INTERFERENCE OF 7-DEHYDROCHOLESTEROL IN ALPHA-TOCOPHEROL DETERMINATION BY HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY - A POSSIBLE SCREENING-TEST FOR THE SMITH-LEMLI-OPITZ-SYNDROME

7-Dehydrocholesterol (7-DHC), a normal cholesterol precursor, accumula tes in plasma and tissues of patients affected by the Smith-Lemli-Opit z (SLO) syndrome, a recessive autosomic disease characterized by menta l retardation and physical malformations. In these patients, we suspec ted that 7-DHC interfered with alpha-tocopherol (alpha-T) determinatio n because this steroid could be coeluted with alpha-tocopherol acetate (alpha-TA), an internal standard commonly used for alpha-T determinat ion, and because the conjugated dienic structure of 7-DHC strongly abs orbs at 292 nm. The aim of this work was (i) to characterize the inter fering material and (ii) to evaluate the possibility of a rapid screen ing test for SLO syndrome identification from 7-DHC determination by r everse-phase chromatography high-performance liquid chromatography (HP LC). Human plasmas (control and SLO syndrome) were extracted with hexa ne after ethanol protein precipitation and addition of ascorbic acid a s protective agent plus alpha-TA or delta-tocopherol (delta-T) as inte rnal standards. The dry hexane extract was used for HPLC, and trimethy lsilyl ethers derivatives of the extracts were submitted to gas chroma tography (GC) and gas chromatography-mass spectrometry (GC-MS). The ma terial interfering in the alpha-T determination was unambiguously iden tified to 7-DHC by retention time (RT) relative to standard, RT relati ve to delta-T, and analysis of the eluted material by GC and by GC-MS. In alpha-T determination, the interference can be eliminated by using delta-T as internal standard instead of alpha-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 suggest s that the rapid HPLC determination of 7-DHC in plasma can be used wit hin the 1-40 mg/dL range, values commonly found in SLO syndrome.