Development and validation of UPLC method for simultaneous quantification of carvedilol and ivabradine in the presence of degradation products using DoE concept

A methodical design-of-experiments were performed by applying quality-by-design concepts to establish a design-space for simultaneous and rapid quantification of Carvedilol and Ivabradine by UPLC in the presence of degradation products. Response-surface, central-composite design, and quadratic model were employed for statistical assessment of experimental data using the Design-Expert software. Response variables such as resolution and retention time were analyzed statistically for chromatographic screening. During DoE study, various plots such as perturbation, contour, 3D and design-space plots were considered for method optimization. The method was developed using C8 [100?×?2.1?mm, 1.8?µ] UPLC column, mobile phase comprising 0.5% triethylamine buffer [pH 6.4] and acetonitrile in the ratio of 50:50 v/v, the flow rate of 0.4?mL minute^?1 and UV detection at 285?nm for both Carvedilol and Ivabradine. The method was developed with a short run time of two minutes. The method was found to be linear in the range of 25.0–199.9?µg?mL^?1 and 8.9–21.3?µg?mL^?1 for Carvedilol and Ivabradine, respectively with a correlation coefficient of 0.9998 in each case. The recovery values were found in the range of 99.7–100.8% and 98.9–100.9% for Carvedilol and Ivabradine, respectively. The method was validated according to ICH Q2 (R1) guidelines.     

Stability-indicating RP-HPLC method for simultaneous quantitation of tramadol and aceclofenac in presence of their major degradation products: Method development and validation

Primary objective of this study was to develop a stability-indicating reverse-phase high-performance liquid chromatography (HPLC) method for simultaneous quantitation of tramadol and aceclofenac in presence of their degradation products. The drugs were subjected to various International Conference on Harmonization recommended stress conditions, such as acid hydrolysis, alkaline hydrolysis, peroxide oxidation, thermolysis, and photolysis. The major degradation products got well resoluted from the analytes in HPLC analysis with a mobile phase composed of a mixture of 0.01?M ammonium acetate buffer (pH 6.5) and acetonitrile (65:35, v/v) through a Phenomenex Gemini C18 (250?mm?×?4.6?mm, 5?µm particle size) column. The method was linear over a range of 15–60?µg/mL for tramadol and 40–160?µg/mL for aceclofenac concentration. The analytes were detected at a wavelength of 270?nm. The method was validated and found to be specific, accurate, precise, stable, and robust for its intended use. The method can be recommended for its future use in routine quality control, accelerated and real-time stability analysis of the formulations containing tramadol and aceclofenac combination.     

QbD-driven development and validation of an efficient bioanalytical UPLC method for estimation of olmesartan medoxomil

The present studies describe quality by design-based development of bioanalytical ultra performance liquid chromatography method of olmesartan medoxomil. Initially, method objectives were defined and critical analytical attributes (CAAs) earmarked. Method optimization was conducted using a central composite design for optimizing mobile phase ratio and injection volume as the critical method parameters (CMPs) identified from risk assessment and factor screening studies, and evaluated for their influence on peak area, theoretical plates, and asymmetry factor as CAAs. Chromatographic separation was achieved using acetonitrile:water solvent system containing 0.1% orthophosphoric acid (54:46, v/v) as the mobile phase with UV detection at 243 nm. Further optimization of bioanalytical extraction process was accomplished using a Box–Behnken design selecting extraction time, centrifugation speed, and centrifugation time as the CMPs identified from failure mode and effect analysis, and evaluated for percent recovery, peak asymmetry, and theoretical plate count as the CAAs. Establishment of calibration curve indicated linearity between concentration range of 100 and 800 ng mL^?1, excellent accuracy and precision with limit of detection and limit of quantification as 6.2 and 19.0 ng mL^?1, respectively. Drug stability studies indicated mean percent recovery ranging between 92.4 and 97.3% under various stress conditions.     

Determination of rosuvastatin in the presence of its degradation products by a stability-indicating LC method

A forced degradation study was successfully applied for the development of a stability-indicating assay method for determination of rosuvastatin Ca in the presence of its degradation products. The method was developed and optimized by analyzing the forcefully degraded samples. Degradation of the drug was done at various pH values. Moreover, the drug was degraded under oxidative, photolytic, and thermal stress conditions. Mass balance between assay values of degraded samples and generated impurities was found to be satisfactory. The proposed method was able to resolve all of the possible degradation products formed during the stress study. The developed method was successfully applied for an accelerated stability study of the tablet formulation. The major impurities generated during the accelerated stability study of the tablet formulation were matches with those of the forced degradation study. The developed method was validated for determination of rosuvastatin Ca, and the method was found to be equally applicable to study the impurities formed during routine and forced degradation of rosuvastatin Ca.     

Development and validation of a UPLC method for quantification of antiviral agent,Acyclovir in lipid-based formulations

PurposeThe objective of the current study is to evaluate the Ultra Performance Liquid Chromatography (UPLC) method for quantification of Acyclovir in lipid-based formulations.MethodA simple, rapid, reliable and precise reversed phase UPLC method has been developed and validated according to the regulatory guidelines, which composed of isocratic mobile phase; 0.25% formic acid (FA) in Milli-Q water with a flow rate of 0.5 ml/min, and column BEH C18 (2.1 × 50 mm, 1.7 μm). The detection was carried out at 254 nm.ResultsThe developed UPLC method was found to be rapid (1.2 min run time), selective with well resoluted Acyclovir peak (0.89 min) from different lipid matrices and sensitive (Limit of Detection (LOD) was 0.3 ppm and Lower Limit of Quantification (LLOQ) was 1 ppm). The accuracy and precision were determined and were perfectly matching with the standard FDA limits.ConclusionThe study showed that the proposed UPLC method can be used for the assessment of drug purity, stability, solubility and lipid-formulation release profile with no interference of excipients or related substances of active pharmaceutical ingredient.     

Simultaneous quantification of abemaciclib and letrozole in rat plasma: method development,validation and pharmacokinetic application

Treatment through a combination of drugs involving cyclin D-dependent kinase inhibitors like abemaciclib and aromatase inhibitor like letrozole proved to be a potential therapeutic regimen and first-line treatment in estrogen receptor-positive breast cancer. In this study, we developed a simple and simultaneous RP-HPLC bioanalytical method for quantifying abemaciclib and letrozole in rat plasma. Abemaciclib and letrozole were separated on Zorbax Eclipse C₁₈ column employing a gradient elution method comprising 10 mM ammonium acetate (pH 5) and acetonitrile as mobile phase. The method was found to have acceptable selectivity, accuracy (97.20–118.17%), precision (1.10–9.39%) and stability in the validation experiment performed as per the US Food and Drug Administration guidelines. The method sensitivity was low at a concentration level of 100 ng/ml. The applicability of the method has been verified through a single-dose oral pharmacokinetic study in rat. The developed method will be useful to quantitate the analytes in rat plasma samples of different preclinical studies including their pharmacokinetic drug–drug interactions in the future. To date, no method has been reported for the quantification of abemaciclib and letrozole simultaneously in any type of biological matrices. Therefore, this study makes a definite significant contribution in the field of bioanalytical research.     

Development of a rapid UPLC-MS/MS method for quantification of saxagliptin in rat plasma and application to pharmacokinetic study

A novel, simple and rapid ultraperformance liquid chromatography/tandem mass spectrometry (UPLC‐MS/MS) assay was established for quantification of saxagliptin in rat plasma. Plasma samples were processed by liquid–liquid extraction with ethyl acetate and chromatographed on a C₁₈ column (2.1 × 50 mm i.d., 1.7 µm). The mobile phase consisted of methanol and 0.1% formic acid (40:60, v/v). Multiple reaction monitoring transitions were performed for detection in positive‐ion mode with an electrospray ionization source. The calibration curve was linear over the concentration range of 0.5–100 ng/mL (R² > 0.99). All accuracy values were between 90.62 and 105.60% relative error and the intra‐ and inter‐day precisions were less than 9.66% relative standard deviation. Extraction recovery was more than 81.01% and the matrix effect ranged from 90.27 to 109.15%. After validation, the method was applied to a pharmacokinetic study where healthy rats were orally given 0.5 mg/kg saxagliptin. Copyright © 2012 John Wiley & Sons, Ltd.     

Development and validation of a stability-indicating HPLC method for the determination of degradation products in dipyridamole injection

Summary The development and subsequent validation of an isocratic high-performance liquid chromatographic (HPLC) procedure employing ultraviolet (UV) detection for the determination of degradation products in Dipyridamole Injection is reported. The development of this assay involved the evaluation of several factors including buffer type, ionic strength, pH, organic composition, and column type. The described method is simple, reproducible, accurate, and selective. The precision, relative standard deviation (RSD), amongst five sample preparations for total degradation products was not more than (NMT) 10.2 %, while the individual degradation products were NMT 12.1%. Intermediate precision, as determined from fifteen sample preparations, generated by two Analysts on different HPLC systems over three days, exhibited an RSD for total and individual degradation products of 8.2 % and NMT 27.5 %, respectively. The mean absolute recovery of dipyridamole using the described method is 102.1±1. 9%, (mean±SD, n=12) over the concentration range of 0.03 % to 5.0 % of its label claim of 5 mg mL⁻¹. The limit of detection and limit of quantitation were 0.1 and 0.3 μg mL⁻¹, respectively. The linearity of the peak response was verified with respect to dipyridamole concentration over a range of 0.3 and 50 μg mL⁻¹ (0.03 % to 5.0 % label claim). The Standard and Assay Preparations are stable for up to 48 hours at room temperature. The selectivity was evaluated by subjecting the finished product (Dipyridamole Injection) to thermal, acidic, basic, oxidative and fluorescent radiation stress conditions. No interference in the analysis of degradation products was observed, showing the method is stability-indicating.     

Risk assessment,development and validation of a GC-ECD-based method for the quantification of cypermethrin from green pea

The present work describes the persistence, dissipation behaviour, half-life, risk assessment and novel gas chromatography method for the residue estimation of cypermethrin in green pea by spraying cypermethrin 10EC at 50 g a.i. ha⁻¹ at fruiting stage followed by another application at a 10 day interval. The sample extraction and cleanup was followed bya modified quick, easy, cheap, effective, rugged, and safe method, and the residues of cypermethrin were determined using a validated gas chromatography method. The initial deposits were found to be 1.21 mg kg⁻¹ following the application of insecticide at 50 g a.i. ha⁻¹. Cypermethrin residues declined to below the detection limit of 0.05 mg kg⁻¹ after 15 days at the recommended dosage. The half-life of cypermethrin was 2.66 days at 50 g a.i. ha⁻¹. For risk assessment studies, the waiting period of 15 days is recommended as safe for consumption for the insecticide. The GC-ECD method was validated according to the SANTE guidelines by various analytical parameters including linearity, accuracy, detection and quantification limits. The developed method is simple, selective and repeatable, and can be used for the standardization of pesticides on fruits and vegetables.     

Development of a validated liquid chromatographic method for quantification of sorafenib tosylate in the presence of stress‐induced degradation products and in biological matrix employing analytical quality by design approach

The current research work envisages an analytical quality by design‐enabled development of a simple, rapid, sensitive, specific, robust and cost‐effective stability‐indicating reversed‐phase high‐performance liquid chromatographic method for determining stress‐induced forced‐degradation products of sorafenib tosylate (SFN). An Ishikawa fishbone diagram was constructed to embark upon analytical target profile and critical analytical attributes, i.e. peak area, theoretical plates, retention time and peak tailing. Factor screening using Taguchi orthogonal arrays and quality risk assessment studies carried out using failure mode effect analysis aided the selection of critical method parameters, i.e. mobile phase ratio and flow rate potentially affecting the chosen critical analytical attributes. Systematic optimization using response surface methodology of the chosen critical method parameters was carried out employing a two‐factor–three‐level–13‐run, face‐centered cubic design. A method operable design region was earmarked providing optimum method performance using numerical and graphical optimization. The optimum method employed a mobile phase composition consisting of acetonitrile and water (containing orthophosphoric acid, pH 4.1) at 65:35 v/v at a flow rate of 0.8 mL/min with UV detection at 265 nm using a C₁₈ column. Response surface methodology validation studies confirmed good efficiency and sensitivity of the developed method for analysis of SFN in mobile phase as well as in human plasma matrix. The forced degradation studies were conducted under different recommended stress conditions as per ICH Q1A (R2). Mass spectroscopy studies showed that SFN degrades in strongly acidic, alkaline and oxidative hydrolytic conditions at elevated temperature, while the drug was per se found to be photostable. Oxidative hydrolysis using 30% H₂O₂ showed maximum degradation with products at retention times of 3.35, 3.65, 4.20 and 5.67 min. The absence of any significant change in the retention time of SFN and degradation products, formed under different stress conditions, ratified selectivity and specificity of the systematically developed method.     

Development of a simple HPLC–MS/MS method to simultaneously determine teriflunomide and its metabolite in human plasma and urine: Application to clinical pharmacokinetic study of teriflunomide sodium and leflunomide

A simple high‐performance liquid chromatography coupled with tandem mass spectrometry method was developed and fully validated to simultaneously determine teriflunomide (TER) and its metabolite 4‐trifluoro‐methylaniline oxanilic acid (4‐TMOA) in human plasma and urine. Merely 50 μL plasma and 20 μL urine were employed in sample preparation using protein precipitation and direct dilution method, respectively. An Agilent Zorbax eclipse plus C₁₈ column was selected to achieve rapid separation for TER and 4‐TMOA within 3 min. Electrospray ionization under multiple reaction monitoring was used to monitor the ion transitions for TER (m/z 269.0 → 159.9), 4‐TMOA (m/z 231.9 → 160.0), internal standard teriflunomide‐d4 (m/z 273.0 → 164.0) and 2‐amino‐4‐trifluoromethyl benzoic acid (m/z 203.8 → 120.1), operating in the negative ion mode. This method proved to have better accuracy and precision over concentration range of 10–5000 ng/mL in plasma as well as 10–10,000 ng/mL in urine. After a full validation, this method was successfully applied in a pharmacokinetic study of teriflunomide sodium and leflunomide in Chinese healthy volunteers.     

Development,stability-indicating assessment,and evaluation of influential method conditions using a full factorial design for the determination of Nintedanib esylate-related impurities

The design of an appropriate analytical method for assessing the quality of pharmaceuticals requires a deep understanding of science, and risk evaluation approaches are appreciated. The current study discusses how a related substance method was developed for Nintedanib esylate. The best possible separation between the critical peak pairs was achieved using an X-Select charged surface hybrid Phenyl Hexyl (150 × 4.6) mm, 3.5 μm column. A mixture of water, acetonitrile, and methanol in mobile phase-A (70:20:10) and mobile phase-B (20:70:10), with 0.1% trifluoroacetic acid and 0.05% formic acid in both eluents. The set flow rate, wavelength, and injection volumes were 1.0 ml/min, 285 nm, and 5 μl, respectively, with gradient elution. The method conditions were validated as per regulatory requirements and United States Pharmacopeia general chapter < 1225 >. The correlation coefficient for all impurities from the linearity experiment was found to be > 0.999. The % relative standard deviation from the precision experiments ranged from 0.4 to 3.6. The mean %recovery from the accuracy study ranged from 92.5 to 106.5. Demonstrated the power of the stability-indicating method through degradation studies; the active drug component is more vulnerable to oxidation than other conditions. Final method conditions were further evaluated using a full-factorial design. The robust method conditions were identified using the graphical optimization from the design space.     

Determination of amitraz and its degradation products and monitoring degradation process in quince and cucumber

Amitraz is a non-systemic acaracide and insecticide. Current maximum residue limits for amitraz are stated as ‘Amitraz including the metabolites containing the 2,4-dimethylaniline moiety’. Therefore, determination of amitraz and its all degradation products are important. In this study, we develop a gas chromatography/mass spectrometry (GC/MS) method for determination of amitraz and its degradation products 2,4 dimethylaniline (DMA), 2,4 dimethylformamidine (DMF) and N-(2,4-dimethyl phenyl)-N’-methylformamidine (DMPF) in cucumber and quince. The mechanism of the degradation process was monitored at different temperatures. Amitraz and its degradation products were extracted using the QuEChERS method. To determine amitraz and its degradation products, we used GC/MS. Quantification was carried out by using selected ion monitoring, and total ion chromatogram was used to monitor additional degradation products. The method was validated by studying linearity, limit of detection (LOD) and limit of quantification (LOQ), recovery and precision. The mechanism of the degradation process was monitored at different temperatures. Degradation of amitraz mainly to three degradation products, namely DMA, DMF and DMPF, increased with temperature. Besides these three main degradation products, two other new degradation products were detected.     

Novel and sensitive UPLC–MS/MS method for quantification of sofosbuvir in human plasma: application to a bioequivalence study

A novel and sensitive LC–MS/MS method was developed and validated for determination of sofosbuvir (SF) using eplerenone as an internal standard. The Xevo TQD LC–MS/MS was operated under the multiple‐reaction monitoring mode using electrospray ionization. Extraction with tert‐butyl methyl ether was used in sample preparation. The prepared samples were chromatographed on Acquity UPLC BEH C₁₈ (50 × 2.1 mm, 1.7 μm) column by pumping 0.1% formic acid and acetonitrile in an isocratic mode at a flow rate of 0.35 mL/min. Method validation was performed as per the US Food and Drug Administration guidelines and the standard curves were found to be linear in the range of 0.25–3500 ng/mL for SF. The intra‐ and inter‐day precision and accuracy results were within the acceptable limits. A very short run time of 1 min made it possible to analyze more than 500 human plasma samples per day. A very low quantification limit of SF allowed the applicability of the developed method for determination of SF in a bioequivalence study in human volunteers. Copyright © 2016 John Wiley & Sons, Ltd.     

Development and Validation of a Stability-Indicating UPLC Method for the Determination of Hexoprenaline in Injectable Dosage Form Using AQbD Principles

A novel and efficient stability-indicating, reverse phase ultra-performance liquid chromatographic (UPLC^®) analytical method was developed and validated for the determination of hexoprenaline in an injectable dosage form. The development of the method was performed using analytical quality by design (AQbD) principles, which are aligned with the future requirements from the regulatory agencies using AQbD principles. The method was developed by assessing the impact of ion pairing, the chromatographic column, pH and gradient elution. The development was achieved with a Waters Acquity HSS T3 (50 × 2.1 mm i.d., 1.8 µm) column at ambient temperature, using sodium dihydrogen phosphate 5 mM + octane-1-sulphonic acid sodium salt 10 mM buffer pH 3.0 (Solution A) and acetonitrile (Solution B) as mobile phases in gradient elution (t = 0 min, 5% B; t = 1 min, 5% B; t = 5 min, 50% B; t = 7 min, 5% B; t = 10 min, 5% B) at a flow rate of 0.5 mL/min and UV detection of 280 nm. The linearity was proven for hexoprenaline over a concentration range of 3.50–6.50 µg/mL (R² = 0.9998). Forced degradation studies were performed by subjecting the samples to hydrolytic (acid and base), oxidative, and thermal stress conditions. Standard solution stability was also performed. The proposed validated method was successfully used for the quantitative analysis of bulk, stability and injectable dosage form samples of the desired drug product. Using the AQbD principles, it is possible to generate methodologies with enhanced knowledge, which can eventually lead to a reduced regulatory risk, high quality data and lower operational costs.     

Development and validation of a RP‐HPLC method for the quantitation of tofacitinib in rat plasma and its application to a pharmacokinetic study

A novel, simple, specific, sensitive and reproducible high‐performance liquid chromatography (HPLC) assay method has been developed and validated for the estimation of tofacitinib in rat plasma. The bioanalytical procedure involves extraction of tofacitinib and itraconazole (internal standard, IS) from rat plasma with a simple liquid–liquid extraction process. The chromatographic analysis was performed on a Waters Alliance system using a gradient mobile phase conditions at a flow rate of 1.0 mL/min and C₁₈ column maintained at 40 ± 1 °C. The eluate was monitored using an UV detector set at 287 nm. Tofacitinib and IS eluted at 6.5 and 8.3 min, respectively and the total run time was 10 min. Method validation was performed as per US Food and Drug Administration guidelines and the results met the acceptance criteria. The calibration curve was linear over a concentration range of 182–5035 ng/mL (r² = 0.995). The intra‐ and inter‐day precisions were in the range of 1.41–11.2 and 3.66–8.81%, respectively, in rat plasma. The validated HPLC method was successfully applied to a pharmacokinetic study in rats. Copyright © 2015 John Wiley & Sons, Ltd.     

A Stability-Indicating Ultra Performance Liquid Chromato-Graphic (UPLC) Method for the Determination of a Mycophenolic Acid-Curcumin Conjugate and Its Applications to Chemical Kinetic Studies

A simple, precise, and accurate reversed-phase ultra-performance liquid chromatographic (UPLC) method was developed and validated for the determination of a mycophenolic acid-curcumin (MPA-CUR) conjugate in buffer solutions. Chromatographic separation was performed on a C18 column (2.1 × 50 mm id, 1.7 µm) with a gradient elution system of water and acetonitrile, each containing 0.1% formic acid, at a flow rate of 0.6 mL/min. The column temperature was controlled at 33 °C. The compounds were detected simultaneously at the maximum wavelengths of mycophenolic acid (MPA), 254 nm, and curcumin (CUR), or MPA-CUR, at 420 nm. The developed method was validated according to the ICH Q2(R1) guidelines. The linear calibration curves of the assay ranged from 0.10 to 25 μg/mL (r² ≥ 0.995, 1/x² weighting factor), with a limit of detection and a limit of quantitation of 0.04 and 0.10 μg/mL, respectively. The accuracy and precision of the developed method were 98.4–101.6%, with %CV < 2.53%. The main impurities from the specificity test were found to be MPA and CUR. Other validation parameters, including robustness and solution stability, were acceptable under the validation criteria. Forced degradation studies were conducted under hydrolytic (acidic and alkaline), oxidative, thermal, and photolytic stress conditions. MPA-CUR was well separated from MPA, CUR, and other unknown degradation products. The validated method was successfully applied in chemical kinetic studies of MPA-CUR in different buffer solutions.     

Determination of newly synthesized lipoic acid–niacin dimer in rat plasma by UPLC/electrospray ionization tandem mass spectrometry: assay development,validation and application to a pharmacokinetic study

A simple, sensitive and specific ultra‐performance liquid chromatography/tandem mass spectrometry (UPLC‐MS/MS) method was developed to determine the newly synthesized compound lipoic acid–niacin dimer (N2L) in plasma. Plasma samples were precipitated by methanol using tetrahydropalmatine as internal standard. Chromatographic separation was achieved on an Acquity BEH C₁₈ (2.1 × 50 mm i.d., 1.7 µm) column; the mobile phase contains methanol and buffer solution (water with 0.5% formic acid and 10 mmol/L ammonium acetate). Multiple reaction monitoring (m/z 353.9 → 148.6 for N2L and m/z 356.0 → 192.0 for internal standard) was performed for detection and quantification. The method was validated to be rapid, specific, accurate and precise over the concentration range of 1–750 ng/mL; N2L was not stable on the bench‐top or during freeze–freeze‐thaw cycles in plasma, but was stable in the stock solution and after preparation in the autosampler for 24 h. The utility of the assay was confirmed by pharmacokinetic study of N2L in rats. Copyright © 2013 John Wiley & Sons, Ltd.     

ESI-MS/MS stability-indicating bioanalytical method development and validation for simultaneous estimation of donepezil, 5-desmethyl donepezil and 6-desmethyl donepezil in human plasma

A bioanalytical method was developed and validated to estimate donepezil, 6‐desmethyl donepezil and 5‐desmethyl donepezil simultaneously in human plasma using galantamine as an internal standard (IS). The chromatographic separation was achieved on a reverse‐phase XTerra RP (150 × 4.6 mm, 5 µm) column without affecting recovery (mean recovery > 60% with CV < 10%) for all analytes. ESI‐MS/MS multiple reaction monitoring in positive polarity was used to detect mass pairs for donepezil (m/z 380.3 → 91.3), 6‐desmethyl donepezil (m/z 366.4 → 91.3), 5‐desmethyl donepezil (m/z 366.4 → 91.3) and galantamine m/z (288.1 → 213.0). The linearity was established over a dynamic range of 0.339–51.870, 0.100–15.380 and 0.103–15.763 ng/mL for donepezil, 6‐desmethyl donepezil and 5‐desmethyl donepezil, respectively. The current method shows that minimal conversion of labile metabolites to parent donepezil in plasma as stability was successfully achieved for 211 days at ?15 °C storage temperature. The method was successfully applied to a clinical study after administration of 10 mg donepezil tablets to healthy male Indian volunteers. Copyright © 2011 John Wiley & Sons, Ltd.     

Development and validation of a RP-HPLC method for Roflumilast and its degradation products

Roflumilast is a phosphodiesterase type 4 inhibitor that is administered orally as a long-term, in the clinical treatment of chronic obstructive pulmonary disease and asthma. Launched in 2010 for the European market, it currently does not have an official monograph. Here, a reproducible gradient RP-HPLC method was developed and validated for the separation and determination of Roflumilast in the presence of its six major degradation products. Separation was performed on a C₁₈ analytical column (250?×?4.6?mm, 5?µm) with a mobile phase-A of ACN and a phase-B of ammonium acetate buffer (5?mM, pH 4.2) containing triethylamine (0.5% v/v). The most effective RP-HPLC gradient program was determined to be 0/80, 35/10, 36/80, 40/80 (time in minutes/% mobile phase-B). The flow rate was 1.0?ml/min and the column temperature was 25°C. The success of separation of the degradation products with different chemical characteristics was obtained by extending the time of the gradient, changing the proportion of the mobile phases and increasing the velocity of the flow. Two detectors were evaluated for the identification of degradation products and Roflumilast: a diode-arrary detector and a charged aerosol detector. The inability of the charged aerosol detector to dectect one of the six degradation products indicated that the method developed with RP-HPLC and the diode-array detector was more suitable for Roflumilast analysis. The method was validated according to specificity, linearity, LOD, LOQ, accuracy, precision and robustness.