Wudy S A, Hartmann M, Svoboda M
Steroid Laboratory, Department of Pediatrics, University of Ulm, Germany.
Horm Res. 2000;53(2):68-71. doi: 10.1159/000023516.
An assay based on stable isotope dilution liquid chromatography-tandem mass spectrometry (ID/LC-MS-MS) was developed for the quantification of 17-hydroxyprogesterone, the most important indicator of 21-hydroxylase deficiency in human plasma. Plasma was extracted using ethyl acetate and Extrelut columns. LC was performed on a reversed-phase C18 column using a water/methanol gradient. A benchtop triple quadrupole mass spectrometer, operating in selected reaction monitoring mode, served as mass detector. The analytical run time was 9 min per sample. The sensitivity was high: 0.06 pmol of 17-hydroxyprogesterone yielded a signal-to-noise ratio of 13. Precision (CV) and accuracy (relative error) derived from the analyses of unspiked and spiked validation samples were 7.4-12.0% and 6.4%, respectively. When analyzing the same samples - median (range), in nanomoles per liter - from neonates and adults independently by ID/LC-MS-MS as well as by ID/gas chromatography (GC)-MS, corresponding results were obtained: neonates (n = 10), ID/LC-MS-MS 3.99 (0.48-16.05), ID/GC-MS 5.39 (1.57-13.02); adults (n = 10), ID/LC-MS-MS 2.66 (1.39-6.15), ID/GC-MS 2.54 (0.51-5.12). The technique permitted reliable detection of classical and nonclassical forms of 21- hydroxylase deficiency. The much simpler sample preparation, the faster analytical run time and the operational ease possible with ID/LC-MS-MS permit a considerable increase of sample testing per day without compromising on analytical sensitivity and specificity. We expect that benchtop tandem mass spectrometry will open new avenues in clinical steroid analysis.
开发了一种基于稳定同位素稀释液相色谱 - 串联质谱法（ID/LC-MS-MS）的检测方法，用于定量检测人血浆中17 - 羟孕酮，这是21 - 羟化酶缺乏的最重要指标。血浆用乙酸乙酯和Extrelut柱进行萃取。液相色谱在反相C18柱上进行，采用水/甲醇梯度洗脱。一台台式三重四极杆质谱仪，以选择反应监测模式运行，作为质量检测器。每个样品的分析运行时间为9分钟。灵敏度很高：0.06 pmol的17 - 羟孕酮产生的信噪比为13。从未加标和加标验证样品分析得出的精密度（CV）和准确度（相对误差）分别为7.4 - 12.0%和6.4%。当分别用ID/LC-MS-MS以及ID/气相色谱（GC）-MS独立分析新生儿和成年人相同的样品（中位数（范围），单位为纳摩尔/升）时，得到了相应的结果：新生儿（n = 10），ID/LC-MS-MS为3.99（0.48 - 16.05），ID/GC-MS为5.39（1.57 - 13.02）；成年人（n = 10），ID/LC-MS-MS为2.66（1.39 - 6.15），ID/GC-MS为2.54（0.51 - 5.12）。该技术能够可靠地检测出经典型和非经典型21 - 羟化酶缺乏症。ID/LC-MS-MS的样品制备更简单、分析运行时间更快且操作简便，使得每天的样品检测量能大幅增加，同时不影响分析的灵敏度和特异性。我们预计台式串联质谱法将为临床类固醇分析开辟新途径。
