高效液相色谱法测定细胞内外博莱霉素-A_2的含量

HPLC method for determination of bleomycin-A_2 in intracellular and extracellular culture

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（4．6mm×250mm，5μm）柱，流动相为甲醇-乙腈-pH4．2的0．05mmol·L^-1醋酸铵（7：7：86），流速1mL·min^-1，检测波长254nm。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一～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24h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 cul- ture fluid was treated by protein precipitation and cell uhrasonification, and analyzed by HPLC under following con- ditions ： a ZORBAX Eclipse C18 （4. 6 mmx 250 mm,5 p,m） was adopted using a mobile phase of methanol -aceto- nitrle -pH 4.2,0. 05 mmol · L^-1 ammonium acetate （7： 7： 86）. The flow rate was 1.0 mL ~ min-1 and the detec- tion wavelength was 254 nm. Results： The results showed that the bleomycin -A2 had a good linearity in the con- centration range of 0. 1 - 1.2 p,g （ 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 A2had a good linearity in the concentration range of 0. 02-0. 8 p,g （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.