Development and application of a rapid and sensitive liquid chromatography-mass spectrometry method for simultaneous analysis of cytarabine, cytarabine monophosphate, cytarabine diphosphate and cytarabine triphosphate in the cytosol and nucleus

  • J Pharm Biomed Anal. 2022 Mar 20:211:114582. doi: 10.1016/j.jpba.2022.114582.
Shenjia Huang  1 ,  Linsheng Liu  2 ,  Xiaoxue Liu  2 ,  Lin Song  1 ,  Chenrong Huang  3 ,  Liyan Miao  4
Affiliations
  • 1. Department of Clinical Pharmacology, the First Affiliated Hospital of Soochow University, Suzhou, China; Department of Clinical Pharmacy, College of Pharmaceutical Science, Soochow University, Suzhou, China; Institute for Interdisciplinary Drug Research and Translational Sciences, Soochow University, Suzhou, China.
  • 2. Department of Clinical Pharmacology, the First Affiliated Hospital of Soochow University, Suzhou, China; Institute for Interdisciplinary Drug Research and Translational Sciences, Soochow University, Suzhou, China.
  • 3. Department of Clinical Pharmacology, the First Affiliated Hospital of Soochow University, Suzhou, China; Department of Clinical Pharmacy, College of Pharmaceutical Science, Soochow University, Suzhou, China; Institute for Interdisciplinary Drug Research and Translational Sciences, Soochow University, Suzhou, China. Electronic address: [email protected].
  • 4. Department of Clinical Pharmacology, the First Affiliated Hospital of Soochow University, Suzhou, China; Department of Clinical Pharmacy, College of Pharmaceutical Science, Soochow University, Suzhou, China; Institute for Interdisciplinary Drug Research and Translational Sciences, Soochow University, Suzhou, China. Electronic address: [email protected].
PMID: 35101802 DOI: 10.1016/j.jpba.2022.114582
Abstract

In this study, a sensitive and rapid ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed for the simultaneous analysis of cytarabine (ara-C), cytarabine monophosphate (ara-CMP), cytarabine diphosphate (ara-CDP) and cytarabine triphosphate (ara-CTP) in the cytosol and nucleus. The separation of analytes and endogenous interferents was achieved in 8 min on a hypercarb column (2.1 mm × 100 mm, 3 µm) by using a gradient elution with 95% acetonitrile and aqueous 5 mM hexylamine with 0.4% (v/v) diethylamine adjusted to pH 10. The analytes were detected with both negative and positive electrospray ionization in multiple reaction monitoring (MRM) mode. The calibration curve demonstrated good linearity ranging from 5 to 750 nM for ara-C, 50-7500 nM for ara-CMP, 20-3000 nM for ara-CDP and 1-150 nM for ara-CTP in the cytosol. In the nucleus, good linearity was achieved over a concentration range of 1-100 nM for ara-C, 5-500 nM for ara-CMP, 2.5-250 nM for ara-CDP and 0.5-50 nM for ara-CTP. Intra- and interbatch accuracies and precisions met the standards of validation. The matrix effect, recovery and stability were also within acceptable ranges. After incubation with 10 μM ara-C for 3 h, the levels of ara-C, ara-CMP, ara-CDP and ara-CTP in the cytosol and nucleus of HL-60 cells and HL-60/ara-C cells were determined. Most of the metabolites were found within the quantitation range. The results showed that the nuclear ara-CTP level was significantly different than the intracellular ara-CTP level between HL-60 and HL-60/ara-C cells.

Keywords
Ara-C; Ara-CTP; Cellular pharmacokinetics; Centrifugal ultrafiltration; Intranuclear level; UHPLC-MS/MS.
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