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Rationale: Despite a wide range of potential applications, magnesium (Mg) isotope composition has been so far sparsely measured in reference materials with a biological matrix, which is important for the quality control of the results. We describe a method enabling the chemical separation of Mg in geological and biological materials and the determination of its stable isotope composition., Methods: Different geological (BHVO-1, BHVO-2, BCR-1, and IAPSO) and biological (SRM-1577c, BCR-383, BCR380R, ERM-CE464, DORM-2, DORM-4, TORT-3, and FBS) reference materials were used to test the performance of a new sample preparation procedure for Mg isotopic analysis. The procedure consisted of a simple three-stage elution method to separate Mg from the matrix. Mg isotopic analyses were performed in two different laboratories and with three different multi-collector inductively coupled plasma mass spectrometry instruments., Results: The biological reference materials show a wide range of δ 26 Mg values (relative to DSM3 standard), spanning over 2‰, from 0.52 ± 0.29‰ (2SD, n = 7) in bovine liver (SRM-1577c) to -1.45 ± 0.20‰ (2SD, n = 5) in tuna fish (ERM-CE464), with an external precision of 0.03‰ (2SD, n = 85)., Conclusions: This study indicates that isotopic measurements of Mg in biological reference materials show good performance, with the results being within the accepted range. We confirmed that δ 26 Mg values in liver are the most positive of all biological materials reported so far., (© 2021 John Wiley & Sons Ltd.)

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., (Copyright © 2011 John Wiley & Sons, Ltd.)

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., (Copyright © 2011 John Wiley & Sons, Ltd.)

Determination of glutathione kinetics using stable isotopes requires accurate measurement of the tracers and tracees. Previously, the precursor and synthesized product were measured with two separate techniques, liquid chromatography/isotope ratio mass spectrometry (LC/IRMS) and gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS). In order to reduce sample volume and minimize analytical effort we developed a method to simultaneously determine (13)C-glutathione as its dimeric form (GSSG) and its precursor [1-(13)C]glycine in a small volume of erythrocytes in one single analysis. After having transformed (13)C-glutathione into its dimeric form GSSG, we determined both the intra-erythrocytic concentrations and the (13)C-isotopic enrichment of GSSG and glycine in 150 microL of whole blood using liquid chromatography coupled to LC/IRMS. The results show that the concentration (range of micromol/mL) was reliably measured using cycloleucine as internal standard, i.e. with a precision better than 0.1 micromol/mL. The (13)C-isotopic enrichment of GSSG and glycine measured in the same run gave reliable values with excellent precision (standard deviation (sd) <0.3 per thousand) and accuracy (measured between 0 and 5 APE). This novel method opens up a variety of kinetic studies with relatively low dose administration of tracers, reducing the total cost of the study design. In addition, only a minimal sample volume is required, enabling studies even in very small subjects, such as preterm infants., (Copyright (c) 2009 John Wiley & Sons, Ltd.)

Under conditions of high isotopic dilution, e.g. in a tracer study, the ability to determine accurately and quantitatively small variations in isotopic enrichments of differently labelled chemical compounds (e.g. (13)C and (15)N in threonine) in a single run by gas chromatography/mass spectrometry (GC/MS) is desirable but remains a technological challenge. Here, we report a new, rapid and simple GC/MS method for simultaneously measuring the isotopic enrichments of doubly labelled threonine ([U(13)C] and (15)N) with isotopic enrichment lower than 1.5 Molar Percent Excess (MPE). The long-term reproducibility measured was around 0.09 MPE for both tracers (throughout a 6 week period). The intra-day repeatability was lower than 0.05 and 0.06 MPE for [U(13)C]-Thr and (15)N-Thr, respectively. To calculate both isotopic enrichments, two modes of calculations were used: one based on work by Rosenblatt et al. in 1992 and the other one using a matrix approach. Both methods gave similar results (ANOVA, P >0.05) with close precision for each mode of calculation. The GC/MS method was then used to investigate the differential utilization of threonine in different organs according to its route of administration in minipigs after administration of both tracers. In plasma samples, the lowest isotopic enrichment measured between two successive time points was at 0.01 and 0.02 MPE for [U(13)C]-Thr and (15)N-Thr, respectively. Moreover, the accuracy of GC/MS (13)C-isotopic enrichment measured was validated by analyzing the same plasma samples by gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS). Statistical analysis showed that both techniques gave the same results (ANOVA, P >0.05). This new GC/MS method offers the possibility to measure (13)C- and (15)N-isotopic enrichments with higher throughput, and using a lower amount of sample, than using GC/C/IRMS.

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.

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 %.

This paper validates the use of high-temperature conversion elemental analyser isotope-ratio mass spectrometry (TC-EA/IRMS) for measuring the [(2)H/H] enrichment of plasma [(2)H(5)]cholesterol. From a molecular point of view, the free cholesterol is initially separated from plasma by thin-layer chromatography (TLC) and then injected onto the TC-EA reactor which converts cholesterol molecules into CO and H(2) gases. The slope of the curve of the experimental mole percent excess (MPE((exp.))) versus MPE((theor.)) was very close to 1, demonstrating that no significant isotopic fractionation was observed during all processing of the samples (i.e., isolation of plasma free cholesterol by TLC and pyrolysis in the TC-EA reactor). Excellent linearity (r(2) = 0.9994, n = 4) of delta ( per thousand ) of [(2)H/H] isotopic measurements versus mole percent (MP) was assessed over the range 0 to 0.1 MP. The precision of the [(2)H/H] measurement, evaluated with two calibration points processed with TLC, was delta(2)H(V-SMOW) = -192.5 +/- 3.4 per thousand and delta(2)H(V-SMOW) = -136.9 +/- 2.9 per thousand. The standard deviations of the within-assay and between-assay repeatabilities of the analytical process, evaluated using the quality control (QC) of plasma samples, were 4.6 and 6.1 per thousand, respectively. Plant sterols are known to reduce cholesterol absorption and therefore were used as a positive control in a clinical study performed with normocholesterolemic volunteers. This present method produces biological results consistent with those already reported in the literature., (Copyright 2004 John Wiley & Sons, Ltd.)