LC-MS/MS法测定大鼠血浆苯甲酰新乌头原碱浓度

DETERMINATION OF BENZOYLNEOACONITINE IN RAT PLASMA BY LIQUID CHROMATOGRAPHY-TANDEM MASS SPECTROMETRY

目的建立高效液相色谱-串联质谱法(LC-MS/MS法)测定大鼠血浆苯甲酰新乌头原碱浓度的方法,并初步研究其在大鼠体内的药代动力学(药动学)行为。方法以甲基叔丁基醚液-液萃取大鼠血浆样品,采用UltimateAQ-C18column(3.0μm,2.1mm×100.0mm)色谱柱进行色谱分离,柱温40℃,以流动相乙腈(A,含体积分数0.001甲酸)-体积分数0.001的甲酸溶液(B)梯度洗脱(0~0.5min,体积分数0.25A;0.5~3.5min,体积分数0.25~0.70A;3.5~4.0min,体积分数0.70A),流量0.4mL/min,进样量10μL,分别以质子数/电荷数的比值(m/z)590.5→540.4和m/z646.7→587.0为待测成分及内标的质谱检测条件。大鼠灌服5、10、20mg/kg苯甲酰新乌头原碱后,分别在0、0.5、1.0、1.5、2.0、2.5、3.0、4.0、6.0、9.0、12.0和24.0h取样,测定其血浆中苯甲酰新乌头原碱的浓度,应用药动学数据处理软件DAS2.0计算药动学参数。结果苯甲酰新乌头原碱质量浓度为0.1~1000.0μg/L范围内线性关系良好,定量下限为0.1μg/L;苯甲酰新乌头原碱和内标的提取回收率均高于93%,日内和日间相对标准差(RSD)均<10.7%;苯甲酰新乌头原碱样品在室温和低温长期放置稳定性、冻融稳定性、预处理后供试液稳定性RSD分别小于10.2%、4.6%、10.9%、3.4%;苯甲酰新乌头原碱在大鼠体内吸收较快,达峰时间约2h,但是吸收较差,绝对生物利用度仅有0.76%。结论本文建立的LC-MS/MS法操作简便,稳定可靠,适用于苯甲酰新乌头原碱的药动学研究。

Objective To establish the liquid chromatography-tandem mass spectrometry(LC-MS/MS) method for determining the concentration of benzoylneoaconitine in rat plasma, and to preliminarily investigate its pharmacokinetic behaviors in rats. Methods Rat plasma samples were extracted with methyl tertiary butyl ether, and chromatographic separation was performed on an Ultimate AQ-C18 column(3.0 μm, 2.1 mm×100.0 mm) with a mobile phase of acetonitrile(A, containing formic acid at a volume fraction of 0.001)-formic acid solution with a volume fraction of 0.001(B) for gradient elution(0-0.5 min, vo-lume fraction 0.25 A;0.5-3.5 min, volume fraction 0.25-0.70 A;3.5-4.0 min, volume fraction 0.70 A) at a column temperature of 40 ℃, a flow rate of 0.4 mL/min, and a sample size of 10 μL. A mass-to-charge ratio(m/z) of 590.5→540.4 and 646.7→587.0 was used as the mass spectrometry conditions for the component to be tested and the internal standard, respectively. After the rats were given benzoylneoaconitine at a dose of 5,10, and 20 mg/kg by gavage, related samples were collected at 0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 4.0, 6.0, 9.0, 12.0, and 24.0 h to determine the concentration of benzoylneoaconitine in plasma, and pharmacokinetic parameters were calculated by DAS 2.0 software. Results Benzoylneoaconitine showed a good linear relationship within the range of 0.1-1 000.0 μg/L, and the lower limit of quantification was 0.1 μg/L. Both benzoylneoaconitine and the internal standard had a recovery rate of >93%, and both intra-day and inter-day relative standard deviations(RSDs) were <10.7%. The RSD of benzoylneoaconitine samples in terms of stability after long-term storage at room temperature, stability after long-term storage at a low temperature, freeze-thaw stability, and test solution stability after pretreatment was less than 10.2%, 4.6%, 10.9%, and 3.4%, respectively. Benzoylneoaconitine was absorbed rapidly in rats, with a time to peak of about 2 h, while the absorption of benzoylneoaconitine was very poor, with an absolute bioavailability of only 0.76%. Conclusion The LC-MS/MS method established in this study is simple, convenient, stable, and reliable. This method can be used for the pharmacokinetic study of benzoylneoaconitine.