How to Couple LC-IRMS with HRMS─A Proof-of-Concept Study

Abstract

Compound-specific stable isotope analysis (CSIA) is a unique analytical technique for determining small variations in isotope ratios of light isotopes in analytes from complex mixtures. A problem of CSIA using gas chromatography (GC) and liquid chromatography-isotope ratio mass spectrometry (LC-IRMS) is that any structural information of the analytes is lost due to the processes involved in determining the isotope ratio. To obtain the isotopic composition of, for example, carbon from organic compounds, all carbon in each analyte is quantitatively converted to CO 2 . For GC-IRMS, open split GC-IRMS-MS couplings have been described that allow additional acquisition of structural information of analytes and interferences. Structural analysis using LC-IRMS is more difficult and requires additional technical and instrumental efforts. In this study, LC was combined for the first time with simultaneous analysis by IRMS and high-resolution mass spectrometry (HRMS), enabling the direct identification of unknown or coeluting species. We have thoroughly investigated and optimized the coupling and showed how technical problems, arising from instrumental conditions, can be overcome. To this end, it was successfully demonstrated that a consistent split ratio between IRMS and HRMS could be obtained using a variable postcolumn flow splitter. This coupling provided reproducible results in terms of resulting peak areas, isotope values, and retention time differences for the two mass spectrometer systems. To demonstrate the applicability of the coupling, we chose to address an important question regarding the purity of international isotope standards. In this context, we were able to confirm that the USGS41 reference material indeed contains substantial amounts of pyroglutamic acid as suggested previously in the literature. Moreover, the replacement material, USGS41a, still has significant amounts of pyroglutamic acid as impurity, rendering some caution necessary when using this material for isotopic calibration.

Insights into amino acid fractionation and incorporation by compound-specific carbon isotope analysis of three-spined sticklebacks

“…Five primary RM were measured on the LC-IRMS system before the start of a four week-long measurement (10 and 5 µL injection volumes, each in triplicate). The measured δ 13 C values of the five RM were calibrated with the AA in-house standards and were in good agreement with the true literature values except for USGS 41, which showed conversion of glutamic acid to pyroglutamic acid and is a known problem even with the newer USGS 41a material 70 . Chromatograms were individually checked for proper background and peak detection.…”

Discovering Nature’s Fingerprints: Isotope Ratio Analysis on Bioanalytical Mass Spectrometers

“…Initial attempts to couple LC with ESI-Orbitrap for IRMS are promising. 82 This approach is particularly attractive for biological samples from systems that…”

“…Initial attempts to couple LC with ESI-Orbitrap for IRMS are promising. 82 This approach is particularly attractive for biological samples from systems that cannot be controlled in a laboratory and in which labeling is prone to artifacts, which are often associated with biological isotope effects. For example, in human nutrition and clinical research, the investigation of unlabeled studies is particularly attractive.…”

“…In metabolism studies, it is often desirable to quantify several analytes in a single analysis, and some trade-off with regards to isotopic accuracy may be acceptable. Initial attempts to couple LC with ESI-Orbitrap for IRMS are promising . This approach is particularly attractive for biological samples from systems that cannot be controlled in a laboratory and in which labeling is prone to artifacts, which are often associated with biological isotope effects.…”

Transformation of Iminodi(methylene phosphonate) on Manganese Dioxides – Passivation of the Mineral Surface by (Formed) Mn2+

“…Investigation of further TPs was conducted using LC-IRMS hyphenated to a high-resolution mass spectrometer (LC-IRMS-HRMS) after Marks et al 24 All instruments and measurement conditions for LC-IRMS and LC-IRMS-HRMS can be found in the SI.…”