高效液相色谱-串联质谱法测定冠心病患者血浆中14种氨基酸的含量

Determination of 14 Amino Acids in Blood Plasma from Patients with Coronary Artery Heart Disease by High Performance Liquid Chromatography Tandem Mass Spectrometry

取血浆样品2 mL,加入体积比89∶10∶1的乙腈-水-甲酸混合液4 mL,涡旋,离心。取上清液4 mL,用200 mg C_(18)和100 mg氨丙基粉净化,涡旋,静置后取2 mL上清液,加入4 mL甲醇混匀,氮吹至近干。残渣用体积比80∶20的1.5%(体积分数)甲酸溶液-乙腈的混合液0.5 mL溶解,振荡、过滤后,采用高效液相色谱-串联质谱法(HPLC-MS/MS)测定滤液中14种氨基酸(脯氨酸、色氨酸、犬尿氨酸、丙氨酸、缬氨酸、甘氨酸、苏氨酸、精氨酸、亮氨酸、异亮氨酸、苯丙氨酸、酪氨酸、天冬酰胺、天冬氨酸)的含量。色谱分析中以InertSustain AQ-C_(18)柱为固定相,以不同体积比的水-乙腈-甲酸的混合液为流动相进行梯度洗脱。质谱分析中采用电喷雾离子源正离子(ESI~+)模式和多反应监测(MRM)模式。结果表明,14种氨基酸的质量浓度在一定范围内与对应的峰面积呈线性关系,测定下限(10S/N)为0.50~2.50μg·L^(-1)。血浆样品在3个浓度水平下进行加标回收试验,回收率为82.3%~102%,测定值的相对标准偏差(n=6)为3.3%~12%。

The blood plasma sample(2 mL) was taken, and 4 mL of a mixture of acetonitrile, water, and formic acid at the volume ratio of 89∶10∶1 was added. After vortex and centrifugation, 4 mL of the supernatant was taken, and purified with 200 mg of C_(18)and 100 mg of ammonia propyl powder. After vortex and settling, 2 mL of the supernatant was taken, mixed well with 4 mL of methanol, and blown to near dryness by nitrogen. The residue was dissolved in 0.5 mL of a mixture of 1.5%(φ) formic acid solution and acetonitrile at the volume ratio of 80∶20. After shaking and filtration, 14 amino acids(proline, tryptophan, kynurenine, alanine, valine, glycine, threonine, arginine, leucine, isoleucine, phenylalanine, tyrosine, asparagine and aspartic acid) in the filtrate were determined by high performance liquid chromatography tandem mass spectrometry(HPLC-MS/MS). In chromatographic analysis, InertSustain AQ-Ccolumn was used as the stationary phase, and a mixture of water, acetonitrile, and formic acid at different volume ratios was used as the mobile phase for gradient elution. Positive ion mode of electrospray ion source(ESI) and multiple reaction monitoring(MRM) mode were used in mass spectrometry analysis. As shown by the results, linear relationships between values of peak area and mass concentration of 14 amino acids were found in definite ranges, with lower limits of determination(10 S/N) in the range of 0.50-2.50 μg·L^(-1). Test for recovery was made on the blood plasma samples at three concentration levels by standard addition method, giving results in the range of 82.3%-102%, with RSDs(n=6) of the determined values in the range of 3.3%-12%.