Tert-butyldimethylsilyl Derivatives Research Articles

Gas chromatography-mass spectrometry-based quantitative method using tert-butyldimethylsilyl derivatization for plasma levels of free amino acids and related metabolites in Japanese Black cattle.

The quantification of amino acid and related metabolite levels is important for evaluating amino acid metabolism and function in animals. However, a useful quantitative method is not enough. In this study, we developed and validated tert-butyldimethylsilyl derivatization method using gas chromatography-mass spectrometry to quantify plasma levels of free amino acids and related metabolites in Japanese Black cattle. Of the 51 metabolites examined, 24, including 20 amino acids, one amine, and three keto acids, could be quantified. Compared with the trimethylsilyl derivatization method using gas chromatography-mass spectrometry, which has been used for untargeted metabolomic analysis, the present method had higher analytical reliability. This method is advantageous for assessing branched-chain amino acid (BCAA) metabolism because it enables the quantification of not only BCAA levels (valine, leucine, and isoleucine) but also their bioactive metabolite keto acid levels (2-ketoisovaleric acid, 2-ketoisocaproic acid, and 2-keto-3-methylvaleric acid) in the plasma. In addition, this method can quantify the plasma levels of not only tryptophan but also its bioactive metabolites kynurenine and serotonin. These results suggest that this quantitative method has the potential to further our understanding of amino acid metabolic processes and their functions in Japanese Black cattle.

СИНТЕЗ УГЛЕВОДНЫХ БЛОКОВ ДЛЯ 2'.3'-ДИДЕЗОКСИНУКЛЕОЗИДОВ ИЗ ЛЕВОГЛЮКОЗЕНОНА

Nucleosides containing five-membered deoxy-carbohydrates in the glycosidic part, such as zalcitabine, 2'.3'-dideoxyuridine, stavudine, are widely used in acquired immunodeficiency syndrome (AIDS). A number of 2'.3'-dideoxynucleosides is an effective inhibitor of type-1 immunodeficiency virus (HIV-1) in humans. Levoglucosenone and cyrene (dihydrolevoglucosenone) are unique compounds, optically pure ketones of carbohydrate nature obtained from renewable bio-raw materials and available by pyrolysis of any cellulose-containing materials. They are promising carbohydrates for the production of modified nucleosides; in addition, they are commercially available. Based on levoglucosenone, we synthesized chiral carbohydrate blocks for dideoxy-nucleosides, namely, tertbutyldimethylsilyl derivatives of (S)-5-(hydroxymethyl)-2.5-dihydrofuran-2-ol and (S)-5-(hydroxymethyl)tetrahydrofuran-2-ol. At the first stage of the synthesis, a one-stage modification of the pyranose form of levoglucosenone and cyrene to γ-butanolides was carried out by Bayer-Williger oxidation using H2O2 in the presence of Amberlyst-15 in water. The reduction of unprotected hydroxymethyl-γ-butanolide obtained from cyrene to lactol was studied. The reduction of γ-butanolide by the action of BH3·SMe2, Red-Al, or LiAlH(OtBu)3 in THF at 0° C leads mainly to triol, the product of complete reduction. The desired lactol was obtained using iBu2AlH at -78°C in CH2Cl2 in good yield. Raising the reaction temperature to 0° C led to a decrease in the yield of lactol. It has been established that (S)-5-(hydroxymethyl)-tetrahydrofuran-2-ol exists in both five-membered and six-membered forms. Due to the fact that the target lactols easily undergo a transition from the furanose to the pyranose form, they are not used to produce nucleosides. Therefore, we have studied the approach to the synthesis of stable five- membered lactols. For this purpose, in lactones obtained by the Bayer-Williger reaction, the primary hydroxyl group was blocked using TBSCl. Subsequent reduction of the esters using iBu2AlH gave lactols that existed exclusively in the furanose form. The total yield of the target (S)-5-(hydroxymethyl)-2.5-dihydrofuran-2-ol obtained from levoglucosenone was 39% and (S)-5-(hydroxymethyl)-tetrahydrofuran-2-ol obtained from cyrene was 65%.

Mass spectral studies of silyl derivatives of partially hydrolyzed products of nitrogen mustards: Important markers of nitrogen mustard exposure.

Nitrogen mustards (NMs) are vesicant class of chemical warfare agents. From the viewpoint of the Chemical Weapons Convention partially hydrolyzed products of nitrogen mustards (pHpNMs) are considered as important markers of nitrogen mustard exposure. The detection of pHpNMs from biological or environmental samples is highly useful for obtaining forensic evidence of exposure to NMs. Gas chromatography interfaced with tandem mass spectrometry (GC/MS/MS) is a widely used tool for the identification and sensitive detection of metabolites of NMs in complex matrices. The pHpNMs were derivatized using silylating agents as they are highly polar and non-amenable to GC. The mass spectral studies of these silyl derivatives of pHpNMs were performed using GC/MS/MS in both electron ionization (EI) and chemical ionization (CI) mode. Various approaches have been proposed to assess the fragmentation pathways of the trimethylsilyl (TMS) and tert-butyldimethylsilyl (TBDMS) derivatives of pHpNMs. All the proposed fragmentation pathways were based on the product and/or precursor ion scanning of corresponding ions in both EI and CI mode. In the case of EI, most of the fragmentation pathways involved either α-cleavage or inductive cleavage. This is the first report on the MS study of the silyl derivatives of pHpNMs. The study of the two different derivatives of pHpNMs using both EI- and CI-MS provides a reliable, unambiguous identification of pHpNMs in complex environmental and biomedical matrices (such as plasma and urine) during any verification activities.

Retraction Note to: Analysis of loxoprofen in tablets, patches, and equine urine as tert-butyldimethylsilyl derivative by gas chromatography-mass spectrometry.

The authors have retracted this article [1] because after publication they became aware that the equine urine samples analysed for loxoprofen in this study were in fact equine plasma samples. Therefore the results and conclusions of this article cannot be relied upon. All authors agree to this retraction.

Simultaneous quantification of 18 saturated and unsaturated fatty acids and 7 sterols as their tert-butyldimethylsilyl derivatives in human saliva using gas chromatography-tandem mass spectrometry

The profiling of fatty acids (FAs) or sterols has been applied in clinical studies, but still needs to be improved to enable their simultaneous quantification. Moreover, little progress has been made in determining the levels of FAs and sterols in human saliva in a single run. In this study, gas chromatography-tandem mass spectrometry (GC–MS/MS) using one-step tert-butyldimethylsilyl (TBDMS) derivatization was developed for comprehensive profiling of 18 FAs (eight saturated, five monounsaturated, and five polyunsaturated FAs) and 7 sterols (cholesterol and its precursors). The TBDMS derivatization process was also optimized in terms of reaction solvent, catalyst, temperature, and reaction time. The optimized conditions resulted in better derivatization efficiency with good chromatographic separation through a high-temperature column within 23 min. The present method provided good linearity (r > 0.993), precision (coefficient of variation, 2.7% to 10.4%), and accuracy (91.5% to 103.4%). The overall recovery ranged from 73.8% to 114.3% for the 18 FAs, and from 68.9% to 79.8% for the 7 sterols. The validated method was applied to characterize FAs and sterols in human saliva samples. This is the first report of a GC–MS/MS method for the simultaneous determination of various FAs and sterols from a small volume (100 μL) of saliva. This approach can be used as a primary screening tool to examine the levels of both FAs and sterols in saliva, providing detailed information about their homeostasis for diagnostic and prognostic purposes.

Analysis of loxoprofen in tablets, patches, and equine urine as tert-butyldimethylsilyl derivative by gas chromatography-mass spectrometry.

Loxoprofen is a non-steroidal anti-inflammatory drug of the 2-arylpropionic acid type, which has used to treat musculoskeletal disorders in the horse racing industry. However, it has also used illicitly to mask clinical signs of inflammation and pain in racehorses. Thus, its accurate analysis has become an important issue in horse doping laboratories. In this study, an analytical method of loxoprofen was developed as tert-butyldimethylsilyl (TBDMS) derivative by gas chromatography-mass spectrometry (GC-MS). Characteristic fragment ions of [M-15], [M-57], and [M-139] permitted the accurate and selective detection of loxoprofen. Under optimal conditions, this method showed good linearity (r ≥ 0.999) in the range of 10-500ng/mL, repeatability (% relative standard deviation = 5.6-8.5), and accuracy (% relative error = - 0.3-0.9) with a detection limit of 1.0ng. When applied to the analysis of loxoprofen in tablet and patch products, loxoprofen was positively identified as TBDMS derivative by GC-MS. The present method provided rapid and accurate determination of loxoprofen in patch and tablet products. Levels of loxoprofen were highest in equine urine at 0.5 and 1h after oral administration with single dose (3mg/kg) to three horses, and then rapidly reduced to below the lower limit of quantification at 24h. Therefore, the present method will be useful for the pharmacokinetic study and doping tests for loxoprofen and other similar acidic drugs in horses.

Metabolic Profiling of Aliphatic, hydroxy, and Methyl-Branched Fatty Acids in Human Plasma by Gas Chromatography–Mass Spectrometry

ABSTRACTAn efficient method for the simultaneous profiling analysis of 44 fatty acids including 24 aliphatic, 12 hydroxyl, and eight methyl-branched fatty acids as tert-butyldimethylsilyl (TBDMS) derivatives was developed by gas chromatography–mass spectrometry. In this study, new mass spectral databases of isolauric, 11-methyllauric, isomyristic, isopentadecylic, isopalmitic, isostearic, 17-methylstearic, and 19-methylarachidic acids were constructed as TBDMS derivatives. Under the optimal conditions, linearity in the range of 0.1–10.0 µg/mL (24 aliphatic fatty acids) and 0.01–1.0 µg/mL (eight methyl-branched and 12 hydroxy fatty acids) showed good correlation coefficients (r ≥ 0.997). The repeatability showed relative standard deviation of less than 11.2% and accuracy (percent relative error) varied from −11.0 to 7.8 for the 44 fatty acids studied. The recoveries ranged from 61.7 to 90.1% with good repeatability. When applied to human plasma sample, 18 aliphatic, three 2-hydroxy, three 3-hydroxy, and five methyl-branched fatty acids were positively identified and quantified. Therefore, the present method will be useful for profiling analysis of various fatty acids in biological samples including plasma.

Analysis of the free fatty acid metabolome in the plasma of patients with systemic lupus erythematosus and fever.

Systemic lupus erythematosus (SLE) is a multifactorial autoimmune disease with heterogeneous clinical manifestations mediated by immune dysregulation. We aimed to analyze the metabolomic differences in free fatty acids (FFAs) between patients with SLE and healthy controls (HCs). In this study, the levels of 24 FFAs, as their tert-butyldimethylsilyl derivatives, in the plasma of 41 patients with SLE and 41 HCs, were investigated using gas chromatography with mass spectrometry in selected-ion monitoring mode. The results showed that patients with SLE and HCs had significantly different levels of 13 of the 24 FFAs. The levels of myristic, palmitoleic, oleic, and eicosenoic acids were significantly higher, whereas the levels of caproic, caprylic, linoleic, stearic, arachidonic, eicosanoic, behenic, lignoceric, and hexacosanoic acids were significantly lower in patients with SLE, than in the HCs. In the partial-correlation analysis of the FFA profiles and markers of disease activity of SLE, several metabolic markers correlated with SLE disease activity. Our results provide a comprehensive understanding of the relationship between FFAs and markers of SLE disease activity. Thus, this approach has promising potential for the discovery of metabolic biomarkers of SLE.

Metabolomic study for monitoring of biomarkers in mouse plasma with asthma by gas chromatography-mass spectrometry.

Asthma is a multifaceted chronic disease caused by an alteration of various genetic and environmental factors that is increasing in incidence worldwide. However, the biochemical mechanisms regarding asthma are not completely understood. Thus, we performed of metabolomic study for understanding of the biochemical events by monitoring of altered metabolism and biomarkers in asthma. In mice plasma, 27 amino acids(AAs), 24 fatty acids(FAs) and 17 organic acids(OAs) were determined by ethoxycarbonyl(EOC)/methoxime(MO)/tert-butyldimethylsilyl(TBDMS) derivatives with GC-MS. Their percentage composition normalized to the corresponding mean levels of control group. They then plotted as star symbol patterns for visual monitoring of altered metabolism, which were characteristic and readily distinguishable in control and asthma groups. The Mann-Whitney test revealed 25 metabolites, including eight AAs, nine FAs and eight OAs, which were significantly different (p<0.05), and orthogonal partial least-squares-discriminant analysis revealed a clear separation of the two groups. In classification analysis, palmitic acid and methionine were the main metabolites for discrimination between asthma and the control followed by pipecolic, lactic, α-ketoglutaric, and linoleic acids for high classification accuracy as potential biomarkers. These explain the metabolic disturbance in asthma for AAs and FAs including intermediate OAs related to the energy metabolism in the TCA cycle.

Analytical Method for Pyrethroid Metabolites in Urine of the Non-Occupationally Exposed Population by Gas Chromatography/Mass Spectrometry.

Pyrethroids are widely being used as household insecticides or mothproof repellents. An analytical method is described for determination of urinary metabolites as biomarkers for monitoring exposure to the pyrethroids. In total, 11 urinary metabolites, 3-(2-carboxyprop-1-enyl)-2,2-dimethylcyclopropanecarboxylic acid, 3-(2-chloro-3,3,3-trifluoroprop-1-enyl)-2,2-dimethylcyclopropanecarboxylic acid, 3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropanecarboxylic acid, 2,2-dimethyl-3-(2-methylprop-1-enyl)cyclopropanecarboxylic acid, 4-fluoro-3-phenoxybenzoic acid, 4-methoxymethyl-2,3,5,6-tetrafluorobenzly alcohol, 2-methyl-3-phenylbenzoic acid, 4-methyl-2,3,5,6-tetrafluorobenzyl alcohol, 3-phenoxybenzoic acid, 2,3,5,6-tetrafluorobenzoic acid and 2,2,3,3-tetramethylcyclopropanecarboxylic acid, were enzymatically hydrolyzed and extracted with toluene. After transformation to their tert-butyldimethylsilyl or trimethylsilyl derivatives, they were analyzed by gas chromatography/mass spectrometry in electron impact ionization mode. The calibration curves for the metabolite were linear over the concentration range of 0-30 μg/L in urine. They could be determined accurately and precisely (detection limits: 0.01-0.12 μg/L, quantification limits: 0.04-0.41 μg/L). The urine samples collected could be stored for up to 1 month at -20°C in a freezer. The proposed method was applied to determine urine samples from several health volunteers. The method was considered to be available for monitoring pyrethroid exposure in the general population.

Target analysis of tert-butyldimethylsilyl derivatives of nerve agent hydrolysis products by selectable one-dimensional or two-dimensional gas chromatography–mass spectrometry

A target analysis method for the sensitive and discriminative determination of the nerve agent hydrolysis products alkyl methylphosphonic acids as their tert-butyldimethylsilyl (TBDMS) derivatives was developed using a combination of selectable one- and two-dimensional (1D/2D) GC–MS, and applied to the analysis of samples with significant interfering matrices. After sample drying, the alkylmethylphosphonic acids and methylphosphonic acid (MPA) were converted to TBDMS derivatives by addition of N-methyl-N-(tert-butyldimethylsilyl)trifluoroacetamide with heating, and subjected to 1D/2D GC–MS. The apparatus consisted of an initial low thermal mass DB-5 column and a second DB-17 column together with an electron ionization quadrupole mass spectrometer, offering simple and flexible switching between one- and two-dimensional GC–MS analysis in a single GC–MS system. Using 1D/2D GC–MS, analytes that do not co-elute with matrix components can be separated using 1D GC mode alone. Only those parts of the chromatogram that are negatively affected by the co-elution of matrix components need to be transferred and separated with 2D GC. Quantitation can be performed by a combination of both separations and mass spectrometric detection. The TBDMS derivatives of ethyl-, isopropyl-, isobutyl-, pinacolyl-, and cyclohexyl-MPA (cHMPA) and MPA itself were well separated within 3min and determined in 1D GC–MS mode with detection limits of around 10ng/ml of reaction mixture (except for the cHMPA derivative, whose mass spectrum contained noisy background peaks). In 2D-GC–MS mode, where each 0.04min elution window from the 1D GC was subjected to heart-cut (H/C) and transferred to the second column after back-flushing the first column, the peak for the cHMPA TBDMS derivative was isolated and afforded a clean mass spectrum within 6min. The recoveries of all the derivatives on 2D GC from 1D GC were estimated to be over 66%, and the detection limits were around 10ng/ml of reaction mixture. In the presence of urine extract, the target compounds were not detected as separated peaks in 1D GC–MS mode (except for isobutyl-MPA), and quantification based on extracted ion monitoring could not be achieved. However, 2D GC–MS of the H/C fractions of the target derivatives gave single peaks with well-defined mass spectra, and the recoveries of the derivatives were over 70% except for cHMPA (31% at 1.25μg/ml). Phosphonic acids could be detected at less than 60ng/ml. Sulfuric acid and phosphoric acid also negatively affected the determination of alkyl methylphosphonic acid TBDMS derivatives in 1D GC–MS, and the MPA-TBDMS-derivative peak was completely obscured by the large sulfuric-acid-derivative peak. However, under 1D/2D GC–MS conditions, baseline separation of the MPA derivative and sulfuric acid derivative was achieved, enabling highly sensitive MPA detection at 20ng/ml.

Analytical method for urinary metabolites as biomarkers for monitoring exposure to phthalates by gas chromatography/mass spectrometry.

Phthalates, widely used as plasticizers, have been detected in indoor air, but there have been few reports on methods of analyzing urinary metabolites as biomarkers to monitor exposure to di-n-pentyl phthalate or di-n-hexyl phthalate. Presented here is a cost-effective and sensitive analytical method for the determination of urinary metabolites of phthalates containing these two compounds. Nine urinary phthalate metabolites were enzymatically hydrolyzed and extracted with toluene: monomethyl phthalate, monoethyl phthalate, monoisobutyl phthalate, mono-n-butyl phthalate, mono-n-pentyl phthalate, mono-n-hexyl phthalate, monocyclohexyl phthalate, monobenzyl phthalate and mono(2-ethyl-5-carboxypentyl) phthalate. After transformation to their tert-butyldimethylsilyl derivatives, they were analyzed by gas chromatography/mass spectrometry in the electron impact ionization mode. The calibration curves for the metabolites were linear at urinary concentrations of up to 30 μg/L, showing that they could be determined accurately and precisely (detection limits 0.1-0.4 μg/L, quantification limits 0.3-1.3 μg/L). The urine samples collected could be stored for up to 1 month at -20°C. The proposed analytical method was used to examine urine samples from seven healthy volunteers. This method should be useful for monitoring phthalate exposure in the general population.

Metabolomic Study on Fatty Acids in Placenta of Preeclamptic Pregnancies by Gas Chromatography-Mass Spectrometry

Preeclampsia is a serious pregnancy-associated complication and can critically affect the health of the mother and fetus. However, the pathogenesis of preeclampsia remains unclear. One of the characteristics of preeclampsia is abnormal lipid metabolism. Thus, profiling analysis of 23 fatty acids (FAs) as tert-butyldimethylsilyl derivative was performed in the placenta of preeclamptic term pregnancies and uncomplicated term pregnancies by gas chromatography-mass spectrometry. The compositions of saturated, monounsaturated and n-3 polyunsaturated FAs were significantly reduced, whereas those of oleic acid among monounsaturated FAs and arachidonic acid among n-6 polyunsaturated FAs were significantly increased in preeclampsia group compared to the normal group. The distorted star pattern of the preeclamptic pregnancy group was different from the tricosagonal shape of the normal group. Thus, the present FA profiling analysis combined with the star symbol plotting method will be useful for the biochemical monitoring of placental abnormalities and visual discrimination between preeclamptic and normal pregnancies.

The Identification and Quantification of Suberin Monomers of Root and Tuber Periderm from Potato (Solanum tuberosum) as Fatty Acyl tert-Butyldimethylsilyl Derivatives.

Protective plant lipophilic barriers such as suberin and cutin, with their associated waxes, are complex fatty acyl derived polyesters. Their precise chemical composition is valuable to understand the specific role of each compound to the physiological function of the barrier. To develop a method for the compositional analysis of suberin and associated waxes by gas chromatography (GC) coupled to ion trap-mass spectrometry (IT-MS) using N-(tert-butyldimethylsilyl)-N-methyl-trifluoroacetamide (MTBSTFA) as sylilating reagent, and apply it to compare the suberin of the root and tuber periderm of potato (Solanum tuberosum). Waxes and suberin monomers from root and periderm were extracted subsequently using organic solvents and by methanolysis, and subjected to MTBSTFA derivatisation. GC analyses of periderm extracts were used to optimise the chromatographic method and the compound identification. Quantitative data was obtained using external calibration curves. The method was fully validated and applied for suberin composition analyses of roots and periderm. Wax and suberin compounds were successfully separated and compound identification was based on the specific (M-57) and non-specific ions in mass spectra. The use of calibration curves built with different external standards provided quantitative accurate data and showed that suberin from root contains shorter chained fatty acyl derivatives and a relative predominance of α,ω-alkanedioic acids compared to that of the periderm. We present a method for the analysis of suberin and their associated waxes based on MTBSTFA derivatisation. Moreover, the characteristic root suberin composition may be the adaptive response to its specific regulation of permeability to water and gases. Copyright © 2016 John Wiley & Sons, Ltd.

Labelled microbial culture as a calibration medium for (13) C-isotope measurement of derivatized compounds: application to tert-butyldimethylsilyl amino acids.

Compound-specific stable carbon isotope analysis by gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) is widely used in studies of environmental or biological functioning. In the case of derivatized molecules, a calibration might be required due to added non-analyte carbon and in some cases non-stoichiometric recovery by the mass spectrometer. Two biological materials of known isotopic composition were produced by microbial cell cultures on either (13) C-labelled glucose or non-labelled glucose as sole source of carbon. Subsequent hydrolyzed amino acids were derivatized as tert-butyldimethylsilyl (tBDMSi) derivatives and analyzed by GC/C/IRMS. The (13) C-enrichment measurements were used as a direct calibration to calculate the original (13) C/(12) C ratios of individual amino acids. We tested this calibration on both known and unknown samples. For the main proteinogenic amino acids we could determine the number of non-analyte added carbon atoms and assess the non-stoichiometrical recovery of tBDMSi carbon atoms, due to their incomplete oxidation in the combustion step of GC/C/IRMS. The calibration enabled the determination of the natural abundances (δ(13) C values) of amino acids with an average accuracy of ±1.1 ‰. We illustrate the application of the calibration to determine the (13) C/(12) C ratios of amino acids, and the associated uncertainty, in biological and plant materials. The analysis of a labelled microbial cell culture offers a straightforward, rapid and reliable estimate of non-analyte carbon contribution to stable isotope composition. We recommend this method as a calibration or a control in artificial or natural (13) C-tracing experiments. Copyright © 2016 John Wiley & Sons, Ltd.

Simultaneous Determination of Plasma Lactate, Pyruvate, and Ketone Bodies following tert-Butyldimethylsilyl Derivatization using GC-MS-SIM

Lactate and ketone bodies are considered biological markers for ketosis and several inherited metabolic disorders. In the current study, the specific ratios of lactate and ketone bodies as analytical tools for differential diagnosis of various lactic acidosis were devised. The study included a protein precipitation step following tert-butyldimethylsilyl derivatisation. Total run time was approximately 30 min including sample preparation and GS/MS analysis. The limits of detection were below 0.1 pg/mL over the targeted 4 analytes. The calibration curve was linear over the concentration range of 0.001~250 μg/mL for pyruvate, beta-hydroxybutyrate, and acetoacetate (R² > 0.99). Inter-day accuracy and precision were 87.7~94.8% with RSD of 2.5~5.7% at 2 levels. Absolute recoveries (%) of target analytes were 87.0~98.4%. The method was validated for the quantification of lactate and ketone bodies for differentiation of lactic acidosis.

Expanded metabolite coverage of Saccharomyces cerevisiae extract through improved chloroform/methanol extraction and tert-butyldimethylsilyl derivatization

We present an improved extraction and derivatization protocol for GC–MS analysis of amino/non-amino acids in Saccharomyces cerevisiae. Yeast cells were extracted with chloroform: aqueous-methanol (1:1, v/v) and the resulting non-polar and polar extracts combined and dried for derivatization. Polar and non-polar metabolites were derivatized using tert-butyldimethylsilyl (t-BDMS) dissolved in acetonitrile. Using microwave treatment of the samples, the derivatization process could be completed within 2 h (from >20 h of the conventional method), providing fully derivatized metabolites that contain multiple derivatizable organic functional groups. This results in a single derivative from one metabolite, leading to increased accuracy and precision for identification and quantification of the method. Analysis of combined fractions allowed the method to expand the coverage of detected metabolites from polar metabolites i.e. amino acids, organic acids and non-polar metabolites i.e. fatty alcohols and long-chain fatty acids which are normally non detectable. The recoveries of the extraction method was found at 88 ± 4%, RSD, N = 3 using anthranilic acid as an internal standard. The method promises to be a very useful tool in various aspects of biotechnological applications i.e. development of cell factories, metabolomics profiling, metabolite identification, 13C-labeled flux analysis or semi-quantitative analysis of metabolites in yeast samples.

Analysis of silyl derivatives of methylphosphonic acid esters by gas chromatography-mass spectrometry with atmospheric pressure photoionization

Using trimethylsilyl (TMS) and tert-butyldimethylsilyl (TBDMS) derivatives of methylphosphonic acid and methylphosphonic acid esters, a comparative study of two ionization methods, namely, atmospheric pressure photoionization (APPhI) and photochemical ionization (APCPhI), in gas chromatography/mass spectrometry has been performed. It has been shown that, in the case of APPhI, the mass spectra of TMS and TBDMS derivatives contained only peaks corresponding to [M + 73]+ and [M + 115]+ (or [M + Si(Me)3]+ and [M + Si(tBu)(Me)2]+ ions, respectively, where M is the molecular mass of a respective TMS or TBDMS derivative of methylphosphonic ester). APCPhI mass spectra recorded in the presence of acetone as a reagent substance consisted of only peaks of [M + H]+ and [M + MA + H]+ ions (MA is acetone molecule). The corresponding detection limits for silylated derivatives in APPhI and APCPhI were 30–125 pg and 1.1–23 pg, respectively (depending on the analyte).

Preliminary study on application of urine amino acids profiling for monitoring of renal tubular injury using GLC-MS.

The early diagnosis of the nephrotoxic effect of xenobiotics and drugs is still an unsolved problem. Recent studies suggest a correlation between the nephrotoxic activity of xenobiotics and increased concentration of amino acids in urine. The presented study was focused on the application of GLC-MS method for amino acids profiling in human urine as a noninvasive method for monitoring of kidney condition and tubular injury level. The analytic method is based on the conversion of the amino acids present in the sample to tert-butyldimethylsilyl (TBDMS) derivatives and their analysis by gas-liquid chromatography-mass spectrometry (GLC-MS). The procedure of urine sample preparation for chromatographic analysis was optimized. The presence of 12 amino acids in most of the tested healthy human urine samples was detected. The significant differences in the levels of particular amino acids between patients with tubular injury and healthy controls were found, especially for lysine, valine, serine, alanine and leucine (on average 30.0, 7.5, 3.6, 2.9 and 0.5 fold respectively). We found that this approach based on GLC-MS detection can be used in nephrotoxicity studies for urine amino acids monitoring in exposure to xenobiotics and drugs.

Simultaneous analysis of seven 2-hydroxy fatty acids as tert-butyldimethylsilyl derivatives in plasma by gas chromatography-mass spectrometry.

An efficient method for the simultaneous analysis of seven 2-hydroxy fatty acids (2-HFAs) as tert-butyldimethylsilyl (TBDMS) derivative was developed by gas chromatography-mass spectrometry in selected ion monitoring mode. New mass spectral data on 2-hydroxycapric, 2-hydroxypalmitic, 2-hydroxystearic and 2-hydroxybehenic acids as di-TBDMS derivatives for hydroxyl and carboxyl groups were built. Under the optimal conditions, the present method showed a good correlation coefficient (r ≥ 0.999) in the range of 0.01-0.5 µg. The precision showed low relative standard deviation of <10%, and the accuracy (percentage relative error) varied from -5.2 to 0.3 for the seven 2-HFAs studied. Recovery rates of all 2-HFAs were ≥ 93.2% with good precision. When applied to normal human plasma, seven 2-HFAs were positively identified. Therefore, the present efficient method will be useful for simultaneous analysis of 2-HFAs in plasma.