Honda Akira, Yamashita Kouwa, Numazawa Mitsuteru, Ikegami Tadashi, Doy Mikio, Matsuzaki Yasushi, Miyazaki Hiroshi
Ibaraki Prefectural Institute of Public Health, Mito, Ibaraki 310-0852, Japan.
J Lipid Res. 2007 Feb;48(2):458-64. doi: 10.1194/jlr.D600032-JLR200. Epub 2006 Nov 8.
We describe a highly sensitive and specific method for the quantification of serum 7alpha-hydroxy-4-cholesten-3-one (C4), which has been used as a biomarker for bile acid biosynthesis. This method is based upon a stable isotope dilution technique by liquid chromatography-tandem mass spectrometry (LC-MS/MS). C4 was extracted from human serum (2-50 mul) by a salting-out procedure, derivatized into the picolinoyl ester (C4-7alpha-picolinate), and then purified using a disposable C(18) cartridge. The resulting picolinoyl ester derivative of C4 was quantified by LC-MS/MS using the electrospray ionization mode. The detection limit of the C4 picolinoyl ester was found to be 100 fg (signal-to-noise ratio = 10), which was approximately 1,000 times more sensitive than the detection limit of C4 with a conventional HPLC-ultraviolet method. The relative standard deviations between sample preparations and between measurements by our method were calculated to be 5.7% and 3.9%, respectively, by one-way layout analysis. The recovery experiments were performed using serum spiked with 20.0-60.0 ng/ml C4 and were validated by a polynomial equation. The results showed that the estimated concentration with 95% confidence limit was 23.1 +/- 2.8 ng/ml, which coincided completely with the observed X(0) +/- SD = 23.3 +/- 1.0 ng/ml with a mean recovery of 93.4%. This method provides highly reliable and reproducible results for the quantification of C4, especially in small volumes of blood samples.
我们描述了一种用于定量血清7α-羟基-4-胆甾烯-3-酮（C4）的高灵敏度和特异性方法，C4已被用作胆汁酸生物合成的生物标志物。该方法基于液相色谱-串联质谱（LC-MS/MS）的稳定同位素稀释技术。通过盐析程序从人血清（2 - 50微升）中提取C4，将其衍生化为吡啶甲酰酯（C4 - 7α-吡啶甲酸酯），然后使用一次性C(18)柱进行纯化。使用电喷雾电离模式通过LC-MS/MS对所得的C4吡啶甲酰酯衍生物进行定量。发现C4吡啶甲酰酯的检测限为100飞克（信噪比 = 10），这比传统高效液相色谱 - 紫外法对C4的检测限灵敏度高约1000倍。通过单向布局分析计算，我们方法的样品制备之间和测量之间的相对标准偏差分别为5.�%和3.9%。使用添加了20.0 - 60.0纳克/毫升C4的血清进行回收率实验，并通过多项式方程进行验证。结果表明，95%置信限的估计浓度为23.1±2.8纳克/毫升，与观察到的X(0)±SD = 23.3±1.0纳克/毫升完全一致，平均回收率为93.4%。该方法为C4的定量提供了高度可靠和可重复的结果，特别是在少量血液样本中。
