Preanalytical and analytical challenges in gas chromatographic determination of cholesterol synthesis and absorption markers

Abstract

Introduction: Cholesterol homeostasis disruption contributes to the development of different pathologies. Non cholesterol sterols (NCSs) serve as cholesterol synthesis markers (desmosterol and lathosterol), and cholesterol absorption surrogate markers (campesterol, stigmasterol and beta-sitosterol). The study aimed to resolve certain new pre-analytical and analytical problems and ensure a reliable and validated method. Materials and methods: Method optimization, validation and stability studies were executed in human serum and plasma. Freeze-thaw cycles were done with and without antioxidant. Gas chromatography-mass spectrometer (GC-MS) was used for NCSs confirmation and plasticizer identification, while GC-flame ionization detector (GC-FID) was used for NCSs quantitation. Results: Infra-and inter-assay variabilities for all NCSs were 2.75-9.55% and 5.80-7.75% for plasma and 3.10-5.72% and 3.05-10.92% for serum, respectively. Recovery studies showed satisfactory percentage errors for all NCSs: 93.4-105.7% in plasma and 87.5-106.9 in serum. Derivatized samples were stable up to 7 days at 20 degrees C and derivatization yield was affected by presence of plasticizers. Fatty acid amids were identified as interfering plastic leachates. Statistically different NCSs concentrations were observed after the 1st freeze-thaw cycle, in antioxidant-free samples, and after the 4th cycle in antioxidant-enriched samples. Conclusions: All of the in-house procedures proved to be useful for minimizing the preanalytical and analytical variations, as proven by the validation results.