Simultaneous determination of oxysterols, phytosterols and cholesterol precursors by high performance liquid chromatography tandem mass spectrometry in human serum

Abstract

A fast and sensitive high performance liquid chromatography with atmospheric pressure chemical ionization tandem mass spectrometry (HPLC-APCI-MS/MS) method to identify and quantify oxysterols, phytosterols and non-cholesterol sterols at the pico-molar concentration level in human serum in only one run was developed in this study. This method allows the simultaneous separation and quantitation of individual phytosterols, cholesterol precursors and oxidized derivatives of cholesterol without a derivatization step in a single run, thus providing a more confident quantitation of sterols in serum. After saponification, solid-phase extraction (SPE) used as a clean-up step and HPLC separation, detection by MS was developed using APCI and multiple ion monitoring modes. This method employs reversed-phase C 18 SPE cartridges and serum calibrators, as well as isotopically labelled cholesterol as an internal standard added before sample processing. The time consumed for a single sample is reduced from the 4 hours of conventional sterol analysis to 1 hour including the chromatographic run time. The method has been evaluated by analyzing a certified cholesterol sample as well as by comparison to other two methods used as reference, based on GC and enzymatic reaction, respectively. Serum from 14 individuals was successfully analyzed. Detection limits for oxysterols, phytosterols and non-cholesterol sterols, all determined in a single run in small serum volumes, were between 0.47 and 1.69 pM. Intra-day precision was <7% for all sterols in inhouse-made lipoprotein-deficient serum. The limits of detection showed high sensitivity with very good intraday-and interday-precision for all sterols. The recoveries of the phytosterols, cholesterol precursors and cholestanol ranged from 77% to 92%. These analytical parameters provide a reliable and reproducible method for the identification and quantitation of human sterols.

Effect of different fat-enriched meats on non-cholesterol sterols and oxysterols as markers of cholesterol metabolism: Results of a randomized and cross-over clinical trial

“…The serum concentrations of cholesterol, lanosterol, desmosterol, sitosterol, campesterol, stigmasterol, sitostanol, 24S-hydroxycholesterol, 27-hydroxycholesterol, and 7a-hydroxy-4-cholesten-3-one were quantified using HPLC eMS/MS in all subjects according to the method previously described [15], and they were expressed as mmol/l as well as normalized to mmol/l of total cholesterol. Briefly, 100 ml of serum was transferred to a screw-capped vial, and 6 ml of deuterium-labeled internal standard, [ …”

“…We hypothesized that SFAs modify not only cholesterol synthesis but also cholesterol absorption and bile acid production. Those changes in cholesterol metabolism will be detected "in vivo" measuring serum non-cholesterol sterols by a high-performance liquid chromatographyetandem mass spectrometry (HPLCeMS/MS) [15]. Hence, the objective of this study was to assess the effect of two diets with different FA composition on cholesterol metabolism.…”

Underivatized oxysterols and nanoLC–ESI-MS: A mismatch

“…Regarding the gross carry-over of native oxysterols in nanoLC, extra-column material surfaces and connections are potent 18 1 mm ID Â 100 mm column using a columns switching system as described in [15] followed by a blank injection. No carry-over was detected.…”

“…This was partly inspired by the study of McDonald et al [10] who could monitor oxysterolssalt complexes (along with a number of other lipids) in biological samples using ESI and an AB Sciex API 5000 triple quadrupole mass spectrometer without derivatization. Other ionization sources such as APPI [16] and APCI [17,18] were not considered as they are not compatible with the low flow used in nanoLC.…”

Analysis of phytosterol by MALDI-TOF mass spectrometry

“…18 Consequently, several efforts to develop other MS techniques, such as liquid chromatography MS (LC-MS) and LC-MS/MS, have been exerted using well-established methods for sterol analysis. 19,20 In practice, atmospheric pressure chemical ionization (APCI) 17,21,22 and electrospray ionization (ESI) 20,23 with LC-MS are the most frequently used methods for sterol analysis. Although both GC-MS and LC-MS are reliable for the accurate determination of sterols, they oen require derivatization and chromatographic separation, which makes these methods time consuming.…”