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目的:建立LC-MS/MS法测定比格犬血浆中沃诺拉赞浓度,并进行毒代动力学研究。方法:40只比格犬按体重随机分为溶媒对照组(0.9%氯化钠注射液)和低(1 mg·kg~(-1)·d~(-1))、中(3 mg·kg~(-1)·d~(-1))、高(10 mg·kg~(-1)·d~(-1))剂量组,每组10只,雌雄各半。静脉推注给药,每天1次,连续4周。首、末次给药分别于给药前0 h以及给药后1/12、0.5、1、2、4、7、10和24 h经犬前肢内侧皮下静脉取血。用蛋白沉淀法处理血浆样品,LC-MS/MS分析。色谱柱:ODS柱(2.1 mm×100 mm,5μm);流动相:0.1%甲酸水溶液(氨水调pH至3.24)-乙腈(60∶40,v/v),流速:0.2 mL·min~(-1);内标:兰索拉唑;MS源:ESI,正离子MRM模式检测;定量分析沃诺拉赞10 mg·kg-1和兰索拉唑的监测离子分别为m/z:346.30→315.20,m/z:370.30→252.30。应用DAS2.1软件计算毒动学参数。结果:沃诺拉赞在5~5 000 ng·mL~(-1)线性范围内线性关系良好(r=0.999 6),定量下限为5 ng·mL~(-1)。批内和批间精密度(RSD)均<7.7%,提取回收率均>91.6%,基质效应在106%~114%间。内标提取回收率为(97.3±0.76)%,基质效应为(100.6±0.90)%。各项稳定性RSD<4.7%。首次给药比格犬低、中、高剂量组的毒代动学参数分别为C_(max):(0.54±0.03)、(1.30±0.20)、(3.51±0.52)μg·L~(-1),AUC_((0-24)):(1.35±0.24)、(5.13±0.94)、(23.74±3.12)mg·h·L~(-1),t_(1/2):(1.77±0.30)、(2.37±0.29)、(3.71±0.39)h;末次给药比格犬低、中、高剂量组的毒代动学参数分别为C_(max):(0.53±0.09)、(1.44±0.26)、(5.05±0.64)μg·L~(-1),AUC_((0-24)):(1.29±0.25)、(4.92±0.97)、(25.25±2.96)mg·h·L~(-1),t_(1/2):(1.56±0.26)、(2.11±0.34)、(3.51±0.33)h。置信区间法评价结果显示,给药范围内沃诺拉赞的AUC_((0-24))、AUC_((0-∞))和C_(max)的置信区间与判断区间部分重叠。结论:本方法灵敏度高,专属性好,稳定性好,结果准确,适用于毒代动力学试验中沃诺拉赞血样的分析。连续给药28 d后药物在比格犬体内无性别差异,无明显药物蓄积,且在1~10 mg·kg-1剂量范围内,药物是否具有线性药代动力学特征尚不能判定。
OBJECTIVE: To establish a LC-MS / MS method for the determination of vinorelazan in beagle dogs plasma and to study its toxicokinetics. Methods: Forty Beagle dogs were randomly divided into three groups according to body weight: vehicle control group (0.9% sodium chloride injection) and low (1 mg · kg -1 · d -1) kg -1 d -1 and 10 mg · kg -1 d -1, with 10 mice in each group. Intravenous bolus administration, 1 day, for 4 weeks. First, the last administration was taken at 0 h before administration and 1 / 12,0.5, 1, 2, 4, 7, 10 and 24 h after administration through the medial subcutaneous vein of dogs. Plasma samples were processed by protein precipitation, LC-MS / MS analysis. Column: ODS column (2.1 mm × 100 mm, 5 μm); mobile phase: 0.1% formic acid in water (adjusted to pH 3.24 with ammonia) 1); internal standard: lansoprazole; MS source: ESI, positive ion MRM mode; quantitative analysis of vonolazan 10 mg · kg-1 and lansoprazole monitoring ions were m / z: 346.30 → 315.20, m / z: 370.30 → 252.30. Toxicokinetic parameters were calculated using DAS 2.1 software. Results: Volanzone showed a good linearity (r = 0.999 6) in the linear range of 5 ~ 5000 ng · mL -1 with a lower limit of quantification of 5 ng · mL -1. The intra- and inter-batch precision (RSD) were <7.7%, the recovery rates were> 91.6%, and the matrix effects were between 106% and 114%. The recovery rate of internal standard was (97.3 ± 0.76)% and the matrix effect was (100.6 ± 0.90)%. The stability of the RSD <4.7%. The toxicokinetic parameters of low, medium and high dose Beagle dogs for the first administration were Cmax (0.54 ± 0.03), (1.30 ± 0.20), (3.51 ± 0.52) μg · L -1 ), AUC (0-24) :( 1.35 ± 0.24), (5.13 ± 0.94), (23.74 ± 3.12) mg · h · L -1 t 1/2: 1.77 ± 0.30 ), (2.37 ± 0.29) and (3.71 ± 0.39) h, respectively. The toxicokinetic parameters of low, medium and high dose Beagle dogs were (0.53 ± 0.09) and (1.44 ± 0.26), (5.05 ± 0.64) μg · L -1, AUC 0-24: 1.29 ± 0.25, 4.92 ± 0.97, 25.25 ± 2.96 mg · h · L -1 -1), t 1/2 (1.56 ± 0.26), (2.11 ± 0.34) and (3.51 ± 0.33) h, respectively. The results of confidence interval method showed that the confidence interval of AUC _ ((0-24)), AUC_ ((0-∞)) and C_max of vinorelazine partially overlap with the judgment interval. Conclusion: The method has high sensitivity, good specificity, good stability and accurate results. It is suitable for the analysis of Wollazane blood samples in toxicokinetic test. After continuous administration for 28 days, the drug had no sex difference in Beagle dogs, no significant drug accumulation, and whether the drug had linear pharmacokinetic characteristics could not be determined within the dose range of 1 ~ 10 mg · kg-1.