L-amino acids (L-AAs) play different important roles in the physiology of all living organisms. Their chiral counterparts, D-amino acids (D-AAs) are increasingly being recognized as essential molecules in many biological systems. Secondary amino acids with cyclic structures, such as prolines, exhibit conformational rigidity and thus unique properties in the structural and protein folding. Despite their widespread occurrence, much less attention was paid to their chiral analysis, particularly when the minor, typically D-enantiomer, is present in low amounts in a complex biological matrix. In this paper, a cost-effective, chiral GC-MS method is described for capillary Chirasil-L-Val separation of nine cyclic secondary amino acid enantiomers with four-, five-, and six-membered rings, involving azetidine-2-carboxylic acid, pipecolic acid, nipecotic acid, proline, isomeric cis/trans 3-hydroxy, 4-hydroxyproline, and cis/trans-5-hydroxy-L-pipecolic acid in the excess of its enantiomeric antipode. The sample preparation involves in-situ derivatization with heptafluorobutyl chloroformate, simultaneous liquid-liquid micro-extraction into isooctane followed by amidation of the arising low-polar derivatives with methylamine, an evaporation step, re-dissolution, and final GC-MS analysis. The developed method was used for analyses of human biofluids, biologically active peptides containing chiral proline constituents, and collagen.

• MeSH
• Fluorocarbons chemistry   MeSH
• Formates chemistry   MeSH
• Imino Acids analysis   chemistry   MeSH
• Calibration MeSH
• Humans MeSH
• Methylamines chemistry   MeSH
• Gas Chromatography-Mass Spectrometry methods   standards   MeSH
• Reproducibility of Results MeSH
• Stereoisomerism MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

Gas chromatography (GC) is a commonly used technique in amino acid analysis (AAA). However, one of the requirements of the application of GC for AAA is a need for the polar analytes to be converted into their volatile, thermally stable derivatives. In the last two decades, alkyl chloroformates (RCFs) have become attractive derivatization reagents. The reagents react immediately with most amino acid functional groups in aqueous matrices, and the process can easily be coupled with liquid-liquid extraction of the resulting less polar derivatives into immiscible organic phase. Here we describe a simple protocol for in situ derivatization of amino acids with heptafluorobutyl chloroformate (HFBCF) followed by subsequent chiral as well as nonchiral GC/MS (mass spectrometric) analysis on a respective nonpolar fused silica and an enantioselective Chirasil-Val capillary column.

• MeSH
• Amino Acids blood   chemistry   isolation & purification   MeSH
• Deuterium analysis   chemistry   MeSH
• Liquid-Liquid Extraction instrumentation   methods   MeSH
• Fluorocarbons chemistry   MeSH
• Formates chemistry   MeSH
• Carbon Isotopes analysis   chemistry   MeSH
• Calibration MeSH
• Humans MeSH
• Gas Chromatography-Mass Spectrometry instrumentation   methods   MeSH
• Stereoisomerism MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

A simple analytical workflow is described for gas chromatographic-mass spectrometry (GC-MS)-based metabolomic profiling of protic metabolites, particularly amino-carboxylic species in biological matrices. The sample preparation is carried out directly in aqueous samples and uses simultaneous in situ heptafluorobutyl chloroformate (HFBCF) derivatization and dispersive liquid-liquid microextraction (DLLME), followed by GC-MS analysis in single-ion monitoring (SIM) mode. The protocol involves ten simple pipetting steps and provides quantitative analysis of 132 metabolites by using two internal standards. A comment on each analytical step and explaining notes are provided with particular attention to the GC-MS analysis of 112 physiological metabolites in human urine.

• MeSH
• Urinalysis methods   MeSH
• Biomarkers urine   MeSH
• Fluorocarbons chemistry   MeSH
• Formates chemistry   MeSH
• Humans MeSH
• Metabolomics methods   MeSH
• Liquid Phase Microextraction methods   MeSH
• Gas Chromatography-Mass Spectrometry methods   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

A simple, sensitive and robust method for simultaneous determination of antiepileptic drugs (gabapentin, pregabalin and vigabatrin) in human serum using GC-MS was developed and validated for clinical toxicology purposes. This method employs an emerging class of derivatization agents - alkyl chloroformates allowing the efficient and rapid derivatization of both the amino and carboxylic groups of the tested antiepileptic drugs within seconds. The derivatization protocol was optimized using the Design of Experiment statistical methodology, and the entire sample preparation requires less than 5 min. Linear calibration curves were obtained in the concentration range from 0.5 to 50.0 mg/L, with adequate accuracy (97.9-109.3%) and precision (<12.1%). The method was successfully applied to quantification of selected γ-aminobutyric acid analogs in the serum of patients in both therapeutic and toxic concentration ranges.

• MeSH
• Amines analysis   blood   MeSH
• Anticonvulsants analysis   blood   MeSH
• Computer-Aided Design MeSH
• Formates chemistry   MeSH
• gamma-Aminobutyric Acid analogs & derivatives   analysis   blood   MeSH
• Calibration MeSH
• Cyclohexanecarboxylic Acids analysis   blood   MeSH
• Humans MeSH
• Gas Chromatography-Mass Spectrometry MeSH
• Pregabalin analysis   blood   MeSH
• Vigabatrin analysis   blood   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

A novel 1,1,1,2,2,3,3-heptafluorobutyl chloroformate reagent (HFBCF) was examined for in-situ derivatization of amino-carboxylic metabolites in human urine. The arising reaction products exhibit greatly reduced polarity which facilitates combining the derivatization and liquid-liquid microextraction (LLME) from an aqueous urine into an isooctane phase and immediate gas chromatographic-mas spectrometric analysis (GC-MS). The sample preparation protocol is simple, proceeds without an alcohol excess and provides cleaner extracts than other urinary GC-MS based methods. Moreover, thiol metabolites bound in disulfide bonds can be released by reduction with tris(3-hydroxypropyl)phosphine (THP) prior to the developed derivatization and LLME step. In order to evaluate potential of the novel method for GC-MS metabolomics, reaction products of 153 urinary metabolites with HFBCF, particularly those possessing amino and carboxyl groups (56 amino acids and their conjugates, 84 organic acids, 9 biogenic amines, 4 other polar analytes) and two internal standards were investigated in detail by GC-MS and liquid chromatography-mass spectrometry (LC-MS). One hundred and twenty metabolites (78%) yielded a single product, 25 (16%) and 2 metabolites (2-methylcitrate, citrate) generated two and more derivatives. From the examined set, analytically applicable products of 5 metabolites were not detected; the derivatives of 3 metabolites were only suitable for LC-MS analysis. Electron ionization (EI) of the examined analytes contained characteristic, diagnostic ions enabling to distinguish related and isomeric structures. The new method was validated for 132 metabolites using two internal standards in artificial urine and with special attention to potential disease biomarker candidates. The developed sample preparation protocol was finally evaluated by means of a certified organic acid standard mixture in urine and by GC-MS analysis of 100 morning urines obtained from healthy patients (50 males and 50 females), where 112 physiological metabolites were quantified in a 25 μL sample aliquot. The quantification data for the set were satisfactory, most metabolites were found within the range reported in the reference human metabolome (HMDB) database and literature. The reported results suggest that the described method has been a novel promising tool for targeted GC-MS based metabolomic analysis in urine.

• MeSH
• Amino Acids analysis   chemistry   metabolism   MeSH
• Urinalysis methods   MeSH
• Fluorocarbons chemistry   MeSH
• Formates chemistry   MeSH
• Indicators and Reagents MeSH
• Carboxylic Acids analysis   chemistry   metabolism   MeSH
• Humans MeSH
• Metabolomics methods   MeSH
• Liquid Phase Microextraction MeSH
• Gas Chromatography-Mass Spectrometry * MeSH
• Check Tag
• Humans MeSH
• Male MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

OBJECTIVES: A simple, cost-effective, and fast gas chromatography method with mass spectrometry detection (GC-MS) for simultaneous measurement of ethylene glycol, 1,2-propylene glycol and glycolic acid was developed and validated for clinical toxicology purposes. DESIGN AND METHODS: Successful derivatization of glycolic acid with isobutyl chloroformate was achieved directly in serum/urine while glycols are simultaneously derivatized by phenylboronic acid. The entire sample preparation procedure is completed within 10 min. RESULTS: The assay was proved to be quadratic in the range of 50 to 5000 mgL(-1) with adequate accuracy (96.3-105.8%) and precision (CV ≤ 8.9%). CONCLUSION: The method was successfully applied to quantify the selected compounds in serum of patients from emergency units and the results correlated well with parallel GC-FID measurements (R(2) 0.9933 for ethylene glycol and 0.9943 for glycolic acid).

• MeSH
• Cost-Benefit Analysis * MeSH
• Time Factors MeSH
• Ethylene Glycol blood   urine   MeSH
• Glycolates blood   urine   MeSH
• Humans MeSH
• Emergencies MeSH
• Gas Chromatography-Mass Spectrometry economics   methods   MeSH
• Toxicology MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

A simple, cost effective, and fast gas chromatography method with mass spectrometry detection (GC-MS) for simultaneous measurement of formic acid, glycolic acid, methoxyacetic acid, ethoxyacetic acid and 2-hydroxyethoxyacetic acid in serum and urine was developed and validated. This multi-analyte method is highly suitable for clinical and emergency toxicology laboratory diagnostic, allowing identification and quantification of five most common acidosis inducing organic acids present in cases of alcohol intoxication. Furthermore, when patients are admitted to emergency unit at late stage of toxic alcohol intoxication, the concentration of parent compound may be already low or not detectable. This new method employs a relatively less used class of derivatization agents - alkyl chloroformates, allowing the efficient and rapid derivatization of carboxylic acids within seconds. The entire sample preparation procedure is completed within 5 min. The optimal conditions of derivatization procedure have been found using chemometric approach (design of experiment). The calibration dependence of the method was proved to be quadratic in the range of 25-3000 mg L(-1), with adequate accuracy (97.3-108.0%) and precision (<12.8%). The method was successfully applied for identification and quantification of the selected compounds in serum of patients from emergency units.

• MeSH
• Acetates blood   urine   MeSH
• Acidosis diagnosis   MeSH
• Urinalysis methods   MeSH
• Blood Chemical Analysis methods   MeSH
• Chemistry Techniques, Analytical MeSH
• Formates blood   urine   MeSH
• Glycolates blood   urine   MeSH
• Calibration MeSH
• Humans MeSH
• Limit of Detection MeSH
• Alcoholic Intoxication blood   urine   MeSH
• Gas Chromatography-Mass Spectrometry methods   MeSH
• Reproducibility of Results MeSH
• Toxicology methods   MeSH
• Check Tag
• Humans MeSH
• Male MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

A new method has been described for efficient derivatization of secondary alicyclic hydroxyl groups in steroids by alkyl chloroformates (RCFs). Cholesterol, an essential human sterol and a steroid precursor in eukaryotic cells, was used as a model for treatment with various RCFs in an aqueous and non-aqueous environment. While the cholesterol hydroxyl group did not react completely with any of the tested RCFs reagents in the former case, trifluoroethyl chloroformate (TFECF) or heptafluorobutyl chloroformate (HFBCF) fully converts cholesterol and related metabolites into the corresponding mixed carbonates under anhydrous conditions in seconds. The acylation reaction was combined with liquid-liquid microextraction (LLME) between isooctane and acetonitrile phase. The sample preparation requires just a stepwise addition of 50μl isooctane with 5μl of a pyridine catalyst, 100μl acetonitrile and 100μl isooctane with dissolved 5μl of the fluoroalkyl chloroformate reagent to a dried sample. The protocol developed in this study was successfully tested for GC-MS analysis of 12 important model steroids and four main tocopherols. Each analyte provided a single peak with excellent GC separation properties, well defined EI spectra containing diagnostic fragment ions suitable for their identification and quantitation. The new method was further validated for the determination of six diagnostic non-cholesterol sterols and four main tocopherols in human serum and in amniotic fluid. Satisfactory data were obtained in terms of calibration, quantitation limits (for sterols and tocopherols, 0.05 and 0.15μg/ml, respectively), within-run precision (0.9-19.5%) and between-run precision (0.2-19.0%), accuracy (82-115%) and recovery (90-110%). The validated method was successfully applied to GC-MS analysis of the analytes in woman sera and amniotic fluids and the results are well-comparable with those reported by other authors. The presented work demonstrates for the first time capability of the RCFs to derivatize alicyclic hydroxyls in steroids and tocopherols metabolites for GC-MS with excellent reaction rates, highest reaction yields, minor reagent consumption and easy conjunction with LLME methods.

• MeSH
• Fluorocarbons chemistry   MeSH
• Formates chemistry   MeSH
• Indicators and Reagents MeSH
• Humans MeSH
• Liquid Phase Microextraction MeSH
• Amniotic Fluid chemistry   MeSH
• Gas Chromatography-Mass Spectrometry MeSH
• Serum MeSH
• Sterols analysis   blood   MeSH
• Pregnancy MeSH
• Tocopherols analysis   blood   MeSH
• Check Tag
• Humans MeSH
• Pregnancy MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Validation Study MeSH

A simple, cost effective, and fast gas chromatography method with flame ionization detection (GC-FID) for simultaneous measurement of ethylene glycol, 1,2-propylene glycol and glycolic acid was developed and validated for clinical toxicology purposes. This new method employs a relatively less used class of derivatization agents - alkyl chloroformates, allowing the efficient and rapid derivatization of carboxylic acids within seconds while glycols are simultaneously derivatized by phenylboronic acid. The entire sample preparation procedure is completed within 10 min. To avoid possible interference from naturally occurring endogenous acids and quantitation errors 3-(4-chlorophenyl) propionic acid was chosen as an internal standard. The significant parameters of the derivatization have been found using chemometric procedures and these parameters were optimized using the face-centered central composite design. The calibration dependence of the method was proved to be quadratic in the range of 50-5000 mg mL(-1), with adequate accuracy (92.4-108.7%) and precision (9.4%). The method was successfully applied to quantify the selected compounds in serum of patients from emergency units.

• MeSH
• Chromatography, Gas methods   MeSH
• Ethylene Glycol blood   poisoning   urine   MeSH
• Glycolates blood   poisoning   urine   MeSH
• Humans MeSH
• Flame Ionization methods   MeSH
• Propylene Glycol blood   poisoning   urine   MeSH
• Serum chemistry   MeSH
• Case-Control Studies MeSH
• Toxicology methods   MeSH
• Emergency Medical Services * MeSH
• Check Tag
• Humans MeSH
• Male MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Comparative Study MeSH

OBJECTIVES: The aim of this study was to develop and validate a gas chromatographic method with flame ionization detection (GC-FID) for the measurement of ibuprofen, naproxen and ketoprofen for clinical toxicology purposes. DESIGN AND METHODS: 100μL of plasma was treated with methyl chloroformate and derivatized analytes were extracted with hexane. Optimal conditions of the derivatization procedure have been found using the experimental chemometric design (face-centered central composite design). The selectivity and efficiency of the procedure was confirmed by GC-MS. RESULTS: The assay was linear in the concentration range of 10-400μgmL(-1), with adequate accuracy and precision for GC-FID (98-106.7%, CV≤9.1%, respectively) and for GC-MS (99.3-105.5%, CV≤9.2%, respectively). CONCLUSION: The entire sample preparation procedure is completed within 5 min and the quantitative results are available within 35 min. The method was successfully applied to quantify the selected compounds in serum of patients from emergency units.

• MeSH
• Chromatography, Gas methods   MeSH
• Ibuprofen blood   MeSH
• Ketoprofen blood   MeSH
• Humans MeSH
• Naproxen blood   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Validation Study MeSH