The development and validation of a simple liquid chromatography–tandem mass spectrometry method for polymyxin B1 and B2 quantification in different matrices

Jenna Covelli, Donna Ruszaj, Robert Straubinger, Jian Li, Gauri G. Rao

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Polymyxin B has resurfaced as a last-line treatment for multi-drug resistant Gram-negative bacteria. Accurate characterization and quantification of polymyxin B components is necessary to optimize this therapy. We developed and validated a robust, straightforward LC–MS/MS method to quantify polymyxin B1 and B2, the primary polymyxin B components, in various matrices (cation-adjusted Mueller-Hinton broth (CAMHB), human and rat plasma). Of sample preparation approaches investigated, two protein precipitation/extraction methods were developed as part of an analytical strategy based upon reverse-phase LC–MS/MS using electrospray ionization in positive multiple-reaction monitoring mode. Both methods were validated over therapeutically and experimentally relevant concentration ranges (CAMHB: 0.1–8.0 μg/mL, rat and human plasma: 0.05–4.0 μg/mL). Quality control samples spanning a relevant concentration range were employed to assess intra- and inter-day accuracy (relative error (%RSD)) and precision (coefficient of variation (CV%)). For polymyxin B1 and B2 in CAMHB, inter-day standard deviations were 1.18–4.59% and 0.777–1.23%, respectively, and accuracies were 94.2–99.3% and 94.4–99.1%. For rat plasma, inter-day standard deviations were 1.53%–5.64% and 4.07%–8.26%. Accuracies were 100.6–108.9% and 96.1–108.1%. For human plasma, inter-day standard deviations were 2.77–7.32% and 1.55–7.29%. Accuracies were 89.6–96.4% and 92.9–102.0%. Extraction recoveries for all matrices were >93.5%. Adsorption, storage, and long-term stability were assessed and were acceptable. Accuracy, precision, and cost-efficiency make this an ideal approach for quantifying polymyxin B in in vitro and in vivo samples including those from rat and human subjects.

Original languageEnglish
Pages (from-to)112-118
Number of pages7
JournalJournal of Chromatography B
Publication statusPublished - 15 Oct 2017


  • LC–MS/MS
  • Mueller-Hinton broth
  • Polymyxin B1
  • Polymyxin B2
  • Protein precipitation extraction method
  • Rat and human plasma

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