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 β-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 β-glucuronidase hydrolysis MDA recovery was 17.1% and 26.5% greater than acid hydrolysis and H. pomatia β-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 β-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.