Accurate Reverse Phase HPLC Research Articles

REVERSED PHASE HPLC DETERMINATION OF TOTAL HOMOCYSTEINE, CYSTEINE, CYSTEINYL GLYCINE, GLUTATHIONE IN PLASMA OF EPILEPTIC PATIENTS

The fast and accurate reversed-phase HPLC method was predicated on qualitative and quantitative estimation of the total concentrations of the essential thiols in the plasma of adults volunteers compared with epileptic patients, which is l-cysteine (Cys) total homocysteine (tHys), cysteinyl glycine (Cys-Gly), glutathione (GSH) and cysteamine (Cstm), respectively. The plasma samples of (50) epileptic patients were classified into three groups; the non-anti-epileptic drug consisted of 20 patients (12 male, eight female) who were mainly diagnosed with idiopathic generalized epilepsy. The mean age was 34 ± 13 (range 19–48) years. The second group consisted of 30 patients receiving two types of anti-epileptic drugs, 18 patients (12 male and six female) receiving Carbamazepine, and 12 patients (7 males and five female) receiving phenytoin. The mean age of the patients was 32 ± 11 Compared with 31 healthy volunteers (20 male and 11 female). The mean age of the controls was 31 ± 9 (range 26–49 years). The mean duration of treatment of patients receiving the anti-epileptic drug was six months). All measurement was done using pre-column derivatization with bromobimane, and the derivative of each standard mixture were baseline separated on C18-DB (50 x 4.6 mm ID) column, 3μm particle size. The regression coefficients for the separated and standard deviation SD within-run ranged from 0.09 to 8.40 μmol/L, and between-run ranged from 0.15to 9.16 μmol/L; the analytical procedure gave good linearity in the range between 1.25 to 20 µmole/ l, the detection limit was 0.1 µmole /L for all the thiol groups. Analytical recovery was 96.9–107.4 %; the mean concentration of plasma cysteine and total homocysteine was slightly higher in males than females; it was 221± 75 for adult males and 190± 44 μmol/L for adult females, while t-homocysteine was (10.55 ±2.45 vs. 9.79 ± 1.88 μmol/L, the results observed that the value of cysteine and homocysteine were significantly higher in epileptic patients using Carbamazepine and phenytoin than in healthy volunteers. Mean values for glutathione were lower, while cysteinyl glycine showed no significant difference in healthy and epileptic patients and no sex- and age-dependent.

Identification of benzydamine and its metabolit in the presence of certain anti-inflammatory non-steroidal drugs

The aim of the work. Currently, a large number of cases of non-medical use of benzydamine hydrochloride have been described. The identification of benzydamine and its metabolite, benzydamine N-oxide, in the presence of some non-steroidal anti-inflammatory drugs, has been insufficiently studied. Therefore, the development of a method for its identification in biological material is an urgent task.
 Materials and methods. The subjects of the study were benzydamine hydrochloride and its metabolite, as well as some non-steroidal anti-inflammatory drugs, which are its analogues in terms of pharmacological action. The studies were carried out by methods of thin layer chromatography and high-performance liquid chromatography.
 Results. At the first stage a screening method for benzydamine identification was studied using the extraction in acidic and basic conditions. It was shown that benzydamine can be isolated in both medias with subsequent development with a solution of iodoplatinate and Dragendorff's reagent according to Munier or with Mandelin reagent respectively. The mobile phase was selected and respective hRf for the target molecule were defined.
 After a preliminary identification of benzydamine a reference method for the final confirmation of the drug that had led to poisoning was proposed. A robust, specific and accurate reversed phase HPLC method was chosen. It was shown that benzydamine exists in biological material mainly in a form of metabolite – benzydamine N-oxide. The selected method was able to separate and determine key analytes in biological samples after a preparative isolation by TLC method. The comparison with UV spectra of the reference standard of benzydamine hydrochloride was proposed to avoid false positive conclusion of drug identification.
 Conclusions. Proposed methodology can be applied for routine identification of benzydamine poisoning in toxicological laboratories

STABILITY INDICATING HPLC METHOD DEVELOPMENT AND VALIDATION FOR SIMULTANEOUS ESTIMATION OF EMTRICITABINE, DOLUTEGRAVIR AND TENOFOVIR ALAFENAMIDE IN THEIR COMBINED PHARMACEUTICAL DOSAGE FORM

A simple, rapid, precise and accurate reversed phase HPLC stability indicating method was developed for simultaneous determination of emtricitabine (ECT), dolutegravir (DOL) and tenofovir alafenamide (TEN) in their combined dosage marketed formulation. The chromatographic separation was achieved on Hypersil BDS C18 (250  4.6 mm, 5.0 µ) column using 0.5 M potassium dihydrogen phosphate buffer: acetonitrile (80: 20, v/v), pH 3.5 ± 0.1 maintained by 0.1 % o-phosphoric acid as mobile phase at a flow rate of 1.0 mL/min and monitored at 260 nm. All three analytes were resolved at retention times of 5.777, 6.783, 8.393 min for ECT, DOL, and TEN respectively. The assay performance of the present developed method was validated for various parameters like system suitability, linearity, accuracy, precision, robustness, limit of detection and quantification. The calibration curves were linear over concentration ranges of 12.5-75.0, 50.0-300, and 6.25-37.5 μg/ml for DOL, ECT, and TEN, respectively with correlation coefficients  0.9995. All three analytes were subjected to various stress conditions of hydrolysis, oxidation, photolysis and thermal degradation. Considerable degradation was found in alkaline condition. The present method demonstrated to be effective stability-indicating by resolution of the analytes from their forced-degradation products. The validated method was successfully applied for the simultaneous estimation three drugs in their combined pharmaceutical dosage form Spegra.

DEVELOPMENT AND VALIDATION OF HPLC METHOD FOR QUANTIFICATION OF DACLATASVIR IN PURE AND SOLID DOSAGE FORM

Daclatasvir is a novel and highly selective NS5A replication inhibitor of HCV. It is stated to offer the potential for interferon free treatment when used in combination with another direct acting antivirals. The current project is aimed to develop and validate an easy to perform, precise, fast and accurate reverse phase HPLC method for Daclatasvir in the solid dosage form by wielding UV detector, in accordance with ICH Q2 (R1) guidelines. The process was carried out for daclatasvir, utilizing C-18 column (25 cm length, 4.6 mm internal diameter, and 5 µm particle size) in isocratic mode, with mobile phase comprising of acetonitrile and phosphate buffer (40:60 v/v). The flow rate was 1 mL/min and the detection wavelength examined out by UV detector was 304 nm. The retention time for daclatasvir was 3.0 ± 0.1 min. RSD (0.65 %), theoretical plates (1118117) and tailing factor (1) complied within acceptable certified limits. The developed method showed the linearity within a range (1 – 5 µg/mL). The accuracy of the developed method was figured out by recovery analysis and it was 99 %. The precision analysis i.e. interday (0.0.790937 %), intraday (0.364517 %) and repeatability (0.229167 %), were within the acceptance criteria. All the validation parameters were within the acceptable limits. The sample solutions of daclatasvir tablets (Dacriva, 60 mg) developed and correlated its chromatograms with working standard solution and perceived the percentage purity of daclatasvir in the tablet, found within recommended acceptable limit (90 – 110 %). It is concluded that the HPLC method is sensitive, stable, rapid and reliable. It can be deployed for the quantification of daclatasvir of standard and brands available in market in the solid dosage form in the pharmaceutical industry.

A Thorough HPLC Assay of Quaternary Veterinary Formulation coupled with Environmental Assessment Tool.

A new and accurate reversed phase HPLC method with UV detection has been established for any veterinarian analyst for simultaneous determination of a veterinary quaternary mixture of sulphadimidine sodium (SDS), sulphaquinoxaline sodium (SQS), diaveridine (DVD) and vitamin K3 (VTK3) in their formulation. The stationary phase was SEA C18 column (250 × 4.6mm i.d., 5μm particle size) at 25°C with an isocratic mode, using a mobile phase containing a mixture of methanol:acetonitrile:distilled water in the ratio of (20:20:60, by volume). The flow rate was 0.8mLmin-1, and UV detection was performed at 230nm. The HPLC assay was coupled with Environmental Assessment Tool (EAT), which represents a simple and proficient approach for profiling the greenness of the method. This takes into consideration the environmental, health and safety issues for all solvents that involved in the chromatographic method and calculates a total score that can be used for comparison of the greenness of different methods. The method was found to be linear over (0.5-30) μg/mL for all cited drugs with mean percentage recoveries (99.56±1.141) for VTK3, (99.56±1.056) for DVD, (99.62±1.482) for SDS and (99.52±1.205) for SQS. The results were statistically compared with those of the official and reported methods; using Student's t-test and F-test, showing no significant difference with respect to accuracy. Specificity of the applied method was assessed by analyzing the laboratory-prepared mixtures. The developed method was validated according to ICH guidelines. The proposed methodology can be applied for rapid routine assay of this combination.

Simultaneous Estimation of Four Preservatives in Pharmaceutical Ointment by RP-HPLC

A simple, specific, precise and accurate reverse phase HPLC method has been developed for the simultaneous determination of preservatives (Methyl Para hydroxybenzoate, Ethyl para hydroxybenzoate, Propyl para hydroxybenzoate and Chlorocresol) in ointment. The chromatographic separation was achieved on Zorbax column, 150 mm × 4.6 mm, 5 µm, using PDA detector. The mobile phase was 50% Methanol (v:v), at a flow rate of 1.0 mL/min. All preservatives were detected at 230 nm, at 30oC temperature for column. The retention times were 2.15 min for Methyl para hydroxybenzoate, 2.70 min for Ethyl para hydroxybenzoate, 3.69 min for Propyl para hydroxybenzoate and 4.01 min for Chlorocresol, with incredibly good resolution between peaks. The method was validated according to the ICH guidelines with respect to specificity, linearity (r2 =0.999; 0.998; 0.999 and 0.998), accuracy (99 to 100.1%), precision (RSD<2%).

Development and Validation of Stability Indicating RP-HPLC Method for Determination of Enzalutamide

A simple, isocratic, and accurate reversed phase HPLC method was developed for the quantitative determination of enzalutamide. The chromatographic separation was achieved on an Qualisil BDS C18 (250 mm x 4.6mm, 5 μm) column using methanol: ammonium acetate buffer pH 4.2 adjusted with glacial acetic acid: (60:40, v/v) as a mobile phase, at a flow rate of 1 ml/min and detection at 236nm. The linear range for enzalutamide were 2.0 to 10 μg/mL was obtained with correlation coefficients ≥ 0.998. The retention time was found to be 6.30min. Enzalutamide was subjected to stress conditions hydrolysis (acid, base) oxidation, photolysis and thermal degradation and the stressed samples were analysed by the developed method. The method was validated for the precision, accuracy, linearity and robustness. The developed stability indicating method for enzalutamide was validated as per ICH guidelines.

Optimization and validation of a new chromatographic method for the assay of veterinary formulation

New, validated and accurate reversed phase HPLC method with UV detection has been established for simultaneous determination of a veterinary binary mixture of doxycycline hydrochloride (DOX) and tylosin tartrate (TYT). The stationary phase was ACE- 126-2546 AQ C-18 (250 × 4.6 mm i.d., 5 μm particle size) column at 25 °C, in an isocratic mode, using mobile phase containing a mixture of methanol: acetonitrile: distilled water in the ratio of 60:20:20 (v:v:v), with 0.01% trichloroacetic acid at the flow rate of 0.8 mL/min and UV detection was performed at 270 nm. The retention times were 4.02±0.01 and 5.62±0.01 mins for DOX and TYT, respectively. Selective determination of the cited veterinary drugs has been developed in their formulation. The method was found to be linear over 1-50 µg/mL for DOX and TYT with mean percentage recoveries 99.62±1.220 and 100.09±1.104%. The method was proven to be accurate, precise and specific. The obtained results were statistically compared with those of the official and reported methods; using Student’s t test, F test and one-way ANOVA, showing no significant difference with high accuracy. Specificity of the applied method was assessed by analysing the laboratory-prepared mixtures and their combined dosage form. The developed method was confirmed according to ICH guidelines. The validated method can be considered as alternative and basic method for the routine determination of this fixed dose combination with minimum sample preparation.

ANALYTICAL METHOD EVELOPMENT AND VALIDATION FOR ANTI ASTHAMATIC DRUG OXYMETAZOLINE HYDROCHLORIDE IN NASAL SPRAY FORMULATIONS BY RP-HPLC

A new, simple, accurate and economic reverse-phase HPLC method has been developed for quantification of Oxymetazoline Hydrochloride in nasal spray formulations. This developed method has been validated according to International Conference on Harmonization (ICH) guideline with respect to system suitability, specificity, precision, linearity, accuracy, and robustness. An isocratic condition of mobile phase Phosphate buffer (pH 3.0): Acetonitrile in a ratio of 60:40, v/v at a flow rate of 1.0 mL/minute over RP C18 (octadecylsilane (ODS), 250 × 4.6 mm, 5 µm, ECLIPSE X DB C-18) column at ambient temperature was maintained. This method is specific and showed excellent linear response with correlation coefficient (R2) values of 0.999, which was within the limit of correlation coefficient (R2 0.995). A simple and accurate reversed-phase HPLC method for the analysis of Oxymetazoline Hydrochloride in nasal spray formulations was developed and validated successfully.

Development and Validation of Stability-indicating Reverse Phase HPLC Assay for Tramadol in Bulk and Tablet Formulations

This manuscript describes the development and validation of a simple, isocratic, and accurate reversed-phase HPLC method for the assay of tramadol in bulk powder form and tablet formulations. The chromato-graphic separation was achieved on an Qualisil BDS C18 (250 mm x 4.6mm, 5μm) column using acetonitrile: methanol: phosphate buffer pH 3.4 adjusted with orthophosphoric acid: (20:10:70, v/v) as a mobile phase, at a flow rate of 1 mL/min and detection at 271 nm. The linear range for tramadol were 2.0 to 10 μg/mL was obtained with correlation coefficients ≥ 0.999. The retention time was found to be 4.47 min for tramadol that was subjected to stress conditions, such as hydrolysis, oxidation, photolysis and thermal degradation, and the stressed samples were analyzed using the above methodology. The method was validated for the precision, accuracy, linearity and robustness. The developed stability-indicating method for tramadol was validated as per ICH guidelines.

The Estimation of Ceftaroline Fosamil in Lyophillized Powder for Injection by RP-HPLC.

A simple, precise, rapid and accurate reverse phase HPLC method developed for the estimation of Ceftaroline Fosamil in sterile powder for injection. An RP C-18 Hypersil BDS C18, 150x4.6 mm i.d, 5 µm partical size, with mobile phase consisting of mixture of 0.03 M ammonium acetate in water (solvent A) and methanol (solvent B) was set in the gradient mode over 30 minutes, was found to be most suitable for gradient mode was used to separate ceftaroline fosamil. The flow rate was kept at 1 ml/min and the effluents were monitored at 205 nm. The retention time was 13.12 min. The detector response was linear in the concentration of 20-240 mcg/ml. The respective linear regression equation being Y=25740x+70940. The limit of detection and limit of quantification was 0.25 and 0.75 mcg/ml respectively. The percentage assay of Ceftaroline Fosamil was 99.75%. The method was validated by determining its accuracy, precision and system suitability. The results of the study showed that the proposed RP-HPLC method is simple, rapid, precise and accurate, which is useful for the routine determination of Ceftaroline Fosamil in bulk drug and in its pharmaceutical dosage form.

DEVELOPMENT AND VALIDATION OF RP-HPLC METHOD FOR PHENYTOIN SODIUM AND PHENOBARBITONE IN BULK AND PHARMACEUTICAL DOSAGE FORM

Objective: To develop an accurate, simple, rapid, precise, and linear RP-HPLC method for the simultaneous estimation of phenytoin sodium and phenobarbitone in tablet dosage form and validated as per ICH guidelines.Methods: The method used was a reverse phase HPLC (RP-HPLC) method using Hypersil BDS C18, (250×4.6 mm, 5 μm) column, mobile phase comprising of methanol: phosphate buffer (pH 5.0) (50:50), flow rate of 1.0 ml/min and a detection wavelength of 215 nm using a UV detector.Results: The retention time for phenytoin sodium and phenobarbitone was found to be 3.97 min and 6.90 min, respectively. The linearity of developed method was achieved in the range of 10-30 μg/ml for phenytoin sodium and 3-9 μg/ml for phenobarbitone. The detection (LOD) and quantitation (LOQ) limits were 1.44 and 4.36 µg/ml for phenytoin sodium and 0.40 and 1.35 µg/ml for phenobarbitone respectively.Conclusion: A simple, accurate, precise, linear and rapid RP-HPLC method was developed for simultaneous quantitative estimation of phenytoin sodium and phenobarbitone in bulk and pharmaceutical formulation. The method was validated as per ICH guidelines. Hence, the method holds good for the routine analysis of phenytoin sodium and phenobarbitone in various pharmaceutical industries as well as in academics.

Development and validation of the HPLC method for varenicline determination in pharmaceutical preparation

AbstractA simple and accurate reverse phase HPLC method has been developed and validated for quantification of varenicline in bulk drug and pharmaceutical dosage forms. Herein, an isocratic LC analysis was carried out on a Chromolith Performance RP18e column with methanol-buffer solution pH 3.5 (a buffer solution containing sodium benzoate (0.5 mmol/l) adjusted to pH 3.5 with trifluoroacetic acid (20 mmol/l) (55:45, V/V)) at a flow rate of 1.2 ml/min. The detection wavelength was set at 320 nm. The calibration curve was linear (r = 0.9999) in the studied range of concentration (0.2-10 μg/ml). The selectivity and sensitivity of the elaborated method were satisfactory, and the limits of detection and quantification was less than 20% of the specification level. Moreover, the inter- and intra-day precisions was found to be less than 3% (RSD), while the recovery values expressing inter- and intra-day accuracy was varied from 99.73 to 101.23. The varenicline solution was stable over a period of 3 days on storage under refrigeration. The utility of the developed method was examined by analysing the tablets containing VAR. As a result, the method was found to be selective, sensitive, precise and accurate.

Reverse phase HPLC method for detection and quantification of lupin seed γ-conglutin.

A simple, selective and accurate reverse phase HPLC method was developed for detection and quantitation of γ-conglutin from lupin seed extract. A linear gradient of water and acetonitrile containing trifluoroacetic acid (TFA) on a reverse phase column (Agilent Zorbax 300SB C-18), with a flow rate of 0.8ml/min was able to produce a sharp and symmetric peak of γ-conglutin with a retention time at 29.16min. The identity of γ-conglutin in the peak was confirmed by mass spectrometry (MS/MS identification) and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The data obtained from MS/MS analysis was matched against the specified database to obtain the exact match for the protein of interest. The proposed method was validated in terms of specificity, linearity, sensitivity, precision, recovery and accuracy. The analytical parameters revealed that the validated method was capable of selectively performing a good chromatographic separation of γ-conglutin from the lupin seed extract with no interference of the matrix. The detection and quantitation limit of γ-conglutin were found to be 2.68μg/ml and 8.12μg/ml respectively. The accuracy (precision and recovery) analysis of the method was conducted under repeatable conditions on different days. Intra-day and inter-day precision values less than 0.5% and recovery greater than 97% indicated high precision and accuracy of the method for analysis of γ-conglutin. The method validation findings were reproducible and can be successfully applied for routine analysis of γ-conglutin from lupin seed extract.

A comparative study of HPLC-UV and UPLC-DAD methods for simultaneous estimation of aspirin and cilostazol in the presence of their related impurities in bulk and capsules

ABSTRACTAspirin (ASP) and cilostazol (CST) are used as a combination in pharmaceutical formulations for treatment of strokes. Salicylic acid (SAL) is considered to be one of the main synthesis impurities and a degradation product of ASP. On the other hand, the main related impurities of CST are CST related A, B, and C (CST-RA, CST-RB, and CST-RC), respectively. Furthermore, as high efficiency and less elution are the basic requirements of high-speed chromatographic separation, so, a comparative study of two simple, precise, and accurate reversed-phase HPLC and UPLC methods was developed and validated for simultaneous estimation of ASP and CST in bulk and capsules in the presence of SAL, CST-RA, CST-RB, and CST-RC. A Eurospher II C18 (250 × 4.6 mm2, 5 µm) for HPLC method and an Agilent Zorbax Eclipse Plus C18 (50 × 2.1 mm2, 1.8 µm) for UPLC method were used. A gradient mobile phase of 20 mM anhydrous KH2PO4 buffer solution (containing 0.2% triethylamine (TEA), v/v) with pH adjusted to 2.9 using orthophosphoric acid (solution A) and acetonitrile (solution B) mixed in different proportions for HPLC and UPLC methods was prepared. Flow rate was set to 1.0 and 0.3 mL min−1 for HPLC and UPLC methods, respectively, and the detection was performed for both methods at 210 nm. It worth noting that the proposed UPLC-DAD assay exhibited relatively much more precision, sensitivity, specificity, and economic and chromatographic separation superiority than proposed HPLC-UV assay. Both developed methods were compared with reference methods to prove its applicability and are suitable for purity assessment of ASP and CST in bulk and capsules.

LIQUID CHROMATOGRAPHIC DETERMINATION OF SOLIFENACIN SUCCINATE, FLAVOXATE HYDROCHLORIDE AND TOLTERODINE TARTRATE IN BULK DRUGS AND THEIR PHARMACEUTICAL DOSAGE FORMS

A simple, precise, specific and accurate reversed phase HPLC (RP-HPLC) method has been developed for the determination of solifenacin succinate (SOLS), flavoxate HCl (FLXHC) and toltoridine tartarate (TOLT) in bulk and pharmaceutical dosage forms. The proposed RP-HPLC method was carried out using Xterra RP-18 column (5 μm practical size, 25 cm x 4.6 mm i.d.). The flow rate, the injection volume and the detection wavelength were 1.0 mL/min, 20 mL and 200 nm, respectively. The mobile phase consisted of 0.05 M pentane sulfonic acid sodium salt (SOLS: pH 3.0±0.05, FLXHC and TOLT: pH 5.5±0.05) and acetonitrile (50:50 v/v). The retention times for SOLS, FLXHC and TOLT drugs were found to be 4.1±0.1 min, 4.3±0.0 min and 5.8±0.1 min, respectively. The calibration was linear over the concentration range of 0.1-100 μg/mL. The mean recoveries for SOLS, FLXHC and TOLT drugs were about 99.80%, 100.43% and 100.00%, respectively. The method was validated according to the ICH guidelines with respect to specificity, linearity, accuracy, precision and robustness.

Simultaneous estimation of Esomeprazole and Tadalafil in pharmaceutical formulations using High Performance Liquid Chromatography

An easily, specific, precise, and accurate reversed-phase HPLC method was developed and validated for simultaneous estimation of esomeprazole ( Nexium®) and tadalafil (Cialis®) in pharmaceutical formulation. The separation was achieved by using Hypersil BDS C18 column (250 mm A— 4.6 mm; 5.0 μm) and acetonitrile: 0.05 M potassium dihydrogen phosphate buffer at pH 6 adjusted with phosphoric acid as a mobile phase at a flow rate of 1 mL/min. Detection was carried out at wavelength 285nm. The retention time of esomeprazole and tadalafil were 3.1, 3.7 min, respectively. The linearity was established over the concentration ranges of 60-180μg/mL and 40-120μg/mL with correlation coefficients 0.9998 and 0.9996 for esomeprazole and tadalafil respectively. The mean recoveries were found to be in the ranges of 98–102% for esomeprazole and tadalafil. The proposed method has been validated as per ICH guidelines and successfully applied to the simultaneous estimation of esomeprazole and tadalafil in pharmaceutical formulation.

Development and Validation of a Simple RP-HPLC Method for Simultaneous Estimation of Metformin Hydrochloride and Rosiglitazone in Pharmaceutical Dosage Forms

The objective of this study was to develop a simple, efficient, precise and accurate reversed phase HPLC (RP-HPLC) method for simultaneous determination of metformin in combination with rosiglitazone in newly formulated tablets. The combination of these drugs are commonly used and prescribed as anti-diabetic drugs in Bangladesh. The method has been developed by using the mobile phase comprising of sodium dihydrogen phosphate (NaH2PO4) buffer (pH 3.5) and acetonitrile (60:40, v/v) at a flow rate of 0.7 ml/min over C18 bonded silica column (ODS) (250 x 4.6 mm, 5 ?) at ambient temperature. The effluents were monitored at 230 nm and retention times were found to be 3.35 min and 11.95 min for metformin and rosiglitazone, respectively. Calibration curves were determined with a range from 0.03125 to 0.50 ?mole/ml of standards and the regression coefficients (r2) were found as 0.999 for metformin and 1.0 for rosiglitazone. The assay was validated for the accuracy, precision etc. according to ICH, USP and FDA guidelines. The proposed method can be useful in routine analysis for quantitative determination of metformin hydrochloride and rosiglitazone in pharmaceutical dosage forms. DOI: http://dx.doi.org/10.3329/dujps.v11i2.14574 Dhaka Univ. J. Pharm. Sci. 11(2): 157-163, 2012 (December)

Optimized Preparation of Celastrol-Loaded Polymeric Nanomicelles Using Rotatable Central Composite Design and Response Surface Methodology

Celastrol has been found to be a potent anti-inflammatory and antitumor plant derivative recently. Herein we established an accurate reverse phase HPLC (RP-HPLC) determination method of celastrol, and prepared an effective nanoscale drug delivery system from the optimized formulations of celastrol-loaded carboxyl functioned poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol)(PEO-block-PPO-block-PEO, Pluronic) polymeric nanomicelles. Rotatable central composite design (RCCD) and response surface methodology (RSM) were applied to improve the celastrol entrapment efficiency (EE), drug loading percentage (DLP), and decrease the particle size. The characteristics of the optimized micelles including particle size distribution, morphology, zeta potential and in vitro release of celastrol from micelles were carried out. Results showed that RP-HPLC method was successfully applied to detect and qualify the celastrol. The drug quantification range of the method was 20-200 microg/mL with a linear correlation coefficient of greater than 0.999. The average accuracy (99.763%), the precision (0.521%) and recovery (99.63%) for this method were good. The optimal conditions for the preparation of celastrol-loaded polymeric nanomicelles were found to be: the celastrol/polymer weight ratio 8.5-9 mg/25 mg, hydration volume 11-17 mL. Under the optimal conditions, the EE was 100.3 +/- 4.3%, DLP was 22.8 +/- 1.0%, the average particle size was 117.3 +/- 1.27 nm and the zeta potential was -2.19 +/- 0.15 mV. Transmission electron micrograph (TEM) showed the micelles to be oval and rodlike shaped, with mean diameter around 20 nm. The in vitro experiments proved that celastrol in polymeric nanomicelles released gradually over the period of 24 h. These results showed that the RCCD and RSM could efficiently be applied for optimized preparation of celastrol-loaded polymeric nanomicelles.

Development and Validation of an HPLC Method for Quantifying Dapiprazole in Bulk Preparations

A simple, precise, rapid and accurate Reverse phase HPLC method was developed for the estimation of Dapiprazole in bulk form. A Thermo hypersil C-18 column (250 mm×4.6 mm 5μm) with mobile phase consisting of mixture of methanol: acetonitrile: and ortho-phosphoric acid in the ratio of 89: 9: 1 (v/v) at pH 5.8 adjusted with ortho-phosphoric acid. The flow rate was 0.8 mL/min and the effluents were monitored at 243 nm. The retention time was 3.725 min. The detector response was linear in the concentration of 20-120 μg/mL. The respective linear regression equation being y=1956.4x+3409. The limit of detection and limit of quantification was 0.5 μg/mL and 1.6 μg/mL respectively. The percentage assay of Dapiprazole was 99.94%. This method has been validated and shown to be specific, sensitive, precise, linear, accurate, rugged, robust and fast. Hence, this method can be useful for the routine determination of Dapiprazole in bulk drug and in its pharmaceutical dosage form.