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Clinical Chemistry 35: 1906-1910, 1989;
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Clinical Chemistry, Vol 35, 1906-1910, Copyright © 1989 by American Association for Clinical Chemistry

Diagnosis of recessive X-linked ichthyosis: quantitative HPLC/mass spectrometric analysis of plasma for cholesterol sulfate

CH Shackleton and S Reid
Children's Hospital Oakland Research Institute, CA 94609.

A specific, accurate liquid-chromatographic/mass-spectrometric (HPLC/MS) method for measurement of cholesterol sulfate in plasma from normal individuals and patients with recessive X-linked ichthyosis (RXLI) is described. The method is superior to previously described techniques because it measures the analyte intact rather than after hydrolysis. Traces of free cholesterol in the sample analyzed do not add to the measured result. We used either [13C2]cholesterol sulfate or [2H6]cholesterol sulfate as internal standards, which we add to plasma before extraction. Use of such standards makes quantitative extraction unimportant. We use a single solid-phase extraction (SPE) C18 cartridge for plasma extraction. After the cartridge is washed with methanol/ammonium acetate solutions, the fraction containing the steroid sulfates is eluted with methanol, evaporated, and subjected to HPLC/MS analysis, wherein the molecular anions of analyte and internal standards are monitored. The peak ratio gives the cholesterol sulfate concentration directly. Using this method, we have diagnosed 24 patients with RXLI. Their concentration of cholesterol sulfate ranged between 41.7 and 185.3 mumol/L (mean 93.85, SD 31.2 mumol/L). In normal individuals (n = 9) the mean cholesterol sulfate concentration was 2.77 mumol/L (SD 0.62, range 2.05-3.95 mumol/L). The instrumentation required is complex, but the assay is simple: sample preparation takes about 30 min; mass spectrometry, 10 min. About 0.1 mL of plasma is required for cholesterol sulfate measurement in RXLI patients and 0.5-1 mL in normal individuals.




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Copyright © 1989 by the American Association for Clinical Chemistry.