Dilution Gas Chromatography-mass Spectrometry Research Articles

Plasma and cerebrospinal fluid nonenzymatic protein damage is sustained in Alzheimer's disease

BackgroundOxidative stress is considered to play an important role in the pathogenesis of Alzheimer's disease (AD). It has been observed that oxidative damage to specific protein targets affecting particular functional networks is one of the mechanisms by which oxidative stress contributes to neuronal failure and consequently loss of cognition and AD progression. Studies are lacking in which oxidative damage is measured at both systemic and central fluid levels and in the same cohort of patients. We aimed to determine the levels of both plasma and cerebrospinal fluid (CSF) nonenzymatic protein damage in patients in the continuum of AD and to evaluate the relation of this damage with clinical progression from mild cognitive impairment (MCI) to AD. MethodsDifferent markers of nonenzymatic post-translational protein modification, mostly from oxidative processes, were detected and quantified in plasma and CSF by isotope dilution gas chromatography‒mass spectrometry using selected ion monitoring (SIM-GC/MS) for 289 subjects: 103 AD, 92 MCI, and 94 control subjects. Characteristics of the study population such as age, sex, Mini-mental state examination, CSF AD biomarkers, and APOE ϵ4, were also considered. ResultsForty-seven (52.8%) MCI patients progressed to AD during follow-up (58 ± 12.5 months). After controlling for age, sex, and APOE ϵ4 allele, plasma and CSF concentrations of protein damage markers were not associated with either diagnosis of AD or MCI. The CSF levels of nonenzymatic protein damage markers were associated with none of the CSF AD biomarkers. In addition, neither in CSF nor in plasma were the levels of protein damage associated with the MCI to AD progression. ConclusionThe lack of association between both CSF and plasma concentrations of nonenzymatic protein damage markers and AD diagnosis and progression suggests that oxidative damage in AD is a pathogenic mechanism specifically expressed at the cell-tissue level, not in extracellular fluids.

Legacy Dichlorodiphenyltrichloroethane (DDT) Pollution in a River Ecosystem: Sediment Contamination and Bioaccumulation in Benthic Invertebrates

Dichlorodiphenyltrichloroethane (DDT) contamination in the Toce River in northern Italy was studied by collecting data from 2016 to 2021 upstream and downstream of a production factory which in the past had discharged technical DDT. Analysis of sediments and of bioaccumulation in different benthic invertebrate taxa (Gammaridae, Diptera, Ephemeroptera Baetidae and Heptageniidae) was carried out to assess the transfer of DDT from sediments to benthic invertebrates and the environmental risk of this legacy pollutant for the river ecosystem. DDT and its metabolites dichlorodiphenyldichloroethylene (DDE) and dichlorodiphenyldichloroethane (DDD), here called DDx, were analyzed by isotope dilution gas chromatography–mass spectrometry (GC-MS/MS). DDx values in sediments in upstream stations (1.14–2.25 ng g−1 1% Organic Carbon) were lower than downstream of the industrial site (5.60–7.60 ng g−1 1% Organic Carbon), often exceeding Sediment Quality Guidelines for total DDx. Peak levels derived from new inputs of parental DDT, as confirmed by fingerprint analysis. Bioaccumulation was higher at downstream sites (up to 5107 ng g−1 lipid weight), confirming the bioavailability of residual DDT as well as active metabolism, with the formation of DDD and DDE. The Biota-Sediment Accumulation Factor evidenced the highest values (over 4.2) for Diptera and Gammaridae, highlighting that invertebrates can transfer contamination from sediments to the trophic chain. Linear regression models were developed to estimate DDx concentrations in benthic invertebrates from DDx concentrations in sediments. However, determination coefficients R2 remained in the range of 0.36–0.51, highlighting the necessity of bioaccumulation analysis to fully estimate environmental risk. The results show that DDT contamination, even if residual, may still represent a risk due to its effective transfer to the trophic chain.

A simple and efficient derivatization strategy combined with switchable solvent liquid-liquid microextraction hydroxychloroquine methyl acetate-d3 -based quadruple isotope dilution gas chromatography mass spectrometry for the determination of hydroxychloroquine sulfate in biological fluids.

A derivatization switchable solvent liquid-liquid microextraction quadruple isotope dilution gas chromatography mass spectrometry (D-SS-LLME-ID4 -GC/MS) method is presented for the determination of hydroxychloroquine sulfate in human biofluids. While mixing type/period and concentration of NaOH were optimized via a univariate optimization approach, a multivariate optimization approach was used to determine optimum values for relatively more important parameters such as volumes of derivatization agent (acetic anhydride), NaOH and switchable solvent. Under the optimum experimental conditions, limit of detection and limit of quantification were calculated as 0.03 and 0.09 mg/kg (mass based), respectively. An isotopically labelled material (hydroxychloroquine methyl acetate-d3 ) was firstly synthesized to be used in ID4 experiments which give highly accurate and precise recovery results. After the application of D-SS-LLME-ID4 , superior percent recovery results were recorded as 99.9 ± 1.6-101.3 ± 1.2 for human serum, 99.9 ± 1.7-99.8 ± 1.8 for urine and 99.6 ± 1.5-101.0 ± 1.1 for saliva samples. The developed D-SS-LLME-ID4 -GC/MS method compensates the complicated matrix effects of human biofluids and provides highly accurate quantification of an analyte with precise results.

Determination of chloride in crude oil using isotope dilution GC–MS: A comparative study

Optimisation and validation of a novel isotope dilution gas chromatography mass spectrometry (GC–MS) method for determination of inorganic chloride (Cl−) in crude oils is presented. The use of stable isotope dilution calibration approach decreases losses and biases and it is considered a higher order measurement approach. Samples were diluted with a 1:1 blend of isopropyl alcohol : toluene with 5% water, mixed with 37Cl− internal standard, water extracted and derivatized with triethyloxonium tetrafluoroborate at room temperature. This reagent converted Cl− into stable ethyl chloride (EtCl) which was detected by headspace GC–MS. A limit of quantitation of 100 µg kg−1 and limit of detection of 30 µg kg−1 were observed in crude oil samples when initial dilution was not required. The performance of this novel method was compared with combustion ion chromatography, ion chromatography with conductivity detection after microwave-induced combustion, and ASTM D3230 method on a set of medium and heavy crude oil samples. Despite the challenges of the measurement, a reasonable level of agreement was observed among methods over the range of 0.38 ± 0.02 to 993 ± 103 mg kg−1 of Cl−. Presented headspace GC–MS method proved to be suitable for analysis of all types of crude oils samples with an excellent reproducibility (RSD < 6%) and high sample throughput (7 min GC–MS run time).

Magnetic C60 nanospheres based solid-phase extraction coupled with isotope dilution gas chromatography–mass spectrometry method for the determination of sixteen polycyclic aromatic hydrocarbons in Chinese herbal medicines

C60-based magnetic nanospheres were synthesized by coating Fe3O4 nanospheres with silica, then modifying with 3-aminopropyltriethoxysilane as a linker and a C60 fullerene stationary phase. The morphologies, magnetic properties, infrared absorption and carbon content of magnetic nanospheres were studied by TEM, VSM, FTIR and carbon and sulfur analyzer. The magnetic nanospheres were employed for the magnetic solid-phase extraction (MSPE) of 16 polycyclic aromatic hydrocarbons (PAHs) in nine Chinese herbal medicines. The analyses were conducted by isotope dilution gas chromatography–mass spectrometry. The main parameters influencing the extraction, including extraction solvent, adsorbent amount, and extraction time were optimized. Method validation showed that the limit of detection (LOD) was 0.02–0.11 µg/kg, and the limit of quantification (LOQ) was 0.07–0.36 µg/kg. The spiked recoveries rates for 16 PAHs in white peony root were 84.7–107.2%. The relative standard deviation (RSD) was 1.7–8.4%. The established method was further used for the determination 16 PAHs in nine Chinese herbal medicines. Total content of 16 PAHs varied from 73.6 µg/kg (fructus lycii) to 2172.6 µg/kg (astragalus root). The results indicate that the pollution of PAHs in Chinese herbal medicines is serious. The established method can effective detect PAHs contamination in Chinese herbal medicines.

Validation by collaborative trial of a method for the determination by GC–MS and LC–MS/MS of boar taint marker compounds in pork tissue

Meat from male pigs may develop an off-flavour, commonly known as boar taint. Castration of male piglets prevents the potential formation of off-flavour.In the suggested method, three marker compounds for boar taint (skatole, androstenone and indole) are quantified in pork fat by isotope dilution gas chromatography mass spectrometry (GC–MS) or by isotope dilution liquid chromatography tandem mass spectrometry (LC–MS/MS).This method was validated by collaborative trial according to ISO 5725-2:1994. The studied concentration ranges included sensorial thresholds. The repeatability relative standard deviation (RSDr) ranges from 3% to 10% and the reproducibility relative standard deviation (RSDR) from 10% to about 30%.The method has proven to be robust and free from matrix interferences.The method performance characteristics are compliant with requirements for official control methods in the area of food contaminants; therefore, the method is regarded as fit for its intended purpose.

Performance of StatSensor Point-of-Care Device for Measuring Creatinine in Patients With Chronic Kidney Disease and Postkidney Transplantation.

Background:The StatSensor is a point-of-care device which measures creatinine in capillary whole blood. Previous studies reported an underestimation of the creatinine measurements at high creatinine concentrations and were performed in the prestandardization era for creatinine.Objective:This accuracy-based study evaluates the use of this device in kidney-transplanted patients and those with chronic kidney disease (CKD).Design:Cross-sectional diagnostic accuracy study.Setting:Nephrology outpatient clinic in an urban tertiary center.Participants:Adults with CKD or a functioning kidney transplant.Measurements:Duplicate StatSensor creatinine measurements were performed on capillary whole blood samples collected by direct fingerstick and SAFE-T-FILL collection device. Results were compared with simultaneous venous blood sampling for serum and plasma creatinine measured by an enzymatic method on the Roche Integra 400 mainframe analyzer with traceability to the ID-GC-MS (isotope dilution gas chromatography mass spectrometry) reference method.Methods:Deming regression, Pearson correlation coefficient, and Bland-Altman analysis were used to assess accuracy and comparability between capillary whole blood measured by StatSensor and plasma creatinine measured by routine analyzer with traceability to the reference method. Estimated glomerular filtration (eGFR) rates were calculated using the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation and concordance with Kidney Disease Improving Global Outcomes (KDIGO) CKD stage classification was evaluated.Results:There were 60 participants (mean age = 61.9 ± 15.0 years, 55% men, 33% transplant, mean plasma creatinine = 137 ± 59 µmol/L). Bland-Altman analysis indicated a positive mean bias of 12.7 µmol/L between StatSensor fingerstick creatinine measurement and plasma creatinine. Comparison of eGFR (CKD-EPI) calculated from the StatSensor fingerstick creatinine versus plasma creatinine showed misclassification across all KDIGO CKD stages. Postanalytical correction of the bias did not improve misclassifications. The use of mean of duplicate StatSensor creatinine results did not improve performance compared with the use of singlet results.Limitations:Single center, limited participant numbers.Conclusions:The results of our study suggest that the limiting characteristics of the StatSensor device are not only bias, but also imprecision. The level of imprecision observed may influence clinical decision-making and limit the usefulness of StatSensor as a CKD screening tool. If choosing to utilize it for either screening for or monitoring CKD, it is essential that clinicians understand the limitations of point-of-care devices and apply this knowledge to test interpretation.

Isotope dilution gas chromatography - mass spectrometry for the development of certified reference material of ethyl carbamate in hydroalcoholic matrix

Ethyl carbamate (EC) is a toxic organic contaminant naturally present in some foods and alcoholic beverages. In order to improve the reliability of EC analysis, certified reference materials (CRM) can be used by the laboratories for quality control and for validation and standardization of analytical methods. This paper presents the development of a CRM of EC in hydroalcoholic matrix. This matrix is comprised by a 43% (v/v) aqueous ethanol solution that simulates distilled alcoholic beverages, which generally have alcoholic degree between 35% and 50%. An isotope dilution gas chromatography–mass spectrometry (ID GC–MS) method was developed, validated and employed to attribute the property value and to carry out the appropriate CRM studies, according to ISO Guide 35. Hydroalcoholic matrix was preferred instead of some distilled beverage because in the natural matrix there are EC precursors, which reacting with ethanol enable EC formation, even along storage under appropriate conditions. The applicability of the developed ID GC–MS method was evaluated by testing 26 samples of whisky, vodka, tequila, pisco, rum and the Brazilian cachaça. No interference was observed showing that it can be employed for EC quality control in distilled beverages. According to the analysis of variance, the CRM was considered homogeneous and stable at (4 ± 2) °C for at least three years. The EC concentration is (149.2 ± 5.7) ng/g.

Preparation and characterization of in-house reference material for four organochlorine pesticides in tea matrix

Production and evaluation of in-house reference material for incurred residues of four organochlorine pesticides, named hexachlorobenzene, dicofol, pp′-DDE, and op′-DDT, in tea sample has been performed. Raw materials (tea samples) were confirmed to contain certain levels of incurred organochlorine pesticide residues by a validated gas chromatography–mass spectrometry method. After the materials had been milled and thoroughly mixed, 400 bottles each having 20 g sample were prepared and their homogeneity (within-bottle and bottle-to-bottle variation) was found to be sufficient. The materials demonstrated satisfying stability of four organochlorine pesticides in both short-term (30 days) and long-term (12 months) stability studies. The reference values were characterized using an accurate isotope dilution gas chromatography–mass spectrometry method. The mass fraction (± expanded uncertainty) of hexachlorobenzene, dicofol, pp′-DDE, and op′-DDT in the reference material was (2.1 ± 0.2) mg kg−1, (513.3 ± 31.3) mg kg−1, (8.3 ± 0.6) mg kg−1, and (14.9 ± 1.0) mg kg−1, respectively. The reference materials have been distributed for use in laboratory quality assurance.

Determination of total dissolved nitrogen in seawater by isotope dilution gas chromatography mass spectrometry following digestion with persulfate and derivatization with aqueous triethyloxonium

In this study, we propose a novel approach for the determination of total dissolved nitrogen (TDN) in seawater combining high-precision isotope dilution GC–MS with persulfate digestion. A 2 mL sample aliquot was digested with an alkaline solution of persulfate to convert nitrogen containing compounds to nitrate. Digested samples were spiked with 15NO3− internal standard and treated with aqueous triethyloxonium to convert the analyte into volatile EtONO2. This derivative was readily separated from the matrix under gaseous form and could be sampled from the headspace before GC–MS analysis. The resulting chromatograms showed a stable flat baseline with EtONO2 as the only eluting peak (retention time 2.75 min on a DB 5.625 column). Such an approach provides specificity and obviates the shortcomings of current detection methods employed to analyze seawater samples after digestion with persulfate. In negative chemical ionization mode, the method reached a detection limit of 0.5 μmol/kg TDN (7 ng/g N) and could be applied to quantify seawater samples with 1–25 μmol/kg TDN. On the upper end of the range, quantitation could be repeated within 1%, whereas on a 6 μmol/kg TDN sample repeatability was 2.3% on eight measurements. The method was employed in two proficiency testing exercises providing results in agreement with consensus values. We investigated the impact of reagent blank and we implemented a blank-matching optimal design to account for such contribution. Finally, we performed a study on the yield of persulfate oxidation for organic and inorganic nitrogen compounds typically present in seawater. Whilst nitrite and ammonium are fully converted to nitrate, more complex organic molecules showed recoveries varying from 70% to 100%.

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.

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.

The metabolism and de-bromination of bromotyrosine in vivo

During inflammation, leukocyte-derived eosinophil peroxidase catalyses the formation of hypobromous acid, which can brominate tyrosine residues in proteins to form bromotyrosine. Since eosinophils are involved in the pathogenesis of allergic reactions, such as asthma, urinary bromotyrosine level has been used for the assessment of children with asthma. However, little is known about the metabolism and disposition of bromotyrosine in vivo. The aim of this study was to identify the major urinary metabolites formed during bromotyrosine metabolism and to develop mass spectrometric methods for their quantitation. Deuterium-labeled bromotyrosine was synthesized by deuterium exchange. [D3]bromotyrosine (500nmole) was injected intraperitoneally into Sprague-Dawley rats and urine was collected for 24h in a metabolic cage. 13C-labeled derivatives of bromotyrosine and its major urinary metabolite were synthesized and used as internal standards for quantitation. Following solid phase extraction, urine samples were derivatized to the pentafluorobenzyl ester, and analyzed using isotope dilution gas chromatography and negative-ion chemical ionization mass spectrometry. A novel brominated metabolite, 3-bromo-4-hydroxyphenylacetic acid (bromo-HPA), was identified as the major brominated metabolite of bromotyrosine. Bromo-HPA only accounted for 0.43±0.04% of infused [D3]bromotyrosine and 0.12±0.02% of infused [D3]bromotyrosine was excreted in the urine unchanged. However, ~1.3% (6.66±1.33nmole) of infused [D3]bromotyrosine was excreted in the urine as the de-brominated metabolite, [D3]4-hydroxyphenylacetic acid, which is also a urinary metabolite of tyrosine in mammals. We also tested whether or not iodotyrosine dehalogenase can catalyse de-bromination of bromotyrosine and showed that iodotyrosine dehalogenase is able to de-brominate free bromotyrosine in vitro. We identified bromo-HPA as the main brominated urinary metabolite of bromotyrosine in rats. However, de-halogenation of bromotyrosine is the major metabolic pathway to eliminate free brominated tyrosine in vivo.

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.

Validation of an isotope dilution gas chromatography–mass spectrometry method for combined analysis of oxysterols and oxyphytosterols in serum samples

We describe the validation of a method for the analysis of oxysterols, i.e. oxycholesterols and oxyphytosterols, in human serum using gas chromatography–mass spectrometry selected ion monitoring (GC–MS-SIM). Concentrations of 7α- and 7β-hydroxy-, and 7oxo-cholesterol, -campesterol, and -sitosterol as well as 4β-hydroxycholesterol and side-chain oxygenated 24S-, 25-, and 27-hydroxycholesterol were determined by isotope dilution methodology.After saponification at room temperature the oxysterols were extracted, separated from their substrates, cholesterol, campesterol, and sitosterol, by solid phase extraction, and subsequently derivatised to their corresponding trimethylsilyl-ethers prior to GC–MS-SIM. In order to prevent artificial autoxidation butylated hydroxytoluene and ethylenediaminetetraacetic acid were added. The validation of the method was performed according to the International Conference on Harmonisation guidance, including limits of detection and quantification, ranges, recovery and precision. Due to improved instrumental settings and work-up procedure, limits of detection and quantification ranged between 8.0–202.0pg/mL and 28.0–674pg/mL, respectively. Recovery data in five calibration points varied between 91.9% and 116.8% and in serum samples between 93.1% and 118.1%. The mean coefficient of variation (CV) for the recovery of all compounds was <10%. Well satisfying CVs for within-day precision (2.1–10.8%) and for between-day precision (2.3–12.1%) were obtained. More than 20 samples could be processed in a single routine day and test series of about 300 samples can be realised without impairment of the validation parameters during a sequence. Comparison of oxysterol and oxyphytosterol content in serum and plasma revealed no difference.A fully validated isotope dilution methodology for the quantification of oxycholesterols and oxyphytosterols from human serum or plasma is presented.

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.

Ultra trace determination of fluorobenzoic acids in reservoir and ground water using isotope dilution gas chromatography mass spectrometry

The accurate ultra-trace analysis of six fluorobenzoic acids (FBAs) via isotope dilution gas chromatography mass spectrometry through their deuterated analogues is described. North Sea reservoir and ground water samples were spiked with six deuterated FBAs (dFBAs), enriched using solid-phase extraction (SPE) and analysed using GC/MS after derivatisation with BF 3· MeOH. All FBAs were enriched and determined simultaneously. SPE allowed a 250-fold enrichment of the acids if 100 mL of sample volume was used. The method enables the determination of FBAs down to the range of 8–37 ng L −1 with recoveries between 66 % and 85 %. It uses low amounts of chemicals and is adaptable to larger and smaller sample volumes.

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.