Peaks of desmosterol (purity ≥84%), 7-dehydrocholesterol (purity ≥95), lathosterol (purity >99%), campesterol (purity ∼65%) and b-sitosterol (purity TraceCERT® grade) were identified by comparison with corresponding HPLC grade analytical standards (Supelco, Bellefonte, PA, USA). Deuterated internal standard (IS) d6-cholesterol (HPLC grade) purchased from Sigma-Aldrich (St. Louis, MO, USA) was used. Cholesterol standard of ≥99% purity was obtained from Sigma Aldrich (St. Louis, MO, USA). KOH was purchased from POCH (Center Valley, PA, USA), and ethanol, methanol, n-hexane and acetonitrile (HPLC grade) from Fisher (Pittsburgh, PA, USA). Bovine serum albumin (BSA) was obtained from Sigma-Aldrich (St. Louis, MO, USA).
Commercial cholesterol HDL precipitating reagent produced by BioSystems (Costa Brava, Barcelona, Spain) contained phosphotungstate (0.4 mmol/L) and magnesium chloride (20 mmol/L).
Human serum samples were obtained from healthy volunteers after the general medical examination with permission from local Ethical committee and following the guidelines defined by the Declaration of Helsinki. Samples were used for method development and clinical verification. All participants signed an informed consent form before the enrolment.
HPLC method development and sample analyses were done using C-18 Porochell 120-EC column (150×4.6 mm×2.7 μm) (Agilent Technologies, USA).
Commercial cholesterol HDL precipitating reagent produced by BioSystems (Costa Brava, Barcelona, Spain) contained phosphotungstate (0.4 mmol/L) and magnesium chloride (20 mmol/L).
Human serum samples were obtained from healthy volunteers after the general medical examination with permission from local Ethical committee and following the guidelines defined by the Declaration of Helsinki. Samples were used for method development and clinical verification. All participants signed an informed consent form before the enrolment.
HPLC method development and sample analyses were done using C-18 Porochell 120-EC column (150×4.6 mm×2.7 μm) (Agilent Technologies, USA).
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