Simple High-performance Liquid Chromatography Research Articles

Validation of HPLC-DAD method for analysis of paracetamol and potassium sorbate in liquid oral formulations and its application to forced degradation study

AbstractDue to the frequent use of paracetamol formulations, it is useful to develop an analytical technique for the determination of intact paracetamol in presence of other drugs and excipients or the degradation products. In this study, a simple, isocratic, fast, specific, accurate and precise stability-indicating high performance liquid chromatography (HPLC) method has been developed and validated for simultaneous quantitative determination of paracetamol (PCM) and potassium sorbate (PS) in oral liquid formulations. The chromatographic separation was achieved on Zorbax SB C18 column (150 × 4.6 mm, 5 µm) with Zorbax SB C18 precolumn (12.5 × 4.6 mm, 5 µm) using distilled water pH 2 with ortho-phosphoric acid and acetonitrile (70:30, v/v) as a mobile phase, and UV detection at 235 nm. The temperature of the column was kept constant at 25 °C. The method was validated according to International Conference on harmonization (ICH) guidelines. The method demonstrated excellent linearity, with a correlation coefficient of 0.9996 and 0.9998 for PCM and PS, respectively, over the concentration ranges of 10–600 μg mL−1 (PCM) and 6–500 μg mL−1 (PS). The retention time was found to be 1.98 and 4.86 min for PCM and PS, respectively. Oral liquid formulation samples were subjected to various stress conditions (acidic and alkaline hydrolysis, as well as oxidative, heat and photolytic degradation) for the purpose of forced degradation study. The major degradation of paracetamol was achieved in acidic and basic stress conditions, while thermal and photolytic degradation generally had the least influence. On the other hand, potassium sorbate was highly susceptible to photolytic degradation. It was also shown that the formulation has strong influence on stability of tested compounds. Forced degradation studies demonstrated the stability-indicating power of the method and can be used to assess the stability of paracetamol and potassium sorbate in oral liquid formulations.

Development and Validation of RP-HPLC Method using UV detection for Simultaneous quantification of Amlodipine besylate and Nebivolol hydrochloride in Fixed-dose combination tablets

Calcium channel blockers and beta blockers are the class of choice of drugs in treatment of hypertension and cardio vascular diseases. Such a fixed dose combination is viable to deal with the patients for whom monotherapy is not sufficient. A simple, responsive and productive high performance liquid chromatography method was designed and approved for simultaneous quantification of Amlodipine Besylate (AMB) and Nebivolol Hydrochloride (NBL) in tablet dosage form. In the developed method mobile phase comprised of a mixture of aqueous buffer (pH 6) and acetonitrile (55:45 v/v, respectively) at a flow rate of 1.5 ml/min. Chromatographic separation was achieved using Reverse-phase Kromasil 60-5-CN column, 250 × 4.6 mm (particle size 5 μm). Method was discovered specific as no obstruction of excipients peaks at the retention time of tablets was observed and demonstrated linearity in concentration range of 30.4-70.8 mcg/ml for AMB and 29.8-69.4 mcg/ml for NBL with R2> 0.999. Accuracy values for AMB and NBL was found to be 98.21-99.49% and 97.91-99.09%, respectively. Inter-day and intra-day precision was carried out and % RSD was found to be less than 2%. Robustness was performed to evaluate the results with slight variations in method and % RSD found to be less than 2%. Solution stability studies were conducted in normal conditions. Force degradation study was carried out using Photo-diode Array (PDA) detector and revealed that acid degradation in AMB and NBL was 7.12% and 1.88%, alkaline degradation was 9.61% and 0.05% and oxidative degradation was 8.50% and 1.59%, respectively.

Validation of an Ion-Pair Reverse Phase High-Performance Liquid Chromatography Method for the Detection of Major Components and Related Substances in Diquafosol Sodium Eye Drops.

A simple, feasible, isocratic elution, and stable reversed-phase high performance liquid chromatography method was established and verified. The chromatographic conditions are as follows: EF-C18H, 4.6 × 250 mm, 5 μm column; column temperature 30 °C; for the mobile phase 27.2 g of KH2PO4 and 8.5 g of tetrabutylammonium hydrogen sulfate were taken, 2500 mL of water was added to dissolve, and the pH was adjusted to 6.7 with phosphoric acid:methanol solution with a ratio of 84:16 (V:V). The flow rate was 1.0 mL/min; the injection volume was 10 μL; and the wavelength was 262 nm. According to the current ICH guidelines, the developed method was verified, and the system suitability, specificity, LOD, LOQ, linearity, range, accuracy, repeatability, durability, and solution stability of the proposed method were verified. The validation results demonstrated that the LOQ for the method was 0.05% and the LOD was 0.02%. The content was detected within the concentration range of 300 to 900 μg/mL. The relationship between concentration and measurement was linear, with an r2 of >0.999. The concentration of impurities ranged from 0.3 to 4.5 μg/mL. A good linear correlation was observed within the range of g/mL, with a coefficient of determination r2 greater than 0.999. The accuracy and repeatability met the specified criteria.

DEVELOPMENT AND VALIDATION OF A NEW RP-HPLC METHOD FOR SIMULTANEOUS DETERMINATION AND QUANTIFICATION OF SOFOSBUVIR AND DACLATASVIR

Background: Sofosbuvir (SOF) and daclatasvir (DCV) are usually prescribed in combination for the treatment of hepatitis C worldwide. Objectives: In the present study a novel, simple, rapid, and cost-effective reversed-phase high performance liquid chromatography (HPLC) method was developed, optimised, and validated for simultaneous analysis of sofosbuvir and daclatasvir in human plasma as well as in solid dosage forms. Methodology: The chromatographic analyses were carried out using Shimadzu HPLC gradient system (LC20), while the other HPLC system used for the peak purity and repeated validation studies was Agilent 1260 Infinity fitted with diode array detector. The column used was C18 (150 × 4.6 mm; 5 µm), from Agela Technologies. The mobile phase consisted of acetonitrile and 0.02 M potassium Hexafluorophosphate buffer (42:58, v/v) at pH 2.7, pumped at the rate of 1.3 mL min−1. The limit of detection and limit of quantification were determined using dilution method (RSD/m formula). Results: SOF and DCV combination therapy is significant for the treatment of hepatitis C. Development of this RP-HPLC method for the simultaneous determination and quantification of SOF and DCV is a part of an extensive project of novel formulation development (fixed dose combination tablets of SOF and DCV). This method is successfully applied for raw materials as well as in combination tablets. This method may also be applied for the quantification of sofosbuvir and daclatasvir in blood samples of patients taking these two antivirals for hepatitis C treatment. Conclusion: The optimized method was successfully applied for the determination of sofosbuvir and daclatasvir in single run without any interference. Hence, the developed method is quite appropriate for the simultaneous determination of SOF and DCV in solid dosage form as well as in human plasma for clinical research studies.

Developed and Validated for the Estimation of Bupropion and Dextromethorphan in a Fixed Dose Combination of the Tablet.

The aim of this study was to develop a simple, accurate, and precise method for the estimation of bupropion and dextromethorphan in a fixed-dose combination of tablets and robust high-performance liquid chromatography for assay analysis of such a fixed combination. Chromatographic analysis was performed and separations were achieved on a Denali C18 150 × 4.6 mm, 5 micron using a mobile phase composition of ortho-phosphoric acid and acetonitrile in the ratio of 600:400 (v/v), flow rate of 1.0 mL/min, injection volume is 10 µL and run time of 6 min in isocratic elution. Ultraviolet (UV) detection was performed at a wavelength of 221 nm. The temperature was maintained at 30 °C. Well-resolved peaks were observed with a high number of theoretical plates, lower tailing factor, and reproducible relative retention time. The method was validated, and all validation parameters were found to be within the acceptance limits. A simple, accurate, and precise method has been developed for estimating bupropion and dextromethorphan in a fixed dose combination of tablets. The optimized method included the following parameters: column temperature of 30 °C, 40% acetonitrile as the mobile phase, and flow rate of 1.0 mL/min. Retention times were 2.25 min and 3.12 min for bupropion and dextromethorphan, respectively. The method was found to be linear in the range of 17.5-105 µg/mL [for R2 < 0.999) and 7.5-45 µg/mL (for R2 > 0.999] for bupropion and dextromethorphan, respectively. Both active pharmaceutical ingredients dissolved more than 90% within 5 min. The current study describes a new, simple, reliable, and economical elution reversed-phase high performance liquid chromatography method for estimating bupropion and dextromethorphan in a fixed combination tablet dosage form. The forced degradation studies were conducted using several degradation conditions such as acidic, alkali, oxidation, thermal, UV, and neutral conditions; the proposed method was effectively employed from the resolution of sample peaks. To the best of our knowledge, no such detailed and stability-indicating method has been reported for a fixed tablet dosage form.

Stability Indicating HPLC Method for In-vitro Determination of Pantoprazole Sodium and its Degradation Products in Simulated Gastric and Intestinal Fluids

Background: A high-performance liquid chromatography (HPLC) method was developed for the determination of Pantoprazole Sodium (PPZ) in the presence of its degradation products. The degradation of PPZ was studied in simulated intestinal fluid (SIF) and simulated gastric fluids (SGF) in various temperature conditions. Aim: This study aimed to establish a simple, sensitive, and rapid RP HPLC method for in-vitro determination of Pantoprazole Sodium and its degradation products in simulated gastric and intestinal fluids. Objective: Pantoprazole is acid labile drug. In order to determine pantoprazole in various oral dosage forms, the stability-indicating assay of PPZ was performed in phosphate buffer (pH 6.8) representing simulated intestinal fluid (SIF) and in 0.1 molars (M) Hydrochloric acid (HCl) as simulated gastric fluid (SGF) at two different temperature conditions, i.e., 25°C and 0°C, respectively. Method: Pantoprazole sodium was obtained from the Akums laboratory in Haridwar. The analysis was performed by high-performance liquid chromatography (HPLC), Shimadzu, equipped with two LC-10 AD VP solvent-delivery modules, a SPD-10A UV–-visible detector, and a manual injector valve with 20 μL sample loop. Phenomenex ODS analytical column (150 mm × 4.6 mm i.d., 5 μm particles) was done under reversed-phase partition chromatographic conditions. The mobile phase was phosphate buffer and acetonitrile (ACN) of pH 7.4, respectively, optimized in a 70:30 (v/v) ratio followed by filtration through a 0.45 μm membrane filter and degassed by ultrasonicator before use. The mobile phase was delivered at the flow rate of 2 mL/min. The various parameters, such as linearity, accuracy and precision of the analytical method, were studied. Result: The standard curve of PPZ was linear (R2&gt;0.99) over the concentration range of 5-30 μg/mL, and the relative standard deviation (RSD) values for intra-day and inter-day variations were in the range of 1.0-1.8%. The range of RSD was within ±2. Conclusion: The stability of PPZ in aqueous solution was pH dependent. The rate of degradation increases with decreasing pH. The pH stability of pantoprazole was studied at the above-mentioned temperature conditions. The PPZ peaks were analyzed by comparing them with fresh samples and were stable in SIF solution after 24 hours elapsed time at pH 6.8. The obtained degraded peaks in SGF (pH 1) were successfully separated from the PPZ.

Simultaneously measure the concentrations of busulfan and phenytoin in human plasma using an HPLC-MS/MS method: Application to the TDM for children underwent hematological stem cell transplantation

AbstractIn this work, a simple and rapid high performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) method was developed and validated to carry out the simultaneous measurement of busulfan (BU) and phenytoin (PHT) in the plasma of children. In this method, plasma sample could be prepared by one-step protein precipitation using 1 mL of methanol/water (1:1, v/v). After centrifugation (14,500 rpm, 5 min, 4 °C), 10 μL of the supernatant was injected into a Hypersil Gold C18 column (150 × 2.1 mm, 5 μm, Thermo Fisher Scientific) for separation by gradient elution. Quantification was carried out using multiple reactions monitoring (MRM) under positive scan mode. In the method verification, the calibration curves of BU and PHT showed satisfactory linearity (r &gt; 0.99) at the concentration ranging from 0.02 to 20 μg mL−1. The accuracy and precision were tested at four concentration levels (including the LLOQ level) with the relative error (RE) ranging from −0.80% to 11.45% and coefficient of variation (CV) between 0.93% and 7.74%. There was no pronounced matrix effect to interfere with the quantitative analysis. Compared to determine BU and PHT using two individual methods, less pre-treatment process, labor and blood sample volume are required in this proposed method. Finally, this method was successfully applied to the therapeutic drug monitoring of BU and PHT for children underwent hematological stem cell transplantation.

ANALYTICAL METHOD DEVELOPMENT AND VALIDATION OF RP-HPLC FOR AMLODIPINE BESYLATE AND LISINOPRIL IN COMBINED TABLET DOSAGE FORM BY USING SIMULTANEOUS ESTIMATION METHOD

&#x0D; &#x0D; &#x0D; The purpose of this study was to develop and validate a simple, sensitive, accurate, and reproducible reverse phase high performance liquid chromatography (RP-HPLC) method for simultaneous estimation of amlodipine besylate and lisinopril in a Tablet dosage form. The chromatographic measurement was performed on a Phenomenex C18 (250 × 4.6 mm, 5um) column with an optimised Acetate buffer mobile phase: Methanol (65:35). The flow rate was one ml/min, and the detecting wavelength was 221 nm. Triethanolamine was used to adjust the pH to 5. In the concentration range of 4-20 ug/ml, amlodipine besylate and lisinopril showed a linear response of the suggested approach. The correlation coefficients ('r' values) for amlodipine besylate and lisinopril were 0.9998 and 0.9995, respectively, and the retention times were 3.279 for amlodipine besylate and 6.124 for lisinopril, respectively. The developed chromatographic technique was validated for specificity, linearity, precision, accuracy, LOD, and LOQ using ICH Q2(R1) criteria. The analysis results have been validated in accordance to ICH guidelines&#x0D; &#x0D; &#x0D;

A Validated RP-HPLC Method for Simultaneous Assay Determination and UV-Spectrophotometric Method for Dissolution Estimation in Film-Coated Tablets Containing Cefixime and Clavulanic Acid

A simple and novel reverse phase high performance liquid chromatography (RP-HPLC) method was developed for the simultaneous assay estimation of film coated tablets incorporating clavulanic acid (CVA) and cefixime (CFX). Furthermore, the same method was also applied in order to assess the dissolution of CVA while a UV spectroscopic method was produced to examine the drug release of CFX. In this research, an analytical column used was C18 (4.6-mm × 25-cm); 5-μm. Moreover, the mobile phase was prepared by completely dissolving 3.408 gm of disodium hydrogen phosphate in 800 mL of water (HPLC grade), 200 mL methanol was added in (20:80 v/v) it and then finally pH was adjusted to 6.0 using orthophosphoric acid. In addition, flow rate, detecting wavelength, and run time were 1.0 mL/min, 220 nm, and 15 minutes, respectively. Furthermore, the maximum absorption of CFX was obtained at 288 nm thereby this wavelength was optimized for the dissolution estimation of CFX by UV spectroscopy. The validation parameters were validated according to the specifications and guidelines of the international conference on harmonization (ICH) prior to evaluating both assay and dissolution of CVA and CFX. In case of assay estimation, the findings of the linearity revealed a linear interrelation of concentration between 0.16 - 0.25 mg/mL for both CVA and CFX. Similarly, the calibration curve was linear between the concentration range of 0.03 – 0.045 mg/mL and 0.004 – 0.006 mg/mL, respectively for dissolution determination. Additionally, the value of LOD and LOQ of CVA for assay evaluation was obtained 0.011 and 0.033 μg, respectively. The accuracy of the developed method was assessed by determining recovery studies and the mean recovery of CVA and CFX obtained 99.35% and 100.50%, respectively at 100% spiked level for assay analysis. The repeatability evaluation for assay and dissolution demonstrated that the optimized method is precise within the appropriate range and the RSD% was observed lower than 2.0%. In addition, results of different validation parameters including study of solution stability were achieved within the suitable limit as well.

Validation of a HPLC method for the determination of diclofenac diethylamine and three of its impurities in a gel pharmaceutic form.

Monitoring impurities in drug products is a principal requirement of pharmaceutical regulatory authorities all over the world to ensure drug safety. For this reason, there is a great need of analytical quality control of dugs products. In this study, a simple, efficient and direct high performance liquid chromatography (HPLC) method was developed for the determination of three impurities of diclofenac. The HPLC method was developed using the mobile phase which consisted of HPLC grade mixture (Acetonitrile and 0.01M phosphoric acid adjusted to pH = 2.3) in the ratio of (25:75 v/v) respectively. The separation was performed in 15 minutes. The calibrations curve of the three impurities were linear; the correlation coefficient was 0.999 at a concentration of (0.00015 - 0.003 µg/mL). The validation of this method shows that this method meets all validation criteria. This shows the reliability of the use of this method for the routine control of diclofenac impurities. The validation of a robust HPLC method for the determination of diclofenac impurities is of great importance for the pharmaceutical industry to control its products.

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.

Analytical Method Development and Validation of Perindopril and Amlodipine as Multicomponent Formulation by HPLC Method

The work aims to develop and validate a simple, precise and accurate HPLC method per ICH guidelines. The linearity, specificity, Precision, and accuracy were within limits specified by the ICH guidelines. Hence the proposed system was found to be robust and precise. The objective of this study was to validate the simultaneous assay of Perindopril and Amlodipine by using High-Performance Liquid Chromatography (HPLC). Perindopril and Amlodipine were estimated with C18 Column (2.5 cm × 4.6 mm and five μm particle size) with a VWD detector. The mobile phase consists of Buffer Solution and Acetonitrile 55:45 v/v with a flow rate of 1 ml/min. The volume injected was 20 μL. The detection wavelength was 215 nm, respectively. The retention time of Perindopril and Amlodipine was found to be 3.40 min and 4.73 min. The system suitability and all essential validation parameters were meticulously completed. The LOD and LOQ for Perindopril were found to be 2.62 μg/ml and 7.95 μg/ml, respectively, and for Amlodipine, 1.06 μg/ml and 3.22 μg/ml, respectively. The analytical curve was linear (R2 = 0.999). The system exhibits sufficient accuracy with a relative standard deviation under 2%. The method showed good duplicability and recovery with % RSD less than 2%. The linearity, specificity, Precision, and accuracy were within limits specified by the ICH guidelines. Hence the proposed system was found to be robust and precise. The method has to be such that it takes less time development and the best accurate and complete results have been obtained. Hence the proposed method for simultaneous estimation of Perindopril and Amlodipine by HPLC was found to be robust and precise.

New method for determination of diclofenac sodium by High Performance Liquid Chromatography

In this study a simple, coast effective and direct simple High Performance Liquid Chromatography (HPLC) method has been developed for the determination of diclofenac sodium in it's pure form and different pharmaceutical preparations. Standard diclofenac sodium and its dosage forms were supplied from Ninava State Company For Drug Industries and Medical Appliances (NDI). HPLC method was developed by using mobile phase which was composed of a mixture of HPLC grade (methanol, acetonitrile and deionized water)in the ratio of (60:20:20) respectively. Separation has been completed within 2 min. Calibration curve was linear, coefficient correlation was found to be 1.00 at a concentration of (0.25-4.0 µg.ml-1). The relative standard deviation (RSD) was found to be &gt;1.2%. The proposed method was successfully applied for the determination of diclofenac sodium in its pure form and different pharmaceutical preparations (injection, tablets, eye drops, suppositories and gel) without using buffer system and there is no inference with additives.

DEVELOPMENT AND VALIDATION OF RP-HPLC METHOD FOR SIMULTANEOUS ESTIMATION OF ROSUVASTATIN AND TENELIGLIPTIN IN THEIR SYNTHETIC MIXTURE

A simple, fast and accurate Reverse Phase High Performance Liquid Chromatography method has been established and validated for estimation of rosuvastatin and teneligliptin in their synthetic mixture. The chromatographic separation was achieved on Agilent Inertsil Octa Decyl Silane (ODS) C18 (250 × 4.6 mm, 5 µm) column using mobile phase composition of methanol: water (pH=3 adjust with 10 % Orthophosphoric acid) (70:30 V/V), in isocratic mode at flow rate of 0.8 mL min-1 at a detection wavelength of 244 nm. In terms of precision, accuracy, limit of detection and limit of quantitation, the method was validated. Rosuvastatin and teneligliptin had retention times of 7.41 and 4.35 minutes, respectively. Rosuvastatin and teneligliptin were reported to have linearity of 5-15 µg mL-1 and 10-30 µg mL-1, respectively, with correlation coefficient (R2 ) value of 0.998 for rosuvastatin and 0.999 for teneligliptin. Method can be employed for study of rosuvastatin and teneligliptin in their mixture.

Development and Validation of a RP-HPLC Method for the determination of Venlafaxine hydrochloride in bulk and Pharmaceutical Dosage Forms

A simple, accurate, precise and rapid reverse phase high performance liquid chromatography method was developed and validated for the analysis of venlafaxine hydrochloride in bulk and marketed pharmaceutical dosage form. The chromatographic separation was carried out using a Phenomenex C18, (250mm × 4.6mm × 5µ) column. The optimum mobile phase consisted of ammonium acetate buffer, methanol (60:40) with pH adjusted to 5. The mobile phase was delivered isocratically at a flow rate of 1mL/min with UV detection at 227nm. Venlafaxine hydrochloride was eluted at 2 minutes. The method was validated for linearity, precision, accuracy, robustness and recovery. The method was linear in the concentration range of 15-165µg/ml with correlation coefficient 0.9997. limit of detection and limit and quantification were found to be 185ng/ml and 563ng/ml respectively.

Simple HPLC-UV Method for Piperacillin/Tazobactam Assay in Human Plasma

Piperacillin (Pip)/tazobactam (Taz) is a broad-spectrum antimicrobial agent that has been commonly used in the intensive care unit for severe and life-threatening infections. Recent evidence suggests that therapeutic drug monitoring (TDM) for Pip could be beneficial in clinical practice to facilitate dose optimization and increase the odds of treatment success. The aim was to develop and validate a sensitive and simple high-performance liquid chromatography (HPLC) method for the simultaneous quantification of Pip and Taz in human plasma. Samples (0.3 mL) were deproteinized with acetonitrile. The supernatant was evaporated and then reconstituted and injected into the HPLC. The chromatographic analysis was carried out by using the C18 column and gradient elution with the acetonitrile:water mobile phase mixture with 0.1% trifluoracetic acid at a flow rate of 0.8 mL/min using a UV detector at 218 nm. The method had acceptable linearity (r2 > 0.99) over the concentration ranges of 0.5-400 μg/mL and 1-100 μg/mL for Pip and Taz, respectively. The method demonstrated acceptable inter- and intra-day precision and accuracy within ±20% with adequate stability results. The developed method is sensitive and simple and utilizes simple sample preparation and elution steps, making it suitable and practical for Pip/Taz TDM.

A Sensitive LC-MS/MS Method for the Simultaneous Determination of Skimmin, a Potential Agent for Treating Postpartum Stroke, and Its Metabolite Umbelliferone in Rat Plasma.

Skimmin, a potential agent for treating postpartum stroke, is one of the most important coumarins extracted from the leaves of skimmia. In this study, a specific, sensitive, and simple high-performance liquid chromatography with tandem mass spectrometry (HPLC-MS/MS) method for the simultaneous determination of skimmin and its metabolite umbelliferone in rat plasma was established and validated. Chromatographic separation was performed by an Inertsil ODS-3 column (50 mm × 4.6 mm, 5 μm) with a mobile phase consisting of 0.1% formic acid in distilled water-acetonitrile at a flow rate of 0.5 mL/min with gradient elution mode. All analytes were detected and quantified in negative multiple reaction monitoring (MRM). All calibration curves showed good linearity (r > 0.995) over the concentration range of 10-10 000 and 2.0-2000 ng/mL for skimmin and umbelliferone, respectively. The selectivity, sensitivity, extraction recovery, matrix effect, and stability met all requirements. The analysis method was successfully applied to the pharmacokinetic study of skimmin and umbelliferone in rats following oral administration of skimmin at the doses of 10, 30, and 90 mg/kg. With the exception of AUC(0-∞) and Cmax, MRT and Cl/F of skimmin had significant statistical difference with the increasing doses. Skimmin might exhibit nonlinear pharmacokinetic characteristics in rats. This was the first study to investigate the pharmacokinetic characteristics of skimmin as a candidate agent for treating postpartum stroke.

HPLC-UV Method Development and Validation to Monitor Difluprednate Synthesis.

During the synthesis of active pharmaceutical ingredients (APIs) there is a need for the development and validation of a simple and rapid high performance liquid chromatography (HPLC) method for the determination and quantification of the synthesized product and related by-products. An HPLC method gives a better understanding of how a synthesis is proceeding. A rapid and easy to use HPLC-UV (ultraviolet) method for the determination of difluprednate and monitoring of impurities generated during synthesis was developed and validated. A Shimadzu VP Series HPLC equipped with a LabSolutions software and UV detector set at 240nm was used for analysis. The mobile phase consisted of phosphate buffer (pH6) and acetonitrile 50:50 (v/v) and was eluted at a flow rate of 1.2mL/min. Separation took place on a reversed-phase Kinetex C18 column (150×4.60mm; 5μm i.d.). Column temperature was set at 40°C. The developed method was found to have good linearity and acceptable accuracy and precision. The developed method may be effectively applied to determine products and by-products formed during synthetic reactions of steroids and to calculate the yield of the products obtained during each step of the synthesis.

A rapid quantification method for simultaneous determination of pendimethalin and metribuzin contents in suspoemulsion formulation

Pendimethalin and metribuzin are combined to a suspoemulsion (SE) formulation that is treated as a pre-emergence and early post emergence herbicide. A simple reverse phase high performance liquid chromatography (RP-HPLC) method has been validated and applied for the simultaneous analysis of metribuzin and pendimethalin contents in suspoemulsion formulation. The mobile phase for the method was acetonitrile: water (65:35v/v) at a flow rate of 1.0 mL/min, and quantification was accomplished at 237 nm. Hypersil ODS column was used for the method's development and validation. Metribuzin's and pendimethalin's RTs were found to be 3.6 and 11.9 min, respectively. The limit of detection was 0.03 mg/l and 0.04 mg/l for Metribuzin and Pendimethalin respectively. The limit of quantification values was 0.09 mg/l for metribuzin and pendimethalin. The linearity of proposed method was investigated in the range of 2.51 to 100.54 mg/l (r2 = 99997) and 0.25 to 9.80 mg/l (r2 = 99997) for Pendimethalin and Metribuzin respectively. The percentage recovery found to be in range from 99.8 to 100.1 % and 99.8 to 100.3 for Pendimethalin and Metribuzin respectively. The percentage RSD values for intraday precision study and inter-day precision study were < 2.22 and < 1.57 for Metribuzin and Pendimethalin respectively as per modified Horwitz equation as requirements by SANCO. The method's performance was validated in terms of selectivity, specificity, precision, linearity, accuracy, LOD, LOQ, and robustness, according to SANCO guidelines. In this study, for the first time, an easy and speedy reverse phase high performance liquid chromatographyapproach for the simultaneous analysis of pendimethalin and metribuzin in SE formulation is presented.

A Validated RP-HPLC Method Development and Validation for The Simultaneous Estimation of Nebivolol and Valsartan in Bulk Drug and Pharmaceutical Dosage Form

A novel, simple, accurate and rapid Reverse Phase High Performance Liquid Chromatography (RP-HPLC) method has been developed and validated for the determination of Nebivolol and Valsartan in bulk and tablet dosage form. Gradient elution chromatography has been developed on C18 column (5µm, 4.6mm* 250mm) with a mobile phase consist of pH 4 acetonitrile,water and Methanol (25:35:40) with a flow rate of 1ml/min at detection of 270 nm wavelength. The retention time for Nebivolol and Valsartan was found to be 4.59 and 10.2 respectively. Chromatography parameters were validated as per ICH guidelines and the method can be applicable for routine quantitative analysis of drugs in combined dosage form.