Tert-butyldimethylsilyl Derivatives Research Articles

Determination of 11 Phenolic Endocrine Disruptors using Gas Chromatography/Mass Spectrometry-Selected Ion Monitoring in Five Selected Wastewater Influents

An efficient method for the simultaneous determination of eleven phenolic endocrine-disrupting chemicals (EDCs) present in wastewater influent samples was described. The 11 phenolic EDCs including alkylphenols, chlorophenols, and bisphenol A were determined by gas chromatography/ mass spectrometry-selected ion monitoring (GC/MS-SIM) following two work-up methods for comparison; isobutoxycarbonyl (isoBOC) derivatization and tert-butyldimethylsilyl (TBDMS) derivatization. The wastewater influent samples containing the 11 EDCs were adjusted to pH 2 with H2SO4 and then cleaned up with n-hexane. Next, they were subjected to solid-phase extraction (SPE) with XAD-4 resin and subsequently converted to isoBOC or TBDMS derivatives for sensitivity analysis with gas chromatography/mass spectrometry-selected ion monitoring (GC/MS- SIM). Following isoBOC derivatization and TBDMS derivatization, the recoveries were 86.6-105.2% and 97.6-142.7%, the limits of quantitation (LOQ) for the 11 phenolic EDCs for SIM was 0.001-0.050 ng/mL and 0.003-0.050 ng/mL, and the SIM responses were linear with the correlation coefficient varying by 0.9717-0.9995 and 0.9842-0.9980, respectively. When these methods were applied to five selected wastewater influent samples, for isoBOC derivatization and TBDMS derivatization the ranges of concentration detected were 0.2-99.6 ng/mL and 0.4-147.4 ng/mL, respectively.

Dispersive liquid–liquid microextraction applied to the simultaneous derivatization and concentration of triclosan and methyltriclosan in water samples

A fast and novel sample preparation procedure for the determination of triclosan (TCS) and methyltriclosan (MTCS) in water samples is presented. Dispersive liquid–liquid microextraction, using a ternary mixture consisting of a disperser, an extractant and N-methyl- N-( tert-butyldimethylsilyl)trifluoroacetamide (MTBSTFA) as derivatization reagent, was used for the simultaneous derivatization, case of TCS, and concentration of both species in different water samples. Analytes were determined by gas chromatography with tandem mass spectrometry (GC–MS/MS). Influence of different factors on the performance of the sample preparation process is thoroughly discussed. Under final working conditions, a mixture of 1 mL of methanol, 40 μL of 1,1,1-trichloroethane and the same volume of MTBSTFA was added to 10 mL of water in a conical bottom glass tube. After centrifugation, the settled phase was injected directly in the chromatographic system. TCS was quantitatively extracted and converted into the corresponding tert-butyldimethylsilyl derivative, whereas for MTCS an extraction yield around 90% was attained. Limits of quantification between 2 and 5 ng L −1 and reproducibility values below 10% were achieved; moreover, the performance of the extraction process was scarcely affected by the type of water sample. Globally, these values are comparable, or even better, to those reported for other approaches applied to the determination of same compounds, with the advantage of a shorter sample preparation step. Analysis of surface and wastewater samples confirmed the ubiquitous presence of TCS in the aquatic environment at levels from 20 to 700 ng L −1.

Gas chromatographic-mass spectrometric analyses of cholesterol and its precursors in rat plasma as tert-butyldimethylsilyl derivatives

Background Cholesterol and its metabolic precursors occurring in metabolic pathways are important biochemical indicators in pathological conditions. Methods A method for the simultaneous determination of cholesterol and its metabolic precursors, such as lanosterol and 7-dehydrocholesterol, in rat plasma is demonstrated. It involves their extraction after saponification, followed by conversion to tert-butyldimethylsilyl (TBDMS) derivatives for analysis by gas chromatography-mass spectrometry (GC-MS) in selected ion monitoring (SIM) mode (GC-SIM-MS). Results The characteristic fragment ions of [M-57], m/z 443, 483, and 441 permitted the accurate and selective detection of cholesterol and its precursors in rat plasma. The whole procedure of TBDMS derivatization, with subsequent GC-SIM-MS analysis, was linear ( r ≥ 0.9994), reproducible (% relative standard deviation = 2.2 to 7.5), and accurate (% relative error = − 5.6 to 7.7), with detection limits of 0.02 to 0.07 ng/ml. Recoveries were measured to be ranged from 89.5 to 95.4%. Conclusion The present method was useful for the quantification of cholesterol and its precursors in rat plasma samples of 1 µl.

Measurement of Hydroxyl-Radical Formation in the Rat Striatum by In Vivo Microdialysis and GC-MS

A GC-MS method was developed for measuring hydroxyl-radical capture products of salicylic acid, a common trapping agent for this reactive oxygen species, in samples obtained by in vivo cerebral microdialysis experiments. The assay employed liquid-liquid extraction followed by derivatization of 2,3- and 2,5-dihydroxybenzoic acid, along with 3,5-dihydroxybenzoic acid added as an internal standard. Due to their simple electron ionization mass spectra featuring [M - 57](+) ions through the loss of tertiary alkyl group from the corresponding molecular ions, tert-butyldimethylsilyl (TBDMS) derivatives afforded straightforward method development based on selected-ion monitoring. In addition, tandem mass spectrometry probing collision-induced dissociation of [M - 57](+) ions obtained from the isomeric tert-butyldimethylsilyl derivatives revealed characteristic differences in the resultant product-ion spectra. Our work has demonstrated the applicability of GC-MS for the assay of microdialysates for 2,3- and 2,5-dihydroxybenzoic acid by confirming that local administration of the excitotoxic glutamate into the rat striatum significantly increased in vivo hydroxyl-radical production in this brain region and that subsequent systemic administration of α-phenyl-tert-butylnitrone reversed glutamate-induced oxidative stress.

Hair analysis of anabolic steroids in connection with doping control—results from horse samples

Doping control of anabolic substances is normally carried out with urine samples taken from athletes and horses. Investigation of alternative specimens, e.g. hair samples, is restricted to special cases, but can also be worthwhile, in addition to urine analysis. Moreover, hair material is preferred in cases of limited availability or complicated collection of urine samples, e.g. from horses. In this work, possible ways of interpretation of analytical results in hair samples are discussed and illustrated by practical experiences. The results demonstrate the applicability of hair analysis to detect anabolic steroids and also to obtain further information about previous abuse. Moreover, the process of incorporation of steroids into hairs is described and the consequences on interpretation are discussed, e.g. on the retrospective estimation of the application date. The chosen examples deal with the detection of the anabolic agent testosterone propionate. Hair samples of an application study, as well as a control sample taken from a racing horse, were referred to. Hair material was investigated by a screening procedure including testosterone, nandrolone and several esters (testosterone propionate, phenylpropionate, decanoate, undecanoate, cypionate; nandrolone decanoate, dodecanoate and phenylpropionate; limits of detection (LODs) between 0.1 and 5.0 pg/mg). Confirmation of testosterone propionate (LOD 0.1 pg/mg) was carried out by an optimised sample preparation. Trimethylsilyl (TMS) and tert-butyl dimethylsilyl derivatives were detected by gas chromatography-high-resolution mass spectrometry (GC-HRMS) and gas chromatography-tandem mass spectrometry (GC-MS/MS).

Simultaneous Determination of C22-C26 Very Long - Chain Fatty Acids Following tert-Butyldimethylsilyl Derivatization by Stable Isotope GC- MS for the Screening of Adrenoleucodystrophy

A rapid analytical method was developed to quantify very long-chain fatty acids (VLCFAs, C22:0, C24:0, C26:0) in human plasma with good sensitivity and specificity using tert-butyldimethylsilyl (TBDMS) derivatization and stable isotope GC-MS selective ion monitoring (GC-MS/SIM). Two-hundred and fifty <TEX>${\mu}L$</TEX> of plasma was fortified with deuterated stable isotope internal standards (d3-C22:0, d3-C24:0, d3-C26:0) and standard mixtures of chloroform and methanol, and then extracted with hexane and acetonitrile. To upper layer of liquid-liquid-extraction, N-(t-Butyldimethylsilyl)-N-methyltrifluoroacetamide was added and then heated to <TEX>$60^{\circ}C$</TEX> for 30 min to produce the TBDMS derivatives. Derivatives of VLCFAs were analyzed by GC-MS/SIM. Calibration curves showed a linear relationship for the target compounds in the concentration range of <TEX>$10^{-4}{\sim}2{\times}10^3\;{\mu}g/mL$</TEX> with the correlation coefficient ranging from 0.996 to 0.999. The limit of quantification for the plasma was <TEX>$10^{-4}{\sim}2{\times}10^{-4}\;{\mu}g/mL$</TEX> (S/N=3). When applied to the plasma specimens of patients with peroxisomal disorder, X-linked adrenoleucodystropy (ALD, Mckusick 202370), the method clearly differentiated normal subjects from ALD patients. The C24:0/C22:0 and C26:0/C22:0 ratios were significantly elevated in the plasma of patients with X-linked ALD compared to normal subjects. The new developed method might be useful for a rapid and sensitive diagnosis of X-linked ALD and other peroxisomal disorders.

Anionic Polymerization of (Meth)acrylates with Trialkylsilyl-protected Lithium N-Benzylamide

The polymerization of methacrylates with lithium N-benzyltrimethylsilylamide (BnTMSNLi) in the presence of aluminum compounds afforded syndiotactic or heterotactic polymethacrylates with narrow MWD quantitatively. The end-group analysis of these polymers revealed that benzylamino end-group was quantitatively introduced at the initiating chain-ends of the polymers. The initiator efficiency of the polymerization was slightly lower than unity (0.8–0.9), suggesting possible side reactions in the initiation process. The crude reaction mixture from methyl methacrylate (MMA) polymerization with BnTMSNLi was found to contain a small amount of N-benzylmethacrylamide, which should be formed through the 1,2-addition (carbonyl addition) of BnTMSNLi to MMA in the initiation step of the polymerization. The in situ formed silyl-substituted methacrylamide was not involved in the further polymerization process, as MALDI-TOF mass analysis of the formed polymer indicated the absence of the respective unit in the polymer chain. The methacrylamide remained intact was transformed to N-benzylmethacrylamide when the polymerization was terminated with acidified methanol. Therefore, 1,4-/1,2-addition selectivity in the initiation step could be estimated by NMR spectroscopic quantification of the benzylamino end-group in the polymer and N-benzylmethacrylamide in the polymerization mixture. A lithium amide with bulky triisopropylsilyl substituent, BnTIPSNLi, completely suppressed the 1,2-addition and thus the methacrylamide formation, while 15% of BnTMSNLi and 2% of BnTBSNLi, tert-butyldimethylsilyl derivative, underwent the 1,2-addition.

Dual Subcellular Localization in the Endoplasmic Reticulum and Peroxisomes and a Vital Role in Protecting against Oxidative Stress of Fatty Aldehyde Dehydrogenase Are Achieved by Alternative Splicing

Fatty aldehyde dehydrogenase (FALDH, ALDH3A2) is thought to be involved in the degradation of phytanic acid, a saturated branched chain fatty acid derived from chlorophyll. However, the identity, subcellular distribution, and physiological roles of FALDH are unclear because several variants produced by alternative splicing are present in varying amounts at different subcellular locations. Subcellular fractionation experiments do not provide a clear-cut conclusion because of the incomplete separation of organelles. We established human cell lines heterologously expressing mouse FALDH from each cDNA without tagging under the control of an inducible promoter and detected the variant FALDH proteins using a mouse FALDH-specific antibody. One variant, FALDH-V, was exclusively detected in peroxisomal membranes. Human FALDH-V with an amino-terminal Myc sequence also localized to peroxisomes. The most dominant form, FALDH-N, and other variants examined, however, were distributed in the endoplasmic reticulum. A gas chromatography-mass spectrometry-based analysis of metabolites in FALDH-expressing cells incubated with phytol or phytanic acid showed that FALDH-V, not FALDH-N, is the key aldehyde dehydrogenase in the degradation pathway and that it protects peroxisomes from oxidative stress. In contrast, both FALDHs had a protective effect against oxidative stress induced by a model aldehyde for lipid peroxidation, dodecanal. These results suggest that FALDH variants are produced by alternative splicing and share an important role in protecting against oxidative stress in an organelle-specific manner.

Patterns of Plasma Fatty Acids in Rat Models with Adenovirus Infection

Adenoviral vectors are among the most promising vectors available for human gene therapy. However, the use of recombinant adenoviral vectors, including replicationcompetent adenovirus (RCA), raises a variety of safety concerns in relation to the development of new therapies based on gene therapy. To examine how organic compounds change in rat plasma following the injection of adenovirus, beta-galactosidase expressing recombinant adenovirus (designated rAdLacZ) or RCA, we investigated the content of fatty acids (FAs), which are important biochemical indicators in pathological conditions. Pattern recognition analysis on the level of FAs in rat plasma is described for the visual discrimination of adenovirus infection groups from normal controls. Plasma FAs from four control rats (normal group), and from four rats with rAdLacZ infection and six rats with RCA infection (the two abnormal groups), were examined by gas chromatography-mass spectrometry in selected ion monitoring modes as their tert-butyldimethylsilyl derivatives. In total, 20 FAs were positively detected and quantified. The results of the Studentos t-test on the normal mean of two abnormal groups, the levels of three FAs (p< 0.05) from rAdLacZ group and eleven FAs (p < 0.05) from RCA group were significantly different. When star symbol plotting was applied to the group mean values of 20 FAs after normalization to the corresponding normal mean values, the resulting eicosagonal star patterns of the two infected groups were distorted into similar shapes, but were distinguishable from each other. Thus, these approaches will be useful for screening and monitoring of diagnostic markers for the effects of infection following the use of adenoviral vectors in gene therapy.

Analytical and On-site Detection Methods for Chemical Warfare Agents

Chemical warfare agents (CWAs) are fast acting and sometimes lethal, even at low levels, and can be classified into nerve gases, blister agents, choking agents, blood agents, vomit agents, tear gases, and incapacitating agents. As countermeasures against CWA terrorism, detection and identification are important. In crisis management, monitoring of CWAs in public places and security checks at territorial borders, big event venues, and executive facilities are performed for protection against terrorism. In consequence management, on-site detection by first responders and laboratory analysis after on-site sampling and transfer are performed for minimization of terrorism damage, leading to personal protection, initial investigation, and emergency lifesaving. In incident management, laboratory analysis is performed to provide evidence at court trials for the prevention of future crimes. Laboratory analysis consists of pretreatment of on-site and casualty samples and instrumental analysis using GC-MS. However, CWAs are easily degraded, and thus are difficult to detect. Instead, it is useful to detect their metabolites and degradation products using tert-butyldimethylsilyl derivatization GC-MS or direct LC-MS. Commercially available chemical detection equipment such as gas detection tubes and ion mobility spectrometers are used for on-site detection. We have evaluated the detection performance of such equipment and found that no equipment fulfills the required perfect performance of CWA detection sensitivity, accuracy, response time, return time, and operation. To overcome the drawbacks, we have adopted the monitoring tape method and counterflow introduction atmospheric pressure chemical ionization mass spectrometry and recommend the combination of commercial detection equipment and these new technologies for simultaneous, rapid detection of all CWAs.

Isoprene-catalysed lithiation: deprotection and functionalisation of imidazole derivatives

The isoprene-catalysed lithiation of different 1-substituted imidazoles ( 1) (such as trityl, allyl, benzyl, vinyl, N, N-dimethylsulfamoyl, para-toluenesulfonyl, tert-butoxycarbonyl, acetyl, trimethylsilyl, tert-butyldimethylsilyl derivatives) leads to the cleavage of the protecting group producing 1 H-imidazole. The use of 1-(diethoxymethyl)imidazole ( 3) in the same lithiation reaction allows the preparation of the corresponding 2-lithio intermediate, which by reacting with different electrophiles leads to 2-functionalised imidazoles 4.

GC and MS Properties of β-Blockers as tert-Butyldimethylsilyl Derivatives and as Ethoxycarbonyl/Trimethylsilyl Derivatives

Fourteen β-blockers in pre-dried free base forms were simultaneously converted into tert-butyldimethylsilyl (TBDMS) derivatives in one-step. As a different approach, β-blockers were directly transformed into N-ethoxycarbonyl (EOC) derivatives in aqueous solution, followed by extraction and trimethylsilyl (TMS) derivatization. The retention index sets measured by DB-5 and DB-17 dual-column GC were characteristic for each β-blocker as TBDMS- and EOC/TMS derivatives. Their characteristic fragmentation patterns obtained by GC–MS facilitated easier peak identification of each β-blocker. The overall EOC/TMS method for assays of 14 β-blockers in aqueous solution was linear with acceptable precision and accuracy.

Measurement of salicylic acid in human serum using stable isotope dilution and gas chromatography–mass spectrometry

A simple, highly selective, and sensitive method using stable isotope dilution and gas chromatography–mass spectrometry has been developed to quantify salicylic acid (SA) at concentrations naturally occurring in biological fluids, such as in the serum of subjects not taking aspirin. After extraction of liquid–liquid with diethyl ether and ethyl acetate and preparation of the tert-butyldimethylsilyl derivative, SA content was detected using deuterated SA as internal standard. The mean recovery of SA from serum was 85 ± 6%. Intra- and interday precision and % relative error were <15% in all cases.With a detection limit of 0.6 ng and a quantification limit of 2 ng, the method is therefore also adequate for population studies because of the small amount of blood necessary to perform the analyses.

Determination of nitrogen mustard hydrolysis products, ethanolamines by gas chromatography–mass spectrometry after tert-butyldimethylsilyl derivatization

A method for determining N-ethyldiethanolamine (EDEA), N-methyldiethanolamine (MDEA) and triethanolamine (TEA), hydrolysis products of nitrogen mustards, in water, urine and blood samples using gas chromatography–mass spectrometry (GC–MS) after derivatization by tert-butyldimethylsilylation (TBDMS) is described. The sample solution was evaporated to dryness, and reacted with N-methyl- N-( tert-butyldimethylsilyl)trifluoroacetamide (MTBSTFA) at 60 °C for 1 h. The TBDMS derivatives were separated on a DB-5 column and detected by electron-ionization MS. The quantitation of EDEA, MDEA and TEA was performed by measuring the respective peak areas on the extracted ion chromatograms of m/ z 216, m/ z 202 and m/ z 346, respectively, using nonadecane (C 19), the peak area of which was measured at m/ z 268, as an internal standard. When the water sample was initially analyzed, considerable loss of EDEA, MDEA and TEA occurred by evaporation. The addition of hydrochloric acid (HCl) to the water sample (final 1 mM), however, permitted quantitative recoveries to be achieved (88%, 88% and 79% for EDEA-(TBDMS) 2, MDEA-(TBDMS) 2 and TEA-(TBDMS) 3, respectively). The limits of detections (LODs, scan mode, S/N = 3) were 2.5, 2.5 and 10 ng/ml for EDEA, MDEA and TEA, respectively. Ethanolamines could be also determined in urine samples (volume 0.1 ml), with reasonable recoveries of 72–100% by the addition of HCl (final 1 mM). For the analysis of serum samples, the sample was precipitated by the addition of perchloric acid (final 3.2%), and the resulting supernatant was neutralized with potassium carbonate, and then acidified by the addition of HCl. The recovery of TBDMS derivatives of ethanolamines was found to rather low (7–31%).

Optimisation of a solid-phase microextraction method for the determination of parabens in water samples at the low ng per litre level

A procedure for the determination of five esters of p-hydroxybenzoic acid (parabens) in water samples is presented. Analytes (methyl, ethyl, propyl, butyl and benzyl paraben) are concentrated on a solid-phase microextraction (SPME) fibre, converted on their tert-butyldimethylsilyl derivatives and selectively determined using gas chromatography in combination with tandem mass spectrometry (GC–MS/MS). Influence of different factors on the efficiency of extraction and on-fibre derivatization steps is described in detail. For all species, the highest enrichment factors were achieved using a polyacrylate (PA) fibre exposed directly to stirred water samples, containing 150 mg/ml of sodium chloride, at room temperature. Performance of the further on-fibre derivatization reaction was also maximum at room temperature, considering a short exposition period of the SPME fibre to vapours of the silylation reagent. Under optimised conditions, the proposed method achieved quantification limits from 0.001 to 0.025 ng/ml and it was free of matrix effects; therefore, external calibration can be used as the quantification technique. From our knowledge, this work describes the first application of SPME and gas chromatography to the determination of parabens in water. The analysis of a limited number of real samples revealed the presence of parabens in raw sewage water at concentrations up to 3 ng/ml.

Optimization of silylation using N-methyl- N-(trimethylsilyl)-trifluoroacetamide, N, O-bis-(trimethylsilyl)-trifluoroacetamide and N-( tert-butyldimethylsilyl)- N-methyltrifluoroacetamide for the determination of the estrogens estrone and 17α-ethinylestradiol by gas chromatography–mass spectrometry

This paper reports an improved silylation procedure for simultaneous determination of the steroid hormones 17α-ethinylestradiol (EE2) and estrone (E1) using gas chromatography–mass spectrometry (GC–MS). This follows a re-assessment of some of the popular silylation procedures using N-methyl- N-trimethylsilyltrifluoroacetamide (MSTFA), N- O-bis-(trimethylsilyl)-trifluoroacetamide (BSTFA) and N-( tert-butyldimethylsilyl)- N-methyltrifluoroacetamide (MTBSTFA), which lead to the formation of trimethylsilyl (TMS) and tert-butyldimethylsilyl (TBS) derivatives. Silylation of EE2 using MSTFA or BSTFA + 1% TMCS in ethyl acetate, acetonitrile and dichloromethane solvents produced multiple peaks corresponding to TMS-E1, and 3-mono-TMS-EE2 and/or 3,17-di-TMS-EE2 in variable proportions depending on the solvent used. When pyridine or dimethyl formamide solvents were used in the silylation of EE2 under the same reaction conditions, only 3,17-di-TMS-EE2 derivative was formed. Derivatization using MTBSTFA reagents using ethyl acetate, acetonitrile, dichloromethane, pyridine and dimethyl formamide resulted in almost 100% conversion of mono-TBS-EE2 to the TBS-E1. Therefore, typical methods used in some previous GC–MS determinations of E1 and EE2 in environmental water and/or sediment samples are subject to speculation. However, we can confirm that any of the TMS reagents can be used with either pyridine or dimethyl formamide under suitable reaction conditions.

Determination of endocrine-disrupting phenols, acidic pharmaceuticals, and personal-care products in sewage by solid-phase extraction and gas chromatography–mass spectrometry

The occurrence, fate, and effects of phenols with endocrine-disrupting properties as well as some pharmaceuticals and personal-care products in the environment have frequently been discussed in recent literature. In many cases, these compounds were determined by using individual methods which can be time-consuming if results for multiple parameters are required. Using a solid-phase extraction procedure with an anion exchanger in this work, we have developed and optimized a multi-residue method for the extraction of 21 phenols and acids in sewage influent and effluent. The phenols and acids were then selectively eluted in separate fractions and were converted into pentafluoropropionyl (PFP) and tert-butyldimethylsilyl (TBDMS) derivatives, respectively, for gas chromatography–mass spectrometric (GC/MS) determination. When applied to the sewage samples under study, the results for nonylphenol, bisphenol A (BPA), triclosan (TCS), 17ß-estradiol (E 2), estrone (E 1), salicylic acid, ibuprofen, naproxen, diclofenac, and a few other acidic drugs were consistent with those determined previously by individual methods. Using the same procedure, we also report, for the first time, the occurrence of 2-phenylphenol and parabens in those sewage samples.

Analysis of tert-butyldimethylsilyl derivatives in heavy gas oil from brazilian naphthenic acids by gas chromatography coupled to mass spectrometry with electron impact ionization

Naphthenic acids, C n H 2 n + Z O 2, are a complex mixture of alkyl-substituted acyclic and cycle-aliphatic carboxylic acids. The content of naphthenic acids and their derivatives in crude oils is very small, which hinders their extraction from matrixes of wide and varied composition. In this work, liquid–liquid extraction, followed by solid phase extraction with an ion exchange resin (Amberlyst A-27) and ultrasound desorption were used to isolate the acid fraction from heavy gas oil of Marlim petroleum (Campos, Rio de Janeiro, Brazil). The analysis was accomplished through gas chromatography coupled to mass spectrometry with electron impact ionization, after derivatization with N-methyl- N-( tert-butyldimethylsilyl)trifluoracetamide (MTBDMSTFA). The results indicate the presence of carboxylic acids belonging to families of alicyclic and naphthenic compounds which contain up to four rings in the molecule.

Simultaneous retention index analysis of urinary amino acids and carboxylic acids for graphic recognition of abnormal state

Simultaneous profiling analysis of urinary amino acids (AAs) and carboxylic acids (CAs) was combined with retention index ( I) analysis for graphic recognition of abnormal metabolic state. The temperature-programmed I values of the AA and CA standards measured as ethoxycarbonyl (EOC)/methoxime (MO)/ tert-butyldimethylsilyl (TBDMS) derivatives were used as the reference I values. Urine samples were subjected to the sequential EOC, MO and TBDMS reactions for the analysis by gas chromatography (GC) and GC–mass spectrometry. The complex GC profiles were then transformed into their respective I patterns in bar graphic forms by plotting the normalized peak area ratios (%) of the identified AAs and CAs against their reference I values as the identification numbers. When the present method was applied to infant urine specimens from normal controls and patients with inherited metabolic diseases such as phenylketonuria, maple syrup urine disease, methylmalonic aciduria or isovaleric aciduria, each I pattern of bar graph more distinctly displayed quantitative abundances of urinary AAs and CAs in qualitative I scale, thus allowing graphic discrimination between normal and abnormal states.

Stable-isotope dilution gas chromatography–mass spectrometric measurement of 3-hydroxyglutaric acid, glutaric acid and related metabolites in body fluids of patients with glutaric aciduria type 1 found in newborn screening

We developed a simple and sensitive stable-isotope dilution method for the quantification of 3-hydroxyglutaric acid (3HGA) and glutaric acid (GA) in body fluids. In our method, tert-butyldimethylsilyl (tBDMS) derivatives of 3HGA and GA were measured with a conventional electron-impact ionization (EI) mode in gas chromatography–mass spectrometry (GC–MS). The control values for 3HGA in nmol/ml were 0.15 ± 0.08 (serum; n = 10) and 0.07 ± 0.03 (CSF; n = 10). In addition, glutarylcarnitine and free carnitine were quantified by electrospray tandem mass spectrometry. Using these methods, we monitored 3HGA, GA, and glutarylcarnitine in the body fluids of three patients with glutaric aciduria type 1 found during newborn screening. None of the patients had experienced neurological strokes, which are possibly caused by the accumulation of 3HGA, at 15–24 months of age under a disease-specific treatment, including carnitine supplementation. Our data showed that 3HGA levels were relatively high in some serum samples with lower glutarylcarnitine and carnitine levels, suggesting that carnitine supplementation may play a role in preventing the accumulation of 3HGA in patients with this disease.