Development and Validation of a New Ultra-Performance Liquid Chromatographic Method for Vancomycin Assay in Serum and Its Application to Therapeutic Drug Monitoring

Abstract

The aim of this study was to develop and validate an ultra-performance liquid chromatographic (UPLC) method with photodiode array detector for the measurement of vancomycin in human serum samples for therapeutic drug monitoring or other applications. The method included the extraction of vancomycin in serum by deproteinization with acetonitrile. The analyses were carried out using an ACQUITY UPLC BEH C(18) column (2.1 × 50 mm, 1.7 μm) using acetonitrile and 0.005 M KH(2)PO(4) buffer (pH 2.5) as the mobile phase at a flow rate of 0.3 mL/min, with photodiode array detection at 230 nm. The method was validated for extraction recovery, inter- and intraday precision (relative standard deviation, RSD%), and accuracy and stability of vancomycin in serum. Both the established UPLC method and fluorescence polarization immunoassay (FPIA) were used to measure the prepared quality control (QC) samples (1.0, 7.0, 35.0, 75.0 mg/L) to validate the accuracy of UPLC. Furthermore, both methods were subsequently used to assay the vancomycin concentration in 172 clinical serum samples collected from patients receiving vancomycin in the hospitals localized in Shanghai (China) and 32 control samples from United Kingdom National External Quality Assessment Service (UK NEQAS). The retention time of vancomycin was 2.6 minutes. The calibration curve for UPLC was linear over the range 1.0-100.0 mg/L (R(2) > 0.999). The method was fully validated in terms of recovery, selectivity, accuracy, precision, and various conditions. The absolute difference% and RSD% of the prepared QC samples assayed by UPLC were all better than the results by FPIA. A paired t test of the results of the prepared QC samples indicated that the results of all the QC samples had significant difference (P < 0.05), except for the 7.0 mg/L QC samples, which suggested that UPLC was more accurate for the samples containing low or high concentration of vancomycin. A correlation with the Deming model provided a good linear relation between the results of the 2 methods applied to 172 samples, with equation of UPLC = 0.99 × FPIA - 0.19 (R(2)= 0.923), and the agreement of the 2 methods was illustrated using Bland-Altman plot with a mean difference (UPLC - FPIA) of -0.428 mg/L and 95% confidence interval of -8.33 to 7.47 mg/L, respectively. A Student t test comparing results obtained by the UPLC method and group mean results of control samples from UK NEQAS were not significant (P = 0.057). A short analysis time, small amount of serum needed, high specificity, and accuracy make the UPLC method developed in this study appropriate and practical for vancomycin therapeutic drug monitoring and could be applied to other nonserum applications or where requiring superior validation parameters such as for pharmacokinetic/pharmacodynamic studies.