Rat Plasma Research Articles

Analysis of correlations between behavioral parameters in the elevated plus maze and the levels of interleukin-1beta in blood plasma in rats

Peripheral cytokines may influence psychoemotional behavior, but the role of interleukin-1beta (IL-1beta) in altering anxiety and motor activity in response to inflammatory activation remains unclear. To clarify this issue, correlations between behavioral parameters in the elevated plus maze (EPM) test and plasma levels of IL-1beta after administration of the proinflammatory stimulus lipopolysaccharide (LPS) in different modes were analyzed in adult male rats. LPS in doses of 0.5 or 5 mg/kg, as well as physiological solution (control), were administered to rats intraperitoneally. The most pronounced behavioral effect 24 hours after a single injection was an endotoxin dose-dependent inhibition of the animals’ motor activity. After a dose of 5 mg/kg, increased anxious behavior was also noted every other day. The behavioral changes caused by the high dose of endotoxin were completely normalized after a week. The behavior of the animals one day after the end of repeated injections of LPS at a lower dose for a week (0.5 mg/kg; once every two days) also did not differ from the control. The inhibition of motor activity after LPS could be due to an increase in the level of IL-1beta in the blood plasma, as indicated by the identified significant negative correlations between IL-1beta and the corresponding behavioral parameters. No significant correlation was found between the peripheral level of IL-1beta and such a classic indicator of anxiety as the percentage of entries into the open arms of the maze. In general, the obtained results allow us to conclude that IL-1beta is an undoubted participant in the mechanism of the transient inhibitory effect of LPS on motor activity.

Metabolic fate of the natural anticancer agent cucurbitacin B: an LC-MS/MS-enabled profiling of its major phase I and II conjugates in vivo.

Cucurbitacin B (CuB) is a natural triterpenoid with diverse pharmacological effects including potent anticancer activity. However, its oral bioavailability is hampered by limited metabolism in vivo. We characterized CuB's in vivo metabolism in rats to uncover bioactive metabolites retaining therapeutic potential, using a robust UHPLC-Q-TOF-MS/MS workflow. This workflow combined molecular networking, fragmentation filtering, and mass defect filtering to identify CuB metabolites in rat urine, plasma, and feces following oral administration. Thirteen metabolites were identified and seven were confirmed. Major phase I transformations involved hydrolysis, reduction, epoxidation, and amination. Phase II conjugation included cysteine, glutathione, glucuronide, and gluconic acid conjugates. Notably, one of the main metabolites formed was the cysteine conjugate CuB-Cys. CuB-Cys maintained similar in vitro antiproliferative activity to CuB on HepG2, MCF-7, and PANC-1 cancer cell lines. However, it demonstrated lower cytotoxicity towards non-cancerous L02 cells, highlighting improved therapeutic selectivity. Mechanistically, CuB-Cys induced greater apoptotic signaling in HepG2 cells than CuB via enhanced caspase activation and disrupted BAX-Bcl-2 balance. This represents the first systematic characterization of CuB's in vivo metabolic pathway. The identification and confirmation of CuB-Cys provide insight for drug development efforts aiming to maintain therapeutic efficacy while reducing toxicity, via metabolite-based approaches. Our findings shed light on strategies for improving CuB's clinical potential.

A systematic UHPLC-Q-TOF-MS/MS-based strategy for analysis of chemical constituents and related in vivo metabolites of Buyang Huanwu decoction

Ethnopharmacological relevanceBuyang Huanwu Decoction (BYHWD), a traditional Chinese medicine, is one of the classic prescriptions for the treatment of ischemic stroke in clinical practice. It has the effects of tonifying qi, activating blood circulation, and promoting meridian circulation. However, its chemical analysis has not been clarified, which greatly hinders its further clinical application. Therefore, it is necessary to clarify the chemical constituents and metabolites profile of BYHWD in vivo. Aim of the studyCharacterizing the chemical basis of BYHWD in vitro, and combing studies of related metabolism in vivo to screen out the potential active components of BYHWD with pharmacological effects in vivo. Materials and methodsTwelve male rats weighed 200 ± 20 each were selected for the experiments. According to the fragmentation of different structural types of components and diagnostic ions, UHPLC-Q-TOF-MS/MS was used to classify and clarify the unknown components of BYHWD and identify the material basis of BYHWD in vitro. Then, rat plasma, tissues, feces, and urine were collected for analysis. Based on the similarity of MS responses (accurate molecular weight and secondary fragmentation) and chromatographic behavior (retention time), the in vivo prototype and metabolites were analyzed. Through the phase I and phase II metabolism law, a metabolite library was established to analyze the prototype-matched metabolites. ResultsA total of 121 in vitro compounds and 55 in vivo prototypes of BYHWD were identified, corresponding to 123 matched prototypes. It was mainly composed of flavonoids, triterpene saponins, nucleosides and lactones both in vitro and in vivo. Quercetin, ligustilide, astragaloside IV, calycosin, paeoniflorin and ferulic acid were the main prototypes and metabolites in plasma and urine. ConclusionQuercetin, ligustilide, astragaloside IV, calycosin, paeoniflorin and ferulic acid were the main active ingredients of BYHWD.

A novel quantitative method for the determination of 10B-carrier boronophenylalanine in rat plasma by UHPLC-MS/MS and comparison with ICP-MS

L-Boronophenylalanine (BPA), a widely used 10B carrier for clinical boron neutron capture therapy (BNCT), was quantified in rat plasma through a simple, effective and stable ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method. Chromatographic separation was performed on an ACQUITY UPLC HSS T3 (100 mm × 2.1 mm, 1.8 μm) column with the mobile phase of 0.5 % formic acid aqueous solution and acetonitrile. For the detector, the m/z ion pairs used for quantification were 209.1→120.1 for BPA and 210.1→120.1 for internal standard in a positive mode by electrospray ionization (ESI) using multiple reaction monitoring (MRM). The method is specific and robust with rare affection by endogenous substances in the matrix. A good linear relationship was observed over 80–80000 ng/mL (r2 = 0.9993). The values of inter- and intra-day accuracy and precision were within the acceptance criteria of ±15 %. BPA was found to be stable under different experimental conditions. This developed method was successfully applied on a pharmacokinetic experiment on Sprague-Dawley rats (intravenous injection, 125 mg/kg) and a comparation between UHPLC-MS/MS and ICP-MS for BPA was carried out.

PEGylation of New Thrombin Inhibitor Peptide Ultravariegin for Prolonged In Vivo Circulation and Enhanced Antithrombotic Effects.

Anticoagulant therapy is commonly used to prevent and treat arterial and venous blood clots in patients with cardiovascular disease, cerebrovascular disease, and cancer. Venous blood clots are the third leading cause of cardiovascular death following acute coronary artery disease and stroke. There is a significant need for effective anticoagulant therapy with minimal risk of bleeding. Variegin and its variants are a new type of antithrombin peptide that has shown promising results in preclinical studies. Variegin and its best variant, ultravariegin (UV), can more effectively inhibit blood clot formation while causing less bleeding than traditional medications such as heparin and bivalirudin. However, the short lifespan of UV remains a limitation for its use in clinical settings. PEGylation, a method of conjugating poly(ethylene glycol) (PEG) chains to peptides or drugs, may help improve the effectiveness of UV by extending its circulation time in the body. In this study, UV was PEGylated using maleimide-PEG5k and 10k. The impact of PEGylation on the antithrombin activity of UV was assessed in vitro and ex vivo in rat and rabbit plasma, showing minimal effects on the efficacy. In vivo studies in rats and rabbits revealed that PEGylated UV had a longer half-life and greater anticoagulant effects than unmodified UV did, especially when it was administered subcutaneously. PEGylation significantly extended the half-life of UV in rabbits, resulting in sustained anticoagulant effects for up to 4 days. This demonstrated that increasing the size of UV and shielding it with PEG could reduce clearance by the kidneys and prolong its circulation time. The improved half-life and antithrombin activity of PEGylated UV make it a more favorable choice for anticoagulant therapy.

Impact of calcium-reinforcement on stability, bioavailability, and bioactivity of quercetin-loaded zein/alginate-pectin nanoparticles.

Cationic calcium ions can crosslink anionic alginate and pectin molecules. It was hypothesized that calcium crosslinking would improve the stability and functionality of biopolymer nanoparticles consisting of zein cores coated by alginate-pectin shells. The effects of calcium ion addition on the structural, physicochemical, and gastrointestinal properties of quercetin-loaded zein/alginate-pectin nanoparticles were therefore investigated. The nanoparticles remained stable to aggregation at calcium ion concentrations of 9 mmol/L or less but aggregated and sedimented at higher concentrations. Calcium ion reinforcement increased the particle dispersion stability even at NaCl concentrations up to 1.4 molL-1. The presence of the calcium ions also reduced quercetin release during the early stages of simulated gastrointestinal digestion but increased its release during the later stages. The relatively high release (56.1%) of quercetin from the calcium-reinforced nanoparticles after digestion resulted in higher intracellular antioxidant activities. The pharmacokinetics of the encapsulated quercetin was measured after its oral administration to rats. The maximal concentration (Cmax) of quercetin in rat plasma for calcium-reinforced nanoparticles was 6.1% higher than non-reinforced nanoparticles; the half-life (t1/2) increased by 17.5%, and the mean retention time (MRT) was 10.0% higher (P < 0.05). These results suggest that calcium ion addition improved the performance and bioavailability of nutraceutical-loaded biopolymer nanoparticles. This might find application in the food and beverage industry. © 2024 Society of Chemical Industry.

Development of a Versatile High-through-put Oligonucleotide LC-MS Method to Accelerate Drug Discovery.

Liquid chromatography-mass spectrometry (LC-MS) is an effective tool for high-throughput quantification of oligonucleotides that is crucial for understanding their biological roles and developing diagnostic tests. This paper presents a high-throughput LC-MS/MS method that may be versatilely applied for a wide range of oligonucleotides, making it a valuable tool for rapid screening and discovery. The method is demonstrated using an in-house synthesized MALAT-1 Antisense oligonucleotide (ASO) as a test case. Biological samples were purified using a reversed liquid-liquid extraction process automated by a liquid handling workstation and analyzed with ion-pairing LC-MS/MS. The assay was evaluated for sensitivity (LLOQ = 2nM), specificity, precision, accuracy, recovery, matrix effect, and stability in rat cerebrospinal fluid (CSF) and plasma. Besides some existing considerations such as column selection, ion-pairing reagent, and sample purification, our work focused on the following four subtopics: 1) selecting the appropriate Multiple Reaction Monitoring (MRM) transition to maximize sensitivity for trace-level ASO in biological samples; 2) utilizing a generic risk-free internal standard (tenofovir) to avoid crosstalk interference from the oligo internal standard commonly utilized in the LC-MS assay; 3) automating the sample preparation process to increase precision and throughput; and 4) comparing liquid-liquid extraction (LLE) and solid-phase extraction (SPE) as sample purification methods in oligo method development. The study quantified the concentration of MALAT-1 ASO in rat CSF and plasma after intrathecal injection and used the difference between the two matrices to evaluate the injection technique. The results provide a solid foundation for further internal oligonucleotide discovery and development.

Development of a UHPLC-MS/MS Method for the Determination of Moxidectin in Rat Plasma and Its Application in Pharmacokinetics.

The aim of the present study was to establish a simple and reliable ultra-high-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method and apply it for the determination of pharmacokinetics of moxidectin-loaded microspheres (MOX-MS) in rats. Plasma samples were processed using a simplified liquid-liquid extraction method and were separated using an Agilent Zorbax Eclipse Plus C18 column (50 mm × 2.1 mm, 1.8 μm) with a mobile phase consisting of a 10 mM ammonium formate solution with 0.1% formic acid (A) and acetonitrile (B) at a flow rate of 0.4 mL/min for 5 min. Avermectin B1a was used as an internal standard (IS). The sample was injected at a volume of 10 μL with a column temperature of 35 °C and detected in a positive ion mode. A good linear response across the concentration range of 1.00-200 ng/mL (r2 > 0.99) and a lower limit of quantification (LLOQ) of 1.00 ng/mL were achieved. The extraction recovery of moxidectin exceeded 94.1%, the matrix effect was between 91.2% and 96.2%, the accuracy ranged from 100.1 to 103.6%, and the relative standard deviation (RSD) did not exceed 15% for the intra- and inter-day accuracy and precision. The pharmacokinetic results showed that MOX-MS significantly decreased Cmax, prolonged T1/2, and improved bioavailability. The developed method significantly reduced the assay volume, shortened detection time, simplified sample processing methods and saved assay costs, which may contribute to the development of the new antiparasitic drug.

Simple simultaneous analysis of various cardiovascular drug mixtures with vincamine: comparative eco-friendly assessment

The development of two eco-friendly analytical methods for the simultaneous determination of eight cardiovascular drugs; hydrochlorothiazide (HCT), captopril (CPL), lisinopril (LSP), valsartan (VAL), atorvastatin (ATR), bisoprolol (BSL), amlodipine (AML) and carvedilol (CVL); alongside with the nutraceutical vincamine (VIC) is essential for sustainable pharmaceutical analysis. This study explores the application of Micellar Electro Kinetic Chromatography (MEKC) and High-Performance Liquid Chromatography (HPLC) for this purpose. In MEKC method, the separation was done using fused silica capillary (41.5 cm × 50 µm id) and a back ground electrolyte consisting of 50 mM borate buffer (pH 9) containing 50 mM sodium lauryl sulphate (SLS) and 10% organic modifier (Acetonitrile). In HPLC method, separation was performed on a ZORBAX Extend-C18 (4.6 × 250 mm, 5 µm) column, using a gradient mobile phase consisting of 50 mM phosphate buffer pH 3 and methanol. Both methods attained good linearity (r ≥ 0.9996) with low values of LOD and LOQ. Both methods were successfully applied in the determination of co-administered single, binary and ternary dosage form of the studied drugs. Moreover, application of various combinations of co-administered dosage forms was achieved in rat plasma, confirming the applicability of these methods in different matrices. The use of micellar solutions in MEKC enhances separation efficiency while reducing the need for organic solvents, aligning with green chemistry principles. HPLC methods were optimized using environmentally benign solvents, ensuring reduced toxicity and waste production. The methodologies were evaluated through green, white, and blue metrics to ensure comprehensive sustainability, considering ecological impact, safety, and practical efficiency. These methods were not only cost-effective and time-saving but achieved high efficiency, sensitivity, and reproducibility making them ideal for routine use in pharmaceutical analysis.

Comparative profiling of the absorbed compounds and metabolites, and pharmacokinetic studies of Danshen-Chuanxiong herb pair in rat plasma and brain using liquid chromatography-tandem mass spectrometry

Danshen-Chuanxiong (DS-CX) was a classic herb pair commonly used to treat ischemic stroke. Nevertheless, the metabolic conversion and pharmacokinetic behavior of DS-CX in vivo remains unclear. This work aimed to reveal the in vivo metabolic behavior of DS-CX through establishing metabolic profiles and performing multicomponent pharmacokinetics analysis. The mass defect filtering (MDF) strategy integrated with UHPLC-QTOF-MS was firstly developed to characterize the metabolites of DS-CX in rats’ plasma and brain. Moreover, a sensitive UHPLC-QQQ-MS method was utilized to perform the comparative pharmacokinetic studies of major active ingredients of DS-CX in rats’ plasma. A total of 111 exogenous compounds (29 prototype compounds and 82 metabolites) were identified in rat biological samples. The major metabolic pathways were hydroxylation, methylation, deoxidation, dehydration, hydrogenation, demethylation, hydrolysis, decarboxylation and glucuronidation binding reactions. According to the results of metabolites profiling, sixteen active compounds (8 phenolic acids, 5 phthalides and 3 tanshinones) were selected as markers for further comparative pharmacokinetics study. Compared with the oral administration of DS or CX alone, the higher Cmax of salvianolic acid B, crytotanshinone and tanshinone IIA; the shorter Tmax of lithospermic acid, rosmarinic acid and tanshinone IIA; as well as the higher AUC0−∞ of ferulic acid, rosmarinic acid, salvianolic acid B, senkyunolide I and crytotanshinone, could be found after co-administration of DS-CX (P < 0.05). This study provided the overall knowledge of metabolites profiling of DS-CX in vivo, which would help to understand the effective material basis and promote the clinical application of DC-CX herb pair.

Determination of the extremolyte ectoine in plasma and a pharmacokinetic study in rats by a validated and BAGI-evaluated UPLC-MS/MS method

Ectoine (ECT) has recently gained considerable interest in the healthcare sector due to its promising therapeutic benefits in a variety of human disorders. This research aimed to quantify the ECT plasma level in rats by creating and optimizing a sensitive and validated UPLC-MS/MS method. Prior to analysis, ECT extraction from the plasma samples was conducted via a protein precipitation procedure, using hydroxyectoine as an internal standard (IS). A 1.7 μm UPLC C8 column (100 mm × 2.1 mm) was selected for the chromatographic separation, using a gradient mobile phase consisting of acetonitrile and 0.05% formic acid. The electrospray ionization mass spectrometry (ESI-MS) was used to detect ECT in the positive ion mode. To determine the specific precursor and the product ions of ECT, multiple reaction monitoring (MRM) methods were carried out. The selected ion pair of ECT was 143.1 > 97 and 159.1 > 113.13 for the IS. The ECT’s linearity range in rat plasma was found to be 1-1000 ng/mL, with a recovery rate of 96.48–97.37%. Consistent with FDA guidelines for bio-analytical method validation, the suggested method was validated. The method was efficiently employed to quantify the studied drug in spiked rat plasma with good accuracy and precision with no significant matrix effects. Furthermore, it was effectively used to investigate the pharmacokinetic behavior of ECT in rats after a single oral dose of 30 mg/kg.

Tumoral Pharmacokinetic-Pharmacodynamic Simulation of Doxorubicin-Encapsulated Polymeric Micelles Using a Tissue-Isolated Tumor Perfusion System.

Pharmacokinetic (PK) elucidation of polymeric micelles delivering anticancer drugs is crucial for accurate antitumor PK-pharmacodynamic (PK-PD) simulations. Particularly, establishing a methodology to quantify the tumor inflow and outflow of anticancer drugs encapsulated in polymeric micelles is an essential challenge. General tumor biodistribution experiments are disadvantageous in that inflow quantification is easy, but outflow quantification is challenging. We addressed this issue by proposing a quantification method that combines a tissue-isolated tumor perfusion system with microdialysis. This method aims to determine tumoral drug inflow and outflow by quantifying the drugs released from the polymeric micelles via a tumor-embedded microdialysis probe and perfusate, respectively. Furthermore, we evaluated the feasibility of this method by perfusing pH-sensitive polyethylene glycol-poly(aspartate-hydrazone-doxorubicin/phenylalanine)n (PPDF-Hyd-DOX) in a tissue-isolated tumor perfusion system, and we quantified tumor inflow and outflow released DOX. Based on the quantitative results, we performed compartmental analyses by incorporating the gamma-distributed delay function and calculated the PK rate constants. These parameters were input into a tumor-bearing rat compartment model for ex vivo-in vivo extrapolation (EVIVE) of the rat plasma PPDF-Hyd-DOX concentrations and simulated intratumorally released DOX concentrations. The simulation profiles demonstrated a good fit with the Walker 256 intratumoral released DOX concentration profiles previously reported. This EVIVE-PK model was coupled with the threshold natural-growth tumor PD model, and PK-PD analysis was performed. This model exhibited a better fit to the tumor weight profile of Walker 256-bearing rats treated with PPDF-Hyd-DOX than that of our previously reported PK-PD model. Thus, EVIVE, based on a tissue-isolated tumor perfusion system with microdialysis, is a promising approach for the PK-PD simulation of polymeric micelle anticancer therapy.

Synthesis, Biological Evaluation and Reversal of Sulfonated Di- and Triblock Copolymers as Novel Parenteral Anticoagulants.

Despite targeting different coagulation cascade sites, all Food and Drug Administration-approved anticoagulants present an elevated risk of bleeding, including potentially life-threatening intracranial hemorrhage. Existing studies have not thoroughly investigated the efficacy and safety of sulfonate polymers in animal models and fully elucidate the precise mechanisms by which these polymers act. The activity and safety of sulfonated di- and triblock copolymers containing poly(sodium styrenesulfonate) (PSSS), poly(sodium 2-acrylamido-2-methylpropanesulfonate) (PAMPS), poly(ethylene glycol) (PEG), poly(sodium methacrylate) (PMAAS), poly(acrylic acid) (PAA), and poly(sodium 11-acrylamidoundecanoate) (PAaU) blocks are synthesized and assessed. PSSS-based copolymers exhibit greater anticoagulant activity than PAMPS-based ones. Their activity is mainly affected by the total concentration of sulfonate groups and molecular weight. PEG-containing copolymers demonstrate a better safety profile than PAA-containing ones. The selected copolymer PEG47-PSSS32 exhibits potent anticoagulant activity in rodents after subcutaneous and intravenous administration. Heparin Binding Copolymer (HBC) completely reverses the anticoagulant activity of polymer in rat and human plasma. No interaction with platelets is observed. Selected copolymer targets mainly factor XII and fibrinogen, and to a lesser extent factors X, IX, VIII, and II, suggesting potential application in blood-contacting biomaterials for anticoagulation purposes. Further studies are needed to explore its therapeutic applications fully.

The Effect of Cinnamomum Burmanii Ethanol Extract on Isoniazid-induced Serum Levels of Serum Glutamate Piruvate Transaminase (SGPT) Wistar Strain Male Rats

Hepatotoxicity can be caused by excessive drug use, triggered by increased oxidative stress which is considered as the initial mechanism of Drug Induced Liver Injury (DILI). One of the causes of DILI is Isoniazid. One of the plants acting as a hepatoprotector in protecting liver cells is cinnamon (Cinamommum burmanii). This study aims to determine the effect of 96% ethanolic cinnamon extract as a hepatoprotector against an increase in Alanine Transaminase (ALT) levels induced by isoniazid. The parameters used to assess liver damage were rat plasma ALT. The sample used in this study was rat plasma, which was taken through the retro orbital sinus. This research is an experimental study with a sample of 25 experimental animals divided into five groups, namely negative control (K1), positive control l (K2), and experimental group with cinnamon ethanolic extract at a dose of 100 mg/kgbw (K3), 200 mg/kgbw(K4), and 400 mg/kgbw(K5). The research used a post-test-only control group design. The results were analyzed using the One-way ANOVA test followed by the post hoc Tukey test. The results proved that cinnamon ethanolic extract at doses of 100 mg/kgbw (P = 0.029), 200 mg/kgbw (P = 0.001), and 400 mg/kgbw (P = 0.000) was effective in reducing plasma SGPT levels in isoniazid-induced rats when compared with the positive control group (K2). The most effective dose was at 200 mg/kgbw (K4). Thus, this proves that all of the doses in experimental groups have a hepatoprotective effect against isoniazid-induced liver damage. Keywords: Alanine Transaminase (ALT), cinnamon ethanol extract, Drug Induced Liver Injury (DILI), isoniazid

Design, Synthesis, and Biological Evaluation of New Improved Ferrostatin-1 Derived Ferroptosis Inhibitors.

Ferrostatin-1 (Fer-1), a first potent ferroptosis inhibitor, faces limitations in clinical use due to its low potency and metabolic instability. This study introduces a series of novel Ferrostatin-1 analogs designed to enhance plasm stability. Our design strategy focused on the modification of the 3-NH2 of Fer-1 with benzenesulfonyl groups, resulting in analogs 9-25. Biological evaluation revealed that compound 18, with an EC50 value of 0.57 µM, outperformed Fer-1 in inhibiting ferroptosis. It reduced intracellular ferrous ion accumulation, lipid peroxidation, and restored glutathione (GSH) and glutathione peroxidase 4 (GPX4) levels effectively. Moreover, compound 18 exhibited favorable solubility and remarkable metabolic stability in rat plasma. These results position compound 18 as a promising candidate for developing therapeutics against ferroptosis-related diseases.

4D-printed shape-programmable [H+]-responsive needles for determination of urea

Four-dimensional printing (4DP) technologies are revolutionizing the fabrication, functionality, and applicability of stimuli-responsive analytical devices. More practically, 4DP technologies are effective in fabricating devices with complex geometric designs and functions, and the degree of shape programming of 4D-printed stimuli-responsive devices can be optimized to become a reliable analytical strategy. Although shape-programming modes play a critical role in determining the analytical characteristics of 4D-printed stimuli-responsive sensing devices, the effect of shape-programming modes on the analytical performance of 4D-printed stimuli-responsive devices remains an unexplored subject. We employed digital light processing three-dimensional printing (3DP) with acrylate-based photocurable resins and 2-carboxyethyl acrylate (CEA)-incorporated photocurable resins for 4DP of the bending, helixing, and twisting needles. Upon immersion in samples with pH values above the pKa of CEA, the electrostatic repulsion among the dissociated carboxyl groups of polyCEA caused swelling of the CEA-incorporated part and [H+]-dependent shape programming. When coupling with the derivatization reaction of the urease-mediated hydrolysis of urea, the decline in [H+] induced shape programming of the needles, offering reliable determination of urea based on the shape-programming angles. After optimizing the experimental conditions, the helixing needles provided the best analytical performance, with the method's detection limit of 0.9 μM. The reliability of this analytical method was validated by determining urea in samples of human urine and sweat, fetal bovine serum, and rat plasma with spike analyses and comparing these results with those obtained from a commercial assay kit. Our demonstration and analytical results suggest the importance of optimizing the shape-programming modes to improve the analytical performance of 4D-printed stimuli-responsive shape-programming sensing devices and emphasize the benefits and applicability of 4DP technologies in advancing the development and fabrication of stimuli-responsive sensing devices for chemical sensing and quantitative chemical analyses.

Pharmacokinetics and related gender difference studies of four active components of Codonopsis Pilosula by LC-MS/MS determination

Ethnopharmacological relevanceCodonopsis pilosula (C. pilosula), commonly known as Dangshen in Chinese, had been used to regulate the immune, digestive, and circulatory systems of human. The reported pharmacokinetic studies on C. pilosula are mainly limited to in vivo profile studies of a single component. It has not been detected simultaneously the in vivo pharmacokinetic profiles of multiple active components as well as related gender difference after oral dosing of the extraction of C. pilosula. Aim of the studyThis study aims to reveal the pharmacokinetic characteristics of the four main active components of C. pilosula after oral dosing of its extraction in rats, and to explain the gender differences in absorption and metabolism. Materials and methodsThe plasma pharmacokinetic characteristics of four main active components of C. pilosula was explored using the established LC-MS/MS method after oral dosing of the extraction of C. pilosula in male and female rats. In vitro intestinal pouch permeability and liver microsome metabolic stability were also observed to classify the possible mechanism of gender difference existed in the pharmacokinetic profiles of the four active components in rats. ResultsFour effective components were absorbed quickly in rats after oral administration of alcoholic extract of C. pilosula (1.36 g/mL, equivalent to 2 g/mL as crude drug), and their exposure order was as follows: Atractylenolide III > Lobetyolin > Tangshenoside I > Syringin. The exposure (AUC) and peak concentration (Cmax) of Atractylenolide III in female rats were much higher than those in male rats, indicating a significant gender difference in pharmacokinetics of Atractylenolide III between female and male animals. With the help of the rat model of intestinal sac in vitro, it was found that Lobetyolin was a hypertonic compound, and both Tangshenoside I and Syringin were compounds with medium permeabiltiy. Notably, the Papp of Atractylenolide III was 3.3 × 10−6 cm/s in male rat intestinal sac assay, while that was 10 × 10−6 cm/s in female rat intestinal sac model, showing a significant gender difference in intestinal permeability (P < 0.05). After the addition of NADPH, the four compounds were reduced in a time-dependent manner, suggesting that CYP450s could catalyze their metabolism. After incubation, the remaining content of Atractylenolide III in the liver microsomes of male and female rats was 27% and 57%, respectively, suggesting slower metabolic rate of in female rat liver microsomes. ConclusionA simple, efficient and reliable LC-MS/MS method for the simultaneous determination of four active index components of C. pilosula, Lobetyolin, Tangshenoside I, Atractylenolide III and Syringin, in rat plasma was established and verified. This method was successfully applied in the pharmacokinetic study after single oral administration of the alcoholic extract of C. pilosula in rats. Gender difference was observed in the pharmacokinetic profile of Atractylenolide III in rats. Intestinal absorption and liver metabolism might be two key factors that resulted in the gender difference in exposure and pharmacokinetics of Atractylenolide III in rats. This study provides supportive data for clinical rational application of C. pilosula in individualized medication therapy.

Flavonoid-rich fraction of Monodora tenuifolia Benth seeds improves antioxidant status in male Wistar rats with streptozotocin-induced Diabetes mellitus

BackgroundOxidative stress is a disruption in the balance between free radicals and the body's natural defenses. This study investigated the modulatory effect of a flavonoid-rich fraction of Monodora tenuifolia seed (FRFMTS) on oxidative stress and antioxidant enzymes in diabetic Wistar rats. The plant seeds were collected, and identified then, hydro-ethanolic extract was obtained with 80 % (v/v) ethanol and partitioned with solvents to obtain the FRFMTS. Diabetes mellitus was induced with a single dose of 40 mg/kg bwt streptozotocin and the rats were treated according to their grouping; group 1: non-diabetic control, groups 2 and 3: non-diabetic treated with 25 mg/kg FRFMTS and 50 mg/kg FRFMTS, respectively. Group 4: diabetic control group, groups 5 and 6: diabetic groups treated with 25 mg/kg and 50 mg/kg FRFMTS, respectively, while group 7: diabetic treated with 6.67 mg/kg metformin. Antioxidant status, oxidative stress biomarkers and activities of antioxidant enzymes were determined in plasma, heart, and kidney tissues using established procedures. ResultsPlasma total antioxidant status (TAS) was increased significantly (p < 0.05) in diabetic rats upon treatment with 25 mg/kg FRFMTS (70.4 %), while 50 mg/kg FRFMTS increased TAS by 70 %. The activities of glutathione peroxidase (GPx), catalase (CAT) and superoxide dismutase (SOD) in the plasma, heart, and kidney of diabetic rats significantly decreased (61.3 %, 58.8 % and 31.4 % respectively) while lipid peroxidation (LPO) was elevated when compared with normal control groups. Flavonoid-rich fraction of M. tenuifolia at 25 mg/kg bwt and 50 mg/kg bwt significantly increased the activity GPx enzyme by 32.3 % and 72 % in plasma, while there was no significant differences in both kidney and heart of diabetic rats when compared with diabetic control groups. The treatment of diabetic rats with 25 mg/kg FRFMTS and 50 mg/kg FRFMTS improved SOD activity in the plasma (51.5 % and 69.8 %), heart (32.3 % and 27.6 %) and kidney (31.4 % and 27.1 %) respectively, and CAT activity in plasma (44.2 % and 57 %), heart (28.6 % and 60 %) and kidney (69.6 % and 76.7 %) respectively. The concentration of malondialdehyde (MDA) in lipid peroxidation assay in the heart and kidney of diabetic rats treated with 25 mg/kg FRFMTS and 50 mg/kg FRFMTS reduced significantly by (62.5 % and 71.1 % in heart), while 50 mg/kg FRFMTS significantly reduced MDA in kidney by 43.8 %. ConclusionsThe results showed that FRFMTS reduces oxidative stress and enhances the activities of antioxidant enzymes in STZ-diabetic Wistar rats. As such, FRFMTS could be useful in the management of oxidative stress-mediated diabetic complications.

B-004 Method Validation of Lactate in the Plasma of Rats

Abstract Background The objective of this study is to validate Lactate (LAC) measurement in rat plasma using the ADVIA 1800 analyzer. Methods LAC was validated in rat plasma using the ADVIA 1800 analyzer. Validation testing included intra-assay and inter-assay precision, accuracy, linearity of dilution, limit of quantitation (LOQ), limit of detection (LOD), reference interval, carry-over, correlation, recovery, and stability. Samples were collected into tubes containing sodium fluoride. Intra-assay precision was determined by analyzing two biological samples and the two quality control levels of Bio-Rad Lyphochek Assayed Chemistry Control 10 consecutive times. Inter-assay precision was determined by analyzing the two quality control levels in duplicate for six days over a 10-day period. Accuracy was calculated using the data obtained from the inter-assay precision testing. For linearity of dilution, Audit MicroControl General Chemistry Linearity Kit and biological samples were diluted and analyzed in duplicate. The LOQ was determined by diluting the calibrator material to produce a value at the low end of the reportable range. Diluted material was tested six times. The LOD was determined by assaying the appropriate blank 10 times. The reference interval was determined by analyzing 21 plasma samples. Correlation between the two ADVIA 1800 analyzers was determined by assaying 10 samples on both analyzers once. The carry-over of the assay was determined by analyzing the high-level quality control material followed by the low-level quality control material. Recovery was determined by analyzing a pair of test samples in duplicate that have been spiked and diluted. The proportional error between the two test samples was calculated. Stability of plasma samples was tested on wet ice, refrigerated, and frozen (at -70°C) for various durations of storage. Results Measurement of LAC in rat plasma met the acceptance criteria for precision, accuracy, linearity of dilution, limit of quantitation, carry-over, recovery, and stability. For intra-assay precision in specimen and control material, the %CV was within ±20%. The inter-assay precision %CV was within ±20%. The total error observed (TEobs) was within ±20% for accuracy and the obtained mean of the control material was within the manufacturer’s acceptable range. For linearity of dilution, the coefficient of determination was ≥ 0.9000, the slope was within 1.00 ± 0.25, and TEobs for each plasma and linearity kit dilution was ≤20%. The LOQ was set at the lowest concentration for which the TEobs was within ±20%. The LOD was calculated by adding the mean concentration obtained and three times the standard deviation. The reference interval was set to the 2.5th to 97.5th percentile interval using Excel software. The carry-over was acceptable at ≤2%. Correlation between the two ADVIA 1800 analyzers was determined to describe any bias in result interpretation. Recovery was acceptable as the proportional error was within ±20%. Stability at all storage conditions was found to be acceptable with a mean %difference within ±20%. Conclusions LAC in rat plasma met the acceptance criteria for all required validation parameters. The ADVIA 1800 analyzer can be used for preclinical studies to test LAC in rat plasma.

Simultaneous Determination of Levo-tetrahydropalmatine and Naltrexone in Rat Plasma by LC-MS/MS and its Application in a Pharmacokinetic Study.

Levo-tetrahydropalmatine and low-dose naltrexone are used in association with reducing cocaine-related cravings, but there are no analytical methods for the quantitative simultaneous analysis of this drug combination. A highly selective and sensitive LC-MS/MS assay was developed and validated to simultaneously quantify l-THP and naltrexone. The analytical method for l-THP offers improved sensitivity compared to previously published methods. The product ion transitions of l-THP and naltrexone were 357.0→193.0 and 342.2→324.1, respectively. Chromatographic separations were performed using a BEH-C18 column by an isocratic elution mode with acetonitrile and 0.1% formic acid in water containing 3 mM ammonium acetate. L-THP and naltrexone were extracted from rat plasma using a liquidliquid extraction method. For l-THP and naltrexone, the assay displayed good linear response over a concentration range of 0.5-1000 ng/mL and 0.25-500 ng/mL, respectively. The intra-day accuracy of the method for l-THP and naltrexone was 93.8-101% with a precision (%CV) of 2.43-8.15% and 93.4-108% with a precision of 3.47-8.22%. The inter-day accuracy for l-THP and naltrexone was 91.2-102% with a CV of 2.46-8.06% and 91.5-97.8% with a CV of 3.29-8.92%, respectively. The assay has been used for pharmacokinetic studies of l-THP and naltrexone in the rat.