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Quantitative analysis of mephedrone using liquid chromatography tandem mass spectroscopy: Application to human hair
Journal of Pharmaceutical and Biomedical Analysis 61 (2012) 64–69
Contents lists available at SciVerse ScienceDirect
Journal of Pharmaceutical and Biomedical Analysis journal homepage: www.elsevier.com/locate/jpba
Quantitative analysis of mephedrone using liquid chromatography tandem mass spectroscopy: Application to human hair Syeda A.B. Shah a , Nawed I.K. Deshmukh a , James Barker a,∗ , Andrea Petróczi b , Paul Cross c , Roland Archer a , Declan P. Naughton b a
School of Pharmacy and Chemistry, Kingston University, London, UK School of Life Sciences, Kingston University, London, UK c School of the Environment, Natural Resources and Geography, Bangor University, UK b
a r t i c l e
i n f o
Article history: Received 12 October 2011 Received in revised form 10 November 2011 Accepted 24 November 2011 Available online 3 December 2011 Keywords: Mephedrone Metabolites 4-Methylephedrine 4-Methylnorephedrine: LC–MS/MS
a b s t r a c t Recent abuse of designer drugs such as mephedrone has presented a requirement for sensitive, reliable and reproducible methods for the detection of these controlled drugs in different matrices. This study focuses on a fully developed validated method for the quantitative analysis of mephedrone and its two metabolites 4-methylephedrine and 4-methylnorephedrine in human hair. The calibration curve was found to be linear in the range 5–100 pg/mg for mephedrone and 10–150 pg/mg for 4-methylephedrine and 4-methylnorephedrine. The method was successfully validated for the intraday precision, interday precision, limit of detection, accuracy and extraction recovery. Five out of 154 hair samples were confirmed to be positive for mephedrone. Due to the structural similarities to other methcathinones and amphetamines, one can propose the metabolism for mephedrone based on a similar pathway that has been previously used for these psychoactive drugs. The outlined method can be valuable for the future detection of mephedrone and its two metabolites in hair. © 2011 Elsevier B.V. All rights reserved.
1. Introduction ‘Legal high’ is a term given to chemicals which possess psychoactive activity in humans, but are not subject to prohibition by law [1]. In the last few years, buying legal highs from the internet has become common practice for drug users. This is, in part, due to their structural similarity to controlled psychoactive substances such as phenethylamines and cathinones [2–4]. Mephedrone, sometimes referred to as ‘meow’, ‘mcat’ and ‘bubbles’ is a -ketoamphetamine and has a structure similar to methcathinone, which in turn is the N-methyl analogue of the natural product cathinone [2]. Cathinone can be extracted from the leaves of an African and Middle Eastern plant Catha edulis or ‘Khat’ [4,5]. The alkaloid content of the fresh leaves is responsible for the psychoactive stimulating effects [4]. Mephedrone has been routinely sold on the internet as plant feed, with an accompanied warning of ‘not suitable for human consumption’. However, in April 2010 it was specifically named and hence controlled in UK under the Misuse of Drugs Act 1971 as a
∗ Corresponding author. Tel.: +44 020 8547 7981; fax: +44 020 8417 7562. E-mail addresses: [email protected] (S.A.B. Shah), [email protected] (N.I.K. Deshmukh), [email protected] (J. Barker), [email protected] (A. Petróczi), [email protected] (P. Cross), [email protected] (R. Archer), [email protected] (D.P. Naughton). 0731-7085/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.jpba.2011.11.026
Class B substance [1,6]. It has also been banned in the majority of other European countries [2]. The use of mephedrone is more common in males than females and it is reported to be one of the most commonly abused psychotropic drugs [1]. The popularity of use of these psychoactive drugs has created a demand for sensitive, robust and reliable analytical methods for their identification and quantification in different matrices. Gas chromatography mass spectrometry (GC–MS) has routinely been used for the detection of mephedrone in matrices such as blood and urine [3,6,7]. Recently, liquid chromatography mass spectroscopy (LC–MS) has also been employed for analysing blood samples for the detection of mephedrone [4]. Compared to urine and blood, hair samples can provide information on the long term history of the individual’s intake of controlled drugs depending on the pharmacokinetic properties of the drug in question [8,9]. Also, blood and urine samples require storage at −20 ◦ C in the presence of preservatives until analysis, for mephedrone [3]. Thus, hair is gaining potential interest for the detection of parent drugs in doping control [8]. The drugs bind to melanin which is the major component of hair [8]. Generally, urinalysis is carried out to determine the presence of metabolites of the controlled drugs and it is often difficult to detect the parent drug in urine. In addition, the matrix effects for urine and blood are much greater than hair [8]. In the scientific literature, there is limited information available on the metabolism of mephedrone in humans. As the structure of
S.A.B. Shah et al. / Journal of Pharmaceutical and Biomedical Analysis 61 (2012) 64–69
O
Table 1 Chromatography conditions for the separation of the compounds.
O
LC run time (min)
HN
Mephedrone OH
HN
4-methylephedrine
65
HN
CD3
Mephedrone – d3
0 3 4 5 10
Acetonitrile (%) 65 100 100 65 65
Water (%) 35 0 0 35 35
OH
NH2
4-methylnorephedrine
Fig. 1. Structures of mephedrone, 4-methylephedrine, 4-methylnorephedrine and mephedrone-d3 (internal standard).
mephedrone is substantially similar to other psychoactive drugs such as methcathinone and methamphetamine, one can tentatively propose a metabolism of mephedrone on a similar basis. There are a number of different pathways by which this drug could be metabolised. The two metabolites analysed in this study are 4-methylephedrine and 4-methylnorephedrine [7,10–13]. The structures of mephedrone and its two metabolites are depicted in Fig. 1. The aim of this study was to develop an analytical method for the quantitative analysis of mephedrone and for two of its metabolites 4-methylephedrine and 4-methylnorephedrine in hair using LC–MS/MS. 2. Materials and methods 2.1. Hair specimen Following ethical approval from two higher education institutions, hair samples were obtained from 154 healthy volunteers (95 male, 59 females) aged 18–56 years old [14]. Blank hair samples were obtained from two healthy individuals. 2.2. Chemicals and consumables HPLC grade methanol, acetonitrile, water, and hexane, along with reagent grade ethyl acetate, ethanol, dichloromethane, chloroform and formic acid were purchased from Fisher Scientific UK Ltd. (Loughborough, UK). Tris HCl buffer and Cleland’s reagent were obtained from VWR (Lutterworth, UK). Proteinase K was purchased from Sigma Aldrich (Dorset, UK). The metabolites 4-methylephedrine hydrochloride and 4-methylnorephedrine hydrochloride were synthesised in-house by borohydride reduction of mephedrone and 4-methylcathinone, respectively, at Kingston University (London, UK). Mephedrone hydrochloride and mephedrone-d3 hydrochloride (internal standard) were purchased from LGC standards (Teddington, UK). An Agilent SB-C18 column (2.1 m × 150 mm × 1.8 m) was purchased from Agilent Technologies UK Ltd. (Wokingham, UK). 2.3. Instrumentation Analyses were performed on a Thermoscientific liquid chromatography–tandem mass spectrometry (LC–MS/MS) system consisted of an Accela UPLC system (Thermo Scientific, UK Ltd., Loughborough, Leicestershire, UK) coupled to a Triple Quadrupole TSQTM mass spectrometer (Thermo Electron Corp., UK). This was operated in selective reaction monitoring (SRM) mode using the
conditions given in Table 1 with detection parameters given in Table 2. The column was maintained at 50 ◦ C and the auto sampler tray was set at 10 ◦ C. The flow rate was 200 L/min and the injection volume was 3.0 L. The capillary temperature was maintained at 300 ◦ C and a voltage of 4000 V was employed. The collision pressure was kept at 1.5 mTorr. The fragmentation pathways of all the analytes are shown in Fig. 2. 2.4. Hair digestion and extraction 2.4.1. Decontamination The hair samples were decontaminated by rinsing with 2 mL dichloromethane for 2 min (twice) at room temperature. Decontamination ensured the removal of contaminants from the surface of hair, which may interfere with the analysis [15]. After decontamination, the hair samples were air dried before cutting into very fine segments (ca. 1 mm length) using scissors. 2.5. Digestion Initially, hair digestion was carried out by incubating the hair samples with 1 mL of 1 M sodium hydroxide solution at 95 ◦ C for 10 min. However, this led to the complete degradation of analytes. Hence, enzymatic digestion was employed alternatively. Enzymatic digestion was carried out by placing the fine hair segments (50 mg) in a glass vial containing Cleland’s reagent (100 mg) and enzyme proteinase K (15 mg). The internal standard mephedroned3 (100 L) at 40 pg/mg was added to the mixture. The mixture was finally incubated with Tris buffer (1 mL) for 2 h at 37.5 ◦ C with constant stirring. 2.6. Extraction Liquid–liquid extraction was optimised for mephedrone using hexane (3 mL), and for the metabolites using a mixture of chloroform, ethanol and diethyl ether (3 mL in total) with the ratio of 3:1:1. The contents were mixed using a vortex mixer and centrifuged at 5 ◦ C for 5 min at 1750 × g. The organic layers were separated and pooled together in a glass tube. The organic layer was then dried using nitrogen gas and reconstituted with 200 L of acetonitrile. 2.7. Method validation Method validation was carried out in order to establish the sensitivity, specificity, selectivity, linearity, limit of detection (LOD), limit of quantification (LOQ), inter and intraday precision and accuracy and percentage recoveries. The metabolites when diluted in methanol gave better sensitivity as compared to acetonitrile, whereas mephedrone gave a similar sensitivity in both solvents. A calibration curve was obtained by spiking blank hair samples with known concentrations of the drugs in the range of 5–100 pg/mg for mephedrone and 10–150 pg/mg for 4-methylephedrine and 4-methylnorephedrine. The internal
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S.A.B. Shah et al. / Journal of Pharmaceutical and Biomedical Analysis 61 (2012) 64–69
Fig. 2. LC–MS/MS chromatogram and CID spectra of (a) mephedrone, (b) 4-methylephedrine and (c) 4-methylnorephedrine in hair at concentrations of 10 pg/mg, 20 pg/mg and 20 pg/mg, respectively.
S.A.B. Shah et al. / Journal of Pharmaceutical and Biomedical Analysis 61 (2012) 64–69
67
Table 2 Ion transitions, retention time (RT) and collision energies used for the analysis of the analytes and the internal standard. Compound
RT (min)
Precursor ions (m/z)
Product ions (m/z)
Collision energy (eV)
Mephedrone
2.07
178.1
4-Methylephedrine
1.93
180.2
4-Methylnorephedrine
1.85
166.2
Mephedrone-d3
1.98
181.2
160.1 145.1 147.2 131.3 115.2 131.2 115.2 90.8 163.2 148.2
10 18 19 18 39 14 33 24 17 20
Table 3 Precision and accuracy for intraday and interday of the three drugs at four different concentrations. Compound
Concentration (pg/mg)
Precision RSD (%)
5 10 20 40 10 20 50 100 10 20 50 100
Mephedrone
4-Methylephedrine
4-Methylnorephedrine
Accuracy %
Intraday N=6+6+6+6
Interday N = 18 + 18 + 18 + 18
0.62 0.4 2.11 2.56 0.6 0.75 0.8 4.92 0.54 0.2 2.91 6.97
0.8 0.7 1.7 4.5 0.9 0.7 4 3.4 1.1 0.7 2 7.3
99.15 106.34 103.16 107.53 114.59 110.76 107.02 98.52 107.98 106.51 97.6 100.67
Table 4 Extraction recoveries of the three drugs at three different concentrations. Compound
Concentration (pg/mg)
Recovery (%) N = 6 + 6 + 6
Regression coefficient
Regression equation
Mephedrone
5 20 40 10 20 100 10 20 100
90.36 110.37 108.10 106.16 98.37 101.87 100.23 96.78 101.32
0.999
y = 0.112x + 0.429
0.990
y = 0.012x + 0.033
0.990
y = 0.008x + 1.389
4-Methylephedrine
4-Methylnorephedrine
standard mephedrone-d3 (100 L) was added to all the standards with a final concentration of 40 pg/mg. The extraction recovery was validated for mephedrone and its metabolites at three different concentrations using six replicates by comparing the extracted samples with non-extracted samples. 3. Results and discussion 3.1. Validation results There were no chromatographic interferences found due to the endogenous substances present in the blank (control) hair that was analysed using the above chromatographic conditions. The precision, accuracy and recoveries lie within the limits set by the FDA guidelines [16]. The validation data are given in Tables 3–5. Table 5 Signal to noise ratios for the LOD and LOQ. Compound
S/N for LOD N = 6
S/N for LOQ N = 6
Mephedrone 4-Methylephedrine 4-Methylnorephedrine
7 5 4
11 10 10
LOD and LOQ values for mephedrone were 2.5 pg/mg and 5 pg/mg, respectively. For 4-methylephedrine and 4methylnorephedrine the LOD was 5 pg/mg and the LOQ 10 pg/mg. Out of the 154 samples analysed, mephedrone was detected in only five samples. However, the metabolites were not detected in any of the samples analysed. However, owing to the method used for data and sample collection [14], it is impossible to link admission of mephedrone use to individuals, or individual samples. Out of the five samples, mephedrone could be successfully quantified in only one sample. It was found to be present at a concentration of 21.11 pg/mg. Fig. 3 shows the chromatogram and mass spectra of the quantified positive sample. The results confirm that the LC–MS/MS method developed for mephedrone and its two metabolites is sensitive and robust. There was no significant change in column performance or the pressure after making several injections. Only 50 mg hair is required for the detection of the mephedrone and its metabolites. Hair analysis is capable of providing a long term history of drug use and any kind of hair treatment such as bleaching cannot affect the incorporation of the drug into hair. Thus, it can be used to compliment urinalysis for identifying routine drug users [17]. In this study, the metabolites could not be detected in the hair samples. In general, high polarity
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S.A.B. Shah et al. / Journal of Pharmaceutical and Biomedical Analysis 61 (2012) 64–69
Fig. 3. LC–MS/MS chromatogram and CID spectra of the positive samples at concentrations of 21.11 pg/mg.
metabolites which are destined to be excreted in urine are difficult to detect in hair. In contrast, the parent drugs are lipophilic and can become incorporated into the hair [8]. It should be noted that as mephedrone is not smoked, the route of entry via environmental exposure would not provide a false positive for hair analyses. There has been very little research carried out on the metabolism of mephedrone. Due to the structural similarities of mephedrone to methcathinones and methamphetamines, the metabolism of mephedrone has been proposed to follow a similar pathway. The pathway involves the hydroxylation of the methylene group and the second metabolite is formed by N-demethylation, thus forming a primary amine. However, there is a possibility of other pathways for the metabolism of mephedrone [7,10–13].
4. Conclusion The present study describes a rapid and replicable method for the quantitative analysis of mephedrone and its two metabolites in human hair. The method is quite sensitive with an LOQ of 5 pg/mg for mephedrone and 10 pg/mg for its two metabolites. The absence of any interference on the chromatograms shows that the method is highly selective. The method is suitable for the future detection and quantification of mephedrone and its metabolites.
Author contributions J.B., A.P. and D.P.N. conceived and designed the study. S.A.B.S. performed the analysis. R.A. synthesised and provided the metabolites. P.C. provided samples. S.A.B.S. and N.I.K.D. drafted the paper. J.B., A.P., D.P.N. and S.A.B.S. interpreted the results. All authors contributed to writing, and approved the final manuscript.
Acknowledgements The authors thank Helen Taft and Christiana Adesanwo for their help with collecting hair samples.
References [1] I. Vardakoua, C. Pistos, C.H. Spiliopouloua, Drugs for youth via internet and the example of mephedrone, Toxicol. Lett. 201 (2011) 191–195. [2] E.Y. Santali, A. Cadogan, N. Daeid, K.A. Savage, O.B. Sutcliffe, Synthesis, full chemical characterisation and development of validated methods for the quantification of (±)-4’-methylmethcathinone (mephedrone): a new legal high, J. Pharm. Biomed. Anal. 56 (2011) 246–255. [3] K.J. Lusthof, R. Oosting, A. Maes, M. Verschraagen, A. Dijkhuizen, A.G.A. Sprong, A case of extreme agitation and death after the use of mephedrone in The Netherlands, Forensic Sci. Int. 206 (2011) e93–e95. [4] L.K. Sørensen, Determination of cathinones and related ephedrines in forensic whole-blood samples by liquid-chromatography–electrospray tandem mass spectrometry, J. Chromatogr. B 879 (2011) 727–736. [5] D.M. Wood, D. Davies, M. Puchnarewicz, J. Button, R. Archer, H. Ovaska, J. Ramsey, T. Lee, D.W. Holt, P.I. Dargan, Recreational use of mephedrone (4methylmethcathinone, 4-MMC) with associated sympathomimetic toxicity, J. Med. Toxicol. 6 (2010) 327–330. Torrance, G. Cooper, The detection of mephedrone (4[6] H. methylmethcathinone) in 4 fatalities in Scotland, Forensic Sci. Int. 202 (2010) e62–e63. [7] M.R. Meyer, J. Wilhelm, F.T. Peters, Beta-keto amphetamines: studies on the metabolism of the designer drug mephedrone and toxicological detection of mephedrone, butylone, and methylone in urine using gas chromatography–mass spectroscopy, Anal. Bioanal. Chem. 397 (2010) 1225–1233. [8] N. Deshmukh, I. Hussain, J. Barker, A. Petroczi, D.P. Naughton, Analysis of anabolic steroids in human hair using LC–MS/MS, Steroids 75 (2010) 710–714. [9] P. Kintz, V. Cirimele, V. Demestre-Toulet, B. Ludes, Doping control for nandrolone using hair analysis, J. Pharm. Biomed. Anal. 24 (2001) 1125–1130. [10] H.T. Kamata, N. Shima, K. Zaitsu, T. Kamata, A. Miki, M. Nishikawa, M. Katagi, H. Tsuchihashi, Metabolism of the recently encountered designer drug, methylone, in human and rats, Xenobiotica 36 (2006) 709–723. [11] E. Pawlik, G. Plasser, H. Mahler, Studies on the phase I metabolism of the new designer drug 3-fluoromethcathinone using rabbit liver slices, Int. J. Legal. Med. (2011), doi:10.1007/s00414-011-0601-6.
S.A.B. Shah et al. / Journal of Pharmaceutical and Biomedical Analysis 61 (2012) 64–69 [12] J. Caldwell, L.G. Dring, R.T. Williams, Metabolism of [14 C] methamphetamine in man, the guinea pig and the rat, Biochem. J. 129 (1972) 11–22. [13] D.A. Williams, T.A. Lemke (Eds.), Foye’s Principles of Medicinal Chemistry, 5th ed., Lippincott, Williams & Wilkins, USA, 2002, p. 227. [14] A. Petróczi, T. Nepusz, P. Cross, H. Taft, S. Shah, N. Deshmukh, J. Schaffer, M. Shane, C. Adesanwo, J. Barker, D.P. Naughton, New nonrandomised model to assess the prevalence of discriminating behaviour: a pilot study on mephedrone, Susbst. Abuse Treat. Prev. Policy 6 (2011) , doi:10.1186/1747-597X-6-20.
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[15] F. Musshoff, B. Madea, New trends in hair analysis and scientific demands on validation and technical notes, Forensic Sci. Int. 165 (2007) 204–215. [16] Guidance for Industry Bioanalytical Method Validation, http://www.fda.gov/ downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/ ucm070107.pdf, 2001 (accessed 16.09.11). [17] R.L. DuPont, W.A. Baumgartner, Drug testing by urine and hair analysis: complementary features and scientific issues, Forensic Sci. Int. 70 (1995) 63–76.
Contents lists available at SciVerse ScienceDirect
Journal of Pharmaceutical and Biomedical Analysis journal homepage: www.elsevier.com/locate/jpba
Quantitative analysis of mephedrone using liquid chromatography tandem mass spectroscopy: Application to human hair Syeda A.B. Shah a , Nawed I.K. Deshmukh a , James Barker a,∗ , Andrea Petróczi b , Paul Cross c , Roland Archer a , Declan P. Naughton b a
School of Pharmacy and Chemistry, Kingston University, London, UK School of Life Sciences, Kingston University, London, UK c School of the Environment, Natural Resources and Geography, Bangor University, UK b
a r t i c l e
i n f o
Article history: Received 12 October 2011 Received in revised form 10 November 2011 Accepted 24 November 2011 Available online 3 December 2011 Keywords: Mephedrone Metabolites 4-Methylephedrine 4-Methylnorephedrine: LC–MS/MS
a b s t r a c t Recent abuse of designer drugs such as mephedrone has presented a requirement for sensitive, reliable and reproducible methods for the detection of these controlled drugs in different matrices. This study focuses on a fully developed validated method for the quantitative analysis of mephedrone and its two metabolites 4-methylephedrine and 4-methylnorephedrine in human hair. The calibration curve was found to be linear in the range 5–100 pg/mg for mephedrone and 10–150 pg/mg for 4-methylephedrine and 4-methylnorephedrine. The method was successfully validated for the intraday precision, interday precision, limit of detection, accuracy and extraction recovery. Five out of 154 hair samples were confirmed to be positive for mephedrone. Due to the structural similarities to other methcathinones and amphetamines, one can propose the metabolism for mephedrone based on a similar pathway that has been previously used for these psychoactive drugs. The outlined method can be valuable for the future detection of mephedrone and its two metabolites in hair. © 2011 Elsevier B.V. All rights reserved.
1. Introduction ‘Legal high’ is a term given to chemicals which possess psychoactive activity in humans, but are not subject to prohibition by law [1]. In the last few years, buying legal highs from the internet has become common practice for drug users. This is, in part, due to their structural similarity to controlled psychoactive substances such as phenethylamines and cathinones [2–4]. Mephedrone, sometimes referred to as ‘meow’, ‘mcat’ and ‘bubbles’ is a -ketoamphetamine and has a structure similar to methcathinone, which in turn is the N-methyl analogue of the natural product cathinone [2]. Cathinone can be extracted from the leaves of an African and Middle Eastern plant Catha edulis or ‘Khat’ [4,5]. The alkaloid content of the fresh leaves is responsible for the psychoactive stimulating effects [4]. Mephedrone has been routinely sold on the internet as plant feed, with an accompanied warning of ‘not suitable for human consumption’. However, in April 2010 it was specifically named and hence controlled in UK under the Misuse of Drugs Act 1971 as a
∗ Corresponding author. Tel.: +44 020 8547 7981; fax: +44 020 8417 7562. E-mail addresses: [email protected] (S.A.B. Shah), [email protected] (N.I.K. Deshmukh), [email protected] (J. Barker), [email protected] (A. Petróczi), [email protected] (P. Cross), [email protected] (R. Archer), [email protected] (D.P. Naughton). 0731-7085/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.jpba.2011.11.026
Class B substance [1,6]. It has also been banned in the majority of other European countries [2]. The use of mephedrone is more common in males than females and it is reported to be one of the most commonly abused psychotropic drugs [1]. The popularity of use of these psychoactive drugs has created a demand for sensitive, robust and reliable analytical methods for their identification and quantification in different matrices. Gas chromatography mass spectrometry (GC–MS) has routinely been used for the detection of mephedrone in matrices such as blood and urine [3,6,7]. Recently, liquid chromatography mass spectroscopy (LC–MS) has also been employed for analysing blood samples for the detection of mephedrone [4]. Compared to urine and blood, hair samples can provide information on the long term history of the individual’s intake of controlled drugs depending on the pharmacokinetic properties of the drug in question [8,9]. Also, blood and urine samples require storage at −20 ◦ C in the presence of preservatives until analysis, for mephedrone [3]. Thus, hair is gaining potential interest for the detection of parent drugs in doping control [8]. The drugs bind to melanin which is the major component of hair [8]. Generally, urinalysis is carried out to determine the presence of metabolites of the controlled drugs and it is often difficult to detect the parent drug in urine. In addition, the matrix effects for urine and blood are much greater than hair [8]. In the scientific literature, there is limited information available on the metabolism of mephedrone in humans. As the structure of
S.A.B. Shah et al. / Journal of Pharmaceutical and Biomedical Analysis 61 (2012) 64–69
O
Table 1 Chromatography conditions for the separation of the compounds.
O
LC run time (min)
HN
Mephedrone OH
HN
4-methylephedrine
65
HN
CD3
Mephedrone – d3
0 3 4 5 10
Acetonitrile (%) 65 100 100 65 65
Water (%) 35 0 0 35 35
OH
NH2
4-methylnorephedrine
Fig. 1. Structures of mephedrone, 4-methylephedrine, 4-methylnorephedrine and mephedrone-d3 (internal standard).
mephedrone is substantially similar to other psychoactive drugs such as methcathinone and methamphetamine, one can tentatively propose a metabolism of mephedrone on a similar basis. There are a number of different pathways by which this drug could be metabolised. The two metabolites analysed in this study are 4-methylephedrine and 4-methylnorephedrine [7,10–13]. The structures of mephedrone and its two metabolites are depicted in Fig. 1. The aim of this study was to develop an analytical method for the quantitative analysis of mephedrone and for two of its metabolites 4-methylephedrine and 4-methylnorephedrine in hair using LC–MS/MS. 2. Materials and methods 2.1. Hair specimen Following ethical approval from two higher education institutions, hair samples were obtained from 154 healthy volunteers (95 male, 59 females) aged 18–56 years old [14]. Blank hair samples were obtained from two healthy individuals. 2.2. Chemicals and consumables HPLC grade methanol, acetonitrile, water, and hexane, along with reagent grade ethyl acetate, ethanol, dichloromethane, chloroform and formic acid were purchased from Fisher Scientific UK Ltd. (Loughborough, UK). Tris HCl buffer and Cleland’s reagent were obtained from VWR (Lutterworth, UK). Proteinase K was purchased from Sigma Aldrich (Dorset, UK). The metabolites 4-methylephedrine hydrochloride and 4-methylnorephedrine hydrochloride were synthesised in-house by borohydride reduction of mephedrone and 4-methylcathinone, respectively, at Kingston University (London, UK). Mephedrone hydrochloride and mephedrone-d3 hydrochloride (internal standard) were purchased from LGC standards (Teddington, UK). An Agilent SB-C18 column (2.1 m × 150 mm × 1.8 m) was purchased from Agilent Technologies UK Ltd. (Wokingham, UK). 2.3. Instrumentation Analyses were performed on a Thermoscientific liquid chromatography–tandem mass spectrometry (LC–MS/MS) system consisted of an Accela UPLC system (Thermo Scientific, UK Ltd., Loughborough, Leicestershire, UK) coupled to a Triple Quadrupole TSQTM mass spectrometer (Thermo Electron Corp., UK). This was operated in selective reaction monitoring (SRM) mode using the
conditions given in Table 1 with detection parameters given in Table 2. The column was maintained at 50 ◦ C and the auto sampler tray was set at 10 ◦ C. The flow rate was 200 L/min and the injection volume was 3.0 L. The capillary temperature was maintained at 300 ◦ C and a voltage of 4000 V was employed. The collision pressure was kept at 1.5 mTorr. The fragmentation pathways of all the analytes are shown in Fig. 2. 2.4. Hair digestion and extraction 2.4.1. Decontamination The hair samples were decontaminated by rinsing with 2 mL dichloromethane for 2 min (twice) at room temperature. Decontamination ensured the removal of contaminants from the surface of hair, which may interfere with the analysis [15]. After decontamination, the hair samples were air dried before cutting into very fine segments (ca. 1 mm length) using scissors. 2.5. Digestion Initially, hair digestion was carried out by incubating the hair samples with 1 mL of 1 M sodium hydroxide solution at 95 ◦ C for 10 min. However, this led to the complete degradation of analytes. Hence, enzymatic digestion was employed alternatively. Enzymatic digestion was carried out by placing the fine hair segments (50 mg) in a glass vial containing Cleland’s reagent (100 mg) and enzyme proteinase K (15 mg). The internal standard mephedroned3 (100 L) at 40 pg/mg was added to the mixture. The mixture was finally incubated with Tris buffer (1 mL) for 2 h at 37.5 ◦ C with constant stirring. 2.6. Extraction Liquid–liquid extraction was optimised for mephedrone using hexane (3 mL), and for the metabolites using a mixture of chloroform, ethanol and diethyl ether (3 mL in total) with the ratio of 3:1:1. The contents were mixed using a vortex mixer and centrifuged at 5 ◦ C for 5 min at 1750 × g. The organic layers were separated and pooled together in a glass tube. The organic layer was then dried using nitrogen gas and reconstituted with 200 L of acetonitrile. 2.7. Method validation Method validation was carried out in order to establish the sensitivity, specificity, selectivity, linearity, limit of detection (LOD), limit of quantification (LOQ), inter and intraday precision and accuracy and percentage recoveries. The metabolites when diluted in methanol gave better sensitivity as compared to acetonitrile, whereas mephedrone gave a similar sensitivity in both solvents. A calibration curve was obtained by spiking blank hair samples with known concentrations of the drugs in the range of 5–100 pg/mg for mephedrone and 10–150 pg/mg for 4-methylephedrine and 4-methylnorephedrine. The internal
66
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Fig. 2. LC–MS/MS chromatogram and CID spectra of (a) mephedrone, (b) 4-methylephedrine and (c) 4-methylnorephedrine in hair at concentrations of 10 pg/mg, 20 pg/mg and 20 pg/mg, respectively.
S.A.B. Shah et al. / Journal of Pharmaceutical and Biomedical Analysis 61 (2012) 64–69
67
Table 2 Ion transitions, retention time (RT) and collision energies used for the analysis of the analytes and the internal standard. Compound
RT (min)
Precursor ions (m/z)
Product ions (m/z)
Collision energy (eV)
Mephedrone
2.07
178.1
4-Methylephedrine
1.93
180.2
4-Methylnorephedrine
1.85
166.2
Mephedrone-d3
1.98
181.2
160.1 145.1 147.2 131.3 115.2 131.2 115.2 90.8 163.2 148.2
10 18 19 18 39 14 33 24 17 20
Table 3 Precision and accuracy for intraday and interday of the three drugs at four different concentrations. Compound
Concentration (pg/mg)
Precision RSD (%)
5 10 20 40 10 20 50 100 10 20 50 100
Mephedrone
4-Methylephedrine
4-Methylnorephedrine
Accuracy %
Intraday N=6+6+6+6
Interday N = 18 + 18 + 18 + 18
0.62 0.4 2.11 2.56 0.6 0.75 0.8 4.92 0.54 0.2 2.91 6.97
0.8 0.7 1.7 4.5 0.9 0.7 4 3.4 1.1 0.7 2 7.3
99.15 106.34 103.16 107.53 114.59 110.76 107.02 98.52 107.98 106.51 97.6 100.67
Table 4 Extraction recoveries of the three drugs at three different concentrations. Compound
Concentration (pg/mg)
Recovery (%) N = 6 + 6 + 6
Regression coefficient
Regression equation
Mephedrone
5 20 40 10 20 100 10 20 100
90.36 110.37 108.10 106.16 98.37 101.87 100.23 96.78 101.32
0.999
y = 0.112x + 0.429
0.990
y = 0.012x + 0.033
0.990
y = 0.008x + 1.389
4-Methylephedrine
4-Methylnorephedrine
standard mephedrone-d3 (100 L) was added to all the standards with a final concentration of 40 pg/mg. The extraction recovery was validated for mephedrone and its metabolites at three different concentrations using six replicates by comparing the extracted samples with non-extracted samples. 3. Results and discussion 3.1. Validation results There were no chromatographic interferences found due to the endogenous substances present in the blank (control) hair that was analysed using the above chromatographic conditions. The precision, accuracy and recoveries lie within the limits set by the FDA guidelines [16]. The validation data are given in Tables 3–5. Table 5 Signal to noise ratios for the LOD and LOQ. Compound
S/N for LOD N = 6
S/N for LOQ N = 6
Mephedrone 4-Methylephedrine 4-Methylnorephedrine
7 5 4
11 10 10
LOD and LOQ values for mephedrone were 2.5 pg/mg and 5 pg/mg, respectively. For 4-methylephedrine and 4methylnorephedrine the LOD was 5 pg/mg and the LOQ 10 pg/mg. Out of the 154 samples analysed, mephedrone was detected in only five samples. However, the metabolites were not detected in any of the samples analysed. However, owing to the method used for data and sample collection [14], it is impossible to link admission of mephedrone use to individuals, or individual samples. Out of the five samples, mephedrone could be successfully quantified in only one sample. It was found to be present at a concentration of 21.11 pg/mg. Fig. 3 shows the chromatogram and mass spectra of the quantified positive sample. The results confirm that the LC–MS/MS method developed for mephedrone and its two metabolites is sensitive and robust. There was no significant change in column performance or the pressure after making several injections. Only 50 mg hair is required for the detection of the mephedrone and its metabolites. Hair analysis is capable of providing a long term history of drug use and any kind of hair treatment such as bleaching cannot affect the incorporation of the drug into hair. Thus, it can be used to compliment urinalysis for identifying routine drug users [17]. In this study, the metabolites could not be detected in the hair samples. In general, high polarity
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Fig. 3. LC–MS/MS chromatogram and CID spectra of the positive samples at concentrations of 21.11 pg/mg.
metabolites which are destined to be excreted in urine are difficult to detect in hair. In contrast, the parent drugs are lipophilic and can become incorporated into the hair [8]. It should be noted that as mephedrone is not smoked, the route of entry via environmental exposure would not provide a false positive for hair analyses. There has been very little research carried out on the metabolism of mephedrone. Due to the structural similarities of mephedrone to methcathinones and methamphetamines, the metabolism of mephedrone has been proposed to follow a similar pathway. The pathway involves the hydroxylation of the methylene group and the second metabolite is formed by N-demethylation, thus forming a primary amine. However, there is a possibility of other pathways for the metabolism of mephedrone [7,10–13].
4. Conclusion The present study describes a rapid and replicable method for the quantitative analysis of mephedrone and its two metabolites in human hair. The method is quite sensitive with an LOQ of 5 pg/mg for mephedrone and 10 pg/mg for its two metabolites. The absence of any interference on the chromatograms shows that the method is highly selective. The method is suitable for the future detection and quantification of mephedrone and its metabolites.
Author contributions J.B., A.P. and D.P.N. conceived and designed the study. S.A.B.S. performed the analysis. R.A. synthesised and provided the metabolites. P.C. provided samples. S.A.B.S. and N.I.K.D. drafted the paper. J.B., A.P., D.P.N. and S.A.B.S. interpreted the results. All authors contributed to writing, and approved the final manuscript.
Acknowledgements The authors thank Helen Taft and Christiana Adesanwo for their help with collecting hair samples.
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