A validated gas chromatographic–electron impact ionization mass spectrometric method for methamphetamine, methylenedioxymethamphetamine (MDMA), and metabolites in mouse plasma and brain
Introduction
The sympathomimetic amines, methamphetamine (MAMP) and its derivative methylenedioxymethamphetamine (MDMA, ecstasy) are psychostimulants widely abused worldwide [1], [2], [3], [4]. MAMP and MDMA can cause serious health abnormalities [5], [6], [7], [8]. Research in humans has shown that MAMP is highly toxic and can produce hyperthermia, heart failure, aggression, psychotic behavior, memory loss and potential brain damage [5]. MAMP caused prolonged depletion of the neurotransmitter dopamine and dopamine transporter protein in brain [5], [7]; additional studies provided evidence of neuronal death in MAMP users’ brains [9].
Although MDMA is structurally similar to MAMP, MDMA's effects differ by possessing both stimulant and hallucinogenic properties [6]. In most species, MDMA is selectively neurotoxic to serotonergic terminals, except in mice MDMA depletion of dopaminergic terminals predominates [7], [10]. Studies have demonstrated long-term impairments of memory and learning in human subjects reporting heavy MDMA use [8].
Transgenic mice are genetically engineered to lack expression of particular proteins and are useful for delineating sympathomimetic amine mechanisms of action and neurotoxicity [11], [12], [13], [14], [15], [16], [17], [18], [19], [20], [21], [22], [23], [24]. Results from transgenic mice can be confounded by compensatory changes in protein expression. Therefore, it is important to identify whether drug metabolism and distribution are altered in the transgenic mice, confounding interpretation of experimental results.
Sympathomimetic amines have been measured in plasma and tissue using gas chromatography-nitrogen-phosphorous detection [25], [26] gas chromatography–mass spectrometry (GC–MS) [27], [28], [29], [30], [31], [32], [33], [34], [35], [36], high pressure liquid chromatography (HPLC)-diode array detection [37], HPLC-fluorescence detection [38], [39], [40] and HPLC–mass spectrometry [41], [42], [43], [44], [45]. Most methods have been developed for measuring AMP and MAMP or MDMA and metabolites in human plasma, although Peters et al. detailed a validated GC–MS method for measuring AMP, MAMP, MDMA and MDA in human plasma [34]. Three validated LC–MS methods for measuring MAMP, MDMA and metabolites in plasma have also been reported [41], [42], [45]. Only a few reports detail measuring MAMP and AMP [16] or MDMA and metabolites [40], [46] in mouse brain. Comprehensive validation of these brain analytical methods was not presented [16], [40], [46]. MAMP- and MDMA-induced depletion of dopaminergic and serotonergic nerve terminals have been identified in multiple regions of the brain, predominantly in striatum and cortex [7], [10], [47], [48]. Therefore, it is important to measure drug concentrations in brain regions most affected by MAMP and MDMA. Measuring drug concentrations in mouse striatum presents a considerable challenge as the brain volume is small, typically weighing 15–20 mg. Collection of blood from mice via cardiac puncture typically yields 100–300 μL of plasma for analysis. Therefore, analytical sensitivity is critical for measuring concentrations of MAMP, MDMA and metabolites in small specimens collected during mice neurotoxicity studies.
This manuscript details the development and validation of a gas chromatographic electron impact ionization mass spectrometric (GC–MS-EI) method for simultaneous quantification of MAMP, its metabolites amphetamine (AMP) and para-hydroxy-methamphetamine (OH-MAMP) and MDMA and its metabolites methylenedioxyamphetamine (MDA), 4-hydroxy-3-methoxy-methamphetamine (HMMA) and 4-hydroxy-3-methoxyamphetamine (HMA) in mouse plasma and brain to support our studies investigating the role of drug metabolism and distribution in MAMP- and MDMA-induced neurotoxicity in transgenic mice. This manuscript details the first validated method capable of simultaneously measuring MAMP, MDMA and metabolites in mouse plasma and striatum and will be applied to the investigation of MAMP and MDMA metabolism and distribution in wild-type and transgenic mice during neurotoxicity studies.
Section snippets
Reagents
Racemic mixtures of MAMP, AMP, MDMA, MDA (1 mg/mL in methanol) and internal standards MAMP-d14, AMP-d11, MDMA-d5, and MDA-d5 (100 μg/mL) were purchased from Cerilliant Corporation (Round Rock, TX, USA). Racemic HMMA and HMA (1 mg/mL in methanol) were obtained from Lipomed Inc. (Cambridge, MA, USA) and p-hydroxy-methamphetamine powder (greater than 98% purity) from Sigma–Aldrich (St. Louis, MO, USA). Racemic MDMA hydrochloride and racemic MAMP hydrochloride administered to mice were from Lipomed
Hydrolysis of conjugated metabolites
Preliminary studies during method development compared assay characteristics obtained employing conditions similar to those validated for human urine [49] using 100 μL HCl and 45 min 100 °C incubation and overnight 37 °C enzyme hydrolysis with β-glucuronidase from H. pomatia as per Pizarro et al. [29] and found that enzyme hydrolysis produced elevated matrix interferences that resulted in 2–5-fold decreases in AMP and MAMP sensitivities compared to sensitivities achieved with acidic hydrolysis.
Discussion
It has been shown that OH-MAMP, HMMA and HMA are highly conjugated being excreted in urine conjugated to sulfate and glucuronide with only small amounts of parent drug present in urine [50], [51]. Shima et al. directly measured HMMA, HMMA-glucuronide and HMMA-sulfate in human urine and found that HMMA-sulfate predominates 3-fold compared to HMMA-glucuronide [51]. Inter-species differences between amounts of OH-MAMP and HMMA glucuronidation and sulfation were noted between humans and rats [50],
Acknowledgements
The authors thank the co-investigators of the MAMP and MDMA neurotoxicity study conducted at the Intramural Research Program, National Institute on Drug Abuse, Jean Lud Cadet and Bruce Ladenheim for generously providing mouse plasma and brain specimens for proof of method. This research was supported by the Intramural Research Program of the National Institute on Drug Abuse, National Institutes of Health.
References (51)
- et al.
Drug Alcohol Depend.
(2002) - et al.
Brain Res. Brain Res. Rev.
(2003) - et al.
Neuron
(1997) - et al.
Neuroscience
(2001) - et al.
Neurosci. Res.
(2002) - et al.
Biol. Psychiatry
(2007) - et al.
J. Chromatogr. B Biomed. Appl.
(1995) - et al.
J. Chromatogr. B Biomed. Sci. Appl.
(1999) - et al.
J. Pharm Biomed. Anal.
(1999) - et al.
J. Pharm. Sci.
(2008)