromatography/mass spectrometry using microwave-accelerated derivatization.

    PubMed

    Bowden, John A; Colosi, Dominic M; Stutts, Whitney L; Mora-Montero, Diana C; Garrett, Timothy J; Yost, Richard A

    2009-08-15

    Derivatization of steroids is typically required before analysis by gas chromatography/mass spectrometry (GC/MS); nevertheless, the derivatization process can often be time-consuming and irreproducible. Although several strategies have been employed to enhance this process, few have the potential of microwave-accelerated derivatization (MAD) to be more efficient than traditional thermal derivatization methods. MAD using a synthesis microwave system was evaluated and compared to traditional thermal derivatization methods in terms of yield, reproducibility, and overall analysis time. Parameters affecting MAD, including reaction temperature, time, and power, were systematically optimized for several silyl reagents (BSTFA with TMCS, MSTFA, and BSA) and other derivatization procedures (MOX reagent and MTBSTFA). MSTFA was found to derivatize best with the microwave, as demonstrated by the enhanced relative response factors (RRFs). BSTFA with TMCS, on the other hand, did not couple as well, but RRF values improved significantly upon addition of polar solvents. The rapid (1 min) derivatization reactions associated with MAD had comparable RRFs for all reagents with those obtained with thermal heating (>30 min). This study highlights the best methods for analyzing a comprehensive variety of steroids and also provides ideal strategies for MAD of steroids on an individual or class level.

  2. Hydrophobically derivatized hyperbranched polyglycerol as a human serum albumin substitute.

    PubMed

    Kainthan, Rajesh K; Janzen, Johan; Kizhakkedathu, Jayachandran N; Devine, Dana V; Brooks, Donald E

    2008-04-01

    There is a huge clinical demand for Human Serum Albumin (HSA), with a world market of approximately $1.5B/year. Concern over prion and viral transmission in the blood supply has led to a need for safer substitutes and offers the opportunity for development of materials with enhanced properties over the presently available plasma expanders. We report here the synthesis and testing of a new synthetic plasma expander that can replace not only the osmotic and volume expansion properties of HSA but, uniquely, its binding and transport properties. We have synthesized several hyperbranched polyglycerols derivatized with hydrophobic groups and short poly(ethylene glycol) (PEG) chains. The hydrophobic groups provide regions for binding fatty acids and other hydrophobic materials while PEG imparts the necessary protection from host defense systems and enhances circulation longevity. These polymers, being hyperbranched, have only a small effect on plasma viscosity. We have shown in vitro that our materials bind 2-3 moles palmitic acid per mole, do not activate the platelet, coagulation or complement systems and do not cause red cell aggregation. In mice these materials are non-toxic with circulation half-lives as high as 34h, controllable by manipulating the molecular weight and the degree of PEG derivatization.

  3. Derivatization of isothiocyanates and their reactive adducts for chromatographic analysis.

    PubMed

    Agerbirk, Niels; De Nicola, Gina Rosalinda; Olsen, Carl Erik; Müller, Caroline; Iori, Renato

    2015-10-01

    Isothiocyanates form adducts with a multitude of biomolecules, and these adducts need analytical methods. Likewise, analytical methods for hydrophilic isothiocyanates are needed. We considered reaction with ammonia to form thiourea derivatives. The hydrophilic, glycosylated isothiocyanate moringin, 4-(α-L-rhamnopyranosyloxy)benzyl isothiocyanate, was efficiently derivatized to the thiourea derivative by incubation with ammonia. The hydrophobic benzyl isothiocyanate was also efficiently derivatized to the thiourea derivative. The thiourea group provided a UV absorbing chromophore, and the derivatives showed expectable sodium and hydrogen adducts in ion trap mass spectrometry and were suitable for liquid chromatography analysis. Reactive dithiocarbamate adducts constitute the major type of reactive ITC adduct expected in biological matrices. Incubation of a model dithiocarbamate with ammonia likewise resulted in conversion to the corresponding thiourea derivative, suggesting that a variety of matrix-bound reactive isothiocyanate adducts can be determined using this strategy. As an example of the application of the method, recovery of moringin and benzyl isothiocyanate applied to cabbage leaf discs was studied in simulated insect feeding assays. The majority of moringin was recovered as native isothiocyanate, but a major part of benzyl isothiocyanate was converted to reactive adducts. Copyright © 2015 Elsevier Ltd. All rights reserved.

  4. Spectrofluorimetric determination of tetrabenazine after photochemical derivatization in basic medium

    NASA Astrophysics Data System (ADS)

    Osório, Ana C. P.; da Cunha, Alessandra L. M. C.; Khan, Sarzamin; Ponciano, Cássia R.; Aucélio, Ricardo Q.

    Photochemical derivatization is proposed for the spectrofluorimetric determination of tetrabenazine (TBZ). A central composite design was used to adjust experimental conditions (60 min of UV in a 0.45 mol L-1 NaOH solution) enabling the improvement of the analyte signal-to-blank ratio of one order of magnitude, when compared to the TBZ original fluorescence. Limit of quantification was 4.7 × 10-8 mol L-1 but the detection power can be improved at least 10 times using solid phase extraction that also allows the separation of the analyte from matrix components, enabling the analysis of biologic fluids. Linear range covered at least three orders of magnitude. The combined uncertainty of the determination (at a 5 × 10-6 mol L-1) was 16%. Recoveries of TBZ in the analyses of a pharmaceutical formulation were in agreement with the ones obtained using a HPLC method. Recovery in saliva (5 × 10-7 mol L-1 of TBZ) was 90 ± 3% (n = 3). The procedure minimizes the use of toxic chemical derivatization reagents and the generation of hazardous waste.

  5. Investigation on enantiomeric separations of fluorenylmethoxycarbonyl amino acids and peptides by high-performance liquid chromatography using native cyclodextrins as chiral stationary phases.

    PubMed

    Tang, Y; Zukowski, J; Armstrong, D W

    1996-09-06

    A systematic study was carried out to investigate enantiomeric separations of fluorenylmethoxycarbonyl (FMOC) amino acids and their peptides. Twenty amino acids were derivatized by 9-fluorenylmethyl chloroformate (FMOC-Cl) and its analogues, FMOC-glycyl-Cl and FMOC-beta-alanyl-Cl. All derivatives were chromatographed on native beta- and gamma-cyclodextrin columns using acetonitrile as the main mobile phase component. The results indicated that glycyl and beta-alanyl groups between FMOC and amino acid moieties enhanced chiral selectivities of amino acid derivatives. The addition of modifiers, triethylamine, acetic acid and methanol, into the mobile phase caused alterations in retention, enantiorecognition and elution order. The structures of amino acids and the type of chiral stationary phase employed exhibited significant impacts on chiral resolutions. It is also found that the number and position of glycyl moieties affect the retentions and enantioselectivities of FMOC derivatized glycyl containing peptides.

  6. Development, validation and comparison of two microextraction techniques for the rapid and sensitive determination of pregabalin in urine and pharmaceutical formulations after ethyl chloroformate derivatization followed by gas chromatography-mass spectrometric analysis.

    PubMed

    Mudiam, Mohana Krishna Reddy; Chauhan, Abhishek; Jain, Rajeev; Ch, Ratnasekhar; Fatima, Ghizal; Malhotra, Ekta; Murthy, R C

    2012-11-01

    The present article reports first time the use of solid-phase microextraction (SPME) and dispersive liquid-liquid microextraction (DLLME) to extract pregabalin (PRG) from urine and pharmaceutical formulations followed by GC-MS analysis after ethyl chloroformate (ECF) derivatization. PRG is an antiepileptic and analgesic drug, which is a structural analogue of γ-amino-butyric acid (GABA). It is approved by Food and Drug Administration (FDA) for the treatment of central nervous system (CNS) disorders and neuropathic pain. Initially PRG was derivatized with ECF in the presence of pyridine at room temperature for 30s. Experimental parameters were investigated for derivatization, SPME and DLLME conditions. The limit of detection (LOD) and limit of quantitation (LOQ) were found to be 0.019 μg/ml and 0.063 μg/ml for SPME and 0.022 μg/ml and 0.075 μg/ml for DLLME respectively. The percentage recovery, in case of SPME was in the range of 83-98% while for DLLME it is in the range of 84-98%. The intra and inter-day precisions were found to be less than 6%. The developed methods after ECF derivatization were found to be simple, fast, efficient and inexpensive. DLLME has several advantages like lesser extraction time and cost effectiveness as compared to SPME. The developed methods may find wide application for the routine determination of PRG in biological as well as in quality control samples of pharmaceutical formulations.

  7. Enantio sensing property of helicin, the derivative of a natural product: Discrimination of amines and amino alcohols

    NASA Astrophysics Data System (ADS)

    Kumari, Divya; Sachin, S. L.; Suryaprakash, N.

    2015-09-01

    The chiral sensing property of helicin (the derivative of natural product obtained by partial oxidation of salicin, extracted from willow tree (Salix helix)) is reported. The use of helicin as a chiral derivatizing agent for the discrimination of amines and amino alcohols is convincingly established using 1H NMR spectroscopy. The large chemical shift separation achieved between the discriminated peaks facilitated the accurate quantification of enantiomeric composition. The consistent trend observed in the shifting of imine proton peak (Δδ) of helicin in all the derivatized molecules might aid the determination of spatial configuration.

  8. Preferential Treatment: Interaction Between Amino Acids and Minerals

    NASA Astrophysics Data System (ADS)

    Crapster-Pregont, E. J.; Cleaves, H. J.; Hazen, R. M.

    2008-12-01

    Amino acids are the building blocks of proteins and are important for some models of the origin of life. Polymerization of amino acids from dilute solution is unlikely without a scaffold or catalyst. The surfaces of early Earth minerals are the most likely candidates for this role. The surface adsorption behavior of 12 amino acids (L-alanine, L-serine, L-aspartic acid, L-proline, L- phenylalanine, L-valine, L-arginine, d-amino valeric acid, glycine, L-lysine, L-isoleucine, and B-alanine) on 21 minerals (quartz, calcite, enstatite, illite, olivine, pyrrhotite, pyrite, alkali basalt, albite, analcime, chlorite, barite, hydroxyl apatite, hematite, magnetite, aluminum hydroxide, kaolin, silica gel, corundum, rutile, and montmorillonite) was determined via batch adsorption experiments. Absorption was determined for concentrations between 10-4M and 10-6M in the presence of 0.1M NaCl, and between pH values of 3 and 9 at 25 degrees C. The equilibrated solutions were centrifuged, filtered, derivatized using a fluorescent amino group tag (dansyl-chloride) and analyzed by HPLC. Adsorption was standardized using BET surface area measurements for each mineral to give the number of mols of each amino acid adsorbed per square meter for each mineral. The results indicate an enormous difference in the adsorption of amino acids between minerals, along with major differences in the adsorption of individual amino acids on the same mineral surface. There is also a change in the absorbance of amino acids as the pH changes. Many previous studies of amino acid concentration and catalysis by minerals have used clay minerals because of their high surface areas, however, this data suggests that the surfaces of minerals such as calcite, quartz and pyrite have even higher affinities for amino acids. The results suggest mineral surfaces that could be optimal locations for the polymerization of molecules linked to the origin of life.

  9. Sample preconcentration with chemical derivatization in capillary electrophoresis. Capillary as preconcentrator, microreactor and chiral selector for high-throughput metabolite screening.

    PubMed

    Ptolemy, Adam S; Britz-McKibbin, Philip

    2006-02-17

    New strategies for integrating sample pretreatment with chemical analyses under a single format is required for rapid, sensitive and enantioselective analyses of low abundance metabolites in complex biological samples. Capillary electrophoresis (CE) offers a unique environment for controlling analyte/reagent band dispersion and electromigration properties using discontinuous electrolyte systems. Recent work in our laboratory towards developing a high-throughput CE platform for low abundance metabolites via on-line sample preconcentration with chemical derivatization (SPCD) is primarily examined in this review, as there have been surprisingly only a few strategies reported in the literature to date. In-capillary sample preconcentration serves to enhance concentration sensitivity via electrokinetic focusing of long sample injection volumes for lower detection limits, whereas chemical derivatization by zone passing is used to expand detectability and selectivity, notably for enantiomeric resolution of metabolites lacking intrinsic chromophores using nanolitre volumes of reagent. Together, on-line SPCD-CE can provide over a 100-fold improvement in concentration sensitivity, shorter total analysis times, reduced sample handling and improved reliability for a variety of amino acid and amino sugar metabolites, which is also amenable to automated high-throughput screening. This review will highlight basic method development and optimization parameters relevant to SPCD-CE, including applications to bacterial metabolite flux and biomarker analyses. Insight into the mechanism of analyte focusing and labeling by SPCD-CE is also discussed, as well as future directions for continued research.

  10. Optimizing sample pretreatment for compound-specific stable carbon isotopic analysis of amino sugars in marine sediment

    NASA Astrophysics Data System (ADS)

    Zhu, R.; Lin, Y.-S.; Lipp, J. S.; Meador, T. B.; Hinrichs, K.-U.

    2014-09-01

    Amino sugars are quantitatively significant constituents of soil and marine sediment, but their sources and turnover in environmental samples remain poorly understood. The stable carbon isotopic composition of amino sugars can provide information on the lifestyles of their source organisms and can be monitored during incubations with labeled substrates to estimate the turnover rates of microbial populations. However, until now, such investigation has been carried out only with soil samples, partly because of the much lower abundance of amino sugars in marine environments. We therefore optimized a procedure for compound-specific isotopic analysis of amino sugars in marine sediment, employing gas chromatography-isotope ratio mass spectrometry. The whole procedure consisted of hydrolysis, neutralization, enrichment, and derivatization of amino sugars. Except for the derivatization step, the protocol introduced negligible isotopic fractionation, and the minimum requirement of amino sugar for isotopic analysis was 20 ng, i.e., equivalent to ~8 ng of amino sugar carbon. Compound-specific stable carbon isotopic analysis of amino sugars obtained from marine sediment extracts indicated that glucosamine and galactosamine were mainly derived from organic detritus, whereas muramic acid showed isotopic imprints from indigenous bacterial activities. The δ13C analysis of amino sugars provides a valuable addition to the biomarker-based characterization of microbial metabolism in the deep marine biosphere, which so far has been lipid oriented and biased towards the detection of archaeal signals.

  11. Optimizing sample pretreatment for compound-specific stable carbon isotopic analysis of amino sugars in marine sediment

    NASA Astrophysics Data System (ADS)

    Zhu, R.; Lin, Y.-S.; Lipp, J. S.; Meador, T. B.; Hinrichs, K.-U.

    2014-01-01

    Amino sugars are quantitatively significant constituents of soil and marine sediment, but their sources and turnover in environmental samples remain poorly understood. The stable carbon isotopic composition of amino sugars can provide information on the lifestyles of their source organisms and can be monitored during incubations with labeled substrates to estimate the turnover rates of microbial populations. However, until now, such investigation has been carried out only with soil samples, partly because of the much lower abundance of amino sugars in marine environments. We therefore optimized a procedure for compound-specific isotopic analysis of amino sugars in marine sediment employing gas chromatography-isotope ratio mass spectrometry. The whole procedure consisted of hydrolysis, neutralization, enrichment, and derivatization of amino sugars. Except for the derivatization step, the protocol introduced negligible isotopic fractionation, and the minimum requirement of amino sugar for isotopic analysis was 20 ng, i.e. equivalent to ~ 8 ng of amino sugar carbon. Our results obtained from δ13C analysis of amino sugars in selected marine sediment samples showed that muramic acid had isotopic imprints from indigenous bacterial activities, whereas glucosamine and galactosamine were mainly derived from organic detritus. The analysis of stable carbon isotopic compositions of amino sugars opens a promising window for the investigation of microbial metabolisms in marine sediments and the deep marine biosphere.

  12. Searching for amino-acid homochirality on Mars with the Mars Organic Molecule Analyzer (MOMA) onboard ExoMars

    NASA Astrophysics Data System (ADS)

    Buch, A.; Freissinet, C.; Sternberg, R.; Brault, A.; Szopa, C.; Claude-Geffroy, C.; Coll, P. J.; Grand, N.; Raulin, F.; Pinick, V.; Goesmann, F.

    2012-12-01

    The joint ESA-Roscosmos Exo-Mars-2018 rover mission plans to seek the signs of a past or a present life on Mars. The Mars Organic Molecule Analyzer (MOMA) experiment onboard theExoMars rover will be a key analytical tool in providing molecular information from Mars solid samples, with a specific focus on the characterization of their organic content. In this purpose, one of MOMA's main instruments is a gas chromatograph-mass spectrometer (GC-MS), which provides a unique ability to characterize a broad range of compounds and allow chemical analyses on volatile and refractory species. The challenge with the analysis of this refractory matter embedded in soil is their primary extraction before their analysis by GC-MS. Since the extraction of organic matter is not possible by liquid solvent extraction, we have developed a method based on the thermodesorption and subsequent derivatization of the organic molecules. The goal of the thermodesorption is to extract the organic matter by heating the sample quickly enough not to degrade its organic content. One of the main focuses is to determine the chirality of this organic matter, notably amino acids. Indeed, on Earth, homochirality of molecules is an indicator for the presence of life. Amino acids appear to bear only the left-handed form (L) in living system. However, other refractory compounds can raise interest: nucleobases, carboxylic acids and PAHs are among molecules supported by life as we know it, and all of them can display chirality. The intrinsic chirality of molecules being thermosensitive, the thermodesorption parameters have been adjusted to occur within a range of temperatures from 150 °C to 300 °C over a period of 30 s to 10 min, depending on the chemical compound. Under these conditions, we have shown that amino acids are not degraded and that their chirality is preserved. Once extracted, refractory molecules with labile hydrogens (e.g. amino acids, nucleobases, carboxylic acids, etc.) are derivatized

  13. Accurate determination of aldehydes in amine catalysts or amines by 2,4-dinitrophenylhydrazine derivatization.

    PubMed

    Barman, Bhajendra N

    2014-01-31

    Carbonyl compounds, specifically aldehydes, present in amine catalysts or amines are determined by reversed-phase liquid chromatography using ultraviolet detection of their corresponding 2,4-dinitrophenylhydrazones. The primary focus has been to establish optimum conditions for determining aldehydes accurately because these add exposure concerns when the amine catalysts are used to manufacture polyurethane products. Concentrations of aldehydes determined by this method are found to vary with the pH of the aqueous amine solution and the derivatization time, the latter being problematic when the derivatization reaction proceeds slowly and not to completion in neutral and basic media. Accurate determination of aldehydes in amines through derivatization can be carried out at an effective solution pH of about 2 and with derivatization time of 20min. Hydrochloric acid has been used for neutralization of an amine. For complete derivatization, it is essential to protonate all nitrogen atoms in the amine. An approach for the determination of an adequate amount of acid needed for complete derivatization has been described. Several 0.2M buffer solutions varying in pH from 4 to 8 have also been used to make amine solutions for carrying out derivatization of aldehydes. These solutions have effective pHs of 10 or higher and provide much lower aldehyde concentrations compared to their true values. Mechanisms for the formation of 2,4-dinitrophenylhydrazones in both acidic and basic media are discussed.

  14. Determination of amphetamines in hair by integrating sample disruption, clean-up and solid phase derivatization.

    PubMed

    Argente-García, A; Moliner-Martínez, Y; Campíns-Falcó, P; Verdú-Andrés, J; Herráez-Hernández, R

    2016-05-20

    The utility of matrix solid phase dispersion (MSPD) for the direct analysis of amphetamines in hair samples has been evaluated, using liquid chromatography (LC) with fluorescence detection and precolumn derivatization. The proposed approach is based on the employment of MSPD for matrix disruption and clean-up, followed by the derivatization of the analytes onto the dispersant-sample blend. The fluorogenic reagent 9-fluorenylmethyl chloroformate (FMOC) has been used for derivatization. Different conditions for MSPD, analyte purification and solid phase derivatization have been tested, using amphetamine (AMP), methamphetamine (MET), ephedrine (EPE) and 3,4-methylenedioxymethamphetamine (MDMA) as model compounds. The results have been compared with those achieved by using ultrasound-assisted alkaline digestion and by MSPD combined with conventional solution derivatization. On the basis of the results obtained, a methodology is proposed for the analysis of amphetamines in hair which integrates sample disruption, clean-up and derivatization using a C18 phase. Improved sensitivity is achieved with respect to that obtained by the alkaline digestion or by the MSPD followed by solution derivatization methods. The method can be used for the quantification of the tested amphetamines within the 2.0-20.0ng/mg concentration interval, with limits of detection (LODs) of 0.25-0.75ng/mg. The methodology is very simple and rapid (the preparation of the sample takes less than 15min).

  15. In situ search for organics by gas chromatography analysis: new derivatization / thermochemolysis approach

    NASA Astrophysics Data System (ADS)

    Geffroy, Claude; Buch, Arnaud; David, Marc; Aissat, Lyes; El Mufleh, Amel; Papot, S.; Sternberg, Robert

    Many organic molecules are present in interstellar clouds and might be carried to the early Earth by comets and meteorites during the heavy bombardment phase in the first few hundred million years of the solar system. It has been suggested that extraterrestrial organic material may well represent an important part of the organic material available for the origin of life. Until samples, brought by future space missions, are available on Earth, in situ measurements are one of the way to get unaltered and non-contaminated samples for analysis. The analytical technique has to be robust, sensitive and non-specific due to the large scope of targets molecules. The only currently flight qualified technique of analysis of organic molecules in space is gas chromatography (Viking, Cassini-Huygens, SAM-MSL, COSAC-Rosetta). The main objective of this work is to present a new approach with multi step analysis using derivatisation and thermochemolysis reagents for a one pot in situ analysis of volatile and refractory organics in surface or sub-surface samples (Mars, comets).Indeed, no single technology enables to identify all organic compounds because naturally occurring molecules have different polarities, molecular weights, being extractible or recalcitrant, bonded trapped or adsorbed on minerals. Thus, we propose to wider the scope of chemical reagent already validated for in situ wet chemistry such as MTBSTFA (Rodier et al. 2001, 2002), DMF-DMA (Rodier et al. 2002), or TMAH (Rodier et al, 2005, Geffroy-Rodier et al; 2009) to analyze enantiomers of amino acids, carbohydrates and lipids in a one pot several steps sub system using a multi reagent and multi step approach. Thus using a new derivatizing agent, we successfully identified twenty one amino acids including twelve of the twenty proteinic amino acids without inhibiting following multi step thermochemolysis. *Geffroy-Rodier C, Grasset L, Sternberg R. Buch A. Amblès A. (2009) Thermochemolysis in search for organics in

  16. Quantitative enantioseparation of amino acids by comprehensive two-dimensional gas chromatography applied to non-terrestrial samples.

    PubMed

    Myrgorodska, Iuliia; Meinert, Cornelia; Martins, Zita; le Sergeant d'Hendecourt, Louis; Meierhenrich, Uwe J

    2016-02-12

    This work presents an improved analytical procedure for the resolution and quantification of amino acid enantiomers by multidimensional gas chromatography. The procedure contains a derivatization step, by which amino acids were transformed into N(O,S)-ethoxycarbonylheptafluorobutyl esters. It was optimized for the resolution of non-proteinogenic amino acids in the matrix of complex non-terrestrial samples. The procedure has proven to be highly sensitive and shows a wide linearity range with 0.005-3 pmol detection limits for quantitative determinations. The developed procedure was tested on a sample of the Murchison meteorite, for which obtained chromatograms show excellent peak resolution, minimal co-elution and peak overlap. We conclude that comprehensive two dimensional chromatography, in combination with the optimized derivatization method is a highly suitable technique for the analysis of samples with very limited quantities and containing potentially prebiotic molecules, such as interstellar ice analogs and meteorites.

  17. Investigation of thiol derivatized gold nanoparticle sensors for gas analysis

    NASA Astrophysics Data System (ADS)

    Stephens, Jared S.

    Analysis of volatile organic compounds (VOCs) in air and exhaled breath by sensor array is a very useful testing technique. It can provide non-invasive, fast, inexpensive testing for many diseases. Breath analysis has been very successful in identifying cancer and other diseases by using a chemiresistor sensor or array with gold nanoparticles to detect biomarkers. Acetone is a biomarker for diabetes and having a portable testing device could help to monitor diabetic and therapeutic progress. An advantage to this testing method is it is conducted at room temperature instead of 200 degrees Celsius. 3. The objective of this research is to determine the effect of thiol derivatized gold nanoparticles based on sensor(s) detection of VOCs. The VOCs to be tested are acetone, ethanol, and a mixture of acetone and ethanol. Each chip is tested under all three VOCs and three concentration levels (0.1, 1, and 5.0 ppm). VOC samples are used to test the sensors' ability to detect and differentiate VOCs. Sensors (also referred to as a chip) are prepared using several types of thiol derivatized gold nanoparticles. The factors are: thiol compound and molar volume loading of the thiol in synthesis. The average resistance results are used to determine the VOC selectivity of the sensors tested. The results show a trend of increasing resistance as VOC concentration is increased relative to dry air; which is used as baseline for VOCs. Several sensors show a high selectivity to one or more VOCs. Overall the 57 micromoles of 4-methoxy-toluenethiol sensor shows the strongest selectivity for VOCs tested. 3. Gerfen, Kurt. 2012. Detection of Acetone in Air Using Silver Ion Exchanged ZSM-5 and Zinc Oxide Sensing Films. Master of Science thesis, University of Louisville.

  18. Accelerated on-column lysine derivatization and cysteine methylation by imidazole reaction in a deuterated environment for enhanced product ion analysis.

    PubMed

    Cindrić, Mario; Cepo, Tina; Skrlin, Ana; Vuletić, Marko; Bindila, Laura

    2006-01-01

    The combination of separation techniques and mass spectrometry (MS) for peptide investigation allows superior sensitivity of detection and richer fragmentation data than available by direct MS analysis of a complex mixture. In this regard, liquid chromatography (LC) coupled to electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) MS have evolved as versatile analytical tools in proteomics. Very often, however, the product ion mass spectrum is either incomplete or overfilled with ions, thus making sequence analysis difficult. Here we report overall ion intensity improvement of C-terminal lysine-containing peptides from Lys-C digest by on-column derivatization of lysines with 2-methoxy-4,5-dihydro-1H-imidazole. The method is simple, fast and exhibits 100% efficiency of the reaction. Additionally, post-source decay carried out on derivatized peptides gave rise almost exclusively to y-series ion formation, at 100% sequence coverage and high intensity. The novelty of the method resides in the side reaction of this derivatization process, namely the methylation of cysteines. This facilitates the estimation of the disulfide bridge position in a protein and the fragmentation of cysteine-containing peptide fragments. Additionally, by using this derivatization procedure, the loss of peptides, their degradation and/or oxidation, usually occurring in digest alkylation procedures, is greatly minimized. The new on-column derivatization protocol is designed to be carried out on C18 Spin Tubes or Cleanup C18 Pipette Tips. We observed that use of buffered D2O solvent prevented unwanted oxidation and degradation reactions with respect to the stationary phase. This may be due to the fact that a deuteron is less polar than a proton, and thus the bonded silica stationary phase saturated with deuterons does not affect the reaction between epsilon-amino or cysteine thiol groups and 2-methoxy-4,5-dihydro-1H-imidazole. Complete tagging of the peptides by on

  19. Derivatization reagents in liquid chromatography/electrospray ionization tandem mass spectrometry.

    PubMed

    Santa, Tomofumi

    2011-01-01

    Liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) is one of the most prominent analytical techniques owing to its inherent selectivity and sensitivity. In LC/ESI-MS/MS, chemical derivatization is often used to enhance the detection sensitivity. Derivatization improves the chromatographic separation, and enhances the mass spectrometric ionization efficiency and MS/MS detectability. In this review, an overview of the derivatization reagents which have been applied to LC/ESI-MS/MS is presented, focusing on the applications to low molecular weight compounds.

  20. Study of the matrix effects and sample dilution influence on the LC-ESI-MS/MS analysis using four derivatization reagents.

    PubMed

    Oldekop, Maarja-Liisa; Herodes, Koit; Rebane, Riin

    2014-09-15

    For liquid chromatographic analysis of amino acids involving derivatization and mass-spectrometric detection, it becomes more important to evaluate the presence of matrix effects in complex samples. This is somewhat complicated for amino acid analysis where analyte free sample matrix is often unavailable. In this work, matrix effects were investigated using post-column infusion method for 9-fluorenylmethyl chloroformate (FMOC-Cl) derivatives of β-Ala, Gly and Phe and diethyl ethoxymethylenemalonate (DEEMM) derivative of β-Ala. While for DEEMM derivatives, the main signal suppression was due to the borate buffer, in case of FMOC-Cl, other FMOC-derivatives caused signal suppression. Analysis of amino acids in tea and honey with DEEMM, FMOC-Cl, p-N,N,N-trimethylammonioanilyl N'-hydroxysuccinimidyl carbamate iodide (TAHS) and dansyl chloride (DNS) showed that amino acid concentrations found with different reagents do not agree well. Sample dilution experiments indicated that the sample matrix affected the analysis results obtained with DEEMM the least, but with FMOC-Cl, TAHS and DNS, sample dilution had an influence on the results. When sample dilution and extrapolative dilution approach were applied on the latter results, an agreement of amino acid concentrations measured with different reagents was achieved within relative standard deviation (RSD) of 22% for most cases.

  1. On-line sample preconcentration by sweeping and poly(ethylene oxide)-mediated stacking for simultaneous analysis of nine pairs of amino acid enantiomers in capillary electrophoresis.

    PubMed

    Lin, En-Ping; Lin, Kai-Cheng; Chang, Chia-Wei; Hsieh, Ming-Mu

    2013-09-30

    This study proposes a sensitive method for the simultaneous separation and concentration of 9 pairs of amino acid enantiomers by combining poly(ethylene oxide) (PEO)-based stacking, β-cyclodextrin (β-CD)-mediated micellar electrokinetic chromatography (MEKC), and 9-fluoroenylmethyl chloroformate (FMOC) derivatization. The 9 pairs of FMOC-derivatized amino acid enantiomers were baseline separated using a discontinuous system, and the buffer vials contained a solution of 150 mM Tris-borate (TB), 12.5% (v/v) isopropanol (IPA), 0.5% (w/v) PEO, 35 mM sodium taurodeoxycholate (STDC), and 35 mM β-CD, and the capillary was filled with a solution of 1.5 M TB, 12.5% (v/v) IPA, 35 mM STDC, and 35 mM β-CD. Based on the difference in viscosity between the sample zone and PEO solution and because of the STDC sweeping, the discontinuous system effectively stacked 670 nL of the 9 pairs of FMOC-derivatized amino acid enantiomers without losing chiral resolution. Consequently, the limits of detection for the 9 pairs of FMOC-derivatized amino acid enantiomers were reduced to 40-60 nM. This method was successfully used to determine d-Tryptophan (Trp), l-Trp, d-Phenylalanine (Phe), l-Phe, d-Glutamic acid (Glu), and l-Glu in various types of beers.

  2. Autistic children exhibit distinct plasma amino acid profile.

    PubMed

    Naushad, Shaik Mohammad; Jain, Jamal Md Nurul; Prasad, Chintakindi Krishna; Naik, Usha; Akella, Radha Rama Devi

    2013-10-01

    In order to ascertain whether autistic children display characteristic metabolic signatures that are of diagnostic value, plasma amino acid analyses were carried out on a cohort of 138 autistic children and 138 normal controls using reverse-phase HPLC. Pre-column derivatization of amino acids with phenyl isothiocyanate forms phenyl thio-carbamate derivates that have a lamba(max) of 254 nm, enabling their detection using photodiode array. Autistic children showed elevated levels of glutamic acid (120 +/- 89 vs. 83 +/- 35 micromol/L) and asparagine (85 +/- 37 vs. 47 +/- 19 micromol/L); lower levels of phenylalanine (45 +/- 20 vs. 59 +/- 18 micromol/L), tryptophan (24 +/- 11 vs. 41 +/- 16 micromol/L), methionine (22 +/- 9 vs. 28 +/- 9 micromol/L) and histidine (45 +/- 21 vs. 58 +/- 15 micromol/L). A low molar ratio of (tryptophan/large neutral amino acids) x 100 was observed in autism (5.4 vs 9.2), indicating lesser availability of tryptophan for neurotransmitter serotonin synthesis. To conclude, elevated levels of excitatory amino acids (glutamate and asparagine), decreased essential amino acids (phenylalanine, tryptophan and methionine) and decreased precursors of neurotransmitters (tyrosine and tryptophan) are the distinct characteristics of plasma amino acid profile of autistic children. Thus, such metabolic signatures might be useful tools for early diagnosis of autism.

  3. Determination of BMAA and three alkaloid cyanotoxins in lake water using dansyl chloride derivatization and high-resolution mass spectrometry.

    PubMed

    Roy-Lachapelle, Audrey; Solliec, Morgan; Sauvé, Sébastien

    2015-07-01

    A new analytical method was developed for the detection of alkaloid cyanotoxins in harmful algal blooms. The detection of the nonproteinogenic amino acid β-N-methylamino-L-alanine (BMAA) and two of its conformation isomers, 2,4-diaminobutyric acid (DAB) and N-(2-aminoethyl) glycine (AEG), as well as three alkaloid cyanotoxins, anatoxin-a (ANA-a), cylindrospermopsin (CYN), and saxitoxin (STX), is presented. The use of a chemical derivatization with dansyl chloride (DNS) allows easier separation with reversed phase liquid chromatography. Detection with high-resolution mass spectrometry (HRMS) with the Q-Exactive enables high selectivity with specific fragmentation as well as exact mass detection, reducing considerably the possibilities of isobaric interferences. Previous to analysis, a solid phase extraction (SPE) step is used for purification and preconcentration. After DNS derivatization, samples are submitted to ultra high-performance liquid chromatography coupled with heated electrospray ionisation and the Q-Exactive mass spectrometer (UHPLC-HESI-HRMS). With an internal calibration using isotopically-labeled DAB-D3, the method was validated with good linearity (R (2)  > 0.998), and method limits of detection and quantification (MLD and MLQ) for target compounds ranged from 0.007 to 0.01 μg L(-1) and from 0.02 to 0.04 μg L(-1), respectively. Accuracy and within-day/between-days variation coefficients were below 15%. SPE recovery values ranged between 86 and 103%, and matrix effects recovery values ranged between 75 and 96%. The developed analytical method was successfully validated with 12 different lakes samples, and concentrations were found ranging between 0.009 and 0.3 μg L(-1) except for STX which was not found in any sample.

  4. High performance liquid chromatography for the determination of glucosamine sulfate in human plasma after derivatization with 9-fluorenylmethyl chloroformate.

    PubMed

    Huang, Tao-min; Deng, Chun-Hui; Chen, Nian-zhu; Liu, Zhen; Duan, Geng-Li

    2006-10-01

    In this study, we developed a simple, rapid, sensitive, and reliable method for the determination of glucosamine sulfate in human plasma, which was based on derivatization with 9-fluorenylmethyl chloroformate (FMOC-Cl) followed by reverse-phase HPLC-FLD. For the first time, FMOC-Cl was introduced into derivatization of glucosamine sulfate in human plasma. The amino groups of glucosamine sulfate and vertilmicin sulfate (the internal standard) were trapped with FMOC-Cl to form glucosamine-FMOC-Cl and vertilmicin-FMOC-Cl adducts, which can be very suitable for HPLC-FLD. Precipitation of plasma proteins by acetonitrile was followed by vortex mixing and centrifugation. Chromatographic separation was performed on a C18 column (DIAMONSIL 150 x 4 mm id, 5 microm) with a mobile phase gradient consisting of acetonitrile and water at a flow-rate of 1 mL/min. The retention times of glucosamine-FMOC-Cl and vertilmicin-FMOC-Cl adducts were 8.9 and 21.2 min, respectively. This method was shown to be selective and sensitive for glucosamine sulfate. The limit of detection was 15 ng/mL for glucosamine sulfate in plasma and the linear range was 0.1-10 mg/mL in plasma with a correlation coefficient (r) of 0.9999. The relative standard deviations (RSDs) of intra-day and inter-day assays were 5.2-8.1% and 6.1- 8.5%, respectively. Extraction recoveries of glucosamine sulfate in plasma were greater than 90%. The validated method was successfully applied to the determination of glucosamine sulfate in human plasma samples.

  5. Analysis of Theanine in Tea (Camellia sinensis) Dietary Ingredients and Supplements by High-Performance Liquid Chromatography with Postcolumn Derivatization: Single-Laboratory Validation, First Action 2016.10.

    PubMed

    Ofitserova, Maria; Nerkar, Sareeta

    2016-11-01

    An HPLC method with postcolumn derivatization was developed and validated for the determination of theanine content in tea dietary ingredients and supplements. A variety of common commercially available supplement forms such as powders, liquid tinctures, tablets, softgels, and gelcaps, as well as three National Institute of Standards and Technology Camellia sinensis Standard Reference Materials were investigated in the study. A simple extraction procedure using citrate buffer at pH 2.2 allowed for the analysis of theanine without additional cleanup or concentration steps, even at low ppm levels. Theanine was separated from other naturally occurring amino acids using a cation-exchange column and detected using a UV-Vis detector after derivatization with ninhydrin reagent. A single-laboratory validation demonstrated that specificity, accuracy, precision, and other method performance parameters have met the requirements set for theanine analysis by the AOAC Stakeholder Panel on Dietary Supplements.

  6. General approach for introduction of various chemical labels in specific RNA locations based on insertion of amino linkers.

    PubMed

    Graifer, Dmitri; Karpova, Galina

    2013-11-25

    Introduction of reporter groups at designed RNA sites is a widely accepted approach to gain information about the molecular environment of RNAs in their complexes with other biopolymers formed during various cellular processes. A general approach to obtain RNAs bearing diverse reporter groups at designed locations is based on site-specific insertion of groups containing primary aliphatic amine functions (amino linkers) with their subsequent selective derivatization by appropriate chemicals. This article is a brief review on methods for site-specific introduction of amino linkers in different RNAs. These methods comprise: (i) incorporation of a nucleoside carrying an amino-linker or a function that can be substituted with it into oligoribonucleotides in the course of their chemical synthesis; (ii) assembly of amino linker-containing RNAs from short synthetic fragments via their ligation; (iii) synthesis of amino linker-modified RNAs using T7 RNA polymerase; (iv) insertion of amino linkers into unmodified RNAs at functional groups of a certain type such as the 5'-phosphates and N7 of guanosine residues and (v) introduction of an amino linker into long highly structured RNAs exploiting an approach based on sequence-specific modification of nucleic acids. Particular reporter groups used for derivatization of amino linker-containing RNAs together with types of RNA derivatives obtained and fields of their application are presented.

  7. Analysis of underivatized amino acids in geological samples using ion-pairing liquid chromatography and electrospray tandem mass spectrometry.

    PubMed

    Liu, De-Ling; Beegle, Luther W; Kanik, Isik

    2008-04-01

    The capability of detecting biomarkers, such as amino acids, in chemically complex field samples is essential to establishing the knowledge required to search for chemical signatures of life in future planetary explorations. However, due to the complexities of in situ investigations, it is important to establish a new analytical scheme that utilizes a minimal amount of sample preparation. This paper reports the feasibility of a novel and sensitive technique, which has been established to quantitate amino acids in terrestrial crust samples directly without derivatization using volatile ion-pairing liquid chromatography and tandem mass spectrometry equipped with an electrospray ionization source. Adequate separation of 20 underivatized amino acids was achieved on a C(18) capillary column within 26 min with nonafluoropentanoic acid (NFPA) as ion-pairing reagent. Each amino acid was identified from its retention time as well as from its characteristic parent-to-daughter ion transition. Using tandem mass spectrometry as a detection technique allows co-elution of some amino acids, as it is more specific than traditional spectrophotometric methods. In the present study, terrestrial samples collected from 3 different locations were analyzed for their water-extractable free amino acid contents, following the removal of metal and organic interferences via ion exchange procedures. This is the first time that amino acids in geological samples were directly determined quantitatively without complicated derivatization steps. Depending on the amino acid, the detection limits varied from 0.02 to 5.7 pmol with the use of a 1 microl sample injection loop.

  8. 15N and13C NMR investigation of hydroxylamine-derivatized humic substances

    USGS Publications Warehouse

    Thorn, K.A.; Arterburn, J.B.; Mikita, M.A.

    1992-01-01

    Five fulvic and humic acid samples of diverse origins were derivatized with 15N-labeled hydroxylamine and analyzed by liquid-phase 15N NMR spectrometry. The 15N NMR spectra indicated that hydroxylamine reacted similarly with all samples and could discriminate among carbonyl functional groups. Oximes were the major derivatives; resonances attributable to hydroxamic acids, the reaction products of hydroxylamine with esters, and resonances attributable to the tautomeric equilibrium position between the nitrosophenol and monoxime derivatives of quinones, the first direct spectroscopic evidence for quinones, also were evident. The 15N NMR spectra also suggested the presence of nitriles, oxazoles, oxazolines, isocyanides, amides, and lactams, which may all be explained in terms of Beckmann reactions of the initial oxime derivatives. INEPT and ACOUSTIC 15N NMR spectra provided complementary information on the derivatized samples. 13C NMR spectra of derivatized samples indicated that the ketone/quinone functionality is incompletely derivatized with hydroxylamine. ?? 1991 American Chemical Society.

  9. Simple determination of L-hydroxyproline in idiopathic pulmonary fibrosis lung tissues of rats using non-extractive high-performance liquid chromatography coupled with fluorescence detection after pre-column derivatization with novel synthetic 9-acetylimidazol-carbazole.

    PubMed

    Ren, Yan; Zhao, Juanjuan; Shi, Yanan; Chen, Caiyun; Chen, Xiangming; Lv, Changjun

    2017-08-05

    L-Hydroxyproline (L-Hyp) is an important biomarker for idiopathic pulmonary fibrosis (IPF). The quantitative methods based on high-performance liquid chromatography coupled with fluorescence detection after pre-column derivatization typically requires complicated derivatization conditions and obtains unstable derivatives. Here, a novel derivatization reagent, 9-acetylimidazol-carbazole, was synthesized for the first time to efficiently and rapidly label the amino groups of L-Hyp. The high-performance liquid chromatography method with pre-column derivatization was performed on an Agilent ZORBAX SB-C18 column (4.6×250mm, 5μm). The product was measured using fluorescence detection at excitation and emission wavelengths of 232 and 370nm, respectively. The method was validated in specificity, linearity, limit of detection (66.7 fmol), limit of quantification (333.3fmol), intra-day precision (0.75%), inter-day precision (3.82%), stability (3.15%), and recovery (90.7-109.4%). The validated method was successfully applied to the determination of L-Hyp in the lung tissues of healthy and IPF rats. The results showed that the concentration of L-Hyp (3.64mg/g) in the IPF model was significantly higher than the concentration (2.33mg/g) in the healthy control group with P<0.01. This is a new method for the determination of L-Hyp and can be used for other amino acid-related studies in the future. Copyright © 2017. Published by Elsevier B.V.

  10. Effect of carbohydrates attached to polystyrene on hepatocyte morphology on sugar-derivatized polystyrene matrices.

    PubMed

    Kim, Sang-Heon; Hoshiba, Takashi; Akaike, Toshihiro

    2003-12-15

    Sugar-carrying polymers have been utilized as artificial matrices for cell adhesion in tissue engineering. We have developed sugar-derivatized polystyrenes (PV-sugars) as artificial matrices, which control hepatocyte adhesion and hepatic function. Hepatocytes adhere to PV-sugar matrices in a receptor-mediated manner. In this study, we designed a new galactose-derivatized PV-sugar, poly-(6-O-p-vinylbenzyl-alpha-D-galactose) (PV6Gal) and evaluated the role of carbohydrate attached to polystyrene (PS) backbone in the morphological difference of hepatocyte cultured on PV-sugar matrices. Hepatocytes spread on monosaccharide-derivatized PV-sugars but not on disaccharide-derivatized PV-sugars. The actin filament remained aggregated in the central area of the cell body on disaccharide-derivatized PV-sugars. Hepatocyte cell bodies fully were spread on collagen, and the actin filament was almost completely reorganized. Hepatocyte spreading on monosaccharide-derivatized PV-sugars, however, was caused by protrusive cell-matrix contact like lamellipodia and the actin filament was not completely reorganized. This indicated that hepatocyte spreading on PV-sugar matrices was restricted compared with ECM-mediated cell spreading. In addition, typical spheroid formation of hepatocytes was promoted on disaccharide-derivatized PV-sugars compared with monosaccharide-derivatized PV-sugars. Although hepatocytes adhered with different affinities to PV-sugar matrices, hepatocyte morphology was not affected by the adhesion affinity. We suggest that the type of carbohydrate attached to the PS backbone governs the morphology of hepatocyte cultured on PV-sugar matrices.

  11. Microwave-accelerated derivatization prior to GC-MS determination of sex hormones.

    PubMed

    Xu, Xu; Zhao, Xin; Zhang, Yupu; Li, Dan; Su, Rui; Yang, Qiuling; Li, Xueyuan; Zhang, Huihui; Zhang, Hanqi; Wang, Ziming

    2011-06-01

    A new microwave-accelerated derivatization method was developed for rapid determination of 13 natural sex hormones in feeds. Sex hormones were isolated from the sample matrix by ultrasonic extraction, followed by solid-phase extraction, derivatized under microwave irradiation, and then analyzed directly by gas chromatography-mass spectrometry (GC-MS) in selective ion monitoring (SIM) mode. The key parameters affecting derivatization efficiency, including microwave irradiation time, microwave power, and reaction solvent were studied. Under microwave power of 360 W and microwave irradiation for 3 min, 13 natural sex hormones were simultaneously derivatized using heptafluorobutyric acid anhydride (HFBA) as derivatization reagent. This method was applied to the determination of 13 natural sex hormones in different feed samples, and the obtained results were compared with those obtained by the traditional thermal derivatization. The recoveries from 58.1 to 111% were obtained at sex hormone concentrations of 10-300 μg/kg with RSDs ≤12.0%. The results showed that the proposed method was fast, simple, efficient and can be applied to the determination of 13 natural sex hormones in different feed samples.

  12. Highly selective detection of histidine using o-phthaldialdehyde derivatization after the removal of aminothiols through Tween 20-capped gold nanoparticles.

    PubMed

    Huang, Chia-Chi; Tseng, Wei-Lung

    2009-08-01

    In this paper, we describe a simple and sensitive method for the selective detection of histidine by combining Tween 20-capped gold nanoparticles (Tween 20-AuNPs) as aminothiol removers and o-phthaldialdehyde (OPA) as the derivatization reagent. We have shown that Tween 20-AuNPs are capable of removing homocysteine (HCys), glutathione (GSH), and gamma-glutamylcysteine (Glu-Cys) at low pH conditions, but they are ineffective in the case of removal of histidine. In contrast, at high pH, Tween 20-AuNPs have strong hydrophobic interactions with the unprotonated imidazole group of histidine. It is observed that 48.0 nM Tween 20-AuNPs can remove 95.7% of HCys, 99.7% of GSH, and 99.5% of Glu-Cys from 40 mM phosphate solution at pH 2.0 in the presence of 0.1 mM cetyltrimethylammonium bromide, whereas only 2.1% of histidine was removed under identical conditions. In addition, OPA is a highly selective fluorogenic reagent for GSH, HCys, Glu-Cys, and histidine. Thus, after the centrifugation of a solution containing Tween 20-AuNPs, histidine, HCys GSH, Glu-Cys, and other amino acids, the selectivity of this method is remarkably high for histidine through OPA derivatization. Under optimum derivatization conditions, the lowest detectable concentration of histidine detected with this method was 5.2 nM. This method has been successfully applied to detect the presence of histidine in urine and serum samples.

  13. Analysis of amino acids and carbohydrates in green coffee.

    PubMed

    Murkovic, Michael; Derler, Karin

    2006-11-30

    The analysis of carbohydrates and amino acids in green coffee is of the utmost importance since these two classes of compounds act as precursors of the Maillard reaction during which the colour and aroma are formed. During the course of the Maillard reaction potentially harmful substances like acrylamide or 5-hydroxymethyl-furfural accrue as well. The carbohydrates were analysed by anion-exchange chromatography with pulsed amperometric detection and the amino acids by reversed phase chromatography after derivatization with 6-amino-quinolyl-N-hydroxysuccinimidyl carbamate and fluorescence detection. Both methods had to be optimized to obtain a sufficient resolution of the analytes for identification and quantification. Sucrose is the dominant carbohydrate in green coffee with a concentration of up to 90 mg/g (mean = 73 mg/g) in arabica beans and significantly lower amounts in robusta beans (mean=45 mg/g). Alanine is the amino acid with the highest concentration (mean = 1200 microg/g) followed by asparagine (mean = 680 microg/g) in robusta and 800 microg/g and 360 microg/g in arabica respectively. In general, the concentration of amino acids is higher in robusta than in arabica.

  14. Amino Acid Metabolism Disorders

    MedlinePlus

    ... this process. One group of these disorders is amino acid metabolism disorders. They include phenylketonuria (PKU) and maple syrup urine disease. Amino acids are "building blocks" that join together to form ...

  15. Development of a liquid chromatography-tandem mass spectrometry method for quantitative analysis of trace d-amino acids.

    PubMed

    Nakano, Yosuke; Konya, Yutaka; Taniguchi, Moyu; Fukusaki, Eiichiro

    2017-01-01

    d-Amino acids have recently attracted much attention in various research fields including medical, clinical and food industry due to their important biological functions that differ from l-amino acid. Most chiral amino acid separation techniques require complicated derivatization procedures in order to achieve the desirable chromatographic behavior and detectability. Thus, the aim of this research is to develop a highly sensitive analytical method for the enantioseparation of chiral amino acids without any derivatization process using liquid chromatography-tandem mass spectrometry (LC-MS/MS). By optimizing MS/MS parameters, we established a quantification method that allowed the simultaneous analysis of 18 d-amino acids with high sensitivity and reproducibility. Additionally, we applied the method to food sample (vinegar) for the validation, and successfully quantified trace levels of d-amino acids in samples. These results demonstrated the applicability and feasibility of the LC-MS/MS method as a novel, effective tool for d-amino acid measurement in various biological samples. Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  16. Measurement of δ13C values of soil amino acids by GC-C-IRMS using trimethylsilylation: a critical assessment.

    PubMed

    Rubino, Mauro; Milin, Sylvie; D'Onofrio, Antonio; Signoret, Patrick; Hatté, Christine; Balesdent, Jérôme

    2014-01-01

    In this study, we evaluated trimethylsilyl (TMS) derivatives as derivatization reagents for the compound-specific stable carbon isotope analysis of soil amino acids by gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS). We used non-proteinogenic amino acids to show that the extraction-derivatization-analysis procedure provides a reliable method to measure δ(13)C values of amino acids extracted from soil. However, we found a number of drawbacks that significantly increase the final total uncertainty. These include the following: production of multiple peaks for each amino acid, identified as di-, tri- and tetra-TMS derivatives; a number of TMS-carbon (TMS-C) atoms added lower than the stoichiometric one, possibly due to incomplete combustion; different TMS-C δ(13)C for di-, tri- and tetra-TMS derivatives. For soil samples, only four amino acids (leucine, valine, threonine and serine) provide reliable δ(13)C values with a total average uncertainty of 1.3 ‰. We conclude that trimethylsilyl derivatives are only suitable for determining the (13)C incorporation in amino acids within experiments using (13)C-labelled tracers but cannot be applied for amino acids with natural carbon isotope abundance until the drawbacks described here are overcome and the measured total uncertainty significantly decreased.

  17. Proteins and Amino Acids

    USDA-ARS?s Scientific Manuscript database

    Proteins are the most abundant substances in living organisms and cells. All proteins are constructed from the same twenty amino acids that are linked together by covalent bonds. Shorter chains of two or more amino acids can be linked by covalent bonds to form polypeptides. There are twenty amino...

  18. A microfluidic device for the automated derivatization of free fatty acids to fatty acid methyl esters.

    PubMed

    Duong, Cindy T; Roper, Michael G

    2012-02-21

    Free fatty acid (FFA) compositions are examined in feedstock for biodiesel production, as source-specific markers in soil, and because of their role in cellular signaling. However, sample preparation of FFAs for gas chromatography-mass spectrometry (GC-MS) analysis can be time and labor intensive. Therefore, to increase sample preparation throughput, a glass microfluidic device was developed to automate derivatization of FFAs to fatty acid methyl esters (FAMEs). FFAs were delivered to one input of the device and methanolic-HCl was delivered to a second input. FAME products were produced as the reagents traversed a 29 μL reaction channel held at 55 °C. A Design of Experiment protocol was used to determine the combination of derivatization time (T(der)) and ratio of methanolic-HCl:FFA (R(der)) that maximized the derivatization efficiencies of tridecanoic acid and stearic acid to their methyl ester forms. The combination of T(der) = 0.8 min and R(der) = 4.9 that produced optimal derivatization conditions for both FFAs within a 5 min total sample preparation time was determined. This combination of T(der) and R(der) was used to derivatize 12 FFAs with a range of derivatization efficiencies from 18% to 93% with efficiencies of 61% for tridecanoic acid and 84% for stearic acid. As compared to a conventional macroscale derivatization of FFA to FAME, the microfluidic device decreased the volume of methanolic-HCl and FFA by 20- and 1300-fold, respectively. The developed microfluidic device can be used for automated preparation of FAMEs to analyze the FFA compositions of volume-limited samples.

  19. Evaluation of multistep derivatization methods for identification and quantification of oxygenated species in organic aerosol.

    PubMed

    Flores, Rosa M; Doskey, Paul V

    2015-10-30

    Two, 3-step methods for derivatizing mono- and multi-functional species with carbonyl (CO), carboxylic acid (-COOH), and alcohol (-OH) moieties were compared and optimized. In Method 1, the CO, -COOH, and -OH moieties were converted (1) to methyloximes (R-CN-OCH3) with O-methylhydroxylamine hydrochloride (MHA), (2) to methyl esters (OC-R-OCH3) with (trimethylsilyl)diazomethane in methanol (TMSD/MeOH), and (3) to trimethylsilyl ethers [R-OSi(CH3)3] with N,O-bis(trimethylsilyl)-trifluoroacetamide (BSTFA) containing 1% trimethylchlorosilane (TMCS), respectively. Steps 1 and 3 of both methods were identical; however, in Step 2 of Method 2, -COOH moieties were derivatized with 10% (v/v) boron trifluoride (BF3) in MeOH or n-butanol (n-BuOH). The BF3/MeOH and BF3/n-BuOH were ineffective at converting species with more than 2-OH moieties. Average standard deviations for derivatization of 36 model compounds by the 3-step methods using TMSD/MeOH and BF3/(MeOH) were 7.4 and 14.8%, respectively. Average derivatization efficiencies for Methods 1 and 2 were 88.0 and 114%, respectively. Despite the lower average derivatization efficiency of Method 1, distinct advantages included a greater certainty of derivatization yield for the entire suite of mono- and multi-functional species and fewer processing steps for sequential derivatization. Detection limits for Method 1 using GC×GC-ToF-MS were 0.3-54pgm(-3). Approximately 100 oxygenated organic species were identified and quantified in aerosol filtered from 39m(3) of air in an urban location. Levels of species were 0.013-17ngm(-3) and were nearly all above the Method 1 limit of detection.

  20. Evaporative Derivatization of Phenols with 2-Sulfobenzoic Anhydride for Detection by MALDI-MS

    PubMed Central

    Yao, Yuanyuan; Wang, Poguang; Giese, Roger

    2014-01-01

    RATIONALE Phenols are an important class of analytes, for example as bioactive environmental contaminants. Towards a goal of improving their detection by MALDI-TOF-MS or MALDI-TOF/TOF-MS, we studied their derivatization with 2-sulfobenzoic anhydride (SBA). We chose SBA for this purpose since it is commercially available, inexpensive, and forms an anionic derivative. METHODS In selected conditions developed here for phenols, a reaction mixture of one or more of such compounds in acetonitrile containing SBA and 4-dimethylaminopyridine (DMAP) is evaporated to a solid, heated at 60°C for 1 h, redissolved in 50% acetonitrile containing matrix, spotted onto a MALDI target, and subjected to negative ion MALDI-TOF/TOF-MS. RESULTS While conventional (solution-phase) reaction of 4-phenylphenol (model analyte) with SBA and DMAP only gave a 47% yield of SBA-tagged 4-phenylphenol, evaporative derivatization as above gave a 96% yield, and 25 pmol (4.3 ng) of 4-phenylphenol could be detected in this way by MALDI-TOF/TOF-MS at S/N = 260, whereas even 1 nmol of the nonderivatized phenol was not detected in the absence of derivatization. A wide range of responses was observed when a mixture of 15 phenols was derivatized, with the higher responses coming from phenols with a pKa value above 9. Without derivatization, phenols with pKa values below 5 were the most readily detected. CONCLUSION Evaporative derivatization with SBA (a convenient reagent) can improve the detection of phenols with relatively high pKa values (above 9) by negative ion MALDI-TOF-MS, and accomplish this in the absence of post-derivatization reaction cleanup. PMID:24519828

  1. High-performance liquid chromatographic assay for catecholamines and metanephrines using fluorimetric detection with pre-column 9-fluorenylmethyloxycarbonyl chloride derivatization.

    PubMed

    Chan, E C; Wee, P Y; Ho, P Y; Ho, P C

    2000-12-01

    A convenient HPLC-fluorescent assay of norepinephrine (NE), epinephrine (E), dopamine (DA) and their 3-O-methylated metabolites, normetanephrine (NM) and metanephrine (MN) was developed. These analytes were coupled to 9-fluorenylmethyloxycarbonyl chloride (FMOC-Cl) before assays. Results showed that using a linear gradient elution, peaks of FMOC-NE, FMOC-E, FMOC-DA, FMOC-NM, FMOC-MN and FMOC-DHBA (3,4-dihydroxybenzylamine, internal standard) were simultaneously resolved within 40 min. Optimization of the chromatographic and derivatization conditions, and validation of the assay were further discussed in the paper. The structures of these derivatives were confirmed by atmospheric pressure chemical ionization mass spectrometry (APCI-MS). The molecular ions [M+H]- of FMOC-NE, FMOC-E, FMOC-DA, FMOC-NM and FMOC-MN were m/z 836, 850, 820, 628 and 642, respectively. Based on these findings, the FMOC-derivatives of metanephrines and catecholamines were confirmed to be bi-substituted and tri-substituted respectively at the amino and catechol functional groups. Finally, the assay was successfully applied to the measurement of urinary E, DA, NM and MN after direct derivatization and simple cleaning.

  2. An Improved Method for Analysis of Polyamines in Plant Tissue by Precolumn Derivatization with o-Phthalaldehyde and Separation by High Performance Liquid Chromatography 1

    PubMed Central

    Corbin, James L.; Marsh, Barry H.; Peters, Gerald A.

    1989-01-01

    An improved high-performance liquid-chromatographic method was developed for estimation of polyamines in crude plant extracts. Polyamines were derivatized with o-phthalaldehyde and mercaptoethanol (OPT). The fluorescent derivatives were eluted from a C18 column with the dimethylcyclohexylamine-phosphate buffer derived by T. Skaaden and T. Greibrokk ([1982] J Chromatogr 247: 111-122) after treatment to remove impurities in the buffer. The method had a sensitivity of 1-2 picomoles and completely resolved nine polyamines (agmatine, spermine, nor-spermidine, spermidine, 3,5-homospermidine, 4,4-homospermidine, 1,3-diaminopropane, putrescine, and cadaverine) in 12 to 14 minutes. An optional ion-exchange step was used to remove less basic amines (including amino acids) and to concentrate the crude extracts. This method was compared with benzoyl chloride derivatization. Use of the benzoyl chloride method vastly under-estimated the amount of polyamine in some plant extracts, a problem not encountered with the OPT procedure. Additionally, the OPT procedure resolved two isomers of homospermidine found in Azolla caroliniana. These two isomers were not resolved with the benzoylation method. Overall, the OPT method described here requires preparation and analysis time similar to other current methods but provides greater sensitivity and selectivity. PMID:16666789

  3. Enantiomer labelling, a method for the quantitative analysis of amino acids.

    PubMed

    Frank, H; Nicholson, G J; Bayer, E

    1978-12-21

    Enantiomer labelling a method for the quntitative analysis of optically active natural compounds by gas chromatography, involves the use of the unnatural enantiomer as an internal standard. With Chirasil-Val, a chiral stationary phase that is thermally stable up to up to 240 degrees, the enantiomers of amino acids and a variety of other compounds can be separated and quantitated. Incomplete recovery from the sample, incomplete derivatization, hydrolysis and thermal decomposition of the derivative and shifting response factors can be compensated for by adding the unnatural enantiomer. The accuracy of amino acid analysis by enantiomer labelling is equal or superior to that of hitherto known methods. The procedure affords a complete analysis of peptides with respect to both amino acid composition and the optical purity of each amino acid.

  4. Direct analysis of underivatized amino acids in plant extracts by LC-MS-MS.

    PubMed

    Thiele, Björn; Stein, Nadine; Oldiges, Marco; Hofmann, Diana

    2012-01-01

    In this chapter, we describe a method for quantification of 20 proteinogenic amino acids as well as 13 (15)N-labeled amino acids by liquid chromatography-mass spectrometry without the need for derivatization and use of organic solvents. Analysis of the underivatized amino acids is performed by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS-MS) in the positive ESI mode. Separation is achieved on a strong cation exchange (SCX) column (Luna 5 μm SCX 100 Å) with 30 mM ammonium acetate in water (A) and 5% acetic acid in water (B). Quantification is accomplished by use of d(5)-phenylalanine as internal standard achieving limits of detection of 0.1-3.0 μM. The method was successfully applied for the determination of proteinogenic and (15)N-labeled amino acids in plant extracts.

  5. [Determination of fatty acids in shark cartilage by GC-MS using microwave-assisted digestion and derivatization].

    PubMed

    Li, G K; He, X Q; Zhang, Z X

    2000-07-01

    A rapid microwave-assisted digestion and derivatization method for the determination of fatty acids in shark cartilage by GC-MS was developed. The optimum conditions for digestion and derivatization were studied in detail using orthogonal design. The digestion and derivatization were accomplished in 4 minutes at 600 W microwave power using HCl-methanol (1:4, V/V) as digestion and derivatization solvent, and the extraction of the target analytes could be carried out simultaneously. This method is rapid, solvent-saving, and particularly suitable for the rapid determination of fatty acids in solid samples.

  6. Analysis of amino acid neurotransmitters from rat and mouse spinal cords by liquid chromatography with fluorescence detection.

    PubMed

    Şanlı, Nurullah; Tague, Sarah E; Lunte, Craig

    2015-03-25

    A RP-LC-FL detection method has been developed to identify and quantitate four amino acid neurotransmitters including glutamic acid, glycine, taurine and γ-aminobutyric acid in rat and mouse spinal cord tissue. 3-(4-carboxybenzoyl)-2-quinolinecarboxaldehyde (CBQCA) was employed for the derivatization of these neurotransmitters prior to RP-LC-FL analysis. Different parameters which influenced separation and derivatization were optimized. Under optimum conditions, linearity was achieved within the concentration ranges of 0.50-50.00 μM for all analytes with correlation coefficients from 0.9912 to 0.9997. The LODs ranged from 0.03 μM to 0.06 μM. The proposed method has been successfully applied to the determination of amino acid neurotransmitters in biological samples such as rat and mouse spinal cord with satisfactory recoveries.

  7. Complementary Cell-Free Translational Assay for Quantification of Amino Acids.

    PubMed

    Jang, Yeon-Jae; Lee, Kyung-Ho; Yoo, Tae Hyeon; Kim, Dong-Myung

    2017-09-19

    In this study, we present a simple and economical method that enables rapid quantification of amino acids based on their polymerization into a signal-generating protein. This method harnesses amino acid-deficient cell-free protein synthesis systems that generate fluorescence signals in response to exogenous amino acids. When premixed with assay samples containing the amino acids in question, incubation of the cell-free synthesis reaction mixture rapidly resulted in the production of sfGFP, the fluorescence intensity of which was linearly proportional to the concentration of the amino acids. The assay method achieved a limit of detection as low as ∼100 nM and was successfully applied to the quantification of disease-related amino acids in biological samples. Compared with standard methods in current use that require chemical derivatization of amino acids and chromatographic equipment, the complementation assay method developed in this work enables the direct translation of amino acid titer into measurable biofluorescence intensity in a much shorter period, providing a more affordable and flexible option for the quantification of amino acids.

  8. Simultaneous analysis of amino acids and amines as their o-phthalaldehyde-ethanethiol-9-fluorenylmethyl chloroformate derivatives in cheese by high-performance liquid chromatography.

    PubMed

    Korös, A; Varga, Zs; Molnár-Perl, I

    2008-09-05

    A high-performance liquid chromatographic method (HPLC) with combined diode array and fluorescence detection of amino acids and amines in various cheese samples is described. The proposal is based on acidic deproteinization, derivatization and gradient optimization studies, resulting in the identification and quantification of 21 amino acids and 9 amines from a single solution, by one injection. The optimized, simple protocol consists of deproteinization (1M perchloric acid), centrifugation, filtration and the subsequent derivatization with the o-phthalaldehyde-ethanethiol-9-fluorenylmethyl chloroformate (OPA-ET-FMOC) reagent. The method can be characterized with a linearity of wide concentration range (6.25-1000 pM/injection), a good chromatographic reproducibility (average: 2.69% RSD) and an excellent recovery (average: 100.2%; average 3.84% RSD). The developed method was successfully applied in the determination of the amino acid and amine contents of port salut cheese, blue cheese and smoked cheese samples.

  9. High-performance liquid chromatography of histamine and 1-methylhistamine with on-column fluorescence derivatization.

    PubMed

    Saito, K; Horie, M; Nose, N; Nakagomi, K; Nakazawa, H

    1992-03-20

    An on-column fluorometric derivatization method was developed for the determination of histamine and 1-methylhistamine (HMs) by high-performance liquid chromatography. The system for the derivatization consisted only of a commercially available single-plunger pump and a reversed-phase C18 column supported on synthetic polymer with a mobile phase of acetonitrile and alkaline borate buffer solution containing o-phthalaldehyde as a derivatization reagent. It required no additional reaction system as for a post-column derivatization method. Injected HMs might be derivatized to a fluorophore on the inlet site of the high-performance liquid chromatographic column, followed by chromatography on the same column. Optimization of the on-column reaction conditions resulted in a simple and sensitive analytical method for the determination of HMs with excellent reproducibility and linearity of 0.05-5 micrograms/ml of both HMs. Application of this method to the determination of HMs in food samples resulted in a limit of quantification of 0.05 mg/100 g and in a greater than 95% overall mean recovery at a fortification of 0.1 mg/g of both HMs. This method was furthermore applicable to the determination of histamine released from rat peritoneal mast cells.

  10. Use of a statistically designed experimental approach to optimize the propylketal derivatization of barbiturates.

    PubMed

    Kushnir, M M; Urry, F M

    2001-04-01

    The derivatization of barbiturates with dimethylformamide dipropylacetal and dimethylformamide diisopropylacetal is studied with respect to the optimization of reaction recovery and reliability. A second-order orthogonal experimental design is utilized in order to obtain regression equations for the reaction recovery dependence on the derivatization solution composition, incubation temperature, and time for amobarbital, butalbital, pentobarbital, phenobarbital, and secobarbital. Regression equations for the effect of incubation temperature and time on the derivative recovery and the optimum conditions for derivatization recoveries are obtained. Differences in the phenomena of the derivative formation are evaluated between the two derivatizing reagents and the barbiturates. Based on the analysis of the obtained equations, it is concluded that the dipropylketal derivative of barbiturates is superior in comparison with diisopropylketal when considering the milder conditions of the reaction, absence of sudden changes in the recovery with a variation in the derivatization parameters, and reliability for the simultaneous testing of the barbiturates. A method for the routine testing of the barbiturates by gas chromatography-mass spectrometry in urine specimens is included.

  11. Determination of amitrole in environmental water samples with precolumn derivatization by high-performance liquid chromatography.

    PubMed

    Sun, Yan; Liu, Peng Fei; Wang, Deng; Li, Jian Qiang; Cao, Yong Song

    2009-06-10

    Amitrole is a nonselective polar herbicide that can easily pollute ground and surface waters because of its high solubility in water. A precolumn derivatization high-performance liquid chromatographic method for amitrole analysis has been developed. Derivatization of amitrole was performed with 4-chloro-3,5-dinitrobenzotrifluoride (CNBF). The derivatization conditions and the influence of elution composition on the separation were investigated. In pH 9.5 H(3)BO(3)-Na(2)B(4)O(7) media, the reaction of amitrole with CNBF was complete at 60 degrees C after 30 min. The stability of the derivative under light irradiation and room temperature in methanol-water samples was demonstrated. The derivatized amitrole was separated on a K C(18) column (250 mm x 4.6 mm, 5 microm) at room temperature, and UV detection was applied at 360 nm. The separation of derivatized amitrole was achieved within 18 min by gradient elution mode. The method correlation coefficient was 0.9995, in concentrations ranging from 1.59 to 318 mg L(-1). The detection limit of amitrole was 0.16 mg L(-1) with a signal-to-noise ratio of 3. The proposed method was applied to the quantitative determination of amitrole in environmental water with recoveries of 92.0-103.0% and RSDs of 2.22-6.26, depending on the sample investigated. This method has good accuracy and repeatability that can be used to quantify amitrole in environmental water.

  12. High performance solid-phase analytical derivatization of phenols for gas chromatography-mass spectrometry.

    PubMed

    Kojima, Miki; Tsunoi, Shinji; Tanaka, Minoru

    2004-07-09

    The solid-phase analytical derivatization of phenols with pentafluoropyridine is performed. Fourteen phenols including chlorophenols and alkylphenols, could be efficiently adsorbed on a strong anion-exchange solid phase, Oasis MAX. The phenols adsorbed on Oasis MAX as phenolate ions were desorbed after derivatization with pentafluoropyridine. After optimization of the adsorption and derivatization, we established a procedure for the determination of the phenols in water samples by means of GC-MS. Under the optimized conditions, calibration curves were linear in the range of 10-1000 ng/l for the alkylphenols (100-10000 ng/l for nonylphenol) and 50-1000 ng/l for the others. By processing 100 ml samples, the method detection limits (MDLs) were in the range of 0.45-2.3 ng/l for the alkylphenols (8.5 ng/l for nonylphenol) and 2.4-16 ng/l for the others. Compared with the biphasic reaction system, the signal-to-noise ratios obtained by the solid-phase analytical derivatization were significantly higher. This is ascribed to the fact that coexisting neutral and acidic compounds are efficiently removed from the sample solution by this solid-phase analytical derivatization system.

  13. Microwave-assisted derivatization: application to steroid profiling by gas chromatography/mass spectrometry.

    PubMed

    Casals, Gregori; Marcos, Josep; Pozo, Oscar J; Alcaraz, José; Martínez de Osaba, María Jesús; Jiménez, Wladimiro

    2014-06-01

    Gas chromatography-mass spectrometry (GC-MS) remains as the gold-standard technique for the study of the steroid metabolome. A main limitation is the need of performing a derivatization step since incubation with strong silylations agents for long periods of time (usually 16 h) is required for the derivatization of hindered hydroxyls present in some steroids of interest. In the present work, a rapid, simple and reproducible microwave-assisted derivatization method was developed. In the method, 36 steroids already treated with methoxyamine (2% in pyridine) were silylated with 50 μl of N-trimethylsilylimidazole by using microwave irradiation, and the formed methyloxime-trimethylsilyl derivatives were analyzed by GC-MS. Microwave power and derivatization time silylation conditions were optimized being the optimum conditions 600 W and 3 min respectively. In order to evaluate the usefulness of this technique, the urine steroid profiles for 20 healthy individuals were analyzed. The results of a comparison of microwave irradiation with the classical heating protocol showed similar derivatization yields, thus suggesting that microwave-assisted silylation is a valid tool for the rapid steroid metabolome study.

  14. Sensitive derivatization methods for the determination of genotoxic impurities in drug substances using hyphenated techniques.

    PubMed

    Raman, Nanduri V V S S; Prasad, Adapa V S S; Reddy, Kura Ratnakar

    2014-02-01

    Six sensitive derivatization methods for the determination of genotoxic impurities in selected drug substances were developed using hyphenated techniques. Some of the raw materials, reagents and reaction intermediates of the selected drug substances were identified as genotoxic impurities through DEREK software for windows. The genotoxic impurities which are amenable for derivatization were selected as substrates. Derivatizing agents were selected based on the functional groups of the genotoxic impurities. The chemistry involved in the derivatization was explained with suitable mechanisms. An appropriate hyphenated technique viz. LC-MS and GC-MS was opted based on the sensitivity and aromaticity of the derivatized genotoxic impurities. All the methods were validated as per International Conference on Harmonization guidelines. Correlation coefficient values were found about 0.99. The obtained % R.S.D values from replicate injections in the range of 2.3-4.8 and % recoveries of the added impurities in the range of 83.7-101.7 ensured the precision and accuracy, respectively.

  15. Enantioselective separation of amino acids as biomarkers indicating life in extraterrestrial environments.

    PubMed

    Pietrogrande, Maria Chiara

    2013-10-01

    Traces of prebiotic amino acids, i.e., the building blocks of proteins, are excellent biomarkers that could provide evidence of extinct or extant life in extra-terrestrial environments. In particular, characterization of the enantiomeric excess of amino acids gives relevant information about the biotic or abiotic origin of molecules, because it is generally assumed that life elsewhere could be based on either L or D amino acids, but not both. The analytical procedures used in in-situ space missions for chiral discrimination of amino acids must meet severe requirements imposed by flight conditions: short analysis time, low energy consumption, robustness, storage for long periods under extreme conditions, high efficiency and sensitivity, automation, and remote-control operation. Such methods are based on gas chromatography, high-pressure liquid chromatography, and capillary electrophoresis, usually coupled with mass spectrometry; of these, gas chromatography-mass spectrometry (GC-MS) is the only such combination yet used in space missions. Preliminary in-situ sample derivatization is required before GC-MS analysis to convert amino acids into volatile and thermally stable compounds. The silylation reagent most commonly used, N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide, is unsuitable for detection of homochirality, and alternative derivatization techniques have been developed that preserve the stereochemical configuration of the original compounds and are compatible with spaceflight conditions. These include the reagent N,N-dimethylformamide dimethylacetal, which has already been used in the Rosetta mission, a mixture of alkyl chloroformate, ethanol, and pyridine, a mixture of perfluorinated anhydrides and perfluoro alcohols, and hexafluoroacetone, the first gaseous derivatizing agent. In all the space instruments, solvent extraction of organic matter and chemical derivatization have been combined in a single automatic and remote-controlled procedure in a

  16. Screening for non-protein amino acids in seeds of the Guam cycad, Cycas circinalis, by an improved GC-MS method.

    PubMed

    Oh, C H; Brownson, D M; Mabry, T J

    1995-02-01

    The non-protein amino acid, beta-N-methyl-amino-L-alanine (L-BMAA) from Cycas circinalis seeds, has been implicated as a causative agent of amyotrophic lateral sclerosis-Parkinsonism dementia complex (ALS-PDC), a disease known from Guam and other areas in the western Pacific. We analyzed C. circinalis seeds for additional free non-protein amino acids by a recently developed GC-MS method. The samples were prepared by water extraction of seed flour. The amino acids present in the extract were derivatized by N(O,S)-isobutyloxycarbonylation of the amine functional groups and then tert-butyldimethylsilylation of the carboxyl functional groups. Peaks for a total of seventeen derivatives of non-protein amino acids were detected by GC-MS. In addition to L-BMAA, four other non-protein amino acids were identified as beta-alanine, gamma-amino-butyric acid, pyroglutamic acid, and alpha-aminoadipic acid.

  17. Extraterrestrial material analysis: loss of amino acids during liquid-phase acid hydrolysis

    NASA Astrophysics Data System (ADS)

    Buch, Arnaud; Brault, Amaury; Szopa, Cyril; Freissinet, Caroline

    2015-04-01

    Searching for building blocks of life in extraterrestrial material is a way to learn more about how life could have appeared on Earth. With this aim, liquid-phase acid hydrolysis has been used, since at least 1970 , in order to extract amino acids and other organic molecules from extraterrestrial materials (e.g. meteorites, lunar fines) or Earth analogues (e.g. Atacama desert soil). This procedure involves drastic conditions such as heating samples in 6N HCl for 24 h, either under inert atmosphere/vacuum, or air. Analysis of the hydrolyzed part of the sample should give its total (free plus bound) amino acid content. The present work deals with the influence of the 6N HCl hydrolysis on amino acid degradation. Our experiments have been performed on a standard solution of 17 amino acids. After liquid-phase acid hydrolysis (6N HCl) under argon atmosphere (24 h at 100°C), the liquid phase was evaporated and the dry residue was derivatized with N-Methyl-N-(t-butyldimethylsilyl)trifluoroacetamide (MTBSTFA) and dimethylformamide (DMF), followed by gas chromatography-mass spectrometry analysis. After comparison with derivatized amino acids from the standard solution, a significant reduction of the chromatographic peak areas was observed for most of the amino acids after liquid-phase acid hydrolysis. Furthermore, the same loss pattern was observed when the amino acids were exposed to cold 6N HCl for a short amount of time. The least affected amino acid, i.e. glycine, was found to be 73,93% percent less abundant compared to the non-hydrolyzed standard, while the most affected, i.e. histidine, was not found in the chromatograms after hydrolysis. Our experiments thereby indicate that liquid-phase acid hydrolysis, even under inert atmosphere, leads to a partial or total loss of all of the 17 amino acids present in the standard solution, and that a quick cold contact with 6N HCl is sufficient to lead to a loss of amino acids. Therefore, in the literature, the reported increase

  18. Amino Acid, Organic Acid, and Sugar Profiles of 3 Dry Bean (Phaseolus vulgaris L.) Varieties.

    PubMed

    John, K M Maria; Luthria, Devanand

    2015-12-01

    In this study, we compared the amino acid, organic acid and sugar profiles of 3 different varieties of dry beans (black bean [BB], dark red bean [DRB], and cranberry bean [CB]). The efficiency of the 2 commonly used extraction solvents (water and methanol:chloroform:water [2.5:1:1, v/v/v/]) for cultivar differentiation based on their metabolic profile was also investigated. The results showed that the BB contained the highest concentration of amino acids followed by DRB and CB samples. Phenylalanine, a precursor for the biosynthesis of phenolic secondary metabolites was detected at low concentration in CB samples and correlated with the reduced anthocyanins content in CB extract as documented in the published literature. Comparing the extractability of 2 extraction solvents, methanol:chloroform:water (2.5:1:1, v/v/v/) showed higher recoveries of amino acids from 3 beans, whereas, sugars were extracted in higher concentration with water. Analytically, gas chromatography detected sugars (9), amino acids (11), and organic acids (3) in a single run after derivatization of the extracts. In comparison, ion chromatography detected only sugars in a single run without any derivatization step with the tested procedure. Bean samples are better differentiated by the sugar content extracted with water as compared to the aqueous organic solvent extracts using partial least-square discriminant analysis. Published 2015. This article is a U.S. Government work and is in the public domain in the USA.

  19. Chromatographic method for determination of the free amino acid content of chamomile flowers

    PubMed Central

    Ma, Xiaoli; Zhao, Dongsheng; Li, Xinxia; Meng, Lei

    2015-01-01

    Objective: To determine the free amino acid contents of chamomile flowers using reverse-phase high-performance column chromatography preceded by pre-column derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC), and to determine the reliability of this method. Materials and Methods: Derivatization with reconstituted AQC was used to prepare the samples and standards for injection into the chromatography column. The peaks were analyzed by fluorescence detection (λ excitation, 250 nm; λ emission, 395 nm. Results: Alanine, proline, and leucine were the most abundant amino acids, whereas tyrosine and methionine were the least abundant. The linearity of the method was found to be good with amino acid concentrations of 0.012-0.36 μM. The precision was 0.05-1.36%; average recovery, 91.12-129.41%; and limit of detection, 0.006-0.058 μM. Conclusion: The method is reliable for determining the free amino acid content of different types of chamomile flowers. PMID:25709230

  20. Enantiomeric derivatization on the Mars Organic Molecule Analyzer (MOMA) experiment aboard ExoMars 2018: how to unravel martian chirality

    NASA Astrophysics Data System (ADS)

    Freissinet, C.; Buch, A.; Szopa, C.; Morisson, M.; Grand, N.; Raulin, F.; Brinckerhoff, W.

    2015-10-01

    The origin of homochirality in life on Earth remains unknown. The answer to this question lies in the study of chirality elsewhere in the Solar System. The Sample Analysis at Mars (SAM) experiment aboard Curiosity established the presence of organic molecules indigenous to a clay-rich sample on Mars [1]. However, SAM does not have the ability to separate between the enantiomers of potential medium- or high- molecular weight organic molecules. One of the wet chemistry experiments to be used in the MOMA instrument of the Exomars mission is designed for the extraction and identification of refractory organic chemical components in solid samples using gas chromatography-mass spectrometry (GCMS), while keeping the chiral center of the molecules intact [2]. This derivatization technique, using dimethylformamide dimethylacetal (DMF-DMA) as a reagent, will allow MOMA to separate the enantiomers of molecules of interest for astrobiology, such as amino acids, sugars or carboxylic acids. We present here the results of laboratory experiments which display the feasability and limitations of the detection of an enantiomeric excess of complex organic molecules in various analog samples, depending on the mineralogy of the Mars analog solid sample.

  1. Chiral separation of amino acids and peptides by capillary electrophoresis.

    PubMed

    Wan, H; Blomberg, L G

    2000-04-14

    Chiral separation of amino acids and peptides by capillary electrophoresis (CE) is reviewed regarding the separation principles of different approaches, advantages and limitations, chiral recognition mechanisms and applications. The direct approach details various chiral selectors with an emphasis on cyclodextrins and their derivatives, antibiotics and chiral surfactants as the chiral selectors. The indirect approach deals with various chiral reagents applied for diastereomer formation and types of separation media such as micelles and polymeric pseudo-stationary phases. Many derivatization reagents used for high sensitivity detection of amino acids and peptides are also discussed and their characteristics are summarized in tables. A large number of relevant examples is presented illustrating the current status of enantiomeric and diastereomeric separation of amino acids and peptides. Strategies to enhance the selectivity and optimize separation parameters by the application of experimental designs are described. The reversal of enantiomeric elution order and the effects of organic modifiers on the selectivity are illustrated in both direct and indirect methods. Some applications of chiral amino acid and peptide analysis, in particular, regarding the determination of trace enantiomeric impurities, are given. This review selects more than 200 articles published between 1988 and 1999.

  2. Optimizing high-performance liquid chromatography method for quantification of glucosamine using 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate derivatization in rat plasma: application to a pharmacokinetic study.

    PubMed

    Wang, Xianhuo; Chen, Xiang; Chen, Lijuan; Wang, Biqin; Peng, Cheng; He, Chunmei; Tang, Minghai; Zhang, Fan; Hu, Jia; Li, Rui; Zhao, Xia; Wei, Yuquan

    2008-11-01

    A sensitive and reliable HPLC method with fluorescence detection based on the precolumn derivatization of glucosamine with 6-aminoquinolyl-N-hydroxylsuccinimidyl carbamate (AQC) was established for the quantitative determination of glucosamine in rat plasma. The plasma protein was precipitated by acetonitrile, followed by vortex mixing and centrifugation. The supernatant was divided into the organic layer and aqueous layer by adding sodium chloride, and then the aqueous layer was derivatized with AQC in 0.2 M borate buffer of pH 8.8 before the HPLC analysis. An amino acid analysis column (3.9 x 150 mm, 4 microm) was applied, with 140 mM sodium acetate buffer (pH = 5.25) and acetonitrile as mobile phase at a flow rate of 1 mL/min. A linear correlation coefficient of 0.9987 was calculated within the range of 0.1-30 microg/mL of the standard curve for glucosamine. The limit of detection was 30 ng/mL. The intra- and inter-day precisions (as RSD) were less than 7.38 and 12.72%, respectively. The intra- and inter-day accuracy ranged from 91.8 to 110.0%. Extraction recoveries of glucosamine in plasma were more than 90%. The validated method was successfully applied for the quantitative determination of glucosamine in rat plasma and evaluation for pharmacokinetic study of glucosamine. It was also possible to be applied for the quantitative determination of other compounds containing amino group in biological samples.

  3. A new derivatization approach with D-cysteine for the sensitive and simple analysis of acrylamide in foods by liquid chromatography-tandem mass spectrometry.

    PubMed

    Lim, Hyun-Hee; Shin, Ho-Sang

    2014-09-26

    A liquid chromatography-tandem mass spectrometry method (LC-MS/MS) was developed in order to determine the amount of acrylamide in foods after derivatization with d-cysteine. The sulfhydryl group of d-cysteine was added at the β-site double bond of acrylamide to form 2-amino-3-(3-amino-3-oxo-propyl)sulfanyl-propanoic acid. Deuterated acrylamide (acrylamide-d3) was chosen as the internal standard (IS) for analyzing the food samples. Acrylamide was extracted from 2.0 g of food sample with 6 mL of methylene chloride, and the organic extract was diluted with 3 mL of hexane, and then the analyte was back-extracted with 0.5 mL of pure water. The derivatization of acrylamide was performed in the water extract. The best reaction conditions (3.0mg of d-cysteine, a pH 6.5, a reaction temperature of 90°C, and a heating time of 50 min) were established by the variation of parameters. The formed derivative was injected into the LC-MS/MS without further extraction or purification procedures. Separation and detection were improved with the use of an ion-pairing reagent of perfluorooctanoic acid. Under the established conditions, the limits of detection and the limits of quantification were 0.04 μg/kg and 0.14 μg/kg, respectively, and the inter-day relative standard deviation was less than 8% at concentrations of 20 and 100 μg/kg. The method was successfully applied to determine the amount of acrylamide in potato chips, French fries, and coffee. Copyright © 2014 Elsevier B.V. All rights reserved.

  4. Application of solid-phase microextraction combined with derivatization to the enantiomeric determination of amphetamines.

    PubMed

    Cháfer-Pericás, C; Campíns-Falcó, P; Herráez-Hernández, R

    2006-03-18

    The utility of combining chiral derivatization and solid-phase microextraction (SPME) for the enantiomeric analysis of primary amphetamines by liquid chromatography has been investigated. Different derivatization/extraction strategies have been evaluated and compared using the chiral reagent o-phthaldialdehyde (OPA)-N-acetyl-l-cysteine (NAC) and fibres with a Carbowax-templated resin coating. Amphetamine, norephedrine and 3,4-methylenedioxyamphetamine (MDA) were used as model compounds. On the basis of the results obtained, a new method is presented based on the derivatization of the analytes in solution followed by SPME of the OPA-NAC derivatives formed. The proposed conditions have been applied to determine the compounds of interest at low ppm levels (

  5. Process for derivatizing carbon nanotubes with diazonium species and compositions thereof

    NASA Technical Reports Server (NTRS)

    Tour, James M. (Inventor); Bahr, Jeffrey L. (Inventor); Yang, Jiping (Inventor)

    2011-01-01

    Methods for the chemical modification of carbon nanotubes involve the derivatization of multi- and single-wall carbon nanotubes, including small diameter (ca. 0.7 nm) single-wall carbon nanotubes, with diazonium species. The method allows the chemical attachment of a variety of organic compounds to the side and ends of carbon nanotubes. These chemically modified nanotubes have applications in polymer composite materials, molecular electronic applications, and sensor devices. The methods of derivatization include electrochemical induced reactions, thermally induced reactions, and photochemically induced reactions. Moreover, when modified with suitable chemical groups, the derivatized nanotubes are chemically compatible with a polymer matrix, allowing transfer of the properties of the nanotubes (such as, mechanical strength or electrical conductivity) to the properties of the composite material as a whole. Furthermore, when modified with suitable chemical groups, the groups can be polymerized to form a polymer that includes carbon nanotubes.

  6. Direct derivatization and rapid GC-MS screening of nerve agent markers in aqueous samples.

    PubMed

    Subramaniam, Raja; Astot, Crister; Juhlin, Lars; Nilsson, Calle; Ostin, Anders

    2010-09-01

    A rapid screening and identification method based on derivatization and gas chromatography mass spectrometry (GC-MS) has been developed for the detection of alkylphosphonic acids (APAs), the degradation products of organophosphorus nerve agents. The novel method described involves rapid (5 min) and direct derivatization of 25 microL aqueous sample using highly fluorinated phenyldiazomethane reagents (e.g., 1-(diazomethyl)-3,5-bis(trifluoromethyl)benzene). The APA derivatives are then screened by GC-MS negative ion chemical ionization (NICI) and identified by electron ionization (EI) mode. The conditions for the derivatization were optimized using statistical experimental design and multivariate data analysis. Method robustness was evaluated using aqueous samples from an official OPCW Proficiency Test and all APAs present in the sample were conclusively identified. Limits of detection for rapid screening using SIM NICI were between 5 and 10 ng/mL APA in aqueous sample, and for identification using full scan EI 100 ng/mL.

  7. Chemical derivatization technique in ToF-SIMS for quantification analysis of surface amine groups

    NASA Astrophysics Data System (ADS)

    Lee, Tae Geol; Kim, Jinmo; Shon, Hyun Kyong; Jung, Donggeun; Moon, Dae Won

    2006-07-01

    A chemical derivatization technique in ToF-SIMS along with principal component analysis (PCA) were used to perform a quantitative study of the surface amine density of the plasma-polymerized ethylenediamine (PPEDA) thin film. We used the scores on principal component (PC) 1 from a PCA of ToF-SIMS data for the PPEDA films and their chemical-derivatized surfaces for comparison with the surface amine densities. These surface amine densities were independently determined by UV-visible spectroscopy. Our work found a good linear relationship between the surface amine densities and the scores on PC 1 from a PCA of the ToF-SIMS data for the chemical-derivatized PPEDA surfaces, but not for the PPEDA thin films themselves. In addition to quantification, our PCA results provided insights into the surface chemical composition of each surface.

  8. New aniline-containing amino alcohols from trans-(R,R)-2-(2-nitrophenyl)-3-phenyloxirane as useful intermediates for the synthesis of chiral ligands, bases, and benzoxazine nucleus.

    PubMed

    Solladié-Cavallo, Arlette; Lupattelli, Paolo; Bonini, Carlo; Ostuni, Valeria; Blasio, Nadia Di

    2006-12-22

    New enantiopure aniline-containing amino alcohols are directly derived from trans-(R,R)-2-(2-nitrophenyl)-3-phenyloxirane, by alternative regioselective double reductions. Subsequent selective alkylation procedures and derivatizations provide a rapid and high-yielding access to different chiral ligands, bases, and benzoxazines, without loss of optical purity.

  9. PEG-derivatized octacosanol as micellar carrier for paclitaxel delivery.

    PubMed

    Chu, Bingyang; Qu, Ying; Huang, Yixing; Zhang, Lan; Chen, Xiaoxin; Long, Chaofeng; He, Yunqi; Ou, Caiwen; Qian, Zhiyong

    2016-03-16

    In this study, PEG-derivatized octacosanol copolymer was successfully developed to improve the anti-tumor activity and eliminate toxicity of the commercial formulation of paclitaxel (PTX). MPEG2K-C28, the conjugation of monomethoxy Poly(ethylene glycol) 2000 and octacosanol, was readily soluble in aqueous solution and self-assembled to form micelles with small sizes (< 20 nm) that are efficient in encapsulating PTX with a drug loading of 9.38 ± 0.18% and an encapsulation efficiency of 93.90 ± 2.12%. Meanwhile, octacosanol is very safe for humans and amazingly exhibits antitumor activity through inhibition activity of matrix metalloproteinases (MMPs) and translocation of the transcription factor (nuclear factor-kappa B, NF-κB) to the nucleus, which may be able to promote synergistic effects with PTX. A sustained and slower in vitro release behavior was observed in the (PTX micelles) than that of Taxol. PTX micelles exhibited more potent cytotoxicity than Taxol in the 4T1 breast cancer cell line. More interestingly, MPEG2K-C28 selectively inhibited the growth of 4T1 cells rather than the normal cells (HEK293 and L929 cell lines), indicating the antitumor activity of octacosanol remained after conjugation with MPEG. Acute toxicity evaluations indicated that MPEG2K-C28 was a safe drug carrier. Pharmacokinetic study revealed that PTX micelles improved the T1/2 and AUC of PTX (compared with Taxol) from 1.910 ± 0.139 h and 13.999 ± 1.109 mg/l × h to 2.876 ± 0.532 h and 76.462 ± 8.619 mg/l × h in vivo, respectively. The maximal tolerated dose (MTD) for PTX micelles (ca. 120 mg PTX/kg) in mice was significantly higher than that for Taxol (ca. 20mg PTX/kg). PTX micelles exhibited slightly better antitumor activity than Taxol but safer in 4T1 breast cancer model in vivo. The cell apoptosis in the immunofluorescent studies and the cell proliferation in the immunohistochemical studies also proved the results. In conclusion, MPEG2K-C28 is a simple, safe and effective

  10. Microfluidic lab-on-a-chip derivatization for gaseous carbonyl analysis.

    PubMed

    Pang, Xiaobing; Lewis, Alastair C; Ródenas-García, Milagros

    2013-06-28

    We present a microfluidic lab-on-a-chip derivatization technique for the analysis of gaseous carbonyl compounds using O-(2,3,4,5,6-pentafluorobenzyl) hydroxylamine (PFBHA) as the derivatizing reagent. The novel microfluidic lab-on-a-chip derivatization technique has been developed to measure nmol per mole (ppbv) mixing ratios of gaseous carbonyl compounds, which are of particular importance to atmospheric chemistry. The technique utilised a planar glass microreactor comprising three inlets and one outlet, gas and fluid splitting and combining channels, mixing junctions, and a 2.0m long, 620μm internal diameter reaction microchannel. The microreactor integrated three functions, providing: (1) a gas and liquid mixer and reactor, (2) reagent heating, and (3) sample pre-concentration. The concentration of derivatization solution, the volumetric flow rates of the incoming gas sample and PFBHA solution, and the temperature of the microreactor were optimised to achieve a near real-time measurement. The enhanced phase contact area-to-volume ratio and the high heat transfer rate in the microreactor resulted in a fast and high efficiency derivatization reaction, generating an effluent stream which was ready for direct introduction to GC-MS. Good linearity was observed for eight carbonyl compounds over the measurement ranges of 1-500ppbv when they were derivatized under optimal reaction conditions. The method detection limits (MDLs) were below 0.10nmolmol(-1) for most carbonyls in this study, which is below or close to their typical concentrations in clean ambient air. The performance of the technique was assessed by applying the methodology to the quantification of glyoxal (GLY) and methylglyoxal (MGLY) formed during isoprene photo-oxidation in an outdoor photoreactor chamber (EUPHORE). Good agreements between GLY and MGLY measurements were obtained comparing this new technique with Fourier Transform InfraRed (FTIR), which provides support for the potential effectiveness of

  11. Distribution of D-amino acids in vinegars and involvement of lactic acid bacteria in the production of D-amino acids.

    PubMed

    Mutaguchi, Yuta; Ohmori, Taketo; Akano, Hirofumi; Doi, Katsumi; Ohshima, Toshihisa

    2013-01-01

    Levels of free D-amino acids were compared in 11 vinegars produced from different sources or through different manufacturing processes. To analyze the D- and L-amino acids, the enantiomers were initially converted into diastereomers using pre-column derivatization with o-phthaldialdehyde plus N-acethyl-L-cysteine or N-tert-butyloxycarbonyl-L-cysteine. This was followed by separation of the resultant fluorescent isoindol derivatives on an octadecylsilyl stationary phase using ultra-performance liquid chromatography. The analyses showed that the total D-amino acid level in lactic fermented tomato vinegar was very high. Furthermore, analysis of the amino acids in tomato juice samples collected after alcoholic, lactic and acetic fermentation during the production of lactic fermented tomato vinegar showed clearly that lactic fermentation is responsible for the D-amino acids production; marked increases in D-amino acids were seen during lactic fermentation, but not during alcoholic or acetic fermentation. This suggests lactic acid bacteria have a greater ability to produce D-amino acids than yeast or acetic acid bacteria.

  12. Matrix influence on derivatization and ionization processes during selenoamino acid liquid chromatography electrospray ionization mass spectrometric analysis.

    PubMed

    Rebane, Riin; Oldekop, Maarja-Liisa; Herodes, Koit

    2014-04-01

    Considering the importance of derivatization in LC/ESI/MS analysis, the objective of this work was to develop a method for evaluation of matrix effect that would discriminate between matrix effect due to the derivatization reaction yield and from the ESI. Four derivatization reagents (TAHS, DEEMM, DNS, FMOC-Cl) were studied with respect to matrix effects using two selenoamino acids and onion matrix as model system. A novel method for assessing matrix effects of LC/ESI/MS analyses involving derivatization is proposed, named herein post-derivatization spiking, that allows evaluating effect of matrix on ESI ionization without derivatization reaction yield contribution. The proposed post-derivatization spiking method allowed to demonstrate that the reason of reduced analytical signal can be signal suppression in ESI (as in case of DNS derivatives with matrix effects 38-99%), alteration of derivatization reaction yield (TAHS, matrix effects 92-113%, but reaction yields 20-50%) or both (FMOC-Cl, matrix effects 28-88% and reaction yields 50-70%). In case of DEEMM derivatives, matrix reduces reaction yield but enhances ESI/MS signal. A method for matrix effect evaluation was developed. It was also confirmed that matrix effects can be reduced by dilution.

  13. Amino acid analysis

    NASA Technical Reports Server (NTRS)

    Winitz, M.; Graff, J. (Inventor)

    1974-01-01

    The process and apparatus for qualitative and quantitative analysis of the amino acid content of a biological sample are presented. The sample is deposited on a cation exchange resin and then is washed with suitable solvents. The amino acids and various cations and organic material with a basic function remain on the resin. The resin is eluted with an acid eluant, and the eluate containing the amino acids is transferred to a reaction vessel where the eluant is removed. Final analysis of the purified acylated amino acid esters is accomplished by gas-liquid chromatographic techniques.

  14. Parenteral Nutrition: Amino Acids.

    PubMed

    Hoffer, Leonard John

    2017-03-10

    There is growing interest in nutrition therapies that deliver a generous amount of protein, but not a toxic amount of energy, to protein-catabolic critically ill patients. Parenteral amino acids can achieve this goal. This article summarizes the biochemical and nutritional principles that guide parenteral amino acid therapy, explains how parenteral amino acid solutions are formulated, and compares the advantages and disadvantages of different parenteral amino acid products with enterally-delivered whole protein products in the context of protein-catabolic critical illness.

  15. Parenteral Nutrition: Amino Acids

    PubMed Central

    Hoffer, Leonard John

    2017-01-01

    There is growing interest in nutrition therapies that deliver a generous amount of protein, but not a toxic amount of energy, to protein-catabolic critically ill patients. Parenteral amino acids can achieve this goal. This article summarizes the biochemical and nutritional principles that guide parenteral amino acid therapy, explains how parenteral amino acid solutions are formulated, and compares the advantages and disadvantages of different parenteral amino acid products with enterally-delivered whole protein products in the context of protein-catabolic critical illness. PMID:28287411

  16. Radiation damage and derivatization in macromolecular crystallography: a structure factor’s perspective

    PubMed Central

    Owen, Robin L.; Sherrell, Darren A.

    2016-01-01

    During, or even after, data collection the presence and effects of radiation damage in macromolecular crystallography may not always be immediately obvious. Despite this, radiation damage is almost always present, with site-specific damage occurring on very short time (dose) scales well before global damage becomes apparent. A result of both site-specific radiation damage and derivatization is a change in the relative intensity of reflections. The size and approximate rate of onset of X-ray-induced transformations is compared with the changes expected from derivatization, and strategies for minimizing radiation damage are discussed. PMID:26960125

  17. Derivatization of small biomolecules for optimized matrix-assisted laser desorption/ionization mass spectrometry.

    PubMed

    Tholey, Andreas; Wittmann, Christoph; Kang, Min-Jung; Bungert, Ditte; Hollemeyer, Klaus; Heinzle, Elmar

    2002-09-01

    Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) is a powerful tool for the measurement of low molecular mass compounds of biological interest. The limitations for this method are the volatility of many analytes, possible interference with matrix signals or bad ionization or desorption behavior of the compounds. We investigated the application of well-known and straightforward one-pot derivatization procedures to circumvent these problems. The derivatizations tested allow the measurement and the labeling of alcohols, aldehydes and ketones, carboxylic acids, alpha-ketocarboxylic acids and amines.

  18. Profiling Amino Acids of Jordanian Scalp Hair as a Tool for Diabetes Mellitus Diagnosis: A Pilot Study.

    PubMed

    Rashaid, Ayat H B; Harrington, Peter de B; Jackson, Glen P

    2015-07-21

    Hair analysis is an area of increasing interest in the fields of medical and forensic sciences. Human scalp hair has attractive features in clinical studies because hair can be sampled easily and noninvasively from human subjects, and unlike blood and urine samples, it contains a chronological record of medication use. Keratin protein is the major component of scalp hair shaft material and it is composed of 21 amino acids. The method used herein for the amino acid determination in hair included keratin protein acid hydrolysis using 6 M hydrochloric acid (HCl), followed by amino acids derivatization using N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA), and the determination of derivatized amino acids by gas chromatography/mass spectrometry (GC/MS). Amino acid profiles of scalp hair of 27 Jordanian subjects (15 diabetes mellitus (DM) type 2 patients and 12 control subjects) were analyzed. A fuzzy rule-building expert system (FuRES) classified the amino acid profiles into diabetic and control groups based on multivariate analyses of the abundance of 14 amino acids. The sensitivity and specificity were 100% for diabetes detection using leave-one-individual-out cross-validation. The areas under the receiver operative characteristics (ROC) curves were 1.0, which represents a highly sensitive and specific diabetes test. The nonessential amino acids Gly and Glu, and the essential amino acid Ile were more abundant in the scalp hair of diabetic patients compared to the hair of control subjects. The associations between the abundance of amino acids of human hair and health status may have clinical applications in providing diagnostic indicator or predicting other chronic or acute diseases.

  19. Chiral resolution of derivatized amino acids using uniformly sized molecularly imprinted polymers in hydro-organic mobile phases.

    PubMed

    Haginaka, Jun; Kagawa, Chino

    2004-04-01

    Uniformly sized molecularly imprinted polymers (MIPs) for Boc-L-Trp were prepared using ethylene glycol dimethacrylate (EDMA) as the cross-linker, and methacylic acid (MAA) and/or 4-vinylpyridine (4-VPY) as the functional monomers or without use of a functional monomer. The MIPs prepared were evaluated using acetonitrile or a mixture of phosphate buffer and acetonitrile as the mobile phase. The Boc-L-Trp-imprinted EDMA polymers can recognize Boc-L-Trp by its molecular shape, and can thus afford the enantioseparation of Boc-Trp. Besides the molecular shape recognition, the hydrophobic interactions with the polymer backbones as well as the hydrogen-bonding interactions of Boc-L-Trp with carboxyl and pyridyl groups in the polymers should work for the retention and recognition of Boc-L-Trp on the imprinted MAA- co-EDMA and 4-VPY- co-EDMA polymers, respectively, in the hydro-organic mobile phase. The hydrogen-bonding interactions seem to become dominant when only acetonitrile is used as the mobile phase. The Boc-L-Trp-imprinted 4-VPY- co-EDMA polymers gave the highest retentivity and enantioselectivity for Boc-Trp among the MIPs prepared. However, the simultaneous use of MAA and 4-VPY was not effective for the enantioseparation of Boc-Trp in a hydro-organic mobile phase. Furthermore, the baseline separation of Boc-Trp enantiomers was attained within 10 min on the Boc-L-Trp-imprinted 4-VPY- co-EDMA polymers under the optimized HPLC conditions.

  20. Determination of Selected Amino Acids in Serum of Patients with Liver Disease.

    PubMed

    Kanďár, Roman; Drábková, Petra; Toiflová, Tereza; Čegan, Alexander

    2016-01-01

    The determination of amino acids can be a reliable approach for extended diagnosis of liver diseases. This is because liver disease can be a cause of impaired amino acid metabolism. Therefore, a method for the determination of serum amino acids, applicable for clinical purposes, is necessary. The aim of this study was to find differences in the levels of selected amino acids between patients with liver disease and a control group. Samples of peripheral venous blood were obtained from a group of patients with liver disease (n = 131, 59 women at an average age of 60 years and 72 men at an average age of 52 years) and a control group (n = 105, 47 women at an average age of 62 years and 58 men at an average age of 58 years). Before the separation, the amino acids were derivatized with naphthalene-2,3-dicarboxaldehyde. For the separation, reverse phase column was used. The effluent was monitored with a fluorescence detector. There were significant differences in the concentrations of some amino acids between the patients and the control group, but also between women and men. Correlations between some amino acids and markers of liver blood tests and lipid metabolism were observed. A simple, relatively rapid and selective HPLC method with fluorescence detection for the determination of selected amino acids in serum has been developed.

  1. Amino Acids Composition of Teucrium Nutlet Proteins and their Systematic Significance

    PubMed Central

    JUAN, R.; PASTOR, J.; MILLÁN, F.; ALAIZ, M.; VIOQUE, J.

    2004-01-01

    • Background and Aims Plant species are considered as a good source of dietary proteins, although the nutritional quality of proteins depends on their amino acid composition. In this work the protein content and amino acid composition of nutlets of 21 Teucrium taxa (Lamiaceae) from Spain were analysed and their nutritional quality was compared with the minimum values established by the Food and Agriculture Organization of the United Nations (FAO). In addition, the amino acid composition was evaluated as a chemical character to clarify the taxonomic complexity in this genus. • Methods Amino acid content of nutlets was determined after derivatization with diethyl ethoxymethylenemalonate by high-performance liquid chromatography. Previously, nutlets samples were hydrolysed and incubated in an oven at 110 °C for 24 h. • Key Results The protein content was variable, ranging from 6·4 % in T. dunense to 43·8 % in T. algarbiense. According to the FAO values all taxa contain satisfactory amounts of leucine, threonine and valine and are deficient in lysine. The similarity analysis of Teucrium taxa using amino acid composition data did not clearly reflect the infrageneric classification of this genus. • Conclusions Annual species, such as T. spinosum, T. aristatum and T. resupinatum showed a better balanced amino acid composition. The dendrogram partly matched with the karyological complexity of Teucrium. No correlation between amino acid composition and habitat has been observed, showing that Teucrium nutlet amino acid composition may not be strongly influenced by the environment. PMID:15329333

  2. Assessment of thiol compounds from garlic by automated headspace derivatized in-needle-NTD-GC-MS and derivatized in-fiber-SPME-GC-MS.

    PubMed

    Warren, Jamie Marcus; Parkinson, Don-Roger; Pawliszyn, Janusz

    2013-01-23

    This study investigates the analysis of thiol compounds using a needle trap device (HS-NTD) and solid-phase microextraction (HS-SPME) derivatized headspace techniques coupled to GC-MS. Thiol compounds and their outgassed products are particularly difficult to monitor in foodstuffs. It was found that with in-needle and in-fiber derivatization, using the derivatization agent N-phenylmaleimide, it was possible to enhance the selectivity toward thiol, which allowed the quantitation of butanethiol, ethanethiol, methanethiol, and propanethiol compounds found in fresh garlic. A side-hole NTD was prepared and packed in house and utilized mixed DVB and Carboxen polymer extraction phases made of 60-80 mesh particles. NTD sampling was accomplished in the exhaustive sampling mode, where breakthrough was negligible. This work demonstrates a new application for a side-hole NTD sampling. A commercial mixed polymer phase of polydimethylsiloxane (PDMS) and divinylbenzene polymer (DVB) SPME fiber was used for SPME extractions. Under optimized derivatization, extraction, and analysis conditions for both NTD-GC-MS and SPME-GC-MS techniques, automated sampling methods were developed for quantitation. Both methods demonstrate a successful approach to thiol determination and provide a quantitative linear response between <0.1 and 10 mg L(-1) (R(2) = 0.9996), with limits of detection (LOD) in the low micrograms per liter range for the investigated thiols. Addition methods using known spiked quantities of thiol analytes in ground garlic facilitated method validation. Carry-over was also negligible for both SPME and NTD under optimized conditions.

  3. Universal solid-phase approach for the immobilization, derivatization, and resin-to-resin transfer reactions of boronic acids.

    PubMed

    Gravel, Michel; Thompson, Kim A; Zak, Mark; Bérubé, Christian; Hall, Dennis G

    2002-01-11

    Boronic acid-containing molecules are employed in a broad range of biological, medicinal, and synthetic applications. These compounds, however, tend to be difficult to handle by solution-phase methods. Herein, this problem is addressed with the development of the first general solid-phase approach for the derivatization of functionalized boronic acids. This approach is based on the use of a diethanolamine resin anchor that facilitates boronic acid immobilization by avoiding the need for exhaustive removal of water in the esterification process. The immobilization of a wide variety of boronic acids onto N,N-diethanolaminomethyl polystyrene (DEAM-PS, 1) can be performed within minutes by simple stirring in anhydrous solvents at room temperature. Evidence for the formation of a bicyclic diethanolamine boronate with putative N-B coordination was shown by (1)H NMR analysis of DEAM-PS-supported p-tolylboronic acid. The hydrolytic cleavage of the same model boronic acid from the DEAM-PS resin was studied by UV spectroscopy. Hydrolysis and attachment were shown to occur under a rapidly attained equilibrium, and a large excess of water (>32 equiv) is required to effect a practically quantitative release of boronic acids from DEAM-PS. Despite their relative sensitivity to water and alcohols, DEAM-PS-bound arylboronic acids functionalized with a formyl, a bromomethyl, a carboxyl, or an amino group can be transformed in good to excellent yields into a wide variety of amines, amides, anilides, and ureas, respectively. Ugi multicomponent reactions on DEAM-PS-supported aminobenzeneboronic acids, derivatization of multifunctional arylboronic acids, and sequential reactions can also be carried out efficiently. These new DEAM-PS-supported arylboronic acids can be employed directly into resin-to-resin transfer reactions (RRTR). This type of multiresin process helps eliminate time-consuming cleavage and transfer operations, thereby considerably simplifying the outlook of combinatorial

  4. Amino Acid Crossword Puzzle

    ERIC Educational Resources Information Center

    Sims, Paul A.

    2011-01-01

    Learning the 20 standard amino acids is an essential component of an introductory course in biochemistry. Later in the course, the students study metabolism and learn about various catabolic and anabolic pathways involving amino acids. Learning new material or concepts often is easier if one can connect the new material to what one already knows;…

  5. Amino Acid Crossword Puzzle

    ERIC Educational Resources Information Center

    Sims, Paul A.

    2011-01-01

    Learning the 20 standard amino acids is an essential component of an introductory course in biochemistry. Later in the course, the students study metabolism and learn about various catabolic and anabolic pathways involving amino acids. Learning new material or concepts often is easier if one can connect the new material to what one already knows;…

  6. GC-MS analysis of amino acid enantiomers as their N(O,S)-perfluoroacyl perfluoroalkyl esters: application to space exploration

    NASA Astrophysics Data System (ADS)

    Zampolli, M.; Sternberg, R.; Szopa, C.; Pietrogrande, M. C.; Buch, A.; Dondi, F.; Raulin, F.

    The search for optical activity in extraterrestrial sample is an important key for the study of the origin of life With this aim detection of chemical biomarkers i e of organic molecules which play an important role in biochemistry will be one of the main goals of future space missions To reach this goal an investigation of a GC-MS method based on the derivatization of amino acids by using a mixture of perfluorinated alcohols and perfluorinated anhydrides has been performed Amino acids are converted in their N O S -perfluoroacyl perfluoroalkyl esters in a single step procedure using different combinations of the derivatization reagents trifluoroacetic anhydride TFAA - 2 2 2-trifluoro-1-ethanol TFE TFAA-2 2 3 3 4 4 4-heptafluoro-1-butanol HFB heptafluorobutyric anhydride HFBA -HFB The obtained derivatives are analyzed using two different chiral columns a Chirasil-L-Val and a gammat cyclodextrin Rt- gamma -DEXsa stationary phases which show different and complementary enantiomeric selectivity The mass spectra of the derivatized compounds are studied and mass fragmentation patterns are proposed significant fragment ions can be identified to detect amino acid derivatives The obtained results are compared in terms of the enantiomeric separation achieved and mass spectrometric response Linearity studies and the measurement of the limit of detection LOD prove that the proposed method is suitable for a quantitative determination of several amino acids enantiomers 1 The use of a PTV Programmed Temperature Vaporiser

  7. Amino acid analysis in micrograms of meteorite sample by nanoliquid chromatography-high-resolution mass spectrometry.

    PubMed

    Callahan, Michael P; Martin, Mildred G; Burton, Aaron S; Glavin, Daniel P; Dworkin, Jason P

    2014-03-07

    Amino acids and their enantiomers in a 360 microgram sample of Murchison meteorite were unambiguously identified and quantified using chemical derivatization and nanoliquid chromatography coupled to nanoelectrospray ionization high resolution orbitrap mass spectrometry techniques. The distribution and abundance of amino acids were similar to past studies of Murchison meteorite but the samples used here were three orders of magnitude lower. The analytical method was also highly sensitive, and some amino acid reference standards were successfully detected at a level of ∼200 attomoles (on column). These results may open up the possibility for investigating other less studied, sample-limited extraterrestrial samples (e.g., micrometeorites, interplanetary dust particles, and cometary particles) for biologically-relevant organic molecules.

  8. Aerosol-Forming Reactions of Glyoxal, Methylglyoxal and Amino Acids in Clouds

    NASA Astrophysics Data System (ADS)

    de Haan, D. O.; Smith, K. W.; Stroik, D. R.; Corrigan, A. L.; Lee, F. E.; Phan, J. T.; Conley, A. C.

    2008-12-01

    Glyoxal and methylglyoxal are two common aldehydes present in fog and cloud water. Amino acids are present in clouds at similar concentrations. Here we present bulk and aerosol mass spectroscopic data demonstrating that irreversible reactions between glyoxal and amino acids, triggered by droplet evaporation, produce N-derivatized imidazole compounds along with deeply colored Maillard reaction products. These reactions can occur in the dark and in the absence of oxidants. Reactions between methylglyoxal and amino acids produce analogous methylated products plus oligomers with masses up to m/z = 1000. These reactions, which go to completion on the 10-min-timescale of cloud processing, could be significant sources of secondary organic aerosol and humic-like substances (HULIS or brown carbon).

  9. Stable isotope labeling by amino acids in cell culture for quantitative proteomics.

    PubMed

    Ong, Shao-En; Mann, Matthias

    2007-01-01

    Mass spectrometry (MS)-based quantitative proteomics is an increasingly popular approach to study changes in protein abundances in biological samples. Stable isotope labeling by amino acids in cell culture (SILAC), one of the more widely used methods for quantitative proteomics, is a metabolic-labeling strategy that encodes whole cellular proteomes. Cells are grown in a culture medium where the natural form of an amino acid is replaced with a stable isotope form, such as arginine bearing six 13C atoms. Incorporation of the "heavy" amino acid occurs through cell growth, protein synthesis, and turnover. SILAC allows "light" and "heavy" proteomes to be distinguished by MS while avoiding any chemical derivatization and associated purification. In this chapter, we provide detailed SILAC protocols and explain how to incorporate SILAC into any experiment.

  10. On-matrix Derivatization for Dynamic Headspace Sampling of Nonvolatile Surface Residues

    SciTech Connect

    Harvey, Scott D.; Wahl, Jon H.

    2012-09-01

    The goal of this study is to extend sampling by the field and laboratory emission cell (FLEC) purge-and-trap technique to applications that target nonvolatile residues. On-matrix derivatization of residues to render analytes stable and more volatile is explored to achieve this goal. Results show that on-matrix derivatizations of nerve agent hydrolysis products (monoalkyl methylphosphonic acids and methylphosphonic acid [MPA]) with diazomethane were successful on glass and painted wallboard (at the 10-µg level). It also was successful on the more difficult concrete (at the 500-µg level) and carpet (at the 20-µg level) substrates that cannot be successfully sampled using swipe techniques. Analysis of additional chemical warfare (CW)-associated residues can be approached by on-matrix derivatization with trifluoroacetic anhydride (TFAA). For example, amines (used as stabilizers or present as decomposition products of the nerve agent VX) or thiodiglycol (hydrolysis product of sulfur mustard) could be sampled as their TFAA derivatives from glass, painted wallboard, and concrete (at the 40-µg level), as well as carpet (at the 80-µg level) surfaces. Although the amine and thiodiglycol are semi-volatile and could be sampled directly, derivatization improves the recovery and chromatographic behavior of these analytes.

  11. On-tissue chemical derivatization of 3-methoxysalicylamine for MALDI-imaging mass spectrometry

    PubMed Central

    Chacon, Almary; Zagol-Ikapitte, Irene; Amarnath, Venkataraman; Reyzer, Michelle L.; Oates, John A.; Caprioli, Richard M.; Boutaud, Olivier

    2011-01-01

    MALDI-imaging mass spectrometry (IMS) has been shown to be a powerful tool to study drug distributions in organ tissue as well as whole animal bodies. Nevertheless, not all drugs are amenable to MALDI while others may be limited by poor sensitivity poor sensitivity. The use of chemical derivatization to improve detection of small molecules by mass spectrometry techniques is well documented. To our knowledge, however, this approach has not been applied to direct tissue analysis of small organic molecules. In this manuscript, we demonstrate the use of on-tissue chemical derivatization of a small organic molecule, 3-methoxysalicylamine (3-MoSA) a scavenger of γ -ketoaldehydes. Derivatization of 3-MoSA with 1,1′-thiocarbonyldiimidazole (TCDI) results in an oxothiazolidine derivative which is detected with much greater sensitivity by MALDI than 3-MoSA itself. TCDI treatment of tissue from mice dosed with 3-MoSA allowed images to be obtained showing its spatial distribution as well as its pharmacokinetic profile in different organs. These images correlated well with results obtained from HPLC-MS/MS analyses of the same tissues. These results provide proof-of-concept that on-tissue chemical derivatization can be used to improve detection of a small organic molecule by MALDI-IMS. PMID:21834023

  12. Liquid chromatographic analysis of aflatoxin using post-column photochemical derivatization: collaborative study.

    PubMed

    Waltking, Arthur E; Wilson, David

    2006-01-01

    Aflatoxin analysis, with post-column derivatization using a photochemical reactor for enhanced detection (PHRED) system for derivatization, has been compared to the officially recognized iodine and Kobra cell derivatization systems. This photochemical system has been extensively used for screening peanuts by some U.S. Department of Agriculture laboratories for many years. From their periodic method checks, using standard spiked samples, an 80 sample series with each of the 3 derivatization methods was statistically analyzed. Paired comparisons, using the same sample extract, were also made between the PHRED and one of the other 2 methods, among laboratories in 4 different countries, on a variety of naturally contaminated commodity products. The differences between the techniques were not significant for peanuts, but for corn the photochemical system consistently gave slightly higher values for aflatoxins B1 and B2 than the Kobra cell method. However, a comparison of all sample results showed no significant differences between methods. The Pearson correlation coefficients for aflatoxin B1 in 102 test samples and aflatoxin B2 in 94 test samples were 0.9994 and 0.9874, respectively. The probability factor was P < 0.0001, and the t-tests were not significantly different except for the corn. These indicated that the PHRED system is equivalent to the iodine and Kobra cell methods for peanuts relative to the current official procedures, but the PHRED system has a slightly high bias for corn compared to the iodine and Kobra cell systems.

  13. Chemical derivatization for electrospray ionization mass spectrometry. 1. Alkyl halides, alcohols, phenols, thiols, and amines

    SciTech Connect

    Quirke, J.M.E.; Adams, C.L.; Van Berkel, G.J. )

    1994-04-15

    Derivatization strategies and specific derivatization reactions for conversion of simple alkyl halides, alcohols, phenols, thiols, and amines to ionic or solution-ionizable derivatives, that is [open quotes]electrospray active[close quotes] (ES-active) forms of the analyte, are presented. Use of these reactions allows detection of analytes among those listed that are not normally amenable to analysis by electrospray ionization mass spectrometry (ES-MS). In addition, these reactions provide for analysis specificity and flexibility through functional group specific derivatization and through the formation of derivatives that can be detected in positive ion or in negative ion mode. For a few of the functional groups, amphoteric derivatives are formed that can be analyzed in either positive or negative ion modes. General synthetic strategies for transformation of members of these five compound classes to ES-active species are presented along with illustrative examples of suitable derivatives. Selected derivatives were prepared using model compounds and the ES mass spectra obtained for these derivatives are discussed. The analytical utility of derivatization for ES-MS analysis is illustrated in three experiments: (1) specific detection of the major secondary alcohol in oil of peppermint, (2) selective detection of phenols within a synthetic mixture of phenols, and (3) identification of the medicinal amines within a commercially available cold medication as primary, secondary or tertiary. 65 refs., 3 figs., 3 tabs.

  14. Derivatization of castor oil based estolide esters: Preparation of epoxides and cyclic carbonates

    USDA-ARS?s Scientific Manuscript database

    Estolides that are based on castor oil and oleic acid are versatile starting points for the production of industrial fluids with new properties. A variety of unsaturated estolides were derivatized by epoxidation with hydrogen peroxide. The epoxidized estolides were further modified using supercritic...

  15. LC-MSn Analysis of Isomeric Chondroitin Sulfate Oligosaccharides Using a Chemical Derivatization Strategy

    PubMed Central

    Huang, Rongrong; Pomin, Vitor H.; Sharp, Joshua S.

    2011-01-01

    Improved methods for structural analyses of glycosaminoglycans (GAGs) are required to understand their functional roles in various biological processes. Major challenges in structural characterization of complex GAG oligosaccharides using liquid chromatography-mass spectrometry (LC-MS) include the accurate determination of the patterns of sulfation due to gas-phase losses of the sulfate groups upon collisional activation and inefficient on-line separation of positional sulfation isomers prior to MS/MS analyses. Here, a sequential chemical derivatization procedure including permethylation, desulfation, and acetylation was demonstrated to enable both on-line LC separation of isomeric mixtures of chondroitin sulfate (CS) oligosaccharides and accurate determination of sites of sulfation by MSn. The derivatized oligosaccharides have sulfate groups replaced with acetyl groups, which are sufficiently stable to survive MSn fragmentation and reflect the original sulfation patterns. A standard reversed-phase LC-MS system with a capillary C18 column was used for separation, and MSn experiments using collision-induced dissociation (CID) were performed. Our results indicate that the combination of this derivatization strategy and MSn methodology enables accurate identification of the sulfation isomers of CS hexasaccharides with either saturated or unsaturated nonreducing ends. Moreover, derivatized CS hexasaccharide isomer mixtures become separable by LC-MS method due to different positions of acetyl modifications. PMID:21953261

  16. On-matrix derivatization for dynamic headspace sampling of nonvolatile surface residues.

    PubMed

    Harvey, Scott D; Wahl, Jon H

    2012-09-21

    The goal of this study is to extend sampling by the field and laboratory emission cell (FLEC) dynamic headspace technique to applications that target nonvolatile residues. On-matrix derivatization of residues to render analytes stable and more volatile is explored to achieve this goal. Results show that on-matrix derivatizations of nerve agent hydrolysis products (monoalkyl methylphosphonic acids and methylphosphonic acid [MPA]) with diazomethane were successful on glass and painted wallboard (at the 10-μg level). It also was successful on the more difficult concrete (at the 500-μg level) and carpet (at the 20-μg level), substrates that cannot be successfully sampled using swipe techniques. Analysis of additional chemical warfare (CW)-associated residues can be approached by on-matrix derivatization with trifluoroacetic anhydride (TFAA). For example, amines (used as stabilizers or present as decomposition products of the nerve agent VX) or thiodiglycol (hydrolysis product of sulfur mustard) could be sampled as their TFAA derivatives from glass, painted wallboard, and concrete (at the 40-μg level), as well as carpet (at the 80-μg level) surfaces. Although the amine and thiodiglycol are semi-volatile and could be sampled directly, derivatization improves the recovery and chromatographic behavior of these analytes.

  17. Reversible derivatization to enhance enzymatic synthesis: Chemoenzymatic synthesis of Doxorubicin-14-O-Esters

    USDA-ARS?s Scientific Manuscript database

    An efficient three-step, chemoenzymatic synthesis of unprotected doxorubicin-14-O-esters from doxorubicin hydrochloride salt is described. The key step is a lipase-catalyzed regioselective transesterification/esterification using commercially-available acyl donors and doxorubicin reversibly derivat...

  18. Recent developments and applications of EMMA in enzymatic and derivatization reactions.

    PubMed

    Zhang, Jie; Hoogmartens, Jos; Van Schepdael, Ann

    2010-01-01

    This review covers the time period of 2007 until mid-2009 and describes new developments in the field of electrophoretically mediated microanalysis. The review is subdivided in two parts dealing with (i) enzymatic and (ii) derivatization or chemical reactions. A compilation of the relevant literature is given for each part.

  19. Extraction and derivatization of Leucaena leucocephala (Lam.) galactomannan: Optimization and characterization.

    PubMed

    Mittal, Neeraj; Mattu, Pooja; Kaur, Gurpreet

    2016-11-01

    Water soluble gums also known as hydrocolloids are increasingly finding applications in the pharmaceutical and food industry due to their versatile functional properties. They possess considerable use in food and pharmaceutical industries as emulsifying, thickening and gelling agents. In the present investigation a heteropolysaccharide galactomannan was extracted from Leucaena leucocephala (Lam.) seeds by an aqueous method, characterized for its compositional analysis (mannose: galactose ratio), physicochemical and functional properties (solubility), and mechanical properties. The extracted gum was derivatized to form its carboxymethyl derivative and the method of its derivatization was optimized by varying the reaction parameters. The native and derivatized gum was characterized by FTIR, XRD, DSC, NMR, SEM and elemental analysis, etc. The yield of Leucaena leucocephala galactomannan (LLG) was found to be 20% (w/w). The optimized parameters for carboxymethylation reaction (degree of substitution 0.805) were found to be 6.0g NaOH, 10.0g MCA, at 60°C for 4h. The physicochemical and functional characteristics of native and derivatized gum suggest its potential role in food and pharmaceutical industries.

  20. Synthesis of 2-substituted tetraphenylenes via transition-metal-catalyzed derivatization of tetraphenylene

    PubMed Central

    Pan, Shulei; Jiang, Hang; Zhang, Yu; Chen, Dushen

    2016-01-01

    Summary A new strategy for the synthesis of 2-substituted tetraphenylenes through a transition-metal-catalyzed derivatization has been developed. Three types of functionalities, including OAc, X (Cl, Br, I) and carbonyl, were introduced onto tetraphenylene, which allows the easy access to a variety of monosubstituted tetraphenylenes. These reactions could accelerate research on the properties and application of tetraphenylene derivatives. PMID:27559378

  1. Determination of memantine in plasma and vitreous humour by HPLC with precolumn derivatization and fluorescence detection.

    PubMed

    Puente, Belen; Hernandez, Esther; Perez, Susana; Pablo, Luis; Prieto, Esther; Garcia, Maria Angeles; Bregante, Miguel Angel

    2011-01-01

    A new HPLC procedure with precolumn derivatization and rimantadine as the internal standard for determining memantine, a candidate agent for the treatment of glaucoma in plasma and vitreous humour, has been developed and validated. Precolumn derivatization was performed with 9-fluorenylmethyl-chloroformate-chloride (FMOC-Cl) as the derivatization reagent and followed by a liquid-liquid extraction with n-hexane. Optimal conditions for derivatization were an FMOC-Cl concentration of 1.5 mM, a reaction time of 20 min, the temperature at 30°C, the borate buffer pH 8.5, and a borate buffer-acetonitrile ratio of 1:1. The derivatives were analyzed by isocratic HPLC with the fluorescence detector λex 260 nm λem 315 nm on a Novapack C(18) reversed-phase column with a mobile phase of acetonitrile-water (73:27, v/v), 40°C, and a flow rate of 1.2 mL/min. The linear range was 10-1000 ng/mL with a quantification limit of ∼ 10 ng/mL for both types of samples. This analytical method may be suitable for using in ocular availability studies.

  2. Derivatization followed by gas chromatography-mass spectrometry for quantification of ethyl carbamate in alcoholic beverages.

    PubMed

    Xu, Xuejiao; Gao, Yihan; Cao, Xiujun; Wang, Xiang; Song, Guoxin; Zhao, Jianfeng; Hu, Yaoming

    2012-04-01

    A sensitive and rapid analytical methodology based on derivatization followed by gas chromatography-mass spectrometry (GC-MS) was developed for the quantitative determination of the toxic contaminant ethyl carbamate (EC, urethane, C(2)H(5)OCONH(2)) in alcoholic samples. EC was extracted using liquid-liquid extraction technique, and then silylated with bis-(trimethylsilyl)trifluoroacetamide, analysed finally by GC-MS. The isopropyl carbamate was used as the internal standard for quantitative analysis of EC in alcoholic samples. In this work, the sample extraction and derivatization reaction conditions were investigated, and the optimal extraction conditions obtained were: pH 9 and solvent of ethyl acetate, and the derivatization conditions were: derivatization reaction temperature of 80°C and time duration of 30 min. With the optimal conditions, the method validations were also studied. In the validation studies, EC exhibited good linearity with a regression coefficient of 0.9999. The limit of detection and limit of quantification were 0.30 and 5.0 μg/kg, respectively. The precision was less than 8.4%. Finally, the proposed technique was successfully applied to the analysis of EC in 35 kinds of alcoholic samples. The experimental results have demonstrated that the proposed technique is a fast, reliable and low-cost method for determination of EC in alcoholic samples. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. Robust analysis of underivatized free amino acids in soil by hydrophilic interaction liquid chromatography coupled with electrospray tandem mass spectrometry.

    PubMed

    Gao, Jiajia; Helmus, Rick; Cerli, Chiara; Jansen, Boris; Wang, Xiang; Kalbitz, Karsten

    2016-06-03

    Amino acids are an important and highly dynamic fraction of organic N in soils and their determination in soil without derivatization is challenging due to the difficulties in separation and detection of trace amounts of these polar analytes. In the present work, we developed an analytical method to quantify 20 free amino acids in aqueous soil extracts without derivatization. The method employed hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS) technique combined with a cation exchange solid phase extraction (SPE). Four stable isotope labelled amino acids were used as internal standards to improve the method performance. Good separation of 20 underivatized amino acids was achieved within 12min. The limit of detection (LODs) and limit of quantification (LOQs) were in the range of 13-384ngg(-1) and 43-1267ngg(-1) (dry soil basis), respectively. The results showed that overall recoveries with high precision were obtained for the extracted free amino acids from ten different soils. The overall recoveries of 18 amino acids were similar for the ten soils used, which differed substantially in organic C content and in other properties as soil texture and pH. For most of the amino acids, the average recoveries from soil extracts were between 74% and 117%, with the exception of Met (31%), Pro (52%) and Arg (68%). Variability was within acceptable limits (relative standard deviations were between 4% and 13%), with the exception of Met (relative standard deviation=90%) and Arg (relative standard deviation=53%). Thus the proposed method with high throughout and high analyte specificity shows great promise for consistent analysis of free amino acids extracted from soils and offers new horizons for the analysis of amino acids in terrestrial and aquatic ecosystem. Copyright © 2016 Elsevier B.V. All rights reserved.

  4. Analysis of neurotoxin 3-N-oxalyl-L-2,3-diaminopropionic acid and its alpha-isomer in Lathyrus sativus by high-performance liquid chromatography with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) derivatization.

    PubMed

    Chen, X; Wang, F; Chen, Q; Qin, X C; Li, Z

    2000-08-01

    A method was developed for the quantitative determination of the neurotoxic nonprotein amino acid, 3-N-oxalyl-L-2,3-diaminopropionic acid (beta-ODAP), and its nontoxic alpha-isomer, 2-N-oxalyl-L-2, 3-diaminopropionic acid (alpha-ODAP), in the plant samples of Lathyrus sativus after derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) by reversed-phase high-performance liquid chromatography (HPLC). Hippuric acid was used as an internal standard. A linear response was recorded in the concentration rang 0.32-32 nmol with r > 0.999. The RP HPLC detection limit for both isomers is 1.8 ng. According to different experimental needs, a ternary gradient system can be used to determine toxin and other nonprotein amino acids. The RP HPLC method and a colorimetric method were compared for measuring ODAP.

  5. Stable isotope coded derivatizing reagents as internal standards in metabolite profiling.

    PubMed

    Bruheim, Per; Kvitvang, Hans Fredrik Nyvold; Villas-Boas, Silas G

    2013-06-28

    Gas chromatography (GC) and liquid chromatography (LC) coupled to mass spectrometric (MS) detection have become the two main techniques for the analysis of metabolite pools (i.e. Metabolomics). These technologies are especially suited for Metabolite Profiling analysis of various metabolite groups due to high separation capabilities of the chromatographs and high sensitivity of the mass analysers. The trend in quantitative Metabolite Profiling is to add more metabolites and metabolite groups in a single method. This should not be done by compromising the analytical precision. Mass spectrometric detection comes with certain limitations, especially in the quantitative aspects as standards are needed for conversion of ion abundance to concentration and ionization efficiencies are directly dependent on eluent conditions. This calls for novel strategies to counteract all variables that can influence the quantitative precision. Usually, internal standards are used to correct any technical variation. For quantitation of single or just a few analytes this can be executed with spiking isotopically labeled standards. However, for more comprehensive analytical tasks, e.g. profiling tens or hundreds of analytes simultaneously, this strategy becomes expensive and in many cases isotopically labeled standards are not available. An alternative is to introduce a derivatizing step where the sample is derivatized with naturally labeled reagent, while a standard solution is separately derivatized with isotopically labeled reagent and spiked into the sample solution prior to analysis. This strategy, named isotope coded derivatization - ICD, is attractive in the emerging field of quantitative Metabolite Profiling where current protocols can easily comprise over hundred metabolites. This review provides an overview of isotopically labeled derivatizing reagents that have been developed for important metabolite groups with the aim to improve analytical performance and precision.

  6. One drop chemical derivatization--DESI-MS analysis for metabolite structure identification.

    PubMed

    Lubin, Arnaud; Cabooter, Deirdre; Augustijns, Patrick; Cuyckens, Filip

    2015-07-01

    Structural elucidation of metabolites is an important part during the discovery and development process of new pharmaceutical drugs. Liquid Chromatography (LC) in combination with Mass Spectrometry (MS) is usually the technique of choice for structural identification but cannot always provide precise structural identification of the studied metabolite (e.g. site of hydroxylation and site of glucuronidation). In order to identify those metabolites, different approaches are used combined with MS data including nuclear magnetic resonance, hydrogen/deuterium exchange and chemical derivatization followed by LC-MS. Those techniques are often time-consuming and/or require extra sample pre-treatment. In this paper, a fast and easy to set up tool using desorption electrospray ionization-MS for metabolite identification is presented. In the developed method, analytes in solution are simply dried on a glass plate with printed Teflon spots and then a single drop of derivatization mixture is added. Once the spot is dried, the derivatized compound is analyzed. Six classic chemical derivatizations were adjusted to work as a one drop reaction and applied on a list of compounds with relevant functional groups. Subsequently, two successive reactions on a single spot of amoxicillin were tested and the methodology described was successfully applied on an in vitro incubated alprazolam metabolite. All reactions and analyses were performed within an hour and gave useful structural information by derivatizing functional groups, making the method a time-saving and efficient tool for metabolite identification if used in addition or in some cases as an alternative to common methods.

  7. Chemical Derivatization of Peptide Carboxyl Groups for Highly Efficient Electron Transfer Dissociation

    NASA Astrophysics Data System (ADS)

    Frey, Brian L.; Ladror, Daniel T.; Sondalle, Samuel B.; Krusemark, Casey J.; Jue, April L.; Coon, Joshua J.; Smith, Lloyd M.

    2013-11-01

    The carboxyl groups of tryptic peptides were derivatized with a tertiary or quaternary amine labeling reagent to generate more highly charged peptide ions that fragment efficiently by electron transfer dissociation (ETD). All peptide carboxyl groups—aspartic and glutamic acid side-chains as well as C-termini—were derivatized with an average reaction efficiency of 99 %. This nearly complete labeling avoids making complex peptide mixtures even more complex because of partially-labeled products, and it allows the use of static modifications during database searching. Alkyl tertiary amines were found to be the optimal labeling reagent among the four types tested. Charge states are substantially higher for derivatized peptides: a modified tryptic digest of bovine serum albumin (BSA) generates ~90% of its precursor ions with z > 2, compared with less than 40 % for the unmodified sample. The increased charge density of modified peptide ions yields highly efficient ETD fragmentation, leading to many additional peptide identifications and higher sequence coverage (e.g., 70 % for modified versus only 43 % for unmodified BSA). The utility of this labeling strategy was demonstrated on a tryptic digest of ribosomal proteins isolated from yeast cells. Peptide derivatization of this sample produced an increase in the number of identified proteins, a >50 % increase in the sequence coverage of these proteins, and a doubling of the number of peptide spectral matches. This carboxyl derivatization strategy greatly improves proteome coverage obtained from ETD-MS/MS of tryptic digests, and we anticipate that it will also enhance identification and localization of post-translational modifications.

  8. Post-column labeling techniques in amino acid analysis by liquid chromatography.

    PubMed

    Rigas, Pantelis G

    2013-10-01

    Amino acid analysis (AAA) has always presented an analytical challenge in terms of sample preparation, separation, and detection. Because of the vast number of amino acids, various separation methods have been applied taking into consideration the large differences in their chemical structures, which span from nonpolar to highly polar side chains. Numerous separation methods have been developed in the past 60 years, and impressive achievements have been made in the fields of separation, derivatization, and detection of amino acids (AAs). Among the separation methods, liquid chromatography (LC) prevailed in the AAA field using either pre-column or post-column labeling techniques in order to improve either separation of AAs or selectivity and sensitivity of AAA. Of the two approaches, the post-column technique is a more rugged and reproducible method and provides excellent AAs separation relatively free from interferences. This review considers current separations combined with post-column labeling techniques for AAA, comparison with the pre-column methods, and the strategies used to develop effective post-column methodology. The focus of the article is on LC methods coupled with post-column labeling techniques and studying the reactions to achieve optimum post-column derivatization (PCD) conditions in order to increase sensitivity and selectivity using various types of detectors (UV-Vis, fluorescence, electrochemical etc.) and illustrating the versatility of the PCD methods for practical analysis.

  9. Derivatization of carbohydrates for GC and GC-MS analyses.

    PubMed

    Ruiz-Matute, A I; Hernández-Hernández, O; Rodríguez-Sánchez, S; Sanz, M L; Martínez-Castro, I

    2011-05-15

    GC and GC-MS are excellent techniques for the analysis of carbohydrates; nevertheless the preparation of adequate derivatives is necessary. The different functional groups that can be found and the diversity of samples require specific methods. This review aims to collect the most important methodologies currently used, either published as new procedures or as new applications, for the analysis of carbohydrates. A high diversity of compounds with diverse functionalities has been selected: neutral carbohydrates (saccharides and polyalcohols), sugar acids, amino and iminosugars, polysaccharides, glycosides, glycoconjugates, anhydrosugars, difructose anhydrides and products resulting of Maillard reaction (osuloses, Amadori compounds). Chiral analysis has also been considered, describing the use of diastereomers and derivatives to be eluted on chiral stationary phases.

  10. A sensitive and simple ultra-high-performance-liquid chromatography-tandem mass spectrometry based method for the quantification of D-amino acids in body fluids.

    PubMed

    Visser, Wouter F; Verhoeven-Duif, Nanda M; Ophoff, Roel; Bakker, Steven; Klomp, Leo W; Berger, Ruud; de Koning, Tom J

    2011-10-07

    D-Amino acids are increasingly being recognized as important signaling molecules in mammals, including humans. D-Serine and D-aspartate are believed to act as signaling molecules in the central nervous system. Interestingly, several other D-amino acids also occur in human plasma, but very little is currently known regarding their function and origin. Abnormal levels of D-amino acids have been implicated in the pathogenesis of different diseases, including schizophrenia and amyotrophic lateral sclerosis (ALS), indicating that D-amino acid levels hold potential as diagnostic markers. Research into the biological functions of D-amino acids is hindered, however, by the lack of sufficiently sensitive, high-throughput analytical methods. In particular, the interference of large amounts of L-amino acids in biological samples and the low concentrations of D-amino acids are challenging. In this paper, we compared 7 different chiral derivatization agents for the analysis of D-amino acids and show that the chiral reagent (S)-NIFE offers outstanding performance in terms of sensitivity and enantioselectivity. An UPLC-MS/MS based method for the quantification of D-amino acids human biological fluids was then developed using (S)-NIFE. Baseline separation (R(s)>2.45) was achieved for the isomers of all 19 chiral proteinogenic amino acids. The limit of detection was <1 nM for all amino acids except d-alanine (1.98 nM), d-methionine (1.18 nM) and d-asparagine (5.15 nM). For measurements in human plasma, cerebrospinal fluid and urine, the accuracy ranged between 85% and 107%. The intra-assay and inter-assay were both <16% RSD for these three different matrices. Importantly, the method does not suffer from spontaneous racemization during sample preparation and derivatization. Using the described method, D-amino acid levels in human cerebrospinal fluid, plasma and urine were measured.

  11. Derivatization of Dextran for Multiply Charged Ion Formation and Electrospray Ionization Time-of-Flight Mass Spectrometric Analysis

    NASA Astrophysics Data System (ADS)

    Tapia, Jesus B.; Hibbard, Hailey A. J.; Reynolds, Melissa M.

    2017-06-01

    We present the use of a simple, one-pot derivatization to allow the polysaccharide dextran to carry multiple positive charges, shifting its molecular weight distribution to a lower m/z range. We performed this derivatization because molecular weight measurements of polysaccharides by mass spectrometry are challenging because of their lack of readily ionizable groups. The absence of ionizable groups limits proton abstraction and suppresses proton adduction during the ionization process, producing mass spectra with predominantly singly charged metal adduct ions, thereby limiting the detection of large polysaccharides. To address this challenge, we derivatized dextran T1 (approximately 1 kDa) by attaching ethylenediamine, giving dextran readily ionizable, terminal amine functional groups. The attached ethylenediamine groups facilitated proton adduction during the ionization process in positive ion mode. Using the low molecular weight dextran T1, we tracked the number of ethylenediamine attachments by measuring the mass shift from underivatized to derivatized dextran T1. Using electrospray ionization time-of-flight mass spectrometry, we observed derivatized dextran chains ranging from two to nine glucose residues with between one and four attachments/charges. Our success in shifting derivatized dextran T1 toward the low m/z range suggests potential for this derivatization as a viable route for analysis of high molecular weight polysaccharides using electrospray ionization time-of-flight mass spectrometry.

  12. Derivatization of Dextran for Multiply Charged Ion Formation and Electrospray Ionization Time-of-Flight Mass Spectrometric Analysis

    NASA Astrophysics Data System (ADS)

    Tapia, Jesus B.; Hibbard, Hailey A. J.; Reynolds, Melissa M.

    2017-10-01

    We present the use of a simple, one-pot derivatization to allow the polysaccharide dextran to carry multiple positive charges, shifting its molecular weight distribution to a lower m/ z range. We performed this derivatization because molecular weight measurements of polysaccharides by mass spectrometry are challenging because of their lack of readily ionizable groups. The absence of ionizable groups limits proton abstraction and suppresses proton adduction during the ionization process, producing mass spectra with predominantly singly charged metal adduct ions, thereby limiting the detection of large polysaccharides. To address this challenge, we derivatized dextran T1 (approximately 1 kDa) by attaching ethylenediamine, giving dextran readily ionizable, terminal amine functional groups. The attached ethylenediamine groups facilitated proton adduction during the ionization process in positive ion mode. Using the low molecular weight dextran T1, we tracked the number of ethylenediamine attachments by measuring the mass shift from underivatized to derivatized dextran T1. Using electrospray ionization time-of-flight mass spectrometry, we observed derivatized dextran chains ranging from two to nine glucose residues with between one and four attachments/charges. Our success in shifting derivatized dextran T1 toward the low m/ z range suggests potential for this derivatization as a viable route for analysis of high molecular weight polysaccharides using electrospray ionization time-of-flight mass spectrometry. [Figure not available: see fulltext.

  13. An HPLC method for the determination of selected amino acids in human embryo culture medium.

    PubMed

    Drábková, Petra; Andrlová, Lenka; Kanďár, Roman

    2017-02-01

    A method for the determination of selected amino acids in culture medium using HPLC with fluorescence detection is described. Twenty hours after intra-cytoplasmic sperm injection, one randomly selected zygote was transferred to the culture medium. After incubation (72 h after fertilization), the culture medium in which the embryo was incubated and blank medium was immediately stored at -80°C. Filtered medium samples were derivatized with ortho-phthalaldehyde (naphthalene-2,3-dicarboxaldehyde), forming highly fluorescent amino acids derivatives. Reverse-phase columns (LichroCART, Purospher STAR RP18e or Ascentis Express C18 ) were used for the separation. The derivatives were analyzed by gradient elution with a mobile phase containing ethanol and sodium dihydrogen phosphate. The analytical performance of this method is satisfactory for all amino acids; the intra-assay coefficients of variation were <10% and quantitative recoveries were between 95.5 and 104.4%. Changes in the levels of selected amino acids before and after human embryo cultivation were observed. After embryo incubation, the levels of all amino acids in the medium were increased, apart from aspartate and asparagine. After the cultivation of some embryos, amino acids which were not part of the medium were detected. Low amino acids turnover was observed in some embryos. Copyright © 2016 John Wiley & Sons, Ltd.

  14. Parent Body Influences on Amino Acids in the Tagish Lake Meteorite

    NASA Technical Reports Server (NTRS)

    Glavin, D. P.; Callahan, M. P.; Dworkin, J. P.; Elsila, J. E.; Herd, C. D. K.

    2010-01-01

    The Tagish Lake meteorite is a primitive C2 carbonaceous chondrite with a mineralogy, oxygen isotope, and bulk chemical. However, in contrast to many CI and CM carbonaceous chondrites, the Tagish Lake meteorite was reported to have only trace levels of indigenous amino acids, with evidence for terrestrial L-amino acid contamination from the Tagish Lake meltwater. The lack of indigenous amino acids in Tagish Lake suggested that they were either destroyed during parent body alteration processes and/or the Tagish Lake meteorite originated on a chemically distinct parent body from CI and CM meteorites where formation of amino acids was less favorable. We recently measured the amino acid composition of three different lithologies (11h, 5b, and 11i) of pristine Tagish Lake meteorite fragments that represent a range of progressive aqueous alteration in order 11h < 5b < 11i as inferred from the mineralogy, petrology, bulk isotopes, and insoluble organic matter structure. The distribution and enantiomeric abundances of the one- to six-carbon aliphatic amino acids found in hot-water extracts of the Tagish Lake fragments were determined by ultra performance liquid chromatography fluorescence detection and time of flight mass spectrometry coupled with OPA/NAC derivatization. Stable carbon isotope analyses of the most abundant amino acids in 11h were measured with gas chromatography coupled with quadrupole mass spectrometry and isotope ratio mass spectrometry.

  15. Amino Acids and Chirality

    NASA Technical Reports Server (NTRS)

    Cook, Jamie E.

    2012-01-01

    Amino acids are among the most heavily studied organic compound class in carbonaceous chondrites. The abundance, distributions, enantiomeric compositions, and stable isotopic ratios of amino acids have been determined in carbonaceous chondrites fi'om a range of classes and petrographic types, with interesting correlations observed between these properties and the class and typc of the chondritcs. In particular, isomeric distributions appear to correlate with parent bodies (chondrite class). In addition, certain chiral amino acids are found in enantiomeric excess in some chondrites. The delivery of these enantiomeric excesses to the early Earth may have contributed to the origin of the homochirality that is central to life on Earth today. This talk will explore the amino acids in carbonaceous chondritcs and their relevance to the origin of life.

  16. Potential of vancomycin for the enantiomeric resolution of FMOC-amino acids by capillary electrophoresis-ion-trap-mass spectrometry.

    PubMed

    Sánchez-Hernández, Laura; Domínguez-Vega, Elena; Montealegre, Cristina; Castro-Puyana, María; Marina, María Luisa; Crego, Antonio L

    2014-05-01

    The potential of the antibiotic vancomycin (VC) as chiral selector for the enantiomeric separation of amino acids by CE-ESI-MS/MS² was investigated for the first time in this work. Derivatization of amino acids with FMOC-Cl was carried out to enable their interaction with VC as well as the formation of precursor ions with larger m/z which were employed in MS² experiments. The partial filling of a coated capillary was employed to avoid the loss in MS sensitivity originated by the introduction of VC in the ionization source. Under optimized conditions, the simultaneous enantiomeric separation and unequivocal identification of 17 amino acids (two of them being nonprotein amino acids) took place in about 20 min with LODs in the micromolar range.

  17. Structural and chemical derivatization of graphene for electronics and sensing

    NASA Astrophysics Data System (ADS)

    Mohanty, Nihar Ranjan

    Graphene - a single atom thick two dimensional sheet of sp 2 bonded carbon atoms arranged in a honeycomb lattice - has shown great promise for both fundamental research & applications because of its unique electrical, optical, thermal, mechanical and chemical properties. Derivatization of graphene unlocks a plethora of novel properties unavailable to their pristine parent "graphene". In this dissertation we have synthesized various structural and chemical derivatives of graphene; characterized them in detail; and leveraged their exotic properties for diverse applications. We have synthesized protein/DNA/ethylenediamine functionalized derivatives of graphene via a HATU catalyzed amide reaction of primary-amine-containing moieties with graphene oxide (GO) -- an oxyfunctional graphene derivative. In contrast to non-specificity of graphene, this functionalization of GO has enabled highly specific interactions with analytes. Devices fabricated from the protein (concanavalin -- A) and DNA functionalized graphene derivatives were demonstrated to enable label-free, specific detection of bacteria and DNA molecules, respectively, with single quanta sensitivity. Room temperature electrical characterization of the sensors showed a generation of ˜ 1400 charge carriers for single bacterium attachment and an increase of 5.6 X 1012 charge carriers / cm2 for attachment of a single complementary strand of DNA. This work has shown for the first time the viability of graphene for bio-electronics and sensing at single quanta level. Taking the bio-interfacing of graphene to the next level, we demonstrate the instantaneous swaddling of a single live bacterium (Bacillus subtilis ) with several hundred sq. micron (˜ 600 mum2) areal protein-functionalized graphene sheets. The atomic impermeability and high yield strength of graphene resulted in hermetic compartmentalization of bacteria. This enabled preservation of the dimensional and topological characteristics of the bacterium against

  18. Simultaneous Solid Phase Extraction and Derivatization of Aliphatic Primary Amines Prior to Separation and UV-Absorbance Detection

    PubMed Central

    Felhofer, Jessica L.; Scida, Karen; Penick, Mark; Willis, Peter A.; Garcia, Carlos D.

    2013-01-01

    To overcome the problem of poor sensitivity of capillary electrophoresis-UV absorbance for the detection of aliphatic amines, a solid phase extraction and derivatization scheme was developed. This work demonstrates successful coupling of amines to a chromophore immobilized on a solid phase and subsequent cleavage and analysis. Although the analysis of many types of amines is relevant for myriad applications, this paper focuses on the derivatization and separation of amines with environmental relevance. This work aims to provide the foundations for future developments of an integrated sample preparation microreactor capable of performing simultaneous derivatization, preconcentration, and sample cleanup for sensitive analysis of primary amines. PMID:24054648

  19. Enhanced responses in matrix-assisted laser desorption/ionization mass spectrometry of peptides derivatized with arginine via a C-terminal oxazolone.

    PubMed

    Nakazawa, Takashi; Yamaguchi, Minoru; Nishida, Kimiko; Kuyama, Hiroki; Obama, Takashi; Ando, Eiji; Okamura, Taka-Aki; Ueyama, Norikazu; Tanaka, Koichi; Norioka, Shigemi

    2004-01-01

    We have developed a novel method for enhancing the response of a peptide in matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) by activating the C-terminal carboxyl group through an oxazolone with which is coupled an amine containing a functional group to help ionize the peptide. The reactions consist of dehydration with acetic anhydride to give an oxazolone, followed by aminolysis with an appropriate amino acid derivative such as arginine methyl ester. The MALDI signal of Ac-Tyr-Gly-Gly-Phe-Leu-Arg-OMe, thus converted from leucine-enkephalin, was detected while completely excluding the responses of arginine-deficient peptides coexisting in the reaction mixture. Some less intense peaks corresponding to a few sequential degradation products, also terminated with the arginine derivative, were also observed. The side-chain groups potentially that are reactive were conveniently protected by acetylation simultaneous with the C-terminal activation, and those that remained unprotected were reduced to virtually negligible proportions when the reaction was conducted in a peptide solution of concentration less than 1 mM. The greatly increased responses of such arginine-terminated peptides could possibly be exploited to discern the C-terminal tryptic peptide of a protein that is otherwise almost insensitive to MALDI-MS in general. The simplicity of the post-source decay spectrum of enkephalin derivatized by arginine methyl ester characteristically accentuated z- and b-type ions, and this should facilitate sequencing of such derivatized peptides. Remaining problems with practical applications of this approach are discussed.

  20. Design of chiral monochloro-s-triazine reagents for the liquid chromatographic separation of amino acid enantiomers.

    PubMed

    Brückner, H; Wachsmann, M

    2003-05-23

    A series of chiral derivatizing reagents (CDRs) was synthesized by nucleophilic replacement of one chlorine atom in cyanuric chloride (2,4,6-trichloro-1,3,5-triazine; s-triazine) by alkoxy (methoxy, butoxy, 1,1,1-trifluoroethoxy) or aryloxy groups (phenoxy, nitrophenoxy, phenylphenoxy, 4-methylcoumaryloxy), and displacement of a second chlorine by L-alanine amide, L-phenylalanine amide, L-proline tert.-butyl ester, or Boc-L-lysine tert.-butyl ester. Further, CDRs were investigated in which two chlorine atoms in cyanuric chloride were substituted consecutively by L-valine amide and L-phenylalanine amide. The resulting CDRs having a remaining reactive chlorine were tested for their capability of derivatizing DL-amino acids followed by liquid chromatographic separation of the resulting diastereomers.

  1. Analysis of steroid estrogens in water using liquid chromatography/tandem mass spectrometry with chemical derivatizations.

    PubMed

    Lin, Ying-Hsuan; Chen, Chia-Yang; Wang, Gen-Shuh

    2007-01-01

    Even in trace amounts, estrogens such as 17beta-estradiol (E2), estrone (E1), estriol (E3), and 17alpha-ethinyl estradiol (EE2) may have adverse effects on humans and the aquatic ecosystem. Therefore, it is essential to be able to measure trace amounts of steroid estrogens in water. To date, most instruments are not sensitive enough to detect these chemicals in small samples of water. Sensitivity, however, may be improved by using appropriate derivatization reagents to modify the structures of these estrogens so that their ionization efficiency is increased, making them more detectable by liquid chromatography/mass spectrometry (LC/MS). This study uses dansyl chloride, 2-fluoro-1-methylpyridinium p-toluenesulfonate (FMPTS), and pentafluorobenzyl bromide (PFBBr) as derivatization reagents to react with the phenolic estrogens to make them more detectable in water. We also test how environmental matrices (wastewater effluent, river water, and drinking water) influence the detectability of these estrogens. Both qualitative and semi-quantitative comparisons of these derivatization methods were made. We found that dansyl chloride derivatives created signal intensities one or two orders of magnitude greater than those normally found in underivatized estrogen standards. The signals derived by FMPTS were analyte-dependent, and the products derived from E1, E2, and EE2 produced 2.19 to 12.1 times the signal intensity of underivatized E1, E2, and EE2. The product derived from E3 produced weaker signals than that produced by underivatized E3. The PFBBr derivatives produced signals that were as much as 5.8 times those found in the underivatized estrogens. When these derivatization methods were applied to river water, drinking water and effluents from a sewage treatment plant (STP), the different matrices were found to significantly suppress the signals if we used electrospray ionization, though this influence became less significant if we used atmospheric pressure chemical

  2. DETERMINATION OF ALIPHATIC AMINES IN WATER USING DERIVATIZATION WITH FLUORESCEIN ISOTHIOCYANATE AND CAPILLARY ELECTROPHORESIS/LASER-INDUCED FLUORESCENCE DETECTION.

    EPA Science Inventory

    Detection-oriented derivatization of aliphatic amines and amine functional groups in coumpounds of environmental interest was studied using fluorescein isothiocyanate (FITC) with separation/determination by capillary electrophoresis/laser-induced fluorescence. Determinative level...

  3. Ultraviolet derivatization of low-molecular-mass thiols for high performance liquid chromatography and capillary electrophoresis analysis.

    PubMed

    Kuśmierek, K; Chwatko, G; Głowacki, R; Kubalczyk, P; Bald, E

    2011-05-15

    Thiols play an important role in metabolic processes of all living creatures and their analytical control is very important in order to understand their physiological and pathological function. Among a variety of methods available to measure thiol concentrations, chemical derivatization utilizing a suitable labeling reagent followed by liquid chromatographic or electrophoretic separation is the most reliable means for sensitive and specific determination of thiol compounds in real world samples. Ultraviolet detection is, for its simplicity, commonly used technique in liquid chromatography and capillary electrophoresis, and consequently many ultraviolet derivatization reagents are in used. This review summarizes HPLC and CE ultraviolet derivatization based methods, including pre-analytical considerations, procedures for sample reduction, derivatization, and separation of the primary biological aminothiols--cysteine, homocysteine, cysteinylglycine and glutathione, and most important thiol-drugs in pharmaceutical formulations and biological samples. Cognizance of the biochemistry involved in the formation of the analytes is taken.

  4. Modification of Amino Groups.

    PubMed

    Geoghegan, Kieran F

    2016-11-01

    Chemical modification of amino groups in proteins serves a diversity of preparative and analytical purposes. The most prominent is to attach nonpeptide groups with useful properties to proteins. Examples of these groups include biotin for affinity capture and fluorescent dyes for detectability. A widely applied chemistry, and one for which many reagents are available, is reaction of the activated ester of a carboxylic acid (often a succinimidyl ester) with amino groups at mildly basic pH. Reductive alkylation using a carbonyl compound and a hydride-donating reducing agent is another valued reaction with multiple applications. Most proteins contain more than one amino group, so the extent of reaction desired must be considered in advance and the result assessed experimentally after the fact. The distinctive environment of the α-amino group of a polypeptide sets it apart from the ϵ-amino groups of lysine side chains, and can afford useful specificity. © 2016 by John Wiley & Sons, Inc. Copyright © 2016 John Wiley & Sons, Inc.

  5. Dansyl chloride derivatization of methamphetamine: a method with advantages for screening and analysis of methamphetamine in urine.

    PubMed

    Yamada, Hideyuki; Yamahara, Ayako; Yasuda, Satsuki; Abe, Masamiki; Oguri, Kazuta; Fukushima, Sunao; Ikeda-Wada, Sachiko

    2002-01-01

    The screening and quantitation of methamphetamine (MP) in urine using dansyl chloride (DNC) as the derivatization reagent were studied. Urinary MP derivatized with DNC could be detected by visual observation of the fluorescence in a solid-phase extraction column such as a Sep-Pak C18 cartridge to which the whole reaction solution was applied. The DNC-derivatized MP was eluted from the cartridge and then identified and quantitated by gas chromatography-mass spectrometry (GC-MS) and high-performance liquid chromatography (HPLC). In the GC-MS analysis with the MS detector in the electron-impact mode, DNC-derivatized MP and amphetamine (AP), exhibited diagnostic molecular ion peaks. The intensities of the molecular ions were 15% (DNC-MP) and 35% (DNC-AP) of the base peak (a fragment ion because of the loss of dimethylnaphthalene from M+), demonstrating that this method of derivatization has a major advantage for confirming APs by GC-MS. MP derivatized with DNC could be determined by HPLC with ultraviolet detection. Because a good correlation (r = 0.95) between the GC-MS and HPLC method for urinary MP was confirmed, both HPLC and GC-MS appear to be useful tools for determining urinary MP. The intensity of the cartridge fluorescence due to DNC-derivatized MP was approximately related to the urinary content of MP determined by HPLC or GC-MS, although a false positive in the visual fluorescence was observed in some urinary specimens from healthy volunteers. From these results, screening and confirmation/determination following DNC derivatization is proposed as a suitable method for the analysis of MP.

  6. Sensitive micellar electrokinetic chromatography-laser-induced fluorescence method to analyze chiral amino acids in orange juices.

    PubMed

    Simó, Carolina; Barbas, Coral; Cifuentes, Alejandro

    2002-09-11

    In this work a new method to detect the existence of chiral amino acids in orange juice is presented. The method employs beta-cyclodextrins and micellar electrokinetic chromatography with laser-induced fluorescence (MEKC-LIF) to separate and detect L- and D-amino acids (L-aa and D-aa) previously derivatized with fluorescein isothiocianate (FITC). A systematic optimization of the chiral-MEKC conditions is done bringing about in less than 20 min a good separation of the main amino acids found in orange juice (i.e., Pro, Asp, Ser, Asn, Glu, Ala, Arg, and the nonchiral GABA, i.e., gamma-aminobutyric acid). Using this procedure, the analysis time reproducibility for the 15 standard compounds (L-aa, D-aa, and GABA) has been determined to be better than 0.2% (n = 5) for the same day and better than 0.7% (n = 15) for three different days. Corrected peak area reproducibility is somewhat lower, providing values better than 3.3% (n = 5) for the same day and 6.9% (n = 15) for three different days. The limit of detection using this procedure was determined to be 0.86 attomoles for L-Arg. The optimized FITC derivatization method allows the easy and straightforward detection of amino acids in orange concentrates and juices (i.e., only centrifugation of diluted samples for 5 min is needed prior to their derivatization). D-Ala, D-Asp, D-Arg, and D-Glu were determined in orange juices and orange concentrates from different geographical origins using this new method. Moreover, the effect of different temperature treatments (50, 92, and 150 degrees C) on the content of D-aa in orange juice was evaluated.

  7. Determination of carbocysteine in human plasma by liquid chromatography/tandem mass spectrometry employing precolumn derivatization.

    PubMed

    Chen, Xiaoyan; Zhong, Dafang; Han, Ying; Xie, Zhiyong

    2003-01-01

    A sensitive liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed to determine carbocysteine in human plasma using 2-pyridylacetic acid as the internal standard (IS). The method employed derivatization with 10 M hydrochloric acid/methanol, which significantly improved the ionization efficiency of carbocysteine. After methanol-induced protein precipitation of plasma samples, carbocysteine and the IS were derivatized and subjected to LC/MS/MS analysis using atmospheric pressure chemical ionization. The method has a lower limit of quantitation of 20 ng/mL for a 0.2-mL plasma aliquot. The intra- and inter-day precision (RSD), calculated from quality control (QC) samples, was less than 7%. The accuracy, determined using QC samples, was within +/- 1%. The method offered increased sensitivity, selectivity and speed of analysis over existing methods. The method was utilized to support clinical pharmacokinetic studies of carbocysteine in volunteers following oral administration.

  8. Isothiocyanates as derivatization reagents for amines in liquid chromatography/electrospray ionization-tandem mass spectrometry.

    PubMed

    Santa, Tomofumi

    2010-09-01

    The applicability of 3-pyridyl isothiocyanate, p-(dimethylamino)phenyl isothiocyanate and m-nitrophenyl isothiocyanate as the derivatization reagents for amines in high-performance liquid chromatography/electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS) was examined. The generated derivatives of amines with these reagents were favorably separated on the reversed-phase column and detected by ESI-MS/MS. The C-N bond of the generated thiourea structure was efficiently cleaved by collision-induced dissociation and gave the single and intense product ion. Among the three reagents, 3-pyridyl isothiocyanate was the most suitable as the derivatization reagent with regard to the reactivity to amines and the detection sensitivity.

  9. Single hollow fiber SLM extraction of polyamines followed by tosyl chloride derivatization and HPLC determination.

    PubMed

    Dziarkowska, Katarzyna; Jönsson, Jan Ake; Wieczorek, Piotr P

    2008-01-14

    Determination of polyamines in biological fluids possesses medical diagnostic relevance. Despite the vast panel of analytical methods developed for polyamines they are not applied in routine clinical usage, mainly due to the time and labor consuming sample preparation step and complicated derivatization procedures. Thus, new simpler methods are needed. This paper describes a single hollow fiber SLM extraction method of polyamines followed by simple pre-column derivatization with tosyl chloride and HPLC-UV analysis. The influence of different parameters such as the extraction time, organic phase composition, acceptor pH, donor pH, acceptor volume, donor volume and stirring speed on the transport parameters and enrichment was studied and discussed. The optimized method was applied to real matrices such as urine and plasma.

  10. Fluorescent derivatization of aromatic carboxylic acids with horseradish peroxidase in the presence of excess hydrogen peroxide.

    PubMed

    Odo, Junichi; Inoguchi, Masahiko; Aoki, Hiroyuki; Sogawa, Yuto; Nishimura, Masahiro

    2015-01-01

    The fluorescent derivatization of aromatic carboxylic acids by the catalytic activity of horseradish peroxidase (HRP) in the presence of excess H2O2 was investigated. Four monocarboxylic acids, nine dicarboxylic acids, and two tricarboxylic acids, all of which are non- or weakly fluorescent, were effectively converted into fluorescent compounds using this new method. This technique was further developed for the fluorometric determination of trace amounts of terephthalic acid (3c) and lutidinic acid (2b), and linear calibration curves for concentrations between 2.5 and 20.0 nmol of terephthalic acid (3c) and 1.0 and 10.0 nmol of lutidinic acid (2b) were demonstrated. Compound III, an intermediate of HRP, played an essential role in this process. Additionally, lactoperoxidase and manganese peroxidase, peroxidases similar to HRP, showed successful fluorescent derivatization of nicotinic acid (1b), lutidinic acid (2b), and hemimellitic acid (4a) in the presence of excess H2O2.

  11. High-performance liquid chromatographic determination of vertilmicin in rat plasma using sensitive fluorometric derivatization.

    PubMed

    Liu, Zhen; Sha, Yunfei; Huang, Taomin; Yang, Bei; Duan, Geng-Li

    2005-12-15

    A sensitive and reliable high-performance liquid chromatographic method was developed for the determination of vertilmicin in rat plasma. Derivatization with 9-fluorenylmethyl chloroformate (FMOC-Cl) followed by C(18) reversed-phase chromatography allowed the fluorimetric detection of vertilmicin. Optimal conditions for the derivatization of vertilmicin are described. The limit of quantification was 0.02 mg/L. The pharmacokinetics of vertilmicin was studied in 24 rats following intramuscular injection (i.m.) of different doses (4, 8, 16, 32 mg/kg of body weight). The pharmacokinetic parameter values were estimated by use of 3P97 program. In this study, we assessed the dose proportionality of vertilmicin after single intramuscular injection doses and obtained new information on the pharmacokinetics of the compound.

  12. Induction of clonally restricted thymus-dependent antibody responses in vitro using phosphorylcholine derivatized sheep erythrocytes.

    PubMed

    Cambier, J C; Neale, M J

    1980-01-01

    Induction of anti-phosphorylcholine responses in vitro using phosphorylcholine derivatized sheep erythrocytes (SRBC) is described. These responses are thymus dependent and restricted to expression of the TEPC 15 idiotype. Optimal B-cell concentrations in cultures were shown to be different for elicitation of direct anti-PC PFC responses than for elicitation of PFC specific for carrier (SRBC) determinants. Similarly, these responses differed in optimal immunogen concentration requirements. These differences presumably relate to the clonality of the respective responses. The methodology described provides an alternative to induction of clonally restricted thymus-dependent responses in vitro using PC derivatized soluble protein antigens. Induction of responses using PC-SRBC has the advantages of the relative case of T-cell priming to SRBC and concurrent stimulation of monoclonal anti-PC responses and easily assayed polyclonal anti-SRBC responses.

  13. Synthesis of the first tellurium-derivatized oligonucleotides for structural and functional studies.

    PubMed

    Sheng, Jia; Hassan, Abdalla E A; Huang, Zhen

    2009-10-05

    We report here the first synthesis of Te-nucleoside phosphoramidites and Te-modified oligonucleotides. We protected the 2'-tellurium functionality by alkylation and found that the Te functionality is compatible with solid-phase synthesis and that the Te oligonucleotides are stable during deprotection and purification. In addition, the redox properties of the Te functionalities have been explored. We found that the telluride and telluoxide DNAs are interchangeable by redox reactions. At elevated temperature, the Te-DNA can also be site-specifically fragmented oxidatively or reductively when 2'-TePh functionality is present, whereas elimination of the nucleobase is observed in the presence of 2'-TeMe. Moreover, the stability of the DNA duplexes derivatized with the Te functionalities has been investigated. Our Te derivatization of nucleic acids provides a novel approach for investigating DNA damage as well as for structure and function studies of nucleic acids and their protein complexes.

  14. On-matrix derivatization extraction of chemical weapons convention relevant alcohols from soil.

    PubMed

    Chinthakindi, Sridhar; Purohit, Ajay; Singh, Varoon; Dubey, D K; Pardasani, Deepak

    2013-10-11

    Present study deals with the on-matrix derivatization-extraction of aminoalcohols and thiodiglycols, which are important precursors and/or degradation products of VX analogues and vesicants class of chemical warfare agents (CWAs). The method involved hexamethyldisilazane (HMDS) mediated in situ silylation of analytes on the soil. Subsequent extraction and gas chromatography-mass spectrometry analysis of derivatized analytes offered better recoveries in comparison to the procedure recommended by the Organization for the Prohibition of Chemical Weapons (OPCW). Various experimental conditions such as extraction solvent, reagent and catalyst amount, reaction time and temperature were optimized. Best recoveries of analytes ranging from 45% to 103% were obtained with DCM solvent containing 5%, v/v HMDS and 0.01%, w/v iodine as catalyst. The limits of detection (LOD) and limit of quantification (LOQ) with selected analytes ranged from 8 to 277 and 21 to 665ngmL(-1), respectively, in selected ion monitoring mode.

  15. Discrimination between arabica and robusta coffee species on the basis of their amino acid enantiomers.

    PubMed

    Casal, Susana; Alves, M Rui; Mendes, Eulália; Oliveira, M Beatriz P P; Ferreira, Margarida A

    2003-10-22

    This work reports the results for the composition of robusta and arabica coffee species in terms of their amino acid enantiomers in the green and roasted states. The analyses were conducted for the free amino acids, as well as for the amino acids obtained after acid hydrolysis. The amino acids were extracted/hydrolyzed and isolated by SPE on strong cation exchange columns, derivatized to their N-ethoxycarbonylheptafluorobutyl esters, and analyzed by gas chromatography/FID on a Chirasil l-Val column. Multivariate analyses applied to the results showed that the free amino acids can be used as a tool for discrimination between coffee species, with a special reference to l-glutamic acid, l-tryptophan, and pipecolic acid. There is also some evidence that these compounds can be used for discrimination between green coffees subjected to different postharvest processes. It is also shown that the amino acid levels observed after acid hydrolysis can be used for the same purposes, although displaying less discriminatory power.

  16. Abiotic synthesis of amino acids and self-crystallization under prebiotic conditions

    PubMed Central

    Jiang, Liying; Dziedzic, Pawel; Spacil, Zdenek; Zhao, Gui-Ling; Nilsson, Lennart; Ilag, Leopold L.; Córdova, Armando

    2014-01-01

    Building on previous research on the origin and homochirality of life, this study focuses on analyses profiling important building blocks of life: the natural amino acids. The spark discharge variation of the iconic Miller experiment was performed with a reducing gas mixture of ammonia, methane, water and hydrogen. Amino acid analysis using liquid chromatography coupled with tandem mass spectrometry after pre-column derivatizaiton revealed the generation of several amino acids including those essential for life. Re-crystallization of the synthetic products and enantiomeric ratio analysis were subsequently performed. Results from liquid chromatography coupled with either fluorescent detector or tandem mass spectrometry after pre-column derivatization with chiral reagent revealed spontaneous and effective asymmetric resolution of serine and alanine. This work describes a useful analytical platform for investigation of hypotheses regarding the origin and homochirality of amino acids under prebiotic conditions. The formation of numerous amino acids in the electric discharge experiment and the occurrence of high enantiomeric ratios of amino acids in re-crystallization experiment give valuable implications for future studies in unraveling fundamental questions regarding origins and evolution of life. PMID:25346284

  17. Simultaneous determination of free amino acids in Pu-erh tea and their changes during fermentation.

    PubMed

    Zhu, Yuchen; Luo, Yinghua; Wang, Pengpu; Zhao, Mengyao; Li, Lei; Hu, Xiaosong; Chen, Fang

    2016-03-01

    Pu-erh ripened tea is produced through a unique microbial fermentation process from the sun-dried leaves of large-leaf tea species (Camellia sinensis (Linn.) var. assamica (Masters) Kitamura) in Yunnan province of China. In this study, the changes of amino acid profiles during fermentation of Pu-erh tea were investigated, based on the improved HPLC-UV method with PITC pre-column derivatization for the simultaneous determination of twenty free amino acids. Results showed that aspartic acid, glutamic acid, arginine, alanine, theanine and tyrosine were the major amino acids in tea samples. Fermentation significantly influenced on the amino acid profiles. The total free amino acid contents significantly decreased during fermentation (p<0.05). Meanwhile, low amount of acrylamide were detected. Its concentration increased after 7-days' fermentation and then decreased gradually. The results provided the useful information for the manipulation of fermentation process according to the changes of amino acids and acrylamide contents in Pu-erh ripened tea.

  18. Abiotic synthesis of amino acids and self-crystallization under prebiotic conditions

    NASA Astrophysics Data System (ADS)

    Jiang, Liying; Dziedzic, Pawel; Spacil, Zdenek; Zhao, Gui-Ling; Nilsson, Lennart; Ilag, Leopold L.; Córdova, Armando

    2014-10-01

    Building on previous research on the origin and homochirality of life, this study focuses on analyses profiling important building blocks of life: the natural amino acids. The spark discharge variation of the iconic Miller experiment was performed with a reducing gas mixture of ammonia, methane, water and hydrogen. Amino acid analysis using liquid chromatography coupled with tandem mass spectrometry after pre-column derivatizaiton revealed the generation of several amino acids including those essential for life. Re-crystallization of the synthetic products and enantiomeric ratio analysis were subsequently performed. Results from liquid chromatography coupled with either fluorescent detector or tandem mass spectrometry after pre-column derivatization with chiral reagent revealed spontaneous and effective asymmetric resolution of serine and alanine. This work describes a useful analytical platform for investigation of hypotheses regarding the origin and homochirality of amino acids under prebiotic conditions. The formation of numerous amino acids in the electric discharge experiment and the occurrence of high enantiomeric ratios of amino acids in re-crystallization experiment give valuable implications for future studies in unraveling fundamental questions regarding origins and evolution of life.

  19. Determination of monosaccharides derivatized with 2-aminobenzoic Acid by capillary electrophoresis.

    PubMed

    Abo, Mitsuru; He, Li-Ping; Sato, Kae; Okubo, Akira

    2013-01-01

    Reducing monosaccharides were derivatized with 2-aminobenzoic acid (2-AA) through reductive amination using sodium cyanoborohydride as a reductant, and the derivatives were separated by capillary zone electrophoresis with UV detection using 50 mM sodium phosphate (pH 5.5) or 150 mM sodium borate-50 mM sodium phosphate (pH 7.0) running buffer. The derivatives of monosaccharides, which are major components of various carbohydrate materials, were completely separated within 25 min.

  20. Application of solid-phase microextraction combined with derivatization to the determination of amphetamines by liquid chromatography.

    PubMed

    Cháfer-Pericás, C; Campíns-Falcó, P; Herráez-Hernández, R

    2004-10-15

    This work evaluates the utility of solid-phase microextraction (SPME) in the analysis of amphetamines by liquid chromatography (LC) after chemical derivatization of the analytes. Two approaches have been tested and compared, SPME followed by on-fiber derivatization of the extracted amphetamines, and solution derivatization followed by SPME of the derivatives formed. Both methods have been applied to measure amphetamine (AP), methamphetamine (MA), and 3,4-methylenedioxymethamphetamine (MDMA), using the fluorogenic reagent 9-fluorenylmethyl chloroformate (FMOC) and carbowax-templated resin (CW-TR)-coated fibers. Data on the application of the proposed methods for the analysis of different kind of samples are presented. When analyzing aqueous solutions of the analytes, both approaches gave similar analytical performance, but the sensitivity attainable with the solution derivatization/SPME method was better. The efficiencies observed when processing spiked urine samples by the SPME/on-fiber derivatization approach were very low. This was because the extraction of matrix components into the fiber coating prevented the extraction of the reagent. In contrast, the efficiencies obtained for spiked urine samples by the solution derivatization/SPME approach were similar to those obtained for aqueous samples. Therefore, the later method would be the method of choice for the quantification of amphetamines in urine.

  1. Detection of methamphetamine in the presence of nicotine using in situ chemical derivatization and ion mobility spectrometry.

    PubMed

    Ochoa, Mariela L; Harrington, Peter B

    2004-02-15

    The detection of methamphetamine in the presence of nicotine has been successfully accomplished using in situ chemical derivatization with propyl chloroformate as the derivatization reagent and ion mobility spectrometry (IMS). The rapid detection of methamphetamine is important for forensic scientists in order to establish a chain of evidence and link criminals to the crime scene. Nicotine is pervasive in clandestine drug laboratories from cigarette smoke residue. It has been demonstrated that nicotine obscures the methamphetamine peaks in ion mobility spectrometers due to their similar charge affinities and ion mobilities, which makes their detection a challenging task. As a consequence, false positive or negative responses may arise. In situ chemical derivatization poses as a sensitive, accurate, and reproducible alternative to remove the nicotine background when detecting nanogram amounts of methamphetamine. The derivatization agent was coated onto the sample disk, and the derivatization product corresponding to propyl methamphetamine carbamate was detected. In the present study, in situ chemical derivatization was demonstrated to be a feasible method to detect methamphetamine hydrochloride as the carbamate derivative, which was baseline-resolved from the nicotine peak. Alternating least squares (ALS) was used to model the datasets. A mixture containing both compounds revealed reduced mobilities of 1.61 cm(2)/V.s and 1.54 cm(2)/V.s for methamphetamine and nicotine, respectively. The reduced mobility of propyl methamphetamine carbamate was found at 1.35 cm(2)/V.s.

  2. Detection and Quantitation of Succinimide in Intact Protein via Hydrazine Trapping and Chemical Derivatization

    PubMed Central

    KLAENE, JOSHUA J.; NI, WENQIN; ALFARO, JOSHUA F.; ZHOU, ZHAOHUI SUNNY

    2014-01-01

    Formation of aspartyl succinimide (Asu) is a common post-translational modification (PTM) of protein pharmaceuticals under acidic conditions. We present a method to detect and quantitate succinimide in intact protein via hydrazine trapping and chemical derivatization. Succinimide, which is labile under typical analytical conditions, is first trapped with hydrazine to form stable hydrazide and can be directly analyzed by mass spectrometry. The resulting aspartyl hydrazide can be selectively derivatized by various tags, such as fluorescent rhodamine sulfonyl chloride that absorbs strongly in the visible region (570 nm). Our tagging strategy allows the labeled protein to be analyzed by orthogonal methods, including HPLC-UV, LC-MS, and SDS-PAGE coupled with fluorescence imaging. A unique advantage of our method is that variants containing succinimide, after derivatization, can be readily resolved via either affinity enrichment or chromatographic separation. This allows further investigation of individual factors in a complex protein mixture that affect succinimide formation. Some additional advantages imparted by fluorescence labeling include, the facile detection of the intact protein without proteolytic digestion to peptides; and high sensitivity, e.g. without optimization 0.41% succinimide was readily detected. As such, our method should be useful for rapid screening, optimization of formulation conditions and related processes relevant to protein pharmaceuticals. PMID:25043726

  3. [Determination of monensin residue in chicken by HPLC with post-column derivatization].

    PubMed

    Chen, X; Shi, X

    1999-01-01

    The monensin residue was extracted from the tissue by homogenization with methanol-water and the extract was filtered and partitioned with dichloromethane. The dichloromethane extract is concentrated and clean up by passing through a silica gel cartridge. The analyte on the cartridge is then eluted with dichloromethane-methanol. The eluate is collected and evaporated to dryness. The residue is dissolved and made to a definite volume with 1 mL methanol and the solution is used for post-column derivatization-HPLC determination. Monensin is separated on mu-Bondapak C18 column (3.9 mm i.d. x 300 mm) with methanol-water-phosphoric acid as a mobile phase and the flow rate was 0.7 mL/min. The eluted monensin was reacted with vaniline under acidic and heated condition in post-column derivatization system then detected at 520 nm and quantitated by external standard method. The derivatization reagent consisted of 20 mL concentrated sulfuric acid, 950 mL methanol and 30 g vaniline. The flow rate was 0.7 mL/min. The reactor was a stainless steel coil (300 cm x 1 mm i.d.) set in a 90 degrees C oven. The response values was linear between 20-200 ng. The recovery was 88.1%-101.3%. The coefficient of variation was 0.1%-0.73%.

  4. Quantification of busulfan in plasma by gas chromatography-mass spectrometry following derivatization with tetrafluorothiophenol.

    PubMed

    Quernin, M H; Poonkuzhali, B; Montes, C; Krishnamoorthy, R; Dennison, D; Srivastava, A; Vilmer, E; Chandy, M; Jacqz-Aigrain, E

    1998-05-08

    A specific and highly sensitive method has been developed for the determination of busulfan in plasma by gas chromatography-mass spectrometry using a deuterium-labeled busulfan (busulfan-d8) as internal standard. Plasma containing busulfan and busulfan-d8 were extracted with ethyl acetate and derivatized with 2,3,5,6-tetrafluorothiophenol prior to the monitoring of specific ions. The limit of quantification of the assay was 20 ng/ml and the calibration curve was linear over the range of 10 to 2000 ng/ml of derivatized busulfan. This method was in good agreement with the GC-MS assay using derivatization with sodium iodide and measuring diiodobutane. In addition, a pharmacokinetic study of busulfan was conducted in six children. The apparent oral clearance was 5.7+/-1.9 ml/kg/min and the volume of distribution was 1.0+/-0.4 l/kg and were similar to those previously reported in pediatric patients.

  5. Process for making polymers comprising derivatized carbon nanotubes and compositions thereof

    NASA Technical Reports Server (NTRS)

    Tour, James M. (Inventor); Bahr, Jeffrey L. (Inventor); Yang, Jiping (Inventor)

    2007-01-01

    The present invention incorporates new processes for blending derivatized carbon nanotubes into polymer matrices to create new polymer/composite materials. When modified with suitable chemical groups using diazonium chemistry, the nanotubes can be made chemically compatible with a polymer matrix, allowing transfer of the properties of the nanotubes (such as mechanical strength) to the properties of the composite material as a whole. To achieve this, the derivatized (modified) carbon nanotubes are physically blended with the polymeric material, and/or, if desired, allowed to react at ambient or elevated temperature. These methods can be utilized to append functionalities to the nanotubes that will further covalently bond to the host polymer matrix, or directly between two tubes themselves. Furthermore, the nanotubes can be used as a generator of polymer growth, wherein the nanotubes are derivatized with a functional group that is an active part of a polymerization process, which would also result in a composite material in which the carbon nanotubes are chemically involved.

  6. LC-MS determination of bioactive molecules based upon stable isotope-coded derivatization method.

    PubMed

    Toyo'oka, Toshimasa

    2012-10-01

    Liquid chromatography (LC) coupled with mass spectrometry (MS) has been widely used for the analyses of various molecules in many research fields. The electrospray ionization of MS has contributed to the advancement of the LC-MS and LC-MS/MS methods. However, the detection sensitivity is not always sufficient in biological samples, in spite of the highly sensitive ionization method. To increase the sensitivity, chemical derivatization, providing ionization enhancement and avoiding the matrix effect, is effective for various functional groups in the target molecules. However, the accuracy and precision by the determination is sometimes insufficient, especially in complex matrices. In such a case, stable isotope-labeled analogs are often used as the internal standards for the determination of the analytes. When the target compound in samples is limited, a high accuracy and precision is usually obtained by the isotope dilution method. However, the use of individual isotope standards is very difficult for the analyses of multiple molecules in complex matrices. Instead of the use of an isotope analog of the analytes, the differential isotope labeling method based upon chemical derivatization (stable isotope-coded derivatization) (ICD) by both reagents possessing different isotopes is realized. The ICD technique utilizing mass-different isotope tags is known to be an efficient means for metabolite profiling analyses. Thus, the present paper reviews the ICD method reported in the past 10 years. The species of the ICD reagents, their features and the applications to biological specimens are also described in this review.

  7. Examination of structurally selective derivatization of vitamin D(3) analogues by electrospray mass spectrometry.

    PubMed

    Weiskopf, A S; Vouros, P; Cunniff, J; Binderup, E; Björkling, F; Binderup, L; White, M C; Posner, G H

    2001-01-01

    The structural specificity of vitamin D derivatization by PTAD (4-phenyl-1,2,4-triazoline-3,5-dione) was probed using synthetic analogues and ion trap mass spectrometry. EB 1089, a vitamin D(3) analogue which contains a second site for Diels--Alder cycloaddition on its side-chain, allowed the examination of derivatization modes and comparisons of ion fragment structures. The origins of a PTAD-vitamin D(3) ion fragment, commonly used in metabolite characterization and quantitation of vitamin D(3) analogues (m/z 314), were established; ion trap mass spectrometry revealed that the PTAD comprises a portion of this diagnostic fragment, and is not lost by a retro-Diels--Alder step. Furthermore, the unique structure of the EB 1089 side-chain also permits facile determination of its side-chain metabolism. Use of PTAD derivatization and detection of metabolite-specific ion fragments identify hydroxylation at the end of the EB 1089 sidechain. It is believed that the results from these studies provide a clearer understanding of the mass spectrometry of triazolinedione derivatives, not only in the specific case of EB 1089, but also in their application to other vitamin D compounds.

  8. Derivatization and diffusive motion of molecular fullerenes: Ab initio and atomistic simulations

    SciTech Connect

    Berdiyorov, G. Tabet, N.; Harrabi, K.; Mehmood, U.; Hussein, I. A.; Peeters, F. M.; Zhang, J.; McLachlan, M. A.

    2015-07-14

    Using first principles density functional theory in combination with the nonequilibrium Green's function formalism, we study the effect of derivatization on the electronic and transport properties of C{sub 60} fullerene. As a typical example, we consider [6,6]-phenyl-C{sub 61}-butyric acid methyl ester (PCBM), which forms one of the most efficient organic photovoltaic materials in combination with electron donating polymers. Extra peaks are observed in the density of states (DOS) due to the formation of new electronic states localized at/near the attached molecule. Despite such peculiar behavior in the DOS of an isolated molecule, derivatization does not have a pronounced effect on the electronic transport properties of the fullerene molecular junctions. Both C{sub 60} and PCBM show the same response to finite voltage biasing with new features in the transmission spectrum due to voltage induced delocalization of some electronic states. We also study the diffusive motion of molecular fullerenes in ethanol solvent and inside poly(3-hexylthiophene) lamella using reactive molecular dynamics simulations. We found that the mobility of the fullerene reduces considerably due to derivatization; the diffusion coefficient of C{sub 60} is an order of magnitude larger than the one for PCBM.

  9. A novel fluorescence derivatization method combined with HPLC for determining the activities of endogenous caspase.

    PubMed

    Liu, Jiachi; Lu, Ye; Liang, Jianying

    2012-11-07

    A novel fluorescence derivatization method combined with HPLC was developed to detect the activity of caspase-3 and -8 in two cell lines (Hela cells and A549 cells) which were activated by low temperature-assisted ultraviolet irradiation (LT-UV), mitomycin C (MMC) and camptothecin during the apoptosis, respectively. Two peptide substrates for either caspase-3 or -8 were designed, of which peptide fragments were obtained by enzymatic modification, followed by fluorescence derivatization. A single fluorescent product was formed when a peptide was heated at 120 °C for 10 min in a neutral aqueous medium (pH 7.0) containing catechol, sodium periodate and sodium borate. Commercial kits for detecting the activity of caspase-3 and -8 were used as a control. The relative activity of the caspases detected by fluorescence derivatization was similar to that obtained by commercial kits, which indicated that the novel method is reliable. The activity assays of recombinant human caspases showed that the novel method provided higher selectivity than that of commercial kits, which proved it to be more accurate for determining the activity of caspases in apoptosis.

  10. Derivatization of hydroxyl functional groups for liquid chromatography and capillary electroseparation.

    PubMed

    Escrig-Doménech, A; Simó-Alfonso, E F; Herrero-Martínez, J M; Ramis-Ramos, G

    2013-06-28

    The derivatization reactions commonly used to enhance the analytical signal in the HPLC and CE determination of compounds with hydroxyl functional groups are revised. Focus is placed on the determination of compounds having aliphatic alcohols and phenols while lacking other reactive functional groups. The derivatization with acyl chlorides, organic anhydrides, isocyanates and a variety of other approaches, including oxidation of primary and secondary alcohols, sulfonation, esterification with carboxylic acids, and the use of azides, sulfonyl chlorides and other reagents having miscellaneous leaving groups, is covered. Reactions mainly addressed to introduce a chromophore or a fluorophore in the analyte molecule, or to introduce a charge to enhance sensitivity in MS detection, or to enable CE separation are included. Applications related to the industrial quality control of raw materials and manufactured products, and to the evaluation of their environmental impact are emphasized. The problem of the different response factors of the derivatives when complex mixtures of oligomers are derivatized, as occurs with non-ionic surfactants (mainly fatty alcohol ethoxylates) and soluble synthetic polymers, is discussed. Other applications related to the biochemical, biomedical, pharmaceutical, nutritional and toxicological fields are also reviewed. The reactions, the criteria to be applied to select the reagent, and the characteristics of the derivatives in relation to separation and detection, are discussed. Copyright © 2013 Elsevier B.V. All rights reserved.

  11. In Situ Derivatization and Quantification of Seven Carbonyls in Cigarette Mainstream Smoke.

    PubMed

    Ding, Yan S; Yan, Xizheng; Wong, Joshua; Chan, Michele; Watson, Clifford H

    2016-01-19

    Carbonyls, especially aldehydes, are a group of harmful volatile organic compounds that are found in tobacco smoke. Seven carbonyls are listed on the FDA's harmful and potential harmful constituents list for tobacco or tobacco smoke. Carbonyls have reactive functional groups and thus are challenging to quantitatively measure in cigarette smoke. The traditional method of measuring carbonyls in smoke involves solvent-filled impinger trapping and derivatization. This procedure is labor-intensive and generates significant volumes of hazardous waste. We have developed a new method to efficiently derivatize and trap carbonyls from mainstream smoke in situ on Cambridge filter pads. The derivatized carbonyls are extracted from the pads and subsequently quantified by ultra-high-pressure liquid chromatography coupled with tandem mass spectrometry. The new method has been validated and applied to research and commercial cigarettes. Carbonyl yields from research cigarettes are comparable to those from other published literature data. With a convenient smoke collection apparatus, a 4 min sample analysis time, and a low- or submicrogram detection limit, this new method not only simplifies and speeds the detection of an important class of chemical constituents in mainstream smoke but also reduces reactive losses and provides a more accurate assessment of carbonyl levels in smoke. Excellent accuracy (average 98%) and precision (14% average relative standard deviation in research cigarettes) ensure this new method's sufficient fidelity to characterize conventional combusted tobacco products, with potential application toward new or emerging products.

  12. Enantiomeric determination of amphetamine and methamphetamine in urine by precolumn derivatization with Marfey's reagent and HPLC.

    PubMed

    Foster, B S; Gilbert, D D; Hutchaleelaha, A; Mayersohn, M

    1998-01-01

    An analytical method was developed for enantiomeric determination of amphetamine and methamphetamine in human urine. The enantiomers were isolated from urine by solid-phase extraction, and diastereomers were formed by derivatization with the chiral Marfey's reagent (1-fluoro-2,4-dinitrophenyl-5-l-aniline amide). The diastereomers were separated by reversed-phase high-performance liquid chromatography in a water/methanol mobile phase and detected by absorbance spectrophotometry at 340 nm. Linear standard curves were obtained for all four enantiomers over a concentration range of 0.16-1.00 mg/L in urine. The detection limit was 0.16 mg/L urine for each enantiomer, and the limit of quantitation was 0.40 mg/L. The urine of 10 decedents was analyzed by this method and by a previously published precolumn derivatization procedure using (-)-1-(9-fluorenyl)ethyl chloroformate (FLEC) as the derivatizing agent and fluorescence detection. Comparison of the results of the two methods by linear regression showed comparable results for both d-amphetamine and d-methamphetamine. Neither method detected the presence of the l-enantiomers in the urine samples.

  13. Oxidized fatty acid analysis by charge-switch derivatization, selected reaction monitoring, and accurate mass quantitation.

    PubMed

    Liu, Xinping; Moon, Sung Ho; Mancuso, David J; Jenkins, Christopher M; Guan, Shaoping; Sims, Harold F; Gross, Richard W

    2013-11-01

    A highly sensitive, specific, and robust method for the analysis of oxidized metabolites of linoleic acid (LA), arachidonic acid (AA), and docosahexaenoic acid (DHA) was developed using charge-switch derivatization, liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI MS/MS) with selected reaction monitoring (SRM) and quantitation by high mass accuracy analysis of product ions, thereby minimizing interferences from contaminating ions. Charge-switch derivatization of LA, AA, and DHA metabolites with N-(4-aminomethylphenyl)-pyridinium resulted in a 10- to 30-fold increase in ionization efficiency. Improved quantitation was accompanied by decreased false positive interferences through accurate mass measurements of diagnostic product ions during SRM transitions by ratiometric comparisons with stable isotope internal standards. The limits of quantitation were between 0.05 and 6.0pg, with a dynamic range of 3 to 4 orders of magnitude (correlation coefficient r(2)>0.99). This approach was used to quantitate the levels of representative fatty acid metabolites from wild-type (WT) and iPLA2γ(-/-) mouse liver identifying the role of iPLA2γ in hepatic lipid second messenger production. Collectively, these results demonstrate the utility of high mass accuracy product ion analysis in conjunction with charge-switch derivatization for the highly specific quantitation of diminutive amounts of LA, AA, and DHA metabolites in biologic systems. Copyright © 2013 Elsevier Inc. All rights reserved.

  14. A validated LC-MS/MS method for the sensitive quantitation of serum 7alpha hydroxy-, 7beta hydroxy- and 7keto-dehydroepiandrosterone using a novel derivatization reagent.

    PubMed

    Ke, Yuyong; Gonthier, Renaud; Simard, Jean-Nicolas; Labrie, Fernand

    2016-04-01

    7alpha hydroxy-, 7beta hydroxy- and 7keto-dehydroepiandrosterone (7α OH-DHEA, 7β OH-DHEA and 7 oxo-DHEA) are oxidized metabolites of dehydroepiandrosterone (DHEA). Their concentrations are low in the circulation, especially in postmenopausal women, thus resulting in a considerable challenge for their reliable measurement. A sensitive and accurate LC-MS/MS method has been developed using a simple sample preparation procedure and a novel derivatization with 1-amino-4-methyl piperazine (MP). The derivatized metabolites are stable in high water content reagents. A 10 pg/mL (0.2 pg on column) for the low limit of quantitation (LLOQ) has been achieved for all three compounds. A proper choice of multiple reaction monitoring (MRM) transitions provides good specificity. The excess amount of reagent can be removed from the sample during the derivatization process. Within the calibration range of 10-2000 pg/mL, a good linearity was obtained with R>0.99 where the weighing factor is 1/X while the bias and coefficient of variance (CV) are within 8% for all levels of QCs and calibration curves. This method has been fully validated according to the FDA guidelines, where the results of the matrix effect meet the acceptance criteria while freeze-thaw stability, short and long term stability in matrix and solution as well as post-processed sample stability meet the requirements. With this method, the concentrations of 7α OH-DHEA, 7β OH-DHEA and 7 oxo-DHEA were measured in premenopausal and postmenopausal serum. The average concentration of 7α OH-DHEA is equivalent to that of 7β OH-DHEA in both types of sera. Copyright © 2016 Elsevier Inc. All rights reserved.

  15. Amino acid analysis by means of MALDI TOF mass spectrometry or MALDI TOF/TOF tandem mass spectrometry.

    PubMed

    Gogichaeva, Natalia V; Alterman, Michail A

    2012-01-01

    Here, we describe two different amino acid analysis protocols based on the application of matrix-assisted laser desorption ionization time-of-flight (MALDI TOF) mass spectrometry (MS). First protocol describes a MALDI TOF MS-based method for a routine simultaneous qualitative and quantitative analysis of free amino acids and protein hydrolysates (Alterman et al. Anal Biochem 335: 184-191, 2004). Linear responses between the amino acid concentration and the peak intensity ratio of corresponding amino acid to internal standard were observed for all amino acids analyzed in the range of concentrations from 20 to 300 μM. Limit of quantitation varied from 0.03 μM for arginine to 3.7 μM for histidine and homocysteine. This method has one inherent limitation: the analysis of isomeric and isobaric amino acids. To solve this problem, a second protocol based on the use of MALDI TOF/TOF MS/MS for qualitative analysis of amino and organic acids was developed. This technique is capable of distinguishing isobaric and isomeric compounds (Gogichayeva et al. J Am Soc Mass Spectrom 18: 279-284, 2007). Both methods do not require amino acid derivatization or chromatographic separation, and the data acquisition time is decreased to several seconds for a single sample.

  16. Brain amino acid sensing.

    PubMed

    Tsurugizawa, T; Uneyama, H; Torii, K

    2014-09-01

    The 20 different amino acids, in blood as well as in the brain, are strictly maintained at the same levels throughout the day, regardless of food intake. Gastric vagal afferents only respond to free glutamate and sugars, providing recognition of food intake and initiating digestion. Metabolic control of amino acid homeostasis and diet-induced thermogenesis is triggered by this glutamate signalling in the stomach through the gut-brain axis. Rats chronically fed high-sugar and high-fat diets do not develop obesity when a 1% (w/v) monosodium glutamate (MSG) solution is available in a choice paradigm. Deficiency of the essential amino acid lysine (Lys) induced a plasticity in rats in response to Lys. This result shows how the body is able to identify deficient nutrients to maintain homeostasis. This plastic effect is induced by activin A activity in the brain, particularly in certain neurons in the lateral hypothalamic area (LHA) which is the centre for amino acid homeostasis and appetite. These neurons respond to glutamate signalling in the oral cavity by which umami taste is perceived. They play a quantitative role in regulating ingestion of deficient nutrients, thereby leading to a healthier life. After recovery from malnutrition, rats prefer MSG solutions, which serve as biomarkers for protein nutrition. © 2014 John Wiley & Sons Ltd.

  17. Microemulsion electrokinetic chromatography with laser-induced fluorescence detection: as tested with amino acid derivatives.

    PubMed

    Jianping, Xie; Jiyou, Zhang; Huanxiang, Liu; Jiaqin, Liu; Jianniao, Tian; Xingguo, Chen; Zhide, Hu

    2004-10-01

    Over a decade ago, microemulsion electrokinetic chromatography was introduced as a novel mode of capillary electrophoresis. However, there has not been publication on the combination of microemulsion electrokinetic chromatography with laser-induced fluorescence detection. In this paper, a preliminary method using microemulsion eletrokinetic chromatography combined with laser-induced fluorescence detection and second derivative electrophoregram was established as a sensitive and selective assay for separation and determination of nine amino acids after derivatization with 4-chloro-7-nitrobenzo-2-oxa-1, 3-diazol. The derivatization and separation conditions were optimized. In the investigated concentration ranges correlation coefficients were better than 0.995. The relative standard deviation (n = 5) of the migration times and peak heights were 0.56-0.76 and 2.21-7.15%, respectively. The detection limits (S/N = 3) were at a neaomolar level (0.32-2.20 nM). The method was applied for the analysis of compound amino acid injection and a Chinese traditional herbal medicine. The recoveries were 95.9-107.9%.

  18. A novel derivatization method for the determination of Fosfomycin in human plasma by liquid chromatography coupled with atmospheric pressure chemical ionization mass spectrometric detection via phase transfer catalyzed derivatization.

    PubMed

    Papakondyli, Theodora A; Gremilogianni, Aikaterini M; Megoulas, Nikolaos C; Koupparis, Michael A

    2014-03-07

    An analytical method employing novel sample preparation and liquid chromatography coupled with atmospheric pressure chemical ionization mass spectrometric detection (LC-APCI/MS) was developed for the determination of fosfomycin in human plasma. Sample preparation involves derivatization through phase transfer catalysis (PTC) which offers multiple advantages due to the simultaneous extraction, preconcentration and derivatization of the analyte. Using a PT catalyst, fosfomycin was extracted from plasma in an organic phase and, then converted to a pentafluorobenzyl ester with the use of pentafluorobenzyl bromide (PFBBr) derivatization reagent. The method was fully optimized by taking into account both PTC and derivatization parameters. Several catalysts, in a wide range of concentrations, with different counter ions and polarities were tested along with different extraction solvents and pH values. Thereafter, the derivatization procedure was optimized by altering the amount of the derivatization reagent, the temperature of the reaction and finally, the derivatization duration. As internal standard (I.S.) ethylphosphonic acid was chosen and underwent the same pretreatment. The derivatives were separated on a pentafluorophenyl (PFP)-C18 analytical column, which provides unique selectivity, using an isocratic elution with acetonitrile-water (70-30, v/v). The method was validated according to US Food and Drug Administration (FDA) guidelines and can be used for a bioequivalence study of fosfomycin in human plasma. The correlation coefficient (r(2)) of the calibration curve of spiked plasma solutions in the range of 50-12000 ng/mL was found greater than 0.999 with a limit of quantitation (LOQ) equal to 50 ng/ml (for 500 μL plasma sample). Copyright © 2014 Elsevier B.V. All rights reserved.

  19. Parallel Synthesis of an Imidazole-4,5-dicarboxamide Library Bearing Amino Acid Esters and Alkanamines

    PubMed Central

    Solinas, Rosanna; DiCesare, John C.; Baures, Paul W.

    2008-01-01

    The imidazole-4,5-dicarboxylic acid scaffold is readily derivatized with amino acid esters and alkanamines to afford compounds with intramolecularly hydrogen bonded conformations that mimic substituted purines and therefore are hypothesized to be potential inhibitors of kinases through competitive binding to the ATP site. In this work, a total of 126 dissymmetrically disubstituted imidazole-4,5-dicarboxamides with amino acid ester and alkanamide substituents were prepared by parallel synthesis. The library members were purified by column chromatography on silica gel and the purified compounds characterized by LC-MS with LC detection at 214 nm. A selection of the final compounds was also analyzed by 1H-NMR spectroscopy. The analytically pure final products have been submitted to the Molecular Library Small Molecule Repository (MLSMR) for screening in the Molecular Library Screening Center Network (MLSCN) as part of the NIH Roadmap. PMID:19078856

  20. Design, synthesis and antiviral activity of 2-(3-amino-4-piperazinylphenyl)chromone derivatives.

    PubMed

    Kim, Mi Kyoung; Yoon, Hyunjun; Barnard, Dale Lynn; Chong, Youhoon

    2013-01-01

    Previously, we have confirmed that the antiviral activities of the chromone derivatives were controlled by the type as well as the position of the substituents attached to the chromone core structure. In the course of our ongoing efforts to optimize the antiviral activity of the chromone derivatives, we have been attempting to derivatize the chromone scaffold via introduction of various substituents. In this proof-of-concept study, we introduced a 3-amino-4-piperazinylphenyl functionality to the chromone scaffold and evaluated the antiviral activities of the resulting chromone derivatives. The synthesized 2-(3-amino-4-piperazinylphenyl)-chromones showed severe acute respiratory syndrome-corona virus (SARS-CoV)-specific antiviral activity. In particular, the 2-pyridinylpiperazinylphenyl substituents provided the resulting chromone derivatives with selective antiviral activity. Taken together, this result indicates the possible pharmacophoric role of the 2-pyridinylpiperazine functionality attached to the chromone scaffold, which warrants further in-depth structure-activity relationship study.

  1. Amino Acid Composition of Breast Milk from Urban Chinese Mothers

    PubMed Central

    Garcia-Rodenas, Clara L.; Affolter, Michael; Vinyes-Pares, Gerard; De Castro, Carlos A.; Karagounis, Leonidas G.; Zhang, Yumei; Wang, Peiyu; Thakkar, Sagar K.

    2016-01-01

    Human breast milk (BM) amino acid (AA) composition may be impacted by lactation stage or factors related to geographical location. The present cross-sectional study is aimed at assessing the temporal changes of BMAA over lactation stages in a large cohort of urban mothers in China. Four hundred fifty BM samples, collected in three Chinese cities covering eight months of lactation were analyzed for free (FAA) and total (TAA) AA by o-phthalaldehyde/ fluorenylmethylchloroformate (OPA/FMOC) derivatization. Concentrations and changes over lactation were aligned with previous reports. Both the sum and the individual TAA values significantly decreased during the first periods of lactation and then generally leveled off. Leucine and methionine were respectively the most and the least abundant indispensable amino acids across all the lactation stages, whereas glutamic acid + glutamine (Glx) was the most and cystine the least abundant dispensable AA. The contribution of FAA to TAA levels was less than 2%, except for free Glx, which was the most abundant FAA. In conclusion, the AA composition of the milk from our cohort of urban Chinese mothers was comparable to previous studies conducted in other parts of the world, suggesting that this is an evolutionary conserved trait largely independent of geographical, ethnic, or dietary factors. PMID:27690094

  2. Amino Acid Composition of Breast Milk from Urban Chinese Mothers.

    PubMed

    Garcia-Rodenas, Clara L; Affolter, Michael; Vinyes-Pares, Gerard; De Castro, Carlos A; Karagounis, Leonidas G; Zhang, Yumei; Wang, Peiyu; Thakkar, Sagar K

    2016-09-28

    Human breast milk (BM) amino acid (AA) composition may be impacted by lactation stage or factors related to geographical location. The present cross-sectional study is aimed at assessing the temporal changes of BMAA over lactation stages in a large cohort of urban mothers in China. Four hundred fifty BM samples, collected in three Chinese cities covering eight months of lactation were analyzed for free (FAA) and total (TAA) AA by o-phthalaldehyde/ fluorenylmethylchloroformate (OPA/FMOC) derivatization. Concentrations and changes over lactation were aligned with previous reports. Both the sum and the individual TAA values significantly decreased during the first periods of lactation and then generally leveled off. Leucine and methionine were respectively the most and the least abundant indispensable amino acids across all the lactation stages, whereas glutamic acid + glutamine (Glx) was the most and cystine the least abundant dispensable AA. The contribution of FAA to TAA levels was less than 2%, except for free Glx, which was the most abundant FAA. In conclusion, the AA composition of the milk from our cohort of urban Chinese mothers was comparable to previous studies conducted in other parts of the world, suggesting that this is an evolutionary conserved trait largely independent of geographical, ethnic, or dietary factors.

  3. Microwave-accelerated derivatization for the simultaneous gas chromatographic-mass spectrometric analysis of natural and synthetic estrogenic steroids.

    PubMed

    Zuo, Yuegang; Zhang, Kai; Lin, Yuejuan

    2007-05-04

    A rapid microwave-accelerated derivatization process for the GC-MS analysis of steroid estrogens, estrone (E1), 17beta-estradiol (E2), estriol (E3), 17alpha-ethynylestradiol (EE2) and mestranol (MeEE2), was developed. Under microwave irradiation, the five estrogenic hormones studied were simultaneously derivatized with N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA)+trimethylchlorosilane (TMCS) in pyridine solution. Effects of irradiation time (15-120 s) and power level (240-800 W) on the yield of the derivatization were investigated. The derivatization under the irradiation of 800 W microwave for 60s produced comparable results when compared with the conventional heating process in a sand bath for 30 min at 80 degrees C in terms of derivatization yield, linearity and precision for all steroid hormones tested. The calibration curves are linear between 3.00 and 3.00 x 10(2) microg mL(-1). The square of the regression coefficients (R(2)) range from 0.979 to 1.000. The applicability of the method was evaluated on spiked river and distilled water samples at two concentrations, 25.0 and 2.00 x 10(2) ng mL(-1). The recoveries obtained by using microwave heating (60s, 800 W) were similar to those by conventional heating. When combined solid-phase extraction (SPE) with the application of the microwave-accelerated derivatization proposed here, the detection limits of 0.02-0.1 ng L(-1) for the steroid hormones have been achieved. The results demonstrated that microwave-accelerated derivatization is an efficient and suitable sample preparation method for the GC-MS analysis of estrogenic steroids.

  4. Differentiation of green, white, black, Oolong, and Pu-erh teas according to their free amino acids content.

    PubMed

    Alcázar, A; Ballesteros, O; Jurado, J M; Pablos, F; Martín, M J; Vilches, J L; Navalón, A

    2007-07-25

    In this paper, the differentiation of green, black, Oolong, white, and Pu-erh teas has been carried out according to their free amino acid contents. Alanine, arginine, asparagine, aspartic acid, glutamic acid, isoleucine, histidine, leucine, phenylalanine, serine, theanine, threonine, and tyrosine have been determined by liquid chromatography with derivatization with o-phthalaldehyde and fluorescence detection. The chromatographic separation was achieved with a Hypersil ODS column and gradient elution. The amino acid contents were used as chemometric descriptors for classification purposes of different tea varieties. Principal component analysis, k-nearest neighbors, linear discriminant analysis, and artificial neural networks were applied to differentiate tea varieties. Using back-propagation multilayer perceptron artificial neural networks, 100% success in the classification was obtained. The most differentiating amino acids were glutamic acid, asparagine, serine, alanine, leucine, and isoleucine.

  5. Investigation of liquid and gas chromatography techniques for separation of diastereomers of beta-(alpha-methylbenzyl) amino isobutyric acid.

    PubMed

    Held, Charles B; Robbins, David K

    2003-09-01

    Cryptophycins are macrolides investigated as potential anticancer agents. These large cyclic molecules are generated via a convergent process, utilizing the coupling of several smaller fragments synthesized individually. During early synthetic development of the beta-amino acid fragment C, analytical methods are necessary for the characterization of products resulting from the various routes being studied. One route being evaluated produces (RR) and (RS) diastereomers of beta-(alpha-methylbenzyl) amino isobutyric acid as intermediates. To measure diastereomeric excess (%de), assay conditions using high-performance liquid chromatography (HPLC) and capillary gas chromatographic (GC) techniques are explored. Derivatization methods using trifluoroacetyl- and silyl-derivatives are investigated for use with capillary GC. The results of the GC investigations are found to be only partially successful. Ion-pair HPLC is determined to be the optimal technique, utilizing pentanesulfonic acid as the counter ion to the amine group of beta-(alpha-methylbenzyl) amino isobutyric acid.

  6. Amino aciduria in weightlessness

    NASA Technical Reports Server (NTRS)

    Leach, C. S.; Rambaut, P. C.; Di Ferrante, N.

    1979-01-01

    Urinary excretion of amino acids by the 9 Skylab crewmen was studied as an indicator of the metabolic effects caused by exposure to the space flight environment. Intake was consistent in quality and quantity throughout the 28, 59 and 84-day flights for each of the crewmen and complete collections were accomplished. The results indicated an increased excretion in most amino acids during the first month of flight which remained elevated in the second and third months but to a lesser extent. Additional indications of change in muscle and skeletal metabolism were observed. These results point to the desirability of obtaining additional indices of alterations in protein synthetic processes in conjunction with future space flights.

  7. Simultaneous determination of 18 D-amino acids in rat plasma by an ultrahigh-performance liquid chromatography-tandem mass spectrometry method: application to explore the potential relationship between Alzheimer's disease and D-amino acid level alterations.

    PubMed

    Xing, Yuping; Li, Xiaoyan; Guo, Xingjie; Cui, Yan

    2016-01-01

    D-Amino acids are increasingly being recognized as important signaling molecules, and abnormal levels of D-amino acids have been implicated in the pathogenesis of Alzheimer's disease. To evaluate the potential relationship between Alzheimer's disease and D-amino acids, a simple, sensitive, and reliable UPLC-MS/MS method with pre-column derivatization was developed and validated for simultaneous determination of 18 D-amino acids in rat plasma. The analytes were extracted from plasma samples by a protein precipitation procedure, and then derivatized with (S)-N-(4-nitrophenoxycarbonyl) phenylalanine methoxyethyl ester [(S)-NIFE]. Chromatographic separation was achieved using an ACQUITY UPLC BEH C18 column (2.1 mm × 50 mm, 1.7 μm) with a mobile phase composed of acetonitrile containing 8 mM ammonium hydrogen carbonate at a flow rate of 0.6 mL min(-1). The analytes were detected by electrospray ionization in positive ion multiple reaction monitoring modes. Under the optimum experimental conditions, all the linear regressions were acquired with r > 0.9932. The limits of quantitation of all derivatized D-amino acids were within 0.05-40.0 ng mL(-1) in rat plasma. The intra- and inter-day precisions, expressed as percentage relative standard deviations (%RSD), were within the range of 12.3 and 10.1%, respectively. The recoveries for all the analytes were observed over the range of 82.8-100.5% with RSD values less than 12.5%. Finally, the proposed method was successfully applied to simultaneous determination of the 18 D-amino acids in plasma from Alzheimer's disease rats and age-matched normal controls. Results showed that the concentrations of D-serine, D-aspartate, D-alanine, D-leucine, and D-proline in Alzheimer's disease rat plasma were significantly decreased compared with those in normal controls, while D-phenylalanine levels increased. It was revealed that some of these D-amino acids would be potential diagnostic biomarkers for Alzheimer's disease.

  8. Capillary electrophoresis method with UV-detection for analysis of free amino acids concentrations in food.

    PubMed

    Omar, Mei Musa Ali; Elbashir, Abdalla Ahmed; Schmitz, Oliver J

    2017-01-01

    Simple and inexpensive capillary electrophoresis with UV-detection method (CE-UV) was optimized and validated for determination of six amino acids namely (alanine, asparagine, glutamine, proline, serine and valine) for Sudanese food. Amino acids in the samples were derivatized with 4-chloro-7-nitro-2,1,3-benzoxadiazole (NBD-Cl) prior to CE-UV analysis. Labeling reaction conditions (100mM borate buffer at pH 8.5, labeling reaction time 60min, temperature 70°C and NBD-Cl concentration 40mM) were systematically investigated. The optimal conditions for the separation were 100mM borate buffer at pH 9.7 and detected at 475nm. The method was validated in terms of linearity, limit of detection (LOD), limit of quantification (LOQ), precision (repeatability) (RSD%) and accuracy (recovery). Good linearity was achieved for all amino acids (r(2)>0.9981) in the concentration range of 2.5-40mg/L. The LODs in the range of 0.32-0.56mg/L were obtained. Recoveries of amino acids ranging from 85% to 108%, (n=3) were obtained. The validated method was successfully applied for the determination of amino acids for Sudanese food samples. Copyright © 2016 Elsevier Ltd. All rights reserved.

  9. Novel renal amino acid transporters.

    PubMed

    Verrey, François; Ristic, Zorica; Romeo, Elisa; Ramadan, Tamara; Makrides, Victoria; Dave, Mital H; Wagner, Carsten A; Camargo, Simone M R

    2005-01-01

    Reabsorption of amino acids, similar to that of glucose, is a major task of the proximal kidney tubule. Various amino acids are actively transported across the luminal brush border membrane into proximal tubule epithelial cells, most of which by cotransport. An important player is the newly identified cotransporter (symporter) B0AT1 (SLC6A19), which imports a broad range of neutral amino acids together with Na+ across the luminal membrane and which is defective in Hartnup disorder. In contrast, cationic amino acids and cystine are taken up in exchange for recycled neutral amino acids by the heterodimeric cystinuria transporter. The basolateral release of some neutral amino acids into the extracellular space is mediated by unidirectional efflux transporters, analogous to GLUT2, that have not yet been definitively identified. Additionally, cationic amino acids and some other neutral amino acids leave the cell basolaterally via heterodimeric obligatory exchangers.

  10. A new derivatizing agent, trimethylammoniopropyl methanethiosulphonate, is efficient for preparation of recombinant brain-derived neurotrophic factor from inclusion bodies.

    PubMed

    Inoue, M; Akimaru, J; Nishikawa, T; Seki, N; Yamada, H

    1998-12-01

    Derivatization with trimethylammoniopropyl methanethiosulphonate (TAPS-sulphonate) enabled brain-derived neurotrophic factor (BDNF) to be prepared efficiently from Escherichia coli inclusion bodies. Reduced BDNF obtained from inclusion bodies solubilized by urea and reduced by dithiothreitol was suggested to form a complex with itself or with other compounds such as lipids. It could hardly be adsorbed on to cation-exchange resin for partial purification prior to a refolding reaction. Reversible derivatization of cysteine residues was tested as a method of dissociating BDNF from such complexes. However, even if a methyl or aminoethyl group was introduced, BDNF could not be dissociated readily. Derivatization with TAPS-sulphonate brought about good dissociation of BDNF, and more than 50% adsorbed on to the cation-exchange resin. BDNF derivatized with TAPS-sulphonate refolded well, and the refolded samples showed the same biological activity as purified BDNF. Derivatization with TAPS-sulphonate would increase the intermolecular repulsion of BDNF, due to the positively charged character of the quaternized amine, and inhibit complex formation. Thus, TAPS-sulphonate is effective for the preparation of BDNF under denatured conditions.

  11. In-capillary derivatization and laser-induced fluorescence detection for the analysis of organophosphorus pesticides by micellar electrokinetic chromatography.

    PubMed

    Zhou, Lei; Luo, Zhi; Wang, Shumin; Hui, Yang; Hu, Zhide; Chen, Xingguo

    2007-05-18

    We developed a rapid and sensitive method using in-capillary derivatization and laser-induced fluorescence (LIF) detection for the fully automated analysis of organophosphorus pesticides (OPPs), including glufosinate, aminomethylphosphonic acid (AMPA) and glyphosate by micellar electrokinetic chromatography (MEKC). The potential of 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) as in-capillary derivatization reagent is described for the first time. The unique feature of this MEKC method is the capillary being used as a small reaction chamber. In in-capillary derivatization, the sample and reagent solutions were injected directly into the capillary by tandem mode, followed by an electrokinetic step to enhance the mixing efficiency of analytes and reagent plugs in accordance with their different electrophoretic mobilities. Standing a specified time for reaction, the derivatives were then immediately separated and determined. Careful optimization of the derivatization and separation conditions allowed the determination of glufosinate, AMPA and glyphosate with detection limits of 2.8, 3.6 and 32.2 ng/mL, respectively. These detection limits were comparable to those of 1.4, 1.9 and 23.8 ng/mL obtained from conventional pre-capillary derivatization. Furthermore, repeatability better than 0.40% for migration time and 3.4% for peak area, as well as shorter migration time, was obtained. The method was successfully applied to the analysis of spiked river water sample with satisfactory results.

  12. Derivatization of haemoglobin with periodate-generated reticulation agents: evaluation of oxidative reactivity for potential blood substitutes.

    PubMed

    Deac, Florina; Iacob, Bianca; Fischer-Fodor, Eva; Damian, Grigore; Silaghi-Dumitrescu, Radu

    2011-01-01

    Periodate modification of the sugar moiety in sugars, including adenosine triphosphate (ATP), has previously been employed in order to prepare dialdehyde-type reagents, which were then utilized in crosslinking reactions on haemoglobin, yielding polymerized material with useful dioxygen-binding properties and hence proposed as possible artificial oxygen carriers ('blood substitutes'). Here, the periodate protocol is shown to be applicable to a wider range of oxygen-containing compounds, illustrated by starch and polyethylene glycol. Derivatization protocols are described for haemoglobin with such periodate-treated crosslinking agents, and the dioxygen-binding properties and redox reactivities are investigated for the derivatized haemoglobins, with emphasis on pro-oxidative properties. There is a general tendency of the derivatization to result in higher autooxidation rates. The peroxide reactivity of the met (ferric) form is also affected by derivatization, as witnessed, among others, by varying yields of ferryl [Fe (IV)-oxo] and free radical generated. In cell, culture tests (human umbilical vein epithelial cells, HUVEC), the derivatization protocols show no toxic effect.

  13. Electrochemical behaviors of amino acids at multiwall carbon nanotubes and Cu2O modified carbon paste electrode.

    PubMed

    Dong, Shuqing; Zhang, Shan; Chi, Langzhu; He, Pingang; Wang, Qingjiang; Fang, Yuzhi

    2008-10-15

    A carbon paste electrode modified with multiwall carbon nanotubes and copper(I) oxide (MWCNT-Cu(2)O CPME) was fabricated, and the electrochemical behaviors of 19 kinds of natural amino acids at this modified electrode were studied. The experimental results showed that the various kinds of amino acids without any derivatization displayed obvious oxidation current responses at the modified electrode. It was also found that the current response values of amino acids were dependent mainly on pH values of buffer solutions. The phenomenon could be explained by the fact that the amino acids suffered complexation or electrocatalytic oxidation processes under different pH values. Six kinds of amino acids (arginine, tryptophan, histidine, threonine, serine, and tyrosine), which performed high-oxidation current responses in alkaline buffers, were selected to be detected simultaneously by capillary zone electrophoresis coupled with amperometric detection (CZE-AD). These amino acids could be perfectly separated within 20 min, and their detection limits were as low as 10(-7) or 10(-8)mol L(-1) magnitude (signal/noise ratio=3). The above results demonstrated that MWCNT-Cu(2)O CPME could be successfully employed as an electrochemical sensor for amino acids with some advantages of convenient preparation, high sensitivity, and good repeatability.

  14. Enhanced amine and amino acid analysis using Pacific Blue and the Mars Organic Analyzer microchip capillary electrophoresis system.

    PubMed

    Chiesl, Thomas N; Chu, Wai K; Stockton, Amanda M; Amashukeli, Xenia; Grunthaner, Frank; Mathies, Richard A

    2009-04-01

    The fluorescent amine reactive probe Pacific Blue succinimidyl ester (PB) is used for the detection of trace amounts of amines and amino acids by microchip capillary electrophoresis on the Mars Organic Analyzer (MOA). The spectral and chemical properties of PB provide a 200-fold increase in sensitivity and improved resolution compared to fluorescamine derivatization. With the use of cross injection and PB labeling, the MOA detected amino acids at concentrations as low as 75 pM (sub-parts-per-trillion). Micellar electrokinetic chromatography (MEKC) which separates PB-labeled amino acids by their hydrophobicity is also demonstrated. The optimized MEKC conditions (45 mM CHAPSO, pH 6 at 5 degrees C) effectively separated amines and 25 amino acids with enantiomeric resolution of alanine, serine, and citrulline. Samples from the Yungay Hills region in the Atacama Desert, Chile, and from the Murchison meteorite are successfully analyzed using both techniques, and amino acids are found in the parts-per-billion range. Abiotic amino acids such as beta-alanine and epsilon-aminocaprioc acid are detected along with several neutral and acidic amino acids in the Murchison sample. The Atacama Desert sample is found to contain homochiral L-alanine and L-serine indicating the presence of extant or recently extinct life.

  15. The influence of extrusion on loss and racemization of amino acids.

    PubMed

    Csapó, J; Varga-Visi, E; Lóki, K; Albert, Cs; Salamon, Sz

    2008-02-01

    The influence of the operation conditions (temperature and residence time) of a thermic treatment on the total amount (free and protein-bound) of amino acid enantiomers of dry fullfat soya was investigated. Total amino acid content was determined using conventional ion-exchange amino acid analysis of total hydrolysates and chiral amino acid analysis was performed by HPLC after precolumn derivatization with o-phthaldialdehyde and 1-thio-beta-D-glucose tetraacetate. Contrary to corn that was investigated previously, notable racemization was detected even at lower temperatures. At 140 degrees C the ratio of the D-enantiomer was 0.87% for glutamic acid, 2.81% for serine, and 1.92% for phenylalanine; at 220 degrees C the ratios of the D-enantiomer of the above amino acids were 1.43, 4.61, and 4.68%, respectively. The concentration of several L-amino acids decreased. At 220 degrees C there was 10% less L-glutamic acid, 17% less L-serine, 5% less L-phenylalanine, 6.6% less L-aspartic, acid and 21% less L-lysine than in the control; their loss can be assigned to different degrees of L - D conversion. While nearly complete transformation of L-phenylalanine can be attributed to racemization, the main cause of the loss of L-lysine is not racemization. The treatments in the same order of magnitude resulted in the formation of more D-amino acids and greater extent of racemization of amino acids in fullfat soya than that of maize.

  16. Synthesis and Crystal Structure Study of 2’-Se-Adenosine-Derivatized DNA

    SciTech Connect

    Sheng, J.; Salon, J; Gan, J; Huang, Z

    2010-01-01

    The selenium derivatization of nucleic acids is a novel and promising strategy for 3D structure determination of nucleic acids. Selenium can serve as an excellent anomalous scattering center to solve the phase problem, which is one of the two major bottlenecks in macromolecule X-ray crystallography. The other major bottleneck is crystallization. It has been demonstrated that the incorporated selenium functionality at the 2'-positions of the nucleosides and nucleotides is stable and does not cause significant structure perturbation. Furthermore, it was observed that the 2'-Se-derivatization could facilitate crystallization of oligonucleotides with fast crystal growth and high diffraction quality. Herein, we describe a convenient synthesis of the 2'-Se-adenosine phosphoramidite, and report the first synthesis and X-ray crystal structure determination of the DNA containing the 2'-Se-A derivatization. The 3D structure of 2'-Se-A-DNA decamer [5'-GTACGCGT(2'-Se-A)C-3']{sub 2} was determined at 1.75 {angstrom} resolution, the 2'-Se-functionality points to the minor groove, and the Se-modified and native structures are virtually identical. Moreover, we have observed that the 2'-Se-A modification can greatly facilitate the crystal growth with high diffraction quality. In conjunction with the crystallization facilitation by the 2'-Se-U and 2'-Se-T, this novel observation on the 2'-Se-A functionality suggests that the 2'-Se moiety is sole responsible for the crystallization facilitation and the identity of nucleobases does not influence the crystal growth significantly.

  17. Simultaneous determination of phosphorus-containing amino acid-herbicides by nonionic surfactant micellar electrokinetic chromatography with laser-induced fluorescence detection.

    PubMed

    Molina, M; Silva, M

    2001-04-01

    The potential of micellar electrokinetic chromatography (MEKC) with laser-induced fluorescence (LIF) detection for the separation and determination of phosphorus-containing amino acid-herbicides (glufosinate and glyphosate), and aminomethylphosphonic acid (the major metabolite of glyphosate), involving derivatization with fluorescein isothiocyanate (FITC) isomer I, was investigated. Different variables that affect derivatization (pH, FITC concentration, time and temperature) and separation (pH and concentration of the buffer, kind and concentration of surfactants and applied voltage) were studied. The analysis was conducted within about 8 min and the use of the nonionic surfactant Triton X-100 improved the selectivity, thus indirectly enhancing sensitivity by shifting of the interfering peaks of the FITC excess. Dynamic ranges of 2.0-3,000 microg/L, limits of detection at microgram or submicrogram-per-liter level, and relative standard deviations from 4.7 to 6.4% were obtained. The ensuing method--nonionic surfactant MEKC-- is a useful choice for the determination of these herbicides as it provides limits of detection similar or lower than those reported by existing chromatographic alternatives without the use of an additional preconcentration technique such as solid-phase extraction. The separation of a mixture of nine FITC-derivatized amino acids, selected as target compounds, was also carried out to assess the discrimination power of the nonionic surfactant MEKC method for the analysis of closely related anionic analytes.

  18. Determination of oxidative DNA base damage by gas chromatography-mass spectrometry. Effect of derivatization conditions on artifactual formation of certain base oxidation products.

    PubMed

    England, T G; Jenner, A; Aruoma, O I; Halliwell, B

    1998-10-01

    GC-MS is a widely used tool to measure oxidative DNA damage because of its ability to identify a wide range of base modification products. However, it has been suggested that the derivatization procedures required to form volatile products prior to GC-MS analysis can sometimes produce artifactual formation of certain base oxidation products, although these studies did not replicate previously-used reaction conditions, e.g. they failed to remove air from the derivatization vials. A systematic examination of this problem revealed that levels of 8-hydroxyguanine, 8-hydroxyadenine, 5-hydroxycytosine and 5-(hydroxymethyluracil) in commercial calf thymus DNA determined by GC-MS are elevated by increasing the temperature at which derivatization is performed in our laboratory. In particular, 8-hydroxyguanine levels after silylation at 140 degrees C were raised 8-fold compared to derivatization at 23 degrees C. Experiments on the derivatization of each undamaged base revealed that the artifactual oxidation of guanine, adenine, cytosine and thymine respectively was responsible. Formation of the above products was potentiated by not purging with nitrogen prior to derivatization. Increasing the temperature to 140 degrees C or allowing air to be present during derivatization did not significantly increase levels of the other oxidized bases measured. This work suggests that artifactual oxidation during derivatization is restricted to certain products (8-hydroxyguanine, 8-hydroxyadenine, 5-hydroxycytosine and 5-[hydroxymethyluracil]) and can be decreased by reducing the temperature of the derivatization reaction to 23 degrees C and excluding as much air possible. Despite some recent reports, we were easily able to detect formamidopyrimidines in acid-hydrolyzed DNA. Artifacts of derivatization are less marked than has been claimed in some papers and may vary between laboratories, depending on the experimental procedures used, in particular the efficiency of exclusion of O2 during

  19. Stable Oligomeric Clusters of Gold Nanoparticles: Preparation, Size Distribution, Derivatization, and Physical and Biological Properties

    PubMed Central

    2015-01-01

    Reducing dilute aqueous HAuCl4 with NaSCN under alkaline conditions produces 2–3 nm diameter yellow nanoparticles without the addition of extraneous capping agents. We here describe two very simple methods for producing highly stable oligomeric grape-like clusters (oligoclusters) of these small nanoparticles. The oligoclusters have well-controlled diameters ranging from ∼5 to ∼30 nm, depending mainly on the number of subunits in the cluster. Our first [“delay-time”] method controls the size of the oligoclusters by varying from seconds to hours the delay time between making the HAuCl4 alkaline and adding the reducing agent, NaSCN. Our second [“add-on”] method controls size by using yellow nanoparticles as seeds onto which varying amounts of gold derived from “hydroxylated gold”, Na+[Au(OH4–x)Clx]−, are added-on catalytically in the presence of NaSCN. Possible reaction mechanisms and a simple kinetic model fitting the data are discussed. The crude oligocluster preparations have narrow size distributions, and for most purposes do not require fractionation. The oligoclusters do not aggregate after ∼300-fold centrifugal-filter concentration, and at this high concentration are easily derivatized with a variety of thiol-containing reagents. This allows rare or expensive derivatizing reagents to be used economically. Unlike conventional glutathione-capped nanoparticles of comparable gold content, large oligoclusters derivatized with glutathione do not aggregate at high concentrations in phosphate-buffered saline (PBS) or in the circulation when injected into mice. Mice receiving them intravenously show no visible signs of distress. Their sizes can be made small enough to allow their excretion in the urine or large enough to prevent them from crossing capillary basement membranes. They are directly visible in electron micrographs without enhancement, and can model the biological fate of protein-like macromolecules with controlled sizes and charges

  20. Recent advances in CE mediated microanalysis for enzymatic and derivatization reactions.

    PubMed

    Ramana, Pranov; Adams, Erwin; Augustijns, Patrick; Van Schepdael, Ann

    2016-01-01

    This review gives an overview of the applications and recent advances in CE mediated microanalysis. As a continuation of earlier reviews, it covers articles published from a period of mid 2013-early 2015. The article contains three main parts. In the first part of the article, different kinds of in-line CE mediated microanalysis are briefly discussed along with relevant papers regarding in-line CE for enzyme analysis. Recent advances in on-capillary derivatization reactions and immobilized enzyme reactors (IMERs) have been summarized along with their purpose and relevance in the second and third part of the paper, respectively.

  1. C-Terminal acetylene derivatized peptides via silyl-based alkyne immobilization.

    PubMed

    Strack, Martin; Metzler-Nolte, Nils; Albada, H Bauke

    2013-06-21

    A new Silyl-based Alkyne Modifying (SAM)-linker for the synthesis of C-terminal acetylene-derivatized peptides is reported. The broad scope of this SAM2-linker is illustrated by manual synthesis of peptides that are side-chain protected, fully deprotected, and disulfide-bridged. Synthesis of a 14-meric (KLAKLAK)2 derivative by microwave-assisted automated SPPS and a one-pot cleavage click procedure yielding protected 1,2,3-triazole peptide conjugates are also described.

  2. Fabrication of an all-polymer battery based on derivatized polythiophenes.

    PubMed

    Gofer, Y; Sarker, H; Killian, J G; Giaccai, J; Poehler, T O; Searson, P C

    1998-01-01

    Conducting polymers that can be switched between an insulating neutral state and a conducting doped state are of interest for charge-storage applications such as secondary batteries. The authors report on the fabrication of an all-polymer battery incorporating derivatized polythiophene films electropolymerized onto graphite-coated polymeric supports and a polymer gel electrolyte film. The cells had discharge voltages of about 2.4 V and capacities of 9.5 to 11.5 mAh g-1. The elimination of any metallic components or liquids and the lightweight and flexible construction provide a unique alternative for secondary battery technology.

  3. Derivatization in gas chromatographic determination of phenol and aniline traces in aqueous media

    NASA Astrophysics Data System (ADS)

    Gruzdev, I. V.; Zenkevich, I. G.; Kondratenok, B. M.

    2015-06-01

    Substituted anilines and phenols are the most common hydrophilic organic environmental toxicants. The principles of gas chromatographic determination of trace amounts of these compounds in aqueous media at concentrations <=0.1 μg litre-1 based on synthesis of their derivatives (derivatization) directly in the aqueous phase are considered. Conversion of relatively hydrophilic analytes into more hydrophobic derivatives makes it possible to achieve such low detection limits and optimize the protocols of extractive preconcentration and selective chromatographic detection. Among the known reactions, this condition is best met by electrophilic halogenation of compounds at the aromatic moiety. The bibliography includes 177 references.

  4. Stable oligomeric clusters of gold nanoparticles: preparation, size distribution, derivatization, and physical and biological properties.

    PubMed

    Smithies, Oliver; Lawrence, Marlon; Testen, Anze; Horne, Lloyd P; Wilder, Jennifer; Altenburg, Michael; Bleasdale, Ben; Maeda, Nobuyo; Koklic, Tilen

    2014-11-11

    Reducing dilute aqueous HAuCl4 with NaSCN under alkaline conditions produces 2-3 nm diameter yellow nanoparticles without the addition of extraneous capping agents. We here describe two very simple methods for producing highly stable oligomeric grape-like clusters (oligoclusters) of these small nanoparticles. The oligoclusters have well-controlled diameters ranging from ∼5 to ∼30 nm, depending mainly on the number of subunits in the cluster. Our first ["delay-time"] method controls the size of the oligoclusters by varying from seconds to hours the delay time between making the HAuCl4 alkaline and adding the reducing agent, NaSCN. Our second ["add-on"] method controls size by using yellow nanoparticles as seeds onto which varying amounts of gold derived from "hydroxylated gold", Na(+)[Au(OH4-x)Clx](-), are added-on catalytically in the presence of NaSCN. Possible reaction mechanisms and a simple kinetic model fitting the data are discussed. The crude oligocluster preparations have narrow size distributions, and for most purposes do not require fractionation. The oligoclusters do not aggregate after ∼300-fold centrifugal-filter concentration, and at this high concentration are easily derivatized with a variety of thiol-containing reagents. This allows rare or expensive derivatizing reagents to be used economically. Unlike conventional glutathione-capped nanoparticles of comparable gold content, large oligoclusters derivatized with glutathione do not aggregate at high concentrations in phosphate-buffered saline (PBS) or in the circulation when injected into mice. Mice receiving them intravenously show no visible signs of distress. Their sizes can be made small enough to allow their excretion in the urine or large enough to prevent them from crossing capillary basement membranes. They are directly visible in electron micrographs without enhancement, and can model the biological fate of protein-like macromolecules with controlled sizes and charges. The ease of

  5. Derivatized nanoparticle coated capillaries for purification and micro-extraction of proteins and peptides.

    PubMed

    Bakry, R; Gjerde, D; Bonn, G K

    2006-06-01

    Various methods are used to enrich or purify a protein of interest from other proteins and components in a crude cell lysate or other sample. One of the most powerful methods is affinity purification, also called affinity chromatography, whereby the proteins of interest are purified by virtue of their specific binding properties to an immobilized ligand. Affinity purification is becoming more widely used for exploring post-translation modifications and protein-protein interactions, especially with a view toward developing new general tag systems and strategies of chemical derivatization on peptides for affinity selection. Our work was aimed to immobilize proteins or ligands for affinity purification of antibodies, fusion-tagged proteins and other proteins and peptides. Selected proteins or peptides are efficiently extracted and enriched using chemically derivatized walls of a fused silica capillary column. In this paper, we present an open tubular capillary, where the inner wall of a fused silica capillary was derivatized by covalent binding of modified polystyrene latex particles. The capillaries were derivatized with iminodiacetic acid and loaded with Fe3+ or Ni2+ for the purification and enrichment of phosphopeptides or His-tagged proteins, respectively. The latex coated capillaries have been successfully applied to enrich phosphopeptides from beta-casein tryptic digest and ovalbumin tryptic digest at a micro volume scale with recoveries ranging from 92 to 95%. The capillaries have been eluted under conditions compatible with MALDI-MS without any prior desalting step. In another approach, concanavalin A (Con A) or Protein G were immobilized on the epoxy modified latex on the inner wall of the fused silica capillary for the purification of glycoproteins and immunoglobulin, respectively. The design of the capillary and the protocols used for purification permits the direct detection of eluted proteins and peptides with gel electrophoresis or with mass spectrometry

  6. Liquid chromatographic enantioseparation of three beta-adrenolytics using new derivatizing reagents synthesized from (S)-ketoprofen and confirmation of configuration of diastereomers.

    PubMed

    Alwera, Shiv; Bhushan, Ravi

    2016-11-01

    Diastereomers of racemic β-adrenolytic drugs [namely (RS)-propranolol, (RS)-metoprolol and (RS)-atenolol] were synthesized under microwave irradiation with (S)-ketoprofen based chiral derivatization reagents (CDRs) newly synthesized for this purpose. (S)-Ketoprofen was chosen for its high molar absorptivity (εo  ~ 40,000) and its availability as a pure (S)-enantiomer. Its -COOH group was activated with N-hydroxysuccinimide and N-hydroxybenzotriazole; these were easily introduced and also acted as good leaving groups during nucleophilic substitution by the amino group of the racemic β-adrenolytics. The CDRs were characterized by UV, IR, (1) H-NMR, HRMS and CHNS. Separation of diastereomers was achieved by RP HPLC and open column chromatography. Absolute configuration of the diastereomers was established with the help of (1) HNMR supported by developing their optimized lowest energy structures using Gaussian 09 Rev. A.02 program and hybrid density functional B3LYP with 6-31G* basis set (based on density functional theory), and elution order was established. RP HPLC conditions for separation were optimized and the separation method was validated. The limit of detection values were 0.308 and 0.302 ng mL(-1) . Copyright © 2016 John Wiley & Sons, Ltd.

  7. A method for the determination of acrylamide in a broad variety of processed foods by GC-MS using xanthydrol derivatization.

    PubMed

    Yamazaki, Kumiko; Isagawa, Satoshi; Kibune, Nobuyuki; Urushiyama, Tetsuo

    2012-01-01

    A novel GC-MS method was developed for the determination of acrylamide, which is applicable to a variety of processed foods, including potato snacks, corn snacks, biscuits, instant noodles, coffee, soy sauces and miso (fermented soy bean paste). The method involves the derivatization of acrylamide with xanthydrol instead of a bromine compound. Isotopically labelled acrylamide (d₃-acrylamide) was used as the internal standard. The aqueous extract from samples was purified using Sep-Pak™ C₁₈ and Sep-Pak™ AC-2 columns. For amino acid-rich samples, such as miso or soy sauce, an Extrelut™ column was used for purification or extraction. After reaction with xanthydrol, the resultant N-xanthyl acrylamide was determined by GC-MS. The method was validated for various food matrices and showed good linearity, precision and trueness. The limit of detection and limit of quantification ranged 0.5-5 and 5-20 µg kg⁻¹), respectively. The developed method was applied as an exploratory survey of acrylamide in Japanese foods and the method was shown to be applicable for all samples tested.

  8. Fluorescence determination of N-acetylaspartic acid in the rat cerebrum homogenate using high-performance liquid chromatography with pre-column fluorescence derivatization.

    PubMed

    Fukushima, Takeshi; Arai, Kotaro; Tomiya, Masayuki; Mitsuhashi, Shogo; Sasaki, Tsukasa; Santa, Tomofumi; Imai, Kazuhiro; Toyo'oka, Toshimasa

    2008-01-01

    N-acetyl-L-aspartic acid (NAA) is an endogenous compound, and its brain concentration is suggested to be altered in neurological disorders. In the present study, a fluorescence determination method for NAA was developed by employing reversed-phase high-performance liquid chromatography (HPLC) with pre-column fluorescence derivatization using 4-N,N-dimethylaminosulfonyl-7-N-(2-aminoethyl)amino-2,1,3-benzoxadiazole (DBD-ED). Using methylsuccinic acid as the internal standard, a linear calibration curve for NAA was constructed in the range 125-1000 microM (n=3). The detection limit on the column was approximately 5.0 fmol (signal-to-noise ratio 3). The proposed HPLC method was applied to determine NAA in the rat cerebrum homogenate. Cerebrum NAA was successfully determined using 10 microL of the homogenate, and the validation data for the proposed HPLC method demonstrated satisfactory results. Intra- and inter-day precision and accuracy were within 1.1-7.0 and -8.1-6.3%, respectively. The concentration of NAA in the male rat cerebrum (13 weeks old) was 84 +/-4.6 nmol/mg protein (n = 3) [corrected]. Copyright (c) 2007 John Wiley & Sons, Ltd.

  9. Synthesis of amino acids

    DOEpatents

    Davis, J.W. Jr.

    1979-09-21

    A method is described for synthesizing amino acids preceding through novel intermediates of the formulas: R/sub 1/R/sub 2/C(OSOC1)CN, R/sub 1/R/sub 2/C(C1)CN and (R/sub 1/R/sub 2/C(CN)O)/sub 2/SO wherein R/sub 1/ and R/sub 2/ are each selected from hydrogen and monovalent hydrocarbon radicals of 1 to 10 carbon atoms. The use of these intermediates allows the synthesis steps to be exothermic and results in an overall synthesis method which is faster than the synthesis methods of the prior art.

  10. Amino acids in Arctic aerosols

    NASA Astrophysics Data System (ADS)

    Scalabrin, E.; Zangrando, R.; Barbaro, E.; Kehrwald, N. M.; Gabrieli, J.; Barbante, C.; Gambaro, A.

    2012-11-01

    Amino acids are significant components of atmospheric aerosols, affecting organic nitrogen input to marine ecosystems, atmospheric radiation balance, and the global water cycle. The wide range of amino acid reactivities suggest that amino acids may serve as markers of atmospheric transport and deposition of particles. Despite this potential, few measurements have been conducted in remote areas to assess amino acid concentrations and potential sources. Polar regions offer a unique opportunity to investigate atmospheric processes and to conduct source apportionment studies of such compounds. In order to better understand the importance of amino acid compounds in the global atmosphere, we determined free amino acids (FAAs) in seventeen size-segregated aerosol samples collected in a polar station in the Svalbard Islands from 19 April until 14 September 2010. We used an HPLC coupled with a tandem mass spectrometer (ESI-MS/MS) to analyze 20 amino acids and quantify compounds at fmol m-3 levels. Mean total FAA concentration was 1070 fmol m-3 where serine and glycine were the most abundant compounds in almost all samples and accounted for 45-60% of the total amino acid relative abundance. The other eighteen compounds had average concentrations between 0.3 and 98 fmol m-3. The higher amino acid concentrations were present in the ultrafine aerosol fraction (< 0.49 μm) and accounted for the majority of the total amino acid content. Local marine sources dominate the boreal summer amino acid concentrations, with the exception of the regional input from Icelandic volcanic emissions.

  11. Amino acids in Arctic aerosols

    NASA Astrophysics Data System (ADS)

    Scalabrin, E.; Zangrando, R.; Barbaro, E.; Kehrwald, N. M.; Gabrieli, J.; Barbante, C.; Gambaro, A.

    2012-07-01

    Amino acids are significant components of atmospheric aerosols, affecting organic nitrogen input to marine ecosystems, atmospheric radiation balance, and the global water cycle. The wide range of amino acid reactivities suggest that amino acids may serve as markers of atmospheric transport and deposition of particles. Despite this potential, few measurements have been conducted in remote areas to assess amino acid concentrations and potential sources. Polar regions offer a unique opportunity to investigate atmospheric processes and to conduct source apportionment studies of such compounds. In order to better understand the importance of amino acid compounds in the global atmosphere, we determined free amino acids (FAAs) in seventeen size-segregated aerosol samples collected in a polar station in the Svalbard Islands from 19 April until 14 September 2010. We used an HPLC coupled with a tandem mass spectrometer (ESI-MS/MS) to analyze 20 amino acids to quantify compounds at fmol m-3 levels. Mean total FAA concentration was 1070 fmol m-3 where serine and glycine were the most abundant compounds in almost all samples and accounted for 45-60% of the total amino acid relative abundance. The other eighteen compounds had average concentrations between 0.3 and 98 fmol m-3. The higher amino acid concentrations were present in the ultrafine aerosol fraction (<0.49 μm) and accounted for the majority of the total amino acid content. Local marine sources dominate the boreal summer amino acid concentrations, with the exception of the regional input from Icelandic volcanics.

  12. β-Isocyanoalanine as an IR probe: comparison of vibrational dynamics between isonitrile and nitrile-derivatized IR probes.

    PubMed

    Maj, Michał; Ahn, Changwoo; Kossowska, Dorota; Park, Kwanghee; Kwak, Kyungwon; Han, Hogyu; Cho, Minhaeng

    2015-05-07

    An infrared (IR) probe based on isonitrile (NC)-derivatized alanine 1 was synthesized and the vibrational properties of its NC stretching mode were investigated using FTIR and femtosecond IR pump-probe spectroscopy. It is found that the NC stretching mode is very sensitive to the hydrogen-bonding ability of solvent molecules. Moreover, its transition dipole strength is larger than that of nitrile (CN) in nitrile-derivatized IR probe 2. The vibrational lifetime of the NC stretching mode is found to be 5.5 ± 0.2 ps in both D2O and DMF solvents, which is several times longer than that of the azido (N3) stretching mode in azido-derivatized IR probe 3. Altogether these properties suggest that the NC group can be a very promising sensing moiety of IR probes for studying the solvation structure and dynamics of biomolecules.

  13. Rapid and sensitive step gradient assays of glutamate, glycine, taurine and gamma-aminobutyric acid by high-performance liquid chromatography-fluorescence detection with o-phthalaldehyde-mercaptoethanol derivatization with an emphasis on microdialysis samples.

    PubMed

    Piepponen, T P; Skujins, A

    2001-06-15

    We developed a rapid step-gradient HPLC method for determination of glutamate, glycine and taurine, and a separate method for determination of gamma-aminobutyric acid (GABA) in striatal microdialysates. The amino acids were pre-column derivatized with o-phthalaldehyde-2-mercaptoethanol by using an automated refrigerated autoinjector. Separation of the amino acids was established with a non-porous ODS-II HPLC column, late-eluting substances were washed out with a one-step low-pressure gradient. Concentrations of the amino acids were determined with a fixed-wavelength fluorescence detector. The detection limit for GABA was 80 fmol in a 15 microl sample, detection limits for glutamate, glycine and taurine were not determined because their concentrations in striatal perfusates were far above their detection limits. Total analysis time was less than 12 min, including the wash-out step. The methods described are relatively simple, sensitive, inexpensive, and fast enough to keep up with the microdialysis sampling.

  14. Gas chromatography/trace analysis of derivatized azelaic acid as a stability marker.

    PubMed

    Alzweiri, Muhammed; Tarawneh, Ruba; Khanfar, Mohammad A

    2013-10-01

    Azelaic acid, a naturally occurring saturated dicarboxylic acid, is found in many topical formulations for its various medical benefits or as a byproduct of the oxidative decomposition of unsaturated fatty acids. The poor volatility of azelaic acid hinders its applicability in GC analysis. Therefore, azelaic acid was derivatized by methylation and silylation procedures to enhance its volatility for GC analysis. Accordingly, dimethyl azelate (DMA) and di(trimethylsilyl) azelate were synthesized and characterized by GC-MS. Subsequently, a GC with flame ionization detection method was developed and validated to analyze trace amounts of azelaic acid in some marketed skin creams. Unlike DMA, di(trimethylsilyl) azelate was chemically unstable and degraded within few hours. Nonane was used as a stable internal standard. Variability due to derivatization and extraction was controlled by a standard addition procedure. DMA analysis was linear in a wide concentration range (100 ng/mL to 100 mg/mL). Moreover, the method was accurate (96.4-103.4%) and precise with inter- and intraday variability <2.0% and LOQ and LOD of 100 and 10 ng/mL, respectively. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. Fabrication of electrolytic cell for online post-column electrochemical derivatization in ion chromatography.

    PubMed

    Wu, Shuchao; Xu, Wei; Yang, Bingcheng; Ye, Mingli; Zhang, Peimin; Shen-Tu, Chao; Zhu, Yan

    2012-07-20

    An electrolytic cell (EC), composed of two ruthenium-plated titanium electrodes separated by cation-exchange membranes, was fabricated and evaluated for online postcolumn derivatization in ion chromatography (IC). Folic acid (FA) and methotrexate (MTX) were preliminarily used as prototype analytes to test the performance of EC. After separation by an anion exchange column, FA and MTX, which emit very weak fluorescence when excited, were electrochemically oxidized online in the anode chamber of the EC. The compounds with strong fluorescence, which are oxidation products, were detected by the fluorescence detector. The phosphate buffer solution (100 mM KH(2)PO(4)) served as an optimal eluent for anion exchange chromatographic separation and a suitable supporting electrolyte for electro-oxidation, leading to ideal compatibility between IC separation and the postcolumn electrochemical derivatization. For the presently proposed method, the linear ranges were from 0.01 mg L(-1) to 5 mg L(-1) for both FA and MTX. The detection limits of FA and MTX were 1.8 and 2.1 μg L(-1), and the relative standard deviations (RSD, n=7) were 2.9% and 3.6%, respectively. The method was applied for the simultaneous determination of FA and MTX in the plasma of patients being treated for rheumatoid arthritis. The determination of MTX in the urine of the patients of diffuse large B cell lymphoma was also demonstrated.

  16. Thiol derivatization of Xanthan gum and its evaluation as a mucoadhesive polymer.

    PubMed

    Bhatia, Meenakshi; Ahuja, Munish; Mehta, Heena

    2015-10-20

    Thiol-derivatization of xanthan gum polysaccharide was carried out by esterification with mercaptopropionic acid and thioglycolic acid. Thiol-derivatization was confirmed by Fourier-transformed infra-red spectroscopy. Xanthan-mercaptopropionic acid conjugate and xanthan-thioglycolic acid conjugate were found to possess 432.68mM and 465.02mM of thiol groups as determined by Ellman's method respectively. Comparative evaluation of mucoadhesive property of metronidazole loaded buccal pellets of xanthan and thiolated xanthan gum using chicken buccal pouch membrane revealed higher ex vivo bioadhesion time of thiolated xanthan gum as compared to xanthan gum. Improved mucoadhesive property of thiolated xanthan gum over the xanthan gum can be attributed to the formation of disulfide bond between mucus and thiolated xanthan gum. In vitro release study conducted using phosphate buffer (pH 6.8) revealed a sustained release profile of metronidazole from thiolated xanthan pellets as compared to xanthan pellets. In conclusion, thiolation of xanthan improves its mucoadhesive property and sustained the release of metronidazole over a prolonged period.

  17. Individual volatile fatty acids determination by chromogenic derivatization coupled to multi-syringe chromatography.

    PubMed

    Robert-Peillard, Fabien; Boudenne, Jean-Luc; Coulomb, Bruno

    2013-10-15

    In this paper, a new multisyringe chromatography (MSC) system is proposed for a simple and accurate measurement of individual volatile fatty acids (VFA) in anaerobic treatment processes. The determination method is based on the derivatization of VFA with N-(1-naphthyl) ethylenediamine (EDAN) followed by the separation of VFA derivatives on an Onyx C18 monolithic column (25 mm × 4.6mm i.d.). Chromatographic separation conditions have been investigated and were found to be optimal with a mixture of acetonitrile and formic acid 0.1% (ratio 35/65), providing good separation of C2-C5 VFA in 8 min. Optimization of the derivatization reaction was also carried out with special attention paid to the buffering capacity of the reaction medium, so as to be able to deal with samples of various characteristics in terms of alkalinity or of VFA concentration range. Individual VFA could be quantified between 0.05-2.5 g L(-1) with LOD of 0.01-0.02 g L(-1). Overall procedure time was about 18 min for one analytical cycle, which fulfils the requirement of real-time monitoring of an anaerobic digester. Validation of the system developed has been assessed by application of the procedure to sludge samples from various origins, and comparative results with gas chromatography analyses showed satisfactory correlation (R²>0.98).

  18. A method for the measurement of atmospheric HONO based on DNPH derivatization and HPLC analysis

    SciTech Connect

    Zhou, X.; Qiao, H.; Deng, G.; Civerolo, K.

    1999-10-15

    A simple measurement technique was developed for atmospheric HONO based on aqueous scrubbing using a coil sampler followed by 2,4-dinitrophenylhydrazine (DNPH) derivatization and high-performance liquid chromatographic (HPLC) analysis. Quantitative sampling efficiency was obtained using a 1 mM phosphate buffer, pH 7.0, as the scrubbing solution at a gas sampling flow rate of 2 L min{sup {minus}1} and a liquid flow rate of 0.24 mL min{sup {minus}1}. Derivation of the scrubbed nitrous acid by DNPH was fast and was completed within 5 min in a derivatization medium containing 300 {micro}M DNPH and 8 mM HCI at 45 C. The azide derivative was separated from DNPH reagent and carbonyl derivatives by reverse-phase HPLC and was detected with an UV detector at 309 nm. The detection limit is {le}5 pptv and may be lowered to 1 pptv with further DNPH purification. Interferences from NO, NO{sub 2} PAN, O{sub 3}, HNO{sub 3}, and HCHO were studied and found to be negligible. Ambient HONO concentration was measured simultaneously in downtown Albany, NY, by this method and by an ion chromatographic technique after sampling using a fritted bubbler. The results, from 70 pptv during the day to 1.7 ppbv in the early morning, were in very good agreement from the two techniques, within {+-} 20%.

  19. Isolation and derivatization of plasma taurine for stable isotope analysis by gas chromatography-mass spectrometry

    SciTech Connect

    Irving, C.S.; Klein, P.D.

    1980-09-01

    A method for the isolation and derivatization of plasma taurine is described that allows stable isotope determinations of taurine to be made by gas chromatography-mass spectrometry. The isolation procedure can be applied to 0.1 ml of plasma; the recovery of plasma taurine was 70 to 80%. For gc separation, taurine was converted to its dimethylaminomethylene methyl ester derivative which could not be detected by hydrogen flame ionization, but could be monitored readily by NH/sub 3/ chemical ionization mass spectrometry. The derivatization reaction occurred partially on-column and required optimization of injection conditions. Using stable isotope ratiometry multiple ion detection, (M + 2 + H)/sup +//(M + H)/sup +/ ion ratio of natural abundance taurine was determined with a standard deviation of less than +-0.07% of the ratio. The (1,2-/sup 13/C)taurine/taurine mole ratios of standard mixtures could be accurately determined to 0.001. This stable isotope gc-ms method is suitable for studying the plasma kinetics of (1,2-/sup 13/C)taurine in infants who are at risk with respect to taurine depletion.

  20. In-capillary derivatization and determination of iodine in sodium chloride solution.

    PubMed

    Nguyen, Bao D Q; Chernov'yants, Margarita S; Burykin, Igor V

    2012-01-21

    A new capillary electrophoretic (CE) method was developed for the simple and selective determination of iodine in 0.5 mol l(-1) NaCl. The proposed method is based on the in-capillary derivatization of iodine with thiosulfate ions using the zone-passing technique and direct photometric detection of the iodide and tetrathionate formed. The optimal conditions for the separation and derivatization reaction were established by varying the concentration of iodine, electrolyte pH and applied voltage. The optimized separations were carried out in phosphate electrolyte (pH 6.86) using direct photometric detection at 253.7 nm. Common photometric detection absorbing anions such as Cl(-), NO(2)(-), S(2)O(3)(2-) did not give any interference. Valid calibration (r(2) = 0.994) is demonstrated in the range 16.5-198.1 mg l(-1) of iodine. The detection limit (calculated according to K. Doerffel, Statistik in der analytischen Chemie, 1990) was 11.53 mg l(-1) (by iodide peak area) and 8.45 mg l(-1) (by tetrathionate peak area). The proposed system was applied to the determination of iodine after oxidation of iodide in underground water.

  1. [Determination of sucralose in foods by HPLC using pre-column derivatization].

    PubMed

    Nojiri, Shuko; Nakazato, Mitsuo; Kasuya, Yoko; Takano, Ichiro; Oishi, Mitsuo; Yasuda, Kazuo; Suzuki, Sukeji

    2002-10-01

    The development of a sensitive pre-column derivatization high-performance liquid chromatography (HPLC) method for determination of sucralose is reported. Sucralose is converted into a strongly ultraviolet (UV)-absorbing derivative, possessing strong absorption at 260 nm, by treatment with p-nitrobenzoyl chloride (PNBCl). Homogenized samples were dialyzed and washed with a Bond Elut ENV cartridge, then the eluate was evaporated to dryness and the residue was derivatized. Subsequently, the sucralose derivative was purified with hexane-ethyl actate (9:1) in a silica cartridge, and then the sucralose derivative was eluted with acetone. HPLC was performed on a phenyl column, using acetonitrile-water (73:27) as a mobile phase with UV detection (260 nm). The calibration curve was linear in the range of 1 microgram/mL to 50 micrograms/mL of sucralose. The recoveries of sucralose from eight kinds of foods spiked at the levels of 0.20 and 0.05 g/kg of sucralose were more than 76.2% with SD values in the range from 0.90% to 4.31%. The quantitative limit of the developed method was 0.005 g/kg for sucralose in samples.

  2. Application of INEPT nitrogen-15 and silicon-29 nuclear magnetic resonance spectrometry to derivatized fulvic acids

    USGS Publications Warehouse

    Thorn, K.A.; Folan, D.W.; Arterburn, J.B.; Mikita, M.A.; MacCarthy, P.

    1989-01-01

    Use of the INEPT experiment has been examined in two derivatization studies of the Suwannee River fulvic acid. In the first study, the fulvic acid was derivatized with 15N enriched hydroxylamine. The quantitative 15N NMR spectrum, acquired with a 45° pulse angle, 2.0 second pulse delay and inverse gated decoupling, showed that oximes (390-340 ppm) were the major derivatives, followed by nitriles (270-240 ppm), hydroxamic acids (170-160 ppm), secondary amides (150-115 ppm), and lactams (115-90 ppm). The INEPT 15N NMR spectrum was acquired using refocussing delays and polarization transfer times optimized for signal enhancement of singly protonated nitrogens. INEPT greatly enhanced the amide and lactam resonances, and showed that resonances downfield of 180 ppm in the quantitative spectrum represented nonprotonated nitrogens. In the second study, the fulvic acid was first methylated with diazomethane and then silylated with hexamethyldisilazane. The 29Si NMR spectra exhibited two major peaks, from approximately 33 to 22 ppm, representing silyl esters of carboxylic acids, and from 22 to 13 ppm, representing silyl ethers of alcohols and phenols. The INEPT 29Si NMR spectrum was virtually identical to the quantitative 29Si spectrum, acquired with a 90° pulse angle, 5.0 second pulse delay, inverse gated decoupling, and relaxation reagent. INEPT therefore can be used for quantitative analysis of trimethylsilyl derivatives of the fulvic acid, saving spectrometer time and eliminating the need for relaxation reagents.

  3. Fixed bed reactor for solid-phase surface derivatization of superparamagnetic nanoparticles.

    PubMed

    Steitz, Benedikt; Salaklang, Jatuporn; Finka, Andrija; O'Neil, Conlin; Hofmann, Heinrich; Petri-Fink, Alke

    2007-01-01

    The functionalization of nanoparticles is conditio sine qua non in studies of specific interaction with a biological target. Often, their biological functionality is achieved by covalent binding of bioactive molecules on a preexisting single surface coating. The yield and quality of the resulting coated and functionalized superparamagnetic iron oxide nanoparticles (SPIONs) can be significantly improved and reaction times reduced by using solid-phase synthesis strategies. In this study, a fixed bed reactor with a quadrupole repulsive arrangement of permanent magnets was assayed for SPION surface derivatization. The magnet array around the fixed bed reactor creates very high magnetic field gradients that enables the immobilization of SPIONs with a diameter as low as 9 nm. The functionalization on the surface of immobilized 25 nm 3-(aminopropyl)trimethoxysilane-coated SPIONs (APS-SPIONs) was performed using fluorescein-isothiocyanate directly, and by the SV40 large T-antigen nuclear localization signal peptide (PKKKRKVGC) conjugated to acryloylpoly(ethylene glycol)-N-hydroxysuccinimide, where the PEG reagent is conjugated first to create a functionalized nanoparticle and the peptide is added to the acryloyl group. We show that the yield of reactant grafted on the surface of the APS-coated SPIONs was higher in solid-phase within the fixed bed reactor compared to conventional liquid-phase chemistry. In summary, the functionalization of SPIONs using a magnetically fixed bed reactor was superior to the liquid-phase reaction in terms of the yield, reaction times required for derivatization, size distribution, and scalability.

  4. Structural determination of nerve agent markers using gas chromatography mass spectrometry after derivatization with 3-pyridyldiazomethane.

    PubMed

    Nyholm, Jenny Rattfelt; Gustafsson, Tomas; Östin, Anders

    2013-07-01

    Nerve agents are a class of organophosphorous chemicals that are prohibited under the Chemical Weapons Convention. Their degradation products, phosphonic acids, are analyzed as markers of nerve agent contamination and use. Because the phosphonic acids are non-volatile and very polar, their identification by GC-MS requires a derivatization step prior to analysis. Standard derivatization methods for gas-chromatography electron-impact mass-spectrometry analysis give very similar spectra for many alkyl phosphonic acid isomers, which complicates the identification process. We present a new reagent, 3-pyridyldiazomethane, for preparing picolinyl ester derivatives of alkyl methylphosphonic acids facilitating the determination of their structure by enhancing predictable fragmentation of the O-alkyl chain. This fragmentation is directed by the nitrogen nucleus of the pyridyl moiety that abstracts hydrogen from the O-alkyl chain, inducing radical cleavage of the carbon-carbon bonds and thereby causing extensive fragmentation that can be used for detailed structure elucidation of the O-alkyl moiety. The separability of related isomers was tested by comparing the spectra of the picolinyl esters formed from twelve hexyl methylphosphonic acid isomers. Spectral library matches and principal component analysis showed that the picolinyl esters were more effectively separated than the corresponding trimethylsilyl derivatives used in the standard operating procedures. The suggested method will improve the unambiguous structural determination process for phosphonic acids. Copyright © 2013 John Wiley & Sons, Ltd.

  5. An alternative derivatization reaction to the determination of doramectin in bovine milk using spectrofluorimetry

    NASA Astrophysics Data System (ADS)

    Maia, Priscila M. S.; de F. Rezende, Flavia B.; Netto, Annibal D. Pereira; de C. Marques, Flávia F.

    The doramectin (DOR), which belongs to the avermectins group (AVM), has a high antiparasitic activity and so it has been widely used in food-producing animals. The DOR shows low fluorescence quantum efficiency and as a consequence, chemical derivatization reactions are necessary to produce derivatives with improved luminescent properties before its determination by fluorimetry. As the presence of this compound in food represents a risk to human health, an easy, clean and low cost derivatization reaction, which is alternative to those usually employed and that enables its spectrofluorimetric determination in milk samples, was developed. Ethanolic solutions of DOR, containing sodium hydroxide at a final concentration of 0.25 mol L-1, after 60 min of heating at 50 °C, produced fluorescent signals 1000 times higher than the original ethanolic solution. Using these optimized conditions, a linear response range that extended from 50.00 to 1000 μg L-1, with a value of (R2) equal to 0.9970, was obtained. Average recovery of DOR was 92.5 ± 1.5% (n = 3) in bovine milk fortified samples submitted to a liquid-liquid extraction at low temperature and pre concentration process, indicating the usefulness and effectiveness of the proposed method. The proposed spectrofluorimetric method is an alternative to high-performance liquid chromatography (HPLC) based methods, allowing rapid and simple detection of doramectin in milk samples.

  6. [Determination of histamine in canned fish by high performance liquid chromatography with pre-column derivatization].

    PubMed

    Jin, Gaowa; Cai, Youqiong; Yu, Huijuan; Qian, Beilei

    2010-11-01

    A pre-column derivatization-high performance liquid chromatographic (HPLC) method has been developed for the determination of histamine in canned fish. The homogenated samples were ultrasonically extracted with perchloric acid aqueous solution, derivatized with dansyl chloride and diluted with acetonitrile to a desired volume. The samples were determined by HPLC with ultraviolet detector and quantified by external standard method. Adopting a C18 column with 1.8 microm stationary phase particles, the analysis time for each sample was smaller than 5 min with the flow rate of 0.3 mL/min. It can decrease the consumption of the mobile phase and save the cost. The linear range was 0.08-8.00 mg/L for histamine. The correlation coefficient was 0.999 98. The average recoveries of histamine at different concentration levels in spiked samples were greater than 96% and the relative standard deviations (RSDs) were smaller than 2.5%. The quantitation limit was 5.00 mg/kg for histamine in canned fish by HPLC. The results indicated that this HPLC method is fast, sensitive, reproducible and practical for the routine analysis of histamine in canned fish.

  7. Optimized synthesis of phosphorothioate oligodeoxyribonucleotides substituted with a 5'-protected thiol function and a 3'-amino group.

    PubMed

    Aubert, Y; Bourgerie, S; Meunier, L; Mayer, R; Roche, A C; Monsigny, M; Thuong, N T; Asseline, U

    2000-02-01

    A new deprotection procedure enables a medium scale preparation of phosphodiester and phosphor-othioate oligonucleotides substituted with a protected thiol function at their 5'-ends and an amino group at their 3'-ends in good yield (up to 72 OD units/micromol for a 19mer phosphorothioate). Syntheses of 3'-amino-substituted oligonucleotides were carried out on a modified support. A linker containing the thioacetyl moiety was manually coupled in two steps by first adding its phosphor-amidite derivative in the presence of tetrazole followed by either oxidation or sulfurization to afford the bis-derivatized oligonucleotide bound to the support. Deprotection was achieved by treating the fully protected oligonucleotide with a mixture of 2,2'-dithiodipyridine and concentrated aqueous ammonia in the presence of phenol and methanol. This proced-ure enables (i) cleavage of the oligonucleotide from the support, releasing the oligonucleotide with a free amino group at its 3'-end, (ii) deprotection of the phosphate groups and the amino functions of the nucleic bases, as well as (iii) transformation of the 5'-terminal S -acetyl function into a dithiopyridyl group. The bis-derivatized phosphorothioate oligomer was further substituted through a two-step procedure: first, the 3'-amino group was reacted with fluorescein isothiocyanate to yield a fluoresceinylated oligo-nucleotide; the 5'-dithio-pyridyl group was then -quantitatively reduced to give a free thiol group which was then substituted by reaction with an N alpha-bromoacetyl derivative of a signal peptide containing a KDEL sequence to afford a fluoresceinylated peptide-oligonucleotide conjugate.

  8. Micro-Detection System for Determination of the Biotic or Abiotic Origin of Amino Acids

    NASA Technical Reports Server (NTRS)

    Bada, Jeffrey L.

    2003-01-01

    The research carried out under this PIDDP involves the development of a breadboard version of a spacecraft-based system for the detection of amino acid chirality (molecular handedness) on solar system bodies. Chirality provides an unambiguous way of distinguishing between abiotic and biotic origins since only one mirror-image form is used in the functional molecules of life. Recent advances in a variety of nano-fabrication technologies have resulted in concepts for enabling miniaturized chemical and biological analytical systems. These are complete application-specific systems that integrate fluid micro handling systems for extracting and reacting target molecules, micro-separation technologies for enhanced sensitivity and resolution, and advanced detection technologies. This effort makes use of a relatively new technology that shows demonstrated promise for spacecraft-based amino acid analysis: microchip-based capillary electrophoresis (muCE). The muCE system is capable of analyzing the type of amino acids present as well as the relative amounts of their mirror image forms. The system we developed will be able to chirally resolve all of the major amino acids found in extraterrestrial material (Gly, Ala, Val, Pro, Asp, Glu, a-aminoisobutyric acid, and isovaline) at sub-part-per-billion levels. The _CE analysis requires that the amino acids be extracted from the sample and derivatized for either optical or electrochemical detection. In our implementation, the amino acids are released from the sample by sublimation and prepared for muCE analysis using a microfluidic circuit. In addition, we have investigated the use of a microfluidic circuit for the release of amino acids from samples in which sublimation has proven to be problematic.

  9. Micro-Detection System for Determination of the Biotic or Abiotic Origin of Amino Acids

    NASA Technical Reports Server (NTRS)

    Bada, Jeffrey L.

    2003-01-01

    The research carried out under this PIDDP involves the development of a breadboard version of a spacecraft-based system for the detection of amino acid chirality (molecular handedness) on solar system bodies. Chirality provides an unambiguous way of distinguishing between abiotic and biotic origins since only one mirror-image form is used in the functional molecules of life. Recent advances in a variety of nano-fabrication technologies have resulted in concepts for enabling miniaturized chemical and biological analytical systems. These are complete application-specific systems that integrate fluid micro handling systems for extracting and reacting target molecules, micro-separation technologies for enhanced sensitivity and resolution, and advanced detection technologies. This effort makes use of a relatively new technology that shows demonstrated promise for spacecraft-based amino acid analysis: microchip-based capillary electrophoresis (muCE). The muCE system is capable of analyzing the type of amino acids present as well as the relative amounts of their mirror image forms. The system we developed will be able to chirally resolve all of the major amino acids found in extraterrestrial material (Gly, Ala, Val, Pro, Asp, Glu, a-aminoisobutyric acid, and isovaline) at sub-part-per-billion levels. The _CE analysis requires that the amino acids be extracted from the sample and derivatized for either optical or electrochemical detection. In our implementation, the amino acids are released from the sample by sublimation and prepared for muCE analysis using a microfluidic circuit. In addition, we have investigated the use of a microfluidic circuit for the release of amino acids from samples in which sublimation has proven to be problematic.

  10. Stable Isotope Peptide Mass Spectrometry To Decipher Amino Acid Metabolism in Dehalococcoides Strain CBDB1

    PubMed Central

    Marco-Urrea, Ernest; Seifert, Jana; von Bergen, Martin

    2012-01-01

    Dehalococcoides species are key players in the anaerobic transformation of halogenated solvents at contaminated sites. Here, we analyze isotopologue distributions in amino acid pools from peptides of Dehalococcoides strain CBDB1 after incubation with 13C-labeled acetate or bicarbonate as a carbon source. The resulting data were interpreted with regard to genome annotations to identify amino acid biosynthesis pathways. In addition to using gas chromatography-mass spectrometry (GC-MS) for analyzing derivatized amino acids after protein hydrolysis, we introduce a second, much milder method, in which we directly analyze peptide masses after tryptic digest and peptide fragments by nano-liquid chromatography-electrospray ionization-tandem mass spectrometry (nano-LC-ESI-MS/MS). With this method, we identify isotope incorporation patterns for 17 proteinaceous amino acids, including proline, cysteine, lysine, and arginine, which escaped previous analyses in Dehalococcoides. Our results confirmed lysine biosynthesis via the α-aminoadipate pathway, precluding lysine formation from aspartate. Similarly, the isotopologue pattern obtained for arginine provided biochemical evidence of its synthesis from glutamate. Direct peptide MS/MS analysis of the labeling patterns of glutamine and asparagine, which were converted to glutamate and aspartate during protein hydrolysis, gave biochemical evidence of their precursors and confirmed glutamate biosynthesis via a Re-specific citrate synthase. By addition of unlabeled free amino acids to labeled cells, we show that in strain CBDB1 none of the 17 tested amino acids was incorporated into cell mass, indicating that they are all synthesized de novo. Our approach is widely applicable and provides a means to analyze amino acid metabolism by studying specific proteins even in mixed consortia. PMID:22661690

  11. GC-MS analysis of amino acid enantiomers as their N(O,S)-perfluoroacyl perfluoroalkyl esters: application to space analysis.

    PubMed

    Zampolli, M; Meunier, D; Sternberg, R; Raulin, F; Szopa, C; Pietrogrande, M C; Dondi, F

    2006-05-05

    The target of the in-situ research of optical activity in extraterrestrial samples stimulated an extended investigation of a GC-MS method based on the derivatization of amino acids by using a mixture of perfluorinated alcohols and perfluorinated anhydrides. Amino acids are converted to their N(O,S)-perfluoroacyl perfluoroalkyl esters in a single-step procedure, using different combinations of the derivatization reagents trifluoroacetic anhydride (TFAA)-2,2,2-trifluoro-1-ethanol (TFE), TFAA-2,2,3,3,4,4,4-heptafluoro-1-butanol (HFB), and heptafluorobutyric anhydride (HFBA)-HFB. The derivatives obtained are analyzed using two different chiral columns: Chirasil-L-Val and gamma-cyclodextrin (Rt-gamma-DEXsa) stationary phases which show different and complementary enantiomeric selectivity. The mass spectra of the derivatives are studied, and mass fragmentation patterns are proposed: significant fragment ions can be identified to detect amino acid derivatives. The obtained results are compared in terms of the enantiomeric separation achieved and mass spectrometric response. Linearity studies and the measurement of the limit of detection (LOD) show that the proposed method is suitable for a quantitative determination of enantiomers of several amino acids. The use of the programmed temperature vaporiser (PTV) technique for the injection of the untreated reaction mixture is a promising method for avoiding manual treatment of the sample and decreasing the LOD.

  12. Amino Acids Generated from Hydrated Titan Tholins: Comparison with Miller-Urey Electric Discharge Products

    NASA Technical Reports Server (NTRS)

    Cleaves, H. James, II; Neish, Catherine; Callahan, Michael P.; Parker, Eric; Fernandez, Facundo M.; Dworkin, Jason P.

    2014-01-01

    Various analogues of Titan haze particles (termed tholins) have been made in the laboratory. In certain geologic environments on Titan, these haze particles may come into contact with aqueous ammonia (NH3) solutions, hydrolyzing them into molecules of astrobiological interest. A Titan tholin analogue hydrolyzed in aqueous NH3 at room temperature for 2.5 years was analyzed for amino acids using highly sensitive ultra-high performance liquid chromatography coupled with fluorescence detection and time-of-flight mass spectrometry (UHPLC-FDToF-MS) analysis after derivatization with a fluorescent tag. We compare here the amino acids produced from this reaction sequence with those generated from room temperature Miller-Urey (MU) type electric discharge reactions. We find that most of the amino acids detected in low temperature MU CH4N2H2O electric discharge reactions are generated in Titan simulation reactions, as well as in previous simulations of Triton chemistry. This argues that many processes provide very similar mixtures of amino acids, and possibly other types of organic compounds, in disparate environments, regardless of the order of hydration. Although it is unknown how life began, it is likely that given reducing conditions, similar materials were available throughout the early Solar System and throughout the universe to facilitate chemical evolution.

  13. Chiral MEKC-LIF of amino acids in foods: analysis of vinegars.

    PubMed

    Carlavilla, Davinia; Moreno-Arribas, M Victoria; Fanali, Salvatore; Cifuentes, Alejandro

    2006-07-01

    The formation of D-amino acids (D-aa's) in many fermented foods depends, among other factors, on the particular fermentation conditions, the action and autolysis of the microorganisms involved. In this sense, the analysis of chiral amino acids is an interesting analytical strategy for food scientists, since these compounds can be used as bacterial markers and can help, e.g., to detect adulterations, microbiological contaminations, etc. In this work, a fast and sensitive method based on MEKC-LIF has been developed to analyze and quantitate L-amino acid (L-aa) and D-aa in vinegars. The chiral MEKC-LIF procedure uses 100 mM sodium tetraborate, 30 mM SDS, and 20 mM beta-CD at pH 9.7 as running buffer, obtaining a good separation of the main vinegar L-/D-aa previously derivatized with fluorescein isothiocianate. Namely, L/D proline, alanine, arginine, glutamic, and aspartic acid, plus the nonchiral amino acid gamma-aminobutyric acid are separated in less than 20 min with high efficiency (up to 720,000 plates/m) and good sensitivity (LODs lower than 16.6 nM were achieved). Several D-aa's were detected and quantified in balsamic, sherry, white wine, and cider vinegars using this MEKC-LIF procedure, observing interesting differences in their L-aa and D-aa profiles and contents.

  14. Amino acids generated from hydrated Titan tholins: Comparison with Miller-Urey electric discharge products

    NASA Astrophysics Data System (ADS)

    Cleaves, H. James; Neish, Catherine; Callahan, Michael P.; Parker, Eric; Fernández, Facundo M.; Dworkin, Jason P.

    2014-07-01

    Various analogues of Titan haze particles (termed ‘tholins’) have been made in the laboratory. In certain geologic environments on Titan, these haze particles may come into contact with aqueous ammonia (NH3) solutions, hydrolyzing them into molecules of astrobiological interest. A Titan tholin analogue hydrolyzed in aqueous NH3 at room temperature for 2.5 years was analyzed for amino acids using highly sensitive ultra-high performance liquid chromatography coupled with fluorescence detection and time-of-flight mass spectrometry (UHPLC-FD/ToF-MS) analysis after derivatization with a fluorescent tag. We compare here the amino acids produced from this reaction sequence with those generated from room temperature Miller-Urey (MU) type electric discharge reactions. We find that most of the amino acids detected in low temperature MU CH4/N2/H2O electric discharge reactions are generated in Titan simulation reactions, as well as in previous simulations of Triton chemistry. This argues that many processes provide very similar mixtures of amino acids, and possibly other types of organic compounds, in disparate environments, regardless of the order of hydration. Although it is unknown how life began, it is likely that given reducing conditions, similar materials were available throughout the early Solar System and throughout the universe to facilitate chemical evolution.

  15. On the reported optical activity of amino acids in the Murchison meteorite

    NASA Astrophysics Data System (ADS)

    Bada, J. L.; Cronin, J. R.; Ho, M.-S.; Kvenvolden, K. A.; Lawless, J. G.; Miller, S. L.; Oro, J.; Steinberg, S.

    1983-02-01

    It is shown that the explanation of terrestrial contamination of the Murchison meteorite is consistent with the analysis of extracts from the meteorite reported by Engel and Nagy (EN) (1982) and is much more probable than their suggestion that the excess of L-enantiomers for several protein amino acids is due to asymmetric synthesis or decomposition. The low abundance of serine and threonine reported by EN may be due to their decomposition during the derivatization procedure, and the absence of methionine, tyrosine, and phenylalanine can be attributed to various causes. The amount of contamination in EN's extracts are estimated from a mass balance of the amino acid enantiomers, and it is found that the amino acids in the HCl could be due entirely to contamination while in the water extract the amount of contamination ranges from about 40 to 97 percent, depending on the amino acid. The argument that contaminants were preferentially extracted by EN's procedure cannot account for the failure to detect methionine, tyrosine, and phenylalanine.

  16. Analysis of amino acids in human vascular endothelial (ECV-304) cells by microchip electrophoresis with fluorescence detection.

    PubMed

    Shi, Baoxian; Huang, Weihua; Cheng, Jieke

    2008-04-01

    A rapid and sensitive method was developed for the analysis of amino acids by microchip electrophoresis with Hg-lamp excitation fluorescence detection. Fluorescein-isothiocyanate (FITC) was chosen to estimate the sensitivity of this system, and the detection limit (S/N = 3) with FITC was 1.7 nM, which showed that the system was sensitive as well as simple. Two derivatizing agents, FITC and ortho-phthalaldehyde (OPA) were employed to label amino acids and were compared in the same fluorescence detection system with an Hg lamp as the excitation source. The separation parameters were optimized in detail. Optimum separation of OPA-labeled amino acids was obtained in less than 200 s with 20 mM borate buffer (pH 9.0) containing 20% acetonitrile and 10 mM beta-cyclodextrin. Detection limits for amino acids (alanine (Ala), taurine (Tau), glycine (Gly), glutamic acid (Glu), and aspartic acid (Asp)) of 0.38-1.0 muM were achieved. The method was successfully applied to analysis of amino acids in human vascular endothelial cells (ECV-304). The average amount of amino acids in single ECV-304 cells is estimated to be 5.84 fmol for Ala, 2.78 fmol for Tau, 1.15 fmol for Gly, 3.10 fmol for Glu, and 1.30 fmol for Asp.

  17. Amino acids in carbonaceous chondrites

    NASA Technical Reports Server (NTRS)

    Lawless, J. G.; Peterson, E.

    1975-01-01

    Studies with the combined gas chromatograph-mass spectrometer were conducted to characterize further the amino acids found in extracts of the Murchison meteorite. With the exception of beta-aminoisobutyric acid, all of the amino acids which were found in previous studies of the Murchison meteorite and the Murray meteorite have been identified. The results obtained lend further support to the hypothesis that amino acids are present in the Murchison meteorite as the result of an extraterrestrial abiotic synthesis.

  18. In situ derivatization and hollow fiber membrane microextraction for gas chromatographic determination of haloacetic acids in water.

    PubMed

    Varanusupakul, Pakorn; Vora-Adisak, Narongchai; Pulpoka, Bancha

    2007-08-13

    An alternative method for gas chromatographic determination of haloacetic acids (HAAs) in water using direct derivatization followed by hollow fiber membrane liquid-phase microextraction (HF-LPME) has been developed. The method has improved the sample preparation step according to the conventional US EPA Method 552.2 by combining the derivatization and the extraction into one step prior to determination by gas chromatography electron captured detector (GC-ECD). The HAAs were derivatized with acidic methanol into their methyl esters and simultaneously extracted with supported liquid hollow fiber membrane in headspace mode. The derivatization was attempted directly in water sample without sample evaporation. The HF-LPME was performed using 1-octanol as the extracting solvent at 55 degrees C for 60 min with 20% Na2SO4. The linear calibration curves were observed for the concentrations ranging from 1 to 300 microg L(-1) with the correlation coefficients (R2) being greater than 0.99. The method detection limits of most analytes were below 1 microg L(-1) except DCAA and MCAA that were 2 and 18 microg L(-1), respectively. The recoveries from spiked concentration ranged from 97 to 109% with %R.S.D. less than 12%. The method was applied for determination of HAAs in drinking water and tap water samples. The method offers an easy one step high sample throughput sample preparation for gas chromatographic determination of haloacetic acids as well as other contaminants in water.

  19. Optimized fluorometric determination of urinary delta-aminolevulinic acid by using pre-column derivatization, and identification of the derivative

    SciTech Connect

    Okayama, A.; Fujii, S.; Miura, R. )

    1990-08-01

    We previously reported the usefulness of a fluorometric method to determine urinary delta-aminolevulinic acid (ALA) concentrations by using post-column derivatization to monitor the effect of lead exposure. We have further improved the method by introducing pre-column derivatization by using reaction of ALA with acetylacetone and formaldehyde. Response of the hematopoietic system to lead exposure can now be easily detected at blood lead concentrations as low as 162 micrograms/L. The fluorescent ALA derivative, a new aromatic product, 2-methylideneamino-3,5-diacetyl-4,6-dimethylphenylpropionic acid, is separated on octadecyl silica column by high-performance liquid chromatography and the fluorescence intensity is detected with a fluorophotometer. Sample recoveries for 12 urine samples from workers exposed to lead and unexposed controls were 91.9-110.2%. The results obtained by the pre-column derivatization method agreed with those by the post-column derivatization method. The new method increases the sensitivity to a detection limit to 10 micrograms of delta-aminolevulinic acid per milliliter of urine and is simple enough to be used for routine monitoring of the biological effect of exposure to low concentrations of lead.

  20. Gas-phase reaction products and yields of terpinolene with ozone and nitric oxide using a new derivatization agent

    NASA Astrophysics Data System (ADS)

    Ham, Jason E.; Jackson, Stephen R.; Harrison, Joel C.; Wells, J. R.

    2015-12-01

    The new derivatization agent, O-tert-butylhydroxylamine hydrochloride (TBOX) was used to investigate the carbonyl reaction products from terpinolene ozonolysis. With ozone (O3) as the limiting reagent, four carbonyl compounds were detected: methylglyoxal (MG), 4-methylcyclohex-3-en-1-one, (4MCH), 6-oxo-3-(propan-2-ylidene) heptanal (6OPH), and 3,6-dioxoheptanal (36DOH). The tricarbonyl 36DOH has not been previously observed. Using cyclohexane as a hydroxyl radical (OHrad) scavenger, the yields of 6OPH and 36DOH were reduced indicating the influence secondary OHrad radicals have on terpinolene ozonolysis products. However, the MG yield increased and the 4MCH yield was unchanged when OHrad radicals were scavenged suggesting they are only made by the terpinolene + O3 reaction. The detection of 36DOH using TBOX highlights the advantages of a smaller molecular weight derivatization agent for the detection of multi-carbonyl compounds. The product yields from terpinolene ozonolysis experiments conducted in the presence of 20 ppb nitric oxide (NO) remained unchanged except for MG which decreased. However, in experiments where O3 was kept constant at 50 ppb and NO was varied (20, 50, 100 ppb) MG, 6OPH, 36DOH decreased with increasing NO while 4MCH increased with increasing NO. The use of TBOX derivatization if combined with other derivatization agents may address a recurring need to simply and accurately detect multi-functional oxygenated species in air.

  1. DMAP-catalyzed esterification of pentaerythritol-derivatized POMs: a new route for the functionalization of polyoxometalates.

    PubMed

    Wu, Pingfan; Xiao, Zicheng; Zhang, Jin; Hao, Jian; Chen, Jiake; Yin, Panchao; Wei, Yongge

    2011-05-21

    We have demonstrated that a pentaerythritol-derivatized hexavanadate can be easily esterified with acid anhydrides using 4-(N,N-dimethylamino)pyridine (DMAP) as a catalyst, which provides a convenient method for the functionalization of polyoxometalates. © The Royal Society of Chemistry 2011

  2. CHARACTERIZATION OF NON-DERIVATIZED PLANT CELL WALLS USING HIGH-RESOLUTION SOLUTION-STATE NMR SPECTROSCOPY

    USDA-ARS?s Scientific Manuscript database

    A recently described plant cell wall dissolution system has been logically modified to utilize perdeuterated solvents to allow direct in-nmr-tube dissolution and high-resolution solution-state NMR of the whole cell wall without derivatization. Finely ground cell wall material dissolves in a solvent ...

  3. Determination of malondialdehyde in biological fluids by high-performance liquid chromatography using rhodamine B hydrazide as the derivatization reagent.

    PubMed

    Li, Pingliang; Ding, Guanglong; Deng, Yufang; Punyapitak, Darunee; Li, Deguang; Cao, Yongsong

    2013-12-01

    Malondialdehyde (MDA) is a biomarker for lipid peroxidation, and studies of sensitive and selective analytical methods for it are very important for pathological research. The aim of this work was to develop and validate a novel HPLC method for the quantification of MDA in biological fluids using rhodamine B hydrazide (RBH) as the derivatization reagent. After pretreatment and derivatization in acid medium at 50 °C for 40 min, the RBH-derivatized MDA was separated on a Kromasil C18 column at 25 °C and detected by a fluorescence detector at excitation wavelength of 560 nm and emission wavelength of 580 nm. The results showed linearity in the range of 0.8-1500.0 nM with a detection limit of 0.25 nM (S/N = 3). The recovery of MDA from plasma and urine was 91.50 to 99.20%, with a relative standard deviation range of 1.45 to 3.26%. In comparison to other methods reported for the determination of MDA, the proposed method showed superiority in simplicity, more sensitivity, shorter derivatization time, and less interference. The developed method was applied to quantification of MDA in human biological fluids collected from five volunteers with a concentration range of 24.62-245.00 nM. Copyright © 2013. Published by Elsevier Inc.

  4. Configuration and racemization determination of cysteine residues in peptides by chiral derivatization and HPLC: application to oxytocin peptides.

    PubMed

    Szabó, S; Szókán, G; Khlafulla, A M; Almás, M; Kiss, C; Rill, A; Schön, I

    2001-06-01

    An improved RP-HPLC method was developed for the determination of the configuration and stereochemical purity of cysteine residues in peptides. The method consists of oxidation of cysteine and cystine residues to cysteic acid, followed by hydrolysis and pre-column chiral derivatization with Val-Marfey's reagent.

  5. Novel high-throughput and widely-targeted liquid chromatography-time of flight mass spectrometry method for d-amino acids in foods.

    PubMed

    Konya, Yutaka; Taniguchi, Moyu; Fukusaki, Eiichiro

    2017-01-01

    Recently, the demand for d-amino acid profiling has been drastically increasing because the significance of d-amino acid in various biological events is suggested. However, the present methodologies for d-amino acid profiling are still unsatisfactory. Therefore, a highly sensitive, robust, high-throughput, and user-friendly method for d-amino acid profiling must be developed. In this paper, we developed a novel method for d-amino acid profiling using a combination of a chiral column and time of flight mass spectrometry (TOFMS). To our knowledge, our approach has the best performance for d-amino acid analysis that includes the shortest analytical time (within 10 min), the highest enantioseparability without derivatization, and the largest coverage for analytical targets (more than one hundred targets including non-proteinogenic amino acids and amines). Thus, our novel profiling method will be instrumental in advancing the d-amino acid research in the future. Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  6. [Separation and identification of oligosaccharides labeled with 3-amino-9-ethylcarbazole using high performance liquid chromatography and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry].

    PubMed

    Mou, Qing; Zhang, Ying; Huang, Linjuan; Wang, Zhongfu

    2009-01-01

    A pre-column derivatization method for the determination of oligosaccharides based ion a labeling reagent 3-amino-9-ethylcarbazole (AEC) was proposed. The enamines were generated by the reaction of the reducing ends of oligosaccharides and the primary amines of AEC, and then reduced to secondary amines by NaBH3CN, making oligosaccharides labeled by AEC. The derivatives were separated by reversed-phase high performance liquid chromatography (RP-HPLC), and then directly analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The HPLC separation was carried out on a Waters Symmetry C18 column (3.9 mm x 150 mm, 5 microm) with a gradient elution (acetonitrile and ammonium acetate as mobile phases at a flow rate of 1 mL/min) and ultraviolet detection at 254 nm. Under the optimized derivatization and HPLC conditions, the derivatized oligosaccharides were separated, and the derivatization with AEC increased the sensitivity of MS detection. The developed method for the analysis of oligosaccharides is satisfactory.

  7. [Determination of total aminothiols and neuroactive amino acids in plasma by high performance liquid chromatography with fluorescence detection].

    PubMed

    Pozdeev, V K; Pozdeev, N V

    2010-01-01

    This paper describes a simple and sensitive reversed-phase HPLC method for the determination of total homocysteine, total cysteine, total glutathione (GSH+GSSG), and neuroactive amino acids (Asp, Glu, Tau, GABA) using precolumn derivatization with ortho-phtaldialdehyde and fluorimetric detection at 360 and 470 nm for emission and excitation, respectively. Derivatization was performed with ortho-phthaldialdehyde in the presence of 2-mercaptoethanol after alkylation of the free sulfhydryl groups with iodoacetic acid. For determination of total aminothiols, the disulfide bonds were reduced and protein-bound thiols were released by addition of dithiothreitol to the plasma sample. The advantage of this method is the simultaneous determination of both homocysteine/cysteine/glutathione and neuroactive amino acids in the sample. The plasma levels of studied compounds were determined in 14 healthy volunteers (20-45 years old) and 55 patients with chronic hepatitis C (20-49 years old) and the resulting numbers were in a good agreement the studies published earlier. The calibration curves were linear over a concentration range of 5-100 microM in plasma (r2 = 0.985-0.996). The intraday and interday coefficients of variation were 3-6% and 4-7%, respectively. The recovery of the standards added to the plasma samples ranged from 94 to 102%. The limits of detection (LOD) were 0.2-0.5 ng per 10 microl injection volume (signal-to-noise ratio of 3).

  8. Individual ((f,t) A)- and ((f,t) C)-Fullerene-Based Nickel(II) Glycinates: Protected Chiral Amino Acids Directly Linked to a Chiral π-Electron System.

    PubMed

    Levitskiy, Oleg A; Grishin, Yuri K; Semivrazhskaya, Olesya O; Ambartsumyan, Asmik A; Kochetkov, Konstantin A; Magdesieva, Tatiana V

    2017-03-01

    Stereoselective electrosynthesis of the first individual ((f,t) A)- and ((f,t) C)-1,4-fullerene derivatives with a non-inherently chiral functionalization pattern is described, as well as the first example of an optically pure protected primary amino acid directly linked to the fullerene through only the chiral α-amino-acid carbon atom. An application of an auxiliary chiral nickel-Schiff base moiety as derivatizing agent allowed separation of ((f,t) A)- and ((f,t) C)-1,4-fullerene derivatives using an achiral stationary phase, a separation which has never been done before.

  9. Enantioseparation of α-amino acids by means of Cinchona alkaloids as selectors in chiral ligand-exchange chromatography.

    PubMed

    Keunchkarian, Sonia; Franca, Carlos A; Gagliardi, Leonardo G; Castells, Cecilia B

    2013-07-12

    A conventional nonchiral column was used for the enantioseparation of several racemic α-amino acids (native and derivatized) through the use of Cinchona alkaloids as chiral selectors along with Cu(II) ions in chiral ligand-exchange chromatography. The mobile phase composition (i.e., the organic modifier content and pH) was studied in order to modulate retention and enantioselectivity. Good enantioseparation of many amino acids was obtained using equimolar amounts of Cu(II) and either cinchonidine, quinine or quinidine as chiral selectors in the mobile phase. The molecular geometry of the diastereomeric complexes formed was modeled and energetic differences between both compounds were calculated by methods based on semi-empirical force-field. Good correlations were obtained between experimental enantioselectivity factors and calculated energetic differences. Copyright © 2013 Elsevier B.V. All rights reserved.

  10. A smog chamber comparison of a microfluidic derivatization measurement of gas-phase glyoxal and methylglyoxal with other analytical techniques

    NASA Astrophysics Data System (ADS)

    Pang, X.; Lewis, A. C.; Richard, A.; Baeza-Romero, M. T.; Adams, T. J.; Ball, S. M.; Daniels, M. J. S.; Goodall, I. C. A.; Monks, P. S.; Peppe, S.; Ródenas García, M.; Sánchez, P.; Muñoz, A.

    2013-06-01

    A microfluidic lab-on-a-chip derivatization technique has been developed to measure part per billion volume (ppbV) mixing ratios of gaseous glyoxal (GLY) and methylglyoxal (MGLY), and the method compared with other techniques in a smog chamber experiment. The method uses o-(2,3,4,5,6-pentafluorobenzyl) hydroxylamine (PFBHA) as a derivatization reagent and a microfabricated planar glass micro-reactor comprising an inlet, gas and fluid splitting and combining channels, mixing junctions, and a heated capillary reaction microchannel. The enhanced phase contact area-to-volume ratio and the high heat transfer rate in the micro-reactor result in a fast and highly efficient derivatization reaction, generating an effluent stream ready for direct introduction to a gas chromatograph-mass spectrometer (GC-MS). A linear response for GLY was observed over a calibration range 0.7 to 400 ppbV, and for MGLY of 1.2 to 300 ppbV, when derivatized under optimal reaction conditions. The method detection limits (MDLs) were 80 pptV and 200 pptV for GLY and MGLY respectively, calculated as 3 times the standard deviation of the S/N of the blank sample chromatograms. These MDLs are below or close to typical concentrations in clean ambient air. The feasibility of the technique was assessed by applying the methodology under controlled conditions to quantify of α-dicarbonyls formed during the photo-oxidation of isoprene in a large scale outdoor atmospheric simulation chamber (EUPHORE). Good general agreement was seen between microfluidic measurements and Fourier Transform Infra Red (FTIR), Broad Band Cavity Enhanced Absorption Spectroscopy (BBCEAS) and a detailed photochemical chamber box modelling calculation for both GLY and MGLY. Less good agreement was found with Proton-Transfer Reaction Time-of-Flight Mass Spectrometry (PTR-ToF-MS) and Solid Phase Microextraction (SPME) derivatization methods for MGLY measurement.

  11. Quantitation of low molecular weight sugars by chemical derivatization-liquid chromatography/multiple reaction monitoring/mass spectrometry.

    PubMed

    Han, Jun; Lin, Karen; Sequria, Carita; Yang, Juncong; Borchers, Christoph H

    2016-07-01

    A new method for the separation and quantitation of 13 mono- and disaccharides has been developed by chemical derivatization/ultra-HPLC/negative-ion ESI-multiple-reaction monitoring MS. 3-Nitrophenylhydrazine (at 50°C for 60 min) was shown to be able to quantitatively derivatize low-molecular weight (LMW) reducing sugars. The nonreducing sugar, sucrose, was not derivatized. A pentafluorophenyl-bonded phase column was used for the chromatographic separation of the derivatized sugars. This method exhibits femtomole-level sensitivity, high precision (CVs of ≤ 4.6%) and high accuracy for the quantitation of LMW sugars in wine. Excellent linearity (R(2) ≥ 0.9993) and linear ranges of ∼500-fold for disaccharides and ∼1000-4000-fold for monosaccharides were achieved. With internal calibration ((13) C-labeled internal standards), recoveries were between 93.6% ± 1.6% (xylose) and 104.8% ± 5.2% (glucose). With external calibration, recoveries ranged from 82.5% ± 0.8% (ribulose) to 105.2% ± 2.1% (xylulose). Quantitation of sugars in two red wines and two white wines was performed using this method; quantitation of the central carbon metabolism-related carboxylic acids and tartaric acid was carried out using a previously established derivatization procedure with 3-nitrophenylhydrazine as well. The results showed that these two classes of compounds-both of which have important organoleptic properties-had different compositions in red and white wines. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. Differentiation of Sialyl Linkage Isomers by One-Pot Sialic Acid Derivatization for Mass Spectrometry-Based Glycan Profiling.

    PubMed

    Nishikaze, Takashi; Tsumoto, Hiroki; Sekiya, Sadanori; Iwamoto, Shinichi; Miura, Yuri; Tanaka, Koichi

    2017-02-21

    Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has been used for high-throughput glycan profiling analysis. In spite of the biological importance of sialic acids on nonreducing ends of glycans, it is still difficult to analyze glycans containing sialic acid residues due to their instability and the presence of linkage isomers. In this Article, we describe a one-pot glycan purification/derivatization method employing a newly developed linkage-specific sialic acid derivatization for MS-based glycan profiling with differentiation of sialyl linkage isomer. The derivatization, termed sialic acid linkage specific alkylamidation (SALSA), consists of sequential two-step alkylamidations. As a result of the reactions, α2,6- and α2,3-linked sialic acids are selectively amidated with different length of alkyl chains, allowing distinction of α2,3-/α2,6-linkage isomers from given mass spectra. Our studies using N-glycan standards with known sialyl linkages proved high suitability of SALSA for reliable relative quantification of α2,3-/α2,6-linked sialic acids compared with existing sialic acid derivatization approaches. SALSA fully stabilizes both α2,3- and α2,6-linked sialic acids by alkylamidation; thereby, it became possible to combine SALSA with existing glycan analysis/preparation methods as follows. The combination of SALSA and chemoselective glycan purification using hydrazide beads allows easy one-pot purification of glycans from complex biological samples, together with linkage-specific sialic acid stabilization. Moreover, SALSA-derivatized glycans can be labeled via reductive amination without causing byproducts such as amide decomposition. This solid-phase SALSA followed by glycan labeling has been successfully applied to human plasma N-glycome profiling.

  13. LC-ESI-MS/MS analysis of testosterone at sub-picogram levels using a novel derivatization reagent.

    PubMed

    Star-Weinstock, Michal; Williamson, Brian L; Dey, Subhakar; Pillai, Sasi; Purkayastha, Subhasish

    2012-11-06

    Testosterone analysis by LC-MS/MS is becoming the analytical method of choice over immunoassays due to its specificity and accuracy. However, neutral steroid hormones possess poor ionization efficiency in MS/MS, resulting in insufficient sensitivity for analyzing samples with trace concentrations of the hormones. The method presented here utilizes a derivatization step involving a novel, permanently charged, quaternary aminooxy (QAO) reagent or MS-tag that reacts to the ketone functionality of testosterone and significantly enhances its ESI-MS/MS sensitivity. This derivatization method enabled quantitation of total testosterone in human serum (200 μL) with a lower limit of quantitation (LLOQ) of 1 pg/mL (3.47 pmol/L), total testosterone in dried blood spots (8-10 μL) with a LLOQ of 40 pg/mL, and free testosterone in serum ultrafiltrate (400 μL) with a LLOQ of 0.5 pg/mL. The linearity of each of the high sensitivity applications was maintained over a broad dynamic range of 1-5000 pg/mL for the serum samples and 40-10,000 pg/mL for the dried blood spots (DBS) with R(2) >0.998. The %CV at the LLOQ was <15 for all applications. The QAO derivatization and sample preparation workflows are quick, simple, and robust. Comparison of the derivatization method with an LC-ESI-MS/MS nonderivatization method yielded high correlation and agreement. The derivatization reagent is universal and reacts with other compounds containing ketone or aldehyde functionality.

  14. Optimization of the derivatization protocol of pentacyclic triterpenes prior to their gas chromatography-mass spectrometry analysis in plant extracts.

    PubMed

    Jemmali, Zaineb; Chartier, Agnes; Dufresne, Christelle; Elfakir, Claire

    2016-01-15

    This paper focuses on the application of a two-level full factorial design to optimize the key derivatization step before the GC-FID and GC-MS analysis of pentacyclic triterpenes. The derivatization reaction was screened for influential factors and statistically significant parameters with a p value less than 0.05. A multi-response optimization based on a desirability function was then applied, while simultaneously considering overall detection enhancement of compounds. Results showed that derivatization using N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) and trimethylchlorosilane (TMCS) in pyridine (22:13:65v/v/v) for 2h at 30°C was the most efficient method of derivatizing all the hydroxyl and carboxylic acid groups contained in the triterpene structures. The validity of the method was demonstrated using GC-MS analyzes of a mixture containing eleven standards (β-amyrin, α-amyrin, lupeol, erythrodiol, uvaol, betulin, oleanolic acid, betulinic acid, ursolic acid, maslinic acid and corosolic acid). These compounds are representative of different classes of terpene compounds bearing different functional groups such as alcohols, diols, and carboxylic acids. The derivatization procedure was then tested on four plant extracts: apple pomace, salvia sclarea (dried leaves and flowers), sea buckthorn (Hyppophae rhammnoides L.) berries, and B. serrata resin. The identification of triterpenes was based on the comparison of their retention time and mass spectra to those of standards. The presence of compounds already identified in the literature was confirmed and new ones such as maslinic and corosolic acids were identified in apples, sea buckthorn and salvia sclarea.

  15. p-Tolyl isocyanate derivatization for analysis of CWC-related polar degradation products by mass spectrometry.

    PubMed

    Karthikraj, R; Sridhar, L; Murty, M R V S; Raju, N P; Vairamani, M; Prabhakar, S

    2014-08-01

    Most of the precursors and/or degradation products related to the Chemical Weapons Convention (CWC) are polar. Identification of these molecules in environmental samples provides clues regarding the alleged usage and/or synthesis of the parent toxic chemicals. Such polar compounds need to be derivatized in order to analyze them by gas chromatography-mass spectrometry (GC-MS). In this study, we developed a new derivatizing reagent, para-tolyl isocyanate (PTI), for derivatization of polar CWC-related compounds. The PTI reagent selectively derivatizes the -OH and/or-SH functional groups with high efficiency, but does not react with carboxylic acid (-COOH) or phosphonic acid (-(O)P(OH)2) groups. The PTI derivatives of dialkyl aminoethanols, dialkyl aminoethanol-N-oxides, and 3-quinuclidinol were successfully eluted through GC, and their electron ionization (EI) mass spectra were distinct and provided the structure information by which the isomeric compounds can be easily distinguished. We also calculated the GC-retention index values that can be used for further confirmation of the target compounds. All the studied PTI derivatives can be analyzed by EI-MS with direct insertion probe and/or by direct electrospray ionization mass spectrometry (ESI-MS) together with the MS-MS data; both sets of data provide full structure information. The PTI reagent was found to be better in some respects than the conventional bistrimethylsilyl trifluoroacetamide (BSTFA), a trimethyl silylating reagent. The PTI reagent is commercially available, and the PTI derivatives are highly stable for months and are not sensitive to moisture. The applicability of the PTI derivatization for trace-level determination of the target CWC-related polar compounds in environmental matrices and in human plasma samples is also evaluated.

  16. Protein and amino acid nutrition

    USDA-ARS?s Scientific Manuscript database

    Dairy cow protein and amino acid nutrition have a significant role in sustainable dairying. Protein, amino acids, and nitrogen are inextricably linked through effects in the rumen, metabolism of the cow, and environmental nutrient management. Feeding systems have been making progress toward emphasiz...

  17. Hydrothermal synthesis of amino acids

    NASA Astrophysics Data System (ADS)

    Marshall, William L.

    1994-05-01

    This study presents further evidence that amino acids can be synthesized rapidly in hydrothermal solutions from reactants that may have been present in primitive environments. Aqueous NH 4HCO 3 solutions were reacted with C 2H 2, H 2, and O 2 (formed in situ from CaC 2, Ca, and H 2O 2) at 200-275°C over 0.2-2 h periods to synthesize several amino acids and abundant amines. These amino acid and amine producing reactions were not observed to occur below 150°C. Amino acids and amines also were synthesized at 210°C from solutions of NH 4OH, HCHO, NaCN, and H 2. When NH 4OH was replaced by NH 4HCO 3, the syntheses predominantly confirmed the recent results of RENNET et al. (1992). Additionally, amino acids and amines were observed to form by reactions among NH 4OH, HCHO, and H 2 at hydrothermal conditions, essentially confirming the results of FOX and WINDSOR (1970). Inclusion of both carbonate and O 2 in these latter solutions greatly enhanced the production rate of amino acids. The amines synthesized hydrothermally could be significant if they are precursors in the amino acid syntheses either at hydrothermal or later at lower temperatures. These observations provide additional input to the current questions of synthesis, stability, and decomposition of amino acids at hydrothermal conditions, and their possible relevance to the origin of life.

  18. Determination of methylmercury in fish samples using GC/AA and sodium tetraethylborate derivatization.

    PubMed

    Fischer, R; Rapsomanikis, S; Andreae, M O

    1993-03-15

    A simple technique is described for the rapid determination of methylmercury in fish tissue. Following simple dissolution in methanolic KOH solution, aqueous phase ethylation by derivatization with NaB(C2H5)4, cryogenic trapping on a packed chromatographic column, and GC separation, volatile mercury species are detected by atomic absorption spectrometry. Absolute detection limits are 4 pg of Hg for CH3Hg+ and 75 pg of Hg for labile Hg2+. Concentration detection limits for this optimized procedure are 4 ng of Hg for CH3Hg+ and 75 ng of Hg for labile Hg2+ per gram of pulverized dried fish tissue. Analysis of standard reference materials demonstrates the accuracy, precision, and reproducibility of the analytical method.

  19. Selective determination of arbutin in cosmetic products through online derivatization followed by disposable electrochemical sensor.

    PubMed

    Zen, Jyh-Myng; Yang, Hsueh-Hui; Chiu, Mei-Hsin; Yang, Chao-Hsun; Shih, Ying

    2011-01-01

    An online derivatization followed by a disposable electrochemical sensor was used for the determination of arbutin (AR) in cosmetic products. The AR was chemically oxidized by MnO2 and subsequently reduced at inexpensive screen-printed carbon electrodes using a low detection potential which improved the selectivity of the method. The effects of various parameters, such as solution pH, detection potential, and flow rate of the mobile phase, were studied in detail. Under optimal conditions [pH 1.6 (0.1 M H3PO4), detection potential 0.0 V (versus Ag/AgCl), flow rate 0.6 mL/min], the linear range for AR was 0.1-1500 ppm (r2 = 0.999) with LOD of 30.06 ppb (S/N = 3). The practical application of the proposed method was demonstrated by the determination of arbutin concentration in commercial cosmetic products.

  20. Hyperbranched polyglycerol-grafted titanium oxide nanoparticles: synthesis, derivatization, characterization, size separation, and toxicology

    NASA Astrophysics Data System (ADS)

    Qin, Hongmei; Maruyama, Kyouhei; Amano, Tsukuru; Murakami, Takashi; Komatsu, Naoki

    2016-10-01

    We have been developing surface functionalization of various nanoparticles including nanodiamond and iron oxide nanoparticles in view of biomedical applications. In this context, TiO2 nanoparticles (TiO2 NP) are functionalized with polyglycerol (PG) to provide water-dispersible TiO2-PG, which is further derivatized through multi-step organic transformations. The resulting TiO2-PG and its derivatives are fully characterized by various analyses including solution-phase 1H and 13C NMR. TiO2-PG was size-tuned with centrifugation by changing the acceleration and duration. At last, no cytotoxicity of TiO2 NP, TiO2-PG, and TiO2-PG functionalized with RGD peptide was observed under dark conditions.

  1. Synthesis, structure and imaging of oligodeoxyribonucleotides with tellurium-nucleobase derivatization

    SciTech Connect

    Sheng, J.; Soares, A.; Hassan, A. E. A.; Zhang, W.; Zhou, J.; Xu, B.; Huang, Z.

    2011-05-01

    We report here the first synthesis of 5-phenyl-telluride-thymidine derivatives and the Te-phosphoramidite. We also report here the synthesis, structure and STM current-imaging studies of DNA oligonucleotides containing the nucleobases (thymine) derivatized with 5-phenyl-telluride functionality (5-Te). Our results show that the 5-Te-DNA is stable, and that the Te-DNA duplex has the thermo-stability similar to the corresponding native duplex. The crystal structure indicates that the 5-Te-DNA duplex structure is virtually identical to the native one, and that the Te-modified T and native A interact similarly to the native T and A pair. Furthermore, while the corresponding native showed weak signals, the DNA duplex modified with electron-rich tellurium functionality showed strong topographic and current peaks by STM imaging, suggesting a potential strategy to directly image DNA without structural perturbation.

  2. Synthesis Structure and Imaging of Oligodeoxyribonucleotides with Tellurium-nucleobase Derivatization

    SciTech Connect

    J Sheng; A Hassan; W Zhang; J Zhou; B Xu; A Soares; Z Huang

    2011-12-31

    We report here the first synthesis of 5-phenyl-telluride-thymidine derivatives and the Te-phosphoramidite. We also report here the synthesis, structure and STM current-imaging studies of DNA oligonucleotides containing the nucleobases (thymine) derivatized with 5-phenyl-telluride functionality (5-Te). Our results show that the 5-Te-DNA is stable, and that the Te-DNA duplex has the thermo-stability similar to the corresponding native duplex. The crystal structure indicates that the 5-Te-DNA duplex structure is virtually identical to the native one, and that the Te-modified T and native A interact similarly to the native T and A pair. Furthermore, while the corresponding native showed weak signals, the DNA duplex modified with electron-rich tellurium functionality showed strong topographic and current peaks by STM imaging, suggesting a potential strategy to directly image DNA without structural perturbation.

  3. Measurement of free carnitine and acylcarnitines in plasma by HILIC-ESI-MS/MS without derivatization.

    PubMed

    Peng, Minzhi; Liu, Li; Jiang, Minyan; Liang, Cuili; Zhao, Xiaoyuan; Cai, Yanna; Sheng, Huiying; Ou, Zhiying; Luo, Hong

    2013-08-01

    Measurement of carnitine and acylcarnitines in plasma is important in diagnosis of fatty acid β-oxidation disorders and organic acidemia. The usual method uses flow injection tandem mass spectrometry (FIA-MS/MS), which has limitations. A rapid and more accurate method was developed to be used for high-risk screening and diagnosis. Carnitine and acylcarnitines were separated by hydrophilic interaction liquid chromatography (HILIC) without derivatization and detected with a QTRAP MS/MS System. Total analysis time was 9.0min. The imprecision of within- and between-run were less than 6% and 17%, respectively. Recoveries were in the range of 85-110% at three concentrations. Some acylcarnitine isomers could be separated, such as dicarboxylic and hydroxyl acylcarnitines. The method could also separate interferent to avoid false positive results. 216 normal samples and 116 patient samples were detected with the validated method, and 49 patients were identified with fatty acid oxidation disorders or organic acidemias.

  4. Improvement of 2,4-dinitrophenylhydrazine derivatization method for carbon isotope analysis of atmospheric acetone.

    PubMed

    Wen, Sheng; Yu, Yingxin; Guo, Songjun; Feng, Yanli; Sheng, Guoying; Wang, Xinming; Bi, Xinhui; Fu, Jiamo; Jia, Wanglu

    2006-01-01

    Through simulation experiments of atmospheric sampling, a method via 2,4-dinitrophenylhydrazine (DNPH) derivatization was developed to measure the carbon isotopic composition of atmospheric acetone. Using acetone and a DNPH reagent of known carbon isotopic compositions, the simulation experiments were performed to show that no carbon isotope fractionation occurred during the processes: the differences between the predicted and measured data of acetone-DNPH derivatives were all less than 0.5 per thousand. The results permitted the calculation of the carbon isotopic compositions of atmospheric acetone using a mass balance equation. In this method, the atmospheric acetone was collected by a DNPH-coated silica cartridge, washed out as acetone-DNPH derivatives, and then analyzed by gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS). Using this method, the first available delta13C data of atmospheric acetone are presented.

  5. Derivatized mesoporous silica beads for MALDI-TOF MS profiling of human plasma and urine.

    PubMed

    Terracciano, Rosa; Pasqua, Luigi; Casadonte, Francesca; Frascà, Stella; Preianò, Mariaimmacolata; Falcone, Daniela; Savino, Rocco

    2009-05-20

    Matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS) is a promising tool for large-scale screening of body fluids for the early detection of human diseases. Proteins, peptides, and metabolites present in cells, tissues, or in body fluids constitute the molecular signatures of individuals. The design and generation of material-based platforms for capturing molecular signatures from body fluids has gained increasing interest in recent years. Highly selective materials are attractive candidates for a wide range of applications in biofluid proteomics. We have therefore developed a procedure based on mesoporous silica particles for the selective binding and enrichment of low molecular weight plasma/serum proteins by MALDI MS analysis ( Terracciano, R., Gaspari, M., Testa, F., Pasqua, L., Cuda G., Tagliaferri, P., Cheng, M. C., Nijdam, A. J., Petricoin, E. F., Liotta, L. A., Ferrari, M., and Venuta, S. ( 2006 ) Selective binding and enrichment for low-molecular weight biomarker molecules in human plasma after exposure to nanoporous silica particles . Proteomics 6, 3243-3250 ). Mesoporous silica beads (MSB) are able to harvest peptides from plasma and serum by means of nanosized porous channels with high surface area, while excluding large size proteins. Moreover, the absorption properties can be modified since the pore walls can be functionalized with different chemical species due to the high concentration of silanol groups at the surface. In this study, we performed derivatization of MSB with different functionalities, and we evaluated the derivatized materials for plasma and urine peptidomic profiling. Aminopropyl, N-(2-aminoethyl)-3-aminopropyl, and N,N,N' tris-carboxymethyl ethylene diamine, have been introduced onto the mesoporous silica surfaces in order to modulate selective peptide enrichment. We also explored various experimental conditions in order to optimize the performance of chemically modified MSB in the peptide

  6. SITS Derivatization of Peptides to Enhance 266 nm Ultraviolet Photodissociation (UVPD)

    NASA Astrophysics Data System (ADS)

    Quick, M. Montana; Mehaffey, M. Rachel; Johns, Robert W.; Parker, W. Ryan; Brodbelt, Jennifer S.

    2017-07-01

    N-terminal derivatization of peptides with the chromogenic reagent 4-acetamido-4-isothiocyanatostilbene-2,2-disulfonic acid (SITS) is demonstrated to enhance the efficiency of 266 nm ultraviolet photodissociation (UVPD). Attachment of the chromophore results in a mass shift of 454 Da and provides significant gains in the number and abundances of diagnostic fragment ions upon UVPD. Activation of SITS-tagged peptides with 266 nm UVPD leads to many fragment ions akin to the a/b/y ions commonly produced by CID, along with other sequence ions ( c, x, and z) typically accessed through higher energy pathways. Extreme bias towards C-terminal fragment ions is observed upon activation of SITS-tagged peptides using multiple 266 nm laser pulses. Due to the high reaction efficiency of the isothiocyanate coupling to the N-terminus of peptides, we demonstrate the ability to adapt this strategy to a high-throughput LC-MS/MS workflow with 266 nm UVPD.

  7. Azide-derivatized gold nanosphere "clicked" to indium and zinc phthalocyanines for improved nonlinear optical limiting

    NASA Astrophysics Data System (ADS)

    Bankole, Owolabi M.; Nyokong, Tebello

    2017-05-01

    We report on the conjugation of azide-derivatized gold nanoparticles (AuNPs) to alkyne moieties of ZnPc and InPc via azide-alkyne Huisgen cycloaddition reaction to form phthalocyanines-AuNPs (MPc-AuNPs) conjugates. The detailed structural characterizations of the composites were in good agreement with the expected results. The nonlinear absorption coefficients and other nonlinear optical limiting parameters were almost two times larger for the conjugates compared to free phthalocyanines. We established direct relationship between improved photophysical characterizations and enhanced nonlinear effects of reverse saturable absorption mechanisms favoured by excited triplet absorption of the phthalocyanines in the presence of AuNPs. The combination of InPc with AuNPs resulted in the lowest limiting intensity value of 0.06 J/cm2, hence the best performance in terms of optical limiting.

  8. SITS Derivatization of Peptides to Enhance 266 nm Ultraviolet Photodissociation (UVPD)

    NASA Astrophysics Data System (ADS)

    Quick, M. Montana; Mehaffey, M. Rachel; Johns, Robert W.; Parker, W. Ryan; Brodbelt, Jennifer S.

    2017-03-01

    N-terminal derivatization of peptides with the chromogenic reagent 4-acetamido-4-isothiocyanatostilbene-2,2-disulfonic acid (SITS) is demonstrated to enhance the efficiency of 266 nm ultraviolet photodissociation (UVPD). Attachment of the chromophore results in a mass shift of 454 Da and provides significant gains in the number and abundances of diagnostic fragment ions upon UVPD. Activation of SITS-tagged peptides with 266 nm UVPD leads to many fragment ions akin to the a/b/y ions commonly produced by CID, along with other sequence ions (c, x, and z) typically accessed through higher energy pathways. Extreme bias towards C-terminal fragment ions is observed upon activation of SITS-tagged peptides using multiple 266 nm laser pulses. Due to the high reaction efficiency of the isothiocyanate coupling to the N-terminus of peptides, we demonstrate the ability to adapt this strategy to a high-throughput LC-MS/MS workflow with 266 nm UVPD.

  9. Ultraclean derivatized monodisperse gold nanoparticles through laser drop ablation customization of polymorph gold nanostructures.

    PubMed

    Bueno-Alejo, Carlos J; D'Alfonso, Claudio; Pacioni, Natalia L; González-Béjar, María; Grenier, Michel; Lanzalunga, Osvaldo; Alarcon, Emilio Isaac; Scaiano, Juan C

    2012-05-29

    We report a novel nanosecond laser ablation synthesis for spherical gold nanoparticles as small as 4 nm in only 5 s (532 nm, 0.66 J/cm(2)), where the desired protecting agent can be selected in a protocol that avoids repeated sample irradiation and undesired exposure of the capping agent during ablation. This method takes advantage of the recently developed synthesis of clean unprotected polymorph and polydisperse gold nanostructures using H(2)O(2) as a reducing agent. The laser drop technique provides a unique tool for delivering controlled laser doses to small drops that undergo assisted fall into a solution or suspension of the desired capping agent, yielding monodisperse custom-derivatized composite materials using a simple technique.

  10. Determination of methotrexate and indomethacin in urine using SPE-LC-DAD after derivatization.

    PubMed

    Michail, Karim; Moneeb, Marwa S

    2011-05-15

    A validated HPLC-DAD assay is presented for the simultaneous quantification of methotrexate and indomethacin in a drug combination which is used synergistically to intervene with tumoral or inflammatory tissue microenvironments. The analytes were isolated from urine via solid phase extraction. The method is based on derivatizing both analytes with a soluble carbodiimide coupler and 2-nitrophenylhydrazine directed to their commonly occurring carboxylate functions. The chromatographic separation was accomplished on an octylsilica column in less than 15 min using acetate buffer (pH 4; 10 mM)-methanol (60:40, v/v) as eluent at 1.5 ml/min and monitored at 400 nm. The selectivity of the method was demonstrated in a pre-dosed rheumatoid arthritis patient. Quality control samples were prepared and analyzed to reveal the validity of the method. Life samples collected from a healthy volunteer were monitored for both drugs and their urinary levels were determined.

  11. Determination of N-nitrosamines in latex by sequential supercritical fluid extraction and derivatization.

    PubMed

    Reche, F; Garrigós, M C; Marín, M L; Jiménez, A

    2002-11-08

    A new method to determine N-nitrosamines in latex products has been developed by combination of supercritical fluids and chemical derivatization. A new design for a liquid trap has been introduced. A factorial fractional design was used in order to evaluate the influence of the different factors affecting the process. Factors such as pressure, temperature, static and dynamic time, restrictor temperature and volume of an hydrobromic acid-acetic anhydride mixture (1:10, v/v) were included in the design. CO2 was used as the extraction fluid. Gas chromatography with nitrogen and phosphorus sensitive detection was employed to achieve good sensitivity attending to the molecular structure of these compounds (N-nitrosamines and their corresponding secondary amines). The obtained results have shown to be useful to increase selectivity and reduce sample handling.

  12. Recent developments and applications of EMMA in enzymatic and derivatization reactions.

    PubMed

    Hai, Xin; Yang, Bao-feng; Schepdael, Ann Van

    2012-01-01

    This review provides systematic coverage of examples in the field of in-capillary electrophorecially mediated microanalysis (EMMA). The recent developments and applications in the time period up to mid 2011 have been described, as well as relevant older papers. The basic principles and modes of in-capillary assays have been demonstrated. An overview is also given of the various injection, separation and detection modes implemented in combination with EMMA. The review is presented in two parts mainly dealing with (i) enzymatic and (ii) derivatization or chemical reactions. Finally, the future trends of CE in performing and monitoring reactions have been drawn. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. Discovery of pyridine-based agrochemicals by using Intermediate Derivatization Methods.

    PubMed

    Guan, Ai-Ying; Liu, Chang-Ling; Sun, Xu-Feng; Xie, Yong; Wang, Ming-An

    2016-02-01

    Pyridine-based compounds have been playing a crucial role as agrochemicals or pesticides including fungicides, insecticides/acaricides and herbicides, etc. Since most of the agrochemicals listed in the Pesticide Manual were discovered through screening programs that relied on trial-and-error testing and new agrochemical discovery is not benefiting as much from the in silico new chemical compound identification/discovery techniques used in pharmaceutical research, it has become more important to find new methods to enhance the efficiency of discovering novel lead compounds in the agrochemical field to shorten the time of research phases in order to meet changing market requirements. In this review, we selected 18 representative known agrochemicals containing a pyridine moiety and extrapolate their discovery from the perspective of Intermediate Derivatization Methods in the hope that this approach will have greater appeal to researchers engaged in the discovery of agrochemicals and/or pharmaceuticals.

  14. Synthesis of the tellurium-derivatized phosphoramidites and their incorporation into DNA oligonucleotides.

    PubMed

    Jiang, Sibo; Sheng, Jia; Huang, Zhen

    2011-12-01

    In this unit, an efficient method for the synthesis of 2'-tellerium-modified phosphoramidite and its incorporation into oligonucleotide are presented. We choose 5'-O-DMTr-2,2'-anhydro-uridine and -thymidine nucleosides (S.1, S.2) as starting materials due to their easy preparation. The 5'-O-DMTr-2,2'-anhydro-uridine and -thymidine can be converted to the corresponding 2'-tellerium-derivatized nucleosides by treating with the telluride nucleophiles. Subsequently, the 2'-Te-nucleosides can be transformed into 3'-phosphoramidites, which are the building blocks for DNA/RNA synthesis. The DNA synthesis, purification, and applications of oligonucleotides containing 2'-Te-U or 2'-Te-T are described in the protocol.

  15. Synthesis, structure and imaging of oligodeoxyribonucleotides with tellurium-nucleobase derivatization.

    PubMed

    Sheng, Jia; Hassan, Abdalla E A; Zhang, Wen; Zhou, Jianfeng; Xu, Bingqian; Soares, Alexei S; Huang, Zhen

    2011-05-01

    We report here the first synthesis of 5-phenyl-telluride-thymidine derivatives and the Te-phosphoramidite. We also report here the synthesis, structure and STM current-imaging studies of DNA oligonucleotides containing the nucleobases (thymine) derivatized with 5-phenyl-telluride functionality (5-Te). Our results show that the 5-Te-DNA is stable, and that the Te-DNA duplex has the thermo-stability similar to the corresponding native duplex. The crystal structure indicates that the 5-Te-DNA duplex structure is virtually identical to the native one, and that the Te-modified T and native A interact similarly to the native T and A pair. Furthermore, while the corresponding native showed weak signals, the DNA duplex modified with electron-rich tellurium functionality showed strong topographic and current peaks by STM imaging, suggesting a potential strategy to directly image DNA without structural perturbation.

  16. Electrochemical oxidation of 243Am(III) in nitric acid by a terpyridyl-derivatized electrode

    SciTech Connect

    Dares, C. J.; Lapides, A. M.; Mincher, B. J.; Meyer, T. J.

    2015-11-05

    A high surface area, tin-doped indium oxide electrode surface-derivatized with a terpyridine ligand has been applied to the oxidation of trivalent americium to Am(V) and Am(VI) in nitric acid. Potentials as low as 1.8 V vs. the saturated calomel electrode are used, 0.7 V lower than the 2.6 V potential for one-electron oxidation of Am(III) to Am(IV) in 1 M acid. This simple electrochemical procedure provides, for the first time, a method for accessing the higher oxidation states of Am in non-complexing media for developing the coordination chemistries of Am(V) and Am(VI) and, more importantly, for separation of americium from nuclear waste streams.

  17. Microwave-accelerated derivatization for capillary electrophoresis with laser-induced fluorescence detection: a case study for determination of histidine, 1- and 3-methylhistidine in human urine.

    PubMed

    Zhou, Lei; Yan, Na; Zhang, Huige; Zhou, Ximin; Pu, Qiaosheng; Hu, Zhide

    2010-06-30

    The feasibility of microwave-accelerated derivatization for capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection was evaluated. The derivatization reaction was performed in a domestic microwave oven. Histidine (His), 1-methylhistidine (1-MH) and 3-methylhistidine (3-MH) were selected as test analytes and fluorescein isothiocyanate (FITC) was chosen as a fluorescent derivatizing reagent. Parameters that may affect the derivatization reaction and/or subsequent CE separation were systematically investigated. Under optimized conditions, the microwave-accelerated derivatization reaction was successfully completed within 150 s, compared to 4-24 h in a conventional water-bath derivatization process. This will remarkably reduce the overall analysis time and increase sample throughput of CE-LIF. The detection limits of this method were found to be 0.023 ng/mL for His, 0.023 ng/mL for 1-MH, and 0.034 ng/mL for 3-MH, respectively, comparable to those obtained using traditional derivatization protocols. The proposed method was characterized in terms of precision, linearity, accuracy and successfully applied for rapid and sensitive determination of these analytes in human urine.

  18. Relative Amino Acid Concentrations as a Signature for Parent Body Processes of Carbonaceous Chondrites

    NASA Technical Reports Server (NTRS)

    Botta, Oliver; Glavin, Daniel P.; Kminek, Gerhard; Bada, Jeffrey L.

    2002-01-01

    Most meteorites are thought to have originated from objects in the asteroid belt. Carbonaceous chondrites, which contain significant amounts of organic carbon including complex organic compounds, have also been suggested to be derived from comets. The current model for the synthesis of organic compounds found in carbonaceous chondrites includes the survival of interstellar organic compounds and the processing of some of these compounds on the meteoritic parent body. The amino acid composition of five CM carbonaceous chondrites, two CIs, one CR, and one CV3 have been measured using hot water extraction-vapor hydrolysis, OPA/NAC derivatization and high-performance liquid chromatography (HPLC). Total amino acid abundances in the bulk meteorites as well as the amino acid concentrations relative to glycine = 1.0 for beta-alanine, alpha-aminoisobutyric acid and D-alanine were determined. Additional data for three Antarctic CM meteorites were obtained from the literature. All CM meteorites analyzed in this study show a complex distribution of amino acids and a high variability in total concentration ranging from approx. 15,300 to approx. 5800 parts per billion (ppb), while the CIs show a total amino acid abundance of approx. 4300 ppb. The relatively (compared to glycine) high AIB content found in all the CMs is a strong indicator that Strecker-cyanohydrin synthesis is the dominant pathway for the formation of amino acids found in these meteorites. The data from the Antarctic CM carbonaceous chondrites are inconsistent with the results from the other CMs, perhaps due to influences from the Antarctic ice that were effective during their residence time. In contrast to CMs, the data from the CI carbonaceous chondrites indicate that the Strecker synthesis was not active on their parent bodies.

  19. Relative Amino Acid Concentrations as a Signature for Parent Body Processes of Carbonaceous Chondrites

    NASA Technical Reports Server (NTRS)

    Botta, Oliver; Glavin, Daniel P.; Kminek, Gerhard; Bada, Jeffrey L.

    2002-01-01

    Most meteorites are thought to have originated from objects in the asteroid belt. Carbonaceous chondrites, which contain significant amounts of organic carbon including complex organic compounds, have also been suggested to be derived from comets. The current model for the synthesis of organic compounds found in carbonaceous chondrites includes the survival of interstellar organic compounds and the processing of some of these compounds on the meteoritic parent body. The amino acid composition of five CM carbonaceous chondrites, two CIs, one CR, and one CV3 have been measured using hot water extraction-vapor hydrolysis, OPA/NAC derivatization and high-performance liquid chromatography (HPLC). Total amino acid abundances in the bulk meteorites as well as the amino acid concentrations relative to glycine = 1.0 for beta-alanine, alpha-aminoisobutyric acid and D-alanine were determined. Additional data for three Antarctic CM meteorites were obtained from the literature. All CM meteorites analyzed in this study show a complex distribution of amino acids and a high variability in total concentration ranging from approx. 15,300 to approx. 5800 parts per billion (ppb), while the CIs show a total amino acid abundance of approx. 4300 ppb. The relatively (compared to glycine) high AIB content found in all the CMs is a strong indicator that Strecker-cyanohydrin synthesis is the dominant pathway for the formation of amino acids found in these meteorites. The data from the Antarctic CM carbonaceous chondrites are inconsistent with the results from the other CMs, perhaps due to influences from the Antarctic ice that were effective during their residence time. In contrast to CMs, the data from the CI carbonaceous chondrites indicate that the Strecker synthesis was not active on their parent bodies.

  20. [Determination of alditols in wine by gas chromatography-mass spectrometry after acetate derivatization].

    PubMed

    Zhou, Hongbin; Xiong, Zhiyu; Yu, Yang; Wan, Rong; Li, Ping; Shen, Bo

    2013-08-01

    The acetate derivatization of alditols for determining alditol level in wine by gas chromatography (GC)-mass spectrometry (MS) has been developed. The wine sample was mixed with pyridine and centrifuged at 5,000 r/min at the temperature of 4 degrees C for 10 min. After filtration with organic phase membrane, the supernatant was derivatized with acetic anhydride, and then dehydrated with anhydrous sodium sulfate. The GC separation was performed on a DB-5MS capillary column. The alditols were determined by MS in selected ion monitoring (SIM) mode and quantified by external standard method. The calibration curves showed good linearities in the range of 0.019 - 1.25 mg/L except for lactitol (0.039 - 2.50 mg/L) with the correlation coefficients greater than 0.99. The limits of quantification (S/N= 10) of erythritol, xylitol, D-mannitol, sorbitol, galactitol and lactitol were 0.17, 0.29, 0.43, 0.46, 0.47 and 2.88 mg/L respectively. The limits of detection (S/N = 3) were 0.05, 0.08, 0.13, 0.14, 0.14 and 1.38 mg/L respectively. The recoveries of alditols spiked in the wine at two levels of 40 mg/L and 80 mg/L were ranged from 80.15% to 108.75% with the relative standard deviations (RSDs) of 2.16% - 6.97%. The sensitivity, accuracy and precision of the method can meet the technical standard. The method can be applied to the rapid determination of alditols in wine.

  1. Automatic analyzer for highly polar carboxylic acids based on fluorescence derivatization-liquid chromatography.

    PubMed

    Todoroki, Kenichiro; Nakano, Tatsuki; Ishii, Yasuhiro; Goto, Kanoko; Tomita, Ryoko; Fujioka, Toshihiro; Min, Jun Zhe; Inoue, Koichi; Toyo'oka, Toshimasa

    2015-03-01

    A sensitive, versatile, and reproducible automatic analyzer for highly polar carboxylic acids based on a fluorescence derivatization-liquid chromatography (LC) method was developed. In this method, carboxylic acids were automatically and fluorescently derivatized with 4-(N,N-dimethylaminosulfonyl)-7-piperazino-2,1,3-benzoxadiazole (DBD-PZ) in the presence of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride by adopting a pretreatment program installed in an LC autosampler. All of the DBD-PZ-carboxylic acid derivatives were separated on the ODS column within 30 min by gradient elution. The peak of DBD-PZ did not interfere with the separation and the quantification of all the acids with the exception of lactic acid. From the LC-MS/MS analysis, we confirmed that lactic acid was converted to an oxytriazinyl derivative, which was further modified with a dimethoxy triazine group of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM). We detected this oxytriazinyl derivative to quantify lactic acid. The detection limits (signal-to-noise ratio = 3) for the examined acids ranged from 0.19 to 1.1 µm, which correspond to 95-550 fmol per injection. The intra- and inter-day precisions of typical, highly polar carboxylic acids were all <9.0%. The developed method was successfully applied to the comprehensive analysis of carboxylic acids in various samples, which included fruit juices, red wine and media from cultured tumor cells.

  2. Characterization of Hydroxyphthioceranoic and Phthioceranoic Acids by Charge-Switch Derivatization and CID Tandem Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Hsu, Fong-Fu

    2016-04-01

    Hydroxyphthioceranoic (HPA) and phthioceranoic (PA) acids are polymethylated long chain fatty acids with and without a hydroxyl group attached to the carbon next to the terminal methyl-branched carbon distal to the carboxylic end of the long-chain fatty acid, respectively. They are the major components of the sulfolipids found in the cell wall of Mycobacterium tuberculosis (M. tuberculosis) strain H37Rv. In this report, I describe CID linear ion-trap MSn mass spectrometric approaches combined with charge-reverse derivatization strategy toward characterization of these complex lipids, which were released from sulfolipids by alkaline hydrolysis and sequentially derivatized to the N-(4-aminomethylphenyl) pyridinium (AMPP) derivatives. This method affords complete characterization of HPA and PA, including the location of the hydroxyl group and the multiple methyl side chains. The study also led to the notion that the hydroxyphthioceranoic acid in sulfolipid consists of two (for hC24) to 12 (for hC52) methyl branches, and among them 2,4,6,8,10,12,14,16-octamethyl-17-hydroxydotriacontanoic acid (hC40) is the most prominent, while phthioceranoic acids are the minor constituents. These results confirm our previous findings that sulfolipid II, a family of homologous 2-stearoyl(palmitoyl)-3,6,6'-tris(hydroxyphthioceranoy1)-trehalose 2'-sulfates is the predominant species, and sulfolipid I, a family of homologous 2-stearoyl(palmitoyl)-3-phthioceranoyl-6,6'-bis(hydroxyphthioceranoy1)-trehalose 2'-sulfates is the minor species in the cell wall of M. tuberculosis.

  3. Profiling of structurally labile oxylipins in plants by in situ derivatization with pentafluorobenzyl hydroxylamine.

    PubMed

    Schulze, Birgit; Lauchli, Ryan; Sonwa, Mesmin Mekem; Schmidt, Annika; Boland, Wilhelm

    2006-01-15

    A GC-MS-based method for the simultaneous quantification of common oxylipins along with labile and highly reactive compounds based on in situ derivatization with pentafluorobenzyl hydroxylamine to the corresponding O-2,3,4,5,6-pentafluorobenzyl oximes (PFB oximes) is presented. The approach covers oxo derivatives such as jasmonic acid (JA), 12-oxophytodienoic acid (OPDA), certain phytoprostanes, unsaturated oxo-acids, oxo-hydroxy acids, and aldehyde fragments from the polar head of fatty acids. In the positive electron impact-MS mode, the PFB oximes display characteristic fragment ions that greatly facilitate the identification of oxylipins in complex matrices. In addition, the fluorinated derivatives allow a highly selective and low-background analysis by negative chemical ionization. Besides showing the general value of the method for the identification of a broad range of oxylipins (18 examples), we also demonstrate sensitivity, linearity, and reproducibility for the quantification of JA, OPDA, 11-oxo-9-undecenoic acid, and 13-oxo-9,11-tridecadienoic acid. The efficiency of the method is demonstrated by differential profiling of these four oxylipins in lima bean leaves after mechanical wounding and feeding by the herbivore Spodoptera littoralis. Caterpillar feeding induced several oxylipins, whereas after wounding only the level of JA increased. The rapid in situ derivatization prevents the isomerization of cis-JA to trans-JA. The resting level of JA in lima beans showed an isomer ratio of 80:20 for trans/cis-JA. After wounding, de novo synthesis of JA alters the ratio to 20:80 in favor of the cis isomer.

  4. Improved sensitivity of serum/plasma 1α,25-dihydroxyvitamin D quantification by DAPTAD derivatization.

    PubMed

    Ishige, Takayuki; Satoh, Mamoru; Ogawa, Shoujiro; Nishimura, Motoi; Matsushita, Kazuyuki; Higashi, Tatsuya; Nomura, Fumio

    2017-10-01

    Although immunoassays have several limitations such as the cross-reactivities of antibodies, such techniques are widely used for serum/plasma 1,25(OH)2D quantification. An accurate method is required for the determination of the 1,25(OH)2D status. We designed a serum/plasma 1,25(OH)2D quantification method using LC-MS/MS. Immunoaffinity extraction (IE) and the recently developed Cookson-type reagent 4-(4'-dimethylaminophenyl)-1,2,4-triazoline-3,5-dione (DAPTAD) were used for sample preparation and derivatization, respectively. Analytical and pre-analytical validations were performed. Serum 1,25(OH)2D3 concentrations were determined in 232 healthy Japanese individuals. The intra- and inter-assay CVs for 1,25(OH)2D3 were 5.2% and 7.0%, respectively. The limit of quantification for 1,25(OH)2D3 was 7.1pg/ml. Rheumatoid factor (RF) at concentrations below 517IU/ml did not affect serum 1,25(OH)2D analysis. No significant differences were observed for various blood collection tubes, repeated freeze-thaw cycles, whole blood standing time, or serum storage time. A strong correlation between LC-MS/MS and radioimmunoassay (RIA) was observed (r=0.786), but serum 1,25(OH)2D concentrations obtained from RIA were 2-fold higher than those obtained from LC-MS/MS. Serum 1,25(OH)2D3 concentrations by LC-MS/MS were 18.7-53.9pg/ml. A highly sensitive and selective LC-MS/MS-based serum/plasma 1,25(OH)2D quantification method was developed using IE and DAPTAD derivatization. This method will enable the accurate determination of serum/plasma 1,25(OH)2D concentrations in the clinical setting. Copyright © 2017 Elsevier B.V. All rights reserved.

  5. ESA mission ROSETTA will probe for chirality of cometary amino acids.

    PubMed

    Thiemann, W H; Meierhenrich, U

    2001-01-01

    New crucial theoretical investigations on the origin of biomolecular chirality are reviewed briefly. With the goal to investigate these theories our team is going to perform the 'chirality-experiment' in the near future with cometary matter. In 2012 the robotical lander RoLand will detach from the orbiter of the ROSETTA spacecraft and set down on the surface of comet 46P/Wirtanen in order to separate and identify cometary organic compounds via GC-MS in situ. Chiral organics will be separated into their enantiomers by application of 3 capillary columns coated with different kinds of stationary phases. Non-volatile compounds like amino acids will be derivatized in especially developed gas phase alkylation steps avoiding reactions in the liquid phase. The results of these preliminary gas phase reactions are presented in this article.

  6. Amino Acid Sequence of Human Cholinesterase

    DTIC Science & Technology

    1985-10-01

    liquid chromatography (HPLC). Activity testing of the aged, DFP-labeled cholinesterase showed that 99.8% of the active sites had been labeled, since...acids were quantitated by ninhydrin at the AAA Labs, or by derivatization with phenylisothiocyanate at the University of Michigan. The latter method

  7. Amino acid composition in eyes from zebrafish (Danio rerio) and sardine (Sardina pilchardus) at the larval stage.

    PubMed

    Falco, Francesca; Barra, Marco; Cammarata, Matteo; Cuttitta, Angela; Jia, Sichao; Bonanno, Angelo; Mazzola, Salvatore; Wu, Guoyao

    2016-01-01

    A comparative study was performed to identify differences in the amino acid composition of the eyes from zebrafish (Danio rerio) and sardine (Sardina pilchardus) larvae and their link to the environmental adaption of the species. Amino acids in the acidic hydrolysates of eyes from 11 zebrafish and 12 sardine were determined with the use of high-performance liquid chromatography involving precolumn derivatization with ortho-phthalaldehyde. Differences in the content of most amino acids were detected between zebrafish and sardine. These amino acids were aspartate, glutamate, serine, glycine, threonine, arginine, methionine, valine, phenylalanine, isoleucine, leucine and lysine. Of particular note, the percentage of methionine in zebrafish eyes was much higher than that in sardine, whereas the opposite was observed for glutamate and glycine. These results indicate that zebrafish and sardine likely have experienced differences in adaptation to environmental changes. We suggest that the amino acid composition of eyes represents a powerful tool to discriminate between species characterized by different lifestyle and inhabiting different environments.

  8. Microalgae amino acid extraction and analysis at nanomolar level using electroporation and capillary electrophoresis with laser-induced fluorescence detection.

    PubMed

    Nehmé, Reine; Atieh, Carla; Fayad, Syntia; Claude, Bérengère; Chartier, Agnès; Tannoury, Mona; Elleuch, Fatma; Abdelkafi, Slim; Pichon, Chantal; Morin, Philippe

    2017-01-01

    Amino acids play a key role in food analysis, clinical diagnostics, and biochemical research. Capillary electrophoresis with laser-induced fluorescence detection was used for the analysis of several amino acids. Amino acid labeling with fluorescein isothiocyanate was conducted using microwave-assisted derivatization at 80°C (680 W) during only 150 s. Good electrophoretic resolution was obtained using a background electrolyte composed of sodium tetraborate buffer (100 mM; pH 9.4) and β-cyclodextrin (10 mM), and the limits of quantification were 3-30 nM. The developed capillary electrophoresis with laser-induced fluorescence method was used to analyze amino acids in Dunaliella salina green algae grown under different conditions. A simple extraction technique based on electroporation of the cell membrane was introduced. A home-made apparatus allowed the application of direct and alternating voltages across the electrochemical compartment containing a suspension of microalgae in distilled water at 2.5 g/L. A direct voltage of 12 V applied for 4 min gave the optimum extraction yield. Results were comparable to those obtained with accelerated-solvent extraction. The efficiency of electroporation in destroying microalgae membranes was shown by examining the algae surface morphology using scanning electron microscopy. Stress conditions were found to induce the production of amino acids in Dunaliella salina cells. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. Novel chiral derivatization reagents possessing a pyridylthiourea structure for enantiospecific determination of amines and carboxylic acids in high-throughput liquid chromatography and electrospray-ionization mass spectrometry for chiral metabolomics identification.

    PubMed

    Nagao, Ryuji; Tsutsui, Haruhito; Mochizuki, Toshiki; Takayama, Takahiro; Kuwabara, Tomohiro; Min, Jun Zhe; Inoue, Koichi; Todoroki, Kenichiro; Toyo'oka, Toshimasa

    2013-06-28

    This paper reports the synthesis and the application of novel derivatization reagents possessing a pyridylthiourea structure for the enantiospecific determination of chiral amines and carboxylic acids in high-throughput LC-ESI-MS/MS. The novel reagents, i.e., (R)-N-(3-pyridylthiocarbamoyl)pyrrolidine-2-carboxylic acid (PyT-C) and (S)-3-amino-1-(3-pyridylthiocarbamoyl)pyrrolidine (PyT-N), were evaluated as chiral derivatization reagents for the enantiomeric determination of chiral amines and carboxylic acids, respectively, in terms of separation efficiency by reversed-phase chromatography and detection sensitivity by ESI-MS/MS. The chiral amines and carboxylic acids were easily labeled with PyT-C and PyT-N, respectively, at 60°C in 60min in the presence of 2,2'-dipyridyl disulfide (DPDS) and triphenylphosphine (TPP) as the activation reagents. The resulting diastereomers were completely separated by reversed-phase chromatography using a small particle (1.7μm) ODS column (Rs=3.54-6.00 for carboxylic acids and Rs=3.07-4.75 for amines). A highly sensitive detection at the sub-fmol level was also obtained from the SRM chromatograms at a single monitoring ion, m/z 137.0 (0.72-1.46fmol for carboxylic acids and 0.55-1.89fmol for amines). The proposed procedure using PyT-C and PyT-N was applied to the determination of chiral amines and carboxylic acids spiked into human saliva, as a model study of chiral metabonomics identification. dl-Amino acid methyl esters and N-acetyl dl-amino acids, which are the representatives as the chiral amines and carboxylic acids, in the saliva were clearly identified by the present method. Because the same product ion at m/z 137.0 was obtained from collision-induced dissociation (CID) of protonated molecular ions of all the derivatives, the proposed procedure using both reagents (i.e., PyT-C and PyT-N) seems to be useful for chiral metabolomics identification having selected functional groups (i.e., amines and carboxylic acids).

  10. α-Amino Acid-Isosteric α-Amino Tetrazoles

    PubMed Central

    Zhao, Ting; Kurpiewska, Katarzyna; Kalinowska-Tłuścik, Justyna; Herdtweck, Eberhardt

    2016-01-01

    The synthesis of all 20 common natural proteinogenic and 4 otherα-amino acid-isosteric α-amino tetrazoles has been accomplished, whereby the carboxyl group is replaced by the isosteric 5-tetrazolyl group. The short process involves the use of the key Ugi tetrazole reaction followed by deprotection chemistries. The tetrazole group is bioisosteric to the carboxylic acid and is widely used in medicinal chemistry and drug design. Surprisingly, several of the common α-amino acid-isosteric α-amino tetrazoles are unknown up to now. Therefore a rapid synthetic access to this compound class and non-natural derivatives is of high interest to advance the field. PMID:26817531

  11. Determination of octopamine and tyramine traces in dietary supplements and phytoextracts by high performance liquid chromatography after derivatization with 2,5-dimethyl-1H-pyrrole-3,4-dicarbaldehyde.

    PubMed

    Gatti, Rita; Lotti, Cinzia; Morigi, Rita; Andreani, Aldo

    2012-01-13

    The use of 2,5-dimethyl-1H-pyrrole-3,4-dicarbaldehyde (DPD) as a pre-column derivatization reagent for HPLC (high performance liquid chromatography) analysis of octopamine (oct) and tyramine (tyr) is proposed. The compound reacts under mild conditions (2 min at ambient temperature) with primary amino groups. The derivatization conditions were optimized by considering different parameters (temperature, time and reagent concentration). The synthesized oct and tyr adducts were characterized by (1)H NMR (nuclear magnetic resonance), ESI-MS (electrospray ionization mass spectrometry), IR (infrared) and UV (ultraviolet). Derivative chromatographic separations were performed on a Sinergy Hydro-RP column (150 mm × 4.6 mm i.d.) using a mobile phase consisting of methanol and triethylammonium phosphate buffer (pH 3; 10mM) at varying composition gradient elution and at a flow rate of 0.8 mL/min. Detection was set at λ=320 nm. The obtained results were compared with those achieved by a validated direct HPLC method with detection at λ=275 nm using a Sinergy Polar-RP column (250 mm × 3 mm i.d.) by isocratic elution conditions with a mobile phase consisting of methanol/acetonitrile/sodium pentanesulphonate (SPS; pH 3; 10mM), 7.5:7.5:85 (v/v/v) at a flow rate of 0.3 mL/min. Derivatization method sensitivity proved to be ten times higher than direct method. Limit of detection of oct and tyr was 0.010 and 0.008 μg/mL, respectively. The reliability of the pre-column method was satisfactory also in terms of linearity (from 0.028 to 1.255 and 0.024 to 1.244 μg/mL for oct and tyr, respectively), precision (relative standard deviation ≤2, without significant differences between intra-day and inter-day data) and recovery (from 98.9 to 101.2%). The proposed method showed to be suitable for a reliable determination of oct and tyr traces in commercially available phytoproducts using the instrumentation usually present in any analytical laboratory.

  12. Amino Acids from a Comet

    NASA Technical Reports Server (NTRS)

    Cook, Jamie Elisla

    2009-01-01

    NASA's Stardust spacecraft returned samples from comet 81P/Wild 2 to Earth in January 2006. Examinations of the organic compounds in cometary samples can reveal information about the prebiotic organic inventory present on the early Earth and within the early Solar System, which may have contributed to the origin of life. Preliminary studies of Stardust material revealed the presence of a suite of organic compounds including several amines and amino acids, but the origin of these compounds (cometary- vs. terrestrial contamination) could not be identified. We have recently measured the carbon isotopic ratios of these amino acids to determine their origin, leading to the first detection of a coetary amino acid.

  13. Amino Acids from a Comet

    NASA Technical Reports Server (NTRS)

    Cook, Jamie Elisla

    2009-01-01

    NASA's Stardust spacecraft returned samples from comet 81P/Wild 2 to Earth in January 2006. Examinations of the organic compounds in cometary samples can reveal information about the prebiotic organic inventory present on the early Earth and within the early Solar System, which may have contributed to the origin of life. Preliminary studies of Stardust material revealed the presence of a suite of organic compounds including several amines and amino acids, but the origin of these compounds (cometary- vs. terrestrial contamination) could not be identified. We have recently measured the carbon isotopic ratios of these amino acids to determine their origin, leading to the first detection of a coetary amino acid.

  14. AMINO ACID ANALYSES OF THE ANTARCTIC CM2 METEORITES ALH 83100 AND LEW 90500 USING LIQUID CHROMATOGRAPHY-TIME OF FLIGHT-MASS SPECTROMETRY

    NASA Technical Reports Server (NTRS)

    Glavin, D. P.; Dworkin, J. P.; Aubrey, A.; Botta, O.; Doty, J. H., III; Bada, J. L.

    2001-01-01

    The investigation of organic compounds in primitive carbonaceous meteorites provides a record of the chemical processes that occurred in the early solar system. In particular, amino acids have been shown to be potential indicators in tracing the nature of carbonaceous chondrite parent bodies [ 13. The delivery of amino acids by carbonaceous chondrites to the early Earth could have been any important source of the Earth's prebiotic organic inventory [2]. Over 80 different amino acids have been detected in the Murchison CM2 meteorite, most of them completely non-existent in the terrestrial biosphere [3]. We have optimized a new liquid chromatography-time-of-flight-mass spectrometry (LC-ToF-MS) technique coupled with OPAMAC derivatization in order to detect amino acids in meteorite extracts by UV fluorescence and exact mass simultaneously. The detection limit of the LC-ToF-MS instrument for amino acids is at least 3 orders of magnitude lower than traditional GC-MS techniques. Here we report on the first analyses of amino acids and their enantiomeric abundances in the CM2 carbonaceous meteorites ALH 83100, LEW 90500, and Murchison using this new LC-ToF-MS instrument configuration. Amino acid analyses of any kind for the CM meteorite ALH 83100 have not previously been reported.

  15. Analysis of plasma free amino acid profiles in canine brain tumors

    PubMed Central

    Utsugi, Shinichi; Azuma, Kazuo; Osaki, Tomohiro; Murahata, Yusuke; Tsuka, Takeshi; Ito, Norihiko; Imagawa, Tomohiro; Okamoto, Yoshiharu

    2017-01-01

    Canine brain tumors are best diagnosed using magnetic resonance imaging (MRI). However, opportunities of MRI examination are restricted due to its limited availability in veterinary facilities; thus, numerous canine brain tumors are diagnosed at an advanced stage. Therefore, development of a noninvasive diagnostic biomarker is required for the early detection of brain tumors. In the present study, plasma free amino acid (PFAA) profiles between dogs with and without brain tumors were compared. A total of 12 dogs with brain tumors, diagnosed based on clinical signs, and on the results of intracranial MRI and/or pathological examination were evaluated. In addition, eight dogs diagnosed with idiopathic epilepsy and 16 healthy dogs were also included. A liquid chromatography system with automated pre-column derivatization functionality was used to measure the levels of 20 amino acids. As a result, the levels of three amino acids (alanine, proline and isoleucine) were increased significantly (1.6-, 1.5- and 1.6-fold, respectively) in the plasma of dogs with brain tumors as compared with the levels in control dogs (all P<0.05). Thus, the PFAA levels of dogs with brain tumors differed from those of healthy dogs. The present study demonstrated that analysis of PFAA levels of dogs with brain tumors may serve as a useful biomarker for the early detection of canine brain tumors. PMID:28357072

  16. Treatment of Amino Acid Metabolism Disorders

    MedlinePlus

    ... Treatment of amino acid metabolism disorders Treatment of amino acid metabolism disorders E-mail to a friend Please ... this page It's been added to your dashboard . Amino acid metabolism disorders are rare health conditions that affect ...

  17. Single-drop microextraction followed by in-syringe derivatization and GC-MS detection for the determination of parabens in water and cosmetic products.

    PubMed

    Saraji, Mohammad; Mirmahdieh, Shiva

    2009-04-01

    A single-drop microextraction (SDME) method followed by in-syringe derivatization and GC-MS determination has been developed for analysis of five parabens, including methyl, ethyl, isopropyl, n-propyl and n-butyl paraben in water samples and cosmetic products. N,O-Bis(trimethylsilyl)acetamide (BSA) was used as derivatization reagent. Derivatization reaction was performed inside the syringe barrel using 0.4 microL of BSA. Parameters that affect the derivatization yield such as temperature and time of the reaction were studied. In addition, experimental SDME parameters such as selection of organic solvent, addition of salt, extraction time and extraction temperature were investigated and optimized. The RSD of the method for aqueous samples varied from 8.1 to 13%. The LODs ranged from 0.001 (n-butyl paraben) to 0.015 (methyl paraben) microg/L, and the enrichment factors were between 23 and 150.

  18. APPLICATION OF DNPH DERIVATIZATION WITH LC/MS TO THE IDENTIFICATION OF POLAR CARBONYL DRINKING WATER DISINFECTION BY-PRODUCTS IN DRINKING WATER

    EPA Science Inventory

    A qualitative method using 2,4-dinitrophenylhydrazine (DNPH) derivatization followed by analysis with liquid chromatography (LC)/negative ion-electrospray mass spectrometry (MS) was developed for analyzing and identifying highly polar aldehydes and ketones in ozonated drinking wa...

  19. APPLICATION OF DNPH DERIVATIZATION WITH LC/MS TO THE IDENTIFICATION OF POLAR CARBONYL DRINKING WATER DISINFECTION BY-PRODUCTS IN DRINKING WATER

    EPA Science Inventory

    A qualitative method using 2,4-dinitrophenylhydrazine (DNPH) derivatization followed by analysis with liquid chromatography (LC)/negative ion-electrospray mass spectrometry (MS) was developed for analyzing and identifying highly polar aldehydes and ketones in ozonated drinking wa...

  20. Amino acids and cell regulation.

    PubMed Central

    Forster, R. P.; Goldstein, L.

    1979-01-01

    Free amino play an important role in regulating cell volume in fishes. Four tissues/cells (skeletal muscle, RBC, brain, and myocardium) of the little skate, Raja erinacea, were selected for detailed study because of their special importance or unique advantage as experimental models. Three particular amino acids, beta-alanine, taurine, and sarcosine play a predominant role in all four tissues. As in higher vertebrates, amino acid uptake in skate brain, heart, and RBC is mediated via a Na+-dependent process. Amino acids leave the skate brain rapidly in response to a sudden decrease in plasma osmolality and/or to a simultaneous drop in extracellular Na+ concentration. However, although amino acids are important for volume regulation in normal brain cells, they do not appear to be likely candidates for the unidentified "idiogenic" osmolytes in mammalian brain cells. The high concentration of taurine in skate myocardium is of special interest because of the special role of this amino acid in myocardial contractility. Thus, unlike beta-alanine and sarcosine, taurine may play a dual role in regulating both cell volume and contractility of myocardial cells. The isolated skate atrium is well suited for in vitro studies of these two processes. PMID:395764

  1. Determination of Diacetyl in Beer by a Precolumn Derivatization-HPLC-UV Method Using 4-(2,3-Dimethyl-6-quinoxalinyl)-1,2-benzenediamine as a Derivatizing Reagent.

    PubMed

    Wang, Ji-Yu; Wang, Xin-Jie; Hui, Xian; Hua, Shui-Hong; Li, Heng; Gao, Wen-Yun

    2017-03-29

    Diacetyl is an important flavoring compound in many foods, especially in beer. In the present study, we developed and validated a new precolumn derivatization HPLC-UV method for the determination of diacetyl using 4-(2,3-dimethyl-6-quinoxalinyl)-1,2-benzenediamine as a novel derivatizing reagent. After derivatization with the reagent at a pH value 4.0 at ambient temperature for 10 min, diacetyl was analyzed on an ODS column and detected at 254 nm. The results show that the correlation coefficient of the method is 0.9991 in the range of 0.10 to 100.0 μM diacetyl, and the limit of detection is 0.02 μM. The method was further evaluated in the analysis of beer samples with the recoveries ranging from 94.4 to 102.6% and RSDs from 1.36 to 3.33%. The concentrations of diacetyl in 8 beer samples were determined in the range of 0.19 to 0.42 μM. The method established in this study may be well suitable for the determination of diacetyl in beer.

  2. Release of free amino acids upon oxidation of peptides and proteins by hydroxyl radicals.

    PubMed

    Liu, Fobang; Lai, Senchao; Tong, Haijie; Lakey, Pascale S J; Shiraiwa, Manabu; Weller, Michael G; Pöschl, Ulrich; Kampf, Christopher J

    2017-03-01

    Hydroxyl radical-induced oxidation of proteins and peptides can lead to the cleavage of the peptide, leading to a release of fragments. Here, we used high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) and pre-column online ortho-phthalaldehyde (OPA) derivatization-based amino acid analysis by HPLC with diode array detection and fluorescence detection to identify and quantify free amino acids released upon oxidation of proteins and peptides by hydroxyl radicals. Bovine serum albumin (BSA), ovalbumin (OVA) as model proteins, and synthetic tripeptides (comprised of varying compositions of the amino acids Gly, Ala, Ser, and Met) were used for reactions with hydroxyl radicals, which were generated by the Fenton reaction of iron ions and hydrogen peroxide. The molar yields of free glycine, aspartic acid, asparagine, and alanine per peptide or protein varied between 4 and 55%. For protein oxidation reactions, the molar yields of Gly (∼32-55% for BSA, ∼10-21% for OVA) were substantially higher than those for the other identified amino acids (∼5-12% for BSA, ∼4-6% for OVA). Upon oxidation of tripeptides with Gly in C-terminal, mid-chain, or N-terminal positions, Gly was preferentially released when it was located at the C-terminal site. Overall, we observe evidence for a site-selective formation of free amino acids in the OH radical-induced oxidation of peptides and proteins, which may be due to a reaction pathway involving nitrogen-centered radicals.

  3. Nanoflow Separation of Amino Acids for the Analysis of Cosmic Dust

    NASA Technical Reports Server (NTRS)

    Martin, M. P.; Glavin, D. P.; Dworkin, Jason P.

    2008-01-01

    The delivery of amino acids to the early Earth by interplanetary dust particles, comets, and carbonaceous meteorites could have been a significant source of the early Earth's prebiotic organic inventory. Amino acids are central to modern terrestrial biochemistry as major components of proteins and enzymes and were probably vital in the origin of life. A variety of amino acids have been detected in the CM carbonaceous meteorite Murchison, many of which are exceptionally rare in the terrestrial biosphere including a-aminoisobutyric acid (AIB) and isovaline. AIB has also been detected in a small percentage of Antarctic micrometeorite grains believed to be related to the CM meteorites We report on progress in optimizing a nanoflow liquid chromatography separation system with dual detection via laser-induced-fluorescence time of flight mass spectrometry (nLC-LIF/ToF-MS) for the analysis of o-phthaldialdehydelN-acetyl-L-cysteine (OPA/NAC) labeled amino acids in cosmic dust grains. The very low flow rates (<3 micro-L/min) of nLC over analytical LC (>0.1 ml/min) combined with <2 micron column bead sizes has the potential to produce efficient analyte ionizations andchromatograms with very sharp peaks; both increase sensitivity. The combination of the selectivity (only primary amines are derivatized), sensitivity (>4 orders of magnitude lower than traditional GC-MS techniques), and specificity (compounds identities are determined by both retention time and exact mass) makes this a compelling technique. However, the development of an analytical method to achieve separation of compounds as structurally similar as amino acid monomers and produce the sharp peaks required for maximum sensitivity is challenging.

  4. Mass spectrometric analysis of free fatty acids in infant milk powders by frozen pretreatment coupled with isotope-labeling derivatization.

    PubMed

    Zhou, Tianxiao; Leng, Jiapeng; Peng, Yaoshan; Zhang, Lei; Guo, Yinlong

    2016-03-01

    In combination with frozen pretreatment and carboxyl group derivatization, a novel workflow was developed for the determination of free fatty acids in milk powder. The workflow showed a significantly enhanced performance for comprehensive free fatty acid analysis owing to a highly efficient frozen extraction method. In addition, the advantages of the workflow also involved high sensitivity and great tolerance to a complex matrix. Characteristic fragment ions of derivatization reagents also provide clear evidence for the qualitative analysis of free fatty acids. Fourteen types of free fatty acids in a number of domestic and overseas infant milk powders have been successfully detected. The content of free fatty acids in the different samples was different, which probably indicates the diverse quality of infant milk powder. The workflow is expected to be a pragmatic tool for the analysis of free fatty acids in intricate matrices. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. Determination of brassinosteroids in the sub-femtomolar range using dansyl-3-aminophenylboronate derivatization and electrospray mass spectrometry.

    PubMed

    Svatos, Ales; Antonchick, Andrey; Schneider, Bernd

    2004-01-01

    A selective and sensitive electrospray ionization (ESI) mass spectrometry based method for detection of brassinosteroids (BS) in plant samples was developed. The limit of detection (LOD) was dramatically reduced over existing analytical methods using a microbore (1.00 mm) C18 column and chemical derivatization of free BS to dansyl-3-aminophenylboronates. The LOD in the selected-ion monitoring (SIM) mode for derivatized BS was 125 attomole (signal-to-noise ratio 3). The practical utility of the method is documented in Arabidopsis thaliana plant transformation of castasterone to brassinolide using a deuterium-labeled precursor. The method could be very useful for the detection of native BS in plant tissue and biosynthetic studies.

  6. A novel derivatization procedure and chiral gas chromatographic method for enantiomeric purity screening of L-carnitine.

    PubMed

    Albreht, Alen; Zupančič, Borut; Vovk, Irena

    2014-01-01

    L-Carnitine is used extensively in functional foods and food supplements; consequently, the control of its enantiomeric purity is of paramount importance. A new derivatization procedure and chiral gas chromatographic method with flame ionization detection, using a cyclodextrin based stationary phase, enables prompt, simple, and inexpensive screening of the enantiomeric ratio of L- and D-carnitine in samples with different matrices. Conversion of carnitine to beta-acetoxy-gama-butyrolactone was optimized for maximum conversion (98% of the desired product lactone was formed and 2% of the side product gama-crotonolactone) and minimum racemization (no changes at the chiral center were detected) and time consumption. As it is shown in this study, a fast gas chromatographic method, with total run time of 7 min, together with the new derivatization procedure enables an effective enantiomeric purity screening of L-carnitine in real samples such as food supplements and L-carnitine raw ingredient.

  7. Ultra trace level determinations of acrylamide in surface and drinking water by GC-MS after derivatization with xanthydrol.

    PubMed

    Lim, Hyun-Hee; Shin, Ho-Sang

    2013-09-01

    A sensitive GC-MS method has been established for the determination of acrylamide in surface and drinking water based on derivatization with xanthydrol. Deuterated acrylamide (acrylamide-d3 ) was chosen as the internal standard for analyzing the water sample. The derivatization of acrylamide was performed directly in water, and the best reaction conditions (xanthydrol of 1.6 mM, HCl concentration of 0.05 M, reaction for 30 min at ambient temperature) were established by variation of parameters. Under the established conditions, the detection and quantification limits were 3.0 and 9.7 ng/L, respectively, and the interday RSD was less than 8% at concentrations of 20 and 100 ng/L. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. Sequencing and characterization of oligosaccharides using infrared multiphoton dissociation and boronic acid derivatization in a quadrupole ion trap.

    PubMed

    Pikulski, Michael; Hargrove, Amanda; Shabbir, Shagufta H; Anslyn, Eric V; Brodbelt, Jennifer S

    2007-12-01

    A simplified method for determining the sequence and branching of oligosaccharides using infrared multiphoton dissociation (IRMPD) in a quadrupole ion trap (QIT) is described. An IR-active boronic acid (IRABA) reagent is used to derivatize the oligosaccharides before IRMPD analysis. The IRABA ligand is designed to both enhance the efficiency of the derivatization reaction and to facilitate the photon absorption process. The resulting IRMPD spectra display oligosaccharide fragments that are formed from primarily one type of diagnostic cleavage, thus making sequencing straightforward. The presence of sequential fragment ions, a phenomenon of IRMPD, permit the comprehensive sequencing of the oligosaccharides studied in a single stage of activation. We demonstrate this approach for two series of oligosaccharides, the lacto-N-fucopentaoses (LNFPs) and the lacto-N-difucohexaoses (LNDFHs).

  9. A rapid and sensitive detection of D-Aspartic acid in Crystallin by chiral derivatized liquid chromatography mass spectrometry.

    PubMed

    Mizuno, Hajime; Miyazaki, Yasuto; Ito, Keisuke; Todoroki, Kenichiro; Min, Jun Zhe; Toyo'oka, Toshimasa

    2016-10-07

    A method for the determination of D-Aspartic acid (D-Asp) and its D/L ratio in peptides and proteins has been developed. This method was carried out with good separation of the D/L chiral peptide pairs by combination of a chiral derivatization and an ADME column separation. Furthermore, a cationic derivatization reagent, DBD-Py-NCS, increased the sensitivity of the ESI-MS/MS detection. To confirm the comprehensive peptide analysis, synthesized α-Crystallin tryptic peptides, which included D-Asp residues, were analyzed. The 5 pairs of D/L-Asp that included peptide diastereomers were well separated. Their peak resolutions were more than 1.5 and the results were reproducible (RSD<0.05, n=5). As an application of this method, we analyzed the α-Crystallin standard and UV irradiated α-Crystallin. After trypsin digestion and DBD-Py-NCS derivatization, the tryptic peptide derivatives were applied to LC-MS/MS. Based on the results of peptide sequence identification, almost all the tryptic peptides of the αA- and αB-Crystallin homologous subunits of α-Crystallin were detected as DBD-Py NCS derivatives. However, there was no D-Asp residue in the standard proteins. In the case of the UV irradiated α-Crystallin, Asp(76) and Asp(84) in the αA-Crystallin and Asp(96) in αB-Crystallin were racemized to D-Asp. These results show that this proposed chiral peptide LC-MS/MS method using chiral derivatization provides a rapid and sensitive analysis for post translational Asp racemization sites in aging proteins.

  10. Determination of azelaic acid in pharmaceuticals and cosmetics by RP-HPLC after pre-column derivatization.

    PubMed

    Ferioli, V; Rustichelli, C; Vezzalini, F; Gamberini, G

    1994-06-01

    This paper reports a RP-HPLC method for the determination in topics of azelaic acid, a keratolytic and anti-comedogenic agent widely used in the treatment of all types of acne. A derivatization step was needed prior to chromatographic analysis because the analyte is lacking in chromophore. A sample clean-up procedure by solid-phase extraction was also developed to analyse azelaic acid in complex matrices, such as pharmaceutical and cosmetic formulations.

  11. A new on-line, in-tube pre-column derivatization technique for high performance liquid chromatographic determination of azithromycin in human serum.

    PubMed

    Bahrami, Gholamreza; Mohammadi, Bahareh

    2006-01-18

    Pre-column derivatization methods for high performance liquid chromatographic assay of specific pharmaceutical agents using 9-fluorenylmethyl chloroformate (FMOC-Cl) have received special attention because highly fluorescent and stable adducts are provided by these methods. However, unlike the post-column on-line techniques, long derivatization time is needed and the reaction cannot be well controlled. A new, sensitive and fast pre-column on-line derivatization technique coupled with high-performance liquid chromatography using FMOC-Cl as labeling agent is described and validated for determination of azithromycin in human serum. After extraction of the drug from serum, the residue was reconstituted in mixture of acetonitrile-phosphate buffer (3:1, v/v; pH 8.5) and directly injected onto the chromatographic system. Continuous on-line derivatization and analysis of the compounds were successfully performed using in-tube elution of FMOC-Cl. The total time needed for derivatization and chromatographic analysis of the drug was 13 min. The assay was reliable and reproducible, with limit of quantification of 10 ng/ml. The described technique may offer significant advantages over existing off-line derivatization methods using FMOC-Cl.

  12. [Applications of multi-micro-volume pressure-assisted derivatization reaction device for analysis of polar heterocyclic aromatic amines by gas chromatography-mass spectrometry].

    PubMed

    Wang, Yiru; Chen, Fangxiang; Shi, Yamei; Tan, Connieal; Chen, Xi

    2013-01-01

    A multi-micro-volume pressure-assisted derivatization reaction device has been designed and made for the silylation derivatization of polar heterocyclic aromatic amines by N-(tert-butyldimethylsilyl )-N-methyl-trifluoroacetamide (MTBSTFA) with 1% catalyst tert-butyldimethylchlorosilane (TBDMCS) at a high temperature. The tert-butyldimethylsilyl derivatives then could be automatically analyzed by gas chromatography-mass spectrometry. Using the pressure-assisted device, the silylation reaction may occur at a temperature higher than the boiling points of the reagents, and several micro-volume samples can be simultaneously pretreated in the same device to shorten the sample-preparation time and to improve the repeatability. The derivatization conditions including the headspace volume of the vial, the evaporative surface area of the reagent, derivatization temperature and time have been discussed for the use of the pressure-assisted device. The experimental results proved that the device is an effective way for the simultaneous derivatization of several micro-volume samples at a high temperature. Compared with a common device, the derivative amounts were obviously increased when using the pressure-assisted device at 90 degrees C. Quantitative derivatization can be achieved even at 150 degrees C while there was no common device could be applied at such a high temperature due to the heavy losses of reagents by evaporation. However, no obviously higher reaction speed has been observed in such a circumstance with a higher temperature and a higher pressure using the pressure-assisted device.

  13. Derivatization of bisphenol A and its analogues with pyridine‐3‐sulfonyl chloride: multivariate optimization and fragmentation patterns by liquid chromatography/Orbitrap mass spectrometry

    PubMed Central

    Regueiro, Jorge; Breidbach, Andreas

    2015-01-01

    Rationale Due to the growing restrictions on the use of bisphenol A (BPA), several other bisphenols are gaining importance as substitutes for BPA in a variety of applications. There is, therefore, a real need for selective and sensitive methods based on mass spectrometry which will allow the human exposure to these new bisphenols to be assessed. Methods Derivatization of BPA and its substitutes with pyridine‐3‐sulfonyl chloride is used to enhance the detection capability of bisphenols by electrospray ionization mass spectrometry. A multivariate experimental design, Box‐Behnken response surface, was used to evaluate the influence of the main variables potentially affecting the derivatization yield. Fragmentation patterns for all the derivatized bisphenols were acquired by high‐resolution/accurate‐mass Orbitrap mass spectrometry. Results Temperature and pH were identified as the most important factors affecting the derivatization yield of bisphenols. Fragmentation of the protonated molecules produced abundant analyte‐specific product ions. Most of the derivatized bisphenols showed significant improvements in their signal‐to‐noise ratios compared with the underivatized forms. The stability of these derivatives was demonstrated through several freeze/thaw cycles, short‐term room temperature and long‐term cold storage. Conclusions Derivatization of BPA and its structural analogues with pyridine‐3‐sulfonyl chloride is proposed as a specific, sensitive, high‐throughput approach to their analysis by liquid chromatography coupled to electrospray ionization mass spectrometry. © 2015 The Authors. Rapid Communications in Mass Spectrometry Published by John Wiley & Sons Ltd. PMID:26212162

  14. Trace analysis of ethosuximide in human plasma with a chemically removable derivatizing reagent and high-performance liquid chromatography.

    PubMed

    Chen, S H; Wu, H L; Shen, M C; Kou, H S

    1999-06-11

    A simple and sensitive liquid chromatographic method is described for the determination of ethosuximide in human plasma, as a highly sensitive derivative. Ethosuximide spiked in plasma was extracted with toluene and derivatized with a chemically removable derivatizing reagent, 2-(2-naphthoxy)ethyl 2-[1-(4-benzyl)piperazyl]ethanesulfonate, in a homogeneous system, using magnesium oxide as base catalyst. The resulting derivative was separated on a LiChrospher diol column with 1.2% isopropanol in n-hexane as the mobile phase and using coumarin as the internal standard. Several parameters affecting the extraction/derivatization of ethosuximide from spiked plasma were investigated. The linear range for the determination of ethosuximide in spiked plasma was over 30-700 nmol/ml. For ethosuximide in plasma, the detection limit (signal-to-noise ratio=3; sample size, 10 microl) was about 9 pmol; the relative standard deviation was 6.4% for intra-day assay (n=6) and 9.2% for inter-day assay (n=6) and the relative recovery was found greater than 94%.

  15. An optimized two-step derivatization method for analyzing diethylene glycol ozonation products using gas chromatography and mass spectrometry.

    PubMed

    Yu, Ran; Duan, Lei; Jiang, Jingkun; Hao, Jiming

    2017-03-01

    The ozonation of hydroxyl compounds (e.g., sugars and alcohols) gives a broad range of products such as alcohols, aldehydes, ketones, and carboxylic acids. This study developed and optimized a two-step derivatization procedure for analyzing polar products of aldehydes and carboxylic acids from the ozonation of diethylene glycol (DEG) in a non-aqueous environment using gas chromatography-mass spectrometry. Experiments based on Central Composite Design with response surface methodology were carried out to evaluate the effects of derivatization variables and their interactions on the analysis. The most desirable derivatization conditions were reported, i.e., oximation was performed at room temperature overnight with the o-(2,3,4,5,6-pentafluorobenzyl) hydroxyl amine to analyte molar ratio of 6, silylation reaction temperature of 70°C, reaction duration of 70min, and N,O-bis(trimethylsilyl)-trifluoroacetamide volume of 12.5μL. The applicability of this optimized procedure was verified by analyzing DEG ozonation products in an ultrafine condensation particle counter simulation system. Copyright © 2016. Published by Elsevier B.V.

  16. Quantitative mass spectrometric analysis of dipeptides in protein hydrolysate by a TNBS derivatization-aided standard addition method.

    PubMed

    Hanh, Vu Thi; Kobayashi, Yutaro; Maebuchi, Motohiro; Nakamori, Toshihiro; Tanaka, Mitsuru; Matsui, Toshiro

    2016-01-01

    The aim of this study was to establish, through a standard addition method, a convenient quantification assay for dipeptides (GY, YG, SY, YS, and IY) in soybean hydrolysate using 2,4,6-trinitrobenzene sulfonate (TNBS) derivatization-aided LC-TOF-MS. Soybean hydrolysate samples (25.0 mg mL(-1)) spiked with target standards were subjected to TNBS derivatization. Under the optimal LC-MS conditions, five target dipeptides derivatized with TNBS were successfully detected. Examination of the standard addition curves, with a correlation coefficient of r(2) > 0.979, provided a reliable quantification of the target dipeptides, GY, YG, SY, YS, and IY, in soybean hydrolysate to be 424 ± 20, 184 ± 9, 2188 ± 199, 327 ± 16, and 2211 ± 133 μg g(-1) of hydrolysate, respectively. The proposed LC-MS assay is a reliable and convenient assay method, with no interference from matrix effects in hydrolysate, and with no requirement for the use of an isotope labeled internal standard.

  17. On-Line Derivatization Gas Chromatography Ion Trap Mass Spectrometry for Determination of Endocrine Disruptors in Surface Water

    SciTech Connect

    Tzing, Shin-Hwa; Chang, Jia-Yaw; Ling, Yong-Chien

    2004-03-31

    A method has been developed for the determination of endocrine disruptors (EDs) (containing hydroxyl groups) in surface water from different sources. The surface water samples from different sites including school and local dormitory sewage effluents, lake water and river water were collected and analyzed. In this method, the pretreated sample is directly analyzed by GC-MS using on-line derivatization, where tetramethylammonium hydroxide (TMA-OH) was used as the derivatizing agent. Use of large-volume direct sample introduction (DSI) and co-injection of the sample and TMAOH avoids external contaminations as observed in conventional derivatization protocols. Additionally, the use of chemical ionization (CI) and CI-MS/MS could enable detection of EDs at lower concentrations and reduce the matrices' interference thereby enhancing detection sensitivity of EDs for quantification. In this work, the use of dichloromethane as CI reagent for EDs is reported for the first time and could detect EDs to concentrations as low as 0.5 pg/mL. The recovery ranged from 74 to 112 % and the relative standard derivations for replicate analyses ranged from 5 to 17 %. We hope that this method will be applicable for routine analysis of EDs with hydroxyl functional groups.

  18. Simultaneous determination of HFBA-derivatized amphetamines and ketamines in urine by gas chromatography-mass spectrometry.

    PubMed

    Lee, Hei Hwa; Lee, Jong Feng; Lin, Sin Yu; Chen, Ping Ho; Chen, Bai Hsiun

    2011-04-01

    To facilitate the analysis of targeted drugs under high sample volume testing environment, an extraction, derivatization and gas chromatographic-mass spectrometric analysis method was developed for simultaneously determination of amphetamine (AMP), methamphetamine (MAMP), 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxymethamphetamine (MDMA), 3,4-methylenedioxyethylamphetamine (MDEA), ketamine, and norketamine in urine. This method utilized solid-phase extraction in conjunction with derivatization using heptafluorobutyric anhydride (HFBA) as the derivatization reagent. Using a 1-mL sample, the limits of quantitation achieved for the analysis of AMP, MAMP, MDA, MDMA, MDEA, ketamine, and norketamine were 25, 15, 60, 60, 70, 25, and 30 ng/mL, respectively. Upper limits of quantitation were 8000 ng/mL for all amphetamines and 6000 ng/mL for ketamine and norketamine. Except for dehydronorketamine (DHNK), within-day and between-day precisions (as expressed in CV%) for quality control samples were ≤ 3.1% and ≤ 4.95%, respectively. Except DHNK, the within-day accuracy ranged between 96.0% and 110.7% and the between-day accuracy ranged between 96.9% and 108.7%. A group of 107 urine samples previously determined to contain the target analytes were analyzed by this new approach. Quantitative data produced by both methods agreed well. With this new approach, we were able to use a single analytical protocol to conduct the confirmation test for samples that preliminarily tested positive (by immunoassay) for amphetamines, ketamine, or both.

  19. [Determination of bisphenol A from toys and food contact materials by derivatization and gas chromatography-mass spectrometry].

    PubMed

    Gao, Yonggang; Zhang, Yanyan; Gao, Jianguo; Zhang, Huiling; Zheng, Lisha; Chen, Jing

    2012-10-01

    A method was developed for the determination of bisphenol A (BPA) in toys and food contact materials. The BPA was extracted with Soxhlet extraction method from the sample and reacted with acetic anhydride. The final product was determined by gas chromatography-mass spectrometry (GC-MS). To achieve the optimum derivatization performance, the derivatization time and dosage of derivatization reagent etc. were investigated. Under the optimized experimental conditions, the final product was stable and the peak shape was good. The linearity of the derivative was good in the range of 0.05 to 50 mg/L with the correlation coefficient (r2) above 0.999. The recoveries ranged from 80% to 93% at the spiked levels of 0.05, 1.00, 10.00 mg/kg with the relative standard deviations (RSDs) less than 3.7%. The limit of detection (S/N = 3) was 10 microg/kg. The method is accurate and has high recovery. The method is suitable for the inspection of bisphenol A in toys and food contact materials.

  20. Novel ethyl-derivatization approach for the determination of fluoride by headspace gas chromatography/mass spectrometry.

    PubMed

    Pagliano, Enea; Meija, Juris; Ding, Jianfu; Sturgeon, Ralph E; D'Ulivo, Alessandro; Mester, Zoltán

    2013-01-15

    We report a novel derivatization chemistry for determination of fluoride based on the batch reaction of fluoride ions with triethyloxonium tetrachloroferrate(III) in a closed vessel to yield fluoroethane. Gaseous fluoroethane was readily separated from the matrix, sampled from the headspace, and determined by gas chromatography/mass spectrometry. The method was validated using rainwater certified reference material (IRMM CA408) and subsequently applied to the determination of fluoride in various matrixes, including tap water, seawater, and urine. An instrumental limit of detection of 3.2 μg/L with a linear range up to 50 mg/L was achieved. The proposed derivatization is a one-step reaction, requires no organic solvents, and is safe, as the derivatizing agent is nonvolatile. Determination of fluoride is affected by common fluoride-complexing agents, such as Al(III) and Fe(III). The effect of large amounts of these interferences was studied, and the adverse effect of these ions was eliminated by use of the method of standard additions.

  1. Diels-Alder derivatization for sensitive detection and characterization of conjugated linoleic acids using LC/ESI-MS/MS.

    PubMed

    Higashi, Tatsuya; Takekawa, Mioko; Min, Jun Zhe; Toyo'oka, Toshimasa

    2012-04-01

    The utility of Diels-Alder derivatization with 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) for liquid chromatography/electrospray ionization tandem mass spectrometry of conjugated linoleic acids (CLAs) was examined. PTAD rapidly reacted with the CLAs, and the resulting derivatives were highly responsive in electrospray ionization mass spectrometry operating in the positive-ion mode. The derivatives produced characteristic product ions during tandem mass spectrometry, which enabled the sensitive detection [limit of detection 18 fmol (signal-to-noise ratio of 5)] and the identification of the conjugated diene position. The PTAD derivatization also significantly increased the reversed-phase liquid chromatography separation selectivity for the most biologically active CLA isomers: cis-9,trans-11-CLA and trans-10,cis-12-CLA. The PTAD derivatization was applied to analyses of food and biological samples; the major CLAs in milk and beef fat samples were successfully identified, and trace amounts of CLAs in human saliva were detected with a simple pretreatment and short analysis time.

  2. Green nondegrading approach to alkyne-functionalized cellulose fibers and biohybrids thereof: synthesis and mapping of the derivatization.

    PubMed

    Mangiante, Gino; Alcouffe, Pierre; Burdin, Béatrice; Gaborieau, Marianne; Zeno, Elisa; Petit-Conil, Michel; Bernard, Julien; Charlot, Aurélia; Fleury, Etienne

    2013-01-14

    Alkyne-functionalized cellulose fibers have been generated through etherification under basic water or hydroalcoholic conditions (NaOH/H(2)O/isopropanol). For a given NaOH content, the medium of reaction and, more particularly, the water/IPA ratio, were shown to be of crucial importance to derivatize the fibers without altering their integrity and their crystalline nature. It was shown that the degree of substitution (DS) of the fibers increases concomitantly with isopropanol weight ratio and that, contrary to water or water-rich conditions, derivatization of fibers under isopropanol-rich conditions induces an alteration of the fibers. Optimization of etherification conditions in aqueous media afforded functionalized cellulose materials with DS up to 0.20. Raman confocal microscopy on derivatized fibers cross sections stressed that alkyne moieties are incorporated all over the fibers. The resulting fibers were postfunctionalized by molecular probes and macromolecules in aqueous or water-rich conditions. The effectiveness of the grafting was strongly impacted by the nature of the coupling agents.

  3. Spectrofluorimetric method for the determination of sulpiride in pharmaceutical preparations and human plasma through derivatization with 2-cyanoacetamide.

    PubMed

    Shah, Jasmin; Jan, M Rasul; Khan, M Naeem; Shah, Sultan

    2013-01-01

    A sensitive and accurate spectrofluorimetric method has been developed for the determination of sulpiride in pharmaceutical preparations and human plasma. The developed method is based on the derivatization reaction of 2-cyanoacetamide with sulpiride in 30% ammonical solution. The fluorescent derivatized reaction product exhibited maximum fluorescence intensity at 379 nm after excitation at 330 nm. The optimum conditions for derivatization reactions were studied and the fluorescence intensity versus concentration plot was found to be linear over the concentration range 0.2-20.0 µg/mL with a correlation coefficient of 0.9985. The limit of detection (LOD) and limit of quantification (LOQ) were found to be 0.82 and 2.73 ng/mL, respectively. The proposed method was validated according to ICH guidelines. The effects of common excipients and co-administered drugs were also studied. The accuracy of the method was checked using the standard addition method and percent recoveries were found to be in the range of 99.00-101.25% for pharmaceutical preparations and 97.00-97.80% for spiked human plasma. The method was successfully applied to commercial formulations and the results obtained for the proposed method were compared with a high-performance liquid chromatography reference method and statistically evaluated using the Student's t-test for accuracy and the variance ratio F-test for precision. A reaction pathway was also proposed.

  4. Developing an on-line derivatization of FAs by microwave irradiation coupled to HPLC separation with UV detection.

    PubMed

    Bravo, Bélgica; Chávez, Gerson; Piña, Nolberto; Ysambertt, Fredy; Márquez, Nelson; Cáceres, Ana

    2004-12-15

    The development of analytical methods for routine simultaneous identification and quantification of carboxylic fatty acids (CFAs) are required in different fields, such as, pharmaceutical cosmetics, food products and formulations of water-microemulsion-oil systems. Determination of CFAs has been developed mainly by gas chromatography (GC). As an alternative to GC, liquid chromatography (LC) has better sensitivity and selectivity. However, most CFAs show no useful absorption in ultraviolet-violet (UV-Vis) region, one of the more used detection technique in high-performance liquid chromatography (HPLC). In order to allow the use of UV-Vis detection, the use of pre-column derivatization has been reported to increase sensitivity and selectivity. Therefore, establishment of a simpler and faster on-line method with complete separation is needed for the screening of large numbers of samples. 2,4-Dinitrophenylhydrazine (2,4-DNPH.), benzoil chloride (BC), and phenylhydrazine (PH) were used for derivatization of different FAs by microwaves radiation (MW). After the on-line derivatization, products were separated and quantified by HPLC. Reactor coil was placed inside of microwaves oven at 450W. Parameters as flow, amount of reagents, irradiation time, and chromatographic conditions were optimized. The continuous analysis using the MW-HPLC-UV system provided high sensitivity and reduced both the amount of reagent used and the analysis times. This proposed method can be used for the routine analysis of FAs contained in water-microemulsion-oil systems, to quantify the total acid fraction in each phase.

  5. In-gel NHS-propionate derivatization for histone post-translational modifications analysis in Arabidopsis thaliana.

    PubMed

    Chen, Jiajia; Gao, Jun; Peng, Maolin; Wang, Yi; Yu, Yanyan; Yang, Pengyuan; Jin, Hong

    2015-07-30

    Post-translational modifications (PTMs) on histone are highly correlated with genetic and epigenetic regulation of gene expression from chromatin. Mass spectrometry (MS) has developed to be an optimal tool for the identification and quantification of histone PTMs. Derivatization of histones with chemicals such as propionic anhydride, N-hydroxysuccinimide ester (NHS-propionate) has been widely used in histone PTMs analysis in bottom-up MS strategy, which requires high purity for histone samples. However, biological samples are not always prepared with high purity, containing detergents or other interferences in most cases. As an alternative approach, an adaptation of in gel derivatization method, termed In-gel NHS, is utilized for a broader application in histone PTMs analysis and it is shown to be a more time-saving preparation method. The proposed method was optimized for a better derivatization efficiency and displayed high reproducibility, indicating quantification of histone PTMs based on In-gel NHS was achievable. Without any traditional fussy histone purification procedures, we succeeded to quantitatively profile the histone PTMs from Arabidopsis with selective knock down of CLF (clf-29) and the original parental (col) with In-gel NHS method in a rapid way, which indicated the high specificity of CLF on H3K27me3 in Arabidopsis. In-gel NHS quantification results also suggest distinctive histone modification patterns in plants, which is invaluable foundation for future studies on histone modifications in plants.

  6. Direct derivatization and gas chromatography-tandem mass spectrometry identification of nerve agent biomarkers in urine samples.

    PubMed

    Subramaniam, Raja; Östin, Anders; Nilsson, Calle; Åstot, Crister

    2013-06-01

    Rapid determination of nerve agent biomarkers at low-ppb levels in urine samples was achieved by direct derivatization and sample analysis using gas chromatography-tandem mass spectrometry. The studied biomarkers were alkylphosphonic acids (APAs), as they are specific hydrolysis products of organophosphorus nerve agents that can be used to verify nerve agent exposure. The sample preparation technique employed involves rapid direct derivatization (5min) of acidified urine samples (25μL) using a highly fluorinated phenyldiazomethane reagent [1-(diazomethyl)-3,5-bis(trifluoromethyl)benzene]. The derivatization conditions were optimized using statistical experimental design and multivariate data analysis. The APA derivatives were analyzed by GC-MS and MS/MS using negative ion chemical ionization. The selectivity and sensitivity of analyses performed by low and high resolution single ion monitoring MS-mode were compared with those performed by multiple reaction monitoring MS/MS-mode. The MS/MS technique offered the greatest sensitivity and selectivity of the tested mass spectrometric techniques, with limits of detection ranging from 0.5 to 1ng APAs/mL of urine. The method's robustness was evaluated using urine samples from the OPCW 2nd biomedical confidence building exercise and all APAs present in the samples were conclusively identified. The method thus offers excellent performance and is viable for the simultaneous trace determination of a wide range of nerve agent markers.

  7. Analysis of ammonium nitrate headspace by on-fiber solid phase microextraction derivatization with gas chromatography mass spectrometry.

    PubMed

    Lubrano, Adam L; Andrews, Benjamin; Hammond, Mark; Collins, Greg E; Rose-Pehrsson, Susan

    2016-01-15

    A novel analytical method has been developed for the quantitation of trace levels of ammonia in the headspace of ammonium nitrate (AN) using derivatized solid phase microextraction (SPME) fibers with gas chromatography mass spectrometry (GC-MS). Ammonia is difficult to detect via direct injection into a GC-MS because of its low molecular weight and extreme polarity. To circumvent this issue, ammonia was derivatized directly onto a SPME fiber by the reaction of butyl chloroformate coated fibers with the ammonia to form butyl carbamate. A derivatized externally sampled internal standard (dESIS) method based upon the reactivity of diethylamine with unreacted butyl chloroformate on the SPME fiber to form butyl diethylcarbamate was established for the reproducible quantification of ammonia concentration. Both of these compounds are easily detectable and separable via GC-MS. The optimized method was then used to quantitate the vapor concentration of ammonia in the headspace of two commonly used improvised explosive device (IED) materials, ammonium nitrate fuel oil (ANFO) and ammonium nitrate aluminum powder (Ammonal), as well as identify the presence of additional fuel components within the headspace.

  8. Pentafluorobenzyl bromide-A versatile derivatization agent in chromatography and mass spectrometry: I. Analysis of inorganic anions and organophosphates.

    PubMed

    Tsikas, Dimitrios

    2017-02-01

    Pentafluorobenzyl bromide (PFB-Br) is a versatile derivatization agent. It is widely used in chromatography and mass spectrometry since several decades. The bromide atom is largely the single leaving group of PFB-Br. It is substituted by wide a spectrum of nucleophiles in aqueous and non-aqueous systems to form electrically neutral, in most organic solvents soluble, generally thermally stable, volatile, strongly electron-capturing and ultraviolet light-absorbing derivatives. Because of these greatly favoured physicochemical properties, PFB-Br emerged an ideal derivatization agent for highly sensitive analysis of endogenous and exogenous substances including various inorganic and organic anions by electron capture detection or after electron-capture negative-ion chemical ionization in GC-MS. The present article attempts an appraisal of the utility of PFB-Br in analytical chemistry. It reviews and discusses papers dealing with the use of PFB-Br as the derivatization reagent in the qualitative and quantitative analysis of endogenous and exogenous inorganic anions in various biological samples, notably plasma, urine and saliva. These analytes include nitrite, nitrate, cyanide and dialkyl organophosphates. Special emphasis is given to mass spectrometry-based approaches and stable-isotope dilution techniques.

  9. Fluorous derivatization and fluorous-phase separation for fluorometric determination of naproxen and felbinac in human plasma.

    PubMed

    Sakaguchi, Yohei; Yoshida, Hideyuki; Hayama, Tadashi; Yoshitake, Makoto; Itoyama, Miki; Todoroki, Kenichiro; Yamaguchi, Masatoshi; Nohta, Hitoshi

    2011-04-28

    Fluorous derivatization followed by fluorous-phase liquid chromatographic (LC) separation exploits the affinity between perfluoroalkyl compounds for highly selective and quantitative isolation of various analytes. However, the applicability of this technique as a simple pretreatment for fluorometric determination in clinical settings has not been fully explored. Here we show the applicability of this technique to the clinical determination of non-steroidal anti-inflammatory drugs (NSAIDs) in human plasma. Naproxen and felbinac, widely used native-fluorescent NSAIDs with a carboxyl group, can have toxic effects at acute doses, and were therefore chosen as representative NSAIDs. Samples were precolumn derivatized with a non-fluorescent fluorous amine, which allowed highly selective retention of only derivatized substances in the fluorous LC column. Thus, subsequently, only the retained fluorous-labeled and fluorescent analytes were detected fluorometrically at appropriate retention times. The detection limits for these two drugs were less than 11fmol on column. Correlation curves were liner over the range of 0.04-10 and 5-250nmol/mL plasma for both two drugs (r>0.999) with good repeatability. Thus, this method offers a simple, sensitive, and selective solution for determination of NSAIDs in clinical settings. Copyright © 2011 Elsevier B.V. All rights reserved.

  10. Drawbacks in the use of unconventional hydrophobic anhydrides for histone derivatization in bottom-up proteomics PTM analysis

    PubMed Central

    Sidoli, Simone; Yuan, Zuo-Fei; Lin, Shu; Karch, Kelly; Wang, Xiaoshi; Bhanu, Natarajan; Arnaudo, Anna M.; Britton, Laura-Mae; Cao, Xing-Jun; Gonzales-Cope, Michelle; Han, Yumiao; Liu, Shichong; Molden, Rosalynn C.; Wein, Samuel; Afjehi-Sadat, Leila; Garcia, Benjamin A.

    2015-01-01

    Mass spectrometry (MS)-based proteomics has become the most utilized tool to characterize histone post-translational modifications (PTMs). Since histones are highly enriched in lysine and arginine residues, lysine derivatization has been developed to prevent the generation of short peptides (3-6 residues) during trypsin digestion. One of the most adopted protocols applies propionic anhydride for derivatization. However, the propionyl group is not sufficiently hydrophobic to fully retain the shortest histone peptides in reversed-phase liquid chromatography, and such procedure also hampers the discovery of natural propionylation events. In this work we tested 12 commercially available anhydrides, selected based on their safety and hydrophobicity. Performance was evaluated in terms of yield of the reaction, MS/MS fragmentation efficiency and drift in retention time by using the following samples: (i) a synthetic unmodified histone H3 tail, (ii) synthetic modified histone peptides and (iii) a histone extract from cell lysate. Results highlighted that 7 of the selected anhydrides increased peptide retention time as compared to propionic, and several anhydrides such as benzoic and valeric led to high MS/MS spectra quality. However, propionic anhydride derivatization still resulted in our opinion as the best protocol to achieve high MS sensitivity and more even ionization efficiency among the analyzed peptides. PMID:25641854

  11. Simple derivatization of aldehydes with D-cysteine and their determination in beverages by liquid chromatography-tandem mass spectrometry.

    PubMed

    Kim, Hyun-Ji; Shin, Ho-Sang

    2011-09-30

    We describe a simple derivatization method to determine aldehydes. This method is based on derivatization with D-cysteine and consecutive liquid chromatography-tandem mass spectrometry (LC-MS/MS). The optimum derivatization conditions of aldehydes with D-cysteine were 10 min at 50°C and pH 7.0. The formed alkyl thiazolidine-4-carboxylic acid derivatives were directly injected in LC-MS/MS. In the established condition, the method was used to detect eight aldehydes in beverages. The limit of detection (LOD) and limit of quantification (LOQ) of the aldehydes were 0.2-1.9 μg L(-1) and 0.7-6.0 μg L(-1) and the relative standard deviation was less than 2.0% at concentrations of 0.1 mg L(-1) and 1.0 mg L(-1) with the exception of octanal. All the beverage samples had detectable levels of methanal (0.033-0.145 mg L(-1)), ethanal (0.085-2.12 mg L(-1)), propanal (ND to 0.250 mg L(-1)), butanal (ND to 0.003 mg L(-1)), pentanal (ND to 0.471 mg L(-1)), hexanal (ND to 0.805 mg L(-1)), heptanal (0.019-3.91 mg L(-1)) and octanal (0.029-0.118 mg L(-1)). Crown Copyright © 2011. Published by Elsevier B.V. All rights reserved.

  12. Capillary electrophoresis separation of a mixture of chitin and chitosan oligosaccharides derivatized using a modified fluorophore conjugation procedure.

    PubMed

    Beaudoin, Marie-Eve; Gauthier, Julie; Boucher, Isabelle; Waldron, Karen C

    2005-08-01

    A capillary electrophoresis (CE) method was developed for the simultaneous analysis of small chitin and chitosan oligosaccharides. For detection purposes, the oligomers were derivatized with 8-aminopyrene-1,3,6-trisulfonic acid (APTS), a well known fluorophore for oligosaccharides analysis. The detection was performed by laser-induced fluorescence (LIF) with an argon ion laser having an excitation wavelength of 488 nm and with emission monitored at 520 nm. Derivatization parameters such as reaction time and conditions were examined. Separation conditions were also varied by testing a range of buffer pHs and concentrations. The best conditions were found using an 80 mM borate buffer at pH 8.4. This CE-LIF optimized method was used for the analysis of an enzymatically produced oligo-chitosan sample composed of a complex mixture and having an average degree of polymerization of 3.7 monomer units and 80% deacetylation. The oligo-chitosan sample was treated with a chitin deacetylase-like enzyme, the products were derivatized with APTS, and then analyzed without purification. The goal was to determine whether the deacetylase-like enzyme could increase the extent of deacetylation of the oligo-chitosan sample.

  13. Electrospray ionization tandem mass spectrometric and electron impact mass spectrometric characterization of mycosporine-like amino acids.

    PubMed

    Whitehead, Kenia; Hedges, John I

    2003-01-01

    Positive-ion mass spectral fragmentations of seven mycosporine-like amino acids (MAAs) are reported and discussed. The MAAs studied are small compounds composed of a cycloheximine ring substituted with amino acid or amino alcohol units. Techniques used include electron impact (EI) and electrospray ionization (ESI) with tandem mass spectrometry (MS/MS). ESI-MS/MS showed unusual small radical losses, generally resulting from the loss of a methyl group with the exception of shinorine and porphyra for which the initial losses were 30 and 44 Da, respectively. As expected from structural similarities, porphyra, shinorine and palythinol displayed similar fragmentation patterns, while palythenic acid and palythene fragmented in a similar manner. Overall, the ESI-MS/MS fragmentations at m/z <200 exhibited a distinctive pattern for all seven MAAs with characteristic ions at m/z 137, 168, 186, and 197 or 199. Several ions were observed for each of the MAAs analyzed, and together provide a useful and potentially diagnostic pattern for identification of MAAs and as an aid in structure elucidation of novel MAAs. For GC/EI-MS analysis, trimethylsilyl (TMS) derivatives were made. The EI-MS fragmentation patterns of TMS-MAAs showed many features typical of TMS-derivatized alpha-amines. The precursor TMS-MAA ion was not detected, but a [M-90](+ radical) ion was the highest-mass intense peak observed for palythine, palythinol and shinorine, while palythene gave a [M-116](+ radical) ion. Besides determining the number of acidic hydrogens, EI-MS of TMS-derivatized MAAs will aid in structure elucidation of novel MAAs. Copyright 2003 John Wiley & Sons, Ltd.

  14. Determination of cyclamate in urine by derivatized gas chromatography-mass spectrometry.

    PubMed

    Idris, Mohd; Middha, Deepak; Rasool, Shaik N; Shukla, Sudhir K; Baggi, Tulsidas R

    2013-01-01

    It is important in toxicological/drug screening work to rule out the possible interfering analytes, to eliminate the false positive or negative results. In this paper, we describe a simple, selective, and sensitive derivatized GC-MS method for the determination of cyclohexylsulfamic acid (cyclamate) in urine. Elite- 5MS capillary column was used for the separation of analytes and detection using GC-MS. The analysis was carried out in selected ion monitoring mode (SIM) in the range of 26 to 200 using m/z values of 57, 30, 55, 41, 44, 67, 82, 98, and 39. The method is based on the conversion of cyclamate into nitroso derivative of cyclamate followed by its gas chromatography-mass spectrometry determination. The limit of detection, limit of quantitation, and linearity range of the proposed method were found to be 0.2 μg/ ml, 0.7 μg/ml, and 1-15 μg/ml, respectively. The recovery of the present method is in the range of 88-94%. The proposed method can be applied for detection and quantification of cyclamate in urine.

  15. Determination of cyclamate in urine by derivatized gas chromatography-mass spectrometry

    PubMed Central

    Idris, Mohd; Middha, Deepak; Rasool, Shaik N.; Shukla, Sudhir K.; Baggi, Tulsidas R.

    2013-01-01

    Aim: It is important in toxicological/drug screening work to rule out the possible interfering analytes, to eliminate the false positive or negative results. In this paper, we describe a simple, selective, and sensitive derivatized GC-MS method for the determination of cyclohexylsulfamic acid (cyclamate) in urine. Materials and Methods: Elite- 5MS capillary column was used for the separation of analytes and detection using GC-MS. The analysis was carried out in selected ion monitoring mode (SIM) in the range of 26 to 200 using m/z values of 57, 30, 55, 41, 44, 67, 82, 98, and 39. Results and Discussion: The method is based on the conversion of cyclamate into nitroso derivative of cyclamate followed by its gas chromatography-mass spectrometry determination. The limit of detection, limit of quantitation, and linearity range of the proposed method were found to be 0.2 μg/ ml, 0.7 μg/ml, and 1-15 μg/ml, respectively. The recovery of the present method is in the range of 88-94%. Conclusion: The proposed method can be applied for detection and quantification of cyclamate in urine. PMID:23559823

  16. Fluorescence derivatization method for sensitive chromatographic determination of zidovudine based on the Huisgen reaction.

    PubMed

    Maeda, Yuki; Kishikawa, Naoya; Ohyama, Kaname; Wada, Mitsuhiro; Ikeda, Rie; Kuroda, Naotaka

    2014-08-15

    A novel pre-column fluorescence derivatization method for chromatographic analysis of azide compounds was developed based on the Huisgen reaction, which is a specific cycloaddition reaction between an alkyne and an azide. We designed and synthesized a fluorescent alkyne, 2-(4-ethynylphenyl)-4,5-diphenyl-1H-imidazole (DIB-ET) as a reagent. DIB-ET has a lophine skeleton carrying an alkyne acting as fluorophore and reactive center, respectively. In order to evaluate the practicality of DIB-ET, a high-performance liquid chromatography with fluorescence detection method was developed for the determination of zidovudine as a model of azide compound. Zidovudine could be reacted with DIB-ET in the presence of copper(II) sulfate and L-ascorbic acid as catalysts, and the formed derivative was detected fluorometrically. The proposed method allows sensitive and selective determination of zidovudine in plasma samples with the detection limit of 0.28ngmL(-1) at a S/N=3. Finally, the proposed method could be applied to determine the zidovudine concentration in rat plasma after administration of zidovudine without interference from biological components.

  17. Spectrofluorimetric method for determination and validation of cefixime in pharmaceutical preparations through derivatization with 2-cyanoacetamide.

    PubMed

    Shah, Jasmin; Jan, M Rasul; Shah, Sultan; Inayatullah

    2011-03-01

    A simple, sensitive and accurate method has been developed for spectrofluorimetric determination of cefixime in pure form and pharmaceutical preparations. The method is based on the reaction of cefixime with 2-cyanoacetamide in the presence of 21% ammonia at 100 °C. The fluorescent reaction product showed maximum fluorescence intensity at λ 378 nm after excitation at λ 330 nm. The factors affecting the derivatization reaction were carefully studied and optimized. The fluorescence intensity versus concentration plot was rectilinear over the range of 0.02 to 4 μg mL(-1) with correlation coefficient of 0.99036. The limit of detection (LOD) and limit of quantification (LOQ) was found to be 2.95 ng mL(-1) and 9.84 ng mL(-1), respectively. The proposed method was validated statistically and through recovery studies. The method was successfully applied for the determination of cefixime in pure and dosage form with percent recoveries from 98.117% to 100.38%. The results obtained from the proposed method have been compared with the official HPLC method and good agreement was found between them.

  18. [Determination of magnesium stearate in pharmaceutical preparations using derivatization with 2-nitrophenylhydrazine and HPLC].

    PubMed

    Arai, Takashi; Hosoi, Yasue

    2005-03-01

    In general, spectrophotometric methods (inductively coupled plasma or atomic absorption spectrophotometry) are used for the assay of magnesium stearate (Mg-St). In this study, a new rapid, selective assay method was developed for Mg-St in pharmaceutical formulations. The method was based on isocratic reverse-phase liquid chromatography using a mobile phase of acetonitrile-water (80:20, v/v) after precolumn derivatization with 2-nitrophenyl hydrazine for sensitive UV detection. Margaric acid was used as an internal standard and the substances were detectable at 230 nm or 400 nm. Using a short (2 cm) HPLC column reduced the analytical time to 5 min. Validation of the newly developed method was performed in accordance with the International Conference on Harmonization guidelines. The linearity range for Mg-St was 0.00-0.04 mg/ml (as the concentration of injected sample solution) and their correlation factor was 0.9998. The determination and detection limits for Mg-St were 6 microg and 2 microg, respectively. The proposed method was successfully applied to the determination of trace amounts of Mg-St in commercially available tablets with a high recovery percentage, good accuracy, and precision.

  19. Immobilization of derivatized dextran nanoparticles on konjac glucomannan/chitosan film as a novel wound dressing.

    PubMed

    Zhang, Hui; Gu, Chun-Hu; Wu, Hong; Fan, Li; Li, Fei; Yang, Fan; Yang, Qian

    2007-01-01

    The aim of this study was to prepare konjac glucomannan (KGM)/chitosan (CS) film containing glycidyl methacrylate derivatized dextran (dex-GMA)/acrylic acid(AAc) nanoparticles loaded with antibacterial agent. In this study, An optimized procedure chosen from three methods was used to prepare Erythromycin (EM)-loaded poly(dex-GMA/AAc) nanoparticles and obtained nanoparticles ranged from 50-200 nm. Film was found to have equilibrium water content (EWC) 99.3% which could prevent exudates on wound bed from accumulating and also have excellent water adsorption 2362.3 +/- 55.2%; the water vapor transmission rate (WVTR) was 2335 +/- 36 gm(-2) day(-1) and evaporative water loss from the film (EWL) was approximately 10% after 1 h and within 6 h it increased to 90%. Drug release of film containing nanoparticles or absent was determined, within 22 h accumulative release was 40.3%, 72.5% respectively. In conclusion, KGM/CS film containing nanoparticles could not only maintain a moist environment over wound bed in moderate to heavily exuding wound but also provide a continuous and sustained release of the antibacterial agent on the wound surface, which could be potential wound dressing.

  20. Simultaneous derivatization and trapping of volatile products from aqueous photolysis of thiamethoxam insecticide.

    PubMed

    Schwartz, B J; Sparrow, F K; Heard, N E; Thede, B M

    2000-10-01

    An aqueous photolysis study was conducted with radiolabeled thiamethoxam, 4H-1,3,5-oxadiazin-2-imine, 3-[(2-chloro-5-thiazolyl)methyl]tetrahydro-5-methyl-N-nitro, to establish the relevance of aqueous photolysis as a transformation process for (14)C-[thiazolyl]-thiamethoxam. (14)C-[thiazolyl]-thiamethoxam was applied to sterile sodium acetate pH 5 buffer solution at a dose rate of approximately 10 ppm. The resulting samples were incubated for up to 30 days at 25 degrees C under irradiated and nonirradiated conditions. The irradiated samples were exposed to a 12-hour-on and 12-hour-off light cycle. Volatile fractions accounted for up to an average of 56.76% of the total dose for the irradiated incubations and <0.08% for the nonirradiated incubations. These fractions were proposed to be a mixture of carbonyl sulfide (COS) and isocyanic acid (CONH). Verification of these components was accomplished by trapping with cyclohexylamine and formation of the thiocarbamate and the isocyanic acid derivatives. A similar method of trapping thiocarbamate metabolites was reported (Chen and Casida, 1978) where filter paper saturated with isobutylamine in methanol was arranged to trap (14)COS and (14)CO(2) under a positive flow of O(2) at 25 degrees C. Mass spectroscopy of the derivatized components confirmed the presence of carbonyl sulfide as the cyclohexylamine thiocarbamate and of isocyanic acid as its cyclohexylamine derivative. Evidence from this study indicates that thiamethoxam degrades significantly under photolytic conditions.

  1. Comparison of MRI properties between derivatized DTPA and DOTA gadolinium-dendrimer conjugates

    PubMed Central

    Nwe, K.; Bernardo, M; Regino, C. A. S.; Williams, M; Brechbiel, M. W.

    2010-01-01

    In this report we directly compare the in vivo and in vitro MRI properties of gadolinium-dendrimer conjugates of derivatized acyclic diethylenetriamine-N,N’,N’,N’’, N’’-pentaacetic acid (1B4M-DTPA) and macrocyclic 1,4,7,10-tetraazacyclododecane-N,N’,N’’,N’’’-tetraacetic acid (C-DOTA). The metal-ligand chelates were pre-formed in alcohol prior to conjugation to the generation 4 PAMAM dendrimer (G4D), and the dendrimer-based agents were purified by Sephadex® G-25 column. The analysis and SE-HPLC data indicated chelate to dendrimer ratios of 30:1 and 28:1 respectively. Molar relaxivity measured at pH 7.4, 22°C, and 3T are comparable (29.5 vs. 26.9 mM−1s−1), and both conjugates are equally viable as MRI contrast agents based on the images obtained. The macrocyclic agent however exhibits a faster rate of clearance in vivo (t1/2 = 16 vs. 29 min.). Our conclusion is that the macrocyclic-based agent is the more suitable agent for in vivo use for these reasons combined with kinetic inertness associated with the Gd(III) DOTA complex stability properties. PMID:20663676

  2. Derivatization patterns among starch chain populations assessed by ion-exchange chromatography: a model system approach.

    PubMed

    Hong, Jung Sun; Huber, Kerry C

    2015-05-20

    Reaction patterns of wheat starch derivatized with a fluorescent probe (model reaction system) were investigated over the course of a reaction period (0.5, 4, 12, or 24h). Debranched derivatives were fractionated into three populations (Unbound, Bound-1, Bound-2) based on charge density via anion-exchange chromatography, with each ion-exchanged fraction further analyzed by size-exclusion chromatography (refractive index [RI] and fluorescence [FL] detection) to assess proportions and patterns of reaction on amylose (AM) and amylopectin (AP) long (LC), medium (MC), and short (SC) branch chains. Approximately 11-12% of the total chains accounted for 63-75% of the FL recovered in the two bound fractions. FL peaks representing AM, AP-LC, AP-MC, AP-SC, and intermediate material (eluted with AM based on molecular size, but reacted more akin to AP-LC) were monitored over the reaction period. Fractionation behaviors varied amongst starch chains, attributable to the impact of the granule structure on molecular reaction patterns.

  3. Amylose and amylopectin branch chain reactivity in a model derivatization system.

    PubMed

    Hong, Jung Sun; Huber, Kerry C

    2015-05-20

    The impact of starch granule structure on amylose (AM) and amylopectin (AP) chain reactivities was investigated over a 24h period in a model reaction system utilizing a fluorescent probe [DTAF, 5-(4,6-dichlorotriazinyl)aminofluorescein] as a reagent. For various reaction time intervals (0.5, 4, 12, or 24h), molecular reactivities of debranched starch derivatives were assessed via size-exclusion chromatography equipped with refractive index and fluorescence detection. For all starch chain fractions, the initial rate of derivatization (0-0.5h) was rapid, but decreased thereafter. Starch chain reactivities followed the general order: AP long chains ≫ AM, AP medium chains>AP short chains, though both AM and AP long chain reactivities were somewhat impacted by the high relative reactivity of starch chains eluting in the AM/AP long chain boundary region. Varied starch chain reactivities were attributed to their relative locale within the granule, corroborating the impact of granule architecture on molecular-level reaction patterns of starch chains.

  4. Utilization of Ionic Liquids in Lignocellulose Biorefineries as Agents for Separation, Derivatization, Fractionation, or Pretreatment.