LC-MS/MS测定药根碱、巴马汀和小檗碱在Caco-2细胞的摄取特性

Determination of Jatrorrhizine,Palmatine and Berberine in Caco-2 Cells by LC-MS/ MS Assay

目的建立药根碱、巴马汀、小檗碱的LC-MS/MS测定法,研究其在Caco-2细胞上的摄取特性。方法色谱条件:Phe-nomenexluna C18柱(4.6mm×250mm,5μm);乙腈-水(4mmol·L-1醋酸铵和0.08%甲酸)为流动相,梯度洗脱;质谱条件:采用电喷雾离子源(ESI),正离子检测,MRM模式。实验考察药物浓度和P-糖蛋白(P-glycoprotein,P-gp)抑制剂环孢素A和维拉帕米对药根碱、巴马汀、小檗碱在Caco-2细胞上摄取的影响。结果药根碱、小檗碱在0.0015～0.30μmol.L-1(r=0.9998、r=1.0000),巴马汀在0.0014～0.28μmol·L-1(r=0.9998)内均呈良好的线性关系;药根碱、巴马汀、小檗碱在Caco-2细胞的摄取在一定范围内呈浓度依赖性,表现为被动扩散;P-gp抑制剂环孢素A和维拉帕米可显著增加药根碱、巴马汀、小檗碱在Caco-2细胞上的摄取。结论该法快速、灵敏、简单。药根碱、巴马汀、小檗碱在Caco-2细胞的摄取存在P-gp的参与,药根碱、巴马汀和小檗碱是P-gp的底物。

OBJECTIVE To establish a LC-MS/MS assay for determining the uptake status of jatrorrhizine,palmatine and berberine in Caco-2 cells. METHODS Chromatographic separation was achieved with gradient elution by a Phenomenex luna C18 column. A API 4000 mass spectrometer system coupled with negative electrospray ionization （ESI） was in the MRM mode. The mobile phase. A was acetonitrile consisted of 4 mmol·L^-1 ammonium acetate and 0.08% methanoic acid and the mobile phase B. The uptakes of jatrorrhizine,palmatine and berberine in Caco-2 cells at various concentration and P-gp inhibitors were measured. RESULTS The linearities of jatrorrhizine,palmatine and berberine concentration curves were 0.001 5-0.30,0.001 4-0.28 and 0.001 5-0.030 μmol·L^-1 respectively. The uptakes of jatrorrhizine,palmatine and berberine in Caco-2 cells showed a passive diffusion in concentration-dependent manner. The uptakes of jatrorrhizine,palmatine and berberine in Caco-2 cells were increased by P-gp inhibitors significantly.CONCLUSION This fast,sensitive and and specific LC-MS/MS assay was applied to determine jatrorrhizine,palmatine and berberine concentrations in Caco-2 cells.Jatrorrhizine,palmatine and berberine showed well uptake in Caco-2 cells,the P-gp inhibitors maybe participate in the uptake. Jatrorrhizine,palmatine and berberine were the substrates of P-gp.