Utility of porous graphitic carbon stationary phase in quantitative liquid chromatography/tandem mass spectrometry bioanalysis: quantitation of diastereomers in plasma

Abstract

A major challenge in selecting an appropriate stationary phase for diastereomeric separation is that it is difficult to predict which of the commercially available stationary phases could achieve the required liquid chromatographic (LC) separation. This work describes the selection and evaluation of a porous graphitic carbon (PGC) column coupled with tandem mass spectrometry (MS/MS) for the simultaneous quantitation of an experimental drug candidate (I), its two diastereomeric metabolites (II and III), and its demethylated metabolite (IV) in rat plasma. In addition, we investigated the PGC column for the separation of another drug candidate (VI), its two diastereomeric metabolites (VII and VIII) and its ketone metabolite (IX). The PGC column showed excellent chromatographic resolution for the two diastereomers II and III, as well as for VII and VIII. In contrast, the required resolution for the diastereomers II and III could not be achieved using silica-bonded C(18), C(30), phenyl, perfluorinated, polar embedded and polar end-capped phases. The PGC column showed ruggedness with excellent reproducibility of retention times, peak symmetry and response over a period of more than 400 injections of a plasma acetonitrile-precipitation extract. Excellent accuracy and precision were achieved, with accuracy of 94-108% and intra- and inter-run precision within 9%. This work indicates that PGC is a valuable addition to the repertoire of LC columns used for quantitative LC/MS/MS bioanalysis, especially where the separation and quantitation of diastereomeric analytes is involved.

A new, validated HPLC‐MS/MS method for the simultaneous determination of the anti‐cancer agent capecitabine and its metabolites: 5′‐deoxy‐5‐fluorocytidine, 5′‐deoxy‐5‐fluorouridine, 5‐fluorouracil and 5‐fluorodihydrouracil, in human plasma

“…The ability of this graphitic material to retain small polar molecules is superior to that of silica-based sorbents (Gamelin et al, 1998;Fleming et al, 1993;Remaud et al, 2005;Ayrton et al, 1995;Gu and Lim, 1990;Cantu et al, 2001;Mazan et al, 2002;Gaudin et al, 2002;Jackson and Carr, 2002;Hanai, 2003;Antonio et al, 2007;Pereira et al, 2007;Merelli et al, 2007;Tachon et al, 2007;Xia et al, 2006;Roy et al, 2006;Bohnstedt et al, 2005;Reepmeyer et al, 2005;Xing et al, 2004;Thiebaut et al, 2006;Vial et al, 2001;Lepont et al, 2001;Holmgren et al, 2005). Moreover, porous graphitic carbon proved to be chemically and pH stable, and an inert stationary phase.…”

Mass spectrometry in the quantitative analysis of therapeutic intracellular nucleotide analogs

“…Moreover, it has shown a remarkable selectivity for structurally similar compounds, which is most likely caused by differences in analyte contact area with the planar carbon sheets (Xia et al, 2006). PGC has therefore been used to separate cytarabine (ara-C) and its MP from their endogenous stereoisomers cytidine and its MP (Gouy et al, 2006).…”

Systematic LC‐MS/MS bioanalytical method development that incorporates plasma phospholipids risk avoidance, usage of incurred sample and well thought‐out chromatography

“…Epimeric, diastereomeric and enantiomeric metabolites (Testa et al, 1993;Xia et al, 2006a) also cause the same kind of interference since such metabolites would obviously interfere with the SRM transition used for the quantitation of the drug.…”

“…The purpose of using the modifi ed chromatographic conditions is to test if an additional peak would appear in the drug SRM channel, which would indicate the presence of a metabolite which is not separated from the drug under the initially used chromatographic conditions. With isomeric metabolites such as diastereomers, epimers and E/Z isomers, unlike metabolites that undergo in-source conversion to produce the drug molecular ion, there are no additional SRM channels that can distinguish the metabolites from the drug and hence chromatographic separation is essential (Xia et al, , 2006aTesta et al, 1993). It is impossible to know the chromatographic conditions or chromatographic run times required to achieve the chromatographic separation of the drug from such metabolites in the absence of authentic reference standards.…”