Anal Bioanal Chem DOI 10.1007/s00216-013-7386-z

RESEARCH PAPER

Simultaneous quantification of 28 synthetic cathinones and metabolites in urine by liquid chromatography-high resolution mass spectrometry Marta Concheiro & Sebastien Anizan & Kayla Ellefsen & Marilyn A. Huestis

Received: 7 August 2013 / Revised: 12 September 2013 / Accepted: 16 September 2013 # Springer-Verlag Berlin Heidelberg (outside the USA) 2013

Abstract Synthetic cathinones are novel stimulants derived from cathinone, with amphetamines or cocaine-like effects, often labeled “not for human consumption” and considered “legal highs”. Emergence of these new designer drugs complicate interpretation of forensic and clinical cases, with introduction of many new analogs designed to circumvent legislation and vary effects and potencies. We developed a method for the simultaneous quantification of 28 synthetic cathinones, including four metabolites, in urine by liquid chromatography coupled to high resolution mass spectrometry (LC-HRMS). These cathinones include cathinone, methcathinone, and synthetic cathinones position-3’-substituted, N-alkyl-substituted, ring-substituted, methylenedioxysubstituted, and pyrrolidinyl-substituted. One mL phosphate buffer pH 6 and 25 μL IStd solution were combined with 0.25 mL urine, and subjected to solid phase cation exchange extraction (SOLA SCX). The chromatographic reverse-phase separation was achieved with a gradient mobile phase of 0.1 % formic acid in water and in acetonitrile in 20 min. We employed a Q Exactive high resolution mass spectrometer, with compounds identified and quantified by target-MSMS experiments. The assay was linear from 0.5–1 to 100 μg/L, with limits of detection of 0.25–1 μg/L. Imprecision (n =20) was 160.0750

50 0 100

Methylone-d3 211.2>163.0943

50 0 100

Ethylcathinone 178.2>131.0730

50 0 100

-PPP 204.1>105.0703

50

0 100

Buphedrone ephedrine 180.1>133.0886

50 0 100

Ethylone 222.2>174.0912

50 0 100

Ethylone-d5 227.2>179.1224

50 0 100

4-Methoxymethcathinone 194.2>161.0833

50 0 100

Buphedrone 178.2>131.0731

50 0 100

Normephedrone 164.1>131.0730

50 0 100

Diethylcathinone 206.1>105.0703

50 0 100

Diethylcathinone-d10 216.1>110.1750

50 0 100

MDPPP 248.2>98.0968

50 0 100

4-Methylephedrine 180.1>147.1041

50 0 100

Butylone 222.2>174.0913

50 0 100

Butylone-d3 225.2>177.1100

50 0 100

Mephedrone 178.2>145.0885

50 0 100

Mephedrone-d3 181.2>148.1073

50 0 100

4-MEC 192.2>145.0886

50 0 100

4-MEC met 194.2>147.1041

50 0 100

MDPBP 262.2>112.1123

50 0 100

Pentedrone 192.2>132.0808

50 0 100

Pentylone 236.2>188.1068

50 0 100

3,4-DMMC 192.1>159.1041

50 0 100

PVP 232.2>91.0548

50 0 100

4-MPBP 232.3>105.0702

50 0 100

MDPV 276.2>126.1278

50 0 100

MDPV-d8 284.2>134.1779

50 0 100

Pyrovalerone y 246.2>105.0703

50 0 0 100

Benzedrone 254.2>91.0547

50 0 100

Naphyrone 282.3>141.0698

50 0 100

Naphyrone-d5 287.3>131.1593

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Simultaneous quantification of 28 synthetic cathinones

ƒFig. 2

LC-MSMS chromatogram of a urine sample fortified at the lower limit of quantification (0.5 μg/L for all compounds, except buphedrone ephedrine at 1 μg/L)

Imprecision (160.0750

50 0 100

Methylone 208.2>132.0807

50 0 100

Pentedrone 192.2>132.0808

50 0 100

Pentedrone 192.2>91.0546 0 100

Pentylone 236.2>188.1068

50 0 100

Pentylone 236.2>175.0624

50 0 100

PVP 232.2>91.0548

50 0 100

PVP 232.2>126.1280

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phase (0.1 % formic acid in water and in acetonitrile) and the gradient described in the materials and methods section, allowed the complete chromatographic separation of these isomers. In a previously published method, ethylone and butylone co-eluted and could not be individually identified [19]. This confirmation method included four metabolites, buphedrone ephedrine (buphedrone metabolite), 4-methylN-ethyl-norephedrine (4-MEC metabolite), and normephedrone and 4-methylephedrine (mephedrone metabolites). For mephedrone, normephedrone was identified as the predominant metabolite in urine [12, 13], and 4methylephedrine was detected in human urine [13]. Although no data are available for buphedrone and 4-MEC metabolism, the reduction of the keto moiety to form buphedrone ephedrine and 4-methyl-N-ethyl-norephedrine, respectively, is expected to occur [12, 17]. Most of the compounds included in the present method (24 out of 28) are parent compounds and not metabolites, mainly because metabolites were not commercially available or metabolism is as yet unknown. Low LOQ (0.5 μg/L) in this method enables detection of synthetic cathinones in urine, even if the drugs are extensively metabolized [14, 31]. The short-term stability of synthetic cathinones in urine fortified at low and high QC concentrations (3 and 300 ng/ mL) was investigated. Urine pH was 7.6 and no preservatives were added. Most compounds, except benzedrone and

naphyrone (up to −33.3 % loss), were stable 72 h at 4 °C and after 3 freeze-thaw cycles. However, losses between −20 % and −67.6 % were observed after 24 h at room temperature. Only metabolites with a hydroxyl instead of a β-keto moiety (4-methylephedrine, buphedrone ephedrine and 4-methyl-N-ethyl-norephedrine), diethylcathinone and the majority of the pyrrolidinyl-derivatives (MDPPP, MDPBP, α-PVP, 4-MPBP and MDPV) were stable under this storage condition. Tsujikawa et al. [32] showed that the substituted group on the benzene ring and the groups attached at the nitrogen atom affect synthetic cathinone stability. Synthetic cathinones with a tertiary amine were more stable than those with primary and secondary amine groups. Also our results are in accordance with Johnson et al. [33], who studied mephedrone and MDPV stability in urine at 1,000 ng/mL at room temperature, 4 °C and −20 °C for 1 to 14 days. Mephedrone exhibited stability problems in urine at room temperature (60 % loss after 14 days), while MDPV was stable under different storage conditions. Sorensen [34] studied the stability of cathinone, methcathinone, ethylcathinone, mephedrone, diethylcathinone, 4-fluoromethcathinone, methedrone, methylone and butylone in blood at 20 and 5 °C under different pH conditions. Cathinones were not stable in blood samples at neutral and basic conditions and room temperature, similarly to the urine in our study. Special attention has to be paid to specimen storage conditions if synthetic cathinones determination is required.

Simultaneous quantification of 28 synthetic cathinones

Conclusion We developed a sensitive and selective LC-HRMS method for the simultaneous determination of 24 synthetic cathinones and four metabolites in urine. Only 0.25 mL were required, and the LOQ was 0.5–1 μg/L. Comprehensive multi-analyte confirmation methods are needed due to the wide spectrum of synthetic cathinones available in the market. The confirmation data will help in the interpretation of the results in forensic and clinical cases, and in the evaluation of the toxicity of these designer drugs. Acknowledgments This research was supported by the Intramural Research Program of the National Institute on Drug Abuse (NIDA), National Institutes of Health (NIH).

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