高效液相色谱-四极杆/静电场轨道阱高分辨率质谱法快速检测大鼠血浆中6种附子生物碱
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篇名: 高效液相色谱-四极杆/静电场轨道阱高分辨率质谱法快速检测大鼠血浆中6种附子生物碱
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摘要: 目的:建立高效液相色谱-四极杆/静电场轨道阱高分辨率质谱法快速测定大鼠血浆中的乌头碱、新乌头碱、次乌头碱、苯甲酰乌头原碱、苯甲酰新乌头原碱、苯甲酰次乌头原碱的方法。方法:血浆样品加入内标氢溴酸高乌甲素,以甲醇沉淀蛋白进行预处理。采用高效液相色谱-四极杆/静电场轨道阱高分辨率质谱法进行样品测定,色谱柱为Sinochrom ODS-BP C18,流动相为乙腈-1%甲酸水溶液(50 ∶ 50,V/V),流速为0.6 mL/min,进样量为10 μL,柱温为25 ℃,自动进样器温度为4 ℃;质谱扫描模式为全离子监测模式,正离子采集,设定检测离子质荷比(m/z)分别为646.32(乌头碱)、632.30(新乌头碱)、616.31(次乌头碱)、604.31(苯甲酰乌头原碱)、590.29(苯甲酰新乌头原碱)、574.30(苯甲酰次乌头原碱)、585.31(内标)。取雄性Wistar大鼠6只,单剂量灌胃附子总生物碱提取物(4 mg/kg),分别于给药前(0 h)和给药后0.5、0.75、1.25、1.5、2、4、6、8、10、24 h时取血,测定血浆药物浓度并以PK-Solver V2.0软件计算药动学参数。结果:血浆中6种附子生物碱质量浓度均在0.1~10 μg/L范围内线性关系良好(r>0.992),定量限均为0.1 μg/L;平均提取回收率均大于75%,日内精密度、日间精密度、基质效应、稳定性试验的RSD均小于15%。大鼠血浆中6种附子生物碱的tmax均分别为1.2 h左右,t1/2均分别为10 h左右,单酯型乌头原碱的cmax均分别高于双酯型乌头碱。结论:本研究所建立的高效液相色谱-四极杆/静电场轨道阱高分辨率质谱法准确、灵敏、简便、快速,可用于6种附子生物碱的血药浓度监测。
ABSTRACT: OBJECTIVE: To establish HPLC-quadrupole/electrostatic field orbitrap high resolution mass spectrometry for rapid determination of aconitine, mesaconitine, hypaconitine, benzoylaconitine, benzoylmesaconine and benzoylhypacoitine in rat plasma. METHODS: Internal standard lappaconitine was added into plasma sample, and methanol precipitated protein was used for pretreatment. HPLC-quadrupole/electrostatic field orbitrap high resolution mass spectrometry was adopted. HPLC condition was as follows as Sinochrom ODS-BP C18 column, mobile phase consisted of acetonitrile-1% formic acid solution (50 ∶ 50,V/V), the flow rate of 0.6 mL/min, sample size of 10 μL, column temperature of 25 ℃, automatic sampler temperature of 4 ℃. Mass spectrum scanning mode was full ion monitoring model, positive ion acquisition, mass charge ratios (m/z) of ion were 646.32(aconitine), 632.30(mesaconitine),616.31(hypaconitine),604.31(benzoylaconitine),590.29 (benzoylmesaconine),574.30(benzoylhypacoitine), 585.31 (internal standard). Six male Wistar rats were collected and given single dose of total alkaloid extract of Aconitum carmichaeli (4 mg/kg)intragastrically. Blood samples were collected before medication (0 h) and 0.5, 0.75, 1.25, 1.5, 2, 4, 6, 8, 10, 24 h after medication. Plasma concentration was determined and pharmacokinetic parameters were calculated by using PK-Solver V2.0 software. RESULTS:The linear range of 6 kinds of aconitum alkaloids in plasma were 0.1-10 μg/L (r>0.992).  The limit of quantitation was 0.1 μg/L. Average recovery was higher than 75%, RSDs of intra-day and inter-day, matrix effects, stability test were all lower than 15%. The tmax of 6 kinds of aconite alkaloids  were about 1.2 h; t1/2 were about 10 h; cmax of monoestertype aconite alkaloids were higher than those of diester-type aconite alkaloids. CONCLUSIONS: Established HPLC-quadrupole/electrostatic field orbitrap high resolution mass spectrometry is accurate, sensitive, simple and rapid, and can be used for plasma concentration monitoring of 6 kinds of aconitum alkaloids.
期刊: 2018年第29卷第6期
作者: 赛那,金蓉,布仁,顾艳丽,林英华,雷雯朴,孙耀
AUTHORS: SAI Na,JIN Rong,BU Ren,GU Yanli,LIN Yinghua,LEI Wenpu,SUN Yao
关键字: 高效液相色谱法;四极杆/静电场轨道阱高分辨率质谱法;附子生物碱;血药浓度;大鼠
KEYWORDS: HPLC; Quadrurpole/electrostatic field orbitrap mass spectrometry; Aconitum alkaloids; Plasma concentration; Rats
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