Separation and Determination of Some of the Main Cholesterol-Related Compounds in Blood by Gas Chromatography-Mass Spectrometry (Selected Ion Monitoring Mode)

Abstract

Oxysterols are metabolites produced in the first step of cholesterol metabolism, which is related to neurodegenerative disorder. They can be detected by testing blood, plasma, serum, or cerebrospinal fluid. In this study, some cholesterol precursors and oxysterols were determined by gas chromatography coupled to mass spectrometry. The selected cholesterol-related compounds were desmosterol, lathosterol, lanosterol, 7α-hydroxycholesterol, 7β-hydroxycholesterol, 24(S)-hydroxycholesterol, 25-hydroxycholesterol, 7-ketocholesterol, and 27-hydroxycholesterol. A powerful method was developed and validated considering various analytical parameters, such as linearity index, detection and quantification limits, selectivity and matrix effect, precision (repeatability), and trueness (recovery factor) for each cholesterol-related compound. 7α-hydroxycholesterol, 7β-hydroxycholesterol, and desmosterol exhibited the lowest detection and quantification limits, with 0.01 and 0.03 µg/mL, respectively, in the three cases. 7-ketocholesterol and lathosterol showed matrix effect percentages between 95.5% and 104.8%, respectively (demonstrating a negligible matrix effect), and very satisfactory repeatability values (i.e., overall performance of the method). Next, the method was applied to the analysis of a very interesting selection of mouse plasma samples (9 plasma extracts of non-transgenic and transgenic mice that had been fed different diets). Although the number of samples was limited, the current study led to some biologically relevant conclusions regarding brain cholesterol metabolism.

GC–MS analysis of oxysterols and their formation in cultivated liver cells (HepG2)

“…It should be noted that surprisingly few studies report values of LOD in the very low nanomolar range using comparable methods and instruments (Husche et al, 2011;Schott & Lutjohann, 2015). Nevertheless, all previous studies consistently describe the most sensitive detection for 7α-and 7β-OH-chol (Chiu et al, 2018;Dzeletovic, Breuer, et al, 1995;Schott & Lutjohann, 2015;Valverde-Som et al, 2018) compared to 5α,6α-epcholestanol showing a high LLOQ (Chiu et al, 2018;Dzeletovic, Breuer, et al, 1995;Menendez-Carreno et al, 2008) which is in line with our observations. Interestingly, a relatively poor sensitivity was described for 7-keto-chol (Chiu et al, 2018;Dzeletovic, Breuer, et al, 1995 Note: Shown are the retention times, the full width at half maximum (FWHM) of the chromatographic peaks, the chromatographic resolution, the limit of detection (LOD, S/N ≥ 3) as well as the lower limit of quantification (LLOQ, S/N ≥ 5, accuracy >20%) in selected ion monitoring mode with a dwell time of 80 ms.…”

“…Selected ions (m/z) 7α-OH-chol 456, 457 (Valverde-Som et al, 2018);546, 531, 456, 441 (Hu et al, 2015) 456 [M-OTMSi] + (Hu et al, 2015;Schott & Lutjohann, 2015;Valverde-Som et al, 2018) 441, 456, 546 19-OH-chol 456, 366, 353 (Son et al, 2014531, 366, 353, 253 (Hu et al, 2015) 353 Hu et al, 2015;Son et al, 2014) 353, 366, 456 7β-OH-chol 546, 456, 366 (Son et al, 2014456, 457 (Valverde-Som et al, 2018);546, 531, 456, 441 (Hu et al, 2015) 456 [M-OTMSi] + (Hu et al, 2015;Schott & Lutjohann, 2015;Son et al, 2014;Valverde-Som et al, 2018) 366, 456, 546 4β-OH-chol 546, 456, 441, 417, 366, 327, 147 (Son et al, 2014) 546, 456, 441, 417, 129 (Son et al, 2014);546, 456, 441, 417, 255, 161, 129 (Valverde-Som et al, 2018) Lutjohann, 2015) 417, 456, 546 7-keto-chol 472, 367, 382 (Valverde-Som et al, 2018472, 382, 367, 457 (Hu et al, 2015) 472 [M] + (Hu et al, 2015;Schott & Lutjohann, 2015) 367, 382, 472…”

“…On the contrary to ring epoxidation, side-chain epoxidation leads to a rather low number of Cholesterol and its derivatives contain several hydrogen atoms leading to a positive mass defect, that is, a monoisotopic/exact mass of 0.5 Da above the nominal mass, for example, m/z 353.5 for [M-OTMSi-TMSi-O + = CH 2 ] + . When analyzing sterols by means of a quadrupole mass analyzer operating in unit resolution using selected ion monitoring of the nominal mass-the most common technique in GC-MS for sterols (Dzeletovic, Breuer, et al, 1995;Hu et al, 2015;Matysik et al, 2012;Valverde-Som et al, 2018)-it is important to ensure that the MS instrument is well calibrated. Otherwise, the majority of target ions are not detected, even using unit resolution.…”

“…Even though most dihydroxylated derivatives eluted later, the retention times of the side-chain hydroxylated cholesterols are longer than those of the dihydroxylated products. In the literature, oxysterol analysis is often carried out using 5%diphenyl/95%-dimethyl polysiloxane as the stationary phase (Dzeletovic, Breuer, et al, 1995;Hu et al, 2015; 12) Matysik et al, 2012;Valverde-Som et al, 2018). Shan et al determined the Kovats indices for oxysterols on two different 5%-diphenyl/95%-dimethyl polysiloxane (Rtx-5 and DB-5) and a trifluoropropyl-methyl polysiloxane (Rtx-200) analytical columns (Shan et al, 2003).…”

“…The gold standard of oxysterol analysis is gas chromatography coupled to mass spectrometry (GC-MS) (Dzeletovic, Breuer, et al, 1995;Schott & Lutjohann, 2015;Shan et al, 2003;Son et al, 2014;Valverde-Som et al, 2018). The MS fragmentation behavior during electron ionization (EI) and the gas chromatographic properties of ring-and side-chain hydroxylated oxysterols were studied in the past (Brooks et al, 1968;Dzeletovic, Breuer, et al, 1995;Husche et al, 2011;Pizzoferrato et al, 1993;Schott & Lutjohann, 2015;Valverde-Som et al, 2018;Won Park & Addis, 1985). However, no data are available for the biologically important dihydroxylated cholesterol derivatives and the side-chain epoxide.…”