Characterization of exogenous testosterone in livestock by gas chromatography/combustion/isotope ratio mass spectrometry: influence of feeding and age

The detection of exogenous testosterone in bovine urine was investigated by using gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS). The carbon isotopic ratio measurement of epitestosterone, etiocholanolone (testosterone metabolite) and DHEA (testosterone precursor) in female bovine urines after testosterone enanthate administration was carried out. An important modification in the 13C/12C ratio of testosterone metabolites was observed, such that significant differences between precursor and metabolites of testosterone occurred until three weeks after intramuscular administration of testosterone enanthate. The factors influencing the 13C/12C of endogenous steroids were studied especially through cattle feeding and age. The DHEA mean delta13C value was found to vary between -25 and -26/1000 when hay and concentrate diet were used for fattening. On the other hand the delta13C value observed when maize silage was used increased to -20/1000. Testosterone metabolites showed the same delta13C increase as their precursor. Moreover, we observed a clear relationship between age and efficiency of misuse determination. Indeed, because of the lower concentration of natural hormones in young animals, the contribution of exogenous molecules increases significantly compared with older subjects. Consequently, demonstration of administration is easier to achieve in calves than in mature animals.     

A rapid screening assay for measuring urinary androsterone and etiocholanolone delta(13)C ( per thousand) values by gas chromatography/combustion/isotope ratio mass spectrometry

A gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) method is described and validated for measurement of delta(13)C values of the acetate derivatives of urinary etiocholanolone and androsterone. The analysis was performed with only 2 mL of urine. The sample preparation consisted of deconjugation with beta-glucuronidase, solid phase extraction, and derivatization with acetic anhydride and pyridine. The within-assay precision of two quality control (QC) urine samples ranged from 0.5 to 2.1 CV%. The between-assay precision in the same QC urines ranged from 1.7 to 3.4 CV%. Administration of testosterone enanthate to a subject resulted in a 6 per thousand decrease in delta(13)C values from -25 per thousand (baseline) to -31 per thousand. Two weeks after testosterone administration was discontinued, the delta(13)C values remained abnormally low while the urine testosterone/epitestosterone (T/E) ratio returned to less than 6. This relatively simple method is useful for rapidly screening a large number of urine samples, including those with T/E <6.     

Development and application of stable carbon isotope analysis to the detection of cortisol administration in cattle

The use of anabolic agents in food-producing animals has been prohibited within the EU since 1988. The control of the illegal use of natural steroid hormones in cattle is still an exciting analytical challenge as no definitive method and nonambiguous analytical criteria are available. We have used gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) to demonstrate the administration of cortisol to cattle. The method consisted of an efficient combination between OASIS HLB solid-phase extraction (SPE), oxidation, SiOH SPE and semi-preparative high-performance liquid chromatography (HPLC) for glucocorticoid purification. By comparison of the (13)C/(12)C isotopic ratio of the oxidised product of cortisol, i.e. 5 beta-androstane-3,11,17-trione (5 beta AAT), with an endogenous reference compound (ERC), dehydroepiandrosterone (DHEA), the differentiation of cortisol metabolite origin, either endogenous or exogenous, has been achieved. After treatment of an animal, the delta(13)C(VPDB) values of 5 beta AAT reached -30 to -32 per thousand, whereas the delta(13)C(VPDB) values of DHEA remained at -25 per thousand. A significant difference in the delta(13)C(VPDB) values between DHEA and 5 beta AAT was measurable over a period of 3 days after a single administration of cortisol to the animal.     

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.     

C and N stable isotope variation in urine and milk of cattle depending on the diet

The stable carbon and nitrogen isotopic composition of urine and milk samples from cattle under different feeding regimes were analysed over a period of six months. The isotope ratios were measured with isotope ratio mass spectrometry (IRMS). The δ ¹³C values of milk and urine were dependent on different feeding regimes based on C₃ or C₄ plants. The δ ¹³C values are more negative under grass feeding than under maize feeding. The δ ¹³C values of milk are more negative compared to urine and independent of the feeding regime. Under grass feeding the analysed milk and urine samples are enriched in ¹³C relative to the feed, whereas under maize feeding the ¹³C/¹²C ratio of urine is in the same range and milk is depleted in ¹³C relative to the diet. The difference between the ¹⁵N/¹⁴N ratios for the two feeding regimes is less pronounced than the ¹³C/¹²C ratios. The δ ¹⁵N values in urine require more time to reach the new equilibrium, whereas the milk samples show no significant differences between the two feeding regimes.     

Determination of the exogenous character of testosterone in bovine urine by gas chromatography-combustion-isotope ratio mass spectrometry.

A new approach was developed in order to control testosterone abuse in animal production. A gas chromatographic-combustion-isotope ratio mass spectrometric (GC-C-IRMS) method was used to distinguish the exogenous character from the endogenous character of the main metabolites of testosterone (epitestosterone and etiocholanolone) in cattle urine. This method is based on a comparison between the carbon isotope ratio (13C/12C) of testosterone metabolites and those of testosterone endogenous precursors. After urinary steroid purification, extracts were acetylated with acetic anhydride and injected into the GC-C-IRMS system. In order to validate the method, testosterone enanthate was administered to a 4 year old cow. The 13C/12C isotope ratios of testosterone exogenous metabolites appeared to be significantly different to the 13C/12C precursor ratios and were detected until 3 weeks after the anabolic administration. These preliminary results appear to be promising for the difficult control of natural hormones in livestock.     

Detection of dehydroepiandrosterone misuse by means of gas chromatography- combustion-isotope ratio mass spectrometry

According to World Anti-Doping Agency (WADA) rules (WADA Technical Document-TD2004EAAS) urine samples containing dehydroepiandrosterone (DHEA) concentrations greater than 100 ng ML(-1) shall be submitted to isotope ratio mass spectrometry (IRMS) analysis. The threshold concentration is based on the equivalent to the glucuronide, and the DHEA concentrations have to be adjusted for a specific gravity value of 1.020. In 2006, 11,012 doping control urine samples from national and international federations were analyzed in the Cologne doping control laboratory, 100 (0.9%) of them yielding concentrations of DHEA greater than 100 ng mL(-1). Sixty-eight percent of the specimens showed specific gravity values higher than 1.020, 52% originated from soccer players, 95% were taken in competition, 85% were male urines, 99% of the IRMS results did not indicate an application of testosterone or related prohormones. Only one urine sample was reported as an adverse analytical finding having 319 ng mL(-1) DHEA (screening result), more than 10,000 ng mL(-1) androsterone and depleted carbon isotope ratio values for the testosterone metabolites androsterone and etiocholanolone. Statistical evaluation showed significantly different DHEA concentrations between specimens taken in- and out-of- competition, whereas females showed smaller DHEA values than males for both types of control. Also a strong influence of the DHEA excretion on different sport disciplines was detectable. The highest DHEA values were detected for game sports (soccer, basketball, handball, ice hockey), followed by boxing and wrestling. In 2007, 6622 doping control urine samples were analyzed for 3alpha,5-cyclo-5alpha-androstan-6beta-ol-17-one (3alpha,5-cyclo), a DHEA metabolite which was described as a useful gas chromatography-mass spectrometry (GC-MS) screening marker for DHEA abuse. Nineteen urine specimens showed concentrations higher than the suggested threshold of 140 ng mL(-1), six urine samples yielded additionally DHEA concentrations higher than 100 ng mL(-1), none of them showing positive IRMS findings. These results should be taken into consideration in future discussions about threshold values for endogenous steroids in doping control.     

Application of stable carbon isotope analysis to the detection of 17beta-estradiol administration to cattle

The use of anabolic agents in food producing animals is prohibited within the EU since 1988 (96/22/EC directive). The control of the illegal use of natural steroid hormones in cattle is still an exciting analytical challenge as far as no definitive method and non-ambiguous analytical criteria are available. The ability of gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) to demonstrate the administration of 17beta-estradiol to bovine has been investigated in this paper. By comparison of 13C/12C isotopic ratio of main urinary estradiol metabolite, i.e. 17alpha-estradiol, with two endogenous reference compounds (ERCs), i.e. dehydroepiandrosterone (DHEA) and 5-androstene-3beta,17alpha-diol, the differentiation of estradiol metabolite origin, either endogenous or exogenous, has been proved to be achievable. After treatment, the delta(13)C(VPDB)-values of 17alpha-estradiol reached -27 per thousand to -29 per thousand, whereas delta13CVPDB-values of DHEA remained between -13 per thousand and -20 per thousand depending on the diet, maize and grass, respectively. A significant difference of delta13CVPDB between ERCs and 17alpha-estradiol was measurable over a period of 2 weeks after estradiol ester administration to the animal.     

Stable carbon isotope analysis of different tissues of beef animals in relation to their diet

As part of a larger experiment, 31 young bulls, divided into three groups, were given different diets containing either C(3) plants or a combination of C(3) and C(4) plant-based feeds in three feeding periods before slaughter. Variation in the proportion of C(4) plant material in the diets was made by including or not maize or maize-derived ingredients, whereas the other dietary constituents were from C(3) plants. Analysis of stable carbon isotope ratios (delta(13)C value) was performed on different tissues taken at slaughter: blood, plasma, liver, kidney fat, hair, muscle and ruminal contents. Blood and plasma samples were also taken at the beginning of each period. A highly significant difference was found in the delta(13)C values of blood and plasma samples taken from animals that had received a diet of only C(3) plants or with 59% C(4) material for 70 days. The delta(13)C values of all different samples taken at slaughter were highly significantly different between the three feeding groups that had received diets with 0, 13.5 or 35% C(4) material for on average 137, 139 and 83 days, respectively. For the three groups, samples of hair, muscle, plasma, whole blood and liver were significantly enriched in (13)C compared with the diet (except for liver in one group), whereas kidney fat was significantly depleted. The proportion of C(4) plant material could be accurately estimated from the delta(13)C values of different tissue samples. Stable carbon analysis of different tissues from beef animals can be used to trace back diets containing variable proportions of C(3) and C(4) plant material.     

Determination of the 13C/12C ratio of ethanol derived from fruit juices and maple syrup by isotope ratio mass spectrometry: collaborative study

A collaborative study of the carbon-13 isotope ratio mass spectrometry (13C-IRMS) method based on fermentation ethanol for detecting some sugar additions in fruit juices and maple syrup is reported. This method is complementary to the site-specific natural isotope fractionation by nuclear magnetic resonance (SNIF-NMR) method for detecting added beet sugar in the same products (AOAC Official Methods 995.17 and 2000.19), and uses the same initial steps to recover pure ethanol. The fruit juices or maple syrups are completely fermented with yeast, and the alcohol is distilled with a quantitative yield (>96%). The carbon-13 deviation (delta13C) of ethanol is then determined by IRMS. This parameter becomes less negative when exogenous sugar derived from plants exhibiting a C4 metabolism (e.g., corn or cane) is added to a juice obtained from plants exhibiting a C3 metabolism (most common fruits except pineapple) or to maple syrup. Conversely, the delta13C of ethanol becomes more negative when exogenous sugar derived from C3 plants (e.g., beet, wheat, rice) is added to pineapple products. Twelve laboratories analyzed 2 materials (orange juice and pure cane sugar) in blind duplicate and 4 sugar-adulterated materials (orange juice, maple syrup, pineapple juice, and apple juice) as Youden pairs. The precision of that method for measuring delta13C was similar to that of other methods applied to wine ethanol or extracted sugars in juices. The within-laboratory (Sr) values ranged from 0.06 to 0.16%o (r = 0.17 to 0.46 percent per thousand), and the among-laboratories (SR) values ranged from 0.17 to 0.26 percent per thousand (R = 0.49 to 0.73 percent per thousand). The Study Directors recommend that the method be adopted as First Action by AOAC INTERNATIONAL.     

Determination of the origin of urinary norandrosterone traces by gas chromatography combustion isotope ratio mass spectrometry

On the one hand, 19-norandrosterone (NA) is the most abundant metabolite of the synthetic anabolic steroid 19-nortestosterone and related prohormones. On the other hand, small amounts are biosynthesized by pregnant women and further evidence exists for physiological origin of this compound. The World Anti-Doping Agency (WADA) formerly introduced threshold concentrations of 2 or 5 ng of NA per ml of urine to discriminate 19-nortestosterone abuse from biosynthetic origin. Recent findings showed however, that formation of NA resulting in concentrations in the range of the threshold levels might be due to demethylation of androsterone in urine, and the WADA 2006 Prohibited List has defined NA as endogenous steroid. To elucidate the endogenous or exogenous origin of NA, (13)C/(12)C-analysis is the method of choice since synthetic 19-nortestosterone is derived from C(3)-plants by partial synthesis and shows delta(13)C(VPDB)-values of around -28 per thousand. Endogenous steroids are less depleted in (13)C due to a dietary mixture of C(3)- and C(4)-plants. An extensive cleanup based on two high performance liquid chromatography cleanup steps was applied to quality control and doping control samples, which contained NA in concentrations down to 2 ng per ml of urine. (13)C/(12)C-ratios of NA, androsterone and etiocholanolone were measured by gas chromatography/combustion/isotope ratio mass spectrometry. By comparing delta(13)C(VPDB)-values of androsterone as endogenous reference compound with NA, the origin of NA in doping control samples was determined as either endogenous or exogenous.     

Influence of dietary composition on the carbon, nitrogen, oxygen and hydrogen stable isotope ratios of milk

The stable isotope ratios ((13)C/(12)C, (15)N/(14)N, (18)O/(16)O, D/H) of animal feed and milk were investigated, considering cows stabled in two farms and fed with diets made up of different kinds of C(3) plants and different amounts of maize. Maize was characterised by delta(13)C, delta(18)O and deltaD values significantly higher than those of the C(3) plants, while, for the C(3) plants, Festuca arudinacea had significantly higher content of (13)C and (15)N. The delta(13)C and delta(18)O values of the overall diet and the delta(13)C of milk casein and lipids were shown to be significantly correlated with the percentage of maize in the animal diet. On the other hand, the delta(18)O values of milk water and the delta(18)O, deltaD and delta(15)N values of casein were shown to be only slightly influenced by the amount of maize in the feed, being probably more closely correlated with the geo-climatic and pedological characteristics of the area of origin and with the presence of fresh plant or silage in the ration. The delta(13)C value of casein was shown to be a suitable parameter for evaluating the amount of maize in the diet: each 10% increase in the maize content corresponded to a shift of 0.7 per thousand to 1.0 per thousand in the delta(13)C of casein. A threshold value of -23.5 per thousand for delta(13)C in milk casein, above which it is not possible to exclude the presence of maize in the diet, was suggested. The results obtained could be useful for determining mislabelling of dairy products declared to have been produced by pastured animals or of PDO cheeses with an established amount of maize in the diet and for verifying the unpermitted addition of exogenous components to milk.     

Practical and theoretical considerations in the gas chromatography/combustion/isotope ratio mass spectrometry delta(13)C analysis of small polyfunctional compounds

Carbohydrates and proteins are among the most abundant naturally occurring biomolecules and so suitable methods for their reliable stable isotope analysis by gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) are required. Due to the non-volatile nature of these compounds they require hydrolytic cleavage to their lower molecular weight subunits and derivatisation prior to GC/C/IRMS analysis. The addition of carbon to the molecules and any kinetic isotopic fractionation associated with derivatisation must be accounted for in order to provide meaningful stable isotope values and estimates of propagated errors. To illustrate these points amino acid trifluoroacetate/isopropyl esters and alditol acetates were prepared from authentic amino acids and monosaccharides, respectively. As predicted from the derivatisation reaction mechanisms, a kinetic isotope effect was observed which precludes direct calculation of delta(13)C values of the amino acids and monosaccharides by simple mass balance equations. This study shows that the kinetic isotope effect associated with derivatisation is both reproducible and robust, thereby allowing the use of correction factors. We show how correction factors can be determined and accurately account for the addition of derivative carbon. As a consequence of the addition of a molar excess of carbon and the existence of a kinetic isotope effect during derivatisation, errors associated with determined delta(13)C values must be assessed. We illustrate how such errors can be quantified (for monosaccharides +/-1.3 per thousand and for amino acids between +/-0.8 per thousand and +/-1.4 per thousand). With the magnitude of the errors for a given delta(13)C value of a monosaccharide or amino acid quantified, it is possible to make reliable interpretations of delta(13)C values, thereby validating the determination of delta(13)C values of amino acids as TFA/IP esters and monosaccharides as alditol acetates.     

Detection of royal jelly adulteration using carbon and nitrogen stable isotope ratio analysis

Stable isotope ratios ((13)C/(12)C and (15)N/(14)N) were measured in royal jelly (RJ) samples by isotope ratio mass spectrometry (IRMS) to evaluate authenticity and adulteration. Carbon and nitrogen isotope contents (given as delta values relative to a standard, delta(13)C, delta(15)N) of RJ samples from various European origins and samples from commercial sources were analyzed. Uniform delta(13)C values from -26.7 to -24.9 per thousand were observed for authentic RJ from European origins. Values of delta(15)N ranged from -1.1 to 5.8 per thousand depending on the plant sources of nectars and pollen. High delta(13)C values of several commercial RJ samples from -20.8 to -13.3 per thousand indicated adulteration with high fructose corn syrup (HFCS) as a sugar source. Use of biotechnologically produced yeast powder as protein source for the adulterated samples was assumed as delta(15)N values were lower, as described for C(4) or CAM plant sources. RJ samples from authentic and from adulterated production were distinguished. The rapid and reliable method is suitable for urgent actual requirements in food monitoring.     

Isotopic fractionation of endogenous anabolic androgenic steroids and its relationship to doping control in sports

The use of gas chromatography (GC)-combustion (C)-isotope ratio mass spectrometry (IRMS) demonstrates that a single oral administration of dehydroepiandrosterone (DHEA, 100 mg) to a male subject significantly lowers the 13C content of etiocholanolone (Et) and androsterone (A) in the subject's urine. The difference in carbon isotope ratio (d13C per thousand) values between Et and A increases from 1.6 per thousand at the time of administration to 5.1 per thousand at 26 h post-administration, indicating preferential metabolism of administered DHEA to form Et in relation to A. Multiple oral administrations of DHEA to a male subject reveals lower d13C values during the excretion period of Et (-31.7 per thousand to -34.6 per thousand) and A (-31.4 per thousand to -33.0 per thousand) to that of the d13C value of the administered DHEA (-31.3 per thousand). Reference distributions of d13C Et and d13C A constructed from normal athlete populations within Australia and New Zealand show a small natural discrimination against 13C in the formation of Et relative to A (mean=0.3 per thousand, n=167, p=0.007). Amplified differences between d13C Et and d13C A, and in vivo 13C depletion measured by GC-C-IRMS are shown to be potentially useful for doping control.     

Stable isotope ratio analysis as a tool to discriminate between rainbow trout (O. mykiss) fed diets based on plant or fish-meal proteins

The use of stable isotope ratio analysis (SIRA) as a rapid analytical tool to characterize and discriminate farmed fish on the basis of the feedstuffs included in the diet formulation is discussed. Two isoproteic (44.8%) and isolipidic (19.6%) extruded diets were formulated: a fish-meal-based diet (FM diet), containing fish meal as the sole protein source; a plant-protein-based diet (PP diet), where pea protein concentrate and wheat gluten meal replaced 80% of fish meal protein. The diets were fed to eight groups of rainbow trout (initial body weight: 106.6g) for 103 days in two daily meals under controlled rearing conditions. Growth performance (final body weight: 318.5 g; specific growth rate: 1.06%) and feed-to-gain ratio (0.79) were not affected by the dietary treatment. The differences in isotopic values of the two diets were clearly reflected in the different carbon and nitrogen isotopic values in rainbow trout fillets. The delta(13)C and delta(15)N values of muscle of farmed rainbow trout showed differences between farmed fish fed a fish-protein-based diet (-20.47 +/- 0.34 and 12.38 +/- 0.57 for delta(13)C and delta(15)N, respectively) and those fed a plant-protein-based diet (-23.96 +/- 0.38 and 7.15 +/- 0.51 for delta(13)C and delta(15)N, respectively). The results suggest that SIRA provides a robust and verifiable analytical tool to discriminate between fish fed on a plant or a fish protein diet.     

Carbon isotope determination for separate components of heterogeneous materials using coupled thermogravimetric analysis/isotope ratio mass spectrometry

A gas-tight thermal analysis system (Netzsch STA 449C Jupiter) has been connected to an isotope ratio mass spectrometer (PDZ Europa 20-20) via an interface containing an oxidizing furnace, water trap, and gas-sampling valve. Using this system, delta(13)C has been measured for CO(2) derived from the thermal decomposition of carbonate and oxalate minerals and organic materials at temperatures that correspond to different decomposition events. There is close agreement between measured and published delta(13)C values for carbonate and oxalate minerals, which have simple decarbonation reactions on heating. Cellulose and lignin-rich materials show much more complex thermal decomposition, reflecting differences in their purity and structure, and measured delta(13)C values vary with the temperature of gas sampling. Provided that measurements are made at temperatures that correspond to the decomposition of cellulose and lignin (indicated by maximum weight loss), internally consistent data can be obtained. However, measurements for cellulose and lignin are systematically enriched in delta(13)C (by up to 1.4 per thousand) with respect to those reported for reference materials, possibly due to the slower combustion kinetics (compared with EA-IRMS). Thermogravimetric analysis/isotope ratio mass spectrometry (TG-IRMS) is ideal for materials and samples for which it is not possible to use other isotopic measurement techniques, for example because of sample heterogeneity.     

Stable isotope ratio measurements of royal jelly samples for controlling production procedures: impact of sugar feeding

The carbon and nitrogen stable ratios of royal jelly (RJ) samples from various origins are determined using an elemental analyser linked online to an isotope ratio mass spectrometer to evaluate authenticity and adulteration. The (13)C/(12)C and (15)N/(14)N stable isotope ratios are measured in more than 500 RJs (domestic, imported and derived from feeding experiments) in order to obtain isotopic measurements that take into account seasonal, botanical and geographical effects. Authenticity intervals are established for traditional beekeeping practices, without feeding, in the range -22.48 to -27.90‰ for δ(13)C. For these samples, the δ(15)N values range from -1.58 to 7.98‰, depending on the plant sources of pollen and nectar. The δ(13)C values of the commercial samples vary from -18.54 to -26.58‰. High δ(13)C values are typical of sugar cane or corn syrups which have distinctive isotopic (13)C signatures because both plants use the C4 photosynthetic cycle, in contrast to most RJs which are derived from C3 plants. These differences in the (13)C-isotopic composition allow the detection of the addition of such sugars. RJs from traditional sources and from industrial production by sugar feeding are thus successfully distinguished.     

Origin differentiation of heroin sample and its acetylating agent with (13)C isotope ratio mass spectrometry

A novel method for deducing the origins of heroin and the reagent used for acetylation was established based on delta(13)C determinations of heroin and its hydrolysate, morphine, using gas chromatography (13)C isotope ratio mass spectrometry (GC-C-IRMS). The alkaline and acid hydrolysis conditions of heroin were optimized. Both yield and purity of morphine produced could meet the requirement for a GC-C-IRMS analysis. Using (2-diethylaminoethyl-2,2- diphenylvalerate) as internal standard the determinations of heroin and morphine contents were performed with a GC method in a linear range of 0.2 to 2.0 mg ml(1) that was required to gain the isotope ratio results. The hydrolysis and synthesis of heroin did not change the delta(13)C value of morphine. The precision for delta(13)C detection of both heroin and morphine was sufficient for origin differentiation of heroin samples. The information about the origins of acetylation reagents could be deduced from the difference of delta(13)C values between heroin and morphine. The results for origin differentiation of 10 heroin samples grouped into different regions and their acetylating agents were satisfactory.     

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.