δD and δ13C analyses of atmospheric volatile organic compounds by thermal desorption gas chromatography isotope ratio mass spectrometry

This paper describes the establishment of a robust method to determine compound specific δD and δ(13)C values of volatile organic compounds (VOCs) in a standard mixture ranging between C(6) and C(10) and was applied to various complex emission samples, e.g. from biomass combustion and car exhaust. A thermal desorption (TD) unit was linked to a gas chromatography isotope ratio mass spectrometer (GC-irMS) to enable compound specific isotope analysis (CSIA) of gaseous samples. TenaxTA was used as an adsorbent material in stainless steel TD tubes. We determined instrument settings to achieve a minimal water background level for reliable δD analysis and investigated the impact of storage time on δD and δ(13)C values of collected VOCs (176 days and 40 days of storage, respectively). Most of the standard compounds investigated showed standard deviations (SD)<6‰ (δD) when stored for 148 days at 4 °C. However, benzene revealed occasionally D depleted values (21‰ SD) for unknown reasons. δ(13)C analysis demonstrated that storage of 40 days had no effect on VOCs investigated. We also showed that breakthrough (benzene and toluene, 37% and 7%, respectively) had only a negligible effect (0.7‰ and 0.4‰, respectively) on δ(13)C values of VOCs on the sample tube. We established that the sample portion collected at the split flow effluent of the TD unit can be used as a replicate sample for isotope analysis saving valuable sampling time and resources. We also applied TD-GC-irMS to different emission samples (biomass combustion, petrol and diesel car engines exhaust) and for the first time δD values of atmospheric VOCs in the above range are reported. Significant differences in δD of up to 130‰ were observed between VOCs in emissions from petrol car engine exhaust and biomass combustion (Karri tree). However, diesel car emissions showed a high content of highly complex unresolved mixtures thus a baseline separation of VOCs was not achieved for stable hydrogen isotope analysis. The ability to analyse δD by TD-GC-irMS complements the characterisation of atmospheric VOCs and is maybe used for establishing further source(s).     

Liquid chromatography/isotope ratio mass spectrometry measurement of δ13C of amino acids in plant proteins

In archaeological studies, the isotopic enrichment values of carbon and nitrogen in bone collagen give a degree of information on dietary composition. The isotopic enrichments of individual amino acids from bone collagen and dietary protein have the potential to provide more precise information about the components of diet. A limited amount of work has been done on this, although the reliability of these studies is potentially limited by fractionation arising through hydrolysis of whole plant tissue (where reaction between amino acids and carbohydrates may occur) and, for certain amino acids, the use of derivatives (particularly trifluoroacetyl derivatives) for gas chromatography/isotope ratio mass spectrometry (GC/IRMS) analysis. The present study takes the approach of extracting the protein components of plant tissues before hydrolysis and using liquid chromatography/isotope ratio mass spectrometry (LC/IRMS), which does not require derivatisation, for measurement of the isotopic enrichment of the amino acids. The protocol developed offers a methodology for consistent measurement of the δ(13)C values of amino acids, allowing isotopic differences between the individual amino acids from different plant tissues to be identified. In particular, there are highly significant differences between leaf and seed protein amino acids (leaf minus grain) in the cases of threonine (-4.1‰), aspartic acid (+3.5‰) and serine (-3.2‰). In addition to its intended application in archaeology, the technique will be of value in the fields of plant sciences, nutrition and environmental food-web studies.     

Comparison of liquid chromatography-isotope ratio mass spectrometry (LC/IRMS) and gas chromatography-combustion-isotope ratio mass spectrometry (GC/C/IRMS) for the determination of collagen amino acid δ13C values for palaeodietary and palaeoecological reconstruction

Results are presented of a comparison of the amino acid (AA) δ(13)C values obtained by gas chromatography-combustion-isotope ratio mass spectrometry (GC/C/IRMS) and liquid chromatography-isotope ratio mass spectrometry (LC/IRMS). Although the primary focus was the compound-specific stable carbon isotope analysis of bone collagen AAs, because of its growing application for palaeodietary and palaeoecological reconstruction, the results are relevant to any field where AA δ(13)C values are required. We compare LC/IRMS with the most up-to-date GC/C/IRMS method using N-acetyl methyl ester (NACME) AA derivatives. This comparison involves the analysis of standard AAs and hydrolysates of archaeological human bone collagen, which have been previously investigated as N-trifluoroacetyl isopropyl esters (TFA/IP). It was observed that, although GC/C/IRMS analyses required less sample, LC/IRMS permitted the analysis of a wider range of AAs, particularly those not amenable to GC analysis (e.g. arginine). Accordingly, reconstructed bulk δ(13)C values based on LC/IRMS-derived δ(13)C values were closer to the EA/IRMS-derived δ(13)C values than those based on GC/C/IRMS values. The analytical errors for LC/IRMS AA δ(13)C values were lower than GC/C/IRMS determinations. Inconsistencies in the δ(13)C values of the TFA/IP derivatives compared with the NACME- and LC/IRMS-derived δ(13)C values suggest inherent problems with the use of TFA/IP derivatives, resulting from: (i) inefficient sample combustion, and/or (ii) differences in the intra-molecular distribution of δ(13)C values between AAs, which are manifested by incomplete combustion. Close similarities between the NACME AA δ(13)C values and the LC/IRMS-derived δ(13)C values suggest that the TFA/IP derivatives should be abandoned for the natural abundance determinations of AA δ(13)C values.     

Systematic comparison of δ13C measurements of testosterone and derivative steroids in a freeze-dried urine candidate reference material for sports drug testing by gas chromatography/combustion/isotope ratio mass spectrometry and uncertainty evaluation using four different metrological approaches

An alternative calibration procedure for use when performing carbon isotope ratio measurements by gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) has been developed. This calibration procedure does not rely on the corrections in-built in the instrument software, as the carbon isotope ratios of a sample are calculated from the measured raw peak areas. The method was developed for the certification of a urine reference material for sports drug testing, as the estimation of measurement uncertainty is greatly simplified. To ensure that the method is free from bias arising from the choice of calibration material and instrument, the carbon isotope ratios of steroids in urine extracts were measured using two different instruments in different laboratories, and three different reference materials (CU/USADA steroid standards from Brenna Laboratory, Cornell University; NIST RM8539 mineral oil; methane calibrated against NIST RM8560 natural gas). The measurements were performed at LGC and the Australian National Measurement Institute (NMI). It was found that there was no significant difference in measurement results when different instruments and reference materials were used to measure the carbon isotope ratio of the major testosterone metabolites androsterone and etiocholanolone, or the endogenous reference compounds pregnanediol, 11- ketoetiocholanolone and 11β-hydroxyandrosterone. Expanded measurement uncertainties at the 95% coverage probability ranged from 0.21‰ to 1.4‰, depending on analyte, instrument and reference material. The measurement results of this comparison were used to estimate a measurement uncertainty of δ(13)C for the certification of the urine reference material being performed on a single instrument using a single reference material at NMI.     

Determination of petitgrain oils landmark parameters by using gas chromatography–combustion–isotope ratio mass spectrometry and enantioselective multidimensional gas chromatography

Gas chromatography–combustion–isotope mass spectrometry was employed for the assessment of the Carbon isotope ratios of volatiles in Italian mandarin and lemon petitgrain oils. In addition, the composition of the whole oil and the enantiomeric distribution of selected chiral compounds were determined for all the samples by using gas chromatography and by multidimensional and conventional enantioselective gas chromatography. The composition of the oils was compared with previous studies. The enantiomeric distribution of lemon petitgrain oils is here reported for the first time. On the composition of mandarin petitgrain oil, the information available in literature, to date, is relative only to one sample from Egypt. Carbon isotope ratio of several terpene hydrocarbons and of their oxygenated derivatives contained in petitgrains was compared with the δ ¹³C_VPDB values of the same compounds present in the corresponding genuine Italian Citrus peel oil. The results prove that the isotopic values obtained for lemon and mandarin petitgrain oils are very close to those relative to the corresponding peel oils determined in previous studies.     

Determination of the natural abundance δ15N of nortropane alkaloids by gas chromatography–isotope ratio mass spectrometry of their ethylcarbamate esters

An important route for the detoxification of tropane alkaloids involves N-demethylation to the nor-compounds followed by further degradation. In order to study the mechanisms of the pertinent reactions, a suitable means to determine the isotope ratios of the substrates and products is required. However, the polarity and functionality of the nortropane compounds makes their analysis as free bases difficult. A method is described which allows both the quantification of nortropane alkaloids and the determination of their natural abundance δ¹⁵N values. The protocol exploits the derivatisation of the alkaloids by reaction with ethyl chloroformate in aqueous medium and the quantitative extraction of the ensuing ethylcarbamate esters. The improved chromatographic properties of these derivatives gives ample separation of the isomeric nortropine and norpseudotropine for measurement of their δ¹⁵N (‰) values by isotope ratio mass spectrometry interfaced to gas chromatography. Adequate separation could not be achieved with the underivatised compounds. Repeatability and precision are sufficient to allow differences in the δ¹⁵N values (∆δ¹⁵N) > 0.8‰ to be measured, with a standard deviation routinely ∼0.3‰. The methodology has been tested by determining the changes in the δ¹⁵N values of nortropine and norpseudotropine during degradation by cell suspension cultures of a Pseudomonas strain expressing a specific capacity for tropine catabolism. The precision and reproducibility are shown sufficient to allow the evolution of the δ¹⁵N values to be followed during the fermentation.     

Evaluation of a bracketing calibration-based isotope dilution liquid chromatography–tandem mass spectrometry candidate reference measurement procedure for 17α-hydroxyprogesterone in human plasma

Analytical and Bioanalytical Chemistry - A candidate reference measurement procedure (RMP) based on isotope dilution coupled with liquid chromatography–tandem mass spectrometry...     

Establishing a chromium-reactor design for measuring δ2H values of solid polyhalogenated compounds using direct elemental analysis and stable isotope ratio mass spectrometry

²H/¹H isotope ratios of polyhalogenated compounds were determined by elemental analysis and isotope ratio mass spectrometry (EA-IRMS). Initial measurements with standard EA-IRMS equipment, which used high-temperature pyrolysis to convert the organic compounds into hydrogen, did not achieve significant signals for polychlorinated pesticides and related compounds, presumably due to the formation of HCl instead of hydrogen. To reverse this problematic reaction, a chromium reactor was incorporated into the element analyzer system, which scavenged Cl, forming chromium chloride and releasing hydrogen again in the form of H₂. The optimized system therefore allowed the δ²H values of polyhalogenated compounds to be determined. A quality assurance program was developed based on several parameters. (i) Each compound was analyzed using a sequence of five injections, where the first measurement was discarded. (ii) Recovery of H (when calculated relative to acetanilide) had to be >90% for all replicates in a sequence. (iii) All δ-values within a sequence had to vary by less than 10‰. (iv) Results had to be reproducible on another day with a different sample scheme. Once this reproducibility had been established, variabilities in the δ²H values of organohalogen standards were investigated using the technique. The highest δ²H value of +75‰ was found for o,p′-DDD, whereas the strongest depletion in deuterium was found for Melipax (–181‰). The most important results for comparable compounds were as follows. DDT-related compounds gave δ²H values of between +59 and +75‰ (technical DDT, o,p′- and p,p′-DDD) or in the range of approximately −1‰, indicative of the different sources/methods of producing this compound. Four HCH isomers from the same supplier showed relatively similar hydrogen isotope distributions, whereas two lindane (γ–HCH) standards from other sources had 39‰ less deuterium. This difference is likely due to different purification steps during the isolation of pure lindane from the technical HCH mixture. An even greater difference was observed between the δ²H values of Toxaphene (US product dating from 1978) and Melipax (product from the former East Germany, dating from 1979), which gave δ²H values of –101‰ and –181‰, respectively, meaning that both products were easily distinguished via δ²H-IRMS. Fractioning of hydrogen isotopes in the atmospheric water cycle was suggested as one reason for the different values. In this theory, the water (which had different δ²H values depending on where it was taken from) was incorporated during the biosynthesis of camphene, which is the natural product used to produce both products. These results indicate that hydrogen isotope-specific analysis can be a valuable tool for tracing the origins of a compound in certain cases.     

Mass measurement errors caused by “local” frequency perturbations in FTICR mass spectrometry

One of the key qualities of mass spectrometric measurements for biomolecules is the mass measurement accuracy (MMA) obtained. FTICR presently provides the highest MMA over a broad m/z range. However, due to space charge effects, the achievable MMA crucially depends on the number of ions trapped in the ICR cell for a measurement. Thus, beyond some point, as the effective sensitivity and dynamic range of a measurement increase, MMA tends to decrease. While analyzing deviations from the commonly used calibration law in FTICR we have found systematic errors which are not accounted for by a "global" space charge correction approach. The analysis of these errors and their dependence on charge population and post-excite radius have led us to conclude that each ion cloud experiences a different interaction with other ion clouds. We propose a novel calibration function which is shown to provide an improvement in MMA for all the spectra studied.     

Ultrasonication extraction and gel permeation chromatography clean-up for the determination of polycyclic aromatic hydrocarbons in edible oil by an isotope dilution gas chromatography–mass spectrometry

An analytical method for the determination of US EPA priority pollutant 16 polycyclic aromatic hydrocarbons (PAHs) in edible oil was developed by an isotope dilution gas chromatography–mass spectrometry (GC–MS). Extraction was performed with ultrasonication mode using acetonitrile as solvent, and subsequent clean-up was applied using narrow gel permeation chromatographic column. Three deuterated PAHs surrogate standards were used as internal standards for quantification and analytical quality control. The limits of quantification (LOQs) were globally below 0.5 ng/g, the recoveries were in the range of 81–96%, and the relative standard deviations (RSDs) were lower than 20%. Further trueness assessment of the method was also verified through participation in international cocoa butter proficiency test (T0638) organised by the FAPAS with excellent results in 2008. The results obtained with the described method were satisfying (z ≤ 2). The method has been applied to determine PAH in real edible oil samples.     

A comprehensive procedure based on gas chromatography–isotope ratio mass spectrometry following high performance liquid chromatography purification for the analysis of underivatized testosterone and its analogues in human urine

The confirmation by GC/C/IRMS of the exogenous origin of pseudo-endogenous steroids from human urine samples requires extracts of adequate purity. A strategy based on HPLC sample purification prior to the GC/C/IRMS analysis of human urinary endogenous androgens (i.e. testosterone, androsterone and/or androstenediols), is presented. A method without any additional derivatization step is proposed, allowing to simplify the urine pretreatment procedure, leading to extracts free of interferences permitting precise and accurate IRMS analysis, without the need of correcting the measured delta values for the contribution of the derivatizing agent. The HPLC extracts were adequately combined to both reduce the number of GC/C/IRMS runs and to have appropriate endogenous reference compounds (ERC; i.e. pregnanediol, 11-keto-etiocholanolone) on each GC–IRMS run. The purity of the extracts was assessed by their parallel analysis by gas chromatography coupled to mass spectrometry, with GC conditions identical to those of the GC/C/IRMS assay. The method has been validated according to ISO17025 requirements (within assay precision below 0.3 ‰ ¹³C delta units and between assay precision below 0.6 ‰ ¹³C delta units for most of the compounds investigated) fulfilling the World Anti-Doping Agency requirements.     

Exploring the “intensity fading” phenomenon in the study of noncovalent interactions by MALDI-TOF mass spectrometry

The difficulties to detect intact noncovalent complexes involving proteins and peptides by MALDI-TOF mass spectrometry have hindered a widespread use of this approach. Recently, "intensity fading MS" has been presented as an alternative strategy to detect noncovalent interactions in solution, in which a reduction in the relative signal intensity of low molecular mass binding partners (i.e., protease inhibitors) can be observed when their target protein (i.e., protease) is added to the sample. Here we have performed a systematic study to explore how various experimental conditions affect the intensity fading phenomenon, as well as a comparison with the strategy based on the direct detection of intact complexes by MALDI MS. For this purpose, the study is focused on two different protease-inhibitor complexes naturally occurring in solution, together with a heterogeneous mixture of nonbinding molecules derived from a biological extract, to examine the specificity of the approach, i.e., those of carboxypeptidase A (CPA) bound to potato carboxypeptidase inhibitor (PCI) and of trypsin bound to bovine pancreatic trypsin inhibitor (BPTI). Our results show that the intensity fading phenomenon occurs when the binding assay is carried out in the sub-muM range and the interacting partners are present in complex mixtures of nonbinding compounds. Thus, at these experimental conditions, the specific inhibitor-protease interaction causes a selective reduction in the relative abundance of the inhibitor. Interestingly, we could not detect any gaseous noncovalent inhibitor-protease ions at these conditions, presumably due to the lower high-mass sensitivity of MCP detectors.     

Quantitative analysis of highly homologous proteins: the challenge of assaying the “CYP-ome” by mass spectrometry

Cytochrome P450 enzymes comprise families of highly homologous proteins. These proteins play a pivotal role in oxidative drug metabolism and are important targets in drug discovery research. Proteomics today is a valuable tool for the analysis of proteins. In the past, qualitative analysis of the proteome was the main focus of research, but in the last few years interest in the mathematical modelling of protein networks has been growing and so has the demand on quantitative proteome analysis. As a thorough understanding of cytochrome P450 dependent metabolism is crucial for drug discovery, it is thus not astounding that cytochrome P450 enzymes are a target for quantitative proteomics research. In this article, we review the techniques available for quantitative proteome analysis and to what extent these techniques have been used for the quantification of cytochrome P450 enzymes and give a brief outlook of the techniques that have promising potential for the analysis of these proteins in the future.     

In vitro stable isotope labeling for discovery of novel metabolites by liquid chromatography–mass spectrometry: Confirmation of γ-tocopherol metabolism in human A549 cell

A general approach for discovering novel catabolic metabolites from a parent biocompound was developed and validated on the metabolism of γ-tocopherol in human A549 cell. The method is based on LC–MS analysis of in vitro stable isotope-labeled metabolites and assumes that a parent compound and its metabolites share a common functional group that can be derivatized by well-documented reagents. In this method, two equal aliquots of extracted metabolites are separately derivatized with isotope-coded (heavy) and non-isotope-coded (light) form of derivatizing reagent, mixed at 1:1 ratio and analyzed using LC–MS. The metabolites with common functional group are then easily recognized by determination of a chromatographically co-eluted pair of isotopomers (MS doublet peaks) with similar peak intensities and mass difference corresponding to the mass difference between heavy and light form of derivatization reagent. The feasibility of this approach was demonstrated and validated by the identification of products of γ-tocopherol catabolism in human A549 cell culture media using N-methyl-nicotinic acid N-hydroxysuccinimide ester (C1-NANHS) and N-methyl-d3-nicotinic acid N-hydroxysuccinimide ester (C1-d3-NANHS) derivatizing reagent. Overall four γ-tocopherol metabolites were identified including 9′-COOH, 11′-COOH, 13′-COOH and 13′-OH. In addition, the developed LC–MS method can also be used for the fast and sensitive quantitative analysis of γ-tocopherol and other forms of vitamin E related compounds.     

“Direct” 13C Hyperpolarization of 13C-Acetate by MicroTesla NMR Signal Amplification by Reversible Exchange (SABRE)

Herein, we demonstrate “direct” ¹³C hyperpolarization of ¹³C-acetate via signal amplification by reversible exchange (SABRE). The standard SABRE homogeneous catalyst [Ir-IMes; [IrCl(COD)(IMes)], (IMes=1,3-bis(2,4,6-trimethylphenyl), imidazole-2-ylidene; COD=cyclooctadiene)] was first activated in the presence of an auxiliary substrate (pyridine) in alcohol. Following addition of sodium 1-¹³C-acetate, parahydrogen bubbling within a microtesla magnetic field (i.e. under conditions of SABRE in shield enables alignment transfer to heteronuclei, SABRE-SHEATH) resulted in positive enhancements of up to ≈100-fold in the ¹³C NMR signal compared to thermal equilibrium at 9.4 T. The present results are consistent with a mechanism of “direct” transfer of spin order from parahydrogen to ¹³C spins of acetate weakly bound to the catalyst, under conditions of fast exchange with respect to the ¹³C acetate resonance, but we find that relaxation dynamics at microtesla fields alter the optimal matching from the traditional SABRE-SHEATH picture. Further development of this approach could lead to new ways to rapidly, cheaply, and simply hyperpolarize a broad range of substrates (e.g. metabolites with carboxyl groups) for various applications, including biomedical NMR and MRI of cellular and in vivo metabolism.     

Dispersive liquid–liquid microextraction combined with high-performance liquid chromatography–tandem mass spectrometry for the identification and the accurate quantification by isotope dilution assay of Ochratoxin A in wine samples

A novel approach for the rapid analysis of ochratoxin A (OTA) in wine samples is presented. Mycotoxin was extracted and concentrated from matrix using dispersive liquid–liquid microextraction (DLLME). The final extract is analyzed by liquid chromatography coupled to positive electrospray ionization tandem mass spectrometry employing [²H₅]-ochratoxin A as internal standard. Some important parameters, such as the nature and volume of extraction solvent and dispersive solvent, and salt effect were investigated and optimized to achieve the best extraction efficiency and higher enrichment factor. Under the optimum extraction condition, the method provided enrichment factor around 80 times and showed a high sensitivity with method detection and quantification limits of 0.005 and 0.015 ng mL⁻¹, respectively. To test the accuracy of the analytical procedure, the optimized method was applied to the analysis of reference material T1755 (naturally contaminated white wine), with excellent results (accuracy of 103%) and showing a good precision with a CV (n = 6) of 5.8%. The proposed method, which is demonstrated to be quick, cheap, accurate and highly selective, was successfully applied to the analysis of Italian wines.