JAC Advance Access originally published online on September 1, 2008
Journal of Antimicrobial Chemotherapy 2008 62(5):1009-1014; doi:10.1093/jac/dkn343
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Original research |
Development and validation of a reversed-phase high-performance liquid chromatography assay for polymyxin B in human plasma
1 Facility for Anti-infective Drug Development and Innovation, Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville, Victoria 3052, Australia 2 Department of Pharmacy, Beijing Hospital, Beijing 100730, P. R. China 3 Centre for Drug Candidate Optimisation, Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville, Victoria 3052, Australia 4 Infectious Diseases Service, Hospital São Lucas da Pontifícia Universidade Católica do Rio Grande do Sul, Brazil 5 Division of Infectious Diseases, Hospital de Clínicas de Porto Alegre, 2350 Ramiro Barcelos Street 90035-903, Porto Alegre, Brazil
Received 6 May 2008; returned 18 June 2008; revised 14 July 2008; accepted 1 August 2008
* Corresponding author. Tel: +61-3-9903-9702; Fax: +61-3-9903-9629; E-mail: jian.li{at}vcp.monash.edu.au
Objectives: The purpose of this study was to develop a specific, sensitive, accurate and reproducible high-performance liquid chromatographic (HPLC) method to measure polymyxin B in human plasma.
Methods: Derivatization of polymyxin B with fluorescent 9-fluorenylmethyl chloroformate (FMOC-Cl) was performed in the same solid-phase extraction C18 cartridge used for the sample pre-treatment. Reversed-phase HPLC was employed with fluorometric detection. The summed peak areas of polymyxin B1 and B2 derivatives were used for quantification. Stability of polymyxin B FMOC derivatives was examined at room temperature for 6 days. Specificity was investigated against seven potentially co-administered antibiotics. Accuracy and reproducibility of the HPLC assay were determined by inter- and intra-day validation.
Results: The derivatives of polymyxin B2 and B1 were well resolved and had retention times of 4.75 and 5.55 min, respectively. Good linearity (r2 > 0.99) was obtained between 0.125 and 4.00 mg/L polymyxin B in human plasma with good accuracy and reproducibility at the limit of quantification (0.125 mg/L). Intra- and inter-day validation demonstrated good accuracy and reproducibility for quality control samples with nominal concentrations of 0.30 and 3.00 mg/L. FMOC derivatives of polymyxin B were stable for at least 3 days at room temperature. None of the possibly co-administered antibiotics tested interfered with the chromatographic analysis of the polymyxin B FMOC derivatives.
Conclusions: A rapid, specific, sensitive, accurate and reproducible HPLC method has been developed and validated to measure polymyxin B in human plasma. The method is suitable for clinical pharmacokinetic studies.
Keywords: derivatization , HPLC , polymyxin B sulphate , 9-fluorenylmethyl chloroformate
Joint senior authors. Both authors contributed equally to the article.
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  V. H. Tam, J. Hou, A. L. Kwa, and R. A. Prince Comment on: Development and validation of a reversed-phase high-performance liquid chromatography assay for polymyxin B in human plasma J. Antimicrob. Chemother., March 1, 2009; 63(3): 627 - 628. [Full Text] [PDF]
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J. Li, R. L. Nation, G. Cao, F. E. A. Ali, F. Chiu, and A. P. Zavascki Development and validation of a reversed-phase high-performance liquid chromatography assay for polymyxin B in human plasma--authors' response J. Antimicrob. Chemother., March 1, 2009; 63(3): 628 - 629. [Full Text] [PDF]
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