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.     

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.     

Isotope ratio mass spectrometry coupled to liquid and gas chromatography for wine ethanol characterization

Two new procedures for wine ethanol 13C/12C isotope ratio determination, using high-performance liquid chromatography and gas chromatography isotope ratio mass spectrometry (HPLC/IRMS and GC/IRMS), have been developed to improve isotopic methods dedicated to the study of wine authenticity. Parameters influencing separation of ethanol from wine matrix such as column, temperature, mobile phase, flow rates and injection mode were investigated. Twenty-three wine samples from various origins were analyzed for validation of the procedures. The analytical precision was better than 0.15 per thousand, and no significant isotopic fractionation was observed employing both separative techniques coupled to IRMS. No significant differences and a very strong correlation (r = 0.99) were observed between the 13C/12C ratios obtained by the official method (elemental analyzer/isotope ratio mass spectrometry) and the proposed new methodology. The potential advantages of the developed methods over the traditional one are speed (reducing time required from hours to minutes) and simplicity. In addition, these are the first isotopic methods that allow 13C/12C determination directly from a liquid sample with no previous ethanol isolation, overcoming technical difficulties associated with sample treatment.     

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.     

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.     

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。     

Microfluidic chip-based liquid chromatography coupled to mass spectrometry for determination of small molecules in bioanalytical applications

The development and integration of microfabricated liquid chromatography (LC) microchips have increased dramatically in the last decade due to the needs of enhanced sensitivity and rapid analysis as well as the rising concern on reducing environmental impacts of chemicals used in various types of chemical and biochemical analyses. Recent development of microfluidic chip-based LC mass spectrometry (chip-based LC-MS) has played an important role in proteomic research for high throughput analysis. To date, the use of chip-based LC-MS for determination of small molecules, such as biomarkers, active pharmaceutical ingredients (APIs), and drugs of abuse and their metabolites, in clinical and pharmaceutical applications has not been thoroughly investigated. This mini-review summarizes the utilization of commercial chip-based LC-MS systems for determination of small molecules in bioanalytical applications, including drug metabolites and disease/tumor-associated biomarkers in clinical samples as well as adsorption, distribution, metabolism, and excretion studies of APIs in drug discovery and development. The different types of commercial chip-based interfaces for LC-MS analysis are discussed first and followed by applications of chip-based LC-MS on biological samples as well as the comparison with other LC-MS techniques.     

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.     

Novel method for measurement of glutathione kinetics in neonates using liquid chromatography coupled to isotope ratio mass spectrometry

A novel analytical method using liquid chromatography coupled to isotope ratio mass spectrometry (LC/IRMS) was developed for measuring the fractional synthesis rate (FSR) of glutathione (GSH) in neonates after infusion of [1-(13)C]-glycine as a tracer. After transformation of GSH into GSSG, its dimeric form, the intra-erythrocytic concentration and (13)C-isotopic enrichment of GSH were determined using 200 microL of blood. The results showed that, using LC/IRMS, the concentration (range of micromol/mL) was reliably measured using norvaline as internal standard with precision better than 0.1 micromol/mL. In addition, the (13)C-isotopic enrichment measured in the same run gave reliable values with excellent precision (with standard deviation (sd) lower than 0.3 per thousand) and accuracy (measured between 0 and 2 Atom % Excess (APE)). The inter-assay repeatability of delta(13)C of norvaline used as internal standard with in vivo samples was assessed at -26.07 +/- 0.28 per thousand with coefficient of variance (CV) at 1.1%. The FSR calculated either with GSH or GSSG showed similar results with slightly higher values for GSSG (41.6 +/- 4.7 and 46.5 +/- 4.4, respectively). The slightly lower FSR of GSH is probably due to interfering compounds in the biological matrix. Successfully used in a clinical study, this rapid and reliable method opens up a variety of kinetic studies with relatively low administration of tracer infusates, reducing the total cost of the study design. The small volume of blood needed enables studies even in extremely small subjects, such as premature infants, as reported in this study.     

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.     

Strong anion‐exchange liquid chromatography coupled with isotope ratio mass spectrometry using a Liquiface interface

The introduction of liquid chromatography coupled with isotope ratio mass spectrometry (LC/IRMS) as an analytical tool for the measurement of isotope ratios in non‐volatile analytes has somewhat simplified the analytical cycle from sample collection to analysis mainly due to the avoidance of the extensive sample processing and derivatisation that were necessary for gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS). Here we test the performance of coupling strong anion exchange to IRMS using only the second commercially available interface; the Liquiface. The system was modified from installation specification to improve peak resolution in the interface and maintain peak separation from the column to the mass spectrometer. The system performance was assessed by the determination of sensitivity, accuracy and precision attained from carbohydrate separations. The system performed satisfactorily after modifications, resulting in maintenance of peak resolution from column to mass spectrometer. The sensitivity achieved suggested that ～150 ng carbon could be analysed with acceptable precision (<0.3‰). Accuracy was maintained in the interface as determined by correlation with offline techniques, resulting in regression coefficient of r² = 0.98 and a slope of 0.99. The average precision achieved for the separation of seven monosaccharides was 0.36‰. The integration of a carbonate removal device limited the effect of background carbon perturbations in the mass spectrometer associated with eluent gradients, and the coupling of strong anion‐exchange chromatography with IRMS was successfully achieved using the Liquiface. Copyright © 2010 John Wiley & Sons, Ltd.     

Review: Current applications and challenges for liquid chromatography coupled to isotope ratio mass spectrometry (LC/IRMS)

High-precision isotope analysis is recognized as an essential research tool in many fields of study. Until recently, continuous flow isotope ratio mass spectrometry (CF-IRMS) was available via an elemental analyzer or a gas chromatography inlet system for compound-specific analysis of light stable isotopes. In 2004, however, an interface that coupled liquid chromatography with IRMS (LC/IRMS) became commercially available for the first time. This brought the capability for new areas of application, in particular enabling compound-specific δ(13)C analysis of non-volatile, aqueous soluble, compounds from complex mixtures. The interface design brought with it several analytical constraints, however, in particular a lack of compatibility with certain types of chromatography as well as limited flow rates and mobile phase compositions. Routine LC/IRMS methods have, however, been established for measuring the δ(13)C isotopic ratios of underivatized individual compounds for application in archeology, nutrition and physiology, geochemistry, hydrology, soil science and food authenticity. Seven years after its introduction, we review the technical advances and constraints, methodological developments and new applications of liquid chromatography coupled to isotope ratio mass spectrometry.     

Quantitative determination of chloramphenicol in milk powders by isotope dilution liquid chromatography coupled to tandem mass spectrometry

A method is described for the determination of residues of the illegal antibiotic chloramphenicol (CAP) in milk powders. The analyte is quantified by liquid chromatography coupled to electrospray ionisation tandem mass spectrometry (LC-ESI-MS-MS) operating in negative ion multiple reaction monitoring mode (MRM) after a liquid-liquid extraction followed by a clean-up step on solid phase extraction (SPE) cartridge. Because of the presence of two chlorine atoms in the CAP molecule, four specific transition reactions of CAP were monitored by MS-MS in selecting m/z 321 --> 257, 321 --> 152 (35Cl2) and m/z 323 --> 257, 323 --> 152 (37Cl35Cl). Two calibration curves were constructed by plotting the area ratio of m/z 321 --> 152 versus 326 --> 157 and m/z 321 --> 257 versus 326 --> 262 against their corresponding amount ratio. Indeed, even if m/z 321 --> 152 was found to give a higher MS-MS response (calibration curve used by default), an interfering chemical substance was sometimes observed for some milk extracts and not for the transition m/z 321 --> 257. The quantitation method was validated according to the European Union (EU) criteria for the analysis of veterinary drug residues at 0.1, 0.2 and 0.5 microg/kg concentration levels using d5-CAP as internal standard. The decision limit (CCalpha) and detection capability (CCbeta) of CAP in milk were calculated for m/z 321 --> 152 at 0.02 microg/kg and 0.03 microg/kg, respectively, and for m/z 321 --> 257 at 0.02 microg/kg and 0.04 microg/kg, respectively. At the lowest fortification level (i.e. 0.1 microg/kg), repeatability and within-laboratory reproducibility were calculated for m/z 321 --> 257 both at 0.02 microg/kg and for m/z 321 --> 152 at 0.03 and 0.05 microg/kg, respectively. Moreover, the measurement of uncertainty of the analytical method was calculated at the same spiking levels and falls within the precision values of the within-laboratory reproducibility. This method can be applied to several types of milk powders (e.g. full cream, skim) and can serve as a monitoring tool to avoid that unacceptable levels of residues of CAP enter the food chain.     

Ultra-performance liquid chromatography coupled to quadrupole-orthogonal time-of-flight mass spectrometry

Ultra-performance liquid chromatography (UPLC) utilizes sub-2 microm particles with high linear solvent velocities to effect dramatic increases in resolution, sensitivity and speed of analysis. The reduction in particle size to below 2 microm requires instrumentation that can operate at pressures in the 6000-15,000 psi range. The typical peak widths generated by the UPLC system are in the order of 1-2 s for a 10-min separation. In the present work this technology has been applied to the study of in vivo drug metabolism, in particular the analysis of drug metabolites in bile. The reduction in peak width significantly increases analytical sensitivity by three- to five-fold, and the reduction in peak width, and concomitant increase in peak capacity, significantly reduces spectral overlap resulting in superior spectral quality in both MS and MS/MS modes. The application of UPLC/MS resulted in the detection of additional drug metabolites, superior separation and improved spectral quality.     

The role of liquid chromatography and flow injection analyses coupled to isotope ratio mass spectrometry for studying human in vivo glucose metabolism

Under most physiological conditions, glucose, or carbohydrate (CHO), homeostasis is tightly regulated. In order to mechanistically appraise the origin of circulating glucose (e.g. via either gluconeogenesis, glycogenolysis or oral glucose intake), and its regulation and oxidation, the use of stable isotope tracers is now a well-accepted analytical technique. Methodologically, liquid chromatography coupled to isotope ratio mass spectrometry (LC/IRMS) can replace gas chromatography coupled to combustion-isotope ratio mass spectrometry (GC/C/IRMS) for carrying out compound-specific (13)C isotopic analysis. The LC/IRMS approach is well suited for studying glucose metabolism, since the plasma glucose concentration is relatively high and the glucose can readily undergo chromatography in an aqueous mobile phase. Herewith, we report two main methodological approaches in a single instrument: (1) the ability to measure the isotopic enrichment of plasma glucose to assess the efficacy of CHO-based treatment (cocoa-enriched) during cycling exercise with healthy subjects, and (2) the capacity to carry out bulk isotopic analysis of labeled solutions, which is generally performed with an elemental analyzer coupled to IRMS. For plasma samples measured by LC/IRMS the data show a isotopic precision SD(δ(13)C) and SD(APE) of 0.7 ‰ and 0.001, respectively, with δ(13)C and APE values of -25.48 ‰ and 0.06, respectively, being generated before and after tracer administration. For bulk isotopic measurements, the data show that the presence of organic compounds in the blank slightly affects the δ(13)C values. Despite some analytical limitations, we clearly demonstrate the usefulness of the LC/IRMS especially when (13)C-glucose is required during whole-body human nutritional studies.     

Determination of halogenated flame retardants by liquid chromatography coupled to mass spectrometry

We present an overview of current analytical methods for selected halogenated flame retardants (HFRs), focusing on instrumental determination using liquid chromatography coupled to mass spectrometry. We based the strategy for literature search on recent articles published in peer-reviewed scientific journals or conference proceedings. We report on selected HFRs and some metabolites and transformation products, and on the analytical performances of different ionization modes, with emphasis on selectivity and sensitivity. Moreover, we compare these parameters with those obtained by gas chromatography.     

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.     

Comprehensive two-dimensional liquid chromatography coupled with mass spectrometry

In this review, instrumental aspects of comprehensive two-dimensional liquid chromatography coupled with mass spectrometry are presented. The milestones of LC×LC are briefly summarized. Instrument configuration, selection of experimental conditions, the different interfaces used in the system and the current applications of LC×LC–MS systems are described.