On-line cleavage of urinary estriol conjugates with immobilized beta-glucuronidase before liquid-chromatographic...

Urinary estriol measurement has been widely accepted as a useful indicator of fetoplacental status. Classically, glucuronide conjugates of estriol have been cleaved with soluble beta-glucuronidase (EC 3.2.1.31) before extraction and measurement. We have developed a system in which urine is injected directly into a "high-performance" liquid chromatograph and the conjugates are cleaved on-line in an immobilized beta-glucuronidase reactor. Estriol is quantitated with fluorescence detection after separation from other interfering species with a reversed-phase column. The stability of the immobilized enzyme under storage conditions and in the presence of the mobile phase are discussed. Only 150 mL/L methanol could be pumped through the reactor and onto the analytical column, but this allowed on-column preconcentration of the free estriol produced. Gradient elution bypassing the immobilized enzyme reactor eluted the compounds of interest without damaging the enzyme. Comparison with radioimmunoassay results yielded a slope of 0.97 (r = 0.996, n = 19).