title: Selection and optimization of hydrolysis conditions for the quantification of urinary metabolites of MDMA.
abstract: 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 beta-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 beta-glucuronidase hydrolysis MDA recovery was 17.1% and 26.5% greater than acid hydrolysis and H. pomatia beta-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 beta-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 degrees C and above. Effective hydrolysis could be conducted in a dry block heater, GC oven, or autoclave at temperatures from 100 to 140 degrees C. Optimal hydrolysis conditions for the measurement of MDMA metabolite conjugates were addition of 100 microL of hydrochloric acid to 1 mL urine and incubation at 120 degrees 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.
authors.affiliation: Chemistry and Drug Metabolism, Intramural Research Program, National Institute on Drug Abuse, National Institutes of Health, 5500 Nathan Shock Drive, Baltimore, Maryland 21224, USA.
authors.forename: Stephane O
authors.initials: SO
authors.lastname: Pirnay
authors.forename: Tsadik T
authors.initials: TT
authors.lastname: Abraham
authors.forename: Ross H
authors.initials: RH
authors.lastname: Lowe
authors.forename: Marilyn A
authors.initials: MA
authors.lastname: Huestis
mesh_terms: D015104:3,4-Methylenedioxyamphetamine / Q000737:chemistry / Q000652:urine; D004298:Dopamine / Q000031:analogs & derivatives / Q000737:chemistry / Q000652:urine; D053593:Forensic Toxicology / Q000379:methods*; D005966:Glucuronidase / Q000737:chemistry; D006213:Hallucinogens / Q000737:chemistry* / Q000652:urine; D006801:Humans; D006851:Hydrochloric Acid / Q000737:chemistry; D006868:Hydrolysis; D008694:Methamphetamine / Q000031:analogs & derivatives / Q000737:chemistry / Q000652:urine; D018817:N-Methyl-3,4-methylenedioxyamphetamine / Q000031:analogs & derivatives / Q000737:chemistry* / Q000652:urine; D015813:Substance Abuse Detection / Q000379:methods*
chemical_list: D006213:Hallucinogens; C079646:4-hydroxy-3-methoxymethamphetamine; C031105:3-O-methyl-alpha-methyldopamine; D008694:Methamphetamine; D015104:3,4-Methylenedioxyamphetamine; D005966:Glucuronidase; D018817:N-Methyl-3,4-methylenedioxyamphetamine; D006851:Hydrochloric Acid; D004298:Dopamine