Determination of cholesterol and four phytosterols in foods without derivatization by gas chromatography-tandem mass spectrometry

摘要

Abstract In this study, a method for determination of cholesterol and four phytosterols by gas chromatography coupled with electron impact ionization mode–tandem mass spectrometry without derivatization in general food was developed. The sample was saponified with 7.5% KOH in methanol. After heating on hot plate and reflux for 60 minutes, the saponified portion was extracted with n -hexane/petroleum ether (50:50, v/v). The extracts were evaporated with rotary evaporator and then redissolved with tetrahydrofuran. The tetrahydrofuran layer was transferred into an injection vial and analyzed by gas chromatography on a 30?m VF-5 column. Limit of quantification was 2?mg/kg. Recoveries of cholesterol and four phytosterols from general food were between 91% and 100%. prs.rt("abs_end"); Keywords cholesterol ; gas chromatography–tandem mass spectrometry ; phytosterol 1. Introduction Sterols are tetracyclic lipid components found in animals, plants, and microorganisms. Several hundred different structures have been identified to date. While cholesterol is the major sterol in animals [1] , the most common representatives in the plant kingdom are β-sitosterol [2] , campesterol [3] , and stigmasterol [3] . Cholesterol is required to build and maintain membranes. Through the interaction with the phospholipid fatty-acid chains, cholesterol increases membrane packing, which reduces membrane fluidity [4] . The structure of the tetracyclic ring of cholesterol contributes to the decreased fluidity of the cell membrane as the molecule is in a trans conformation, making all but the side chain of cholesterol rigid and planar [5] . In this structural role, cholesterol reduces the permeability of the plasma membrane to neutral solutes [6] , protons (positive hydrogen ions), and sodium ions [7] . Phytosterols are plant compounds that have similar chemical structure and biological functions as cholesterol [8] . Phytosterols contain an extra methyl group, ethyl group, or double bond. The suggested daily dietary intake of phytosterols is from 160?mg to 400?mg for different races of humans [9] , [10] , [11] , [12] , [13] , [14] , [15] and [16] . Phytosterols are known to have hypocholesterolemic properties. Phytosterols analogs are suggested to lower cholesterol absorption and the lower the serum cholesterol level, leading to cardiologic health benefits [1] and [7] . Cholesterol, corresponding precursors, and phytosterols in human blood have been determined by gas chromatography (GC)–mass spectrometry (MS) [17] . GC-MS is also executed to analyze cholesterol, corresponding precursors and phytosterols in cultured cells [18] . Rocco and Fanali [19] tried to determine phytosterols by nanoliquid chromatography–MS. AOAC published an official method 994.10 as analysis of cholesterol in foods by GC–flame ionization detector after saponification and derivatization with trimethylchlorosilane [20] . However, determination of cholesterol has been treated with derivatization during sample preparation in past reports, and there was no research to show an assay of cholesterol and phytosterols by GC–tandem MS (MS/MS). In this study, we aimed to develop a method of determination of cholesterol and four phytosterols without derivatization by GC-MS/MS in 20 minutes. 2. Methods 2.1. Apparatus The GC–electron impact-MS/MS (GC-EI-MS/MS) system consisted of a Bruker456-GC system (Bruker, Singapore) connected to a Scion TQ series triple-stage quadrupole mass spectrometer (Bruker, Philadelphia, PA, USA). GC analysis was performed on a VF-5ms (30?m?×?0.25?mm, film thickness?=?0.25?μm; Agilent Technologies, Amstelveen, The Netherlands) at 280°C. N2 was applied as carrier gas. Total running time was 20 minutes. The injection volume was 1?μL. The MS detection system included an electron impact ionization. Its energy was fixed at 70?eV. Temperatures of ion source and transfer line were set at 200°C and 300°C, respectively. Argon was used as the collision-induced dissociation gas at a pressure of 1.5 mTorr. Heating plates contain heat controls. Rotary evaporator with glass condenser flask between concentration flask and metal shaft were applied. Glassware used included 250-mL Erlenmeyer flasks, 250-mL separatory funnel, volumetric flasks, pipets, 250-mL Rohrig extraction tubes, glass funnels, and graduated cylinders. 2.2. Reagents and solutions Cholesterol standard (purity??99%) was purchased from Sigma–Aldrich (St Louis, MO, USA). Brassicasterol (purity??92.5%), stigmasterol (purity??89%), and β-sitosterol (purity??92%) were supplied by ChromaDEX (Irvine, CA, USA). Campesterol (purity??99%) was purchased from Sigma–Aldrich (Munich, Germany). As an internal standard, 5α-cholestane (purity??97%) was provided by Sigma–Aldrich (USA). Individual stock standard solutions were prepared at a concentration of 1000?mg/L in tetrahydrofuran (THF; stable for 3 months), apart from 5α-cholestane, which was prepared in n -heptane at ?25°C. Intermediate si