Measurement of plasma renin activity with use of HPLC-electrospray-tandem mass spectrometry.
Measurement of plasma renin activity with use of HPLC-electrospray-tandem mass spectrometry.
复制标题
使用 HPLC-电喷雾串联质谱法测量血浆肾素活性。
作者：
V. Fredline;E. Kovács;P. Taylor;A. G. Johnson
背景
肾素活性的测量由于难以定量血管紧张素1（Ang 1）（肾素催化反应的产物）而变得复杂。我们报告了一种高效液相色谱-电喷雾-串联质谱（HPLC-ESI-MS/MS）的方法定量血管生成素1作为血浆肾素活性（PRA）的措施。
方法
孵育（37 ℃，3或18 h）后，使用C18固相萃取制备样品。[瓦尔] 5Ang 1用作内标物（IS）。采用C18色谱柱，以200 mL/L醋酸铵缓冲液-800 mL/L甲醇为移动的流动相。流速为150微升/分钟，色谱运行时间为5分钟/样品。质谱检测以正离子化模式进行，选择反应监测（Ang 1 m/z 649.0->784.0; IS m/z 641.9->770.4）。
结果
该测定在2.5-500 ng Ang 1/mL范围内呈线性，这对应于PRA的检测限（信噪比为3：1）为0.14 ng Ang 1。mL-1。h-1。PRA值为26.1、13.5、3.2和0.78 ng Ang 1时测定的不精密度（CV）。mL-1。h-1分别为7.0%、7.0%、15%和11%。绝对回收率分别为92.3%（Ang 1）和87.4%（IS）。在PRA测定中，3小时与18小时的孵育时间在PRA <2 ng Ang 1时得到良好的一致性。mL-1。h ~（-1），但PRA为2-5 ng Ang 1. mL-1。h ~（-1）孵育18 h后的PRA结果低于孵育3 h后的PRA结果。我们比较了HPLC-ESI-MS/MS法和RIA法测定PRA，PRA孵育时间分别为3 h和1.5 h。Ang 1的RIA法测定结果高于HPLC-ESI-MS/MS法测定结果。
结论
HPLC-ESI-MS/MS方法可灵敏、特异地测定PRA。用RIA方法测得的较高活性突出了其高估PRA的可能性。
BACKGROUND
The measurement of renin activity is complicated by difficulties in the quantification of angiotensin 1 (Ang1), the product of the renin-catalyzed reaction. We report an HPLC-electrospray-tandem mass spectrometry (HPLC-ESI-MS/MS) method for the quantification of Ang1 as a measure of plasma renin activity (PRA).
METHODS
After incubation (37 degrees C for 3 or 18 h), samples were prepared using C18 solid-phase extraction. [Val]5Ang1 was used as the internal standard (IS). Chromatography was performed on a C18 column, using 200 mL/L ammonium acetate buffer-800 mL/L methanol as the mobile phase. The flow rate was 150 microL/min, with a chromatographic run time of 5 min/sample. Mass spectrometric detection was in the positive ionization mode with selected reaction monitoring (Ang1 m/z 649.0-->784.0; IS m/z 641.9-->770.4).
RESULTS
The assay was linear over the range 2.5-500 ng Ang1/mL, which corresponded to a limit of detection (signal-to-noise ratio of 3:1) of PRA of 0.14 ng Ang1. mL-1. h-1. The imprecision (CV) of the assay at PRA values of 26.1, 13.5, 3.2, and 0.78 ng Ang1. mL-1. h-1 was 7.0%, 7.0%, 15%, and 11%, respectively. Absolute recoveries were 92.3% (Ang1) and 87.4% (IS). Incubation times of 3 h vs 18 h in the PRA assay gave good agreement at PRA <2 ng Ang1. mL-1. h-1, but samples with a PRA of 2-5 ng Ang1. mL-1. h-1 gave lower PRA results after incubation for 18 h than after 3 h. We compared the HPLC-ESI-MS/MS assay and an RIA for the determination of PRA, with PRA incubation times of 3 h and 1.5 h, respectively. The mean PRA based on RIA of Ang1 was higher than that obtained using HPLC-ESI-MS/MS.
CONCLUSION
The HPLC-ESI-MS/MS method allows sensitive and specific measurement of PRA. The higher activities measured with the RIA method highlight its potential for overestimation of PRA.
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