HPLC-MS/ESI同时测定人血浆中米屈肼和左卡尼汀

Simultaneous HPLC-MS/ESI determination of mildronate and levocarnitine in human plasma

目的:建立一种快速、准确、灵敏的高效液相色谱-质谱(HPLC-MS)法同时测定人血浆中米屈肼和左卡尼汀的浓度。方法:以卡巴胆碱作为内标,血浆样品经蛋白沉淀法处理,用氰基色谱柱(Ultimate XB-CN,4.6 mm×250 mm,5μm)进行分离,以乙腈-30 mmol·L^(-1)醋酸铵水溶液(含0.08%甲酸)(80:20,v/v)为流动相,流速1.0 mL·min^(-1),柱温40℃,柱后分流比为4:1,HPLC-MS/ESI^+法选择性监测准分子离子峰[M+H]^+(米屈肼m/z 147.1,左卡尼汀m/z 162.1,内标卡巴胆碱m/z147.1)。结果:米屈肼、左卡尼汀及内标卡巴胆碱在氰基柱上保留较好,分离完全;米屈肼、左卡尼汀线性范围分别为0.1～30.0μg·mL^(-1)(r=0.9985)和0.4～12.8μg·mL^(-1)(r=0.9994);最低定量限分别为0.1μg·mL^(-1)和0.4μg·mL^(-1);平均萃取回收率均大于80%,方法回收率为92%～114%;日内日间RSD均小于12%。结论:本方法简便准确,可用于同时测定人血浆中米屈肼和左卡尼汀的浓度,考察服用米屈肼对血浆中内源性物质左卡尼汀浓度的影响。

Objective：To establish a high - performance liquid chromatography - electrospray ionization mass spectrometry （HPLC -MS/ESI） method for simultaneous determination of mildronate and levocarnitine in human plasma. Methods：Carbachol was used as the internal standard（IS）.The compound and internal standard were exreacted by protein precipitation using acetonitrile.The HPLC separation of the analytes was performed on a Ultimate XB - CN（4.6 mm×250 mm,5μm） column with a column temperature of 40℃.The mobile phase was acetonitrile-water （ammonium acetate：30 mmol·L^-1;formic acid：0.08%）（80：20,v/v）.The flow rate was 1.0 mL·min^-1, and the postcolumn splitting ratio was 4：1.Electrospray ionization mass spectrometry（ESI/MS） was used for determination, with positive ion SIM detection of mildronate（[M ＋ H]^＋,m/z 147.1）,levocarnitine（[M ＋ H]^＋,m/z 162.1） and carbachol（[M ＋ H]^＋,m/z 147.1）.Results：Mildronate,levocarnitine and IS all could be retained well and separated completely on the Ultimate XB - CN column.The calibration curves were linear in the ranges of 0.1 - 30.0μg·mL^-1（r = 0.9985 ） for mildronate and 0.4 - 12.8μg·mL^-1（r = 0.9994 ） for levocarnitine;The limits of detection（LOD） were 0.1μg·mL^-1 for mildronate and 0.4μg·mL^-1 for levocarnitine;The average extraction recoveries for all the two analytes were above 80%;The methodology recoveries were 92%- 114%;The intra - day and inter - day relative standard deviations were less than 12%.Conclusion：The method is accurate and simple for simultaneous determination of mildronate and levocarnitine in human plasma,and can be used to investigate the effect of administed mildronate on the plasma concentration of levocarnitine in human.