Mixed-mode chromatography/isotope ratio mass spectrometry y James S. O. McCullagh * Chemistry Research Laboratory, University of Oxford, Mansfield Rd, Oxford OX1 3TA, UK Received 4 August 2009; Revised 30 September 2009; Accepted 2 October 2009 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 d 13 C measurements to be applied to individual analytes in aqueous mixtures creating new opportunities for IRMS appli- cations, 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 separ- ations 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. Development of commercially available interfaces between liquid chromatography and isotope ratio mass spectrometry (LC/IRMS) has provided a novel way to measure carbon isotope ratios in aqueous mixtures. 1 The IsoLink (Thermo Fisher, Bremen, Germany) and the LiquiFace, a more recent development (Isoprime, Cheadle Hulme, UK), enable a conventional high-performance liquid chromatography (HPLC) system to be coupled with an isotope ratio mass spectrometer such that a mixture of analytes injected into the HPLC system can be separated, oxidized to CO 2 in the interface and carbon isotope ratios measured, in a continuous process (Fig. 1). Without a chromatographic column inline the HPLC instrument can be used as a liquid injector providing elemental analysis (EA) where single compounds in solution can be injected and carbon isotopes measured. The precision, accuracy and linearity of the LC-IsoLink have been reported, in a number of publications, for several different compounds. 1,2,5,6 The recent introduction of LC/IRMS has provided new research opportunities in a wide range of fields including chemical archaeology, biochemistry, food adulteration, medi- cine, physiology and soil biochemistry. 2–12 Compounds that are strongly polar, thermally non-labile or otherwise unamen- able to analysis by gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) can now be subject to compound-specific isotope measurement in aqueous mixtures; however, the chromatographic separation of analytes prior to oxidation requires baseline resolution for accurate isotopic measurement. This, in particular, provides one of the key challenges facing the development of new applications. It should be noted that baseline resolution is far more important for LC/IRMS measurements than for conventional LC/MS (liquid chromatography coupled to molecular mass spectrom- etry). For LC/IRMS integration of CO þ 2 peaks (produced by the oxidation and ionization of chromatographically separated analyte peaks) is used to determine the carbon isotope ratio; hence, co-eluting peaks would lead directly to imprecise isotopic measurements. Mass measurements using RAPID COMMUNICATIONS IN MASS SPECTROMETRY Rapid Commun. Mass Spectrom. 2010; 24: 483–494 Published online in Wiley InterScience (www.interscience.wiley.com) DOI: 10.1002/rcm.4322 *Correspondence to: J. S. O. McCullagh, Chemistry Research Laboratory, University of Oxford, Mansfield Rd, Oxford OX1 3TA, UK. E-mail: James.McCullagh@chem.ox.ac.uk y Presented at SIMSUG 2009, held 14–15 January 2009 at the University of Glasgow. Copyright # 2010 John Wiley & Sons, Ltd.