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A simple HPLC–DAD method for the detection and quantification of psychotropic mitragynine in Mitragyna speciosa (ketum) and its products for the application in forensic investigation
Forensic Science International 226 (2013) 183–187
Contents lists available at SciVerse ScienceDirect
Forensic Science International journal homepage: www.elsevier.com/locate/forsciint
A simple HPLC–DAD method for the detection and quantification of psychotropic mitragynine in Mitragyna speciosa (ketum) and its products for the application in forensic investigation Suhanya Parthasarathy a, Surash Ramanathan a,*, Vikneswaran Murugaiyah b, Mohammad Razak Hamdan a, Mohd Ikram Mohd Said c, Choon-Sheen Lai a, Sharif Mahsufi Mansor a a
Centre for Drug Research, Universiti Sains Malaysia, 11800 Penang, Malaysia School of Pharmaceutical Sciences, Universiti Sains Malaysia, 11800 Penang, Malaysia c School of Chemical Sciences and Food Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 21 March 2012 Received in revised form 31 October 2012 Accepted 7 January 2013 Available online 4 February 2013
Mitragyna speciosa, a native plant of Thailand and Malaysia known as ‘ketum’, is a plant of considerable interest. It exhibits strong antinociceptive effect and yet, acts like a psychostimulant. Due to the affordability and its ease of availability, the abuse of this plant as a substitute for other banned narcotics has become a major concern in many societies. In countries such as Thailand, Myanmar, Australia and Malaysia, the use of ketum is illegal. However, for a person to be charged for possessing or selling ketum, a reliable analytical method is needed in order to detect and identify the plant and its products. Mitragynine is the major alkaloid of ketum. This compound manifests its antinociceptive effects by acting on the opioid receptors. Since M. speciosa contain large quantity of mitragynine and it is exclusive to the species, the present analytical method is developed and validated for the purpose of screening ketum products based on this unique compound as the analytical marker. The method uses a HPLC–DAD system with Inertsil C8 (4.6 mm 150 mm, 5 mm) as the column and a mixture of acetonitrile and formic acid, 50:50 (v/v), as the mobile phase. This method not only detects mitragynine, it can also be used to quantify the amount of mitragynine in the sample. The limit of detection is 0.25 mg/ml, while the limit of quantification is 0.50 mg/ml. The method is quick, simple and reliable with an accuracy of 97.27– 101.74% and coefficient of variations of between 0.91 and 3.96%. The method has been tested and found suitable for the identification and quantification of mitragynine in dried plants, a variety of ketum extracts, as well as ketum drink obtained from the market. ß 2013 Elsevier Ireland Ltd. All rights reserved.
Keywords: Mitragyna speciosa Mitragynine Ketum extracts Ketum drink HPLC–DAD
1. Introduction Mitragyna speciosa Korth (Rubiaceae) is a tropical plant indigenous to the Northern region of Peninsular Malaysia as well as the Central and South Thailand. The plant is known as ‘‘biakbiak’’ or ‘‘ketum’’ in Malaysia, and as ‘‘kratom’’ in Thailand. The physiological effect of ketum is dose dependent. At lower concentrations, ketum appears to be stimulating, producing a coca-like and euphoric effect but at higher concentrations, the plant acts like opium in that it is able to suppress pain and is used to mitigate opioid withdrawal syndromes [1]. Ketum is often a more economical choice compared to other opioids as it is available at a very low price [2]. In Malaysia, 1 kg of fresh ketum leaves can be obtained at a price of RM 24 (USD 8), while a glass of ketum drink is sold only for RM 2–4 (USD 0.70–1.30). Ketum
* Corresponding author. Tel.: +60 4 6533783; fax: +60 4 6568669. E-mail address: [email protected] (S. Ramanathan). 0379-0738/$ – see front matter ß 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.forsciint.2013.01.014
products can also be purchased over the internet in various forms such as dried leaves, powdered leaves, extracts and tinctures. The ease of availability and its extremely low price have attracted many young people to seek ketum as an alternative to other banned narcotics such as heroin or cannabis. This trend has been growing and is now a serious concern in the country. Although ketum has been used traditionally to help reduce the dependency on other drugs, it itself causes addiction and abstinence from it results in deleterious withdrawal effects [3,4]. Moreover, ketum is often sold adulterated with other substances such as cough mixtures, traditional herbs and even synthetic pyrethroid from mosquito coil, which may lead to more serious complications [5]. As a mean to curb ketum abuse, the Malaysian government has called on a ban to selling and possessing ketum. Effective from August 2003, a person if convicted, can be fined up to RM 10,000 or jailed up to 4 years, or both, under section 30 (5) of the Poisons Act 1952 [6]. Ketum is also illegal in other countries like Thailand, Myanmar and Australia, while in Japan, it is listed as ‘designated
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substances’ [7]. Since ketum can be sold in several forms, it is difficult to determine the presence of such material merely by visual observation, especially when it is in the form of packet drink, dried leaf or plant extract. Hence, a rapid and reliable analytical method is required for the detection and quantification of ketum and its preparations. Mitragynine (Fig. 1) is the major active alkaloid of ketum. It accounts for 12% (w/w) of the total alkaloid content in Malaysian ketum and 66% (w/w) in Thai ketum on dry weight basis [8]. Mitragynine is found exclusively in M. speciosa but not in any other species of Mitragyna [9]. As such it can be used as the marker compound for identification of ketum. To date, few methods on the analysis of mitragynine (MG) in biological fluids and its application in pharmacokinetic studies have been reported [10–13]. However, there is still a lack of suitable analytical methods for the detection and quantification of MG in raw materials, extracts and ketum preparations for routine analysis. A GC method has been described by Chan et al. However, this method is only applicable for qualitative detection of MG and it has not been evaluated and validated for quantitative studies. Moreover, the detection sensitivity of this method is also not reported [14]. Another method which has been reported is based on LC–PDA–ESI-MS. This method simultaneously detects MG, 7hydroxymitragynine and other alkaloids in ketum and its product. The total run time of each analysis is 55 min, with MG appearing at approximately 20 min. After the analysis of each sample, another 11 min is needed to prepare the system for the next injection [7]. Although the method has the advantage of detecting several ketum alkaloids simultaneously, the lengthy analysis time and high capital expenditure on the instrument limit its application and availability for routine use. Hence, there is a need for the development of a faster and more economical method for routine detection of ketum and its products based on its unique marker, mitragynine. This report describes the validation of a HPLC–DAD method which is simple, rapid and reliable for the detection and routine quantification of MG in ketum raw materials, alkaloid extracts and ketum drinks.
2. Methodology 2.1. Plant material Fresh leaves of M. speciosa Korth. (Rubiaceae) were collected from the state of Perak, Malaysia. A voucher specimen was deposited at the herbarium of Universiti Kebangsaan Malaysia (Specimen No. UKMB06509).
Fig. 1. Chemical structure of mitragynine (MG).
2.2. Chemicals Solvents used for chromatographic separation were of HPLC grade. Formic acid, methanol (MeOH) and acetonitrile (ACN) were purchased from Fisher Scientific (Loughborough, UK), while NaOH was purchased from R&M Chemicals (Essex, UK). Petroleum ether (PE), chloroform (CHCl3) and MeOH used for extracting the plant material were of analytical grade purchased from Merck (Darmstadt, Germany). Mitragynine (94.3% purity) was purchased from Chromadex Inc. (CA, USA). Deionized water for HPLC was prepared using an Elga Classic UVF ultrapure water purifier system (Elgastat, Bucks, UK). 2.3. Chromatographic conditions The analytical method was developed on an Agilent 1200 series HPLC system which was coupled to a photodiode array detector (Agilent, CA, USA). Chromatographic separation was achieved at room temperature (25 8C) on a C8 reversed-phase Inertsil column (4.6 mm 150 mm, 5 mm) (GL Sciences Inc., Tokyo, Japan) which was protected with a guard column (4.0 mm 20 mm, 5 mm). Mobile phase was a mixture of ACN and 0.05% formic acid (adjusted to pH 5 with NaOH), 50:50 (v/v) running at an isocratic mode at flow rate 1.0 ml/min. The sample injection volume was 20 ml and the total analytical run time was 6 min with mitragynine (MG) eluting at 5.5 min. Detection was carried out at 200–400 nm, while the UV signal at 223 nm was extracted for quantification purposes. Identification of MG was done by comparing the HPLC retention time and UV spectrum of the analyte with that of MG standard. Peak purity test was also performed using the ChemStation LC3D software to ensure method selectivity for reliable quantification of MG. 2.4. Analytical method 2.4.1. Validation of the HPLC method A 50 mg/ml stock solution of MG reference standard was prepared in MeOH. From the stock solution, working standard solutions of 0.5, 1.0, 2.5, 5.0, 7.5, and 10.0 mg/ml were prepared. Calibration curve was constructed on each day of analysis. To evaluate the within-day and between-day precision, as well as the accuracy of the method, five replicates of MG at six different concentrations were analysed on five consecutive days. Extraction recovery was carried out by spiking MG standards to blank samples. Extraction was then carried out as described in Section 2.4.2 and the recovery value was expressed in terms of percentage of spiked MG obtained from the extraction over that of an equivalent amount of MG in the pure standards. 2.4.2. Preparation and analysis of M. speciosa extracts The analytical method was developed and tested with authentic samples prepared from M. speciosa. Fresh leaves were washed with clean water and dried in the oven at 45 8C until a constant weight was obtained. Following that, the dried leaves were pulverised with a mill grinder and extracted using various means of extraction methods. To produce water extract, 5 kg of the dried leaves were boiled in 8 L of water for 2 h. The extract was then removed and the plant residue was extracted again with the same amount of water for another 2 h. Following that, both extracts were combined and dried using a freeze-dryer. Methanol extract of the plant was prepared by macerating dried leaves (10 g) in MeOH (50 ml) at room temperature for 5 days. The maceration procedure was repeated twice with fresh MeOH to ensure that the materials were fully extracted. Solvent was then evaporated under vacuum to yield the MeOH extract. The alkaloid-rich extract was obtained by extracting the alkaloids from the MeOH extract of M. speciosa. The
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MeOH extract was mixed with 90% acetic acid in aqueous at a ratio of 1:35 (w/v) and the content was partitioned between aqueous and petroleum ether. Following that, the aqueous layer was separated and basified with Na2CO3 to pH 9 and the alkaloids were subsequently extracted into CHCl3. The extract was washed with water to remove excess alkali and dried over Na2SO4 anhydrous prior to removing the solvent. To prepare the samples for analysis, 100 mg of each extract was dissolved in 20 ml MeOH with the assistance of ultrasound to produce a solution of 5 mg/ml. The samples were filtered through a 0.45 mm PTFE syringe filter (Whatman, Maidstone, England) and diluted accordingly prior to injecting into the HPLC system. Quantification of mitragynine (MG) was carried out based on the calibration curve of MG standard.
Fig. 2. UV spectrum of: (A) MG standard; (B) the analyte in ketum drink sample which has a retention time that corresponds to MG peak. Each subfigure shows an overlay of three spectra (represented by blue, green and purple colour lines) obtained at different sections of the peak. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
2.4.3. Analysis of ketum drink Ketum drink was obtained from eight different locations of Northern Peninsular Malaysia, namely Teluk kumbar, Gua Nangka, Chuping, Jelutong, Jitra, Lembah Raja, Sungai Batu and Balik Pulau. These samples were kept at 70 8C prior to analysis. On the day of analysis, the samples were thawed to room temperature, following which an aliquot of 1 ml was taken and centrifuged at 3000 r.p.m. for 10 min. The supernatant was carefully removed from the centrifuge tube and diluted accordingly with methanol prior to HPLC analysis. 2.5. Stability of MG The stability of MG at concentrations 5.0 and 50.0 mg/ml in MeOH, was evaluated at storage temperature (4 8C) over a period of 4 weeks.
Fig. 3. Representative HPLC chromatograms of: (A) MG standard; (B) ketum drink; (C) alkaloid extract; (D) methanol extract; (E) water extract; (F) blank, obtained on an Inertsil C8 column (150 mm 4.6 mm, 5 mm) using a mobile phase of ACN-0.05% formic acid (pH 5), 50:50 (v/v).
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Table 1 Accuracy and precision of the analysis of mitragynine. Actual concentration (mg/ml)
0.50 1.00 2.00 5.00 7.50 10.00
Within-day analysis (n = 5)
Between-day analysis (n = 5)
Mean obtained concentration (mg/ml)
Accuracy (%)
Precision (CV) (%)
Mean obtained concentration (mg/ml)
Accuracy (%)
Precision (CV) (%)
0.50 1.02 1.96 5.00 7.42 9.73
100.41 101.74 97.97 100.05 98.95 97.27
2.96 2.14 0.91 1.73 1.86 1.75
0.49 1.02 2.01 5.08 7.56 9.83
97.44 101.51 100.69 100.68 100.77 99.32
3.96 1.56 2.67 2.18 2.06 2.01
CV, coefficient of variation.
3. Results and discussion 3.1. Optimisation of the chromatographic conditions Janchawee et al. (2007) reported the characteristic of MG on various chromatographic conditions, particularly on the selection of mobile phase. With >80% methanol in the mobile phase, the authors found MG to elute quickly but interfere with other peaks in the sample. With the composition of MeOH < 80%, MG was well separated from the polar interferences but broader peaks were observed. The use of short column (150 mm) resulted in a run time of <10 min but poor resolution, as such, the analysis was carried out using long column (250 mm) with a total run time of 30 min [11]. In the present study, a variety of HPLC columns and mobile phase systems were evaluated. Among the chromatographic columns evaluated, i.e. Gemini C18, 4.6 mm 250 mm, 5 mm (Phenomenex, USA), Symmetry Shield C18, 4.6 mm 150 mm, 5 mm (Waters, MA, USA) and Inertsil C8, 4.6 mm 150 mm, 5 mm (GL Sciences, Tokyo, Japan), Inertsil C8 provided the best chromatographic separation. Several combinations of solvents with acetonitrile were also evaluated, i.e. 0.1% tetrahydrofuran, ammonium formate, ammonium acetate and formic acid. Based on the results, ACN-0.05% formic acid (adjusted to pH 5 with NaOH), 50:50 (v/v), was found to be the optimum mobile phase because it gave the best resolution and peak shape, while the total run time was only 6 min. 3.2. Method validation The method was found to be selective, in which the chromatographic peak of MG was free from interferences originating from the sample matrix. This was confirmed through the peak purity test using ChemStation LC3D which evaluates the
Table 2 Extraction recovery of mitragynine. Concentration (mg/ml)
Alkaloid-rich extract 2.5 5.0 8.0 Methanol extract 2.5 5.0 8.0 Water extract 2.5 5.0 8.0
Recovery, % (n = 5) Mean
CV
99.2 99.5 99.9
0.8 0.5 1.5
101.1 100.1 99.7
95.4 97.4 101.2
1.5 0.25 0.12
1.65 0.72 2.61
consistency of UV spectra at different sections of the peak: (1) peak start, (2) peak maximum, and (3) peak end. Identification was done by comparing the UV spectrum of the analyte with that of MG standard within 210–400 nm (Fig. 2). A representative chromatogram of the standard and each type of samples are given in Fig. 3. The calibration curve of MG was found to be linear over the concentration range of 0.5–10.0 mg/ml with a mean equation of y = 28.71x + 2.087 and r2 > 0.999. The lower limit of detection and quantification were 0.25 and 0.5 mg/ml, respectively. Although the sensitivity was lower compared to the method reported by Moraes et al. [10] and Lu et al. [12] for the analysis of MG in biological samples, the sample preparation procedure involved in the present method is less laborious. The quantification limit and range of the present study was also found to be sufficient to quantify the presence of MG in the extracts as well as that in the ketum drink. Moreover, the short analytical run time in this study is an advantage for routine quantification of MG. The precision and accuracy of the method were found to be satisfactory with the results given in Table 1. The extraction recovery of MG at >96% (Table 2) indicates that the technique employed could effectively extract MG into the solvent system. Results of the stability study showed that MG was stable in MeOH at 4 8C over a period of one month (Table 3). 3.3. Sample analysis The method was applied to study MG content in water, MeOH and alkaloid-rich extracts of M. speciosa leaf. Water extract was found to contain the least amount of MG, while the MeOH and alkaloid-rich extracts were found to contained MG 6 and 30 times higher, respectively (Table 4). The low quantity of MG found in the water extract was mainly due to its poor solubility in water, while on the other hand, MG that solubilised well in MeOH was found in much higher concentration in the MeOH extract. The concentration of MG further increased when it was extracted using the acid–base extraction technique. This technique converts MG and other alkaloids into their salt form which solubilises in water and then back extracts the compounds into the organic layer after neutralising them. The end product was a more concentrated extract which contained mainly the alkaloids of M. speciosa. As there are a variety of ketum extracts available
Table 3 Stability of mitragynine in methanol at 4 8C. Concentration (mg/ml)
% MG remained after storage Day 0
5.0 50.0
Week 1
Week 4
Mean
CV (%)
Mean
CV (%)
Mean
CV (%)
95.4 97.9
0.3 0.3
101.0 101.0
0.7 0.2
101.0 98.5
0.5 0.7
S. Parthasarathy et al. / Forensic Science International 226 (2013) 183–187 Table 4 Mitragynine content in leaf extracts of Mitragyna speciosa and ketum drink obtained from several locations in the Northern Peninsular of Malaysia. M. speciosa extract
MG content SD, mg/g (n = 3)
Ketum drink sample
MG content SD, mg/ml (n = 3)
Alkaloid-rich MeOH Water
24.72 0.67 4.77 1.30 0.80 0.11
Teluk Kumbar Gua Nangka Chuping Jelutong Jitra Sungai Batu Lembah Raja Balik Pulau
292.97 4.56 259.2 1.27 245.83 3.33 408.29 1.16 330.03 2.81 444.23 3.55 30.95 1.61 255.00 9.06
187
Conflict of interest None declared. Acknowledgements The authors would like to thank Mr. Asokan Muniandy and Mr. Salam Abdullah for their technical assistance. This project was funded by Ministry of Science, Technology and Innovation (MOSTI) and Universiti Sains Malaysia Research University grant. Suhanya Parthasarathy was supported by Universiti Sains Malaysia fellowship scheme from Institute for Postgraduate Studies, Universiti Sains Malaysia. References
commercially which are priced according to their potency and taste (which are largely dependent on how the materials were extracted and the types of additives added), the data presented here can therefore be used as a reference to compare the type of extracts available in the market. Apart from ketum extracts, the applicability of the present method was also extended to study the amount of MG in ketum drinks. Samples obtained from eight different sources in the Northern Peninsular of Malaysia were evaluated. Results of the analysis showed that MG content varied greatly from among the different sources, i.e. approximately 31–444 mg/ml, as shown in Table 4. The amount of MG was found to be highest in the sample from Sungai Batu, while the sample from Lembah Raja contained the least amount of MG. The reasons to these variations may be attributed to geographical difference, maturity of the plant, method of preparation and the concentration of the drink. 4. Conclusion A rapid and reliable HPLC method has been developed and validated for the detection and quantification of MG in M. speciosa and its products. This method has advantage over the existing methods in terms of the simplicity of sample preparation, short analytical run time and is inexpensive compared to LC–MS. It is suitable for routine screening of M. speciosa products in the market including crude extracts, alkaloid enriched extracts and ketum drink. The amount of MG in the plant extracts obtained from the study ranged from 0.8 to 25 mg/g, while the amount of MG in ketum drink ranged from 31 to 444 mg/ml.
[1] K.M. Babu, C.R. McCurdy, E.W. Boyer, Opioid receptors and legal highs: Salvia divinorum and Kratom, Clin. Toxicol. 46 (2008) 146–152. [2] I.H. Burkill, A Dictionary of the Economic Products of the Malay Peninsula, vol. II, Crown Agents for the Colonies, London, 1935. [3] K.R. Jansen, C.J. Prast, Ethnopharmacology of kratom and the Mitragyna alkaloids, J. Ethnopharmacol. 23 (1988) 115–119. [4] V. Balasingam, S. Narayanan, T.B. Goh, S.M. Mansor, The informal use of ketum (Mitragyna speciosa) for opioid withdrawal in the northern states of peninsular Malaysia and implications for drug substitution therapy, Int. J. Drug Policy 21 (2010) 283–288. [5] O. Bothiphon, S. Prutipanlai, B. Janchawee, 35th Congress on Science and Technology of Thailand, Chonburi, Thailand, (2009), pp. 1–6. [6] A. Simon, It’s Illegal Now to Sell, Possess Ketum Leaves, New Straits Times, Jitra, Malaysia, 2011, 11 August. [7] R. Kikura-Hanajiri, M. Kawamura, T. Maruyama, M. Kitajima, H. Takayama, Y. Goda, Simultaneous analysis of mitragynine, 7-hydroxymitragynine, and other alkaloids in the psychotropic plant ‘‘kratom’’ (Mitragyna speciosa) by LC–ESI-MS, Forensic Toxicol. 27 (2009) 67–74. [8] H. Takayama, Chemistry and pharmacology of analgesic Indole alkaloids from the rubiaceous plants, Mitragyna speciosa, Chem. Pharm. Bull. 52 (8) (2004) 916–928. [9] E.J. Shellard, Ethnopharmacology of kratom and the Mitragyna alkaloids, J. Ethnopharmacol. 25 (1) (1989) 123–124. [10] N.V. de Moraes, R.A.C. Moretti, E.B. Furr III, C.R. McCurdy, V.L. Lanchote, Determination of mitragynine in rat plasma by LC–MS/MS: application to pharmacokinetics, J. Chromatogr. B 877 (2009) 2593–2597. [11] B. Janchawee, N. Keawpradub, S. Chittrakarn, A high-performance liquid chromatographic method for determination of mitragynine in serum and its application to a pharmacokinetic study in rats, Biomed. Chromatogr. 21 (2007) 176–183. [12] S. Lu, B.N. Tran, J.L. Nelsen, K.M. Aldous, Quantitative analysis of mitragynine in human urine by high performance liquid chromatography–tandem mass spectrometry, J. Chromatogr. B 877 (2009) 2499–2505. [13] A.A. Philipp, D.K. Wissenbach, S.W. Zoerntlein, Studies on the metabolism of mitragynine, the main alkaloid of the herbal drug Kratom, in rat and human urine using liquid chromatography–linear ion trap mass spectrometry, J. Mass Spectrom. 44 (2009) 1249–1261. [14] K.B. Chan, C. Pakiam, R.A. Rahim, Psychoactive plant abuse: the identification of mitragynine in ketum and in ketum preparations, Bulletin on Narcotics LVII (1/2) (2005) 249–256.
Contents lists available at SciVerse ScienceDirect
Forensic Science International journal homepage: www.elsevier.com/locate/forsciint
A simple HPLC–DAD method for the detection and quantification of psychotropic mitragynine in Mitragyna speciosa (ketum) and its products for the application in forensic investigation Suhanya Parthasarathy a, Surash Ramanathan a,*, Vikneswaran Murugaiyah b, Mohammad Razak Hamdan a, Mohd Ikram Mohd Said c, Choon-Sheen Lai a, Sharif Mahsufi Mansor a a
Centre for Drug Research, Universiti Sains Malaysia, 11800 Penang, Malaysia School of Pharmaceutical Sciences, Universiti Sains Malaysia, 11800 Penang, Malaysia c School of Chemical Sciences and Food Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 21 March 2012 Received in revised form 31 October 2012 Accepted 7 January 2013 Available online 4 February 2013
Mitragyna speciosa, a native plant of Thailand and Malaysia known as ‘ketum’, is a plant of considerable interest. It exhibits strong antinociceptive effect and yet, acts like a psychostimulant. Due to the affordability and its ease of availability, the abuse of this plant as a substitute for other banned narcotics has become a major concern in many societies. In countries such as Thailand, Myanmar, Australia and Malaysia, the use of ketum is illegal. However, for a person to be charged for possessing or selling ketum, a reliable analytical method is needed in order to detect and identify the plant and its products. Mitragynine is the major alkaloid of ketum. This compound manifests its antinociceptive effects by acting on the opioid receptors. Since M. speciosa contain large quantity of mitragynine and it is exclusive to the species, the present analytical method is developed and validated for the purpose of screening ketum products based on this unique compound as the analytical marker. The method uses a HPLC–DAD system with Inertsil C8 (4.6 mm 150 mm, 5 mm) as the column and a mixture of acetonitrile and formic acid, 50:50 (v/v), as the mobile phase. This method not only detects mitragynine, it can also be used to quantify the amount of mitragynine in the sample. The limit of detection is 0.25 mg/ml, while the limit of quantification is 0.50 mg/ml. The method is quick, simple and reliable with an accuracy of 97.27– 101.74% and coefficient of variations of between 0.91 and 3.96%. The method has been tested and found suitable for the identification and quantification of mitragynine in dried plants, a variety of ketum extracts, as well as ketum drink obtained from the market. ß 2013 Elsevier Ireland Ltd. All rights reserved.
Keywords: Mitragyna speciosa Mitragynine Ketum extracts Ketum drink HPLC–DAD
1. Introduction Mitragyna speciosa Korth (Rubiaceae) is a tropical plant indigenous to the Northern region of Peninsular Malaysia as well as the Central and South Thailand. The plant is known as ‘‘biakbiak’’ or ‘‘ketum’’ in Malaysia, and as ‘‘kratom’’ in Thailand. The physiological effect of ketum is dose dependent. At lower concentrations, ketum appears to be stimulating, producing a coca-like and euphoric effect but at higher concentrations, the plant acts like opium in that it is able to suppress pain and is used to mitigate opioid withdrawal syndromes [1]. Ketum is often a more economical choice compared to other opioids as it is available at a very low price [2]. In Malaysia, 1 kg of fresh ketum leaves can be obtained at a price of RM 24 (USD 8), while a glass of ketum drink is sold only for RM 2–4 (USD 0.70–1.30). Ketum
* Corresponding author. Tel.: +60 4 6533783; fax: +60 4 6568669. E-mail address: [email protected] (S. Ramanathan). 0379-0738/$ – see front matter ß 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.forsciint.2013.01.014
products can also be purchased over the internet in various forms such as dried leaves, powdered leaves, extracts and tinctures. The ease of availability and its extremely low price have attracted many young people to seek ketum as an alternative to other banned narcotics such as heroin or cannabis. This trend has been growing and is now a serious concern in the country. Although ketum has been used traditionally to help reduce the dependency on other drugs, it itself causes addiction and abstinence from it results in deleterious withdrawal effects [3,4]. Moreover, ketum is often sold adulterated with other substances such as cough mixtures, traditional herbs and even synthetic pyrethroid from mosquito coil, which may lead to more serious complications [5]. As a mean to curb ketum abuse, the Malaysian government has called on a ban to selling and possessing ketum. Effective from August 2003, a person if convicted, can be fined up to RM 10,000 or jailed up to 4 years, or both, under section 30 (5) of the Poisons Act 1952 [6]. Ketum is also illegal in other countries like Thailand, Myanmar and Australia, while in Japan, it is listed as ‘designated
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substances’ [7]. Since ketum can be sold in several forms, it is difficult to determine the presence of such material merely by visual observation, especially when it is in the form of packet drink, dried leaf or plant extract. Hence, a rapid and reliable analytical method is required for the detection and quantification of ketum and its preparations. Mitragynine (Fig. 1) is the major active alkaloid of ketum. It accounts for 12% (w/w) of the total alkaloid content in Malaysian ketum and 66% (w/w) in Thai ketum on dry weight basis [8]. Mitragynine is found exclusively in M. speciosa but not in any other species of Mitragyna [9]. As such it can be used as the marker compound for identification of ketum. To date, few methods on the analysis of mitragynine (MG) in biological fluids and its application in pharmacokinetic studies have been reported [10–13]. However, there is still a lack of suitable analytical methods for the detection and quantification of MG in raw materials, extracts and ketum preparations for routine analysis. A GC method has been described by Chan et al. However, this method is only applicable for qualitative detection of MG and it has not been evaluated and validated for quantitative studies. Moreover, the detection sensitivity of this method is also not reported [14]. Another method which has been reported is based on LC–PDA–ESI-MS. This method simultaneously detects MG, 7hydroxymitragynine and other alkaloids in ketum and its product. The total run time of each analysis is 55 min, with MG appearing at approximately 20 min. After the analysis of each sample, another 11 min is needed to prepare the system for the next injection [7]. Although the method has the advantage of detecting several ketum alkaloids simultaneously, the lengthy analysis time and high capital expenditure on the instrument limit its application and availability for routine use. Hence, there is a need for the development of a faster and more economical method for routine detection of ketum and its products based on its unique marker, mitragynine. This report describes the validation of a HPLC–DAD method which is simple, rapid and reliable for the detection and routine quantification of MG in ketum raw materials, alkaloid extracts and ketum drinks.
2. Methodology 2.1. Plant material Fresh leaves of M. speciosa Korth. (Rubiaceae) were collected from the state of Perak, Malaysia. A voucher specimen was deposited at the herbarium of Universiti Kebangsaan Malaysia (Specimen No. UKMB06509).
Fig. 1. Chemical structure of mitragynine (MG).
2.2. Chemicals Solvents used for chromatographic separation were of HPLC grade. Formic acid, methanol (MeOH) and acetonitrile (ACN) were purchased from Fisher Scientific (Loughborough, UK), while NaOH was purchased from R&M Chemicals (Essex, UK). Petroleum ether (PE), chloroform (CHCl3) and MeOH used for extracting the plant material were of analytical grade purchased from Merck (Darmstadt, Germany). Mitragynine (94.3% purity) was purchased from Chromadex Inc. (CA, USA). Deionized water for HPLC was prepared using an Elga Classic UVF ultrapure water purifier system (Elgastat, Bucks, UK). 2.3. Chromatographic conditions The analytical method was developed on an Agilent 1200 series HPLC system which was coupled to a photodiode array detector (Agilent, CA, USA). Chromatographic separation was achieved at room temperature (25 8C) on a C8 reversed-phase Inertsil column (4.6 mm 150 mm, 5 mm) (GL Sciences Inc., Tokyo, Japan) which was protected with a guard column (4.0 mm 20 mm, 5 mm). Mobile phase was a mixture of ACN and 0.05% formic acid (adjusted to pH 5 with NaOH), 50:50 (v/v) running at an isocratic mode at flow rate 1.0 ml/min. The sample injection volume was 20 ml and the total analytical run time was 6 min with mitragynine (MG) eluting at 5.5 min. Detection was carried out at 200–400 nm, while the UV signal at 223 nm was extracted for quantification purposes. Identification of MG was done by comparing the HPLC retention time and UV spectrum of the analyte with that of MG standard. Peak purity test was also performed using the ChemStation LC3D software to ensure method selectivity for reliable quantification of MG. 2.4. Analytical method 2.4.1. Validation of the HPLC method A 50 mg/ml stock solution of MG reference standard was prepared in MeOH. From the stock solution, working standard solutions of 0.5, 1.0, 2.5, 5.0, 7.5, and 10.0 mg/ml were prepared. Calibration curve was constructed on each day of analysis. To evaluate the within-day and between-day precision, as well as the accuracy of the method, five replicates of MG at six different concentrations were analysed on five consecutive days. Extraction recovery was carried out by spiking MG standards to blank samples. Extraction was then carried out as described in Section 2.4.2 and the recovery value was expressed in terms of percentage of spiked MG obtained from the extraction over that of an equivalent amount of MG in the pure standards. 2.4.2. Preparation and analysis of M. speciosa extracts The analytical method was developed and tested with authentic samples prepared from M. speciosa. Fresh leaves were washed with clean water and dried in the oven at 45 8C until a constant weight was obtained. Following that, the dried leaves were pulverised with a mill grinder and extracted using various means of extraction methods. To produce water extract, 5 kg of the dried leaves were boiled in 8 L of water for 2 h. The extract was then removed and the plant residue was extracted again with the same amount of water for another 2 h. Following that, both extracts were combined and dried using a freeze-dryer. Methanol extract of the plant was prepared by macerating dried leaves (10 g) in MeOH (50 ml) at room temperature for 5 days. The maceration procedure was repeated twice with fresh MeOH to ensure that the materials were fully extracted. Solvent was then evaporated under vacuum to yield the MeOH extract. The alkaloid-rich extract was obtained by extracting the alkaloids from the MeOH extract of M. speciosa. The
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MeOH extract was mixed with 90% acetic acid in aqueous at a ratio of 1:35 (w/v) and the content was partitioned between aqueous and petroleum ether. Following that, the aqueous layer was separated and basified with Na2CO3 to pH 9 and the alkaloids were subsequently extracted into CHCl3. The extract was washed with water to remove excess alkali and dried over Na2SO4 anhydrous prior to removing the solvent. To prepare the samples for analysis, 100 mg of each extract was dissolved in 20 ml MeOH with the assistance of ultrasound to produce a solution of 5 mg/ml. The samples were filtered through a 0.45 mm PTFE syringe filter (Whatman, Maidstone, England) and diluted accordingly prior to injecting into the HPLC system. Quantification of mitragynine (MG) was carried out based on the calibration curve of MG standard.
Fig. 2. UV spectrum of: (A) MG standard; (B) the analyte in ketum drink sample which has a retention time that corresponds to MG peak. Each subfigure shows an overlay of three spectra (represented by blue, green and purple colour lines) obtained at different sections of the peak. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
2.4.3. Analysis of ketum drink Ketum drink was obtained from eight different locations of Northern Peninsular Malaysia, namely Teluk kumbar, Gua Nangka, Chuping, Jelutong, Jitra, Lembah Raja, Sungai Batu and Balik Pulau. These samples were kept at 70 8C prior to analysis. On the day of analysis, the samples were thawed to room temperature, following which an aliquot of 1 ml was taken and centrifuged at 3000 r.p.m. for 10 min. The supernatant was carefully removed from the centrifuge tube and diluted accordingly with methanol prior to HPLC analysis. 2.5. Stability of MG The stability of MG at concentrations 5.0 and 50.0 mg/ml in MeOH, was evaluated at storage temperature (4 8C) over a period of 4 weeks.
Fig. 3. Representative HPLC chromatograms of: (A) MG standard; (B) ketum drink; (C) alkaloid extract; (D) methanol extract; (E) water extract; (F) blank, obtained on an Inertsil C8 column (150 mm 4.6 mm, 5 mm) using a mobile phase of ACN-0.05% formic acid (pH 5), 50:50 (v/v).
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Table 1 Accuracy and precision of the analysis of mitragynine. Actual concentration (mg/ml)
0.50 1.00 2.00 5.00 7.50 10.00
Within-day analysis (n = 5)
Between-day analysis (n = 5)
Mean obtained concentration (mg/ml)
Accuracy (%)
Precision (CV) (%)
Mean obtained concentration (mg/ml)
Accuracy (%)
Precision (CV) (%)
0.50 1.02 1.96 5.00 7.42 9.73
100.41 101.74 97.97 100.05 98.95 97.27
2.96 2.14 0.91 1.73 1.86 1.75
0.49 1.02 2.01 5.08 7.56 9.83
97.44 101.51 100.69 100.68 100.77 99.32
3.96 1.56 2.67 2.18 2.06 2.01
CV, coefficient of variation.
3. Results and discussion 3.1. Optimisation of the chromatographic conditions Janchawee et al. (2007) reported the characteristic of MG on various chromatographic conditions, particularly on the selection of mobile phase. With >80% methanol in the mobile phase, the authors found MG to elute quickly but interfere with other peaks in the sample. With the composition of MeOH < 80%, MG was well separated from the polar interferences but broader peaks were observed. The use of short column (150 mm) resulted in a run time of <10 min but poor resolution, as such, the analysis was carried out using long column (250 mm) with a total run time of 30 min [11]. In the present study, a variety of HPLC columns and mobile phase systems were evaluated. Among the chromatographic columns evaluated, i.e. Gemini C18, 4.6 mm 250 mm, 5 mm (Phenomenex, USA), Symmetry Shield C18, 4.6 mm 150 mm, 5 mm (Waters, MA, USA) and Inertsil C8, 4.6 mm 150 mm, 5 mm (GL Sciences, Tokyo, Japan), Inertsil C8 provided the best chromatographic separation. Several combinations of solvents with acetonitrile were also evaluated, i.e. 0.1% tetrahydrofuran, ammonium formate, ammonium acetate and formic acid. Based on the results, ACN-0.05% formic acid (adjusted to pH 5 with NaOH), 50:50 (v/v), was found to be the optimum mobile phase because it gave the best resolution and peak shape, while the total run time was only 6 min. 3.2. Method validation The method was found to be selective, in which the chromatographic peak of MG was free from interferences originating from the sample matrix. This was confirmed through the peak purity test using ChemStation LC3D which evaluates the
Table 2 Extraction recovery of mitragynine. Concentration (mg/ml)
Alkaloid-rich extract 2.5 5.0 8.0 Methanol extract 2.5 5.0 8.0 Water extract 2.5 5.0 8.0
Recovery, % (n = 5) Mean
CV
99.2 99.5 99.9
0.8 0.5 1.5
101.1 100.1 99.7
95.4 97.4 101.2
1.5 0.25 0.12
1.65 0.72 2.61
consistency of UV spectra at different sections of the peak: (1) peak start, (2) peak maximum, and (3) peak end. Identification was done by comparing the UV spectrum of the analyte with that of MG standard within 210–400 nm (Fig. 2). A representative chromatogram of the standard and each type of samples are given in Fig. 3. The calibration curve of MG was found to be linear over the concentration range of 0.5–10.0 mg/ml with a mean equation of y = 28.71x + 2.087 and r2 > 0.999. The lower limit of detection and quantification were 0.25 and 0.5 mg/ml, respectively. Although the sensitivity was lower compared to the method reported by Moraes et al. [10] and Lu et al. [12] for the analysis of MG in biological samples, the sample preparation procedure involved in the present method is less laborious. The quantification limit and range of the present study was also found to be sufficient to quantify the presence of MG in the extracts as well as that in the ketum drink. Moreover, the short analytical run time in this study is an advantage for routine quantification of MG. The precision and accuracy of the method were found to be satisfactory with the results given in Table 1. The extraction recovery of MG at >96% (Table 2) indicates that the technique employed could effectively extract MG into the solvent system. Results of the stability study showed that MG was stable in MeOH at 4 8C over a period of one month (Table 3). 3.3. Sample analysis The method was applied to study MG content in water, MeOH and alkaloid-rich extracts of M. speciosa leaf. Water extract was found to contain the least amount of MG, while the MeOH and alkaloid-rich extracts were found to contained MG 6 and 30 times higher, respectively (Table 4). The low quantity of MG found in the water extract was mainly due to its poor solubility in water, while on the other hand, MG that solubilised well in MeOH was found in much higher concentration in the MeOH extract. The concentration of MG further increased when it was extracted using the acid–base extraction technique. This technique converts MG and other alkaloids into their salt form which solubilises in water and then back extracts the compounds into the organic layer after neutralising them. The end product was a more concentrated extract which contained mainly the alkaloids of M. speciosa. As there are a variety of ketum extracts available
Table 3 Stability of mitragynine in methanol at 4 8C. Concentration (mg/ml)
% MG remained after storage Day 0
5.0 50.0
Week 1
Week 4
Mean
CV (%)
Mean
CV (%)
Mean
CV (%)
95.4 97.9
0.3 0.3
101.0 101.0
0.7 0.2
101.0 98.5
0.5 0.7
S. Parthasarathy et al. / Forensic Science International 226 (2013) 183–187 Table 4 Mitragynine content in leaf extracts of Mitragyna speciosa and ketum drink obtained from several locations in the Northern Peninsular of Malaysia. M. speciosa extract
MG content SD, mg/g (n = 3)
Ketum drink sample
MG content SD, mg/ml (n = 3)
Alkaloid-rich MeOH Water
24.72 0.67 4.77 1.30 0.80 0.11
Teluk Kumbar Gua Nangka Chuping Jelutong Jitra Sungai Batu Lembah Raja Balik Pulau
292.97 4.56 259.2 1.27 245.83 3.33 408.29 1.16 330.03 2.81 444.23 3.55 30.95 1.61 255.00 9.06
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Conflict of interest None declared. Acknowledgements The authors would like to thank Mr. Asokan Muniandy and Mr. Salam Abdullah for their technical assistance. This project was funded by Ministry of Science, Technology and Innovation (MOSTI) and Universiti Sains Malaysia Research University grant. Suhanya Parthasarathy was supported by Universiti Sains Malaysia fellowship scheme from Institute for Postgraduate Studies, Universiti Sains Malaysia. References
commercially which are priced according to their potency and taste (which are largely dependent on how the materials were extracted and the types of additives added), the data presented here can therefore be used as a reference to compare the type of extracts available in the market. Apart from ketum extracts, the applicability of the present method was also extended to study the amount of MG in ketum drinks. Samples obtained from eight different sources in the Northern Peninsular of Malaysia were evaluated. Results of the analysis showed that MG content varied greatly from among the different sources, i.e. approximately 31–444 mg/ml, as shown in Table 4. The amount of MG was found to be highest in the sample from Sungai Batu, while the sample from Lembah Raja contained the least amount of MG. The reasons to these variations may be attributed to geographical difference, maturity of the plant, method of preparation and the concentration of the drink. 4. Conclusion A rapid and reliable HPLC method has been developed and validated for the detection and quantification of MG in M. speciosa and its products. This method has advantage over the existing methods in terms of the simplicity of sample preparation, short analytical run time and is inexpensive compared to LC–MS. It is suitable for routine screening of M. speciosa products in the market including crude extracts, alkaloid enriched extracts and ketum drink. The amount of MG in the plant extracts obtained from the study ranged from 0.8 to 25 mg/g, while the amount of MG in ketum drink ranged from 31 to 444 mg/ml.
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