R-/S-HPABA大鼠体内排泄规律的立体选择性

Stereoselectivity of the Excretion of R-/S-HPABA in Rats

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,1.7μm),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0.002~5μg·mL-1内具有良好的线性关系(r=0.9924~0.9976),定量下限均为0.002μg·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(3963.0±345.2)和(4771.8±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。

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,1.7μm)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 0.002-5μg·mL-1 and the lower limit of quantification(LLOQ)was 0.002μg·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(3963.0±345.2)and(4771.8±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.