Metabolic profiles of dioscin in rats revealed by ultra‐performance liquid chromatography quadrupole time‐of‐flight mass spectrometry

Dioscin (DIS), one of the most abundant bioactive steroidal saponins in Dioscorea sp., is used as a complementary medicine to treat coronary disease and angina pectoris in China. Although the pharmacological activities and pharmacokinetics of DIS have been well demonstrated, information regarding the final metabolic fates is very limited. This study investigated the in vivo metabolic profiles of DIS after oral administration by ultra‐performance liquid chromatography quadrupole time‐of‐flight mass spectrometry method. The structures of the metabolites were identified and tentatively characterized by means of comparing the molecular mass, retention time and fragmentation pattern of the analytes with those of the parent compound. A total of eight metabolites, including seven phase I and one phase II metabolites, were detected and tentatively identified for the first time. Oxidation, deglycosylation and glucuronidation were found to be the major metabolic processes of the compound in rats. In addition, a possible metabolic pathway on the biotransformation of DIS in vivo was proposed. This study provides valuable and new information on the metabolism of DIS, which will be helpful for further understanding its mechanism of action. Copyright © 2015 John Wiley & Sons, Ltd.     

Chemical UPLC‐ESI‐MS/MS profiling of aconitum alkaloids and their metabolites in rat plasma and urine after oral administration of Aconitum carmichaelii Debx. Root extract

In this paper, an ultra high performance liquid chromatography tandem mass spectrometric (UPLC‐ESI‐MS/MS) method in positive ion mode was established to systematically identify and to compare the major aconitum alkaloids and their metabolites in rat plasma and urine after oral administration of Fuzi extract. A total twenty‐nine components including twenty‐five C19‐diterpenoid alkaloids and four C20‐diterpenoid alkaloids were identified in Fuzi extract. Thirteen of the parent components and five metabolites were detected in rat plasma and sixteen parent compounds and six metabolites in urine. These parent components found in rat plasma and urine were mainly C19‐diterpenoid alkaloids. All of the metabolites in vivo were demethylated metabolites (phase I metabolites), which suggested that demethylation was the major metabolic pathway of aconitum alkaloids in vivo. A comparison of the parent components in rat plasma and urine revealed that 3‐deoxyacontine was found in plasma but not in urine, while kalacolidine, senbusine and 16‐β‐hydroxycardiopetaline existed in urine but not in plasma, which indicated that most alkaloids components were disposed and excreted in prototype form. This research provides some important information for further metabolic investigations of Fuzi in vivo.     

Characterization of ginsenoside compound K metabolites in rat urine and feces by ultra‐performance liquid chromatography with electrospray ionization quadrupole time‐of‐flight tandem mass spectrometry

Ginsenoside compound K (CK) is an active metabolite of ginsenoside and has been shown to have ameliorative property in various diseases. However, the detailed in vivo metabolism of this compound has rarely been reported. In the present study, a method using liquid chromatography quadrupole time‐of‐flight tandem mass spectrometry together with multiple data processing techniques, including extracted ion chromatogram, multiple mass defect filter and MS/MS scanning, was developed to detect and characterize the metabolites of CK in rat urine and feces. After oral administration of CK at a dose of 50 mg/kg, urine and feces were collected for a period of time and subjected to a series of pretreatment. A total of 12 metabolites were tentatively or conclusively identified, comprising 11 phase I metabolites and a phase II metabolite. Metabolic pathways of CK has been proposed, including oxidation, deglycosylation, deglycosylation with sequential oxidation and dehydrogenation and deglycosylation with sequential glucuronidation. Relative quantitative analyses suggested that deglycosylation was the main metabolic pathway. The result could offer insights for better understanding of the mechanism of its pharmacological activities.     

Characterization of metabolites of sweroside in rat urine using ultra‐high‐performance liquid chromatography combined with electrospray ionization quadrupole time‐of‐flight tandem mass spectrometry and NMR spectroscopy

Sweroside, a major active iridoid in Swertia pseudochinensis Hara, is recognized as an effective agent in the treatment of liver injury. Based on previous reports, the relatively short half‐life (64 min) and poor bioavailability (approximately 0.31%) in rats suggested that not only sweroside itself but also its metabolites could be responsible for the observed hepato‐protective effect. However, few studies have been carried out on the metabolism of sweroside. Therefore, the present study aimed at identifying the metabolites of sweroside in rat urine after a single oral dose (100 mg/kg). With ultra‐high‐performance liquid chromatography coupled with electrospray ionization quadrupole time‐of‐flight tandem mass spectrometry (UHPLC/Q‐TOF‐MS), the metabolic profile revealed 11 metabolites in rat urine, including phase I, phase II and aglycone‐related products. The chemical structures of metabolites were proposed based on accurate mass measurements of protonated or deprotonated molecules and their fragmentation patterns. Our findings showed that the aglycone of sweroside (M05) and its glucuronide conjugate (M06) were principal circulating metabolites in rats. While several other metabolic transformations, occurring via reduction, N‐heterocyclization and N‐acetylation after deglycosylation, were also observed. Two metabolites (M05 and M06) were isolated from the rat urine for structural elucidation and identifcation of reaction sites. Both M05 and M06 were characterized by ¹H, ¹³C and two‐dimensional nuclear magnetic resonance (NMR) spectroscopy. UHPLC/Q‐TOF‐MS analysis has provided an important analytical platform to gather metabolic profile of sweroside. Copyright © 2014 John Wiley & Sons, Ltd.     

UPLC‐MS/MS method for the determination of tobacco‐specific biomarker NNAL,tamoxifen and its main metabolites in rat plasma

Cigarette smoke is known to interact with tamoxifen‐metabolizing enzymes and transporters and potentially affect its treatment outcome. 4‐(N‐ nitrosomethylamino)‐1‐(3‐pyridyl)‐1‐butanol (NNAL) is an important metabolite of 4‐(methylnitro‐samino)‐1‐(3‐pyridyl)‐1‐butanone (NNK) because it is frequently used as a biomarker to assess human smoke exposure. In order to study the potential pharmacokinetic interaction between cigarette smoke and tamoxifen in rats a UPLC‐MS/MS method for the simultaneous determination of NNAL and tamoxifen along with its metabolites in rat plasma has been developed and validated. Analytes were extracted with methanol and separated on a HSS T3 column by a gradient elution with the mobile phase consisting of acetonitrile and water. The lower limits of quantitation ranged from 0.05 to 0.62 ng/mL. Precisions showed RSD <15.8% and accuracy in the range 80.6–116.0%. Mean analyte recoveries ranged from 76.9 to 108.4%. The method was successfully applied to study the effects of cigarette smoke condensate (CSC), NNK and benzo(a)pyrene pre‐treatment on the pharmacokinetics of tamoxifen and its metabolites in rats. Significant effects of CSC, NNK, benzo(a)pyrene were observed on pharmacokinetics of tamoxifen and its metabolites. We also found that plasma NNAL levels are statistically significant correlated with plasma 4‐hydroxy‐tamoxifen and endoxifen.     

Identification and structural characterization of in vivo metabolites of ketorolac using liquid chromatography electrospray ionization tandem mass spectrometry (LC/ESI‐MS/MS)

In vivo metabolites of ketorolac (KTC) have been identified and characterized by using liquid chromatography positive ion electrospray ionization high resolution tandem mass spectrometry (LC/ESI‐HR‐MS/MS) in combination with online hydrogen/deuterium exchange (HDX) experiments. To identify in vivo metabolites, blood urine and feces samples were collected after oral administration of KTC to Sprague–Dawley rats. The samples were prepared using an optimized sample preparation approach involving protein precipitation and freeze liquid separation followed by solid‐phase extraction and then subjected to LC/HR‐MS/MS analysis. A total of 12 metabolites have been identified in urine samples including hydroxy and glucuronide metabolites, which are also observed in plasma samples. In feces, only O‐sulfate metabolite and unchanged KTC are observed. The structures of metabolites were elucidated using LC‐MS/MS and MSⁿ experiments combined with accurate mass measurements. Online HDX experiments have been used to support the structural characterization of drug metabolites. The main phase I metabolites of KTC are hydroxylated and decarbonylated metabolites, which undergo subsequent phase II glucuronidation pathways. Copyright © 2012 John Wiley & Sons, Ltd.     

Metabolic profiles of the Flos Abelmoschus manihot extract by intestinal bacteria from the normal and CKD model rats based on UPLC‐Q‐TOF/MS

Flos Abelmoschus manihot is a traditional herbal medicine widely used in clinical practice to tackle chronic kidney disease (CKD) for thousands of years. Nowadays, many studies indicate that gut bacteria are closely related to the progression of CKD and CKD‐related complications. In this study, a UPLC‐Q‐TOF/MS method coupled with the MetaboLynx™ software was established and successfully applied to investigate the metabolites and metabolic profile of Flos A. manihot extract by intestinal bacteria from normal and CKD rats. Eight parent components and eight metabolites were characterized by their protonated ions. Among these compounds, 15 were detected in the two group samples while M16 was only determined in the CKD model samples. Compared with the quercetin‐type glycosides, fewer myricetin‐type and gossypetin‐type metabolites were obtained in the two group samples. These metabolites suggested that deglycosylation and methylation are the major metabolic pathways of Flos A. manihot extract. Few differences of metabolite classes were observed in the two group samples. However, the concentrations of aglycones such as quercetin, myricetin and gossypetin in the normal samples were notably higher than those in the CKD model samples. The results are important in unravelling the pharmacological effects of A. manihot and clarifying its mechanism of action in vivo .     

Development and validation of a UPLC‐MS/MS method for the determination of cucurbitacin B in rat plasma and application to a pharmacokinetic study

Cucurbitacin B (CuB), one of the most abundant forms of cucurbitacins, is a promising natural anticancer drug candidate. Although the anticancer activity of CuB has been well demonstrated, information regarding the pharmacokinetics is limited. A rapid, selective and sensitive UPLC‐MS/MS for CuB was developed and validated using hemslecin A (HeA) as internal standard (IS). Plasma samples were pre‐treated by liquid–liquid extraction with dichloromethane. Separation was achieved on a reversed‐phase C₁₈ column (50 × 4.6 mm, 5 µm) at 35°C using isocratic elution with water–methanol (25:75, v/v) at a flow rate of 0.3 mL/min. The analytes were monitored by a triple quadrupole tandem mass spectrometer with positive electrospray ionization mode. The calibration curve was linear (r > 0.995) in a concentration range of 0.3–100 ng/mL with a limit of quantification of 0.3 ng/mL. Intra‐ and inter‐day accuracy and precision were validated by percentage relative error and relative standard deviation, respectively, which were both lower than the limit of 15%. This assay was successfully applied to a pharmacokinetic study of CuB in Wistar rats. Copyright © 2015 John Wiley & Sons, Ltd.     

In vivo metabolic investigation of silodosin using UHPLC–QTOF–MS/MS and in silico toxicological screening of its metabolites

Silodosin (SLD) is a novel α1‐adrenoceptor antagonist which has shown promising clinical efficacy and safety in patients with benign prostatic hyperplasia (BPH). However, lack of information about metabolism of SLD prompted us to investigate metabolic fate of SLD in rats. To identify in vivo metabolites of SLD, urine, feces and plasma were collected from Sprague–Dawley rats after its oral administration. The samples were prepared using an optimized sample preparation approach involving protein precipitation followed by solid‐phase extraction and then subjected to LC/HR‐MS/MS analysis. A total of 13 phase I and six phase II metabolites of SLD have been identified in rat urine which includes hydroxylated, N‐dealkylated, dehydrogenated, oxidative, glucosylated, glucuronide and N‐sulphated metabolites, which are also observed in feces. In plasma, only dehydrogenated, N‐dealkylated and unchanged SLD are observed. The structure elucidation of metabolites was done by fragmentation in MS/MS in combination with HRMS data. The potential toxicity profile of SLD and its metabolites were predicted using TOPKAT software and most of the metabolites were proposed to show a certain degree of skin sensitization and occular irritancy. Copyright © 2016 John Wiley & Sons, Ltd.     

Determination of a hydrophilic paclitaxel derivative, 7‐xylosyl‐10‐deacetylpaclitaxel in rat plasma by LC–MS/MS

A LC‐MS/MS method for the determination of a hydrophilic paclitaxel derivative 7‐xylosyl‐10‐deacetylpaclitaxel in rat plasma was developed to evaluate the pharmacokinetics of 7‐xylosyl‐10‐deacetylpaclitaxel in the rats. 7‐Xylosyl‐10‐deacetylpaclitaxel and docetaxel (IS for 7‐xylosyl‐10‐deacetylpaclitaxel) were extracted from rat plasma with acetic ether and analyzed on a Hypersil C₁₈ column (4.6 × 150 mm i.d., particle size 5 µm) with the mobile phase of ACN/0.05% formic acid (50:50, v/v). The analytes were detected using an ESI MS/MS in the multiple reaction monitoring mode. The standard curves for 7‐xylosyl‐10‐deacetylpaclitaxel in plasma were linear (r >0.999) over the concentration range of 2.0–1000 ng/mL with a weighting of 1/concentration². The method showed a satisfactory sensitivity (2.0 ng/mL using 50 µL plasma), precision (CV ≤ 10.1%), accuracy (relative error ?12.4 to 12.0%), and selectivity. This method was successfully applied to the pharmacokinetic study of 7‐xylosyl‐10‐deacetylpaclitaxel in rat plasma after intravenous administration of 7‐xylosyl‐10‐deacetylpaclitaxel to female Wistar rats. Copyright © 2008 John Wiley & Sons, Ltd.     

Liquid chromatography/quadrupole time‐of‐flight mass spectrometry in combination with online hydrogen/deuterium exchange technique for structural elucidation of phase I metabolites of iso‐phenylcyclopentylamine in rat bile

MS/MS experiment and accurate mass measurement are powerful tools in metabolite identification. However, sometimes these data do not provide enough information to assign an unambiguous structure to a metabolite. In combination with MS techniques, hydrogen/deuterium (H/D) exchange can provide additional information for structural elucidation by determination of the number of exchangeable hydrogen atoms in a structure. In this study, the principal phase I metabolites of iso‐phenylcyclopentylamine in rat bile were identified by high‐performance liquid chromatography with electrospray ionization quadrupole time‐of‐flight mass spectrometry (ESI‐Q‐TOF‐MS). Since N‐oxidation may occur because of the existence of the primary amino group in the structure, it was difficult to differentiate the hydroxylated metabolites from N‐oxides by ESI‐Q‐TOF‐MS alone. Therefore, online H/D exchange technique was applied to solve this problem. Finally, 25 phase I metabolites were detected and structurally described, in which 11 were confirmed to be N‐oxides. This study demonstrated the effectiveness of high‐resolution mass spectrometry in combination with an online H/D exchange technique in rapid identification of drug metabolites, especially in discriminating hydroxylated metabolites from N‐oxides. Copyright © 2014 John Wiley & Sons, Ltd.     

Identification of metabolites of isopropyl 3‐(3,4‐dihydroxyphenyl)‐2‐hydroxypropanoate in rats by high‐performance liquid chromatography combined with electrospray ionization quadrupole time‐of‐flight tandem mass spectrometry

Isopropyl 3‐(3,4‐dihydroxyphenyl)‐2‐hydroxypropanoate (IDHP) is an investigational new drug having the capacity for treating ailments in the cardiovascular and cerebrovascular system. In this work, a rapid and sensitive method using high‐performance liquid chromatography coupled with electrospray ionization quadrupole time‐of‐flight tandem mass spectrometry (HPLC‐ESI‐Q‐TOF‐MS) was developed to reveal the metabolic profile of IDHP in rats after oral administration. The method involved pretreatment of the samples by formic acid–methanol solution (v/v, 5:95), chromatographic separation by an Agilent Eclipse XDB‐C₁₈ column (150 × 4.6 mm i.dx., 5 μm) and online identification of the metabolites by Q‐TOF‐MS equipped with electrospray ionizer. A total of 16 metabolites from IDHP, including four phase I metabolites and 12 phase II metabolites, were detected and tentatively identified from rat plasma, urine and feces. Among these metabolites, Danshensu (DSS), a hydrolysis product of IDHP, could be further transformed to 11 metabolites. These results indicated that DSS was the main metabolite of IDHP in rats and the major metabolic pathways of IDHP in vivo were hydrolysis, O‐methylation, sulfation, glucuronidation and reduction. The results also demonstrated that renal route was the main pathway of IDHP clearance in rat. The present study provided valuable information for better understanding the efficacy and safety of IDHP. Copyright © 2015 John Wiley & Sons, Ltd.     

Metabolic profile of phillyrin in rats obtained by UPLC‐Q‐TOF‐MS

Forsythia suspensa Vahl (Oleaceae) is an important original plant in traditional Chinese medicine. The air‐dried fruits of Forsythia suspensa have long been used to relieve respiratory symptoms. Phillyrin is one of the main chemical constituent of Forsythia suspensa. A clear understanding of the metabolism of phillyrin is very important in rational clinical use and pharmacological research. In this study, the metabolism of phillyrin in rat was investigated for the first time using an ultra‐high‐performance liquid chromatography quadrupole time‐of‐flight mass spectrometry (UPLC‐Q‐TOF‐MS) method. Bile, urine and feces were collected from rats after single‐dose (10 mg/kg) orally administered phillyrin. Liquid–liquid extraction and ultrasonic extraction were used to prepare samples. UPLC‐Q‐TOF‐MS analysis of the phillyrin samples showed that phillyrin was converted to a major metabolite, M26, which underwent deglucosidation, further dehydration and desaturation. A total of 34 metabolites were detected including 30 phase I and four phase II metabolites. The conjugation types and structure skeletons of the metabolites were preliminarily determined. Moreover, 28 new metabolites were reported for the first time. The main biotransformation route of phillyrin was identified as hydrolysis, oxidation and sulfation. These findings enhance our understanding of the metabolism and the real active structures of phillyrin. Copyright © 2015 John Wiley & Sons, Ltd.     

Development and validation of an LC‐ESI‐MS/MS approach to determine a highly hydrophobic drug,norcantharidin palmitate,and apply to a preliminary pharmacokinetic study in rats

In order to investigate the pharmacokinetics of norcantharidin palmitate (NCTD‐PAL) in rats, we developed and validated an LC‐ESI‐MS/MS method. The NCTD‐PAL and internal standard (triamcinoloneacetonide palmitate, TAP) were separated on a Phenomenex Kinetex®XB C₁₈ column, and the mobile phase was composed of tetrahydrofuran (THF)–acetonitrile (20/80, v /v) and an aqueous phase containing 0.2% ammonium hydroxide at a flow rate of 0.3 mL/min. The ESI interface operated in positive mode was used to acquire the mass spectrometric data, and the transition ions were m /z 635.50 → 168.95 and 673.65 → 397.13 for NCTD‐PAL and IS, respectively. The method had a linear range of 10–2000 ng/mL with a correlation coefficient of >0.99. The accuracy (RE, %) was within ±10.1%, and the intra‐ and inter‐day precisions (RSD, %) were 10.9 and 13.8%, respectively. The extraction recovery of NCTD‐PAL at different concentrations ranged from 89.3 to 102.0%. The validated approach was efficaciously applied to a pharmacokinetic study of NCTD‐PAL in rats via intravenous injection. Based on these results obtained, this method is practical and suitable for a wide range of applications.     

Identification of metabolites of Helicid in vivo using ultra‐high performance liquid chromatography‐quadrupole time‐of‐flight mass spectrometry

Helicid is an active natural aromatic phenolic glycoside ingredient originating from a well‐known traditional Chinese herbal medicine and has the significant effects of sedative hypnosis, anti‐inflammatory analgesia and antidepressant. In this study, we analyzed the potential metabolites of Helicid in rats by multiple mass defect filter and dynamic background subtraction in ultra‐high‐performance liquid chromatography–quadrupole time‐of‐flight mass spectrometry (UHPLC‐Q‐TOF‐MS). Moreover, we used a novel data processing method, ‘key product ions’, to rapidly detect and identify metabolites as an assistant tool. MetabolitePilot™ 2.0 software and PeakView™ 2.2 software were used for analyzing metabolites. Twenty metabolites of Helicid (including 15 phase I metabolites and five phase II metabolites) were detected by comparison with the blank samples. The biotransformation route of Helicid was identified as demethylation, oxidation, dehydroxylation, hydrogenation, decarbonylation, glucuronide conjugation and methylation. This is the first study simultaneously detecting and identifying Helicid metabolism in rats employing UHPLC‐Q‐TOF‐MS technology. This experiment not only proposed a method for rapidly detecting and identifying metabolites, but also provided useful information for further study of the pharmacology and mechanism of Helicid in vivo. Furthermore, it provided an effective method for the analysis of other aromatic phenolic glycosides metabolic components in vivo.     

The application of HPLC‐MS/MS to studies of pharmacokinetics and interconversion of isoliquiritigenin and neoisoliquiritigenin in rats

A specific and sensitive HPLC‐MS/MS method was developed and validated for the simultaneously quantification of isoliquiritigenin (ISL) and neoisoliquiritin (NIS) in rat plasma by oral administration. Analytes were analyzed on an Agilent 6460 LC‐MS/MS system (Agilent, USA) using an Agilent Zorbax SB‐C₁₈ column (4.6 × 150 mm, 5 μm). Gradient elution was applied for the analyte separation using a mobile phase composed of 0.1% formic acid aqueous solution and methanol at a flow rate of 1.0 mL/min with a total running time of 12 min. The calibration curves for ISL and NIS showed good linearity in the concentrations ranging from 0.001 to 4.000 μg/mL with correlation coefficients >0.998. The precision, accuracy, recovery and stability were deemed acceptable. The method was applied to the pharmacokinetics study of ISL and NIS in rats by single and combination administration. The result showed that C_max and AUC_0→t of ISL were markedly increased from 0.53 to 1.20 μg/mL, and from 69.63 to 200.74 min μg/mL by combination administration. The mean t_1/2 value was also prolonged from 64.55 to 203.74 min in the combination group. These results indicated that NIS may have been metabolized to ISL which increased the absorption and extended the elimination of ISL. However, little difference was found for NIS pharmacokinetics parameters between single NIS and the combination group, which suggested that there was no significant biotransformation of ISL to NIS. Copyright © 2015 John Wiley & Sons, Ltd.     

Metabolite analysis in Curcuma domestica using various GC‐MS and LC‐MS separation and detection techniques

The metabolic profile of polar (methanol) and non‐polar (hexane) extracts of Curcuma domestica, a widely used medicinal plant, was established using various different analytical techniques, including GC‐FID, GC‐MS, HR‐GC‐MS and analytical HPLC‐ESI‐MS/MS by means of LTQ‐Orbitrap technology. The major non‐volatile curcuminoids curcumin, demethoxycurcumin and bisdemethoxycurcumin were identified when their chromatographic and precursor ion masses were compared with those of authentic standard compounds. In this paper we describe for the first time a GC/MS‐based method for metabolic profiling of the hydrophilic extract. We also identified 61 polar metabolites as TMS derivatives. Copyright © 2009 John Wiley & Sons, Ltd.     

Metabolic profiling of tenacigenin B,tenacissoside H and tenacissoside I using UHPLC‐ESI‐Orbitrap MS/MS

Marsdenia tenacissima, which is widely used as an anticancer herb in traditional Chinese medicine, has been shown to possess anticancer activity. However, its metabolic profile is poorly investigated. Tenacigenin B is the major steroidal skeleton of C‐21 steroids in M. tenacissima. Tenacissoside H and Tenacissoside I are detected at relatively high levels in M. tenacissima. Therefore, we studied their metabolic characteristics in human liver microsomes by ultra‐high‐performance liquid chromatography coupled with high‐resolution mass spectrometry. Fourteen metabolites were tentatively identified by accurate mass measurement and MS/MS fragmentation behavior. It was found that hydroxylation reactions were the major metabolic pathway of Tenacissoside H and Tenacissoside I in human liver microsomes, whereas the metabolic pathway of Tenacigenin B involved dehydrogenation reactions. This is the first time that the metabolic profile of C‐21 steroids from M. tenacissima has been explored in human liver microsomes, which is of great significance for subsequent pharmacokinetic and interaction research. Biotransformation in vivo or in vitro may influence the structure of a compound and change its activity. Identification of their fragmentation behaviors and metabolites provides valuable and new information for further understanding the anti‐tumor activity of M. tenacissima. Copyright © 2016 John Wiley & Sons, Ltd.     

Studies on the metabolism of the Δ9‐tetrahydrocannabinol precursor Δ9‐tetrahydrocannabinolic acid A (Δ9‐THCA‐A) in rat using LC‐MS/MS,LC‐QTOF MS and GC‐MS techniques

In Cannabis sativa, Δ9‐Tetrahydrocannabinolic acid‐A (Δ9‐THCA‐A) is the non‐psychoactive precursor of Δ9‐tetrahydrocannabinol (Δ9‐THC). In fresh plant material, about 90% of the total Δ9‐THC is available as Δ9‐THCA‐A. When heated (smoked or baked), Δ9‐THCA‐A is only partially converted to Δ9‐THC and therefore, Δ9‐THCA‐A can be detected in serum and urine of cannabis consumers. The aim of the presented study was to identify the metabolites of Δ9‐THCA‐A and to examine particularly whether oral intake of Δ9‐THCA‐A leads to in vivo formation of Δ9‐THC in a rat model. After oral application of pure Δ9‐THCA‐A to rats (15 mg/kg body mass), urine samples were collected and metabolites were isolated and identified by liquid chromatography‐mass spectrometry (LC‐MS), liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) and high resolution LC‐MS using time of flight‐mass spectrometry (TOF‐MS) for accurate mass measurement. For detection of Δ9‐THC and its metabolites, urine extracts were analyzed by gas chromatography‐mass spectrometry (GC‐MS). The identified metabolites show that Δ9‐THCA‐A undergoes a hydroxylation in position 11 to 11‐hydroxy‐Δ9‐tetrahydrocannabinolic acid‐A (11‐OH‐Δ9‐THCA‐A), which is further oxidized via the intermediate aldehyde 11‐oxo‐Δ9‐THCA‐A to 11‐nor‐9‐carboxy‐Δ9‐tetrahydrocannabinolic acid‐A (Δ9‐THCA‐A‐COOH). Glucuronides of the parent compound and both main metabolites were identified in the rat urine as well. Furthermore, Δ9‐THCA‐A undergoes hydroxylation in position 8 to 8‐alpha‐ and 8‐beta‐hydroxy‐Δ9‐tetrahydrocannabinolic acid‐A, respectively, (8α‐Hydroxy‐Δ9‐THCA‐A and 8β‐Hydroxy‐Δ9‐THCA‐A, respectively) followed by dehydration. Both monohydroxylated metabolites were further oxidized to their bishydroxylated forms. Several glucuronidation conjugates of these metabolites were identified. In vivo conversion of Δ9‐THCA‐A to Δ9‐THC was not observed. Copyright © 2009 John Wiley & Sons, Ltd.     

Studies on the metabolism and toxicological detection of glaucine,an isoquinoline alkaloid from Glaucium flavum (Papaveraceae), in rat urine using GC‐MS,LC‐MSn and LC‐high‐resolution MSn

Glaucine ((S)‐5,6,6a,7‐tetrahydro‐1,2,9,10‐tetramethoxy‐6‐methyl‐4H‐dibenzo [de,g]quinoline) is an isoquinoline alkaloid and main component of Glaucium flavum (Papaveraceae). It was described to be consumed as recreational drug alone or in combination with other drugs. Besides this, glaucine is used as therapeutic drug in Bulgaria and other countries as cough suppressant. Currently, there are no data available concerning metabolism and toxicological analysis of glaucine. To study both, glaucine was orally administered to Wistar rats and urine was collected. For metabolism studies, work‐up of urine samples consisted of protein precipitation or enzymatic cleavage followed by solid‐phase extraction. Samples were afterwards measured by liquid chromatography (LC) coupled to low or high‐resolution mass spectrometry (HR‐MS). The phase I and II metabolites were identified by detailed interpretation of the corresponding fragmentations, which were further confirmed by determination of their elemental composition using HR‐MS. From these data, the following metabolic pathways could be proposed: O‐demethylation at position 2, 9 and 10, N‐demethylation, hydroxylation, N‐oxidation and combinations of them as well as glucuronidation and/or sulfation of the phenolic metabolites. For monitoring a glaucine intake in case of abuse or poisoning, the O‐ and N‐demethylated metabolites were the main targets for the gas chromatography‐MS and LC‐MSⁿ screening approaches described by the authors. Both allowed confirming an intake of glaucine in rat urine after a dose of 2 mg/kg body mass corresponding to a common abuser's dose. Copyright © 2013 John Wiley & Sons, Ltd.