Mixed‐mode chromatography/isotope ratio mass spectrometry

Liquid chromatography coupled to molecular mass spectrometry (LC/MS) has been a standard technique since the early 1970s but liquid chromatography coupled to high‐precision isotope ratio mass spectrometry (LC/IRMS) has only been available commercially since 2004. This development has, for the first time, enabled natural abundance and low enrichment δ¹³C measurements to be applied to individual analytes in aqueous mixtures creating new opportunities for IRMS applications, particularly for the isotopic study of biological molecules. A growing number of applications have been published in a range of areas including amino acid metabolism, carbohydrates studies, quantification of cellular and plasma metabolites, dietary tracer and nucleic acid studies. There is strong potential to extend these to new compounds and complex matrices but several challenges face the development of LC/IRMS methods. To achieve accurate isotopic measurements, HPLC separations must provide baseline‐resolution between analyte peaks; however, the design of current liquid interfaces places severe restrictions on compatible flow rates and in particular mobile phase compositions. These create a significant challenge on which reports associated with LC/IRMS have not previously focused. Accordingly, this paper will address aspects of chromatography in the context of LC/IRMS, in particular focusing on mixed‐mode separations and their benefits in light of these restrictions. It aims to provide an overview of mixed‐mode stationary phases and of ways to improve high aqueous separations through manipulation of parameters such as column length, temperature and mobile phase pH. The results of several practical experiments are given using proteogenic amino acids and nucleosides both of which are of noted importance in the LC/IRMS literature. This communication aims to demonstrate that mixed‐mode stationary phases provide a flexible approach given the constraints of LC/IRMS interface design and acts as a practical guide for the development of new chromatographic methods compatible with LC/IRMS applications. Copyright © 2010 John Wiley & Sons, Ltd.     

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

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

Improved isotope ratio measurement performance in liquid chromatography/isotope ratio mass spectrometry by removing excess oxygen

A low dead volume oxygen scrubbing system was introduced in a commercially available liquid chromatography/isotope ratio mass spectrometry (LC/IRMS) interface to enhance the analytical capability of the system. In the LC/IRMS interface carbon from organic samples is converted into CO(2) inside the mobile phase by wet chemical oxidation using peroxodisulfate (Na(2)S(2)O(8)). After passing the hot reaction zone, surplus oxygen (O(2)) remains dissolved in the liquid phase. Both CO(2) and O(2) diffuse through a transfer membrane into the helium carrier and are transferred to the mass spectrometer. The presence of O(2) in the ion source may have detrimental effects on measurement accuracy and precision as well as on filament lifetime. As a remedy, a new on-line O(2)-removing device has been incorporated into the system.The new O(2) scrubber consists of two parallel hot copper reduction reactors (0.8 mm i.d., active length 120 mm) and a switch-over valve between them. One reactor is regenerated using He/H(2) while the other is actively scavenging O(2) from the gas stream. The capacity of each reduction reactor, expressed as usage time, is between 40 and 50 min. This is sufficient for a single LC run for sugars and organic acids. A further increase of the reduction capacity is accompanied by a peak broadening of about 100%. After switching to a freshly reduced reactor the oxygen background and the delta(13)C values of the reference gas need up to 500 s to stabilize. For repeated injections the delta(13)C values of sucrose remain constant (+/-0.1 per thousand) for about 3000 s. The long-term stability for measurements of sucrose was 0.11 per thousand without the reduction oven and improved slightly to 0.08 per thousand with the reduction oven. The filament lifetime improved by more than 600%, thereby improving the long-term system stability and analytical efficiency. In addition the costs per analysis were reduced considerably.     

A new concept for isotope ratio monitoring liquid chromatography/mass spectrometry

A new interface for the on-line coupling of a liquid chromatograph to a stable isotope ratio mass spectrometer has been developed and tested. The interface is usable for (13)C/(12)C determination of organic compounds, allowing measurement of small changes in (13)C abundance in individual analyte species. All of the carbon in each analyte is quantitatively converted into CO(2) while the analyte is still dissolved in the aqueous liquid phase. This is accomplished by an oxidizing agent such as ammonium peroxodisulfate. The CO(2) is separated from the liquid phase and transferred to the mass spectrometer. It is shown that the whole integrated process does not introduce isotope fractionation. The measured carbon isotope ratios are accurate and reproducible. The sensitivity of the complete system allows isotope ratio determination down to 400 ng of compound on-column. By-passing the high-performance liquid chromatography (HPLC) separation allows bulk isotopic analysis with substantially lower sample amounts than those required by conventional elemental analyzers. The results of the first applications to amino acids, carbohydrates, and drugs, eluted from various types of HPLC columns, are presented. The wide range of chromatographic methods enables the analysis of compounds never before amenable to isotope ratio mass spectrometry techniques and may lead to the development of many new assays.     

Applied gas chromatography coupled to isotope ratio mass spectrometry.

Compound-specific isotope analysis (CSIA) by isotope ratio mass spectrometry (IRMS) following on-line combustion (C) of compounds separated by gas chromatography (GC) is a relatively young analytical method. Due to its ability to measure isotope distribution at natural abundance level with great accuracy and high precision, GC-C-IRMS has increasingly become the method of choice in authenticity control of foodstuffs and determination of origin in archaeology, geochemistry, and environmental chemistry. In combination with stable isotope labelled compounds, GC-C-IRMS is also used more and more in biochemical and biomedical application as it offers a reliable and risk-free alternative to the use of radioactive tracers. The literature on these topics is reviewed from the advent of commercial GC-C-IRMS systems in 1990 up to the beginning of 1998. Demands on sample preparation and quality of GC separation for GC-C-IRMS are discussed also.     

High-temperature reversed-phase liquid chromatography coupled to isotope ratio mass spectrometry

Compound‐specific isotope analysis (CSIA) by liquid chromatography coupled to isotope ratio mass spectrometry (LC/IRMS) has until now been based on ion‐exchange separation. In this work, high‐temperature reversed‐phase liquid chromatography was coupled to, and for the first time carefully evaluated for, isotope ratio mass spectrometry (HT‐LC/IRMS) with four different stationary phases. Under isothermal and temperature gradient conditions, the column bleed of XBridge C₁₈ (up to 180 °C), Acquity C₁₈ (up to 200 °C), Triart C₁₈ (up to 150 °C), and Zirchrom PBD (up to 150 °C) had no influence on the precision and accuracy of δ¹³C measurements, demonstrating the suitability of these columns for HT‐LC/IRMS analysis. Increasing the temperature during the LC/IRMS analysis of caffeine on two C₁₈ columns was observed to result in shortened analysis time. The detection limit of HT‐RPLC/IRMS obtained for caffeine was 30 mg L^–1 (corresponding to 12.4 nmol carbon on‐column). Temperature‐programmed LC/IRMS (i) accomplished complete separation of a mixture of caffeine derivatives and a mixture of phenols and (ii) did not affect the precision and accuracy of δ¹³C measurements compared with flow injection analysis without a column. With temperature‐programmed LC/IRMS, some compounds that coelute at room temperature could be baseline resolved and analyzed for their individual δ¹³C values, leading to an important extension of the application range of CSIA. Copyright © 2011 John Wiley & Sons, Ltd.     

Determination of phenylalanine isotope ratio enrichment by liquid chromatography/time- of-flight mass spectrometry

The application of time-of-flight mass spectrometry to isotope ratio measurements has been limited by the relatively low dynamic range of the time-to-digital converter detectors available on commercial LC/ToF-MS systems. Here we report the measurement of phenylalanine isotope ratio enrichment by using a new LC/ToF-MS system with wide dynamic range. Underivatized phenylalanine was injected onto a C18 column directly with 0.1% formic acid/acetonitrile as the mobile phase. The optimal instrument parameters for the time-of-flight mass spectrometer were determined by tuning the instrument with a phenylalanine standard. The accuracy of the isotope enrichment measurement was determined by the injection of standard solutions with known isotope ratios ranging from 0.02% to 9.2%. A plot of the results against the theoretical values gave a linear curve with R2 of 0.9999. The coefficient of variation for the isotope ratio measurement was below 2%. The method is simple, rapid, and accurate and presents an attractive alternative to traditional GC/MS applications.     

Carbon isotope ratio measurements of glyphosate and AMPA by liquid chromatography coupled to isotope ratio mass spectrometry

The interest in compound-specific isotope analysis for product authenticity control and source differentiation in environmental sciences has grown rapidly during the last decade. However, the isotopic analysis of very polar analytes is a challenging task due to the lack of suitable chromatographic separation techniques which can be used coupled to isotope ratio mass spectrometry. In this work, we present the first method to measure carbon isotope compositions of the widely applied herbicide glyphosate and its metabolite aminomethylphosphonic acid (AMPA) by liquid chromatography coupled to isotope ratio mass spectrometry. We demonstrate that this analysis can be carried out either in cation exchange or in reversed-phase separation modes. The reversed-phase separation yields a better performance in terms of resolution compared with the cation exchange method. The measurement of commercial glyphosate herbicide samples show its principal applicability and reveals a wide range of δ¹³C values between ?24 and ?34 ‰ for different manufacturers. The absolute minimum amounts required to perform a precise and accurate determination of carbon isotope compositions of glyphosate and AMPA were in the sub-microgram range. The method proposed is sensitive enough to further perform the experiments that are necessary to better understand the carbon isotope fractionation associated to the natural degradation of glyphosate into AMPA. Furthermore, it can be used for contaminant source allocation and product authenticity as well.     

Position‐specific carbon isotope analysis of trichloroacetic acid by gas chromatography/isotope ratio mass spectrometry

Trichloroacetic acid (TCAA) is an important environmental contaminant present in soils, water and plants. A method for determining the carbon isotope signature of the trichloromethyl position in TCAA using gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) was developed and tested with TCAA from different origins. Position‐specific isotope analysis (PSIA) can provide direct information on the kinetic isotope effect for isotope substitution at a specific position in the molecule and/or help to distinguish different sources of a compound. The method is based on the degradation of TCAA into chloroform (CF) and CO₂ by thermal decarboxylation. Since thermal decarboxylation is associated with strong carbon isotope fractionation (ε = ?34.6 ± 0.2‰) the reaction conditions were optimized to ensure full conversion. The combined isotope ratio of CF and CO₂ at the end of the reaction corresponded well to the isotope ratio of TCAA, confirming the reliability of the method. A method quantification limit (MQL) for TCAA of 18.6 µg/L was determined. Samples of TCAA produced by enzymatic and non‐enzymatic chlorination of natural organic matter (NOM) and some industrially produced TCAA were used as exemplary sources. Significant different PSIA isotope ratios were observed between industrial TCAA and TCAA samples produced by chlorination of NOM. This highlights the potential of the method to study the origin and the fate of TCAA in the environment. Copyright © 2011 John Wiley & Sons, Ltd.     

A versatile method for stable carbon isotope analysis of carbohydrates by high-performance liquid chromatography/isotope ratio mass spectrometry

We have developed a method to analyze stable carbon isotope ((13)C/(12)C) ratios in a variety of carbohydrates using high-performance liquid chromatography/isotope ratio mass spectrometry (HPLC/IRMS). The chromatography is based on strong anion-exchange columns with low strength NaOH eluents. An eluent concentration of 1 mM resulted in low background signals and good separation of most of the typical plant neutral carbohydrates. We also show that more strongly bound carbohydrates such as acidic carbohydrates can be separated by inclusion of NO(3) (-) as an inorganic pusher ion in the eluent. Analyses of neutral carbohydrate concentrations and their stable carbon isotope ratios are shown for plant materials and marine sediment samples both at natural abundance and for (13)C-enriched samples. The main advantage of HPLC/IRMS analysis over traditional gas chromatography based methods is that no derivatization is needed resulting in simple sample treatment and improved accuracy and reproducibility.     

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.     

Measurement of insulin sensitivity indices using 13C-glucose and gas chromatography/combustion/isotope ratio mass spectrometry

Important aspects of glucose metabolism can be quantified by using the minimal model of glucose kinetics to interpret the results of intravenous glucose tolerance tests. The power of this methodology can be greatly increased by the addition of stable isotopically labelled tracer to the glucose bolus dose. This allows the separation of glucose disposal from endogenous glucose production and also increases the precision of the estimates of the physiological parameters measured. Until now the tracer of choice has been deuteriated glucose and the analytical technique has been gas chromatography/mass spectrometry (GC/MS). The consequence of this choice is that nearly 2 g of labelled material are needed and this makes the test expensive. We have investigated the use of (13)C-labelled glucose as the tracer in combination with gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) as the analytical technique. This methodology offers superior analytical precision when compared with the conventional method and so the amount of tracer used, and hence the cost, can be reduced considerably. Healthy non-obese male volunteers were recruited for a standard intravenous glucose tolerance test (IVGTT) protocol but 6,6-(2)H-glucose and 1-(13)C-glucose were administered simultaneously. Tracer/tracee ratios were derived from isotope ratio measurements of plasma glucose using both GC/MS and GC/C/IRMS. The results of these determinations indicated that the two tracers behaved identically under the test protocol. The combination of these results with plasma glucose and insulin concentration data allowed determination of the minimal model parameters S*g and S*i. The parameter relating to insulin-assisted glucose disposal, S*i, was found to be the same in the two techniques, but this was not the case for the non-insulin-dependent parameter S*g.     

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.     

Forensic isotope ratio mass spectrometry of packaging tapes

Pressure sensitive adhesive tape (brown parcel tape) is employed in a great many criminal activities such as the restraint of individuals during robbery and offences against the person, the enclosure of explosive devices and the packaging and concealment of controlled drugs. Packaging materials are ubiquitous in modern society and are produced in such vast quantities that it is increasingly difficult to distinguish between different products or to link materials to a common source. This study demonstrates the potential of stable isotope ratio mass spectrometry to characterise parcel tapes based on a number of properties. The carbon isotopic signature, derived from the substrate polymer, associated additives and adhesive is highly characteristic of a particular tape and allows samples from different sources to be readily distinguished. Further discrimination may be achieved by the incorporation of deuterium and oxygen isotopic data and by analysis of the isolated backing polymer. Recovery of intact tape from simulated forensic samples proved straightforward and the isotopic signature of the tape did not appear to be affected by adverse storage conditions.     

液相色谱-同位素比质谱技术发展及应用研究进展

液相色谱-同位素比质谱(LC-IRMS)是一种特征化合物同位素分析技术,该技术利用LC IsoLink接口设备实现液相色谱与同位素比质谱的联用,通过检测目标物质的稳定碳同位素比(δ¹³C),实现样品的产地来源与品质真实性鉴定。该文总结了IRMS与LC-IRMS技术的概况,以及过去20年LC-IRMS的发展历程;归纳整理了LC-IRMS在食品安全、生态与环境、生命科学及考古学等领域的应用情况;评述了LC-IRMS面临的技术局限、挑战及其未来的发展趋势。     

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 %.