Identification of Metabolites of Epimedin A in Rats Using UPLC/Q–TOF–MS

Herba Epimedii (Epimedium) is a kind of tonic herb, widely used in China. Epimedin A is a major component of Herba Epimedii with bioactivities. Analysis of the metabolic profile in vivo plays a pivotal role in understanding how traditional Chinese medicine works. And the metabolites of epimedin A might influence the effects of Herba Epimedii. Moreover, the metabolic routes of epimedin A provide an important basis for safety evaluation. Until now, little has been known about the metabolism of epimedin A. The current study was designed to characterize the metabolic pathways of epimedin A in vivo. The metabolites in rat plasma, bile, feces, and urine were identified by UPLC/Q–TOF–MS analysis. A total of 27 metabolites from epimedin A were detected or tentatively identified. The major metabolic processes were hydrolysis, hydrogenation, hydroxylation, dehydrogenation, demethylation, and conjugation with glucuronic acid and different sugars. The present study revealed the metabolic pathways of epimedin A in rat for the first time, and epimedin A could undergo extensive phase I and phase II metabolism in rat. These findings would provide an important basis for the further study and clinical application of epimedin A. In addition, the results of this work have shown the feasibility of the UPLC/Q–TOF–MS approach for rapid and reliable characterization of metabolites.

     

Urinary Metabolites of Isoliquiritigenin in Wistar Rats using UHPLC–TOF–MS-based Xenometabolomics

Isoliquiritigenin, a chalcone found in licorice root and many other plants, has shown potential antioxidant, estrogenic and antitumor activities. The present study was to investigate urinary metabolism of isoliquiritigenin in Wistar rats by ultra-high pressure liquid chromatography coupled to electrospray ionization TOF–MS (UHPLC–TOF–MS)-based xenometabolomics. Urine samples were collected before and after oral administration of isoliquiritigenin, and analyzed by UHPLC–TOF–MS. After deconvolution, the resulting data matrices were subjected to multivariate data analysis. Projection to latent structures discriminant analysis was performed to screen the metabolites. Fifteen urinary metabolites of isoliquiritigenin were screened out and 13 of them were further identified by Agilent MassHunter Software. The results of this work show that UHPLC–TOF–MS-based xenometabolomics was able to comprehensively identify the metabolites of phytochemicals and may represent a valuable tool for monitoring the food consumption.

     

Study on Pharmacokinetics of Liguzinediol and Four Metabolites in Rats by UFLC–MS/MS

A rapid and sensitive UFLC–MS/MS method was developed for the simultaneous determination of liguzinediol and its four primary metabolites (M1, M2, M3, and M4) in rat plasma using antipyrine as internal standards. The analytes were separated on an XR-ODS column (50 mm × 2.0 mm, 2.2 μm) using 0.1 % formic acid–methanol gradient elution at a flow rate of 0.5 mL·min⁻¹. The detection was performed on a triple quadrupole tandem mass spectrometer in a multiple reaction monitoring mode with positive electrospray ionization. Method validation was performed as per the Food and Drug Administration guidelines. The resulting calibration curves offered satisfactory linearity (r > 0.9993) with the set ranges. The limits of quantification for liguzinediol, M1, M2, M3, and M4 were 20, 20, 21, 27, and 10 ng mL⁻¹, respectively. The recovery rates in different matrices ranged from 91.2 to 114.1 %, and the inter-day and intra-day precisions were all less than 13.4 % for the target analytes. After validation, this method was successfully applied to further study pharmacokinetics profiles of liguzinediol and its metabolites after intravenous administration of 10 mg·kg⁻¹ in male and female rats.

     

Using Nuclear Magnetic Resonance and MS/MS Spectroscopy for the Identification of Brodimoprim Metabolites in Rat Urine

Brodimoprim,a trimethoprim analogue diaminopyrimidine, 2,4-diamino-5-(4'-bromo-3',5'-dimethoxybenzyl) pyrimidine, is a new inhibitor of bacterial dihydro folate reductases (OHFRs), it shows activity against a broad spectrum of Gram-positive and negative-bacteria. Recently we reported the results of the study on the metabolites of brodimoprim in vivo with SPE-NMR method (solid phase extraction coupled with nuclear magnetic resonance), five metabolites were detected in rat urine1. They were …     

Identification of In Vitro Metabolites of Amoxicillin in Human Liver Microsomes by LC–ESI/MS

Amoxicillin (AMOX) metabolism in human liver microsomes was studied in vitro using liquid chromatography–mass spectrometry (LC/MS). Amoxicillin was incubated with human liver microsomes along with NADPH, and the reaction mixture was analyzed by LC/MS to obtain the specific metabolic profile of the studied antibiotic drug. Positive electrospray ionization was employed as the ionization source. An ACE C18-column (4.6 mm × 150 mm, 3 μm) was implemented with acetonitrile and water (+0.1 % formic acid) in isocratic mode as the mobile phase at the flow 0.4 mL min^?1. The chemical structures of metabolites were proposed on the basis of the accurate mass measurement of the protonated molecule as well as their main product. Six phase I and one phase II metabolites were detected and structurally described. The metabolism of AMOX occurred via oxidation, hydroxylation and oxidative deamination, as well as through combination of these reactions. Compound M7, with glucuronic acid was also observed as phase II metabolite. Neither sulfate nor glutathione conjugates were detected. This study presents novel information about the chemical structure of the potential AMOX metabolites and provides vital data for further pharmacokinetic and in vivo metabolism studies.     

Forensic Identification of Dyes in Ballpoint Pen Inks Using LC–ESI–MS

The aim of this work was to develop a new technique using flow injection analysis combined with LC–ESI–MS which allows identification of dyes in ballpoint pen inks. A sample preparation procedure for the extraction of dyes from ballpoint pen strokes has been developed. The characteristic group of ions for each sample of 21 studied ballpoint pen inks corresponding to the present dyes has been determined using flow injection method. LC separation conditions for identified dyes have been optimized on reversed-phase sorbent based on silica gel. The best composition of the mobile phase for the dyes mixture LC separation was 0.1% aqueous formic acid and acetonitrile. Detection of dyes was carried out using mass spectrometry with electrospray ionization in positive and negative modes after reversed-phase liquid chromatography separation. Dye composition of ink was additionally confirmed comparing the data obtained from the literature. Flow injection analysis allows obtaining intensive ions of unknown strokes. It is difficult to get this information using only chromatographic methods, because dyes peak intensity can be low and noise of basic line high. Flow injection method allows distinguishing the analyzed 21 ballpoint pens by determining a characteristic set of dyes. The developed flow injection technique is very simple and quick. As a result, a novel approach for the identification of dyes in the ballpoint pen inks by flow injection analysis with LC–ESI–MS and UV detection without using standard dye samples has been established. It can be an effective alternative to the existing LC–DAD–MS and IR spectroscopy methods.     

Identification of the absorptive constituents and their metabolites in vivo of Ziziphi Spinosae Semen by UPLC–ESI–Q-TOF–MS/MS

In this research, ultra-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry (UPLC–ESI–Q-TOF–MS/MS) was used for detection and identification of the absorptive constituents and their metabolites in rat plasma, urine and feces following oral administration of Ziziphi Spinosae Semen alcohol extract. After structure elucidation, a total of 12 compounds in rat plasma, comprising seven prototypes and five metabolites, 28 compounds in urine, comprising 17 prototypes and 11 metabolites, and 23 compounds in feces, comrpising 17 prototypes and six metabolites, have been tentatively identified by comparison with standard compounds and reference literature information. To the best of our knowledge, this is the first comprehensive and systematical metabolic study on the seed. Mostly importantly, we propose that gastric acid could convert jujubosides into an absorbable form of ebelin lactone oligosaccharides, which may be responsible for the low bioavailability and specific bioactivities of these compounds. Additionally, we deduced that the absorption site of ebelin lactone oligosaccharides is located in the stomach, and that the ebelin lactone form of jujubosides may be more suitable for absorption than its hydrolysis product. Our investigation will be helpful to narrow the scope for potentially active ingredients of the seed, and pave the way for determination of the pharmacological mechanism of the seed.     

Identification of the Major Constituents in Zhimu–Huangqi Herb-Pair Extract and Their Metabolites in Rats by LC–ESI-MSn

The Zhimu–Huangqi herb-pair is a famous Chinese herbal formula with a combination of Rhizoma Anemarrhenae (Zhimu in Chinese) and Radix Astragali (Huangqi in Chinese). This work describes a sensitive and specific LC–ES-MSⁿ methodology for identification of the major constituents in Zhimu–Huangqi herb-pair extract and their metabolites in rats after oral administration. A total of 30 compounds have been identified or tentatively characterized from the herb-pair extract, and 13 of them were unambiguously identified by comparing the retention times and mass spectra with those of reference standards, while the other 17 compounds were tentatively identified on the basis of their MSⁿ fragmentation behaviors and exact mass information from literature. Moreover, the metabolites in vivo were also identified. The Zhimu–Huangqi herb-pair extract was actively metabolized in rats, including four parent compounds and 8 metabolites in serum and seven parent compounds and 23 metabolites in urine. This study proposed a good example for the rapid identification of major constituents in complex systems such as herbal extract or traditional Chinese medicine formula, which facilitated the clarification of the metabolic pathway of the herbs in the body to better understand the action mechanism.

     

Identification of the Major Constituents in Zhimu–Huangqi Herb-Pair Extract and Their Metabolites in Rats by LC–ESI-MSn

The Zhimu–Huangqi herb-pair is a famous Chinese herbal formula with a combination of Rhizoma Anemarrhenae (Zhimu in Chinese) and Radix Astragali (Huangqi in Chinese). This work describes a sensitive and specific LC–ES-MSⁿ methodology for identification of the major constituents in Zhimu–Huangqi herb-pair extract and their metabolites in rats after oral administration. A total of 30 compounds have been identified or tentatively characterized from the herb-pair extract, and 13 of them were unambiguously identified by comparing the retention times and mass spectra with those of reference standards, while the other 17 compounds were tentatively identified on the basis of their MSⁿ fragmentation behaviors and exact mass information from literature. Moreover, the metabolites in vivo were also identified. The Zhimu–Huangqi herb-pair extract was actively metabolized in rats, including four parent compounds and 8 metabolites in serum and seven parent compounds and 23 metabolites in urine. This study proposed a good example for the rapid identification of major constituents in complex systems such as herbal extract or traditional Chinese medicine formula, which facilitated the clarification of the metabolic pathway of the herbs in the body to better understand the action mechanism.     

Identification of Acepromazine and Its Metabolites in Horse Plasma and Urine by LC–MS/MS and Accurate Mass Measurement

Acepromazine maleate (Sedalin^®) was administered orally to six thoroughbred horses at a dose of 0.15 mg kg⁻¹. Urine and blood samples were collected up to 412 h post-administration. Plasma and urine were hydrolysed; plasma samples were then processed using liquid–liquid extraction and urine samples using solid-phase extraction. A sensitive tandem mass spectrometric method was developed in this study, achieving a lower limit of quantification for acepromazine of 10 pg mL⁻¹ in plasma and 100 pg mL⁻¹ in urine. Acepromazine, hydroxyethylpromazine, hydroxyacepromazine, hydroxyethylpromazine sulphoxide, hydroxyethylhydroxypromazine, dihydroxyacepromazine and dihydroxyhydroxyethylpromazine were detected in the post-administration samples. The parent drug and its metabolites were identified using a combination of UPLC–MS/MS and accurate mass measurement. Separation of the structural isomers hydroxyethylpromazine sulphoxide and hydroxyethylhydroxypromazine was another significant outcome of this work and demonstrated the advantages to be gained from investing in chromatographic method development.

     

Identification of Acepromazine and Its Metabolites in Horse Plasma and Urine by LC–MS/MS and Accurate Mass Measurement

Acepromazine maleate (Sedalin^?) was administered orally to six thoroughbred horses at a dose of 0.15?mg?kg^?1. Urine and blood samples were collected up to 412?h post-administration. Plasma and urine were hydrolysed; plasma samples were then processed using liquid–liquid extraction and urine samples using solid-phase extraction. A sensitive tandem mass spectrometric method was developed in this study, achieving a lower limit of quantification for acepromazine of 10?pg?mL^?1 in plasma and 100?pg?mL^?1 in urine. Acepromazine, hydroxyethylpromazine, hydroxyacepromazine, hydroxyethylpromazine sulphoxide, hydroxyethylhydroxypromazine, dihydroxyacepromazine and dihydroxyhydroxyethylpromazine were detected in the post-administration samples. The parent drug and its metabolites were identified using a combination of UPLC–MS/MS and accurate mass measurement. Separation of the structural isomers hydroxyethylpromazine sulphoxide and hydroxyethylhydroxypromazine was another significant outcome of this work and demonstrated the advantages to be gained from investing in chromatographic method development.     

Stress Studies and Identification of Degradation Products of Cephalexin Using LC–PDA and LC–MS/MS

A stability indicating RP-HPLC method for cephalexin has been developed and validated to identify and characterize potential degradation products. Drug was subjected to hydrolytic (acidic, basic, and neutral), oxidative, thermal, and photolytic stresses as per ICH guidelines Q1A (R2) and Q1B. Chromatographic separation was achieved on C8 column with mixture of ammonium acetate buffer pH 4.5 and acetonitrile in gradient mode as a mobile phase with PDA detection. Specificity of the method was established by peak purity studies. Method was validated as per ICH guideline Q2 (R1) for accuracy, precision, linearity, sensitivity, and robustness. Kinetics for each degradation condition was studied with respect to order of reaction and rate constant. Method was found to comply with acceptance criteria of validation parameters with respect to specificity (peak purity greater than 0.999) linearity (r ² greater than 0.99), accuracy (% recovery in the range of 98–102%), and precision (% RSD not more than 2). A total of six degradation products were generated in different stress conditions; these were identified and structures were proposed using LC–MS/MS. Cephalexin undergoes degradation in almost all the conditions. The developed stability indicating method is suitable for analysis of stability samples as it adequately separates all degradation products. Degradation products generated in photolytic and oxidative conditions are reported for the first time.     

High-throughput serum metabolomics analysis of gouty arthritis rat treated by total saponins of Rhizoma Dioscoreae Makino by UPLC–Q/TOF–MS

Rhizoma Dioscoreae Makino (RDM) is effective in treating gouty arthritis (GA) and hyperuricacidemia, especially in promoting uric acid excretion and reducing the inflammatory reaction. Bioactive constituents in RDM are mainly steroidal saponins such as dioscin, trillin, protodioscin and protogracillin. However, the mechanism of its anti-GA action is still unclear, owing to the complex pathological and physiological characteristics of GA, and integration of RDM with multiple components, multiple targets and multiple pathways. Herein, a GA rat model was induced with monosodium urate (MSU), and RDM reduced inflammation of rat synovium tissue. Through metabolomics analysis, 35 potential biomarkers with significant changes involved in the pathogenesis of GA induced by MSU were identified, and perturbations were restored after RDM treatment. The most correlated pathways involved in d -galactose, d -mannose, d -glucose, myoinositol, Phosphatidylcholine (PC) (16:0/16:0), LysoPC (15:0), phosphatidic acid (PA) [18:1(9Z)/18:1(11Z)] and glutathione induced by MSU were galactose metabolism, inositol phosphate metabolism, glycerophospholipid metabolism and glutathione metabolism, and the derivations of all those biomarkers could be regulated by RDM treatment. RDM has a therapeutic effect on GA by intervening in changes in endogenous metabolisms and the related metabolic pathways.     

Analysis of Linarin and Its Metabolites in Rat Urine by LC–MS/MS

Liquid chromatography–ion trap mass spectrometry was employed to investigate the metabolism of linarin in rats. Identification and structural elucidation of the metabolites were performed by comparing the differences in molecular masses, retention times, and full scan MS ⁿ spectra between linarin and its metabolites. Six metabolites (acacetin, apigenin, acacetin glucuronide, apigenin glucuronide, acacetin sulfate, apigenin sulfate) were detected in rat urine after oral administration of linarin at the dose of 50 mg kg^?1. Furthermore, a selective and sensitive liquid chromatography–triple quadruple mass spectrometry assay was developed and validated for the simultaneous determination of linarin and acacetin (the major metabolite of linarin) in rat urine. Chromatographic separation was carried out on a C₁₈ column, and mass spectrometric detection was performed using a triple-quadrupole mass spectrometer coupled with an electrospray ionization source in the positive ion mode. Quantitation of linarin and acacetin was performed using selected reaction monitoring of precursor–product ion transitions at m/z 593 → 285 for linarin, 285 → 242 for acacetin, and 303 → 153 for hesperitin (internal standard), respectively. The assay exhibited good linearity (r > 0.9900) for both linarin and acacetin. The intra- and inter-day precisions were <13.4 % and the accuracy was between ?8.1 and 3.1 %. The method was successfully applied to the urinary excretion study of linarin in rats after oral administration of linarin.     

LC–MS and UPLC–Quadrupole Time-of-Flight MS for Identification of Photodegradation Products of Aflatoxin B1

UV irradiation of a solution of aflatoxin B₁ in acetonitrile resulted in three major degradation products which have been identified by LC–MS. Accurate masses and proposed molecular formulas of the degradation products—315.0868 (C₁₇H₁₅O₆), 285.0758 (C₁₆H₁₃O₅), and 275.0553 (C₁₄H₁₁O₆)—were obtained with low mass error and high matching property by ultra-performance liquid chromatography–quadrupole time-of-flight mass spectrometry (UPLC–Q-TOF MS). Structural formulas of the photodegradation products, and the degradation pathways leading to the compounds, are proposed on the basis of the molecular formulas and MS–MS spectra. UPLC–Q-TOF MS has been recognized as a powerful analytical tool for qualitative analysis of trace materials and degradation products.     

HPLC–MS Profiling and Structural Identification of [14C]-Diclofenac Metabolites in Mouse Bile

Biliary metabolites present at 6 h post-dose following a single oral dose of [¹⁴C]-diclofenac (10 mg kg^?1) to male bile duct-cannulated C57BL/6 J mice were profiled and identified. Over the 6 h duration of the study ~19.5 % of the administered radioactivity was excreted into the bile as either [¹⁴C]-diclofenac or metabolites. When profiled using HPLC with online radiodetection, the presence of at least 13 radiolabelled components was indicated. These compounds were shown, by consecutive reaction mass spectrometry, to comprise a range of hydroxylated metabolites conjugated to either taurine, glucose and/or glucuronic acid. Both phenolic and acylglucuronide-containing metabolites were observed. The confirmation of the presence of these glucuronide conjugates in mouse bile may have important consequences in the light of emerging theories concerning the role of bacterial glucuronidases for the GI-tract toxicity of NSAIDs such as diclofenac.     

The Rapid Analysis of Antibiotics in Animal Meat and Egg Using a Novel SEP Method and UPLC–MS/MS

A simple and sensitive method for the determination of 39 antibiotics, including sulfonamides and quinolones, in pork, chicken, fish tissues and eggs, has been developed. The sample preparation included ultrasound-assisted extraction (UAE) with 0.1% formic acid in 90:10 acetonitrile/water (v:v) and a final clean-up with Oasis PRiME HLB, a new reversed phase SPE without traditional pre-equilibration and washing steps before eluting SPE. Analysis was performed by ultra-performance liquid chromatography coupled to tandem quadrupole mass spectrometry (UPLC–MS/MS). The positive ionization in multiple reaction monitoring mode (MRM) and product ion confirmation scan (PICs) were used in the method. The PICs provides additional confirmation for compound identification through acquisition of MS/MS spectra in the same injection and a means of verifying that the signal from the MRM peak is from the compound of interest. In particular, single test is simultaneously able to gain both quantitative MRM and qualitative full-scan MS/MS data without the need for long analysis times or repeat injection. All solvent and matrix-matched calibration curves showed excellent correlation coefficient >0.990, with the dynamic range 0.2–100 ng mL^?1. For over 90% of the analytes, the recoveries were between 60 and 120% in all matrices studied at three spiked levels of low, medium, and high concentrations, with the intra-day precision values in the range of 2.7–20.0% and the inter-day precision values in the range of 6.2–21.3%. The limits of detection (LODs) and limits of quantification (LOQs) of all drugs were 0.05–2.6 and 0.12–5.6 μg kg^?1, respectively. A weak matrix effect was observed for most of the compounds in four complex samples. The proposed method was proven very simple, fast, sensitive, and selective and has been successfully applied in real samples from local markets and farms.