LC-MS Method Research Articles

Construction of an electrochemical aptamer-based sensors for rapid quantification of the anticancer drug imatinib in blood to improve drug bioavailability at microdoses

Imatinib (Ima), as a commonly used anticancer drug for the clinical treatment of leukemia and gastrointestinal mesenchymal stromal tumour, requires timely monitoring of patients' blood concentration to ensure efficacy while reducing complications and achieving precision medicine due to its narrow therapeutic window (1–5 μM) and the varying sensitivity and resistance of different patients to Ima. However, traditional assays are slow and cumbersome, so improved and innovative platforms for monitoring Ima in the clinic are necessary. In this work, a nanoporous electrochemical aptamer-based (E-AB) sensor was designed for the detection of Ima and imatinib mesylate (Ima-Mes) in blood. Apt-37, a high-affinity and conformationally variable aptamer, was screened by molecular docking simulation calculations and circular dichroism (CD) for the construction of the E-AB sensor. The sensor detected Ima and Ima-Mes in the range of 0.1 μM-1 mM, and the recoveries in spiked blood samples were in the range of 70.7 %-104.6 % and 74.8 %-113.9 %, respectively. The precision and accuracy of the E-AB sensor for measuring Ima-Mes concentration in blood was similar to the standard LC-MS method. These results demonstrate that the developed E-AB sensor is an effective tool for rapid monitoring of Ima and Ima-Mes in blood.

Comparative Analysis on Polyphenolic Composition of Different Olive Mill Wastewater and Related Extra Virgin Olive Oil Extracts and Evaluation of Nutraceutical Properties by Cell-Based Studies.

This study reports a comparative analysis of the polyphenolic composition and nutraceutical properties of different olive mill wastewater (OMWW) and corresponding extra virgin olive oil (EVOO) extracts. Specifically, four OMWWs and corresponding EVOOs from cultivars Frantoio (A) and Leccino (B) obtained from different crushing seasons (early-stage (A1 and B1) and later-stage (A2 and B2)) were analyzed. Employing HPLC-DAD and LC-MS methods, the primary polyphenol content was identified and quantified. Overall, OMWW extracts showed a greater polyphenolic content compared to corresponding EVOO extracts, with OMWW B1 displaying the highest levels of polyphenols. The antiradical properties of extracts towards radical species (DPPH, ABTS, O2-, and HOCl-) were demonstrated in vitro, revealing a correlation with polyphenolic content. In fact, OMWW B1 and B2 demonstrated the strongest antiradical activity. Exploring nutraceutical properties of OMWWs, the intestinal permeation of the main polyphenols in a co-culture model (Caco-2 and HT29-MTX cell lines) was assessed, with tyrosol achieving a permeation of almost 60%. Furthermore, the involvement in the inflammation process has been evaluated in cell studies on THP1-derived macrophages by immunocytochemistry, demonstrating that OMWW B1 may exert an anti-inflammatory effect by modulating specific phenotype expression on macrophages. In conclusion, this study provides evidence supporting the reuse of OMWWs as a source of polyphenols with nutraceutical properties.

Sensitive and rapid detection of bisphenol A using signal amplification nanoparticles loaded with anti-bisphenol A monoclonal antibody

Bisphenol A has been reported to be a ubiquitous contaminant, and exposure to this compound can lead to adverse effects in human health. In the study, monoclonal antibody against BPA (anti-BPA mAb) with high affinity (3.74 × 109 L/mol) secreted by cell line 2E3 was successfully screened. Inspired by the signal amplification of nanoparticles, anti-BPA mAbs were labeled with nano-materials including colloidal gold (AuNP) and gold nanoflowers (AuNF) for preparation of immunoprobes and AuNP-/AuNF-based test strips. The developed AuNP- and AuNF-based test strips achieved the rapid and sensitive detection of BPA within 10 min, with the limit of detection (LOD) of 25 μg/mL and 3.125 μg/mL, respectively. The detection result in BPA spiked samples measured by the proposed methods was consistent with that detected by LC-MS method. The preparation process of as-prepared test strip is time-saving and considered as ideal candidates method for rapid screening BPA in real samples.

Prostaglandin Metabolites Analysis in Urine by LC-MS/MS.

The analysis of prostaglandin urinary metabolites is valuable for assessing physiological processes and identifying disease biomarkers. These metabolites, derived from the breakdown of prostaglandins, offer a noninvasive means to gauge prostaglandin production and its potential impact on various biological functions. We report an efficient LC-MS method of four commonly analyzed prostaglandin urinary metabolites including tetranor-PGEM (derived from PGE2), tetranor-PGDM, 11β-PGF2α, and 2,3-dinor-11β-PGF2α (derived from PGD2). Each metabolite possesses distinct characteristics and clinical applications, collectively contributing to our understanding of prostaglandin-mediated pathways.

Increasing plant protein in the diet induces changes in the plasma metabolome that may be beneficial for metabolic health. A randomized crossover study in males

Background & aimDietary shifts replacing animal protein (AP) with plant protein (PP) sources have been associated with lowering cardiometabolic risk (CMR), but underlying mechanisms are poorly characterized. This nutritional intervention aims to characterize the metabolic changes induced by diets containing different proportions of AP and PP sources in males at CMR. DesignThis study is a 4-week, crossover, randomized, controlled-feeding trial in which 19 males with CMR followed two diets providing either 36% for the control diet (CON-D) or 64% for the flexitarian diet (FLEX-D) of total protein intake from PP sources. Plasma nontargeted metabolomes (LC-MS method) were measured in the fasted state and after a high-fat challenge meal at the end of each intervention arm. Lipogenesis and protein synthesis fluxes, flow-mediated dilatation (FMD) and gluco-lipidic responses were assessed after the challenge meal. Data were analyzed with mixed models, and univariate and multivariate models for metabolomics data. ResultsIn both arms CMR improved with time, with decreased body weight (-0.9%), insulin resistant (-34%, HOMA-IR, Homeostatic Model Assessment for Insulin Resistance) and low-density lipoproteins (LDL)-cholesterol (-11%). Diet had no effect on FMD or metabolic fluxes, but a trend was observed for a stronger decrease in HOMA-IR and lower postprandial glucose after FLEX-D vs CON-D. The abundance of 21 and 37 metabolites differed between diets at fasted and fed states, respectively, including food intake biomarkers of AP (methylhistidine, eicosapentaenoic acid, hydroxyprolines) and PP sources (trigonelline, N-acetyl-ornithine). In fasted or fed states, indole acrylic acid and indole propionic acid, both products of tryptophan catabolism, were higher after FLEX-D vs CON-D, while the indispensable amino acids-related metabolites alpha-aminoadipic acid, hydroxymethylbutyric acids and propionylcarnitine were lower. In the postprandial state only, the ω-oxidation products dodecanedioic, tetradecanedioic and hexadecanedioic acids were higher after FLEX-D vs CON-D. ConclusionsDespite little changes in risk factors after 4 wk, this study evidenced subtle metabolic adaptations in amino acids and lipid metabolism and gut microbiota activity occurring after higher PP source intake that may be beneficial to CMR. ClinicalTrials.gov study identifierNCT04236518 Clinical Trial RegistryNCT04236518 on ClinicalTrials.gov

Phytochemical Analysis and In Vitro Effects on Isolated Murine Lymphocytes and Macrophages of Polymeric Micelles Loaded with Cycloartane Saponin

Triterpenoid saponins from the Astragalus species possess valuable effects (cytotoxic, adjuvant, hepatoprotective, neuroprotective, antiviral, etc.). Some also have immunomodulatory activities. Astragalus glycyphyllos is distributed in Bulgaria and mainly accumulates cycloartane saponins. From the overground parts of the species, a triterpenoid cyloartane-type saponin (AGOS3) was isolated by different chromatographic techniques. A quantitative LC-MS method for the determination of the saponin was developed and validated. Further, the saponin was loaded in copolymeric micelles based on triblock copolymers of polyethylene oxide and polypropylene oxide (Pluronics). The LC-MS method was applied on the developed micelles to determine their loading degrees. Afterwards, the possible pharmacological effects of free and encapsulated in polymeric nanoparticles of triterpenoid saponin (1, 5, 10, 25, 50, and 100 µg/mL) were evaluated in isolated murine macrophages and lymphocytes in vitro. Free AGOS3 stimulated proliferation only at the highest tested concentrations (50–100 µg/mL), and the effect was more evident in isolated macrophages. Interestingly, AGOS3-loaded polymeric micelles caused concentration dependency and statistically significant increases in the proliferation of both isolated lymphocytes and macrophages, even at a lower concentration (10 µg/mL). These results could serve as the basis for further research on the immunomodulatory effect of this saponin.

Quantitation of trace polyfunctional thiols in wine by liquid chromatography quadrupole Orbitrap high-resolution mass spectrometry in parallel reaction monitoring

Polyfunctional thiols are key contributors to wine aroma due to their extremely low odor thresholds, and their quantitative analysis remains challenging as a result of their ultratrace concentrations and high reactivity. This work presents the first method based on ultra-high-performance liquid chromatography (UHPLC) coupled to quadrupole Orbitrap high-resolution mass spectrometry (HRMS) in parallel reaction monitoring (PRM) mode for quantifying thiols at nanograms per liter (ng/L) levels in wine. Thiols in wine were derivatized using 4,4′-dithiodipyridine and isolated by liquid-liquid extraction. This protocol allowed rapid sample preparation with minimum labor input and low consumable expenses. Instrumental analysis was conducted using UHPLC–quadrupole Orbitrap HRMS in PRM mode. Twenty thiol analytes, including literature-known, recently identified, and novel thiols were selected and validated by the optimized method in three wine matrices. The overall analytical performances demonstrated by this method were equivalent, and in most cases, greater than many previously developed GC–MS or LC–MS methods. The validated method was applied to analyze a selection of wines in which 12 thiols were quantified.

Mitigating In-Column Artificial Modifications in High-Temperature LC-MS for Bottom-Up Proteomics and Quality Control of Protein Biopharmaceuticals.

Elevating the column temperature is an effective strategy for improving the chromatographic separation of peptides. However, high temperatures induce artificial modifications that compromise the quality of the peptide analysis. Here, we present a novel high-temperature LC-MS method that retains the benefits of a high column temperature while significantly reducing peptide modification and degradation during reversed-phase liquid chromatography. Our approach leverages a short inline trap column maintained at a near-ambient temperature installed upstream of a separation column. The retentivity and dimensions of the trap column were optimized to shorten the residence time of peptides in the heated separation column without compromising the separation performance. This easy-to-implement approach increased peak capacity by 1.4-fold within a 110 min peptide mapping of trastuzumab and provided 10% more peptide identifications in exploratory LC-MS proteomic analyses compared with analyses conducted at 30 °C while maintaining the extent of modifications close to the background level. In the peptide mapping of biopharmaceuticals, where in-column modifications can falsely elevate the levels of some critical quality attributes, the method reduced temperature-related artifacts by 66% for N-terminal pyroGlu and 63% for oxidized Met compared to direct injection at 60 °C, thus improving reliability in quality control of protein drugs. Our findings represent a promising advancement in LC-MS methodology, providing researchers and industry professionals with a valuable tool for improving the chromatographic separation of peptides while significantly reducing the unwanted modifications.

Development and validation of a LC-MS method for the quantification of microcystins in fish for their monitoring in environmental and food context

Cyanobacterial blooms in aquatic ecosystems can result in the accumulation of cyanotoxins in other organisms. Fish can potentially accumulate large amounts of cyanotoxins through their direct interaction with contaminated water and the ingestion of cyanobacterial cells. As a human food source, this can result in a potential health risk. Investigating and monitoring this risk requires analytical methods sufficiently robust to be suitable for different fish species. This study, therefore, aimed to optimize and validate an UHPLC-MS/MS method for the quantification of eight microcystin congeners and nodularin in five different fish species. The limits of detection and quantification were 1 and 3 μg kg−1, respectively. The linearity of the method was very good, with calculated R2 values above 0.99. The mean recovery of the method was within the acceptable range of 70.0%–120.0 %, the repeatability was not higher than 12.6 %, and the highest intra-laboratory reproducibility was 18.7 %. The estimated range for the expanded measurement uncertainty was between 29 % and 58 %. Moreover, multiple fish from Flemish waters were analyzed to show the method's applicability. Multiple microcystin congeners were detected (up to 88.3 μg kg−1 sum of analyzed microcystins) in the liver and viscera of perch and sander. Small amounts of MC-LR and MC-YR (up to 6.1 μg kg−1) were also detected in perch muscle tissue.

Standardization via Post Column Infusion-A Novel and Convenient Quantification Approach for LC-MS/MS.

Mass spectrometry (MS) is a widely used analytical technique including medical diagnostics, forensic toxicology, food and water analysis. The gold standard for quantifying compounds involves using stable isotope-labeled internal standards (SIL-IS). However, when these standards are not commercially available, are prohibitively expensive, or are extremely difficult to synthesize, alternative external quantification techniques are employed. We hereby present a novel, convenient and cheap quantification approach-quantification via post column infusion (PCI). As a proof of concept, we demonstrated PCI quantification for the immunosuppressant tacrolimus in whole blood using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The validation results met the criteria according to the guideline on bioanalytical method validation of the European Medicine Agency (EMA), achieving imprecisions and inaccuracies with coefficient of variation and relative bias below 15%. Anonymized and leftover whole blood samples from immunosuppressed patients receiving tacrolimus were used for method comparison (PCI quantification vs. conventional internal standard (IS) quantification). Both methods showed strong agreement with a Pearson correlation coefficient of r = 0.9532. This novel PCI quantification technique (using the target analyte itself) expands the quantification options available in MS, providing reliable results, particularly when internal standards are unavailable or unaffordable. With the current paper, we aim to demonstrate that our innovative PCI technique has great potential to overcome practical issues in quantification and to provide guidance on how to incorporate PCI in existing or new LC-MS methods. Moreover, this study demonstrated a more convenient method for correcting matrix effects in comparison to alternative PCI techniques.

Cultivated Winter-Type Lunaria annua L. Seed: Deciphering the Glucosinolate Profile Integrating HPLC, LC-MS and GC-MS Analyses, and Determination of Fatty Acid Composition.

Lunaria annua L. (Brassicaceae) is an ornamental plant newly identified in Europe as a promising industrial oilseed crop for its valuable very-long-chain monounsaturated fatty acids (MUFAs), especially erucic acid (EA) and nervonic acid (NA). L. annua seeds were obtained from annual winter-type plants selected and cultivated in Northern France. Using a systematic multiple-method approach, we set out to determine the profile and content of glucosinolates (GSLs), which are the relevant chemical tag of Brassicaceae. Intact GSLs were analyzed through a well-established LC-MS method. Identification and quantification were performed by HPLC-PDA of desulfo-GSLs (dGLs) according to the official EU ISO method. Moreover, GSL structures were confirmed by GC-MS analysis of the related isothiocyanates (ITCs). Seven GSLs were identified, directly or indirectly, as follows: 1-methylethyl GSL, (1S)-1-methylpropyl GSL, (Rs)-5-(methylsulfinyl)pentyl GSL, (Rs)-6-(methylsulfinyl)hexyl GSL, (2S)-2-hydroxy-4-pentenyl GSL, 2-phenylethyl GSL, and 1-methoxyindol-3-ylmethyl GSL. In other respects, the FA composition of the seed oil was determined. Results revealed cultivated L. annua seed to be a source of NA-rich oil, and presscake as a valuable coproduct. This presscake is indeed rich in GSLs (4.3% w/w), precursors of promising bioactive molecules for agricultural and nutraceutical applications.

The inhibition of DPP-4 by Plantago asiatica L. and the discovery of active compounds

The objective of this study was to investigate the inhibitory effects of Plantago asiatica L. on DPP-4 and to determine the active compounds responsible for this inhibition. Extracts were prepared from the root and aerial portions of the plant using various polar solvents. The DPP-4 inhibition of these extracts was analyzed in an extracellular assay and then assigned to either the high or low inhibition group. The chemical profiles of these extracts were analyzed using an LC-MS method. The ion fragments that were potent in the high inhibition extracts were selected through OPLS-DA and correlation analysis, and then identified and obtained for verification of DPP-4 inhibition in both the extracellular assay and the Caco-2 cell line. The extracts showed various inhibitory activities with an average inhibition of 28.4 %. A total of 20 compounds were selected from the extracts with higher inhibitory activity and identified. Six of them, baicalein, scutellarein, calceolarioside B, isoacteoside, verbascoside, and plantamajoside, exhibited DPP-4 inhibition with IC50 values of 44.32, 59.90, 112.71, 157.02, 154.58, and 195.07 μg/mL, respectively, in extracellular assays. They also showed inhibition of DPP-4 on the Caco-2 cell line, with IC50 values of 31.56, 34.71, 20.29, 33.29, 24.52, and 87.88 μg/mL, respectively. The extracts from PAL showed inhibitory activity towards DPP-4, and the related compounds were identified and verified.

Erythrina subumbrans (Hassk) Merr. (Fabaceae) Inhibits Insulin Resistance in the Adipose Tissue of High Fructose-Induced Wistar Rats.

The twigs and roots of Erythrina subumbrans (Hassk). Merr. Was reported to possess antidiabetic activity by reducing the activity of α-glucosidase and α-amylase. TNF-α is a pro-inflammatory cytokine in obesity and diabetes mellitus (DM). It inhibits the action of insulin, causing insulin resistance. Adiponectin is an anti-inflammatory peptide synthesized in white adipose tissue (WAT) and its high levels are linked with a decreased risk of DM. However, information about the effect of Erythrina subumbrans (Hassk). Merr. on insulin resistance are still lacking. To obtain the effects of the ethanol extract of E. subumbrans (Hassk) Merr. leaves (EES) in improving insulin resistance conditions. The leaves were collected at Ciamis, West Java, Indonesia, and were extracted using ethanol 96%. The effects of EES were studied in fructose-induced adult male Wistar rats by performing the insulin tolerance test (ITT) and assessing blood glucose, TNF-α, adiponectin, and FFA levels. The number of WAT and BAT of the adipose tissues was also studied. The total phenols and flavonoids in EES were determined by the spectrophotometric method and the presence of quercetin in EES was analyzed using the LC-MS method. EES significantly reduced % weight gain, TNF-α levels, and increased adiponectin levels in fructose-induced Wistar rats. EES significantly reduced the FFA levels of fructose-induced Wistar rats and significantly affected the formation of BAT similar to that of metformin. All rats in EES and metformin groups improved insulin resistance as proven by higher ITT values (3.01 ± 0.91 for EES 100 mg/kg BW; 3.01 ± 1.22 for EES 200 mg/kg BW; 5.86 ± 3.13 for EES 400 mg/kg BW; and 6.44 ± 2.58 for metformin) compared with the fructose-induced group without treatment (ITT = 2.62 ± 1.38). EES contains polyphenol compounds (2.7638 ± 0.0430 mg GAE/g extract), flavonoids (1.9626 ± 0.0152 mg QE/g extract), and quercetin 0.246 µg/mL at m/z 301.4744. Erythrina subumbrans (Hassk). Merr. extract may have the potential to be further explored for its activity in improving insulin resistance conditions. However, further studies are needed to confirm its role in alleviating metabolic disorders.

Bridging Targeted (Zeno MRM-HR) and Untargeted (SWATH) LC-HRMS in a Single Run for Sensitive Exposomics.

Traditionally, chemical exposure has been assessed by low-resolution mass spectrometry via targeted approaches due to the typically extremely low concentration of such compounds in biological samples. Nevertheless, untargeted approaches are now becoming a promising tool for a broader investigation of the exposome, covering additional compounds, their biotransformation products, and possible metabolic alterations (metabolomics). However, despite broad compound coverage, untargeted metabolomics still underperforms in ultratrace biomonitoring analysis. To overcome these analytical limitations, we present the development of the first combined targeted/untargeted LC-MS method, merging MRM-HR and SWATH experiments in one analytical run, making use of Zeno technology for improved sensitivity. Multiple reaction monitoring transitions were optimized for 135 highly diverse toxicants including mycotoxins, plasticizers, PFAS, personal care products ingredients, and industrial side products as well as potentially beneficial xenobiotics such as phytohormones. As a proof of concept, standard reference materials of human plasma (SRM 1950) and serum (SRM 1958) were analyzed with both Zeno MRM-HR + SWATH and SWATH-only methodologies. Results demonstrated a significant increase in sensitivity represented by the detection of lower concentration levels in spiked SRM materials (mean value: 2.2 and 3 times lower concentrations for SRMs 1950 and 1958, respectively). Overall, the detection frequency was increased by 68% (19 to 32 positive detections) in the MRM-HR + SWATH mode compared to the SWATH-only. This work presents a promising avenue for addressing the outstanding key challenge in the small-molecule omics field: finding a balance between high sensitivity and broad chemical coverage. It was demonstrated for exposomic applications but might be transferred to lipidomics and metabolomics workflows.

Polar metabolomics using trichloroacetic acid extraction and porous graphitic carbon stationary phase

IntroductionAccurately identifying and quantifying polar metabolites using untargeted metabolomics has proven challenging in comparison to mid to non-polar metabolites. Hydrophilic interaction chromatography and gas chromatography–mass spectrometry are predominantly used to target polar metabolites.ObjectivesThis study aims to demonstrate a simple one-step extraction combined with liquid chromatography–mass spectrometry (LC–MS) that reliably retains polar metabolites.MethodsThe method involves a MilliQ + 10% trichloroacetic acid extraction from 6 healthy individuals serum, combined with porous graphitic carbon liquid chromatography–mass spectrometry (LC–MS). The coefficient of variation (CV) assessed retention reliability of polar metabolites with logP as low as − 9. QreSS (Quantification, Retention, and System Suitability) internal standards determined the method's consistency and recovery efficiency.ResultsThe method demonstrated reliable retention (CV < 0.30) of polar metabolites within a logP range of − 9.1 to 5.6. QreSS internal standards confirmed consistent performance (CV < 0.16) and effective recovery (70–130%) of polar to mid-polar metabolites. Quality control dilution series demonstrated that ~ 80% of annotated metabolites could be accurately quantified (Pearson’s correlation coefficient > 0.80) within their concentration range. Repeatability was demonstrated through clustering of repeated extractions from a single sample.ConclusionThis LC–MS method is better suited to covering the polar segment of the metabolome than current methods, offering a reliable and efficient approach for accurate quantification of polar metabolites in untargeted metabolomics.

A comparative metabolomics analysis of Açaí (Euterpe oleracea Mart.) fruit, food powder, and botanical dietary supplement extracts.

Euterpe oleracea Mart. (açaí) is a botanical of interest to many who seek functional foods that provide antioxidant and anti-inflammatory properties. Cancer patients are increasingly taking botanical dietary supplements containing açaí to complement their conventional therapeutics, which may lead to serious adverse events. Before testing our açaí extracts in vitro for botanical-drug interactions, the goal is to chemically characterize our extracts for compounds whose biological activity in açaí is unknown. The objective of this work was to develop a chemical fingerprinting method for untargeted characterization of açaí samples from a variety of sources, including food products and botanical dietary supplement capsules, made with multiple extraction solvents. An optimized LC-MS method was generated for in-depth untargeted fingerprinting of chemical constituents in açaí extracts. Statistical analysis models were used to describe relationships between the açaí extracts based on molecular features found in both positive and negative mode ESI. In an attempt to elucidate the differences in metabolites among açaí extracts from different cultivars, we identified or tentatively identified 173 metabolites from the 16 extracts made from 6 different sources. Of these compounds, there are 138 reported in açaí for the first time. Statistical models showed similar yet distinct differences between the extracts tested based on the polarity of compounds present and the origin of the source material. A high-resolution mass spectrometry method was generated that allowed us to greatly characterize 16 complex extracts made from different sources of açaí with different extraction solvent polarities.

A simple and sensitive LC-MS/MS method for the determination of loganic acid in rat plasma and its pharmacokinetic analysis.

Loganic acid is an iridoid compound extracted from Gentianaceae plant Gentiana macrophylla Pall. It can effectively inhibit inflammation and tumor migration and has antioxidant activity. In this paper, we establish a simple, fast, sensitive and validated LC-MS method with the purpose of quantification of loganic acid in rat plasma with gliclazide as an internal standard (IS). Methanol was used to precipitate the protein in the plasma sample, and a C18 column (2.1 × 50 mm, 1.7μm) was used for the separation of the target compound. Meanwhile, 0.1% formic acid water-methanol was employed as the mobile phase. Multiple reaction monitoring detection mode was adopted in detection with m/z 375.1 > 213.2 for loganic acid and m/z 322.1 > 169.9 for the IS, respectively, in negative ion scan mode. The linear range of calibration curve was 5.77-11,540.00 ng/ml, and the lower limit of detedtion was 2.89 ng/ml. The inter-day and intra-day precision and accuracy were <15% for lower limit of quantitation, low, middle and high quality control samples. This method was successfully used for the pharmacokinetic study of loganic acid in rat plasma at a dose range of 50-150 mg/kg for oral administration and 2mg/kg for intravenous administration. The pharmacokinetic results showed that the oral bioavailability of loganic acid was low (2.71-5.58%).

Development of a Stability-Indicating Purity Method for Ubrogepant Through Stress Degradation Analysis, Extraction, and Characterization of Unidentified Degradation Products Using Flash Chromatography, NMR, IR, and LC–MS

Abstract Background Ubrogepant is a prescription medication used to prevent migraine headaches. It is currently available in tablet form. Objective The goal of this work is to investigate the drug degradation profile of ubrogepant, as well as to isolate and characterize undiscovered ubrogepant degradation products by utilizing LC–MS, NMR, and IR spectroscopic analytical techniques and, furthermore, to develop a high-resolution, sensitive, stability-indicating analytical approach for detecting and quantifying ubrogepant degradation products in its pharmaceutical formulation. Methods To identify and quantify the degradation products of ubrogepant in pharmaceutical products, a novel gradient reverse-phase HPLC (RP-HPLC) technique with a photo diode array (PDA) detector was developed by utilizing a C18 stationary phase column. The eluent comprised a mixture of acetonitrile and water with 0.1% (v/v) ortho-phosphoric acid. To establish the intrinsic stability of the ubrogepant pharmaceutical product, it was stress-tested under various degradation conditions, including water, alkaline, acid hydrolysis, photolytic, oxidative, and thermal. Flash chromatography was used to isolate the two major degradants, and the structures were determined using NMR (1H, 13C, distortionless enhancement by polarisation transfer-DEPT-135), IR, and LC–MS methods. Results The ubrogepant medication was relatively more degradable in alkaline and acidic conditions, and two unique degradation products were discovered. Based on spectroscopic and chromatographic evidence, it was conclusively demonstrated that these unique compounds were ubrogepant hydrolysis products. All degradation products were separated with a resolution greater than 2.0. The peak purity data showed that the ubrogepant peak in all of the stress samples examined was pure. Under all stress environments, ubrogepant achieved a minimum mass balance of 95%. The validated approach developed was sensitive enough to quantify ubrogepant degradation products at 0.03% of the ubrogepant test concentration. Conclusion The proposed method was found to be stability-indicating since it fits all of the regulatory authorities' typical requirements. This method is highly efficient for detecting and quantifying impurities in ubrogepant drug substances and drug products in QC laboratories. Highlights Two new degradation products of ubrogepant were successfully extracted and characterized using NMR, IR, and LC–MS spectroscopic methods. The proposed HPLC method can accurately quantify the degradation products of ubrogepant in pharmaceutical products and is sensitive enough to detect degradation products of ubrogepant as low as 0.17 µg/mL.

Analgesic and anti-inflammatory activities of NPK 500 capsules, a Cassia sieberiana DC. - Based herbal analgesic medicine used to treat dysmenorrhea and peptic ulcer, is mediated through the inhibition of PGE2 and iNOS

Ethnopharmacological relevancePain and inflammation are the most frequent reasons for which people seek medical care. Currently available analgesics against these conditions produce fatal adverse effects. NPK 500 capsules is an alternative herbal analgesic employed to treat dysmenorrhea, peptic ulcer and pain. NPK 500 is produced from Cassia sieberiana. A plant used in traditional medicine to treat pain and inflammation. Aim of the studyThis study reports the analysis, phytochemical characterization and mechanism of analgesic and anti-inflammatory activities of two NPK 500 capsules, called old and new NPK500 capsules (ONPK500 and NNPK500) respectively. Materials and methodsPhysicochemical, organoleptic, GC-MS and LC-MS methods were employed to analyze the NPK 500 capsules. Analgesic activity was evaluated using tail immersion, Randall-Selitto and acetic acid induced writing tests. Anti-inflammatory activity was evaluated using carrageenan-induced rat paw inflammation. Additionally, pro-inflammatory mediators such as prostaglandin E2 (PGE2), inducible nitric oxide synthase (iNOS), cyclooxygenase 1 and 2 (COX-2 and COX-1) were quantified in the sera of the rats using Enzyme Linked Immunosorbent Assay (ELISA) kits. ResultsThirteen major compounds were characterized in the NNPK 500 capsules via the GC-MS and LC-MS spectroscopies. Kaempferol was the major compound characterized in addition to physcion, β-sitosterol 3-O-β-D-glucopyranoside, betulinic acid and nine others. Physicochemical and organoleptic indices of the capsules were also derived for its authentication and quality control. Furthermore, NNPK 500 0.5–1.5 mg/kg p.o. produce significant (P < 0.5) analgesic activity (160–197%) higher than that of ONPK500 (109.8%) and Morphine (101%) in the tail immersion test. The analgesic activity of NNPK 500 0.5–1.5 mg/kg p.o. (171.0–258.3%) and ONPK 500 (179.5%) were also significant (P < 0.01) and higher than that of Aspirin (103.00%) in the Randall-Selitto test. Both capsules also demonstrated significant (P < 0.5) analgesic and anti-inflammatory activities in the acetic acid-induced writhing and carrageenan-indued paw edema tests respectively. The two NPK500 capsules also, significantly (P < 0.5) inhibited PGE2 and iNOS but not COX-2 and COX-1 in the carrageenan-indued paw edema test. ConclusionThese results show that NNPK 500 and ONPK 500 capsules possessed potent analgesic and anti-inflammatory activities via inhibition of PGE2 and iNOS as a result of their chemical constituents. NPK500 capsules thus, relief acute pain and inflammation without causing gastrointestinal, renal or hepatic injuries, since they did not inhibit COX-1.

Magneto-fluorescent nanobiosensor with tetrahedral framework nucleic acid-confined aptamer for trace adenosine triphosphate detection

Accurate quantitation of trace-level ATP biomarkers often suffers from interference of sample matrix and remains a challenge. Herein, a novel magnetic biological nanosensor (MBNS) integrating tetrahedral framework nucleic acid (tFNA)-confined aptamers and ultrasensitive laser-induced fluorescence (LIF) was tailored and evaluated. The fluorescent aptamer nanoprobe hybridized with FAM-labelled cDNA was particularly incorporated into the inner cavity of tFNA, enabling a dual-mode mechanism that integrates molecular size selection and aptamer affinity recognition. By this strategy, the benefits of tFNA molecular sieve, the super sensitive properties of LIF, and the specific recognition of “aptamer gene codes” were integrated, as triggered the superior response of ATP. The structures, properties and reaction mechanism of tFNA, as well as tFNA modified with aptamer probe at different sites, were characterized in detail. A high level of anti-interference was achieved, shielding against non-specific adsorption even in matrices with high protein and analogue content, 103∼105 times higher than ATP. The release of fluorescent beacon from aptamer probe was facilely driven by specific binding of ATP, and an ultra-sensitive detection of 75 pmol/L ATP was obtained without amplification. It was 2–3 orders of magnitude more sensitive than most fluorescent aptasensors. Applied to serum samples, the stability and reproducibility of the resultant aptasensor have been validated with the intra- and inter-assay RSD in 1.6–6.4 % and -1.5–8.4 %, respectively. Acceptable recoveries of 96.5–106.8 % were achieved, consistent with the LC-MS method. It was expected to open a simple and efficient protocol for highly specific and ultrasensitive ATP detection in clinical diagnostics.