DEVELOPMENT AND VALIDATION OF A STABILITY-INDICATING RP-HPLC METHOD FOR THE DETERMINATION OF XIPAMIDE IN PURE AND DOSAGE FORMS

Objective: A simple, selective, precise and stability-indicating RP-HPLC-method was developed and validated for the determination of xipamide (XIP).Methods: Stability tests were done through exposure of the analyte solution to thermal, photolytic, hydrolytic and oxidative stress conditions. The chromatographic separation was carried out in less than five min on a RP stainless-steel C-18 analytical column (150 mm ×4.6 mm ID, 5 µm) with an isocratic elution system of 0.023 M orthophosphoric acid of pH 2.6 and acetonitrile as the mobile phase in the ratio of 60: 40 at 1.5 ml/min flow rate at room temperature. A diode array UV was used at 220 nm for detection.Results: The degradation products were well separated from the pure drug. The elution time of XIP was found to be 4.561±0.024 min. The method was validated in terms of linearity, accuracy, precision, limit of detection (LOD), limit of quantitation (LOQ) and robustness. Good linearity was found in the concentration range of 1–100 µg/ml with a correlation coefficient of 0.9999. Intraday and interday precision were within 1.4%. LOD and LOQ were 0.088 μg/ml and 0.267 μg/ml, respectively and percentage recovery of XIP was found to be 99.92±1.02 %. Conclusion: The proposed method was successfully applied to the determination of XIP in pure form and in its pharmaceutical preparation without interference from its degradation products.Keywords: Xipamide, Stability indicating RP-HPLC, Stress degradation, Pure form, Dosage form

High performance liquid chromatography for simultaneous determination of xipamide, triamterene and hydrochlorothiazide in bulk drug samples and dosage forms

A novel, simple and robust high-performance liquid chromatography (HPLC) method was developed and validated for simultaneous determination of xipamide (XIP), triamterene (TRI) and hydrochlorothiazide (HCT) in their bulk powders and dosage forms. Chromatographic separation was carried out in less than two minutes. The separation was performed on a RP C-18 stationary phase with an isocratic elution system consisting of 0.03 mol L–1orthophosphoric acid (pH 2.3) and acetonitrile (ACN) as the mobile phase in the ratio of 50:50 at 2.0 mL min–1 flow rate at room temperature. Detection was performed at 220 nm. Validation was performed concerning system suitability, limits of detection and quantitation, accuracy, precision, linearity and robustness. Calibration curves were rectilinear over the range of 0.195–100 µg mL–1 for all the drugs studied. Recovery values were 99.9, 99.6 and 99.0 % for XIP, TRI and HCT, respectively. The method was applied to simultaneous determination of the studied analytes in their pharmaceutical dosage forms

Development of High-Throughput Method for Measurement of Vascular Nitric Oxide Generation in Microplate Reader

Background: Despite the importance of nitric oxide (NO) in vascular physiology and pathology, a high-throughput method for the quantification of its vascular generation is lacking. Objective: By using the fluorescent probe 4-amino-5-methylamino-2′,7′-difluorofluorescein (DAF-FM), we have optimized a simple method for the determination of the generation of endothelial nitric oxide in a microplate format. Methods: A nitric oxide donor was used (3-morpholinosydnonimine hydrochloride, SIN-1). Different factors affecting the method were studied, such as the effects of dye concentration, different buffers, time of reaction, gain, and number of flashes. Results: Beer’s law was linear over a nanomolar range (1–10 nM) of SIN-1 with wavelengths of maximum excitation and emission at 495 and 525 nm; the limit of detection reached 0.897 nM. Under the optimized conditions, the generation of rat aortic endothelial NO was measured by incubating DAF-FM with serial concentrations (10–1000 µM) of acetylcholine (ACh) for 3 min. To confirm specificity, Nω-Nitro-l-arginine methyl ester (l-NAME)—the standard inhibitor of endothelial NO synthase—was found to inhibit the ACh-stimulated generation of NO. In addition, vessels pre-exposed for 1 h to 400 µM of the endothelial damaging agent methyl glyoxal showed inhibited NO generation when compared to the control stimulated by ACh. Conclusions: The capability of the method to measure micro-volume samples makes it convenient for the simultaneous handling of a very large number of samples. Additionally, it allows samples to be run simultaneously with their replicates to ensure identical experimental conditions, thus minimizing the effect of biological variability

Development and validation of an eco-friendly HPLC–UV method for determination of atorvastatin and vitamin D3 in pure form and pharmaceutical formulation

Abstract Statin-associated muscle symptoms are considered as obvious adverse effects of prolonged statin therapy such as myopathy, myalgia, and rhabdomyolysis. These side effects are associated with vitamin D3 deficiency and can be adjusted by amendment of serum vitamin D3 level. Green chemistry aims to decrease the harmful effects of analytical procedures. Here we have developed a green and eco-friendly HPLC method for the determination of atorvastatin calcium and vitamin D3. The two drugs were separated in less than 10 min on Symmetry column C18 (100 × 4.6 mm, 3.5 µm) using a mixture consisting of 0.1% ortho-phosphoric acid (OPA) (pH = 2.16) and ethanol as the mobile phase in gradient manner. We have used Green Analytical Procedure Index (GAPI) tools and the Analytical GREEnness Metric Approach (AGREE) for assessment of the greenness of our proposed method. The method proved linearity over concentration ranges of (5–40) and (1–8) µg/ml with low limit of detection of 0.475 and 0.041 µg/ml for atorvastatin calcium and vitamin D3 respectively. The method was successfully validated in accordance with ICH instructions and utilized for determination of the drugs of interest either in pure form or in their pharmaceuticals

Determination of clemastine hydrogen fumarate, desloratadine, losartan potassium and moxepril HCl through binary complex formation with eosin

A simple and sensitive spectrophotometric method has been established for the determination of clemastine hydrogen fumarate (I), desloratadine (II), losartan potassium(III) and moxepril HCl(IV) based on binary complex formation with eosin. The method does not involve solvent extraction through the use of a non-ionic surfactant (methylcellulose). The color of the produced complex was measured at 552, 549 nm for (I), (II) while was measured at 540 nm for (III) and (IV). Appropriate conditions were established for the color reaction between eosin and the studied drugs to obtain maximum sensitivity. Under the proposed conditions, the method is applicable over concentration range of 1.25–11.25, 0.31–2.81, 2.5–20 and 1.25–15 μg/ml for (I), (II), (III) and (IV), respectively. The molar absorptivity (ε), sandell sensitivity, detection (LOD) and quantitation limits (LOQ) are calculated. Unlike other reported ion-pair techniques, the suggested methods have the advantage of being applicable for the determination of the four drugs in their pharmaceutical dosage forms without prior extraction with excellent recoveries

Multi-Steps Fragmentation-Ion Trap Mass Spectrometry Coupled to Liquid Chromatography Diode Array System for Investigation of Olaparib Related Substances

A high-performance liquid chromatography-diode array-mass spectrometric (LC-DAD-MS) method was developed and validated to investigate the related substances of olaparib (OLA) in bulk form. OLA was exposed to acid⁻base hydrolysis, boiling, oxidation with hydrogen peroxide, and UV light followed by LC-DAD-MS analysis. OLA and OLA-related substances were simultaneously and quantitatively monitored by DAD at 278 nm and triple quadrupole mass spectrometry (QQQ-MS). The investigated compounds were auto-scanned by an ion trap MS which applied positive and negative modes separately. The fragmentation pathway was confirmed by applying multi-steps fragmentation to identify the resulted cleaved ions and their parent ion. OLA was found to be sensitive to the alkaline hydrolysis and less sensitive to UV light. Two major hydrolytic degradation products, including the protonated molar ions m/z 299 and m/z 367, were identified. Three potential impurities were also characterized. The LC-MS limit of detection (LOD) and limit of quantification (LOQ) were 0.01 and 0.05 ng/µL, respectively. The quantitative results obtained by LC-DAD was comparable with that of LC-QQQ-MS. The proposed method shows good intra-day and inter-day precision with relative standard deviation (RSD) <2%

High performance liquid chromatography for simultaneous determination of xipamide, triamterene and hydrochlorothiazide in bulk drug samples and dosage forms

A novel, simple and robust high-performance liquid chromatography (HPLC) method was developed and validated for simultaneous determination of xipamide (XIP), triamterene (TRI) and hydrochlorothiazide (HCT) in their bulk powders and dosage forms. Chromatographic separation was carried out in less than two minutes. The separation was performed on a RP C-18 stationary phase with an isocratic elution system consisting of 0.03 mol L−1 orthophosphoric acid (pH 2.3) and acetonitrile (ACN) as the mobile phase in the ratio of 50:50, at 2.0 mL min−1 flow rate at room temperature. Detection was performed at 220 nm. Validation was performed concerning system suitability, limits of detection and quantitation, accuracy, precision, linearity and robustness. Calibration curves were rectilinear over the range of 0.195–100 μg mL−1 for all the drugs studied. Recovery values were 99.9, 99.6 and 99.0 % for XIP, TRI and HCT, respectively. The method was applied to simultaneous determination of the studied analytes in their pharmaceutical dosage forms

Rp-HPLC Determination of Quercetin in a Novel D-α-Tocopherol Polyethylene Glycol 1000 Succinate Based SNEDDS Formulation: Pharmacokinetics in Rat Plasma

Despite its proven efficacy in diverse metabolic disorders, quercetin (QU) for clinical use is still limited because of its low bioavailability. D-α-Tocopherol polyethylene glycol 1000 succinate (TPGS) is approved as a safe pharmaceutical adjuvant with marked antioxidant and anti-inflammatory activities. In the current study, several QU-loaded self-nanoemulsifying drug delivery systems (SNEDDS) were investigated to improve QU bioavailability. A reversed phase high performance liquid chromatography (RP-HPLC) method was developed, for the first time, as a simple and sensitive technique for pharmacokinetic studies of QU in the presence of TPGS SNEDDS formula in rat plasma. The analyses were performed on a Xterra C18 column (4.6 × 100 mm, 5 µm) and UV detection at 280 nm. The analytes were separated by a gradient system of methanol and phosphate buffer of pH 3. The developed RP-HPLC method showed low limit of detection (LODs) of 7.65 and 22.09 ng/mL and LOQs of 23.19 and 66.96 ng/mL for QU and TPGS, respectively, which allowed their determination in real rat plasma samples. The method was linear over a wide range, (30–10,000) and (100–10,000) ng/mL for QU and TPGS, respectively. The selected SNEDDS formula, containing 50% w/w TPGS, 30% polyethylene glycol 200 (PEG 200), and 20% w/w pumpkin seed oil (PSO), showed a globule size of 320 nm and −28.6 mV zeta potential. Results of the pharmacokinetic studies showed 149.8% improvement in bioavailability of QU in SNEDDS relative to its suspension. The developed HPLC method proved to be simple and sensitive for QU and TPGS simultaneous determination in rat plasma after oral administration of the new SNEDDS formula

Rp-HPLC Determination of Quercetin in a Novel D-α-Tocopherol Polyethylene Glycol 1000 Succinate Based SNEDDS Formulation: Pharmacokinetics in Rat Plasma

Despite its proven efficacy in diverse metabolic disorders, quercetin (QU) for clinical use is still limited because of its low bioavailability. D-α-Tocopherol polyethylene glycol 1000 succinate (TPGS) is approved as a safe pharmaceutical adjuvant with marked antioxidant and anti-inflammatory activities. In the current study, several QU-loaded self-nanoemulsifying drug delivery systems (SNEDDS) were investigated to improve QU bioavailability. A reversed phase high performance liquid chromatography (RP-HPLC) method was developed, for the first time, as a simple and sensitive technique for pharmacokinetic studies of QU in the presence of TPGS SNEDDS formula in rat plasma. The analyses were performed on a Xterra C18 column (4.6 × 100 mm, 5 µm) and UV detection at 280 nm. The analytes were separated by a gradient system of methanol and phosphate buffer of pH 3. The developed RP-HPLC method showed low limit of detection (LODs) of 7.65 and 22.09 ng/mL and LOQs of 23.19 and 66.96 ng/mL for QU and TPGS, respectively, which allowed their determination in real rat plasma samples. The method was linear over a wide range, (30–10,000) and (100–10,000) ng/mL for QU and TPGS, respectively. The selected SNEDDS formula, containing 50% w/w TPGS, 30% polyethylene glycol 200 (PEG 200), and 20% w/w pumpkin seed oil (PSO), showed a globule size of 320 nm and −28.6 mV zeta potential. Results of the pharmacokinetic studies showed 149.8% improvement in bioavailability of QU in SNEDDS relative to its suspension. The developed HPLC method proved to be simple and sensitive for QU and TPGS simultaneous determination in rat plasma after oral administration of the new SNEDDS formula