Stability-indicating LC Method Research Articles

Validation of stability-indicating LC method, degradation study, and impact on antioxidant and antifungal activities of Eugenia uniflora leaves extract.

The aim of this study is to demonstrate the stability-indicating capacity of an analytical method for Eugenia uniflora, enhance understanding of the stability of myricitrin, and assess the effect of degradation of spray-dried extract (SDE) on antioxidant and antifungal activities. Validation of the stability-indicating method was carried out through a forced degradation study of SDE and standard myricitrin. The antioxidant and antifungal activities of SDE were evaluated both before and after degradation. The quantification method described was found to be both accurate and precise in measuring myricitrin levels in SDE from E. uniflora, with excellent selectivity that confirmed its stability-indicating capability. The forced degradation study revealed that the marker myricitrin is sensitive to hydrolysis, but generally stable under other stress conditions. By contrast, the standard myricitrin displayed greater susceptibility to degradation under forced degradation conditions. Analysis of the antioxidant activity of SDE before and after degradation showed a negative impact in this activity due to degradation, while no significant effect was observed on antifungal activity. The method described can be a valuable tool in the quality control of E. uniflora, and the findings can assist in determining the optimal conditions and storage of products derived from this species.

Advancing Quality and Environmental Responsibility: A Stability-Indicating LC Method Development for Lenvatinib Through QbD and Green Chemistry

Advancing Quality and Environmental Responsibility: A Stability-Indicating LC Method Development for Lenvatinib Through QbD and Green Chemistry

Empagliflozin: Validation of Stability-Indicating LC Method and in silico Toxicity Studies.

A new stability-indicating liquid chromatography method was developed for the quantification of empagliflozin and two synthetic impurities. The chromatographic conditions included Spherisorb® RP-18 column (150 × 4.6mm, 5μm) with a PDA detector, using acetonitrile and formic acid (pH4.0) as mobile phase in gradient elution and flow-rate of 1.2mL·min-1. The gradient increasing from 51 to 100% acetonitrile until 11.00min, followed by decreasing the solvent from 100% to the initial ratio from 11.01 to 15.00min. The method was validated according to International Council of Harmonization guidelines. The LOD and LOQ values for impurities A and B were 35 and 15ng·mL-1, respectively, (for LOD) and 115 and 35ng.mL-1, respectively (for LOQ). The method was linear in the range of 80-140, 115-1150 and 35-350ng·mL-1 for EMPA, impurities A and B, respectively, and the correlation coefficient were > 0.999 in all situations, indicating the method good linearity. The developed method showed a good recovery for empagliflozin and added impurities. The method has proven to be precise, demonstrated values less than 2.0% to empagliflozin and 5.0% to synthetic impurities, robust and selective with no interference from other products in the determination of analytes. The in silico toxicity prediction suggested that the impurities do not present any toxicity risk for the parameters evaluated.

Stability-indicating LC Method for Quantification of Azelnidipine: Synthesis and Characterization of Oxidative Degradation Product

In the work presented here, the degradation behavior of azelnidipine under diverse forced degradation conditions was studied. A stability-indicating liquid chromatographic method was established which could separate and resolve azelnidipine from its degradation products. Further, chemical kinetics under acidic and alkaline conditions were studied, and validation studies were performed. Using reversed-phase chromatography, azelnidipine and its formed degradants were resolved using phosphate buffer (pH 3.0) and methanol in a mixture of 10:90% v/v as a mobile phase at a flow rate of 1.0 mL/min. All eluents were detected at a wavelength of 256 nm. Azelnidipine was degraded under acid, alkali, wet heat, and oxidized environment. The pH-dependent rate of hydrolysis of azelnidipine was studied under acidic and alkaline conditions and chemical kinetics were determined. Further, the oxidative degradation product of azelnidipine was synthesized and characterized as 3-(1-benzhydrylazetidin-3-yl) 5-isopropyl 2-amino-6-methyl-4-(3-nitrophenyl) pyridine-3,5-dicarboxylate (dehydro-AZD). The susceptibility of azelnidipine to hydrolysis was attributed to the presence of ester at 3 and 5 positions of 1,4 dihydropyridine. Further, under oxidative conditions, the aromatization of 1,4 dihydropyrinine resulted in dehydro-AZD. Azelnidipine followed the first-order reaction under acid and alkali hydrolysis, and was more susceptible to degradation under acidic conditions. The synthesized and confirmed dehydro-AZD was found as one of the metabolites and impurities of azelnidipine. The evaluated validation parameters ascertained the practicality of the method for the quantification of azelnidipine tablets.

Chemometric Assisted Development and Validation of a Stability-indicating LC Method for Determination of Related Substances in Haloperidol Decanoate Injection

Abstract: Aim: Haloperidol decanoate injection is a phenyl butyl piperadine derivative with antipsychotic, neuroleptic, antiemetic effects and has multiple related substances as process and degradant impurities. Objectives: This study focuses on chemometric assisted liquid chromatographic approach to develop a stability indicating impurity profile of Haloperidol decanoate injection. Methodology: Dual experimental designs (combined mixture I-optimal design and response surface historical data design) were employed to resolve all thirteen known impurities of Haloperidol decanoate. Chromatographic separation was achieved on a Hypersil BDS C18 (100 x 4.0 mm) 3-μm column to attain separation of related compounds. Results: The optimum conditions for the chromatographic system resulted in a mobile phase consisting of tetra butyl ammonium hydrogen sulphate/ 1-decane Sulphonate sodium buffer solution and acetonitrile with linear gradient elution at a flow rate of 1.4 mL/min. Selectivity, forced degradation, linearity, accuracy and precision were demonstrated in a range of 0.75-30.0 μg/mL. Conclusion: The optimized method was able to resolve the ghost peak observed because of gradient change of mobile phase and is able to separate both polar and non-polar impurities within 60 min, in single method, with resolution of more than 2.0 between adjacent impurities. The inter-day precision for all impurities and haloperidol decanoate were evaluated and found to have a % RSD of less than 10. Key words: Design of Experiments, Haloperidol decanoate Injection, Stability indicating method, DoE aided chromatographic method development, Artifact optimization.

Assessment of ceftolozane/tazobactam stability in elastomeric devices and suitability for continuous infusion via outpatient parenteral antimicrobial therapy.

ObjectivesTo investigate the stability of ceftolozane/tazobactam 5 mg/mL and 20 mg/mL solutions for infusion in two elastomeric devices: FOLFusor LV10 (Baxter Healthcare) and Easypump® II (B. Braun Medical Ltd) and determine if an extended shelf life of up to 8 days storage at 2–8°C plus 24 h ‘in use’ at 32°C was achievable.MethodsTesting was as per the latest NHS Pharmaceutical Quality Assurance Committee Yellow Cover Document (YCD) requirements. A stability-indicating LC method was used for assessing the stability of solutions of ceftolozane/tazobactam at 5 mg/mL and 20 mg/mL (combined concentration of both actives) respectively, tested in two batches in triplicate (n = 3) at five timepoints according to the requirements of the YCD.ResultsCeftolozane/tazobactam, diluted in 0.9% w/v sodium chloride at 5 mg/mL and 20 mg/mL, degraded during in-use storage at 32°C with <95% remaining after 18 h for some device/concentration combinations and all device/concentration combinations at 24 h, respectively. The data does support extended storage of up to 8 days at 2–8°C plus 12 h at 32°C ‘in-use’ when using either FOLFusor LV10 or Easypump® II devices and is compliant with YCD.ConclusionsSolutions of ceftolozane/tazobactam can be administered in outpatient parenteral antimicrobial therapy (OPAT) services following refrigerated storage for up to 8 days, when limited to a 12 h infusion at in-use temperature of 32°C. For UK OPAT services where twice daily dosing is feasible, our data provides another treatment option for challenging infections. In countries where a 10% loss of ceftolozane/tazobactam is acceptable, a 24 h infusion is supported by the data.

Luliconazole: Stability-indicating LC method, structural elucidation of major degradation product by HRMS and in silico studies

Aim: A new stability-indicating liquid chromatography method was developed and validated for the quantitative determination of luliconazole. Materials and methods: Preliminary forced degradation study demonstrated an additional peak of the degradation product at the same retention time to the drug, due to this, the method was developed optimizing the chromatographic conditions to provide sufficient peak resolution (R ≥ 2). The experimental design was evaluated to assess the robustness and the best chromatographic conditions to be used for the validation. Methodology: Luliconazole solutions were exposed to various stress conditions to evaluate the method indication stability, in which the degradation product (DP-1) formed was isolated, identified, and evaluated in silico to predict degradation pathway and toxicity. The procedure was validated by robustness, selectivity, linearity, precision, and accuracy. Liquid chromatography was performed in a Phenomenex® RP-18 column with a mixture of acetonitrile and 0.3% (v/v) triethylamine solution as a mobile phase in isocratic elution. Results and conclusions: The method demonstrated robustness, good recovery, precision, linear response over a range from 5.0 to 40.0 μg.mL-1, and to be stability indicating. The alkaline stress condition resulted in the formation of DP-1. hrms studies identified this product as an hydroxyacetamide derivative, and in silico studies did not show toxic potential.

Development and Validation of Stability-Indicating HPLC Method for the Quantification of Levetiracetam in Bulk and Oral Solution: Application to Chemical Kinetics

In the present study, a simple, accurate, precise and specific stability-indicating LC method was developed and validated for the quantification of levetiracetam (LEV) in bulk and oral solution. LEV and its degradation products were separated and resolved successfully on C18 column using a methanol:water (30:70 %, v/v) mixture in the isocratic mode at a flow rate of 1 mL/min. All eluents were detected at 205 nm. In forced degradation experiments, LEV was found to degrade significantly in acid, alkali, wet heat, peroxide mediated oxidation, and photolytic conditions, and found stable under dry heat conditions. Validation experiments showed acceptable accuracy, precision, and specificity of the developed method. Assay of the oral solution was in good agreement with the amount of LEV as per the label claim. The method was applied to investigate chemical kinetics under acid and alkali hydrolysis and the pH rate profile of LEV within a range of pH 2 – 12.

LC-MS/MS Characterization of Forced Degradation Products of Repaglinide Quality by Design Approach for Stability-Indicating LC Method Development and Validation for Degradation Products

The current study demonstrates the characterization of unspecified degradation impurities of repaglinide by liquid chromatography-tandem mass spectrometry and development of stability-indicating test procedure for quantification of repaglinide and their organic impurities by application of Qbd approach. The use of QbD guidelines has proved to be realistic in the amplification of analytical methods. Identified analytical target profile (ATP) and critical analytical attributes (CAA). Based on the initial assessment, the important factors were finely tuned using Box-Behnken design with the help of numerical and graphical optimization. Evaluated stress studies under different types of conditions. The repaglinide was subjected to different forced degradation conditions such as acid, base, oxidation, thermal, and UV light to discover the stability-indicating competence of the proposed method. Significant degradation was detected during the oxidative stress condition. The repaglinide was well resolved from its impurities produced during the various stress conditions. The chromatographic optimization was attained on an acquity BEH; Shield-RP-C18100 mm x 2.1 mm; 1.7 μm with gradient elution mode at a flow rate of 0.3 ml min-1. The peaks were detected at 214 nm. The developed UHPLC method was validated as per the current ICH guidelines. The unspecified impurities were exemplified by comparing their collision-induced dissociation mass spectral data with that of repaglinide and the most probable degradation and fragmentation pathways were discussed.

Stability-Indicating LC Method for Estimation of Nimodipine in Dosage Form and Spiked Serum Using Systematized Design of Experiments (DoE) Approach

A simple and new liquid chromatographic method was developed and validated for the estimation of nimodipine in formulations and spiked serum samples using an analytical quality by design approach. This approach helped to define the analytical target profile and related critical analytical attributes such as retention time, theoretical plates, and tailing factor. Critical method variables such as methanol %, the flow rate of the mobile phase, and pH were identified and optimized using Box-Behnken experimental design to develop a robust method and further control strategies were developed. Chromatography for the purpose constituted of methanol: water (pH 3.5 maintained by o-phosphoric acid), 80:20, v/v as mobile phase flowing at 1.0 mL/min on a ShimPack GWS C18 column. The photodiode array detection was performed at 355 nm. Results for method validation parameters viz. linearity (0.5-80 μg/mL), accuracy (>98 %) and precision (<1 %) advocated method reliability. Chromatographic method specificity was ensured by degrading the drug forcefully. Nimodipine revealed its susceptibility to applied acid, alkali, and photolysis stress conditions, calling for adequate preventive measures. Further, the method was applied to determine suitability for the estimation of the target analyte in spiked biological fluids. Overall the method was reliable and can be applied in routine and bio-analytical purposes.

Stability-indicating LC method for the simultaneous determination of methyl paraben, propyl paraben, butylated hydroxytoluene and alpha-tocopherol contents in marijuana capsules

The current study is a simple liquid chromatographic method developed and validated for the simultaneous determination of methyl paraben, propyl paraben, butylated hydroxytoluene and alpha-tocopherol in marijuana (delta-8-tetra hydro cannabinol (or) dronabinol) soft gelatin capsules by using high-performance liquid chromatographic technique. The chromatographic separation was achieved on a Waters Xterra RP18 (150 × 4.6)-mm 5-µm column with a mobile phase contained gradient mixtures, which constitutes of purified water and acetonitrile in the ratio of 700:300 (Port-A) and acetonitrile (Port-B). The optimized flow rate of 2.0 mL/min and UV detection was made at 210 nm and column temperature of 40 °C. The resolution among MP, PP, BHT and ATP was found to be > 4. The optimized method was validated as per the guidelines prescribed by the international conference on harmonization. Regression analysis confers a correlation coefficient higher than 0.999 for MP, PP, BHT and ATP peaks. The % RSD for the interday and intraday precisions for MP, PP, BHT and ATP was estimated and found to be < 5.0. The proposed HPLC method could be suitable for the estimation of these drugs in different types of pharmaceutical drug products. Schematic diagram of the procedure for the simultaneous determination of MP, PP, BHT and ATP contents in marijuana capsules

Degradation studies of quetiapine fumarate by liquid chromatography-diode array detection and tandem mass spectrometry methods.

Quetiapine fumarate (QUE) is an antipsychotic agent with a chemical structure that is susceptible to degradation; therefore, it is important to study its stability using appropriate analytical tools. Knowledge of the stability profile of a drug is important because chemical degradation of its active component often results in a loss of potency, affecting its efficacy and safety. This current work reports degradation studies of QUE as drug substance, under different stress conditions such as oxidation, hydrolysis, heat, humidity and photolysis, by a stability-indicating LC method. The chemical stability was evaluated using a simple HPLC/diode array detection method, with a core-shell C18 column under isocratic conditions, which allows the separation of all primary degradation products (DPs) in a short run time. QUE was mainly degraded under oxidative and hydrolytic conditions, with the formation of three and two DPs, respectively, which were identified by electrospray ionization-tandem mass spectrometry. The method was properly validated in terms of linearity, accuracy, precision, selectivity, robustness and quantitation limit. Commercial tablets containing 25 mg of QUE were quantified, with results obtained within the United States Pharmacopeia limits. The proposed method is suitable to assess the stability and perform routine analysis of QUE in pharmaceutical samples.

Biological Safety Studies and Simultaneous Determination of Linagliptin and Synthetic Impurities by LC-PDA.

A stability-indicating LC method was developed for quantification of linagliptin (LGT) and three synthetic impurities. The method utilizes a Thermo Scientific® RP-8 column (100 mm × 4.6 mm; 5 μm) with the PDA detector for quantitation of impurities. A mixture of 0.1% formic acid with pH 3.5 (A) and acetonitrile (B) was used as the mobile phase at a flow rate of 0.6 mL·min−1 with gradient elution. The percentage of mobile phase B increases from 30% to 70% over 5 min and decreases from 70% to 30% between 5 and 8 min. The method was validated according to International Council for Harmonization (ICH) guidelines. The LOD values obtained were 0.0171 μg·mL−1 and 0.015 μg·mL−1 for LGT and impurities, respectively. The LOQ values were 0.06 μg·mL−1 for LGT and impurities. In all cases, the correlation coefficients of LGT and impurities were >0.999, showing the linearity of the method. The % recovery of the LGT and added impurity were in the range of 92.92–99.79%. The precision of the method showed values less than 1.47% for LGT and less than 4.63% for impurities. The robustness was also demonstrated by small modifications in the chromatographic conditions. The selectivity was evidenced because the degradation products formed in stress conditions did not interfere in the determination of LGT and impurities. Toxicity prediction studies suggested toxicity potential of the impurities, which was confirmed using biological safety studies in vitro.

Delapril and Indapamide: Development and Validation of a Stability-Indicating Core-Shell LC Method and Its Application for Simultaneous Tablets Assay.

Background: The combination of delapril (DEL) and indapamide (IND) may be regarded as an optimal drug treatment for hypertensive patients. However, there is no published study concerning the suitable stability conditions and evaluation of drugs in the raw material and commercial product. Objective: The aim of the present study was to develop an innovative, high-throughput, and stability-indicating LC method for the simultaneous analysis of DEL and IND in combined dosage form. Methods: Analyses were performed using a core-shell C18 column (100 mm × 4.6 mm id, 2.6 μm) at 45°C using isocratic elution for the mobile phase composed of triethylamine solution (0.3%, pH 5.0)-acetonitrile-methanol (58 + 35 + 7, v/v/v). The separation was obtained within 3.5 min at a flow rate of 1.0 mL/min and UV detection set at 213 nm. Results: The specificity and stability-indicating capability of the method were proven through degradation studies, which also showed that there is no interference of the formulation excipients, showing that the peak is free from any co-eluting peak. Conclusions: The method showed adequate precision, with relative standard deviation values lower than 1.85%. Excellent values of accuracy were obtained, with a mean value of 98.64% for IND and 98.65% for DEL. Experimental design was used during validation to calculate and prove the method robustness. Highlights: The proposed LC method was successfully validated according to International Conference on Harmonisation requirements and applied for the simultaneous determination of DEL and IND in tablets, presenting suitability for stability studies and contributing to improve the QC of pharmaceuticals.

STABILITY-INDICATING LC METHOD FOR THE QUANTIFICATION OF MIDAZOLAM ACTIVE PHARMACEUTICAL INGREDIENT AND IN PHARMACEUTICAL FORMULATIONS

A stability-indicating LC method was validated for the quantification of midazolam (MDZ) active pharmaceutical ingredient (API) and in pharmaceutical formulations. Isocratic chromatography was performed on C18 column with mobile phase containing methanol/acetonitrile/water (45:35:20 v/v/v) with 0.4% of triethylamine pH 6.5. The validation included specificity, linearity, accuracy, precision and robustness. In specificity, after acid, basic, neutral, oxidant and thermal degradation, it was found that the concentration of MDZ decreased substantially, with the appearance of peaks representatives of the degradation products, proving the stability-indicating power of the method. The response was linear in the range 50.0 – 250.0 µg.mL-1, with 11.73 µg.mL-1 and 3.87 µg.mL-1 as LOQ and LOD, respectively. Recoveries ranged between 98.68 and 100.41%. The relative standard deviation values for intra and interday precision were 1.11%, 0.82% and 1.47%, respectively. The tablets and injections containing MDZ were approved in the assay and content uniformity. The method can be adopted by pharmacopeias and for routine quality control for analysis of MDZ API, tablets and injection.

Quantitative estimation of mebeverine hydrochloride in sustained-release dosage form using an analytical lifecycle management oriented stability-indicating LC method

A new liquid chromatographic method with stability-indicating potential was developed and validated for estimation of mebeverine hydrochloride in formulations using lacidipine as internal standard. A contemporary approach of analytical life-cycle management was followed to develop a robust and reliable chromatographic method. Scouted method variables such as % acetonitrile, flow rate and pH were optimized using design of experiment approach and their effect on critical quality attributes was studied. The attributes viz. resolution and theoretical plates of mebeverine, were highly influenced by % acetonitrile and flow rate of mobile phase. Chromatographic method specificity was ensured by degrading the drug forcefully. Validation studies postulated method acceptability and suitability for estimating mebeverine in both bulk as well as tablet formulation. Results for parameters viz. linearity (0.5–150 µg/mL), accuracy (>98%) and precision (<2%) advocated method reliability. Overall the method was reliable and of optimum quality and, possess the potential of application in routine and bio-analytical purposes.

Simultaneous Determination of Curcumin and Quinine Co-Encapsulated in Nanoemulsion by Stability-Indicating LC Method

Simultaneous Determination of Curcumin and Quinine Co-Encapsulated in Nanoemulsion by Stability-Indicating LC Method

Stability Study of Finasteride: Stability-Indicating LC Method, In Silico and LC-ESI-MS Analysis of Major Degradation Product, and an In Vitro Biological Safety Study.

Stability studies of the pharmaceutically important compound finasteride were conducted in order to evaluate decomposition of the drug under forced degradation conditions. A simple stability-indicating liquid chromatography method was developed and validated for the evaluation of finasteride and degradation products formed in pharmaceutical preparations and the raw material. Isocratic LC separation was achieved on a C18 column using a mobile phase of o-phosphoric acid (0.1% v/v), adjusted to pH 2.8 with triethylamine (10% v/v) and acetonitrile (52:48 v/v), with a flow rate of 1.0 mL min-1. The alkaline degradation kinetics of the drug were also evaluated and could be best described as second-order kinetics under the experimental conditions applied for the tablets and raw material. Based on in silico studies and molecular weight confirmation, a comprehensive degradation pathway for the drug and the identity of its major product could be suggested without complicated isolation or purification processes. Furthermore, a biological safety study was performed to evaluate the effect of the degraded sample in relation to the intact molecule. The results showed that the degraded sample affected the cell proliferation. Therefore, these studies show that special care must be taken during the manipulation, manufacture and storage of this pharmaceutical drug.

Application of Quality by Design to optimize a stability-indicating LC method for the determination of ticagrelor and its impurities

Simultaneous analysis of drug compounds and their impurities of degradation and synthesis became constant in the modern pharmaceutical analysis. Likewise, analytical techniques must improve sensitivity and selectivity for the monitoring of pharmaceutical products, allowing a full assessment of impurities in drug products and, therefore, ensure safety and efficacy of pharmacological treatments. The application of Quality by Design (QbD) principles has proved to be feasible on the elaboration of analytical methods, allowing the comprehensive evaluation and measurement of different analytical parameters and their effects on critical properties of the methodology in development. QbD approach was applied to the development of a fast and selective HPLC method for the analysis of the antiplatelet aggregation drug ticagrelor and its degradation products in presence of three impurities of synthesis. Fractional factorial resolution V was the screening experimental design applied to five method parameters. Response surface methodology was carried by central composite star face design on the two critical method parameters selected. Analytical design space, established after the application of Monte-Carlo simulations, verified whether predicted results were in accordance with critical quality attributes. The developed and validated HPLC method with DAD detection at 225 nm was able to resolve eight related compounds in less than three minutes.

Development and Validation of a Stability-Indicating LC Method for Determination of Bexarotene in Softgel Dosage Formulation

This study focuses on a novel liquid chromatographic approach that has been developed and approved for the quantitative determination of bexarotene (BXT), its potential impurities in drug substances and drug products. Chromatographic separation was developed on a Symmetry C8 (150 × 4.6) mm 5-µm column with a mobile phase containing an isocratic mixture of acetonitrile:DI water:glacial acetic acid (650:350:7.5) v/v/v at a flow rate of 1.2 mL min−1, and quantitation was carried out using ultraviolet detection at 262 nm for BXT and 290 nm for BHA with a column temperature of 35 °C. The resolution among butylated hydroxyanisole (BHA), BXT and its process-related impurity-A was found to be greater than 5. Regression analysis confers an R value (correlation coefficient) higher than 0.998 for BHA, BXT and impurity-A. The detection level for BXT impurities was found at a level below 0.03% (0.18 µg mL−1). The inter- and intra-day precisions for BHA, BXT and impurities were evaluated and found to have a %RSD of less than 3.0.