High-performance liquid chromatography with electrochemical detection applied to the analysis of 3,4-dihydroxymethamphetamine in human plasma and urine.
Metabolic activation in the disposition of 3,4-methylenedioxymethamphetamine (MDMA, "ecstasy") has been implicated in some of its pharmacological and toxicological effects, with the major metabolite 3,4-dihydroxymethamphetamine (HHMA) as a putative toxicant through the formation of thioether adducts. We describe the first validated method for HHMA determination based on acid hydrolysis of plasma and urine samples, further extraction by a solid-phase strong cation-exchange resin (SCX, benzenesulfonic acid), and analysis of extracts by high-performance liquid chromatography with electrochemical detection. The chromatographic separation was performed in an n-butyl-silane (C4) column and the mobile phase was a mixture of 0.1 M sodium acetate containing 0.1 M 1-octanesulphonic acid and 4 mM EDTA (pH 3.1) and acetonitrile (82:18, v/v). Compounds were monitored with an electrochemical cell (working potentials 1 and 2, +0.05 and +0.35 V, respectively, gain 60 microA). A mobile phase conditioning cell with a potential set at +0.40 V was connected between the pumping system and the injector. Calibration curves were linear within the working concentration ranges of 50-1000 microg/L for urine and plasma. Limits of detection and quantification were 10.5 and 31.8 microg/L for urine and 9.2 and 28.2 microg/L for plasma. Recoveries for HHMA and DHBA (3,4-dihydroxybenzylamine, internal standard) were close to 50% for both biological matrices. Intermediate precision and inter-day accuracy were within 3.9-6.5% and 7.4-15.3% for urine and 5.0-10.8% and 9.2-13.4% for plasma.
Segura
M
M
Pharmacology Research Unit, Institut Municipal d' Investigació Medica, Barcelona, Spain.
Ortuño
J
J
McLure
J A
JA
Pujadas
M
M
Pizarro
N
N
Farré
M
M
Llebaria
A
A
Joglar
J
J
Segura
J
J
de la Torre
R
R
D002138:Calibration; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003846:Deoxyepinephrine / Q000031:analogs & derivatives* / Q000032:analysis* / Q000097:blood / Q000652:urine; D004563:Electrochemistry / Q000379:methods*; D012015:Reference Standards; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
C069512:alpha-methylepinine; D003846:Deoxyepinephrine