Bioanalytical method development and validation of a liquid chromatography-tandem mass spectrometry method for determination of β-lapachone in human plasma

William C. Putnam, Raja Reddy Kallem, Indhumathy Subramaniyan, M. Shaalan Beg, Vindhya Edpuganti

Research output: Contribution to journalArticle

Abstract

The purpose of this work was to develop and validate a rapid, sensitive and robust liquid chromatography tandem mass spectrometric method for the quantification of β-lapachone in human plasma and to use that method to analyze human clinical samples. Sample preparation for the developed method involved liquid-liquid extraction using ethyl acetate for extraction of β-lapachone and cryptotanshinone (internal standard) from human plasma. Chromatographic resolution was achieved on a Kinetex C18 column using a gradient elution and a chromatographic flow rate of 0.5 mL/min. The retention times of β-lapachone and cryptotanshinone were 1.98 and 2.28 min, respectively, and the method had a total run time of 4 min. Bioanalytical method validation was conducted in accordance with the United States Food and Drug Administration regulatory guidelines. The method was validated over 2 calibration ranges in order to support high- and low-dose clinical studies. Calibration curve-1 covered the range of 0.25–50 ng/mL and calibration curve-2 covered the range of 50–2000 ng/mL. The method was determined to be accurate (percent relative errors between −1.07 to 5.36 %), precise (percent relative standard deviations less than 7.4), and sensitive (LLOQ 0.25 ng/mL). β-lapachone was determined to be stable (% change from time = 0 between −11.6 and 12.6 %) across the autosampler, benchtop, freeze/thaw and long-term (63 days) stability studies. The validated bioanalytical method was employed to determine β-lapachone concentrations in human plasma samples from a clinical study.

Original languageEnglish (US)
Article number113466
JournalJournal of Pharmaceutical and Biomedical Analysis
Volume188
DOIs
StatePublished - Sep 5 2020

Keywords

  • Bioanalysis
  • Clinical study
  • Human plasma
  • LC–MS/MS
  • NAD(P)H:quinone oxidoreductase 1
  • Pharmacokinetics
  • β-Lapachone

ASJC Scopus subject areas

  • Analytical Chemistry
  • Pharmaceutical Science
  • Drug Discovery
  • Spectroscopy
  • Clinical Biochemistry

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