OBJECTIVE: To establish the HPLC method for the content determination of hyperoside (Hyp) in L02 cells.METHODS: HPLC was used to determine the concentrations of Hyp in L02 cells, after co-cultured with Hyp at the concentration of80 μg·mL-1 for 0, 3, 6, 12, 24 h.HPLC analysis was performed on a KROMASIL C18(250 mm×4.6 mm,5 μm) column at flow rate of 1.0 mL·min-1 using methanol-0.4％ phosphoric acid (45:55) as mobile phase with UV detection at 360 nm.The column temperature was 30 ℃.RESULTS: The linear range of extracellular Hyp was 2～200 μg· mL-1 and that of intracellular Hyp was 0.1～10 μg·mL-1(r=0.999 9).The standard curves of extracellular Hyp was Y=35.759X-7.422 and that of intracellular Hyp was Y= 35.412X- 8.586.Average recovery rates of extracellular and intracellular Hyp were (96.00％ ± 2.43％ ) and (97.18％ ±4.62％ ).The RSD of intra-day and inter-day were 1.75％ and 2.86％, respectively.The treated samples were kept at room temperature for 22 hours and untreated samples were kept at -20 ℃ for 6 days.The average RSD was below 5％.CONCLUSION: The results show that established HPLC method is simple, accurate and sensitive for the content determination of Hyp in L02 cells.The wide liner range, satisfied precision and good mean recovery guarantee it can be used for further study of antioxidative effect of Hyp.%目的:建立测定人肝细胞L02内、外液中金丝桃苷(Hyp)含量的方法.方法:以80μg·mL-1的Hyp与L02共孵育0、3、6、12、24 h后,采用高效液相色谱(HPLC)法测定细胞内、外液中金丝桃苷的含量.色谱柱为KROMASIL C18(250 mm×4.6mm,5 μm),流动相为甲醇-0.4%磷酸(45:55),柱温为30℃,流速为1.0 mL·min-1,紫外检测波长为360 nm.结果:细胞外液中Hyp的标准曲线为y=35.412X-8.586(r=0.999 9),表明Hyp检测浓度在2～200μg·mL-1范围内与峰面积积分值呈良好线性关系;细胞内液中Hyp的标准曲线为y=35.759X一7.422(r=0.999 9),表明Hyp检测浓度在0.1～10μg·mL-1范围内与峰面积积分值呈良好线性关系.细胞外、内液中Hyp的平均回收率分别为(96.00±2.43)%、(97.18±4.62)%,平均日内RSD分别为1.98%、4.36%,平均日间RSD分别为1.75%、2.86%.样品溶液处理后室温放置22h,未处理样品溶液于一20℃放置6 d,平均RSD均＜5%.结论:本方法简单、准确、线性范围宽、灵敏度高、精密度和回收率良好,可为进一步研究Hyp的抗氧化作用与机制奠定方法学基础.
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