Mobile phase-A Research Articles

Development and Validation of RP-UPLC Method for the Determination of Process and Degradant Impurities Present in Dabigatran Etexilate Mesylate Capsules Using High Strength Silica-T3 Sorbent Column

A Reverse phase-Ultra Performance liquid chromatography (RP-UPLC) method was developed and validated for the quantification of process and degradant impurities present in Dabigatran etexilate mesylate capsules. Degradation data in each condition shows that peaks are pure and method is found to be stability indicating in nature. Impurities are well separated at satisfactory level in Acquity High strength silica (HSS)-T3, 100 mm length x 2.1 mm ID, Octadecyl column with 1.8μ particle size. Mobile phase-A consists of 0.1% Orthophosphoric acid in water adjusted the pH to 3.5 with triethyl amine. Acetonitrile used as Mobile phase-B. Gradient elution mode has been chosen to elute all impurities of Dabigatran etexilate which are having different polarities. Finalized flow rate was 0.18 mL.min‒1 with a quantification wavelength of 290 nm. The developed method was successfully validated for specificity, linearity, precision, accuracy, determination of LOD, LOQ and robustness as per International conference on harmonization (ICH) general guidelines requirement. The validated method is useful for quantitative determination of impurities of Dabigatran etexilate in Dabigatran etexilate capsules dosage form, which enables to identify the therapeutic efficacy at reduced run time.

LC–MS/MS and NMR characterization of forced degradation products of mirabegron

ABSTRACTA rapid, precise, and reliable liquid chromatography tandem mass spectrometry (LC–MS/MS) method has been developed for the characterization of stressed degradation products of mirabegron. It is used in the treatment of overactive bladder and administered to treat urinary symptoms such as urgency or frequency and incontinence. It also works by relaxing the muscles around bladder.Mirabegron was subjected to hydrolysis (acidic, alkaline, and neutral) and peroxidation, as per ICH-specified conditions. The drug showed degradation under stress conditions. However, it was stable to neutral conditions. A total of seven degradation products were observed and the chromatographic separation of the drug and its degradation products was achieved on X-TerraRP-8 (250 mm × 4.6 mm, i.d., 5 µm) column using 0.01 M ammonium acetate as mobile phase-A and 60:40 ratio of acetonitrile (ACN):water as mobile phase-B. The degradation products were characterized by LC–MS/MS and its fragmentation pathways were proposed. Probable possible structures were drawn based on parent and daughter molecular ions. One peroxide degradant impurity was isolated using preparative LC and characterized using liquid chromatography–mass spectrometry and NMR data.

Development and Validation of Liquid Chromatography Method for the Determination and Quantification of Impurities in Imiquimod

New stability indicating reverse phase high performance liquid chromatography (HPLC) method is developed for the determination of Imiquimod and its impurities. Separation is achieved on a C18 column (Inertsil-ODS3 4.6 × 250 mm, 5 µm) using gradient elution mode with mobile phase-A having disodium hydrogen phosphate buffer (10 mM) with 0.1% v/v triethyl amine and pH adjusted to 6.0 with ortho-phosphoric acid while mobile phase-B consist of an equal mixture of methanol and acetonitrile. The flow rate was optimized to 1.2 mL/min and column oven temperature 30°C. Detection was carried out at wavelength 226 nm. This developed method is then validated as per International Council for Harmonisation (ICH) guideline and found out to be linear, accurate, specific, selective, precise, and robust. The drug is also subjected to forced degradation using stress conditions of acid-base hydrolysis, oxidation, photolysis and thermal degradation. Considerable degradation was found out only in harsh condition of oxidative degradation where degradation impurity is also predicted. All degradation products were well separated. Test solution was found to be stable for 24 hrs. The method can be successfully applied for the determination of Imiquimod and its impurities in routine and stability samples.

LC–MS/MS Characterization of Forced Degradation Products of Tetrabenazine

A rapid, precise and reliable LC-MS/MS method has been developed for the identification and characterization of stressed degradation products of Tetrabenazine. Tetrabenazine is a drug, for the symptomatic treatment of hyperkinetic movement disorder. Tetrabenazine works mainly as a VMAT inhibitor. It promotes the early metabolic degradation of monoamines, in particular the neurotransmitter dopamine. Tetrabenazine was subjected to hydrolysis acidic, alkaline, neutral peroxidation, light and thermal stress conditions as per ICH-specified conditions. The drug showed degradation under peroxidation, thermal, acid and base hydrolysis stress conditions. However, it was stable to neutral stress conditions and light degradation. A total of 4 degradation products were observed and the chromatographic separation of the drug and its degradation products were achieved on Inertsil ODS-3V 150 mm × 4.6 mm, i.d., 5 μm column using 0.01 M ammonium acetate and ACN in the ratio of 640:360 as mobile phase-A and 900:100 ratio of ACN:Water as mobile phase –B. The degradation products were characterized by LC–MS/MS and its fragmentation pathways were proposed. Probable possible structures were drawn based on parent and daughter molecular ions.

Gradient RP-HPLC method for the determination of potential impurities in dabigatran etexilate in bulk drug and capsule formulations

A selective RP-HPLC method for separation and determination of potential related impurities (starting materials and by-products of synthesis, and degradants) of dabigatran etexilate (DAB) drug substance has been developed and validated. The separation was accomplished on a Inertsil ODS 3V (250mm×4.6mm, 5μm) column connected to a photodiode array (PDA) detector using 20mM ammonium formate with 0.1% of triethylamine (pH: 5.0 adjusted with formic acid) as mobile phase-A and acetonitrile as mobile phase-B, under gradient elution. Two unknown impurities found in dabigatran etexilate industrial batch stability condition at levels more than 0.1% in HPLC analysis were characterized preliminarily by ESI-MS/MS studies. The major unknown (unknown-1) was enriched and isolated by preparative LC and structure was evidenced by 1H and 13CNMR spectroscopy, mass spectrometry and FT-IR. Another unknown (unknown-2) found as secondary degradant and structure was proposed by ESI-MS/MS study. This method can be used for the quality control of both drug substance and drug product. The performance of the method was validated according to the ICH guide lines for specificity, limit of detection, limit of quantification, linearity, accuracy, precision, ruggedness and robustness.

Development and validation of a novel stability-indicating HPLC method for the quantitative determination of eleven related substances in ezetimibe drug substance and drug product

Ezetimibe is a novel lipid-lowering agent that inhibits intestinal absorption of dietary and biliary cholesterol. In the present work, a simple, sensitive and reproducible gradient reverse phase high performance liquid chromatographic (RP-HPLC) method for separation and determination of the related substances of ezetimibe was developed and validated. Eleven potential process-related impurities (starting materials, (3S,4S,3'S)-isomer, degradants and byproducts) were identified in the crude samples. Tentative structures for all the impurities were assigned primarily based on comparison of their retention time and mass spectrometric data with that of available standards and references. This method can be applied to routine analysis in quality control of both bulk drugs and commercial tablets. Separation of all these compounds was performed on a Phenomenex Luna Phenyl-Hexyl (100mm×4.6mm, 5μm) analytical column. The mobile phase-A consists of acetonitrile–water (pH adjusted to 4.0 with phosphoric acid)–methanol at 15:75:10 (v/v/v), and mobile phase-B contains acetonitrile. The eluted compounds were monitored at 210nm. Ezetimibe was subjected to hydrolytic, acid, base, oxidative, photolytic and thermal stress conditions as per ICH serves to generate degradation products that can be used as a worst case to assess the analytical method performance. The drug showed extensive degradation in thermal, acid, oxidative, base and hydrolytic stress conditions, while it was stable to photolytic degradation conditions. The main degradation product formed under thermal, acid, oxidative, base and hydrolytic stress conditions corresponding to (2R,3R,6S)-N, 6-bis(4-fluorophenyl)-2-(4-hydroxyphenyl)-oxane-3-carboxamide (Ezetimibe tetrahydropyran impurity) was characterized by LC-MS/MS analysis. The degradation products were well resolved from the main peak and its impurities, thus proved the stability-indicating power of the method. The developed method was validated as per international conference on harmonization (ICH) guidelines with respect to specificity, linearity, limit of detection (LOD), limit of quantification (LOQ), accuracy, precision and robustness.

Development and Validation of Stability Indicating RP-HPLC Method on Core Shell Column for Determination of Degradation and Process Related Impurities of Apixaban—An Anticoagulant Drug

A rapid, specific, sensitive, and precise reverse-phase HPLC method for the quantitative determination of process related and degradation impurities of Apixaban, an anticoagulant drug is described. The developed RP-HPLC method was successfully applied to the analysis of both Apixaban drug substance and drug product. The chromatographic separation was achieved on a Sigma-Aldrich’s Ascentis Express® C18 (4.6 mm × 100 mm, 2.7 μ) HPLC column with a runtime of 40 min. Mobile phase-A and mobile phase-B were phosphate buffer and acetonitrile respectively. The column oven temperature was set at 35°C and photodiode array detector was set at 225 nm. Nine process related impurities (Imp-1 to Imp-9) have been detected in test sample of Apixaban by using newly developed RP-HPLC method. Forced degradation study was carried out under acidic, alkaline, oxidative, photolytic and thermal conditions to demonstrate the stability-indicating nature of the developed RP-HPLC method. The developed method was validated as per ICH guideline and found to be specific, precise, sensitive and robust.

Development and Validation of Stability-Indicating RP-UPLC Method for Simultaneous Determination of Related Substances of S(−)Amlodipine and S(−)Metoprolol Succinate in Fixed Dose Combination Tablet Dosage Form

A novel, rapid, accurate, sensitive, precise, and stability-indicating reverse-phase ultra-performance liquid chromatographic (RP-UPLC) method was developed and validated for determination of related substances of S(−)Amlodipine and S(−)Metoprolol Succinate in fixed dose combination tablet dosage form. The chromatographic separation was achieved with the use of Acquity UPLC HSS T3, 1.8 μm, 2.1 × 100 mm analytical column at 45°C employing a gradient elution. Mobile phase consisting of mobile phase-A (solution containing 5.0 gm of sodium dihydrogen phosphate monohydrate per liter of water and Acetonitrile in the ratio of 95 : 5) and mobile phase-B (Acetonitrile) was used at a flow rate of 0.5 mL min−1 with injection volume of 10 μL and the detection was done at 232 nm using UV detector. The retention times of S(−)Metoprolol Succinate and S(−)Amlodipine were found to be 2.8 minutes and 8.1 minutes, respectively. During method validation all the parameters were evaluated as per ICH guidelines, which remained well within acceptable limits. This method can be used for the estimation of related substances of S(−)Amlodipine and S(−)Metoprolol Succinate in fixed dose combination tablet dosage form.

Development of a kinetic reaction rate limited study on degradation of impurity profile for the estimation of acetaminophen in acidic aqueous solution for pharmaceutical dosage form by High Performances Liquid Chromatography (HPLC).

A reaction-limited model for impurity profile is developed for the acetaminophen which consists of degradation impurity as 4- Amino phenol including ten other potential impurities. As many ways of the synthetic routes for acetaminophen it has been identified the impurities are of eleven. Out of which many degrading impurities, 4-Amino phenol is one of the major degradant. And remaining Organic Impurities that may appear in acetaminophen preparations are process-related impurities. Impurity evolution process is considered from Reverse-phase liquid chromatography (RPLC) which is the method of choice for the analysis of basic compounds. The chromatographic behavior of acidic and basic substances is closely related to their structure and is easily influenced by many factors, Such as pH, molarity and Composition of the Mobile Phase. Determination of kinetic reaction for acetaminophen and its main degradation of impurity as 4- aminophenol was developed and method have been validated for the same through the High Performances Liquid Chromatography for all potential eleven impurities. Chromatographic separation has been obtained on an Intersil ODS 3V using gradient elution of having mobile phase A&B. In Mobile phase-A of 0.01M Di-potassium hydrogen phosphate adjusted pH 7.0 diluted with ortho-phosphoric acid and Mobile phase- B is the mixture of 800ml methanol and 200ml of 0.01M Di-potassium hydrogen phosphate adjusted pH 7.0 diluted with ortho-phosphoric acid. Analysis time did not exceed 75minutes and good resolution peak shapes and asymmetric have retained. The method has been successfully obtained for the study of kinetic degradation of acetaminophen with respect to its degradation impurity of the 4-aminophenoland other process related impurities. A kinetic investigation of acidic hydrolysis of acetaminophen was carried out in hydrochloride solutions of 0.3N, 0.5N and 1.0N by monitoring the parent compound itself with respect to its main degradant. The order of acetaminophen followed pseudo-first order kinetics. The activation energy could be extended from the Arrhenius plot and it was found to be 21.2Kcal/mol.

New Stability Indicating Method for Quantification of Impurities in Amlodipine and Valsartan Tablets by Validated HPLC

A stability indicating LC method was developed for simultaneous determination of amlodipine and valsartan in pharmaceutical dosage form. Efficient chromatographic separation was achieved on C8 stationary phase with simple combination of mobile phase-A (70 : 20 : 10 v/v/v of water : acetonitrile : methanol with 2 mL of Octylamine adjusted the pH to 2.50 + 0.05 with orthophosphoric acid) and mobile phase-B (Acetonitrile) delivered in gradient mode. Quantification was carried out using ultraviolet detection at 240 nm at flow rate of 1.0 mL/min with Injection Volume of 100 μL and ambient column temperature. This method was capable to detect both the drug components of Amlodipine and Valsartan in presence of their degradation products (Amlodipine Imp-A and Valsartan Impurity-B) with the detection level of 0.05%. Amlodipine/Valsartan and their combination drug product were exposed to thermal studies, photolytic, hydrolytic and oxidative stress conditions, and samples analysed. Peak homogeneity data of Amlodipine and Valsartan is obtained using PDA detector, demonstrating the specificity. The method shows excellent linearity over range of 0.05–2.0% for Amlodipine; Amlodipine Impurity-A and 0.05–1.0% for Valsartan and Valsartan Impurity-B. The correlation coefficient for Amlodipine and Valsartan are 0.9999. The proposed method was found to be suitable and accurate for quantitative determination and the stability study of Amlodipine and Valsartan in pharmaceutical preparations.

Development and Validation of a Specific Stability Indicating High Performance Liquid Chromatographic Methods for Related Compounds and Assay of Solifenacin Succinate

Gradient, reverse phase liquid chromatographic methods were developed separately for the related compounds and solifenacin succinate, an active pharmaceutical ingredient used for the treatment of overactive bladder. Gradient LC method was employed for related compounds. The mobile phase‐A contains a 0.01 M phosphate buffer pH: 3.5 ± 0.05 with orthophosphoric acid (88%) and mobile phase‐B contains a mixture of acetonitrile and water in the ratio of 90 : 10(v/v). The flow rate was 1.0 mL/minute, column temperature was kept at 35°C, and detection was monitored at 220 nm. In the developed HPLC method the resolution between solifenacin succinate and its closely eluting impurity, that is, solifenacin N‐oxide was found to be greater than 3.0. The drug was subjected to stress conditions such as hydrolysis, oxidation, photolysis, and thermal degradation. Considerable degradation was found to occur in only oxidative stress condition. Degradation product formed during oxidative stress condition was found to be impurity‐C and it can be identified by LC‐MS. The stress samples were assayed against a qualified reference standard and the mass balance was found close to 99.5%. The developed RP‐LC method was validated as per ICH guidelines. We also developed LC‐MS/MS method for determination and identification of these impurities in solifenacin succinate.

Quantitative Determination of Azithromycin in Human Plasma by Liquid Chromatography–Mass Spectrometry and its Application in Pharmackokinetic Study

Azithromycin is an effective and well-known antimicrobial agent. In the present study, a simple, sensitive and specific LC/MS/MS method has been developed and validated for the quantification of Azithromycin in human serum samples using Clarithromycin as internal standard. Azithromycin was extracted from biological matrix by using solid phase extraction process. The chromatographic separation was performed on Luna C18 (3 ?, 2x150 mm) column with a mobile phase consisting of 35 mM ammonium acetate buffer (mobile phase-A) and acetonitrile and methanol in ratio of 90:10 ( as mobile phase-B) at a flow rate of 0.25 mL/min. The method was validated over a linear concentration range of 0.5?50.0 ng/mL and limit of quantification (LOQ) was 0.5 ng/mL with a coefficient of correlation (r2) = 0.9998. The intra-day and inter-day precision expressed as relative standard deviation were 1.64% – 8.43% and 2.32% – 9.92%, respectively. The average recovery of azithromycin from serum was 98.11%. The method was successfully applied to a pharmacokinetic study after oral administration of Azithromycin 200 mg/5 ml suspension in healthy Bangladeshi volunteers. DOI: http://dx.doi.org/10.3329/dujps.v11i1.12488 Dhaka Univ. J. Pharm. Sci. 11(1): 55-63, 2012 (June)

Development and Validation of a Stability Indicating LC Method for the Assay and Related Substances Determination of a Proteasome Inhibitor Bortezomib

A novel, simple, sensitive, stability indicating HPLC method was developed and validated for quantification of impurities (process related and degradants) and assay determination of bortezomib. Stability indicating power of the method was established by forced degradation experiments and mass balance study. The chromatographic separation was achieved with Waters SymmetryShield RP18 column using gradient elution using the mobile phase-A consists of a mixture of water-acetonitrile-formic acid (715 : 285 : 1, v/v/v) and the mobile phase-B consists a mixture of methanol-water-formic acid (800 : 200 : 1, v/v/v), respectively. The developed method is validated for parameters like precision, accuracy, linearity, LOD, LOQ, and ruggedness. Central composite experimental design (CCD) was applied to check the robustness of the method. The stability tests were also performed on drug substances as per ICH norms.

SEPARATION, IDENTIFICATION AND QUANTITATION OF DEGRADANTS OF TOPOTECAN AND ITS RELATED IMPURITIES IN TOPOTECAN INJECTION BY RP-HPLC AND LC-MS

A simple, rapid, accurate, robust, sensitive, and stability-indicating reversed-phase high-performance liquid chromatographic (RP-HPLC) analytical method has been developed for the separations of related impurities and degradants of topotecan in topotecan injection. Forced degradation studies performed on topotecan injection formulation revealed that the compound (topotecan) is extremely susceptible to all hydrolytic, oxidation, light exposure, and thermal stress. The method was tested in the presence of commonly used formulation excipients such as mannitol and tartaric acid. Separation of topotecan from its degradation products and three related impurities (Imp-A, Imp-B, Imp-C) as process impurities was achieved on ACE® C18, 150 × 4.6 mm i.d, 3 µm, stainless steel analytical column using a gradient elution. The elution of impurities employed multi-step gradient programmed mobile phase consisting of 0.02 M ammonium acetate (pH, 4.2) as mobile phase-A and a mixture of methanol and isopropyl alcohol in the ratio of 750:250 (v/v), respectively, as mobile phase-B. The mobile phase flow rate was maintained at 0.8 mL min−1. The column eluent was monitored at 267 nm. The column oven temperature was 25°C. The same method was also extended to LC-MS with the same gradient conditions used to identify the degradation products observed under forced degradation studies. The method was validated according to FDA, USP (general chapter 1225), and ICH guidelines, and statistical analysis of the data proved to be suitable for stability testing at quality control. The linearity of the proposed method for all the aforementioned related impurities and topotecan was investigated in the range of LOQ to 4.9 µg mL−1, respectively. The method was shown to separate topotecan well from the degradants and commonly used excipients. The specificity of the method was established through peak purity testing using a photodiode array detector.

Determination of Alliin and Allicin in different types Garlic using High Performance Liquid Chromatography

Alliin and Allicin products were measured and determined by ion-pair reversed-phase liquid chromatography (RP-LC) with UV detection at 210 nm. These two Compounds were extracted from various types of garlic with methanol / ethyl acetate and chromatographed on octadecyl silane column [ODS C18 (250 x 4.6 mm id)] with gradient elution from 0.01M phosphate buffer (PH=2.5) with 5M heptanslfonic acid (mobile phaseA) to 0.01M phosphate buffer (PH=2.5) acetonitrile(1:1) (mobile phase B). Allicin was eluted after Alliin. The results observed show that the concentration differs between the different types of garlic. The aqueous Iraqi garlic extract has the highest concentration of Alliin and Allicin (17.9 ppm, 0.9%),(23.94 ppm, 1.2%) respectively. But the lowest concentration of allicin was found in French garlic extract (0.56 ppm, 0.03%) while the lowest level of Alliin was (4.3 ppm, 0.22%) in Chinese garlic extract.

DEGRADATION STUDIES OF HIGHLY POTENT AND LIFE THREATENING HUMAN BIRTH DEFECT DRUG—LENALIDOMIDE BY HPLC AND LC-MS

Due to the potential toxicity of lenalidomide, a highly potent and severe life threatening human birth defect, a lot of precaution should to be taken in handling such kind of drugs. Lenalidomide drug-excipient blend from the capsule pharmaceutical dosage form was subjected to different ICH prescribed stress conditions of thermal stress, pH hydrolysis, oxidation and photolysis. The drug was found to be stable only at photolysis and thermal stress, while it was extremely susceptible to other stressing conditions especially it showed extensive degradation under alkali conditions. For establishment of stability-indicating method, the rate of degradation in solutions in which different degradation products were formed were monitored at different time intervals, by using different concentrations of stressing reagents, and the separations was optimized by varying the LC conditions. An acceptable separation was achieved through a multi-step gradient elution using an ACE® C18, 150 × 4.6 mm i.d, 3 μm, stainless steel analytical column and a mobile phase comprising of 0.01 M phosphate buffer (pH, 2.0 ± 0.1) as mobile phase-A, and a mixture of water and acetonitrile in the ratio of 200:800 (v/v) as mobile phase-B, with a flow rate and detection wavelength being 1.0 mL min−1 and 220 nm respectively. The major degradation products appeared at relative retention times (RRT) of 0.75, 0.86, 0.96, 1.33, 1.52, 1.99, 2.04, 2.62 and 2.66 respectively.

LC and LC–MS/MS study of forced decomposition behavior of anastrozole and establishment of validated stability-indicating analytical method for impurities estimation in low dose anastrozole tablets

Anastrozole tablets were subjected to different ICH prescribed stress conditions of thermal, hydrolysis, humidity, photolysis and oxidation stress. The drug was found to be stable for all the stressed conditions except for oxidation. Separation of anastrozole from its potential impurities, degradation products and five anastrozole related compounds as main impurities were achieved on Inertsil ODS-3V, 250 mm × 4.6 mm i.d, 5 μm analytical column using reversed phase high performance liquid chromatography (RP-HPLC). The elution of impurities employed time dependent gradient programmed mobile phase consisting of water as mobile phase-A and acetonitrile as mobile phase-B at column flow rates of 1 ml/min and at 215 nm UV detection. The same method was also extended to LC–MS/MS studies which were carried out to identify the degradation product. The method developed was established to have sufficient intermediate precision as similar separation was achieved on another instrument handled by a different operator. The LOQ for anastrozole related compound-A (RC-A), related compound-B (RC-B), related compound-C (RC-C), related compound-D (RC-D), related compound-E (RC-E) and anastrozole were 0.05, 0.03, 0.03, 0.06, 0.06 and 0.06 μg ml −1 respectively. The linearity of the proposed method for all the above related compounds was investigated in the range of LOQ to 0.600 μg ml −1 respectively. The specificity was established through peak purity testing using a photo-diode array detector. Method was validated according to ICH guidelines and statistical analysis of the data proved to be suitable for stability testing at quality control.