Metabolite Fingerprinting Using 1H-NMR Spectroscopy and Chemometrics for Classification of Three Curcuma Species from Different Origins

Curcuma longa, Curcuma xanthorrhiza, and Curcuma manga have been widely used for herbal or traditional medicine purposes. It was reported that turmeric plants provided several biological activities such as antioxidant, anti-inflammatory, hepatoprotector, cardioprotector, and anticancer activities. Authentication of the Curcuma species is important to ensure its authenticity and to avoid adulteration practices. Plants from different origins will have different metabolite compositions because metabolites are affected by soil nutrition, climate, temperature, and humidity. ¹H-NMR spectroscopy, principal component analysis (PCA), and orthogonal projections to latent structures-discriminant analysis (OPLS-DA) were used for authentication of C. longa, C. xanthorrhiza, and C. manga from seven different origins in Indonesia. From the ¹H-NMR analysis it was obtained that 14 metabolites were responsible for generating classification model such as curcumin, demethoxycurcumin, alanine, methionine, threonine, lysine, alpha-glucose, beta-glucose, sucrose, alpha-fructose, beta-fructose, fumaric acid, tyrosine, and formate. Both PCA and OPLS-DA model demonstrated goodness of fit (R² value more than 0.8) and good predictivity (Q² value more than 0.45). All OPLS-DA models were validated by assessing the permutation test results with high value of original R² and Q². It can be concluded that metabolite fingerprinting using ¹H-NMR spectroscopy and chemometrics provide a powerful tool for authentication of herbal and medicinal plants.     

细胞代谢组学用于羽扇豆醇干预人乳腺癌细胞MCF-7的机理探究

应用基于气相色谱-质谱联用（GC-MS）的代谢组学方法结合细胞周期实验,研究羽扇豆醇体外抑制人乳腺癌细胞MCF-7增殖的作用机理。代谢组学的研究结果表明：通过正交偏最小方差判别分析（OPLS-DA）可以很好地区分羽扇豆醇作用的MCF-7细胞代谢谱与对照组细胞代谢谱,模型参数为：R²Ycum=0.988,Q²Ycum=0.964。VIP（variable importance in the projection）值大于1的差异代谢物进一步用t检验进行单位分析,选择t<0.05（VIP>1）的代谢物作为羽扇豆醇作用组的生物标志物,得到琥珀酸、磷酸、亮氨酸、异亮氨酸等11种代谢差异物。结合羽扇豆醇将细胞周期抑制在G1期这一现象,推测羽扇豆醇可能是主要抑制了三羧酸循环中的琥珀酰辅酶A的生成和底物磷酸化生成ATP的反应来抑制MCF-7细胞的增殖。本实验从代谢组学角度为乳腺癌抗肿瘤机制提供新的线索。     

1H NMR Combined with Multivariate Statistics for Discrimination of Female and Male Flower Buds of Populus tomentosa

¹H Nuclear Magnetic Resonance (¹H NMR) combined with multivariate statistics was adopted to discriminate female and male flower buds of Populus tomentosa in the study. Samples of 11 female and 16 male flower buds of P. tomentosa were collected in Beijing, China. ¹H NMR spectra were acquired on a 400 MHz spectrometer. In total, 30 chemical compounds were identified with standards and literature according to chemical shifts, peak areas, and multiplicity. Principal component analysis (PCA), hierarchical clustering analysis (HCA), and supervised orthogonal partial least squares-discriminant analysis (OPLS-DA) were applied to discriminate female and male flower buds. An apparent grouping trend (R²X, 0.809; Q², 0.903) between female and male groups was exhibited with PCA and HCA. The two groups were also well discriminated with OPLS-DA (R²X, 0.808; R²Y, 0.976; Q², 0.960). Combined with variable importance in projection (VIP) > 1.0 and p < 0.05 of OPLS-DA, it was found that the content of daucosterol, β-sitosterol, ursolic acid, and betulonic acid in male group was higher than that in female, which should be the key differences of chemical constituents in female and male flower buds of P. tomentosa. The study demonstrated that ¹H NMR combined with multivariate statistics could be used to discriminate female and male plants and clarify differences, which provided a novel method to identify the gender of dioecious plants.     

GC-MS-based urine metabolic profiling of autism spectrum disorders

Autism spectrum disorders (ASD) are a group of neurodevelopmental disorders resulting from multiple factors. Diagnosis is based on behavioural and developmental signs detected before 3 years of age, and there is no reliable biological marker. The purpose of this study was to evaluate the value of gas chromatography combined with mass spectroscopy (GC-MS) associated with multivariate statistical modeling to capture the global biochemical signature of autistic individuals. GC-MS urinary metabolic profiles of 26 autistic and 24 healthy children were obtained by liq/liq extraction, and were or were not subjected to an oximation step, and then were subjected to a persilylation step. These metabolic profiles were then processed by multivariate analysis, in particular orthogonal partial least-squares discriminant analysis (OPLS-DA, R ²Y(cum) = 0.97, Q ²(cum) = 0.88). Discriminating metabolites were identified. The relative concentrations of the succinate and glycolate were higher for autistic than healthy children, whereas those of hippurate, 3-hydroxyphenylacetate, vanillylhydracrylate, 3-hydroxyhippurate, 4-hydroxyphenyl-2-hydroxyacetate, 1H-indole-3-acetate, phosphate, palmitate, stearate, and 3-methyladipate were lower. Eight other metabolites, which were not identified but characterized by a retention time plus a quantifier and its qualifier ion masses, were found to differ between the two groups. Comparison of statistical models leads to the conclusion that the combination of data obtained from both derivatization techniques leads to the model best discriminating between autistic and healthy groups of children.     

Determination of polyamines in urine via electrospun nanofibers–based solid-phase extraction coupled with GC-MS and application to gastric cancer patients

The simultaneous determination of polyamines and their metabolites in urine samples was achieved by gas chromatography–mass spectrometry in the selected ion monitoring mode. After conjugating with the ion-pair reagent bis-2-ethylhexylphosphate in the aqueous phase, the polyamines in the samples were extracted with polystyrene nanofiber-based packed-fiber solid-phase extraction followed by a derivatization step using pentafluoropropionyl anhydride. With optimal conditions, all analytes were separated well. For analytes of putrescine, cadaverine, N-acetylputrescine, and N-acetylcadaverine, the linearity was good in the range of 0.05–500 μmol/L (R² ≥ 0.993). While for spermidine, spermine, acetylspermidine, N⁸-acetylspermidine, and N-acetylspermine, the linearity was good in the range of 0.5–500 μmol/L (R² ≥ 0.990). The recoveries of three spiked concentrations (0.5, 5, 300 μmol/L) were 85.6%–108.4%, and relative standard deviations for intra- and interday were in the range of 2.9%–13.4% and 4.5%–15.1%, respectively. The method was successfully applied to the analysis of urine samples of gastric cancer patients. The results showed that the levels of most polyamines and N-acetylated polyamines from the patient group were significantly higher than those from the control group. The altered concentrations of the above-mentioned metabolites suggest their role in the pathogenesis of gastric cancer, and they should be further evaluated as potential markers of gastric cancer.     

Nuclear magnetic resonance-based metabolomics for prediction of gastric damage induced by indomethacin in rats

Non-steroidal anti-inflammatory drugs (NSAIDs) have side effects including gastric erosions, ulceration and bleeding. In this study, pattern recognition analysis of the ¹H-nuclear magnetic resonance (NMR) spectra of urine was performed to develop surrogate biomarkers related to the gastrointestinal (GI) damage induced by indomethacin in rats. Urine was collected for 5 h after oral administration of indomethacin (25 mg kg⁻¹) or co-administration with cimetidine (100 mg kg⁻¹), which protects against GI damage. The ¹H-NMR urine spectra were divided into spectral bins (0.04 ppm) for global profiling, and 36 endogenous metabolites were assigned for targeted profiling. The level of gastric damage in each animal was also determined. Indomethacin caused severe gastric damage; however, indomethacin administered with cimetidine did not. Simultaneously, the patterns of changes in their endogenous metabolites were different. Multivariate data analyses were carried out to recognize the spectral pattern of endogenous metabolites related to indomethacin using partial least square-discrimination analysis. In targeted profiling, a few endogenous metabolites, 2-oxoglutarate, acetate, taurine and hippurate, were selected as putative biomarkers for the gastric damage induced by indomethacin. These metabolites changed depending on the degree of GI damage, although the same dose of indomethacin (10 mg kg⁻¹) was administered to rats. The results of global and targeted profiling suggest that the gastric damage induced by NSAIDs can be screened in the preclinical stage of drug development using a NMR based metabolomics approach.     

Metabonomics study of atherosclerosis rats by ultra fast liquid chromatography coupled with ion trap-time of flight mass spectrometry

An ultra fast liquid chromatography coupled with IT-TOF mass spectrometry (UFLC/MS-IT-TOF) metabonomic approach was employed to study the plasma and urine metabolic profiling of atherosclerosis rats. Acquired data were subjected to principal component analysis (PCA) for differentiating the atherosclerosis and the control groups. Potential biomarkers were screened by using S-plot and were identified by the accurate mass and MSⁿ fragments information obtained from UFLC/MS-IT-TOF analysis. 12 metabolites in rat plasma and 8 metabolites in urine were identified as potential biomarkers. Concentrations of leucine, phenylalanine, tryptophan, acetylcarnitine, butyrylcarnitine, propionylcarnitine and spermine in plasma and 3-O-methyl-dopa, ethyl N2-acetyl-l-argininate, leucylproline, glucuronate, t6A N(6)-(N-threonylcarbonyl)-adenosine and methyl-hippuric acid in urine decreased in atherosclerosis rats. Ursodeoxycholic acid, chenodeoxycholic acid, LPC (C16:0), LPC (C18:0) and LPC (C18:1) in plasma and hippuric acid in urine were in higher levels in atherosclerosis rats. The alterated metabolites demonstrated abnormal metabolism of phenylalanine, tryptophan, bile acids and amino acids. This research proved that metabonomics is a promising tool for disease research.     

核磁共振技术测试不同性别大鼠尿液代谢物的研究

采用核磁共振波谱技术测试不同性别大鼠尿液代谢物,分析性别因素对大鼠尿液代谢成分的影响.大鼠尿液核磁共振氢谱（1HNMR谱）结果采用主成分分析（principal component analysis,PCA）和正交偏最小二乘判别分析（orthogonal to partial least squares discriminant analysis,OPLS-DA）方法分析,得到不同性别大鼠尿液中的差异性代谢物.PCA分析结果显示2组尿液代谢成分有明显的差异,进一步进行OPLS-DA分析可以判别出2组尿液中具有差异性的代谢物.结果显示,雌性大鼠尿液中的丙氨酸、缬氨酸、鸟氨酸等氨基酸类以及乙酸、硫胺、氨基马尿酸、苯乙胺、氧氨嘧啶等代谢物含量高于雄性大鼠,差异有统计学意义（p〈0.05）.雄性大鼠尿液中的甲胺、二甲胺、三甲胺、肌酸酐、尿囊素、延胡索酸、甲酸等代谢物则明显高于雌性大鼠,差异有统计学意义（p〈0.05）.性别因素对大鼠尿液中的代谢成分有一定的影响.     

Development and validation of a gas chromatography/mass spectrometry method for the metabolic profiling of human colon tissue

In this study, a gas chromatography/mass spectrometry (GC/MS) method was developed and validated for the metabolic profiling of human colon tissue. Each colon tissue sample (20 mg) was ultra‐sonicated with 1 mL of a mixture of chloroform/methanol/water in the ratio of 20:50:20 (v/v/v), followed by centrifugation, collection of supernatant, drying, removal of moisture using anhydrous toluene and finally derivatization using N‐methyl‐N‐trifluoroacetamide (MSTFA) with 1% trimethylchlorosilane (TMCS). A volume of 1 µL of the derivatized mixture was injected into the GC/MS system. A total of 53 endogenous metabolites were separated and identified in the GC/MS chromatogram, all of which were selected to evaluate the sample stability and precision of the method. Of the identified endogenous metabolites 19 belonging to diverse chemical classes and covering a wide range of the GC retention times (Rt) were selected to investigate the quantitative linearity of the method. The developed GC/MS method demonstrated good reproducibility with intra‐ and inter‐day precision within relative standard deviation (RSD) of ±15%. The metabolic profiles of the intact tissue were determined to be stable (100 ± 15%) for up to 90 days at ?80°C. Satisfactory results were also obtained in the case of other stability‐indicating studies such as freeze/thaw cycle stability, bench‐top stability and autosampler stability. The developed method showed a good linear response for each of the 19 analytes tested (r² > 0.99). Our GC/MS metabolic profiling method was successfully applied to discriminate biopsied colorectal cancer (CRC) tissue from their matched normal tissue obtained from six CRC patients using orthogonal partial least‐squares discriminant analysis [two latent variables, R²Y = 0.977 and Q² (cumulative) = 0.877]. Copyright © 2009 John Wiley & Sons, Ltd.     

Rapid structural characterization of in vivo and in vitro metabolites of tinoridine using UHPLC–QTOF–MS/MS and in silico toxicological screening of its metabolites

Tinoridine is a nonsteroidal anti‐inflammatory drug and also has potent radical scavenger and antiperoxidative activity. However, metabolism of tinoridine has not been thoroughly investigated. To identify in vivo metabolites, the drug was administered to Sprague–Dawley rats (n = 5) at a dose of 20 mg kg^?1, and blood, urine and feces were collected at different time points up to 24 h. In vitro metabolism was delved by incubating the drug with rat liver microsomes and human liver microsomes. The metabolites were enriched by optimized sample preparation involving protein precipitation using acetonitrile, followed by solid‐phase extraction. Data processes were carried out using multiple mass defects filters to eliminate false‐positive ions. A total of 11 metabolites have been identified in urine samples including hydroxyl, dealkylated, acetylated and glucuronide metabolites; among them, some were also observed in plasma and feces samples. Only two major metabolites were formed using liver microsomal incubations. These metabolites were also observed in vivo. All the 11 metabolites, which are hitherto unknown and novel, were characterized by using ultrahigh‐performance liquid chromatography–quadrupole time‐of‐flight tandem mass spectrometry in combination with accurate mass measurements. Finally, in silico toxicological screening of all metabolites was evaluated, and two metabolites were proposed to show a certain degree of lung or liver toxicity. Copyright © 2015 John Wiley & Sons, Ltd.     

黄芪口服液降低大鼠顺铂毒性作用机制的尿液代谢组学研究

采用基于液相色谱-飞行时间质谱联用(LC-TOF-MS)技术的代谢组学方法,分析大鼠尿液内源性代谢物的变化,研究黄芪口服液(HO)降低大鼠顺铂(CDDP)毒性的作用机制.采用低剂量多次腹腔注射CDDP的方法建立CDDP染毒大鼠模型,并连续给予16天HO.于第18天收集正常对照(Control)组、顺铂模型(CDDP)组和黄芪口服液(HO)组大鼠的24 h尿液, 进行LC-TOF-MS分析,以获取尿液代谢物组数据集,对所得数据进行主成分分析(PCA)和正交偏最小二乘法-判别分析(OPLS-DA)等多元统计分析,以筛选潜在生物标志物.于第20天采集大鼠血清测定肌酐和尿素氮水平.血清指标测定结果表明, HO可以显著降低CDDP染毒大鼠的肌酐和尿素氮水平(p<0.05).PCA得分图显示,3组可分别聚类,HO组位于Control组和CDDP组中间,表明HO可部分改善CDDP所致大鼠尿液代谢产物的异常变化.综合OPLS-DA分析、t检验和倍数变化分析结果,最终共筛选并初步鉴定出35个尿液代谢产物作为HO减毒相关的潜在生物标记物.代谢通路分析结果表明,HO可通过纠正体内氨基酸代谢、能量代谢和核苷酸代谢等通路的紊乱,降低CDDP所致机体毒性.     

Der Metabolismus des Retinoids Ro 10-9359. Isolierung und Identifizierung der Hauptmetaboliten aus Plasma,Urin und Faeces des Menschen sowie aus der Galle der Ratte Synthese von drei Urinmetaboliten

The Metabolism of the Retinoid Ro 10-9359. Isolation and Identification of the Major Metabolites in Human Plasma, Urine and Feces Synthesis of Three Urinary Metabolites After oral administration of therapeutic doses of the ³H-labelled aromatic retinoic acid analog (retinoid) Ro 10-9359 (ethyl all-trans-9-(4-methoxy-2,3,6-trimethyl-phenyl)-3,7-dimethyl-2,4,6,8-nonatetraenoate) to humans 75 and 15% of the ³H-dose were excreted within the first five days in the feces and the urine, respectively. Using chromatographic procedures including high pressure liquid chromatography 18 metabolites could be isolated from human urine. Their structures were elucidated by mass spectrometry and FT–¹H-NMR. spectroscopy. In these urinary metabolites the tetraene side chain of the parent compound Ro 10-9359 is shortened. The radioactivity of the identified urinary metabolites accounted for about 11% of the dose. Three urinary metabolites were synthesized. The main part of the radioactivity excreted within the first five days in the feces consisted of unchanged drug (60% of the dose). A smaller (amount 15% of the dose) could not be identified. The unchanged drug and a major metabolite, the corresponding acid, were found in human plasma. In an experiment with bile-duct cannulated rats the radioactively labelled retinoid Ro 10-9359 was injected intravenously. About 70% of the ³H-dose was excreted in the bile, within the first 48 hours. The whole radioactivity of the rat bile consisted of polar metabolites. No unchanged drug could be found. After enzymatic hydrolysis of the bile conjugates three metabolites were isolated. The main metabolite (49% of the i.v. dose) was a conjugate of the corresponding acid of the parent drug, already found as free compound in human plasma. The other bile metabolites (9 and 7% of the i.v. dose) had an intact side chain, too. An enterohepatic recycling of the bile metabolites was observed in the rat.     

基于超高效液相色谱-质谱分析的代谢组学在小檗碱治疗多囊卵巢综合征研究中的应用

多囊卵巢综合征（polycystic ovary syndrome,PCOS）是妇女最常见的内分泌和代谢紊乱性疾病,合理的药物治疗非常重要。本研究中,超重/肥胖的多囊卵巢综合征患者经过小檗碱治疗3个月,采集治疗前后的血样进行分析,利用超高效液相色谱-质谱联用技术（UHPLC-MS）对治疗前后血清中的内源性物质进行了相对含量测定。正交信号校正的偏最小二乘模式识别分析结果显示治疗前后有明显的区分。多变量分析结合非参数检验找出的与小檗碱治疗相关的差异代谢物均与脂类代谢有关。临床生化数据结合代谢组学数据显示,小檗碱治疗超重/肥胖型的多囊卵巢综合征患者可以增加机体胰岛素敏感性、改善脂类代谢。上述研究结果表明代谢组学是研究中药治疗疾病效果的有效工具之一。     

Dimethocaine,a synthetic cocaine analogue: studies on its in-vivo metabolism and its detectability in urine by means of a rat model and liquid chromatography–linear ion-trap (high-resolution) mass spectrometry

Dimethocaine (DMC, larocaine), a synthetic derivative of cocaine, is a widely distributed “legal high” consumed as a “new psychoactive substance” (NPS) without any safety testing, for example studies of metabolism. Therefore, the purpose of this work was to study its in-vivo and in-vitro metabolism by use of liquid chromatography–(high resolution) mass spectrometry (LC–HRMS ⁿ ). DMC was administered to male Wistar rats (20 mg kg^?1) and their urine was extracted either by solid-phase extraction after enzymatic cleavage of conjugates or by use of protein precipitation (PP). The metabolites were separated and identified by LC–HRMS ⁿ . The main phase I reactions were ester hydrolysis, deethylation, hydroxylation of the aromatic system, and a combination of these. The main phase II reaction was N-acetylation of the p-aminobenzoic acid part of the unchanged parent compound and of several phase I metabolites. The metabolites identified were then used for identification of DMC in rat urine after application of a common user’s dose. By use of GC–MS and LC–MS ⁿ standard urine-screening approaches (SUSAs), DMC and its metabolites could be detected in the urine samples.     

Identification of the metabolites of gigantol in rat urine by ultra‐performance liquid chromatography combined with electrospray ionization quadrupole time‐of‐flight tandem mass spectrometry

Gigantol is a typical bibenzyl compound isolated from Dendrobii Caulis that has been widely used as a medicinal herb in China for the treatment of diabetic cataract, cancer and arteriosclerosis obliterans and as a tonic for stomach nourishment, saliva secretion promotion and fever reduction. However, few studies have been carried out on its in vivo metabolism. In the present study, a rapid and sensitive method based on ultra‐performance liquid chromatography/electrospray ionization quadrupole time‐of‐flight tandem mass spectrometry (UPLC‐Q/TOF‐MS) in positive ion mode was developed and applied to identify the metabolites of gigantol in rat urine after a single oral dose (100 mg/kg). Chromatographic separation was performed on an Acquity UPLC HSS T3 column (100 × 2.1 mm i. d., 1.8 µm) using acetonitrile and 0.1% aqueous formic acid as mobile phases. A total of 11 metabolites were detected and identified as all phase II metabolites. The structures of the metabolites were identified based on the characteristics of their MS, MS² data and chromatographic retention times. The results showed that glucuronidation is the principal metabolic pathway of gigantol in rats. The newly identified metabolites are useful to understand the mechanism of elimination of gigantol and, in turn, its effectiveness and toxicity. As far as we know, this is the first attempt to investigate the metabolic fate of gigantol in vivo. Copyright © 2014 John Wiley & Sons, Ltd.     

In vivo metabolism study of (R)‐bambuterol in humans using ultra high performance liquid chromatography with tandem mass spectrometry

(R)‐Bambuterol, a selective β2‐adrenoceptor agonist, has been approved as a new drug for the treatment of asthma and chronic obstructive pulmonary disease by the China Food and Drug Administration and is currently under phase I clinical trials. In this study, a combined method based on ultra high performance liquid chromatography with triple quadrupole mass spectrometry and ultra high performance liquid chromatography with quadrupole time‐of‐flight mass spectrometry was employed for the identification of the major metabolites of (R)‐bambuterol in human plasma and urine after an oral dose of 10 mg. The metabolites were separated by gradient elution program and different sample preparation methods were compared. Totally, 12 metabolites of (R)‐bambuterol were identified, including four metabolites in plasma and all 12 metabolites in urine. Among these, four metabolites are reported for the first time. The possible metabolic pathways of (R)‐bambuterol were subsequently proposed. The results indicated that (R)‐bambuterol was metabolized via hydrolysis, demethylation, oxygenation, glucuronidation, and sulfation pathways in vivo. This study revealed that this combined method was accurate and sensitive to identify the possible metabolites and to better understand the metabolism of (R)‐bambuterol in vivo.     

LC–MS for Identification and Elucidation of the Structure of In-Vivo and In-Vitro Metabolites of Atropine

In-vivo and in-vitro metabolism of atropine has been investigated by use of a highly specific and sensitive LC–MS ⁿ method. Feces, urine, and plasma samples were collected separately after ingestion of 25 mg kg⁻¹ atropine by healthy rats. Rat feces and urine samples were cleaned by liquid–liquid extraction and by solid-phase extraction (on C₁₈ cartridges), respectively. Methanol was added to rat plasma samples to precipitate plasma proteins. Atropine was incubated, in vitro, with homogenized liver and with intestinal flora from rats. The metabolites in the incubation solution were extracted with ethyl acetate. These pretreated samples were then analyzed by reversed-phase high-performance liquid chromatography on a C₁₈ column with methanol–ammonium acetate (2 mm, adjusted to pH 3.5 with formic acid), 70:30 (v/v), as mobile phase. Detection was by on-line MS ⁿ . Identification and elucidation of the structure of the metabolites were achieved by comparing molecular mass (ΔM), retention-times, and full-scan MS ⁿ spectra with those of the parent drug. Ten new metabolites (aponoratropine, apoatropine, hydroxymethoxyatropine, trihydroxyatropine, dimethoxyatropine, dihydroxymethoxyatropine, hydroxydimethoxyatropine, trihydroxymethoxyatropine, dihydroxydimethoxyatropine, and tropic acid) were identified in rat urine after ingestion of atropine. Nine metabolites (nortropine, tropine, aponoratropine, apoatropine, noratropine, hydroxyatropine, hydroxyatropine N-oxide, hydroxymethoxyatropine, and tropic acid) and the parent drug were detected in rat feces. Five metabolites (nortropine, tropine, tropic acid, apoatropine, and hydroxyatropine) and the parent drug were detected in rat plasma. Only two metabolites (apoatropine and noratropine) were detected in the homogenized liver incubation mixture. The hydrolyzed metabolites (tropine and tropic acid) and dehydrated metabolite apoatropine were found in the rat intestinal flora incubation mixture.     

Metabolism studies of the <Emphasis Type="Italic">Kratom</Emphasis> alkaloid speciociliatine,a diastereomer of the main alkaloid mitragynine,in rat and human urine using liquid chromatography-linear ion trap mass spectrometry

Mitragyna speciosa (Kratom) is currently used as a drug of abuse. When monitoring its abuse in urine, several alkaloids and their metabolites must be considered. In former studies, mitragynine (MG), its diastereomer speciogynine (SG), and paynantheine and their metabolites could be identified in rat and human urine using LC-MSⁿ. In Kratom users' urines, besides MG and SG, further isomeric compounds were detected. To elucidate whether the MG and SG diastereomer speciociliatine (SC) and its metabolites represent further compounds, the phase I and II metabolites of SC were identified first in rat urine after the administration of the pure alkaloid. Then, the identified rat metabolites were screened for in the urine of Kratom users using the above-mentioned LC-MSⁿ procedure. Considering the mass spectra and retention times, it could be confirmed that SC and its metabolites are so far the unidentified isomers in human urine. In conclusion, SC and its metabolites can be used as further markers for Kratom use, especially by consumption of raw material or products that contain a high amount of fruits of the Malaysian plant M. speciosa.