The use of equilin as an internal standard to quantitate estriol in pregnancy urine

The assay of pregnancy urine estriol by gas chromatography is most often carried out by adding cholesterol as an internal standard at the end of the extraction procedure. The reason for this is that cholesterol does not contain a phenolic ring, and would be lost during the extraction procedure for estriol. This does not allow for corrections of recovery loss, which would be the case if the internal standard was a phenolic steroid. This paper describes the utilization of equilin (delta 1,3,5,7-estratetraen-3-01-17-one) as the internal standard. The sample is hydrolyzed to remove sulfate and glucuronide groups and then the equilin is added. If equilin is added before hydrolysis, variable destruction takes place. Derivative studies indicate that room temperature silylation can be used with bis (trimethylsilyl)-trifluoroacetamide/trichlorosilane/trimethylsilylimidazole, 3:3:2 by weight. Results on control material containing a low, critical pregnancy concentration of estriol were as follows--listed value 5.0 mg/1, found 5.3 mg/1, co-efficient of variation %, 8.9% for 17 runs on different days.