Abstract
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.
Highlights
We describe a highly sensitive and specific method for the quantification of key regulatory oxysterols in biological samples
Seven oxysterols were converted into the corresponding picolinyl ester derivatives and positive electrospray ionization (ESI)-MS, MS/MS, TABLE 1
We have developed a new method for the enhancement of the ionization efficiency by derivatizing into picolinyl esters [23, 27]
Summary
We describe a highly sensitive and specific method for the quantification of key regulatory oxysterols in biological samples. The oxysterol 24S,25-epoxycholesterol is not derived from cholesterol but is produced de novo from acetyl-CoA via a shunt in the mevalonate pathway [8] These oxysterols are important molecules for preserving lipid homeostasis in the body. GC-MS is still not an ideal method, especially for the analysis of 24S,25-epoxycholesterol, because this epoxycholesterol does not survive the temperature required for GC analysis [16] Another approach to quantifying oxysterols in biological samples was HPLC with ultraviolet (UV) detection after derivatization to the D4-3ketones [16,17,18,19].
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