Impact of Biotin Supplementation on Human Chorionic Gonadotropin Immunoassays Utilizing Biotin-Streptavidin Binding Methods in Urine

Abstract Background Human chorionic gonadotropin (hCG) detection is indicative of pregnancy and can be indicative of some forms of cancerous tumors. The hCG drug itself, however, is a performance enhancing substance used by male athletes to increase testosterone production. Antidoping testing for hCG is conducted in urine, often on immunoanalyzer platforms, many of which utilize biotin-streptavidin dependent immunoassays in which the presence of biotin in samples is a known confounding factor. While biotin interference in serum has been well-studied, the extent of biotin interference in urine has not. Methods Ten active male individuals underwent a 2-week hCG administration protocol concurrent with supplementation with biotin (20 mg/day) or placebo. Urine and serum samples were collected throughout the study and analyzed for hCG and biotin concentrations. Results Urinary biotin levels in the hCG + biotin group increased 500-fold over baseline and 29-fold over corresponding serum biotin levels after biotin supplementation. When using a biotin-dependent immunoassay, the hCG + placebo group produced hCG-positive results (hCG ≥ 5 mIU/mL) in 71% of urine samples, while the hCG + biotin group produced positive results in only 19% of samples. Both groups had elevated hCG values in serum measurements by a biotin-dependent immunoassay and in urine when using a biotin-independent immunoassay. Urinary hCG measurements and biotin levels from the hCG + biotin group showed a negative correlation (Spearman r = −0.46, P < 0.0001) when measured using a biotin-dependent immunoassay. Conclusions Biotin supplementation can severely suppress urinary hCG values in assays utilizing biotin-streptavidin binding methods and therefore these types of assays are not recommended for use in urine samples containing high levels of biotin. Clinicaltrials.gov Registration Number: NCT05450900