LC-MS-MS method for mitragynine and 7-hydroxymitragynine in hair and its application in authentic hair samples of suspected kratom abusers.

Kratom is a natural psychoactive product known primarily in Southeast Asia, including Thailand, Malaysia, etc. It is also known as krathom, kakuam, ithang, thom (Thailand), biak-biak, ketum (Malaysia) and mambog (Philippines) and is sometimes used as an opium substitute. It is stimulant at doses of 1-5 g, analgesic at doses of 5-15 g and euphoric and sedative at doses of >15 g. Mitragynine is the most abundant indole compound in kratom (Mitragyna speciosa) and is metabolized in humans to 7-hydroxymitragynine, the more active metabolite. Adverse effects include seizures, nausea, vomiting, diarrhea, tachycardia, restlessness, tremors, hallucinations and death. There are few studies on the analytical method for the detection of mitragynine and 7-hydroxymitragynine in hair. Therefore, this study proposes a liquid chromatography-tandem mass spectrometry (LC-MS-MS) method for the analysis of kratom in hair. Hair samples were first weighed to ∼10 mg and washed with methanol. Then the washed hair samples were cut into pieces and incubated in methanol with stirring and heating (16 h/38℃). Extracts were then analyzed by LC-MS-MS. This method was validated by determining the limit of detection (LOD), limit of quantification, linearity, intra- and inter-day accuracy and precision, recovery and matrix effects. The intra- and inter-day precision (CV%) and accuracy (bias%) were within ±20%, which was considered acceptable. Using this newly developed LC-MS-MS method, the simultaneous detection of mitragynine and 7-hydroxymitragynine in six authentic hair samples was achieved to provide the direct evidence of kratom use in the past. Mitragynine concentrations ranged from 16.0 to 2,067 pg/mg (mean 905.3 pg/mg), and 7-hydroxymitragynine concentrations ranged from 0.34 to 15 pg/mg (mean 7.4 pg/mg) in six authentic hair samples from kratom abusers. This may be due to the higher sensitivity of the LOD in this study, with values of 0.05 pg/mg for mitragynine and 0.2 pg/mg for 7-hydroxymitragynine in hair.

Kratom (Mitragyna speciosa) Validation: Quantitative Analysis of Indole and Oxindole Alkaloids Reveals Chemotypes of Plants and Products.

Many consumers are turning to kratom (Mitragyna speciosa) to self-manage pain and opioid addiction. In the United States, an array of capsules, powders, and loose-leaf kratom products are readily available. Additionally, several online sites supply live kratom plants. A prerequisite to establishing quality control and quality assurance standards for the kratom industry, or understanding how alkaloid levels effect clinical outcomes, is the identification and quantitation of major and minor alkaloid constituents within available products and preparations. To this end, an ultra-high performance liquid chromatography-high resolution mass spectrometry method was developed for the analysis of 8 indole alkaloids (7-hydroxymitragynine, ajmalicine, paynantheine, mitragynine, speciogynine, isopaynantheine, speciociliatine, and mitraciliatine) and 6 oxindole alkaloids (isomitraphylline, isospeciofoleine, speciofoline, corynoxine A, corynoxeine, and rhynchophylline) in US-grown kratom plants and commercial products. These commercial products shared a qualitatively similar alkaloid profile, with 12 - 13 detected alkaloids and high levels of the indole alkaloid mitragynine (13.9 ± 1.1 - 270 ± 24 mg/g). The levels of the other major alkaloids (paynantheine, speciociliatine, speciogynine, mitraciliatine, and isopaynantheine) and the minor alkaloids varied in concentration from product to product. The alkaloid profile of US-grown M. speciosa "Rifat" showed high levels of the indole alkaloid speciogynine (7.94 ± 0.83 - 11.55 ± 0.18 mg/g) and quantifiable levels of isomitraphylline (0.943 ± 0.033 - 1.47 ± 0.18 mg/g). Notably, the alkaloid profile of a US-grown M. speciosa seedling was comparable to the commercial products with a high level of mitragynine (15.01 ± 0.20 mg/g). This work suggests that there are several M. speciosa chemotypes.

Unnatural spirocyclic oxindole alkaloids biosynthesis in Uncaria guianensis.

Spiro-oxindole scaffolds have been studied due to their promising therapeutic potential. In the Amazon rainforest there are two important Uncaria species known as "cat's claw", which biosynthesize spirocyclic oxindole alkaloids; Uncaria tomentosa (Willd. ex Schult.) DC. and Uncaria guianensis (Aublet) Gmell. We carried out a precursor-directed biosynthesis approach with U. guianensis and successfully obtained oxindole alkaloid analogues with molecular mass corresponding to the addition of a methyl or fluorine group on the oxindole ring using tryptamine analogue precursors. Two of these novel oxindole alkaloid analogues (3b-7-methyl-isomitraphylline and 3c-6-fluoro-isomitraphylline) were isolated and characterized by NMR spectroscopy and ESI-QTOF-MS/MS. Having established a substrate feeding protocol for these plantlets, the biosynthetic route for mitraphylline (1), rhynchophylline (2), isomitraphylline (3) and isorhynchophylline (4) was also investigated using 13C-precursors (1-13C-D-glucose, 2-13C-tryptophan, 1-13C-DL-glyceraldehyde, and methyl-13C-D-methionine).

[Effects of light intensity on growth and content of active components of Uncaria rhynchophyll].

The aim of this study was to study the effects of different light intensity on the growth,biomass accumulation and distribution,chlorophyll content and effective components of Uncaria rhynchophylla seedlings,and explore the suitable light intensity conditions for artificial cultivation of U. rhynchophylla seedlings. Three-year-old U. rhynchophylla seedlings were used as experimental materials. Four light intensity levels( 100%,70%-75%,30%-35%,5%-10%) were set up with different layers of black shading net. With the decrease of light intensity,the results showed that the plant height,basal diameter and biomass( root,stem,leaf and hook) of U.rhynchophylla seedlings exhibited the trend of " increase-decrease". Under 70%-75% light intensity,the plant height,basal diameter,biomass( root,stem,leaf,hook) of U. rhynchophylla seedlings were significantly higher than those of other treatments( P< 0. 05);under 5%-10% light intensity,the plant height,basal diameter and biomass( root,hook) of U. rhynchophylla seedlings were significantly lower than those of other treatments( P<0. 05). With the decrease of light intensity,the chlorophyll content of U. rhynchophylla seedlings increased gradually: under 100% light intensity,the chlorophyll content of U. rhynchophylla seedlings were the smallest,while under 100% light intensity,its chlorophyll content was the highest. With the decrease of light intensity,the contents of rhynchophylline and isorhynchophylline in different organs of U. rhynchophylla seedlings varied: under 30%-35% light intensity,the contents of rhynchophylline and isorhynchophylline in hooks and rhynchophylline content in stems were the highest; under 5%-10% light intensity,the contents of rhynchophylline and isorhynchophylline in leaves and stems of U. rhynchophylla were the highest. In conclusion,70%-75% light intensity is suitable for seedling growth and biomass accumulation,and 30%-35% light intensity is suitable for the accumulation of effective components in U. rhynchophylla seedlings.

Effects of light intensity on growth and content of active components of Uncaria rhynchophyll / 中国中药杂志

The aim of this study was to study the effects of different light intensity on the growth,biomass accumulation and distribution,chlorophyll content and effective components of Uncaria rhynchophylla seedlings,and explore the suitable light intensity conditions for artificial cultivation of U. rhynchophylla seedlings. Three-year-old U. rhynchophylla seedlings were used as experimental materials. Four light intensity levels( 100%,70%-75%,30%-35%,5%-10%) were set up with different layers of black shading net. With the decrease of light intensity,the results showed that the plant height,basal diameter and biomass( root,stem,leaf and hook) of U.rhynchophylla seedlings exhibited the trend of " increase-decrease". Under 70%-75% light intensity,the plant height,basal diameter,biomass( root,stem,leaf,hook) of U. rhynchophylla seedlings were significantly higher than those of other treatments( P< 0. 05);under 5%-10% light intensity,the plant height,basal diameter and biomass( root,hook) of U. rhynchophylla seedlings were significantly lower than those of other treatments( P<0. 05). With the decrease of light intensity,the chlorophyll content of U. rhynchophylla seedlings increased gradually: under 100% light intensity,the chlorophyll content of U. rhynchophylla seedlings were the smallest,while under 100% light intensity,its chlorophyll content was the highest. With the decrease of light intensity,the contents of rhynchophylline and isorhynchophylline in different organs of U. rhynchophylla seedlings varied: under 30%-35% light intensity,the contents of rhynchophylline and isorhynchophylline in hooks and rhynchophylline content in stems were the highest; under 5%-10% light intensity,the contents of rhynchophylline and isorhynchophylline in leaves and stems of U. rhynchophylla were the highest. In conclusion,70%-75% light intensity is suitable for seedling growth and biomass accumulation,and 30%-35% light intensity is suitable for the accumulation of effective components in U. rhynchophylla seedlings.

Genetic structure and chemical diversity in natural populations of Uncaria guianensis (Aubl.) J.F.Gmel. (Rubiaceae).

Uncaria guianensis is native to the Amazon and is used traditionally as an anti-inflammatory. Natural populations of the species have declined markedly in recent times because of strong anthropic pressure brought about by deforestation and indiscriminate collection. The aim of the present study was to assess the genetic and chemical diversity among eight natural populations of U. guianensis located in the Brazilian states of Acre, Amapá and Amazonas. A set of four primer combinations was employed in sequence-related amplified polymorphism (SRAP) amplifications of leaf DNA, and the fragments were analyzed in an LI-COR model 4300 DNA Analyzer. Genetic variability within the populations (81%) was substantially greater than that detected between them (19%). The highest percentage of polymorphic loci (90.21%) and the largest genetic variability were observed in the population located in Mazagão, Amapá. Genetic differentiation between populations was high (Fst = 0.188) and the studied populations formed three distinct genetic groups (K = 3). The population located in Assis Brasil, Acre, presented the highest average content of the mitraphylline (0.60 mg/g dry weight,). However, mitraphylline and isomitraphylline not detected in most individuals in the studied populations, and it is questionable whether they should be considered as chemical markers of the species. The genetic data confirm the urgent need for conservation programs for U. guianensis, and for further studies aimed at ascertaining the genetic basis and heritability of alkaloid accumulation.