Conjugates of p-hydroxymethamphetamine and 4-hydroxy-3-methoxymethamphetamine in blood obtained from methamphetamine and 3,4-methylenedioxymethamphetamine users: analysis by LC-MS-MS

A detailed procedure for simultaneous analysis of methamphetamine (MA), 3,4-methylenedioxymethamphetamine (MDMA) and their metabolites including the conjugates in whole blood by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS-MS) has been established. The methods were well validated and applied to users' blood specimens. The blood concentrations of MA users (n = 11) ranged from < 1.0 to 131 ng/ml (from < 6.1 to 794 nM) for p-hydroxymethamphetamine (p-OHMA), from < 2.0 to 46.7 ng/ml (from < 5.9 to 137 nM) for p-OHMA-glucuronide (p-OHMA-Glu), and from 3.8 to 495 ng/ml (from 15.5 to 2020 nM) for p-OHMA-sulfate (p-OHMA-Sul). The molar conjugation percentage [(p-OHMA-Sul + p-OHMA-Glu)/(p-OHMA + p-OHMA-Sul + p-OHMA-Glu) x 100%] for every specimen except one ranged from 64.9% to 100% [average +/- standard deviation (SD): 85.0% +/- 12.0%, n = 10]. The blood concentrations of MDMA users (n = 3) ranged from < 1.0 to 84.2 ng/ml (from < 5.1 to 432 nM) for 4-hydroxy-3-methoxymethamphetamine (HMMA), from 78.1 to 508 ng/ml (from 211 to 1370 nM) for HMMA-glucuronide (HMMA-Glu), and from 190 to 610 ng/ml (from 691 to 2220 nM) for HMMA-sulfate (HMMA-Sul). The molar conjugation percentage [(HMMA-Sul + HMMA Glu)/(HMMA + HMMA-Sul + HMMA-Glu) x 100%] ranged from 89.3% to 100% (average +/- SD: 95.2% +/- 5.4%, n = 3). The present study clearly showed the presence of conjugated forms of p-OHMA and HMMA in human blood; the levels of sulfate conjugates were much higher than those of glucuronide conjugates. The utility of the p-OHMA-Sul and HMMA-Sul remains to be explored.