Purpose: Pharmacological, pharmacokinetics: To present a "validated method for HHMA determination in plasma and urine by strong cation exchange solid-phase extraction and HPLC analysis with electrochemical detection (ED)." (p. 1204).

Design: Not explicitly stated, probably a between-subjects / within-subjects design, with blood and urine samples collected over time as within-subjects variables, and with all participants receiving 100 mg MDMA. Previous reports have collected samples from people participating in a randomized, double blind, placebo-controlled study.

Subjects: 4 MDMA-experienced volunteers (4 men, no women), mean age 25.5 ± 5.0 years, with no information provided on manner of recruitment. Recruitment in prior studies used "word of mouth."

Criteria for Inclusion - Male, having used ecstasy at least once (and perhaps at least 5 times) in the past, and extensive metabolizers of MDMA as assessed via dextromethorphan probe. No information presented on health or medical conditions of volunteers, but previous studies included individuals on the basis of health as assessed through medical examination, laboratory tests, urinalysis and ECG.

Measures: Blood - Blood samples were collected at baseline or prior to drug administration, 20, 40, 60 and 90 min post-drug and 2, 3, 4, 6, 8, 10 and 24 h after drug administration. Concentrations of MDMA, MDA, HMMA, and HHMA (DHMA) in blood were measured with HPLC with ED. HPLC detection was also validated by using standardized methods that employed solid phase extraction and GC-MS detection.

Urine - Urine samples were collected pre-drug (basal), at 0-4, 4-8, 8-12, and 12-24 h post-drug. Concentration of MDMA, MDA, HMMA and HHMA (DHMA) were measured in urine via HPLC, and validated with methods employing solid phase extraction and GC-MS detection.

Analyses: Tmax, Cmax, Ka (formation rate constant), Kd and T1/2 were all calculated for MDMA and metabolites with PKCALC software. AUC calculated via the linear trapezoidal rule.

Results: HHMA (DHMA) was successfully detected in urine and plasma samples. Plasma - Concentration of HHMA peaked in plasma at 1.2 ± 0.3 h, compared with 1.9 ± 0.2 h for MDMA and 2 ± 0.0 for HMMA (Tmax), and peak concentration was 154.5 ± 76.6, compared with 181.6 ± 24.5 ng/mL). T1/2 for HHMA was 13.4 ± 8.1 h (compare 7.1 ± 1.3 h for MDMA 8.3 ± 1.3 for HMMA.) AUC for HHMA was 1990.94 ± 647.1 ng/mL h; AUC for MDMA was 1465.8± 705.1 ng/mL/h, and for HMMA 3190.9 ± 714.5 ng/mL/h. Urine - Urinary excretion of HHMA is as follows (in micromoles): 0-4 h 30.9 ± 10.2, at 4-8 h, 21.4 ± 9.7, at 8-12 h, 12.8 ± 6.2 and at 12-24 h, 26.6 ± 9.39. The 24 h urinary recovery of HHMA accounted for 17.7% of the MDMA dose administered, raising total drug recovery (includes unmetabolized drug, MDA, HMA, HMMA and HHMA) to 58%.

Overall Effects: The MDMA metabolite HHMA (DHMA) was successfully detected in plasma and urine samples collected from 4 male volunteers given 100 mg MDMA. Plasma values were close to those for MDMA itself, and urinary recovery over 24 h accounts for 17.7% of drug recovery. Peak concentrations of HHMA appeared slightly earlier than peak concentrations of MDMA, but at around the same time (1 to 2 h post-drug). While no statistical comparisons were made between concentrations of metabolites, plasma and urinary HMMA is highest, followed by HHMA, then MDA.

Adverse Effects: None reported in this paper.

Comments: This is the first reported quantifiable detection of the MDMA metabolite HHMA (DHMA) in human plasma and urine. HHMA is highly reactive, causing it to readily conjugate and making it difficult to extract from biofluids (blood, urine, etc).. Previously, HHMA had been shown to form in vitro and had been reported in human biofluids on several occasions. However, pharmacokinetics for HHMA had not been characterized, and it was suspected that this metabolite might be rapidly transformed to HMMA. This paper is important in that it describes a method for extracting HHMA and establishes that HHMA is a major metabolite of MDMA, with peak plasma concentrations of HHMA being comparable to those of MDMA when 100 mg MDMA is administered. The pharmacological and toxicological significance of HHMA is not clear. Although HHMA does not produce neurotoxicity when infused into the rat brain, Monks and colleagues have conducted a series of experiments that suggest some glutathione conjugates of dihydroxyamphetamine (and, by implication, HHMA) may induce selective serotonergic neurotoxicity in low concentrations. The current study did not measure specific conjugates of HHMA, but the extensive formation of HHMA makes it plausible that significant amounts of glutathione conjugates of HHMA might form in humans. This report is also one of several indicating that MDA is only a minor metabolite of MDMA compared with either HMMA or HHMA. An important limitation of the study concerns the single dose level employed. MDMA kinetics appear to be dose-dependent. This group has previously shown that formation of HMMA is probably dose independent over a range of commonly administered doses (50-15 0 mg). Because HMMA is thought to form from HHMA, it is possible that HHMA formation is also dose-independent over the same dose range. The sample size used in this paper is very small and consists of only male volunteers. Future examinations of HHMA excretion may wish to administer several dose levels of MDMA to larger samples of both male and female volunteers.