A Chemometrical Approach to Optimization and Validation of an HPLC Assay for Rizatriptan and its Impurities in Tablets

Abstract

An isocratic reversed‐phase high‐performance liquid chromatographic method was developed and validated for the analysis of a novel antimigraine drug, rizatriptan benzoate, in a dosage form along with its two impurities, L‐749.019 and L‐783.540. The method used a C₁₈ XTerra? (150×3.9 mm), 5 µm column. The mobile phase consisted of a mixture of methanol, TEA (1%) and 10 mM KH₂PO₄ (5:9.5:85.5 v/v) at a flow rate of 1.2 ml min^?1 (pH of the water phase was adjusted to 5.5 with 85% orthophosphoric acid). Column temperature was 20°C and the detection was performed at 225 nm. The central composite design technique and the response surface method were used in the robustness test considerations. The method was applied satisfactorily to the analysis of commercial rizatriptan formulation.     

Development of a Simple,Rapid, and Robust Isocratic Liquid Chromatographic Method for the Determination of Pyrimethamine and its Synthetic Impurities in Bulk Drugs and Pharmaceutical Formulations

Pyrimethamine is an important antiparasitic drug in the treatment of malaria and toxoplasmosis and is often used in combination with either sulfadoxine, sulfalene, or sulfadiazine. Determining the content of pyrimethamine and investigating the related substances is currently possible applying either a compendial monograph utilizing thin layer chromatography as well as liquid chromatographic methods used by the respective manufacturers. To provide a simple method which is capable of determining the content of pyrimethamine and of resolving four of its potential synthetic impurities a very simple, cheap, precise, and accurate isocratic RP-HPLC method was developed. All analytes can be separated within a total runtime of 30 min and the method was linear within the concentration ranges of 0.12–0.740, 0.104–0.621, 0.120–0.710, 2.0–11.8, and 1.01–5.80 µg mL^?1 for pyrimethamine, impurity A, impurity B, impurity C, and impurity D, respectively. These substances were separated by employing a Eurospher-II C₁₈H column (250 × 4.6 mm, 5 µm particle size), a mobile phase being a mixture of a 0.05 M KH₂PO₄ buffer solution (pH 2.6) and methanol in the ratio 40:60 (v/v). The analysis was carried out at 30 °C, applying a flow rate of 1.2 mL min^?1, and a detection wavelength of λ = 215 nm. The coefficients of determinations (R ²) for the five analytes were greater than 0.994 for pyrimethamine and all impurities. Results of recovery studies were within the range of 89.1–105.1% for all substances. In all tested genuine batches of pyrimethamine raw material impurities within the specified limits were present which is concurrent with results obtained from using the present manufacturer’s method.     

Development and Validation of a Novel Stability Indicating UPLC <Emphasis Type="SmallCaps">M</Emphasis>ethod for Analysis of Related Compounds and Assay of Bexarotene,an Anti-Cancer Agent

A novel liquid chromatographic method for the analysis of four potential impurities in the anti-cancer agent Bexarotene has been developed and validated using efficient chromatographic separation, achieved on a C₁₈ column (50 mm × 2.1 mm, 1.7-μm particles) with a simple isocratic mobile phase at a flow rate of 0.5 mL min^?1. The mobile phase consisted of a 70:30:0.1 (v/v/v) mixture of acetonitrile, water and trifluoroacetic acid, and quantification was achieved by use of ultraviolet detection at 260 nm. Resolution between Bexarotene and its four potential impurities was greater than 2.0. Regression analysis showed the r value (correlation coefficient) was >0.999 for Bexarotene and its four impurities. The method was capable of detecting all four impurities of Bexarotene at levels below 0.10 μg in a Bexarotene test concentration of 0.5 mg mL^?1 using an injection volume of 5 μL. The recovery for Bexarotene in assay method at a 100% level was observed to be 99.1 ± 0.32% with % RSD value of 0.5% for drug substance and 98.9 ± 0.46% for Bexarotene capsules with % RSD value of 0.4%. Recovery of imp-1, imp-2, imp-3, and imp-4 from bulk drug samples ranged from 96.3 to 102.0%. Recovery of imp-1, imp-2, imp-3, and imp-4 from Bexarotene Capsule samples ranged from 97.2 to 101.4%. A solution of Bexarotene in ethanol was stable for 48 h. The drug was also subjected to stress conditions as prescribed by ICH Guidelines. Degradation was found to occur under acidic and basic hydrolysis stress conditions; however, the drug was stable to oxidative, photolytic, and thermal stress. Assay of the stressed samples was calculated relative to a qualified reference standard and the mass balance was found to be close to 99.8%. The method was validated for linearity, accuracy, precision, and robustness.     

Development and validation of a novel RP-HPLC method for stability-indicating assay of Abiraterone acetate

Abiraterone acetate is a prodrug of Abiraterone widely used for the treatment of metastatic castration resistant prostate cancer. In this study, a simple, sensitive, and rapid stability-indicating reverse phase HPLC method was developed and validated for the determination of Abiraterone acetate in bulk and its pharmaceutical formulation. The method was developed by HPLC using a Hypersil ODS C-18 (150 mm × 4.6 mm, 5 µm) column in a isocratic mode with mobile phase constituted by potassium phosphate buffer and acetonitrile (40:60, v/v%) flow rate was 1.0 mL min^?1, column temperature of 30°C, UV detection wavelength 235 nm, and injection volume of 20 µL. The validated parameters were in accordance with FDA and ICH specifications, assay exhibited a linear range of 25–250 µg mL^?1 with regression (r²) coefficient 0.9998. The limits of detection and quantification were 0.23 and 0.70 µg mL. Accuracy was between 99.34 and 100.07%. The drug was subjected to various stress conditions like acidic, base hydrolysis, oxidation, thermal, and photolytic degradation. Stress study Abiraterone acetate was found susceptible to degrade under hydrolytic (acid and base) conditions. The proposed method has stability indicating the resolution of the main peak from their degradation peaks. The validated method is suitable for quality control application and reduced analysis time.     

Development and Validation of Rapid Resolution RP-HPLC Method for Simultaneous Determination of Atorvastatin and Related Compounds by Use of Chemometrics

Abstract

A Rapid Resolution Reversed Phase High-Performance Liquid Chromatography (RR RP-HPLC) method has been developed and validated for the simultaneous determination of atorvastatin and seven related compounds specified as impurities. Experimental design was used during method optimization (full factorial 3² design) and robustness testing (central composite design). Chromatography was performed with mobile phase containing phosphate buffer pH 3.5 and a mixture of 10% (v/v) tetrahydrofuran in acetonitrile as organic modifier. A Zorbax Eclipse XDB C18 Rapid Resolution HT 4.6 mm × 50 mm, 1.8 µm particle size column was used. The developed method allowed determination of Atorvastatin Calcium (ATV Ca) purity and level of impurities in drug substances.     

Development and Validation of a New LC Method for Analysis of Brimonidine Tartrate and Related Compounds

A novel liquid chromatographic method for analysis three potential impurities in brimonidine tartrate drug substance has been developed and validated. Efficient chromatographic separation was achieved on a C₈ column (250 mm × 4.6 mm, 5-μm particles) with a simple mobile-phase gradient at a flow rate of 1.0 mL min⁻¹. Quantification was achieved by use of ultraviolet detection at 248 nm. Resolution between brimonidine tartrate and its three potential impurities was greater than 3.0. Regression analysis showed the r value (correlation coefficient) was >0.999 for brimonidine and its three impurities. The method was capable of detecting all three impurities of brimonidine tartrate at levels below 0.07 μg in a test concentration of brimonidine tartrate of 1.0 mg mL⁻¹ and for an injection volume of 10 μL. A solution of brimonidine tartrate in acetonitrile–water 2:8 (v/v) was stable for 48 h. The drug was subjected to stress conditions as prescribed by the ICH. Degradation was found to occur slightly under oxidative stress conditions but the drug was stable to aqueous, acidic, and basic hydrolysis, and photolytic and thermal stress. The assay of the stressed samples was calculated relative to a qualified reference standard and the mass balance was found close to 99.8%. The method was validated for linearity, accuracy, precision, and robustness.

     

Development and Validation of a New LC Method for Analysis of Brimonidine Tartrate and Related Compounds

A novel liquid chromatographic method for analysis three potential impurities in brimonidine tartrate drug substance has been developed and validated. Efficient chromatographic separation was achieved on a C₈ column (250 mm × 4.6 mm, 5-μm particles) with a simple mobile-phase gradient at a flow rate of 1.0 mL min^?1. Quantification was achieved by use of ultraviolet detection at 248 nm. Resolution between brimonidine tartrate and its three potential impurities was greater than 3.0. Regression analysis showed the r value (correlation coefficient) was >0.999 for brimonidine and its three impurities. The method was capable of detecting all three impurities of brimonidine tartrate at levels below 0.07 μg in a test concentration of brimonidine tartrate of 1.0 mg mL^?1 and for an injection volume of 10 μL. A solution of brimonidine tartrate in acetonitrile–water 2:8 (v/v) was stable for 48 h. The drug was subjected to stress conditions as prescribed by the ICH. Degradation was found to occur slightly under oxidative stress conditions but the drug was stable to aqueous, acidic, and basic hydrolysis, and photolytic and thermal stress. The assay of the stressed samples was calculated relative to a qualified reference standard and the mass balance was found close to 99.8%. The method was validated for linearity, accuracy, precision, and robustness.     

Comparison of Full Factorial Design,Central Composite Design,and Box-Behnken Design in Chromatographic Method Development for the Determination of Fluconazole and Its Impurities

This paper presents the development and optimization of a liquid chromatographic method for the determination of fluconazole and its impurities by experimental design methodology. Four experimental design types were applied: two-level full factorial design, central composite design, Box-Behnken design, and three-level full factorial design. The advantages and drawbacks of each design are described and detailed statistical evaluation of mathematical models was performed. The central composite design and three-level full factorial design created significantly better models comparing to the other methods. As the central composite design requires a smaller number of experiments, its models were used for theoretical examination of experimental space. Multiobjective optimization aiming to achieve maximal separation of all investigated substances and minimal analysis duration was performed by a grid point search. The defined optimal separation was achieved on a C₁₈ (125 mm × 4 mm, 5 µm particle size) column with a mobile phase consisting of acetonitrile and water (5 mM ammonium formate) (15:85, v/v); a column temperature of 25°C; a flow rate of 1.2 mL min^?1; and a detection wavelength of 260 nm.     

Comparison of Ion-Pairing and Reversed Phase Liquid Chromatography in Determination of Sulfamethoxazole and Trimethoprim

Abstract

Two simple, rapid, and sensitive HPLC methods have been developed for the simultaneous determination of sulfamethoxazole and trimethoprim in their pure and dosage forms, one utilizing reversed phase HPLC and the other ion-pair HPLC. In the reversed phase HPLC method (A) the mobile phase consists of 0.05% aqueous solution of formic acid with pH adjusted to 4.5±0.2 with triethylamine : acetonitrile:tetrahydrofuran 50 : 49 : 1 (v/v), and the mobile phase pumped at flow rate of 1.0 ml min^?1. An Appolo LC18 column (5.0 µm), 250 mm length × 4.6 mm diameter, was utilized as the stationary phase. Detection was affected spectrophotometrically at 254 nm. In the ion-pair HPLC method (B) the mobile phase consisted of methanol : buffer 35 : 65 (v/v) with the buffer composed of potassium dihydrogen phosphate 0.3 M and sodium heptan sulfonic acid 5.0 mM. To 500 ml of buffer was added 2.0 ml triethylamine, and then the pH was adjusted to 5.0 with phosphoric acid, and the mobile phase was pumped at a flow rate of 1.2 ml min^?1. A Hypersil C₁₈ column (5.0 µm), 150 mm length × 4.6 mm diameter, was utilized as the stationary phase. Detection was affected spectrophotometrically at 254 nm. Linearity ranges for sulfamethoxazole and trimethoprim were 1.0–110 and 1.5–98 µg ml^?1, respectively, with method A and 0.5–100 and 1.0–125 µg ml^?1, respectively, with method (B). Minimum detection limits obtained were 0.1969 and 0.3451 µg ml^?1 for sulfamethoxazole and trimethoprim, respectively, with method A, and 0.1377 and 0.2454 µg ml^?1 with method (B). The proposed methods were further applied to the analysis of tablets containing the two drugs, and the results were satisfied.     

Validation of an HPLC Analytical Method for Determination of Pancuronium Bromide in Pharmaceutical Injections

Abstract

Pancuronium bromide is used with general anesthesia in surgery for muscle relaxation and as an aid to intubation. A high performance liquid chromatographic method was fully validated for the quantitative determination of pancuronium bromide in pharmaceutical injectable solutions. The analytical method was performed on an amino column (Luna® 150 mm × 4.6 mm, 5 µm). The mobile phase was composed of acetonitrile:water containing 50 mmol L^?1 of 1-octane sulfonic acid sodium salt (20:80 v/v) with a flow rate of 1.0 mL min^?1 and ultraviolet (UV) detection at 210 nm. The proposed analytical method was compared with that described in the British Pharmacopoeia.     

LC Determination of Lidocaine and Prilocaine Containing Potential Risky Impurities and Application to Pharmaceuticals

A liquid chromatographic method for the determination of lidocaine (LID), prilocaine (PRL) and their impurities 2,6-dimethylaniline (DMA) and o-toluidine (TOL) has been developed. The analysis was performed on a reversed phase C18 Hypersil BDS column at ambient temperature. A mobile phase consisting of Briton-Robinson buffer, pH 7—methanol—acetonitrile (40: 45: 15 v/v/v) was used at a flow rate of 1.2 mL min^?1. Detection was achieved at 225 nm using benzophenone as internal standard over the concentration range 1.25–80 μg mL^?1 for all analytes. The relative standard deviations RSD (n = 7) for the assay were less than 0.95%. Limit of detection values were found to be 0.346, 0.423, 0.112 and 0.241 μg mL^?1 for LID, PRL, DMA and TOL, respectively. The intraday and the inter-days RSD % indicated the precision of the procedure. The method proved to be suitable for the quality control of LID and PRL in pharmaceuticals.     

A Stability-Indicating LC Method for Analysis of Balsalazide Disodium in the Bulk Drug and in Pharmaceutical Dosage Forms

A novel, sensitive, stability-indicating gradient RP-LC method has been developed for quantitative analysis of balsalazide disodium and its related impurities both in the bulk drug and in pharmaceutical dosage forms. Efficient chromatographic separation was achieved on a C₁₈ stationary phase with a simple mobile-phase gradient prepared from methanol and phosphate buffer (10 mm potassium dihydrogen orthophosphate monohydrate, adjusted to pH 2.5 by addition of orthophosphoric acid). The mobile-phase flow rate was 1.0 mL min^?1. Quantification was achieved by use of ultraviolet detection at 240 nm. Under these conditions resolution of balsalazide disodium from its three potential impurities was greater than 2.0. Regression analysis resulted in a correlation coefficient greater than 0.99 for balsalazide disodium and all three impurities. This method was capable of detecting the three impurities at 0.003% of the test concentration of 0.3 mg mL^?1, using an injection volume of 10 μL. Inter-day and intra-day precision for all three impurities and for balsalazide disodium was within 2.0% RSD. Recovery of balsalazide disodium from the bulk drug (99.2–101.5%) and from pharmaceutical dosage forms (99.8–101.3%), and recovery of the three impurities (99.1–102.1%) was consistently good. The test solution was found to be stable in 70:30 (v/v) methanol–water for 48 h. When the drug was subjected to hydrolytic, oxidative, photolytic, and thermal stress, acidic and alkaline hydrolysis and oxidizing conditions led to substantial degradation. The RP-LC method was validated for linearity, accuracy, precision, and robustness.     

Ion-Pair RP-LC of Tegaserod Maleate and its Impurities in Pharmaceutical Formulations and in Dissolution Studies

A simple ion-pair reversed-phase high-performance liquid chromatographic (RP-HPLC) method has been developed for determination of tegaserod maleate and related impurities in tablet dosage forms. The mobile phase was 60:40 (v/v) acetonitrile-25 mmol L^?1 sodium dodecyl sulfate, adjusted to pH 2.6 with glacial acetic acid. A C18 column was used as stationary phase and UV detection was at 314 nm. The method was optimized and validated. Response was linearly dependent on concentration between 0.1 and 100 µg mL^?1 with a limit of quantification (LOQ) of 0.1 µg mL^?1 for tegaserod maleate (S/N = 10). Under optimum conditions, tegaserod maleate was successfully separated from related substances, including 5-methoxyindole-3-carboxaldehyde remaining after synthesis and other impurities possibly resulting from oxidization and decomposition. The excipients did not interfere with assay of tegaserod maleate in tablet dosage forms. It is suggested that the proposed method can be used for routine quality control and dosage-form assay of tegaserod maleate.     

Reversed-Phase Liquid Chromatographic Method for Determination of Daclatasvir Dihydrochloride and Study of Its Degradation Behavior

A simple and selective reversed-phase stability-indicating liquid chromatographic method has been developed and validated for the determination of daclatasvir in drug substance and drug product. Daclatasvir was subjected to acidic, alkaline, oxidative, thermal and photo-degradation study. The LC method was based on isocratic elution of daclatasvir and its degradation products on a reversed-phase C₁₈ Hypersil column using a mobile phase consisting of phosphate buffer (10 mM, 1 mL triethylamine L^?1): acetonitrile (60:40 v/v) at a flow rate of 2 mL min^?1. Quantitation was achieved with UV detection at 312 nm. Linearity, accuracy, and precision were found to be acceptable over the concentration range of 0.75–120 μg mL^?1, with regression coefficient value of 0.9999, and with limit of detection and quantitation of 0.148 and 0.447 μg mL^?1, respectively. Peak purity was checked for principle drug and its alkali induced degradation product, and the pathway of alkaline hydrolysis of daclatasvir was suggested by LC/MS.     

Identification and Determination of Related Substances in Diosmin Bulk Drug and Pharmaceutical Formulations by HPLC and HPLC–MS

Six related substances were detected in diosmin bulk drug substances and products by a newly developed gradient reverse-phase high performance liquid chromatography (RP-HPLC) with UV detection. The chromatographic system consisted of an Intersil Wondasil TM ODS (C 18) column (250 × 4.6 mm; 5 μm). The mobile phase consisted of water/acetic acid 66:6 v/v (solvent A) and methanol (solvent B) using a gradient program at a flow rate of 0.8 mL min^?1 with 345 nm detection and an injection volume of 20 μL. In addition, the linearity, quantitation limit (QL), accuracy, selectivity, robustness and precision were determined. Good linearity was obtained over the concentration range 0.5–200 μg mL^?1 with the coefficient of determination (r ²) of 0.999. The QL was 0.125 μg mL^?1 (relative standard deviation <2.0 %). The major impurities have been resolved and identified using two analytical systems, HPLC and HPLC/electrospray ionization-mass spectrometry operated in a negative ion mode. One of these impurities marked as 7-hexopyranosidal diosmetin was unknown and has not been reported previously. Based on mass spectrometry data the structure of the new impurity was proposed as 5-hydroxy-2-(3-hydroxy-4-methoxyphenyl)-4-oxo-4H-chromen-7-yl hexopyranoside. The newly developed RP–LC method for quantitative determination of diosmin-related substances was found to be precise, accurate, robust and specific. It has been successfully employed for the quality evaluation of different sources of raw material and generic formulations of diosmin.     

A Stability Indicating LC Method for Deferasirox in Bulk Drugs and Pharmaceutical Dosage Forms

A novel, sensitive, stability indicating RP-LC method has been developed for the quantitative determination of deferasirox, its related impurities in both bulk drugs and pharmaceutical dosage forms. Efficient chromatographic separation was achieved on a C18 stationary phase with simple mobile phase combination delivered in an isocratic mode and quantitation was by ultraviolet detection at 245 nm. The mobile phase consisted of buffer, acetonitrile and methanol (50:45:5, v/v) delivered at a flow rate of 1.0 mL min^?1. Buffer consisted of 10 mM potassium dihydrogen orthophosphate monohydrate, pH adjusted to 3.0 by using orthophosphoric acid. In the developed LC method the resolution (R _s ) between deferasirox and its four potential impurities was found to be greater than 2.0. Regression analysis showed an r value (correlation coefficient) greater than 0.999 for deferasirox and its four impurities. This method was capable to detect all four impurities of deferasirox at a level of 0.002% with respect to test concentration of 0.5 mg mL^?1 for a 10 μL injection volume. The inter- and intra-day precision values for all four impurities and for deferasirox was found to be within 2.0% RSD. The method showed good and consistent recoveries for deferasirox in bulk drugs (98.3–101.1%), pharmaceutical dosage forms (100.2–103.1%) and for its all the four impurities (99.7–102.1%). The test solution was found to be stable in methanol for 48 h. The drug was subjected to stress conditions of hydrolysis, oxidation, photolysis and thermal degradation. Considerable degradation was found to occur in acid stress hydrolysis. The stress samples were assayed against a qualified reference standard and the mass balance was found close to 99.95%. The developed RP-LC method was validated with respect to linearity, accuracy, precision and robustness.     

Determination of Abacavir, Lamivudine and Zidovudine in Pharmaceutical Tablets, Human Serum and in Drug Dissolution Studies by HPLC

A simple, accurate, precise and fully automated method for the simultaneous determination of abacavir, lamivudine and zidovudine in pharmaceutical tablets, human serum samples and drug dissolution studies has been developed. Separation was performed on a 5 μm Zorbax^® C₁₈ column (150 × 4.6 mm ID) with methanol:water:phosphate buffer at pH 5.65 (80:10:10; v/v/v) isocratic elution in less than 7 min with a flow rate of 0.6 mL min^?1.Good sensitivity for all analytes was observed with UV detection at 275 nm. The method allowed quantitation over the 500–3,000 ng mL^?1 range for abacavir and 500–5,000 ng mL^?1 range for lamivudine and zidovudine. The method has been applied, without any interference from excipients or endogenous substances, for the simultaneous determination of these three compounds in tablets. Human serum and drug dissolution studies.     

Forced Degradation Study to Develop and Validate Stability-Indicating RP-LC for Quantification of Ropinirole Hydrochloride in Its Modified Release Tablets

A forced degradation study on ropinirole hydrochloride in bulk and in its modified release tablets was conducted under the conditions of hydrolysis, oxidation and photolysis in order to develop an isocratic stability-indicating LC-UV method for quantification of the drug in tablets. An impurity peak in standard solution was found to increase under acidic and neutral hydrolytic conditions while another degradation product was formed under alkaline condition. The drug and its degradation products were optimally resolved on a Hypersil C₁₈ column with mobile phase composed of diammonium hydrogen orthophosphate (0.05 M; pH 7.2), tetrahydrofuran and methanol (80:15:5% v/v) at a flow rate of 1.0 mL min^?1 at 30 °C using 250 nm as detection wavelength. The method was linear in the range of 0.05–50 μg mL^?1 drug concentrations. The %RSD of inter- and intra-day precision studies was <1. The system suitability parameters remained unaffected during quantification of the drug on three different LC systems. Excellent recoveries (101.59–102.28%) proved that the method was sufficiently accurate. The LOD and LOQ were found to be 0.012 and 0.040 μg mL^?1, respectively. Degradation behaviour of the drug in both bulk and tablets was similar. The drug was very unstable to hydrolytic conditions but stable to oxidative and photolytic conditions. The method can be used for rapid and accurate quantification of ropinirole hydrochloride in tablets during stability testing. Based on chemical reactivity of ropinirole in different media, the degradation products were suspected to be different from the known impurities of the drug.     

Chemometrically Assisted Development and Validation of LC for Simultaneous Determination of Carbamazepine and Its Impurities Iminostilbene and Iminodibenzyl in Solid Dosage Form

A chemometrical approach was applied to develop a reversed-phase liquid chromatographic method for simultaneous determination of carbamazepine and its impurities iminostilbene and iminodibenzyl in solid dosage form. According to contemporary literature, no method was developed for simultaneous determination of carbamazepine and these impurities by chemometrical approach. The fractional factorial design was used for selection of variables significantly influencing the chromatographic separation of the investigated substances. The investigated variables were: temperature of the column, the percentage of organic modifier, the acetate buffer concentration and pH of water phase. The first three variables were proved to be significant and were optimized by face centered, central composite design. Investigation was performed using C18 XBridge Shield analytical column (50 mm × 4.6 mm i.d., particle size 3.5 µm). The optimal conditions for the separation were established with the mobile phase composition of methanol–10 mM acetate buffer (pH adjusted to 2.21 with glacial acetic acid) (50:50, v/v) at a flow rate of 1.5 mL min^?1, 25 °C column temperature and detection at 260 nm. Total analysis time was shortened to about 8 min. Finally, the method was successfully validated and subsequently applied to the analysis of commercially available carbamazepine tablets.     

Analysis of Temocillin and Impurities by Reversed Phase Liquid Chromatography: Development and Validation of the Method

A robust, specific, precise and sensitive high-performance liquid chromatographic method has been described for purity control of temocillin. Chromatographic separation was achieved using a Symmetry C₁₈ (150 × 4.6 mm, 5 µm) column kept at 30 °C. The mobile phase consisted of a gradient mixture of mobile phases A (5 g/L solution of Na₂HPO₄·2H₂O, pH 7) and B (ACN-MeOH-H₂O, 50:10:40 v/v/v) pumped at a flow rate of 1.0 mL/min. UV detection was performed at 235 nm. The developed method was validated according to the ICH guidelines for its robustness, selectivity, sensitivity, precision and linearity. An experimental design was applied for the robustness study. Linearity was assessed both at impurity level in the range from LOQ to 10 % and assay level from 25 % to 150 % (0.6 mg/mL = 100 %). It is the first liquid chromatographic method described for the separation of temocillin and its potential impurities. It was possible to identify four degradation products from the forced degradation studies. The degradants do not interfere with the main peak and other known impurities showing that the method is specific and stability-indicating.