Highly sensitive quantification of key regulatory oxysterols in biological samples by LC-ESI-MS/MS.
We describe a highly sensitive and specific method for the quantification of key regulatory oxysterols in biological samples. This method is based upon a stable isotope dilution technique by liquid chromatography-tandem mass spectrometry (LC-MS/MS). After alkaline hydrolysis of human serum (5 microl) or rat liver microsomes (1 mg protein), oxysterols were extracted, derivatized into picolinyl esters, and analyzed by LC-MS/MS using the electrospray ionization mode. The detection limits of the picolinyl esters of 4beta-hydroxycholesterol, 7alpha-hydroxycholesterol, 22R-hydroxycholesterol, 24S-hydroxycholesterol, 25-hydroxycholesterol, 27-hydroxycholesterol, and 24S,25-epoxycholesterol were 2-10 fg (5-25 amol) on-column (signal-to-noise ratio = 3). Reproducibilities and recoveries of these oxysterols were validated according to one-way layout and polynomial equation, respectively. The variances between sample preparations and between measurements by this method were calculated to be 1.8% to 12.7% and 2.9% to 11.9%, respectively. The recovery experiments were performed using rat liver microsomes spiked with 0.05 ng to 12 ng of oxysterols, and recoveries of the oxysterols ranged from 86.7% to 107.3%, with a mean recovery of 100.6%. This method provides reproducible and reliable results for the quantification of oxysterols in small amounts of biological samples.
Honda
Akira
A
Center for Collaborative Research, Tokyo Medical University, Kasumigaura Hospital, Ami, Ibaraki 300-0395, Japan.
Yamashita
Kouwa
K
Hara
Takashi
T
Ikegami
Tadashi
T
Miyazaki
Teruo
T
Shirai
Mutsumi
M
Xu
Guorong
G
Numazawa
Mitsuteru
M
Matsuzaki
Yasushi
Y
D000818:Animals; D002138:Calibration; D002853:Chromatography, Liquid / Q000379:methods*; D006801:Humans; D006888:Hydroxycholesterols / Q000032:analysis*; D008862:Microsomes, Liver / Q000737:chemistry; D051381:Rats; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods*
D006888:Hydroxycholesterols; C099828:cholest-5-ene-3,4-diol; C003585:22-hydroxycholesterol; C044563:24-hydroxycholesterol; C011724:cholest-5-en-3 beta,7 alpha-diol; C076996:27-hydroxycholesterol; C007997:25-hydroxycholesterol