Choice of dietary protein of vegetarians and omnivores is reflected in their hair protein 13C and 15N abundance

Stable isotopic (15N, 13C) composition of tissues depends on isotopic pattern of food sources. We investigated whether the isotopic compositions of human hair protein and amino acids reflect the habitual dietary protein intake. Hair samples were analyzed from 100 omnivores (selected randomly out of the 1987-1988 German nutrition survey VERA), and from 15 ovo-lacto-vegetarians (OLV), and from 6 vegans recruited separately. Hair bulk and amino acid specific isotopic compositions were analyzed by isotope-ratio mass spectrometry (EA/IRMS and GC/C/IRMS, respectively) and the results were correlated with data of the 7 day dietary records. Hair bulk 15N and 13C abundances clearly reflect the particular eating habits. Vegans can be distinguished from OLV and both are significantly distinct from omnivores in both 15N and 13C abundances. 15N and 13C abundances rose with a higher proportion of animal to total protein intake (PAPI). Individual proportions of animal protein consumption (IPAP) were calculated using isotopic abundances and a linear regression model using animal protein consumption data of vegans (PAPI = 0) and omnivores (mean PAPI = 0.639). IPAP values positively correlated with the intake of protein, meat, meat products, and animal protein. Distinct patterns for hair amino acid specific 15N and 13C abundances were measured but with lower resolution between food preference groups compared with bulk values. In conclusion, hair 13C and 15N values both reflected the extent of animal protein consumption. Bulk isotopic abundance of hair can be tested for future use in the validation of dietary assessment methods.     

Carbon isotope analysis of bulk keratin and single amino acids from British and North American hair

The reconstruction of ancient diets using isotopic measurements of bone collagen, and other tissues, which survive in archaeological contexts, relies on known isotopic relationships between diet and body tissues. Examination of these relationships often requires the study of modern human and animal subjects. While hair keratin can act as a useful proxy for bone collagen in isotopic studies on living humans, where it is inappropriate to sample tissues such as collagen, it can, in addition, act as a chronological indicator of dietary change. This study investigates hair keratin delta13C values from current residents of the UK and the USA. Residents in the USA showed a clear bulk hair delta13C enrichment of approximately 3 per thousand over UK individuals, attributed to an elevated C4 dietary input from maize fed to livestock in North America. The keratin delta13C of subjects who moved between the UK and USA showed a pronounced change after relocation, taking approximately 4 months to reach isotopic equilibrium. To investigate these differences further, we measured delta13C values of dispensable and indispensable amino acids as a group, and selected individual amino acids. As a group, enrichment of dispensable amino acids compared with indispensable amino acids occurred in samples from both continents, averaging 7.2 per thousand in the UK and 7.9 per thousand in the USA. Dispensable and indispensable amino acids, as well as all individual amino acids measured, were enriched in samples from the USA compared with those from the UK.     

Nitrogen balance and delta15N: why you're not what you eat during pregnancy

Carbon (13C/12C) and nitrogen (15N/14N) stable isotope ratios were longitudinally measured in human hair that reflected the period from pre-conception to delivery in 10 pregnant women. There was no significant change in the delta13C results, but all subjects showed a decrease in delta15N values (-0.3 to -1.1 per thousand) during gestation. The mechanisms causing this decrease in hair delta15N have not been fully elucidated. However, since the delta15N values of dietary nitrogen and urea nitrogen are significantly lower compared to maternal tissues, it is hypothesized that the increased utilization of dietary and urea nitrogen for tissue synthesis during pregnancy resulted in a reduction of the steady state diet to a body trophic level effect by approximately 0.5-1 per thousand. An inverse correlation (R2 = 0.67) between hair delta15N and weight gain was also found, suggesting that positive nitrogen balance results in a reduction of delta15N values independent of diet. These results indicate that delta15N measurements have the ability to monitor not only dietary inputs, but also the nitrogen balance of an organism. A potential application of this technique is the detection of fertility patterns in modern and ancient species that have tissues that linearly record stable isotope ratios through time.     

Two new organic reference materials for delta13C and delta15N measurements and a new value for the delta13C of NBS 22 oil

Analytical grade L-glutamic acid is chemically stable and has a C/N mole ratio of 5, which is close to that of many of natural biological materials, such as blood and animal tissue. Two L-glutamic acid reference materials with substantially different 13C and 15N abundances have been prepared for use as organic reference materials for C and N isotopic measurements. USGS40 is analytical grade L-glutamic acid and has a delta13C value of -26.24 per thousand relative to VPDB and a delta15N value of -4.52 per thousand relative to N2 in air. USGS41 was prepared by dissolving analytical grade L-glutamic acid with L-glutamic acid enriched in 13C and 15N. USGS41 has a delta13C value of +37.76 per thousand and a delta15N value of +47.57 per thousand. The delta13C and delta15N values of both materials were measured against the international reference materials NBS 19 calcium carbonate (delta13C=+1.95 per thousand ), L-SVEC lithium carbonate (delta13C=-46.48 per thousand ), IAEA-N-1 ammonium sulfate (delta15N=0.43 per thousand ), and USGS32 potassium nitrate (delta15N=180 per thousand ) by on-line combustion continuous-flow and off-line dual-inlet isotope-ratio mass spectrometry. Both USGS40 and USGS41 are isotopically homogeneous; reproducibility of delta13C is better than 0.13 per thousand, and that of delta15N is better than 0.13 per thousand in 100-microg amounts. These two isotopic reference materials can be used for (i) calibrating local laboratory reference materials, and (ii) quantifying drift with time, mass-dependent fractionations, and isotope-ratio-scale contraction in the isotopic analysis of various biological materials. Isotopic results presented in this paper yield a delta13C value for NBS 22 oil of -29.91 per thousand, in contrast to the commonly accepted value of -29.78 per thousand for which off-line blank corrections probably have not been quantified satisfactorily.     

Serial analysis of stable nitrogen and carbon isotopes in hair: monitoring starvation and recovery phases of patients suffering from anorexia nervosa

Stable nitrogen and carbon isotopic ratios of hair strands of six patients suffering from anorexia nervosa were measured to monitor a dietary change from near starvation to recovery. This paper presents the results of a first-time study of nitrogen and carbon balance of the patients prior to and after admittance to a hospital and therapy. Sequential analysis of the isotopic ratios of hair strands of all patients could be related to the respective body mass index (BMI) of each patient. Our hypothesis concerning the diachronic change in delta15N and delta13C during therapy was met: The delta15N values were inversely related to the BMI, indicating a slow-down in catabolism of bodily protein due to the process of gluconeogenesis during the starvation phase. In contrast, the delta13C values and BMI were in phase: an increase in BMI resulted in an increase in the delta13C values. This rise in delta13C ratios is best interpreted by an increased supply of protein in the diet. Furthermore, delta15N and delta13C were inversely related. We conclude that hair, which is easily and non-traumatically sampled, is an adequate monitor that reflects dietary change and nitrogen balance within days. This isotopic method may also be applied in forensic studies with regard to cases of deprivation, and starvation, and may be a method for investigating starvation in historic populations.     

Alteration of the carbon and nitrogen stable isotope composition of beef by substitution of grass silage with maize silage

This study investigated the effect of substituting grass silage (C3 photosynthetic plant product) with maize silage (C4 photosynthetic plant product) on the natural abundance carbon (delta13C) and nitrogen (delta15N) stable isotope composition of bovine muscle tissue. Forty-five continental crossbred heifers were assigned to one of three diets consisting of 3 kg of a barley-based concentrate plus grass silage, maize silage or an equal mixture (dry matter basis) of grass silage and maize silage, fed ad libitum, for 167 days. Substitution resulted in less negative delta13C values (P<0.001) in lipid-free muscle and in lipid, and also a lower delta15N (P<0.001) in lipid-free muscle. Feeding of maize silage was clearly reflected in the delta13C of muscle, with each 10% difference in the dietary C4 carbon intake resulting in a 0.9 to 1.0 per thousand shift of delta13C in lipid-free muscle and a 1.0 to 1.2 per thousand in lipid. Minimum detectable mean differences (95% confidence, power 0.80, n=15) in this experiment were about 0.5 per thousand and 1.0 per thousand for delta13C of lipid-free muscle and lipid, respectively, and about 0.5 per thousand for delta15N of lipid-free muscle. The power analysis presented here is useful for estimating minimum isotopic differences that can be detected between any two groups of beef samples with a given number of replicates. It is concluded that carbon stable isotope ratio analysis of meat can be used to quantify C3/C4 dietary constituents in beef production.     

Effects of temperature on isotopic enrichment in Daphnia magna: implications for aquatic food-web studies

Laboratory experiments were conducted with Daphnia magna and Hyalella sp. grown on a single food source of known isotopic composition at a range of temperatures spanning the physiological optima for each species. Daphnia raised at 26.5 degrees C were enriched in delta(13)C and delta(15)N by 3.1 and 2.8 per thousand, respectively, relative to diet. Daphnia raised at 12.8 degrees C were enriched 1.7 and 5.0 per thousand in delta(13)C and delta(15)N, respectively. Results imply a significant negative relationship between the delta(13)C and delta(15)N of primary consumers when a temperature gradient exists. Similar responses were observed for Hyalella. Results indicate a general increase in delta(13)C enrichment and decrease in delta(15)N enrichment as temperature rises. Deviations from the commonly applied isotopic enrichment values used in aquatic ecology were attributed to changes in temperature-mediated physiological rates. Field data from a variety of sources also showed a general trend toward delta(13)C enrichment with increasing temperature in marine and lacustrine zooplankton. Multivariate regression models demonstrated that, in oligotrophic and mesotrophic lakes, zooplankton delta(13)C was related to lake-specific POM delta(13)C, lake surface temperature and latitude. Temperature-dependent isotopic separation (enrichment) between predator and prey should be taken into consideration when interpreting the significance of isotopic differences within and among aquatic organisms and ecosystems, and when assigning organisms to food-web positions on the basis of observed isotope values.     

Stable isotope (13C, 15N and 34S) analysis of the hair of modern humans and their domestic animals

Relationships between dietary status and recent migration were examined by delta(13)C, delta(15)N and delta(34)S analysis of hair samples from 43 modern humans living in a rural community in SW England. The isotopic content of 38 'local' hair samples was compared with that of five recently arrived individuals (from Canada, Chile, Germany and the USA). Hair samples from domestic animals (i.e. mainly cats, dogs, cows and horses) were analysed to examine the difference in delta(13)C, delta(15)N and delta(34)S values between herbivores and carnivores. Generally, modern human hair data from the triple stable isotope (delta(13)C, delta(15)N and delta(34)S) provided enough information to confirm the dietary status and origin of the individual subjects. The dietary intake was generally reflected in the animal hair delta(15)N and delta(13)C values, i.e. highest in the carnivores (cats). However, a non-local origin of food sources given to domesticated omnivores (i.e. dogs) was suggested by their hair delta(34)S values.     

Multiple stable isotope (18O, 13C, 15N and 34S) analysis of human hair to identify the recent migrants in a rural community in SW England

Relationships between recent migration and hair delta(18)O values were examined for 40 people living in a rural community in SW England. The isotopic contents of 35 'local' hair samples were compared with those of 5 recently arrived individuals (from Australia, Canada, Chile, Germany and the USA). The hair delta(18)O values of these 'visitors' were +7.9 (Omaha, USA), +11.2 (Jena, Germany), +12.1 (Osorno, Chile), +12.6 (Montreal, Canada) and +14.3 per thousand (Adelaide, Australia). The hair value for the USA visitor (+7.9 per thousand) fell outside the range for the 33 local adult residents, +10.5 to +14.3 per thousand (+12.7 +/- 0.8 per thousand). Hair delta(18)O values did not identify the individuals from Adelaide, Montreal and Osorno as 'visitors', but hair delta(13)C or delta(34)S data did. Combining the hair delta(18)O, delta(13)C and delta(34)S values using principal components analysis (two components explained 89% of the overall variation among the 40 subjects) helped to more clearly distinguish European from non-European individuals, indicating the existence of global overall isotope (geo-origin) relationships.     

Stable isotopes to discriminate lambs fed herbage or concentrate both obtained from C(3) plants

This study was aimed at determining whether isotopic ratio mass spectrometry (IRMS) enables us to discriminate between lambs fed herbage or concentrate, both obtained from C(3) plants, and those fed a concentrate obtained from C(4) plants. Thirty-four Comisana male lambs (age 45 days) were assigned to three feeding treatments. Fourteen lambs were fed vetch (Vicia sativa) ad libitum. Another fourteen lambs received a barley-based concentrate. The remaining six lambs were fed a maize-based concentrate. After 60 days of experimental treatment the animals were slaughtered and the wool, perirenal fat and muscle longissimus dorsi were sampled. The delta(13)C and delta(15)N values of the muscle, wool and feed were measured by continuous flow elemental analysis (CF-EA)-IRMS. The delta(13)C of the fat was determined likewise. The isotopic composition of the tissues reflected that of the three diets. For the lambs which were fed herbage the muscle delta(13)C values were higher (P < 0.0005) and delta(15)N values were lower (P < 0.0005) than those of the lambs receiving concentrates. The delta(15)N and delta(13)C values in the muscle and delta(13)C values in the adipose tissue allowed perfect discrimination between the lambs fed the three different diets. The regression between the delta(13)C values measured in muscle and in wool of lambs was linear (R(2) = 0.99; P < 0.0005). This result shows that delta(13)C measured in the wool can predict muscle delta(13)C distribution, suggesting that wool is a valuable matrix for meat authentication.     

Clinical-scale investigation of stable isotopes in human blood: delta13C and delta15N from 406 patients at the Johns Hopkins Medical Institutions

Objective chemical biomarkers are needed in clinical studies of diet-related diseases to supplement subjective self-reporting methods. We report on several critical experiments for the development of clinically legitimate dietary stable isotope biomarkers within human blood. Our examination of human blood revealed the following: (1) Within blood clot and serum from anonymous individuals (201 males, 205 females) we observed: mean serum delta13C = -19.1 +/- 0.8 per thousand (standard deviation, SD); clot, -19.3 +/- 0.8 per thousand (SD); range = -15.8 per thousand to -23.4 per thousand. Highly statistically significant differences are observed between clot and serum, males and females for both clot and serum. For 15N (n = 206), mean serum = +8.8 +/- 0.5 per thousand (SD); clot +7.4 +/- 0.4 per thousand (SD); range = +6.3 per thousand to +10.5 per thousand. Blood serum is enriched in 15N relative to blood clot by +1.4 per thousand on average, which may reflect differing protein amino acid content. Serum nitrogen is statistically significantly different for males and females, however, clot shows no statistical difference. (2) Relative to clot, capillary blood is marginally different for 13C, but not 15N. Clot 13C is not significantly different from serum; however, it is depleted in 15N by 1.5 per thousand relative to serum. (3) We assessed the effect of blood additives (sodium fluoride and polymerized acrylamide resin) and laboratory process (autoclaving, freeze drying) commonly used to preserve or prepare venous blood. On average, no alteration in delta13C or delta15N is detected compared with unadulterated blood from the same individual. (4) Storage of blood with and without the additives described above for a period of up to 115 days exhibits statistically significant differences for 13C and 15N for sodium fluoride. However, storage for unadulterated blood and blood preserved with polymerized acrylamide resin does not change the delta13C or delta15N isotopic composition of the blood in a significant way. With these experiments, we gain a clinical context for future development of a stable isotope based dietary biomarker.     

Isotope ratio monitoring of small molecules and macromolecules by liquid chromatography coupled to isotope ratio mass spectrometry

In the field of isotope ratio mass spectrometry, the introduction of an interface allowing the connection of liquid chromatography (LC) and isotope ratio mass spectrometry (IRMS) has opened a range of new perspectives. The LC interface is based on a chemical oxidation, producing CO2 from organic molecules. While first results were obtained from the analysis of low molecular weight compounds, the application of compound-specific isotope analysis by irm-LC/MS to other molecules, in particular biomolecules, is presented here. The influence of the LC flow rate on the CO2 signal and on the observed delta13C values is demonstrated. The limits of quantification for angiotensin III and for leucine were 100 and 38 pmol, respectively, with a standard deviation of the delta13C values better than 0.4 per thousand. Also, accuracy and precision of delta13C values for elemental analyser-IRMS and flow injection analysis-IRMS (FIA-LC/MS) were compared. For compounds with molecular weights ranging from 131 to 66,390 Da, precision was better than 0.3 per thousand, and accuracy varied from 0.1 to 0.7 per thousand. In a second part of the work, a two-dimensional (2D)-LC method for the separation of 15 underivatised amino acids is demonstrated; the precision of delta13C values for several amino acids by irm-LC/MS was better than 0.3 per thousand at natural abundance. For labelled mixtures, the coefficient of variation was between 1% at 0.07 atom % excess (APE) for threonine and alanine, and around 10% at 0.03 APE for valine and phenylalanine. The application of irm-LC/MS to the determination of the isotopic enrichment of 13C-threonine in an extract of rat colon mucosa demonstrated a precision of 0.5 per thousand, or 0.001 atom %.     

Stable (2)H isotope analysis of modern-day human hair and nails can aid forensic human identification

Continuous-flow isotope ratio mass spectrometry (CF-IRMS) was used to compare (2)H isotopic composition at natural abundance level of human scalp hair and fingernail samples collected from subjects worldwide with interpolated delta(2)H precipitation values at corresponding locations. The results showed a strong correlation between delta(2)H values of meteoric water and hair (r(2) = 0.86), while the corresponding correlation for nails was not as strong (r(2) = 0.6). Offsets of -180 per thousand and -127 per thousand were observed when calculating solutions of the linear regression analyses for delta(2)H vs. delta(18)O correlation plots of hair and nail samples, respectively. Compared with the +10 per thousand offset of the global meteoric water line equation these findings suggested that delta(18)O data from hair and nail would be of limited diagnostic value. The results of this pilot study provide for the first time tentative correlations of (2)H isotopic composition of human hair and nails with local water. Linear regression analyses for measured delta(2)H values of human hair and nails vs. water yielded delta(2)H(hair) = 0.49 x delta(2)H(water) - 35 and delta(2)H(nails) = 0.38 x delta(2)H(water) - 49, respectively. The results suggest that (2)H isotopic analysis of hair and nail samples can be used to provide information regarding an individual's recent geographical life history and, hence, location. The benefit of this technique is to aid identification of victims of violent crime and mass disasters in circumstances where traditional methods such as DNA and fingerprinting cannot be brought to bear (or at least not immediately).     

Effects of hydrolysis on the delta13C values of individual amino acids derived from polypeptides and proteins

This study investigates the effects of hydrolysis on the delta13C values of individual amino acids (IAAs) derived from polypeptide standards, and modern and ancient bone collagen. All IAAs were derivatised to their trifluoroacetyl/isopropyl (TFA/IP) esters for delta13C determination using gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS). Firstly, authentic single poly amino acid standards (SPAAs; n = 5) were hydrolysed for 4, 10, 24 and 48 h. As expected, IAA yields increased as a function of hydrolysis time. Significantly, it was only after 24 h of hydrolysis that IAA delta13C values were statistically identical to bulk SPAA values for all five standards. The accuracy of IAA delta13C values was thus shown to be a function of yield; however, poly phenylalanine demonstrated accurate IAA delta13C values with yields of only 1.4 and 4.3%, after 24 and 48 h of hydrolysis time, respectively. Authentic mixed poly amino acid standards (MPAAs; n = 5) comprising two different amino acids were then hydrolysed for 24 h. Percentage recoveries ranged from 36-95%. Estimates of bulk MPAA delta13C values calculated from measured IAA delta13C values agreed within experimental error with measured bulk MPAA values for three out of the five standards. Finally, the experimental procedure was applied to modern rat (MBCs; n = 20) and ancient ovi-caprine and bovine (ABCs; n = 27) bone collagen samples where the delta13C values of 12 out of its 18 constituent amino acids were determined. Estimated bulk MBC and ABC delta13C values were calculated from constituent amino acid delta13C values using mass balance. With the exclusion of three ABC samples, calculated bulk bone collagen delta13C values (delta13C(BCcal)) were shown to correlate extremely well with measured bone collagen values (delta13C(BCmes)) for both modern and ancient samples, where R2 = 0.91 and 0.84, respectively. Significantly, the variation between calculated and measured bone collagen values (Delta13C(BCcal-BCmes)) exhibited similar ranges for both MBC (from -2.6 to +1.2 per thousand ) and ABC (from -2.7 to +2.2 per thousand ) samples, providing evidence for the preservation of intact collagen in the ancient samples. These results demonstrate that the experimental procedures employed in the acid hydrolytic cleavage of peptides or proteins to their constituent amino acids does not involve significant isotopic fractionation.     

Enhancing the understanding of earthworm feeding behaviour via the use of fatty acid delta13C values determined by gas chromatography-combustion-isotope ratio mass spectrometry

Litter-dwelling (epigeic) Lumbricus rubellus and soil-dwelling (endogeic) Allolobophora chlorotica earthworms were observed aggregating under C(3) (delta(13)C = -31.3 per thousand; delta(15)N = 10.7 per thousand) and C(4) (delta(13)C = -12.6 per thousand; delta(15)N = 7.5 per thousand) synthetic dung pats applied to a temperate grassland (delta(13)C = -30.3 per thousand; delta(15)N = 5.7 per thousand) in an experiment carried out for 372 days. Bulk delta(13)C values of earthworms collected from beneath either C(3) or C(4) dung after 28, 56, 112 and 372 days demonstrated that (i) L. rubellus beneath C(4) dung were significantly (13)C-enriched after 56 days (delta(13)C = -23.8 per thousand) and 112 days (delta(13)C = -22.4 per thousand) compared with those from C(3) dung treatments (56 days, delta(13)C = -26.5 per thousand; 112 days, delta(13)C = -27.0 per thousand), and (ii) A. chlorotica were 2.1 per thousand (13)C-enriched (delta(13)C = -24.2 per thousand) relative to those from C(3) dung (delta(13)C = -26.3 per thousand) treatments after 372 days. Bulk delta(15)N values did not suggest significant uptake of dung N by either species beneath C(3) or C(4) dung, but showed that the endogeic species (total mean delta(15)N = 3.3 per thousand) had higher delta(15)N values than the epigeic species (total mean delta(15)N = 5.4 per thousand). Although the two species exhibited similar fatty acid profiles, individual fatty acid delta(13)C values revealed extensive routing of dietary C into body tissue of L. rubellus, but minor incorporation into A. chlorotica. In particular, the direct incorporation of microbial biomarker fatty acids (iC(17:0), aC(17:0)) from (13)C-labelled dung in situ, the routing of dung C into de novo synthesised compounds (iC(20:4)(omega)(6),C(20:5)(omega)(3), and the assimilation of essential fatty acids ((C(18:1)(omega)(9), C(18:1)(omega(7), C(18:2)(omega(6), C(18:3)(omega)(3)) derived from dung, were determined.     

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.     

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.     

Isolation of ammonium-N as 1-sulfonato-iso-indole for measurement of delta15N

The conversion of ammonium (NH(4) (+)) to 1-sulfonato-iso-indole has been examined as a method for natural abundance measurement of delta(15)N of NH(4) (+). The reaction is complete within 2 h and is based on the derivatisation of NH(4) (+) by o-phthaldialdehyde and sodium sulfite at a high pH, 11.2. The product is readily concentrated from dilute solutions by reverse-phase solid-phase extraction (SPE). The method is compound-specific despite partial derivatisation of potentially interfering amino acids, as their derivatives are not extracted by SPE. delta(15)N values of NH(4) (+) in KCL soil extracts can be measured within 48 h by automated continuous-flow IRMS with a precision of 0.23 per thousand (1 SD). Parallel measurements of NH(4) (+) standards of known delta(15)N are made to allow correction for the isotopic dilution by non-sample NH(4) (+). The practicality of this method is demonstrated by measuring the changes in NH(4) (+) concentration and delta(15)N following the addition of urea as a nitrogen source to inorganic N-depleted soil.     

The effect of acidification on the determination of organic carbon, total nitrogen and their stable isotopic composition in algae and marine sediment

We investigated the effects of sample acidification on the stable carbon and nitrogen isotopic composition (delta13C and delta15N), as well as the organic carbon (OC) and total nitrogen (TN) composition, of an algal culture and a marine sediment. Replicate measurements of untreated and acid-treated samples were made using 1 M, 2 M and 6 M HCl, 6% H2SO3 and 1 M H3PO4. For all treatments the precision of the analysis for the acid-treated sample was equal to or less than that in the non-acidified sample. For the algae, analysis of variance (ANOVA) indicated no significant differences in the mean OC and TN concentration, or delta13C and delta15N composition, between any acid treatment and non-acidified samples. For the sediment sample a comparison could only be made between the different acid treatments because the untreated contained significant amounts ( approximately 30%) of carbonate carbon. ANOVA indicated that the mean OC determined in sediment samples after the 1 M HCl treatment and the mean delta13C values after the 6% H2SO3 and 1 M H3PO4 treatments were significantly different (p < 0.013 and < .05, respectively) from all other treatments. Mass balance calculations indicate that in some instances delta13C values were biased due to a contribution from unreacted carbonate carbon. There were no significant differences in the mean TN between any acid-treated and non-acidified samples. The mean delta15N values after 6 M HCl, 6% H2SO3 and 1 M H3PO4 treatments were significantly different from the untreated sediment sample (p < 0.044). Based on the significant bias observed for the delta15N and delta13C values, a weak (1-2 M) HCl solution is confirmed as the most appropriate acid for the removal of inorganic carbon from natural materials requiring elemental and isotopic analysis.     

<Superscript>15</Superscript>N isotopic analyses: a powerful tool to establish links between seized 3,4-methylenedioxymethamphetamine (MDMA) tablets

In this study the (15)N/(14)N isotopic ratios of 43 samples of 3,4-methylenedioxymethamphetamine (MDMA) samples were measured using Gas Chromatography-Combustion-Isotope-Ratio Mass Spectrometry (GC-C-IRMS). The results show a large discrimination between samples with a range of delta(15)N values between -16 and +19 per thousand. Comparison between delta(15)N values and other physical and chemical parameters shows a strong relationship between delta(15)N and brand logo or composition. Thus, it could be assumed that tablets from different seizures probably originated from the same clandestine manufacturing source. Hence, (15)N isotopic parameters provide an important additional tool to establish common origins between seizures of clandestine synthetic drugs.