Determination of free and total meropenem levels in human plasma and its application for the consistency evaluation of generic drugs.

Abstract

The consistency evaluation of generic drugs is important for the overall reformation of drug registration in China. In this study, we used meropenem as a model drug to explore the key techniques for clinical consistency evaluation by studying the plasma protein binding ratio of different preparations. Because the free portion of drug is the effective part in vivo, it is essential to measure the free drug concentration in the circulatory system. Therefore, in this study, a fast and accurate HPLC-MS/MS method was developed to determine the total and free concentrations of meropenem in human plasma. Simple protein precipitation procedures were used for the sample processing assay, and ultrafiltration was implemented for the separation of free drugs. Liquid chromatography separation was performed using hydrophilic interaction liquid chromatography (HILIC) silica (2.1 × 50 mm, 3 μm) column. The mobile phase and sample preparation procedures were optimized. Factors affecting the measurement of free drug concentration were also explored. Nonspecific binding of the ultrafiltration membrane was negligible because the recovery rate for post-ultrafiltration was greater than 96%. Under optimal conditions, the drug concentrations were linear from 0.5 to 50 μg/mL for both total and free drug concentrations. The plasma protein binding (PPB) ratio was calculated based on the free and total drug concentrations. The PPB of meropenem varied from 1.4% to 24.2% in different subjects. The validated method was applied to evaluate PPB of four preparations, and the results varied from 6.57 ± 3.19% to 10.40 ± 8.31%. ANOVA showed no significant differences between the four preparations. We established a rapid, robust, and reliable method for the determination of total and free meropenem concentrations using LC-MS/MS with ultrafiltration techniques. The method provided a new perspective for the clinical consistency evaluation of generic drugs.