目的 建立一种高效液相色谱串联质谱(UPLC-MS/MS)方法测定大鼠尿、粪和胆汁中的R-/S-2-(2-羟基)丙酰氨基-苯甲酸 (R-/S-HPABA)的浓度,并应用于R-/S-HPABA在大鼠体内的排泄研究。方法 色谱柱为Thermo Syncronis C18(2.1 mm×50 mm,1.7 μm),流动相为A-甲醇,B-0.1%甲酸水溶液,梯度洗脱,流速为0.4 mL·min-1,采用乙酸乙酯液液萃取法处理生物样品,多反应监测模式检测大鼠尿、粪和胆汁中R-/S-HPABA的浓度。结果 R-/S-HPABA质量浓度在0.002~5 μg·mL-1内具有良好的线性关系(r=0.992 4~0.997 6),定量下限均为0.002 μg·mL-1,日内和日间精密度均小于13.0%,准确度在-7.5%~4.2%内,平均提取回收率为(87.3±3.4)%~(104.4±7.0)%。给药后0~48 h时间段,R-HPABA和S-HPABA在大鼠尿液中的累积排泄量分别为(536.1±29.7)和(771.7±38.6) μg,分别约占给药剂量的4.9%和7.0%;给药后0~48 h时间段,R-HPABA和S-HPABA在大鼠粪便中的累积排泄量分别为 (3 963.0±345.2)和(4 771.8±355.0) μg,分别约占给药剂量的36.0%和43.4%;给药后0~12 h时间段,R-HPABA和S-HPABA在大鼠胆汁中的累积排泄量分别为(150.6±30.3)和(747.7±89.2) μg,分别约占给药剂量的1.4%和6.8%。大鼠通过尿液、粪便和胆汁这3种途径排泄R-HPABA和S-HPABA的总量分别约占给药剂量的42.3%和57.2%。S-HPABA在体内以原型药物被排泄的量显著大于R-HPABA。结论 该法适用于R-/S-HPABA在大鼠体内的排泄研究,且两对映体在大鼠体内的排泄规律存在显著的立体选择性差异。
Abstract
OBJECTIVE To develop an UHPLC-MS/MS method for the determination of R-/S-2-(2-hydroxypropanamido) benzoic acid (R-/S-HPABA) in rat urine, feces and bile and apply the method to study the excretion of R-/S-HPABA in rats. METHODS After liquid-liquid extraction by ethyl acetate,the separation was achieved on a Thermo Syncronis C18 column (2.1 mm×50 mm, 1.7 μm) using mobile phase consisting of solvent A (methanol) and solvent B (0.1% formic acid) at a flow rate of 0.4 mL·min-1. The concentration of R-/S-HPABA after single dose oral administration of 50 mg·kg-1 R-/S-HPABA to rats was detected by UHPLC-MS/MS method. RESULTS The calibration curve was linear over the range of 0.002-5 μg·mL-1 and the lower limit of quantification (LLOQ) was 0.002 μg·mL-1. The intra-and inter-day RSDs were less than 13.0% and the accuracy (relative error) of R-/S-HPABA was within -7.5%-4.2%. The average recoveries were (87.3±3.4)%-(104.4±7.0)%. After intragastric administration of R-HPABA and S-HPABA at a dose of 50 mg·kg-1, the cumulative amounts of R-HPABA and S-HPABA excreted in the urine (0-48 h) were (536.1±29.7) and (771.7±38.6) μg, the urinary excretion amounted to 4.9% and 7.0% of the dosage, respectively; the cumulative amounts of R-HPABA and S-HPABA excreted in feces (0-48 h) were (3 963.0±345.2) and (4 771.8±355.0) μg, the fecal excretion amounted to 36.0% and 43.4% of the dosage, respectively; the cumulative amounts of R-HPABA and S-HPABA excreted in bile (0-12 h) were (150.6±30.3) and (747.7±89.2) μg, the biliary excretion amounted to 1.4% and 6.8% of the dosage, respectively; the summation of urinary, fecal and biliary excretion amounted to 42.3% and 57.2% of the dosage in rats for R-HPABA and S-HPABA, respectively. The cumulative amounts of S-HPABA excreted in the urine, feces and bile were higher than those of R-HPABA. CONCLUSION This UHPLC-MS/MS method is suitable for the study of the excretion of R-/S-HPABA in rats. The excretion of the two enantiomers in rats shows significant stereoselectivity difference.
关键词
手性药物 /
R-/S-2-(2-羟基)丙酰氨基苯甲酸 /
高效液相色谱串联质谱 /
立体选择性 /
排泄
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Key words
chiral drug /
R-/S-HPABA /
UHPLC-MS/MS /
stereoselectivity /
excretion
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中图分类号：
R969
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