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Published:
Journal of Analytical Toxicology,
ISSN 0146-4760,
Volume 30, Number 8, October 2006,
pp.563-569
Selection and Optimization of Hydrolysis Conditions for
the Quantification of Urinary Metabolites of MDMA
Stephane O. Pirnay, Tsadik T. Abraham, Ross H. Lowe, and Marilyn A. Huestis
Chemistry and Drug Metabolism, Intramural Research Program, National Institute
on Drug Abuse, National Institutes of Health, 5500 Nathan Shock Drive, Baltimore,
Maryland 21224
Recovery of 3,4-methylenedioxymethamphetamine (MDMA) urinary metabolites
requires optimization of the hydrolysis of 4-hydroxy-3-methyoxymethamphetamine
(HMMA), 4-hydroxy-3-methoxyamphetamine (HMA), and 3,4-methylenedioxyamphetamine
(MDA) conjugates prior to chromatographic analysis. Acidic and enzymatic hydrolysis
with b-glucuronidase from Escherichia coli and Helix pomatia were evaluated.
Acid hydrolysis yielded 40.0% and 39.3% higher HMA recovery compared to E. coli
and H. pomatia hydrolysis, respectively (SE = 9.8 and 11.4%). E. coli b-glucuronidase
hydrolysis MDA recovery was 17.1% and 26.5% greater than acid hydrolysis and
H. pomatia b-glucuronidase recovery (SE = 3.3 and 6.1%), respectively. HMMA
recovery by acid hydrolysis was 336.1% and 159.8% greater than E. coli and H.
pomatia b-glucuronidase (SE = 72.8 and 31.6%), respectively. The effects of
temperature, time, and acid amount on metabolite recovery were also evaluated.
HMA and HMMA acid hydrolysis recoveries were improved at 100°C and above.
Effective hydrolysis could be conducted in a dry block heater, GC oven, or autoclave
at temperatures from 100 to 140°C. Optimal hydrolysis conditions for the
measurement of MDMA metabolite conjugates were addition of 100 µL of hydrochloric
acid to 1 mL urine and incubation at 120°C in a GC oven for 40 min. Therefore,
based on HMMA, HMA, and MDA recoveries, time efficiency, availability of instrumentation,
and cost, acid hydrolysis was preferred to enzyme hydrolysis.
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