Detection of exogenous intake of natural corticosteroids by gas chromatography/combustion/isotope ratio mass spectrometry: application to misuse in sport

A detailed procedure for the analysis of exogenous hydrocortisone and cortisone in urine by gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) is proposed. As urinary levels of hydrocortisone are rather low for GC/C/IRMS analysis, the focus is on the main corticosteroid metabolites, tetrahydrocortisone (THE) and tetrahydrocortisol (THF). Following different solid phase extraction purifications, THE and THF are oxidized to 5beta-androstanetrione before analysis by GC/C/IRMS. Significant differences in delta(13)C per thousand values of synthetic natural corticosteroids and endogenous human corticosteroids have been observed. Therefore, a positive criterion, to detect exogenous administration of synthetic corticosteroids in anti-doping control, is proposed.     

A multiresidue pesticide monitoring procedure using gas chromatography/mass spectrometry and selected ion monitoring for the determination of pesticides containing nitrogen, sulfur, and/or oxygen in fruits and vegetables

A procedure for the analysis of over 100 pesticides that only contain combinations of carbon, hydrogen, nitrogen, sulfur, and oxygen (i.e., no phosphorous or halogen atoms) has been developed. The procedure employs gas chromatography with a mass selective detector (GC/MSD), electron impact ionization, and selected ion monitoring. This GC/MSD procedure provided lower limits of quantitation and increased confirmational data compared to the traditional element-selective GC procedures that are commonly used for the detection of this "class" of pesticides. These analytical improvements were demonstrated by 26 pesticides that were detected at ng/g levels in a variety of fruit and vegetable matrixes using this procedure; these pesticides were missed by the traditional element-selective GC procedures. Validation of the procedure was performed using acetone extraction with solid-phase extraction cleanup. Twenty representative target pesticides were used to demonstrate repeatability and linearity of the chromatographic response and recovery from fruit and vegetable matrixes.     

Studies about the desorption of volatile halocarbons from activated carbon by application of static headspace gas chromatography

The analysis of volatile halocarbons (VHCs) in air or ground air is often performed after their adsorption and enrichment on activated carbon. The current procedure for their subsequent determination is based on their extraction from the activated carbon with a volatile organic solvent such as n-pentane, followed by gaschromatographic (GC) analysis. In order to avoid extraction steps, the static headspace method in combination with GC analysis using diphenylmethane as a desorption agent has been applied. Satisfactory desorption rates for the chloromethanes, for 1,1,1-trichloroethane, trichloroethene and for tetrachloroethene have been obtained after a sample equilibration of 45 min at 120 degrees C in the presence of diphenylmethane. The results have shown a higher recovering rate especially of the unsaturated VHCs compared to the extraction with n-pentane, whereby a potential loss of analytes by the latter procedure has been avoided.     

Studies about the desorption of volatile halocarbons from activated carbon by application of static headspace gas chromatography

The analysis of volatile halocarbons (VHCs) in air or ground air is often performed after their adsorption and enrichment on activated carbon. The current procedure for their subsequent determination is based on their extraction from the activated carbon with a volatile organic solvent such as n-pentane, followed by gaschromatographic (GC) analysis. In order to avoid extraction steps, the static headspace method in combination with GC analysis using diphenylmethane as a desorption agent has been applied. Satisfactory desorption rates for the chloromethanes, for 1,1,1-trichloroethane, trichloroethene and for tetrachloroethene have been obtained after a sample equilibration of 45 min at 120° C in the presence of diphenylmethane. The results have shown a higher recovering rate especially of the unsaturated VHCs compared to the extraction with n-pentane, whereby a potential loss of analytes by the latter procedure has been avoided.     

Determination of 112 halogenated pesticides using gas chromatography/mass spectrometry with selected ion monitoring

A procedure for the analysis of 112 halogenated pesticides that do not contain phosphorus has been developed. The procedure uses gas chromatography with a mass selective detector (GC-MSD), electron impact ionization, and selected-ion monitoring. This GC-MSD procedure provided lower limits of quantitation and provided increased confirmational data compared to the traditional element-selective GC procedures that are commonly used for the detection of this class of pesticides. These analytical improvements were demonstrated by the 25 pesticides that were detected at < or =50 ng/g levels in a variety of fruit and vegetable matrixes using this procedure that were missed by the traditional element selective GC procedures. Validation of the procedure was performed using 20 representative target pesticides with an acetone extraction and a solid-phase extraction cleanup. These target pesticides were used to demonstrate repeatability and linearity of the chromatographic response and recovery from fruit and vegetable matrixes.     

Optimisation of derivatisation procedures for the determination of delta13C values of amino acids by gas chromatography/combustion/isotope ratio mass spectrometry

Compound-specific stable carbon isotope analysis of amino acids by gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) is a highly selective and sensitive method for probing the biosynthetic/diagenetic pathways, pool size and turnover rates of proteins, previously intractable to bulk isotope analyses. However, amino acids are polyfunctional, non-volatile compounds which require derivatisation prior to GC analysis. While a wide range of derivatives exist for the GC analysis of amino acids only a handful have been utilised for their GC/C/IRMS analysis. Significantly, none of those derivatives currently employed appear completely satisfactory and a thorough assessment of their relative utility is lacking. Seven derivatives (three previously reported and four novel) for obtaining delta(13)C values of amino acids via GC/C/IRMS analysis were compared. More specifically, standard mixtures of 15 protein amino acids were converted into N-acetylmethyl (NACME) esters, N-acetyl n-propyl (NANP) esters, N-acetyl i-propyl (NAIP) esters, N-trifluoroacetyl-i-propyl (TFA-IP) esters, N-pivaloyl methyl (NPME) esters, N-pivaloyl n-propyl (NPNP) esters and N-pivaloyl i-propyl (NPIP) esters. Each derivative was assessed with respect to its applicability to carbon isotope determinations of all the common alpha-amino acids, reaction yield, chromatographic resolution, stability, analyte-to-derivative carbon ratio, kinetic isotope effects and errors associated with their carbon isotope determinations. The NACME derivative was concluded to be the preferred derivative mainly due to the highest analyte-to-derivative carbon ratio being achieved, resulting in the lowest analytical errors for amino acid delta(13)C value determinations, ranging from +/-0.6 per thousand for phenylalanine, leucine and isoleucine to +/-1.1 per thousand for serine and glycine.     

Analysis of isotope ratios in vitamin K1 (phylloquinone) from human plasma by gas chromatography/mass spectrometry

Vitamin K(1) is a fat-soluble vitamin required for the gamma-carboxylation of vitamin K-dependent proteins. Recent work has suggested an important role for vitamin K(1) in bone health beyond its more established function in the control and regulation of blood coagulation. However, current UK recommended intakes do not reflect this recent evidence. The use of stable isotopes provides a powerful tool to investigate vitamin K kinetics, turnover and absorption in man, although published methods have reported difficulties in the extraction and analysis of isotope ratios of vitamin K in human plasma. In this paper, we report a new methodology for the extraction and measurement of isotope ratios in vitamin K(1). Sample clean-up is achieved with liquid-liquid extraction, enzyme hydrolysis with lipase and cholesterol esterase, and solid-phase extraction. Isotopic analysis of the pentafluoropropionyl derivative of vitamin K(1) is performed by gas chromatography/mass spectrometry (GC/MS). The limit of quantitation is equivalent to at least 0.3 nmol/L and the method is demonstrated to be linear over a range of enrichments. This method provides a robust alternative to previous work requiring the use of semi-preparative high-performance liquid chromatography (HPLC).     

Urinary 19-norandrosterone purification by immunoaffinity chromatography: application to gas chromatography/combustion/isotope ratio mass spectrometric analysis

The detection of exogenous 19-norandrosterone (19-NA) in urines was investigated by using gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS). 19-NA is, for the first time to our knowledge, isolated from urinary matrix by specific immunoaffinity chromatography (IAC) before analysis. The sample preparation consisted of a preliminary purification of urine by solid-phase extraction after hydrolysis by beta-glucuronidase. Unconjugated 19-NA was thus isolated by IAC and directly analysed by GC/C/IRMS. Optimisation of IAC purification was achieved and the reliability of the technique for anti-doping control is discussed.     

A single glucose derivative suitable for gas chromatography/mass spectrometry and gas chromatography/combustion/isotope ratio mass spectrometry

The incorporation of stable isotopes improves the assessment of glucose metabolism and, with some researchers using two tracers, (2)H-glucose assessed by gas chromatography/mass spectrometry (GC/MS) and (13)C-glucose by gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS), a common derivative for both is advantageous. The most commonly used derivatives for GC/MS are inappropriate for GC/C/IRMS as additional functional groups dilute the label. We therefore considered the suitability of six derivatives for both GC/MS and GC/C/IRMS. Glucose alkylboronates were prepared by adding the appropriate alkylboronic acid (butyl- or methylboronic acid) in pyridine to desiccated glucose. The derivatisation was completed by reacting this with either (a) acetic anhydride or trifluoroacetic anhydride (acetate derivatives) or (b) bis(trimethylsilyl)trifluoroacetamide BSTFA (TMS derivatives). All six derivatives were assessed using GC/MS and (13)C GC/C/IRMS.Neither TMS derivative exhibited any signal intensity in the molecular ion, although a M-15 ion showed good agreement between experimental and theoretical data and, whilst still low in intensity, could be suitable for isotope work. Similarly, none of the acetate derivatives showed any intensity at the molecular ion although three key fragmentation series were identified. The most attractive sequence, initiated by the loss of 1,2 cyclic boronate, resulted in the main fragment ion of interest, m/z 240, corresponding to the fluorinated methylboronate derivate. Minimal carbon and hydrogen atoms are added to this derivative making it an excellent choice for stable isotope work, while proving suitable for analysis by both GC/MS and GC/C/IRMS.     

固相微萃取/气相色谱/质谱联用测定水中的2,4-二硝基苯酚

研究了水中痕量2,4-二硝基苯酚的固相微萃取（SPME）方法,得到了2,4-二硝基苯酚的SPME最佳萃取条件：水溶液调pH2,并用NaCl他和,在室温下直接萃取30min,气相色谱／质谱分析时,纤维探针在270℃下脱附3min。所建立的方法适于快速、方便地测定水中2,4-二硝基苯酚,不需浓缩和预处理。     

Analysis of exogenous dehydroepiandrosterone excretion in urine by gas chromatography/combustion/isotope ratio mass spectrometry.

A detailed procedure for the analysis of exogenous dehydroepiandrosterone (DHEA) in urine by gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) has been established for detecting doping with DHEA. The average delta-value (parts per thousand difference of (13)C/(12)C ratio from the isotope ratio standard) of 26 synthetic steroids commercially available was -30.1 +/- 2.6, and was significantly lower than that of human endogenous DHEA in urine of the world class athletes who had participated in the XVIIth Olympic Winter Games (-20.3 +/- 2.1, n = 446). Although large inter-individual variations of urinary DHEA excretion were observed following a single oral administration of 50 mg of DHEA, no significant inter-individual difference was found when the excretion of exogenous DHEA was monitored in terms of delta-values using GC/C/IRMS; the minimum delta-values were observed around 6-8 h after the administration, and the values returned to the base level at over 72 h after the dosing. Thus, the deviations in delta-values of DHEA and its diol metabolites are considered to be conclusive evidence for detecting doping with DHEA. Some successful cases of detection of doping with DHEA from athletes are also reported.     

Isolation of free phenolic compounds from arboreal leaves by use of the Florisil/C<Subscript>18</Subscript> system

In studies of the phenolic compounds present in leaves and needles, GC and GC–MS have so far been applied only sporadically. This is probably because of the greater difficulties encountered in preparing the samples for this method than those used for liquid chromatography. When preparing a sample for gas chromatography the analyst is faced with two difficult stages—separation of the compound from the matrix without losses (stage 1) so that the final sample can be derivatized to make it suitable for analysis on a non-polar capillary column of the gas chromatograph (stage 2). This paper presents a procedure for extraction of phenolic compounds from the matrix by means of a Florisil/C₁₈ sorbent system and their analysis by GC. After passage through the adsorbents the recovery ranges from 32% for ferulic acid to 88% for gentisic acid. It was found that this extraction method and the GC analysis are very precise (particularly for samples of a mass <1 g) and can be used for quantification. The high-precision quantification of 15 phenolic acids, shikimic acid, and six other compounds present in pine needles has been achieved. The conditions used for GC analysis and construction of calibration curves for quantitative determination are given.     

Application of microwave-assisted desorption/headspace solid-phase microextraction as pretreatment step in the gas chromatographic determination of 1-naphthylamine in silica gel adsorbent

Pretreatment of silica gel sample containing 1-naphthylamine by microwave-assisted desorption (MAD) coupled to in situ headspace solid phase microextraction (HS-SPME) has been investigated as a possible alternative to conventional methods prior to gas chromatographic (GC) analysis. The 1-naphthylamine desorbs from silica gel to headspace under microwave irradiation, and directly absorbs onto a SPME fiber located in a controlled-temperature headspace area. After being collected on the SPME fiber, and desorbed in the GC injection port, 1-naphthylamine is analyzed by GC-FID. Parameters that influence the extraction efficiency of the MAD/HS-SPME, such as the extraction media and its pH, the microwave irradiation power and irradiation time as well as desorption conditions of the GC injector, have been investigated. Experimental results indicate that the extraction of a 150 mg silica gel sample by using 0.8 ml of 1.0 M NaOH solution and a PDMS/DVB fiber under high-powered irradiation (477 W) for 5 min maximizes the extraction efficiency. Desorption of 1-naphthylamine from the SPME fiber in GC injector is optimal at 250 °C held for 3 min. The detection limit of method is 8.30 ng. The detected quantity of 1-naphthylamine obtained by the proposed method is 33.3 times of that obtained by the conventional solvent extraction method for the silica gel sample containing 100 ng of 1-naphthylamine. It provides a simple, fast, sensitive and organic-solvent-free pretreatment procedure prior to the analysis of 1-naphthylamine collected on a silica gel adsorbent.     

Optimization of solid-phase microextraction for the gas chromatography/mass spectrometry analysis of persistent organic pollutants

In this work, solid-phase microextraction (SPME) has been applied as an alternative for the selective extraction of 3 polybrominated diphenyl ethers (PBDEs), 2,2',4,4'-tetrabromodiphenyl ether (PBDE-47); 2,2',4,4',5-pentabromodiphenyl ether (PBDE-99); and 2,2',4,4',6-pentabromodiphenyl ether (PBDE-100), and 2 alkylphenols, 4-tert-OP and 4-NP, prior to their analysis by gas chromatography/mass spectrometry (GC/MS). The advantages of this technique are mainly its simplicity, cost-effectiveness, and time-saving sample preparation, as well as being a solvent-free technique. With the aim of optimizing the conditions for an efficient extraction of the studied compounds, different fiber coatings and the main parameters affecting the extraction procedure have been evaluated. The results obtained showed a good linearity in the range of concentrations investigated, and adequate relative standard deviation values were found according to the range accepted for SPME. Recovery values were in the range of 78-108%, and good detection and quantitation limits at ppt levels were obtained for both methods, allowing the determination of the selected compounds in samples at trace levels. The results obtained clearly show the potential of SPME for efficient concentration of the target compounds and also demonstrate the reliability of this extraction technique for their GC/MS analysis.     

色谱／质谱定量分析技术在纺织品禁用偶氮染料测试中的应用

建立了偶氮染料印染的纺织品中有害芳胺的ＧＣ／ＭＳ定量分析方法。     

Measurement of 13C and 15N isotope labeling by gas chromatography/combustion/isotope ratio mass spectrometry to study amino acid fluxes in a plant-microbe symbiotic association

We have developed a method based on a double labeling with stable isotopes and gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) analyses to study amino acid exchange in a symbiotic plant-microbe association. Isotopic precision was studied for 21 standards including 15 amino acid derivatives, three N-protected amino acid methyl esters, three amines and one international standard. High correlations were observed between the δ(13)C and δ(15)N values obtained by GC/C/IRMS and those obtained by an elemental analyzer (EA) coupled to an isotope ratio mass spectrometer (R(2) = 0.9868 and 0.9992, respectively). The mean precision measured was 0.04‰ for δ(13)C and 0.28‰ for δ(15)N (n = 15). This method was applied in vivo to the symbiotic relationship between alfalfa (Medicago sativa L.) and N(2)-fixing bacteria. Plants were simultaneously labeled over 10 days with (13)C-depleted CO(2) ((12)CO(2)), which was assimilated through photosynthesis by leaves, and (15)N(2) fixed via nodules. Subsequently, the C and N isotope compositions (i.e. δ(13)C and δ(15)N) of free amino acids were analyzed in leaves and nodules by GC/C/IRMS. The method revealed the pattern of C and N exchange between leaves and nodules, highlighting that γ-aminobutanoic acid and glycine may represent an important form of C transport from leaves to the nodules. The results confirmed the validity, reliability and accuracy of the method for assessing C and N fluxes between plants and symbiotic bacteria and support the use of this technique in a broad range of metabolic and fluxomic studies.     

Extraction and purification procedures for simultaneous quantification of phenolic xenoestrogens and steroid estrogens in river sediment by gas chromatography/ion trap mass spectrometry

A sensitive and simple method based on ultrasonication extraction with a hexane/acetone (2:1, v/v) mixture, followed by clean up of the extract by solid-phase extraction (SPE) and gas chromatography/mass spectrometry (GC/MS) detection, has been developed and validated for the analysis of 20 estrogenic endocrine-disrupting chemicals (EEDCs) including phenolic xenoestrogens, synthetic and natural estrogens in river sediment. After extraction and purification, analytes are derivatised with a BSTFA/TMCS/pyridine (49:1:50, v/v/v) mixture and quantified by GC/MS. The GC/MS method involves switching between electron ionisation (EI) and chemical ionisation (CI); it also switches between selected ion storage and tandem mass spectrometry detection. The applicability of the method has been demonstrated by analysing extracts of French river sediments for which bioanalytical tests (in vitro) had already shown that they were impacted by estrogenic endocrine disrupters. The biological contribution of all the products detected in each sediment extract was compared to the estrogenic activity measured by bioassays.     

Achieving traceable chemical measurements: inter-laboratory evaluation of a simplified technique for isotope dilution mass spectrometry. Part 2. Methodology for high accuracy analysis of organic analytes

A high accuracy measurement procedure developed and validated at LGC has been transferred to a number of expert UK laboratories, and their experience in applying the technique has been evaluated by inter-laboratory comparisons. It is an “exact matching” calibration procedure for analysis of organic analytes using isotope dilution mass spectrometry (IDMS). This calibration procedure uses a calibration blend and a sample blend with closely matched isotope amount ratios, and is an iterative process, culminating in the calibration blend and sample blend having identical isotope amount ratios. It is capable of high accuracy, since systematic errors in the determination of the isotope amount ratios are cancelled out. A series of four inter-laboratory comparisons of increasing difficulty were carried out involving a number of expert laboratories. The first three comparisons used gas chromatography mass spectrometry (GC–MS) analysis of the pesticide metabolite (pp′-dichlorodiphenyl) dichloroethylene (pp′-DDE), involving both conventional calibration and IDMS exact matching procedures for pp′-DDE in a solvent and a complex liquid matrix (corn oil). The fourth comparisons utilised liquid chromatography mass spectrometry (LC–MS) and involved the analysis of sulphamethazine (4-amino-N-(4,6 dimethyl-2 pyrimidinyl) benzenesulphonamide) in solvent using IDMS and conventional calibration techniques. Following the first trial, a workshop for participants was held on the use of the exact matching procedure together with a short course on uncertainty estimation. The results of the comparisons clearly showed the superior accuracy of using IDMS with the exact matching procedure for both GC–MS and LC–MS applications. These comparisons and the workshop have enabled the methodology to be transferred to UK industry, helping to improve UK measurement capability.     

Achieving traceable chemical measurements: inter-laboratory evaluation of a simplified technique for isotope dilution mass spectrometry. Part 2. Methodology for high accuracy analysis of organic analytes

A high accuracy measurement procedure developed and validated at LGC has been transferred to a number of expert UK laboratories, and their experience in applying the technique has been evaluated by inter-laboratory comparisons. It is an “exact matching” calibration procedure for analysis of organic analytes using isotope dilution mass spectrometry (IDMS). This calibration procedure uses a calibration blend and a sample blend with closely matched isotope amount ratios, and is an iterative process, culminating in the calibration blend and sample blend having identical isotope amount ratios. It is capable of high accuracy, since systematic errors in the determination of the isotope amount ratios are cancelled out. A series of four inter-laboratory comparisons of increasing difficulty were carried out involving a number of expert laboratories. The first three comparisons used gas chromatography mass spectrometry (GC–MS) analysis of the pesticide metabolite (pp′-dichlorodiphenyl) dichloroethylene (pp′-DDE), involving both conventional calibration and IDMS exact matching procedures for pp′-DDE in a solvent and a complex liquid matrix (corn oil). The fourth comparisons utilised liquid chromatography mass spectrometry (LC–MS) and involved the analysis of sulphamethazine (4-amino-N-(4,6 dimethyl-2 pyrimidinyl) benzenesulphonamide) in solvent using IDMS and conventional calibration techniques. Following the first trial, a workshop for participants was held on the use of the exact matching procedure together with a short course on uncertainty estimation. The results of the comparisons clearly showed the superior accuracy of using IDMS with the exact matching procedure for both GC–MS and LC–MS applications. These comparisons and the workshop have enabled the methodology to be transferred to UK industry, helping to improve UK measurement capability.

     

Site-specific carbon isotope analysis of aromatic carboxylic acids by elemental analysis/pyrolysis/isotope ratio mass spectrometry

Site-specific carbon isotope composition of organic compounds can provide useful information on their origin and history in natural environments. Site-specific isotope analyses of small amounts of organic compounds (sub-nanomolar level), such as short-chain carboxylic acids and amino acid analogues, have been performed using gas chromatography/pyrolysis/isotope ratio mass spectrometry (GC/pyrolysis/IRMS). These analyses were previously limited to volatile compounds. In this study, site-specific carbon isotope analysis has been developed for non-volatile aromatic carboxylic acids at sub-micromolar level by decarboxylation using a continuous flow elemental analysis (EA)/pyrolysis/IRMS technique. Benzoic acid, 2-naphthylacetic acid and 1-pyrenecarboxylic acid were pyrolyzed at 500-1000 degrees C by EA/pyrolysis/IRMS to produce CO2 for delta13C measurement of the carboxyl group. These three aromatic acids were most efficiently pyrolyzed at 750 degrees C. Conventional sealed-tube pyrolysis was also conducted for comparison. The delta13C values of CO2 generated by the continuous flow technique were within 1.0 per thousand of those performed by the conventional technique, indicating that the new continuous flow technique can accurately analyze the carbon isotopic composition of the carboxyl group in aromatic carboxylic acids. The new continuous flow technique is simple, rapid and uses small sample sizes, so this technique will be useful for characterizing the isotopic signature of carboxyl groups in organic compounds.