Online solid-phase extraction–liquid chromatography–mass spectrometry to determine free sterols in human serum

• The method allows the analysis of 2 cholesterol precursors and 3 phytosterols.
• The organic phase from the Bligh–Dyer extraction was injected in the online system.
• The method avoids the time-consuming evaporation and reconstitution steps.
• A water cross-flow solved the problem of having two reversed phases in the system.
• The method is able to process 25 samples per day with 2 hours of sample handling.

An automated method for analyzing free non-cholesterol sterols in human serum using online solid phase extraction–liquid chromatography–mass spectrometry is proposed herein. The method allows the determination of three phytosterols (sitosterol, stigmasterol and campesterol) and two cholesterol precursors (desmosterol and lanosterol). The analysis of sterols in human serum is critical in the study of cholesterol-related disorders, such as inherited familial hypercholesterolemias. Special effort was made to isolate the analytes from the serum lipoproteins, their natural conveyance through the bloodstream. The sample treatment consisted of a Bligh–Dyer extraction followed by dilution of the extract. This treatment allowed the sample to be injected into the online system and ensured the correct detection of the analytes, while avoiding the matrix effects commonly related to serum samples.The analytical performance showed linear ranges that covered two orders of magnitude, with correlation coefficients above 0.99. Limits of detection and quantification ranged from 0.2 ng/mL to 13 ng/mL and from 1.0 ng/mL to 43 ng/mL, respectively. Recovery when spiking serum with a half or a tenth of the average concentration reported in human serum ranged from 99% to 111% and from 102% to 120%, respectively. Intra-day precision and inter-day precision were below 20%.

Figure optionsDownload as PowerPoint slide