Accurate Reversed-phase High-performance Liquid Chromatography Research Articles

Development and Validation of an RP-HPLC Method for the Simultaneous Estimation of Azelnidipine and Telmisartan in Pharmaceutical Tablet Dosage Form

A simple, precise, and accurate reverse-phase high-performance liquid chromatography (RP-HPLC) method to estimate Azelnidipine (AZL) and Telmisartan (TEL) in bulk and pharmaceutical dosage forms has been developed. A reverse-phase chromatography C18 column (250cm × 4.6mm × 5µm) with a mobile phase consisting of 0.2% triethylamine: Acetonitrile (20:80 V/V) having pH 3.0 adjusted using orthophosphoric acid. The flow rate was 1.0mL min and the effluents were monitored at 226nm. The retention time was found to be 6.0min for Azelnidipine and 3.8min for Telmisartan. The linearity of the drug was obtained in the range of Azelnidipine 1-5ml and Telmisartan 5-25ml. The results of the analysis have been validated according to the ICH guideline.

Accurate and Precise RP-HPLC Quantification of Piperacillin and Tazobactum Combination in Dosage Forms: A Quality Control Perspective

A simple, precise and accurate reverse phase high performance liquid chromatography (RP-HPLC) method has been developed and validated for the simultaneous estimation of piperacillin and tazobactum in pharmaceutical formulations. Separation was achieved on a C18 column using a mixture of potassium dihydrogen orthophosphate buffer (pH 3.5) and acetonitrile in the ratio of 60:40 v/v as the mobile phase at a flow rate of 1 ml/min. Detection was carried out at 226 nm. The retention times of piperacillin and tazobactum were about 2.5 and 4.3 min respectively. The method was validated as per ICH guidelines with respect to linearity, accuracy, precision, limit of detection, limit of quantification, robustness and ruggedness. The linearity ranges were found to be 5-15 μg/ml and 10-30 μg/ml for piperacillin and tazobactum respectively with correlation coefficients greater than 0.996. Accuracy, expressed as percentage recovery, was within 98-102%. The method also showed good precision and selectivity for the analysis of the drugs. The limits of detection and quantification for both analytes were adequate. In conclusion, the developed method was found to be suitable for the routine quality control analysis of piperacillin and tazobactum in pharmaceutical dosage forms.

An improved and sensitive method for nitrotyrosine estimation by reverse phase-high performance liquid chromatography

Reactive nitrogen species such as peroxynitrite anion (ONOO-), nitrogen oxide radical (NO.) may lead to nitrotyrosine formation by oxidative nitration of tyrosine. Nitrotyrosine is considered as an important marker to estimate the severity of biological oxidative damage. Therefore, in the present study, we evaluated a reverse phase-high performance liquid chromatography (RP-HPLC) method to improve the sensitivity and specificity of the nitrotyrosine estimation. The mobile phase used in the present study was methanol (pH 4.7) and 0.1% phosphoric acid (60:40%). The standard serial dilutions were injected onto the HPLC column (C-18) at a flow rate of 1 ml/min mobile phase with a time window of 6 min. The results were monitored on the Diode Array detector at wavelength 210 nm. The evaluation and quantification of output signals were made on the Chromelion software version 6.80 which controls the whole liquid chromatography system. The same standard serial dilutions were monitored spectrophotometrically at 210nm. We found the standard curves were linear over the concentration range of 0.3 to 20 ng/ml for both RP-HPLC with UV detector (r2 = 0.997) and spectrophotometric (r2 = 0.999) methods. In spectrophotometric method, only four standard points were detected out of seven and represented on a straight line, this shows the spectrophotometric method was unable to measure less than 2.5 ng/ml concentration. On the other hand, in RP-HPLC method, all the seven standards were lying on a straight line and HPLC machine can detect up to 1ng/ml or 1pg/µl. The present study established the highly specific, sensitive, accurate and cost-effective RP-HPLC with UV detector method for nitrotyrosine estimation.

Development and Validation for the Estimation of Fenofibrate in Pharmaceutical Dosage form by Reversed-phase High-performance Liquid Chromatography

Very simple, exact, precise and accurate reversed-phase high-performance liquid chromatography (RP-HPLC) methods used to estimate the amount of fenofibrate in both the bulk and tablet formulations. The reversed-mobile phase buffer and ACN in the proportion (40:60) v/v at a flow rate of 1.0 mL/min were used to create the Princeton (C18) (250 mm x 4.6 mm, 5) column. At 240 nm wavelength, the detection procedure was approved. The RT was established to be 3.905 minutes under fenofibrate’s optimum circumstances. The calibration curve had a range of 87 to 232 g/mL and a correlation coefficient of 0.9994. All-important parameters’ RSD values were below 2.0%. A 99.13 and a 100.44% recovery rate, respectively, were shown to exist. In accordance with ICH requirements, the established technique was evaluated for linearity, specificity, precision, accuracy, and system applicability. The feasibility and repeatability of the suggested technique for determining the amount of the commercially available dosage forms of fenofibrate in tablet and bulk form were also demonstrated.

Response Surface Method for the Simultaneous Estimation of Atorvastatin and Olmesartan

The simultaneous estimation of Atorvastatin Calcium (ATS) and Olmesartan Medoxomil (OLM) in bulk and pharmaceutical dosage forms, a new, quick, and cost-effective Reverse Phase High Performance Liquid Chromatography (RP-HPLC) method has been developed. The experimental design was used to achieve multivariate optimization of the RP-HPLC experimental conditions. Three independent variables were used to create mathematical models: Acetonitrile content in the mobile phase composition, buffer pH, and flow rate. Here, the applied model was central composite design (CCD) to research the response surface methodology and study the effects of independent factors. The Shimadzu (LC 20 AT VP) HPLC system with Spinchrom software has been used. Zodiac, C18 (250×4.6 ID) 5μm column, phosphate buffers, and acetonitrile were used as mobile phase in the ratio 40:60 v/v with a flow rate 1.15 mL/min. The eluent was monitored at 212 nm using the Prominence UV-Visible detector. The retention time for OLM and ATS was 2.673 and 3.717, respectively. The optimized procedure was validated as per ICH guidelines. The correlation coefficient of OLM and ATS was 0.9869 and 0.9832. The % of recovery was 98.59, 99.68 %. OLM had a LOD of 17.568 μg/mL, while ATS had a LOD of 12.88 μg/mL. OLM had a LOQ of 53.24 μg/mL, while ATS had a LOQ of 39.04 μg/mL. The pH aqueous phase, solvent composition, and flow rate were the most stringent variables affecting the responses, according to the 3D response surface graphs. A new accurate and precise RP-HPLC approach has been developed and validated and used to regularly analyse OLM and ATS.
 HIGHLIGHTS
 
 A new, quick, and cost-effective RP-HPLC method for the simultaneous estimation of Atorvastatin Calcium and Olmesartan Medoxomil in bulk and pharmaceutical dosage forms
 RP-HPLC method developed by experimental design and response surface method
 Developed method was validated as per ICH guidelines
 
 GRAPHICAL ABSTRACT

Total Content of Piperidine Analysis in Artane by RP-HPLC Using Pre-Column Derivatization with 4-Toluene Sulfonyl Chloride.

A simple, sensitive and accurate reversed-phase high-performance liquid chromatography (RP-HPLC) method was established for the determination of piperidine and piperidine hydrochloride in artane, using pre-column derivatization with 4-toluenesulfonyl chloride. The RP-HPLC method was carried out on a Inertsil C18 column (250 × 4.6mm I.D.) maintained at 30°C. The mobile phase consisted of water with 0.1% phosphoric acid (phase A) and acetonitrile (phase B) (32:68, V:V) at a flow rate of 1.0mL/min. Linearity of piperidine was found in the range of 0.44-53.33μg/mL with R2 = 0.9996. The limit of detection was estimated to be 0.15μg/mL, and the limit of quantitation was 0.44μg/mL. The average recovery was 101.82% with relative standard deviations of 0.6% at three spiked levels. The developed method using HPLC-ultraviolet system was a rapid tool for routine analysis of piperidine in the bulk form with good accuracy.

SIMULTANEOUS ESTIMATION OF LORNOXICAM AND PARACETAMOL IN COMBINED TABLET DOSAGE FORM USING REVERSE PHASE HIGH- PERFORMANCE LIQUID CHROMATOGRAPHY METHOD

Using simple, precise, and accurate reversed-phase high-performance liquid chromatography (RP-HPLC) method, the synchronous of lornoxicam (LOR) and paracetamol (PCM) in the unite dosage form in the tablet has been established and confirmed. Acetonitrile: methanol: water is the mobile phase of the RP-HPLC method (pH adjusted with orthophosphoric acid), pH 3.8 (50:30:20 v/v/v), a diode array detector tuned to 290 nm was used to detect the signal. For LOR and PCM, chromatographic technique linearity was obtained in the concentration, ranges of 2-40 and 8–150 g/mL, respectively. In HPLC techniques, the recoveries were in the range of 99.85 0.0642 percent for LOR and 99.73 0.187 percent for PCM. Both approaches may be used to analyze the medicines in a pharmaceutical formulation regularly. The results of the analysis were statistically verified. KEYWORDS: linearity, validation, oxicam, N-methyl Aspartate

New and validated RP-HPLC Method for Quantification of Safinamide Mesylate in Presence of Its Basic Degradate, Levodopa and Ondansetron: Application to Human Plasma.

A simple, precise, rapid and accurate reversed-phase high-performance liquid chromatography (RP-HPLC) method was developed and validated for analysis of safinamide mesylate (SAF) in presence of its basic degradate, and co-administered drugs levodopa and ondansetron. The mobile phase consisted of acetonitrile and 20 mM potassium dihyrogen orthophosphate buffer having pH=5 (40: 60v/v). Quantification was achieved with ultraviolet detector at 226nm. The linear range was 0.5-10μg/mL with mean recovery ± SD of 99.72±1.59. The peak purity of SAF in pharmaceutical preparation spiked with its degradate and co-administered drugs revealed symmetry factor (999.8) within the calculated threshold (>998.1). The suggested method was validated in compliance with the International Conference on Harmonization (ICH) guidelines and statistically compared with the manufacturer HPLC method with no significant difference in terms of accuracy and precision. The assay method was successfully used to estimate SAF in tablets with good percentage recoveries. The high sensitivity (lower than Cmax of the drug 0.65μg/mL) of the proposed HPLC method enabled the determination of SAF in presence of its basic degradate and co-administered drug, ondansetron in human plasma with acceptable accuracy. The suggested HPLC method could be used in Quality Control (QC) lab for analysis of the studied drug in pharmaceutical preparation.

A Quantitative, Sensitive and Rapid Validated Analytical RP-HPLC Method for the Estimation of Dapagliflozin in Bulk and Pharmaceutical Dosage Formulations

The present study is aimed to develop a linear, precise, and accurate RP-HPLC (Reverse Phase High-Performance Liquid Chromatography) method for the determination of dapagliflozin in the formulation. The method was accomplished on a C18 column (250×4.6mm; 5µm), & Samples were eluted using acetonitrile: water (40:60%v/v) delivered at a flow rate of 1.0ml/min with a chromatographic run time of 10 min. The eluents were observed utilizing a UV detector with a wavelength set at 277nm. The method that was developed resulted in the retention of dapagliflozin at 7.029minutes. Dapagliflozin through current method has shown linearity (r2 > 0.999) over the concentration range of 1-16 µg/ml. The percentage recovery was observed to be within the limits of 98-102%, demonstrating the accuracy of the method. Limit of detection (LOD) and limit of quantification (LOQ) were qualified at 0.049µg/ml and 0.1485µg/ml, respectively. A Linear, precise, accurate, simple, and rapid RP-HPLC method has been developed and validated for the evaluation of dapagliflozin in bulk drug and tablet dosage forms (5mg &10mg) according to ICH Q2(R1) rules. Additionally, the proposed method could be of use in quality control tests of dapagliflozin in pharmaceutical industries.

Novel Reverse-Phase High-Performance Liquid Chromatography (RP-HPLC) Method Development and Validation of Atorvastatin and Fenofibrate in Tablets

A novel, simple, selective, precise, and accurate reverse-phase high-performance liquid chromatography (RP-HPLC) gradient method was developed for the simultaneous estimation of atorvastatin and fenofibrate in the combined formulation. The drugs atorvastatin calcium and fenofibrate were separated in the presence of their impurities atorvastatin related compound H, fenofibrate related compound A, and fenofibrate related compound B. The drugs and related compounds were separated on Kromasil C18 (250 x 4.6, 5μ) with reverse-phase gradient elution. Water adjusted pH 4.0 with phosphoric acid used as a buffer in pump A and acetonitrile used as a solvent in pump B as a mobile phase with gradient elution. The flow rate was 2.0 mL/min. 254 nm was the detection wavelength. The retention times were about 4.6 minutes for fenofibrate related compound A, 5.2 minutes for atorvastatin calcium, 5.7 minutes for fenofibrate related compound B, 8.7 minutes for atorvastatin related compound H, and 17.6 minutes for fenofibrate. The linearity ranges for atorvastatin calcium and fenofibrate were 5.00 to 15.00 and 80 to 240 mcg/mL, respectively, with correlation coefficient 0.999 for both. The proposed method validated statistically with respect to system suitability, specificity, linearity, precision, accuracy, range, robustness, and ruggedness. The method was accurate, linear, precise, specific, selective, and rapid suitable for the quantitative estimation of atorvastatin and fenofibrate in tablets.

Novel Isocratic RP-HPLC Method Development and Validation of Rosuvastatin and Fenofibrate in Tablets

A novel, simple, selective, precise, and accurate reverse-phase high-performance liquid chromatography (RP-HPLC) gradient method was developed for the simultaneous estimation of atorvastatin and fenofibrate in the combined formulation. The drugs atorvastatin calcium and fenofibrate were separated in the presence of their impurities atorvastatin related compound H, fenofibrate related compound A, and fenofibrate related compound B. The drugs and related compounds were separated on Kromasil C18 (250 x 4.6, 5μ) with reverse-phase gradient elution. Water adjusted pH 4.0 with phosphoric acid used as a buffer in pump A and acetonitrile used as a solvent in pump B as a mobile phase with gradient elution. The flow rate was 2.0 mL/min. 254 nm was the detection wavelength. The retention times were about 4.6 minutes for fenofibrate related compound A, 5.2 minutes for atorvastatin calcium, 5.7 minutes for fenofibrate related compound B, 8.7 minutes for atorvastatin related compound H, and 17.6 minutes for fenofibrate. The linearity ranges for atorvastatin calcium and fenofibrate were 5.00 to 15.00 and 80 to 240 mcg/mL, respectively, with correlation coefficient 0.999 for both. The proposed method validated statistically with respect to system suitability, specificity, linearity, precision, accuracy, range, robustness, and ruggedness. The method was accurate, linear, precise, specific, selective, and rapid suitable for the quantitative estimation of atorvastatin and fenofibrate in tablets.

METHOD DEVELOPMENT AND VALIDATION FOR SIMULTANEOUS ESTIMATION OF LAMIVUDINE AND ZIDOVUDINE IN TABLET BY REVERSE-PHASE HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

Objective: The objective of the study was to develop and validate reverse-phase high-performance liquid chromatography (RP-HPLC) method and apply method to tablet dosage form.
 Methods: A simple, rapid, economical, precise, and accurate RP-HPLC method for simultaneous estimation of lamivudine and zidovudine in their combined dosage form has been developed.
 Results: A RP-HPLC method was developed for the simultaneous estimation of lamivudine and zidovudine. In their combined dosage form has been developed. The separation was achieved by LC-C18 column (150 mm ×4.6 mm, 5 μm) and water: methanol (65:35v/v) as mobile phase, at a flow rate of 0.8 ml/min. Detection was carried out at 272 nm. Retention time of lamivudine and zidovudine was found to be 3.007 min and 4.647, respectively. The method has been validated for linearity, accuracy, and precision. The assay method was found to be linear from 50% to 150% for lamivudine and zidovudine.
 Conclusion: Developed method was found to be accurate, precise, and rapid for simultaneous estimation of lamivudine and zidovudine in their combined dosage form.

Novel HPLC method for simultaneous estimation of gallic acid, protocatechuic acid and mangiferin in mango

Mango (Mangifera indica) is the main tropical fruit of Asia and has developed its prominence all over the world. Mango wastes such as peels and seeds generated from fruit are a good source of functional ingredients out of which important ones are phenolic compounds with potent antioxidant properties. The quantification of gallic acid (GA), protocatechuic acid (PCA) and mangiferin (MGF) in the seed and peel extracts of three different varieties of mango fruit was performed by a novel, simple, specific, linear, precise, accurate and economic RPHPLC (reversed-phase high- performance liquid chromatography) method. The chromatographic separation was achieved on the C18 analytical column with a flow rate of 1.2 mL/min in an isocratic mode using 0.2% orthophosphoric acid and acetonitrile in the ratio 90:10 as the mobile phase. The column temperature was maintained at 40 ℃ and observations were recorded at 242 nm. The retention time of gallic acid, protocatechuic acid and mangiferin was observed at 3.41, 5.50, and 14.6 min respectively. The developed method when validated as per ICH guidelines was found to be specific, linear, precise, robust and accurate

Development of a RP-HPLC method for analysis of docetaxel in tumor-bearing mice plasma and tissues following injection of docetaxel-loaded pH responsive targeting polymeric micelles.

Background and purpose:A simple, rapid, and sensitive reversed-phase high performance liquid chromatography (RP-HPLC) method based on liquid-liquid extraction was developed and validated for determination of docetaxel (DTX) in plasma and homogenate tissues of tumor-bearing mice.Experimental approach:Samples were spiked with celecoxib as the internal standard and separation was achieved on a μ-Bondapak C18 HPLC column. The mobile phase consisted of a mixture of acetonitrile/water (40/60 v/v) at flow rate of 1.2 mL/min and the effluent was monitored at 230 nm.Results:Calibration curves were linear over the concentration range of 0.1-10 μg/mL of DTX in plasma and 0.25-50 μg/mL in tissue homogenates with acceptable precision and accuracy. The mean recoveries of the drug from plasma extraction was 94.6 ± 1.44% while those of tissue homogenates ranged from 73.5 ± 3.2 to 85.3 ± 2.8% depending on the type of tissues examined. DTX was stable in biological samples with no evidence of degradation during 3 freeze-thaw cycles and two months of storage at -70 ± 15 °C. The developed HPLC method was applied to quantify DTX in the mouse plasma and tissues after intravenous administration of 7.5 mg equivalent DTX/kg dose of DTX-loaded folic acid-polyethylene glycol-heparin-tocopherol (FA-PEG-HEP-CA-TOC) micelle formulation to female Balb/c mice.Conclusion:A simple, sensitive, rapid, accurate, and prudent RP-HPLC method was developed, validated, and applied for DTX determination in plasma and tissues.

SIMULTANEOUS ESTIMATION OF CURCUMIN AND GEFITINIB IN BULK AND TISSUE SAMPLES (PLASMA AND BRAIN HOMOGENATE) BY RP-HPLC: APPLICATION TO A DISTRIBUTION STUDY

The present study was aimed at developing a Reversed-Phase High-Performance Liquid Chromatography (RP-HPLC) method for simultaneous determination of curcumin (CRM) and gefitinib (GFT) in bulk, plasma and brain homogenate. hydrochlorothiazide was used as an internal standard (IS). A new simple, rapid, selective, precise and accurate RP-HPLC method has been developed. The separation was achieved by using C-18 column (Qualisil BDS C18, 250 mm x 4.6 mm I.D.) coupled with a guard column of silica, mobile phase consisted of acetonitrile: water with 0.1% formic acid (30:70 v/v). The flow rate was 0.2 ml/min and the drug was detected using PDA detector at the wavelength of 242 nm. The experimental conditions, including the diluting solvent, mobile phase composition, column saturation and flow rate, were optimised to provide high-resolution and reproducible peaks. The method was developed and tested for linearity range of 10-60 μg/mL for bulk analysis and 200-800 ng/mL for plasma and brain homogenate. The developed method was validated as per ICH guidelines, in terms of linearity, application of the proposed method to bulk sample, recovery, precision, repeatability, ruggedness, sensitivity (LOD and LOQ) and robustness and stability study (short and long-term stabilities, freeze/thaw stability, post-preparative). The low value of % RSD showed that the method was precise within the acceptance limit of 2%. The developed method was successfully applied for the analysis of the drug in bulk as well as various marketed formulation and drug in plasma and brain distribution studies.

A NEW REVERSED-PHASE HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY METHOD FOR THE SIMULTANEOUS ESTIMATION OF SERRATIOPEPTIDASE AND DICLOFENAC SODIUM IN BULK AND TABLET DOSAGE FORM

Objective: The objective is to study the development of a simple, rapid, specific, precise, and accurate reversed-phase high-performance liquid chromatography (RP-HPLC) method for the simultaneous estimation of serratiopeptidase (SER) and diclofenac (DC) sodium in bulk and tablet formulation.Methods: RP-HPLC method was developed for the simultaneous estimation of SER and DC sodium in tablet formulation. The separation was achieved by Kromasil C18 column (250 mm × 4.6 mm, 5 μm particle size) with phosphate buffer pH-7 and o-phosphoric acid:methanol:acetonitrile (5:4:1% v/v/v). Flow rate was maintained at 1 mL/min and UV detection was carried at 270 nm.Result: For RP-HPLC method, the retention time for SER and DC sodium was found to be 3.3833 min and 8.1667 min, respectively. The method was validated for accuracy, precision, and specificity. Linearity for SER and DC sodium was in the range of 5–50 μg/ml.Conclusion: The developed RP-HPLC method is simple, accurate, rapid, sensitive, precise, and economic. Hence, this method can be employed successfully for the estimation of SER and DC sodium in both bulk and tablet dosage forms.

A NEW REVERSED-PHASE HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY METHOD FOR THE SIMULTANEOUS ESTIMATION OF SERRATIOPEPTIDASE AND DICLOFENAC SODIUM IN BULK AND TABLET DOSAGE FORM

Objective: The objective is to study the development of a simple, rapid, specific, precise, and accurate reversed-phase high-performance liquid chromatography (RP-HPLC) method for the simultaneous estimation of serratiopeptidase (SER) and diclofenac (DC) sodium in bulk and tablet formulation.Methods: RP-HPLC method was developed for the simultaneous estimation of SER and DC sodium in tablet formulation. The separation was achieved by Kromasil C18 column (250 mm × 4.6 mm, 5 μm particle size) with phosphate buffer pH-7 and o-phosphoric acid:methanol:acetonitrile (5:4:1% v/v/v). Flow rate was maintained at 1 mL/min and UV detection was carried at 270 nm.Result: For RP-HPLC method, the retention time for SER and DC sodium was found to be 3.3833 min and 8.1667 min, respectively. The method was validated for accuracy, precision, and specificity. Linearity for SER and DC sodium was in the range of 5–50 μg/ml.Conclusion: The developed RP-HPLC method is simple, accurate, rapid, sensitive, precise, and economic. Hence, this method can be employed successfully for the estimation of SER and DC sodium in both bulk and tablet dosage forms.

DEVELOPMENT AND VALIDATION OF A STABILITY INDICATING RP-HPLC METHOD FOR THE DETERMINATION OF POTENTIAL DEGRADATION PRODUCTS OF DIFLUPREDNATE IN OPHTHALMIC EMULSION

Objective: The objective of the current study was to develop and validate a simple, robust, precise and accurate RP-HPLC (reverse phase-high performance liquid chromatography) method for the quantitative determination of potential degradation products of Difluprednate (DIFL) in the ophthalmic emulsion.Methods: Chromatographic separation was achieved on the YMC pack ODS-AQ (150× 4.6) mm, 3μm column with a mobile phase containing a gradient mixture of mobile phase A (0.02M Ammonium formate buffer pH 4.5 adjusted with formic acid) and Acetonitrile as mobile phase B, at flow rate of 1.5 ml/min and with UV detection at 240 nm.Results: The peak retention time of DIFL was found at about 17.2 min, the RRT of degradation product-1 (DP-1), degradation product-2 (DP-2), and degradation product-3 (DP-3), were found to be about 0.49, 0.65 and 0.79 respectively (calculated with respect to Difluprednate). Stress testing was performed in accordance with an ICH (international council for harmonisation) guideline Q1A (R2) [1]. The method was validated as per ICH guideline Q2 (R1)[2]. The calibration curve was found to be linear in the concentration range of 0.1 to 0.75 µg/ml for Difluprednate, DP-1, DP-2 and DP-3. The LOD (Limit of detection) was found to be 0.1µg/ml and LOQ (Limit of quantification) of 0.15µg/ml for Difluprednate, DP-1, DP-2 and DP-3 respectively. The recovery from LOQ to 150% was within 90-110%. The forced degradation data confirms the stability indicating the nature of the method.Conclusion: A simple, robust, precise and accurate RP-HPLC method for the quantitative determination of potential degradation products of Difluprednate in the ophthalmic emulsion was developed and validated.

A VALIDATED REVERSED-PHASE HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY METHOD FOR SIMULTANEOUS DETERMINATION OF FIVE ANTIEPILEPTIC DRUGS USED IN THE TREATMENT OF LENNOX–GASTAUT SYNDROME IN THEIR PHARMACEUTICAL DOSAGE FORMS

Objective: Lennox–Gastaut syndrome (LGS) is mainly treated with antiepileptic drugs (AEDs) but using one AED is not sufficient to relieve all or even most patients. A combination of agents is usually preferred. In the current study, an isocratic, selective, sensitive, precise, and accurate reversed-phase high-performance liquid chromatography (RP-HPLC) method was developed for the simultaneous determination of rufinamide (RUF), lamotrigine (LAM), clonazepam (CLO), valproic acid (VAL), and diazepam (DIA) which are commonly used in the management of LGS in their dosage forms using lacosamide as internal standard.Methods: The method depends on using RESTEK C18 column (5 μm, 250 mm × 4.6 mm) and a mobile phase composed of acetonitrile:water (55: 45, v/v), pH = 3.3 adjusted with phosphoric acid. The method was conducted in an isocratic mode with a flow rate of 1ml/min and ultraviolet detection at 210 nm.Results: The linearity range was 2–40 μg/ml for RUF and DIA, 0.5–40 μg/ml for LAM and CLO, and 36–180 μg/ml for VAL.Conclusion: Statistical analysis revealed no significant difference between the results obtained and the official or reported ones for each cited drug. The method is simple to be easily implemented in quality control studies of the mentioned drugs in their pharmaceutical preparations.

STABILITY INDICATING RP-HPLC METHOD FOR THE ESTIMATION OF DIETHYLCARBAMAZINE CITRATE, GUAIPHENESIN AND CHLORPHENIRAMINE MALEATE

Objective: The present work describes the development and subsequent validation of a simple, precise and stability–indicating reversed-phase high-performance liquid chromatography (RP-HPLC) method for the simultaneous estimation of diethylcarbamazine citrate, guaiphenesin and chlorpheniramine maleate in tablet dosage forms.Methods: A simple, accurate, precise and robust RP-HPLC method was developed and validated for the estimation of diethylcarbamazine citrate, guaiphenesin and chlorpheniramine maleate. The chromatographic separation of all the three active components was achieved by using luna phenyl-hexyl column (250 mmx4.6 mm, dp=5 µm) with a mobile phase consisting of isocratic method with 0.1% triethylamine as buffer along with orthophosphoric acid adjusted to PH 2.5: acetonitrile (50:50v/v) at a flow rate 1.0 ml/min and ultraviolet detection at 210 nm.Results: The retention time of chlorpheniramine maleate, guaiphenesin and diethylcarbamazine citrate were 2.86, 4.89 and 7.76 min respectively. Validation of the proposed method was carried out according to an international conference on harmonization (ICH) guidelines. The established method was linear in the range of 1-15, 0.6-9, 0.02-0.3 µg/ml and correlation coefficient was 0.999, 0.9991, and 0.993 for diethylcarbamazine citrate, guaiphenesin and chlorpheniramine maleate respectively.Conclusion: The proposed method can be used for the quantitative analysis of diethylcarbamazine citrate, guaiphenesin and chlorpheniramine maleate.