TY - JOUR
T1 - Derivatization-independent cholesterol analysis in crude lipid extracts by liquid chromatography/mass spectrometry
T2 - Applications to a rabbit model for atherosclerosis
AU - Shui, Guanghou
AU - Cheong, Wei Fun
AU - Jappar, Ignasius A.
AU - Hoi, Aina
AU - Xue, Yangkui
AU - Fernandis, Aaron Z.
AU - Tan, Benny Kwong Huat
AU - Wenk, Markus R.
PY - 2011/7/15
Y1 - 2011/7/15
N2 - Direct measurement of various sterols in crude lipid extracts in a single experiment from limited biological samples is challenging. Current mass spectrometry (MS) based approaches usually require chemical derivatization before subjecting to MS analysis. Here, we present a derivatization-independent method for analyzing various sterols, including cholesterol and its congeners, using liquid chromatography and atmospheric pressure chemical ionization mass spectrometry. Based on the specific tandem mass spectrometry pattern of cholesterol, multiple reaction monitoring (MRM) transitions were used to quantify free cholesterol and its fatty acyl esters. Several cholesterol oxidation products could also be measured using the upfront liquid chromatography separation and specific MRM transitions. The method was validated alongside established enzymatic assays in measuring total cholesterol. As a proof of concept, we analyzed plasma sterols in rabbits administrated with a high cholesterol diet (HCD) which is a classical atherosclerotic model. Free cholesterol, cholesterol esters, 7-hydroxycholesterol, and 7-ketocholesterol were elevated in plasma of rabbits on HCD. This method could also serve as an excellent tool for quantitative analysis of other sterols such as ergosterol and sitosterol in other organisms beside mammalian. In Saccharomyces cerevisiae, our results indicated dramatic increases of the ratio of ergosterol esters to free ergosterol in both yeh2Δ and tgl1Δ cells, which are consistent with the function of the respective enzymes.
AB - Direct measurement of various sterols in crude lipid extracts in a single experiment from limited biological samples is challenging. Current mass spectrometry (MS) based approaches usually require chemical derivatization before subjecting to MS analysis. Here, we present a derivatization-independent method for analyzing various sterols, including cholesterol and its congeners, using liquid chromatography and atmospheric pressure chemical ionization mass spectrometry. Based on the specific tandem mass spectrometry pattern of cholesterol, multiple reaction monitoring (MRM) transitions were used to quantify free cholesterol and its fatty acyl esters. Several cholesterol oxidation products could also be measured using the upfront liquid chromatography separation and specific MRM transitions. The method was validated alongside established enzymatic assays in measuring total cholesterol. As a proof of concept, we analyzed plasma sterols in rabbits administrated with a high cholesterol diet (HCD) which is a classical atherosclerotic model. Free cholesterol, cholesterol esters, 7-hydroxycholesterol, and 7-ketocholesterol were elevated in plasma of rabbits on HCD. This method could also serve as an excellent tool for quantitative analysis of other sterols such as ergosterol and sitosterol in other organisms beside mammalian. In Saccharomyces cerevisiae, our results indicated dramatic increases of the ratio of ergosterol esters to free ergosterol in both yeh2Δ and tgl1Δ cells, which are consistent with the function of the respective enzymes.
KW - Cholesterol
KW - Cholesterol esters
KW - Cholesterol oxidation products
KW - Multiple reaction monitoring (MRM)
UR - https://www.scopus.com/pages/publications/79959327578
U2 - 10.1016/j.chroma.2011.05.011
DO - 10.1016/j.chroma.2011.05.011
M3 - Article
C2 - 21621788
AN - SCOPUS:79959327578
SN - 0021-9673
VL - 1218
SP - 4357
EP - 4365
JO - Journal of Chromatography A
JF - Journal of Chromatography A
IS - 28
ER -