LC-MS/MS液质联用快速同时分析血浆中新橙皮苷与新橙皮苷二氢查耳酮的含量

A Rapid Determination for Neohesperidin and Neohesperidin Dihydrochalcone in Plasma via Liquid Chromatography-Tandem Mass Spectrometry Method

目的建立简单快速同时测定大鼠血浆中新橙皮苷（Neohesperidin,NH）与新橙皮苷二氢查耳酮（Neohesperidin Dihydrochalcone,NHDC）浓度的液相色谱-串联质谱（LC-MS/MS）的方法。方法血浆样本100μL加入一定量的柚皮素（内标）,用乙酸乙酯-正丁醇（4∶1）沉淀蛋白同时进行液-液萃取后,离心取上清液,氮气挥干复溶后直接进样分析,以甲醇-水（70∶30,v/v）为流动相,采用ESI负离子源以及多离子反应监测（MRM）对血浆中新橙皮苷、新橙皮苷二氢查耳酮和柚皮素进行定量分析,新橙皮苷、新橙皮苷二氢查耳酮和柚皮素（内标物）离子对分别为m/z609.18→301.1,m/z611.2→303.1,m/z 273.1→153.1。结果新橙皮苷与新橙皮苷二氢查耳酮的线性范围均为0.64-400 ng/mL（r2=0.999）,最低定量限（LLOQ）均为0.64 ng/mL。2种待测物在低（3.2 ng/mL）,中（40 ng/mL）,高（200 ng/mL）3个质控浓度的提取回收率分别为85.22%±6.15%,88.37%±4.99%及86.05%±6.01%,以及81.43%±4.85%,83.43%±6.32%,82.11%±5.27%。日间和日内精密度差异均小于10%。经验证,本方法准确度、精密度、稳定性和介质效应均符合方法学分析要求。结论本实验建立同时测定新橙皮苷、新橙皮苷二氢查耳酮的方法灵敏度高,专属性强,样品处理过程简单快捷,适用于检测血浆中新橙皮苷、新橙皮苷二氢查耳酮的浓度。

OBJECTIVE To establish a simple and rapid liquid chromatography-tandem mass spectrometry（ LC-MS / MS） method for quantifying Neohesperidin and Neohesperidin dihydrochalcone in rat plasma. METHODS After protein precipitation of 100 μL sample plasma by ethyl acetate（4∶ 1）,Neohesperidin,Neohesperidin dihydrochalcone and naringinin（ internal standard） were analyzed by LC-MS / MS. The mobile phase consisted of methanol and ultrapure water（ 70 ∶ 30,v / v）. Detection was performed with multiple reactions monitoring（ MRM） in an electrospray ionization source（ ESI）. The precursor / product ion transitions of Neohesperidin,Neohesperidin dihydrochalcone and naringinin（ internal standard） were monitored at m / z609. 18→301. 1,m / z611. 2→303. 1,m / z 273.1→153. 1. RESULTS The calibration curves were linear in the concentration range of 0. 64-400 ng / mL（ r2= 0. 999）. The lowest limit of quantification（ LLOQ） reached 0. 64 ng / mL. The extraction recoveries of Neohesperidin and Neohesperidin dihydrochalcone were 85. 22% ± 6. 15%,88. 37% ± 4. 99% versus 86. 05% ± 6. 01%,and 81. 43% ± 4. 85%,83. 43% ± 6. 32%,82. 11% ±5. 27% for 3 QC concentration levels（3. 2,40,200 ng / mL）. The relative standard deviation（ RSD） of intra- and inter-day precision was below 10%. The method was validated by determining accuracy, precision, stability and matrix effects parameters.CONCLUSION The method was proved to be suitable for detection of Neohesperidin and Neohesperidin dihydrochalcone in plasma with the advantages of simplicity,high sensitivity and specificity.