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目的:建立灵敏、简便、准确的自动采血系统采集大鼠血浆样本,高效液相色谱-三重四级杆质谱联用技术(HPLC-QQQ-MS)测定血浆中青藤碱含量的方法,并应用于其体内药代动力学研究。方法:SD大鼠颈总静脉插管,待清醒后按40 mg·kg-1腹腔注射青藤碱,应用自动采血系统在大鼠自由活动状态下采集样品。血浆样品中加入内标(磷酸可待因),乙腈沉淀蛋白,流动相乙腈-水(含0.1%甲酸铵)梯度洗脱。质谱采用电喷雾电离源(ESI),扫描方式为多反应离子监测模式(MRM),定量分析离子对分别为青藤碱m/z 330.0,磷酸可待因m/z 300.0。利用Win Nonlin软件计算药代动力学参数。结果:血浆中青藤碱在5.0~500.0μg·L-1线性关系良好,定量下限1μg·L-1,样品处理方法的提取回收率>88%,日内及日间准确度101.08%~111.08%,RSD均<10%,稳定性良好。血浆中青藤碱的达峰时间(tmax),药峰浓度(Cmax),药时曲线下面积(AUC0-24 h),表观分布容积(Vz/F),清除率(CL/F),平均驻留时间(MRT0-24 h)分别为(1.65±1.48)h,(210.43±96.35)μg·L-1,(1 053.80±427.69)h·μg·L-1,(252.65±57.36)L·kg-1,(30.37±11.85)L·h-1·kg-1,(5.15±2.47)h。结论:建立的方法有效控制了采血过程对药动学分析结果的影响,样品用量少,简便、准确、可靠,能够满足自由活动大鼠体内青藤碱连续定量分析和药代动力学研究。
OBJECTIVE: To establish a sensitive, simple and accurate automatic blood collection system for collecting plasma samples from rats and using HPLC-QQQ-MS to determine the content of sinomenine in plasma. In vivo Pharmacokinetics Study. Methods: Sprague-Dawley rats were cannulated with common carotid artery. After sober, they were injected intraperitoneally with sinomenine at a dose of 40 mg · kg-1. Samples were collected from rats free-moving with automatic blood sampling system. Plasma samples were spiked with an internal standard (codeine phosphate), acetonitrile precipitated protein and mobile phase of acetonitrile-water (containing 0.1% ammonium formate). The mass spectrometry was electrospray ionization source (ESI). The scanning mode was multi-reactive ion monitoring (MRM). The quantitative analysis ion pair was sinomenine m / z 330.0 and codeine phosphate m / z 300.0 respectively. Pharmacokinetic parameters were calculated using Win Nonlin software. Results: The linearities of sinomenine in plasma ranged from 5.0 to 500.0 μg · L-1 with a lower limit of quantitation of 1 μg · L-1. The recovery rate of samples was 88%, and the intra-and inter-day accuracy was 101.08% -111.08% , RSD <10%, good stability. Plasma sinomenine peak time (tmax), peak drug concentration (Cmax), drug area under the curve (AUC0-24 h), apparent volume of distribution (Vz / F), clearance rate (CL / F) The average residence time (MRT0-24 h) were (1.65 ± 1.48) h, (210.43 ± 96.35) μg · L-1, (1053.80 ± 427.69) h · μg · L-1 and (252.65 ± 57.36) L · Kg -1, (30.37 ± 11.85) L · h -1 · kg -1, (5.15 ± 2.47) h. CONCLUSION: The established method can effectively control the influence of blood sampling process on pharmacokinetic analysis. The sample dosage is small, simple, accurate and reliable, which can meet the continuous quantitative analysis and pharmacokinetic study of sinomenine in free-moving rats.