Gas chromatography coupled to mass spectrometry‐based metabolomic to screen for anabolic practices in cattle: identification of 5α‐androst‐2‐en‐17‐one as new biomarker of 4‐androstenedione misuse

The use of anabolic steroids as growth promoters for meat‐producing animals is banned within the European Union. However, screening for the illegal use of natural steroid hormones still represents a difficult challenge because of the high interindividual and physiological variability of the endogenous concentration levels in animals. In this context, the development of untargeted profiling approaches for identifying new relevant biomarkers of exposure and/or effect has been emerging for a couple of years. The present study deals with an untargeted metabolomics approach on the basis of GC‐MS aiming to reveal potential biomarkers signing a fraudulent administration of 4‐androstenedione (AED), an anabolic androgenic steroid chosen as template. After a sample preparation based on microextraction by packed sorbent, urinary profiles of the free and deglucurono‐conjugates urinary metabolites were acquired by GC‐MS in the full‐scan acquisition mode. Data processing and chemometric procedures highlighted 125 ions, allowing discrimination between samples collected before and after an administration of 4‐AED. After a first evaluation of the signal robustness using additional and independent non‐compliant samples, 17 steroid‐like metabolites were pointed out as relevant candidate biomarkers. All these metabolites were then monitored using a targeted GC‐MS/MS method for an additional assessment of their capacity to be used as biomarkers. Finally, two steroids, namely 5α‐androstane‐3β,17α‐diol and 5α‐androst‐2‐en‐17‐one, were concluded to be compatible with such a definition and which could be finally usable for screening purpose of AED abuse in cattle. Copyright © 2012 John Wiley & Sons, Ltd.     

Analysis of the Volatile Components in the Leaves of Cinna-momum camphora by Static Headspace Gas Chromatography Mass Spectrometry Combined with Accurate Weight Measurement

The volatile components in the leaves of C. camphora were analyzed by static headspace‐gas chromatography/mass spectrometry (HS‐GC‐MS) combined with accurate weight measurement. Accurate weight measurement obtained by Time‐of‐Flight mass spectrometry (TOF‐MS) helped to confirm the identification of volatiles in the analysis. 59 volatile components in the leaves of C. camphora were identified, which mainly included cis‐3‐hexen‐1‐ol (5.6%), 3‐hexen‐1‐ol, acetate (Z) (11.1%), β‐caryophyllene (15.4%), bicyclogermarene (8.4%), trans‐nerolidol (19.5%) and 9‐oxofarnesol (7.7%). The results show that method using HS‐GC‐MS combined with accurate weight measurement achieves reliable identification and has extensive application in the analysis of volatile components present in complex samples.     

Development of single‐drop microextraction and simultaneous derivatization followed by GC‐MS for the determination of aliphatic amines in tobacco

In this work, for the first time, headspace (HS) single‐drop microextraction and simultaneous derivatization followed by GC‐MS was developed to determine the aliphatic amines in tobacco samples. In the HS extraction procedure, the mixture of derivatization reagent and organic solvent was employed as the extraction solvent for HS single‐drop microextraction and in situ derivatization of aliphatic amine in the samples. Fast extraction and simultaneous derivatization of the analytes were performed in a single step, and the obtained derivatives in the microdrop extraction solvent were analyzed by GC‐MS. The optimized experiment conditions were: sample preparation temperature of 80°C and time of 30 min, HS extraction solvent (the mixture of benzyl alcohol and 2,3,4,5,6‐pentafluorobenzaldehyde) volume of 2.0 μL, extraction time of 90 s. With the optimal conditions, the method validations were also studied. The method has good linearity (R² more than 0.99), accepted precision (RSD less than 13%), good recovery (98–104%) and low limit of detection (0.11–0.97 μg/g). Finally, the proposed technique was successfully applied to the analyses of aliphatic amines in tobacco samples of seven different brands. It was further demonstrated that the proposed method offered a simple, low‐cost and reliable approach to determine aliphatic amines in tobacco samples.     

Comparison of GC–MS and MEKC methods for caffeine determination in preworkout supplements

In this study, GC–MS‐ and MEKC‐based methods for determination of caffeine (CAF) in preworkout supplements were developed and validated. The proposed protocols utilized minimal sample preparation (simple dilution and syringe filtration). The developed methods achieved satisfactory validation parameters, i.e. good linearity (R² > 0.9988 and R² > 0.9985 for GC–MS‐ and MEKC‐based method, respectively), satisfactory intra‐ and interaccuracy (within 92.6–100.7% for method utilizing GC–MS and 92.1–110.3% for protocol based on MEKC) and precision (CV < 15.9% and CV < 6.3% for GC–MS‐ and MEKC‐based method, respectively) and recovery (within 100.1–100.8% for method utilizing GC–MS and 101.5–106.2% for protocol based on MEKC). The LOD was 0.03 and 3 μg/mL for method utilizing GC–MS and MEKC, respectively. The CAF concentrations determined by GC–MS‐ and MEKC‐based methods were found to be in the range of 8.53–11.23 and 8.20–11.61 μg/mL, respectively. Taking into consideration information on the labels, the investigated supplements were found to contain from 110.0 to 167.3% of the declared CAF content, which confirmed the literature reports on incompatibility of the declared product compositions with real ones. Nevertheless, the consumption of examined supplements as recommended by producers did not lead to exceeding the CAF safe limit of 400 mg per day. Additionally, the MEKC‐based method allowed for detection and identification of vitamin B3 and B6 in all of the investigated supplement samples, which demonstrated that MEKC‐based protocols may be an appropriate assays for simultaneous determination of CAF and vitamins.     

Comparative analysis of volatile constituents between recipe jingfangsan and its single herbs by GC‐MS combined with alternative moving window factor analysis method

Comparative analysis of volatile constituents between recipe jingfangsan and its single herbs was performed by GC‐MS combined with alternative moving window factor analysis (AMWFA), a new chemometric resolution method. Identification of the compounds was also assisted by comparison of temperature‐programmed retention indices (PTRIs) on the OV‐1 column with authentic samples. In total, 36, 29, and 42 volatile components in essential oil of Herba schizonepetae (HS), Radix saposhnikoviae (RS), and the recipe were respectively determined qualitatively and quantitatively, accounting for 81.80, 82.62 and 85.98% total contents of volatile oil of HS, RS, and the recipe respectively. Analysis by the method of AMWFA indicates that there are 22 common volatile constituents between the recipe and single herbal medicine HS, and 14 common volatile constituents between the recipe and single herb medicine RS. The experimental results also show that the volatile components of the recipe in number are almost addition of that of two single herbal medicines HS and RS, and are mainly from the single herbal medicine HS.     

Separation and fragmentation study of isocoproporphyrin derivatives by UHPLC‐ESI‐exact mass MS/MS and identification of a new isocoproporphyrin sulfonic acid metabolite

Isocoproporphyrin and its derivatives are commonly used as biomarkers of porphyria cutanea tarda, heavy metal toxicity and hexachlorobenzene (HCB) intoxication in humans and animals. However, most are isobaric with other porphyrins and reference materials are unavailable commercially. The structural characterisation of these porphyrins is important but very little data is available. We report here the separation and characterisation of isocoproporphyrin, deethylisocoproporphyrin, hydroxyisocoproporphyrin and ketoisocoproporphyrin, isolated in the faeces of rats fed with a diet containing HCB, by ultra high performance liquid chromatography‐exact mass tandem mass spectrometry (UHPLC‐MS/MS). Furthermore, we report the identification and characterisation of a previously unreported porphyrin metabolite, isocoproporphyrin sulfonic acid isolated in the rat faeces. The measured mass‐to‐charge ratio (m/z) of the precursor ion was m/z 735.2338, corresponding to a molecular formula of C₃₆H₃₉N₄O₁₁S with an error of 0.3 ppm from the calculated m/z 735.2336. The MS/MS data was consistent with an isocoproporphyrin sulfonic acid structure, derived from dehydroisocoproporphyrinogen by sulfonation of the vinyl group. The metabolite was present in a greater abundance than other isocoproporphyrin derivatives and may be a more useful biomarker for HCB intoxication. Copyright © 2014 John Wiley & Sons, Ltd.     

Integrated LC/MS and GC/MS Metabolomics Data for the Evaluation of Protection Function of Fructus Ligustri Lucidi on Mouse Liver

A comprehensive metabolomic strategy, integrating GC/MS and LC/MS data, had been developed to study the protection function of herbal medicine, Fructus Ligustri Lucidi, on mouse liver. Mouse plasma samples were analyzed by GC/MS and LC/MS in conjunction with multiblock multivariate analysis method, multiblock partial least squares discriminant analysis (MBPLS-DA), to supply more important and distinct information of metabolomic biomarkers. Then, the biological pathway analysis was carried out to help further understanding the mechanism of liver protection of Fructus Ligustri Lucidi.     

Investigation of bacterial viability from incubated saliva by application of flow cytometry and hyphenated separation techniques

The aim of the study was determination of bacterial viability in saliva samples and finding a correlation between microbiological and volatile profiles of saliva depending on incubation time. Bacteria colonizing healthy oral cavities were also identified. Twelve healthy adults donated unstimulated saliva samples. Flow cytometry, optical density measurements and colony‐forming unit (CFU) counting method were employed for analyses of native and inoculated saliva after 0, 1, 2, 24, and 48 h of incubation. Volatile profiles were acquired using headspace‐solid phase microextraction‐gas chromatography/mass spectrometry (HS‐SPME‐GC/MS). Oral bacteria were the most viable within 2 h after collection of saliva. Extension of incubation time to 48 h caused considerable decrease in live bacteria counts and sharp increase in dead bacteria counts. The most prevalent strain was Sphingomonas paucimobilis (26.67%). The number of volatiles raised from 5 to 27 with incubation time and most of them were putrefaction products, such as methanethiol, indole and pyrrole. HS‐SPME‐GC/MS method is insufficient for volatile profiling of “fresh” saliva and should be directed rather to investigation of bacterial metabolites.     

Speciation of inorganic and tetramethyltin by headspace solid‐phase microextraction and gas chromatography–mass spectrometry

A headspace solid‐phase microextraction (HS‐SPME) method coupled to GC‐MS was developed in order to determine trace levels of tetramethyltin (TeMT) and inorganic tin (iSn) after ethylation to tetraethyltin (TeET) in various matrices. The derivatization of iSn and the extraction of both TeMT and iSn as TeET were performed in one step. Sodium tetraethylborate (NaBEt₄) was used as derivatization agent and the volatile derivatives were absorbed on a PDMS‐coated fused silica fiber. The conditions for the HS‐SPME procedure were optimized in order to gain in repeatability and sensitivity. Several critical parameters of GC‐MS were also studied. The detection of TeMT and iSn as TeET peaks was performed by the SIM mode. The precision of the proposed method is satisfactory providing RSD values below 10% for both tin species and good linearity up to 10 μg/L. The developed method was successfully applied to the determination of tin species in several samples like canned fish, fish tissues, aquatic plants, canned mineral water and sea water. The proposed HS‐SPME‐GC‐MS method was proved suitable to monitor the concentration levels of toxic tin compounds in environmental and biological samples.     

Overview of Analytical Methodologies for Dioxin Analysis

Abstract

Poly(chlorinated) dibenzo‐p‐dioxins and dibenzofurans (PCDDs/PCDFs) are persistent organic pollutants which are globally distributed in practically all environmental compartments and which exert a broad spectrum of toxic and biochemical effects. Humans are exposed to these compounds mainly through the diet with food of animal origin usually being the predominant source.

Multiple step isolation and clean up procedures are necessary to determine trace levels of these analytes in complex environmental and biological samples. This article reviews some of the recent developments in the extraction procedures, such as SFE, PLE, HS‐SPME, MAE, SCWE, ASE; clean‐up areas and instrumental analysis of dioxins in biological/environmental samples. Due to its specificity and sensitivity gas chromatography coupled with high resolution mass spectrometry (GC–HRMS), high‐resolution gas chromatography high‐resolution mass spectrometry (HRGC‐HRMS), or GC‐MS/GC techniques have been extensively applied to environmental, medicinal, and biological studies.     

Measurement of S-methylcysteine and S-methyl-mercapturic acid in human urine by alkyl-chloroformate extractive derivatization and isotope-dilution gas chromatography-mass spectrometry

S‐methylcysteine (SMC) is a minor amino acid naturally excreted in human urine, a protective agent against oxidative stress and a biotransformation product of the fumigant biocide methyl bromide and of nicotine. A metabolic source of SMC is catabolism of the repair catalytic protein MGMT (EC 2.1.1.37), which specifically removes the methyl group from the modified DNA nucleotide O‐6‐methyl‐guanine to revert the normal GC base pairing. To assess the value of SMC and of S‐methylmercapturic acid (SMMA) as candidate biomarkers of proliferative phenomena, a sensitive analytical method by GC‐MS was applied in a pilot study of healthy subjects to assess their urinary elimination and the intra‐ and inter‐individual variability. Extractive alkylation with butylchloroformate‐n‐butanol‐pyridine (Husek technique) was employed for sample derivatization and isotope dilution GC‐MS with S‐[CD₃]‐SMC and ‐SMMA was applied for specific and sensitive detection. To resolve the target analytes from the main coeluting interferents in the derivatized urine extract a medium‐polarity stationary phase was employed. SMMA was not detected in the morning urine of three healthy fertile‐age women followed for one month above the minimum detectable level of approx. 500 µg/L while SMC concentrations were in the 0.02–0.7 µg/mL range (n = 61) with large inter‐day and inter‐individual variations. In a young healthy male urine samples taken throughout a few days yielded concentrations in the same 90–810 µg/L range (n = 11). These preliminary results points at SMC as a candidate biomarker for the study of methylation turnover in several biochemical processes. Copyright © 2010 John Wiley & Sons, Ltd.     

Determination of human muscle protein fractional synthesis rate: an evaluation of different mass spectrometry techniques and considerations for tracer choice

In the present study, different MS methods for the determination of human muscle protein fractional synthesis rate (FSR) using [ring‐¹³C₆]phenylalanine as a tracer were evaluated. Because the turnover rate of human skeletal muscle is slow, only minute quantities of the stable isotopically labeled amino acid will be incorporated within the few hours of a typical laboratory experiment. GC combustion isotope ratio MS (GC‐C‐IRMS) has thus far been considered the ‘gold’ standard for the precise measurements of these low enrichment levels. However, advances in liquid chromatography‐tandem MS (LC‐MS/MS) and GC‐tandem MS (GC‐MS/MS) have made these techniques an option for human muscle FSR measurements. Human muscle biopsies were freeze dried, cleaned, and hydrolyzed, and the amino acids derivatized using either N‐acetyl‐n‐propyl, phenylisothiocyanate, or N‐methyl‐N‐(tert‐butyldimethylsilyl)trifluoroacetamide (MTBSTFA) for GC‐C‐IRMS, LC‐MS/MS, and GC‐MS/MS analysis, respectively. A second derivative, heptafluorobutyric acid (HFBA), was also used for GC‐MS/MS analysis as an alternative for MTBSTFA. The machine reproducibility or the coefficients of variation for delta tracer‐tracee‐ratio measurements (delta tracer‐tracee‐ratio values around 0.0002) were 2.6%, 4.1%, and 10.9% for GC‐C‐IRMS, LC‐MS/MS, and GC‐MS/MS (MTBSTFA), respectively. FSR determined with LC‐MS/MS compared well with GC‐C‐IRMS and so did the GC‐MS/MS when using the HFBA derivative (linear fit Y = 1.08 ± 0.10, X + 0.0049 ± 0.0061, r = 0.89 ± 0.01, P < 0.0001). In conclusion, (1) IRMS still offers the most precise measurement of human muscle FSR, (2) LC‐MS/MS comes quite close and is a good alternative when tissue quantities are too small for GC‐C‐IRMS, and (3) If GC‐MS/MS is to be used, then the HFBA derivative should be used instead of MTBSTFA, which gave unacceptably high variability. Copyright © 2014 John Wiley & Sons, Ltd.     

E,E-α-Farnesene rich essential oil of Saraca asoca (Roxb.) Wilde flower

Saraca asoca (Roxb.) Wilde (Fabaceae) commonly known as ‘Ashoka’ is a highly valued medicinal plant categorised ‘vulnerable’ by International Union for Conservation of Nature. The hydro-distilled essential oil from the flowers of S. asoca was investigated using gas chromatography equipped with a flame ionisation detector (GC-FID) and gas chromatography coupled with a mass spectrometry (GC/MS). Twenty-eight compounds representing 95.8% of the total oil were identified. The major constituents of the essential oil were E,E-α-farnesene (41.2%), hexadecanoic acid (15.3%), methyl salicylate (9.5%) and Z-lanceol (6.6%). The oil was found to be rich in sesquiterpene hydrocarbon-type constituents.     

QSRR Study of GC Retention Indices of Volatile Compounds Emitted from Mosla chinensis Maxim by Multiple Linear Regression

The volatile compounds emitted from Mosla chinensis Maxim were analyzed by headspace solid‐phase microextraction (HS‐SPME) and headspace liquid‐phase microextraction (HS‐LPME) combined with gas chromatography‐mass spectrometry (GC‐MS). The main volatiles from Mosla chinensis Maxim were studied in this paper. It can be seen that 61 compounds were separated and identified. Forty‐nine volatile compounds were identified by SPME method, mainly including myrcene, α‐terpinene, p‐cymene, (E)‐ocimene, thymol, thymol acetate and (E)‐β‐farnesene. Forty‐five major volatile compounds were identified by LPME method, including α‐thujene, α‐pinene, camphene, butanoic acid, 2‐methylpropyl ester, myrcene, butanoic acid, butyl ester, α‐terpinene, p‐cymene, (E)‐ocimene, butane, 1,1‐dibutoxy‐, thymol, thymol acetate and (E)‐β‐farnesene. After analyzing the volatile compounds, multiple linear regression (MLR) method was used for building the regression model. Then the quantitative structure‐retention relationship (QSRR) model was validated by predictive‐ability test. The prediction results were in good agreement with the experimental values. The results demonstrated that headspace SPME‐GC‐MS and LPME‐GC‐MS are the simple, rapid and easy sample enrichment technique suitable for analysis of volatile compounds. This investigation provided an effective method for predicting the retention indices of new compounds even in the absence of the standard candidates.     

Comparison of different mass spectrometry techniques in the measurement of L‐[ring‐13C6]phenylalanine incorporation into mixed muscle proteins

Precise measurement of low enrichment of stable isotope labeled amino‐acid tracers in tissue samples is a prerequisite in measuring tissue protein synthesis rates. The challenge of this analysis is augmented when small sample size is a critical factor. Muscle samples from human participants following an 8 h intravenous infusion of L‐[ring‐¹³C₆]phenylalanine and a bolus dose of L‐[ring‐¹³C₆]phenylalanine in a mouse were utilized. Liquid chromatography tandem mass spectrometry (LC/MS/MS), gas chromatography (GC) MS/MS and GC/MS were compared to the GC‐combustion‐isotope ratio MS (GC/C/IRMS), to measure mixed muscle protein enrichment of [ring‐¹³C₆]phenylalanine enrichment. The sample isotope enrichment ranged from 0.0091 to 0.1312 molar percent excess. As compared with GC/C/IRMS, LC/MS/MS, GC/MS/MS and GC/MS showed coefficients of determination of R² = 0.9962 and R² = 0.9942, and 0.9217 respectively. However, the precision of measurements (coefficients of variation) for intra‐assay are 13.0%, 1.7%, 6.3% and 13.5% and for inter‐assay are 9.2%, 3.2%, 10.2% and 25% for GC/C/IRMS, LC/MS/MS, GC/MS/MS and GC/MS, respectively. The muscle sample sizes required to obtain these results were 8 µg, 0.8 µg, 3 µg and 3 µg for GC/C/IRMS, LC/MS/MS, GC/MS/MS and GC/MS, respectively. We conclude that LC/MS/MS is optimally suited for precise measurements of L‐[ring‐¹³C₆]phenylalanine tracer enrichment in low abundance and in small quantity samples. Copyright © 2013 John Wiley & Sons, Ltd.     

SPME – A valuable tool for investigation of flower scent

A novel Headspace Solid Phase Microextraction (HS‐SPME) protocol is proposed for the analysis of floral scent. Volatile compounds emitted from the flower are collected on a Carboxen/PDMS fiber for 1 hour, transferred to the GC, and analyzed by GC/MS. The method completely eliminates the use of organic solvents, does not require special instrumentation, and may readily be performed in the field without access to mains electricity and other energy supplies. The method is robust, sensitive, and reduces the sampling stress on the investigated plant. Since enzymatic reactions in living flowers may cause changes in the composition of emitted fragrance, dried rosemary (Rosmarinus officinalis L.) was used as a stable standard for the method development and optimization. In addition, grape wine was also suggested as homogeneous, bio‐compatible, and relatively stable standard of pronounced and typical scent for the same purpose. The optimized method was used for the comparative investigation of the fragrances emitted by two different species – Lathyrus vernus (L.) and Orchis pallens (L.). Several monoterpenes (C10 compounds) were found as the main fragrance components of lathyrus, while sesquiterpenes (C15 compounds) were typical for the orchid.     

In situ ethylation of organolead,organotin and organomercury species by bromomagnesium tetraethylborate prior to GC‐ICP‐MS analysis

An analytical method for simultaneous in situ ethylation, of organolead, organotin and organomercury compounds in aqueous samples was developed using a new derivatisation agent, bromomagnesium tetraethylborate (BrMgEt₄B). The determination of lead, tin and mercury compounds was done by species‐specific isotope dilution, derivatisation and GC–inductively coupled plasma MS (GC‐ICP‐MS) or by GC‐MS. The recovery and accuracy of the derivatisation were evaluated. The effect of pH and the relative quantity of derivatisation agent were studied.     

Solid-phase extraction and purification for the quantification of polycyclic aromatic hydrocarbon metabolites in fish bile

An analytical protocol including solid-phase extraction and purification is described for the individual quantification of polycyclic aromatic hydrocarbon metabolites (hydroxylated PAHs) in liquid biological matrices such as plasma and bile. The method consists in an enzymatic deconjugation followed by a solid-phase extraction on a C₁₈ cartridge and by a cleanup on an NH₂ cartridge. Extracts are then submitted to a derivatization step before gas chromatography/mass spectrometry (GC/MS) analysis. The quantification of PAH metabolites is ensured by adding an internal standard, 1-hydroxypyrene deuterated, at the beginning of the protocol. Recoveries obtained for the entire protocol were for the major part of the compounds between 96 and 70%. However, recoveries were not so satisfying concerning 2-hydroxybiphenyl and especially 3-hydroxybenzo(a)pyrene, with 62 and 36% respectively. Finally, the protocol was applied to different fish bile samples and showed its good applicability to fish bile samples. The NH₂ cleanup step has been proved to be a very selective purification step, necessary to remove most of the bile pigments before GC/MS injection. Different PAH metabolites could be detected in those natural samples and their quantification allowed us to distinguish different levels of fish exposure.