Simultaneous quantification of ten key Kratom alkaloids in Mitragyna speciosa leaf extracts and commercial products by ultra-performance liquid chromatography–tandem mass spectrometry

Abstract

Kratom (Mitragyna speciosa) is a psychoactive plant popular in the United States for the self-treatment of pain and opioid addiction. For standardization and quality control of raw and commercial kratom products, an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed and validated for the quantification of ten key alkaloids, namely: corynantheidine, corynoxine, corynoxine B, 7-hydroxymitragynine, isocorynantheidine, mitragynine, mitraphylline, paynantheine, speciociliatine, and speciogynine. Chromatographic separation of diastereomers, or alkaloids sharing same ion transitions, was achieved on an Acquity BEH C18 column with a gradient elution using a mobile phase containing acetonitrile and aqueous ammonium acetate buffer (10mM, pH 3.5). The developed method was linear over a concentration range of 1–200 ng/mL for each alkaloid. The total analysis time per sample was 22.5 minutes. The analytical method was validated for accuracy, precision, robustness, and stability. After successful validation, the method was applied for the quantification of kratom alkaloids in alkaloid-rich fractions, ethanolic extracts, lyophilized teas, and commercial products. Mitragynine (0.7%–38.7% w/w), paynantheine (0.3%–12.8% w/w), speciociliatine (0.4%–12.3% w/w), and speciogynine (0.1%–5.3% w/w) were the major alkaloids in the analyzed kratom products/extracts. Minor kratom alkaloids (corynantheidine, corynoxine, corynoxine B, 7-hydroxymitragynine, isocorynantheidine) were also quantified (0.01%–2.8% w/w) in the analyzed products; however mitraphylline was below the lower limit of quantification in all analyses.

1 |. INTRODUCTION

Kratom, Mitragyna speciosa (Korth.) Havil. (Rubiaceae), is a tropical plant in the same family as coffee. Tea brewed from its freshly harvested leaves has been consumed traditionally by the natives of Southeast Asia for centuries to treat fatigue and opium withdrawal. Kratom tea is also consumed for recreational purposes in Thailand, Malaysia, and Indonesia.^1,2 Kratom is popular among the American population and has been reported to be self-prescribed for the treatment of pain and opioid addiction. Traditionally, freshly harvested leaves from Mitragyna speciosa (kratom/ketum) are brewed or chewed, but in the United States, kratom preparations (made from dried leaves) are available in gas stations, local specialty shops, and online shopping portals in the form of tablets, capsules, powders, energy drinks, and dried leaves.^3,4 Although the importation of kratom is banned in the USA, it is still abundantly available in the marketplace.⁵ In many cases, available kratom products are manufactured at unregulated facilities with no standardization processes. However, some vendors do produce products under good manufacturing practice (GMP) regulations for dietary supplements. Yet, in the currently unregulated marketplace, various kratom products have been suspected to be adulterated by some suppliers with additional psychoactive substances leading to a number of emergency admissions and causalities in consumers.^3,6 A number of marketed kratom products do not have any manufacturing or expiration dates, or manufacturer addresses on their labels. Given the wide discrepancies of kratom products in the marketplace, there is an urgent need for standardization in the industry. The first step would be a proper way to identify and quantify the multiple kratom alkaloids that are present in each product and traditional preparations. There are multiple analytical and bioanalytical methods available in the literature for the quantification of kratom alkaloids.^3,7–14 Most of the reported methods were only able to quantify mitragynine and/or 7-hydroxymitragynine except the method reported by Kikura-Hanajiri et al⁹ which is able to quantify five kratom alkaloids (mitragynine and 7-hydroxymitragynine along with speciogynine, speciociliatine, and paynantheine), but there is no method available which can quantify 10 kratom alkaloids simultaneously. For this purpose, an ultra-performance liquid chromatography-mass spectrometry (UPLC-MS/MS) method was developed for the quantification of ten alkaloids in kratom products: corynantheidine, corynoxine, corynoxine B, 7-hydroxymitragynine, isocorynantheidine, mitragynine, mitraphylline, paynantheine, speciociliatine, and speciogynine (Figure 1). Mitragynine, the major kratom alkaloid, is considered to be primarily responsible for the pharmacological activities. Mitragynine is known to be a partial agonist of μ-opioid receptors (EC₅₀, 203 ± 13 nM) but does not recruit β-arrestin-2 mediated pathways like DAMGO (μ-opioid receptors; EC₅₀, 19 ± 7 nM). The β-arrestin-2 mediated pathways are thought to be responsible for the opioid mediated side effects (eg, respiratory depression, constipation, physical dependence, and tolerance).¹⁵ In addition, mitragynine exhibits polypharmacology in that it interacts with multiple central nervous system (CNS) drug targets.⁴ Thus, given these unique pharmacological mechanisms of action, mitragynine is considered an “atypical opioid.” Importantly, mitragynine has recently been reported to reduce morphine or heroin self-administration without any abuse or addiction potential in rodent models.^16,17 However, a minor, but potent μ-opioid receptor agonist kratom alkaloid, 7-hydroxymitragynine, exerts actions through β-arrestin-2 mediated pathways and produces tolerance and cross-tolerance to morphine, in addition to physical dependence, which most likely contributes to the liabilities associated with kratom use.^15,16 Interestingly, another minor kratom alkaloid, corynantheidine, acts as a functional antagonist at μ-opioid receptor and can reverse morphine-induced inhibition of twitch contraction in pig ileum.^18–20 Variance in the composition of alkaloids in kratom products, where there would be multiple alkaloids present including functional antagonist (corynantheidine) with partial (mitragynine) or full agonist (7-hydroxymitragynine), could potentially affect the μ-opioid receptor mediated therapeutic outcomes. A recent study reported that the phytochemicals present in both the organic fraction and lyophilized tea of kratom are able to affect the pharmacokinetics of mitragynine. The observed oral bioavailability of mitragynine was higher in rats dosed with the organic fraction or lyophilized tea, than in those dosed with mitragynine alone.²¹ Based on the traditional use of kratom for centuries and the available literature, kratom could be considered a potential treatment for opioid withdrawal and pain,¹ but this may be anecdotal. Systematic pre-clinical and clinical studies are warranted to understand the efficacy and abuse potential of kratom. In order to understand the pharmacological activities associated with the kratom alkaloids, it is requisite to know the composition of all the major and minor alkaloids in a given product, either commercially available or traditionally utilized. Therefore, a simple and sensitive analytical method was developed and validated for the simultaneous quantification of 10 major alkaloids. To the best of our knowledge, this is the first analytical method that is able to simultaneously quantify 10 key kratom alkaloids in plant material, extracts, and marketed products.

FIGURE 1.

Chemical structures of kratom alkaloids

2 |. MATERIALS AND METHODS

2.1 |. Chemicals and reagents

Corynantheidine, corynoxine, corynoxine B, 7-hydroxymitragynine, isocorynantheidine, mitragynine (as mitragynine hydrochloride salt), mitraphylline, paynantheine, speciociliatine, and speciogynine (purity ≥98%) were extracted and purified from the dried leaves of Mitragyna speciosa.²² Additional synthesis of 7-hydroxymitragynine (purity ≥98%, to increase material for our studies) was performed in-house using a method reported by Ponglux et al.²³ The alkaloids were subjected to proton nuclear magnetic resonance (¹H NMR), carbon nuclear magnetic resonance (¹³C NMR), high-performance liquid chromatography-photometric diode array (HPLC-PDA), and ultra-performance liquid chromatography-quadrupole time of flight (UPLC-QTOF), and found to be pure (≥ 98%) (Figures S1–S32). Phenacetin (purity ≥98%; internal standard) was purchased from Sigma Aldrich (St Louis, MO, USA). LC-MS grade acetonitrile, acetic acid, ammonium acetate, and water were purchased from Fisher Scientific (Fair Lawn, NJ, USA). Dried leaves of Mitragyna speciosa were purchased from Pure Land Ethnobotanicals, (Madison, WI, USA). Kratom commercial products [OPMS Gold; Choice Organic, Los Angeles, CA, USA); BLR (Urban Ice Organics, North Las Vegas, NV, USA); MDG (Urban Ice Organics, North Las Vegas, NV, USA); Plantation Red MD (Gaia Ethnobotanical, Jacksonville, FL, USA); Medicine Man Lone Wolf White Vein Maeng Da, Medicine Man Black Jaguar, and Supernatural Sun Red Label (all from Free River Distributing)] were either purchased from a local market or provided gratis.

2.2 |. Instrumentation and analytical conditions

A Waters Acquity Class I UPLC coupled with an Xevo TQ-S Micro triple quadrupole mass spectrometer (Milford, MA, USA) was used for the quantitative analysis of kratom alkaloids. Chromatographic separation of kratom alkaloids sharing monoisotopic masses (M + H⁺, (m/z) was achieved on a Waters Acquity BEH C18 column (1.7 μm, 2.1 × 100 mm) with a slow gradient using a mobile phase consisting of aqueous ammonium acetate buffer (10mM, pH 3.5; A) and acetonitrile (B). The flow rate of the mobile phase was held at 0.35 mL/min and a gradient was started with pump A and B supplying 80 and 20% of the mobile phase components up to 1 minute. The composition of component A in the mobile phase was decreased to 72% reaching 19.5 minutes. The composition of component A in the mobile phase was further decreased to 10% up to 20.5 minutes and was kept constant until 21.5 minutes, followed by a linear increase to 80% by 21.5 minutes, and was maintained up to 22.5 minutes. The sample injection volume was 2 μL. The strong needle wash consisted of acetonitrile, water, and isopropyl alcohol (70:10:20, %v/v). The weak needle wash comprised acetonitrile, water, and methanol (1:1:1, %v/v). The column oven and autosampler temperatures were maintained at 50 and 10°C, respectively. The detection of kratom alkaloids was achieved using a multiple reaction monitoring (MRM) method with electrospray ionization (ESI) in positive mode. The compound parameters (ion transitions, cone voltage, and collision energy) of the analyzed kratom alkaloids and the internal standard are shown in Table 1. The source parameters, such as capillary voltage, source temperature, desolvation temperature, desolvation gas, and cone gas flow rates, were held at 500 V, 150°C, 450°C, 900 L/h, and 50 L/h, respectively. The dwell time was set at 21 milliseconds to assure at least 10 data points per peak were recorded by the detector. Nitrogen was used as the source gas while argon was applied as the collision gas. Acquisition and analysis of the UPLC-MS/MS system was controlled by MassLynx software version 4.1.

TABLE 1.

Source parameters for kratom alkaloids and internal standard (phenacetin)

S. No.	Alkaloid	Ion Transition (m/z)	Cone Voltage (V)	Collision Energy (V)
1.	Corynantheidine	369.16 > 144.12	46.0	32.0
2.	Corynoxine	385.19 > 132.09	26.0	44.0
3.	Corynoxine B	385.19 > 132.09	26.0	44.0
4.	7-Hydroxymitragynine	415.19 > 190.09	42.0	30.0
5.	Isocorynantheidine	369.16 > 144.12	46.0	32.0
6.	Mitragynine	399.25 > 174.16	66.0	32.0
7.	Mitraphylline	369.22 > 160.12	2.0	32.0
8.	Paynantheine	397.16 > 174.10	58.0	30.0
9.	Speciociliatine	399.25 > 174.16	66.0	32.0
10.	Speciogynine	399.25 > 174.16	66.0	32.0
11.	Phenacetin	180.12 > 110.03	34.0	20.0

2.3 |. Preparation of calibration and quality control standards

Primary stock solutions of the kratom alkaloids (corynantheidine, corynoxine, corynoxine B, 7-hydroxymitragynine, isocorynantheidine, mitragynine, mitraphylline, paynantheine, speciociliatine, and speciogynine) were prepared separately by dissolving an accurately weighed quantity of each reference standard in an appropriate volume of methanol to achieve a concentration of 1 mg/mL. The primary stock solutions of kratom alkaloids were mixed together (200 μL, each) to prepare a combined stock (2 mL) of ten alkaloids containing 100 μg/mL of each alkaloid. The combined stock of kratom alkaloids was further diluted with acetonitrile to prepare combined working stocks containing 0.05, 0.1, 0.25, 1.25, 2.5, 4.5, 7.5, and 10 μg/mL of each analyte. Calibration standards containing 1, 2, 5, 25, 50, 90, 150, and 200 ng/mL were prepared by diluting 4 μL of the respective working stock to 200 μL of reconstitution solution. The reconstitution solution consisted of acetonitrile and water (50: 50, %v/v) containing the IS (10 ng/mL). Quality control standards were set at four concentrations [1 (LLOQ; lower limit of quantification), 2.5 (LQC; low quality control), 100 (MQC; medium quality control), and 180 ng/mL (HQC; high quality control)] of each analyte and were prepared using the same method as described for calibration standards except, the combined stocks of the kratom alkaloids (0.05. 0.125, 5, and 9 μg/mL) were prepared from separately weighed reference standards. A working stock solution containing 250 ng/mL of the internal standard was prepared in acetonitrile. All stock solutions were stored at −20°C and vortex mixed for 10 minutes prior to use.

2.4 |. Validation

The developed analytical method for the quantitative analysis of the kratom alkaloids was validated to establish the sensitivity, linearity, accuracy, precision, dilution integrity, robustness, and stability following the Food and Drug Administration (FDA) guidelines for the analysis of drugs and biologics.²⁴ The limit of detection (LOD) and LLOQ were defined as the spiked lowest concentrations of each alkaloid in acetonitrile and water (1:1, %v/v) which yielded a signal-to-noise (S/N) ratio of ≥6:1 for LOD and ≥ 10:1 with an accuracy and precision within 20% for LLOQ. A calibration curve for all ten alkaloids was comprised of a blank, a zero (blank with IS), and eight non-zero calibration standards (1, 2, 5, 25, 50, 90, 150, and 200 ng/mL with the IS, each alkaloid). A separate calibration curve for each alkaloid was prepared by plotting the peak area ratio of the analyte and the IS against the corresponding nominal concentrations applying a weighted (1/x) least-square regression model. Data analysis was performed using the TargetLynx application (Version 4.1). Accuracy and precision of the analytical method for each analyte was assessed by replicate analysis of the QC standards at four different concentrations [1 (LLOQ), 2.5 (LQC), 100 (MQC), and 180 (HQC) ng/mL, each N = 6] against the calibration standards on three different days. Accuracy and precision (intra- and inter-day) were calculated as %bias [(observed concentration – nominal concentration)/nominal concentration *100)] and percentage relative standard deviation (%R.S.D.), respectively. For the validation of the method in terms of accuracy and precision, an acceptance limit of ≤20% for LLOQ and ≤ 15% was applied for other quality control standards (LQC, MQC, and HQC).²⁴ For the analysis of the test samples above the linearity range, a dilution integrity test was performed. Dilution integrity quality control (DIQC) samples of high concentrations (5000 and 10 000 ng/mL, each alkaloid) were diluted (50x and 100x) and analyzed against freshly prepared calibration standards. The DIQC test was considered acceptable when the levels of analytes in the DIQC samples were ± 15% of nominal concentrations. The robustness of the analytical method was established. Low and high QC samples (LQC and HQC, each N = 3) were analyzed with freshly prepared calibration standards after changing the column oven temperature (47.5 and 52.5°C), mobile phase flow rate (0.33 and 0.37 mL/min), and pH of the ammonium acetate buffer (3.3 and 3.7), and the effects on peak properties (peak area and retention time) were assessed. A standard addition method was applied to understand the effect of other phytoconstituents and pharmaceutical excipients available in the analyzed marketed formulation. A formulation was spiked with a combined stock to add 50 ng/mL equivalent of each alkaloid (N = 3). Both samples were analyzed with freshly prepared calibration standards and concentrations of each kratom alkaloid were compared in pre- and post-spiked formulations. The chemical stability of the analytes and the IS in stock solutions and autosampler were assessed at −20°C for 1 week and 10°C for 48 hours, respectively. For stock solution stability, analytical standards prepared from stored stock solutions (on day 8) were analyzed against calibration standards obtained from freshly prepared stock solutions. To assure the stability of analytes in the autosampler during the analysis, QC samples of low and high (LQC and HQC, each N = 6) concentrations were analyzed once, and then stored in the auto-sampler (maintained at 10°C) for 48 hours. The stored QC samples were reanalyzed after 48 hours against freshly prepared calibration standards and concentrations of the analytes in the QC standards were compared.

2.5 |. Processing and analysis of kratom leaf alkaloid fraction, lyophilized tea, and commercial products

Kratom users in the United States typically consume it by making a tea in boiling water, or as organic/alcoholic extracts formulated in commercial products. Therefore four types of kratom samples (LKT, ethanolic extract, alkaloid fraction, and commercial products) were selected for the quantitative analysis.³ For the preparation of the ethanolic extract, dry leaves of Mitragyna speciosa (900 g) were extracted with ethanol (95%v/v), three times (1.5 L each), over three days. After the first cycle, the sample was filtered and fresh ethanol was added. The ethanol portions were combined and dried under reduced pressure to obtain an ethanolic extract (80 g). The alkaloid fraction was obtained from the ethanolic extract by acid–base extraction using the procedure described by Ponglux et al and Ali et al.^23,25 Briefly, dried ethanol extract (78 g) was dissolved in methanol and water (20%v/v) and 10% aqueous hydrochloric acid was added until pH was in the range of 2–3. Ethyl acetate (two times, 300 mL each) was added and partitioned through a separation funnel. The aqueous layer was separated and carefully basified (pH 8–9) using a 10% ammonia solution. The basified mixture was extracted with methylene chloride, (three times, 300 mL each) and the emulsion formed was further treated with brine. Combined organic layers were washed with water. After washing, the organic layer was dried over sodium sulfate (Na₂SO₄) and further concentrated under reduced pressure to yield the alkaloid fraction (4.8 g). The commercial kratom samples were extracted with ethanol (95%v/v), three times, over three days. After the first cycle, the sample was filtered and fresh ethanol was added. The ethanol portions were combined and dried under reduced pressure to obtain an ethanolic extract. The method described by Avery et al was implemented for aqueous extraction (tea).²¹ Dry leaves of Mitragyna speciosa (200 g) were placed in a Fernbach conical flask with 2 L of water and boiled for 20 minutes, the sample was filtered and the filtrate was dehydrated using a lyophilizer (Labconco^® 2.5 L free zone) to yield lyophilized kratom tea. Accurately weighed (2–5 mg), dried alkaloidal fractions of kratom, ethanolic extracts of commercial kratom products, and lyophilized kratom teas were dissolved in methanol. Diluted samples were vortex mixed and sonicated according to the method described by Kikura-Hanajiri et al.⁹ Samples containing solid particles were centrifuged. Test samples/supernatant (4 μL) were further diluted to 200 μL with the reconstitution solution and analyzed with freshly prepared calibration and quality control samples using the UPLC-MS/MS method.

3 |. RESULTS AND DISCUSSION

3.1 |. UPLC-MS/MS method development and validation

Method development started with the identification of ion transitions and optimization of compound parameters for the 10 of the kratom alkaloids. Therefore, analytical standards (50 ng/mL) were prepared from the corresponding reference standards of the various kratom alkaloids and infused through the inbuilt syringe pumps of the Waters Xevo TQ-S Micro mass spectrometer. The ionization of alkaloids was performed in both positive and negative ionization modes using an ESI source, but all studied alkaloids exhibited better response in positive rather than negative ionization mode. The IntelliStart application of MassLynx was used for the optimization of compound parameters (ion transitions, cone voltage, and collision energy) for each alkaloid. As shown in Table 1, the most sensitive and selective ion transitions containing product ions (m/z) generated from collision induced dissociation of precursor ions (m/z) were selected for the quantification of the kratom alkaloids (Figure S33).

A number of bioanalytical and analytical methods have been reported for the quantification of kratom alkaloids individually and in combination.^9,12–14,21 For the identification of appropriate chromatographic conditions, mobile phases and columns reported in earlier analytical methods of mitragynine and 7-hydroxymitragynine were evaluated first, but importantly, none of the methods were able to achieve adequate peak resolution between diastereomers or alkaloids sharing the same ion transitions (mitragynine, speciociliatine, and speciogynine; corynantheidine and isocorynantheidine; corynoxine and corynoxine B). A method reported by Kikura-Hanajiri et al was able to separate mitragynine from its diastereomers (speciociliatine and speciogynine) but it was long (38 minutes) and unable to separate other alkaloids sharing the same ion transitions.⁹ It is necessary to separate alkaloids sharing the same ion transitions chromatographically to assure their peak purity; otherwise, samples may be inappropriately over quantified for certain alkaloids. For example, mitragynine might have been over quantified along with its three other co-eluting diastereomers by earlier reported isocratic or short gradient methods.^9,12–14,21 Therefore, various mobile phase compositions (mobile phase A: aqueous ammonium acetate, water containing acetic acid, aqueous ammonium formate, or water containing formic acid; pH 3.0–6.8, mobile phase B: acetonitrile and methanol) and columns (Acquity UPLC BEH C18, CSH C18, Cyano, and HILIC) were tested to achieve at least a resolution of ≥2 between alkaloids sharing the same ion transitions with Gaussian peak shapes. Adequate peak separation with an appropriate peak shape was achieved with a combination of mobile phase containing acetonitrile and aqueous ammonium acetate buffer (10mM, pH 3.5) using an Acquity BEH C18 column via a slow gradient method at a flow rate of 0.35 mL/min. Retention times of corynantheidine, corynoxine, corynoxine B, 7-hydroxymitragynine, isocorynantheidine, mitragynine, mitraphylline, paynantheine, speciociliatine, speciogynine, and the IS were 11.06, 9.47, 7.06, 4.75, 14.66, 13.77, 5.15, 13.52, 18.03, 15.45, and 6.02 minutes, respectively (Figure 2). Total analysis time per sample was 22.5 minutes (Figure 2). Strong (800 μL) and weak (2400 μL) needle washes were applied in between the samples to diminish the effect of carryover between the samples, if any. The absence of autosampler carryover was determined by analyzing a blank sample after an analytical standard containing 200 ng/mL (each) of all the alkaloids and the IS (10 ng/mL). To assure no crosstalk was present between the alkaloids, analytical standards of individual alkaloids were prepared from reference standards and analyzed using the aforementioned method with no significant crosstalk between the analytes observed.

FIGURE 2.

Chromatograms of A, corynantheidine; B, corynoxine; C, corynoxine B; D, 7-hydroxymitragynine; E, isocorynantheidine; F, mitragynine; G, mitraphylline; H, paynantheine; I, speciociliatine; J, speciogynine at LLOQ (1 ng/mL, each); and K, internal standard (10 ng/mL)

After successful development, the analytical method was validated for sensitivity, linearity, accuracy, precision, dilution integrity, robustness, and stability.²⁴ Due to the narrow and sharp peaks, LLOQ of all the analytes were set at 1 ng/mL where accuracy and precision values were within 20% (N = 6, each analyte) and the S/N ratio was always ≥10:1. An eight-point calibration curve was established to check the linearity of the method and was found to be linear for a concentration range of 1–200 ng/mL (correlation coefficient ≥ 0.99, Figure S34). For the confirmation of reproducibility and reliability of the method, accuracy and precision (intra- and inter-day) of the method were calculated as %bias and %RSD at four different concentrations (LLOQ, LQC, MQC, and HQC) for each analyte. As shown in Table 2, intra- and inter-day accuracy values for kratom alkaloids were within the acceptable limits.²⁴ The studied alkaloids were stable in stock solutions at storage of −20°C for seven days in amber colored microcentrifuge tubes. Total analysis time per sample was 22.5 minutes, therefore it was essential to ensure the stability of the kratom alkaloids in the autosampler during analysis. All of the kratom alkaloids were found stable (−10.3–10.4; %deviation) after storage in autosampler up to 48 hours at 10°C as the deviation between the concentrations of analytes after storage was within the acceptable limit (±15%; Table 3). Analyte concentrations in DIQC samples after dilution (50X and 100X) were found within the acceptable limit (−5.2%–12.7%) which supported that the test samples containing alkaloids ≤10 000 ng/mL could be analyzed after dilution up to 100 X. The variations in chromatographic conditions (pH, flow rate, and column oven temperature) had significant effects on the retention times of the kratom alkaloids, but resolution between diastereomers, or alkaloids sharing same ion transitions, was adequate except for the mobile phase containing ammonium acetate buffer of higher (3.7) pH. However, %RSD values of analyte concentrations for low and high QC samples were within the acceptable limits (≤ 15%) to establish the robustness of the method. Effects of other phytoconstituents and pharmaceutical excipients on the analysis of any kratom alkaloid were not observed and the deviation of equivalent analyte concentration between pre- and post-spiked formulation was −1.6%-5.8%.

TABLE 2.

Accuracy and precision of the analytical method for the quantification of kratom alkaloids

Alkaloid	Concentration (ng/mL)	Accuracy (%Bias)		Precision (%RSD)
		Intra-day	Inter-day	Intra-day	Inter-day
Corynantheidine	1	−2.8	0.7	4.0	6.4
	2.5	−1.5	−1.6	3.9	4.2
	100	3.8	3.5	4.3	5.8
	180	3.2	−0.2	3.3	9.8
Corynoxine	1	−6.0	−3.7	4.3	7.6
	2.5	−3.0	−5.1	5.2	4.8
	100	−0.7	2.5	4.6	3.1
	180	3.0	0.5	3.2	7.9
Corynoxine B	1	−6.4	−6.5	4.3	7.9
	2.5	−0.6	−0.5	5.0	5.6
	100	6.0	4.8	4.6	6.4
	180	5.6	1.5	3.8	10.7
7-Hydroxymitragynine	1	5.2	11.5	5.6	17.1
	2.5	−0.1	0.3	3.3	5.6
	100	6.0	3.8	4.2	6.0
	180	5.4	1.6	3.1	6.6
Isocorynantheidine	1	3.2	10.2	3.1	7.3
	2.5	−0.7	0.1	1.4	3.9
	100	5.7	3.7	4.0	6.3
	180	5.2	1.2	3.2	7.7
Mitragynine	1	5.6	6.7	4.6	6.3
	2.5	−2.2	−3.6	2.3	4.1
	100	4.9	3.2	4.2	5.9
	180	4.0	1.6	2.8	6.3
Mitraphylline	1	−5.1	−7.8	4.7	7.8
	2.5	−5.3	−4.1	2.4	5.9
	100	1.0	−0.7	4.3	5.5
	180	2.5	1.2	2.9	5.2
Paynantheine	1	0.6	3.1	4.0	5.7
	2.5	−2.0	−1.4	1.5	3.0
	100	5.2	4.0	4.0	5.8
	180	4.5	1.6	3.2	6.9
Speciociliatine	1	3.3	4.3	3.7	5.5
	2.5	−4.2	−4.7	2.5	3.3
	100	5.3	4.0	4.5	6.3
	180	4.8	2.6	3.3	6.8
Speciogynine	1	−0.4	1.6	4.5	5.0
	2.5	−3.0	−2.7	1.7	3.6
	100	5.4	3.5	4.2	6.1
	180	4.4	2.5	3.2	6.6

TABLE 3.

Autosampler stability of kratom alkaloids at 10 °C for 48 hours

Alkaloid	Concentration (ng/mL)	Variation (% Deviation)
Corynantheidine	2.5	0.7
	180	−0.1
Corynoxine	2.5	−2.4
	180	−0.2
Corynoxine B	2.5	6.1
	180	4.8
7-Hydroxymitragynine	2.5	10.4
	180	0.4
Isocorynantheidine	2.5	4.2
	180	3.7
Mitragynine	2.5	−1.0
	180	−1.2
Mitraphylline	2.5	−10.3
	180	−4.6
Paynantheine	2.5	−1.0
	180	0.4
Speciociliatine	2.5	2.8
	180	1.6
Speciogynine	2.5	0.4
	180	0.6

3.2 |. Quantitative analysis of kratom alkaloidal fraction, ethanolic extract, lyophilized tea, and commercial products

After appropriate validation, the developed method was applied for the quantitative analysis of 10 major and minor alkaloids in the kratom leaf alkaloidal fraction, ethanolic extract, lyophilized tea, and commercial products (capsule and dried leaf). The alkaloidal fraction, ethanolic extract, and lyophilized tea were prepared in-house from authenticated kratom plant leaves and analyzed for alkaloid content. The results of the analysis indicated that the alkaloidal fraction, ethanolic extract, and lyophilized tea contained 33.6, 6.2, and 0.7% of mitragynine, which is less than the mitragynine content reported in earlier publications.^21,26 There is a possibility that earlier reported studies did not separate the four mitragynine diastereomers, resulting in over-quantification of mitragynine in these methods. Mitragynine, paynantheine, speciociliatine, and speciogynine are the major alkaloids present in both ethanolic extract and alkaloidal fraction of Mitragyna speciosa dried leaves. Mitraphylline was below LLOQ in all the analyzed samples. The potent kratom alkaloid, 7-hydroxymitragynine, content ranged from 0.01 to 0.04% which is equivalent to the earlier reported levels (0.01%–0.59%) in kratom products³ except in the in-house alkaloid rich fraction of Mitragyna speciosa where it has higher content (0.75%). The percent mitragynine content of the capsule formulations ranged from 4.7 to 8.7% except OPMS capsules which contained 38.7% of mitragynine. The possibility exists that most of the capsules were filled with the alcoholic extract of kratom, except OPMS Gold, which appears to contain the alkaloid-rich fraction of kratom. Empty packages of OPMS capsules were also found near a deceased person in Florida, and the higher alkaloid content in the OPMS Gold capsules and concomitant administration of other illicit substances (CYP3A4 and CYP2D6 inhibitors) can possibly account for the high systemic availability of mitragynine.^27–29 Corynoxine (2.75%) and its isomer corynoxine B (1.44%) were also identified in the OPMS capsules. The content of corynantheidine in the analyzed samples ranged from 0.02 to 1.16%. Corynantheidine is reported to be an opioid antagonist, so varying concentrations of corynantheidine in kratom products could affect the overall therapeutic effects of kratom products.¹⁸

Traditionally, freshly harvested kratom leaves are brewed to make tea and consumed by the natives of Southeast Asia. In this respect, kratom tea was prepared and lyophilized to quantify 10 key alkaloids present. One important caveat of this work is that the leaves that were utilized to produce the kratom tea that was analyzed were from dried leaf material and therefore, may not be directly comparable to the native decoction. Nonetheless, the 10 kratom alkaloids analyzed in this method were detected in the lyophilized kratom tea, but corynoxine and mitraphylline were found below LLOQ. Mitragynine (0.74%, w/w), speciociliatine (0.35%, w/w), and paynantheine (0.25%, w/w) were the major alkaloids detected in the lyophilized kratom tea (Table 4). The content of mitragynine in commercial products, especially capsules available in the United States, is much higher than the total mitragynine consumed from brewed tea preparations, similar to those used traditionally by the natives of Southeast Asia. Two causalities associated with kratom also support this claim, as the mitragynine plasma concentrations during the autopsy procedures were 3500 and 1800 ng/mL which were higher than controlled clinical studies in Thailand.^28,30 The measured mitragynine plasma concentration in a deceased individual from Florida was found to be 1800 ng/mL, which is 17.1 to 97.3 times higher than the peak plasma concentration (C_max) (18.5–105.0 ng/mL) measured in regular kratom users followed by a booster dose (6.25–23.00 mg, mitragynine equivalent dose, oral) of kratom tea on day 8 after reaching the steady state.^27,28,30 Due to the great variation in the range of alkaloid content between kratom samples and commercial products, it is necessary to standardize the dose and dosage regimen of kratom products and the developed analytical method can be helpful towards this goal.

TABLE 4.

Alkaloid content in kratom alkaloidal fraction, ethanolic extract, lyophilized tea, and commercial products

	Dry Powder/Extract Contains (%w/w)
Sample	Corynantheidine	Corynoxine	Corynoxine B	7-Hydroxymitragynine	Isocorynantheidine	Mitragynine	Mitraphyline	Paynantheine	Speciociliatine	Speciogynine
Ethanol extract of Kratom	-	-	-	-	-	6.24	-	2.39	3.66	1.18
Alkaloid fraction of Kratom	1.16	0.77	-	0.75	-	33.59	-	10.43	8.79	4.05
Lyophilized Kratom tea	0.02	-	0.03	0.01	0.02	0.74	-	0.25	0.35	0.10
OPMS Gold	0.88	2.75	1.44	-	-	38.74	-	12.76	12.29	5.29
BLR Kelly’s	0.08	-	0.09	0.01	0.07	4.71	-	1.81	1.36	0.87
MDG Kelly’s	0.08	-	0.07	0.01	0.05	4.83	-	1.81	1.60	0.89
Plantation red MD	0.08	0.03	0.03	0.04	0.08	5.35	-	2.09	2.13	0.99
Medicine man lone wolf white vein Maeng Da	0.10	0.04	0.04	0.02	0.08	6.25	-	2.49	2.70	1.10
Medicine man black jaguar	0.09	0.07	0.06	0.01	0.07	5.55	-	2.11	2.64	0.96
Supernatural sun red label	0.12	0.43	0.46	0.01	0.13	8.72	-	3.43	4.10	1.42

4 |. CONCLUSION

An accurate and precise method was developed for the quantification of ten major and minor kratom alkaloids (corynantheidine, corynoxine, corynoxine B, 7-hydroxymitragynine, isocorynantheidine, mitragynine, mitraphylline, paynantheine, speciociliatine, and speciogynine) in various kratom products. There is a possibility that previously reported analytical methods were not able to chromatographically resolve the diastereoisomers present in kratom products or biological samples, and thus the amount of mitragynine present in the samples analyzed by these methods might have overestimated the levels of mitragynine, including those utilized for determination of mitragynine content in human subjects. There is an urgent need to understand the selectivity issues associated with the natural products during the quantitative analysis. The developed method reported herein, was able to separate various combinations of diastereoisomers (corynantheidine and isocorynantheidine; corynoxine and corynoxine B; mitragynine, speciociliatine, and speciogynine) with adequate resolution (≥ 2), which will be beneficial for future kratom research and analysis. The present method provides a significant advantage for the characterization of various kratom products for their alkaloid composition. This method is now regularly used for the analysis of kratom extracts and products intended for the pharmacokinetic and pharmacodynamics studies in our laboratory. In association with pre-clinical and clinical studies, this method can be a stepping stone toward the characterization of standardized kratom preparations; and identification of doses and dosage regimens of kratom alkaloids to treat opioid dependence or pain.