LC-MS/MS法测定比格犬血浆中红景天苷和酪醇的浓度及其药动学研究

Concentration determination of salidroside and p-tyrosol in Beagle dogs by LC-MS/MS method and related pharmacokinetics study

目的:建立LC-MS/MS法测定比格犬血浆中红景天苷及其代谢物酪醇的浓度,并应用于药动学研究。方法:血浆样品经氯仿、苯酚和异戊醇混合溶剂萃取,Shimadzu Shim-pack Velox PFPP色谱柱(100 mm×2.1 mm,1.8μm)分离,以水(A相)-含20%乙腈的甲醇溶液(B相)为流动相,梯度洗脱(0~1 min,12%B;1~2.5 min,12%~85%B;2.5~3.5 min,85%B;3.5~3.6 min,85%~12%B;3.6~5 min,12%B)5 min,流速为0.3 ml/min,柱温40℃。采用电喷雾离子源(ESI),负离子多反应监测模式(MRM)进行质谱监测,离子对:红景天苷m/z 299.1→118.9、酪醇m/z 137.0→105.9、内标天麻素m/z 285.1→122.9。测定比格犬口服和静脉注射红景天苷后的主要药动学参数。结果:红景天苷在10~10000 ng/ml,酪醇在1~1000 ng/ml范围内线性良好,日内和日间精密度RSD均<12.9%。口服给药后,红景天苷和酪醇的AUC0~t分别为(19600±4870)和(946±188)ng·h·ml^(-1),AUC_(0~∞)分别为(19700±4880)和(2280±1780)ng·h·ml^(-1),c_(max)分别为(8890±2650)和(132±44.3)ng/ml,t_(1/2)分别为(1.85±0.69)和(40.3±45.1)h;静脉注射给药后,红景天苷和酪醇的AUC0~t分别为(5960±882)和(127.0±32.4)ng·h·ml^(-1),AUC_(0~∞)分别为(6000±887)和(157.0±31.6)ng·h·ml^(-1),c_(max)分别为(9210±1900)和(42.0±12.3)ng/ml,t_(1/2)分别为(0.976±0.156)和(4.46±2.42)h。结论:本方法简单、高效,专属性好,灵敏度高,可用于红景天苷及其代谢物酪醇在比格犬体内的药动学研究。

Objective:To establish a LC-MS/MS method for simultaneous determination of the concentrations of salidroside and its metabolite p-tyrosol in Beagle dog plasma and also to study their pharmacokinetics.Methods:The plasma samples were extracted by a mixed solution of chloroform,phenol and isoamyl alcohol.Then,they were separated on Shimadzu Shim-pack Velox PFPP column(100 mm×2.1 mm,1.8μm).Water(phase A)and methanol containing 20%acetonitrile(phase B)was used as mobile phase,the flow rate was 0.3 ml/min with a gradient elution(0-1 min,12%B;1-2.5 min,12%-85%B;2.5-3.5 min,85%B;3.5-3.6 min,85%-12%B;3.6-5 min,12%B),and the column temperature was 40℃.Detections were made by using an electrospray ionization(ESI)source and negative ion multiple reaction monitoring(MRM)of m/z 299.1→118.9(salidroside),m/z 137.0→105.9(p-tyrosol)and m/z 285.1→122.9(internal standard gastrodin).Main pharmacokinetic parameters of salidroside and p-tyrosol were calculated after oral and intravenous injection of salidroside.Results:The calibration curves of salidroside and p-tyrosol showed good linearity within the ranges of 10-10000 ng/ml and 1-1000 ng/ml.The intra-day and inter-day RSD were all less than 12.9%.After oral administration,the AUC0-t of salidroside and p-tyrosol were respectively(19600±4870)and(946±188)ng·h·ml^(-1),AUC_(0-∞)were respectively(19700±4880)and(2280±1780)ng·h·ml^(-1),c_(max)were(8890±2650)and(132.0±44.3)ng/ml,t_(1/2)were(1.85±0.69)and(40.3±45.1)h.After intravenous injection,AUC0-t of salidroside and p-tyrosol were respectively(5960±882)and(127.0±32.4)ng·h·ml^(-1),AUC_(0-∞)were respectively(6000±887)and(157.0±31.6)ng·h·ml^(-1),c_(max)were(9210±1900)and(42.0±12.3)ng/ml,t_(1/2)were(0.976±0.156)and(4.46±2.42)h,respectively.Conclusion:The method is simple,sensitive and efficient with high specificity and could be used for pharmacokinetics study of salidroside and its metabolite p-tyrosol in Beagle dogs.