LC-MS/MS法测定大鼠血浆中丹参酮ⅡA磺酸钠及其毒代动力学研究

LC-MS/MS Analysis of Sodium Tanshinone ⅡA Sulfonate in Rat Plasma and Study on Its Toxicokinetics

目的建立测定大鼠血浆中丹参酮ⅡA磺酸钠浓度的LC-MS/MS方法,并用于毒代动力学研究。方法 30只大鼠随机分为低[10mg/(kg·d)-1]、中[20mg/(kg·d)-1]、高[40mg/(kg·d)-1]剂量组,每组10只,雌雄各半。静脉推注给药,一日1次,连续28天。分别于D1、D28的给药前及给药后0.083,0.25,0.5,1,2,4,8,12h采集血样;用蛋白沉淀法处理血浆,采用Agilent1260-6430三重四极杆串联质谱仪测定浓度,离子化方式为电喷雾-负离子,监测方式为MRM,监测离子:丹参酮ⅡA磺酸钠:m/z 373.07→357,色谱柱:Inertsil ODS-SP色谱柱,流动相:乙腈-水,梯度洗脱。结果丹参酮ⅡA磺酸钠血药浓度线性范围为100~10000 ng/ml(r=0.9932),定量限为100 ng/ml,批内和批间准确度与精密度良好,准确度93.3%~105.6%,精密度RSD≤9.3%,专属性良好。注射用丹参酮ⅡA磺酸钠连续28天给药后在大鼠体内未见性别差异及明显药物蓄积现象。结论该法重现性好、灵敏度高、简单快速,适用于大鼠血浆中丹参酮ⅡA磺酸钠的含量测定及毒代动力学研究。

Objective To establish a LC-MS/MS method for the determination of sodium tanshinone ⅡA sulfonate in rat plasma and study its toxicokinetic characteristics. Methods 30 rats were randomly divided into low-dose group [10 mg/(kg·d)-1], medium-dose group [20 mg/(kg·d)-1] and high-dose group [40 mg/(kg·d)-1], 10 rats in each group, half male and half female. The drug was given by intravenous injection, once daily for consecutive 28 days. Blood samples were collected at 0.083, 0.25, 0.5, 1, 2, 4, 8 and 12 h before and after administration on Day 1 and Day 28, respectively. The protein in plasma was precipitated with acetonitrile. The Agilent1260-6430 triple quadrupole electrospray tandem mass spectrometry was used to determine the concentration of sodium tanshinone ⅡA sulfonate. Negative-ion electrospray ionization and multi reaction monitoring(MRM) were adopted. The monitoring ion pair was m/z 373.07→357. The separation was achieved on an Inertsil ODS-SP column, using acetonitrile-water as mobile phase of gradient elution. Results Sodium tanshinone ⅡA sulfonate showed good linearity in the range of 100-10 000 ng/ml(r=0.9932). The limit of quantitation was 100 ng/ml. The intra-and inter-assay accuracy and precision were good: the RSDs of precision test were less than 9.3 %, and the accuracy was within the range of 93.3 %-105.6 %. Good specificity of the method was also confirmed. Endogenous substance in plasma had no effect on the results. The results showed no gender difference and no significant accumulation in rats after repeated dosing for 28 days. Conclusion The method is reproducible, sensitive, simple and rapid. It can be used to study the toxicokinetics of sodium tanshinone ⅡA sulfonate in rats.