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Efficient Cleavage of Conjugates of Drugs or Poisons by Immobilized ß-Glucuronidase and Arylsulfatase in Columns

Department of Toxicology, Institute of Pharmacology and Toxicology, University of Saarland, D-66421 Homburg (Saar), Germany. Portions of this work were published in the proceedings of the 34th International TIAFT Meeting, August 11–15, 1996, Interlaken, Switzerland (Toennes SWH, Maurer HH. Immobilization of ß-glucuronidase and arylsulfatase: which are the advantages of column packed immobilizate for the cleavage of conjugates in analytical toxicology? In: Sachs H, Bernhard W, Jeger A, eds. Proceedings of the 34th International TIAFT Meeting in Interlaken. Leipzig, Germany: Molinapress, 1997:92–6) and the 35th International TIAFT Meeting, August 24–28, 1997, Padova, Italy (Toennes SWH, Maurer HH. Column packed immobilized ß-glucuronidase and arylsulfatase for the cleavage of conjugates—stability, reusability and application in toxicological routine analysis. In: Ferrara SD, ed. Proceedings of the 35th International TIAFT Meeting in Padova. Padova, Italy: Centre of Behavioural and Forensic Toxicology, 1997:227–40).
^a Author for correspondence. Fax 49-6841-16-6051; e-mail Hans.Maurer{at}med-rz.uni-sb.de

Background: Cleavage of conjugates is an important step in toxicological analysis, especially of urine samples. The aim of this study was to combine the advantages and to reduce the disadvantages of acid hydrolysis and conventional enzymatic hydrolysis procedures.

Methods: ß-Glucuronidase (GRD; EC 3.2.1.31) and arylsulfatase (ARS; EC 3.1.6.1) were purified and coimmobilized on an agarose gel matrix and packed into columns.

Results: In columns packed with GRD and ARS, the test conjugates 4-nitrophenyl glucuronide and 4-nitrophenyl sulfate added into urine could be completely cleaved within 25 min. Even the relatively stable morphine conjugates could be completely hydrolyzed within 60 min in authentic urine samples. Therefore, an incubation time of 1 h is recommended. Enzyme inhibition by matrix or by rather high concentrations of acetaminophen conjugates was tested and found to be up to 50%. However, a large excess of GRD and ARS was used. The immobilizate columns could be reused for at least 70 incubations and had a storage stability of at least 12 weeks. Carryover of analytes in reused columns could be avoided by rinsing with 200 mL/L methanol in acetate buffer. Thus, five drugs known to be contaminants added in very high concentrations into urine could be completely removed from the columns. A study on the applicability in systematic toxicological analysis showed that 120 different drugs and/or their metabolites could be detected in 35 different authentic urine samples.

Conclusions: Use of immobilized and column-packed GRD and ARS is an efficient alternative for the cleavage of urinary conjugates in clinical toxicology.

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