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J Thai Trad Alt Med Vol. 21 No. 2 May-Aug 2023 415

Development and Method Validation of Mitragynine Contents in Mitragyna
Speciosa (Korth.) Havil. Leaves by Ultra High Performance Liquid
Chromatography

Aussavashai Shuayprom , Siriwan Chaisomboonpan, Sakwichai Ontong, Peradhama Thiemthieprat,
*
Sayan Ruengkhet, Thanawat Thongchin
Medicinal Plant Research Institute, Department of Medical Sciences, Tiwanon Road, Talat Khwan Subdistrict,
Mueang District, Nonthaburi 11000, Thailand

* Corresponding author: aussavashai.s@dmsc.mail.go.th

Abstract

Introduction and Objective: Kratom (Mitragyna speciosa) plant has been removed from the list of
controlled substances in Category 5, making it legally permissible for possession and consumption. Additionally,
kratom is now recognized for its economic value. The primary active ingredient in kratom leaves is mitragynine,
which induces pleasurable effects and provides pain relief. The objectives of this study were to develop a method
for analyzing the quantity of mitragynine in kratom leaves using ultra-high performance liquid chromatography
(UHPLC) and to test the validity of the developed analysis method.
Methods: The study was divided into three steps: developing the UHPLC method for analyzing mitragy-
nine in kratom leaves, testing the validity of the analysis method, and analyzing the quantity of mitragynine in 17
kratom leaf samples.
Results: The appropriate conditions for the analysis were using an ARC-18 stationary phase measuring 4.6
5 150 millimeters with a particle size of 2.7 micrometers and a mobile phase consisting of 0.1% ortho-phosphoric
acid in water and 0.1% ortho-phosphoric acid in acetonitrile at a flow rate of 1.0 milliliter per minute. The detection
wavelength was set at 222 nanometers, with an average retention time of 7.19 minutes. The applicability test of
the analysis method showed that the calibration curve was linear in the concentration range of 0.30–30.00 micro-
grams/milliliter, with a correlation coefficient (r) of 0.9998. The mean recovery percentage ranged from 99.72% to
100.88%, and the HORRAT values were in the range of 0.21–0.51. The precision test showed a relative standard
deviation of 0.53 for samples analyzed on the same day and 0.78 for samples analyzed over three consecutive days.
The limit of detection (LOD) was found to be 0.19 micrograms/milliliter, and the limit of quantification (LOQ) was
0.64 micrograms/milliliter. Using this developed UHPLC method for analysis, the study determined the quantity
of mitragynine in the 17 kratom leaf samples, with concentrations ranging from 0.99% to 1.89% w/w.
Discussion: The method developed for analyzing mitragynine in raw kratom leaves using UHPLC involves
the extraction of dried kratom leaves with ethanol as the solvent through the reflux method. The results of the
validity test of the analysis method were all within acceptable limits. This developed analysis method requires less
time for analysis and involves a simple, convenient, and rapid preparation of the mobile phase.
Conclusion and Recommendation: The developed UHPLC method for analyzing mitragynine in kratom
leaves was found to be accurate, precise, and suitable for determining the quantity of mitragynine in raw kratom
leaves and kratom leaf extracts. It can be used for routine analysis.

Key words: Mitragyna speciosa (Korth.) Havil., mitragynine, UHPLC