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Development and validation of an assay for the quantitative determination of cladribine nucleotides in MDCKII cells and culture medium using weak anion‐exchange liquid chromatography coupled with tandem mass spectrometry
Author(s) -
Jansen Robert S.,
Rosing Hilde,
de Wolf Cocky J. F.,
Beijnen Jos H.
Publication year - 2007
Publication title -
rapid communications in mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.528
H-Index - 136
eISSN - 1097-0231
pISSN - 0951-4198
DOI - 10.1002/rcm.3318
Subject(s) - chemistry , chromatography , tandem mass spectrometry , mass spectrometry , electrospray , liquid chromatography–mass spectrometry , analyte , lysis , biochemistry
The development and validation of an assay for the quantitative analysis of cladribine mono‐, di‐ and triphosphate (2‐chloro, 2′‐deoxyadenosine 5′‐mono‐, di‐ and triphosphate or 2CdAMP, 2CdADP and 2CdATP) in culture medium (Optimem) and cell lysate is described. Cladribine mono‐ and diphosphate reference compounds were obtained by thermal degradation of cladribine triphosphate. The reference compounds were characterized using ion‐pairing reversed‐phase high‐performance liquid chromatography with ultraviolet detection. The bioanalytical assay for 2CdAMP, 2CdADP and 2CdATP is based on weak anion‐exchange liquid chromatography coupled with tandem mass spectrometry in the positive ion mode (WAXLC/MS/MS). A fused‐silica electrospray capillary was used instead of a stainless steel electrospray capillary to minimize adsorption of analytes and thus decrease variation in the analyte signals. Dynamic ranges of 1.11–27.7, 0.550–55.0 and 1.31–52.3 nM for 2CdAMP, 2CdADP and 2CdATP, respectively, were validated in culture medium and cell lysate. Optimem samples required stabilization with 30% methanol to prevent conversion of 2CdATP into 2CdAMP and 2CdADP. All intra‐ and interday accuracies and precisions were within ±20%. The stability of the compounds was assessed under various analytically relevant conditions. The method was successfully used to investigate cladribine nucleotide transport in vitro in Madin‐Darby canine kidney II (MDCKII) cells. Copyright © 2007 John Wiley & Sons, Ltd.