Quantitative Charge-Tags for Sterol and Oxysterol Analysis

Abstract

BACKGROUND:Global sterol analysis is challenging owing to the extreme diversity of sterol natural products, the tendency of cholesterol to dominate in abundance over all other sterols, and the structural lack of a strong chromophore or readily ionized functional group. We developed a method to overcome these challenges by using different isotope-labeled versions of the Girard P reagent (GP) as quantitative chargetags for the LC-MS analysis of sterols including oxysterols.

Oxidized cholesterol as the driving force behind the development of Alzheimer’s disease

“…Of these cholesterol metabolites, that exported in the largest quantities is 24-OH. Two other cholesterol metabolites, 7α, 25-dihydroxycholest-4-en-3-one and 7α, (25R)26-hydroxycholest-4-en-3-one, were reported to be exported from the brain (Crick et al, 2015b). Oxysterols and cholestenoic acids have also been identified and quantified in mouse cerebrospinal fluid (CSF), and the findings compared with concentrations of the same metabolites found in the plasma, in order to clarify cholesterol metabolism.…”

7β-Hydroxysitosterol crosses the blood–brain barrier more favored than its substrate sitosterol in ApoE−/− mice

“…Supplementation of foods with 60 plant sterols and daily intake of 2 g plant sterol esters leads to a 61 nearly 10% lowering of cholesterol in the blood and a slight 62 increase of the serum levels of these plant sterols [1,3] [10]. The same group recently reported on flux of unesteri-83 fied 7a-25-and 7a-(25R)26-dihydroxycholesterol from the brain 84 into the periphery [11] from the same material. 85 In addition to cholesterol plant sterols can be oxidized and oxy-86 phytosterols can either be absorbed from the diet or endogenously 87 produced from their substrates [12] within the mammalian organ-88 ism.…”

Thiyl Radical-Based Charge Tagging Enables Sterol Quantitation via Mass Spectrometry

“…Established derivatization methods either target the hydroxyl function group via forming an ester bond with a charge tag (e.g., sulfate, 6 phosphonium, 7 picolinyl esters 8 ) or convert the hydroxyl to ketone first and then use carbonyl chemistry to link a charge tag to sterols (e.g., forming Girard P hydrazones). 9,10 These methods significantly improved sterol analysis via ESI with the limit of detection (LOD) achieved at nanomolar to sub-nanomolar. 6–10 The derivatization reactions typically require several hours or even days to accomplish.…”

“…9,10 These methods significantly improved sterol analysis via ESI with the limit of detection (LOD) achieved at nanomolar to sub-nanomolar. 6–10 The derivatization reactions typically require several hours or even days to accomplish. Several derivatization reagents need to be synthesized in-house, further curbing wide accessibility to these methods.…”

“…LOD was achieved at 0.5 nM (S/N ratio > 3, Figure S5, Supporting Information), comparable to those reported by other charge derivatization approaches, such as sulfation (0.2 nM), 6 phosphonium labeling (0.05 nM), 7 N -alkylpyridinium quaternization (54 nM), 24 and Girard P reagent (0.1 nM). 10 Most importantly, the whole process of TGA tagging and subsequent MS analysis is significantly shortened to less than 2 min per run. Although CO 2 loss is a facile fragmentation channel for unsaturated fatty acids, 25 their potential interference for sterol analysis via 44 Da NLS should be limited due to their appearance at lower m / z range than the commonly observed sterols.…”