Spectrophotometric UV Research Articles

Comparative evaluation of dissolution profile of drug in its formulation by UV spectrophotometry

The aim of the research work was to develop a method for comparative evaluation of dissolution profile of two different brands of Teneligliptin hydrobromide hydrate drug in its formulations containing using UV Spectrophotometer. Simple, precise and accurate UV-spectrophotometric method was developed for Teneligliptin hydrobromide hydrate using optimized dissolution parameters like as 900mL of Phosphate buffer pH 6.8 as a dissolution medium and paddle (type II) apparatus at a stirring rate of 100 rpm. The drug release was evaluated by UV spectrophotometric method using 243.2nm as detection wavelength. Developed method obeyed Beer’s-Lambert’s law in the concentration range of 0.5-25 ?g/mL, with correlation coefficient value less than 1. The percent drug amount released estimated by proposed method was nearly 100%, found to be in good agreement with label claim of marketed tablet formulation. The proposed method were validated as per ICH guidelines with respect to accuracy, precision, LOD, LOQ and found to be within limits. The proposed method can be adopted for routine quality control test for estimation of drug in formulation. Also the statistical data analysis of percent drug release of brand 1 and 2 were compared with preexisting dissolution data of literature by using F-test and t-test. Keywords: Teneligliptin hydrobromide, Spectrophotometric method.

Chemometric Approach to Quantify 5-Hydroxymethylfurfural and Furfural Obtained from Sugarcane Bagasse and Peanut Hull by UV

5-hydroxymethylfurfural (HMF) is considered a platform chemical used for producing industrial purpose carbon-based compounds. During HMF synthesis, the compound furfural (FF) is formed as a byproduct. Using spectrophotometric UV is not feasible because HMF and FF overlap around 280 nm, demanding separation techniques as high-performance liquid chromatography (HPLC). This work aimed at applying a synthetic route for HMF production by using wastes such as sugarcane bagasse and peanut hull, and developing a quantification method of HMF and FF compounds using UV spectra and pseudo-univariate calibration based on multivariate curve resolution with alternating least squares. HMF synthesis was studied using inorganic acids in a closed reflux system followed by a biphasic extraction with n-butanol as an organic solvent. When comparing their respective biomass, sugarcane bagasse provides a higher amount of HMF, reaching levels of 1.65 × 10−2 mol L−1 after the synthesis with HCl at 0.50 mol L−1. Regarding yield, each ton of peanut hull produces 572.6 g of HMF, while each ton of sugarcane bagasse produces 10 kg of HMF. The calibration study using pseudo-univariate calibration models was validated by using the HPLC with relative errors lower than 15%, showing that it is possible quantify HMF and FF in the medium by using UV spectrophotometer.

Development and Validation of UV Spectrophotometric and RP-HPLC Methods for the Estimation of Gallic Acid in Herbal Formulation of Amalaki

In this study, two analytical methods viz. UV spectrophotometry and RP-HPLC were developed for the evaluation of Amalaki in the marketed herbal formulation. The amount of gallic acid estimated in the marketed formulation complies with the standard (not less than 1% w/w of gallic acid) specified in the official monograph of Amalaki. The developed methods showed good linearity, accuracy, precision, ruggedness, robustness, specificity, LOD and LOQ. Results of validation studies were found satisfactory with % RSD values of less than 2% indicating good specificity, validity and reliability of the developed methods. Both analytical methods are claimed to be simple, accurate and precise. Present methods can, therefore, be applied widely for the routine analysis of marketed formulations or any crude traditional preparations of Amalaki based on the quantitative determination of gallic acid at a reasonable cost with simple analytical set up.

Salvia virgata L. Leaves Extract: Qualitative and Quantitative Phenolic Compounds and Antioxidant Activity

Plants' materials are a rich source of phenolic compounds (flavonoids, tannins and anthocyanin) that are the most important natural antioxidants. Antioxidants in the diet are very important for health and protected the body for oxidative stress. This research has been done for measurement of quantitative and qualitative of phenolic compounds and antioxidant activity of Salvia virgata L. as an alternative to synthesis antioxidants. In this work, the phytochemical study was performed on Salvia virgata L., and the amount of phenol and flavonoids were measured from three extract using spectrophotometric UV method. The antioxidant activity of the different extract was evaluated in different concentrations using (DPPH). Data analysis and variance analysis were done with SPSS software 16th version The results showed that total phenolic compounds of aqueous, ethanol and methanol extracts were 35.5, 34, 32.5 GAE/g dry samples respectively, and the total flavonoids content of aqueous, ethanol and methanol extracts were 28.12, 23.40, 26.50 mgQE/g dry sample The concentration inhibition of 50% ethereal extract (IC50) =5.4±0.6 and phytochemical results indicate the presence of flavonoids, tannins and anthocyanin in the extract. The results showed that the different extracts are rich withphenolic compounds and also present a high level of antioxidant properties, therefore, Salvia virgata L. could be used as a source of antioxidant compounds in food and as medicinal plant.

UV Spectrophotometric and Stability Indicating RP-HPLC Assay Methods for the Estimation of Etodolac in Bulk and Tablet Dosage Form

Objectives: The present article involved the development of sensitive and validated reverse phase liquid chromatographic and UV-spectrophotometric method for the determination of etodolac in bulk and pharmaceutical dosage form. Methods: The presents UV method is based on measurement of absorption at maximum at 226 nm using methanol and water (70:30) as a solvent. The stock solution of etodolac was prepared and subsequent suitable dilution was prepared in distilled water to obtained standard curve. The standard solution of Etodolac shows absorption maxima at 226 nm. Results: The drug obeyed beer lambert’s law in the concentration range of 2-20 μg/ml with regression coefficient 0.9998 at 226 nm. The chromatographic method was optimised using Phenomenex Luna C18, 100A, 5mm, 250mm x 4.6mm i.d. column with UV detection at 226 nm and Acetonitrile: Methanol in 65:35 ratio at a flow rate of 1.0 ml/ min. The proposed method was successfully applied to the determination of Etodolac in bulk and pharmaceutical dosage form. The method was found linear over the range of 0 – 48 μg/ml. The recovery was observed in the range of 98% to 102% and limit of detection and limit of quantification were found to be 0.08 μg/ml and 0.26 μg/ml. Different analytical performance parameters such as precision, accuracy, limit of detection, limit of quantification and robustness were determined and found satisfactory according to International Conference on Harmonization (ICH) guidelines. Conclusion: The developed methods were found reliable, easy and validated for the estimation of etodolac in bulk and tablet dosage form.

Development and Validation of Absorption Correction UV-Spectrophotometry Method for Simultaneous Estimation of Ranitidine HCL and Dicyclomine Hydrochloride in Their Marketed Formulation

A Simple, selective, precise and rapid absorption correction Spectrophotometric method has been developed and validated as per ICH guideline for the simultaneous estimation of Ranitidine Hydrochloride and Dicyclomine Hydrochloride in tablet dosage form. Method is base on UV Spectrophotometric for determination of two drug, by using Methanol as a solvent and diluted the same with 0.1N NaOH, solution. In this UV method, the two wavelength were selected, 311.4 nm and at 217 nm for RANTD and DICY, respectively. this method was validated according to ICH guideline and Linearity range, was found to be 7.5-37.5 µg/ml and 1-5 µg/ml for RANTD and DICY, respectively. The method was successfully applied to assay drugs in tablet.

VALIDATION OF GAS CHROMATOGRAPHIC AND UV SPECTROMETRIC METHODS OF QUANTITATIVE DETERMINATION OF BUPRENORPHINE ACCORDING TO REQUIREMENTS OF ISO/IEC 17025:2005 STANDARD

The article is considered to the validation of gas-chromatographic and UV spectrometric methods for the quantitative determination of buprenorphine, which is a partial agonist of opioid receptors and having analgesic properties, according to requirements of ISO/IEC 17025:2005. Authors highlight the fact that during the quantitative calculations of the detected narcotic, psychotropic substances, precursors, drugs, virulent and drastic toxic substances in investigated objects submitted for examination, in order to obtain more accurate and reliable data, there is a need for their reliable quantitative determination in accordance with the requirements of ISO/IEC 17025:2005.
 Results of quantitative calculation and constructing of calibration curves of narcotic containing buprenorphine are presented as an example. The validation of gas chromatography and UV spectrometry methods was carried according to requirements of the international standard ISO/IEC 17025:2005. Validation of developed methods is executed in according to following parameters: peculiarity (specificity), linearity, accuracy, stability and reproducibility. This article presents calibration dependencies, their linearity is represented, which makes possible to calculate the coefficient k, used in future in calculating the value of uncertainty measurements.
 Obtained results of the research and calculations on made allowed to perform quantitative calculations (determinations) of relevant components in the submitted samples for examination on the basis of the methodology developed in the Department of Physical, Technical and Chemical Research of the National Bureau of Expertise of the Republic of Armenia using the obtained values of measurement uncertainty according to requirements of the international standard ISO/IEC 17025:2005. Their practical implementation ensures reliable performance of quantitative determination of buprenorphine are demonstrated. Thus, based on experimental results and validation procedures, forensic expert practice department introduced two methods of analysis: gas-chromatographic and UV Spectrophotometric, providing reliable execution of quantitative determination of buprenorphine according to requirements of the international standard ISO/IEC 17025:2005.

SECOND-ORDER DERIVATIVE UV SPECTROPHOTOMETRIC AND RP-HPLC METHODS FOR THE ANALYSIS OF VILDAGLIPTIN AND APPLICATION FOR DISSOLUTION STUDY

This study describes two analytical methods, by second-order derivative UV spectrophotometric by HPLC, for determination of vildagliptin, a drug used for treatment of type 2 Diabetes Mellitus that belongs to a therapeutic class called inhibitors of dipeptidyl peptidase 4. The methods were validated in accordance with ICH and USP requirements. Analyses by UV derivative method were performed at 220 nm, which was the zero crossing point of excipient solutions. HPLC was optimized and the analysis was carried out using a Zorbax Eclipse Plus RP-C8 column (150 mm × 4.6 mm, 5 μm), detection at 207 nm, and potassium phosphate buffer solution pH 7.0 : acetonitrile (85:15, v/v) as mobile phase. In dissolution test, the conditions used were 0.01 mol L-1 hydrochloric acid in 900 mL of dissolution medium, USP apparatus 2 (paddle) and 50 rpm stirring speed. Both methods were successfully applied for analysis of dissolution samples from marketed vildagliptin tablets.

UV and First Derivative Spectrophotometric Methods for the Estimation of Atorvastatin in Pharmaceutical Preparations

Objective: In this study, new, rapid UV spectrophotometry (UV) and first-order derivative spectrophotometry (1D) methods were developed for the determination of atorvastatin in pure and tablets. Methods: The solvent system and wavelength of detection were optimized in order to maximize the sensitivity of the proposed methods. Parameters such as linearity, precision, accuracy, specificity, stability, limit of detection and limit ofquantification were studied according to the International Conference on Harmonization Guidelines. Results: Calibration curve was linear between the concentration range of 5-20 μg ml-1. Within- and between-day precision values for atorvastatinwere less than 4.57%, and accuracy (relative error) was better than 3.17%. The mean recovery value of atorvastatinwas 100.1% for pharmaceutical preparations. Conclusion: The developed methods were successfully applied to tablet formulations and the results were compared statistically with each other.

Determination of Flavonoid, Tannin and Vitamin C Content from Methanol Extract Wrapping Stone Banana (Musa brachycarpa), Ketip Banana (Musa Paradisiaca Forma Typiaca) and Kepok Banana (Musa acuminata)

Research has been done to investigate the levels of flavanoid, tannin, and vitamin C of methanol extract of Stone banana (Musa brachycarpa), Ketip banana (Musa paradisiaca forma typiaca ), and Kepok banana (Musa acuminata). Jukut Ares is a Balinese traditional cuisine frequently served in any events of celebrations. The use of young stems of such banana varieties as the main raw materials in the process of making jukut ares has not been scientifically elucidated. Antioxidants are compounds with ability to inhibit oxidation-related reactions due to free radicals that cause disturbance of saturated fatty acids of cell membrane, blood vessels, DNA, as well as adipose tissues which lead to occurrence of various diseases. Plants components will have antioxidant activities if they contain compounds (phenol and flavanoid) with ability to scavenge free radicals. Tannin is a water based phenolic compound and vitamin C is also a water based vitamin that play important roles in preventing us from various diseases. Extraction was conducted by applying maceration method and to investigate the levels of flavanoid, spectrophotometric UV vis with aluminum chloride (AlCl3) was applied. The level of vitamin C of the materials was conducted by Iodine titration method, while the level of tannin was determind by applying spectrophotometri. The results showed that methanol extract of Ketip banana stem contained the highest level of flavanoid with a quantity of 53.13 mg/100gr QE, and this was followed by stone banana (36.28) and kepok banana (32.07) The highest vitamin C level was indicated in the extract of ketip banana (418.32 mg/100gr), which is followed by kapok banana and stone banana. In the calculation of tannin content, pisang ketip showed the highest level, while pisang batu contained the lowest level of tannin.

Effect of Honey bee venom against Intensive care units environmental pathogens

The objective of this investigation was to focus on the environmental surface contamination with pathogenic organisms contribute to the development of health care-associated infections (HAI) and evaluate effect of bee venom from Apis mellifera pure Carniolan as the antibacterial activity against the Gram-negative bacterial strains of medical importance including Escherichia coli, Acinetobacter baumannii, and Pseudomonas aeruginosa. The microbial burden (MB) associated with commonly touched surfaces in intensive care units (ICUs) was determined by sampling all exposed objects (such as: IV stand, Patient bed rails, Over-bed table, Visitor chair (arms), Nurse call button, Computer monitors, Data input device, etc.). All pathogenic samples were defined biochemically and assigned to their genera and species using RT- PCR technique. Antimicrobial activity of bee venom in vitro created 10, 12, and 10 mm of inhibition zones compared to control (16, 13, and 15 mm) against the previous gram-negative bacteria respectively. On the other hand, in vitro minimum inhibitory concentration (MIC) activity of the pathogenic microbial was 50.36, 39.22 and 62.5 mg/ml of E. coli, A. baumannii, and P. aeruginosa respectively. These findings strongly suggest that whole bee venom could be effective antimicrobial agents for bacteria. Spectrophotometric studies, elementary analysis, UV, IR, and MS spectrum of the venom was indicated the presence of higher absorbance of 0.223 at wavelength 399.40 nm. The chemical components of the bee venom were supported the presence of aromatic chain attached to the function groups of amine (NH), carboxylic (COH), and carbonyl (CO). These results indicate that BV inhibits the growth and survival of bacterial strains and that BV may be a useful complementary antimicrobial agent against pathogenic bacteria even if bee venom collected by different methods.

Panel test and chemical analyses of commercial olive oils: a comparative study

BackgroundThe quality grade of an olive oil is defined according to the results of analytical and organoleptic examinations.The increasing attention towards both olive oil quality and quality verification methods prompted us to undertake a “critical” analysis of analytical and sensory data supplied by an International Certificated Body (ICB), relative to commercial olive oils produced in Mediterranean areas and purchased in Italy and in USA.MethodsICB data included chemical analyses namely free acidity, peroxide index, spectrophotometric UV evaluation, fatty acid ethyl esters and stigmadiens content and organoleptic evaluations carried out by nine official International Olive Council labs according to EEC Regulation 2568/91.ResultsThe results of the chemical analyses, except the fatty acid ethyl ester content, obtained from the nine labs were consistent giving rise to the same quality grade. In nearly all samples, the fatty acid ethyl ester content was close to the threshold established for extra virgin olive oils indicating a non-excellent quality of the olive oils. Organoleptic evaluations, commonly called panel test, given by the nine labs were not consistent.ConclusionsThe EEC Regulation 2568/91 does not give any indication on the way to report the uncertainty of the results, and in the case of extra virgin olive oils with a borderline value, the way to report the fatty acid ethyl ester content, with or without the uncertainty, can create confusion in defining the olive oil quality grade. Panel test seemed to work well only in the case of extremely good olive oils, whereas, in commercial extra virgin olive oils with borderline value of fatty acid ethyl ester content, a different sensory sensibility seems to be in the different IOC labs.Graphical abstractPanel test and chemical analyses of commercial olive oils: a comparative study.

DEVELOPMENT AND VALIDATION OF SIMPLE UV SPECTROPHOTOMETRIC METHOD FOR THE ESTIMATION OF DEXTROMETHORPHAN HYDROBROMIDE IN BULK AND MARKETED DOSAGE FORMULATIONS

A simple, rapid and economic UV spectrophotometric method has been developed and validated using a solvent 0.1N HCl to determine Dextromethorphan hydrobromide content in bulk and two different pharmaceutical solid dosage formulations, lozenges and chewable tablets. At the pre-determined λmax of 278 nm, it was proved linear in the range of 5.0-30.0 µg/ml and exhibited good correlation coefficient (R2=0.9993) and excellent mean recovery (101.37-100.76%) and (100.66-101.17%) for lozenges and chewable tablets respectively. This method was successfully applied to the determination of Dextromethorphan hydrobromide content in lozenges and chewable tablets and the results were in good agreement with the label claim. The method was validated as per ICH guidelines for linearity, precision, accuracy, specificity, LOD and LOQ. The obtained results proved that the method can be employed for the routine analysis of Dextromethorphan hydrobromide in bulks as well as in the pharmaceutical formulations.

Stability-Indicating Liquid Chromatography–Spectrophotometric UV Method for the Simultaneous Determination of Marbofloxacin, Dexamethasone and Clotrimazole in a Liquid Pharmaceutical Dosage Form

A novel, simple and reliable reversed-phase liquid chromatography (LC)–spectrophotometric UV stability-indicating method was developed and validated for the simultaneous assay of marbofloxacin, clotrimazole and dexamethasone acetate in the presence of their impurities and degradation products in a pharmaceutical formulation for veterinary use. A C18 (75 × 4.6 mm, 4 µm) column was used with an acetonitrile–ammonium acetate mixture as mobile phase delivered with gradient elution. A diode-array detection was used in the 200–400 nm range and the detection wavelength was set at 260 nm. Validation carried out on the pharmaceutical dosage form, according to Veterinary International Conference on Harmonization guidelines, demonstrated excellent specificity, linearity, precision, accuracy and robustness. Excellent specificity with respect to vehicle and degradation products obtained after forced degradation (i.e., oxidation, acid, alkaline and thermal degradation) was demonstrated. As for linearity, the LC–UV assay method is applicable in the 0.180–0.420 mg mL−1 concentration range for marbofloxacin (r 2 = 0.99), 0.060–0.140 mg mL−1 for dexamethasone acetate (r 2 = 0.97) and 0.600–1.400 mg mL−1 for clotrimazole (r 2 = 0.98). Very good repeatability (RSD < 0.8 %) and inter-day precision (RSD < 2.5 %) were observed for all analytes. Accuracy was in the 93–104 %, 98–111 % and 99–108 % confidence interval (95 %) for marbofloxacin, dexamethasone acetate and clotrimazole, respectively. The variations (±20 %) of mobile phase flow rate and pH, and oven column temperature did not exhibit an impact on the analyte content accuracy, demonstrating the robustness of the method. The LC–UV method here developed and validated may be used routinely for quality control.

Liquid Chromatographic Method for the Determination of Content Uniformity of Tetrabenazine

The study is a proposition of the application of high performance liquid chromatography (HPLC) with a spectrophotometric UV range detector to analyse the chemical purity and assay of tetrabenazine in bulk drug and tablet formulation and also to apply the proposed method to determine the content uniformity according to USP specification. During computer assisted literature search only a few publications were found about tetrabenazine, so the ultimate objective of the present investigation was aimed to develop stability-indicating LC method and the development of the newer analytical method was achieved under optimised chromatographic conditions by using mobile phase composed of potassium phosphate buffer: acetonitrile in the ratio (40: 60v/v). The seperation was achieved by using isocratic elution mode with a flow rate of 1.0 mL/min. The effluent was monitored on a UV detector at 284 nm. The retention time of tetrabenazine was found to be at 5.05min and the standard calibration curve was linear over a concentration range of 6.25–37.5 μg/mL with r value of 0.999. The LOD and LOQ were found to be 0.562 and 1.704 μg/mL respectively. The recovery studies were performed and the percentage recoveries were found to be in the range of 98.64–98.88%. The drug was also subjected to acidic, basic, oxidative and photolytic degradation as per the ICH guidelines. The developed method allows to separate all degradation from tetrabenazine and to quantitate the tetrabenazine amount. As there is no official monograph in the pharmacopeias about tetrabenazine, the performed full validation procedure makes the method ready to use in routine analysis.

Determination of ibuprofen and paraben in pharmaceutical formulations using flow-injection and derivative spectrophotometry

A flow-injection UV spectrophotometric method for the determination of ibuprofen in tablets was developed. The proposed method was achieved by using a flow cell which was located in a UV spectrophotometer and a peristaltic pump helped maintaining the carrier solvent, 0.1 M NaOH. During the study, flow rate, loop volume and the number of injections per hour were 15 mL/min, 193 μL and 100, respectively. The analytical signal of ibuprofen was linear in the concentration range of 400–1200 μg/mL. The detection limit and limit of quantification were determined as 27 and 91 μg/mL, respectively. Results for the determination of ibuprofen in tablets, 600 ± 2 mg/tablet (mean ± SD), were in good agreement with the labeled quantities, 600 mg/tablet. A relatively high recovery value, 100.4 ± 0.6%, proves the accuracy of the proposed method. The spectro-photometric method and reference HPLC method were shown to be comparable when Student’s t-test and Fisher test were applied to both methods. In addition, a fourth order derivative UV spectrophotometric method (at 268.8 nm and n = 9) for the determination of ibuprofen and a first order (at 271 nm and n = 1) derivative UV spectrophotometric method for the determination of paraben in pediatric syrups were developed. Recovery values for the determination of ibuprofen and paraben were 102.4 ± 1.1% and 100.2 ± 2.3%, respectively.

Comparative artificial neural network and partial least squares models for analysis of Metronidazole, Diloxanide, Spiramycin and Cliquinol in pharmaceutical preparations

Metronidazole (MNZ) is a widely used antibacterial and amoebicide drug. Therefore, it is important to develop a rapid and specific analytical method for the determination of MNZ in mixture with Spiramycin (SPY), Diloxanide (DIX) and Cliquinol (CLQ) in pharmaceutical preparations. This work describes simple, sensitive and reliable six multivariate calibration methods, namely linear and nonlinear artificial neural networks preceded by genetic algorithm (GA-ANN) and principle component analysis (PCA-ANN) as well as partial least squares (PLS) either alone or preceded by genetic algorithm (GA-PLS) for UV spectrophotometric determination of MNZ, SPY, DIX and CLQ in pharmaceutical preparations with no interference of pharmaceutical additives. The results manifest the problem of nonlinearity and how models like ANN can handle it. Analytical performance of these methods was statistically validated with respect to linearity, accuracy, precision and specificity. The developed methods indicate the ability of the previously mentioned multivariate calibration models to handle and solve UV spectra of the four components’ mixtures using easy and widely used UV spectrophotometer.

Validation of HPLC Method for Quantitative Determination of Pirimiphos Methyl

The objective of this research to optimise the HPLC method was developed for quantitative determination of Pirimiphos-methyl. Chromatographic separation was achieved on a 250 x 4.6 mm i.d. reversed phase column Qualisil BDS 5u C18, Using deionized acetonitrile:water in the ratio of 85:15 v/v respectively as mobile phase. The eluent was monitored at 254 nm. A sharp peak was obtained for the Pirimiphos-methyl at 9.29 min. The UV Spectrophotometric method performed at 254 nm after full scan analysis using methanol as a solvent. The result revealed that both methods are suitable to carry out routine analysis of Pirimiphos methyl, However HPLC results showed high precise, accurate and sensitive than the UV Spectrophotometer. Hence HPLC method is suitable for trace analysis of Pirimiphos methyl in environmental samples.

Validation of HPLC Method for Quantitative Determination of Pirimiphos Methyl

The objective of this research to optimise the HPLC method was developed for quantitative determination of Pirimiphos-methyl. Chromatographic separation was achieved on a 250 x 4.6 mm i.d. reversed phase column Qualisil BDS 5u C18, Using deionized acetonitrile:water in the ratio of 85:15 v/v respectively as mobile phase. The eluent was monitored at 254 nm. A sharp peak was obtained for the Pirimiphos-methyl at 9.29 min. The UV Spectrophotometric method performed at 254 nm after full scan analysis using methanol as a solvent. The result revealed that both methods are suitable to carry out routine analysis of Pirimiphos methyl, However HPLC results showed high precise, accurate and sensitive than the UV Spectrophotometer. Hence HPLC method is suitable for trace analysis of Pirimiphos methyl in environmental samples.

Development and validation of high performance liquid chromatography with a spectrophotometric detection method for the chemical purity and assay of nepafenac

The study is a proposition of the application of high performance liquid chromatography (HPLC) with a spectrophotometric UV range detector to analyze the chemical purity and assay of nepafenac, an active pharmaceutical ingredient (API). During literature search only a few publications were found about nepafenac. HPLC UV methods were mainly presented in patent documents about nepafenac synthesis and chemical purity. The presented method allows to separate all potential related compounds from nepafenac and to quantitate the nepafenac amount. As there is no official monograph in the pharmacopeias about nepafenac, the performed full validation procedure makes the method ready to use in routine analysis. The composition of the mobile phase (10mM ammonium formate, pH 4.1) and the HPLC column (Phenomenex Gemini-NX C18) were selected during the development step. Presented data confirm the benefits of the developed method. Four of the most potential impurities were validated as for the quantitative test and the rest of impurities were validated as for the limit test – according to ICH Q2(R1). The accuracy/recovery results for the chemical purity method are within 90–108%, in the case of assay studies from 99% to 101%; the limit of detection is as low as 15–30ng/mL. The linearity passes all statistical tests.