Objective  To develop a simple and sensitive LC-MS/MS method for the determination of polymyxin B in human serum.

Methods  Polymyxin E₂ was used as an internal standard and acetonitrile (0.1% formic acid) was used for protein precipitation. Chromatographic separation was performed on a Hypersil GOLD^TM C₁₈ column. Mobile phase A was 0.1% formic acid in water, and mobile phase B was methanol. The flow rate was 0.4 mL·min^-1, with gradient elution. The column temperature was 40℃. The injection volume was 2 μL and the analysis time was 3.3 min. Multiple reaction monitoring and electrospray ion source positive ion mode were applied for quantitative analysis. The quantitative analysis ion pairs were m/z 402.10>101.10 (polymyxin B₁), m/z 397.45>101.10 (polymyxin B₂), m/z 578.50>101.10 (polymyxin E₂).

Results  The linear relationship of polymyxin B₁ was at 22-15 000 ng·mL^-1 and B₂ was at 5-1 700 ng·mL^-1 in human serum, which achieved excellent linearity, and the correlation coefficient was higher than 0.999 0. The lower limit of quantification of polymyxin B₁ and B₂ were 22 ng·mL^-1 and 5 ng·mL^-1, respectively. The accuracies were 95.98%-104.60% for polymyxin B₁, 98.11%-105.59% for polymyxin B₂, respectively. The RSDs of intra- and inter-day precision were less than 15%. The recoveries of polymyxin B₁ and B₂ were 97.26%-103.31% and 95.81%-101.22%, respectively, and the matrix effects were 96.52%-109.54% and 93.29%-109.95%, respectively. The RSDs of the samples were all less than 15%. The mean AUC_0- 24h was (72.85±17.87) mg·h·L^-1 in six patients.

Conclusion  The established LC-MS/MS method for determining polymyxin B in human serum meets the requirements of biological sample analysis. It is suitable for determining polymyxin B in human serum.

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