UPLC-Orbitrap-HRMS application for analysis of plasma sterols

• Published in: Analytica chimica acta 2024-04, Vol.1296, p.342347-342347, Article 342347
• Main Authors: van der Ham, Maria, Gerrits, Johan, Prinsen, Berthil, van Hasselt, Peter, Fuchs, Sabine, Jans, Judith, Willems, Anke, de Sain-van der Velden, Monique
• Format: Article
• Language: English
• Subjects: Cholesterol intermediates; Inherited metabolic diseases; Oxysterols; Phytosterols; PTAD derivatization; UPLC-Orbitrap-HRMS
• ISSN: 0003-2670; 1873-4324
• DOI: 10.1016/j.aca.2024.342347

Correct identification and quantification of different sterol biomarkers can be used as a first-line diagnostic approach for inherited metabolic disorders (IMD). The main drawbacks of current methodologies are related to lack of selectivity and sensitivity for some of these compounds. To address this, we developed and validated two sensitive and selective assays for quantification of six cholesterol biosynthesis pathway intermediates (total amount (free and esterified form) of 7-dehydrocholesterol (7-DHC), 8-dehydrocholesterol (8-DHC), desmosterol, lathosterol, lanosterol and cholestanol), two phytosterols (total amount (free and esterified form) of campesterol and sitosterol) and free form of two oxysterols (7-ketocholesterol (7-KC) and 3β,5α,6β-cholestane-triol (C-triol). For quantification of four cholesterol intermediates we based our analytical approach on sterol derivatization with 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD). Quantification of all analytes is performed using UPLC coupled to an Orbitrap high resolution mass spectrometry (HRMS) system, with detection of target ions through full scan acquisition using positive atmospheric pressure chemical ionization (APCI) mode. UPLC and MS parameters were optimized to achieve high sensitivity and selectivity. Analog stable isotope labeled for each compound was used for proper quantification and correction for recovery, matrix effects and process efficiency. Precision (2.4%–12.3% inter-assay variation), lower limit of quantification (0.027 nM–50.5 nM) and linearity (5.5 μM (R2 0.999) – 72.3 μM (R2 0.997)) for phyto- and oxysterols were determined. The diagnostic potential of these two assays in a cohort of patients (n = 31, 50 samples) diagnosed with IMD affecting cholesterol and lysosomal/peroxisomal homeostasis is demonstrated. [Display omitted] •Quantification of sterols in plasma is important in the field of IMD.•UPLC-Orbitrap-HRMS methods were developed and validated.•PTAD derivatization was used for quantification of desmosterol, lanosterol, 7/8-DHC.•Developed methods are highly sensitive, selective and need 100–125 μL plasma.•190 control samples and 50 samples from patients with IMD were studied.