LC-ESI-MS/MS quantification of 4β-hydroxycholesterol and cholesterol in plasma samples of limited volume

J Pharm Biomed Anal. 2013 Nov:85:145-54. doi: 10.1016/j.jpba.2013.07.016. Epub 2013 Jul 25.


A liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) assay was developed and qualified for analyzing 4β-hydroxycholesterol and cholesterol in 5 μl of human and mouse plasma. Stable isotope-labeled d7-analogs of both analytes were used as internal standards and 4.2% (w/v) human serum albumin in phosphate-buffered saline was used as the surrogate matrix for preparation of calibration curves and QCs. The assay is capable of quantification of 4β-hydroxycholesterol and cholesterol from 5 to 500 ng/ml and 50 to 2000 μg/ml, respectively, with acceptable accuracy and precision following evaluation of recovery of analytes, autosampler stability and potential contribution of chemical oxidation to the formation of 4β-hydroxycholesterol. The final reconstituted solution was diluted for quantification of cholesterol typically present at 1000 fold higher concentration than 4β-hydroxycholesterol in the same samples used for 4β-hydroxycholesterol quantification. The successful quantification using a low plasma volume was achieved by quantification of total forms (free and conjugated) of both analytes after alkaline hydrolysis, followed by derivatization to form electrospray ionization-sensitive picolinyl esters, which upon collision-induced dissociation gave high mass precursor-product ion pair for selective detection by multiple reaction monitoring. In addition, chromatographic separation using a 16-min reversed phase gradient elution on a 1.9 μm particle size, C18 column, overcame interference from other isobaric plasma oxysterols during detection by multiple-reaction monitoring. This assay was compared to an orthogonal enzymatic assay for cholesterol and all samples, but one, provided values that were within 10% of each other. In addition, this assay passed the incurred sample tests for both analytes in human and mouse plasma samples according to reported acceptance criteria for incurred sample reanalysis. The quantification of both analytes permitted the determination of 4β-hydroxycholesterol compared to its ratio to cholesterol as an endogenous biomarker for CYP3A4/5 activity. The LC-ESI-MS/MS assay was also successfully applied to quantification of 4β-hydroxycholesterol and cholesterol in plasma samples from untreated human and mice including FRG™ KO C57Bl/6 chimeric mice with humanized livers. The preliminary data indicated that the plasma 4β-hydroxycholesterol concentrations or their ratio to cholesterol from mice including chimeric mice were higher than those from human.

Keywords: 4β-Hydroxycholesterol and cholesterol; CYP3A4/5 endogenous biomarker; FRG KO C57Bl/6 chimeric mice; LC–ESI-MS/MS; Stable isotope-labeled internal standard; Surrogate matrix.

MeSH terms

  • Animals
  • Cholesterol / blood*
  • Chromatography, High Pressure Liquid / methods*
  • Humans
  • Hydroxycholesterols / blood*
  • Mice
  • Spectrometry, Mass, Electrospray Ionization / methods*
  • Tandem Mass Spectrometry / methods*


  • Hydroxycholesterols
  • cholest-5-ene-3,4-diol
  • Cholesterol