|Thursday, August 28||Analytical Procedures|
COLUMN PACKED IMMOBILIZED ß-GLUCURONIDASE AND ARYLSULFATASE FOR THE CLEAVAGE OF CONJUGATES - STABILITY, REUSABILITY AND APPLICATION IN TOXICOLOGICAL ROUTINE ANALYSIS|
Toennes S.W.H., Maurer H.H.
Column packed immobilized ß-glucuronidase and arylsulfatase allowed the cleavage of nitrophenyl glucuronide and sulfate spiked into urine within 15 min and even of morphine conjugates within 60 min in authentic urine samples (Tiaft Meeting, Interlaken, 1996). Studies on the stability, reusability and routine application of column packed immobilized ß-glucuronidase and arylsulfatase for the cleavage of drug conjugates are described here.
Methods. Purified ß-glucuronidase and arylsulfatase were co-immobilized on an agarose gel matrix and packed into 3 mL columns (for experimental details see the Proceedings of Interlaken).
Results. Our further studies showed that such columns were stable during a storage time of at least 6 weeks and that they could be used for at least 70 incubations without relevant decrease of the hydrolysis yields. The carry-over of analytes after incubation and elution of the urine sample could be avoided by rinsing the column with a tenfold column volume of 20% methanol-acetate buffer (0.1 M containing 0.5 M NaCl, pH 5.2). Known contaminants like dihydrocodeine, ibuprofen, paracetamol, perazine or phenobarbital spiked in very high concentrations into urine could completely be removed from the column. Field studies on efficiency of this procedure in routine analysis are now in progress and the results will also be presented.
Conclusions. Cleavage of conjugates by column packed immobilized ß-glucuronidase and arylsulfatase may be an effective and convenient alternative for the cleavage of conjugates and may be useful in automation of sample preparation.
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