Determination of polymyxin B in dried blood spots using LC-MS/MS for therapeutic drug monitoring

Abstract

Polymyxin B, a last line antibiotic for extensively drug resistant gram-negative bacteria, therapeutic drug monitoring (TDM) is recommended to minimize its nephrotoxicity and improve efficacy. In the present study, we developed a novel quantification method of polymyxin B in dried blood spots (DBS) using liquid chromatography coupled with mass spectrometry (LC-MS/MS), which was performed on a Shimadzu Prominence HPLC system coupled with a 4500 triple quadrupole mass spectrometer. An aliquot of 50 μL whole blood sample was spotted on Whatman 903® paper cards. Each DBS sample was cut off into a 6 mm diameter disc and extracted by acetonitrile in water (30% in volume, containing 6% formic acid, v/v). Both intra and inter-batch accuracy was in the range of 92.6%–111.0% for polymyxin B1 and 91.5%–111.5% for polymyxin B2. The precision was in the range of 5.2%–12.2% for polymyxin B1 and 4.9%–13.1% for polymyxin B2. The matrix effects for polymyxin B1 and polymyxin B2 at low, medium and high concentrations were ranged from 102.2%–107.9% and 99.8%–106.2%, respectively. The extraction recoveries were >85.4%. Stability results showed that DBS cards can be transported at room temperature within 2 days and was stable in sealed plastic bags for 38 days at −70 °C. Bland-Altman analysis demonstrated that concentrations of polymyxin B measured in DBS and plasma methods were in moderate agreement with 95.1% samples within the 95% confidence interval of limits of agreement. The DBS method was successfully applied in clinic for TDM of polymyxin B, which can be an alternative approach in clinic.