高效液相色谱法测定细胞内外博莱霉素-A₂的含量

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，5 μm） 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0.1～1.2 μg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0.02～0.8 μg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 an HPLC method for content determination of bleomycin-A₂in cells so as to study the effect of hydrolase for bleomycin on the metabolism of bleomycin in cell culture. Methods: The cell culture fluid was treated by protein precipitation and cell ultrasonification,and analyzed by HPLC under following conditions:a ZORBAX Eclipse C₁₈ (4.6 mm×250 mm,5 μm) was adopted using a mobile phase of methanol-acetonitrle-pH 4.2,0.05 mmol·L^-1 ammonium acetate (7:7:86).The flow rate was 1.0 mL·min^-1 and the detection wavelength was 254 nm. Results: The results showed that the bleomycin-A₂ had a good linearity in the concentration range of 0.1-1.2 μg (r=0.9994,n=5).The average recoveries were 102.2% (RSD=2.8%),101.3% (RSD=1.5%),99.8% (RSD=2.2%) for bleomycin-A₂ at high,medium and low concentrations.In intracellular fluid,the bleomycin A₂ had a good linearity in the concentration range of 0.02-0.8 μg (r=0.9998,n=5).The average recoveries were 100.2% (RSD=1.2%),99.6% (RSD=1.3%),101.3% (RSD=1.1%) for bleomycin-A₂ at high,medium and low concentrations.In addition,bleomycin-A₂ was stable for at least 24 h in both the cell culture fluid (RSD=2.8%) and the intracellular fluid (RSD=1.5%). Conclusion: A reliable HPLC method was established to measure the content of bleomycin-A₂ in intracellular and extracellular culture.

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