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An HPLC-GC/MS reference method for serum total cholesterol with control for ester hydrolysis
1Department of Clinical Biochemistry, Rigshospitalet, Risskov, Denmark.
Clinical Biochemistry
|June 1, 1994
Summary
This study introduces a new method to ensure complete cholesterol ester hydrolysis in serum cholesterol testing. The validated technique enhances the accuracy of total cholesterol measurements using isotope-dilution mass spectrometry.
Area of Science:
- Clinical Chemistry
- Analytical Chemistry
- Biochemistry
Background:
- Current isotope-dilution mass spectrometry methods for serum total cholesterol lack control for cholesterol ester hydrolysis completeness.
- Incomplete hydrolysis can lead to inaccurate total cholesterol quantification.
Purpose of the Study:
- To develop and validate a method to monitor and ensure complete hydrolysis of cholesterol esters in serum.
- To improve the accuracy and reliability of serum total cholesterol measurements.
Main Methods:
- Development of a combined high-pressure liquid chromatography (HPLC) and gas chromatography/mass spectrometry (GC/MS) method.
- Use of labeled internal standards (13C3-cholesterol and cholesteryl [1-14C] oleate) for monitoring hydrolysis and quantitation.
- Quantification of non-hydrolyzed cholesteryl oleate via radioactivity counting.
Main Results:
- The developed method accurately quantifies serum total cholesterol with < 1% deviation from NIST target values.
- The coefficient of variation for the method was found to be <= 1.0%.
- The non-hydrolyzed fraction of cholesterol esters was consistently less than 0.1%.
Conclusions:
- The developed reference method ensures complete hydrolysis of cholesterol esters, significantly improving serum total cholesterol assay accuracy.
- This method provides a reliable control for the critical hydrolysis step in cholesterol analysis.
- The combined HPLC-GC/MS approach offers a robust solution for accurate clinical cholesterol testing.