Analysis of biological samples by gas chromatography–mass spectrometry without a reference standard: measurement of urinary 18-hydroxytetrahydro-11-dehydrocorticosterone excretion rate in human subjects

Reference standards for some minor urinary steroid metabolites are sometimes unavailable. We describe a novel procedure to quantitate a urinary steroid metabolite of known structure and mass spectrum, using as a standard a compound which produces ions in common with it and has a similar retention time in gas chromatography–mass spectrometry. The steroid of interest was 18-hydroxy-11-dehydrotetrahydrocorticosterone (18-OH-THA), the major urinary metabolite of 18-hydroxycorticosterone (18-OH-B), a putative intermediate in the conversion of 11-deoxycorticosterone to aldosterone. The steroid used as an alternative to the authentic 18-OH-THA standard was β-cortol which, like 18-OH-THA, produces a fragmentation ion at m/z 457. Allo-tetrahydrodeoxycorticosterone (5α-THDOC) was used as the internal standard. β-Cortolone also has the fragmentation ion at m/z 449 (in common with β-cortol) and an authentic standard is available commercially. To validate the procedure, we quantitated β-cortolone urinary excretion rate against this alternative standard and also against authentic β-cortolone standards. Both methods produced similar results (adjusted R2: 0.998, P<0.001). The method was then used to measure urinary excretion of 18-OH-THA rate in healthy volunteers. The reference range obtained was 20–204 μg/24 h (n=32). This is similar to the few results available by conventional assay. Method performance was also similar to other assays of urinary steroids. This procedure could be generally applicable for assays when authentic standards are not available but mass spectra are known or can be predicted.