Screening of Psychoactive Components in Fresh Khat Using Direct Analysis in Real Time-Time of Flight-Mass Spectrometry

Abstract

Background: 

Khat (Catha edulis Forsk) is a shrub endemic to East Africa and the Arabian Peninsula that is widely consumed for its psychoactive properties. Due to the increase of illicit drugs consumption in the world, there is a need for fast investigation techniques in order to rapidly characterize the huge number of seized samples. In the recent years, direct analysis in real-time mass spectrometry (DART-MS) proved to be a useful and convenient method which allows recording mass spectra of various samples without any preparation.

Objectives: 

The present work describes a convenient procedure for rapid characterization of fresh Khat samples by identification of cathinone and cathine which are the most active constituents in different parts of the plant.

Materials and Methods: 

A fresh sample of C. edulis was cut into small pieces with separation of leaves and stems; each part was investigated using DART-time-of-flight (ToF)-MS. The fresh samples were submitted to a stream of hot helium as vaporization and ionization gas, without any extraction or prior treatment.

Results: 

The obtained spectra showed many peaks corresponding to the mass range from 100 to 600 Da. Cathinone and cathine which are known to be the main psychoactive components in C. edulis were found in all parts of the fresh samples; their protonated peaks appeared at 150 and 152 Da, respectively. The peak intensity of these two characteristic alkaloids was notably different between leaves and stems; it roughly decreased from lower to upper leaves, while their highest content was obtained in the upper stem.

Conclusion: 

The present work showed that DART-ToF-MS is a powerful tool for fast investigation of real samples, particularly in the field of forensic sciences.

INTRODUCTION

Khat (Cathaedulis Forsk) is an evergreen wild shrub that grows at high altitudes in East Africa and the Arabian Peninsula. Khat consumption has been a traditional and social habit of millions of people in these regions for centuries.[¹] Although the use of khat in the past was limited to this geographical area, nowadays, this plant is widely consumed in various regions of the world, as a result of increasing global migration routes.[²] Fresh leaves are generally chewed in collective sessions for hours, and they have marked psychostimulation effects with euphoria and excitement. However, addiction to khat has also many serious health and social effects. This habit induces a considerable time-wasting which affects the social and economic development of these regions by reducing productivity and performance with increasing absenteeism.[³⁴] One of the biggest problems for consumers is the financial burden that addiction imposes.[⁵] Another negative impact is the replacement of traditional agricultural crops by khat production.[⁴]

The widespread use of khat as a recreational drug leads to appetite suppression, psychiatric, cardiovascular, dental, and gastrointestinal problems.[¹²⁶] Additional negative health effects include nervousness, agitation, constipation, anorexia, stomatitis, esophagitis, gastritis, paralytic ileus, tachycardia, heart palpitations, hypertension, myocardial insufficiency, and cerebral hemorrhage.[³⁵⁷] Moreover, Corkery et al. have established that mortality was associated with addiction to C.edulis.[⁸] Despite the well-established harmful effects of khat consumption, its legal status in the world is not uniform. While using it is permitted in some countries such as the UK, the Netherlands, and Australia, it is prohibited in others like the USA, Canada, and France.[⁹]

Many previous articles have established that more than forty chemical constituents are present in khat. Among them, the main psychostimulant components are cathinone, cathine, and norephedrine, which are phenylpropylamino alkaloids.[⁶¹⁰] Some authors have reported that the active constituents of khat have neurophysiological effects similar to that of amphetamine because they share the same chemical skeleton.[¹⁵¹¹] Cathine, cathinone, and amphetamine have a propyl chain with a phenyl group in position 1 and an amino group attached to carbon 2. Among these phenylalkylamines which are the main active alkaloids of C.edulis, S-(-)-cathinone is the major psychoactive component. Other alkaloids identified in the leaves are cathine (1S,2S-(+) norpseudoephedrine) and 1R,2S-(-) norephedrine which are detected in older leaves and result from reduction of cathinone during drying and storage.[⁹] In addition, other classes of alkaloids such as cathedulins were observed in the fresh plant.[¹⁹] In the last few years, several synthetic psychoactive substances derived from cathinone were developed, produced, and introduced into the drug market. Among these derivatives, 4-methylmethcathinone also called mephedrone, as well as 4-fluoromethcathinone (flephedrone), bromomethcathinone, and 4-methoxymethcathinone (methedrone) are used with increasing frequency.[¹²]

For characterization of khat active constituents, various analytical techniques have been used, such as thin-layer chromatography, ultraviolet-visible spectroscopy, infrared spectroscopy, gas chromatography (GC), mass spectrometry (MS), high-performance liquid chromatography (HPLC), and capillary electrophoresis.[^{13141516171819}] The essential oil constituents of khat leaves were investigated by GC-MS. Seventy-seven compounds were identified, and its antioxidant activity was evaluated.[²⁰] The distribution of alkaloids in the different parts of the khat shrub was also studied by HPLC. It showed that the concentration of cathinone was highest in the leaves and in the younger parts of the plant.[²¹] The main active principles of C.edulis have been determined in various seized samples using GC-MS after derivatization of the alkaloids.[²²] The crude methanolic extracts of fresh khat leaves have been investigated by LC-MS/MS, and this study resulted in the identification of 62 alkaloids derived from cathedulin.[²] Furthermore, capillary electrophoresis was used for separation and quantitation of the main active principles of C.edulis (cathinone, cathine, and phenylpropanolamine).[¹⁶]

In the field of forensic analyses, the development of fast techniques is a challenge for rapid detection of illicit substances. In recent years, ambient ionization MS techniques have been developed, such as direct analysis in real-time (DART), desorption electrospray ionization (MS), and desorption atmospheric pressure photoionization.[²³] Among these ambient ionization methods, DART-MS, which was developed about 15 years ago, has found wide applicability.[²⁴] It allows a rapid and easy screening of various samples, including complex matrices, without sample extraction or pretreatment. Thus, because of its simplicity and flexibility, DART-MS has been used in many investigations for characterization of pharmaceuticals and natural products.[¹³²⁵] Lesiak et al. used high-resolution (HR)-DART-MS to characterize the psychoactive components of Mitragynaspeciosa by direct investigation of its leaves. They showed that this technique allows a rapid and easy identification of the plant material. Thus, it can be useful for forensic analysis of seized samples without the need for prior extraction or sample preparation.[²⁶]

The present study aims to investigate the distribution of the main components in fresh khat. The fresh sample of C.edulis was characterized using DART ionization followed by time-of-flight (TOF) MS, without any prior extraction or sample treatment, by identification of its main constituents in different parts of the plant. This work could be a contribution to the application of DART-MS in the field of forensic analysis.

MATERIALS AND METHODS

Fresh samples of C. edulis were obtained from Jazan (Saudi Arabia) and stored at 4°C, before their investigation by DART-ToF-MS. The different parts of the selected plant were cut into small pieces with separation of leaves and stems as shown in Figure 1. Each small piece was then directly submitted to the DART ion source without any treatment as shown in Figure 2. The distance between the DART ion source exit and the mass spectrometer inlet was about 25 mm. The mass spectra were recorded using the experimental conditions given below. The instrument used was an AccuTOF LC-Plus from JEOL (Tokyo, Japan) including a DART ion source running under atmospheric pressure (IonSense, Saugus, MA, USA) and a ToF mass spectrometer. The volatile components of each fresh part were evaporated in a stream of helium heated at 350°C, then ionized by the excited metastable helium atoms, before entering the ion source inlet of the TOF mass spectrometer. The analyses were performed in positive mode, in which the molecules are mainly protonated without fragmentation. The potentials of orifices 1 and 2 were set at 20 and 5 V, respectively. The ring lens potential was set to 13 V and the temperature of orifice 1 was 80°C. The radiofrequency ion guide potential was 300 V. High purity helium was used as ionization gas at 4.0 L/min flow heated to 350°C for sample evaporation. The potential on the discharge needle electrode of the DART source was set to 3.0 kV, while the perforated and grid electrodes were at 100 and 250 V potential respectively. The mass spectra acquisition range was 50– 1000 Da and the mass resolution was 3760 full width at half maximum. Before recording each spectrum, mass calibration was performed using a 200 μg/mL solution of polyethylene glycol (PEG) 600 (Sigma-Aldrich, Darmstadt, Germany) in methanol. The elemental composition of selected peaks was determined using the MassCentre JEOL software (version 1.3.8e, algorithm, JEOL USA, Inc., Peabody, USA).

Figure 1: Catha edulis fresh sample

Figure 2: Direct introduction of a khat leaf in direct analysis in the real-time ion source

Experimental

Fresh samples of C.edulis were obtained from Jazan (Saudi Arabia) and stored at 4°C, before their investigation by DART-ToF-MS. The different parts of the selected plant were cut into small pieces with separation of leaves and stems as shown in Figure 1. Each small piece was then directly submitted to the DART ion source without any treatment as shown in Figure 2. The distance between the DART ion source exit and the mass spectrometer inlet was about 25 mm. The mass spectra were recorded using the experimental conditions given below. The instrument used was an AccuTOF LC-Plus from JEOL (Tokyo, Japan) including a DART ion source running under atmospheric pressure (IonSense, Saugus, MA, USA) and a ToF mass spectrometer. The volatile components of each fresh part were evaporated in a stream of helium heated at 350°C, then ionized by the excited metastable helium atoms, before entering the ion source inlet of the TOF mass spectrometer. The analyses were performed in positive mode, in which the molecules are mainly protonated without fragmentation. The potentials of orifices 1 and 2 were set at 20 and 5 V, respectively. The ring lens potential was set to 13 V and the temperature of orifice 1 was 80°C. The radiofrequency ion guide potential was 300 V. High purity helium was used as ionization gas at 4.0 L/min flow heated to 350°C for sample evaporation. The potential on the discharge needle electrode of the DART source was set to 3.0 kV, while the perforated and grid electrodes were at 100 and 250 V potentials, respectively. The mass spectra acquisition range was 50–1000 Da and the mass resolution was 3760 full width at half maximum. Before recording each spectrum, mass calibration was performed using a 200 μg/mL solution of polyethylene glycol (PEG) 600 (Sigma-Aldrich, Darmstadt, Germany) in methanol. The elemental composition of selected peaks was determined using the MassCentre JEOL software (version 1.3.8e, algorithm, JEOL USA, Inc., Peabody, USA).

RESULTS AND DISCUSSION

To detect the distribution of psychoactive components in various plant parts, DART-HRMS was used to analyze plant samples directly. As an example, the HR mass spectrum of the Leaf 1 sample cut from a fresh sample of C.edulis is shown in Figure 3. It should be noted that as DART-MS experiment was performed under soft ionization conditions (i.e., orifice 1 = 20 V), there was little to no fragmentation, and therefore, each peak in the mass spectrum represents one constituent in the protonated form.[²⁷]

Figure 3: Mass spectrum of Leaf 1 sample from fresh Catha edulis (a) full range spectrum, (b) enlarged range m/z 132-156 Da

Interpretation of the HR mass spectrum was done by determination of the accurate mass of each peak and assigning its molecular formula. The difference between the experimental and calculated masses was <2 mDa for most peaks. This means that the mass accuracy was >10 ppm in the investigated mass range. The main observation from the spectrum shown in Figure 3a is the presence of two intense peaks at m/z 150 and 152. They correspond to the protonated ions of cathinone and cathine, respectively. Panel B represents a magnification of the mass range m/z 132–156 area of the spectrum. The peaks of protonated cathinone and cathine are observed at the accurate masses of m/z 150.09059 (C₉H₁₂ NO) and 152.10527 (C₉H₁₄ NO), respectively.[²⁸]

In addition to these protonated (M+H)⁺ peaks observed at m/z 150 and 152, the M+2 peaks of cathinone and cathine correspond to the same molecular formula including a carbon 13 isotope, as observed in Figure 3b. The isotope contributions in HRMS can be very useful for confirmation of the proposed molecular formula.[²⁵]

In Figure 3b, beside the characteristic peaks of cathinone and cathine which are the main psychoactive components in khat leaves, another intense peak is present at m/z 134.09477 corresponding to the formula C₉H₁₂N. A compound with this formula has not been reported as a constituent of C.edulis in any previous report. It could be deduced that this peak at m/z 134 is due to an artifact resulting from cathine thermal degradation. This observation confirms that previously discussed by Wabe who mentioned the probable decomposition of cathines which results either in dimer or other smaller fragments.[²⁹] Furthermore, as reported in many previous papers, since cathinone and cathine are the main constituents responsible of the psychoactive properties of C.edulis, this interesting result corroborates that khat consumers prefer chewing fresh leaves.[⁷³⁰] On the other hand, it was also mentioned that these two psychostimulating compounds degrade rather quickly, meaning that the plant should be consumed as fresh as possible.[³¹]

The HR mass spectra of the other six samples cut from the same fresh khat plant (i.e., leaves 2–5 and stems 1 and 2) were recorded under identical conditions and showed a profile similar to that of Leaf 1 [Figure 3a]. The chart shown in Figure 4 indicates the intensity of the main components in the leaves and stems of C.edulis. Although the same main compounds are present in all the parts of the plant, some constituents show different intensities in the samples. The spectra of the five fresh leaves show that cathine is the main compound, followed by cathinone. While we observed cathine to be of highest abundance relative to the other compounds present, several previous studies found cathinone to be the main psychoactive component in khat,[³²³³³⁴] with cathine (norpseudoephedrine), a product of the reduction of cathinone, with only 10% of the activity of cathinone to be major.[³³] Figure 4 shows that cathinone is present in all leaves at similar levels, whereas its intensity is notably higher in the young part of the stem which corresponds to the upper part of the fresh sample [stem 2 in [Figure 4]. This result confirms the quantitative measurements reported in previous studies which were made using other techniques such as GC-MS[³⁰] and imaging mass spectroscopy.[³²] Similar to cathinone, it was also observed that the abundance of cathine decreases in the leaves by age (i.e., from the older [leaf 1] to the younger [leaf 5]), while it is the most abundant in stem 2.

Figure 4: Main peaks in the mass spectra of Catha edulis parts

The relative abundance of cathinone and cathine which are the main psychoactive constituents in the different parts of fresh C.edulis is shown in Figure 5. The content of these two compounds decreases gradually from older to younger leaves, whereas it is much higher in the upper stem of the plant. This important observation aligns with reports that khat consumers prefer chewing the upper part of this plant, due to its perceived stronger psychostimulating effects. However, according to our results, this effect is due to the higher level of cathinone in stem and not in leaf as mentioned in some previous studies.[²¹³⁰] Beside cathinone and cathine which are the main active components in khat, several other compounds were characterized by HR DART-ToF-MS in the different fresh samples. The peak at m/z 177 could correspond to merucathine with the formula C₁₁H₁₅ NO which was mentioned in several previous papers, as well as N-formylnorephedrine (C₁₀H₁₃ NO₂) observed at m/z 180.[¹⁴¹⁶³³] It was also reported that dimerization of cathine results in the two derivatives 3,6-dimethyl-2,5-diphenylpyrazine (C₁₈H₁₆N₂) and C₁₈H₁₈N₂ which have masses consistent with m/z 261and 263, respectively.[²⁹³⁵]

Figure 5: Spatial distribution of cathine and cathinone in fresh Catha edulis

CONCLUSION

Different parts of a fresh sample of C. edulis were investigated by DART-ToF-MS without any sample treatment. The accurate masses of each component as protonated ions were observed, enabling determination of the corresponding formulas. Two prominent peaks (consistent with the presence of cathinone and cathine) were observed. The abundance profile of these two characteristic compounds was quite different in leaves and stems. While their content in leaves decreased from older to younger leaves, it was much higher in the upper stem of the fresh plant. This result is consistent with previous reports, as well as consumer preferences. The observations showed that DART-HRMS could be very useful for rapid characterization of plants by profiling their characteristic small molecule components without the need to perform any sample pretreatment or extraction.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.