LC-MS/MS法同时测定人血浆中谷氨酸和精氨酸

目的: 建立测定人血浆中谷氨酸和精氨酸的方法,以应用于人体药代动力学研究. 方法: 血浆样本经与丹酰氯衍生化反应后,选用Synergi 4μ Hydro-RP 80A柱(150 mm×2.0 mm,4 μm),以甲醇-10 mmol·L^-1乙酸铵(含0.1%甲酸)(50: 50)为流动相,流速0.3 mL·min^-1,采用API3200型三重四极杆串联质谱仪的多重反应监测(MRM)扫描方式进行监测,电喷雾离子化源,正离子方式,选择监测离子反应分别为m/z 381.0→170.2(谷氨酸丹酰氯衍生化产物)、m/z 408.0→170.3(精氨酸丹酰氯衍生化产物)和m/z 353.0→157.1(内标D-高丝氨酸丹酰氯衍生化产物). 结果: 谷氨酸、精氨酸和内标D-高丝氨酸的丹酰氯衍生化产物的保留时间分别为4.62、3.21和4.39 min;血浆中谷氨酸和精氨酸的线性范围均为3.00~300 μg·mL^-1(r>0.99),定量下限(LLOQ)均为3.00 μg·mL^-1;批内、批间精密度(RSD)均小于15%;准确度(RE)均在±15%的范围以内;谷氨酸和精氨酸平均提取回收率分别为(85.6±3.3)%和(79.9±3.9)%;平均基质效应因子分别为(116.3±4.9)%和(102.4±7.3)%;稳定性试验中,在各种贮存条件下血浆中谷氨酸和精氨酸均较稳定. 结论: 该方法适用于谷氨酸精氨酸注射液人体药代动力学研究.

Objective: To develop an LC-MS/MS method for determination of glutamate and arginine in human plasma for human pharmacokinetic studies. Methods: After precolumn derivatization with dansyl chloride,the analytes and internal standard(I.S.)were separated on a Synergi 4 μm Hydro-RP 80A analytical column(150 mm×2.0 mm,4 μm)using the mobile phase of methanol and 10 mmol·L^-1 ammonium acetate containing 0.1% formic acid(50: 50)at a flow rate of 0.3 mL·min^-1. Detection was carried out by electrospray positive ionization mass spectrometry in multiple reaction monitoring(MRM)mode. The MRM transitions of m/z 381.0→170.2,m/z 408.0→170.3 and m/z 353.0→157.1 were applied to quantify the derivatized products of glutamate,arginine and Dhomoserine(I.S.),respectively. Results: The derivatized products of glutamate,arginine and I.S. were eluted at 4.62 min,3.21 min and 4.39 min,respectively. The calibration curves were linear over the concentration range of 3.00-300 μg·mL^-1(r>0.99)with the lower limit of quantitation(LLOQ)3.00 μg·mL^-1. Both Intra-and inter-batch relative standard deviations(RSD)were below 15% and the relative errors(RE)were in the range of ±15%. The mean extraction recovery of glutamate and arginine was(85.6±3.3)% and(79.9±3.9)%,and the mean matrix effect factor was(116.3±4.9)% and(102.4±7.3)%. In the stability studies,glutamate and arginine were found to be stable in plasma under various storage conditions. Conclusion: The established method can be used for the clinical pharmacokinetic study of glutamate and arginine injection.