UPLC-MS/MS法测定猪体内烯丙孕素的血药浓度

Determination of Altrenogest Concentration in Porcine Plasma with UPLC-MS/MS

建立猪血浆中烯丙孕素含量的UPLC-MS/MS测定法。血浆中烯丙孕素以液液萃取法提取,采用Inertsil C4(3.0×150mm,5μm)色谱柱和梯度洗脱对待测样品中各成分进行分离,三重四级杆质谱仪的多重反应监测(MRM)模式进行定量分析,内标法定量。流动相A为5mmol/L甲酸铵水溶液,B为乙腈,流速为0.4mL/min,进样量为10μL。建立的烯丙孕素检测方法在1ng/mL^100ng/mL浓度范围内呈良好的线性关系,相关系数大于0.999 2。烯丙孕素3个空白血浆添加浓度样品(1、50、100ng/mL)的批间回收率分别为93.1%±3.98%、92.7%±5.75%、88.9%±3.69%,日内变异系数分别为3.50%~14.35%、3.43%~6.20%和2.88%%~4.15%,日间变异系数在5.11%~8.86%之间。方法的检测限(LOD)和定量限(LOQ)分别为0.5ng/mL和1ng/mL。基质效应对烯丙孕素的含量测定有增强作用,烯丙孕素在低浓度(1ng/mL)为114.65%,中浓度(50ng/mL)为109.23%,高浓度(100ng/mL)为113.59%。待测样品预处理后在样品室(4℃)放置8h的时间内,低、中、高3个添加浓度水平的变异系数均小于11%,表明药物在样品室测定期间有较高的稳定性。将样品反复冻融3次后,低、中、高3个浓度添加水平的变异系数均小于9%,表明血浆样品中烯丙孕素经3个循环冻融后仍可保持较高的稳定性。该方法可用于准确检测猪血浆中烯丙孕素的含量。

In order to establish a UPLC-MS/MS method for the determination of the content of altrenogest in porcine plasma,extraction of plasma was conducted with altrenogest liquid liquid extraction method,using Inertsil C4（3×150 mm,5 m）to test the separation of each component in the sample column and gradient elution,multiple reaction monitoring three level four pole mass spectrometer（MRM）model for quantitative analysis,internal standard method.The mobile phase A was 5 mmol/L ammonium formate solution,B was acetonitrile,the flow rate was 0.4 ml/min,the sample size was 10μL.In this method,the linear relationship between the concentration of 1 ng/mL-100 ng/mL and the correlation coefficient was0.999 2.Altrenogest three blank plasma concentration samples（1 ng/mL,50 ng/mL,100 ng/mL）of the batch recovery rate were 93.1%±3.98%,92.7%±5.75%,88.9%±3.69%,respectively,the intra day coefficients of variation were 3.50%-14.35%,3.43%-6.20% and 2.88%-4.15%,respectively,the coefficient of variation between 5.11%-8.86% in the daytime.The detection limits（LOD）and the quantitation limit（LOQ）of this method were 0.5 ng/mL and 1 ng/mL,respectively.The effect of matrix on the determination of the content of altrenogest was enhanced,while the low concentration（1 ng/mL）was 114.65%,the concentration of medium（50 ng/mL）was about 109.23%,and the high concentration（100 ng/mL）was113.59%.When the sample was pretreated,the coefficients of variation of the low,middle and high concentration levels were less than 11%in the sample room（8 h）for 4℃.After repeated freezing and thawing for three times,the coefficients of variation of the low,middle and high concentration levels were less than 9%,which indicated that the plasma samples could maintain high stability after being frozen and thawed for 3 cycles.The method can be used for the accurate determination of the content of altrenogest in porcine plasma.