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Clinical Chemistry 54: 379-387, 2008. First published December 18, 2007; 10.1373/clinchem.2007.096800
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(Clinical Chemistry. 2008;54:379-387.)
© 2008 American Association for Clinical Chemistry, Inc.

Drug Monitoring and Toxicology

Two-Dimensional Gas Chromatography/Electron-Impact Mass Spectrometry with Cryofocusing for Simultaneous Quantification of MDMA, MDA, HMMA, HMA, and MDEA in Human Plasma

Erin A. Kolbrich, Ross H. Lowe and Marilyn A. Huestisa

Chemistry and Drug Metabolism, Intramural Research Program, National Institute on Drug Abuse, National Institutes of Health, Baltimore, MD.

aAddress correspondence to this author at: Chemistry and Drug Metabolism, Intramural Research Program, National Institute on Drug Abuse, National Institutes of Health, 5500 Nathan Shock Dr., Baltimore, MD 21224. Fax 410-550-2971; e-mail mhuestis{at}intra.nida.nih.gov.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: 3,4-Methylenedioxymethamphetamine (MDMA, or Ecstasy) is a popular recreational drug. Analysis of MDMA and metabolites in human plasma, particularly in pharmacokinetic studies, requires low limits of quantification. Two-dimensional GC/MS with cryofocusing is a chromatographic technique recognized for its increased selectivity and resolution.

Methods: This method simultaneously quantifies 3,4-methylenedioxyethylamphetamine (MDEA), MDMA, and its metabolites, 3,4-methylenedioxyamphetamine (MDA), 4-hydroxy-3-methoxymethamphetamine (HMMA), and 4-hydroxy-3-methoxyamphetamine (HMA) in human plasma. With hydrochloric acid, we hydrolyzed 1 mL plasma, fortified with internal standard. Analytes were subjected to solid-phase extraction, derivatized with heptafluorobutyric acid anhydride, and quantified using cryofocused 2-dimensional GC/MS operated in electron-impact selected ion-monitoring mode.

Results: Limits of quantification were 1.0 µg/L for MDA and 2.5 µg/L for MDEA, MDMA, HMMA, and HMA. Calibration curves were linear to 100 µg/L for MDA and HMA and to 400 µg/L for MDEA, MDMA, and HMMA, with r2 > 0.997. At 3 concentrations spanning the linear dynamic range of the assay, mean overall extraction efficiencies from plasma were ≥85% for all compounds of interest. Recoveries were 85.6% to 107.2% of target, and intra- and interassay imprecision (CV) was <8.5% for all drugs at 3 concentrations within the range of the assay. None of the 66 exogenous compounds tested interfered with analyte quantification.

Conclusions: This GC/MS assay provides low limits of quantification for simultaneous determination of MDEA, MDMA, and metabolites MDA, HMMA, and HMA in human plasma. The 2D chromatographic system should be suitable for application to other analytes and to other complex matrices.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
3,4-Methylenedioxymethamphetamine (MDMA),1 commonly known as Ecstasy, is an illicit drug ingested for its stimulatory and hallucinogenic effects (1)(2). MDMA is an entactogen, literally meaning "a touching within," for its ability to elicit affective responses including enhanced communication, closeness to others, insight, and self-acceptance. It has gained popularity in the rave club subculture and migrated to mainstream use with street names like "e," "Adam," "hug," "beans," and "love drug." Approximately 0.5 million Americans ages 12 years and older (3) and more than 1 million Europeans ages 15–64 (4) have used Ecstasy in the past month. A national survey noted that Ecstasy use had an annual prevalence of 4.5% in 10th graders and 6.5% in 12th graders for 2006 (5). Combination use with cannabis is common (6), and MDMA is popular with other recreational psychedelics such as lysergic acid diethylamide (LSD) ("candy flipping" or "trolling"), mushrooms ("flower flipping"), and LSD and mushrooms ("triple flipping"). Although overdoses are uncommon, health risks include hypertension, dehydration, and risk of dependency. Animal research indicates that MDMA is neurotoxic, with demonstrated toxic effects on serotonergic neurons and memory (7)(8).

After oral administration, MDMA is enzymatically metabolized to 5 primary metabolites (9)(10). The major metabolic route involves conversion to 3,4-dihydroxymethamphetamine (HHMA), an unstable intermediate, and then to 4-hydroxy-3-methoxymethamphetamine (HMMA) and 4-hydroxy-3-methoxyamphetamine HMA. The minor pathway produces 3,4-methylenedioxyamphetamine (MDA), 3,4-dihydroxyamphetamine (HHA), and HMA. Whereas MDMA and MDA are in plasma primarily in free form, the 4 metabolites carrying one or more free hydroxyl groups are conjugated with both glucuronide and sulfate. Conjugated HMMA is the major metabolite in human plasma (9).

The majority of published methods for quantification of MDMA in human plasma focus on MDMA and its minor metabolite, MDA (11)(12)(13)(14). 3,4-Methylenedioxy-N-ethylamphetamine (MDEA) has not been included previously in conjunction with MDMA, MDA, HMMA, and HMA in a human plasma analytical method. This analysis includes MDEA owing to its potential presence in illicit Ecstasy, known for its poor purity (15)(16)(17)(18), and because toxicology applications frequently require testing for this drug.

Although 2-dimensional GC/MS was first developed nearly 50 years ago (19), forensic toxicology applications began only recently (20)(21)(22)(23)(24). The advantage of 2D-GC/MS is a high degree of chromatographic resolution (25). Components first are separated on a nonpolar primary column. "Cuts" are collected around peaks of interest and diverted to a secondary polar column through use of a pneumatic Deans switch (Fig. 1 ). Additional sensitivity due to peak sharpening is gained by cryofocusing analytes at the entrance to the second column. Increased sensitivity, selectivity, and resolution, as well as decreased routine maintenance and extended column life, are all advantages of 2D-GC/MS.


Figure 1
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  		Figure 1. Schematic of a 2-dimensional gas chromatography system with a Deans switch and cryotrap configured with a flame ionization detector (FID) and mass selective detector (MSD).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
chemicals and reagents
We obtained human plasma from the National Institutes of Health blood bank. Each lot was analyzed in this laboratory by 2D-GC/MS to ensure absence of MDEA, MDMA, and metabolites. Racemic mixtures of MDMA, MDA, MDEA, HMMA, MDMA-d5, MDA-d5, and MDEA-d6 were obtained from Cerilliant Corp. (±) HMA was purchased from Lipomed, Inc., p-hydroxymethamphetamine (pholedrine) from Sigma, and Styre ScreenTM DBX solid-phase extraction (SPE) columns from United Chemical Technologies, Inc. ACS reagent-grade monobasic and dibasic potassium phosphate, hydrochloric acid (HCl), sodium hydroxide, acetic acid, isopropanol, ammonium hydroxide, and ethyl acetate (HPLC grade) were purchased from Mallinckrodt Baker. HPLC-grade methanol was obtained from Fisher Scientific and heptafluorobutyric acid anhydride (HFAA) from Pierce Chemical Co.

calibrators and quality control solutions
We combined 1 mL of 1 g/L MDMA, MDA, MDEA, HMMA, and HMA standards in a 10-mL volumetric flask to obtain a 0.1 g/L stock solution. We prepared 10, 1.0, and 0.1 mg/L standard solutions by dilution in methanol. Appropriate amounts of standard solutions were added to plasma to produce 1.0, 2.5, 5.0, 10, 50, 100, and 400 µg/L working calibrators.

We used stock solutions from a different lot to prepare MDMA, MDA, MDEA, and HMMA quality control (QC) solutions. As only a single lot of HMA was available from the vendor, we used a separate vial of the same lot to make QC solutions. Three QC concentrations were prepared: the low QC contained 3.0 µg/L MDA and HMA and 7.5 µg/L MDMA, HMMA, and MDEA; the medium QC, 15 µg/L MDA and HMA and 40 µg/L MDMA, HMMA, and MDEA; and the high QC, 75 µg/L MDA and HMA and 200 µg/L MDMA, HMMA, and MDEA. We prepared a working mixed internal standard solution (working IS) in methanol to deliver 25 ng MDMA-d5, MDEA-d6, and pholedrine and 15 ng MDA-d5 in a 25-µL aliquot. Calibrator, control, and IS stock solutions were stored at –80 °C and working solutions at –20 °C.

specimen hydrolysis and extraction
We pipetted 1 mL blank or participant plasma into glass 16- by 100-mm screw-cap culture tubes. Calibrators and QCs were fortified as described above. We added 25 µL working IS and 1 mL 0.5 mol/L HCl; tubes were capped, vortex-mixed, and incubated at 100 °C for 40 min. After cooling, we added 1 mL of 0.1 mol/L phosphate buffer [pH 6.0] and 50 µL of 10 mol/L NaOH to adjust the pH to 6. Samples were centrifuged for 10 min at 1855g. Polymeric Styre Screen DBX SPE columns functionalized with benzenesulfonic acid and C18 were preconditioned with 1.5 mL methanol, 1.5 mL deionized H2O, and 0.5 mL 0.1 mol/L phosphate buffer, pH 6.0. We decanted the sample supernatants onto conditioned SPE columns and washed the columns sequentially with 1.5 mL deionized H2O, 0.5 mL 0.1 mol/L acetic acid and 1.5 mL methanol, followed by drying for 15 min. Elution of analytes was achieved with 2 750-µL aliquots of freshly prepared ethyl acetate:isopropanol:ammonium hydroxide (90/6/4, vol/vol/vol).

derivatization
Derivatization and back-extraction procedures were performed as described by Scheidweiler and Huestis (26). To reduce loss of analytes, we added 15 µL of 0.12 mol/L methanolic HCl to the SPE eluates before evaporation under nitrogen at 35 °C. Extract residues were reconstituted with 100 µL 0.5 mol/L triethylamine in heptane, vortex-mixed, and derivatized with 10 µL HFAA at 60 °C for 20 min. After cooling, samples were back-extracted with 200 µL Tris buffer, pH 7.4. Tubes were vortex-mixed and centrifuged at 1855g, and the top organic layers containing analytes were transferred into autosampler vials.

2-dimensional gc/ms
For specimen analysis, we used an Agilent 6890 gas chromatograph configured with a microfluidic Deans switch and flame ionization detector (FID) and interfaced to an Agilent 5973 mass selective detector (MSD). The GC also was equipped with a compressed air–cooled cryogenic focusing trap (Joint Analytical Systems). The microfluidic Deans switch enables 2D-GC with 2 capillary chromatographic columns in series. The pneumatic switch system directs output of the primary column to either the FID or the inlet of the secondary column. The inlet end of the secondary column was inserted through the cryogenic trap and the outlet directed to the MSD. The air-cooled cryogenic trap focuses time-programmed "cuts" from the primary capillary column at the head of the secondary capillary column, then revaporizes trapped eluent with rapid heating. Operating parameters are listed in Table 1 .


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  		Table 1. Gas chromatograph, Deans switch, flame ionization detector, and cryotrap parameters for the detection and simultaneous quantification of MDMA, MDA, HMMA, HMA, and MDEA in human plasma.

We injected 3 µL derivatized extract in splitless mode. Two-dimensional chromatographic separation (Fig. 2 ) was achieved with a primary DB-1MS capillary column (15 m length by 0.25 mm i.d. by 0.25 µm film thickness; Agilent Technologies) and secondary ZB-50 capillary column (30 m length by 0. 32 mm i.d. by 0.25 µm film thickness; Phenomenex).


Figure 2
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  		Figure 2. Chromatograms illustrating 2-dimensional separation of MDMA, MDMA-d5, MDA, MDA-d5, HMMA, HMA, MDEA, MDEA-d6, and pholedrine in a blank plasma sample fortified with 25 ng of each analyte and internal standard, except MDA-d5, which was fortified at 15 ng.

(A), Flame ionization detector (FID) signal of eluent from the primary DB-1 column showing time-cut diversion to secondary ZB-50 column. (B), Mass selective detector total ion chromatogram (TIC) of MDMA, MDA, HMMA, HMA, and MDEA when analytes are resolved on the ZB-50 secondary column.

The GC oven temperature was ramped, at 35 °C/min, from 70 to 150 °C, held for 0.50 min, then increased at 10 °C/min to 195 °C and held for 0.11 min. At this point, analytes of interest were cut from the first column and cryogenically captured at the inlet of the second column. The GC oven temperature was decreased at 50 °C/min to 100 °C, the cryogenic trap was ramped at 800 °C/min to 275 °C to release analytes, and a final GC oven temperature ramp at 10 °C/min to 190 °C resolved analytes on the second column. A complete list of instrument parameters is presented in Table 1Up .

The temperatures of the quadrupole, source, and MSD interface were 150, 230, and 280 °C, respectively. One target ion and 2 qualifier ions were monitored for analytes, MDMA, MDA, MDEA, HMA, and HMMA. One qualifier was monitored for each internal standard, MDMA-d5, MDA-d5, and MDEA-d6. Specific ions were as follows (quantitative ion italicized): MDMA 254, 210, 162; MDMA-d5 258, 213; MDA 162, 135, 375; MDA-d5 167, 380; MDEA 268, 240, 162; MDEA-d6 274, 244; HMMA 210, 254, 360; HMA 240, 163, 360; and pholedrine 254, 210.

data analysis
We identified analytes by comparing retention time (±2%) and relative abundance of qualifier ions to corresponding average values (±20%) of calibrators assayed in the same run. Concentrations of unknowns were calculated from the equation of the regression line using peak area ratios of analyte to internal standard vs concentration. A 1/x weighting scheme was used to account for heteroscedasticity or unequal variance across the linear range. Requirements for acceptable calibration curves were coefficient of determination (r2) >0.985 and individual calibrator concentrations within ±20% of target.

hydrolysis conditions
We evaluated acidic hydrolysis at 3 temperatures, 100, 120, and 140 °C; 3 volumes of acid (0.5 mol/L HCl), 1, 1.5, and 2 mL; and 3 incubation times, 20, 40, and 60 min. Plasma from one participant dosed with MDMA as part of a controlled administration study was used to compare conditions. The clinical study was approved by the National Institute on Drug Abuse Institutional Review Board, and the participant provided written informed consent. We pooled plasma specimens from multiple time points following the MDMA dosing session and used 1-mL aliquots for evaluation of hydrolysis parameters. Each condition was tested in triplicate.

validation experiments
Method validation included determination of dynamic linear range of the assay, limit of detection (LOD), limit of quantification (LOQ), recovery, imprecision, specificity, extraction efficiency, dilution integrity, stability, and carryover. We used low, medium, and high QC samples for each analyte to evaluate each parameter.

We assayed 4 unique calibration curves. Linearity was evaluated by least-squares regression (1/x weighting) and expressed as r2. MDMA-d5, MDA-d5, and MDEA-d6 were used as internal standards for their respective nondeuterated analytes. Analytes with free hydroxyl groups were not commercially available in deuterated form; MDA-d5 was evaluated for use as an internal standard for HMA, MDMA-d5 for HMMA, and pholedrine for both.

We spiked blank plasma with decreasing concentrations of analytes in triplicate to determine LOD and LOQ. LOD was defined as the lowest concentration with a signal-to-noise ratio ≥3:1 for target and qualifier ions, ion ratios within 20% of average qualifier ion ratios of calibrators, retention time within ±2%, and shape of gaussian peaks. At the LOQ, all LOD criteria must be met, signal-to-noise ratio of the target ion is 10:1, and the calibrator must quantify within 20% of target concentration. With these definitions, it is possible for LOD to equal LOQ.

We evaluated imprecision and recovery over the dynamic range at low, medium, and high QC concentrations. Five replicates of each concentration were included in 4 separate analytical runs (ntotal = 20). We determined recovery by comparing mean measured concentration to target, expressed as mean percent of target; results were acceptable if ≤20% of target. Interassay imprecision was expressed as percent coefficient of variation of 20 individual values, equally weighted over 4 batches. Intraassay imprecision was evaluated from 5 determinations per concentration over 4 batches. Coefficients of variation ≤20% were required for a precise method. We used 1-way ANOVA to evaluate interassay variability at each QC concentration.

To test specificity of the analytical method, we added 1000 µg/L of each potential interfering compound (n = 66, Supplementary Data Table 1Up ) to low QC samples. If analytes met identification criteria and quantified within 20% of target, no interference was noted. Six different blank plasma pools were also evaluated for endogenous interference.

To assess extraction efficiency for each analyte, we added analyte control solution to blank matrix at low, medium, and high control concentrations before solid-phase extraction and to a second set after extraction, but before the derivatization step. We calculated relative extraction efficiency at each QC concentration by comparing mean analyte peak area of each compound in the first set with appropriate mean analyte peak area (n = 5) in the second.

To determine dilution integrity, we diluted plasma samples spiked at high QC concentrations with 0.1 mol/L phosphate buffer (pH 6.0). Samples (n = 5) were prepared, and a 75% dilution with buffer was performed. Measured concentrations were compared to one-fourth of mean undiluted QC concentration and percent difference calculated to evaluate dilution integrity. Sample integrity was unaffected by dilution if results were within 20% of target.

To investigate analyte stability, we stored 3 replicates of low, medium, and high QC samples in capped tubes at 3 different temperatures. Short-term stability was evaluated at room temperature for 12 h or at 4 °C for 48 h. Freeze/thaw stability was determined by subjecting samples to 3 24-h freeze/thaw cycles before internal standard fortification and analysis. Concentrations of stability samples were compared to routinely prepared control samples; results were expressed as a percent difference from mean QC concentration.

We evaluated stability of derivatized extracts in triplicate experiments by reinjection of low, medium, and high controls 24 and 48 h after initial injection. To restore sufficient volume of solvent, 50 µL heptane was added to each vial before the 48-h injection. Concentrations of all samples were quantified using the original calibration curve and compared to initial assay concentration. Percent differences between means of each group (n = 3/concentration) were calculated.

To investigate carryover, we assayed low QC samples after a plasma sample was spiked with 1000 µg/L of each analyte. If low QC sample concentrations were within 20% of the mean of low QC samples and ion ratios were acceptable, no carryover occurred. A negative plasma sample was assayed following the highest calibrator to evaluate carryover in each assay run. Analyte abundance in the negative sample was required to be < LOD.


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
internal standard evaluation
Structural similarities led to investigation of MDMA-d5 as an internal standard for HMMA, MDA-d5 for HMA, and pholedrine for both analytes. When pholedrine was used as internal standard for both HMMA and HMA in plasma, linearity improved, as evidenced by higher r2 values and interpolated calibrator concentrations closer to target. Therefore, we selected pholedrine as internal standard for HMMA and HMA. It should be noted that, although pholedrine is appropriate for analysis of samples from controlled pharmacokinetic studies with MDMA, it presents a problem in other forensic applications. Pholedrine (p-hydroxymethamphetamine) is a metabolite of methamphetamine and the designer drug p-methoxymethamphetamine (PMMA).

hydrolysis conditions
Acidic hydrolysis was selected for its speed, low cost, and reported efficiency over basic and enzymatic hydrolysis (27)(28). No significant difference in recovery was observed between the 3 hydrolysis temperatures (results not shown). As a result, the lowest temperature of 100 °C was selected as the working hydrolysis temperature. We chose an incubation time of 40 min and 1 mL HCl (each tested independently) for routine analysis because these parameters provided higher, albeit nonsignificant, abundances for HMMA and HMA.

validation
We constructed calibration curves (n = 4) with 6 concentrations, with the exception of HMA (5 concentrations). MDA was linear from 1 to 100 µg/L, HMA from 2.5 to 100 µg/L, and MDMA, HMMA, and MDEA from 2.5 to 400 µg/L. The LOQ of each analyte was the lowest concentration on the calibration curve. MDA and HMA are found in considerably lower concentrations than MDMA and HMMA; thus, an upper limit of quantification of 100 µg/L was sufficient. Coefficients of determination were ≥0.997 for all analytes in each of 4 curves. Curve characteristics are listed in Table 2 .


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  		Table 2. Simultaneous quantification of MDMA, MDA, HMMA, HMA, and MDEA in human plasma by 2D GC/MS with cryotrapping.

The highest CV for an intraassay batch was 8.4% (Table 3 ). CVs for interassay imprecision at all QC concentrations were ≤6.7%. One-way ANOVA revealed significant interassay variation for low QC HMA and HMMA and medium and high QC MDMA, HMMA, and MDEA concentrations. However, differences in daily mean QC concentrations were ≤8.2%, and therefore, although statistically significant, variation was clinically insignificant.


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  		Table 3. Imprecision, recovery, and extraction efficiency results from a 2D GC/electron-impact MS method with cryotrapping developed for the simultaneous quantification of MDMA, MDEA, and metabolites in human plasma.

MDMA is often not the sole drug ingested, due to frequent abuse of cannabis and other drugs and the poor purity of illicit Ecstasy (16)(18)(29). As this method could be used in analysis of clinical and forensic toxicology cases, interference from other illicit and licit drugs was assessed to reduce risk of false-positive or -negative results. None of 66 tested exogenous interfering compounds caused the concentration of the 5 analytes to vary more than 20% from mean low QC concentration. No endogenous interference was noted from 6 different blank plasma pools.

Extraction efficiency and recovery were ≥85% at all tested concentrations (Table 3Up ), which allowed attainment of low LOQs and provided assurance that the majority of each analyte was successfully extracted.

Although concentrations previously reported from controlled dosing studies have suggested that calibration curves in this method have sufficient upper limits of quantification, MDMA or HMMA may exceed 400 µg/L. In addition, 1 mL plasma may not always be available. In both situations, dilution can be performed to produce quantifiable results. A 75% dilution with phosphate buffer (pH 6.0) resulted in mean concentrations within ±20% of target for all analytes.

All analytes were stable (±20% of mean fresh QC) under short-term room-temperature and refrigeration conditions and 3 freeze/thaw cycles, except HMA at lower concentrations. HMA displayed instability at low and medium QC concentrations after storage at room temperature for 12 h and at low QC concentration after 3 freeze/thaw cycles. Lowered concentrations may be a result of analyte degradation or adhesion to the tube. An additional experiment, 1 freeze/thaw cycle at low QC concentration, was performed because 1 frozen storage cycle is often unavoidable. Mean HMA concentration after 1 freeze/thaw cycle was within 11% of mean fresh low QC concentration. These results demonstrate that to obtain accurate HMA concentrations, plasma specimens should be removed from the freezer and immediately analyzed after thawing and mixing or placed in the refrigerator for up to 48 h before analysis. Other conditions may result in inaccurate HMA quantification.

Mean concentrations of QCs reinjected 24 and 48 h after original injection were within 8.3 and 5.8% of the mean for original values, respectively. These results demonstrate accurate quantification up to 2 days after initial extract preparation.

No carryover was present in 2 low QC samples injected sequentially after a 1000 µg/L sample. Concentrations of all analytes quantified ±20% of target, and ion ratios were within ±20% of the mean for calibrators assayed in the run. Carryover was not detected (< LOD) in negative samples assayed immediately after the high calibrator.

As proof of method, a plasma specimen was collected 7 h after a participant ingested 105.7 mg MDMA. Quantitative analysis of the plasma specimen yielded 80.9 µg/L MDMA, 110.3 µg/L HMMA, 6.7 µg/L MDA, 4.1 µg/L HMA, and no detectable MDEA. Merged ion chromatograms demonstrating 2D separation of analytes are shown in Fig. 3 . Results met all identification and quantification criteria.


Figure 3
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  		Figure 3. Merged extracted ion chromatogram (target ion) of a plasma specimen collected 7 h after the administration of 105.7 mg MDMA.

Specimen contains 4.1 µg/L of HMA (A); 110.3 µg/L HMMA and 6.7 µg/L MDA (B); and 80.9 µg/L MDMA (C).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Two-dimensional GC/MS proved an excellent methodology for analysis of MDEA, MDMA, and metabolites in human plasma. Low LOQs were achieved, with stable ion ratios across extended linear dynamic ranges. Inlet liner changes were the only routine maintenance required.

2D-GC offers greater separation capability than standard 1-dimensional gas chromatography. The 2D capillary chromatography system, in combination with cryofocusing and mass spectrometry selectivity, proved to be an effective tool for selective analyte detection and quantification. Choice of column phases, column dimensions, linear velocity, and temperature parameters provides flexible opportunities for resolution of drug analyte from complex matrices such as plasma, blood, and oral fluid. This method should be applicable for quantification of MDMA analytes in matrices other than plasma. Additionally, the method uses relatively inexpensive upgrades to readily available GC/MS hardware and is cost-effective compared with tandem MS instrumentation.

The primary limitation of the method was the inability to include HHMA and HHA. Also, although run time was 18.8 min, resolution of 5 closely related analytes was achieved without interference from exogenous compounds.

To our knowledge, this is the first 2D-GC/MS method for analysis of amines and the first to combine these 5 analytes in 1 human plasma analytical method. Method applicability was demonstrated by analysis of a plasma specimen obtained during controlled MDMA administration. The 2D chromatographic system should be suitable for application to other analytes and to other complex matrices.


   Acknowledgments
 
Grant/funding Support: This research was supported by the Intramural Research Program of the National Institute on Drug Abuse (NIDA/IRP).

Financial Disclosures: None declared.

Acknowledgments: The authors thank Robert Goodwin, David Gorelick, Robert Hayes, Elliot Stein, Thomas Ross, Loretta Spurgeon, and David Darwin.


   Footnotes
 
1 Nonstandard abbreviations: MDMA, 3,4-methylenedioxymethamphetamine (Ecstasy); LSD, lysergic acid diethylamide; HHMA, 3,4-dihydroxymethamphetamine; HMMA, 4-hydroxy-3-methoxymethamphetamine; HMA, 4-hydroxy-3-methoxyamphetamine; MDA, 3,4-methylenedioxyamphetamine; HHA, 3,4-dihydroxyamphetamine; MDEA, 3,4-methylenedioxy-N-ethylamphetamine; SPE, solid-phase extraction; QC, quality control; LOD, limit of detection; LOQ, limit of quantification.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Nichols DE. Differences between the mechanism of action of MDMA, MBDB, and the classic hallucinogens. Identification of a new therapeutic class: entactogens. J Psychoactive Drugs 1986;18:305-313.[Web of Science][Medline] [Order article via Infotrieve]
  2. Greer G, Tolbert R. Subjective reports of the effects of MDMA in a clinical setting. J Psychoactive Drugs 1986;18:319-327.[Web of Science][Medline] [Order article via Infotrieve]
  3. . SAMHSA. Results from the 2005 National Survey on Drug Use and Health: National Findings. Office of Applied Studies. NSDUH Series H-30 2005:1-268 DHHS Rockville, MD. .
  4. . European Monitoring Centre for Drugs and Drug Addiction. The state of the drugs problem in Europe. Annual Report 2006:1-104.
  5. . National Institute on Drug Abuse. Monitoring the future: national results on adolescent drug use 2006:1-72 National Institutes of Health, U.S. Department of Health and Human Services .
  6. . National Household Survey on Drug Abuse. Ecstasy use. The NHSDA Report 2003:1-3.
  7. Schmued LC. Demonstration and localization of neuronal degeneration in the rat forebrain following a single exposure to MDMA. Brain Res 2003;974:127-133.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  8. Heffernan TM, Jarvis H, Rodgers J, Scholey AB, Ling J. Prospective memory, everyday cognitive failure and central executive function in recreational users of Ecstasy. Hum Psychopharmacol 2001;16:607-612.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  9. de la Torre R, Farre M, Roset PN, Lopez CH, Mas M, Ortuno J, et al. Pharmacology of MDMA in humans. Ann N Y Acad Sci 2000;914:225-237.[Web of Science][Medline] [Order article via Infotrieve]
  10. Segura M, Ortuno J, Farre M, McLure JA, Pujadas M, Pizarro N, et al. 3,4-Dihydroxymethamphetamine (HHMA): a major in vivo 3,4-methylenedioxymethamphetamine (MDMA) metabolite in humans. Chem Res Toxicol 2001;14:1203-1208.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  11. Peters FT, Schaefer S, Staack RF, Kraemer T, Maurer HH. Screening for and validated quantification of amphetamines and of amphetamine- and piperazine-derived designer drugs in human blood plasma by gas chromatography/mass spectrometry. J Mass Spectrom 2003;38:659-676.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  12. Peters FT, Samyn N, Lamers CT, Riedel WJ, Kraemer T, de Boeck G, Maurer HH. Drug testing in blood: validated negative-ion chemical ionization gas chromatographic-mass spectrometric assay for enantioselective measurement of the designer drugs MDEA, MDMA, and MDA and its application to samples from a controlled study with MDMA. Clin Chem 2005;51:1811-1822.[Abstract/Free Full Text]
  13. Samyn N, De Boeck G, Wood M, Lamers CTJ, De Waard D, Brookhuis KA, et al. Plasma, oral fluid and sweat wipe concentrations in controlled and real life conditions. Forensic Sci Int 2002;128:90-97.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  14. Fallon JK, Kicman AT, Henry JA, Milligan PJ, Cowan DA, Hutt AJ. Stereospecific analysis and enantiomeric disposition of 3,4-methylenedioxymethamphetamine (Ecstasy) in humans. Clin Chem 1999;45:1058-1069.[Abstract/Free Full Text]
  15. Murphy PN, Fisk JE, Wareing M. Ecstasy: sorting out the facts. Psychologist 2002;:15.
  16. Cole JC, Bailey M, Sumnall HR, Wagstaff GF, King LA. The content of Ecstasy tablets: implications for the study of their long-term effects. Addiction 2002;97:1531-1536.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  17. Pham JV, Puzantian T. Ecstasy: dangers and controversies. Pharmacotherapy 2001;21:1561-1565.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  18. Baggott M, Heifets B, Jones RT, Mendelson J, Sferios E, Zehnder J. Chem analysis of Ecstasy pills. JAMA 2000;284:2190.[Free Full Text]
  19. Venkatramani CJ, Phillips JB. Comprehensive two-dimensional gas chromatography applied to the analysis of complex mixture. J Microcolumn Sep 1993;5:511-516.[CrossRef][Web of Science]
  20. Moore C, Coulter C, Rana S, Vincent M, Soares J. Analytical procedure for the determination of the marijuana metabolite 11-nor-{Delta}9-tetrahydrocannabinol-9-carboxylic acid in oral fluid specimens. J Anal Toxicol 2006;30:409-412.[Web of Science][Medline] [Order article via Infotrieve]
  21. Moore C, Rana S, Coulter C, Feyerherm F, Prest H. Application of two-dimensional gas chromatography with electron capture chemical ionization mass spectrometry to the detection of 11-nor-{Delta}9-tetrahydrocannabinol-9-carboxylic acid (THC-COOH) in hair. J Anal Toxicol 2006;30:171-177.[Web of Science][Medline] [Order article via Infotrieve]
  22. Fritch DF, Quimby BD. Confirmation of THC in oral fluids using high-resolution 2-D GC/MS. Agilent Technologies. Application Note 2006;:1-10.
  23. Scurlock RD, Ohlson GB, Worthen DK. The detection of {Delta}9-tetrahydrocannabinol (THC) and 11-nor-9-carboxy-{Delta}9-tetrahydrocannabinol (THCA) in whole blood using two-dimensional gas chromatography and EI-mass spectrometry. J Anal Toxicol 2006;30:262-266.[Web of Science][Medline] [Order article via Infotrieve]
  24. Lowe RH, Karschner EL, Schwilke EW, Barnes AJ, Huestis MA. Simultaneous quantification of {Delta}-9-tetrahydrocannabinol (THC), 11-hydroxy-{Delta}-9-tetrahydrocannabinol (11-OH-THC), and 11-nor-{Delta}-9-tetrahydrocannabinol-9-carboxylic acid (THCCOOH) in human plasma using two-dimensional gas chromatography, cryofocusing, and electron impact-mass spectrometry. J Chromatogr A 2007;1163:318-327.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  25. Hinshaw JV. Comprehensive two-dimensional gas chromatography. GC Connect 2004;:1-7.
  26. Scheidweiler KB, Huestis MA. A validated gas chromatographic-electron impact ionization mass spectrometric method for methylenedioxymethamphetamine (MDMA), methamphetamine and metabolites in oral fluid. J Chromatogr B 2006;835:90-99.[CrossRef]
  27. Helmlin H, Bracher K, Bourquin D, Vonlanthen D, Brenneisen R. Analysis of 3,4-methylenedioxymethamphetamine (MDMA) and its metabolites in plasma and urine by HPLC-DAD and GC-MS. J Anal Toxicol 1996;20:432-440.[Web of Science][Medline] [Order article via Infotrieve]
  28. Pirnay SO, Abraham TT, Lowe RH, Huestis MA. Selection and optimization of hydrolysis conditions for the quantification of urinary metabolites of MDMA. J Anal Toxicol 2006;30:563-569.[Web of Science][Medline] [Order article via Infotrieve]
  29. Parrott AC. MDMA in humans: factors which affect the neuropsychobiological profiles of recreational Ecstasy users, the integrative role of bioenergetic stress. J Psychopharmacol (Oxf) 2006;20:147-163.[CrossRef]




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