Simple Liquid Chromatographic Method Research Articles

Box-Behnken design in optimization of the green liquid chromatographic method for the quantification of Afatinib in drug product: AQbD approach

The aim of this research work was to quantitatively determine the tyrosine kinase inhibitor, Afatinib, in tablets using an eco-friendly chromatographic method. The development of a green liquid chromatographic method using analytical quality-by-design (AQbD) approach principles to estimate Afatinib via Box-Behnken design. A simple and effective green liquid chromatographic method was developed and validated. The analysis was done on the Zodiac C18 column by isocratic elution mode. Mobile phase composed of pH 3.0 potassium dihydrogen phosphate buffer and ethanol in the ratio of 65:35%V/V. The flow rate was set to 0.6 mL/min. with an injection volume of 10 µL and at detection wavelength of 254 nm. The optimized method was found to be specific, linear, precise, accurate, and robust. Established linearity in the range of 24.6 to 73.9 µg/mL. The developed method exhibited excellent greenness. Based on the greenness assessment tools, the analytical eco-score value of 94 and the pictograms of NEMI, Modified NEMI, GAPI, Complex GAPI, AGREE, and AGREE prep expressed the method’s greenness. Hence, the AQbD approach based green method and the validation data indicate that the method can be employed in routine analysis for the determination of Afatinib in pharmaceutical dosage form in quality control laboratory use.

RP-HPLC Method Development and Validation for Simultaneous Determination of Sofosbuvir and Ledipasvir: A Pharmaceutical Application Study

The objective of the present work was to develop a simple, precise, accurate, reproducible, and specific reversed-phase high performance liquid chromatographic method for the simultaneous determination of sofosbuvir and ledipasvir in pharmaceutical dosage form. The HPLC system (Shimadzu, LC-2030) consisted of Hypersil ODS - C18 (250 mm, 4.6 mm, 5 μm- Thermo Scientific) was used for the isocratic elution using mobile phase composed of sodium phosphate buffer (pH 3.5) and acetonitrile in the ratio of 40:60% (v/v). Both sofosbuvir and ledipasvir were detected at 260 nm with a flow rate of 1 mL/min. The mean retention time of sofosbuvir was 3.094, whereas ledipasvir was 6.22 minutes. A linear relationship was found in the concentration ranged from 0.54 to 162 μg/mL for sofosbuvir (r2 = 0.9993) and 0.14 to 42 μg/mL for ledipasvir (r2 = 0.9994). The RSD values for recovery test were found 0.99% for sofosbuvir and 0.22% for ledipasvir. The lower limit of detection (LOD) were 0.054 mcg/mL and 0.012 mcg/mL, and the limit of quantification (LOQ) were 0.135 mcg/mL and 0.030 mcg/mL for sofosbuvir and ledipasvir, respectively. The method was successfully applied to the assay and in-vitro dissolution studies of sofosbuvir and ledipasvir in tablet dosage form and all the results were found within the acceptance limits.

A simple and sensitive high-performance liquid chromatographic method combined with fluorescence detection for bioanalysis of scopoletin in rat plasma: Application to a pharmacokinetic study.

Scopoletin, a coumarin class natural phytoalexin, is present in medicinal plants such as noni (Morinda citrifolia). It exhibits diverse pharmacological properties, including antioxidant, anti-hyperuricemic, and anti-inflammatory effects. The objective of this study was to develop a novel HPLC-fluorescence (HPLC-FL) method for the quantitative analysis of scopoletin in the plasma and to investigate its pharmacokinetics in rats. Sample preparation involved a methanol-based protein precipitation method, and chromatographic separation was conducted using a C18 column with an isocratic mobile phase composed of water and acetonitrile containing 0.1% trifluoroacetic acid. The eluent was detected using an FL detector set to optimized excitation/emission wavelengths of 337/453 nm. Method validation encompassed assessments of selectivity, linearity (1-500 ng/mL), precision, accuracy, recovery, matrix effect, and stability in accordance with the prevailing Food and Drug Administration (FDA) guidelines. The developed method was successfully applied for pharmacokineticstudy in rats. To the best of our knowledge, this study is the first application of a simple and sensitive HPLC-FL method for the quantification of scopoletin in a pharmacokinetic study. This method offers a promising alternative for preclinical pharmacokinetic investigations with appropriate modifications and validations and holds potential for clinical applications.

APPLICATION OF VALIDATED RP-HPLC METHOD FOR SIMULTANEOUS DETERMINATION OF METAXALONE AND DICLOFENAC POTASSIUM IN PLASMA

Objective: The present investigation demonstrates a simple, sensitive and accurate high-pressure liquid chromatographic (HPLC) method for simultaneous determination of metaxalone (MTX) and Diclofenac potassium (DIC) in plasma by using Valsartan (VSN) as internal standard. Methods: The chromatographic separation was achieved within 10 min by using methanol: potassium dihydrogen phosphate buffer pH 4.5 adjusted with orthophosphoric acid (60:40) as mobile phase on Altima Grace Smart C-18 column (5μ; 250×4.6 mm) at flow rate of 1.0 ml/min with injection volume 25µl. The drug was extracted from plasma by liquid-liquid extraction using methanol as a solvent. The retention times of drugs (MTX and DIC) and internal standard were found to be 5.83, 9.65 and 11.79 min, respectively. This method was validated as per United States Food and Drug Administration (US-FDA) guidelines. Results: The results of the validation parameters were found to be within the acceptance limits. The method was linear in the concentration range from 25-1000 ng/ml (r2= 0.9998) and the extraction recovery was found to be 77.06% for MTX and 78.37% for DIC. The lower limit of quantification was found to be 25ng/ml and the stability of recovered samples at different conditions were found to be more than 95% for both the drugs. Conclusion: The developed method possesses good selectivity specificity, there was no interference found in the plasma blanks at retention times of MTX and DIC. We found good correlation between the peak area and concentration of the drug under prescribed conditions. Furthermore, the method can also be used to estimate the pharmacokinetic parameters of MTX and DIC simultaneously.

A validated stability‐indicating reversed‐phase‐UPLC method for simultaneous estimation of promethazine hydrochloride, methylparaben, propylparaben and sodium benzoate assay of cough suppressant and antihistamine liquid oral dosage forms

A quick, simple, sensitive, efficient and stability-indicating reverse-phase ultraperformance liquid chromatographic method for the estimation of propylparaben, methylparaben and sodium benzoate in a pharmaceutical liquid oral formulation was developed. A Waters Acquity UPLC BEH C18, 50 × 2.1 mm, 1.7 μm i.d. column was used to perform chromatographic separation with a 0.1% perchloric acid mobile phase used as solvent A and a mixture of 0.1 % perchloric acid and methanol in the ratio 20:80 (v/v), respectively, as solvent B. The experiments were carried out at a flow rate of 0.4 ml/min and the detection wavelength was 240 nm. The compartment temperature of the column was set at 40°C and the injection volume was set at 2 μl. The main aim of the research was to develop a single UPLC assay method for promethazine (active ingredient) and preservatives in the oral solution of promethazine HCl and dextromethorphan HBr that contains promethazine (active ingredient) and methylparaben, propylparaben and sodium benzoate (preservatives). An assay of dextromethorphan HBr was developed and validated by another HPLC method. The drug and preservatives were eluted at retention times of 19.3 min for promethazine HCl, 9.3 min for methylparaben, 18.9 min for propylparaben and 8.9 min for sodium benzoate. Validation of the developed method was carried out as stated by the International Conference on Harmonization guidelines ICH Q2B and under USP<1225>. The analytical parameters verified specificity/selectivity, linearity, accuracy, ruggedness and robustness. The linearity ranges of promethazine HCL, methylparaben, propylparaben and sodium benzoate were 10-100, 10-80, 1.0-8.0 and 10-80 μg/ml, respectively, with a correlation coefficient of active ingredients and preservatives of 1.00. Percentage recoveries of promethazine, propylparaben, methylparaben, and sodium benzoate were 100.0-100.2, 99.0-100.3, 99.5-98.0 and 99.0-100.0%. The validated analytical method proves that the method is specific, precise, linear, accurate, sensitive, rugged and stable, indicating the quantification of the active ingredient and all preservatives in liquid oral formulations.

Development and Validation of New HPLC Method for the Determination of Imidazolidinyl Urea in Topical Formulation

Introduction: In this study, a new, simple, selective, and fast liquid chromatographic method has been developed and validated for the determination of imidazolidinyl urea used as an anti-microbial agent in topical formulation. Methods: The developed HPLC method using a diode array detector for the determination of imidazolidinyl urea was applied to the topical formulation. Imidazolidinyl urea in the sample was analyzed in the cyano column (250 x 4,6 mm, 5 μm i.d.) under chromatographic conditions, where the flow rate was determined as 1.0 mL/min. The column oven was 40.0°C, and imidazolidinyl urea was detected at 210 nm. Isocratic application of acetonitrile-water (25:75, v/v) was used as the mobile phase system. The validation of the developed method was performed according to the International Conference on Harmonisation guidelines Q2 (R1). Results: The linearity range of the imidazolidinyl urea was 0.050-0.150 mg/mL, and the limits of detection and quantification were calculated to be 62.5x10-6 mg/mL and 125x10-6 mg/mL, respectively. Assay recovery and precision of imidazolidinyl urea from topical formulation at 0.050, 0.100, and 0.125 mg/mL concentrations were evaluated. The mean recoveries for imidazolidinyl urea in the topical formulation were calculated as 98.857-104.560%. Conclusion: The validated method was successfully applied to the determination of imidazolidinyl urea in a topical formulation. The proposed method is reproducible and reliable and can be used safely for routine analysis.

STABILITY BASED HPLC METHOD FOR CYCLOPHOSPHAMIDE RELATED SUBSTANCES IN FINISHED DRUG PRODUCTS: DEVELOPMENT AND VALIDATION

Objective: The current study aimed to develop a simple, sensitive, and precise high-performance liquid chromatographic (HPLC) method for estimating cyclophosphamide and its related substances, as well as to implement the developed method in a capsule product. Methods: Method development was performed using various solvent and buffer-solvent ratios at different flow rates for better resolution and to decrease the run time. The developed method was validated in accordance with the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) guidelines. The developed method was implemented to estimate the amount of cyclophosphamide and its related substances. Results: Chromatographical conditions were optimised, and the best chromatographical conditions with adequate resolution for cyclophosphamide and its related substances were achieved using the enable X-Bridge C18 column, using a mobile phase combination of phosphate buffer pH 7.0, water, methanol, and acetonitrile at a flow rate of 0.5 ml/min. The detection was monitored at a wavelength of 200 nm. The developed method was validated for system suitability, specificity, limit of detection (LOD), limit of quantitation (LOQ), linearity, precision, accuracy and robustness. The results indicate that the method was sensitive and could detect and quantify lower levels of cyclophosphamide and its related substances. The linearity range was from LOQ to 150 %, and a correlation coefficient ranging from 0.9987 to 0.9999 indicates that at this concentration range, substances were highly linear. This was further supported by satisfactory forced degradation studies. Conclusion: The developed analytical method is simple, precise, and reproducible and thus can be used for the stability-indicating analysis of cyclophosphamide and its related substances in pharmaceutical formulations.

A SIMPLE RP-HPLC METHOD DEVELOPMENT AND VERIFICATION FOR THE QUANTITATIVE ESTIMATION OF SODIUM BUTYRATE IN TABLETS

Objective: To develop a simple, accurate, precise, and linear reverse-phase high-performance liquid chromatographic (RP-HPLC) method and verify the quantitative estimation (assay) of sodium butyrate in tablets. Methods: The optimized RP-HPLC method uses a reverse phase stationary phase which is YMC Triart C18 column, having specifications of 250×4.6 mm; 5 μm, a mobile phase composition of pH 8.0 sodium dihydrogen phosphate buffer and acetonitrile in the proportion of 92:8 v/v, flow rate of 0.5 ml/min, injection volume of 10 μL, and detection wavelength of 210 nm using a UV/PDA detector. Results: The developed method gave sodium butyrate eluting at about 6 min. Sodium butyrate exhibited linearity in the range of 119.56–1195.6 μg/ ml. The precision was exemplified by a relative standard deviation of 0.30%. The percentage of individual recovery was found to be in the range of 97.0 and 103.0 during accuracy studies. Conclusion: A simple, specific, accurate, precise, and linear RP-HPLC method was developed and verified for the quantitative estimation (assay) of sodium butyrate in tablets and hence this method can be explored for the analysis of sodium butyrate in tablets in various pharmaceutical industries.

HPLC Method Development and Validation for the Determination of Apixaban and Clopidogrel in Novel Fixed-Dose Combination Tablets

A simple, fast, and accurate high-performance liquid chromatographic (HPLC) method is developed, optimized, and validated for a fixed-dose combination of apixaban (APX) and clopidogrel (CLOP) tablets according to ICH guidelines. Chromatographic separation of the drugs was performed on a BDS Hypersil C18 (4.6 ∗ 150 mm, 5 μm), with acetonitrile (ACN) and trifluoroacetic acid (TFA) in the ratio 48 : 52 (v/v) as a mobile phase, at a flow rate of 0.9 ml/min., injection volume of 5 μL, and column temperature 45°C. The proposed method was linear over the level 25–200% for a concentration of APX 5 μg/ml and CLOP 75 μg/ml (R2 &gt; 0.999). The detection limit for APX and CLOP was found to be 0.3465 and 3.8496 μg/ml, whereas the quantification limit was 1.0499 and 11.6656 μg/ml, respectively. The recovery was more than 99% using the standard addition method. The developed method was found to be specific, accurate, precise, and robust against changes in column temperature (±5°C) and mobile phase composition (±5% ACN); hence, it can be used for the determination of APX and CLOP in the fixed-dose combination tablets.

Origanum majorana L. polyphenols: in vivo antiepileptic effect, in silico evaluation of their bioavailability, and interaction with the NMDA receptor.

Introduction: Epilepsy is a chronic brain disease characterized by repeated seizures and caused by excessive glutamate receptor activation. Many plants are traditionally used in the treatment of this disease. This study aimed to evaluate the bioavailability of a polyphenolic extract obtained from Origanum majorana L. (OMP) leaves, as well as its antiepileptic activity and its potential mechanism of action. Methods: We have developed and validated a simple, rapid, and accurate stability-indicating reversed-phase liquid chromatographic method for the simultaneous determination of caffeine and quercetin in rat plasma. The OMP antiepileptic effect was evaluated with pilocarpine-induced seizures, and a docking method was used to determine the possible interaction between caffeic acid and quercetin with the N-methyl-D-aspartate (NMDA) receptor. Results and Discussion: Both compounds tested showed low bioavailability in unchanged form. However, the tested extract showed an anticonvulsant effect due to the considerably delayed onset of seizures in the pilocarpine model at a dose of 100mg/kg. The molecular docking proved a high-affinity interaction between the caffeic acid and quercetin with the NMDA receptor. Taken together, OLP polyphenols demonstrated good antiepileptic activity, probably due to the interaction of quercetin, caffeic acid, or their metabolites with the NMDA receptor.

A SIMPLE RP-HPLC METHOD DEVELOPMENT AND VERIFICATION OF THE DISSOLUTION OF BROMELAIN-A COMPLEX MIXTURE OF PROTEOLYTIC ENZYMES, IN DELAYED-RELEASE TABLETS

Objective: To develop a simple, accurate, precise and linear Reverse Phase High-Performance Liquid Chromatographic (RP-HPLC) method and verify for the quantitative estimation (Dissolution) of Bromelain in delayed-release tablets. Methods: The optimized RP-HPLC method for both acid and buffer stage dissolutions of delayed-release tablets uses Zorbax 300 SB-C8 column (150 mm X 4.6 mm; 3.5μ), a mobile phase-A of 0.1% trifluoroacetic acid in water and a mobile phase-B of 0.1% Trifluoroacetic acid in Acetonitrile in the gradient proportion, flow rate of 1.0 ml/min, injection volume of 25 µl, detection wavelength of 280 nm using a UV/PDA detector, column temperature of 40 °C, sample tray/compartment temperature of 5 °C and a run time of 20 min. Results: The developed method gave Bromelain eluting at about 6 min. Bromelain exhibited linearity in the range 53.4-800.6 μg/ml (r2=0.99992). The precision is exemplified by relative standard deviation of 1.3 and 2.3% for acid and buffer stages, respectively. Percentage recovery of the drug was found to be between 90.0 and 110.0 during accuracy studies. Conclusion: A simple, accurate, precise, and linear RP-HPLC method was developed and verified for the quantitative estimation (Dissolution) of Bromelain in tablets and hence this method can be explored for the analysis of Bromelain in tablets in various pharmaceutical industries.

Development and validation of an HPLC method for the simultaneous estimation of salbutamol, theophylline and ambroxol in tablet dosage form

A simple, accurate and precise reverse phase high performance liquid chromatographic (RP-HPLC) method has been developed and validated for the simultaneous estimation of Salbutamol, Theophylline and Ambroxol in pharmaceutical formulations. The chromatographic separation was achieved on a Inertsil ODS-3V (250 × 4.6 mm, 5μ) column using a mobile phase consisting of phosphate buffer (pH 3.0): acetonitrile in the ratio of 55:45 v/v at a flow rate of 1 mL/min. Detection was carried out at 225 nm. The retention times for Salbutamol, Theophylline and Ambroxol were found to be 2.317, 3.808 and 5.863 min respectively. The proposed method was validated as per the ICH guidelines and was found to be linear in the concentration range of 0.5-3.0 μg/mL, 25-150 μg/mL and 7.5-45 μg/mL for Salbutamol, Theophylline and Ambroxol respectively with correlation coefficients greater than 0.999. The developed method was found to be precise, accurate, robust and specific. Forced degradation studies proved the stability-indicating capability of the developed HPLC method. The developed method can be successfully applied for the routine analysis of Salbutamol, Theophylline and Ambroxol in pharmaceutical dosage forms.

A simple liquid chromatography method running in dual modes for quantification of short and medium chain fatty acids

Sample preparation is often a rate-limiting step in quantification of short chain and medium chain fatty acids (SFAs and MFAs) in biological samples. A novel liquid chromatography (LC) method with simple sample preparation is introduced in this report. The method is performed on a column packed with polyvinyl alcohol sorbents. In separation of a mixture of bovine serum albumin (BSA) with SFAs and MFAs, the protein is eluted in size-exclusion mode in the first part of the chromatogram. SFAs and MFAs are then eluted in the second part of the chromatogram in reverse-phase mode where fatty acids with longer carbon lengths are eluted at longer retention times. SFAs and MFAs are well resolved from each other without pre-column derivatization. The size-exclusion mode affords good tolerance to macromolecules in sample matrixes. The dual mode separation allows sample injections without complicated sample preparation steps, such as derivatization, extraction, evaporation, and reconstitution.

Concepts and principles for new rapid simple liquid chromatography method for quantification of antioxidants resveratrol, vitamin E, and coenzyme Q10 in capsules with high-performance liquid chromatography with a photo-diode array detector.

Principles and problems in the development of simultaneous liquid chromatography (LC) analytical methods for potent antioxidative molecules resveratrol, tocopherol, and coenzyme Q10 in capsules, have been investigated and systematically compared and summarized. For these purposes, experiments within the full polarity spectrum of LC techniques. were tested and recorded. The whole range of polarities included: Alkyl C18 bonded reversed phase, phenyl, cyanopropyl, diol, and the most polar base silica-filled column matrixes have been used. The summarized results concluded that all mentioned LC techniques could be used for the determination of the mentioned group of the three analytes with different run characteristics and efficiency. These successes could be achieved after careful analyses of molecular physicochemical data of analytes. They are especially organic solubilities. The ultraviolet spectral absorption characteristics of each analyte and the mobile phase constituents for appropriate separation were very important to be known. The ultimate targets were the development method with the isocratic mode of separation yielding symmetrical peak shapes for the best sensitivity and accuracy, with the shortest run time and best reproducibility. From an analytical point of view important for LC, the three analytes have quite distinct characteristics that contribute to successful method development. These features are their organic solvent and water solubility, molecular polarities, and ultraviolet-absorption characteristics, like spectra and absorptivities. All these mentioned parameters were taken into account for solving complications appearing in the development of rapid LC methods for the simultaneous determination of three antioxidant molecules.

Simultaneous Quantification of Mometasone Furoate and Formoterol Fumarate in Bulk and Formulations by RP-HPLC Method

A simple and robust reversed-phase high pressure liquid chromatographic method was developed and validated for the quantification of Mometasone and Formoterol in bulk and fixed dosage forms. The column utilized for study was Inertsil C18, ODS was chosen for good peak shape. Ambient temperature was found to be suitable for the nature of drug solution. The mobile phase was streamlined with a ratio of 35:65 Methanol: Water was optimized for symmetrical peaks and good resolution. The flow rate was streamlined at 1.0 ml/min because of good peak area, satisfactory retention time and good resolution. Common λ-max was found to be at 278 nm and Injection volume was 20 µl which gave a good peak area. The percentage recovery was found to be 98.0-101.50 was linear and precise over the same range. Both system and method precision was found to be accurate and well within the acceptable limits. Detection limit was found to be 0.25 Mometasone and 0.34 for Formoterol. The analytical method was found linearity over the range of 20-80 ppm of the target concentration for both the drugs. The LOD and LOQ values for Mometasone, was observed to be 0.25 and 0.77 and for Formoterol was in the range of 0.34 and 1.05 consecutively. The analytical method passed both intermediate precision and robustness tests. On both cases, relative standard deviation was well satisfactory. The validation experiments met ICH standards. It inferred the method found to be simple, accurate, precise and linear. The method was found to be having suitable application in routine laboratory analysis with high degree of accuracy and precision.

A Validated RP-HPLC Method for Simultaneous Estimation of Benidipine and Telmisartan in Bulk and Tablet Dosage Form

Background: In this work, a novel, simple and accurate reverse phase high-performance liquid chromatographic method with UV detection is presented for the simultaneous estimation of Benidipineand Telmisartan in API and marketed formulations. Objective: To develop and validate this method in accordance to regulatory guidelines i.e. ICH Q2B-R1 for analytical parameters. Methods: In RP-HPLC the separation was achieved using a Xbridge C18 (250x 4.6 i.d; 5µm) column with isocratic elution using a mobile phase consisting of methanol: acetonitrile (80:20v/v/ with 0.1% v/v TEA) adjusted to pH 5.2 with glacial acetic acid at 1mL/min flow for 10min. Benidipine and Telmisartan, were detected at dual wavelength at 237 and 297nm with retention time (Rt) 6.891 and 8.997 minutes respectively. Results: Our new methods (RP-HPLC) were found to (r2&gt;0.999) for both Benidipine and Telmisartan. The developed methods are highly selective, precise and reproducible with %RSD &lt; 2%, Moreover the methods are accurate with recovery more than 99% in both methods. Conclusion: The RP-HPLC method results are efficient, simple and easy. These highly sensitive methods were successfully applied for assay determination of both Benidipine and Telmisartan at fixed dose combination.

A NEW RP-HPLC METHOD FOR ESTIMATION OF VERICIGUAT IN BULK DRUG AND PHARMACEUTICAL DOSAGE FORM

A simple, rapid, specific and accurate reverse phase high performance liquid chromatographic method has beendeveloped for the validation of Vericiguat in bulk as well as in marketed pharmaceutical dosage form. This separation wasperformed on a Symmetry ODS C18 (4.6×250mm, 5μm) column with Methanol:Phosphate Buffer (55:45) v/v asmobile phase at a flow rate of 1.0 mL min−1 with UV detection at 225 nm; the constant column temperature wasambient. The run time under these chromatographic conditions was less than 8 min. The retention time of Vericiguatwas found to be 2.252. The calibration plot was linear over the concentration range of 6 - 14 μg mL−1 with limits ofdetection and quantification values of 1.2 and 3.6 μg mL−1 respectively. The mean % assay of marketed formulation wasfound to be 99.86%, and % recovery was observed in the range of 98-102%. Relative standard deviation for theprecision study was found &lt;2%. The developed method was simple, precise, specific, accurate and rapid, making itsuitable for estimation of Vericiguat in bulk and marketed pharmaceutical dosage form dosage form.

Stability-indicating method development and validation for quantitative estimation of organic impurities of the antidiabetic drug glipizide in drug substance and pharmaceutical dosage form using HPLC.

Glipizide is an antidiabetic drug used for the treatment of type 2 diabetes. A simple, reliable and robust reverse-phase liquid chromatographic method (RP-HPLC) was developed and validated as per International Conference on Harmonization Q2(R1) for estimating the impurities of glipizide in pharmaceutical formulations. The chromatographic separation was carried out on a Phenomenex Luna C18 (2), 250 × 4.6mm, 5μm with a binary solvent delivery system [MP-A, a homogenous mixture of water and acetonitrile in a ratio of 90:10 (v/v) and 1ml of orthophosphoric acid; and MP-B, a homogenous mixture of water and acetonitrile in a ratio of 10:90 (v/v) and 1ml of orthophosphoric acid] with a detection wavelength of 225 nm, a column temperature of 30°C, a flow rate of 1.5ml/min, and an injection volume of 20 μl. All process, degradant and unknown impurities were separated well with a resolution >2.2 and were estimated accurately without any interference. The recovered values and regression values were 98.7-100.5% and R2 > 0.9999, respectively. The recovery and linearity studies covered the quantitation limit to 150% of the specification limit. The stability-indicating properties of the developed RP-HPLC method was assessed from the forced degradation studies. The developed method was successfully applied for real-time sample analysis of the glipizide dosage form.

Development and validation of analytical procedure for analysis of Amoxiciline, Metronidazole and Omeprazole, used as anti- Helicobacter pylori agents alone and in mixture

Background: The contemporary treatment of ulcerogenic diseases and gastroesophageal reflux disease is related usually to application of a combination of imidazole-based antibacterial, antibiotic and proton pump inhibitor. In the current study, the three most common representatives Amoxicilline (AMO), Metronidazole (MET) and Omeprazole (OME), respectively, are subjected to analysis through classical analytical procedure, providing high level accuracy, sensitivity and good separation abilities. As such a UV/VIS method was applied as a well known identification and quantitation technique for analyses in various samples. Furthermore, this technique is known to be a good detection method in combination with chromatographic systems. Purpose: A simple, specific, accurate and precise reverse phase-high performance liquid chromatographic method has been developed for the simultaneous determination of Amoxicillin Trihydrate (AMO), Metronidazole (MET) and Omeprazole (OME) in synthetic mixture. Materials and methods: Some important parameters like pH of the mobile phase, concentration of the acid or buffer solution, percentage and type of the organic modifier, etc. were tested for a good chromatographic separation. The sample was analyzed using a mobile phase of Acetonitrile: Phosphate buffer (pH=7.6±0.1) (40:60 v/v). The flow rate was 1.0 mL/ min with detection at 280 nm. Results: The retention time for AMO, MET and OME was found to be 1.67, 2.86 and 5.99 min respectively, and the recoveries in the synthetic mixture were between 98 and 102%. The validated method was linear over the concentration range of 25 to 200 μg/mL for AMO, 12.5 to 100 μg/mL for MET and 5–40 µg/mL for OME, with a correlation coefficient &amp;gt; 0.999. Conclusion: The developed method has been validated in accordance with the International Conference on Harmonization (ICH) guidelines and showed excellent linearity, accuracy, precision, specificity, robustness, as well as system suitability results within the acceptance criteria.

Using the AQbD Approach, Development and Validation of a Simple, Rapid Stability Indicating Chromatographic Method for Quantification of Related Impurities of Apixaban.

Apixaban is a new oral anticoagulant that has been approved by the United States Food and Drug Administration for the prevention of stroke and other cardiovascular complications in people with non-valvular atrial fibrillation. Then, later, it was given the green light to treat deep venous thrombosis and pulmonary embolism. The biggest challenge in the development of pharmaceutical drugs is comprehending science- and risk-based techniques for developing and maintaining analytical procedures appropriate for evaluating the quality of drug substances and drug products. The improved technique provides a methodical strategy to acquire and improve understanding of an analytical method. The current study discusses the related substances method development for an apixaban drug substance employing a regulated authority refined and approved approach of the quality-by-design concept. For the quantification of impurities, and apixaban drug substance, a simple, quick and stability-indicating reverse-phase liquid chromatographic method was developed using a full factorial design. The separation between apixaban and its nine impurities was accomplished using an Zorbax Stable Bond Phenyl, 150 × 4.6mm, 5μm column. The mobile-phase components for gradient elution at a flow rate of 1.2mL/min were chosen to be a mixture of water, acetonitrile, methanol and perchloric acid in various ratios with a total run time of 15min. Chromatograms were extracted at 278nm after a 5μL solution injection. According to regulatory requirements, the developed method has been validated for its intended purpose.