Highly sensitive quantification of 7alpha-hydroxy-4-cholesten-3-one in human serum by LC-ESI-MS/MS.
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.
Honda
Akira
A
Ibaraki Prefectural Institute of Public Health, Mito, Ibaraki 310-0852, Japan. akihonda-gi@umin.ac.jp
Yamashita
Kouwa
K
Numazawa
Mitsuteru
M
Ikegami
Tadashi
T
Doy
Mikio
M
Matsuzaki
Yasushi
Y
Miyazaki
Hiroshi
H
D002138:Calibration; D002783:Cholestenones / Q000097:blood*; D002853:Chromatography, Liquid / Q000379:methods*; D006801:Humans; D008958:Models, Molecular; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods*
D002783:Cholestenones; C002656:7 alpha-hydroxy-4-cholesten-3-one