BIOMEDICAL CHROMATOGRAPHY Biomed. Chromatogr.

20: 13801385 (2006) Published online 2 November 2006 in Wiley InterScience 1380 ORIGINAL RESEARCH (www.interscience.wiley.com) DOI: 10.1002/bmc.716

M. Tomita et al. ORIGINAL RESEARCH

A simple and sensitive HPLC-uorescence method for quantication of MDMA and MDA in blood with 4-(4,5-diphenyl-1H-imidazol-2-yl)benzoyl chloride (DIB-Cl) as a label
Mamoru Tomita,1,2 Mihoko N. Nakashima,1 Mitsuhiro Wada1 and Kenichiro Nakashima1*
1

Department of Clinical Pharmacy, Course of Pharmaceutical Sciences, Graduate School of Biomedical Sciences, Nagasaki University, 1-14 Bunkyomachi, Nagasaki 852-8521, Japan 2 Forensic Science Laboratory, Nagasaki Prefectural Police Headquarters, 4-8 Manzai-machi, Nagasaki 850-8548, Japan Received 25 May 2006; revised 14 June 2006; accepted 14 June 2006

ABSTRACT: A sensitive high-performance liquid chromatographic method with uorescence detection to determine 3,4methylenedioxymethamphethamine (MDMA) and 3,4-methylenedioxyamphethamine (MDA) in human and rat whole blood or plasma samples was developed by using 4-(4,5-diphenyl-1H-imidazol-2-yl)benzoyl chloride (DIB-Cl) as a label. MDMA and MDA in a small amount of blood sample (ca 100 L) were extracted by liquidliquid extraction with ethyl acetate, and were derivatized with DIB-Cl under mild conditions (10 min at room temperature). A good separation of DIB-derivatives could be achieved within 45 min using a commercially available ODS column with an isocratic eluent of 10 mM citric acid20 mM Na2HPO4 aqueous buffer (pH 4.0)CH3CNCH3OH (50:45:5, v/v/v %). The calibration curves prepared with 1-methyl-3-phenylpropylamine (MPPA) as an internal standard showed good linearity (r = 0.999) with 0.360.83 ng/mL detection limit at a signal-to-noise ratio of 3. MDMA and MDA in rat whole blood could be monitored for 6 h after a single administration of MDMA (2.2 mg/kg, i.p.). The pharmacokinetic parameters for MDMA and MDA obtained by triplicate measurements were 426 23 and 39 6 ng/mL (Cmax), 20 5 and 100 10 min (Tmax), respectively. Copyright 2006 John Wiley & Sons, Ltd. KEYWORDS: 3,4-methylenedioxymethamphethamine (MDMA); 3,4-methylenedioxyamphethamine (MDA); HPLC with uorescence detection; 4-(4,5-diphenyl-1H-imidazol-2-yl)benzoyl chloride (DIB-Cl); blood

INTRODUCTION
Recently the number of drug conscations of 3,4-methylenedioxymethamphethamine (MDMA) and/or 3,4-methylenedioxyamphethamine (MDA), amphetamine-type stimulants (ATS), has been increasing remarkably worldwide. Moreover, the ATS tablets are especially abused by young people because of fashion and simplicity of ingestion (Katagi and Tsuchihashi, 2002; Makino et al., 2003; Christophersen, 2000). In addition to MDMA and MDA, the ATS tablets include some components such as 3,4-methylenedioxyethylamphethamine (MDEA),

*Correspondence to: K. Nakashima, Department of Clinical Pharmacy, Course of Pharmaceutical Sciences, Graduate School of Biomedical Sciences, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki 852-8521, Japan. E-mail: naka-ken@nagasaki-u.ac.jp Abbreviations used: ATS, amphetamine-type stimulants; DIB-Cl, 4-(4,5-diphenyl-1H-imidazol-2-yl)benzoyl chloride; MA, methamphetamine; MDA, 3,4-methylenedioxyamphethamine; MDEA, 3,4-methylenedioxyethylamphethamine; MDMA, 3,4methylenedioxymethamphethamine. Copyright 2006 John Wiley & Sons, Ltd.

methamphetamine (MA), ketamine, ephedrine and caffeine (Katagi and Tsuchihashi, 2002; Makino et al., 2003). Some compounds may be present as impurities in formulation, while others may be added with the expectation of additional effects. The regular use of these tablets has potential for multi-drug abuse. Therefore, pharmacokinetic and pharmacodynamic studies of MDMA in the case of concomitant use of these compounds are very important as well as in forensic studies. Moreover, small amounts of various forms of blood samples such as whole blood, plasma, serum and clotted blood are handled in the eld of forensic toxicology, so a versatile, sensitive and selective determination method for MDMA and its metabolite MDA in various types of blood samples is required for forensic analysis. The determination of MDMA and MDA in blood has usually been performed by gas chromatography with nitrogen phosphorus detection (GC-NPD; Ortuo et al., 1999) and gas chromatography-mass spectrometry (GC-MS; Peters et al., 2003). These are sensitive and powerful techniques and thus have been used in forensic analysis. However, these methods require a large amount of plasma (1 mL). HPLC methods with UV
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and uorescence (FL) detection have been used for the determination of MDMA and MDA (Soares et al., 2004; Sadeghipour and Veuthy, 1997). Although the HPLC-UV method is simple, this is less sensitive and specic for biological samples (Soares et al., 2004). The sensitivity of the HPLC-FL method with weak native uorescence of MDMA and MDA was relatively low (Sadeghipour and Veuthy, 1997). To improve the sensitivity, HPLC-FL methods with uorescence labeling have been used. Among the uorescent labeling reagents, 4-(4,5-diphenyl-1Himidazol-2-yl)benzoyl chloride (DIB-Cl) gives intense uorescence derivatives, which react with compounds having a primary or secondary amino group and phenol group. The sensitive determination methods of MA and fenuramine in plasma and hair samples have been reported (Nakashima et al., 2003; Kaddoumi et al., 2001). In this study, a simple and sensitive HPLC-FL method for the simultaneous determination of MDMA and its metabolite MDA in blood samples was developed using DIB-Cl as a uorescent label. The derivatization reaction of MDA and MDMA with DIBCl is shown in Fig. 1. Furthermore, following a single administration of MDMA to rats, blood samples were monitored for MDMA and its metabolite MDA to estimate the compounds pharmacokinetic parameters.

6 m, Wako Pure Chemical Ind.), an 821-FP uorescence detector set at 330 (ex) and 440 (em) nm (Jasco, Tokyo), and a U-228-2P-500 recorder (Pantos, Kyoto, Japan). DIBderivatives were separated isocratically with 10 mM citric acid20 mM Na2HPO4 buffer (pH 4.0)CH3CNCH3OH (50:45:5, v/v/v %). The ow rate of the mobile phase was set at 1.5 mL/min. Pretreatment of blood samples. To 100 L of blood samples (human whole blood, human plasma and rat whole blood), 10 L of IS (200 or 2000 ng/mL) and 200 L of borate buffer (100 mM, pH 10.0) were added. MDMA, MDA and IS were extracted with 1.0 mL of ethyl acetate by vortex-mixing. After centrifugation (2000g) for 10 min at 6C, the organic layer (0.8 mL) was spiked with 5 L of acetic acid, mixed and evaporated to dryness with N2 gas. The residue was applied to derivatization with DIB-Cl. Human whole blood and plasma were obtained from healthy volunteers, with the approval of the Ethics Committee of School of Pharmaceutical Sciences, Nagasaki University (no. 001). Derivatization with DIB-Cl. To the residue, described above, 10 L of 10 mM carbonate buffer (pH 9.0) and 180 L of 0.1 mM DIB-Cl in CH3CN were added, vortex-mixed and then allowed to stand at room temperature for 10 min. The reaction was stopped by adding 10 L of aqueous ammonia (25%). The resulting solution was applied to the HPLC system. Method validation. The validation of the proposed method was performed using human whole blood, human plasma and rat whole blood spiked with known concentrations of standard MDMA and MDA. Calibration curves were prepared by an internal standard method using the peak height ratio of MDMA and MDA to IS. The detection limits of MDMA and MDA were calculated as the peak height at a signal-to-noise (S/N) ratio of 3. The repeatability of intra- and inter-day assays was assessed by triplicate measurements of samples. The precision was calculated as the relative standard deviation (RSD). The reliability of the proposed method was examined. Plasma samples spiked with standard MDA and MDMA ranging from 1 to 60 ng/mL (MDA) and from 2 to 120 ng/mL (MDMA) were prepared blindly by another laboratory worker. The results obtained by the proposed method were compared with those of the GC-MS method involving solid-phase extraction reported by Ortuo et al. (1999) and derivatization with triuoroacetic anhydride. Monitoring of MDMA and MDA in rat blood. Male Wistar rats (290340 g, Otsubo Experimental Animals,

MATERIALS AND METHODS

Materials. MDMA hydrochloride, MDA hydrochloride and MDMA tablets were obtained as narcotics for research from the Ministry of Health, Labour and Welfare (Tokyo, Japan). MPPA was obtained from Aldrich Chemical Company (Milwaukee WI, USA). DIB-Cl was synthesized in our laboratory (Nakashima et al., 1995). Stock solutions of standards were prepared at 1 mg/mL with CH3OH, and stored at 20C. Ethyl acetate, CH3CN and CH3OH were obtained from Wako Pure Chemical Ind. (Osaka, Japan). Other reagents used were of analytical reagent grade. Water was deionized and distilled by an Aquarius GSR-500 automatic water distillation apparatus (Advantec, Tokyo). Apparatus. An HPLC system consisted of a CCPM liquid chromatographic pump (Tosoh, Tokyo), a Rheodyne 7125 injector with a 20 L sample loop (Rheodyne, Cotati, CA, USA), a Wakopak Handy ODS column (150 4.6 mm, i.d.,

Figure 1. Derivatization reaction of MDA and MDMA with DIB-Cl.

Copyright 2006 John Wiley & Sons, Ltd. Biomed. Chromatogr. 20: 13801385 (2006) DOI: 10.1002/bmc

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Nagasaki, Japan) were used for experiments. Rats were anaesthetized with ethyl carbamate (1.5 g/kg) before 1 h of sampling. MDMA tablets were crushed and suspended in saline. After ltration with a membrane lter the solution was administered to rats at 2.2 mg/kg MDMA (i.p.). Blood samples were collected via arteria femoralis in tubes containing EDTA. Sampling was performed at 5, 15, 30, 45, 60, 90, 120, 180, 240 and 360 min after administration. All animal procedures and care in this experiment were permitted by Nagasaki University Animal Care and Use Committee (no. 0503290416).

RESULTS AND DISCUSSION

The separation of DIB-MDA and MDMA in blood samples was performed by an ODS column with a mobile phase of 10 mM citric acid20 mM Na2HPO4 buffer (pH 4.0)CH3CNCH3OH (50:45:5, v/v/v %). The retention times of MDA, MDMA and IS were 18.2, 22.2 and 31.4 min, respectively. Typical chromatograms of control human blood sample (A) and sample spiked with standards (B) are shown in Fig. 2. Method validation Calibration curves for MDA and MDMA were prepared with human and rat blood samples spiked with known concentrations of standards. Good linear relationships between the concentration and peak height ratio were obtained up to 250 (MDA) and 500 (MDMA) ng/mL. The correlation coefcients (r) of MDA and MDMA were larger than 0.999. Parameters of calibration curves for the proposed method are summarized in Table 1. The detection limits for MDA and MDMA at a S/N ratio of 3 were 0.50 ng/mL (5.0 pg on column) and 0.75 ng/mL (7.5 pg on column), human whole blood and plasma, respectively. The sensitivity for MDMA and MDA in blood samples by the proposed method is a few times higher than that using native uorescence detection (Sadeghipour and Veuthy, 1997) and GC-NPD (Ortuo et al., 1999). Moreover,

Figure 2. Chromatograms of (A) human blank blood and (B) spiked blood with 25 ng/mL for MDA, 50 ng/mL for MDMA, and 20 ng/mL for IS.

MDMA and MDA in human whole blood could be sensitively determined at the same level of plasma without any extra treatment by the proposed method. The intra- and inter-day repeatability of the proposed method was assessed using blood samples spiked

Table 1. Calibration curve and detection limits for the proposed method Concentration (ng/mL) Human whole blood MDA MDMA Human plasma MDA MDMA Rat whole blood MDA MDMA
a

Regression equationa y = 0.065x 0.10 y = 0.022x 0.04 y = 0.066x 0.09 y = 0.022x 0.03 y = 0.067x 0.11 y = 0.022x 0.03

Detection limitsc (ng/mL) (pg/inj.) 0.50 0.75 0.50 0.75 0.36 0.83 5.0 7.5 5.0 7.5 3.6 8.3

1.0250 2.0500 1.0250 2.0500 5.0250 10.0500

b

0.999 0.999 0.999 0.999 0.999 0.999

x, sample concentration, ng/mL; y, peakIS ratio;

r = correlation coefcient; c detection limit at S/N ratio = 3. Biomed. Chromatogr. 20: 13801385 (2006) DOI: 10.1002/bmc

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Table 2. Repeatability and recovery for the proposed method Spiked concentration (ng/mL) Human blood MDA MDMA Human plasma MDA MDMA Rat blood MDA MDMA 5 100 10 200 5 100 10 200 5 100 10 200 RSD (%) (n = 3) Intra-day 1.9 2.8 5.4 4.0 2.5 2.0 12.6 1.2 Inter-day 9.5 5.8 9.9 8.9 6.3 1.4 16.6 2.4 5.1 1.7 11.5 8.7 Recovery (%) 93 96 99 96 102 93 99 90 100 105 119 109

Figure 3. Correlation between concentrations of (A) MDA and (B) MDMA determined by GC-MS and HPLC-FL methods (n = 7).

with MDA and MDMA standards. Table 2 shows precision expressed as RSD and recovery of spiked MDA and MDMA for the proposed method. For intraday assay, RSD ranged from 1.2 to 12.6% (n = 3), while the inter-day assay RSD ranged from 1.4 to 16.6% (n = 3). The recoveries obtained for MDA and MDMA ranged from 90 to 119%. By using an IS method and a simple liquidliquid extraction, reproducible and precise measurement could be accomplished. Figure 3 shows correlation between the analysis data obtained by the proposed method and the GC-MS method for the same plasma samples. Correlation coefcients between the concentrations of MDA and MDMA determined by our method and the GC-MS method were 0.987 and 0.997, respectively. Moreover the factors of MDA and MDMA between two methods were 0.97 and 0.91, respectively. These results show that the proposed method is reliable for determination of MDA and MDMA in plasma samples. Furthermore,
Copyright 2006 John Wiley & Sons, Ltd.

this method enables quantication of MDA and MDMA using only 100 L of sample, while the GC-MS method needs 1000 L of plasma sample. It is advantageous for MDA and MDMA monitoring in small animals due to the reduction of blood sample volume. Determination of MDMA and MDA in rat blood To evaluate the applicability of the method, MDMA and its metabolite MDA in rat blood were monitored after a single administration of MDMA (2.2 mg/kg, i.p.). Figure 4 shows typical chromatograms obtained from rat whole blood, before (A) and after (B) 180 min of MDMA administration. The peaks of MDA and MDMA represent 17.0 and 22.6 ng/mL, respectively. Figure 5 shows the time course of MDMA and MDA concentrations in rat blood after a single administration of MDMA. The concentrations for MDMA and MDA ranged from 6.5 to 417 and from 2.5 to
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Table 3. Pharmacokinetic parameters of MDMA and MDA in rat blood after a single i.p. administration of MDMA (2.2 mg/kg) Pharmacokinetic parameters Cmax (ng/mL) Tmax (min) T1/2 (min) AUC0360 (g min/mL) MRT0360 (min) CL (mL/min/kg) MDMA (n = 3) (mean SEM) 427 20 57 38 75 58 23 5 4 4 8 7 MDA (n = 3) (mean SEM) 39 6 100 10 90 12 7.8 1.5 146 7

Figure 4. Chromatograms of rat blood samples. Sample, (A) control blood before administration and (B) blood at 180 min after MDMA administration to rat.

38.5 ng/mL, respectively. Both compounds could still be detected after 360 min of administration. Pharmacokinetic parameters for MDMA and MDA are listed in Table 3. The peak concentration (Cmax) of MDMA was 427 23 ng/mL. The concentration peak time (Tmax) and the elimination half-life (T1/2) of MDMA were 20 5 and 57 4 min, respectively. Kikura et al. (1997) reported that the concentration of MDMA in dark agouti rat plasma after administration of MDMA was 5 mg/kg, i.p., the average Cmax of MDMA was more than 1000 ng/mL 15 min after administration and the T1/2 of MDMA in the plasma was 69.4 9.3 min. These results are in good agreement with those of our study. In conclusion, the HPLC-FL detection with DIBlabeling successfully demonstrated the determination of MDMA and its metabolite MDA. MDMA and MDA in various blood samples (human whole blood, human plasma and rat whole blood) could be sensitively

Figure 5. MDMA and MDA concentration-time proles in rat blood after a single administration of MDMA. Dose, 2.2 mg/kg; data are expressed as mean SEM (n = 3).
Copyright 2006 John Wiley & Sons, Ltd. Biomed. Chromatogr. 20: 13801385 (2006) DOI: 10.1002/bmc

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determined using a small amount of sample with a simple liquidliquid extraction. The proposed method might be a powerful tool for pharmacokinetic interaction studies of MDMA with other drugs in small animals.

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Copyright 2006 John Wiley & Sons, Ltd.

Biomed. Chromatogr. 20: 13801385 (2006) DOI: 10.1002/bmc