Rapid Liquid Chromatographic Method Research Articles

A new analytical RP-HPLC method development and validation for the estimation of Nilutamide in bulk form and marketed pharmaceutical dosage form

A rapid and precise reverse phase high performance liquid chromatographic method has been developed for the validated of Nilutamide, in its pure form as well as in tablet dosage form. Chromatography was carried out on a Symmetry C18 (4.6 x 150 mm, 5 µm) column using a mixture of Methanol and water (45: 55 % v/v) as the mobile phase at a flow rate of 0.8 ml/min, the detection was carried out at 260 nm. The retention time of the Nilutamide was 2.379 ± 0.02 min respectively. The method produce linear responses in the concentration range of 24-120 mg/ml of Nilutamide. The method precision for the determination of assay was below 2.0 % RSD. The method is useful in the quality control of bulk and pharmaceutical formulations. The method was validated for accuracy, precision, linearity, robustness, ruggedness and LOD & LOQ of standard solution. The developed RP-HPLC method was found to be accurate, precise, linear, and robust and was successful applied to a pharmaceutical tablet formulation for qualitative estimation of Nilutamide in Bulk form and Marketed Pharmaceutical Dosage forms.

A Novel Reversed Phase HPLC Assay Method for Simultaneous Estimation of Glucose, Sodium Citrate and Chlorides in Pharmaceutical Formulations and Drug Solution for Oral Rehydration

A rapid, straightforward, sensitive, efficient, and cost-effective reverse-phase high-performance liquid chromatographic method was employed to simultaneously determine Glucose, Sodium Citrate, and chloride in a drug solution for oral solution. Glucose, Sodium Citrate, and Chlorides are all listed in the USP monograph. While various assay methods are available, titrimetric methods are ineffective for trace-level quantification. Although IC, AAS, and ICP-MS offer high accuracy, they are expensive and often inaccessible to many testing facilities. When selecting methods, it’s crucial to consider quality control requirements and user-friendly techniques. A simple proprietary HPLC method has been developed to simultaneously quantify Chlorides, Glucose, and Sodium Citrate, with a shorter run time. The separation was carried out using a Shim-pack SCR-102(H) ion exclusion analytical column (7.9 mm x 300 mm, 7 µm) with a flow rate of 0.6 mL/min. The column compartment was kept at 40°C, and the injection volume was 10 µL, with detection at 200 nm. All measurements were performed in a 0.1% phosphoric acid solution. The analytical curves showed excellent linearity (r > 0.9999) within the concentration ranges of 0.74 to 1.74 mg/mL for Sodium Citrate (SC), 1.0 to 2.5 mg/mL for Sodium Chloride (SC), and 3.0 to 7.3 mg/mL for Glucose. The method was validated according to the guidelines of the International Conference on Harmonization (ICH Q2B) and USP<1225>. The method demonstrated precision, robustness, accuracy, and selectivity. During accelerated stability testing over six months, no significant variations were observed in organoleptic analysis and pH. Consequently, the developed method is considered suitable for routine quality control analyses, enabling the simultaneous determination of Sodium Citrate, Chloride, and Glucose in pharmaceutical formulations and solutions for oral rehydration.

A rapid and accurate liquid chromatographic method for hemp compliance testing

A liquid chromatography ultraviolet (LC-UV) method, which used methanol as the organic solvent for both extraction and separation, was developed for hemp compliance testing. While published methods occasionally used methanol for separation, all of them used gradient elution which would cause baseline drifts and a subsequent high limit of quantification (LOQ), and only eight or less cannabinoids were included. Our method was characterized by a baseline separation of Δ9-tetrahydrocannabinol (Δ9-THC) and Δ9-tetrahydrocannabinolic acid (Δ9-THCA) among nineteen cannabinoids in ten minutes and a calibration between 0.04 to 50 µg/mL, making it the most effective method validated so far. However, a reproducible separation of Δ9-THCA could not be achieved with the common formate buffer system, presumably due to its adsorption onto the C18 column which could be prevented by either using acetonitrile in the organic solvent that was widely undertaken by published methods or adding 0.1 % (v/v) trifluoroacetic acid in the aqueous solvent that was achieved only in this study. While similar problems were not reported by published methods, it was speculated that the problem could be common for all C18 columns, more or less, as the stationary phase in this study was di-isobutyl-n-octadecyl-silane without any polar imbedded moieties. Additionally, while quantification of Δ9-THC at 230 nm achieved good specificity, quantification of Δ9-THCA required 269 nm for acceptable specificity, which was verified by electrospray ionization time-of-flight mass spectrometry (ESI/TOFMS), indicating no false quantification and identification of Δ9-THC and Δ9-THCA, despite the observation of ten matrix peaks in the LC-UV chromatograms and identification of three untargeted/unknown cannabinoids by ESI/TOFMS in ten samples.

Response surface experimental design for simultaneous chromatographic determination of two antiviral agents “Favipiravir and Remdesivir” in pharmaceuticals and spiked plasma samples

AbstractThe antiviral agents, Favipiravir (FAV) and Remdesivir (REM), were introduced in the last few years alone or as combination regimen for successful management of the rapidly spreading CORONA virus pandemic. A newly developed rapid and sensitive high performance liquid chromatographic method (HPLC) has been developed for the simultaneous determination of their mixture. Firstly, one factor at a time optimization (OFAT) has been applied. Afterwards, quality by design approach (QbD) has been utilized using Box Behnken experimental design (BBD) for the development of an experimental design of four independent and nine dependent variables for much better refining of the optimized parameters. The established model has given an optimum resolution at; acetonitrile percentage of 52.66, mobile phase of pH 2.91, percentage of triethylamine of 0.15 and 1.30 mL/min flow rate. The proposed method has been validated according to the USP 31 NF 26 guidelines. Good linearity ranges have been obtained from 5.00 up to 50.00 μg/mL for FAV and from 2.00 up to 60.00 μg/mL for FAV and REM, respectively. Excellent relative standard deviation values (not more than 1.40) were obtained upon investigation of accuracy, precision and robustness. The developed method has succeeded in analysis of investigated drugs in their pharmaceutical formulations and spiked plasma samples with good recoveries of 99.00 and up to 106.00%. The proposed method is considered eligible for the quality control laboratories as well as in‐vivo determinations of both analytes.

Analytical Method Development and Validation of Brivaracetam in API and Marketed Formulation by RP-HPLC

A rapid and precise reverse phase high performance liquid chromatographic method has been developed for the validated of brivaracetam in its pure form as well as in tablet dosage form. Chromatography was carried out on a Symmetry ODS RP C18,5mm, 15mm x 4.6mm column using a mixture of phosphate buffer: methanol: acetonitrile (30:35:35% v/v) as the mobile phase at a flow rate of 1.0ml/min, the detection was carried out at 285 nm. The retention time of the brivaracetam was 2.183. The method produces linear responses in the concentration range of 60-140μg/ml of brivaracetam. The method precision for the determination of assay was below 2.0%RSD. The method was found to be sensitive, accurate and precise useful in the quality control of bulk and pharmaceutical formulations.

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.

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.

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 <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.

Analytical method development and validation for simultaneous estimation of loratadine and pseudoephedrine in bulk and tablet dosage form by RP HPLC method

A rapid and precise reverse phase high performance liquid chromatographic method has been developed for the validated of Pseudoephedrine and Loratadine , in its pure form as well as in tablet dosage form. Chromatography was carried out on a Phenomenex Luna C18 (4.6×150mm, 5µ) column using a mixture of Acetonitrile: Water (10:90% v/v) as the mobile phase at a flow rate of 0.9ml/min, the detection was carried out at 240nm. The retention time of the Pseudoephedrine and Loratadine was 1.933, 3.396±0.02min respectively. The method produce linear responses in the concentration range of 16.5-82.5mg/ml of Pseudoephedrine and 5-25mg/ml of Loratadine . The method precision for the determination of assay was below 2.0%RSD. The method is useful in the quality control of bulk and pharmaceutical formulations.

Development and validation of analytical method for determination of amlodipine and benazepril in bulk and pharmaceutical dosage form by RP-HPLC

A rapid and precise reverse phase high performance liquid chromatographic method has been developed for the validated of Amlodipine and Benazepril, in its pure form as well as in capsule dosage form. Chromatography was carried out on an Altima C18 (4.6 x 150 mm, 5µm) column using a mixture of Methanol: TEA Buffer pH 4.5: Acetonitrile (50:25:25) as the mobile phase at a flow rate of 1.0 mL/min, the detection was carried out at 225 nm. The retention time of the Amlodipine and Benazepril was 2.102, 3.537 ±0.02 min respectively. The method produce linear responses in the concentration range of 5-25 mg/ml of Amlodipine and 20-100 mg/ml of Benazepril. The method precision for the determination of assay was below 2.0 %RSD. The method is useful in the quality control of bulk and pharmaceutical formulations.

Method development and validation of lamivudine and stavudine in bulk and its tablet dosage form by reverse phase high performance liquid chromatography method

A rapid and precise reverse phase High Performance Liquid Chromatographic method has been developed for the validation of Stavudine and Lamivudine, in its pure form as well as in tablet dosage form. Chromatography was carried out on a Phenomenex Gemini C18 (4.6×250mm) 5µ column using a mixture of Methanol: TEA Buffer (65:35 v/v) as the mobile phase at a flow rate of 1.0ml/min, the detection was carried out at 230nm. The retention time of the Stavudine and Lamivudine was 2.121, 3.643 ±0.02min respectively. The method produce linear responses in the concentration range of 5-25µg/ml of Stavudine and 18.75-93.75µg/ml of Lamivudine. The method precision for the determination of assay was below 2.0%RSD. The method is useful in the quality control of bulk and pharmaceutical formulations.

Development of a new chromatographic method for estimation of paracetamol and metoclopramide hcl in bulk and pharmaceutical dosage form by HPLC

A new simple, accurate, economic, rapid and precise reverse phase high performance liquid chromatographic method has been developed for the validated of Paracetamol and Metoclopramide HCl, in its pure form as well as in pharmaceutical dosage form. Chromatography was carried out on X bridge C18 (4.6×150mm) 5 µ column using a mixture of Methanol: Phosphate Buffer pH-3.6 (30:70v/v) as the mobile phase at a flow rate of 1.0ml/min, the detection was carried out at 260nm. The retention time of the Paracetamol and Metoclopramide HCl was 2.669, 3.855±0.02min respectively. The method produce linear responses in the concentration range of 10-50µg/ml of Paracetamol and 10-50µg/ml of Metoclopramide HCl. The method precision for the determination of assay was below 2.0%RSD. The method is useful in the quality control of bulk and pharmaceutical formulations.

Inclusive study for sustainable enantioseparation of racemic chlorpheniramine and caffeine by HPLC using dual cyclodextrin system as chiral mobile phase additive: Assessment with AGREE and Complex-GAPI approaches

A rapid, sustainable green, isocratic, and sensitive liquid chromatographic method was applied successfully for chlorpheniramine (CP) enantioseparation in the presence of common additive caffeine (CAF) in their synthetic mixtures and pharmaceutical preparations. The analytical separation was achieved in 17 min using a C8 column and a UV detector at 254 nm. An aqueous phosphoric acid (10 mM) containing dual-cyclodextrin 10 mM SβCD, and 10 mM βCD: ACN (75:25, v/v, pH 3.0) was utilized as a mobile phase. The proposed method was linear in the range of 15.5–250 μg/mL for CAF and 6.25–100.0 μg/mL for both d-chlorpheniramine (d-CP) and l-chlorpheniramine (l-CP). The proposed methods are rapid and exhibited good repeatability, accuracy, and sensitivity (RSD less than 2.0 %). The detection limits were 1.98 μg/mL (CAF), 0.58 μg/mL (d-CP), and 1.05 μg/mL (l-CP). The limits of quantitation were 5.99 μg/mL (CAF), 1.75 μg/mL (d-CP), and 3.17 μg/mL (l-CP). In accordance with the recommendations of the International Conference on Harmonization (ICH), the technique is validated. Sustainable chemistry, commonly referred to as green chemistry, aims to prevent or minimize the harmful effects that chemicals have on the environment. The AGREE and Complex GAPI metric techniques were used to test the suggested methodology's greenness, and the findings revealed a satisfactory level of greenness with a 0.62 score for AGREE and 11 green patches for Complex GAPI.

A simple and rapid reverse-phase high-performance liquid chromatographic method for the determination of propineb and tricyclazole pesticides from the wettable powder formulation

A simple and rapid reverse-phase high-performance liquid chromatographic method for the determination of propineb and tricyclazole pesticides from the wettable powder formulation

Stability Indicating RP-HPLC Method for Quantitative Estimation of Lenalidomide and Its Impurities in Solid Dosage Form

The main aim and objectives of the research are to develop an effective, sensitive, economical, and simple reverse-phase HPLC method developed for determining and quantifying Lenalidomide impurities in Lenalidomide solid dosage formulations. The lack of research work and no compendial methods available for estimating this drug influenced the current research investigation to give a simple, sensitive, rapid, precise, accurate and robust gradient high-performance liquid chromatographic method for the determination and quantification of Lenalidomide and its impurities. Samples are analyzed using reverse phase (RP-HPLC) using stationary phase an Inertsil ODS-3V (150 x 4.6 mm, 3μm), and the mobile phase consists of two channels A and B. channel-A: pH 3.0 phosphate buffer and Channel-B: Acetonitrile: water (90:10 v/v) in the proportion of gradient elution. The flow rate is 1.0 mL/min. The column temperature was maintained at 40°C, and the sample cooler temperature was maintained at 5°C, injection volume of 20 μL, and wavelength of 210 nm. The developed HPLC method was validated concerning specificity, and the chromatograms were recorded for blank, placebo, standard, sample, and spiked sample solutions of Lenalidomide and its related substances. Specificity studies reveal that the peaks are well separated from each other. For precision, the results were found to be within acceptable limits. The limit of detection (LOD) and limit of quantitation (LOQ) for impurity-A 0.1124 μg/mL and 0.0371μg/mL, Impurity-B 0.2247μg/mL and 0.0742μg/mL, respectively. The linearity results for Lenalidomide and all the impurities in the specified concentration range are satisfactory, with a correlation coefficient greater than 0.99. The accuracy studies were shown as % recovery for Lenalidomide and its impurities at the specification level; the results obtained were within limits. Solution stability parameter was established; standard, sample, and spiked sample solutions are stable up to 48 hrs on a bench top at the refrigerator. Filter validation parameters were established, and the filtered spiked sample solutions are compatible with both 0.45 μm PVDF & 0.45 μm Nylon filters.

QbD based RP-HPLC method for simultaneous determination of a emtricitabine, tenofovir diproxil fumarate and efavirenz in tablet dosage form-an application to stability indicating assay

The current studies entail Quality by Design (QbD)-enabled development of a simple, rapid, sensitive and cost-effective high-performance liquid chromatographic method for estimation of emtricitabine (EMT), tenofovir diproxil fumarate (TEN), and efavirenz (EFA) in combined dosage form. Systematic optimization was performed employing Box-Behnken design by selecting the flow rate, buffer molarity, and acetonitrile volume as the critical method parameters (CMPs) identified from screening studies, thus evaluating the critical analytical attributes (CAAs), namely, retention duration, theoretical plates, resolution, and asymmetry factor as the parameters of method robustness. The optimal chromatographic separation was achieved using acetonitrile and phosphate buffer (pH 3.0) 74.1:25.9 v/v as the mobile phase with a flow rate 0.91 mL/min, and UV detection at 260 nm. The method was validated as per the ICH recommended conditions, which revealed high degree of linearity, accuracy, precision, sensitivity and robustness over the existing liquid chromatographic methods of the drug. Studies on forced degradation under acid, basic, oxidative, photolytic, and thermal conditions revealed the drug’s well-resolved peak and the degradation products’ peaks as well.

HPLC Analytical Method Development and Validation for Estimation of Cytarabine and Daunorubicin in API and Pharmaceutical Formulation

A rapid and precise reverse phase high performance liquid chromatographic method has been developed for the validated of Cytarabine and Daunorubicin, in its pure form as well as in pharmaceutical dosage form. Chromatography was carried out on an Altima C18 (4.6mm x 150mm, 5µm) column using a mixture of ACN, Methanol and Phosphate buffer pH-4.6 (10:25:65 v/v) as the mobile phase at a flow rate of 1.0ml/min, the detection was carried out at 265nm. The retention time of the Cytarabine and Daunorubicin was 2.088, 6.068±0.02 min respectively. The method produces linear responses in the concentration range of 10-50mg/ml of Cytarabine and 20-100mg/ml of Daunorubicin. The method precision for the determination of assay was below 2.0%RSD. The method is useful in the quality control of bulk and pharmaceutical formulations.

Quantitative Estimation of Roxithromycin and Ambroxol in Bulk and Tablet Dosage Forms by RP-HPLC Method

A Rapid and Precise Reverse Phase High Performance Liquid Chromatographic method has been developed for the validated of Roxithromycin and Ambroxol, in its pure form as well as in tablet dosage form. Chromatography was carried out on Altima C18 (4.6 x 150mm, 5µm) column using a mixture of ACN, Methanol and Phosphate buffer pH4.6 (10:25:65 v/v) as the mobile phase at a flow rate of 1.0ml/min, the detection was carried out at 215nm. The retention time of the Roxithromycin and Ambroxol was 2.344, 3.286±0.02min respectively. The method produce linear responses in the concentration range of 10-50mg/ml of Roxithromycin and 2.5-12.5mg/ml of Ambroxol. The method precision for the determination of assay was below 2.0 %RSD. The method is useful in the quality control of bulk and pharmaceutical formulations.

Validated RP-HPLC Method for Simultaneous Estimation of Perphenazine and Amitriptyline in Bulk and Tablet Dosage form

A new, simple, precise, rapid, selective and stability reversed-phase high performance liquid chromatographic (RP-HPLC) method has been developed and validated for the simultaneous quantification of Perphenazine and Amitriptyline in pure form and its pharmaceutical dosage form. The method is based on Phenomenex Gemini C18 (4.6×250mm) 5µ column. The separation is achieved using isocratic elution by Methanol: TEA Buffer in the ratio of 65:35% v/v, pumped at flow rate 1.0mL/min and UV detection at 230nm. The column is maintained at 40°C throughout the analysis. The total run time is about 6min. The method is validated for specificity, accuracy, precision and linearity, robustness and ruggedness, system suitability, limit of detection and limit of quantitation as per International conference of harmonization (ICH) Guidelines. The method is accurate and linear for quantification of Perphenazine, Amitriptyline between 10 - 50µg/mL and 20 - 100µg/mL respectively. Further, satisfactory results are also established in terms of mean percent- age recovery (100.37% for Perphenazine and 100.34% for Amitriptyline, intra-day and inter-day precision (<2%) and robustness. The advantages of this method are good resolution with sharper peaks and sufficient precision. The results indicate that the method is suitable for the routine quality control testing of marketed tablet formulations.

A Validated RP-HPLC Method for Quantitative Estimation of Guaifenesin in Bulk and Pharmaceutical Dosage Forms

A novel approach was used to develop and validate a rapid, specific, accurate and precise reverse phase high performance liquid chromatographic (HPLC) method for the estimation of Guaifenesin in bulk and pharmaceutical dosage forms. The chromatographic separation was achieved by Cosmosil packed (5C18-MS-II, 4.6 internal diameter 250mm lengths) column, using mobile phase ratio of Acetonitrile: Phosphate buffer PH 5(70:30). The flow rate was 1mL/min and the detection wavelength was 260nm. The limit of detection (LOD) for guaifenesin was 0.23µg/ml and the limit of quantification (LOQ) for guaifenesin was 0.78µg/ml. This method was validated concerning linearity, accuracy, precision, specificity and robustness. The method was also found to be stability-indicating.