Methanol Buffer Research Articles

A novel voltammetry offline coupled MALDI/TOF MS characterization of electrochemical reaction products and the voltammetric determination of febuxostat in human plasma

A simple offline coupling voltammetry-MALDI/TOF MS procedure is presented for studying electrochemical reactions. It was utilized for the characterization of the electro-reduction products of febuxostat in methanolic acetate buffer (0.1 M, pH 5). The MS analysis reveals that the carboxylic and nitrile groups are the electro-reducible groups at −0.9338 and −1.5503 V with the conversion to aldehydic and amino groups, respectively. The developed voltammetric method was validated and applied successfully for the drug determination in pharmaceutical tablets and real plasma samples within the linearity ranges 0.03–2 and 0.4–5 μg mL−1, respectively.

Controlled microwave derivatization reaction for reproducible trace analysis of budesonide in human plasma

Microwave reactors with on-line controlled reaction conditions have been recently innovated to improve reaction kinetics and reproducibility. Herein, a modern microwave reactor was dedicated to develop a new, sensitive and reproducible approach for trace analysis of budesonide (BUD) in human plasma. The method was based on fast microwave reaction of BUD with dansyl hydrazine (DNS-HZ) reagent under controlled conditions coupled with high-performance liquid chromatography (HPLC)-fluorescence detection. The microwave irradiation and dansylation conditions were optimized for the best sensitivity and selectivity. The controlled microwave derivatization reaction (CMDR) decreased the reaction time, amplified the reaction yield and enhanced product purities by reducing the unwanted side reactions. The chromatographic separation was attained by isocratic elution on reversed phase column via a mobile phase consisted of methanol and phosphate buffer (10 mM, pH 7.0) at ratio 80:20 (v/v). The fluorescence detector was set at 500 nm after excitation at 330 nm. Betamethasone dipropionate (BDP) was used as an internal standard. CMDR-HPLC method validation was performed in agreement with bioanalytical method validation guidelines by the US food and drug administration (US-FDA). The obtained linearity range was 0.2–100 ng mL−1 with correlation coefficient 0.9991 and the lower limit of quantitation (LLOQ) in human plasma was 0.21 ng mL−1. The developed CMDR -HPLC method was applied successfully for assessment of plasma levels of BUD in allergic rhinitis patients after intranasal administration of the micronized BUD.

DEVELOPMENT AND VALIDATION OF LIQUID CHROMATOGRAPHIC METHOD FOR SIMULTANEOUS ESTIMATION OF STRYCHNINE AND GALLIC ACID

An accurate, precise and robust RP-HPLC method was developed for the simultaneous estimation of strychnine and gallic acid. The markers were resolved using HiQ C18HS column with methanol and potassium dihydrogen phosphate buffer (10 mM, pH 3) (60:40 V/V) as mobile phase at flow rate of 1 mL per minute and run time of 6 minutes. Retention times of strychnine and gallic acid were 3 and 4.7 minutes, respectively at 264 nm. The linearity range for strychnine and gallic acid was found to be 0.5-3 μg/mL and 1-3.5 µg/mL, respectively with coefficient of linear regression greater than 0.99 for both the markers. The developed method was validated as per ICH Q2 (R1) guidelines and the results obtained were satisfactory. The method was applied for quantification of markers in marketed and In-house formulations of Agnitundi Vati, a polyherbal ayurvedic formulation. The developed method thus could be used for standardization of Agnitundi Vati and other herbal preparations containing these two markers.

Changes to Extender, Cryoprotective Medium, and In Vitro Fertilization Improve Zebrafish Sperm Cryopreservation.

Sperm cryopreservation is a highly efficient method for preserving genetic resources. It extends the reproductive period of males and significantly reduces costs normally associated with maintenance of live animal colonies. However, previous zebrafish (Danio rerio) cryopreservation methods have produced variable outcomes and low post-thaw fertilization rates. To improve post-thaw fertilization rates after cryopreservation, we developed a new extender and cryoprotective medium (CPM), introduced quality assessment (QA), determined the optimal cooling rate, and improved the post-thaw in vitro fertilization process. We found that the hypertonic extender E400 preserved motility of sperm held on ice for at least 6 h. We implemented QA by measuring sperm cell densities with a NanoDrop spectrophotometer and sperm motility with computer-assisted sperm analysis (CASA). We developed a CPM, RMMB, which contains raffinose, skim milk, methanol, and bicine buffer. Post-thaw motility indicated that the optimal cooling rate in two types of cryogenic vials was between 10 and 15°C/min. Test thaws from this method produced average motility of 20% ± 13% and an average post-thaw fertilization rate of 68% ± 16%.

ANALYTICAL METHOD DEVELOPMENT AND VALIDATION FOR THE ESTIMATION OF IMATINIB MESYLATE AND ITS DIMER IMPURITY IN PHARMACEUTICAL FORMULATION BY REVERSE-PHASE HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

Objective: The present study is to develop a simple, specific, and validated reverse-phase high-performance liquid chromatography (HPLC) method for the determination of imatinib mesylate and its dimer impurity in pharmaceutical dosage form.Methods: A HPLC instrument incorporated with column HiQ Sil C18 (250 mm × 4.6 mm, 5 μm), mobile phase as methanol and acetate buffer pH 3.5 in the ratio of 80:20 v/v was used for the determination of the imatinib mesylate and its dimer impurity. The detection wavelength was set at 273 nm. The flow rate of the mobile phase was 1.0 mL/min.Results: The retention time for imatinib mesylate was 8.060, and for dimer impurity, it was 11.398. The calibration plot was linear (R2=0.9971) and the % mean recoveries for imatinib mesylate were in the range of 99.83–101.57, and for dimer impurity, it was in the range of 98.16–99.18. The limit of detection concentration was found to be 0.570 μg/ml for imatinib mesylate and 0.033 μg/ml dimer impurity and limit of quantification concentration was 1.728 μg/ml for imatinib mesylate and 0.099 μg/ml dimer impurity.Conclusion: The projected method was validated and successfully functional for the estimation of imatinib mesylate and dimer impurity in formulations. It can be adopted apparently for routine quality control and research tests.

A Novel UPLC-PDA Method for the Simultaneous Determination of Lamivudine, Zidovudine and Nevirapine in Bulk and Tablet Dosage Form

This present paper reports about the development of a novel UPLC method for the estimation of Lamivudine (LVD), Zidovudine (ZVD) and Nevirapine (NVP) simultaneously in bulk and tablet dosage form. Excellent chromatographic separation was achieved by isocratic mode with the mixture of methanol and phosphate buffer (PH-5) in the ratio 70:30% v/v as a mobile phase with Waters HPLC system (Aquity 2996 UPLC) and RP C-18 column (100×2.1 mm x 1.8 μm; make: Waters) as a stationary phase. The detection was performed at 260 nm. Retention time of LVD, ZVD and NVP were found 1.76, 2.24 and 3.75 minutes respectively. The detection and quantitation limits were found 0.023 μg/mL and 0.066 μg/mL for LVD. 0.027 μg/mL and 0.092 μg/mL for ZVD and 0.019 μg/mL and 0.066 μg/mL for NVP. The present method was found suitable for the analysis of marketed formulation, where as the % assay were found within 99-100.6% for all three analytes. Hence the present work was found suitable and reliable for the analysis of LVD, ZVD and NVP in bulk and tablet dosage form.

A Validated RP-HPLC Method for Simultanious Determination of Metformin and Canagliflozin in Pharmaceutical Formulation

The proposed study a new validated RP-HPLC method has been developed for estimation of metformin and canagliflozin in bulk and pharmaceutical dosage form. The present method was a sensitive, precise and RP-HPLC method for analysis of metformin and canagliflozin. To optimize the mobile phase various combinations of organic solvents were used on Primesil C18 250 column, Then the mobile phase containing a mixture of Methanol and phosphate buffer in the ratio of 70: 30% v/v was selected at a flow rate of 1.0ml/min for developing the method and the peak with a good shape and resolution were found resulting in short retention time baseline stability and minimum noise. The retention time metformin and canagliflozin found to be 2.413 and 3.47 min respectively, quantitative linearity was obeyed in the concentration range of 50–250 μg/mL for metformin and 5–25 μg/mL for canagliflozin. The limit of detection and limit of quantitation were found to be 2.1μg/ml and 0.0372μg/ml (metformin), 4.13μg/ml and 0.112μg/ml (canagliflozin) respectively, which indicates the sensitivity of the method. The high percentage recovery indicates that the proposed method is highly accurate no interfering peaks were found in the chromatogram indicating that excipients used in tablet formulation didn't interfere with the estimation of the drug by the proposed HPLC method.

SPECTROPHOTOMETRIC AND REVERSED-PHASE HIGH-PERFORMANCE LIQUID CHROMATOGRAPHIC METHODS FOR SIMULTANEOUS DETERMINATION OF ATORVASTATIN AND PIOGLITAZONE IN COMBINED TABLET DOSAGE FORM

Simple, accurate, precise, and sensitive ultraviolet spectrophotometric and reversed-phase high-performance liquid chromatographic (RP-HPLC) methods for simultaneous estimation of Atorvastatin (ATOR) and Pioglitazone (PIO) in combined tablet dosage form have been developed and validated. The spectroscopic methods employs formation and solving of simultaneous equation at 247 nm and 267 nm as 2 wavelengths for estimation of ATOR and PIO respectively (method 1) While method 2 involves formation of Q- absorbance equation at 233 nm (isoabsorptive point) and 267 nm (I» max of Pioglitazone) with methanol as solvent. Beer’s law is obeyed in the concentration range of 5.0–50.0 mcg/mL for ATOR and PIO, respectively. The RP-HPLC method uses HPLC system with a Phenomenex Luna C18 (5 mm x 25cm x 4.6mm i.d) using Methanol, acetonitrile and potassium dihydrogen phosphate buffer, pH 2.5 adjusted with orthophosphoric acid (60:20:20 v/v) at a flow rate of 1.0 mL/min at ambient temperature as the mobile phase. The detection was carried out using an ultraviolet detector set at 233 nm. For the HPLC method, Beer’s law is obeyed in the concentration range of 5.0-50.0 µg/ml for ATOR and PIO, respectively. LOD values for ATOR and PIO were found to be 57.12µg/ml and 12.01 µg /ml respectively. All the methods have been successfully applied for the analysis of the drugs in a pharmaceutical formulation. Results of analysis were validated statistically and by recovery studies.1-4

Determination of degradation products and process related impurities of asenapine maleate in asenapine sublingual tablets by UPLC

For determination of process related impurities and degradation products of asenapine maleate in asenapine sublingual Tablets, a reversed phase, stability indicating UPLC method was developed. Acetonitrile, methanol and potassium dihydrogen phosphate buffer with tetra-n- butyl ammonium hydrogen sulphate as ion pair (pH 2.2; 0.01 M) at flow rate of 0.2 ml/min were used in gradient elution mode. Separation was achieved by using acquity BEH Shield RP18 column (1.7 μm, 2.1 mm×100 mm) at 35 ºC. UV detection was performed at 228 nm. Subsequently the liquid chromatography method was validated as per ICH. The drug product was exposed to the stress conditions of acid hydrolysis, base hydrolysis, water hydrolysis, oxidative, thermal, and photolytic. In oxidative stress and thermal stress significant degradation was observed. All the degradation products were well separated from analyte peak and its impurities. Stability indicating nature of the method was proved by demonstrating the peak purity of Asenapine peak in all the stressed samples. The mass balance was found >95% for all the stress conditions. Based on method validation, the method was found specific, linear, accurate, precise, rugged and robust.

SIMULTANEOUS EQUATION METHOD FOR THE ESTIMATION OF PALBOCICLIB AND LETROZOLE BY UV-VISIBLE SPECTROPHOTOMETRY

A simple, rapid, accurate, economic and precise UV/VIS method has been developed and validated. Choice of a common solvent were essential so various solvent ranges including methanol, ethanol, acetonitrile and phosphate buffer and various concentrations ranges of various buffers were analyzed. methanol was selected as a solvent for the proposed method. Palbociclib and letrozole showed maximum absorbance at 220 and 240 nm respectively. Both drugs obey Beer Lambert’s law in the concentration range of 2-10 μg mL-1 for palbociclib and letrozole respectively. The LOD and LOQ were found to be 0.0782 μg mL-1and 0.2142μg mL-1for palbociclib, respectively. For Letrozole, the LOD and LOQ values were found to be 0.0585 μg mL-1and 1.6510μg mL-1 respectively. The method was quantitatively evaluated in terms of linearity, precision, precision, LOD, LOQ and recovery. The method is simple, convenient and suitable for the analysis of palbociclib and letrozole in bulk drug.

Validated RP-HPLC Method for the Determination of Ivabradine Hydrochloride in Pharmaceutical Formulation

A simple, rapid, precise, and accurate RP-HPLC method for the estimation of Ivabradine Hydrochloride an anti-anginal agent, both as a bulk drug and in pharmaceutical formulation was developed. The chromatographic separation was achieved on a Thermosil C18 150 × 4.5 mm, 5μm column by using a mobile phase containing a mixture of methanol and phosphate buffer pH 6.5 in the ratio of 65:35 % v/v at a flow rate of 1ml/min and at an ambient temperature. The detection was monitored at a wavelength of 265nm. A clear chromatographic peak was identified with the retention time of 4.36 min and tailing factor of 1.23. The developed method was validated according to ICH guidelines with respect to specificity, linearity, accuracy, precision and robustness. The method shows a good linear relationship with correlation co-efficient of more than 0.992 in the concentration range of 30μg-150μg. The method showed mean % Recovery of 100.4% and %RSD for repeatability and intermediate precision was less than 2%. The proposed method can be used successfully for the quantitative determination of Ivabradine HCL in pharmaceutical dosage forms.

A Rapid and Simple High-Performance Liquid Chromatographic Method for Determination of Levofloxacin in Human Plasma

A simple and rapid high-performance liquid chromatography method was developed and validated for quantifying LEV in human plasma. Chromatographic separation was performed under isocratic elution on a Luna Phenomenex® C18 (150 × 4.6 mm, 5 µm) column. The mobile phase was comprised of acetonitrile, methanol, and phosphate buffer 25 mM pH 3.0 (13:7:80 v/v/v) and pumped at a flow rate of 1.5 mL/min. Detection was performed by UV detector at a wavelength of 280 nm. Samples were pre-treated with acetonitrile followed by centrifugation, evaporation, and reconstitution step. The method proved linear (r = 0.995), sensitive (LLOQ and LOD was 1.8 and 0.6 µg/mL respectively), accurate (% error above LLOQ ≤ 12% and LLOQ ≤ 20%), precise (RSD ≤ 9%), robust in the ranges of 1.8-28.8 µg/mL, rapid (separation time not more than 10 min), and simple (use no organic additive in mobile phase). The method was showed reliable for quantifying LEV in human plasma.

Simple LC Isocratic Methods Development, Validation, and Application in the Analysis of Poor Quality Antimalarial Medicines

Liquid chromatographic methods in isocratic mode for the analysis of poor quality medicines are privileged due to their simplicity and facility in methods development. They are generally fast; do not need to be re-equilibrated between sample injections; have larger flexibility with acceptable changes on different column dimensions; and are applicable to LC systems equipped with simple or high developed pumps. In this study, we focused on developing simple isocratic methods using classical mobile phase composed by methanol and ammonium formate buffer for the analysis of most common antimalarial medicines marketed in malaria endemic countries and susceptible of being counterfeit/falsified, substandard and degraded. The selected medicines were quinine and related cinchona alkaloids in tablets and injectable forms; artemether/lumefantrine tablets; and artemisinin compounds (arteether, artemether, and artesunate) in injectable forms. The current methods were developed thanks to simple methodological approach consisting in sequential isocratic runs through adjustment or adaptation of existing methods to obtain optimal analytical conditions without complex design of experiments that might be long and costly. Then, the new methods presented shorter analysis time; allowed increase of sample analysis throughput; and obviously consumed little mobile phase solvents on classical analytical columns: 50 - 250 mm of length (L), 4.6 mm of internal diameter (I.D.), and 3.5 - 5.0 μm of particle size (dp).

Simultaneous HPLC assay for pretomanid (PA-824), moxifloxacin and pyrazinamide in an inhaler formulation for drug-resistant tuberculosis

A simple and sensitive reversed phase HPLC method has been developed for the simultaneous quantitation of pretomanid (PA-824), moxifloxacin and pyrazinamide in a combination spray-dried powder formulation for inhalation, without any use of an internal standard. Good resolution of the analytes was achieved on a Luna C18 (2), 150×4.6mm, 5μm, 100Å column using gradient elution with a mobile phase containing methanol and triethylamine phosphate buffer (pH 2.5) at a flow rate of 1.0mL/min in a total run time of 25min. Pyrazinamide, moxifloxacin and pretomanid (PA-824) were detected at wavelengths (retention times) of 269nm (3.80min), 296nm (7.94min) and 330nm (17.46min), respectively. The assay was linear for all analytes in the concentration range 2.5–100μg/mL (correlation coefficients >0.999) with LODs and LLOQs (μg/mL) of pretomanid (PA-824) 0.51 and 1.56, moxifloxacin 0.06 and 0.19 and pyrazinamide 0.35 and 1.06, respectively. Recoveries of the three drugs were 99.6–106.8% with intra- and inter-day precisions (as relative standard deviation) of <7%. The method was successfully applied to an evaluation of content uniformity and freedom from interference by l-leucine of a spray-dried combination powder for inhalation.

Development and Validation of a Cost-Effective HPLC-FLD Method for Routine Analysis of Fumonisins B1 and B2 in Corn and Corn Products

Fumonisins, a type of mycotoxins produced by Fusarium species, can cause fatal diseases in animals and humans. Therefore, regulatory limits for food and feed were established in many countries. Herein, a sensitive, valid, cost-effective, and easily transferable analytical method for quantitative determination of the relevant fumonisins B1 (FB1) and B2 (FB2) in corn and corn products was developed. The final method is based on reversed-phase high-performance liquid chromatography with fluorescence detection (RP-HPLC-FLD). Derivatization of analytes was simplified by establishing an automated precolumn derivatization method by using an autosampler. Strong anion exchange (SAX) cartridges were used instead of widespread immunoaffinity chromatography (IAC) approaches. Analytes and matrix signals were chromatographically separated in a total run time of 25 min by means of a C8 column and a gradient elution using methanol and phosphate buffer. The limits of detection (LODs) for FB1 and FB2 were 29.2 and 17.5 μg/kg, respectively. Obtained recoveries ranged from 79.4 to 94.5 % for FB1 and from 79.9 to 98.0 % for FB2. Positive confirmation of the developed method was achieved by participating in an interlaboratory proficiency testing. Nineteen samples of sweet corn and corn products were analyzed to demonstrate the applicability of the method.

Development and validation of a quantitative assay for the determination of cinacalcet and its main metabolites in human plasma using RP-HPLC method

A novel and sensitive methodology for the quantitative determination of cinacalcet (CIN), and its main metabolites (M2a-Glu, M2b-Glu, M5, and M6) in plasma sample is suggested. The analytical assay is based on reversed-phase high-performance liquid chromatography (RP-HPLC) coupled with a fluorescence detector. The method has been fully validated at range covering the plasma concentrations in patients receiving therapeutic dosages of CIN. In this investigation, 2-(2,3-naphthalimino)ethyl trifluoromethanesulfonate (NI-ETFMS) was used as a pre-column derivation agent according to interaction of amine and carboxylate groups of CIN and its metabolites with NI-ETFMS. The sample pretreatment consists of protein precipitation with acetonitrile using only 100μL of plasma, and chromatographic separation was achieved in isocratic mode on Kinetex C18 100Å column with mobile phase of acetonitrile, methanol, and 50mM phosphate buffer (40:20:40v/v). The analytical method was fully validated in terms of selectivity, linear range (0.2 to 5.5ngmL−1), linearity (r2>0.997), sensitivity (LOD=0.05 to 0.75ngmL−1 and LOQ=0.36 to 2.52ngmL−1), intra and inter-days accuracy (−4.6 to 8.1%), precision (<7.4%), and robustness (<3.5%). As results, the proposed method can be useful in the routine analysis for the determination of CIN and its main metabolites in human plasma samples.

Determination of carnosic acid and carnosol in Rosmarinus officinalis L. by high-performance capillary electrophoresis

ABSTRACTCarnosol and carnosic acid in Rosmarinus officinalis L. were separated by high-performance capillary electrophoresis using a 50 cm capillary, a 12 mmol/L 80:20 pH 9.9 borax:methanol buffer using 25 kV of applied voltage at 30°C with detection at 280 nm. Rosmarinus officinalis L. was extracted with methanol with ultrasound at 170 W and 50 kHz for 20 min. The precision, repeatability, and recovery of the method were evaluated. The calibration relationships for carnosol and carnosic acid were y = 2.331x + 5.92 from 0.01 to 0.160 mg/mL and y = 7.980x + 4.32 from 0.005 to 0.080 mg/mL, respectively. The established method allows the separation and quantification of carnosic acid and carnosol in R. officinalis from various locations in China.

9,10-Phenanthrenequinone as a mass-tagging reagent for ultra-sensitive liquid chromatography–tandem mass spectrometry assay of aliphatic aldehydes in human serum

9,10-Phenanthrenequinone (PQ) was successfully used as a new mass-tagging reagent for sensitive labeling of aliphatic aldehydes (C3-C10) prior liquid chromatography-electrospray ionization–tandem mass spectrometry (LC/ESI–MS/MS). This reagent could overcome the drawbacks of previous amine or hydrazine-based reagents, such as lower sensitivity, formation of two stereoisomeric reaction products for each single analyte, need for longer derivatization time, and poor reactivity with aliphatic aldehydes. The PQ-aldehyde derivatives exhibited intense [M+H]+ and a common product ion with ESI in the positive-ion mode. The derivatives were monitored at the transition of [M+H]+→m/z 231.9 with detection limits from 4.0 to 100 pM (signal to noise ratio=3). 3-Phenylpropanal was used as an internal standard (IS) and the separation of the eight aldehydes and IS was achieved in less than 10min employing gradient elution with methanol and ammonium formate buffer (20mM, pH 4.0). The method employed salting out liquid–liquid extraction for aliphatic aldehydes form serum for the first time with excellent recoveries (92.6–110.8%). The developed method was validated and applied for quantification of the target aldehydes in serum of healthy volunteers (n=14).

A novel approach for HPLC determination of 2-cynaoacetamide using derivatization procedure with 2-hydroxyacetophenone as a new useful derivatization reagent

A novel and sensitive derivatization procedure for the determination of 2-cynaoacetamide in pharmaceutical samples using liquid chromatography with the fluorescence detection was discovered. The method is based on derivatization of 2-cynaoacetamide using 2-hydroxyacetophenone as a new derivatization reagent. The product of derivatization reaction was isolated and characterized using spectroscopic techniques namely LC–MS, NMR and IR. The structure of 2-cyanoacetamide derivative was unambiguously assigned as a 2-amino-4-phenylfuran-3-carboxamide.Two derivatization systems were optimized in terms of reaction temperature, reaction time, pH and concentration of 2-hydroxyacetophenone, and a new pre- and post-derivatization HPLC methods were developed. The separations on HPLC with pre-column derivatization were accomplished using stationary phase based on a XBridge C18 column (100×4.6, 3.5μm) and isocratic elution using the mobile phase acetonitrile – 0.1% formic acid (30:70, v/v). The separations on the HPLC with post-column derivatization were performed on stationary phase on a TSKgel Amide-80 column (150×4.6mm, 3μm). The mobile phase was a mixture of acetonitrile, methanol and 10mM sodium formate buffer at pH=4.5 in ratio 3:2:95 (v/v).Both HPLC methods were fully validated in terms of linearity, sensitivity (limit of detection and limit of quantification), accuracy and precision according to ICH guidelines. The pre-column derivatization method was linear in the range 1.1–2000μg/l with method accuracy≥98.2% and method precision RSD≤4.8%. The post-column derivatization method was linear in the range 12–2000μg/l. Method accuracy≥96.3% and method precision RSD≤3.5%. Proposed new methods were proved to be highly sensitive, simple and rapid, and were successfully applied to the determinations of 2-cynaoacetamide in pregabalin.

BOX-BEHNKEN SUPPORTED DEVELOPMENT AND VALIDATION OF ROBUST RP-HPLC METHOD: AN APPLICATION IN ESTIMATION OF PRAVASTATIN IN BULK AND PHARMACEUTICAL DOSAGE FORM

Pravastatin, a natural bioactive compound inhibits cholesterol biosynthesis which is isolated from fungus by fermentation process. A rapid, sensitive and robust novel reversed phase-HPLC method with UV detection set at 238 nm was developed and validated for quantification of pravastatin in pure drug form and pharmaceutical formulation (Pravachol). A liquid-liquid extraction technique was applied for the preparation samples and chromatographic separation was performed on Symmetry® C 18 (5μm, 3.9 × 150 mm) column with a retention time of 1.95 min using isocratic elution of mobile phase. The mobile phase used in this work consisted of a mixture of methanol and phosphate buffer (70:30%, v/v, pH 4) which was delivered at a flow rate at 1.5 ml/min. Developed HPLC-UV method was validated for linearity ( R 2 = 0.999), accuracy, precision, sensitivity, system suitability, selectivity, and robustness. Box-Behnken experimental design was applied in the validation of robustness. Three independent variables such as the composition of mobile phase, flow rate and pH of mobile phase were investigated. The results indicated that a slight change in the mobile phase composition and flow rate affects the responses, while pH had no significant effect on the response. Overall, the proposed HPLC-UV method was found to be simple, sensitive, and highly robust for routine analysis of pravastatin.