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目的:建立细胞内外博莱霉素-A2的HPLC含量测定方法,用于博莱霉素水解酶对博莱霉素代谢在细胞培养中的研究。方法:细胞培养液经过蛋白沉淀和细胞超声破壁预处理后,采用ZORBAX Eclipse plus C18(4.6 mm×250 mm,5μm)柱,流动相为甲醇-乙腈-pH 4.2的0.05 mmol·L-1醋酸铵(7∶7∶86),流速1 mL·min-1,检测波长254 nm。结果:研究结果表明培养液中博莱霉素-A2进样量在0.1~1.2μg范围内线性关系良好(r=0.9994,n=5),高、中、低3个浓度的平均回收率分别为102.2%(RSD=2.8%)、101.3%(RSD=1.5%)、99.8%(RSD=2.2%);细胞内液中博莱霉素-A2进样量在0.02~0.8μg范围内线性关系良好(r=0.9998,n=5),高、中、低3个浓度的平均回收率分别为100.2%(RSD=1.2%)、99.6%(RSD=1.3%)、101.3%(RSD=1.1%)。样品溶液在24 h内稳定性良好,博莱霉素-A2在培养液中的峰面积的RSD=2.8%,在细胞内液中的峰面积的RSD=1.5%。结论:该方法测定结果准确可靠,适用于博莱霉素-A2在细胞培养中细胞内外的含量测定。
OBJECTIVE: To establish a HPLC method for the determination of bleomycin-A2 in and out of cells for the study of the metabolism of bleomycin by bleomycin hydrolase in cell culture. Methods: The cell culture medium was pretreated by protein precipitation and sonication. The cells were separated on a ZORBAX Eclipse Plus C18 (4.6 mm × 250 mm, 5 μm) column with a mobile phase of methanol-acetonitrile-pH 4.2 at 0.05 mmol·L -1 acetic acid Ammonium (7:7:86), flow rate 1 mL · min-1, detection wavelength 254 nm. Results: The results showed that the linear regression of bleomycin-A2 in the range of 0.1-1.2 μg was good (r = 0.9994, n = 5), and the average recoveries of high, middle and low concentrations were 102.2% (RSD = 2.8%), 101.3% (RSD = 1.5%) and 99.8% (RSD = 2.2%). The intracytoplasmic bleomycin-A2 injection showed good linearity in the range of 0.02-0.8 μg (RSD = 1.3%) and 101.3% (RSD = 1.1%), respectively. The average recoveries of high, middle and low concentrations were 100.2% (RSD = 1.2%) and 99.6% (RSD = 1.3%). The stability of the sample solution was good within 24 h. The peak area of bleomycin-A2 in the culture medium was 2.8% RSD, and the peak area in the intracellular solution was 1.5% RSD. Conclusion: The method is accurate and reliable. The method is suitable for the determination of intracellular and extracellular levels of bleomycin-A2 in cell culture.