Development and validation of a high-performance liquid chromatography-fluorescence detection method for the accurate quantification of colistin in human plasma

Divyabharathi Chepyala, I. Lin Tsai, Hsin Yun Sun, Shu Wen Lin, Ching Hua Kuo

Research output: Contribution to journalArticle

16 Citations (Scopus)

Abstract

Recently, colistin has become one of the most important drugs for treating infections caused by multidrug-resistant Gram-negative bacteria. Therapeutic drug monitoring is recommended to ensure the safety and efficacy of colistin and to improve clinical outcomes. This study developed an accurate and sensitive high-performance liquid chromatography-fluorescence detection (HPLC-FLD) method for the quantification of colistin in human plasma. The sample preparation included protein precipitation using trichloroacetic acid (TCA) and methanol, followed by in-solid phase extraction (In-SPE) derivatization with 9-fluorenylmethyl chloroformate (FMOC-Cl). A Poroshell 120 EC-C18 2.1×100mm (2.7μm) column was used in the HPLC method with a mobile phase composed of acetonitrile (ACN), tetrahydrofuran (THF), and deionized (DI) water (82%, 2%, 16% (v/v), respectively). Polymyxin B1 was used as the internal standard. The total analysis time was 22min under optimal separation conditions. The HPLC-FLD method was validated over a therapeutic range of 0.3-6.0μgmL-1. The intra-day and inter-day precisions for colistin A and colistin B were below 9.9% and 4.5% relative standard deviations, respectively. The accuracy test results were between 100.2 and 118.4%. The extraction recoveries were between 81.6 and 94.1%. The method was linear over the test range, with a 0.9991 coefficient of determination. The limit of detection was 0.1μgmL-1. The validated HPLC-FLD method was successfully applied to quantify the colistin concentrations in 2 patient samples for therapeutic drug monitoring.

Original languageEnglish
Pages (from-to)48-54
Number of pages7
JournalJournal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences
Volume980
DOIs
Publication statusPublished - Feb 1 2015
Externally publishedYes

Keywords

  • Colistin
  • FMOC-Cl
  • HPLC-FLD
  • Human plasma
  • Therapeutic drug monitoring

ASJC Scopus subject areas

  • Biochemistry
  • Analytical Chemistry
  • Cell Biology
  • Clinical Biochemistry
  • Medicine(all)

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