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Updated: Jul 2, 2026
Multiplex Therapeutic Drug Monitoring by Isotope-dilution HPLC-MS/MS of Antibiotics in Critical Illnesses
Published on: August 30, 2018
Development and validation of a reversed-phase high-performance liquid chromatography assay for polymyxin B in human
Guoying Cao1, Feda' Emad Atta Ali, Francis Chiu
1Facility for Anti-infective Drug Development and Innovation, Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville, Victoria 3052, Australia.
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 (r(2) > 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.
