Eckfeldt J H, Lewis L A, Belcher J D, Singh J, Frantz I D
Department of Laboratory Medicine and Pathology Medical School, University of Minnesota, Minneapolis 55455.
Clin Chem. 1991 Jul;37(7):1161-5.
We developed an isotope dilution mass spectrometric cholesterol method with [25,26,27-13C]cholesterol as internal standard and a benchtop gas chromatograph/mass spectrometer (GC/MS) that is much easier and less time consuming than previously described Reference and Definitive Methods for cholesterol. The internal standard, cholesterol standards, and unknown specimen are delivered volumetrically with an automated dilutor and the saponifying reagent. After saponification, extraction, and derivatization, specimens are injected into a benchtop quadrupole MS with an autosampler. Unknown cholesterol concentrations are calculated automatically by comparing the peak area ratio of the m/z = 368, 371 ion pair with the ratios for the cholesterol standards (0 to 12.93 mmol/L). We found within-run and day-to-day (overall) imprecision of 0.44% and 0.95%, respectively, when specimens were assayed singly. In several lyophilized and frozen Standard Reference Material (SRM) pools, cholesterol results with our GC/MS method averaged 0.4% less than the National Institute for Standards and Technology definitive GC/MS result performed about three years earlier. Our GC/MS results averaged 1.3% and 2.0% less than results by the National Reference System (NRS) Abell-Levy-Brodie-Kendall (ALBK) results from clinical specimens and the SRM pools, respectively. These results are consistent with the previously reported bias between the NRS Reference and Definitive Methods and the 0.1% per year decrease in cholesterol concentrations in SRM pools as determined by GC/MS analysis. These results further emphasize the small but consistent bias between cholesterol results by isotope dilution mass spectrometry and the ALBK Reference Method, the latter being the basis for the National Cholesterol Education Program guidelines and population reference values.
我们开发了一种以[25,26,27 - 13C]胆固醇为内标的同位素稀释质谱胆固醇测定方法，并使用了台式气相色谱/质谱仪（GC/MS），该方法比先前描述的胆固醇参考方法和决定性方法更简便、耗时更少。内标、胆固醇标准品和未知样本通过自动稀释器和皂化试剂进行定量输送。皂化、萃取和衍生化后，样本通过自动进样器注入台式四极杆质谱仪。通过比较m/z = 368、371离子对的峰面积比与胆固醇标准品（0至12.93 mmol/L）的峰面积比，自动计算未知胆固醇浓度。我们发现单独检测样本时，批内不精密度和日间（总体）不精密度分别为0.44%和0.95%。在几个冻干和冷冻的标准参考物质（SRM）混合样本中，我们的GC/MS方法测得的胆固醇结果平均比大约三年前美国国家标准与技术研究院的决定性GC/MS结果低0.4%。我们的GC/MS结果分别比临床样本和SRM混合样本的美国国家参考系统（NRS）阿贝尔 - 利维 - 布罗迪 - 肯德尔（ALBK）结果平均低1.3%和2.0%。这些结果与先前报道的NRS参考方法和决定性方法之间的偏差以及通过GC/MS分析确定的SRM混合样本中胆固醇浓度每年下降0.1%一致。这些结果进一步强调了同位素稀释质谱法测得的胆固醇结果与ALBK参考方法之间虽小但一致的偏差，后者是美国国家胆固醇教育计划指南和人群参考值的基础。
