微流控芯片毛细管电泳法在酶法工艺青霉素类产品中残留蛋白检测的应用探讨

Objective: The residual protein was one of critical quality attributes influencing semi-synthetic antibiotics produced by enzymatic process, which not only affects the quality of drugs but also threatens the safety of drug application. In this study, a microfluidic chip-based capillary electrophoresis (MCCE) method was established for the determination of residual proteins in penicillin products. Several effect factors and detecting accuracy for MCCE method were also investigated. Methods: Taking amoxicillin-one of typical penicillins-as an example, Agilent high sensitivity protein 250 kit was adopted for the preparation of penicillin G acylase (PGA) and amxocillin. Solutions were loaded into the protein chips (5 cm×5 cm×0.8 cm).Agilent Bioanalyzer 2100 was used for the analysis and detection. The detection range of protein molecular weight were 10~250 kDa. The analysis time was 30 min and the sample volume was 9 μL. Results: The linear range of bovine serum albumin (BSA) was 5~500 ng•mL^-1 (r=0.99). The accuracy of protein sizing was 100.6% (n=4×7). There was no significant difference (P=0.28>0.05, n=4) between the standard curves of BSA in amoxicillin test solution and BSA in water. The limits of detection and quantitation were 3 ng•mL^-1 (1.5 ppm) and 10 ng•mL^-1 (5.0 ppm), respectively. The quantitation RSD of inter- and intra-day was less than 15% (n=4). BSA in amoxicillin test solution was basically stable within 6 hours (RSD<3.4%). Conclusion: The MCCE method developed in this study, which is simple, fast, highly-sensitive and accurate, could be utilized for the determination of residual proteins in amoxicillin produced by enzymatic process. It provided a detection method with different principles for the detection of residual proteins in the semi-synthetic antibiotics produced by enzymatic process.