Method For Simultaneous Quantification Research Articles

Development and Validation of Stability‐Indicating HPLC Method for Simultaneous Quantification of Vardenafil and Dapoxetine in Bulk and in Combined Dosage Form

AbstractAn understanding of drug product degradation issues is needed to advise stable formulation and provide a reasonable shelf‐life evaluation for all drug products. For this purpose, we have developed and validated a novel HPLC method for assessing the stability of vardenafil and dapoxetine in a combined pharmaceutical dosage form. The chromatographic separation was accomplished on a ProntoSIL C18 KROMA PLUS column (250×4.6 mm, 5 μm). The column was run under isocratic conditions with a 2.0 mL/min flow rate. The mobile phase was made up of methanol and 20 mM potassium dihydrogen phosphate at 45 : 55 (v/v), which was adjusted to pH 3.0 with phosphoric acid; these two drugs were eluted at retention times of 5.33 and 9.91 min for vardenafil and dapoxetine, respectively. The proposed method shows a linearity of 1–50 ug/ml with a regression of 0.9999 for both drugs. The method was validated for accuracy, precision, linearity, specificity, and sensitivity. The developed HPLC method has been successfully implemented to analyse dapoxetine and vardenafil in tablets. Furthermore, the stress degradation experiments were conducted on the bulk powder of vardenafil and dapoxetine following the International Council for Harmonization (ICH) recommendations.

Capillary Electrophoresis Method for Simultaneous Quantification of Hypocholesterolemic Drugs in Binary Mixture Formulation: Fast, Green, and Cost-Effective Alternative to HPLC

Capillary Electrophoresis Method for Simultaneous Quantification of Hypocholesterolemic Drugs in Binary Mixture Formulation: Fast, Green, and Cost-Effective Alternative to HPLC

Validation of a liquid chromatography-tandem mass spectrometry method for simultaneous quantification of N,N-dimethylacetamide and N-monomethylacetamide in pediatric plasma.

N,Na-dimethylacetamide is an excipient used in intravenous busulfan formulations, a drug used in hematopoietic stem cell transplantation conditioning. The aim of this study was to develop and validate a liquid chromatography-tandem mass spectrometry method for simultaneous quantification of N,N-dimethylacetamide, and its metabolite N-monomethylacetamide in plasma from children receiving busulfan. A 4μl aliquot of patient plasma was extracted using 196μl 50% methanol solution and quantified against calibrators prepared in the extraction solvent given negligible matrix effects across three concentrations. 9 [H2 ]-N,N-dimethylacetamide was used as an internal standard. Separation of N,N-dimethylacetamide and N-monomethylacetamide was achieved using a Kinetex EVO C18 stationary phase (100mm × 2.1mm × 2.6μm) running an isocratic mobile phase of 30% methanol containing 0.1% formic acid at a flow of 0.2ml/min over 3.0 min. The injection volume was1μl. Calibration curves for N,N-dimethylacetamide and N-monomethylacetamide were linear up to 1200 and 200μg/L, respectively, with a lower limit of quantification 1μg/L for both analytes. Calibrator accuracy and precision were within±10% of the test parameters across four concentration levels. Analytes were stable over 14 days at three different storage conditions. This method was successfully applied to measure N,N-dimethylacetamide and N-monomethylacetamide concentrations in a total of 1265 plasma samples from 77 children.

High-throughput quantitation of serological dimethylarginines by LC/MS/MS: Potential cardiovascular biomarkers for rheumatoid arthritis

Rheumatoid arthritis (RA) is a chronic autoimmune disorder characterized by systemic inflammation of the joints and extra-articular tissues. The incidence of cardiovascular disease (CVD) remains the main cause of morbidity and mortality in patients with RA. Despite the development of new therapeutics targeting the articular manifestations, the relief of the cardiovascular burden is still an unmet medical need during the management of RA. So, the early prognosis of RA-associated CVD plays a crucial role in improving the clinical outcomes of RA patients. Recently, circulating dimethylarginines have gained attention as potential biomarkers for CVDs. Here, we present the development and validation of a high-throughput liquid chromatography-tandem mass spectrometric (LC/MS/MS) method for simultaneous quantification of creatinine, arginine, and dimethylarginines in human serum within 2 mins by isotope dilution mass spectrometry. This method employed a protein precipitation method for rapid sample preparation, trichloroacetic acid (TCA)-based ion pairing chromatography for fast analyte separation, and multiple reaction monitoring (MRM) with stable isotope-labeled internal standards (ISs) for simultaneous quantitation. To assure the quality, our method was validated against the FDA guidelines for lower limit of quantitation (0.2 µM), linearity (square of coefficient correlation>0.99), precision (intra-&inter-assay imprecision < 10 %), accuracy (intra-&inter-assay inaccuracy < 10 %), sample preparation recovery (recovery ≥ 90 %), stability (instability < 10 %), matrix effect (signal suppression < 55 %), and carryover ( < 0.01 %). Afterward, we applied the validated method to a retrospective cross-sectional study. We aimed to evaluate the utility of serological dimethylarginines as potential cardiovascular biomarkers in the development of RA-associated CVD. Our results revealed that the serological ratio of asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA), an indicator of physiological arginine methylation status, was significantly elevated in patients with RA. This finding might provide value in detecting CVD to improve clinical outcomes in RA management.

Development of a Rapid LC-MS/MS Method for Simultaneous Quantification of Donepezil and Tadalafil in Rat Plasma: Its Application in a Pharmacokinetic Interaction Study after Oral Administration in Rats.

This study aimed to establish a simple and sensitive analytical method to simultaneously quantify donepezil (DPZ) and tadalafil (TAD) in rat plasma using lansoprazole (LPZ) as an internal standard (IS) by using liquid chromatography tandem mass spectrometry. The fragmentation pattern of DPZ, TAD, and IS was elucidated using multiple reaction monitoring in electrospray ionization positive ion mode for the quantification of precursor to production at m/z 380.1 → 91.2 for DPZ, m/z 390.2 → 268.1 for TAD, and m/z 370.3 → 252.0 for LPZ. The extracted DPZ and TAD from plasma using acetonitrile-induced protein precipitation was separated using Kinetex C18 (100 × 2.1 mm, 2.6 µm) column with a gradient mobile phase system consisting of 2 mM ammonium acetate and 0.1% formic acid in acetonitrile at a flow rate of 0.25 mL/min for 4 min. The selectivity, lower limit of quantification, linearity, precision, accuracy, stability, recovery, and matrix effect of this developed method was validated according to the guidelines of the U.S. Food and Drug Administration and the Ministry of Food and Drug Safety of Korea. The established method achieved acceptance criteria in all validation parameters, ensuring reliability, reproducibility, and accuracy, and was successfully implemented in a pharmacokinetic study on the co-administration of DPZ and TAD orally in rats.

Development of a simultaneous quantification method for the gut microbiota-derived core nutrient metabolome in mice and its application in studying host-microbiota interaction

The gut microbiota interacts with the host via production of various metabolites of dietary nutrients. Herein, we proposed the concept of the gut microbiota-derived core nutrient metabolome, which covers 43 metabolites in carbohydrate metabolism, glycolysis, tricarboxylic acid cycle and amino acid metabolism, and established a quantitative UPLC-Q/TOF-MS method through 3-nitrophenylhydrazine derivatization to investigate the influence of obesity on the gut microbiota in mice. All metabolites could be simultaneously analyzed via separation on a BEH C18 column within 18 min. The lower limits of quantification of most analytes were less than 1 μM. Validation results demonstrated suitability for the analysis of mouse fecal samples. The method was then applied to detect the gut microbiota-derived nutrient metabolome in the feces of high-fat diet induced obese (DIO) and ob/ob (leptin-deficient) mice, as well as obesity-prone (OP) and obesity-resistant (OR) mice. Compared to the control groups, there were 13, 23 and 10 differentially abundant metabolites detected in ob/ob, DIO and OP groups, respectively. Among them, amino acids including leucine, isoleucine, glycine, methionine, tyrosine and glutamine were co-downregulated in the obese or OP mice and exhibited inverse association with body weight. 16S rDNA analysis revealed that the genera Lactobacillus and Dubosiella were also inversely associated with body weight and positively correlated with fecal amino acids. Collectively, our work provides an effective and simplified method for simultaneous quantifying the gut microbiota-derived core nutrient metabolome in mouse feces, which could assist various future studies on host-microbiota metabolic interaction.

Method development for simultaneous estimation of Amlodipine Besylate and Perindopril Tertbutyl amine in fixed-dose

The fixed-dose combination of Amlodipine Besylate (ADB) with Perindopril Tertbutylamine (PTBA) drug is used to treat patients with mild-to-moderate hypertension. In recent times researchers are interested to find the efficient analytical method development and validation for the simultaneous determination of ADB and PTBA in a fixed-dose, film-coated tablet. Therefore, the current study was performed with a reverse-phase liquid chromatography method developed to simultaneously analyze ADB and PTBA in film-coated tablets as fixed-dose combinations. The linearity of the proposed method was calculated by preparing six different mixtures of both ADB and PTBA in the mobile phase. The concentration of both the analytes was analyzed at 56mg/100 mL to 84mg/100 mL and 32mg/100 mL to 48mg/100 mL, respectively. The ratio of acetonitrile and phosphate buffer was 35:65. The flow rate was adjusted to 1.5 ml per minute to reduce the retention time. The validation study was performed for the parameters specificity, linearity, precision, range, limit of detection, limit of quantification, accuracy/biasness, and robustness. The relative percentage standard deviation for Perindopril Tertbutyl amine was 0.148%, and for Amlodipine is 0.312%. These results show that the advanced analysis method for simultaneous analysis of fixed-dose is precise. The theoretical IR spectra were also calculated by Gaussian 9.2 by employing the B3LYP functional at density functional theory (DFT) level study. All these parameters studied in this work authenticate the effectiveness of the developed validation method and ensure its repeatability/reproducibility accordingly. To the best of our knowledge, this is the first time to develop a new fast, and easy method for simultaneous identification and quantification of ADB and PTBA by high-performance liquid chromatography (HPLC) with a time-efficient and cost-effective approach.

Development of a Simultaneous Liquid Chromatography-Tandem Mass Spectrometry Analytical Method for Urinary Endogenous Substrates and Metabolites for Predicting Cytochrome P450 3A4 Activity.

CYP3A4, which contributes to the metabolism of more than 30% of clinically used drugs, exhibits high variation in its activity; therefore, predicting CYP3A4 activity before drug treatment is vital for determining the optimal dosage for each patient. We aimed to develop and validate an LC-tandem mass spectrometry (LC-MS/MS) method that simultaneously measures the levels of CYP3A4 activity-related predictive biomarkers (6β-hydroxycortisol (6β-OHC), cortisol (C), 1β-hydroxydeoxycholic acid (1β-OHDCA), and deoxycholic acid (DCA)). Chromatographic separation was achieved using a YMC-Triart C18 column and a gradient flow of the mobile phase comprising deionized water/25% ammonia solution (100 : 0.1, v/v) and methanol/acetonitrile/25% ammonia solution (50 : 50 : 0.1, v/v/v). Selective reaction monitoring in the negative-ion mode was used for MS/MS, and run times of 33 min were used. All analytes showed high linearity in the range of 3-3000 ng/mL. Additionally, their concentrations in urine samples derived from volunteers were analyzed via treatment with deconjugation enzymes, ignoring inter-individual differences in the variation of other enzymatic activities. Our method satisfied the analytical validation criteria under clinical conditions. Moreover, the concentrations of each analyte were quantified within the range of calibration curves for all urine samples. The conjugated forms of each analyte were hydrolyzed to accurately examine CYP3A4 activity. Non-invasive urine sampling employed herein is an effective alternative to invasive plasma sampling. The analytically validated simultaneous quantification method developed in this study can be used to predict CYP3A4 activity in precision medicine and investigate the potential clinical applications of CYP3A4 biomarkers (6β-OHC/C and 1β-OHDCA/DCA ratios).

Inter-laboratory study on simultaneous quantification of ten trichothecenes in feed.

An inter-laboratory study was performed in eight laboratories to evaluate the simultaneous quantification method for HT-2 toxin (HT-2), T-2 toxin (T-2), diacetoxyscirpenol (DAS), neosolaniol (NES), 3-acetyldeoxynivalenol (3-AcDON), 15-acetyldeoxynivalenol (15-AcDON), deoxynivalenol (DON), deoxynivalenol-3-glucoside (D3G), nivalenol (NIV), and fusarenon-X (FUS-X) in feed. The mycotoxins in the samples were extracted with hydrous acetonitrile, purified using a multifunctional column (InertSep® VRA-3) and a phospholipid removal column (Hybrid SPE®-Phospholipid), and then quantified using liquid chromatography-tandem mass spectrometry (LC-MS/MS) with atmospheric pressure chemical ionisation mode. The mean recovery, repeatability, reproducibility, and Horwitz ratio from the inter-laboratory validation study were 99.8-109%, 3.1-9.8%, 4.3-9.8%, and 0.19-0.45, respectively, for type A trichothecenes (HT-2, T-2, DAS, and NES). Those values for type B trichothecenes (3-AcDON, 15-AcDON, DON, NIV, and FUS-X) were 89.9-116%, 3.4-9.1%, 5.6-14%, and 0.25-0.70, and the values for modified mycotoxin (D3G) were 78.2-96.7%, 3.5-6.4%, and 13-22%, respectively.

LC-MS/MS method for simultaneous quantification of osilodrostat and metyrapone in human plasma from patients treated for Cushing’s Syndrome

Steroidogenesis inhibitors such as metyrapone (MTP) and osilodrostat (ODT) have a key role in the medical treatment of endogenous Cushing’s Syndrome (ECS). Both drugs are characterized by a high inter-individual variability of response and require a dose-titration period to achieve optimal control of cortisol excess. However, PK/PD data remain scarce for both molecules and a pharmacokinetically guided approach could help reaching eucortisolism more rapidly. We aimed to develop and validate a liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the simultaneous quantification of ODT and MTP in human plasma. After addition of isotopically labeled internal standard (IS), plasma pretreatment consisted in protein precipitation with acetonitrile including 1% formic acid (v/v). Chromatographic separation was performed on Kinetex® HILIC (4.6 × 50 mm; 2.6 µm) analytical column with an isocratic elution during the 2.0-min run time. The method was linear from 0.5 to 250 ng/mL for ODT and from 2.5 to 1250 ng/mL for MTP. Intra- and inter-assay precisions were < 7.2%, with an accuracy ranging from 95.9% to 114.9%. The IS-normalized matrix effect ranged from 106.0% to 123.0% (ODT) and from 107.0% to 123.0% (MTP) and the range of the IS-normalized extraction recovery was 84.0–101.0% for ODT and 87.0–101.0% for MTP. The LC-MS/MS method was successfully applied in patients’ plasma samples (n = 36), trough concentration of ODT and MTP ranged from 2.7 ng/mL to 8.2 ng/mL and from 10.8 ng/mL to 27.8 ng/mL, respectively. Incurred sample reanalysis exhibits less than 14% difference between the first and the second analysis for both drugs. This accurate and precise method, meeting all validation criteria, can therefore be used for plasma drug monitoring of ODT and MTP within the dose-titration period.

Determination of Tea Aroma Precursor Glycosides: An Efficient Approach via Liquid Chromatography-Tandem Mass Spectrometry.

Tea aroma components are often stored as glycosidically bound forms in the tea plant (Camellia sinensis). However, the determination of these glycosides in tea samples is far from optimal. In the present study, we developed a high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method for simultaneous quantification of eight primary aroma glycosides within 10 min. After systematic optimization of multiple reaction monitoring (MRM) parameters, the proposed method was highly sensitive and accurate. Optimization of the method permitted the efficient extraction of aroma glycosides. The developed method was applied to analyze the contents of aroma glycosides in different organs of tea plants, including the bud, leaves, and stem. Contents of aroma glycosides in the harvested 'Shaancha 1' ranged from 36.1 to 40454.4 μg kg-1. Geranyl glucoside and primeveroside mainly accumulated in young leaves, while other glycosides mainly accumulated in mature leaves. The findings document a rapid, reliable, and efficient analysis method. This method will be helpful in elucidating the biosynthesis and biotransformation mechanism of tea aroma glycosides and in promoting the development of the tea industry using advanced technological control approaches during the cultivation of tea plants and tea manufacture.

Low-volume plasma sampling for determination of dextromethorphan and dextrorphan in rat plasma: LC-MS/MS method and its application in pharmacokinetic study.

Dextromethorphan (DM) and its metabolite dextrorphan (DX) continue to draw the attention of researchers owing to their diverse pharmacodynamics. Thus, there are possibilities for repurposing DM. Most of the pharmacodynamics of DM needs further validation in different preclinical models. Also, it is necessary to correlate the pharmacodynamics with relevant pharmacokinetics data. Multiple bioanalytical techniques developed for this purpose primarily use a high sample processing volume. Since sample volume is a limiting factor for many preclinical models, an effort was taken to develop an alternative method suitable for handling low sample processing volumes. An efficient solid-phase extraction technique, robust liquid chromatographic (LC) separation and highly sensitive tandem mass spectrometric detection (MS/MS) showed suitability for use of a 30 μl sample processing volume. This led to the development of a highly specific, selective, accurate and precise-bio-analytical method for simultaneous quantification of DM and DX in rat plasma. The validated method was linear in the range of 0.196-403.356 ng/ml for DM and 0.102-209.017ng/ml for DX. The application of the method was demonstrated through the estimation of pharmacokinetic parameters that showed good congruence with earlier studies.

A Selective and Accurate LC-MS/MS Method for Simultaneous Quantification of Valsartan and Hydrochlorothiazide in Human Plasma

The fixed dose combination of valsartan (VAL) and hydrochlorothiazide (HCTZ) is the most commonly prescribed medicine for the effective treatment of hypertension. In this study, a simple sensitive and accurate liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the simultaneous quantitation of VAL and HCTZ in human plasma by using irbesartan (IRB) and hydroflumethiazide (HFMZ) as their specific internal standards (ISs). HLB cartridge-based solid-phase extraction was used for the extraction of analytes and ISs. The chromatographic separation was achieved on Lichrocart RP Select (125 × 4 mm), 5 nm with the mobile phase composition of acetonitrile: 10 mM ammonium acetate buffer: 95:05, v/v, at flow rate of 0.5 mL/min. The turbo ion electrospray ionization in negative mode was used as ion source for the sample ionization. The precursor to product ion transitions were 434.10 &gt; 179.10 (VAL), 295.70 &gt; 204.90 (HCTZ), 427.10 &gt; 192.90 (IRB), and 329.90 &gt; 302.40 (HFMZ) for detection and quantification of analytes and their ISs. The retention times of VAL and HCTZ were 1.90 min and 2.30 min, respectively. The range for the calibration curves of VAL and HCTZ were 50.2–6018.6 ng/mL and 1.25–507.63 ng/mL, respectively, with good linearity having correlation coefficient values of ≥0.995 for both VAL and HCTZ. All validation parameter results (selectivity, precision and accuracy, matrix effects and stabilities) were within the acceptable range as per USFDA guideline for bioanalytical method validation. The intra-day and inter-day accuracy data for VAL were within the range of 105.68–114.22% and 98.41–108.16%, respectively, whereas for HCTZ they were 87.01–101.18% and 95.16–99.37%, respectively. The ion suppression effects produced for VAL and ion enhancement effects produced for HCTZ were insignificant according to the proposed sample cleanup procedure. The developed LC-MS/MS method was successfully applied to bioequivalence study on healthy volunteers.

Ultra-trace detection and efficient adsorption removal of multiple water-soluble volatile organic compounds by fluorescent sensor array.

Due to the extremely low concentration, complex composition and easy to be converted into each other in water and air of water-soluble volatile organic compounds (VOCs), it is a great challenge to the traditional detection technology, pollution control and traceability, etc. Therefore, developing a convenient, swift and on-site detection method for simultaneous quantification of multiple VOCs is highly anticipated. In this paper, a multifunctional sensor array with adsorption and sensing of VOCs has been constructed by four fluorescence channels of small-sized Eu@Uio-66 and Tb@Uio-66. Due to the obvious cross-reactive characteristics between 4 fluorescence channels and VOCs, the sensor array could detect 8 VOCs simultaneously with all detection limits as low as ppb level. In addition, the detection results of sensor array for actual water samples coexisting with multiple VOCs confirmed that it has strong anti-interference performance and could be used for simultaneous detection of multiple VOCs in real water. The construction of sensor array with VOC adsorption function not only helps to reduce the detection limit of VOCs benefiting from the pre-concentration of materials, but also has significant value to reduce the harmfulness of pollutants. Predictably, this work is of great significance for VOC traceability, analysis of ecotoxicological effects and monitoring of pollution distribution characteristics.

Simultaneous Quantification of Avermectins in Six Aquatic Foods by UHPLC/FLD with Precolumn Derivatization

In this study, a fast, concise and reliable ultra-high performance liquid chromatography-fluorescence (UHPLC/FLD) detection method for simultaneous quantification of avermectins (AVMs), including avermectin (AVM), ivermectin (IVM), emamectin (EMM), moxidectin (MOX) and doramectin (DOR) in six aquatic foods was established. Based on the QuEChERS pretreatment method, the samples were extracted with 0.2% (v/v) ammonia acetonitrile. N-methyl imidazole mixed with acetonitrile (1:1, v/v) and trifluoroacetic anhydride with acetonitrile (1:2, v/v) were used as derivatization reagents. The mobile phase consists of acetonitrile and water with a flow rate of 1.0 mL/min. An Infinity Lab Poroshell 120 EC-C18 column was used for optimum chromatographic separation of target analytes at 40 °C; the excitation and emission wavelengths were set at 365 nm and 465 nm, respectively. In six kinds of aquatic foods, the limits of detection (LODs) of AVM, IVM, EMM, MOX, and DOR were 2.7 μg/kg, 1.8 μg/kg, 2.1 μg/kg, 1.2 μg/kg, and 2.7 μg/kg, respectively, and the limits of quantification (LOQs) of AVM, IVM, EMM, MOX, and DOR were 5 μg/kg, 4.5 μg/kg, 4.5 μg/kg, 3.5 μg/kg and 5.0 μg/kg, respectively. The recoveries were all above 85.38% when the samples were spiked with the target compounds at the concentration level of 5, 10, 50, and 100 μg/kg. The intra-day and inter-day relative standard deviations (RSDs) were all less than 15%. This method considers the requirements of sensitivity, accuracy, and economics of the instrument.

Simple HPLC method for simultaneous quantification of nicotine and cotinine levels in rat plasma after exposure to two different tobacco products

AbstractPurposeDevelopment and validation of a selective analytical method to accurately and precisely quantify nicotine and cotinine levels in rat's plasma after exposure to tobacco cigarettes and tobacco water-pipe.MethodsAn easy HPLC-Photodiode-Array Detection (PDA) method was developed and validated for simultaneous determination of nicotine and cotinine levels in plasma of 15 rats (10 rats after tobacco products exposure and 5 control rats). Nicotine and cotinine were extracted in one step from plasma using acetonitrile and concentrated to lowest volume using nitrogen stream.ResultsThe developed method offered a rapid analysis time of 14 min with single step of analytes extraction from rat's plasma with recovery percentage range between 93 and 95% and excellent linearity with correlation factor more than 0.994 with analytical range between 50 and 1000 ng mL−1 and LOD of 25 ng mL−1 and 23 ng mL−1 for nicotine and cotinine, respectively. The analysis of rat's plasma after 28 days of exposure to tobacco cigarettes and tobacco water-pipe revealed that the average concentrations of 376 ng mL−1 for cotinine and 223 ng mL−1 for nicotine were obtained after tobacco cigarettes exposure, and 220 ng mL−1 for cotinine and 192 ng mL−1 for nicotine after tobacco water-pipe exposure.ConclusionHigher nicotine and cotinine levels were found in plasma after tobacco cigarettes exposure than water-pipe exposure which may have potential undesirable effects on passive smokers in both cases.

Analytical quality by design-based RP-HPLC method for quantification of pioglitazone and candesartan cilexetil in bilayer tablet and its forced degradation studies

Aim: The current project involves developing an RP-HPLC method for simultaneous quantification of Candesartan Cilexetil and Pioglitazone based on analytical quality by design (AQbD). Materials and methods: When analysed in the Design Expert application, the critical method parameters were systematically refined using Central Composite Design and contours were derived for significant variables. A contour plot has been used to discover the technique operable design region that governs response variation, which is then empirically tested. Results: Successful chromatographic separation of title analytes was achieved on kromasil C18 (150 × 4.6 mm, 5 µm) column at 30 °C with mobile phase comprising 60% 20 Mm Potassium dihydrogen orthophosphate and 40% acetonitrile (v/v), isocratic elution pattern, 0.9 mL/min flow rate, and UV detection at 220 nm. The linear model for Candesartan Cilexetil was from 4 to 24 µg/ mL and Pioglitazone at 7.5–45 µg/ mL, respectively. Conclusion: The method met all the ICH Q2 (R1) validation criteria. The current approach aided for analysing simultaneous drugs can be expanded into quantifying drugs in biological matrix predominance with maximum recovery.

Prinsepia utilis Royle leaf extract: Ameliorative effects on allergic inflammation and skin lesions in allergic contact dermatitis and polyphenolic profiling through UPLC–MS/MS coupled to chemometric analysis

Ethnopharmacological relevanceAllergic contact dermatitis (ACD) is a common allergic inflammatory disease that is concomitant with skin swelling, redness, dry itching, and relapses. Prinsepia utilis Royle, a Chinese and Indian folk medicine, is rich in polyphenols with potential anti-inflammatory and skin-protective activities. However, the underlying mechanism of P. utilis leaf (PUL) in the treatment of ACD and its functional basis remains unclear. Aim of the studyThis study is aimed to explore and reveal the active substances and mechanism of PUL against ACD. Materials and methodsHyaluronidase inhibitory assay and fluorescein isothiocyanate (FITC)-induced ACD mouse model were performed to assess the antiallergic effects of PUL in vitro and in vivo. Different solvents were applied to obtain multiple PUL extracts. The extracts were further tested for total phenolic content (TPC) and total flavonoid content (TFC) by using spectrophotometric assays. Polyphenolic profiles were analyzed by using ultrahigh-performance liquid chromatography coupled with time-of-flight mass spectrometry (UPLC-QTOF-MS/MS), and a simultaneous quantification method was established using UPLC-QTrap-MS/MS through multiple reaction monitoring (MRM) and applied to analyze the pharmacokinetics of the multiple major polyphenols of PUL in mice. ResultsThe water extract of PUL with the highest TPC/TFC exhibited the strongest antihyaluronidase effect (IC50 = 231.93 μg/mL). In vivo assays indicated that the oral administration of PUL water extract dose-dependently attenuated ACD-like symptoms by decreased interleukin (IL)-4, IL-5, IL-13, IL-33, thymic stromal lymphopoietin, and IgE production, suppressed eosinophil and basophil secretion, and increasing the expression of tight junction (TJ) proteins (claudin-1 [CLDN-1] and occludin). Concomitantly, UPLC–QTOF–MS/MS analysis enabled the identification of 60 polyphenols and the pharmacokinetic parameters of seven quantified constituents of PUL were characterized. Four compounds, trans-p-coumaric acid 4-O-β-D-glucopyranoside (11), vicenin-2 (21), isoschaftoside (31), and kaempferol 3-O-(2″,6″-di-O-α-L-rhamnopyransoyl)-β-D-glucopyranoside (38) which displayed satisfactory pharmacokinetic features, were considered as potential effective substances in PUL. ConclusionsPUL water extract ameliorated the allergic inflammation of ACD by repairing the epithelial barrier and alleviating Th2-type allergic inflammation. The anti-allergic effect of PUL is closely related to its phenolic substances, and compounds 11, 21, 31, and 38 were the active substances of PUL. It revealed that P. utilis could be developed as a new source of antiallergic agents for ACD therapy.

New method for morphological identification and simultaneous quantification of multiple tetracyclines by a white fluorescent probe.

The threat of tetracycline antibiotics to the environment and human health is attracting widespread attention. The development of morphological analysis and quantitative techniques of multiple tetracyclines is of great significance for the evaluation of biochemical toxicity, wide-spectrum antibacterial property and degradation cycle between different tetracyclines. In this study, the white fluorescent Eu/Tb@CDs was synthesized and applied successfully to the identification and detection of the most widely used tetracycline antibiotics (tetracycline (TC), oxytetracycline (OC), chlortetracycline (CC) and doxycycline (DC)) with detection limits all below 1nM. For the actual water samples with coexistence of the above 4 tetracyclines, their simultaneous morphology identification and accurate quantitative detection can also be realized through simple spectrometric measurement. In addition, the selective and competitive experiments have been carried out on the pollutants widely present in water, and the results have also confirmed that other pollutants could not interfere with the detection of the above 4 tetracyclines. It is undeniable that this work will conveniently and visually reveal the existence information and geographical distribution characteristics of different tetracycline antibiotics in the environment and their action mechanism on organisms.

Simultaneous estimation of voriconazole, moxifloxacin, and pirfenidone in rabbit lacrimal matrix using LC-MS/MS: an application to preclinical ocular pharmacokinetics.

Dynamic emergence of microbial keratitis (MK) requires a promising therapeutic arsenal of antifungal and antibacterial agents like voriconazole (VCZ) and moxifloxacin (MOXI), respectively. Parallelly, another paradigm of MK associated with ulcerative wounds cannot be left unnoticed and requires antifibrotic remedy (pirfenidone, PIR) as an authalic antimicrobial to retain the primordial vision. For designing an effective clinical cure, a combination of these three agents is required at a therapeutic dosage regimen. Following the quest, we have developed a simple and sensitive LC-MS/MS bioanalytical method for simultaneous quantification of VCZ, MOXI, and PIR in rabbit lacrimal fluid. The method was validated as per US-FDA norms using ketoconazole as an internal standard for linearity, accuracy-precision, matrix effect, dilution integrity, selectivity, and stability. The five minutes chromatographic set-up includes isocratic elution with a C18 column using MeOH (80%, v/v) and ultrapure water containing 0.2% formic acid (20%, v/v), respectively. The MS-based analyte detection was achieved in ESI+ multiple reaction monitoring mode. The sample extraction was performed using the protein precipitation method with minimal sample size. The validated methodology was employed to determine the ocular pharmacokinetics profile of marketed formulations containing VCZ, MOXI, and PIR in rabbit lacrimal matrix.