Simultaneous and rapid determination of 6 primary bile acids in plasma by high-performance liquid chromatography-tandem mass spectrometry
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摘要:
目的 建立高效液相色谱 – 串联质谱法快速检测血浆中6种初级胆汁酸的含量。 方法 血浆经甲醇沉淀蛋白后,用Thermo Accucore C18色谱柱(100 mm × 4.6 mm,2.6 μm)进行色谱分离,流动相为甲醇 – 水(0.1 %甲酸 + 5 mmol/L乙酸铵),梯度洗脱。负离子模式下多反应监测模式检测。实测6名健康成年人和6只成年SD大鼠样本。 结果 6种初级胆汁酸在6 min内实现基线分离,在5~2 000 μg/L范围内线性良好,相关系数>0.995,检出限为0.25~0.45 μg/L,定量限为0.84~1.49 μg/L。日内、日间加标回收率分别为86.3 %~111.5 %和87.7 %~104.9 %;日内、日间相对标准偏差分别为5.2 %~11.9 %和6.6 %~14.3 %。健康成年人和成年SD大鼠血浆中初级胆汁酸的浓度显著不同。 结论 该方法简单、快速、灵敏、准确,可用于临床检验和动物试验中初级胆汁酸的快速检测。 -
关键词:
- 高效液相色谱-串联质谱法 /
- 初级胆汁酸 /
- 血浆
Abstract:Objective To develop a rapid and accurate high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method for the simultaneous determination of 6 primary bile acids in plasma. Methods Plasma was separated on a Thermo Accucore C18 column (100 mm × 4.6 mm; 2.6 μm) after being extracted by protein precipitation using methanol. The 6 primary bile acids were separated by using the mobile phase of methanol-water (0.1% formic acid + 5 mmol/L ammonium acetate) running in gradient elution. The mass spectrometer was operated on an electrospray ionization source in negative ion mode for the multiple reaction monitoring analysis. Plasma from 6 healthy adults and 6 adult Sprague-Dawley (SD) rats was detected. Results Baseline separation of 6 primary bile acids was achieved within 6 min. The linearity of the calibration curves of the 6 primary bile acids was excellent in the range of 5 – 2000 μg/L with a correlation coefficient higher than 0.995. The limits of detection and limits of quantitation were in the range of 0.25 – 0.45 μg/L and 0.84 – 1.49 μg/L, respectively. The recoveries of intra-day and inter-day were 86.3% – 111.5% and 87.7% – 104.9%, with relative standard deviations of 5.2% – 11.9% and 6.6% – 14.3%, respectively. The concentrations of primary bile acids in plasma of healthy adults and adult SD rats were significantly different. Conclusion This method is simple, rapid, sensitive, and accurate, and can meet the needs for rapid detection of primary bile acids in clinical tests and animal experiments. -
Key words:
- HPLC-MS/MS /
- primary bile acid /
- plasma
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表 1 6种初级胆汁酸及内标的母离子、子离子、去簇电压和碰撞电压
物质 母离子(m/z) 子离子(m/z) 去簇电压/(V) 碰撞电压/(V) CA 407.9 407.9 a – 141 – 17 343.3 – 47 CDCA 391.3 391.3 a – 151 – 21 373.3 – 47 TCA 514.3 514.3 a – 120 – 10 80.0 – 120 TCDCA 498.2 498.2 a – 90 – 13 80.0 – 120 GCA 464.3 464.3 a – 140 – 10 402.4 – 50 GCDCA 448.3 448.3 a – 110 – 10 368.2 – 46 CA-d4 411.2 411.2 a – 125 – 10 347.4 – 48 TCA-d4 518.3 518.3 a – 120 – 10 80.0 – 120 GCA-d4 468.3 468.3 a – 127 – 30 404.6 – 48 注:a 为定量离子。 表 2 6种初级胆汁酸的线性方程、相关系数、检出限和定量限
物质 内标 线性方程 相关系数 检出限/(μg/L) 定量限/(μg/L) CA CA-d4 y = 1.13 × 10 – 3x + 1.22 × 10 – 3 0.998 3 0.45 1.49 CDCA CA-d4 y = 4.45 × 10 – 3x + 1.25 × 10 – 2 0.998 7 0.39 1.30 TCA TCA-d4 y = 6.19 × 10 – 3x + 9.46 × 10 – 3 0.995 1 0.35 1.17 TCDCA TCA-d4 y = 7.38 × 10 – 3x + 8.33 × 10 – 3 0.995 6 0.38 1.26 GCA GCA-d4 y = 8.09 × 10 – 3x + 3.02 × 10 – 2 0.997 0 0.38 1.28 GCDCA GCA-d4 y = 1.61 × 10 – 2x + 5.18 × 10 – 2 0.995 3 0.25 0.84 表 3 血浆中6种初级胆汁酸的加标回收率及相对标准偏差
物质 加标量(μg/L) 日内 日间 回收率(%) 相对标准偏差(%) 回收率(%) 相对标准偏差(%) CA 6 94.1 8.7 98.6 14.1 800 108.0 9.4 102.9 10.4 1600 95.7 5.2 98.2 7.7 CDCA 6 101.8 11.9 101.0 6.6 800 101.0 11.0 102.9 10.2 1600 91.4 7.9 95.8 9.8 TCA 6 100.1 7.8 99.9 14.0 800 101.8 6.5 96.5 10.9 1600 95.4 11.2 91.5 8.2 TCDCA 6 99.8 10.1 104.9 12.1 800 100.8 7.0 94.1 8.4 1600 99.2 7.8 87.7 7.3 GCA 6 92.4 7.0 97.6 9.6 800 102.0 11.1 101.6 11.1 1600 97.0 9.7 99.2 14.3 GCDCA 6 86.3 8.5 94.1 11.7 800 111.5 10.7 102.6 11.4 1600 102.3 11.4 96.2 12.8 表 4 6种初级胆汁酸在健康成年人和SD大鼠血浆中的含量(n = 6)
物质 检出量/(μg/L) P 值 健康成年人 SD大鼠 CA 9.7~270.0 248.0~467.0 0.031 CDCA 43.4~406.0 106.0~405.0 0.893 TCA 20.0~222.0 65.1~600.0 0.139 TCDCA 129.0~697.0 59.0~572.0 0.139 GCA 184.0~1020.0 5.9~128.0 0.005 GCDCA 1150.0~3144.0 ND~9.8 0.005 注: ND 为未检出。 -
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