phase-II Metabolites Research Articles

A Workflow for the Identification of Mycotoxin Metabolites Using Liquid Chromatography-Ion Mobility-Mass Spectrometry.

The structural identification of phase-I and phase-II metabolites of mycotoxins is a difficult task, mostly due to the lack of standards and because of the large number of isomeric forms. Here, we describe the use of ion mobility-mass spectrometry to analyze cereal extracts and how structural information on newly discovered mycotoxins metabolites could be obtained.

Binding and activity of polybrominated diphenyl ether sulfates to thyroid hormone transport proteins and nuclear receptors.

Polybrominated diphenyl ethers (PBDEs) can be metabolized to hydroxylated PBDEs (OH-PBDEs), which play important roles in their disruption effects on the thyroid hormone (TH) system. Recently, multiple in vitro studies suggested that OH-PBDEs might be further metabolically transformed to PBDE sulfates. However, information about the bioactivity of PBDE sulfate metabolites is limited. In the present study, we explored the possible disruption effects of PBDE sulfates to the TH system by studying their binding and activity towards TH transport proteins and nuclear receptors. We found PBDE sulfates could bind to two major TH transport proteins (thyroxine-binding globulin and transthyretin). Besides, PBDE sulfates could also bind to two subtypes of TH nuclear receptors (TRα and TRβ) and showed agonistic activity towards the subsequent signaling pathway. Moreover, the PBDE sulfates showed higher binding potency to TH transport proteins and TRs compared with their corresponding OH-PBDE precursors. Molecular docking results showed that replacement of hydroxyl groups with sulfate groups might lead to more hydrogen bond interactions with these proteins. Overall, our study suggested that PBDE sulfates might disturb the TH system by binding to TH transport proteins or TRs. Our finding indicated a possible mechanism for the TH system disruption effects of PBDEs through their sulfate metabolites.

Biotransformation and bioconcentration of 6:2 and 8:2 polyfluoroalkyl phosphate diesters in common carp (Cyprinus carpio): Underestimated ecological risks

Polyfluoroalkyl phosphates esters (PAPs) are widely used in a variety of commercial products, and have been detected in many aquatic organisms. In this study, common carps (Cyprinus carpio) were administered with 6:2 and 8:2 diPAP in water to investigate their bio-accumulation and transformation in fish. Several degradation products, including fluorotelomer unsaturated carboxylic acids (6:2 and 8:2 FTUCA), 5:3 and 7:3 fluorotelomer carboxylic acids (5:3 and 7:3 FTCA), perfluoroalkyl carboxylates (PFCAs) were identified in the carp liver. In addition, several phase-II metabolites, such as glutathione- and glucuronide-conjugated compounds were detected in the carp bile. 8:2 diPAP displayed lower accumulation potential than 6:2 diPAP probably due to its relatively large molecular size. However, 8:2 diPAP experienced more extensive transformation (transformation rate 6.78–14.6 mol%) and produced more phase I metabolites than 6:2 diPAP (0.49–0.66 mol%). The in vitro incubation with the liver S9 fraction confirmed that biotransformation of 6:2 and 8:2 diPAP took place in the carp liver. Further analyses of enzyme activities indicated that acid phosphatase (ACP) could be involved in mediating phase I while glutathione S-transferase (GST) involved in phase II metabolism of 6:2 and 8:2 diPAP in carp.

Identification of phase-II metabolites from human serum samples after oral intake of a willow bark extract

BackgroundWillow bark (Salicis cortex) is a herbal medicinal drug used to treat fever and pain, such as headaches and lower back pain. Until now, it has not been fully understood which compounds are responsible for the efficacy of the drug. PurposeAlthough salicylic acid is known as a metabolite of salicylic alcohol derivatives of willow bark in vivo, it has been shown in previous studies that its concentration is too low to account for the overall efficacy of Salicis cortex. The aim this study was to broaden the knowledge regarding phenolic phase-II metabolites after oral intake of a willow bark extract. Study design/methodsSerum samples of a human pharmacokinetic study (Salicis cortex extract intake corresponding to 240 mg of total salicin, 10 volunteers, 12 h fasting time, controlled diet low in phenolics, and 12 blood withdrawals over a period of 24 h) were analyzed by LC-ESI-MS. A library of 142 possible metabolites associated with salicylic alcohol derivatives, flavonoids, and proanthocyanidins was used to characterize possible metabolization products. Their structures were confirmed by LC-ESI-MS experiments with reference compounds after a cleavage reaction using glucuronidase and sulfatase as well as by LC-MS/MS experiments. ResultsIn the serum samples, phase-II metabolites of naringenin (2x glucuronides, 2x sulfates, 2x mixed glucuronide-sulfates), eriodictyol (3x glucuronides, 1x sulfate), taxifolin (1x sulfate), catechin (1x sulfate, 1x mixed glucuronide sulfate), ferulic acid (1x sulfate), hydroxyphenyl-propionic acid (1x sulfate), dihydroxyphenyl-valerolactone (1x sulfate), saligenin (1x glucuronide, 1x sulfate), salicylic acid (1x sulfate, 1x unconjugated, 1x salicyluric acid), and catechol (1x glucuronide, 1x sulfate) were characterized. Because taxifolin, dihydroxyphenyl-valerolactone, ferulic acid, and hydroxyphenyl-propionic acid could not be detected in the willow bark preparation, they could be metabolization products of genuine flavanones and flavan-3-ols as well as coumaric acid or C-ring cleavage products of flavonoids, which were present in the extract. No phase-II metabolites of procyanidins and no genuine flavonoid glycosides were detected in all serum samples. ConclusionThis is the first study to identify human metabolites of flavonoids, proanthocyanidins and salicylic alcohol derivatives of Salicis cortex beside salicylic acid or catechol. For the most characterized metabolites, anti-inflammatory activity has been described in the literature, and the present results are an important step in understanding the anti-inflammatory efficacy of willow bark in vivo.

Intestinal phase-II metabolism of quercetin in HT29 cells, 3D human intestinal tissues and in healthy volunteers: a qualitative comparison using LC-IMS-MS and LC-HRMS

Flavonoids are a large class of dietary molecules, among which quercetin is the most ubiquitous, which undergo an extensive intestinal phase-II metabolism. We compared the in vivo metabolism of quercetin in healthy volunteers with two in vitro models, HT29 cells and 3 D human intestinal tissues. Supernatants of the in vitro experiments and the human intestinal fluids (HIF) were analyzed by LC-IMS-MS and LC-HRMS in a qualitative way.Quercetin glucuronides, sulfates and their methyl conjugates were detected in all three systems. The metabolic profiles were found to be different, both in terms of the metabolites produced and their relative proportions. In particular, quercetin sulfates were almost absent in supernatants from HT29 cells incubations while they were a major metabolite in HIF and also found in 3 D intestinal tissues incubations.IMS provided structural information as well as a third dimension of characterization, while HRMS brought increased sensitivity and MS/MS confirmation. HT29 cells are a useful tool to generate phase-II metabolites but do not represent the in vivo situation. 3 D intestinal tissues appear as a more relevant tool to study the intestinal phase-II metabolism of flavonoids.

Intestinal disposition of quercetin and its phase-II metabolites after oral administration in healthy volunteers.

Quercetin is one of the main dietary flavonoids and undergoes a substantial intestinal phase-II metabolism. Quercetin conjugates have been detected in plasma and in urine, but their presence in the small intestine has not been assessed. This study aimed to investigate the intestinal metabolism and metabolite excretion of quercetin by the human small intestinal wall after oral dosing. Six healthy volunteers were given a capsule of 500 mg of quercetin with 240 ml of water. Duodenal fluids were collected using the intraluminal sampling technique for 4 h and analysed by LC-MS/MS. Phase-II metabolites of quercetin were detected and quantified in aspirated intestinal fluids. Metabolites appeared almost immediately after administration, indicating an intestinal metabolism and apical excretion into the lumen. Quercetin-3'-O-glucuronide was found to be the main intestinal metabolite. Our results could not conclude on the enterohepatic recycling of quercetin or its metabolites, although several individual profiles showed distinctive peaks. This study highlights the intestinal metabolism and excretion of quercetin and its conjugates in humans and gives insights into the relevant concentrations which should be used to investigate potential food-drug interactions in vitro.

Dynamics of paralytic shellfish toxins and their metabolites during timecourse exposure of scallops Chlamys farreri and mussels Mytilus galloprovincialis to Alexandrium pacificum

New C-11 hydroxyl metabolites of paralytic shellfish toxins (PSTs) have been reported in shellfish. To gain further information on these metabolites, as well as the potential for formation of phase-II metabolites and acyl esters of PSTs, bivalves were fed with the PSTs-producing dinoflagellate Alexandrium pacificum (strain ATHK). Through independent experiments, scallops (Chlamys farreri) were fed for 9 days and mussels (Mytilus galloprovincialis) for 5 days plus an additional 5 days of depuration, with representative samples taken throughout. Several common PSTs (C1-4, GTX1-6 and NEO) and metabolites including M1, M3, M5, M7, M9, M2 and M8 were detected in the hepatopancreas of scallops during toxin accumulation and in the hepatopancreas of mussels during both toxin accumulation and elimination periods. The relative molar ratio of metabolites to precursor molecules was used to estimate relative metabolic conversion rates. Conversion rates of C1/2 and GTX2/3 were higher than those of C3/4 and GTX1/4, in scallops and mussels. The first metabolites observed in both bivalve species investigated were M1/3, which are formed from C1/2. However, the conversion of GTX2/3 to M2 was more complete than other biotransformation reactions in both mussels and scallops. In general, metabolic conversion of PSTs was observed after a shorter time and to a greater extent in mussels than in scallops in the exposure period. No acyl esters or conjugation products of PSTs with glucuronic acid, glutathione, cysteine and taurine were detected by liquid chromatography with high resolution tandem mass spectrometry in the samples investigated. Additionally, only GTX1/4 and GTX2/3 were detected in the kidney of scallops, which demonstrates that PSTs are mainly metabolized through the hepatic metabolism pathway in bivalves. This work improves the understanding of PST metabolism during toxin accumulation and depuration in commercially harvested shellfish.

Polyphenol Phase-II Metabolites are Detectable in Human Plasma after Ingestion of 13 C Labeled Spinach-a Pilot Intervention Trial in Young Healthy Adults.

After intrinsic labeling of spinach (Spinacia oleracea L., Chenopodiaceae) with 13 CO2 , we investigated if labeled polyphenol metabolites were detectable in human plasma. In a pilot intervention trial, five healthy men consumed 5g freeze-dried 13 C labeled spinach, including a total amount of 160μmol methoxyflavonols, including 70μmol 5,3',4'-trihydroxy-3-methoxy-6,7-methylendioxyflavone-4'-glucuronide. Plasma samples of all subjects were analyzed with regard to their 13 C/12 C ratio. Additionally, 13 C labeled metabolites of patuletin, spinacetin, and 5,3',4'-trihydroxy-3-methoxy-6,7-methylendioxyflavone (TMM) were analyzed in plasma samples in a subgroup of three subjects. TMM-glucuronide, TMM-sulfate, and spinacetin-glucuronide-sulfate, the latter as 12 C113 C16 and 13 C17 isotopologs, were tentatively identified. Plasma concentration of TMM-glucuronide and TMM-sulfate reached cmax from 19.1-54.3 and 22.5-125.5nmol L-1 , respectively, 7-9h post-ingestion. It seems likely that 13 C labeled TMM-glucuronide and TMM-sulfate are phase-II metabolites which were converted after colonic transformation. Variations in plasma kinetics were observed for these two metabolites and may be attributed to the individual composition of the microbiota. We conclude that 13 C labeled polyphenol metabolites are detectable and quantifiable in human plasma.

Development of a Suspect Screening Strategy for Pesticide Metabolites in Fruit and Vegetables by UPLC-Q-Tof-MS

A multi-pesticide metabolite screening method using liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-Q-Tof-MS) was developed for the detection of pesticide metabolites in fruit and vegetable samples. Based on a retrospectively created accurate mass compound database, a suspect screening approach was established for pesticides of high concern applied to a wide scope of plant-derived commodities. For each matrix, an individual scope of characteristic pesticides was selected and a scientific library of corresponding metabolites generated. The metabolite database contained a total of 648 pesticide metabolites originating from 58 active compounds. In 500 samples from daily routine analysis, 96 samples with positive detects for a total of 26 pesticides were re-analyzed for the occurrence of corresponding metabolites. Forty-seven different phase-I and phase-II metabolites were identified, respectively. The developed metabolite database can be applied for a suspect screening approach for pesticide metabolites identification in all kinds of fruits and vegetables. By the means of the results of the suspect screening workflow, a targeted screening method by UHPLC-MS/MS was established for individual pesticide metabolites. Positive detects for metabolites may give an unequivocal evidence for an illegal application of plant protection products, even if the active compound is not detectable anymore.

Metabolism of five diterpenoid lactones from Dioscorea bulbifera tubers in zebrafish.

Diterpenoid lactones (DLs) have been reported to be the main hepatotoxic constituents in Dioscorea bulbifera tubers (DBT), a traditional Chinese medicinal herb. The acquisition of early information regarding its metabolism is critical for evaluating the potential hepatotoxicity of DLs. We investigated, for the first time, the main metabolites of diosbulbin A (DIOA), diosbulbin C (DIOC), diosbulbin (DIOG), diosbulbin (DIOM) and diosbulbin (DIOF) in adult zebrafish. By using ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-QTOF MS), 6, 2, 7, 5 and 4 metabolites of DIOA, DIOC, DIOF, DIOM and DIOG were identified in the zebrafish body and the aqueous solution, respectively. Both phase-I and phase-II metabolites were observed in the metabolic profiles and the metabolic pathways involved in hydroxyl reduction, glucuronidation, glutathione conjugation and sulfation. The above results indicated that hepatocytic metabolism might be the major route of clearance for DLs. This study provided important information for the understanding of the metabolism of DLs in DBT.

Identification of phase-II metabolites of flavonoids by liquid chromatography-ion-mobility spectrometry-mass spectrometry.

Flavonoids are a class of natural compounds with a broad range of potentially beneficial health properties. They are subjected to an extensive intestinal phase-II metabolism, i.e., conjugation to glucuronic acid, sulfate, and methyl groups. Flavonoids and their metabolites can interact with drug transporters and thus interfere with drug absorption, causing food-drug interactions. The site of metabolism plays a key role in the activity, but the identification of the various metabolites remains a challenge. Here, we developed an analytical method to identify the phase-II metabolites of structurally similar flavonoids. We used liquid chromatography-ion-mobility spectrometry-mass spectrometry (LC-IMS-MS) analysis to identify phase-II metabolites of flavonols, flavones, and catechins produced by HT29 cells. We showed that IMS could bring valuable structural information on the different positional isomers of the flavonols and flavones. The position of the glucuronide moiety had a strong influence on the collision cross section (CCS) of the metabolites, with only minor contribution of hydroxyl and methyl moieties. For the catechins, fragmentation data obtained from MS/MS analysis appeared more useful than IMS to determine the structure of the metabolites, mostly due to the high number of metabolites formed. Nevertheless, CCS information as a molecular fingerprint proved to be useful to identify peaks from complex mixtures. LC-IMS-MS thus appears as a valuable tool for the identification of phase-II metabolites of flavonoids. Graphical abstract Structural identification of phase-II metabolites of flavonoids using LC-IMS-MS.

Metabolite identification of AZD8055 in Sprague-Dawley rats after a single oral administration using ultra-performance liquid chromatography and mass spectrometry

AZD8055 is an ATP-competitive specific dual mTOR inhibitor and exhibited potent antitumor activity on several types of solid tumors. However, the metabolism of AZD8055 in the body still remains unknown. In this study, metabolite identification of AZD8055 was performed using ultra high-performance liquid chromatography-ion trap mass spectrometry (UHPLC-IT-MS) through both in vitro and in vivo approaches using rat liver microsomes (RLMs) and rat plasma, urine and feces, respectively. A total of eight putative metabolites (five phase I and three phase II) were identified, and a tentative metabolic pathway was suggested for the first time. Considering the accurate mass and mass fragmentations of the detected metabolites, their plausible structures were suggested. Demethylation, hydroxylation, oxidation and morpholine ring opening were the major biotransformation processes for the phase-I metabolism, while phase-II metabolites were merely generated by the glucuronide conjugation reaction. The cumulative excretion of AZD8055 in urine and feces was 0.13% and 1.11% of the dose, respectively. When the semi-quantitative analysis of the metabolites was performed using UHPLC–MS/MS (ultra-performance liquid chromatography tandem mass spectrometry) to evaluate the overall trend of metabolites formation and excretion, AZD8055 was excreted more in the form of the metabolites than itself and their formation was very fast. Therefore it was presumed that biotransformation was playing a crucial role in its elimination. Ultimately, this study provides novel insights regarding the in vitro and in vivo biotransformations of AZD8055. Further investigations of metabolites of this potent anti-cancer compound could be beneficial for the antitumor drug design and development process.

Flavanol plasma bioavailability is affected by metabolic syndrome in rats

Flavanols, which exert several health benefits, are metabolized after ingestion. Factors such as the host physiological condition could affect the metabolism and bioavailability of flavanols, influencing their bioactivities. This study aimed to qualitatively evaluate whether a pathological state influenced flavanol plasma bioavailability. Standard and cafeteria (CAF) diet fed rats, a robust model of metabolic syndrome (MeS), were administered 1000mg/kg of flavanol enriched grape seed polyphenol extract (GSPE). Flavanols and their metabolites were quantified by HPLC-MS/MS in plasma before and at 2, 4, 7, 24, and 48h after GSPE ingestion. Results showed that in CAF administered rats the maximum time of plasma flavanol concentration was delayed and these animals presented higher levels of plasma phase-II metabolites as well as altered microbial metabolites. In conclusion, this study demonstrated that MeS pathological state modified flavanol bioavailability, supporting the hypothesis that flavanol metabolism, and therefore flavanol functionality, depend on the organism’s state of health.

Reductive transformation of carbamazepine by abiotic and biotic processes

The antiepileptic drug carbamazepine (CBZ) is ubiquitously present in the anthropogenic water cycle and is therefore of concern regarding the potable water supply. Despite of its persistent behavior in the aquatic environment, a redox dependent removal at bank filtration sites with anaerobic aquifer passage was reported repeatedly but not elucidated in detail yet. The reductive transformation of CBZ was studied, using abiotic systems (catalytic hydrogenation, electrochemistry) as well as biologically active systems (column systems, batch degradation tests). In catalytic hydrogenation CBZ is gradually hydrogenated and nine transformation products (TPs) were detected by liquid chromatography high-resolution mass spectrometry. 10,11-Dihydro-CBZ ((2H)-CBZ) was the major stable product in these abiotic, surface catalyzed reduction processes and turned out to be not a precursor of the more hydrogenated TPs. In the biotic reduction processes the formation of (2H)-CBZ alone could not explain the observed CBZ decline. There, also traces of (6H)-CBZ and (8H)-CBZ were formed by microbes under anaerobic conditions and four phase-II metabolites of reduced CBZ could be detected and tentatively identified. Thus, the spectrum of reduction products of CBZ is more diverse than previously thought. In environmental samples CBZ removal along an anaerobic soil passage was confirmed and (2H)-CBZ was determined at one of the sites.

Identification and characterization of in vitro and in vivo generated metabolites of the adiponectin receptor agonists AdipoRon and 112254

Peroxisome proliferator-activated receptors (PPARs), peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α), sirtuin 1 (SIRT1) and adenosine monophosphate-activated protein kinase (AMPK) are regulators of transcriptional processes and effects of exercise and pseudo-exercise situations. Compounds occasionally referred to as endurance exercise mimetics such as AdipoRon and 112254, both adiponectin receptor agonists, can be used to simulate the physiology of endurance exercise via pathways including these transcriptional regulators. Adiponectin supports fatty acid utilization and triglyceride-content reduction in cells and increases both the mitochondrial biogenesis and the oxidative metabolism in muscle cells. In routine doping control analysis, knowledge about phase-I and -II metabolic products of target analytes is essential. Hence, in vitro- and in vivo-metabolism experiments are frequently employed tools in preventive doping research to determine potential urinary metabolites for sports drug testing purposes, especially concerning new, (yet) unapproved compounds. In the present study, in vitro assays were conducted using human liver microsomal and S9 fractions, and rat in vivo experiments were performed using both AdipoRon and 112254. For AdipoRon, obtained samples were analyzed using liquid chromatography-high resolution/high accuracy (tandem) mass spectrometry with both electrospray ionization or atmospheric-pressure chemical ionization techniques. Overall, more than five phase I-metabolites were found in vitro and in vivo, including particularly monohydroxylated and hydrogenated species. No phase II-metabolites were found in vitro; conversely, signals suggesting the presence of glucuronic acid or other conjugates in samples collected from in vivo experiment were observed, the structures of which were however not conclusively identified. Also for 112254, several phase-I metabolites were found in vitro, e.g. monohydroxylated and demethylated species. Here, no phase II-metabolites were observed neither using in vitro nor in vivo samples. Based on the generated data, the implementation of metabolites and unmodified drug candidates into routine doping control protocols is deemed warranted for comprehensive sports drug testing programs until human elimination study data are available.

Hesperetin and its sulfate and glucuronide metabolites inhibit TNF-α induced human aortic endothelial cell migration and decrease plasminogen activator inhibitor-1 (PAI-1) levels.

Epidemiological, clinical and preclinical studies have reported the protection offered by citrus consumption, mainly orange, against cardiovascular diseases, which is primarily mediated by the antiatherogenic and vasculoprotective effects of the flavanone hesperetin-7-O-rutinoside (hesperidin). However, flavanone aglycones or glycosides are not present in the bloodstream but their derived phase-II metabolites could be the actual bioactive molecules. To date, only a few studies have explored the effects of circulating hesperetin-derived metabolites (glucuronides and sulfates) on endothelial cells. Herein, we describe for the first time the effects of hesperetin 3'-O-glucuronide, hesperetin 7-O-glucuronide, hesperetin 3'-O-sulfate, hesperetin 7-O-sulfate and hesperetin on human aortic endothelial cell (HAEC) migration upon pro-inflammatory stimuli as an essential step to angiogenesis. Hesperetin and its derived metabolites, at physiologically relevant concentrations (1-10 μM), significantly attenuated cell migration in the presence of the pro-inflammatory cytokine TNF-α (50 ng mL(-1)), which was accompanied and perhaps mediated by a significant decrease in the levels of the thrombogenic plasminogen activator inhibitor-1 (PAI-1). However, hesperetin metabolites did not counteract the TNF-α-induced production of pro-inflammatory interleukin-6 (IL-6) and IL-8. We also study here for the first time, the metabolism of hesperetin and its derived metabolites by HAEC with and without a pro-inflammatory stimulus. All these results reinforce the concept according to which circulating phase-II hesperetin metabolites are critical molecules contributing to the cardioprotective effects upon consumption of citrus fruits such as orange.

Mitochondrial toxicity of diclofenac and its metabolites via inhibition of oxidative phosphorylation (ATP synthesis) in rat liver mitochondria: Possible role in drug induced liver injury (DILI)

Diclofenac is a widely prescribed NSAID, which by itself and its reactive metabolites (Phase-I and Phase-II) may be involved in serious idiosyncratic hepatotoxicity. Mitochondrial injury is one of the mechanisms of drug induced liver injury (DILI). In the present work, an investigation of the inhibitory effects of diclofenac (Dic) and its phase I [4-hydroxy diclofenac (4′-OH-Dic) and 5-hydroxy diclofenac (5-OH-dic)] and Phase-II [diclofenac acyl glucuronide (DicGluA) and diclofenac glutathione thioester (DicSG)] metabolites, on ATP synthesis in rat liver mitochondria was carried out. A mechanism based inhibition of ATP synthesis is exerted by diclofenac and its metabolites. Phase-I metabolite (4′-OH-Dic) and Phase-II metabolites (DicGluA and DicSG) showed potent inhibition (2–5 fold) of ATP synthesis, where as 5-OH-Dic, one of the Phase-I metabolite, was a less potent inhibitor as compared to Dic. The calculated kinetic constants of mechanism based inhibition of ATP synthesis by Dic showed maximal rate of inactivation (Kinact) of 2.64±0.15min−1 and half maximal rate of inactivation (KI) of 7.69±2.48μM with Kinact/KI ratio of 0.343min−1μM−1. Co-incubation of mitochondria with Dic and reduced GSH exhibited a protective effect on Dic mediated inhibition of ATP synthesis. Our data from this study strongly indicate that Dic as well as its metabolites could be involved in the hepato-toxic action through inhibition of ATP synthesis.

Identification of in vitro metabolites of ethylphenidate by liquid chromatography coupled to quadrupole time-of-flight mass spectrometry

Ethylphenidate is a new potent synthetic psychoactive drug, structurally related to methylphenidate. Using human liver microsomes and cytosol, we have investigated for the first time the Phase-I and Phase-II in vitro metabolism of ethylphenidate. The structure of the metabolites was elucidated by hybrid quadrupole time-of-flight mass spectrometry. Overall, seven Phase-I, but no Phase-II metabolites were detected. Ethylphenidate underwent hydroxylation forming two primary mono-hydroxylated metabolites and, subsequently, dehydration and ring opening with an additional hydroxylation, forming secondary metabolites. The involvement of different human cytochrome P450 (CYP) enzymes in the formation of ethylphenidate metabolites was investigated using a panel of human recombinant CYPs (rCYPs). rCYP2C19 was the most active recombinant enzyme involved in the formation of all seven ethylphenidate metabolites detected, although other rCYPs (rCYP1A2, rCYP2B6, rCYPC9, rCYP2D6, and rCYP3A4, but not rCYP2E1) played a role in the metabolism of ethylphenidate. All metabolites identified in the present study can be considered as potential specific biomarkers of ethylphenidate in toxicological studies. Additionally, ritalinic acid and methylphenidate were formed by non-enzymatic hydrolysis and trans-esterification, and, therefore, they cannot be considered as (oxidative) metabolites of ethylphenidate. The presence of methylphenidate and ritalinic acid cannot be exclusively associated to the use of ethylphenidate, since methylphenidate is a drug itself and ritanilic acid can be formed from both ethylphenidate and methylphenidate.

Qualitative and quantitative analysis of felbinac and its major metabolites in human plasma and urine by liquid chromatography tandem mass spectrometry and its application after intravenous administration of felbinac trometamol injection.

We present a method for the qualitative and quantitative analysis of felbinac and its major metabolites in human plasma and urine by HPLC-MS/MS and its application. Qualitative analysis through LC-Triple-TOF-MS/MS indicated that oxidization was the main phase-I metabolic pathway of felbinac in human, conjugation with sulfate and glucuronide groups produced at least 7 phase-II metabolites. Quantitative analysis through HPLC-MS/MS in MRM mode was developed and validated for the quantification of felbinac and its major metabolite (4'-hydroxyfelbinac) in human plasma and urine. Linear calibration curves were obtained for felbinac and 4'-hydroxyfelbinac in plasma and urine (r>0.996); intra- and inter-day precision values (RSD%) obtained were ranged from 1.13 to 6.49%, and the accuracy were between 95.9% and 108.6% for the two analytes. The pharmacokinetics and excretion analysis showed that the t1/2 of 4'-hydroxyfelbinac (8.25 ± 4.15 h) is a litter longer than that of felbinac (6.13 ± 2.01 h), but the mean AUC(0-t) value of felbinac was about 20 times higher than that of 4'-hydroxyfelbinac; excretion of felbinac and 4'-hydroxyfelbinac reached their peak values at about 3-6h after intravenous administration of felbinac trometamol in human.

Nutrikinetic modeling reveals order of genistein phase II metabolites appearance in human plasma.

Genistein from foods or supplements is metabolized by the gut microbiota and the human body, thereby releasing many different metabolites into systemic circulation. The order of their appearance in plasma and the possible influence of food format are still unknown. This study compared the nutrikinetic profiles of genistein metabolites. In a randomized cross-over trial, 12 healthy young volunteers were administered a single dose of 30 mg genistein provided as a genistein tablet, a genistein tablet in low fat milk, and soy milk containing genistein glycosides. A high mass resolution LC-LTQ-Orbitrap FTMS platform detected and quantified in human plasma: free genistein, seven of its phase-II metabolites and 15 gut-derived metabolites. Interestingly, a novel metabolite, genistein-4'-glucuronide-7-sulfate (G-4'G-7S) was identified. Nutrikinetic analysis using population-based modeling revealed the order of appearance of five genistein phase II metabolites in plasma: (1) genistein-4',7-diglucuronide, (2) genistein-7-sulfate, (3) genistein-4'-sulfate-7-glucuronide, (4) genistein-4'-glucuronide, and (5) genistein-7-glucuronide, independent of the food matrix. The conjugated genistein metabolites appear in a distinct order in human plasma. The specific early appearance of G-4',7-diG suggests a multistep formation process for the mono and hetero genistein conjugates, involving one or two deglucuronidation steps.