Stable Isotope Dilution Gas Chromatography-mass Research Articles

Improved Quantification of Geosmin and 2-Methylisoborneol in Farmed Fish Using Stable Isotope Dilution Gas Chromatography-Mass Spectrometry.

A requisite to improving the taste and odor attributes of farmed fish is the availability of accurate and practical analytical methods to quantify 2-methylisoborneol (MIB) and geosmin (GSM). Solid-phase microextraction (SPME) enables reliable measurement of nanogram per liter quantities of MIB and GSM in water. In contrast, direct headspace (HS)-SPME of biological matrices with variable proximate compositions can increase bias and uncertainty in off-flavor determinations. Analytical recovery plays a crucial role in the accurate determination of MIB and GSM in fish, and this study investigates strategies to maximize and account for this recovery factor. MIB and GSM values in off-flavor catfish and trout were measured using direct HS-SPME and distillation as sample preparation techniques. Trout samples prepared by distillation yielded 10-fold higher GSM recoveries than those from direct HS-SPME (31% versus 3%). A stable isotope dilution method (SIDM) was implemented by routinely spiking samples with known quantities of deuterium-labeled MIB and GSM, allowing for the correction of sample-to-sample recovery deviations. SIDM-determined GSM values generated recoveries of 106 and 95% for direct HS-SPME and distilled trout, respectively. Aspects of the strategies and techniques presented can be incorporated into existing analytical methods to improve the accuracy and sample throughput. Particularly, routine inclusion of SIDM in the evaluation of MIB and GSM can facilitate identification of reliable practices to control off-flavors in aquaculture.

Asymmetric dimethylation and citrullination of proteinic arginine and homoarginine synthesis in human Helicobacter pylori infection.

The importance of L-arginine (Arg) and relatives, including L-homoarginine (hArg) and asymmetric dimethylarginine (ADMA), in humans infected with Helicobacter pylori (Hp) is little understood. ADMA is produced by asymmetric dimethylation of the guanidine group of Arg residues in certain proteins and is released by proteolysis. High concentrations of circulating free ADMA are considered a risk factor for morbidity and mortality in adults. This risk is considered to arise from the inhibition of the synthesis of nitric oxide (NO), which is a potent vasodilator and inhibitor of platelet aggregation. In the present study, we quantified by stable isotope dilution gas chromatography-mass spectrometry (GC-MS) the concentration of free (f) and total (t) ADMA, Arg, hArg, lysine (Lys) and the sum of citrulline (Cit) and ornithine (Orn) (6M HCl, 20h, 110°C) in serum samples of apparently healthy elderly subjects (n = 27; age, 31-105years) who were tested for Hp infection. Nine subjects (5 males, 4 females) were found to be Hp seropositive (Hp+) and 18 subjects (8 males, 9 females) were found to be Hp seronegative (Hp‒). Proteinic (p) concentrations were determined by difference. fADMA (0.493 ± 0.068 vs 0.466 ± 0.081µM, P = 0.382), pADMA (113 ± 73 vs 76 ± 59nM, P = 0.169) and tADMA (0.606 ± 0.126 vs 0.543 ± 0.121µM, P = 0.280) serum concentrations were found not to differ between the Hp+ and Hp- subjects. Serum concentrations of fArg (162 ± 30 vs 177 ± 36µM, P = 0.471), fhArg (1.600 ± 0.638 vs 1.831 ± 0.742µM, P = 0.554), and fLys (388 ± 170 vs 395 ± 149µM, P = 0.700) also did not differ statistically between Hp+ and Hp- subjects. tArg (12.4 ± 1.49 vs 13.0 ± 1.33mM, P = 0.190), tLys (23.0 ± 2.65 vs. 23.9 ± 2.66mM, P = 0.456) and tCit + Orn (2.53 ± 0.76 vs 2.63 ± 0.85mM, P = 0.817) did not differ between Hp+and Hp‒ subjects as well. phArg concentration was close to the limit of quantitation of the method (Hp+: 30 ± 210nM; Hp-: 42 ± 205nM), suggesting that hArg is virtually absent in serum proteins of the investigated subjects. pCit + Orn did not differ between infected and non-infected subjects. Our study suggests that Hp infection is not associated with elevated asymmetric dimethylation and citrullination of Arg proteins present in the serum or with the hArg synthesis from free Arg in elderly subjects. However, asymmetric Arg dimethylation was found to correlate inversely with Arg citrullination in Hp- (r2 = 0.408, P = 0.004) but not in Hp+ (r2 = 0.065, P = 0.506), with Arg citrullination decreasing and Arg asymmetric dimethylation increasing with subjects' age.

Improved quantification of γ-aminobutyric acid in rice using stable isotope dilution gas chromatography–mass spectrometry

An accurate method for the analysis of γ-aminobutyric acid (GABA) in rice grain was developed using trimethylsilyl (TMS) derivatization and stable isotope dilution gas chromatography-mass spectrometry. When this method was used with GABA‑d6 as an internal standard (IS), the observed GABA concentration was maintained at 100% of the initial concentration with increasing storage time of the vial in the autosampler. In contrast, when using ribitol as an IS and multiple injections from one vial or single injections from different vials, the observed GABA concentration was 85 and 113% of the initial concentration upon increased storage time, respectively. The improved method recoveries at two different spike levels were between 93.3 and 97.8%, with relative standard deviations of less than 3.3%. The GABA content of resveratrol-enriched transgenic rice was compared with that of its non-transgenic counterpart from two field sites, and statistically non-significant differences were observed between the two grains.

Quantification of Arbutin in Plant Extracts by Stable Isotope Dilution Gas Chromatography–Mass Spectrometry

Arbutin is a skin-whitening agent found in plants such as the Ericaceae species. Herein, we report the development and validation of a method based on gas chromatography–mass spectrometry for the analysis of arbutin in plant extracts. Prior to analysis, silylation with trimethylsilyl (TMS) reagent produced 5TMS and 4TMS derivatives, for which the peak area ratios varied significantly prior to sample injection. However, the use of [d4]-arbutin as an internal standard overcame this issue, with little variation in the observed peak area ratios. Method validation confirmed the excellent linearity (r2 = 0.9989), recovery (accuracy 101.4–105.7%), precision (relative standard deviation ≤ 3.43%), limit of detection (0.03 μg mL−1), and limit of quantitation (0.08 μg mL−1) of this novel method. Finally, our method was applied to the detection of arbutin in various plant species. To the best of our knowledge, arbutin was detected and measured in Prismatomeris tetrandra and Phyllanthus reticulatus samples for the first time.

Common food antimicrobials: effects on cellular inflammation and oxidative damage and their estimated occurrence in Singapore.

The prevalence and potential health effects of common food antimicrobials in processed foods and beverages are relatively unknown in Singapore. The occurrence of chemical antimicrobials in processed foods and beverages and their effects on inflammation and oxidative stress in vitro were examined. The occurrence of antimicrobials in 1605 processed food and 359 beverage items were examined by surveying the ingredients on the product labels. Human neutrophils were exposed to physiologically relevant concentrations of common antimicrobials. Established markers of inflammation, l Leukotriene B4 and oxidative stress, F2-isoprostanes were measured using stable-isotope dilution gas chromatography-mass spectrometry. Antimicrobials were added to 23.2% of the processed foods and beverages. Sorbic, benzoic, lactic, propionic and acetic acids accounted for 84.8% of the added antimicrobials in the processed foods and beverages. 92.5% of the bread contained propionic acid. Lactic acid was the most common antimicrobial (44.4%) in cheeses. Sorbic acid was added to 63.2% of the margarines selected. Sauces (31.5%), energy drinks (50.0%), soft drinks (70.7%) and fruit cordials (66.6%) contained added benzoic acid. Benzoic and propionic acids at physiologically relevant concentrations augmented leukotriene B4formation (benzoicacid, EC50 = ~100 μg L-1and propionic acid, >200 μg L-1). Lactic and sorbic acids dose-dependently inhibited the F2-isoprostanes production (IC50 values ~100 μg L-1) and myeloperoxidase activity (IC50values ~100 μg L-1). Our results demonstrate that Singapore consumers are significantly exposed to food antimicrobials, and these molecules, in physiologically relevant concentrations, exert significant and differential effects in vitro.

Quantitative analysis of menthol in human urine using solid phase microextraction and stable isotope dilution gas chromatography-mass spectrometry.

To accurately measure menthol levels in human urine, we developed a method using gas chromatography/electron ionization mass spectrometry with menthol-d4 stable isotope internal standardization. We used solid phase microextraction (SPME) headspace sampling for collection, preconcentration and automation. Conjugated forms of menthol were released using β-glucuronidase/sulfatase to allow for measuring total menthol. Additionally, we processed the specimens without using β-glucuronidase/sulfatase to quantify the levels of unconjugated (free) menthol in urine. This method was developed to verify mentholated cigarette smoking status to study the influence of menthol on smoking behaviour and exposure. This objective was accomplished with this method, which has no carryover or memory from the SPME fiber assembly, a method detection limit of 0.0017μg/mL, a broad linear range of 0.002-0.5μg/mL for free menthol and 0.01-10μg/mL for total menthol, a 7.6% precision and 88.5% accuracy, and an analysis runtime of 17min. We applied this method in analysis of urine specimens collected from cigarette smokers who smoke either mentholated or non-mentholated cigarettes. Among these smokers, the average total urinary menthol levels was three-fold higher (p<0.001) among mentholated cigarette smokers compared with non-mentholated cigarette smokers.

Study on the dietary intake level for indicator polychlorinated biphenyls in China

To obtain representative data on levels of indicator polychlorinated biphenyls (PCBs) in foods consumed by the general population and to estimate the dietary intake of indicator PCBs in China. The food samples were collected during the fifth China Total Diet Study (2009-2013). Based on the geographical location and dietary habits, China was divided into the south area and the north area, and 10 province regions from each area were chosen. In each province region, one urban site and two rural sites were selected to collect food samples. Considering the food consumption level and the PCBs contaminate rule, a total of 160 samples including meat, eggs, fish, milk, cereals, beans, potatoes and vegetables were selected. The concentration of 7 indicator PCBs in food were determined by stable isotope dilution gas chromatography-mass, and combined with food consumption to calculate the dietary intake of indicator PCBs. The concentration of indicator PCBs in 8 categories of food were in the range of 0.8-1 300.1 pg/g. The levels of indicator PCBs were significantly higher in the aquatic products, averaging (307.8±302.4) pg/g, followed by eggs at (76.6±92.1) pg/g and meat at (63.0±54.9) pg/g. The daily dietary intake of indicator PCBs varied from province to province, ranging from 0.13 ng·kg(-1)·d(-1) to 3.58 ng·kg(-1)·d(-1), averaging (0.67±0.77) ng·kg(-1)·d(-1). Fujian had the highest level (3.58 ng·kg(-1)·d(-1)) , followed by Shanghai (1.48 ng·kg(-1)·d(-1)) and Zhejiang (1.09 ng·kg(-1)·d(-1)) . Compared with the minimum risk level (MRL) value (20 ng·kg(-1)·d(-1)) proposed by US Agency for Toxic Substances and Disease Registry, the highest dietary intake level was only 17.9% MRL, the average dietary intake level was 3.4%MRL. Aquatic products was still the major contributor to the dietary intake of indicator PCBs in China, 48% of average dietary intake level (0.32 ng·kg(-1)·d(-1)/0.67 ng·kg(-1)·d(-1)) . The dietary intake of indicator PCBs in China was at a low level, and showing a declining trend.

Evaluation of the metabolic chiral inversion of d-selenomethionine in rats by stable isotope dilution gas chromatography–mass spectrometry

The stereoselective pharmacokinetics of selenomethionine enantiomers in rats has been studied to evaluate the chiral inversion of d-selenomethionine to the l-enantiomer. After bolus intravenous administration of d- or l-selenomethionine to rats, the plasma concentrations of these two enantiomers were determined by stereoselective gas chromatography–mass spectrometry with selected ion monitoring. This method involved derivatization of selenomethionine enantiomers with HCl in methanol to form methyl ester followed by N-acylation with (+)-α-methoxy-α-trifluoromethylphenylacetyl chloride to form the diastereomeric amide, and separation of the diastereomer on GC with an achiral column. Plasma concentrations of administered d- and l-selenomethionine declined with terminal half-lives of 96±17min and 91±6min, respectively. l-Selenomethionine appeared rapidly in plasma after administration of d-selenomethionine, whereas d-selenomethionine was not detected in plasma after administration of l-selenomethionine. The fraction of conversion of d-selenomethionine to l-selenomethionine was estimated to be 61.3±14.5%. The present method evaluates the stereoselective pharmacokinetics of selenomethionine enantiomers, including the estimation of the metabolic chiral inversion.

Rapid analysis of heterocyclic acetals in wine by stable isotope dilution gas chromatography–mass spectrometry

Heterocyclic acetals from acetaldehyde and glycerol have been proposed as markers of age and oxidation in fortified wines. Here the analysis of these acetals (cis- and trans-5-hydroxy-2-methyl-1,3-dioxane and cis- and trans-4-hydroxymethyl-2-methyl-1,3-dioxolane) by liquid–liquid extraction and gas chromatography–mass spectrometry was optimized for rapid monitoring of these compounds in aged and oxidized table wines. The method provides a wide range of linearity and reproducibility below 3.0% RSD, while the detection limits, 2.85–13.5 μg/L, were significantly lower than typical levels measured in wines. Stable isotope dilution improved the accuracy of the method. Oxidation of red wines was shown to correlate with increased levels of the acetals. The accuracy and speed of the new method will allow for quantifying oxidation in large experiments such as wine aging or micro-oxygenation trials, and to assess the potential value of these substances as markers of wine oxidation.

Stable isotope dilution gas chromatography-mass spectrometry for quantification of thymoquinone in black cumin seed oil.

Black cumin seed (Nigella sativa L.) is a widely used spice and herb, where thymoquinone (2-isopropyl-5-methyl-1,4-benzoquinone) is the major bioactive compound. Here, a stable isotope dilution (SID) gas chromatography-mass spectrometry (GC-MS) technique was developed for the quantification of thymoquinone. A doubly deuterated thymoquinone ([(2)H2]-thymoquinone) was synthesized for the first time with more than 93% deuteration degree shown by mass spectrometry and proton nuclear magnetic resonance ((1)H NMR). This compound was used as an internal standard for the quantification of thymoquinone using a SID GC-MS method. The validation experiment showed a recovery rate of 99.1 ± 1.1% relative standard deviation (RSD). Standard addition and external calibration methods have also been used to quantify thymoquinone, which cross-validated the developed stable isotope dilution assay (SIDA). In comparison to external calibration and standard addition methods, the SIDA method is robust and accurate. The concentration of thymoquinone in five marketed black cumin seed oils ranged between 3.34 and 10.8 mg/mL by use of SID GC-MS.

Alkylpyrazine contents of coffee beverages using stable isotope dilution gas chromatography–mass spectrometry

In the presented study the effects of key brewing parameters (temperature, holding time and storage conditions of coffee powder) on 12 alkylpyrazines in coffee beverages were investigated. The results show that the alkylpyrazine profiles of coffee beverages are similar to those of the coffee used. In total, 70–82% of the alkylpyrazines (113.3 ± 3.5 to 132.6 ± 0.6 mg/kg), depending on the brewing method, passed into beverages during the brewing process. At temperatures higher than 50 °C the alkylpyrazine contents of coffee beverages were almost independent of the brewing temperature. During a 30 min holding time at room temperature the alkylpyrazine contents of a freshly brewed cup of coffee decreased by about 10%. Furthermore, storing coffee in closed systems for up to 29 days at +4 °C, −20 °C or room temperature (+22 °C) had minor effects on the alkylpyrazine contents of beverages produced from the coffee. The results show that under customary conditions more than 70% of the alkylpyrazines present in ground coffee are transferred to beverages during the brewing process. Estimated total amounts of alkylpyrazines ingested by the consumer per cup of coffee beverage are in the milligram range, sufficient to have significant potential health effects.

Determination of the Alkylpyrazine Composition of Coffee Using Stable Isotope Dilution–Gas Chromatography–Mass Spectrometry (SIDA-GC-MS)

A stable isotope dilution analysis based on gas chromatography-mass spectrometry analysis (SIDA-GC-MS) was developed for the quantitative analysis of 12 alkylpyrazines found in commercially available coffee samples. These compounds contribute to coffee flavor. The accuracy of this method was tested by analyzing model mixtures of alkylpyrazines. Comparisons of alkylpyrazine-concentrations suggested that water as extraction solvent was superior to dichloromethane. The distribution patterns of alkylpyrazines in different roasted coffees were quite similar. The most abundant alkylpyrazine in each coffee sample was 2-methylpyrazine, followed by 2,6-dimethylpyrazine, 2,5-dimethylpyrazine, 2-ethylpyrazine, 2-ethyl-6-methylpyrazine, 2-ethyl-5-methylpyrazine, and 2,3,5-trimethylpyrazine, respectively. Among the alkylpyrazines tested, 2,3-dimethylpyrazine, 2-ethyl-3-methylpyrazine, 2-ethyl-3,6-dimethylpyrazine, and 2-ethyl-3,5-dimethylpyrazine revealed the lowest concentrations in roasted coffee. By the use of isotope dilution analysis, the total concentrations of alkylpyrazines in commercially available ground coffee ranged between 82.1 and 211.6 mg/kg, respectively. Decaffeinated coffee samples were found to contain lower amounts of alkylpyrazines than regular coffee samples by a factor of 0.3-0.7, which might be a result of the decaffeination procedure.

Simultaneous determination of selenomethionine enantiomers in biological fluids by stable isotope dilution gas chromatography–mass spectrometry

A method for the stereoselective determination of d- and l-enantiomers of selenomethionine in mouse plasma was developed using gas chromatography–mass spectrometry with selected-ion monitoring (GC–MS-SIM). dl-[ 2H 3, 82Se]selenomethionine was used as analytical internal standard to account for losses associated with the extraction, derivatization and chromatography. Selenomethionine enantiomers in mouse plasma were purified by cation-exchange chromatography using BondElut SCX cartridge and derivatized with HCl in methanol to form methyl ester followed by subsequent N-acylation with optically active (+)-α-methoxy-α-trifluoromethylphenylacetyl chloride to form diastereomeric amide. Quantification was performed by SIM of the molecular-related ions of the diastereomers on the chemical ionization mode. The intra- and inter-day precision for d- and l-selenomethionine spiked to mouse plasma gave good reproducibility with relative standard deviation of 3% and 3% for d-selenomethionine and 6% and 3% for l-selenomethionine, respectively. The estimated amounts were in good agreement with the actual amounts spiked, the intra- and inter-day relative error being 5% and 2% for d-selenomethionine and 2% and 1% for l-selenomethionine, respectively. The present method is sensitive enough to determine pharmacokinetics of selenomethionine enantiomers.

Equivalent lipid oxidation profiles in advanced atherosclerotic lesions of carotid endarterectomy plaques obtained from symptomatic type 2 diabetic and nondiabetic subjects

Type 2 diabetes (T2D) increases the risk for cardiovascular disease and is thought to be associated with increased oxidative stress, a contributor to atherogenesis. Surprisingly, however, there is little direct evidence that T2D-associated oxidative stress results in increased lipid oxidation and/or decreased antioxidant capacity in human atherosclerotic lesions. The aim of this study was to measure vascular lipid oxidation and antioxidants in T2D. The arterial content of oxidized lipid and antioxidants in carotid endarterectomy specimens obtained from diabetic and normoglycemic patients was determined using high-performance liquid chromatography (HPLC), stable isotope dilution gas chromatography–mass spectrometry (GC/MS), and gas–liquid chromatography techniques. The concentrations of hydroxyoctadecanoic acid, F 2-isoprostanes, and 7-ketocholesterol, as well as α-tocopherol, ascorbate, and urate were not different in the two patient groups, whether expressed per unit protein or as a ratio per parent compound. Unexpectedly, a significant decrease in the level of arterial lipid hydroperoxide was found in diabetic patients. Our results do not support the notion that advanced atherosclerotic lesions from T2D patients contain more oxidized lipids than corresponding lesions from nondiabetic subjects.

Bromotyrosines in sputum proteins and treatment effects of terbutaline and budesonide in asthma

Inhaled corticosteroids are widely used in the treatment of persistent asthma, usually combined with inhaled beta2-agonists. Previous research suggests that short-acting beta2-agonists (SABAs) may downregulate the anti-inflammatory effects of inhaled corticosteroids, thereby increasing asthma morbidity. To determine whether 3-bromotyrosine and 3,5-dibromotyrosine levels, specific markers of eosinophil activation, reflect treatment effects on airway inflammation of inhaled corticosteroids and SABAs and support previous conclusions. Levels of 3-bromotyrosine and 3,5-dibromotyrosine were measured in sputum supernatants using stable isotope dilution gas chromatography-mass spectrometry in a randomized, placebo-controlled, crossover study of treatment with terbutaline, budesonide, and their combination in patients with persistent asthma. Thirty-four individuals were randomized, and 28 completed the study. Treatment with budesonide lowered median 3-bromotyrosine levels compared with treatment with placebo, terbutaline, and budesonide-terbutaline (0.24 vs 0.64, 0.62, and 0.43 3-bromotyosine/tyrosine [mmol/mol]; P < .05) and lowered median 3,5-dibromotyrosine levels compared with placebo and terbutaline treatments (0.04 vs 0.11 and 0.07 3,5-dibromotyrosine/ tyrosine [mmol/mol], P < .05). Unlike eosinophil numbers, 3-bromotyrosine and 3,5-dibromotyrosine levels did not increase with terbutaline treatment compared with placebo treatment but were significantly raised when terbutaline was added to budesonide treatment. 3-Bromotyrosine levels correlated significantly with eosinophil cationic protein levels in all groups. 3-Bromotyrosine and 3,5-dibromotyrosine levels reflect treatment effects in asthma and support previous findings that SABAs impair the anti-inflammatory effects of inhaled corticosteroids. In addition to eosinophil numbers and eosinophil cationic protein levels, these modified tyrosine residues provide useful information about the inflammatory state of the airways.

Quantification of Cyanamide Contents in Herbaceous Plants

Cyanamide (NH2CN) is found in nature, although it has long been recognized as an industrial product. Distribution of cyanamide in the plant kingdom was investigated using a direct quantitative determination method to detect and measure cyanamide by stable isotope dilution gas chromatography-mass spectrometry (the SID-GC-MS method). The SID-GC-MS method proved to be a robust way to quantify cyanamide contents in the extracts of 101 species of herbaceous plants. The average recovery of cyanamide from all plants tested was 55.6+/-20.3%. Vicia villosa and V. cracca contained cyanamide at 369-498 microg/gFW and 3,460-3,579 microg/gFW respectively, while the other 99 species contained no detectable cyanamide (<1 microg/gFW). This result suggests that distribution of cyanamide in the plant kingdom is limited and uneven.

Direct quantitative determination of cyanamide by stable isotope dilution gas chromatography–mass spectrometry

Cyanamide is a multifunctional agrochemical used, for example, as a pesticide, herbicide, and fertilizer. Recent research has revealed that cyanamide is a natural product biosynthesized in a leguminous plant, hairy vetch ( Vicia villosa). In the present study, gas chromatography–mass spectrometry (GC–MS) equipped with a capillary column for amines was used for direct quantitative determination of cyanamide. Quantitative signals for ( 14N 2)cyanamide, ( 15N 2)cyanamide (internal standard for stable isotope dilution method), and m-(trifluoromethyl)benzonitrile (internal standard for correcting errors in GC–MS analysis) were recorded as peak areas on mass chromatograms at m/ z 42 ( A 42), 44 ( A 44), and 171 ( A IS), respectively. Total cyanamide content, ( 14N 2)cyanamide plus ( 15N 2)cyanamide, was determined as a function of ( A 42 + A 44)/ A IS. Contents of ( 14N 2)cyanamide and ( 15N 2)cyanamide were then calculated by multiplying the total cyanamide content by A 42/( A 42 + A 44) and A 44/( A 42 + A 44), respectively. The limit of detection for the total cyanamide content by the GC–MS analysis was around 1 ng. The molar ratio of ( 14N 2)cyanamide to ( 15N 2)cyanamide in the injected sample was equal to the observed A 42/ A 44 value in the range from 0.1 to 5. It was, therefore, possible to use the stable isotope dilution method to quantify the natural cyanamide content in samples; i.e., the natural cyanamide content was derived by subtracting the A 42/ A 44 ratio of the internal standard from the A 42/ A 44 ratio of sample spiked with internal standard, and then multiplying the resulting difference by the amount of added ( 15N 2)cyanamide (SID-GC–MS method). This method successfully gave a reasonable value for the natural cyanamide content in hairy vetch, concurring with the value obtained by a conventional method in which cyanamide was derivatized to a photometrically active compound 4-cyanimido-1,2-naphthoquinone and analyzed with reversed-phase HPLC (CNQ-HPLC method). The determination range of cyanamide in the SID-GC–MS method was almost the same as that in the CNQ-HPLC method; however, the SID-GC–MS method was much simpler than the CNQ-HPLC method.

Creatine and guanidinoacetate: diagnostic markers for inborn errors in creatine biosynthesis and transport

In this study, measurements of guanidinoacetate (GAA) and creatine (Cr) in urine, plasma, and cerebrospinal fluid (CSF) were performed using stable isotope dilution gas chromatography–mass spectrometry. Both compounds were analyzed in a single analysis. Reference values were established for GAA and Cr. These values were age dependent. No differences with gender were observed. Eight guanidinoacetate methyltransferase (GAMT) deficient patients and eight creatine transporter SLC6A8 deficient patients were investigated. In urine, plasma, and CSF of GAMT deficient patients increased levels of GAA are present. The SLC6A8 deficient patients all show increased creatine/creatinine (Cr/Crn) ratio in urine demonstrating the importance of the Cr/Crn ratio as a pathognomonic marker of the SLC6A8 deficiency.

Improving headspace-solid-phase microextraction of 3-isobutyl-2-methoxypyrazine by experimental design with regard to stable isotope dilution gas chromatography–mass spectrometric analysis of wine

To solve problems of sensitivity, repeatability and multi-step extraction related to 3-isobutyl-2-methoxypyrazine (IBMP) determination in wines, a simple method based on the novel combination of solid-phase microextraction and stable isotope dilution assay is presented. Among the parameters that affect this type of extraction, five of them have been optimised since the other parameters have common values or do not require optimisation (e.g. addition of sodium chloride at saturated concentration) and so were fixed. Vial volume, sample volume/vial volume ratio, pH, adsorption time and temperature have been optimised by means of two experimental designs. After extraction, quantification was performed by stable isotope dilution with gas chromatography-tandem mass spectrometry ([ 2 H 2 ]-IBMP as internal standard). The final procedure allowed quantification far below IBMP’s sensory threshold (1 ng l −1 versus 15 ng l −1) with a 4% standard deviation. This method has been applied to experimental Fer servadou wines. Comparison of IBMP contents confirmed the efficiency of some viticultural and enological techniques on the herbaceous flavour decrease, such as prior fermentation maceration at high temperature (70 °C) and the use of a reflective carpet on viticultural soil.

Simplified Method for Detection and Quantification of 2-Methoxy-3-isobutylpyrazine in Wine

A simplified method was developed to quantify rapidly and accurately 2-methoxy-3-isobutylpyrazine by combining previous techniques of stable isotope dilution gas chromatography-mass spectrometry with headspace solid-phase microextraction.