The in vivo absorbed constituents and metabolites of Danshen decoction in rats identified by HPLC with electrospray ionization tandem ion trap and time‐of‐flight mass spectrometry

Danshen, the dried root and rhizome of Salvia miltiorrhiza Bunge, is widely used for the treatment of cardiovascular and cerebrovascular diseases. This research focuses on the in vivo metabolism of Danshen decoction (DSD) in rats. After oral administration of DSD, the absorptive constituents and their metabolites in urine and plasma were analyzed by HPLC coupled with a photodiode array detector and electrospray ionization hybrid ion trap and time‐of‐flight mass spectrometry. Samples were separated on a C₁₈ column by gradient elution using 0.1% (v/v) aqueous formic acid and acetonitrile. As a result, 93 compounds from urine and 38 compounds from plasma were identified. Among them, lipo‐soluble diterpenoids (24 in urine and 15 in plasma) were reported for the first time as in vivo metabolites of DSD. According to the quantities and contents of the identified compounds, tanshinone IIA, cryptotanshinone and tanshinone I were deduced to be the major absorptive diterpenoids of DSD. Moreover, nine water‐soluble phenolics (caffeic acid, ferulic acid, danshensu, etc.) were proved to be the major absorptive constituents as reported. Most of the absorbed constituents underwent sulfation, glucuronidation, hydrogenation and hydroxylation in vivo. This investigation provided scientific evidence to obtain a more comprehensive metabolic profile of DSD. Copyright © 2014 John Wiley & Sons, Ltd.     

Identification of ilaprazole metabolites in human urine by HPLC‐ESI‐MS/MS and HPLC‐NMR experiments

Ilaprazole is a new proton pump inhibitor designed for the treatment of gastric ulcers, and limited data is available on the metabolism of the drug. In this article, the structural elucidation of urinary metabolites of ilaprazole in human was described by HPLC‐ESI‐MS/MS and stopped‐flow HPLC‐NMR experiments. Urinary samples were precipitated by sodium carbonate solution, and then extracted by liquid–liquid extraction after adding ammonium acetate buffer solution. The enriched sample was separated using a C₁₈ reversed‐phase column with the mobile phase composed of acetonitrile and 0.05 mol/L ammonium acetate buffer solution in a gradient solution, and then directly coupled to ESI‐MS/MS detection in an on‐line mode or ¹H‐NMR (500 MHz) spectroscopic detection in a stopped‐flow mode. As a result, four sulfide metabolites, ilaprazole sulfide (M1), 12‐hydroxy‐ilaprazole sulfide (M2), 11,12‐dihydroxy‐ilaprazole sulfide (M3) and ilaprazole sulfide A (M4), were identified by comparing their MS/MS and NMR data with those of the parent drug and available standard compounds. The main biotransformation reactions of ilaprazole were reduction and the aromatic hydroxylation of the parent drug and its relative metabolites. The result testified that HPLC‐ESI‐MS/MS and HPLC‐NMR could be widely applied in detection and identification of novel metabolites. Copyright © 2010 John Wiley & Sons, Ltd.     

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.     

In vivo metabolism study of bergenin in rats by HPLC‐QTOF mass spectrometry

Bergenin is the major component of Ardisia creanta sims and Rodgersia sambucifolia hemsl with many biological activities. Although bergenin has been used to treat human diseases in China for man years, there is no report regarding its metabolism. This is the first report to separate and identify the metabolites of bergenin in vivo. In the study, HPLC/Q‐TOF‐MS/MS was used to investigate the metabolites of bergenin in vivo by analyzing the rat body fluid and feces samples. Three metabolites of bergenin were finally identified by the TIC chromatograms, and the structures were also confirmed by their MS² spectra. Copyright © 2013 John Wiley & Sons, Ltd.     

Structural elucidation of in vitro metabolites of emodin by liquid chromatography-tandem mass spectrometry

Liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) was employed to investigate the in vitro metabolism of emodin. Emodin was incubated with rat liver microsomes in the presence of a NADPH-generating system, followed by extraction with ethyl acetate. After separation on a reversed-phase C18 analytical column with a linear gradient elution of methanol and 0.1% formic acid in water, negative electrospray ionization tandem mass spectrometry experiments were performed. As a result, the parent drug and its six metabolites were detected from rat liver microsomal incubations. The identification of the metabolites and elucidation of their structure were performed by comparing the changes in molecular masses (DeltaM), retention times and MS(2) spectral patterns of metabolites with those of parent drug. Besides three mono-hydroxylated metabolites (omega-hydroxyemodin, 2-hydroxyemodin, 4-hydroxyemodin), three other metabolites were identified, which were emodic acid, 3-carbomethoxy-6-methoxy-1,8-dihydroxyanthraquinone and physcion, respectively.     

In vitro and in vivo metabolism studies of dimethazine

The use of anabolic steroids is prohibited in sports. Effective control is done by monitoring their metabolites in urine samples collected from athletes. Ethical objections however restrict the use of designer steroids in human administration studies. To overcome these problems alternative in vitro and in vivo models were developed to identify metabolites and to assure a fast response by anti‐doping laboratories to evolutions on the steroid market. In this study human liver microsomes and an uPA^+/+‐SCID chimeric mouse model were used to elucidate the metabolism of a steroid product called ‘Xtreme DMZ’. This product contains the designer steroid dimethazine (DMZ), which consists of two methasterone molecules linked by an azine group. In the performed stability study, degradation from dimethazine to methasterone was observed. By a combination of LC‐High Resolution Mass Spectrometry (HRMS) and GC‐MS(/MS) analysis methasterone and six other dimethazine metabolites (M1–M6), which are all methasterone metabolites, could be detected besides the parent compound in both models. The phase II metabolism of dimethazine was also investigated in the mouse urine samples. Only metabolites M1 and M2 were exclusively detected in the glucuro‐conjugated fraction; all other compounds were also found in the free fraction. For effective control of DMZ misuse in doping control samples, screening for methasterone and methasterone metabolites should be sufficient. Copyright © 2016 John Wiley & Sons, Ltd.     

Measurement of caffeine and its three primary metabolites in human plasma by HPLC‐ESI‐MS/MS and clinical application

Caffeine is a mild stimulant with significant potential for abuse, being consumed in larger doses with the widespread availability of energy drinks and by novel routes of administration such as inspired powder, oral sprays and electronic cigarettes. How these recent changes in caffeine consumption affecting caffeine disposition and abuse potential is of growing concern. In the study of caffeine disposition in humans, it is common to only measure the caffeine concentration; however, caffeine's three major metabolites (paraxanthine, theobromine and theophylline) retain central nervous system stimulant activity that may contribute to the overall pharmacological activity and toxicity. Therefore, it would be scientifically more rigorous to measure caffeine and its major metabolites in the evaluation of caffeine disposition in human subjects. Herein, we report a method for the simultaneous quantification of caffeine and its three major metabolites in human plasma by high‐performance liquid chromatography coupled to electrospray tandem mass spectrometry (HPLC‐ESI‐MS/MS). Human plasma samples were treated by simple protein precipitation and the analytes were separated using a 6 min gradient program. Precision and accuracy were well within in the 15% acceptance range. The simple sample preparation, short runtime, sensitivity and the inclusion of caffeine's major metabolites make this assay methodology optimal for the study of caffeine's pharmacokinetics and pharmacodynamics in human subjects.     

LC/MS/MS for identification of in vivo and in vitro metabolites of jatrorrhizine

The in vivo and in vitro metabolism of jatrorrhizine has been investigated using a specific and sensitive LC/MS/MS method. In vivo samples including rat feces, urine and plasma collected separately after dosing healthy rats with jatrorrhizine (34 mg/kg) orally, along with in vitro samples prepared by incubating jatrorrhizine with rat intestinal flora and liver microsome, respectively, were purified using a C(18) solid-phase extraction cartridge. The purified samples were then separated with a reversed-phase C(18) column with methanol-formic acid aqueous solution (70:30, v/v, pH3.5) as mobile phase and detected by on-line MS/MS. The structural elucidation of the metabolites was performed by comparing their molecular weights and product ions with those of the parent drug. As a result, seven new metabolites were found in rat urine, 13 metabolites were detected in rat feces, 11 metabolites were detected in rat plasma, 17 metabolites were identified in intestinal flora incubation solution and nine metabolites were detected in liver microsome incubation solution. The main biotransformation reactions of jatrorrhizine were the hydroxylation reaction, the methylation reaction, the demethylation reaction and the dehydrogenation reaction of parent drug and its relative metabolites. All the results were reported for the first time, except for some of the metabolites in rat urine.     

Toxicological evaluation in silico and in vivo of secondary metabolites of Cissampelos sympodialis in Mus musculus mice following inhalation

The ethanolic extract of the leaves of Cissampelos sympodialis showed great pharmacological potential, with inflammatory and immunomodulatory activities, however, it showed some toxicological effects. Therefore, this study aims to verify the toxicological potential of alkaloids of the genus Cissampelos through in silico methodologies, to develop a method in LC-MS/MS verifying the presence of alkaloids in the infusion and to evaluate the toxicity of the infusion of the leaves of C. sympodialis when inhaled by Swiss mice. Results in silico showed that alkaloid 93 presented high toxicological potential along with the products of its metabolism. LC-MS/MS results showed that the infusion of the leaves of this plant contained the alkaloids warifteine and methylwarifteine. Finally, the in vivo toxicological analysis of the C. sympodialis infusion showed results, both in biochemistry, organ weights and histological analysis, that the infusion of C. sympodialis leaves presents a low toxicity.     

In vivo metabolite profiles of isoimperatorin and phellopterin in rats analyzed using HPLC coupled with diode array detector and electrospray ionization ion trap time‐of‐flight mass spectrometry technique

Isoimperatorin (IP) and phellopterin (PP) are two furocoumarins existing in Angelicae Dahuricae Radix. There is an isopentenyloxyl substituted at C‐5 in IP, and an isopentenyloxyl and a methoxyl substituted at C‐8 and C‐5, respectively, in PP. To elucidate the in vivo metabolic characteristics of PP and IP, HPLC coupled with diode array detector and electrospray ionization ion trap time‐of‐flight mass spectrometry technique was used. In total, 111 metabolites, including 53 new ones, were identified from the urine and plasma samples of rats after oral administration of IP and PP, respectively. The metabolites were formed through eight reactions on IP and PP: oxidation, hydroxylation–hydrogenation, carboxylation on the isopentenyloxyl, O‐dealkylation, hydroxylation on the furocoumarin nucleus, ring‐opening reaction on the furan ring and reduction or ring‐opening reaction on the lactone ring. Among these, hydroxylation on the furocoumarin nucleus was found for the first time for in vivo metabolites of PP and IP, and the ring‐opening reaction on the furan ring or lactone ring was found for the first time for in vivo metabolites of isopentenyloxyl furocoumarins. The research gave us a new insight into the in vivo metabolic profiles of IP and PP, which could help us better understand their important roles as two active constituents of Angelicae Dahuricae Radix.     

Systematic screening and characterization of absorbed constituents and in vivo metabolites in rats after oral administration of Rhizoma coptidis using UPLC-Q-TOF/MS

Rhizoma coptidis has been used for a long time in China owing to its anti-bacterial, anti-diabetes, anti-hyperlipidemia and anti-obesity activities. However, the in vivo biotransformation of Rhizoma coptidis is still unclear to date. In this study, a three-step strategy using UPLC-Q-TOF/MS was applied to clarify the in vivo absorbed constituents and metabolites in rats after oral administration of Rhizoma coptidis. First, alkaloids in Rhizoma coptidis extract were identified. Second, six abundant alkaloids (berberine, palmatine, coptisine, epiberberine, jatrorrhizine, and columbamine) were selected as representative prototypes and the metabolic fates of them in rats were investigated to obtain a database of Rhizoma coptidis-derived metabolites. Finally, the metabolic profiles of Rhizoma coptidis were fully elucidated based on the above-mentioned results. In summary, 29 alkaloids were identified in Rhizoma coptidis, and a database of Rhizoma coptidis-derived metabolites was obtained with 144 characterized metabolites. A total of 89 xenobiotics including 12 absorbed constituents and 77 metabolites were identified in dosed rat biosamples. Major metabolic pathways of Rhizoma coptidis were hydroxylation, reduction, methylation, demethylation, demethylenation, desaturation, glucuronidation and sulfation. This is the first systematic study on the in vivo absorbed constituents and metabolic profiling of Rhizoma coptidis and will be beneficial for its further studies.     

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.     

Intestinal metabolism of Polygonum cuspidatum in vitro and in vivo

Rhizoma et Radix Polygoni Cuspidati (RRPC) is commonly prescribed for the treatment of amenorrhea, arthralgia, jaundice and abscess in traditional Chinese medicine. Previous pharmacological studies have indicated that polyphenols are the main pharmacological active ingredients in RRPC. Meanwhile, the poor bioavailability of polyphenols in RRPC implies that those components are probably metabolized by intestinal bacteria before absorption. However, there is rather limited information about RRPC''s metabolites produced by intestinal bacteria and the intestinal absorbed constituents. In the present study, the metabolites were characterized after the aqueous extract of RRPC was incubated with the crude enzyme of human intestinal bacteria in vitro. The metabolic characteristics of glycosides in RRPC were figured out by comparing the metabolic profiles of emodin‐8‐O‐β‐d ‐glucopyranoside and polydatin between aqueous extract of RRPC and equivalent amounts of these two glycosides. The transitional constituents absorbed into blood were investigated in rats via intraduodental administration and portal vein intubation. A total of 38 prototype components and 43 metabolites were detected and characterized in vivo. The overall results demonstrated that the intestinal bacteria played an important role in the metabolism of RRPC, and the main metabolic pathways were hydrolysis in vitro, glucuronidation and sulfation in vivo.     

Investigating the in vitro stereoselective metabolism of m‐nisoldipine enantiomers: characterization of metabolites and cytochrome P450 isoforms involved

m‐Nisoldipine, as a novel 1,4‐dihydropyridine calcium ion antagonist, was presented as a couple of enantiomers [(?), (+)‐m‐nisoldipine]. In this report, the in vitro metabolism of m‐nisoldipine enantiomers was investigated in rat liver microsomes (RLM) by the combination of two liquid chromatography mass spectrometric techniques for the first time. The metabolites were separated and assayed by ultra‐high performance liquid chromatography coupled to quadrupole time‐of‐flight mass spectrometry and further identified by comparison of their mass and chromatographic behaviors with reference substances. A total of 18 metabolites of (?)‐m‐nisoldipine and 16 metabolites of (+)‐m‐nisoldipine were detected, respectively, which demonstrated that (+)‐m‐nisoldipine is more metabolically stable than (?)‐m‐nisoldipine. In addition, the identified metabolic pathways of m‐nisoldipine enantiomers were involved in dehydrogenation, oxidation and ester hydrolysis. Afterwards, based on high‐performance liquid chromatography coupled to triple quadrupole linear ion trap mass spectrometry, various selective cytochrome P450 (CYP) enzyme inhibitors were employed to evaluate CYP isoforms. The results indicated that the inhibitors of CYP1A1/2, CYP2B1/2, 2D and 2C11 had no obvious inhibitory effects, yet the inhibitor of CYP 3A had a significant inhibitory effect on metabolism of m‐nisoldipine enantiomers. This showed that CYP 3A might primarily metabolize m‐nisoldipine in RLM. Copyright © 2015 John Wiley & Sons, Ltd.     

Modification of collagen-chitosan membrane by oxidation sodium alginate and in vivo/ in vitro evaluation for wound dressing application

Collagen-chitosan (COL-CS) membranes materials without a cross-linking agent have poor mechanical properties. In this paper, COL-CS membranes were modified by a novel naturally-derived crosslinker, alginate dialdehyde (ADA) with different oxidation degree, and the COL-CS-ADA films were obtained. COL-CS-ADA films were characterized by Fourier transform attenuation total reflection infrared spectroscopy (ATR-FTIR), differential calorimetric scanning (DSC), thermogravimetric analysis (TG), tensile testing, and cross-link density testing. The modification of ADA exhibited positive effects on mechanical properties, the thermal stability of COL-CS membranes. The cross-linking degree between ADA and COL-CS membranes increased significantly with an increase in the oxidation degree. COL-CS-ADA films showed no cytotoxicity toward L929 fibroblasts and had good biocompatibility. The animal experiments showed that COL-CS-ADA film could promote wound healing.     

Determination of cnidilin and its two metabolites in rat bile and stool after oral administration by HPLC/electrospray ionization tandem mass spectrometry

A simple, fast and sensitive method for the simultaneous determination of cnidilin and its two metabolites (M1 and M2) in rat bile and stool using HPLC coupled with electrospray ionization tandem mass spectrometry (HPLC‐ESI‐MS/MS) has been developed. The sample pretreatment was simple, because methanol was the only additive used for dilution of bile and ultrasound of stool. Pimpinellin was used as internal standard (IS). The separation was performed on a reverse phase C₁₈ column with gradient elution consisting of 0.5‰ aqueous formic acid and methanol (containing 0.5‰ formic acid). The detection was in the multiple‐reaction monitoring mode within 7 min. All the analytes were in accordance with the requirement of the validation of the method in vivo (linearity, precision, accuracy, limit of detection and limit of quantification). After oral administrating 24 mg/kg of the prototype drug cnidilin, M1 and M2 were determined in bile within 36 h, and in stool within 60 h. Cnidilin in bile was completely excreted in 24 h, and the main excretive amount of cnidilin was 80% in the first 6 h, but the drug recovery in bile within 24 h was <1.95%. In stool, the main excretive amount of cnidilin was 95.8% in the first 24 h, and the drug recovery within 48 h was lower than 1.48%. Copyright © 2012 John Wiley & Sons, Ltd.     

GC/MS characterization of urinary metabolites of doxylamine succinate: Identification of the aglycones formed from intestinal microflora metabolism of the polar glucuronide metabolites

This study describes the use of high performance liquid chromatography (HPLC) and capillary gas chromatography/mass spectrometry (GC/MS) in the characterization of polar glucuronide conjugates of doxylamine and their subsequent aglycones following deconjugation. Rat urinary extracts which contained doxylamine and both nonconjugated and conjugated doxylamine metabolites, were examined by HPLC before and after incubation with rat intestinal microflora. The subsequent deconjugated urinary metabolites and the nonconjugated products remaining in the urinary extracts were then isolated, acetylated, and assayed by GC/MS. Incubation with the intestinal microflora indicated that anaerobic bacteria were capable of effecting hydrolytic cleavage of these polar O-glucuronide metabolites of doxylamine and its demethylated products to their subsequent aglycones. GC/MS analysis was performed using a fused silica DB-5 GC column and was utilized for the identification of these deconjugated metabolites.     

Bioanalysis of pentoxifylline and related metabolites in plasma samples through LC‐MS/MS

Analytical aspects related to the assay of pentoxifylline (PTX), lisofylline (M1) and carboxypropyl dimethylxanthine (M5) metabolites are discussed through comparison of two alternative analytical methods based on liquid chromatography separation and atmospheric pressure electrospray ionization tandem mass spectrometry detection. One method is based on a ‘pure’ reversed‐phase liquid chromatography mechanism, while the second one uses the additional polar interactions with embedded amide spacers linking octadecyl moieties to the silicagel surface (C‐18 Aqua stationary phase). In both cases, elution is isocratic. Both methods are equally selective and allows separation of unknowns (four species associated to PTX, two species associated to M1) detected through specific mass transitions of the parent compounds and owning respective structural confirmation. Plasma concentration–time patterns of these compounds follow typical metabolic profiles. It has been advanced that in‐vivo formation of conjugates of PTX and M1 is possible, such compounds being cleaved back to the parent ones within the ion source. The first method was associated with a sample preparation procedure based on plasma protein precipitation by strong organic acid addition. The second method used protein precipitation by addition of a water miscible organic solvent. Both analytical methods were fully validated and used to assess bioequivalence between a prolonged release generic formulation and the reference product, under multidose and single dose approaches. Copyright © 2009 John Wiley & Sons, Ltd.     

PAM-microfabricated polyurethane scaffolds: in vivo and in vitro preliminary studies

Polymeric scaffolds were realised with linear degradable PU in the form of square, hexagonal and octagonal grids. They were characterised in terms of their mechanical properties. Analysis shows that the mechanical properties of the scaffolds depend on their geometries which are easily modulated using PAM. In vitro biological assays showed that PU promotes the adhesion and proliferation of fibroblast cells and that cell activities are better on PU scaffolds than on PU films. In vivo implantation of PU and PLGA scaffolds and PU films demonstrated that the scaffolds are completely resorbed after three months with a slight inflammatory response, while the PU film was still present after six months with an intense granulomatous reaction.     

Structural characterization of in vitro rat liver microsomal metabolites of antihistamine desloratadine using LTQ-Orbitrap hybrid mass spectrometer in combination with online hydrogen/deuterium exchange HR-LC/MS

In vitro drug metabolism study is an integral part of drug discovery process. In this report, we have described the application of LTQ-Orbitrap hybrid mass spectrometer in conjunction with online hydrogen (H)/deuterium (D) exchange high resolution (HR)-LC/MS for structural characterization of in vitro rat liver microsomal metabolites of antihistamine desloratadine. Five metabolites M1--M5 have been identified, including three hydroxylated metabolites M1--M3, one N-oxide M4 and one uncommon aromatized N-oxide M5. Accurate mass data have been obtained in both full scan and MSn mode support assignments of metabolite structures with reported mass errors less than 3 ppm. Online H/D exchange HR-LC/MS experiments provide additional evidence in differentiating hydroxylated metabolites from N-oxides. This study demonstrates the effectiveness of this approach in structural characterization of drug metabolites.