Skip to main content
SpringerLink
Log in

Anti-mitragynine monoclonal antibody-based ELISA for determination of alkaloids in the kratom cocktail

  • Short Communication
  • Published:
Forensic Toxicology Aims and scope Submit manuscript

Abstract

Kratom cocktail or the fatal 4 × 100 formula is defined as a mixture of boiled kratom leaves, cola drink, and cough syrup. In the present study, we focused on application of the indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) using the anti-mitragynine (MG) monoclonal antibody (anti-MG mAb) to the kratom cocktail. The ic-ELISA is a rapid method for quantification of the major kratom alkaloids including MG, paynantheine, and speciogynine in kratom cocktails. Because some matrices or additives may influence the binding affinity between the alkaloids and the anti-MG mAb, a liquid–liquid extraction method using chloroform was used to clean-up samples and minimize any cross-reactivity with anti-MG mAb. The anti-MG mAb showed slight cross-reactivity to caffeine, codeine, morphine, tramadol, and dextromethorphan (<0.5 %), which are also commonly added to a kratom cocktail. When applied to eight different kratom cocktail samples, the ic-ELISA using the anti-MG mAb allowed the determination of the combined kratom alkaloid content in the range of 0.083–576 mg/L, and these values were in agreement with the results of the high-performance liquid chromatography method (R 2 = 0.9689). To our knowledge, this is the first report for the quantification of total amounts of kratom alkaloids including MG in kratom cocktail by a simple immunoassay. Because of the sharp rise in kratom cocktail abuse in the world, this method will be a useful tool for detection of kratom cocktail consumption.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2

References

  1. Grewal KS (1932) Observations on the pharmacology of mitragynine. J Pharmacol Exp Ther 46:251–257

    CAS  Google Scholar 

  2. Suwanlert S (1975) A study of kratom eaters in Thailand. Bull Narcotics 27:21–27

    CAS  Google Scholar 

  3. Jansen KLR, Prast CJ (1988) Ethnopharmacology of kratom and the Mitragyna alkaloids. J Ethnopharmacol 23:115–119

    Article  CAS  PubMed  Google Scholar 

  4. Shellard EJ (1974) The alkaloids of Mitragyna with special reference to those of M. speciosa Korth. Bull Narcotics 26:41–55

    CAS  Google Scholar 

  5. Ponglux D, Wongseripipatana S, Takayama H, Kukuchi M, Kukihara M, Kitayama M, Aimi N, Sakai S (1994) A new indole alkaloid, 7 α-hydroxy-7H-mitragynine, from Mitragyna speciosa in Thailand. Planta Med 60:580–581

    Article  CAS  PubMed  Google Scholar 

  6. Matsumoto K, Mizowaki M, Thongpradichote S, Takayama H, Sakai S-L, Aimi N, Watanabe H (1996) Central antinociceptive effects of mitragynine in mice: contribution of descending noradrenergic and serotonergic pathways. Eur J Pharmacol 371:75–81

    Article  Google Scholar 

  7. Matsumoto K, Mizowaki M, Thongpradichote S, Takayama H, Sakai S-L, Aimi N, Watanabe H (1996) Antinociceptive action of mitragynine in mice: evidence for the involvement of supraspinal opioid receptors. Life Sci 59:1149–1155

    Article  CAS  PubMed  Google Scholar 

  8. Watanabe K, Yano S, Horie S, Yamamoto LT (1997) Inhibitory effect of mitragynine, an alkaloid with analgesic effect from Thai medicinal plant, on electrically stimulated contraction of isolated guinea-pig ileum through the opioid receptor. Life Sci 60:933–942

    Article  CAS  PubMed  Google Scholar 

  9. Thongpraditchote S, Matsumoto K, Tohda M, Takayama H, Aimi N, Sakai SI, Watanabe H (1998) Identification of opioid receptor subtypes in antinociceptive actions of supraspinally-administered mitragynine in mice. Life Sci 62:1371–1378

    Article  Google Scholar 

  10. Tsuchiya S, Miyashita S, Horie S, Yamamoto M, Takayama H, Aimi N, Sakai SI, Watanabe K (2002) Effect of mitragynine, derived from Thai folk medicine, on gastric acid secretion through opioid receptor in anesthetized rats. Eur J Pharmacol 443:185–188

    Article  CAS  PubMed  Google Scholar 

  11. Purintrapiban J, Keawpradub N, Kansenalak S, Chittrakarn S, Janchawee B, Sawangjaroen K (2011) Study on glucose transport in muscle cells by extracts from Mitragyna speciosa (Korth) and mitragynine. Nat Prod Res 25:1379–1387

    Article  CAS  PubMed  Google Scholar 

  12. Utar Z, Majid MIA, Adenan MI, Jamil MFA, Lan TM (2011) Mitragynine inhibits the COX-2 mRNA expression and prostaglandin E2 production induced by lipopolysaccharide in RAW264.7 macrophage cells. J Ethnopharmacol 136:75–82

    Article  CAS  PubMed  Google Scholar 

  13. Idayu NF, Hidayat MT, Moklas MAM, Sharida F, Nurul Raudzah AR, Shamima AR, Apryani E (2011) Antidepressant-like effect of mitragynine isolated from Mitragyna speciosa Korth. in mice model of depression. Phytomedicine 18:402–407

    Article  PubMed  Google Scholar 

  14. Hassan Z, Muzaimi M, Navaratnam V, Yusoff NHM, Suhaimi FW, Vadivelu R, Vicknasingam BK, Amato D, Hörsten S, Ismail NIW, Jayabalan N, Hazim AI, Mansor SM, Müller CP (2013) From kratom to mitragynine and its derivatives: physiological and behavioral effects related to use, abuse and addiction. Neurosci Biobehav R 37:138–151

    Article  CAS  Google Scholar 

  15. Assanangkornchai S, Aramrattana A, Perngparn U, Kanato M, Kanika N, Ayudhya A (2008) Current situation of substance-related problems in Thailand. J Psychiatr Assoc Thailand 53:S24–S36

    Google Scholar 

  16. Tanguay P (2011) Kratom in Thailand, decriminalization and community control? Legis Reform Drug Policies 13:1–15

    Google Scholar 

  17. Janchawee B, Keawpradub N, Chittrakarn S, Prasettho S, Wararatananurak P, Sawangjareon K (2007) A high-performance liquid chromatographic method for determination of mitragynine in serum and its application to a pharmacokinetic study in rats. Biomed Chromatogr 21:176–183

    Article  CAS  PubMed  Google Scholar 

  18. de Moraes NV, Moretti RAC, Fur EB III, McCurrdy CR, Lanchote VL (2009) Determination of mitragynine in rat plasma by LC-MS/MS: application to pharmacokinetics. J Chromatogr B 877:2593–2597

    Article  Google Scholar 

  19. Kikura-Hanajiri R, Kawamura M, Maruyama T, Kitajima M, Takayama H, Goda Y (2009) Simultaneous analysis of mitragynine, 7-hydroxymitragynine, and other alkaloids in the psychotropic plant “kratom” (Mitragyna speciosa) by LC-ESI-MS. Forensic Toxicol 27:67–74

    Article  CAS  Google Scholar 

  20. Lu S, Tran BN, Nelsen JL, Aldous KM (2009) Quantitative analysis of mitragynine in human urine by high performance liquid chromatography-tandem mass spectrometry. J Chromatogr B 877:2499–2505

    Article  CAS  Google Scholar 

  21. Parthasarathy S, Ramanathan S, Ismail S, Adenan MI, Mansor SM, Murugaiyah W (2010) Determination of mitragynine in plasma with solid-phase extraction and rapid HPLC-UV analysis, and its application to a pharmacokinetic study in rat. Anal Bioanal Chem 397:2023–2030

    Article  CAS  PubMed  Google Scholar 

  22. Oliveira AS, Fraga S, Carvalho F, Arajúo AM, Pereira CC, Teixeira JP, de Lourdes BM, de Pinho PG (2016) Chemical characterization and in vitro cyto- and genotoxicity of ‘legal high’ products containing Kratom (Mitragyna speciosa). Forensic Toxicol 34:213–226

    Article  CAS  Google Scholar 

  23. Wang M, Carrell EJ, Ali Z, Avula B, Avonto C, Parcher JF, Khan IA (2014) Comparison of three chromatographic techniques for the detection of mitragynine and other indole and oxindole alkaloids in Mitragyna speciosa (kratom) plants. J Sep Sci 37:1411–1418

    Article  CAS  PubMed  Google Scholar 

  24. Chan KB, Pakiam C, Rahim RA (2005) Psychoactive plant abuse: the identification of mitragynine in ketum and in ketum preparations. Bull Narcotics 57:249–256

    CAS  Google Scholar 

  25. Chittrakarn S, Penjamras P, Keawpradub N (2012) Quantitative analysis of mitragynine, codeine, caffeine, chlorpheniramine and phenylephrine in a kratom (Mitragyna speciosa Korth.) cocktail using high-performance liquid chromatography. Forensic Sci Int 217:81–86

    Article  CAS  PubMed  Google Scholar 

  26. Parthasarathy S, Ramanathan S, Murugaiyah V, Hamdan MR, Said MIM, Lai CH, Mansor SM (2013) 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 Sci Int 226:183–187

    Article  CAS  PubMed  Google Scholar 

  27. Limsuwanchote S, Wungsintaweekul J, Keawpradub N, Putalun W, Morimoto S, Tanaka H (2014) Development of indirect competitive ELISA for quantification of mitragynine in kratom (Mitragyna speciosa (Roxb.) Korth.). Forensic Sci Int 244:70–77

    Article  CAS  PubMed  Google Scholar 

  28. Keawpradub N (1990) Alkaloids from the fresh leaves of Mitragyna speciosa. Master Thesis, Graduate School, Chulalongkorn University, Bangkok

  29. Weiler EW, Zenk MH (1976) Radioimmunoassay for the determination of digoxin and related compounds in Digitalis lanata. Phytochemistry 15:1537–1545

    Article  CAS  Google Scholar 

  30. Adkins JE, Boyer EW, McCurdy CR (2011) Mitragyna speciosa, a psychoactive tree from Southeast Asia with opioid activity. Curr Top Med Chem 11:1165–1175

    Article  CAS  PubMed  Google Scholar 

  31. Tungtananuwat W, Lawanprasert S (2010) Fatal 4×100; home-made kratom juice cocktail. J Health Res 24:43–47

    Google Scholar 

Download references

Acknowledgments

The authors are grateful to the Royal Golden Jubilee Ph. D Program, the Thailand Research Fund (PHD/0311/2550), the National Research Council of Thailand (PHA560010S), and the budget revenue (PHA580949S). The Postdoctoral Fellowship from Prince of Songkla University granted to S.L. is also acknowledged.

Author information

Authors and Affiliations

  1. Department of Pharmacognosy and Pharmaceutical Botany, Faculty of Pharmaceutical Science, Prince of Songkla University, Hat Yai, Songkhla, 90112, Thailand

    Supattra Limsuwanchote, Niwat Keawpradub & Juraithip Wungsintaweekul

  2. Faculty of Pharmaceutical Sciences, Khon Kaen University, Khon Kaen, 40002, Thailand

    Waraporn Putalun

  3. Department of Pharmacognosy, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka, 812-8582, Japan

    Hiroyuki Tanaka & Satoshi Morimoto

Authors
  1. Supattra Limsuwanchote
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Waraporn Putalun
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Niwat Keawpradub
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Hiroyuki Tanaka
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Satoshi Morimoto
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Juraithip Wungsintaweekul
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Juraithip Wungsintaweekul.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Limsuwanchote, S., Putalun, W., Keawpradub, N. et al. Anti-mitragynine monoclonal antibody-based ELISA for determination of alkaloids in the kratom cocktail. Forensic Toxicol 35, 167–172 (2017). https://doi.org/10.1007/s11419-016-0332-y

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11419-016-0332-y

Keywords

Search

Navigation