Interference of 7‐dehydrocholesterol in α‐tocopherol determination by high‐performance liquid chromatography: A possible screening test for the smith‐lemli‐opitz syndrome

Abstract

Abstract7‐Dehydrocholesterol (7‐DHC), a normal cholesterol precursor, accumulates in plasma and tissues of patients affected by the Smith‐Lemli‐Opitz (SLO) syndrome, a recessive autosomic disease characterized by mental retardation and physical malformations. In these patients, we suspected that 7‐DHC interfered with α‐tocopherol (α‐T) determination because this steroid could be coeluted with α‐tocopherol acetate (α‐TA), an internal standard commonly used for α‐T determination, and because the conjugated dienic structure of 7‐DHC strongly absorbs at 292 nm. The aim of this work was (i) to characterize the interfering material and (ii) to evaluate the possibility of a rapid screening test for SLO syndrome identification from 7‐DHC determination by reverse‐phase chromatography high‐performance liquid chromatography (HPLC). Human plasmas (control and SLO syndrome) were extracted with hexane after ethanol protein precipitation and addition of ascorbic acid as protective agent plus α‐TA or δ‐tocopherol (δ‐T) as internal standards. The dry hexane extract was used for HPLC, and trimethylsilyl ethers derivatives of the extracts were submitted to gas chromatography (GC) and gas chromatography‐mass spectrometry (GC‐MS). The material interfering in the α‐T determination was unambiguously identified to 7‐DHC by retention time (RT) relative to standard, RT relative to δ‐T, and analysis of the eluted material by GC and by GC‐MS. In α‐T determination, the interference can be eliminated by using δ‐T as internal standard instead of α‐TA. Rapid detection and evaluation of 7‐DHC in plasma appear to be possible by HPLC under the conditions described; comparison with the GC reference method suggests that the rapid HPLC determination of 7‐DHC in plasma can be used within the 1–40 mg/dL range, values commonly found in SLO syndrome.