Fast LC-MS/MS analysis of free oxysterols derived from reactive oxygen species in human plasma and carotid plaque

Abstract

Background: A rapid liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed and validated for the quantification of reactive oxygen species (ROS) derived free oxysterols and cholesterol in human plasma and atherosclerotic plaque.

Method: In vitro autoxidation of cholesterol during sample pretreatment was avoided by applying only one protein precipitation and re-concentration step using 80 μl plasma. For preparation of 10mg atherosclerotic plaques an additional liquid-liquid extraction was included. Free 7-keto-, 7-α/ß-hydroxy-, 5,6-α-epoxy-, 5,6-β-epoxycholesterol, cholestane-3ß,5α,6ß-triol and cholesterol were separated within 7 min on a monolithic column. An API 4000 tandem mass spectrometer was applied in positive ionization mode using atmospheric pressure chemical ionization.

Results: The detection limit was 0.1 ng/ml and the linearity ranged from 0.5 to 0.75 to 2000 ng/ml for the oxysterols and from 50 to 1000 μg/ml for cholesterol. Recovery was between 80.9 and 107.9%. Between-run imprecision ranged from 7.9 to 11.7%. Analysis of plasma samples from additional 50 middle-aged volunteers revealed a large inter-individual variability (e.g. 7-ketocholesterol 2.63-30.47 ng/ml). Oxysterol concentrations normalized to cholesterol were about 43 times higher in carotid plaque compared to plasma (n=5).

Conclusion: This rapid LC-MS/MS method enables reliable quantification focused on especially ROS-derived oxysterols in human plasma and atherosclerotic plaque samples under high-throughput conditions.

Keywords: -OHC; -hydroxycholesterol; 5,6-α-EC; 5,6-α-epoxycholesterol; 5,6-β-EC; 5,6-β-epoxycholesterol; 7-KC; 7-ketocholesterol; 7-α- and 7-β-hydroxycholesterol; 7-α/β-OHC; APCI; Atherosclerotic plaque; BHT; CE; CEA; CID; CV; CXP; DP; EP; Fast chromatography; Free oxysterol; GC-MS; HCl; HPLC; HQC; LC; LC–MS/MS; LLE; LLOQ; LOD; LQC; MQC; MRM; NQC; Na(2)SO(4); NaOH; Oxysterol; PQC; QC; ROS; Reactive oxygen species; SPE; Tandem mass spectrometry; Triol; atmospheric pressure chemical ionization; butylated hydroxytoluene; carotid endarterectomy; cholestane-3β,5α,6β-triol; coefficient of variation; collision energy; collision exit potential; collision induced dissociation; declustering potential; extensions potential; gas chromatography mass spectrometry; high concentrated quality control; high-performance liquid chromatography; hydrochloric acid; limit of detection; liquid chromatography; liquid chromatography tandem mass spectrometry; liquid–liquid extraction; low concentrated quality control; lower level of quantification; medium concentrated quality control; multiple reacting monitoring; native quality control; pathological concentrated quality control; quality controls; reactive oxygen species; s/n; signal-to-noise ratio; sodium hydroxide; sodium sulfate; solid phase extraction.