UPLC-MS/MS法测定血浆中新型酪氨酸酶抑制剂UP302的含量

目的: 建立超高效液相色谱-串联质谱测定血浆中新型酪氨酸酶抑制剂UP302的方法,并用于研究UP302在大鼠、狗、猴和人血浆中的稳定性。 方法: 血浆中加入内标大豆苷元经2倍体积甲醇沉淀后进行分析。采用Hypersil Gold C₁₈(50 mm×2.1 mm,1.9 μm)色谱柱,流动相为甲醇(A)-5 mmol·L^-1甲酸铵水溶液(B),梯度洗脱,梯度流速恒定为0.2 mL·min^-1,柱温为30 ℃,整个分析时间为6 min。采用负离子电喷雾离子化电离源和选择反应监测模式进行检测。 结果: 在5～2000 ng·mL^-1的浓度范围内,标准曲线线性关系良好(r=0.9998);血浆中UP302最低定量下限为5 ng·mL^-1;本方法日内日间准确度在99.2%～107.3%,日内日间精密度均小于9.3%。 结论: 本方法灵敏度高,重现性好,操作简便,可用于血浆中UP302浓度的测定。

Objective: To establish an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS)method for the determination of tyrosinase inhibitor UP302 in plasma, and study the stability of UP302 in rat, dog, monkey and human plasma. Methods: The plasma samples were precipitated by methanol using daidzein as an internal standard. On Hypersil Gold C₁₈ column (50 mm × 2.1 mm, 1.9 μm), separation was achieved by a gradient mobile phase of methanol and 5 mmol·L^-1 ammonium formate solution at a flow rate of 0.2 mL·min^-1 at 30 ℃. The total analysis time was 6 min. Selection-reaction monitoring mode in negative-mode electrospray ionization was used to detect UP302 in plasma. Results: Calibration curve was linear in a range from 5-2000 ng·mL^-1 with a correlation coefficient of 0.9998. The lower limit of quantification was 5 ng·mL^-1. The intra-and inter-day accuracy was between 99.2% and 107.3%, and precision was less than 9.3%. Conclusion: The UPLC-MS/MS method is sensitive and reproducible. It can be used for the determination of UP302 in plasma.

[1] Kobayashi T,Vieira WD,Potterf B,et al.Modulation of melanogenic protein expression during the switch from eu-to phenomelanogenesis.J Cell Sci,1995,108(Pt 6):2301
[2] Kubo I,Yokokawa Y,Kinst-Hori I.Tyrosinase inhibitors from Bolivian medicinal plants.J Nat Prod,1995,58(5):739
[3] DING Da-peng(丁大鹏).Development of tyrosinase inhibitors(酪氨酸酶抑制剂的研究进展).J Pract Med(实用医学杂志),2005,21(12):1364
[4] ZOU Xian-wei(邹先伟),JIANG Zhi-sheng(蒋志胜).Advances in studies on botanical inhibitors of tyrosinase(植物源酪氨酸酶抑制剂研究进展).Chin Tradit Herb Drugs(中草药),2004,35(6):702
[5] PIAO Xiang-lan(朴香兰),PIAO Xiang-shu(朴香淑).Rapid bioassay-liked LC/MS of tyrosinase inhibiters from Sophora flavescens(利用生物活性-液质联用跟踪方法快速分析苦参的酪氨酸酶抑制成分).Chin J Pharm Anal(药物分析杂志),2009,29(9):1407
[6] Nesterov A,Zhao J,Minter D,et al.1-(2,4-dihydroxyphenyl)-3-(2,4-dimethoxy-3-methylphenyl)propane, a novel tyrosinase inhibitor with strong depigmenting effects.Chem Pharm Bull,2008,56(9):1292
[7] Mammone T,Muizzuddin N,Declercq L,et al.Modification of skin discoloration by a topical treatment containing an extract of Dianella ensifolia:a potent antioxidant.J Cosmet Dermatol,2010,9(2):89