Related Experiment Videos
Optimizing the hydrolysis of codeine and morphine glucuronides in urine
L Peter Hackett1, Leon J Dusci, Kenneth F Ilett
1Clinical Pharmacology and Toxicology Laboratory, Western Australian Center for Pathology and Medical Research Nedlands Australia. peter.hackett@health.wa.gov
Therapeutic Drug Monitoring
|September 28, 2002
Summary
Inter-laboratory variations in codeine quantification are likely due to incomplete hydrolysis of codeine glucuronide (C6G). An optimized acid hydrolysis method significantly improves the accuracy of codeine and metabolite measurements in patient samples.
Area of Science:
- Analytical Chemistry
- Forensic Toxicology
- Pharmacology
Background:
- Quality assurance data reveal significant inter-laboratory variability in codeine quantification, particularly in biological samples.
- Codeine glucuronide (C6G) and morphine glucuronides (M3G, M6G) are frequently co-present in urine, complicating accurate analysis.
Purpose of the Study:
- To investigate and compare different hydrolysis procedures for C6G, M3G, and M6G.
- To identify an optimized hydrolysis method for improving the accuracy of codeine and morphine metabolite analysis.
Main Methods:
- Comparison of two beta-glucuronidase sources and various hydrochloric acid concentrations for hydrolysis.
- Solid phase extraction for sample concentration.
- Derivatization followed by gas chromatography-mass spectrometry (GC-MS) with selective ion monitoring for quantification.
Main Results:
- Beta-glucuronidase efficiently hydrolyzed M3G (90-95%) but showed lower efficacy for M6G (60-85%) and poor hydrolysis for C6G (45-58%).
- An optimized method using 50% HCl at 120°C for 1.5 hours yielded reproducible results for C6G hydrolysis, approaching spiked concentrations.
- Findings were validated using urine samples from individuals who had consumed codeine, morphine, or heroin.
Conclusions:
- Incomplete C6G hydrolysis is the probable cause of erratic inter-laboratory codeine quantification results.
- The developed optimized hydrolysis method enhances the reproducibility and accuracy of measuring codeine and its metabolites in clinical and forensic toxicology.