A validated stability-indicating RP-HPLC assay method for Amsacrine and its related substances

A validated specific stability indicating reversed-phase high-performance liquid chromatography method was developed for the quantitative determination of Amsacrine as well as its related substances determination in bulk samples, in presence of degradation products, and its process related impurities. Forced degradation studies were performed on bulk samples of Amsacrine as per International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human use (ICH) prescribed stress conditions using acid, base, oxidative, thermal stress, and photolytic degradation to show the stability indicating power of the method. Significant degradation was observed during basic hydrolysis, slight degradation was observed in oxidative and thermal stress, and no degradation was observed in other stress conditions. The chromatographic method was optimized using the samples generated from forced degradation studies and the impurity spiked solution. Good resolution between the peaks corresponds to process-related impurities and degradation products from the analyte were achieved on Inertsil ODS column using the mobile phase consists a mixture of 1.0% triethyl amine in 20 mM potassium dihydrogen orthophosphate, with pH adjusted to 6.5, with ortho phosphoric acid in water and acetonitrile using a simple linear gradient. The detection was carried out at wavelength 248 nm. The mass balance in each case was in between 99.4% to 99.9%, indicating that the developed method was stability-indicating. Validation of the developed method was carried out as per ICH requirements. The developed method was found to be suitable to check the quality of bulk samples of Amsacrine at the time of batch release and also during its stability studies.     

Development of stability-indicating assay method and liquid chromatography-quadrupole-time-of-flight mass spectrometry-based structural characterization of the forced degradation products of alpelisib

The US Food and Drug Administration and the European Medicines Agency approved alpelisib in 2019 for the treatment of metastatic breast cancer. A thorough literature review revealed that a stability-indicating analytical method (SIAM) is not available for the quantification of alpelisib and its degradation products (DPs). In this study, per the comprehensive stress study recommended by the International Council for Harmonisation (ICH), alpelisib was exposed to hydrolysis, oxidation, photolysis, and thermal stress. Degradation of the drug was observed under hydrolysis, oxidative, and photolysis conditions, whereas the drug was stable under thermal stress condition. We developed a SIAM for the separation of alpelisib and its major DPs that were formed under different stress conditions. The validation of the developed method was performed per ICH Q2(R1) guidelines. Five DPs were identified and characterized. Structure elucidation of all DPs was performed with the modern characterization tool of liquid chromatography-quadrupole time-of-flight mass spectrometer (LC-Q-TOF-MS/MS). The degradation pathway of the drug and its mechanisms were outlined, and in silico toxicity prediction was performed using the ProTox-II tool.     

A simple and sensitive stability-indicating RP-HPLC assay method for the determination of aceclofenac

The aim of the present study is to develop a stability-indicating assay method for the determination of aceclofenac after being subjected to different International Conference on Harmonization prescribed stress conditions, such as hydrolysis, oxidation, heat, and photolysis. Aceclofenac (2-[2-[2-(2,6-dichlorophenyl)aminophenyl]acetyl]oxyacetic acid) is decomposed under hydrolytic stress (neutral, acidic, and alkaline) and also on exposure to light (in solution form). The compound is stable to oxidative stress, heat, and photolytic stress (in solid form). The major degradation product is diclofenac, which is confirmed through comparison with the standard. Separation of the drug from major and minor degradation products is achieved on a C-18 column using methanol-0.02% of ortho phosphoric acid in a ratio of 70:30. The method is linear over the concentration range of 17-100 microg/mL (r(2) = 0.9988). The detection wavelength is 275 nm. The method is validated for linearity, range, precision, accuracy, specificity, and selectivity.     

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 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.     

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.     

Kinetic study of the degradation of forskolin in aqueous systems by stability-indicating HPLC method and identification of its degradation products

The degradation kinetics of forskolin in aqueous solution was investigated qualitatively and quantitatively. Two degradation products were isolated and identified as isoforskolin and forskolin D by liquid chromatography–tandem mass spectrometry (LC–MS/MS) and nuclear magnetic resonance (NMR) spectroscopy. A stability-indicating high-performance liquid chromatography (HPLC) method was developed and validated for the quantification of forskolin and its degradation products. Chromatographic separation was performed on a Luna C₁₈ column with acetonitrile–water (65:35, v/v) as the mobile phase. The flow rate was kept at 1 mL/min, and the detection wavelength was 210 nm. The kinetic study of forskolin was carried out in aqueous solutions of pH 1.5–8.5 at 37, 50, 65, and 80°C. The degradation rate of forskolin increases with increasing temperature. Forskolin is relatively stable in the pH range 3.5–6.5, but its stability decreases when the pH is outside this range. In the pH range 6.5–8.5, the forskolin degradation follows pseudo-first-order kinetics. Based on the structural identification and quantitative analysis of the degradation products, a possible pathway for forskolin degradation is proposed. Forskolin can be converted to isoforskolin rapidly, and both forskolin and isoforskolin can further decompose to forskolin D.     

Optimization and validation of a stability-indicating RP-HPLC method for determination of azithromycin and its related compounds

A validated stability-indicating HPLC method was developed for the analysis of azithromycin (AZ) and its related compounds in raw materials, capsule, and suspension using an Xterra RP C18 column at 50 degrees C with UV detection at 215 nm. Isocratic elution was employed using the mobile phase 14 mM disodium hydrogen phosphate (pH 10.5, adjusted by 1 M NaOH)-methanol-acetonitrile-tetrahydrofuran (40.0 + 30.0 + 30.0 + 0.1, v/v/v/v). AZ and 14 of its related compounds were separated and quantified. The described method was linear over the range of 2-1800 microg/mL AZ with (r = 0.9999). The stability of AZ was studied under accelerated acidic, alkaline, and oxidative conditions. The proposed method was used to investigate the kinetics of acidic and alkaline hydrolysis process of AZ at different temperatures, and the apparent pseudo first-order rate constant, half-life, and activation energy were calculated. The major peak detected from the degradation of AZ in alkaline and acidic conditions was decladinosylazithromycine, while azithromycin N-oxide was detected from the oxidative degradation. Long-term stability studies for capsule and oral suspension were carried out. The proposed stability-indicating method was completely validated according to the U.S. Food and Drug Administration requirements.     

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.     

Isolation and characterization of degradation products of citalopram and process-related impurities using RP-HPLC

A reversed-phase high-performance liquid chromatographic method for simultaneous separation and determination of citalopram hydrobromide and its process impurities in bulk drugs and pharmaceutical formulations was developed. The separation was accomplished on an Inertsil ODS 3V (250x4.6 mm; particle size 5 mum) column using 0.3% diethylamine (pH = 4.70) and methanol/acetonitrile (55:45 v/v) as mobile phase in a gradient elution mode. The eluents were monitored by a photodiode array detector set at 225 nm. The chromatographic behavior of all the related substances was examined under variable conditions of different solvents, buffer concentrations, and pH. The method was validated in terms of accuracy, precision, and linearity. The method could be of use not only for rapid and routine evaluation of the quality of citalopram in bulk drug manufacturing units but also for the detection of its impurities in pharmaceutical formulations. Three unknown impurities were consistently observed during the analysis of different batches of citalopram. Forced degradation of citalopram was carried out under thermal, photo, acidic, alkaline, and peroxide conditions. The degradation products and unknown impurities were isolated and characterized by ESI-MS/MS, (1)H NMR, and FT-IR spectroscopy.     

Quality-by-design-based development and validation of a stability-indicating UPLC method for quantification of teriflunomide in the presence of degradation products and its application to in-vitro dissolution

A systematic design-of-experiments was performed by applying quality-by-design concepts to determine design space for rapid quantification of teriflunomide by the ultraperformance liquid chromatography (UPLC) method in the presence of degradation products. Response surface and central composite quadratic were used for statistical evaluation of experimental data using a Design-Expert software. The response variables such as resolution, retention time, and peak tailing were analyzed statistically for the screening of suitable chromatographic conditions. During this process, various plots such as perturbation, contour, 3D, and design space were studied. The method was developed through UPLC BEH C18 2.1?×?100?mm, 1.7-µ column, mobile phase comprised of buffer (5?mM K₂HPO₄ containing 0.1% triethylamine, pH 6.8), and acetonitrile (40:60 v/v), the flow rate of 0.5?mL?min^?1 and UV detection at 250?nm. The method was developed with a short run time of 1?min. Forced degradation studies revealed that the method was stability-indicating, suitable for both assay and in-vitro dissolution of a drug product. The method was found to be linear in the range of 28–84?µg?mL^?1, 2.8–22.7?µg?mL^?1 with a correlation coefficient of 0.9999 and 1.000 for assay and dissolution, respectively. The recovery values were found in the range of 100.1–101.7%. The method was validated according to ICH guidelines.     

Development and validation of a stability-indicating RP-HPLC method for determination of atomoxetine hydrochloride in tablets

This paper describes the development of a stability-indicating RP-HPLC method for the determination of atomoxetine hydrochloride (ATX) in the presence of its degradation products generated from forced decomposition studies. The drug substance was subjected to stress conditions of acid, base, oxidation, wet heat, dry heat, and photodegradation. In stability tests, the drug was susceptible to acid, base, oxidation, and dry and wet heat degradation. It was found to be stable under the photolytic conditions tested. The drug was successfully separated from the degradation products formed under stress conditions on a Phenomenex C18 column (250 x 4.6 mm id, 5 microm particle size) by using acetonitrile-methanol-0.032 M ammonium acetate (55 + 05 + 40, v/v/v) as the mobile phase at 1.0 mL/min and 40 degrees C. Photodiode array detection at 275 nm was used for quantitation after RP-HPLC over the concentration range of 0.5-5 microg/mL with a mean recovery of 100.8 +/- 0.4% for ATX. Statistical analysis demonstrated that the method is repeatable, specific, and accurate for the estimation of ATX. Because the method effectively separates the drug from its degradation products, it can be used as a stability-indicating method.     

High-performance liquid chromatographic assay of betamethasone 17-valerate and its degradation products

A high-performance liquid chromatographic assay of betamethasone 17-valerate is described. The procedure may be use for quantitative assay of the degradation products, betamethasone 21-valerate and betamethasone, and the application to the analysis of ointments is described. The method is also suitable for the determination of the kinetics of decomposition from one experimental run, and the determination of rate constants from a four-compartment sequential reaction is described. The procedure is also applicable to other corticosteroids, and hydrocortisone 17-butyrate, hydrocortisone 21-butyrate, and hydrocortisone may similarly be determined without modification to the method.     

Determination of methylparaben,propylparaben, clotrimazole and its degradation products in topical cream by RP-HPLC

Summary Reversed-phase, high-performance liquid chromatographic (RP-HPLC) methods with UV detection were developed and validated for determination of compounds in a topical cream. The first method describes determination of the active component clotrimazole and two preservatives present in the cream; methylparaben and propylparaben. The second method describes determination of two degradation products of clotrimazole, imidazole and (2-chlorophenyl) diphenylmethanol, in a topical cream after long-term stability tests. Chromatographic separation was on a Purospher RP-18e column; the mobile phase in Method1 for separation of clotrimazole, methylparaben and propylparaben comprises acetonitrile and water (70:30 v/v). For determination of degradations products-imidazole and (2-chlorophenyl) diphenylmethanol—the optimum composition of mobile phase in Method2 was acetonitrile and water (75:25 v/v) apparent pH^* 2.7. Analysis time was <10 min for both methods. The methods were found to be applicable for routine analysis of the active compound clotrimazole, preservatives and degradation products in the pharmaceutical product: topical cream 1% Clotrimazol Cream. Presented at Balaton Symposium '01 on High-Performance Separation Methods, Siófok, Hungary, September 2–4, 2001     

The ICH guidance in practice: stress degradation studies on stavudine and development of a validated specific stability-indicating HPTLC assay method

A sensitive, selective, precise, and stability-indicating high-performance thin-layer chromatographic (HPTLC) method for the analysis of stavudine both as a bulk drug and in formulations is developed and validated. The solvent system consisted of toluene-methanol-chloroform-acetone (7.0:3.0:1.0:1.0, v/v/v/v). Densitometric analysis of stavudine is carried out in the absorbance mode at 270 nm. This system is found to give compact spots for stavudine (retention factor value of 0.45 +/- 0.05) following development of chromatoplates with the mobile phase. Stavudine is subjected to acid and alkali hydrolysis, oxidation, dry-heat and wet-heat treatment, and photo and UV degradation. The drug undergoes degradation under acidic and basic conditions, oxidation, and wet-heat degradation. Linearity is found to be in the range of 30-1000 ng/spot with a significantly high value of correlation coefficient. The linear regression analysis data for the calibration plots show a good linear relationship with r2 = 0.9997 +/- 0.05 in the working concentration range of 300 to 1000 ng/spot. The mean value of slope and intercept are 0.10 +/- 0.06 and 22.12 +/- 1.08, respectively. The method is validated for precision, robustness, and recovery. The limits of detection and quantitation are 10 and 30 ng/spot, respectively. The proposed HPTLC method is utilized to investigate the kinetics of the acid degradation process. Arrhenius plot is constructed and activation energy is calculated.     

Validated stability indicating LC method for carprofen: characterization of degradation products by MS

A simple, sensitive, and selective stability indicating high performance liquid chromatographic method has been developed and validated for quantitative analysis of carprofen (CPF) in presence of its degradation products. All degradation products in acid hydrolysis and photolysis were separated, identified by mass spectroscopic method and probable structures were elucidated. The forced degradation studies were performed on a bulk sample of CPF by using various methods like 0.1 M hydrochloric acid, 0.1 M sodium hydroxide, 0.33% hydrogen peroxide (H(2)O), heating at 60°C and exposure to UV light at 254 nm. A 5 μm particle octa desyl silane (ODS) column (150 mm × 4.6 mm) was used with acetonitrile-ammonium acetate (100 mM, pH-6.7) 40:60 (v/v) as a mobile phase at flow rate of 1.2 mL/min. Column oven temperature was maintained at 30°C and quantitation was achieved at 239 nm on the basis of peak area. The linear range and correlation coefficient (r(2)) was found 0.5-60 μg/mL and 0.9999 respectively. The limit of detection (LOD) and limit of quantitation (LOQ) were obtained 0.066 μg/mL and 0.20 μg/mL respectively . The proposed method was found to be suitable and accurate for quantitative analysis, stability study and characterisation of degradation product of CPF.     

Development and validation of a stability-indicating reversed-phase high performance liquid chromatography method for assay of betamethylepoxide and estimation of its related compounds

Betamethylepoxide (16beta-methyl-Delta(1,4)-pregnadiene-9beta-11beta-oxide-17alpha,21-diol-3,20-dione) is a key intermediate for the synthesis of various active pharmaceutical ingredients (APIs) of steroid compounds. A stability-indicating reversed-phase HPLC method for assay of betamethylepoxide and estimation of its related compounds has been developed and validated. This method can accurately quantitate betamethylepoxide in the presence of numerous structurally related compounds (including the alpha-epimer, known as alphamethylepoxide). This method can also adequately separate most of the impurities from each other and estimate their quantities in betamethylepoxide samples. The stability-indicating capability of this method has been demonstrated by adequate separation of the degradation products from betamethylepoxide in stress degraded and aged stability samples. The HPLC column used in the method was a 5 cm YMC Hydrosphere C(18) column (4.6 mm I.D.) and the mobile phase consisted of (A) water and (B) acetonitrile:methanol (8:25, v/v).     

Development and validation of a stability-indicating LC method for the assay of lodenafil carbonate in tablets

A stability-indicating liquid chromatographic method has been developed for the quantitative determination of lodenafil carbonate in tablets. The method employs a Synergi Fusion C18 column (250 × 4.6 mm, i.d., 4 μm particle size), with mobile phase consisting of a mixture of methanol-acetic acid 0.1% pH 4.0 (65:35, v/v) and UV detection at 290 nm, using a photodiode array detector. A linear response (r = 0.9999) was observed in the range of 10-80 μg/mL. The method showed good recoveries (average 100.3%) and also intra and inter-day precision (RSD < 2.0%). Validation parameters as specificity and robustness were also determined. Specificity analysis showed that no impurities or degradation products were co-eluting with the lodenafil carbonate peak. The method was found to be stability-indicating and due to its simplicity and accuracy can be applied for routine quality control analysis of lodenafil carbonate in tablets.