Liquid Chromatographic Assay Method Research Articles

A practical approach to estimate analytical method variability from routine testing

The performance of analytical test methods is critical to ensure decisions that affect efficacy and quality of pharmaceutical products are based on accurate and reliable results. As described in USP <1220> and advocated for in ICH Q14, continued verification of critical method attributes linked to bias and precision is essential to ensure method performance throughout the lifecycle of an analytical test method. As continued verification programs for analytical methods within the pharmaceutical industry mature, additional monitoring tools are required to deliver robust and cost-effective verification programs. Herein, a novel methodology is presented to evaluated analytical method variability directly from results generated during routine method execution. The implementation of the methodology is demonstrated for a small molecule liquid chromatographic assay method utilizing a single-point external reference calibration. Approaches to reduce the required data to be collected and broaden the applicability of the methodology to a wide range of analytical methods is described. Finally, the application of the methodology to method development activities is discussed to aid in the identification of variability sources and effectively select replication strategies, thus allowing a holistic understanding of method variability throughout the entirety of the method lifecycle.

A Practical Approach to Estimate Analytical Method Variability from Routine Testing

The performance of analytical test methods is critical to ensure decisions that affect efficacy and quality of pharmaceutical products are based on accurate and reliable results. As described in USP <1220> and advocated for in ICH Q14, continued verification of critical method attributes linked to bias and precision is essential to ensure method performance throughout the lifecycle of an analytical test method. As continued verification programs for analytical methods within the pharmaceutical industry mature, additional monitoring tools are required to deliver robust and cost-effective verification programs. Herein, a novel methodology is presented to evaluated analytical method variability directly from results generated during routine method execution. The implementation of the methodology is demonstrated for a small molecule liquid chromatographic assay method utilizing a single-point external reference calibration. Approaches to reduce the required data to be collected and broaden the applicability of the methodology to a wide range of analytical methods is described. Finally, the application of the methodology to method development activities is discussed to aid in the identification of variability sources and effectively select replication strategies, thus allowing a holistic understanding of method variability throughout the entirety of the method lifecycle.

Development and validation of stability indicating ultra‐high‐performance liquid chromatographic method for assay and impurities of mexiletine hydrochloride and mexiletine hydrochloride capsules

AbstractMexiletine (MEX) is an antiarrhythmic drug used to treat acute and chronic ventricular arrhythmias. A simple isocratic, rapid, specific, stability‐indicating, and sensitive reverse‐phase ultra‐high‐performance liquid chromatography (UHPLC) method was developed and successfully validated to quantify the assay and impurities of MEX hydrochloride. The new UHPLC method showed the optimum separation of MEX and its seven impurities in 20 min by using sodium acetate buffer (0.14 mM) with 0.3% of glacial acetic acid (pH 4.8) and methanol in the ratio of 40:60 (v/v) as mobile phase. The method used an Acquity HSS T3 (100 × 3.0 mm, 1.8 µm) chromatographic column at a 0.2 mL/min flow rate. The injection volume is 2 µL, and 254 nm is the detection wavelength. Note that, 25°C is the column oven temperature. The method is linear over the concentration range of the quantification limit (0.03%) to 150% (0.3%) of the specification level (0.2%) for the impurities and 50%–150% for the assay method. The quality control lab of bulk drugs and finished dosage form successfully applied the new UHPLC method for quantification of assay and impurities of MEX hydrochloride.

Development and validation of RP-HPLC method for estimation of anti- hypothyroidism drug in capsule dosage form

A simple, accurate, sensitive and precise Reverse Phase-High Performance Liquid Chromatographic assay method for estimation of Levothyroxine sodium in dosage form was successfully developed. The chromatographic separation was performed on Welch Ultisil XB-CN, 150 x 4.6 mm, 5µm. column. The mobile phase consists of 0.05% OPA Buffer solution, and Acetonitrile in the ratio of 72:28, v/v. Mobile phase was delivered at a flow rate of 1.0 ml/min. Samples were injected 50 μL the column temperature was kept at 40 0C and sample temperature 150C. The wavelength 221 nm were selected for the evaluation of the chromatogram. The retention time of the drug was found to be 3.7 min. The developed method was found to be linear in a concentration range of 5-15 μg/ml of the drug (r2= 0.999). The low value of % RSD indicates reproducibility of the method. Thus this method can be used for routine analysis of pomalidomide in formulation and to check the stability of bulk samples.

Development and Validation of Stability Indicating RP-HPLC Method for Quantitative Estimation of Enzalutamide in Enzalutamide Capsules Dosage Form

Objectives: A precise, accurate and selective stability-indicating reverse phase high performance liquid chromatographic assay method has been developed for the quantitative estimation of Enzalutamide in Enzalutamide capsules dosage form. Materials and Methods: The separation was achieved by using a stationary phase Waters X-Bridge Shield RP18 (150 x 4.6 mm, 3.5μ) and the mobile phase consisted of perchloric acid buffer and acetonitrile in the proportion of (20:80 volume/volume). The run velocity was 1.2 mL/min. Enzalutamide was identified using UV detector at the wavelength of 210 nm. Column oven temperature 25°C and sample cooler temperature 25°C and infused quantity 20 μL, run time was 15 min. Results: As there is no meddling flanked by blank and placebo at the retention time of Enzalutamide. Degradation study results were shown significant degradation was observed in acid and oxidation (peroxide) stress condition. Hence it can be concluded that Enzalutamide is sensitive to acid and oxidation. To obtain system precision, a study was conducted with six replicate injections. %RSD was estimated from the peak areas of Enzalutamide establish to be 0.55% correspondingly. The relative standard deviation for method exactitude was establish to be 0.55%. The suggested HPLC technique was linear over the range of 100.6-301.8 μg/mL, the correlation coefficient was establish to be 0.9999. The accuracy studies were shown as % recovery for Enzalutamide 50% to150% level. The limit of % recovered revealed is in the assortment of 98 and 102% and the consequences obtained were establish to be within the limits. Hence the technique was establish to be accurate. The solution steadiness of the standard and samples are stable upto 48 hr on a bench top and refrigerator (2-8°C). The method is robust for changes like flow rate and column oven temperature. Performed the filter validation for sample solution 0.45 μm PVDF and 0.45 μm Nylon filterers are suitable for filtration. The method has validated as per ICH guidelines and all the validation parameters are satisfy the ICH Q2 specification acceptance limits. Conclusion: The developed method was validated for an assortment of parameters as per ICH guidelines like accuracy, precision, linearity, specificity, system suitability, solution stability and robustness. The consequences obtained were within the acceptance criteria. So, it can be concluded that the urbanized technique is simple, precise, cost-effective, eco-friendly, and safe and can be successfully employed for the routine analysis of Enzalutamide in bulk and pharmaceutical dosage forms.

Development and Validation of New RP-HPLC Method for Estimation of Pramipexole Dihydrochloride in Bulk and Pharmaceutical Formulation

A novel high-performance liquid chromatographic assay method was developed and validated for the quantitative determination of the anti-Parkinson agent pramipexole dihydrochloride monohydrate in bulk and its tablet dosage form. In this perspective, the chromatographic separation was accomplished on Eclipse XDB-12 C18 (150 mm x 4.6 mm, 5 μm particle size) column using UV detection at 263 nm. The mobile phase consisted of distilled water: acetonitrile (10: 90 v/v), run at a flow rate of 1.0 mL/min with isocratic elution. The method was validated in accordance with ICH guidelines by evaluating the system suitability, linearity, limits of detection (LOD) and quantitation (LOQ), precision, accuracy, specificity, selectivity and short-term stability. Our findings revealed that retention time for pramipexole dihydrochloride was found to be 5.2 minutes. The linearity range was established between 6.25-225.0 μg/mL with a mean recovery of 101.26 % ± 0.56. The limits of detection and quantification were determined to be 4.18 μg/mL and 12.66 μg/mL, respectively, indicating that the method is very sensitive. Intra and inter-day precision were within acceptable limits (RSD

Development and Validation of Fast and Robust Stability Indicating RP-HPLC Method for Simultaneous Estimation of Tolperisone Hydrochloride and Etoricoxib in Pharmaceutical Dosage Form

Objectives: A precise, accurate and selective stability-indicating reverse phase high performance liquid chromatographic assay method has been developed for the simultaneous quantitative determination of Tolperisone hydrochloride and Etoricoxib in tablets. Methods: The chromatographic separation of drugs was attained by using Eclipse plus C18 (150 mm × 4.6 mm, Agilent 5µm) column at room temperature. The composition of mobile phase was mixture of 0.035M triethylamine (pH 3.0 adjusted with orthophosphoric acid) and acetonitrile in ratio of 70:30 v/v and flow rate of mobile phase 1.0 ml/min with isocratic elution. The signal of eluents was observed at 290 nm by using diode array detector. Results: The retention time of Tolperisone hydrochloride and Etoricoxib were found to be 2.826 min and 7.566 min, respectively. The linearity ranges for both drugs were found to be 5-15 μg/ml and the percent recoveries were found to be 99.39% and 99.15% for Tolperisone hydrochloride and Etoricoxib respectively. Various forced degradation conditions, such as alkali hydrolysis, acid hydrolysis, thermal degradation, photolytic degradation, and oxidative degradation, were applied to the drug ingredients and drug formulation. The degradants were efficiently separated from the drugs by using optimized chromatographic conditions. The developed method was validated as per recommendation parameters of International council on harmonization guidelines Q2(R1). Conclusion: The validation parameters indicate that the drug substances were efficiently separated from its degradants and developed method can be routinely applied for the simultaneous quantitative determination of Tolperisone hydrochloride and Etoricoxib in combined tablet formulation in the quality control laboratory.

Development and Validation of a Stability-Indicating Reversed-Phase Ultra High Pressure Liquid Chromatography Method for Assay of Delmopinol Hydrochloride and Estimation of Its Related Substances in Commercial Bulk Batches.

Delmopinol hydrochloride is widely used as an active ingredient (AI) in many human oral hygiene products. To the best of our knowledge (via literature search), there is no stability-indicating liquid chromatography method available in the public domain for assay and estimation of related substances of Delmopinol hydrochloride. A fast stability-indicating Reversed-Phase Ultra High-Performance Liquid Chromatography (RP-UHPLC) method has been developed and validated for identification, assay and estimation of related substances in commercial bulk batches of this AI. The major peak of the AI and its related compounds are adequately separated in 6min by a gradient elution on a hybrid silica-based C18 column (Waters Acquity UPLC® BEH C18, 50mm × 2.1mm I.D., 1.7μm particle size) maintained at 50°C. Mobile phase-A is composed of aqueous 10mM NH4OH and mobile phase-B is ACN. The AI and its related compounds are detected with UV detection at 220nm and quantitated by an external Delmopinol hydrochloride reference standard. The quantitation limit of the method is 0.1% of the target analytical concentration. This UHPLC method is fast and green and has been demonstrated to be specific, accurate, linear, precise, sensitive and robust as per ICH guidelines.

A NOVEL VALIDATED HIGH-PERFORMANCE LIQUID CHROMATOGRAPHIC STABILITY-INDICATING ASSAY METHOD IN REVERSE PHASE FOR ESTIMATION OF HYDROXYPROGESTERONE CAPROATE IN AN INJECTABLE FORMULATION.

A high-performance liquid chromatographic stability-indicating assay method in reverse-phase has been developed and validated for the quantitative measurement of Hydroxyprogesterone corporate in injectable formulation prepared in castor oil base containing excipient benzyl benzoate as preservative. The reverse-phase method was developed by using Sunfire C18 column having dimensions (150 x 4.6 mm x 5μm) with a mobile phase[A] comprised of water, acetonitrile, and Methanol mixed in 40:30:30 v/v ratio and mobile phase [B] comprised of water, acetonitrile, and Methanol mixed in 20:40:40 v/v ratio pumped gradient program at a flow rate of 1.0 ml/min where the thermostat of the column oven controlled at 30°C and sample compartment at 5°C. The injection volume is set at 20μL. Hydroxyprogesterone corporate has a retention time of 10 minutes with UV detection at 240 nm. The detector was showing linear response in a range of 0.25 μg/ml to 150 μg/ml for Hydroxyprogesterone corporate. The guidelines of the International Conference on Harmonization on guidance for industry (2005) validation of analytical procedures, text, and methodology Q2(R1) were followed for the validation of this novel method. For quantification of Hydroxyprogesterone corporate the developed method was found to be genuinely exact precise, accurate, linear, robust, rugged, stable. and cost-effective.

Development and Validation of a Stability-Indicating Reversed-Phase High-Performance Liquid Chromatography Method for Assay of Doramectin and Estimation of its Related Substances in Commercial Batches of Doramectin Drug Substance.

Doramectin is a broad-spectrum antiparasitic drug used in veterinary medicine. It belongs to the family of avermectins, which possess a macrocyclic lactone structure, and is widely used for the treatment of parasites. This study aimed to develop a stability-indicating reversed-phase (RP) HPLC method for the assay and identification of doramectin including identification and estimation of its related substances in commercial batches of doramectin drug substance. Methanol was used to dissolve and prepare doramectin samples. Doramectin and its related substances were separated on a HALO C8 (100 mm × 4.6 mm i.d., 2.7 µm particle size) column maintained at 40 °C using an isocratic HPLC method with a mobile phase composed of acetonitrile-water (70 + 30, v/v). Analytes were detected with UV detection at 245 nm and quantitated against a single point external reference standard of doramectin. The LOQ of the method is 0.1% of the target concentration as described in the method. The HPLC method can separate all analytes of interest by an isocratic elution within 10 min. The method was validated according to the guidelines described in the International Conference on Harmonization Q2(R1). The HPLC method for assay of doramectin and estimation of its related substances was successfully developed, validated, and demonstrated to be accurate, robust, specific, and stability-indicating. This is the first known paper to report an HPLC method for assay of doramectin and estimation of its related substances in commercial batches of doramectin drug substance.

Development and Validation of Q-Absorbance Ratio by UV-Spectrophotometric Method for Simultaneous Estimation of Metformin and Empagliflozin in Bulk and Combined Dosage Form

Advantages of simultaneous stability studies are the identification of new degradation products, to understand mutual induction and/or inhibition of rates of degradation and to analyze the degradation products of both drugs. Various ultraviolet spectroscopic and high performance liquid chromatographic assay methods were reported for the estimation of metformin, sitagliptin, pioglitazone, glimepiride and simvastatin individually and in combination with other drugs. All the above reported methods were based on the estimation of metformin, sitagliptin, pioglitazone, glimepiride and simvastatin alone or in combination with other drugs. The degradation products were generated and successfully separated by the developed and validated high performance liquid chromatographic methods for the estimation of the selected anti-diabetic drug combinations. The aim of the study was to develop and validate of Q-Absorbance Ratio UV-Spectrophotometric Method for Simultaneous Estimation of Metformin and Empagliflozin in Bulk and Combined Dosage Form.&#x0D; Keywords: Metformin, Method Development, Validation, Empagliflozin, UV-Spectrophotometer.

Approved stability indicating reversed-phase-high performance liquid chromatographic assay method for Palonosetron HCl in formulations

Approved stability indicating reversed-phase-high performance liquid chromatographic assay method for Palonosetron HCl in formulations

Application of prediction intervals to the interpretation of analytical methods robustness studies

Since the emergence of the Analytical Quality by Design concept and the announcement of the future ICH Q14 “Analytical Procedure Development” guideline [1], a significant revival of the interest of the laboratories for the robustness study of their methods can be seen.Design of Experiments is a well-established tool used for this task, because of its ability to study the individual effect of a great number of factors in a minimal number of experiments.However, when assessing the robustness of an analytical method the analysis of the individual effect of each factor is not sufficient on its own. Indeed, the role of this study is also to verify, regardless of the very significance of the factors, that the chosen variations of the operating parameters of the method do not modify the experimental variance measured under the operating conditions for which the parameters are strictly defined.We therefore propose, in addition to the analysis of the individuals effects of the factors, an original tool which makes it possible to verify that the studied factors, when they vary together, do not lead to a significant variation in the measured responses. This tool is based on the prediction interval of the matrix experimental results, for each of the studied responses.This concept of prediction interval is already used in the context of analytical methods accuracy study. In a robustness design of experiments, it can represent the interval in which should be found, with a given probability, the response value if complementary experiments are conducted. It therefore represents the variation induced simultaneously by the different factors and can be easily compared to a maximum acceptable deviation of the considered response.We will detail this proposal and illustrate it with an example from a robustness study carried out on a liquid chromatography assay method.

Stability-indicating high-performance liquid chromatographic assay methods for drugs in pharmaceutical dosage forms: Part I

A review of applications of stability-indicating high-performance liquid chromatographic (HPLC) assay methods for drugs in various pharmaceutical dosage forms is presented. A survey of articles primarily on the significant developments in this field is covered in this paper. Selected substances are grouped to integrate drugs that share similar uses or actions. Papers selected by the authors are briefly summarized, and HPLC systems developed in these papers for stability testing of pharmaceuticals are tabulated to assist readers in selecting an appropriate system for a given pharmaceutical. The information given in the table includes the drug names, materials, column types, mobile-phase compositions, detections, internal standards used and the literature references.

Stability-indicating High-Performance Liquid Chromatographic assay methods for drugs in pharmaceutical dosage forms: Part II

Stability-indicating High-Performance Liquid Chromatographic assay methods for drugs in pharmaceutical dosage forms: Part II

Separation and Simultaneous Quantitation of Montelukast Sodium and Ebastine in Tablets by using Stability-Indicating Liquid Chromatographic Method

Objectives: A precise, accurate and selective stability-indicating reverse&nbsp;phase high performance liquid chromatographic assay method has been&nbsp;developed for the simultaneous quantitative determination of Montelukast&nbsp;sodium and Ebastine in tablets. Methods: The chromatographic separation&nbsp;of drugs was attained by using Hypersil octadecyl silane C18 (250 x 4.6&nbsp;mm, 5μm) column at room temperature. The composition of mobile phase&nbsp;was methanol, acetonitrile and 0.02M ammonium acetate buffer (pH 5.5&nbsp;adjusted with dilute acetic acid) in the ratio of 80:15:05 v/v/v and flow rate&nbsp;of mobile phase 1.0 ml/min with isocratic elution. The signal of eluents&nbsp;was observed at 244 nm by using diode array detector. Results: The retention&nbsp;time of Montelukast sodium and Ebastine were found to be 4.28 min&nbsp;and 6.63 min, respectively. The linearity ranges for both drugs were found&nbsp;to be 10-50 μg/ml and the percent recoveries were found to be 99.16%&nbsp;and 100.12% for Montelukast sodium and Ebastine respectively. The drug&nbsp;substances and drug products were treated with various forced degradation&nbsp;conditions like acid hydrolysis, alkali hydrolysis, oxidative degradation,&nbsp;thermal degradation and photolytic degradation. The degradants were efficiently&nbsp;separated from the drugs by using optimized chromatographic conditions.&nbsp;The developed method was validated as per recommendation parameters&nbsp;of International conference on harmonization guidelines Q2(R1).&nbsp;Conclusion: The validation parameters indicate that the drug substances&nbsp;were efficiently separated from its degradants and developed method can&nbsp;be routinely applied for the simultaneous quantitative determination of&nbsp;Montelukast sodium and Ebastine in combined tablet formulation in the&nbsp;quality control laboratory.

Stability Indicating RP-UPLC Photo-Diode Array based Method for Determination of Edoxaban Tosylate

The objective of the current study was to develop a specific, precise, accurate and robust gradient stability indicating reversed-phase ultra performance liquid chromatography (RP-UPLC-PDA) assay method and validated for determination of edoxaban tosylate in API. Gradient separation was achieved on an acquity UPLC BEH C18 column (50 mm, 2.1 mm and 1.7 μm) column using mobile phase of acetoitrile:20 mM potassium dihydrogen phosphate, pH 3.0 ± 0.05 adjust with OPA at flow rate of 0.6 mL/min, the injection volume was 1 μL and the detection was carried out of 289 nm by using photodiode array detector. The drug was subjected to oxidation, hydrolysis, photolysis, and heat to apply stress condition. The method was linear in the drug concentration range of 100-300 μg/mL with correlation coefficient of 0.999. Degradation products produced as a result of stress studies did not interfere with detection of edoxaban tosylate and the assay, thus developed stability indicating method can be used for routine analysis in pharmaceutical industry.

Reversed-Phase High Performance Liquid Chromatographic Analysis of Three First Line Anti-Tuberculosis Drugs

Active pharmaceutical ingredients such as isoniazid, pyrazinamide and rifampicin are among the most important first-line anti-tuberculosis drugs. A simple, rapid and sensitive reversed phase-high performance liquid chromatographic assay method for the simultaneous determination of isoniazid, pyrazinamide and rifampicin has been developed. Separation of the interest compounds was achieved in a 10 min chromatographic run in gradient elution mode on a Zorbax SB-C18 stainless steel column (150 � 4 mm, 5 mm) using a guard column containing the same stationary phase. The gradient elution was carried out with a mobile phase of 10% CH3CN aqueous solution for channel A and 50% CH3CN in pH = 6.8 phosphate buffer (20 mM), to which 1.5 mL triethylamine were added for channel B. Quantification of the analyzed substances was carried out spectrophotometrically at 269 nm. Detection limits of 0.48 mg/L for isoniazid, 0.52 mg/L for pyrazinamide and 0.48 mg/L for rifampicin were established for the developed assay method. The present work showed that the proposed analysis method was advantageous for simple and rapid analysis of the active pharmaceutical ingredients in pharmaceuticals and biological fluids.

Development of a Validated Reverse Phase Liquid Chromatographic Assay-Method for determination of Tofacitinib in pure form and in Physical Admixtures

Aim: The aim is to develop simple validated analytical method for analysis of Tofacitinib by RP HPLC in pure and laboratory prepared physical admixtures. Method: Tofacitinib was estimated by RP HPLC using methanol , water mixture as mobile phase. Linearity range was found to be 10-50 mcg/ml. The correlation coefficient was 0.9998. The limit of detection and quantification were found to be 0.053 and 1.63 µg /ml respectively. The drug solutions were scanned in UV spectrophotometry and the maximum absorption was found to be 285.9 nm and the same was fixed as detecting lambda by RP HPLC method. The Column used was C18.with the dimensions of 150 mm X 4.6 mm and 5 micron particles. Results and Conclusions: The proposed method was successfully applied for the determination of tofacitinib in pure and laboratory prepared physical mixtures. The % RSD value of Tofacitinib in bulk and physical admixture was calculated at different time intervals for recovery , precision (Iintraday and Interday experiments) and quantification studies were found to be less than 2 %.

A validated stability-indicating HPLC assay method for Meclizine HCl in bulk drug

An isocratic reversed phase stability-indicating high-performance liquid chromatographic (HPLC) assay method was developed and validated for quantitative determination of Meclizine HCl in bulk drugs. An isocratic, reversed phase HPLC method was developed to separate the drug from the degradation products, using a Water Nova Pack C18 (250 x 4.6) mm, 5? column and the mobile phase containing 1.0 gm Sodium dihydrogen phosphate and 1.0 gm 1-octaneSulfonic acid salt in 1000ml water filter and mixed. Prepare a homogenous mixture of buffer, methanol and acetonitile. The detection was carried out at wavelength 264 nm. The developed method was validated with respect to linearity, accuracy (recovery), precision, system suitability, selectivity, robustness prove the stability indicating ability of the method.