Steroid glucuronides, detection

Using immobilized -glucuronidase reactors, estriol and estradiol glucuronides have been determined in urine by a column-switching technique (270, 271). Both glucuronides were hydrolyzed by the immobilized enzyme at pH 7. The steroid mixture was subsequently separated by gradient elution on a reversed-phase column, to be finally detected by UV absorbance at 280 nm. In this procedure, the activity of enzyme did not alter even after 150 h continuous run and exposure to a mobile phase containing 10% methanol. When a separate reversed-phase precolumn was inserted in the LC system, additional sample purification and shorter analysis time could be attained (272). 

In veterinary medicine, boldenone, a synthetic anabolic steroid (Figure 2.1), is commercially available, hence it is a concern in the horseracing industry. Pu et al. (2004) used ion-trap LC-MS analysis to detect boldenone conjugates (sulfate and glucuronide) and their 17-epimers in horse mine after intramuscular administration of boldenone undecylenate. Soon afterwards. Ho et al. (2004) reported the occurrence of endogenous boldenone sulfate in the urine of uncastrated male horses, and quantitated it by quadrupole time-of-flight (Q-TOF) LC-MS-MS. 

The steroid moiety can be detected once again with sulphuric acid or, for 17-ketosteroid conjugates, with m-dinitrobenzene for example. The acid component can be characterised by either the Folin-Ciocalteu reagent (No. 122) or pyridylazonaphthol reagent (No. 215) for glucuronides and methylene blue (Rgt. No. 162) for sulphates [44]. 

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