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 β-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: Intra- 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 7days at -20°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.
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 β-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: Intra- 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 7days at -20°C and derivatization yield was affected by presence of plasticizers. Fatty acidamids 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.