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

- A robust, simple, accurate, and reproducible analytical strategy for polymyxin B1 & B2.
- A method suited for quantifying polymyxin in matrices of therapeutic and experimental relevance.
- A efficient protein precipitation extraction method with high recoveries was developed.

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.