Validation of pentaacetylaldononitrile derivative for dual 2H gas chromatography/mass spectrometry and 13C gas chromatography/combustion/isotope ratio mass spectrometry analysis of glucose

A reference method to accurately define kinetics in response to the ingestion of glucose in terms of total, exogenous and endogenous glucose is to use stable‐isotope‐labelled compounds such as ²H and ¹³C glucose followed by gas chromatography/mass spectrometry (GC/MS) and gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) analysis. The use of the usual pentaacetyl (5Ac) derivative generates difficulties in obtaining accurate and reproducible results due to the two chromatographic peaks for the syn and anti isomers, and to the isotopic effect occurring during acetylation. Therefore, the pentaacetylaldononitrile derivative (Aldo) was validated for both isotopes, and compared with the 5Ac derivative. A correction factor including carbon atom dilution (stoichiometric equation) and the kinetic isotopic effect (KIE) was determined. Analytical validation results for the ²H GC/MS and ¹³C GC/C/IRMS measurements produced acceptable results with both derivatives. When ²H enrichments of plasma samples were ≤1 mol % excess (MPE), the repeatability (RSD_{Aldo Intra assay and Intra day} <0.94%, RSD_{5Ac Intra assay and Intra day} <3.29%), accuracy (Aldo <3.4%, 5Ac <29.0%), and stability of the derivatized samples were significantly better when the Aldo derivatives of the plasma samples were used (p < 0.05). When the glucose kinetics were assessed in nine human subjects, after glucose ingestion, the plasma glucose ²H enrichments were identical with both derivatives, whereas the ¹³C enrichments needed a correction factor to fit together. Due to KIE variation, this correction factor was not constant and had to be calculated for each batch of analyses, to obtain satisfactory results. Mean quantities of exogenous glucose exhibit marked difference (20.9 ± 1.3g (5Ac) vs. 26.7 ± 2.5g (Aldo)) when calculated with stoichiometric correction, but fit perfectly when calculated after application of the correction factor (22.1 ± 1.3g (5Ac) vs. 22.9 ± 1.9g (Aldo)). Finally, the pentaacetylaldononitrile derivative, used here in GC/C/IRMS for the first time, enables measurement of ²H and ¹³C enrichments in plasma glucose with a single sample preparation. Copyright © 2009 John Wiley & Sons, Ltd.     

Kinetics and branching ratio studies of the reaction of C2H5O2 + HO2 using chemical ionisation mass spectrometry

The overall rate coefficient for the reaction of C(2)H(5)O(2) with HO(2) was determined using a turbulent flow chemical ionization mass spectrometer (TF-CIMS) system over the pressure range of 75 to 200 Torr and temperatures between 195 and 298 K. The temperature dependence of the overall rate coefficient for the reaction between C(2)H(5)O(2) and HO(2) was fitted using the following Arrhenius expression: k(T) = (2.08) x 10(-13) exp [(864 +/- 79)/T] cm(-3) molecule(-1) s(-1). The upper limits for the branching ratios for reactive channels leading to O(3) and OH production were quantified for the first time. A tropospheric model has been used to assess the impact of the experimental error of the rate coefficients determined in this study on predicted concentrations of a number of key species, including O(3), OH, HO(2), NO and NO(2). In all cases it is found that the propagated error is very small and will not in itself be a major cause of uncertainty in modelled concentrations. However, at low temperatures, where there is a wide discrepancy between existing kinetic studies, modelling using the range of kinetic data in the literature shows a small but significant variation for [C(2)H(5)O(2)], [C(2)H(5)OOH], [NO(x)] and the HO(2) : OH ratio. Furthermore, a structure-activity relationship (SAR) was developed to rationalise the reactivity of the reaction between RO(2) and HO(2).     

Investigation of the feeding effect on the 13C/12C isotope ratio of the hormones in bovine urine using gas chromatography/combustion isotope ratio mass spectrometry

The effect of the feeding on the 13C/12C isotope ratio of four endogenous steroid hormones testosterone (T), epi-testosterone (epi-T), dehydroepiandrosterone (DHEA) and etiocholanolone (ETIO) in bovine urine was investigated. An analytical method to determine the accurate isotope ratio was developed including an extensive clean up followed by enrichment of the analytes in two steps of HPLC fractionation. Feeding experiments with four young animals were performed using C3 and C4 plants (grass, maize silage, hay, etc.) over a time period of about 280 days. One cattle was used as a control animal with no change of its diet over the full period. The detection of the 13C/12C isotope ratio of the acetylated extracts was performed by gas chromatography/combustion isotope ratio mass spectrometry. After the first change of the feeding from C4 to C3 plants significant changes of the delta 13C % values were observed from the -19 to -23% level to the -24 to -32% level for etiocholanolone and epi-testosterone in urine of three animals, whereas the DHEA values remained under the level of the two metabolites. Testosterone could not be detected with GC-C-IRMS due to its low concentration in young animals. After the second change of the diet from C3 to C4 plants (after 222 days), the measured delta 13C % values have been stabilised at the original level. The results show that in case of the feeding with only C3 plants the endogenous delta values of -32% can be reached. In this case the contribution of exogenous material with a delta value of -32% could not be detected independently of the concentration. If the diet contains C4 plants the difference or the ratio of the delta 13C % values becomes the determinant in the discriminatory power. For validation of the method a human and a cattle were treated with testosterone and the delta 13C % values were measured in incurred human and cattle urine.     

Analysis of the 13C natural abundance of CO2 gas from sparkling drinks by gas chromatography/combustion/isotope ratio mass spectrometry

A simple and rapid method to measure naturally occurring delta(13)C values of headspace CO(2) of sparkling drinks has been set up, using direct injections on a gas chromatograph coupled to an isotope ratio mass spectrometer, through a combustion interface (GC/C/IRMS). We tested the method on CO(2) gas from several origins. No significant isotopic fractionation was observed nor influences by secondary compounds eventually present in the gas phase. Standard deviation for these measurements was found to be <0.1 per thousand.     

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.     

Isotope ratio mass spectrometry: delta13C and delta15 N analysis for tracing the origin of illicit drugs

Gas chromatography/combustion/mass spectrometry (GC-C-MS) and elemental analysis/mass spectrometry (EA-MS) techniques are proposed to estimate delta(13)C and delta(15)N values in heroin, morphine, cocaine and hemp leaves, for the purposes of tracing the geographical origins of seized drugs. The values of isotope ratios for pure drugs and drugs with impurities were compared. It was demonstrated that large samples (up to 3 x 10(-6) g C) were combusted completely, so that the results obtained were valid. The data are considered to be an essential supplement to a wide-scale database designed specifically for the delta(13)C and delta(15)N values of drugs. The potential forensic and academic significance of the results is discussed.     

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.     

Flow injection of the lock mass standard for accurate mass measurement in electrospray ionization time-of-flight mass spectrometry coupled with liquid chromatography

A method of flow injection of the lock mass for accurate mass measurement using electrospray ionization time-of-flight mass spectrometry is described. The reference compound is introduced in the chromatographic effluent via a six-port valve placed post-column, prior to the split connector. Flow injection is performed in such a way that the reference elution peak is superimposed in the total ion current and partially overlaps that of the investigated analyte, allowing independent ionization of the two compounds and thus accurate mass measurement with no ion suppression effects. Different lock mass molecules can be injected in a single analytical run to target various analytes. The performance of this methodology is demonstrated in both isocratic and gradient liquid chromatography modes. The molecular ion of the flow-injected lock mass could also be used as a reference for mass measurement of the in-source fragments of the analytes. Good mass accuracy, within 4 mDa of the theoretical values, was obtained.     

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 rhizosphere 13C pulse signals in soil thin sections by laser ablation isotope ratio mass spectrometry

In grassland ecosystems, soil animals act as key soil engineers and architects. The diversity of soil animals is also a regulator of ecosystem carbon flow. However, our understanding of the link between soil animals, carbon fluxes and soil physical organisation remains poor. An integrated approach based on soil micromorphology and laser ablation stable isotope ratio mass spectrometry (LA-IRMS) was developed to provide spatially distributed data of pulse-derived (13)C tracer from roots in the soil environment. This paper describes the development and testing of a LA-IRMS (13)C/(12)C analytical method on soil thin sections as a means to determine the fate of root carbon derived from photosynthesis into soil. Results from this work demonstrated (1) that micro-scale delta(13)C (per thousand) analysis could be made on targeted features located within a soil thin section and (2) that LA-IRMS delta(13)C (per thousand) measurements made on samples obtained from (13)CO(2) pulse labelled plant-soil blocks confirmed the presence of recent photosynthates in the rhizosphere (1 and 4 weeks post-pulse).     

Practical considerations in the gas chromatography/combustion/isotope ratio monitoring mass spectrometry of 13C-enriched compounds: detection limits and carryover effects

This paper describes a methodological investigation of the use of gas chromatography/combustion/isotope ratio monitoring mass spectrometry (GC/C/IRMS) for the compound-specific stable isotope analysis of 13C-enriched compounds. Analysis of two 13C-enriched fatty acid methyl esters, possessing delta13C values of approximately 500 per thousand, at a range of concentrations, demonstrated that detectable responses, i.e. chromatographic peaks, could be observed in the 45/44 output even when the compound was present in such low abundance that no peak was observed in the m/z 44 ion chromatogram. A limit of detection, defined as the point at which the signal-to-background ratio was equal to 3, was calculated for two compounds and for both ion chromatograms. The limit of detection in the 45/44 chromatogram was found to be ca. 30 pg injected for methyl 13C-hexadecanoate and ca. 20 pg injected for methyl 13C-octadecanoate, whilst, in the m/z 44 ion chromatogram, detection limits were approximately 180 and approximately 200 pg, respectively. The delta13C value recorded for the analytes was found to be both inaccurate and imprecise below 5 ng of each component injected, although this would not represent a significant drawback in qualitative tracer-type experiments. In a further study of co-injected mixtures of labelled (approximately 500 per thousand) and unlabelled (natural abundance, -20 to -30 per thousand ) fatty acid methyl esters a significant within-run carryover effect was observed, where the isotope values recorded for compounds eluting immediately after enriched components were significantly affected. Whilst this would not affect qualitative results, quantitative data for mixtures containing enriched compounds should be considered with caution. The standards employed in this investigation were enriched to approximately 500 per thousand in 13C; however, these effects would probably be accentuated at higher levels of labelling and with other elements. The limit of detection work demonstrated the potential of GC/C/IRMS as a highly sensitive and selective detector with many possible applications.     

Stopped-flow electrospray ionization mass spectrometry: a new method for studying chemical reaction kinetics in solution

In this work a new mass spectrometry based method for monitoring the kinetics of chemical reactions in solution is described. A stopped-flow mixing instrument is coupled to an electrospray ionization (ESI) mass spectrometer via a novel type of interface. Chemical reactions are initiated by rapid mixing of two reactant solutions. The mixture is instantaneously transferred to a reaction tube where the kinetics can be monitored in real-time by ESI mass spectrometry. With the current setup, a time window from 2.5 to 36 seconds after mixing of the reactants can be monitored. The experimental setup is used to study the kinetics of acetylcholine hydrolysis under alkaline conditions as a function of pH. The intensities of reactant (acetylcholine) and product (choline) ions are monitored simultaneously as a function of time. The reaction is carried out under pseudo-first-order conditions and the intensity-time curves are well described by single exponentials. The rate constants determined from these fits compare favorably with previous data from the literature.     

Flow injection on-line dilution for multi-element determination in human urine with detection by inductively coupled plasma mass spectrometry

A simple flow injection on-line dilution procedure with detection by inductively coupled plasma mass spectrometry (ICP-MS) was developed for the determination of copper, zinc, arsenic, lead, selenium, nickel and molybdenum in human urine. Matrix effects were minimized by employing a dilution factor of 16.5 with on-line standard addition, and (103)Rh was used as internal standard to compensate for signal fluctuation. The procedure was validated by the analysis of two standard reference materials SRM 2670 (NIST) and Seronormtrade mark Trace Elements in Urine. Recovery experiments were performed by spiking the reference materials as well as artificial urine. The detection limits (mug l(-1)) were 0.12,0.96,0.30,0.09,0.45,0.08,0.09, and the precisions (RSD,%) were 2.6,2.3,3.0,3.7,3.7,4.9,2.8 for Cu, Zn, As, Pb, Se, Ni and Mo, respectively. The procedure was applied to the analysis of 41 human urine samples. No correlations between the concentrations of the elements were observed.     

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.     

13C-IRIS: an improved method to detect the addition of low levels of C4-derived sugars to juices.

A method to improve the detection of cane or corn sugar addition to fruit juices has been developed. This involves the isolation of the individual sugars and measurement of their carbon isotope ratios. Using the Isotopic Relations of Individual Sugars approach, the additions of cane sucrose or cane invert as low as 4% of the total sugars can be detected in orange juice. This method offers a significant improvement over the conventional carbon isotopic method that has a detection limit of about 10%.     

Application of high-performance liquid chromatography with isotope-ratio mass spectrometry for measuring low levels of enrichment of underivatized materials

Combining HPLC separations with an isotope-ratio mass spectrometric (IRMS) detection produces a device capable of measuring very low alterations in 13C abundance from analyte species that cannot be volatilized. Examples are presented showing proteins, carbohydrates, and nucleotides that are eluted from varying types of HPLC columns (reversed-phase, normal-phase, ion-exchange and size-exclusion). This wide range of chromatographic methods enables the analysis of compounds never before amenable to IRMS techniques and may lead to the development of many new assays.     

Cluster calibration in mass spectrometry: laser desorption/ionization studies of atomic clusters and an application in precision mass spectrometry

For accurate mass measurements and identification of atomic and molecular species precise mass calibration is mandatory. Recent studies with laser desorption/ionization and time-of-flight analysis of cluster ion production by use of fullerene and gold targets demonstrate the generation of atomic clusters for calibration purposes. Atomic ion results from the Penning trap mass spectrometer ISOLTRAP, in which a carbon cluster ion source has recently been installed, are presented as an application in the field of precision mass spectrometry.     

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.     

Determination of trace levels of total fluorine in water using combustion ion chromatography for fluorine: a mass balance approach to determine individual perfluorinated chemicals in water

Perfluorinated compounds (PFCs) such as perfluorooctanesulfonate (PFOS) and perfluorooctanoate (PFOA) have received worldwide attention because of their environmental persistence and widespread distribution. Because of the lack of robust analytical methods and standards to detect all of the PFCs, and their precursors and metabolic intermediates, a mass balance approach involving the determination of total fluorine (TF), followed by fractionation of samples to separately determine inorganic and organic fluorine, is needed. In this study, we have developed a method to determine low microg/L levels of total fluorine (TF) in seawater samples. Further, seawater samples were fractionated into organic and inorganic fractions by extraction with organic solvents, which were then analyzed for TF, extractable organic fluorine (EOF) and inorganic fluorine (IF; i.e., fluoride). Concentrations of known perfluorinated compounds (PFCs) including PFOS and PFOA were also determined in water samples by liquid chromatography-tandem mass spectrometry (LC-MS/MS) to enable calculation of the fraction of fluorine that is contributed by PFCs to TF. A major proportion of fluorine in seawater was in the form of fluoride (>90% in locations not affected by direct discharges). Nevertheless, within the organofluorine fraction, a major percentage (60-90%) of fluorine still remains unknown in water samples, suggesting the occurrence of other fluorinated acids in addition to known perfluorinated acids. Further studies are needed to identify and quantify the unidentified organofluorines in seawater. Mass balance analysis of total organic fluorine (TOF) and EOF is important, if we are to understand transport and fate of fluorinated compounds in the environment, and if we are to identify the sources of unidentified fluorinated compounds.