tandem mass spectrometry method for rapid detection, quantification and confirmation of cathinone-derived designer drugs for doping control in equine plasma.

Research Article Received: 18 September 2013

Revised: 29 October 2013

Accepted: 31 October 2013

Published online in Wiley Online Library

Rapid Commun. Mass Spectrom. 2014, 28, 217–229 (wileyonlinelibrary.com) DOI: 10.1002/rcm.6778

Sensitive hydrophilic interaction liquid chromatography/tandem mass spectrometry method for rapid detection, quantification and confirmation of cathinone-derived designer drugs for doping control in equine plasma Xiaoqing Li1, Cornelius E. Uboh1,2*, Lawrence R. Soma1, Ying Liu1, Fuyu Guan1, Craig R. Aurand3, David S. Bell3, Youwen You1, Jinwen Chen1 and George A. Maylin4 1

University of Pennsylvania, School of Veterinary Medicine, Department of Clinical Studies, New Bolton Center Campus, Kennett Square, PA 19348, USA 2 PA Equine Toxicology and Research Center, Department of Chemistry, West Chester University, 220 East Rosedale Avenue, West Chester, PA 19382, USA 3 Sigma-Aldrich Corp, Pharmaceutical and Bioanalytical Research, 595 North Harrison Rd., Bellefonte, PA 16823, USA 4 New York Drug Testing and Research Program, Morrisville University, 777 Warren Road, Ithaca, NY 14850, USA RATIONALE: Cathinone derivatives are new amphetamine-like stimulants that can evade detection when presently available

methods are used for doping control. To prevent misuse of these banned substances in racehorses, development of a liquid chromatography/tandem mass spectrometry (LC/MS/MS) method became the impetus for undertaking this study. METHODS: Analytes were recovered via liquid-liquid extraction using methyl tert-butyl ether. Analyte separation was achieved on a hydrophilic interaction column using liquid chromatography and mass analysis was performed on a QTRAP mass spectrometer in positive electrospray ionization (ESI) mode with multiple reaction monitoring (MRM). Analyte identification was carried out by screening for a specified MRM transition. Quantification was conducted using an internal standard. Confirmation was performed by establishing a match in retention time and ion intensity ratios comparison. RESULTS: The method was linear over the range 0.2–50 ng/mL. The specificity was evaluated by analysis of six different batches of blank plasma and those spiked with each analyte (0.2 ng/mL). The recovery of analytes from plasma at three different concentrations was >70%. The limits of detection, quantification and confirmation were 0.02–0.05, 0.2–1.0 and 0.2–10 ng/mL, respectively. The matrix effect was insignificant. The intra-day and inter-day precision were 1.94–12.08 and 2.58–13.32%, respectively. CONCLUSIONS: The method is routinely employed in screening for the eleven analytes in post-competition samples collected from racehorses in Pennsylvania to enforce the ban on the use of these performance-enhancing agents in racehorses. The method is sensitive, fast, effective and reliably reproducible. Copyright © 2013 John Wiley & Sons, Ltd.

Living a daily life without the bad news of deadly incidents resulting from the use of one designer drug or another is becoming very rare. Designer drugs are produced with the sole intent of by-passing existing drug laws and detection methods. These agents are usually manufactured by modifying the chemical structures of already existing and legally approved drugs to render their detection difficult to impossible. They are commonly assigned fancy street names such as ’bath salts’ and ’legal highs’ to distract the attention of law enforcements and the public. Rapid technological advancements, cheap organic synthesis, increased loss of jobs resulting in the availability of ’basement chemists’, easier

Rapid Commun. Mass Spectrom. 2014, 28, 217–229

Copyright © 2013 John Wiley & Sons, Ltd.

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* Correspondence to: C. E. Uboh, PA Equine Toxicology and Research Center, Department of Chemistry, West Chester University, 220 East Rosedale Avenue, West Chester, PA 19382, USA. E-mail: [email protected]

availability of precursor chemicals, and increased use of the internet for marketing and distributing new illegal drugs have led to the uncontrollable increase in the availability and access to these designer drugs. Between 2009 and 2010, a significant increase in the abuse of synthetic cathinones was reported in western Europe and Japan.[1–4] Cathinone stimulates the central nervous system in a similar way to amphetamine. Cathinone is the main psychoactive constituent of khat (Cathis edulis), a plant leaf commonly chewed by inhabitants of the Horn of Africa and Arabian Peninsula.[5] It is estimated that there are nearly 10 million users of khat leaves worldwide.[6,7] Both psycho-stimulating and hallucinogenic properties are attributed to cathinones. Cathinone was first isolated from the fresh leaves of the khat shrub in 1975.[8] In 1990, methylone was marketed as an anti-depressant and again as an anti-Parkinson drug in 1996. In 2002, mephedrone (4-methylmethcathinone) was identified by the street name of ’explosion’ and became the first cathinone derivative distributed via the internet. By 2008, its popularity had grown

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worldwide leading to its ban by some countries while sparking greater interest in others.[6–8] A select number of derivatives of cathinone comprise methcathinone, butylone, mephedrone, flephedrone (4-fluoromethcathinone), ethylone, methedrone, buphedrone, 3,4-methylenedioxypyrovalerone (MDPV), 3-fluoromethcathinone, methylone and naphyrone which have been synthesized and are widely and freely distributed through secret outlets. Flephedrone (4-fluoromethcathinone) was introduced in 2009 and its demand peaked in 2010 as was the attributed increase in seizures in Europe.[9] These compounds are mainly used for their psychoactive effects. The use of all preparations containing psychoactive compounds in the horse during competition in Pennsylvania is banned by the Pennsylvania (PA) Racing Commissions. These compounds function as norepinephrine-dopamine re-uptake inhibitors which act to increase postsynaptic concentration of neurotransmitters. Interaction of neurotransmitters with post neuronal receptors results in sustained stimulatory effect, similar to the effect of amphetamines for which reason cathinones are referred to as ’natural amphetamines’.[10,11] Thus, these drugs are potential candidates for doping of horses to enhance performance during competition. These agents also have a profound adverse effect on the ability of users to operate machinery and, thus, are blamed for many car accidents.[12] It is only reasonable to conclude that the same would be true in racehorses doped with cathinone derivatives. Chewing of khat leaves has been used for treating respiratory conditions because cathinone in khat leaves relaxes airway smooth muscle, and for this reason it may become an instant substitute for clenbuterol in racehorses.[13–15] However, unlike new legal drugs, which are extensively studied in controlled clinical setting, ’designer drugs’ are marketed without any regard for safety and guidelines for their safe use in humans. The first case of near fatal exposure to ’bath salts’ in the US was reported in 2010. Within a very short period of their appearance on the black market in the USA, more than 1400 cases of misuse and abuse of these agents were reported in 47 out of 50 states.[16] The increasing number of cases reported on the use of ’bath salts’ has captured public attention and that of law enforcements even in light of the current lack of pharmacological, toxicological and analytical data on these new psychoactive compounds. For this reason, in October, 2011, The United States Drug Enforcement Agency (DEA) published an Order in the Federal Register exercising its emergency scheduling authority to control only three synthetic stimulants (mephedrone, 3,4-methylenedioxypyrovalerone (MDPV) and methylone ) out of many that are used and sold as ’bath salts’.[17] In July 2011, the Drug Testing Standards and Practices Committee of the Racing Commissioners International (RCI) approved the addition of MDPV to its list of Class I Prohibited Substances in the horse racing industry. This regulatory action by RCI demonstrating strong disapproval for the use of ’bath salts’ in the racehorse industry is to warn violators of the impending stiff penalties. Gas chromatography (GC) coupled to mass spectrometry (MS) is the accepted analytical method used in most forensic analyses for the qualitative analysis of synthetic cathinones due to the volatility of these agents. Strano-Rossi et al. developed a GC/MS method for screening for MDPV in urine.[18] The method was used to study the phase I and II metabolism of MDPV in isolated human liver microsomes and S9 cellular fractions. Meyer et al. reported on a systematic toxicological analytical procedure to detect mephedrone, butylone, methylone

wileyonlinelibrary.com/journal/rcm

and their metabolites in rat urine.[19] Fluorinated and brominated cathinone derivatives are not readily metabolized although other cathinone derivatives are readily metabolized to norephedrine and norpseudoephedrine derivatives.[20–23] Additional GC/MS-based papers on the determination of different cathinones in biological matrices were recently published.[24–26] However, most of these methods employ time-consuming sample preparation procedures, involving the use of hazardous and expensive derivatizing reagents, all of which render the technology less attractive for the routine analysis of a large number of samples for anti-doping analysis. Liquid chromatography/mass spectrometry (LC/MS) technology has become the ’gold standard’ in modern doping control analyses because it is fast, sensitive, robust, specific and suitable for high-throughput analysis. An impressive number of studies involving the use of LC/MS for screening and quantification of ’bath salts’ have been published.[27–33] Most of the published studies used reversed-phase LC for separation. However, in our hands, the reversed-phase LC method did not meet the desired sensitivity and resolution of all eleven analytes in the present study. For this reason, we investigated the use of hydrophilic interaction liquid chromatography (HILIC) as a possible alternative to reversedphase chromatography. HILIC is the perfect separation technique when coupled to electrospray ionization (ESI) MS because the acetonitrile-rich eluents used provide favorable conditions for efficient droplet formation and desolvation within the mass spectrometer source resulting in improved ionization efficiency and sensitivity.[34–36] HILIC was initially employed in the separation of polar compounds, such as sugars and oligosaccharides, and, in the past decade, has been widely applied to the separation of proteins and peptides, biomarkers, and complex carbohydrates as well as small molecules.[37–41] In humans, cathinones increase alertness, euphoria, heart rate, perspiration, respiration, blood pressure and seizure.[9,12,42,43] To date, there is no information to the contrary in horses. Thus, to protect the welfare, health and safety of the horse and support personnel in the racehorse industry and to enforce the ban on the use of these agents in racehorses competing in PA, a fully validated method for detection, quantification and confirmation of the presence of the eleven analytes was needed. The current study presents evidence of an efficient and selectively robust HILIC-LC/MS/MS method for the high-throughput detection, quantification and confirmation of eleven ’bath salts’ in equine plasma. This is a sensitive method for the simultaneous detection, quantification and confirmation of the presence of the eleven ’bath salts’ in equine plasma. The limit of detection (LOD) was 0.2 ng/mL in equine plasma. The method has been successfully applied to routine screening of post-race samples in Pennsylvania and in the analysis of ’B’ samples from other racing jurisdictions in the USA.

EXPERIMENTAL Chemicals and reagents All stock solutions of the reference standards (1.0 mg/mL) as well as that of mephedrone-d3 (100 μg/mL; internal standard, IS) were purchased from Cerilliant Corp. (Round Rock, TX, USA). Methyl tert-butyl ether (MTBE), formic acid (98–100%), and ammonium hydroxide (29.5%) were obtained from EMD



Analysis of bath salts in equine plasma using LC/MS/MRM

All standard stock solutions (1 mg/mL in methanol) were stored at 20 °C. A mixture of the eleven analytes (10 μg/mL of each) was prepared by adding 100 μL of each stock solution (1 mg/mL) to 8900 μL methanol, for a total of 10 mL. Working standard solutions of 10, 25, 50, 100, 250, 500 and 1000 ng/mL were prepared by serial dilution of the mixed standard solution of the analytes (10 μg/mL) using mobile phase (5 mM ammonium formate/acetonitrile; 5:95, v/v) mixture and were stored at 4 °C. A working solution of mephedrone-d3 (IS) (500 ng/mL) was prepared by diluting 25 μL of stock solution (100 μg/mL) in 4.975 mL methanol. Each drug standard solution was discarded after 6 months. A stock solution of ammonium formate buffer comprising 1.0 M ammonium formate and 1.0 M formic acid was prepared by adding 15.4 mL formic acid and 13.5 mL ammonium hydroxide to 171 mL H2O. Preparation of 1 L ammonium formate buffer (5 mM) was achieved by adding 5 mL of the above stock solution of ammonium formate buffer to 995 mL optima grade water.

(5:95; v/v) mixture which was delivered at 300 μL/min. The oven temperature was set at 30 °C while that of the autosampler was 15 °C. For mass analysis, a 4000 QTRAP mass spectrometer (AB Sciex, Foster City, CA, USA) equipped with a turbo ionspray interface was used. Electrospray ionization (ESI) in positive mode was used for all experiments. All the analytes produced [M + H]+ ions which were then used as the precursor ions for the MS/MS experiments. Source-dependent parameters were optimized using flow injection analysis (FIA) of each analyte. The ionspray voltage was set at 5000 V and the source temperature was 350 °C; the curtain gas, gas 1, gas 2 pressures were 25, 40 and 40 psi of high-purity N2, respectively. Compound-dependent parameters such as declustering potential (DP), collision energy (CE) and collision cell exit potential (CXP) were manually tuned and optimized (Table 1). The entrance potential (EP) for all analytes was 10 V. A multiple reaction monitoring (MRM) scan was employed for screening and quantification, with a dwell time of 100 ms. An enhanced product ion (EPI) scan was used as a dependent scan with instant library search for compound identification. In EPI mode, the scan rate was 4000 m/z units/s, the linear ion trap (LIT) fill time was 20 ms, collisionally activated dissociation (CAD) was set to high, the CE was 35 eV and the collision energy spread (CES) was 15 eV. Data acquisition and analysis were accomplished with Analyst software (version 1.5.1; AB Sciex).

Preparation of calibration samples

Method validation

To prepare calibration samples, 10 μL of the standard drugs working mixture solution (10, 25, 50, 100, 500, 1000, 2500 ng/mL) was added to 0.5 mL blank equine plasma to prepare 0.2, 0.5, 1, 2, 10, 20, 50 ng/mL calibrators.

Specificity

Chemical, Inc. (Gibbstown, NJ, USA). Optima grade water and acetonitrile (LC/MS grade) were purchased from Honeywell Burdick & Jackson (Muskegon, MI, USA) and methanol was purchased from Fisher Scientific (Fair Lawn, NJ, USA). Standard stock solutions

Extraction of analytes from equine plasma Analytes were recovered from equine plasma by liquid-liquid extraction (LLE) using MTBE. First, 10 μL of IS solution (mephedrone-d3, 500 ng/mL) was added to 0.5 mL plasma sample in a 16 125 mm test tube and the contents were mixed using a vortex device prior to adding 5 mL MTBE. The test tube was capped and mixed on a rotorack device for 10 min prior to centrifugation (1610 g for 10 min). The resulting organic layer (top) was transferred to a pre-labeled culture tube (16 100 mm) and evaporated to dryness at 50 °C (Techni Dri-Block DB-3, Duxford, UK) under a steady stream of air or nitrogen. The dried extract was reconstituted in 100 μL of mobile phase (5 mM ammonium formate/ acetonitrile, (5:95, v/v)) mixture and transferred into a 200 μL insert (Target PP Polyspring, National Scientific Company, Rockwood, TN, USA) from which 10 μL was used for LC/MS analysis. LC/MS/MS conditions


Matrix effect The matrix effect was evaluated at three separate concentrations (0.5, 10 and 50 ng/mL) of each analyte in six replicates. Blank equine plasma (0.5 mL) was fortified with 10 μL of a solution mixture of the analytes at the above three different concentrations, mixed and then extracted as described above under sample preparation. The dried residue was reconstituted in 100 μL mobile phase. Water was used as an ideal matrix to compare the matrix effect contributed by plasma with that by water. For this purpose, the same volume (10 μL) of the standard solution of analytes was added to 0.5 mL water, mixed and similarly extracted as in plasma matrix, evaporated, and the dried residue was also reconstituted in 100 μL mobile phase mixture. The matrix effect was calculated by comparing the chromatographic peak area of each drug standard spiked into water with that spiked into blank plasma according to the following equation: Matrix effect ð%Þ ¼ Aplasma –Awater =Awater 100

(1)

where Aplasma is the chromatographic peak area of the analyte standard solution spiked into blank plasma, and Awater is that of an analyte standard solution spiked into water.



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The liquid chromatography (LC) system consisted of a 20A liquid chromatograph with a SIL-HTc autosampler (Shimadzu Scientific Instruments, Columbia, MD, USA). Separation of the analytes was achieved on an Ascentis® Express HILIC column (50 mm 2.1 mm i.d., 2.7 μm particle size; Supelco/Sigma-Aldrich, Bellefonte, PA, USA). All LC separations were performed using isocratic elution. The mobile phase comprised 5 mM ammonium formate/acetonitrile

The specificity of the method was determined by analyzing six different batches of blank equine plasma, plasma spiked with IS and that spiked with 0.2 ng/mL (lower limit of quantification) of each analyte to determine if there were any endogenous compounds eluting at the same retention time as the analytes that might interfere with the resolution of any of the target analytes.

X. Li et al. Table 1. LC/MS/MS/MRM parameters of the eleven analytes tR (min)

Precursor (m/z)

DP (V)

Product ions (m/z)

CE (eV)

CXP (V)

Mephedrone

6.53

178

46

Buphedrone

3.51

178

39

4-Fluoromethcathinone

4.91

182

41

3-Fluoromethcathinone

3.67

182

41

3Methoxymethcathinone

5.40

194

41

Methedrone

8.24

194

46

Methylone

7.04

208

55

Ethylone

5.65

222

52

Butylone

4.34

222

53

MDPV

4.87

276

64

Naphyrone

3.60

282

70

145 144 91 131 132 91 149 148 123 149 148 77 161 146 118 161 146 118 160 132 117 174 146 91 174 191 146 126 205 175 141 211 155

30 43 48 31 25 32 29 44 31 29 44 56 27 37 50 29 40 54 26 46 53 29 39 52 27 19 38 39 26 32 36 28 41

10 11 6 10 10 14 11 11 9 12 11 12 12 11 9 12 10 8 12 10 8 16 11 5 10 15 10 10 13 16 10 4 11

Analyte

tR = retention time; DP = declustering potential; CE = collision energy; CXP = collision cell exit potential

Linearity The linearity was evaluated by analyzing six calibration curves each consisting of seven calibrators at different concentrations ranging from 0.2/0.5 to 50 ng/mL plasma. Each calibration curve was generated using the chromatographic peak area ratio of the analyte versus the IS and the concentration of the analyte. The linearity was assessed by determining the line of best fit with different weighting factors (no weight, 1/x and 1/x2), and the least-squares linear regression model.

to the blank plasma extract before drying. All samples were dried at 50 °C under a steady stream of air and the dried extract was reconstituted in 100 μL mobile phase. The recovery was determined by comparing chromatographic peak area between the samples spiked with the standard solution before and after extraction (Eqn. (2)). The accuracy was calculated by comparing the mean observed concentration of the samples spiked in blank plasma before extraction and the nominal concentration (Eqn. (3)). Recovery ð%Þ ¼ ASB =ASA 100

(2)

Accuracy ð%Þ ¼ CSB =Cnominal 100

(3)

Recovery and accuracy

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The recovery and accuracy were assessed by analyzing samples at three different concentrations (0.5, 10, and 50 ng/mL) in six replicates. For each concentration, 10 μL of the mixed standard solution of the eleven analytes was added to blank plasma (0.5 mL), and the samples were then extracted as described above. Another set of blank plasma samples (six replicates) was also extracted. Following extraction, the mixed standard solution (10 μL) was added


where ASB is the chromatographic peak area of an analyte standard spiked in plasma before extraction; ASA is that of an analyte standard spiked in extracted blank plasma. CSB is the detected concentration of the analyte spiked in blank plasma before extraction and Cnominal is the nominal concentration of the analyte.



Analysis of bath salts in equine plasma using LC/MS/MRM Intra-day and inter-day precision The intra-day precision was investigated by analyzing samples at three different concentrations (0.5, 10, and 50 ng/mL) in six replicates in a single day. As for inter-day precision, three replicates of plasma samples at three different concentrations were analyzed in three consecutive days. The precision of the assay was expressed as the relative standard deviation (RSD) expressed as a percentage of the standard deviation (SD) divided by the mean observed concentrations (Xmean): % RSD ¼ SD=Xmean 100

(4)

Stability It has been shown that the cathinone content in fresh khat leaves is not constant whereas that in the dried leaves (’graba’) is more stable.[44] Thus, it is important to determine the stability of these analytes because post-race samples are not delivered to the laboratory immediately after the race. In the case of a positive finding, the split (B) sample under the security of the Commission at the racetrack may not be sent for independent analysis until after 15–30 days following the initial filing of a positive finding with the regulatory authority. The stability of the analytes was evaluated over short- and long-term intervals under different temperature conditions (room temperature, 4 °C, –20 °C, –70 °C ) as well as during freeze/thaw cycles and during the analysis of samples in the autosampler. Three different concentrations (0.2, 10, 50 ng/mL) in equine plasma were used. To assess any changes in the stability of the analytes, chromatographic peak areas averaged from triplicate samples were compared with those from samples stored for 0 h at room temperature and other samples at different temperature conditions to determine the percentage change of the concentrations of the analytes.

The various temperature conditions were chosen based on sample collection at the race tracks, and the handling, shipment and storage conditions to which the samples are subjected before and after receipt of the sample at the laboratory. The spiked plasma samples were allowed to remain on top of a laboratory bench at room temperature (25 °C) until the specific time (2, 4, 8 and 24 h) interval for analysis was attained. The analyte concentration in a control sample (0 h) was considered to be the point of reference (100%). For short-term stability, the post-race plasma samples are stored at 4 °C for a maximum of 7 days before they are transferred to 20 °C if analyses are not completed; the samples were analyzed at 1, 3, 5 and 7 days post-storage. For long-term storage stability, the plasma samples were stored at 20 °C and 70 °C for a maximum of 4 and 24 weeks, respectively. The temperature conditions represent short, intermediate long-term and long-term storages after the completion of analysis or the storage of research samples. The freeze/thaw cycle stability was also evaluated using three cycles. The plasma samples were thawed at room temperature for 2 h after they had been stored at 20 °C for 24 h. The samples were refrozen at 20 °C for 24 h. The process was repeated three times to determine the effect of freeze/thaw cycles on the stability of the analytes.

RESULTS AND DISCUSSION Liquid chromatography Three different stationary-phase columns, C18, phenyl and HILIC, were compared by chromatographic peak shape, retention time and resolution of the eleven target analytes. All three separation columns are capable of producing good chromatographic peak shapes using appropriate mobile




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Figure 1. Chemical structures of analytes studied. The m/z values given are for the [M + H]+ ions of the compounds.

X. Li et al.

Figure 2. LC/MS/MRM total ion current (top panel) and extracted ion chromatograms of 11 analytes separated on a C18 column (bottom panel).

Figure 3. LC/MS/MRM total ion current (top panel) and extracted ion chromatograms of 11 analytes resolved on a HILIC column (bottom panel).

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phase mixture. Cathinone, ephedrine and methylone are structurally analogous to amphetamine, methamphetamine and methylenedioxymethamphethamine (MDMA),


respectively.[45] Included in these analytes are four pairs of isomers comprising butylone and ethylone, buphedrone and mephedrone, 4-fluoromethcathinone (flephedrone) and



Analysis of bath salts in equine plasma using LC/MS/MRM 3-fluoromethcathinone, and 4-methoxymethcathinone (methedrone) and 3-methoxymethcathinone (Fig. 1). Some of the isomers share similar chemical structures; some are differentiated from each other by the position of substitution on the benzene ring. Thus, these compounds have the same precursor ion, same product ions and same fragmentation pattern. For these reasons, it is difficult to differentiate these isomers by using only results obtained from mass spectrometric analysis. However, chromatography provides another separation dimension through variable stationary-phase chemistry and optimization of mobile phase conditions to achieve resolution of one isomer from another. The C18 and phenyl columns did not separate all eleven analytes with adequately distinct resolution. In contrast, the hydrophilic interaction liquid chromatography (HILIC) column provided baselineresolution of each of the analytes. The term HILIC was first introduced by Alpert in 1990 during his evaluation of the hydrophilic partitioning between variable stationary phases and water.[46] Hydrophilic interaction chromatography is similar to normal-phase chromatography, and water content higher than 5% was used as a stronger eluting solvent. The

mechanism of HILIC involves partitioning, hydrogen bonding and electrostatic interaction.[47–49] Pure or functionalized silica is mostly used as the stationary phase in HILIC to create a ’water-enriched’ layer and render it highly hydrophilic. The water layer allows analytes to partition between the stationary and mobile phases, based on their polarity and electrostatic interaction. Thus, the more polar the compound, the more it associates with the aqueous phase, and the later it elutes (retention is increased). The organic-aqueous mixture for HILIC separation promotes free ionized silanols leading to a negatively charged surface which enhances the interaction resulting in increased retention of cationic and the repulsion of anionic analytes, which, in contrast, reduce retention.[32] The pH of the mobile phase and the ionic concentration influence the retention and selectivity of separation of the analytes from one another because they affect the charge state of the analyte and, thus, modify the strength of hydrophilic partitioning and ionic interaction. As the pH of the mobile phase is increased over the acidic range, surface silanols become negatively charged resulting in increased polarity of the stationary phase. Increase in molar strength

Table 2. Recovery, matrix effect, precision and accuracy Precision (%) Analyte Mephedrone Buphedrone 4-Fluoromethcathinone (flephedrone) 3-Fluoromethcathinone 3-Methoxymethcathinone Methedrone Methylone Ethylone Butylone MDPV Naphyrone

Concentration (ng/mL)

Recovery (%)

Matrix Effect (%)

Intra-day (n = 6)

Inter-day (n = 6)

Accuracy (%)

0.5 10 50 0.5 10 50 0.5 10 50 0.5 10 50 0.5 10 50 0.5 10 50 0.5 10 50 0.5 10 50 0.5 10 50 0.5 10 50 0.5 10 50

85.6 82.2 80.6 79.1 71.0 84.2 81.3 78.5 81.7 67.8 88.1 79.3 79.0 75.8 80.8 82.0 80.8 76.3 88.4 84.6 77.6 86.6 87.5 80.2 83.7 86.4 82.5 99.4 95.5 87.9 100.9 97.9 84.3

5.21 0.27 4.62 16.6 7.3 10.2 13.8 0.66 3.52 17.4 2.20 4.48 1.69 0.35 6.20 14.9 11.2 4.23 20.3 10.9 0.84 14.8 10.4 1.17 14.9 5.65 4.02 15.3 7.81 3.82 16.2 9.72 0.72

3.73 2.89 2.31 12.7 4.18 3.93 4.17 3.20 2.79 11.3 4.71 2.92 11.5 4.54 7.71 9.08 3.89 3.71 9.87 7.62 4.47 10.5 6.71 4.54 10.1 4.68 6.05 7.92 8.41 7.09 11.3 8.41 5.81

2.82 6.67 2.56 4.77 11.3 9.18 4.51 14.1 10.4 5.84 11.8 13.5 3.87 12.3 10.9 2.87 5.96 2.79 4.33 8.58 4.31 7.63 9.95 7.17 3.39 10.9 9.12 4.49 9.77 7.12 9.93 8.74 6.29

95.8 101 98.1 103 107 93.9 101 109 92.7 107 102 91.3 96.3 95.0 95.9 104 95.8 96.8 97.9 101 99.3 105 99.2 94.9 103 109 110 101 97.2 96.6 103 101 109



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ME = matrix effect, ’-’ represents ion suppression, ’ + ’ represents ion enhancement


X. Li et al. of the mobile phase results in increased retention of acidic compounds.[48,50–52] Since the mechanism of the HILIC mode is orthogonal to reversed-phase LC, the order of elution of the chromatograms of the target compounds on the C18 column (Fig. 2) was different from that of HILIC (Fig. 3). Naphyrone eluted earlier on the HILIC column than on the C18 column (3.60 min vs 7.58 min), and methedrone eluted later (8.24 min) on the HILIC than on the C18 column (4.39 min). Two pairs of the isomers, buphedrone-mephedrone and butylone-ethylone, were well resolved by both columns, but the resolution on the HILIC column was better than on the C18 column in terms of peak shape and distinction in the retention times (Figs. 2 and 3). However, methedrone and 3-methoxymethcathinone, 4-fluoromethcathinone and 3-fluoromethcathinone were baseline-resolved only by HILIC (Fig. 3).

spiked with each analyte (0.2 ng/mL) were compared to determine if any endogenous compounds co-eluted with the analytes. The results showed the absence of any interfering component from blank plasma eluting at the retention time of each of the analytes. Thus, LLE employed in the extraction of the analytes from equine plasma provided sufficiently clean sample extracts and all target analytes were wellresolved from any endogenous components in plasma. Thus, the method has high specificity and selectivity for the analysis of the eleven analytes in equine plasma.

Mass spectrometry All applicable MS parameters for all the analytes are shown in Table 1. Most cathinones are either straight-chain secondary amines or contain a pyrrolidine ring. A characteristic feature of these cathinones is the presence of a carbonyl group, which is located at the first carbon atom adjacent to the benzene ring. Thus, water loss with further α-cleavage is the most commonly observed fragmentation. However, the water loss transition (loss of 18 Da) was not chosen for screening or confirmation because it is not compound-specific. The product ions of the analytes in this study are shown in Table 1. The most intense precursorproduct ion transition of each analyte was used in screening for the specific compound whereas the three most intense MRM transitions of each specific analyte were used for confirmation of the presence of each specific analyte in equine plasma. Method validation High specificity is essential for a reliable screening and confirmation method to be employed in forensic analysis. Six different batches of blank plasma and blank plasma

Figure 4. Ion ratio comparison between the authentic standard (3,4-methylenedioxypyrovalerone (MDPV)) and the suspect sample. Top panel is an overlay of the three ion transitions of the QC sample spiked into blank plasma at 2.0 ng/mL, bottom panel is that of the three transitions of the suspect post-race plasma sample. The three MRM ion transitions for confirmation were m/z 276 → 126, m/z 276 → 205, and m/z 276 → 175.

Table 3. Linearity, LOD, LOQ, and LOC

Analyte Mephedrone Buphedrone 4-Fluoromethcathinone 3-Fluoromethcathinone 3-Methoxymethcathinone Methedrone Methylone Ethylone Butylone MDPV Naphyrone

Linear range (ng/mL)

LOD (ng/mL)

LOQ (ng/mL)

LOC (ng/mL)

0.2–50 0.2–50 0.2–50 1.0–50 0.2–50 0.2–50 0.2–50 0.2–50 0.5–50 0.2–50 0.2–50

0.05 0.05 0.05 0.5 0.05 0.02 0.05 0.02 0.2 0.05 0.02

0.2 0.2 0.2 1.0 0.2 0.2 0.2 0.2 0.5 0.2 0.2

0.2 0.2 0.2 1.0 0.2 0.2 0.2 0.2 0.5 0.2 0.2

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LOD = limit of detection; LOQ = limit of quantification; LOC = limit of confirmation




Analysis of bath salts in equine plasma using LC/MS/MRM The results of the extraction recovery are listed in Table 2. The average recovery of the analytes at three different concentrations was >70%, indicating that the LLE procedure effectively recovered all the analytes from the equine plasma. The matrix effect represents the effect of co-eluting residual matrix components on the ionization of the target analytes, resulting in either under-estimation (suppression) or overestimation (enhancement) of quantification results. For this reason, the matrix effect must be fully addressed to ensure accurate quantification results. Each analyte was spiked into blank plasma before and after LLE and the matrix effect was calculated according to Eqn. (1) (Table 2). Negative and positive values indicated that plasma induced minor ion suppression and enhancement, respectively. The contribution

to ion suppression/enhancement from equine plasma was relatively low, less than 20% for all the analytes studied, suggesting that the matrix effect on the analysis of all eleven analytes was negligible and, thus, insufficient to disqualify or diminish quantification results from the present method. The accuracy is the similarity of the measured concentration of a test sample to the nominal concentration; it can also be expressed by bias. The precision describes the degree of agreement among individual determinations of an analyte in multiple samplings of a homogeneous biological matrix. The precision was expressed as the relative standard deviation (RSD) calculated as a percentage of the standard deviation (SD) divided by the mean of the observed concentration. The results (Table 2) indicated that the method was accurate with

Table 4. Similarity of product ion intensity ratio* for confirmation of the presence of MDPV in equine plasma

Analyte

Ion transitions

Ion intensity ratio Standardsa (n = 18)

Ion intensity ratio Unknownb (x = 3)

Ion ratio Similarityc (%)

MDPV

m/z 276 → m/z 126 m/z 276 → m/z 175 m/z 276 → m/z 205

1.00 0.823 ± 0.023 0.820 ± 0.031

1.00 0.843 ± 0.027 0.823 ± 0.024

100.0 102.4 100.4

*Product ion intensity ratio = Ion intensity of the ion transition/ion intensity of the most intense ion transition Mean ion intensity ratio of the drug standards was determined by 18 calibrators and QCs of MDPV at different concentrations (0.2 ~ 50 ng/mL). b Mean ion intensity ratio of unknown sample was determined by 3 replicates of an unknown sample. c Ion ratio similarity (%) = chromatographic peak ion intensity ratio of unknown sample / chromatographic peak ion intensity ratio of the reference drug standard 100. a




225

Figure 5. Identification of MDPV by library searching: (A) EPI spectrum of an unknown substance in a post-race plasma sample; (B) that of the reference drug standard, MDPV, its EPI spectrum was previously stored in the inhouse library; (C) the hit list from the library search; (D) the hit compound information that clearly identifies MDPV as the culprit in the racehorse plasma sample.

X. Li et al. an acceptance limit of ±15% of the theoretical values; both intraday (within day) and inter-day (between-day) precision at three different concentrations did not exceed ±15%. Based on FDA guidance for bioanalytical method validation, the accuracy and precision of this method are acceptable.[53] All the analytes in the present study demonstrated good linearity within a compound-specific range of concentrations (Table 3). A linear regression model with a weighting factor of 1/x was used in describing the regression relationship since it generated the line of best fit. The correlation of determination (r2) for all analytes was ≥0.995. The limit of detection (LOD) was defined as the lowest concentration at which a compound in a test sample could be detected but not necessarily quantified. It can be estimated at a signal-to-noise ratio (S/N) ≥3. The LOD for the target analytes was 0.02–0.05 ng/mL, except for butylone and 3-fluoromethcathinone, where they were 0.2 ng/mL and 0.5 ng/mL, respectively (Table 3). The limit of quantification (LOQ) is the lowest quantity of an analyte that can be quantitatively determined with suitable precision (RSD) and accuracy (expressed by bias, the percentage deviation from the nominal reference value). The acceptable criteria for these

two parameters at LOQ are 20% for RSD and ±20% for bias. Based on these criteria, the LOQ for these analytes was 0.2 ng/mL except for butylone and 3-fluoromethcathinone where they were 0.5 ng/mL and 1.0 ng/mL, respectively. The limit of confirmation (LOC) was defined as the lowest concentration at which the product ions were sufficiently abundant to produce a stable product ion intensity ratio for the confirmation of each analyte. The LOC was 0.2–1.0 ng/mL depending on the analyte (Table 3). Screening for all eleven analytes in equine plasma In screening for each target analyte, one of the most intense precursor-product ion transitions specific to the compound of interest was chosen for MRM analysis (Table 1). In order to reduce the risk of false positives, an information-dependent acquisition (IDA) experiment was used to combine the MRM scan with an EPI scan; the spectrum obtained was compared with that of an authentic drug standard by searching the inhouse library for spectral match. A predefined criterion of 5000 counts per second was set for the activation of an IDA experiment. An in-house library was built based on three

Table 5. Stability of analytes in equine plasma at different temperature conditions and duration of storage Temperature and duration of storage Analyte

Conc. added (ng/mL)

0h

25 °C 24 h

4 °C 7 days

20 °C 30 days

70 °C 6 months

Freeze/thaw 3 cycles

0.5 10 50 0.5 10 50 0.5 10 50 0.5 10 50 0.5 10 50 0.5 10 50 0.5 10 50 0.5 10 50 0.5 10 50 0.5 10 50 0.5 10 50

0.48 9.59 51.7 0.46 10.1 47.9 0.46 9.97 43.5 0.44 10.1 48.0 0.45 9.05 50.0 0.46 10.2 50.6 0.47 9.92 49.1 0.48 10.4 51.2 0.47 9.13 51.6 0.48 9.52 50.2 0.40 9.36 50.4

0.42 9.23 44.0 0.36 9.9 37.1 0.22 4.67 22.5 0.32 2.5 15.9 0.18 4.09 33.3 0.46 11.0 50.2 0.45 10.1 46.2 0.47 10.82 47.2 0.45 10.4 47.8 0.5 10.1 49.7 0.37 7.42 41.5

0.37 7.53 36.9 0.47 10.3 40.2 0.44 6.08 27.2 0.34 3.37 24.9 0.39 4.91 29.2 0.37 10.2 43.8 0.38 10.2 44.5 0.35 10.1 44.5 0.38 9.54 44.2 0.39 8.91 45.6 0.35 8.30 42.1

0.51 9.29 48.2 0.47 11.6 46.0 0.42 9.61 45.0 0.42 8.56 44.9 0.44 8.93 48.0 0.54 12.1 59.9 0.56 10.6 59.8 0.48 11.3 57.7 0.57 10.8 55.7 0.51 10.2 56.0 0.48 8.67 51.1

0.50 9.87 49.7 0.45 10.5 45.7 0.44 10.1 44.8 0.42 10.7 45.6 0.42 9.25 46.0 0.45 11.6 53.3 0.46 10.6 49.8 0.53 11.3 51.7 0.50 11.2 55.1 0.51 10.0 49.6 0.53 9.99 49.4

0.46 11.2 53.2 0.50 10.7 52.1 0.51 10.3 51.5 0.35 9.10 43.4 0.41 8.24 42.7 0.42 10.8 47.5 0.41 8.70 46.5 0.42 10.7 46.2 0.42 10.4 45.7 0.44 10.1 45.7 0.52 11.1 52.1

Mephedrone Buphedrone 4-Fluoromethcathinone (Flephedrone) 3-Fluoromethcathinone 3-Methoxymethcathinone Methedrone Methylone Ethylone Butylone MDPV Naphyrone

226 wileyonlinelibrary.com/journal/rcm



Analysis of bath salts in equine plasma using LC/MS/MRM different collision energies achieved by collision energy (CE) and collision energy spread (CES), which were set at 35 eV and 15 eV, respectively. It has previously been demonstrated that by using a CES instead of three single CEs, the cycle time is reduced while providing richer information in one spectrum than in three single CEs.[54]

Confirmation of an analyte in a test sample Confirmation of the presence of an analyte in a test sample is required in doping control to demonstrate that the ’chemical fingerprints’ of the analyte in the sample are the same as those of an authentic drug standard. Since in most cases reported there was only one analyte present in each sample, a confirmation method of each compound was developed for each of the eleven analytes in the present study. For confirmation using a triple quadrupole instrument, it is generally accepted that chromatographic peaks detected by three MRM ion transitions at the same retention time are sufficient for confirmation of the presence of a target compound in a test sample. Retention time was also used as criterion for confirmation of these compounds. The results indicated that the retention time for each bath salt was reproducible, with less than 0.06 min variation (result was based on 150 calibrations and quality control (QC) samples for consecutive 10-day analyses). The ion intensity ratio is commonly used for the confirmation of compounds in an unknown sample when analyzed on a triple quadrupole instrument. Usually, the three most intense precursor-product ion transitions are chosen for calculating the ion intensity ratio. The product ions employed in confirmation of similarity in ion intensity ratios are shown in Table 1. The similarity in ion ratio was calculated according to the following equation:

matched that of the calibrator and a library search result showed an indisputable match for MDPV. To avoid any confusion, MDPV has an ethyl group substitution at the R4 position instead of a methyl group at the same position in 3,4methylenedioxy-α-pyrrolidinobutiophenone,[55] a synthetic cathinone that was developed in 1960. To date, MDPV has been reported in post-race samples in two jurisdictions in North America using the present method. Thus, the criteria for the confirmation of the presence of the eleven analytes in equine plasma were defined as: (1) the retention time of the target compound in an unknown sample must be within ±0.06 min of those of QC samples; (2) chromatographic peaks should be present on three ion transitions of the target compound at the same retention time; and (3) the similarity in ion intensity ratio between the unknown sample and the QC samples must be within 90–110%. In addition, the use of full product ion spectra as additional evidence in support of the established criteria for confirmation is highly recommended (Fig. 5). Stability The eleven bath salts in the present study were stable during short-term and long-term storage as well as during three freeze/thaw cycles (Table 5). They were stable for at least

Ion intensity ratio similarity ð%Þ ¼ Runknown =Rstandard 100 (5)


Figure 6. Stability of three analytes (3-fluoromethcathinone, 4-fluoromethcathinone (flephedrone) and 3-methoxymethcathinone). (A) Stability at room temperature (≈25 °C) stored for up to 24 h; (B) stability at 4 °C for up to 168 h (7 days). Y-axis is the percentage concentration compared with 0 h, X-axis is the duration of storage in hours. 0 h represents the concentration of 100%.



227

where Runknown represents the ion intensity ratio for an unknown sample and Rstandard is the ratio of an authentic drug standard (calibrators and QC samples). Figure 4 and Table 4 show ion intensity ratio comparison of MDPV which was present in a test sample collected from a post-race sample with that of its reference standard. The result obtained showed that the unknown compound in the test sample and the reference drug standard had similar ion ratios and, therefore, the two were confirmed as being the same compound, MDPV. A full product ion spectrum was used as an additional criterion for the confirmation of each analyte in a test sample. Although a triple quadrupole instrument is not known for producing reproducible full product ion spectra, in some instruments, using hybrid technology such as the QTRAP, Q3 can either be operated in classical triple quadrupole mode, or used as a sensitive linear ion trap to provide an opportunity to acquire a full product ion spectrum for analyte identification. An MRM-triggered enhanced product ion (EPI) scan was performed and the EPI spectrum that resulted was compared with that of the authentic reference drug standard in the in-house spectral library. Spectral comparison of a postrace plasma sample that tested positive for the presence of MDPV was performed with an MDPV drug standard (Fig. 5). The spectrum of the ’real world’ sample unequivocally

X. Li et al. 6 months at 70 °C, 30 days at 20 °C and after 3 freeze/ thaw cycles. A good number of the analytes were stable when stored at room temperature. However, it should be noted that three of the analytes, 4-fluoromethcathinone, 3-fluoromethcathinone and 3-methoxymethcathinone, showed a declining trend in concentration after storage at room temperature for 24 h. Approximately 30% of the original concentration of 3-fluoromethcathinone was detected and the same trend was applicable when the analyte was stored at 4 °C for 7 days. It is important to emphasize the need to pay closer attention to these three analytes because of their profound instability in plasma (Fig. 6). In a published study, Sørensen showed that the same trend was observed when a blood sample was preserved with NaF/potassium oxalate.[56] Thus, post-race plasma samples should be analyzed for cathinone derivatives upon receipt by the laboratory; otherwise, they should be stored at 20 °C pending analysis within 7–30 days post-collection.

CONCLUSIONS A highly selective and sensitive HILIC-LC/MS/MS method for the simultaneous screening, quantification and confirmation of eleven cathinone derivatives in equine plasma was developed and validated. For quantification, good linearity, precision and accuracy were achieved. Full product ion spectra provided additional evidence to similarity in ion intensity ratio and retention time as criteria for confirmation of the presence of the analytes in equine plasma. The method is routinely used in the screening of postcompetition plasma samples obtained from racehorses in Pennsylvania. The method is fast, robust, sensitive, selective and reliably reproducible.

Acknowledgements The authors thank the PA Racing Commissions for providing financial support for this study. The PA Harness Horsemen Association at Pocono and Harrah’s Philadelphia, Meadows Standardbred Owners Association and Horsemen Benevolent and Protective Association at Penn National and Presque Isles Downs also made financial contributions and to them the authors are grateful.

REFERENCES

228

[1] D. M. Wood, S. Davies, S. L. Greene, J. Button, D. W. Holt, J. Ramsey, P. I. Dargan. Case series of individuals with analytically confirmed acute mephedrone toxicity. Clin. Toxicol. (Phila.) 2010, 48, 924. [2] P. D. Maskell, G. D. Paoli, C. Seneviratne, D. J. Pounder. Mephedrone (4-methylmethcathinone)-related deaths. J. Anal. Toxicol. 2011, 35, 188. [3] I. Vardakou, C. Pistos, C. Spiliopoulou. Spice drugs as a new trend: mode of action, identification and legislation. Toxicol. Lett. 2010, 197, 157. [4] H. Torrance, G. Cooper. The detection of mephedrone (4-methylmethcathinone) in 4 fatalities in Scotland. Forensic Sci. Int. 2010, 202, e62.


[5] P. Nencini. Cathinone, active principle of the khat leaf: its effects on in vivo and in vitro lipolysis. Pharmacol. Res. Commun. 1980, 12, 855. [6] M. Odenwald, N. Warfa, K. Buhl, T. Elbert. The stimulant khat – another door in the wall? A call for overcoming the barriers. J. Ethnopharmacol. 2010, 132, 615. [7] A. Klein, P. Metal. A good chew or good riddance – how to move forward in the regulation of khat consumption. J. Ethnopharmacol. 2010, 132, 584. [8] K. Szendrei. The chemistry of khat. Bull. Narcotics 1980, 32, 5. [9] European Monitoring Center for Drugs and Drug Addiction (EMCDDA), Synthetic cathinones. http://www.emcdda, europa.eu/publications/drug.profiles/synthetic.cathinones (accessed May 2, 2013). [10] N. V. Cozzi, M. K. Sievert, A. T. Shulgin, P. Jacob III, A. E. Ruoho. Inhibition of plasma membrane monoamine transporters by beta-ketoamphetamines. Eur. J. Pharmacol. 1999, 381, 63. [11] J. M. Prosser, S. Nelson. The toxicology of bath salts: a review of synthetic cathinones. J. Med. Toxicol. 2012, 8, 33. [12] D. Kebede, A. Alem, G. Mitike, F. Enquselassie, F. Berhane, Y. Abebe, R. Ayele, W. Lemma, T. Assefa, T. Gebremichael. Khat and alcohol use and risky sex behavior among in-school and out-of-school youth in Ethiopia. BMC Public Health 2005, 5, 109. [13] V. C. Freund-Michel, M. A. Birrell, H. J. Patel, I. M. MurrayLyon, M. G. Belvisi. Modulation of cholinergic contractions of airway smooth muscle by cathinone; potential beneficial effects in airway diseases. Eur. Respir. J. 2008, 32, 579. [14] C. D. Murphy, C. W. Le Roux, A. V. Emmanuel, J. M. Halkaet, A. M. Przyborrowska, M. A. Kamm, I. M. Murray-Lyon. The effect of khat (Catha edulis) as an appetite suppressant is independent of ghrelin and PYY secretion. Appetite 2008, 51, 747. [15] S. Gardos, J. O. Cole. Evaluation of pyrovalerone in chronically fatigued volunteers. Curr. Ther. Res. Clin. Exp. 1971, 13, 631. [16] H. A. Spiller, M. L. Ryan, R. G. Weston, J. Jansen. Clinical experience with and analytical confirmation of ’bath salts’ and ’legal highs’ (synthetic cathinones) in the United States. Clin. Toxicol. (Phila.) 2011, 49, 499. [17] Department of Justice, Drug Enforcement Administration. 21 CFR Part 1308. Fed. Reg. 2011, 76, 204. [18] S. Strano-Rossi, A. B. Cadwallader, X. de la Torre, F. Botrè. Toxicological determination and in vitro metabolism of the designer drug methylenedioxypyrovalerone (MDPV) by gas chromatography/mass spectrometry and liquid chromatography/quadrupole time-of-flight mass spectrometry. Rapid Commun. Mass Spectrom. 2010, 24, 2076. [19] M. R. Meyer, C. Vollmar, A. E. Schwaninger, E. Wolf, H. H. Maurer. New cathinone-derived designer drugs 3-bromomethcathinone and 3-fluoromethcathinone: studies on their metabolism in rat urine and human liver microsomes using GC-MS and LC-high-resolution MS and their detectability in urine. J. Mass Spectrom. 2012, 47, 253. [20] R. Brennelsen, H. U. Fisch, U. Koelbing, S. Geischuler, P. Kalix. Amphetamine-like effects in humans of the khat alkaloid cathinone. Br. J. Clin. Pharmacol. 1990, 30, 825. [21] P. Widler, K. Mathys, R. Brennelsen, P. Kalix, H. U. Fisch. Pharmacodynamics and pharmacokinetics of khat: a controlled study. Clin. Pharmacol. Ther. 1994, 55, 556. [22] A. N. Guantai, C. K. Maitai. Metabolism of cathinone to d-norpseudoephedrine in humans. J. Pharm. Sci. 1983, 72, 1217. [23] K. Mathys, R. Brennelsen. Determination of (S)-()cathinone and its metabolites (R,S)-()-norephedrine and (R,R)-()-norpseudoephedrine in urine by high-performance liquid chromatography with photoiode-array detection. J. Chromatogr. 1992, 593, 79.



Analysis of bath salts in equine plasma using LC/MS/MRM [24] G. Frison, M. Gregio, L. Zamengo, F. Zancanaro, S. Frasson, R. Sciarrone. Gas chromatography/mass spectrometry determination of mephedrone in drug seizures after derivatization with 2,2,2-trichloroethyl chloroformate. Rapid Commun. Mass Spectrom. 2011, 25, 387. [25] S. D. Brandt, S. Freeman. Analysis of NRG ’legal highs’ in the UK: identification and formation of novel cathinones. Drug Test. Anal. 2011, 3, 569. [26] D. Zuba. Identification of cathinones and other active components of ’legal highs’ by mass spectrometric methods. Trends Anal. Chem. 2012, 32, 15. [27] P. Jankovics, A. Váradi, L. Tölgyesi, S. Lohner, J. NémethPalotás, H. Kőszegi-Szalai. Identification and characterization of the new designer drug 4’- methylethcathinone (4-MEC) and elaboration of a novel liquid chromatography-tandem mass spectrometry (LC-MS/MS) screening method for seven different methcathinone analogs. Forensic Sci. Int. 2011, 210, 213. [28] C. Bell, C. George, A. T. Kickman, A. Traynor. Development of a rapid LC-MS/MS method for direct urinalysis of designer drugs. Drug Test. Anal. 2011, 3, 496. [29] S. Strano-Rossi, L. Anzillotti , E. Castrignanò, F. S. Romolo, M. Chiarotti. Ultra high performance liquid chromatographyelectrospray ionization-tandem mass spectrometry screening method for direct analysis of designer drugs, "spice" and stimulants in oral fluid. J. Chromatogr. A 2012, 1258, 37. [30] D. Ammann, M. J. McLaren, D. Gerostamoulos, J. Beyer. Detection and quantification of new designer drugs in human blood: Part 2 – Designer cathinones. J. Anal. Toxicol. 2012, 36, 381. [31] R. E. Jamt, A. Gjelstad, L. E. Eibak, E.L. Øiestad, A. S. Christophersen, K. E. Rasmussen, S. Pedersen-Bjergaard. Electromembrane extraction of stimulating drugs from undiluted whole blood. J. Chromatogr. A 2012, 1232, 27. [32] J. Beyer, F. T. Peters, T. Kraemer, H. H. Maurer. Detection and validated quantification of nine herbal phenalkylamines and methcathinone in human blood plasma by LC-MS/MS with electrospray ionization. J. Mass Spectrom. 2007, 42, 150. [33] S. R. Needham, P. R. Brown, K. Duff, D. Bell. Optimized stationary phases for the high-performance liquid chromatography–electrospray ionization mass spectrometric analysis of basic pharmaceuticals. J. Chromatogr. A 2000, 869, 159. [34] T. M. Brunt, A. Poortman, R. J. Neisink, W. van den Brink. Instability of the ecstasy market and a new kid on the block: mephedrone. J. Psychopharmacol. 2011, 25, 1543. [35] The state of the drugs problem in Europe. EMCDDA, Lisbon, Portugal, 2009. http://www.emcdda.europa.eu/ attachements.cfm/att_93236_EN_EMCDDA_AR2009_EN. pdf (accessed May 2, 2013). [36] J. DeRuiter, L. Hayes, A. Valaer, C. R. Clark. Methcathinone and designer analogues: synthesis, stereochemical analysis, and analytical properties. J. Chromatogr. Sci. 1994, 32, 552. [37] T. Yoshida, T. Okada. Peptide separation in normal-phase liquid chromatography. Study of selectivity and mobile phase effects on various columns. J. Chromatogr. A 1999, 840, 1. [38] K. Buck, P. Voehringer, B. Ferger. Rapid analysis of GABA and glutamate in microdialysis samples using high performance liquid chromatography and tandem mass spectrometry. J. Neurosci. Methods 2009, 182, 78.

[39] A. J. Alpert, A. K. Shukala, L. R. Zieskw, S. W. Yuen, M. A. J. Ferguson, A. Mehlert, M. Pauly, R. J. Orlando. Hydrophilic-interaction chromatography of complex carbohydrates. J. Chromatogr. A 1994, 676, 191. [40] W. Jian, R. W. Edom, Y. Xu, N. Weng. Recent advances in application of hydrophilic interaction chromatography for quantitative bioanalysis. J. Sep. Sci. 2010, 33, 681. [41] P. Li, W. D. Beck, P. M. Callahan, A. V. Terry Jr, M. G. Bartlett. Quantitation of cotinine and its metabolites in rat plasma and brain tissue by hydrophilic interaction chromatography tandem mass spectrometry (HILIC-MS/MS). J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 2012, 907, 117. [42] P. Nencini, A. M. Ajmed. Khat consumption: a pharmacological review. Drug Alcohol Depend. 1989, 23, 19. [43] N. B. Patel. Mechanism of action of cathinone: the active ingredient of khat (Catha edulis). East Afr. Med. J. 2000, 77, 329. [44] J. S. Chappell, M. M. Lee. Cathinone preservation in khat evidence via drying. Forensic Sci. Int. 2010, 195, 108. [45] S. Gibbons, M. Zloh. An analysis of the ’legal high’ mephedrone. Bioorg. Med. Chem. Lett. 2010, 20, 4135. [46] A.J. Albert. Hydrophilic-interaction chromatography for the separation of peptides, nucleic acids and other polar compounds. J. Chromatogr. A 1990, 499, 177. [47] P. Hemstrom, K. Irgum. Hydrophilic interaction chromatography. J. Sep. Sci. 2006, 29, 1784. [48] Y. Guo, S. Gaiki. Retention behavior of small polar compounds on polar stationary phases in hydrophilic interaction chromatography. J. Chromatogr. A 2005,1074,71. [49] P. Jandera. Stationary phases for hydrophilic interaction chromatography, their characterization and implementation into multidimensional chromatography concepts. J. Sep. Sci. 2008, 31, 1421. [50] D. V. McCalley. Is hydrophilic interaction chromatography with silica columns a viable alternative to reversed-phase liquid chromatography for the analysis of unstable compounds? J. Chromatogr. A 2007, 1171, 46. [51] N. Weng. Bioanalytical liquid chromatography tandem mass spectrometry methods on underivatized silica columns with aqueous/organic mobile phases. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 2003, 796, 209. [52] M. Storton, J. Exarchalkkis, T. Waters, Z. Hao, B. Parker, M. Knapp. Lactic acid quantitation in hand dishwashing liquid using an HILIC-UV methodology. J. Sep. Sci. 2010, 33, 982. [53] U.S. Food and Drug Administration. Guidance for Industry Bioanalytical Method Validation. Available: http://www. fda.gov/downloads/Drugs/Guidances/ucm070107.pdf. [54] Y. Liu, C. E. Uboh, L. R. Soma, X. Li, F. Guan, Y. You, J. W. Chen. Efficient use of retention time for the analysis of 302 drugs in equine plasma by liquid chromatographyMS/MS with scheduled multiple reaction monitoring and instant library searching for doping control. Anal. Chem. 2011, 83, 6834. [55] F. Westphal., T. Junge, P. Pösner, F. Sönnichsen, F. Schuster. Mass and NMR spectroscopic characterization of 3,4methylenedioxypyrolvalerone a designer drug with a pyrrolidinophenone structure. Forensic Sci. Int. 2009, 190, 1. [56] L. K. Sørensen. Determination of cathinones and related ephedrines in forensic whole-blood samples by liquidchromatography-electrospray tandem mass spectrometry. J. Chromatogr. B 2011, 879, 727.

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