DEVELOPMENT AND VALIDATION OF HPLC METHOD FOR THE SIMULTANEOUS DETERMINATION OF ENALAPRIL MALEATE IN PRESENT OF THEIR IMPURITIES: APPLICATION TO TABLET ANALYSIS

Objective: A simple, rapid, economical, and highly sensitive stability-indicating HPLC method was developed and fully validated for determination of enalapril maleate in presence of its related substances namely enalaprilat dihydrate and diketopiperazine.Methods: Chromatographic separation was achieved on Grace Platinumр C8 EPS column (4.6 mm i.d. X 250 mm, 5 μm) at room temperature. The mobile phase consisted of acetonitrile: 20 mmol phosphate buffer adjusted to pH 2.2 (25:75 v/v) isocratically pumped at a flow rate 2 ml/min and UV-detection was monitored at 215 nm.Results: The proposed method was validated according to ICH guidelines with total run time less than 9 min. The correlation coefficient (r2) was noted as 0.99981 which states that the method was good linear to the concentration versus peak area responses. The developed method found to be high sensitivity with LOD and LOQ of 0.021 and 0.062 %; respectively. The developed, validated method was successfully applied for the determination of enalapril maleate in presence of their impurities in tablet dosage form.Conclusion: A rapid, economical, simple and sensitive HPLC method was developed and validated for the determination of enalapril maleate in tablet dosage form in presence of their impurities. The developed method can help research studies, quality control and routine analysis with lesser resources available. Therefore, the proposed validated method is fast and reliable and can be used for routine quantitative analysis as well as quality control of enalapril maleate in pharmaceutical formulation

HPLC METHOD DEVELOPMENT FOR THE ANALYSIS OF BISOPROLOL IN COMBINED DOSAGE FORM CONTAINING BISOPROLOL AND ENALAPRIL AND IN VITRO DISSOLUTION STUDIED

Objective: A simple, rapid and reproducible HPLC method was developed for the determination of bisoprolol in experimental combined dosage forms containing bisoprolol and enalapril and for drug dissolution studies. Methods: A C18 column (Hi Qsil C18, 5 μm, 4.6х250 mm) and a mobile phase methanol: phosphate buffer solution (65:35, v/v) mixture were used for separation and quantification. Analyses were run at a flow rate of 1.0 ml/min and at ambient temperature. The injection volume was 300 μL and the ultraviolet detector was set at 225 nm. The method was validated as per ICH guidelines. Results: Under these conditions, bisoprolol was eluted at 4.75 min. Total run time was shorter than 6 min. A linear relationship between the concentration and the area of ​​chromatographic peaks of bisoprolol in the range of 0.625 mg/ml-5.000 mg/ml (3.750 mg/ml at pH 1.2) has been established. In the medium with pH 1.2 release of bisoprolol from tablets in 5 min is 38.42%, and after 15 min-85.51%, in medium with pH 4.5 the release of bisoprolol from tablets in 5 min makes 59.78%, and after 15 min-103.71%; in a medium with pH 6.8, the release of bisoprolol from tablets in 5 min is 61.29%, and after 15 min-85.90%. Conclusion: The developed method was applied successfully for quality control assay of bisoprolol in experimental tablets and in vitro dissolution studies

ASSOCIATION OF OBESITY WITH RS1421085 AND RS9939609 POLYMORPHISMS OF FTO GENE WITH T2DM IN EGYPTIAN FEMALES

Objective: Obesity has been described as a worldwide increasing health problem and risk factor of various disorders including type 2 diabetes mellitus (T2DM). So, our study aim to determine of common variants of fat mass and obesity associated gene polymorphisms rs1421085 and rs 9939609; confers risk of obesity and type 2 diabetic mellitus in Egyptian females.Methods: In this population rs1421085 and rs9939609 polymorphisms of fat mass and obesity (FTO) gene were genotyped in 105 obese patients and 100 healthy controls with ages 14-60 y were collected from Medicine Specialized Hospital, Mansoura University, Egypt during the period between Jul.-Oct. 2016, genotyping of SNPs was performed by restriction fragment length polymorphism (RFLP) assay, fasting blood glucose, homeostasis model assessment of insulin resistance (HOMA IR), body mass index (BMI), waist-to-hip ratio (WHR) lipid profile was determined.Results: There was the significantly higher frequency of the AA compared to controls p=0.0001) of genotypers9939609. Also, cases have shown a significantly higher frequency of the C allele, p<0.00001) of rs1421085 genotype polymorphisms increased the risks of obesity. On the other hand, there were no significant correlations between genotypes and obesity-related (anthropometric body composition) parameters. Only the fasting blood glucose was significantly higher in the TA p=0.004).Conclusion: The FTO rs9939609 and rs1421085 single nucleotide polymorphisms (SNPs) was associated with increased risk of obesity in type 2 diabetic populations on Egyptian females

Biosimilars: Review of regulatory, manufacturing, analytical aspects and beyond

Biologics have more complex production processes compared to small-molecule drugs. They may even prove labile when drifting from batch-to-batch or in different production locations. The development of new similar biological product was regulated early to face the relevant challenges of this industry. As a result, since 2006 biosimilars were introduced to biotechnology arena with a massive competition in pharmaceutical industry. In this review, the aspects related to similarity testing of biosimilars to the original biological products are discussed involving manufacturing challenges to ensure the quality, safety, and efficacy of these products to the patient health. Immunogenicity studies are highlighted as an important part of the safety assessments. Additionally, several analytical methods that are usually used to evaluate biosimilars in comparison to their reference biologic are summarized and categorized in terms of the intended physicochemical and biological characterization. On the other hand, the international efforts of several regulatory agencies including the European Medicines Agency, World Health Organization and United States Food and Drug Administration for biosimilar development are discussed according to updated revised guidelines

Two different synchronous spectrofluorimetric approaches for simultaneous determination of febuxostat and ibuprofen

Two green, simple and sensitive synchronous spectrofluorimetric methods were developed for the first time for the simultaneous estimation of febuxostat (FEB) and ibuprofen (IBU). Method I is constant-wavelength synchronous spectrofluorimetry where FEB and IBU were recorded at 329 and 258 nm, respectively, using Δλ of 40 nm. Method II is constant-energy synchronous spectrofluorimetry using a wavenumber interval of −4000 cm−1. All measurements were carried out in a borate buffer of pH 7 and distilled water for dilution which increased the methods' greenness. The two methods were rectilinear over concentration ranges of 30.0–700.0 ng ml−1 and 0.5–9.0 µg ml−1 in the first method and 20.0–500.0 ng ml−1 and 0.1–8.0 µg ml−1 in the second method for FEB and IBU, respectively. High sensitivity was attained for the two drugs with limits of quantitations (LODs) down to 0.41 and 5.51 ng ml−1 in the first method and 0.25 and 3.32 ng ml−1 in the second method for FEB and IBU, respectively. Recovery percentages were in the range of 97.3–101.9% after extraction from spiked human plasma samples, demonstrating high bioanalytical applicability. The two methods were further applied to tablet dosage forms with good recovery results. The methods' greenness was assessed according to the analytical Eco-Scale and Green Analytical Procedure Index guidelines

The Antihypertensive Effect of Quercetin in Young Spontaneously Hypertensive Rats; Role of Arachidonic Acid Metabolism

Hypertension affects almost 50% of the adult American population. Metabolites of arachidonic acid (AA) in the kidney play an important role in blood pressure regulation. The present study investigates the blood pressure-lowering potential of quercetin (QR), a naturally occurring polyphenol, and examines its correlation to the modulation of AA metabolism. Spontaneously hypertensive rats (SHR) were randomly divided into four groups. Treatment groups were administered QR in drinking water at concentrations of 10, 30, and 60 mg/L. Blood pressure was monitored at seven-day intervals. After a total of seven weeks of treatment, rats were killed and kidney tissues were collected to examine the activity of the two major enzymes involved in AA metabolism in the kidney, namely cytochrome P450 (CYP)4A and soluble epoxide hydrolase (sEH). Medium- and high-dose QR resisted the rise in blood pressure observed in the untreated SHR and significantly inhibited the activity of the CYP4A enzyme in renal cortical microsomes. The activity of the sEH enzyme in renal cortical cytosols was significantly inhibited only by the high QR dose. Our data not only demonstrate the antihypertensive effect of QR, but also provide a novel mechanism for its underlying cardioprotective properties

Development and Validation of a Versatile UPLC-PDA Method for Simultaneous Determination of Paracetamol, Tizanidine, Aceclofenac, and Nimesulide in Their New Combinations

A simple, rapid, and validated UPLC method was developed for the simultaneous quantitation of paracetamol (PAR), tizanidine (TIZ), aceclofenac (ACF), and nimesulide (NIM) either in pure forms or in their different tablet dosage forms. Chromatographic separation was attained on an ACQUITY UPLC™ BEH C18 column (100 mm × 2.1 mm, 1.7 μm) with a mobile phase consisting of 20 mM phosphate buffer (pH 7.0) : acetonitrile in the proportion (60 : 40 v/v) isocratically pumped at a flow rate of 1.25 mL·min−1, and detection was monitored at 305 nm. All analytes were separated simultaneously at a retention time (tr) of 1.42, 2.31, 3.63, and 5.62 min for PAR, TIZ, ACF, and NIM, respectively, with a total run time less than 6.0 min. The proposed method was validated according to ICH guidelines with respect to accuracy, precision, linearity, limit of detection, limit of quantitation, and robustness. Linearity was obtained over a concentration range of 81.25–487.5, 0.5–3.5, 25–150, and 25–150 µg·mL−1 for PAR, TIZ, ACF, and NIM, respectively. The development method can be successfully employed in QC laboratories for the routine analysis of the investigated drugs in their new combination

Development and Validation of High-Throughput Bioanalytical Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) Method for the Quantification of Newly Synthesized Antitumor Carbonic Anhydrase Inhibitors in Human Plasma

In the present study, a sensitive and fully validated bioanalytical high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed for the quantitative determination of three newly synthesized carbonic anhydrases inhibitors (CAIs) with potential antitumor activity in human plasma. The analytes and the internal standard (IS) were extracted using 1.5 mL acetonitrile from only 450 µL aliquots of human plasma to achieve the desired protein precipitation. Chromatographic separations were achieved on Phenomenex Kinetex® C18 column (100 × 4.6 mm, 2.6 µm) using a binary gradient elution mode with a run time of less than 6 min. The mobile phase consisted of solvent (A): 0.1% formic acid in 50% methanol and solvent B: 0.1% formic acid in acetonitrile (30:70, v/v), pumped at a flow rate of 0.8 mL/min. Detection was employed using triple quadrupole tandem mass spectrometer (API 3500) equipped with an electrospray ionization (ESI) source in the positive ion mode. Multiple reaction monitoring (MRM) mode was selected for quantitation through monitoring the precursor-to-parent ion transition at m/z 291.9 → 173.0, m/z 396.9 → 225.1, m/z 388.9 → 217.0, and m/z 146.9 → 91.0 for AW-9a, WES-1, WES-2, and Coumarin (IS), respectively. Linearity was computed using the weighted least-squares linear regression method (1/x2) over a concentration range of 1–1000, 2.5–800, and 5–500 ng/mL for AW-9a, WES-1, and WES-2; respectively. The bioanalytical LC-MS/MS method was fully validated as per U.S. Food and Drug Administration (FDA) guidelines with all respect to linearity, accuracy, precision, carry-over, selectivity, dilution integrity, and stability. The proposed LC-MS/MS method was applied successfully for the determination of all investigated drugs in spiked human plasma with no significant matrix effect, which is a crucial cornerstone in further therapeutic drug monitoring of newly developed therapeutic agents

Analytical quality-by-design approach for development and validation of HPLC method for the simultaneous estimation of omarigliptin, metformin, and ezetimibe: application to human plasma and dosage forms

Abstract A simple, selective, and sensitive RP-HPLC method was proposed for the simultaneous determination of two co-administered antidiabetic drugs (omarigliptin and metformin) with an anti-hyperlipidemic drug (ezetimibe) in a medicinally-recommended ratio of 2.5:50:1, respectively. The proposed procedure was optimized by adopting a quality-by-design approach. The influence of different factors on chromatographic responses was optimized by applying the two-level full factorial design (25). The optimum chromatographic separation was achieved using Hypersil BDS C18 column at 45 °C, and the mobile phase pumped isocratically composed of methanol: potassium dihydrogen phosphate buffer (6.6 mM; pH 7, 67:33% v/v) at a flow rate of 0.814 mL/min using 235 nm as a detection wavelength. The developed method was capable of separating this novel mixture in less than 8 min. The calibration plots of omarigliptin, metformin, and ezetimibe showed acceptable linearity over the ranges of 0.2-2.0, 0.5–25.0, and 0.1-2.0 µg/mL with quantitation limits of 0.06, 0.50, and 0.06 µg/mL, respectively. The proposed method was successfully applied to determine the studied drugs in their commercial tablets with high % recoveries (96.8-102.92%) and low % RSD values (less than 2%). The applicability of the method was extended to the in-vitro assay of the drugs in spiked human plasma samples with high % recoveries (94.3-105.7%). The suggested method was validated in accordance with ICH guidelines