目的 建立一種簡(jiǎn)便、快速、靈敏的測(cè)定大鼠多柔比星血藥濃度的超高效液相-質(zhì)譜聯(lián)用（UPLC-MS/MS）法，并將其應(yīng)用于注射用鹽酸多柔比星大鼠體內(nèi)毒代動(dòng)力學(xué)實(shí)驗(yàn)。方法 采用ACQUITY UPLC^® BEH C₁₈（50 mm×2.1 mm，1.7 μm）色譜柱，流動(dòng)相為0.1%甲酸（含2 mmol/L甲酸銨）水溶液-乙腈，梯度洗脫。體積流量為0.4 mL/min，進(jìn)樣量為10 μL。采用電噴霧離子源（ESI），多反應(yīng)監(jiān)測(cè)（MRM）方式掃描，以正離子方式進(jìn)行檢測(cè)，蛋白沉淀法提取樣品。用于定量分析的離子對(duì)分別為多柔比星m/z 544.43→m/z 397.08，內(nèi)標(biāo)地西泮m/z 285.02→154.40。SD大鼠30只，按體質(zhì)量隨機(jī)分為3組，分別單次iv 52.2、61.4、72.3 mg/m²鹽酸多柔比星后測(cè)定血藥濃度，并用DAS 3.1.4軟件計(jì)算毒代參數(shù)。結(jié)果 血漿中內(nèi)源性物質(zhì)不干擾待測(cè)物和內(nèi)標(biāo)的測(cè)定，多柔比星在0.5～100 ng/mL范圍內(nèi)線性關(guān)系良好，定量下限為0.5 ng/mL。多柔比星在0.5、1、20、80 ng/mL 4個(gè)濃度的批內(nèi)批間精密度RSD值為3.21%～12.79%。多柔比星在1、80 ng/mL的提取回收率和基質(zhì)效應(yīng)分別為102.00%～103.75%和79.27%～89.34%。SD大鼠分別單次iv給予注射用鹽酸多柔比星52.2、61.4、72.3 mg/m²后，多柔比星在大鼠體內(nèi)的AUC_0-t分別為（2 318.78±282.65）、（3 203.11±829.41）和（3 326.96±546.04） ng·h/mL，C_0.083h分別為（1 720.50±851.19）、（3 363.00±1 458.84）和（2 156.50±919.90）ng/mL。結(jié)論 建立的UPLC-MS/MS分析方法靈敏度高、樣品處理方法簡(jiǎn)單、樣品分析時(shí)間短，可以應(yīng)用于大鼠多柔比星毒代動(dòng)力學(xué)試驗(yàn)中。

Objective A simple, rapid, and sensitive ultra high-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method has been developed to detect doxorubicin in rat plasma. The method was also validated for its application in toxicokinetics. Methods The separation was carried out on ACQUITY UPLC^® BEH C₁₈ column (50 mm×2.1 mm, 1.7 μm). The mobile phase consisted of 0.1% formic acid with 2 mmol/L ammonium formate and acetonitrile with gradient elution. The flow rate was 0.4 mL/min, and volume of injection was 10 μL. Electrospray ionization (ESI) source and multiple-reaction monitoring (MRM) was performed in the positive ion mode. The samples were prepared by protein precipitation. The ion pairs of m/z 544.43→m/z 397.08 and m/z 285.02→m/z 154.40 were used to doxorubicin and internal standard diazepam. A total of 30 SD rats were randomly divided into three groups according to body weight. The concentration of doxorubicin in rat plasma after intravenous administration of doxorubicin hydrochloride injection of 52.5, 61.4, and 72.3 mg/m² were determined, and the toxicokinetic parameters were calculated by using DAS 3.1.4. Results Endogenous substance in plasma had no effect on results. The linear ranges of doxorubicin were 0.5 — 100 ng/mL, with the lower limit of quantification of 0.5 ng/mL. Under concentrations of 0.5, 1, 20, and 80 ng/mL, the RSD value of intra and inter-day were 3.21%—12.79%. The extraction recovery and matrix effect of doxorubicin at 1 and 80 ng/mL were 102.00% — 103.75% and 79.27% — 89.34%, respectively. After intravenous administration of doxorubicinhydrochloride injection of 52.5, 61.4, and 72.3 mg/m², the toxicokinetic parameters were as follows: AUC_0-t (2 318.78 ±282.65), (3 203.11 ±829.41) and (3 326.96 ±546.04) ng·h/mL; C_0.083h (1 720.50 ±851.19), (3 363.00 ±1 458.84) and (2 156.50 ±919.90) ng/mL, separately. Conclusion The established UPLC-MS/MS method has high sensitivity, simple sample preparation method and short sample analysis time. It can be applied to the toxicokinetic test of doxorubicin in rats.

R969.1

國(guó)家科技重大專項(xiàng)（2015ZX09501004）