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J Chromatogr B Analyt Technol Biomed Life Sci. 2013 Aug 1;932:40-9. doi: 10.1016/j.jchromb.2013.06.006. Epub 2013 Jun 13.

A rapid and sensitive assay to quantify valproyl 1-O-acyl glucuronide in supernatants of sandwich-cultured rat hepatocytes using ultra-high performance liquid chromatography-tandem mass spectrometry.

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  • 1Faculty of Pharmaceutical Sciences, The University of British Columbia, Vancouver, British Columbia, Canada.

Abstract

A rapid and sensitive ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed and validated for the determination of valproyl-1-O-acyl glucuronide (VPA-G) levels in hepatocyte culture medium. Chromatographic separation was achieved using a Waters Acquity UPLC(®) BEH C18 column (1.7μm, 2.1mm×50mm) with gradient elution and a total run time of 4min. [(2)H6]-VPA-G was used as internal standard (IS). Quantification was performed in the multiple reaction monitoring (MRM) mode using the total ion current of the MRM transition pairs m/z 319.1→142.7 and m/z 319.1→175.2 for VPA-G, and m/z 325.1→149.3 and m/z 325.1→174.9 for the IS under negative electrospray ionization mode. The assay was linear over the VPA-G concentrations of 0.5-500ng/mL, with a r(2) value of 0.995±0.002 (mean±SD). The intra- and inter-day accuracy (% deviation) ranged from -10.2% to 11.1%, whereas the intra- and inter-day precision (% RSD) were ≤7.43%. The method was applied successfully to the quantification of VPA-G levels in culture supernatants of sandwich-cultured rat hepatocytes treated with valproic acid (VPA). No significant difference in the levels of VPA-G over a culture period of 6 days was observed in an experiment that investigated the effect of the age of hepatocyte culture on the extent of VPA glucuronidation. The method presented here for the direct quantification of VPA-G is an improvement of existing methods in the literature and offers a shorter run time and greater sensitivity that enables the use of small volumes of sample. To the best of our knowledge, this is the first validated UHPLC-MS/MS method applied to the quantification of VPA-G in cell culture supernatants.

Copyright © 2013 Elsevier B.V. All rights reserved.

KEYWORDS:

IS; MRM; Rat hepatocytes; SIM; TIC; UHPLC–MS/MS; VPA; VPA-G; Valproic acid; Valproyl 1-O-acyl glucuronide; internal standard; multiple reaction monitoring; selected ion monitoring; total ion current; ultra-high performance liquid chromatography–tandem mass spectrometry; valproic acid; valproyl 1-O-acyl glucuronide

PMID:
23827518
[PubMed - indexed for MEDLINE]
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