Metabolites In Mouse Plasma Research Articles

Effects of ethanol extract from senna leaf (EESL) on inflammation and oxidative stress in mice: A non-targeted metabolomic study.

Senna leaf is a commonly used medication for treating constipation, and long-term use can cause damage to the intestinal mucosa and lead to drug dependence. But the exact mechanism remains unclear. Using non-targeted metabolomics technology to study the mechanism of senna leaf ethanol extract (EESL) inducing inflammation and oxidative stress in mice and causing side effects. EESL was administered to mice by gavage to detect inflammation and oxidative stressrelated factors in mice, and the EESL components and differential metabolites in mouse plasma were analyzed using non-targeted metabolome techniques. 23 anthraquinone compounds were identified in the EESL, including sennoside and their derivatives. Administration of EESL to mice resulted in a significant increase in pro-inflammatory factors, IL-1β, and IL-6 in the plasma, while the levels of IgA significantly decreased. The levels of oxidative stress significantly increased, and the intestinal mucosal integrity was impaired. 21 endogenous in plasma metabolites were identified as differential metabolites related with taurine and taurine metabolism, glycerophospholipid metabolism, arachidonic acid metabolism, tryptophan metabolism, and sphingolipid metabolism. These metabolic pathways are related to oxidative stress and inflammation. Senna leaf can inhibit the expression of tight junction proteins in the intestinal mucosa and disrupt intestinal mucosal barrier integrity, exacerbating oxidative stress and inflammation induced by bacterial LPS entering the bloodstream. In addition, the impact of Senna leaf on tryptophan metabolism may be linked to the occurrence of drug dependence.

Cellulose based molecularly imprinted polymer by hydroxyl functional allocation strategy with dual selectivity of boric acid affinity and molecular structure limitation to bortezomib: Preparation strategy, selectivity exploration, and metabolism study

Bortezomib is widely recognized as an effective drug for the treatment of multiple myeloma, but its metabolism in vivo has not been reported. There are extremely high requirements for selectivity and enrichment ability of pretreatment methods due to the complex biological matrix and trace metabolites. Urgently, a novel magnetic porous cellulose molecularly imprinted polymers (B-MPCMIP) based on bortezomib was developed for the first time using the magnetic porous cellulose (MPC) as the both carrier and functional monomer by the hydroxyl functional allocation strategy of cellulose, which showed the dual selectivity towards bortezomib through the boric acid affinity and molecular structure limitation. Firstly, the cis dihydroxy groups of cellulose was occupied by phenylboronic acid; then, another hydroxyl group in cellulose was modified by silane reagent to introduce reaction site for grafting MIP; subsequently, the phenylboronic acid was removed to release the cis dihydroxy groups of cellulose as the functional monomer for MIP; finally, MIP was prepared on the surface of MPC with the MPC as the both carrier and functional monomer for obtaining B-MPCMIP, which can recognize the bortezomib through the boric acid affinity molecular structure limitation. A series of characterization studies showed that B-MPCMIP had a porous structure, good thermal stability, ultra-high magnetism, mesoporous structure, and superhydrophilicity. B-MPCMIP showed good adsorption capacity (Qmax: 4.4 mg/g) and fast adsorption effect (adsorption equilibrium time: 30 min). Adsorption capacity of B-MPCMIP were also inspected under different temperature and pH conditions. The strong pH adsorption dependence indicates that B-MPCMIP mainly adsorbs bortezomib through the boric acid affinity, as the affinity between the cis dihydroxy groups in B-MPCMIP and the boric acid group in bortezomib is strongest at pH 8.5. Furthermore, B-MPCMIP exhibited excellent selectivity towards bortezomib with the imprinting factor, selectivity coefficient, and relative selectivity coefficient up to 8.6, 17, and 14, respectively. B-MPCMIP had outstanding reusability and stability, which remained 96 % of adsorption capacity after multiple recycling. B-MPCMIP-HPLC and B-MPCMIP-UPLC-MS/MS methods were successfully applied to enrich and detect bortezomib and its metabolites in mouse plasma and liver. The methods showed good selectivity, sensitivity, and accuracy. The maximum concentration of bortezomib in plasma and liver was detected as 34.3 μg/mL and 84.2 μg/mL. And six metabolites of bortezomib through oxidative deboronation were triumphantly detected and identified by B-MPCMIP-UPLC-MS/MS method.

Disorders of fatty acid metabolism and imbalance in the ratio of monounsaturated fatty acids promote the development of pulmonary fibrosis

ObjectiveAlthough some studies suggested that metabolic abnormalities may contribute to the development of pulmonary fibrosis, there are no studies that have reported a clear causal relationship between them, and the aim of this study was to explore the causal relationship between plasma metabolites and pulmonary fibrosis using Mendelian randomization (MR) combined with metabolomics analysis. MethodsFirstly, we explored the causal relationship between 1400 metabolites and pulmonary fibrosis using MR analysis, and detected plasma metabolites in mice with pulmonary fibrosis using metabolomics technology, thus validating the results of MR analysis. In addition, we again used MR to explore the causal relationship between the results of the differential metabolite KEGG in metabolomics and pulmonary fibrosis. ResultsA total of 52 metabolites were screened for association with pulmonary fibrosis in the MR analysis of 1400 plasma metabolites with pulmonary fibrosis, based on P < 0.05 for the IVW method, with consistent OR directions for all methods. Four of them were validated in the plasma of mice with pulmonary fibrosis, namely carnitine c18:2 levels (negative correlation), Glutamine degradant levels (positive correlation), Propionylcarnitine (c3) levels (negative correlation), carnitine to palmitoylcarnitine (c16) ratio (negative correlation). In addition, KEGG analysis of plasma differential metabolites revealed that the signaling pathway of biosynthetic of unsaturated fatty acids was most affected in mice with pulmonary fibrosis, and MR analysis showed that imbalance in the ratio of monounsaturated fatty acids was significantly associated with pulmonary fibrosis. ConclusionsOur study suggests that abnormal fatty acid levels due to reduced levels of carnitine-like metabolites, and an imbalance in the ratio of monounsaturated, promote the development of pulmonary fibrosis. This study reveals the marker metabolites and metabolic pathways affecting the development of pulmonary fibrosis to provide a basis for the development of new drugs for the treatment of pulmonary fibrosis.

Lingguizhugan Decoction Improved Obesity by Modulating the Gut Microbiota and its Metabolites in Mice.

The global obese population is rapidly increasing, urgently requiring the development of effective and safe weight-loss medications. The classic Chinese medicine formulation Lingguizhugan De-coction has exerted a significant anti-obesity effect. However, the underlying mechanism is still unclear. This study aimed to explore the mechanism of LGZGD in the treatment of obesity based on the gut microbiota and its metabolites. Three different dosages of LGZGD were gavaged to ob/ob mice for 8 weeks. Body mass and visceral fat mass were evaluated. Additionally, the changes in gut microbiota, fecal and plasma metabolites in mice after LGZGD treatment were analyzed by metagenomics and non-targeted metabolomics. The results demonstrated a significant anti-obesity effect of LGZGD treatment in ob/ob mice. Fur-thermore, the metagenomic analysis revealed that LGZGD reduced the ratio of Firmicutes / Bacteroidetes (F to B) in the gut, restored gut microbiota diversity, and identified 3 enriched KEGG pathways, including energy metabolism, lipid metabolism, and energy production and conversion pathways. Based on non-targeted metab-olomics analysis, 20 key metabolites in the feces and 30 key metabolites in the plasma responding to LGZGD treatment were identified, and the levels of Eicosapentaenoic acid (EPA) and Myristoleic acid (MA) might be the metabolites related to gut microbiota after LGZGD treatment. Their biological functions were mainly re-lated to the metabolism pathway. These findings suggested that LGZGD had therapeutic potential for obesity. The mechanism of LGZGD alleviating obesity was associated with improving dysbiosis of the gut microbiota. LDZGD affected gut microbiota-derived metabolites of EPA and MA and may act on energy metabolism pathways.

Simultaneous determination of NTB-3119, a novel anti-tuberculosis agent, and its major metabolites in mouse plasma by LC-MS/MS and its application in preclinical pharmacokinetics study

NTB-3119, a novel benzothiopyranone derivative, has been developed as a potential anti-tuberculosis(TB) drug with strong activity. In this study, three major metabolites of NTB-3119 were firstly identified in vitro and in vivo. A sensitive and specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was established for the quantitative analysis of NTB-3119 and its major metabolites NTB-3190, NTB-3202 and NTB-3204 in mouse plasma. The plasma samples were processed by protein precipitation with organic solvent. NTB-3119, NTB-3190, NTB-3202, NTB-3204 and NTB-4A (Internal Standard, IS) were separated by a Zorbax-SB C18 column (100 mm × 2.1 mm, 3.5 µm) with a gradient mobile phase of acetonitrile/water at a flow rate of 0.25 mL/min. The analytes were detected by electrospray positive ion mode in Parallel Reaction Monitoring (PRM) mode on a high resolution mass spectrum (HRMS, Thermo Q Executive). The monitored transitions were m/z 456.15632 → 360.06137 for NTB-3119, m/z 426.18214 → 246.01891 for NTB-3190, m/z 472.15124 → 360.06143 for NTB-3202, m/z 442.17706 → 246.01903 for NTB-3204 and m/z 337.13691 → 163.02081 for NTB-4A, respectively. Good linearity was conducted in the range of 5–2000 ng/mL for NTB-3119, NTB-3202 and NTB-3204 as well as 2.5–1000 ng/mL for NTB-3190. The inter- and intra-batch precision (RSD%) were both lower than 13.3 %, with the accuracy ranged from 88.0 % to 108.1 %. The analytes were proved to be stable during all samples storage, preparation and analytic procedures. The validated method was successfully applied to study the pharmacokinetics and bioavailability of NTB-3119 after oral treatment in Balb/c mouse.

Developmental arsenic exposure induces dysbiosis of gut microbiota and disruption of plasma metabolites in mice

Arsenic is a notorious environmental pollutant. Of note, developmental arsenic exposure has been found to increase the risk of developing a variety of ailments later in life, but the underlying mechanism is not well understood. Many elements of host health have been connected to the gut microbiota. It is still unclear whether and how developmental arsenic exposure affects the gut microbiota. In the present study, we found that developmental arsenic exposure changed intestinal morphology and increased intestinal permeability and inflammation in mouse pups at weaning. These alterations were accompanied by a significant change in gut microbiota, as evidenced by considerably reduced gut microbial richness and diversity. In developmentally arsenic-exposed pups, the relative abundance of Muribaculaceae was significantly decreased, while the relative abundance of Akkermansia and Bacteroides was significantly enhanced at the genus level. Metabolome and pathway enrichment analyses indicated that amino acid and purine metabolism was promoted, while glycerophospholipid metabolism was inhibited. Interestingly, the relative abundance of Muribaculaceae and Akkermansia showed a strong correlation with most plasma metabolites significantly altered by developmental arsenic exposure. These data indicate that gut microbiota dysbiosis may be a critical link between developmental arsenic exposure and metabolic disorders and shed light on the mechanisms underlying increased susceptibility to diseases due to developmental arsenic exposure.

Lactulose Improves Neurological Outcomes by Repressing Harmful Bacteria and Regulating Inflammatory Reactions in Mice After Stroke.

Background and ObjectiveGut microbiota dysbiosis following stroke affects the recovery of neurological function. Administration of prebiotics to counteract post-stroke dysbiosis may be a potential therapeutic strategy to improve neurological function. We aim to observe the effect of lactulose on neurological function outcomes, gut microbiota composition, and plasma metabolites in mice after stroke.MethodsMale C57BL/6 mice (20–25 g) were randomly divided into three groups: healthy control, photothrombotic stroke + triple-distilled water, and photothrombotic stroke + lactulose. After 14 consecutive days of lactulose administration, feces, plasma, and organs were collected. 16S rDNA sequencing, plasma untargeted metabolomics, qPCR, flow cytometry and Elisa were performed.ResultsLactulose supplementation significantly improved the functional outcome of stroke, downregulated inflammatory reaction, and increased anti-inflammatory factors in both the brain and gut. In addition, lactulose supplementation repaired intestinal barrier injury, improved gut microbiota dysbiosis, and partially amended metabolic disorder after stroke.ConclusionLactulose promotes functional outcomes after stroke in mice, which may be attributable to repressing harmful bacteria, and metabolic disorder, repairing gut barrier disruption, and reducing inflammatory reactions after stroke.

Hybrid organic/inorganic hybrid surface technology for increasing the performance of LC/MS(MS)-based drug metabolite identification studies: Application to gefitinib and metabolites in mouse plasma and urine

The detection, identification and quantification of drug metabolites plays a key role in drug discovery and development. Liquid chromatography (LC) coupled to mass spectrometry (MS) has become the primary technology for these studies due to its sensitivity and specificity. However, the presence of transition metals in the chromatography system and columns can result in non-specific and unwanted interactions with the drug and/or its metabolites, via electron-pair donation, leading to poor chromatography and analyte loss. The use of a hybrid organic/inorganic surface applied to the metal surfaces of the chromatography system and column has been demonstrated to reduce or eliminate these effects. When employed for the analysis of mouse urine, derived from the oral dosing of mice with the EGFR inhibitor gefitinib, we observed more symmetrical LC peaks. This resulted in a 33 % improvement in peak capacity for a 10 min reversed – phase gradient separation, a two-fold increase in MS response, cleaner MS spectra and improved peak response reproducibility. This hybrid surface barrier appears to offer significant advantages in the analysis of low-concentration metabolites, potentially facilitating the accurate determination of the elimination phase of the pharmacokinetic (PK) curve and detection of drug metabolites in microdosing or microsampling studies.

Method Development and Validation with Statistical Analysis to Access Raspberry Ketone Metabolism in Mice by UHPLC-QqQ-MS/MS

Abstract Objectives Raspberry ketone (4-(4-hydroxyphenyl)-2-butanone) (RK) is the major aroma compound of red raspberries and used commercially as flavoring agent. It is now gaining attention as a weight loss supplement. This work aimed to establish atargeted ultra-high performance liquid chromatography triple quadrupole tandem mass spectrometry (UHPLC-QqQ-MS/MS) method for analysis of raspberry ketone and its 25 associated metabolites in mouse plasma and brain. Methods Dispersion and projection analysis and central composite design were used for LC-MS method optimization. Random effect analysis of variance was applied for validation inference and variation partition. Within this framework, repeatability, a broader sense of precision, was calculated as fraction of accuracy variance, reflecting instrumental imprecision, compound degradation and carry-over effects. Multivariate correlation analysis and principle component analysis were conducted, revealing underlying interlinkage and association among the manifold of method traits. R programming was engaged in streamlined statistical analysis and data visualization. Results The developed and validated method for RK and 25 metabolites had accuracy, matrix effect, recovery and processing efficiency bound mostly within the 80∼120% range, precision error below 5%, limit of quantification at 0.4∼6.0 ng/mL and linear range across three orders of magnitude. Two particular phenomena, the analytes’ background existence in the enzyme solution used for phase II metabolites deconjugation, and the noted liability of analytes in pure solvent at 4 °C vs. elevated stability in biomatrices, were found critical to method development and validation. Conclusions The UHPLC-QqQ-MS/MS method developed and validated provided an important tool for further investigation of RK metabolism and bioavailability. Funding Sources National Institutes of Health.

UHPLC-QqQ-MS/MS method development and validation with statistical analysis: Determination of raspberry ketone metabolites in mice plasma and brain

Raspberry ketone (RK) (4-(4-hydroxyphenyl)-2-butanone) is the major compound responsible for the characteristic aroma of red raspberries, and has long been used commercially as a flavoring agent and recently as a weight loss supplement. A targeted UHPLC-QqQ-MS/MS method was developed and validated for analysis of RK and 25 associated metabolites in mouse plasma and brain. Dispersion and projection analysis and central composite design were used for method optimization. Random effect analysis of variance was applied for validation inference and variation partition. Within this framework, repeatability, a broader sense of precision, was calculated as fraction of accuracy variance, reflecting instrumental imprecision, compound degradation and carry-over effects. Multivariate correlation analysis and principle component analysis were conducted, revealing underlying association among the manifold of method traits. R programming was engaged in streamlined statistical analysis and data visualization. Two particular phenomena, the analytes’ background existence in the enzyme solution used for phase II metabolites deconjugation, and the noted lability of analytes in pure solvent at 4 ℃ vs. elevated stability in biomatrices, were found critical to method development and validation. The approach for the method development and validation provided a foundation for experiments that examine RK metabolism and bioavailability.

Metabolic profiling of mice plasma, bile, urine and feces after oral administration of two licorice flavonones

Ethnopharmacological relevanceLicorice is an ancient food and medicinal plant. Liquiritigenin and liquiritin, two kinds of major flavonoes in licorice, are effective substances used as antioxidant, anti-inflammatory and tumor-suppressive food, cosmetics or medicines. However, their in vivo metabolites have not been fully explored. Aim of studyTo clarify the metabolism of liquiritigenin and liquiritin in mice. Materials and methodsIn this study, we developed a liquid chromatography coupled with quadrupole/time-of-flight mass spectrometry approach to determine the metabolites in mice plasma, bile, urine and feces after oral administration of liquiritigenin or liquiritin. The structures of those metabolites were tentatively identified according to their fragment pathways, accurate masses, characteristic product ions, metabolism laws or reference standard matching. ResultsA total of 26 and 24 metabolites of liquiritigenin or liquiritin were respectively identified. The products related with apigenin, luteolin or quercetin were the major metabolites of liquiritigenin or liquiritin in mice. Seven main metabolic pathways including (de)hydrogenation, (de)hydroxylation, (de)glycosylation, (de)methoxylation, acetylation, glucuronidation and sulfation were summarized to tentatively explain their biotransformation. ConclusionThis study not only can provide the evidence for in vivo metabolites and pharmacokinetic mechanism of liquiritigenin and liquiritin, but also may lay the foundation for further development and utilization of liquiritigenin, liquiritin and then licorice.

Simultaneous determination of a promising anti-brain tumor agent CAT3 and its two major metabolites in mouse plasma and brain by a LC-MS/MS method

A rapid and reproducible method with high selectivity was developed for simultaneous determination of a promising anti-brain tumor agent CAT3 and its two metabolites PF403 and GLU-PF403 in mouse plasma and brain. An economic deproteinization with septuple acetonitrile (v/v) was applied to pretreat the samples in this study. All analytes were well retained and separated on a CAPCELL CORE PC (2.7 μm, 2.1 mm I.D. × 150 mm, SHISEIDO Technologies) column with an eluting solvent of acetonitrile /water containing 0.1 % formic acid (v/v) at the flow rate of 0.2 mL per minute. The detection was carried out on a Q Exactive high resolution mass spectrometer equipped with a HESI ion source in parallel reaction monitoring (PRM) mode. The corresponding transitions for quantitation were 434.23→ 70.07 for CAT3, 350.17→70.07 for PF403, 526.21→70.07 for GLU-PF403, 364.19→70.07 for IS-1 and 625.18→317.07 for IS-2, respectively. A well-linear fit curve was achieved among the range of 0.1∼50 ng/mL for CAT3, 0.2∼100 ng/mL for PF403 and 2.5∼600 ng/mL for GLU-PF403 both in mouse plasma and brain homogenate. The intra-/inter-day accuracies of three analytes were within ±14.5 % and precisions were below to 13.44 %. The mean values of recovery of three compounds in mouse plasma and brain homogenate were among 98.06 ∼ 118.63 % and 81.04∼108.69 %. The analytes in NaF-treated ice cold blood of mouse was stable within tested 30 min. Plasma and brain homogenate samples had no obvious changes during all storage, sample treatment and analytic process of mouse plasma sample. The reproducible and reliable method was well employed to the research of CAT3 pharmacokinetic characteristics in mouse plasma and brain after a single intragastric administration at dose of 10 mg/kg.

A novel UHPLC-HRMS-based metabolomics strategy enables the discovery of potential neuroactive metabolites in mice plasma, following i.p. administration of the main Crocus sativus L. bioactive component

Trans-crocin 4 (TC4) is an important carotenoid constituent of saffron showing potential activity against Alzheimer’s Disease (AD) due to its antioxidant and antiamyloidogenic properties. Metabolomics is an emerging scientific field that enhances biomarker discovery and reveals underlying biochemical mechanisms aiming towards the early subclinical diagnosis of diseases. To date, there are no reports on the changes induced to mice plasma metabolome after TC4 administration. We report a novel untargeted UHPLC-ESI HRMS metabolomics strategy to determine the alteration of the metabolic fingerprint following i.p. administration of TC4 in male and female mice. Blood samples from fiftysix mice treated with TC4 as well as from control animals were analyzed with UHPLC-ESI HRMS. Statistical evaluation of the results was achieved by multivariate analysis (MVA), i.e., principal component analysis (PCA), Partial Least Squares–Discriminant Analysis (PLS-DA) in order to discover the variables that contributed to the discrimination between treated and untreated groups which were identified by online database searching (e.g., Metlin, HMDB, KEGG) aided by chemometric processing, e.g., covariance searching etc. Due to the high variability imposed by various factors, e.g., sex of the animals participating in the study, administration dose and time-points of sacrifice, multilevel sparse PLS-DA analysis, e.g., splitting variation to each individual component, has been employed as a more efficient approach for such designs.This methodology allowed the identification of the time sequence of metabolome changes due to the administration of TC4, whereas a sex-related effect on the metabolome is indicated, denoting that the administration in both sexes is indispensable in order to acquire safe conclusions as reliable metabolome pictures. The results demonstrated a number of annotated metabolites playing a potential role in neuroprotection while they are closely related to AD. Moreover, five additional annotated metabolites were involved in the steroid biosynthesis pathway while two of them may be considered as putative neuroprotective agents.

Simultaneous determination of the pharmacokinetics of A-type EGCG and ECG dimers in mice plasma and its metabolites by UPLC-QTOF-MS

A-type epigallocatechin-3-gallate (EGCG) and epicatechin-3-O-gallate (ECG) dimers have multiply biological activities. In this study, the pharmacokinetics of them were investigated in mice after a single dose intravenous administration, and the metabolites in mice plasma and urine were investigated by ultra-performance liquid chromatography-Quadrupole-time of flight mass spectrometer (UPLC-QTOF-MS). Our results showed that the half-life (t1/2) of A-type EGCG and ECG dimers were 116.37 min and 33.04 min, respectively, and the maximal concentration in plasma was 32.81 μg/mL and 55.59 μg/mL, respectively. It was found that two dimers were firstly experienced by quinone methide (QM) fission to form the EGCG and ECG analogue, and the phase II metabolites were generated subsequently. The main metabolites in plasma and urine were glucuronidation and sulphation derivatives. In addition, small molecule weight of phenolic acids were detected in urine.

RP-HPLC-UV Method for Simultaneous Quantification of Second Generation Non-Steroidal Antiandrogens Along with their Active Metabolites in Mice Plasma: Application to a Pharmacokinetic Study.

A simple, specific and reproducible high-performance liquid chromatography (HPLC) assay method has been developed and validated for the quantitation of second generation antiandrogens and their active metabolites namely apalutamide, enzalutamide, N-desmethylenzalutamide (active metabolite of enzalutamide), darolutamide and ORM-15341 (active metabolite of darolutamide) in mice plasma. The method involves extraction of apalutamide, enzalutamide, N-desmethylenzalutamide, darolutamide and ORM-15341 along with internal standard (IS) from 100 µL mice plasma through a simple protein precipitation process. The chromatographic analysis was performed on a Waters Alliance HPLC system using a gradient mobile phase (comprising 10 mM ammonium acetate and acetonitrile in a flow-gradient) and X-Terra Phenyl column. The UV detection wave length was set at λmax 250 nm. Apalutamide, enzalutamide, N-desmethylenzalutamide, darolutamide and ORM-15341 and the IS eluted at 13.6, 11.4, 9.68, 6.11, 6.93 and 4.69 min, respectively with a total run time of 15 min. Method validation was performed as per regulatory guidelines and the results met the acceptance criteria. The calibration curve was linear over a concentration range of 209 - 5215 ng/mL (r 2=0.998). The intra- and inter-day precisions were in the range of 0.56-13.5 and 1.04-13.9%, respectively. The validated HPLC method was successfully applied to a pharmacokinetic study in mice.

Development and validation of an analytical method for regorafenib and its metabolites in mouse plasma

An analytical method was developed for measuring the effect of OATP1B2 deficiency on plasma levels of the kinase inhibitor regorafenib and its metabolites regorafenib-N-oxide, N-desmethyl-regorafenib-N-oxide, and regorafenib-N-β-glucuronide (RG) in mice. Compounds were separated by liquid chromatography and monitored by a triple quadrupole mass spectrometer in the selected reaction monitoring mode after positive electrospray ionization. All calibration curves were linear in the selected concentration range (R2 ≥ 0.99). The lower limit of quantification was 5 ng/mL for the four analytes. Within-day precisions, between-day precisions, and accuracies were 2.59–6.82%, 3.97–11.3%, and 94.5–111%, respectively. The identification and structure elucidation of RG, isolated from human urine, was performed by NMR. Compared with wild-type mice given regorafenib (10 mg/kg), deficiency of the drug transporter OATP1B2 in vivo had minimal effects on plasma levels of parent drug and the metabolite regorafenib-N-oxide, and N-desmethyl-regorafenib-N-oxide. However, the area under the curve and peak levels of RG were increased by 5.6-fold and 5.1-fold, respectively, in OATP1B2-knockout mice. In conclusion, our analytical method allowed accurate and precise quantitation of regorafenib and its main metabolites in mouse plasma, and is suitable for evaluation of transporter-dependent pharmacokinetic properties of these agents in vivo.

A UPLC-MS/MS method for simultaneous quantification of pairs of oleanene- and ursane-type triterpenoid saponins and their major metabolites in mice plasma and its application to a comparative pharmacokinetic study.

Ilexhainanoside D (IhD) and Ilexsaponin A1 (IsA) are a pair of oleanene- and ursane-type triterpenoid saponins, which are also the main bioactive pharmaceutical ingredients of Ilex hainanensis Merr. with great potential to treat non-alcoholic fatty liver disease (NAFLD). The pharmacokinetics of four representative triterpenoids in mice were investigated in this study, which were IhD, IsA and their major metabolites 3β, 19α-dihydroxyolean-12-ene-24, 28-dioic acid (ID) and Ilexgenin A (IA). A sensitive and accurate UPLC-MS/MS method was developed and validated for the simultaneous quantitative determination of IhD, IsA, ID and IA in control and NAFLD mice plasma after oral administration of the total saponins of I. hainanensis (the contents of IhD and IsA were 41.6% and 54.4%, respectively). The results revealed that the pharmacokinetic behaviors could be changed in NAFLD mice compared with control mice. The area under the plasma drug concentration–time curve and maximum plasma concentrations of IhD and IsA were greatly decreased in the NAFLD mice. However, the main residence time of ID and IA were greatly increased in the NAFLD mice. The results revealed that this method could be used to analyze two pairs of triterpenoid isomers in biological samples.

Simultaneous determination of tilianin and its metabolites in mice using ultra-high-performance liquid chromatography with tandem mass spectrometry and its application to a pharmacokinetic study.

Tilianin is an active flavonoid glycoside found in many medical plants. Data are lacking regarding its pharmacokinetics and disposition in vivo. The objective of this study was to develop a sensitive, reliable and validated ultra-high-performance liquid chromatography with tandem mass spectrometry (UHPLC-MS/MS) method to simultaneously quantify tilianin and its main metabolites and to determine its pharmacokinetics in wild-type and breast cancer resistance protein knockout (Bcrp1-/-) FVB mice. Chromatographic separation was accomplished on a C18 column by utilizing acetonitrile and 0.5 mm ammonium acetate as the mobile phase. Mass spectrometric detection was performed using electrospray ionization in both positive and negative modes. The results showed that the precision, accuracy and recovery, as well as the stability of tilianin and its metabolites in mouse plasma, were all within acceptable limits. Acacetin-7-glucuronide and acacetin-7-sulfate were the major metabolites of tilianin in mouse plasma. Moreover, systemic exposure of acacetin-7-sulfate was significantly higher in Bcrp1 (-/-) FVB mice compared with wild-type FVB mice. In conclusion, the fully validated UHPLC-MS/MS method was sensitive, reliable, and was successfully applied to assess the pharmacokinetics of tilianin in wild-type and Bcrp1 (-/-) FVB mice. Breast cancer resistance protein had a significant impact on the elimination of the sulfated metabolite of tilianin in vivo.

Analysis on metabolites with small molecule of serum in bone marrow suppression model mice with metabolomics method

Bone marrow suppression is a common symptom in patients with malignant tumor after chemotherapy. Studying the changes of metabolites caused by bone marrow depression can provide insights for the diagnosis of bone marrow suppression disease and for the development of drug therapy. Male BalB/C mice were injected with cyclophosphamide to establish a bone marrow suppression model. Gas chromatography-mass spectrometry (GC-MS) with fingerprinting was used to analyze the normal and model mice blood metabolites. Principal component analysis and orthogonal to partial least squares discriminant analysis (OPLS-DA) on metabolomics for data multidimensional statistical analysis was also used. Compared to the normal group in terms of the metabolic profile of bone marrow suppression mice, there were 15 endogenous metabolites in mouse plasma, nine of which were statistically significantly different, including glucose-1-phosphate, 4-nitrophenol, acetanilide, cortisone, nicotinamide, loganin, caffeic acid, linoleic acid and oleic acid (P<0.05). These results indicate that metabolite can be used as an important marker in bone marrow suppression, which can help to reveal the pathogenesis of bone marrow suppression induced by chemotherapy and determine the disease development stage and the effectiveness of follow-up treatment.

Simultaneous determination of gefitinib and its major metabolites in mouse plasma by HPLC-MS/MS and its application to a pharmacokinetics study.

Gefitinib (Iressa) is the first oral EGFR tyrosine kinase inhibitor and it brings benefits to non-small cell lung cancer patients with EGFR mutation. In this study, a simple, rapid and credible high performance liquid chromatography-tandem mass spectrometry method was established and validated for the simultaneous quantification of gefitinib and its main metabolites M523595, M537194, M387783 and M608236 in NSCLC tumor-bearing mouse plasma. Sample extraction was done by protein precipitation using acetonitrile containing dasatinib as the internal standard. The chromatography run time was 6min using an Agilent RRHD SB-C18 column with a gradient of acetonitrile and water (0.1% formic acid, v/v). The mass analysis was performed by a triple quadrupole mass spectrometry in positive multiple reaction monitoring mode. The calibration range was 0.5-100ng/mL for M608236 and 1-200ng/mL for other analytes with the correlation coefficients (r(2))≥0.99. For quality control samples, inter- and intra-assay precision was less than 15% and accuracies ranged from 92.6% to 107.58% for all analytes. The extraction recoveries were in the range of 86-105% and no significant matrix effect was observed. This simple and reproducible high-throughput method was successfully applied to the pharmacokinetic study of gefitinib and its major metabolites in mouse.