Determination of chiral cathinone in fresh samples of Catha edulis

Journal Pre-proof Determination of Chiral Cathinone in Fresh Samples of Catha Edulis Abdulrhman M. Dhabbah (Conceptualization) (Methodology) (Data curation) (Writing - original draft) (Visualization) (Investigation) (Validation) (Writing - review and editing)

PII:

S0379-0738(19)30517-1

DOI:

https://doi.org/10.1016/j.forsciint.2019.110105

Reference:

FSI 110105

To appear in:

Forensic Science International

Received Date:

4 September 2019

Revised Date:

30 November 2019

Accepted Date:

2 December 2019

Please cite this article as: Dhabbah AM, Determination of Chiral Cathinone in Fresh Samples of Catha Edulis, Forensic Science International (2019), doi: https://doi.org/10.1016/j.forsciint.2019.110105

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Determination of Chiral Cathinone in Fresh Samples of Catha Edulis

Abdulrhman M. Dhabbah

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Department of Forensic Sciences, King Fahad Security College, Riyadh, Kingdom of Saudi Arabia

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e-mail: [email protected]

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Highlights

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 Derivatization of S and R cathinone with menthyl chloroformate  Full separation of S and R cathinone diastereomers by GC-MS 

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 Determination of S and R cathinone in leaves and stems of fresh Khat Both S and R cathinone detected in all samples

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 S-cathinone concentration higher in stems but lower in leaves

Abstract

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The main psychoactive compound in Khat is cathinone which consists of two enantiomers, S-(-)cathinone being more stimulant than its R antipode. This study aimed to the enantioseparation and determination of these two stereoisomers in different parts of fresh Catha edulis. The samples were solvent extracted and cathinone was derivatized with menthyl chloroformate. The separation of the two diastereomeric derivatives was carried out by gas chromatography and showed an excellent resolution, while their structure was confirmed by mass spectrometry. The quantitative determination of both enantiomers showed a different distribution in various investigated parts of the plant, as shown in their enantiomeric excess. Unlike the results published in some previous articles, the current study confirmed the presence of both S and R cathinone in all parts of the fresh plant. The concentration of S-cathinone was higher in stems while its values were lower in leaves. The obtained concentrations

were in the ranges 0.081-0.290 and 0.087-0.211 mg/g for S and R antipodes, respectively. Also, Scathinone which is the most psychoactive stereoisomer showed an increasing concentration from lower to upper stems of the plant. The present study is the first quantitative investigation of the two cathinone enantiomers in different parts of fresh Catha edulis.

Keywords: Catha edulis, S/R-cathinone, Derivatization, menthyl chloroformate,

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Enantioseparation, GC-MS

Introduction

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Several psychoactive plants are well-known in different regions of the world and traditionally used for centuries by local populations. Among these species, Catha edulis Forsk, also known as Khat is an evergreen wild shrub or tree cultivated at high altitudes, and widely used in East Africa and Arabian Peninsula. While its consumption was in the past limited to these regions, nowadays it is widespread in more countries, as a result of increasing worldwide migration routes [1,2]. Khat is a seedless and robust plant which grows in various environments. Its leaves have a characteristic aroma with an astringent and sweet taste [3].

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The young parts of Khat fresh buds and leaves are traditionally chewed in groups during several hours to reach the desired state of excitement and euphoria [4]. This social habit induces a strong addiction to Khat consumption and it has several negative effects on consumers health. Moreover, the widespread use of this euphoriant plant also severely affects the economic and social development of these areas, as a result of decreasing both productivity and performance [5]. Indeed, several previous studies have established a link between Khat chewing and its severe economic consequences such as increasing unemployment, poverty and criminality [4]. In spite of its harmful health effects, addiction to Khat forces its consumers to spend all their income in the acquisition of this plant. In addition, the growing demand leads to the replacement of traditional agricultural production by the cultivation of Catha edulis [5,6]. While the consumption of Khat was in the past limited to Arabic Peninsula and East African countries, it is now available in Europe and America, particularly for populations originating from those regions. Thus the increasing use of Catha edulis induced higher incidence of mental health problems [2]. The extent of this phenomenon appears in the number of Khat consumers in these affected regions which is estimated to several millions, corresponding to about 60 to 80% of the adult population [5]. The most common ingestion technique consists of chewing the fresh plant in a group

session, while the dried leaves can also be smoked or used as drink. The plant material is slowly chewed and kept in mouth to allow continuous releasing of the active compounds [3]. Similarly to amphetamine and its related synthetic stimulants which have well established appetite suppression effects, Khat consumption induces chronic malnutrition with low body weight [7]. This anorexia increases the occurrence of infections, particularly tuberculosis. In addition, chewing Catha edulis leaves can cause several adverse other psychiatric and health effects such as cardiovascular, dental and gastrointestinal diseases [4]. Among the various effects related to Khat addiction, several papers reported depression, irritability, insomnia, lethargy, constipation, stomatitis, hypertension and myocardial insufficiency [4,6,8].

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In biological systems stereochemistry has a great influence on the activity of chiral compounds and their pharmacodynamic and pharmacokinetic behavior [9]. The use of chiral analysis in forensic toxicology has rapidly increased; it proved to be useful in many fields such as characterization of illicit drugs as well as the determination of cause of death [10,11]. Since drugs of abuse are now considered as emerging environmental pollutants, their chirality is regarded as a major parameter which greatly influences their toxicity towards living organisms [12]. Several previous studies reported that the S enantiomer of amphetamine and its related compounds exhibit higher psychoactive effects than the R antipode [13–15]. However, there is no agreement about the magnitude of the physiological activities of the two enantiomers [16]. Similarly, many reports have established that cathinone is the main psychoactive component in Khat and that its S enantiomer is more stimulant that the R antipode [13,17–19]. This difference of the pharmacological effects between the two enantiomers was established for many amphetamine related compounds where the S form was found to be more active than the R antipode [17]. On the other hand, S-(-)-cathinone was detected only in Catha edulis so, its presence is characteristic of this plant species [20].

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Several techniques were used for enantioseparation and determination of chiral drugs, such as gas chromatography [10,13], liquid chromatography [21–23] and supercritical fluid chromatography [24]. GC-MS is the most popular method for investigation of chiral bioactive compounds in various samples. It is generally achieved by indirect chiral separation after derivatization of the enantiomers using an enantiomeric pure reagent. This step transforms the two active antipodes into diastereomers which can be resolved on common non-chiral stationary phases [22].

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Among the available chiral derivatization agents, R-(-)-menthyl chloroformate (MCF) was used for successful enantioseparation of various amphetamine derivatives [22]. This method proved to have several advantages such as a low detection limit and a wide linear range [25]. (R)-(+)-α-methoxy-αtrifluoromethylphenylacetic acid (MTPA) was also used for easy derivatization of many amphetamine related compounds, followed by their efficient gas chromatographic enantioseparation on a conventional capillary column [22,26]. Trifluoroacetyl-L-prolyl chloride (L-TPC) is another reagent which allowed convenient derivatization and indirect chiral separation of many amphetamine and cathinone derivatives by GC-MS [13]. Similarly to that of amphetamine, the structure of cathinone and its derivatives consists of a propyl chain with a phenyl group in position 1 and an amino

function in position 2. Since cathinone and its synthetic derivatives are optically active, the biological activities of the two enantiomers are different. Many previous reports indicated that the S(-) enantiomer has a higher psychoactive effect than the R antipode [27]. This stronger stimulant potency of S-(-)-cathinone was explained by its better affinity to the receptor sites [28]. Effectively, several studies showed that S-(-)-cathinone is the main stereoisomer present in Khat, while R-(+)-cathinone is present in smaller amount [7].

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Although several studies investigated the enantioseparation of S and R cathinone and their derivatives [13,24], no previous study mentioned the quantitative determination of these enantiomers in Catha edulis samples. The present work aims to the chiral separation and determination of S and R cathinone in different parts of fresh Khat samples by GC-MS after their derivatization using MCF.

2. Experimental 2.1. Extraction from Catha edulis samples

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A fresh sample of Catha edulis was obtained from Jazan (south of Saudi Arabia). This branch was cut into 8 parts by separation of leaves (L1 to L4) and stems (S1 to S4), as shown in Figure 1. Two other samples named LM and SM were composed of mixed parts of leaves and stems from another branch. Each of the ten fresh samples was weighed, then finely cut using a mixer and soaked in 20 mL of methanol for 24 h. The extraction procedure was first optimized using 10, 20 and 30 mL of methanol, while several extraction times were also used (6, 12 and 24 hours). The highest extraction yield was obtained 20 mL of methanol during 24 h. The mixture was then stirred at ambient temperature for 20 min. and centrifuged at 3000 rpm for 5 min. Each methanol extract was then filtered using 0.45 µm syringe filter and stored at 4ºC. In the present work, the proposed procedure consisted of extraction of fresh Khat samples without drying; also it did not use any acid or basic treatment to avoid keto amino tautomerization which could result in cathinone racemization [29].

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2.2. Chemicals and derivatization procedure

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S-(-)-cathinone (purity: 98%), R-(-)-menthyl chloroformate (MCF) (purity: 99%), methanol (purity ≥ 99.9%) and triethylamine (purity ≥ 99%) were purchased by Sigma Aldrich (Merck KGaA, Darmstadt, Germany). A series of standard solutions of S-(-)-cathinone in methanol were prepared with the following concentrations: 1, 10, 25, 50, 75 and 100µg.mL-1. The derivatization of cathinone was carried out using the following procedure which was previously described [22]. A 500µL sample of each standard solution or extract was mixed with 20µL of triethylamine and 10µL of R-(-)menthylchloroformate. The solution was stirred for 10 min. at ambient temperature, then injected into GC-MS instrument three times.

2.3. Gas chromatography-mass spectrometry conditions

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The standard samples and extracts were analyzed by GC/MS on a Agilent 6890N GC system equipped with a Agilent 6890 Series Injector autosampler, a split-splitless injection system and hyphenated to a 5973 mass selective detector. The separation was carried out on a HP-5 MSI capillary column (30 m length, 0.25 mm i.d., and 0.25 µm film thickness). The injector temperature was set at 250°C and helium was used as carrier gas with a constant flow of 1.0 mL/min. The sample volume was 1 µL and it was injected in splitless mode. The column temperature was programmed as follows: 160°C for 5 min, then heated to 230°C at a rate of 2°/min and held at 230°C for 1 min. The mass spectra were recorded in electron impact mode with ionization energy of 70 eV, whereas the ion source temperature was 230 °C with a scan range from 50 to 550 Da.

3. Results and Discussion

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Several previously published reports have established that the S-isomers of cathinone and amphetamine, as well as their related compounds exhibit notably stronger stimulating effects on the central nervous system than their R enantiomers [13,17,18]. Although all previous studies agreed that S-(-)-cathinone is the most potent compound in Khat, the presence of its R enantiomer in fresh plant is still controversial. Some authors mentioned the presence of both enantiomers, sometimes as racemic mixture; this indicates that the naturally occurring cathinone may undergo a racemization in the plant [7,20]. In contrast, it was also reported in other papers that Khat samples contain only the S active form of cathinone, while the R-(+)-enantiomer was not detected [29–31]. The current study aims to clarify this point by investigating the presence of both cathinone antipodes in Catha edulis.

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Figure 2A shows the chromatogram of standard S-cathinone after derivatization with menthyl chloroformate. The peak observed at 20.902 min. corresponds to the derivatization agent in excess (MCF) while the S-cathinone derivative was eluted at 31.976 min. The small peak which appeared at 32.564 min. was clearly due to the diastereomer product resulting from the optical impurity S-(+)menthyl chloroformate present in MCF. This result agrees with the optical purity 98.5% of MCF indicated by the supplier. Also, the chromatogram does not show any presence of the R-cathinone derivative, this result confirms first the high optical purity of S-cathinone standard. It means also that the derivatization process does not induce any racemization of the S enantiomer under the selected experimental conditions. The mass spectra shown in Figures 2B, 2C and 2D correspond to menthyl chloroformate, S-cathinone derivative and its diastereomer formed with S-(+)-menthyl chloroformate impurity, respectively. The two mass spectra 2C and 2D are very similar since the derivatization with MCF yields two diastereomers of S-cathinone resulting from its reaction with R-(-)-MCF and S-(+)MCF impurity.

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A series of standard solutions of S-cathinone with concentrations ranging from 1 to 100µg/mL were prepared in methanol from a 1000µg/mL stock solution. It should be highlighted that the two diastereomers resulting from cathinone derivatization with MCF are completely separated with an excellent resolution RS of 5.15. The retention time obtained with S-(-)-cathinone standard derivatized with MCF corresponded to that of S-(-)-cathinone in the different extracts. In addition, the mass spectra of both cathinone diastereomers observed in all Khat extracts were similar to that of pure S(-)-cathinone derivative. The obtained calibration curve showed a good linearity in the investigated range with a correlation coefficient of 0.995. The limit of detection (LOD) was estimated to 0.15 µg/mL while the limit of quantitation (LOQ) was 0.5 µg/mL. Repeatability of the results was checked by injecting each standard sample and extract in triplicate in the same day. Figure 3A shows the chromatogram of leaf extract L4 after derivatization with menthyl chloroformate; the three main peaks observed refer to: MCF (20.938 min.), S-cathinone derivative (31.857 min.) and R-cathinone derivative (33.887 min.). Their corresponding mass spectra obtained in electron impact mode are shown in Figures 3B, 3C and 3D. The two mass spectra of S and R cathinone diastereomers show the same main fragments at m/z 83, 226, 105, 182 and 139. As it was

observed in the chromatogram of standard S-cathinone-MCF derivative (Figure 2), Figure 3A also shows two small peaks at 32.585 and 34.198 min which correspond to the diastereomers of S and Rcathinone derivatives formed with the S(+) impurity of MCF. The chromatogram in Figure 4A shows the separation of stem extract S1 after its derivatization. The main peaks identified in this chromatogram are eluted at 20.890, 31.839 and 33,772 min., they correspond to the excess of MCF reagent, S and R-cathinone derivatives, respectively. Their mass spectra are reported in Figures 4B, 4C and 4D.

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The concentrations of S and R cathinone enantiomers in all investigated leaf and stem samples were calculated, their values are mentioned in Table 1. It should be first highlighted that both enantiomers are present in all parts of freshly cut Catha edulis samples, while some authors mentioned that Scathinone occurs only in young leaves and not in the fully developed parts of the plant [29]. Also, this important result confirms those reported by some previously published articles which mentioned the presence of both S and R cathinone antipodes in Khat [7,20]. Another interesting feature deduced from Table 1 is that the concentrations of S-cathinone, the most active enantiomer, are in the ranges 0.138-0.290 mg/g and 0.081-0.124 mg/g in stems and leaves respectively, the highest value being observed in the upper stem S4. This result corroborates the previously reported observation that Khat consumers prefer to chew the younger parts of plant which are more stimulating [8,32]. Nevertheless, Krizevski et al. mentioned that the higher acitivity of the upper portion of Catha edulis plant is mainly due to the greater concentration of S-cathinone in leaves while the present results established that its content is notably higher in stems [29].

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On the other hand, comparison of the R-cathinone values in stems and leaves shows that they are in the intervals 0.087-0.186 mg/g and 0.105-0.211 mg/g, respectively. The highest concentration of Rcathinone corresponds to the upper leaf L4, while it shows a low content of the S(-) antipode. The profile of the two enantiomers in the different parts of the fresh plant is remarkably different; while S-cathinone is more abundant in all stem samples, the R antipode content is higher in all leaf samples. Comparison of the total concentration of cathinone shows that it ranges between 0.225 and 0.476 mg/g in stems, whereas its value in leaves is between 0.186 and 0.318 mg/g.

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According to Table 1, the total concentration of cathinone found in the present study show values in the range 0.225-0.476 mg/g in stems and 0.186-0.318 mg/g in leaves. These values found in fresh samples can be compared with those reported in some previous articles. In a paper published in 2009, the total concentration of cathinone in four cultivars of Catha edulis obtained by Krizevski et al. using GC-MS was between 0.085 and 0.188 mg/g (fresh material), these results being in the same range as ours [29]. In another report published in 1993 by Mathys and co-authors, the S-(-)-cathinone concentration measured by HPLC-DAD and TLC varied between 0.002182 and 0.004506 mg/g of dried material in khat originating from Kenya [33]. The concentrations of S(-)-cathinone mentioned in this article are much lower than the levels measured in the present study as well as in other investigations. Roda et al. in 2013 used capillary electrophoresis for determination of active principles in Catha edulis. They obtained the following results for cathinone in three dried bundles (in mg/g):

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0.277, 0.909 and 1.080, these values being higher than those given in Table 1 which were given for fresh samples [34]. The same research group investigated the main active components of Khat using gas chromatography after derivatization with N-methyl-N-trimethylsilyl-trifluoroacetamide (MSTFA). The cathinone weight percent in dried samples was found in range between 0.0100 and 0.119% [35]. On the other hand, determination of cathinone in fresh Khat was also carried out by Lausmann and Meier-Giebing in 2010 by cation-exchange liquid chromatography; they obtained a value of 1.042 mg/g which is higher than the results found in the present research [36]. The concentration of cathinone in leaves and stems of fresh Catha edulis seizures was determined by Gambaro et al. in 2012 using GC-MS. They results were in the following ranges: 0.390-0.995 and 0.236-0.641 mg/g of fresh leaves and stems, respectively [37].

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Since most synthetic and natural drugs are chiral, their stimulating potency depends mainly on the enantiomeric ratio of their active compounds [9]. In order to differentiate the different parts of fresh Khat according to the ratio between the two cathinone enantiomers, the enantiomeric excess (ee%) was calculated for each sample. The ee% value is given by the following equation:

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ee% = ([S] – [R])/([S] + [R])

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4. Conclusion

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where [S] and [R] are the concentrations of S and R cathinone, respectively. The values obtained for ee% are reported in Figure 5. They show that both cathinone enantiomers were detected in all parts of the studied plant. As it was noticed from the values in Table 1, the ee% results are positive in all stem samples whereas the leaf samples correspond to negative ee% values. This finding confirms that the most psychoactive S-enantiomer is mainly present in stems.

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S and R-cathinone enantiomers were derivatized with menthyl chloroformate and the obtained diastereomers were separated with an excellent resolution using gas chromatography hyphenated to mass spectrometry. While several previous papers discussed the total content of cathinone in Khat, the present study shows the first quantitative determination of both S and R cathinone in different pieces of freshly cut Catha edulis sample. Both enantiomers were present in all investigated parts of the plant, while several previous studies mentioned that only the more active S-cathinone was detected in this plant. Moreover, the distribution of the two enantiomers in Khat sample was quite different: the concentration of S-cathinone was in the ranges 0.081-0.124 and 0.138-0.290 mg/g in leaves and stems, respectively. R-cathinone content was between 0.105 and 0.211 mg/g in leaves, whereas it was between 0.087 and 0.186 mg/g in stems. These results showed that the concentration of S-cathinone which is the main active component in this euphorising plant is notably higher in stems than in leaves. On the other hand, S-cathinone concentration increased from lower to upper parts of the plant. Comparison of the present results with previously reported data obtained using different techniques showed a good agreement.

CRediT authorship contribution statement

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Abdulrhman M Dhabbah has done All things in this paper such as conceptualization, methodology, data curation, Writing- Original draft preparation, visualization, investigation, validation, writing- reviewing and editing.

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Figures

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Figure 1: Khat branch with the different separated leaves and stems

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Figure 2: A- Chromatogram of S-cathinone after derivatization with MCF; B, C and D- mass spectra of the main components (B: MCF, C: S-cathinone derivative, D: diastereomer due to R-(-)-menthyl chloroformate optical impurity of MCF)

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Figure 3: A- Chromatogram of leaf extract L4 after derivatization with MCF; B, C and D- mass spectra of the main components (B: MCF, C: S-cathinone derivative, D: R-cathinone derivative)

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Figure 4: A- Chromatogram of stem extract S1 after derivatization with MCF; B, C and D- mass spectra of the main components (B: MCF, C: S-cathinone derivative, D: R-cathinone derivative)

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Figure 5: enantiomeric excess of S and R cathinone in the different parts of fresh Catha edulis (S: stem samples, L: leaf samples)

Table 1: Concentration of S and R-cathinone in the different parts of fresh Khat sample

S3 S4 SM L1 L2 L3 L4

0.138 ± 0.020 0.239 ± 0.008 0.235±0.011 0.290±0.015 0.209±0.009 0.081±0.004 0.124±0.006 0.119±0.007 0.107±0.005 0.097±0.009

0.087±0.009 0.137±0.008 0.157±0.010 0.186±0.012 0.153±0.006 0.105±0.011 0.170±0.013 0.168±0.010 0.211±0.016 0.150±0.019

0.225±0.029 0.376±0.016 0.393±0.021 0.476±0.027 0.362±0.015 0.186±0.015 0.294±0.018 0.287±0.017 0.318±0.021 0.247±0.028

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LM

Total cathinone (mg/g)

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R-cathinone (mg/g)

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S1

S-cathinone (mg/g)