Development and validation of a UPLC–MS/MS method for simultaneous quantification of polymyxins and caspofungin in human plasma for therapeutic drug monitoring

Abstract

Objective

To develop a rapid, convenient, accurate, and low-residual-effect ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) method for the determination of polymyxin B sulfate and colistin sulfate in the blood of patients with multidrug-resistant bacterial infections, as well as caspofungin acetate in the blood of patients with fungal infections, thus facilitating the rational use of antibiotics in clinical applications.

Methods

All analytes were diluted with 0.2 % aqueous formic acid, and plasma proteins were precipitated using acetonitrile. The selected reaction monitoring (SRM) mode was used for measurement. Separation of all analytes was completed on a Hypersil GOLD C18 column (100 × 2.1 mm, 3.0 µm). They were quantitatively analyzed using electrospray ionization on a triple quadrupole mass spectrometer in the positive ion mode. The mobile phase consisted of water (containing 0.1 % formic acid) and acetonitrile, which was delivered by gradient elution at a flow rate of 0.3 ml/min. The internal standard was bacitracin zinc (BcZn), and the column temperature was maintained at 25 °C. The runtime for each analysis was 3.5 min.

Results

The procedure was validated following the recommendations of the U.S. Food and Drug Administration, which included measurements of accuracy (ranging from 83.27 % to 105.86 % for within-run and between-run accuracy), precision (with coefficients of variation from 2.50 % to 16.51 % for within-run precision and between-run precision), and matrix effects (ranging from 88.65 % to 103.94 %). The extraction recoveries ranged from 38.01 % to 42.76 for polymyxin B1 (PMB1), polymyxin B2 (PMB2), polymyxin E1 (PME1), polymyxin E2 (PME2), and 88.65 % to 89.84 % for caspofungin (CPF). Plasma samples were stable under various storage conditions, including three freeze–thaw cycles at −80 °C, 24-hour periods at room temperature and 4 °C, and 30 days of freezing at both −20 °C and −80 °C, with relative standard deviations (RSD) of less than 15 %.

Conclusion

In this study, a UPLC–MS/MS method was developed to simultaneously quantify PMB1, PMB2, PME1, PME2, and CPF in human plasma. The method was validated in blood samples from patients with multidrug-resistant bacteria combined with fungal infections and is suitable for therapeutic drug monitoring.