Forced Degradation Samples Research Articles

New stability indicating RP-HPLC methods for the determination of related substances and assay of trametinib acetic acid: a mass balance approach.

New stability indicating related substances and assay methods for trametinib acetic acid by RP-HPLC have been developed and validated through forced degradation and mass balance studies for the first time. In related substances (RS) method, trametinib acetic acid was successfully separated from its six related substances namely, cyclopropanamide impurity, desiodo trametinib, desacetyl trametinib, trione acetamide intermediate, trione intermediate, and trione PTSA intermediate using YMC-Triart C18 (150 × 4.6mm, 3µm) column. Orthophosphoric acid (0.15%) in water was used as buffer. Gradient elution was programmed using mobile phase-A (buffer and acetonitrile mixture in 80:20 v/v ratio) and mobile phase-B (buffer and acetonitrile mixture in 20:80 v/v ratio). Acetonitrile and methanol mixture (1:1 v/v) was used as diluent. Flow rate, injection volume, column temperature, and wavelengths were kept as 0.8mL/min, 10µL, 55°C, and 240nm, respectively. Desacetyl trametinib and cyclopropanamide impurity were identified as potential degradation impurities in acid and base degradation conditions, respectively. Assay method specific to above six related substances was also developed. Assay of forced degradation samples was also determined, and the mass balance was established by adding total impurities formed in RS method to assay of trametinib acetic acid.

Development and validation of aggregates analysis method in analytical similarity assessment of HLX04 vs Avastin®

Aggregate of therapeutic antibodies is usually considered as one of the most important critical quality attributes (CQA). The propensity of aggregates formation for bevacizumab is higher than other monoclonal antibody (mAb) drugs due to its tendency of self-association via the non-covalent interaction between the Fab arm of one bevacizumab molecule and the K445 residue on the heavy chain of another bevacizumab molecule. HLX04 has been developed as a biosimilar to bevacizumab (Avastin®) by Shanghai Henlius Biotech. To perform a head-to-head similarity evaluation with respect to aggregates or higher molecular weight species (HMWS) between HLX04 and Avastin®, we developed a robust high performance liquid chromatography (SEC-HPLC) method for aggregates analysis. Our characterization data indicated that HMWS of bevacizumab were mainly composed of dimers, and the dimer formation-dissociation equilibrium was influenced by protein concentration and storage temperature. Based on the characterization data of aggregates, we optimized the key parameters for SEC-HPLC based aggregates analysis method including mobile phase components and pH, autosampler temperature, as well as incubation conditions for sample pretreatment. The developed method was applied in HLX04 and Avastin® aggregates assessment and the similarity were confirmed among HLX04, China-sourced, and Europe-sourced Avastin® using both the pharmaceutical dosage forms and forced degradation samples. The method was also validated per ICH Q2 (R1) guidelines by challenging the parameters including specificity, accuracy, precision, linearity, range, limit of quantitation, and robustness. The validated method was applied in release test and stability study of HLX04 samples generated from commercial manufacturing process.

Isobaric Tandem Mass Tag Multiplexed Post-Translational Modification Quantitation of Biopharmaceuticals by Targeted High-Resolution Mass Spectrometry.

Peptide mapping coupled with liquid chromatography-mass spectrometry (LC-MS) has become an essential analytical technique to quantify the quality attributes (e.g., post-translational modifications [PTMs]) of monoclonal antibodies (mAbs) during drug development. However, the traditional label-free approach for relative quantitation of PTMs requires a great amount of instrument time for LC-MS data acquisition of individual digested samples, which limits the efficiency of peptide mapping when there is an increasing demand for protein characterization. Here, we developed a tandem mass tag (TMT)-based approach in combination with targeted mass spectrometry for multiplexed site-specific PTM quantitation of monoclonal antibodies to overcome this limitation. This approach enables the simultaneous quantitation of quality attributes (e.g., PTMs) for multiple samples in a single LC-MS run. By adjusting higher-energy collision dissociation (HCD) normalized collisional energies (NCEs) from 35 to 90, different types of PTMs were quantified with percentages comparable to those obtained using the conventional approach. The TMT overlabeling on the off-target amino acid residues serine, threonine, and tyrosine was observed to pose a challenge for this targeted MS/MS-based PTM quantitation. However, we inhibited this off-target overlabeling by adding a small-molecule additive during the TMT labeling as a decoy reagent to deplete the excess amount of TMT reagent. The PTM quantitative performance of this approach demonstrated high sensitivity and reproducibility of PTM quantitation with levels as low as 1.0%. Finally, this approach has been utilized to quantify the PTMs for forced degradation samples, comparability samples, and trisulfide standards of monoclonal antibodies.

RP-HPLC Stability Indicating Method Development and Validation of Pseudoephedrine Sulfate and Related Organic Impurities in Tablet Dosage Forms, Robustness by QbD Approach

Pseudoephedrine is used for the temporary relief of stuffy nose and sinus pain/pressure caused by infection such as the common cold and flu. Here we were reporting a simple and precise validated stability indicating RP-HPLC method for estimation of Pseudoephedrine Sulfate and its related compounds in solid oral dosage forms. The stability indicating nature of current method has been demonstrated by a forced degradation study. All process related impurities and degradation products from forced degradation samples were well resolved from each other. The chromatographic separation of impurities was achieved with a Hypersil phenyl column (25 cm x 4.6 mm i.d., particle size 5 μ) using buffer and acetonitrile in the ratio of 95:5 v/v respectively as the mobile phase. The buffer used in mobile phase contained 10 mM potassium dihydrogen phosphate and its pH was adjusted to 3.0 ±0.05 with diluted orthophosphoric acid. The flow rate of HPLC was 1.0 mL min−1 and the wavelength of UV detector at 210 nm. The method was shown a linear response over range of 0.125 μg mL−1 to 0.750 μg mL−1 PES and impurities. The correlation coefficient was more than 0.999. Performed the robustness by using design experiments.

Quadrupole Dalton-Based Controlled Proteolysis Method for Characterization of Higher Order Protein Structure.

The higher order structure (HOS) of proteins plays a critical role in the efficacy and stability of biological drugs. Perturbation of the regional structure of proteins can affect biological activity and cause instability. Characterization of HOS has become an integral part of biological drug development and is expected from regulatory agencies. The commonly used techniques for HOS characterization, such as circular dichroism, Fourier-transform infrared, differential scanning calorimetry, intrinsic fluorescence, and hydrogen-deuterium exchange mass spectrometry, have their limitations ranging from lack of sensitivity and specificity to the need of high-level expertise and poor access to instrumentation due to high cost. In this study, we demonstrated a novel controlled proteolysis-based LC-QDa method for the detection of HOS change. By digesting proteins directly without denaturation and reduction, the HOS information can be revealed through the digested peptides. After optimizing the digestion conditions and the detection procedures, we identified 13 signature peptides that can monitor various antibody domains for any HOS changes caused by external stress. By comparing the peptide peak areas between unknown samples and a native control sample, any regional structural changes in unknown samples can be detected. The method was subsequently applied to a wide range of forced degradation samples to demonstrate higher sensitivity compared to the near-UV CD method that is frequently used for monitoring tertiary structural changes. By further reducing the number of signature peptides to five and optimizing liquid chromatography gradient duration, a streamlined, high-throughput, and controlled proteolysis method was successfully established. This method can be used to support process and formulation development as well as potentially for stability testing.

Quantitative determination of clobetasone butyrate in bulk and cream formulation by a validated stability-indicating reversed-phase HPLC method

A simple isocratic reversed-phase HPLC method for quantification of clobetasone in bulk and cream dosage forms has been developed. Chromatographic analysis was accomplished on an C18 column utilizing a mixture of methanol and water (84:16 v:v, pH = 6.0) as mobile phase. An effluent flow rate of 1 mL/min was adjusted and the detection was made at 240 nm wavelength. The method was evaluated according to ICH guidelines Q2 R1 for linearity, specificity, sensitivity, precision and accuracy. The method exhibited good linearity with correlation coefficient (r2) of 0.9993 over the concentration range from 5 to 50 μg/mL. The recoveries of the test drug from the cream sample was found to be 98.56 to 99.51% and the limit of detection and quantification were calculated as 0.85 and 2.83 μg/mL, respectively, suggesting the accuracy and sensitivity of the developed method. The precision was demonstrated by a low percentage of relative standard deviation (<1%) from six independent assay analysis performed for the cream formulation. Stability indicating property of the proposed method was demonstrated by performing the analysis of forced degradation samples. The developed method can be used for estimation of the clobetasone butyrate in bulk and pharmaceutical formulations for routine analysis in the quality control laboratories.

Enantioselective analysis for L-pidolic acid in ertugliflozin drug substance and drug product by chiral gas chromatography with derivatization

L-pidolic acid is being used as a coformer for ertugliflozin, a sodium-glucose cotransport 2 inhibitor. A sensitive and rapid two-step achiral derivatization combined with gas chromatography with flame ionization detection or gas chromatography with mass spectroscopic detection was developed and validated for the enantiomeric purity determination of L-pidolic acid in the drug substance and drug product, respectively. The method was used to analyze ertugliflozin drug substance forced degradation samples and showed no racemization of pidolic acid in any of the solid or solution stress samples. Analysis of ertugliflozin drug product stability samples showed no significant levels of D-pidolic acid in the drug product indicating that no significant racemization of pidolic acid occurs in the drug product under normal storage conditions. Based on the data generated, a chiral control for pidolic acid is not necessary for drug substance or drug product, but rather can be controlled in the purchase of L-pidolic acid.

HPLC Assay Method Development and Validation for Quantification of Capecitabine in Tablets and Forced Degradation Samples

A simple, rapid, accurate and stability indicating RP-HPLC method was developed for the determination of Capecitabine in pure and tablet dosage form. The mobile phase consisting of 0.1% aqcetic acid, methanol and acetonitrile in the ratio of 35:60:5 v/v. The Inertsil ODS (octadecyl silane), C18, 3V, 250 x 4.6 mm, 5μm with UV detection 304 nm was used. The retention time was found to be 6.4 minutes. The method was statistically validated for accuracy, linearity, precision, robustness, specificity and range. The method was found linear over the concentration range of 50-150 μg/ml. The recovery studies of dosage form were also carried out and analyzed; the % relative standard deviation (RSD) from recovery studies was found to be within limits. The specificity of the method was ascertained by forced degradation studies by acid, alkali hydrolysis and oxidation. About 35% of drug is degraded in acidic medium and less than that in alkaline and oxidative conditions. Due to simplicity, rapidity and accuracy of the method, the method will be useful for routine analysis and checking purity of capecitabine tablet. The method further can be investigated for pharmacokinetic and biopharmaceutical analysis.

HPLC Assay Method Development and Validation for Quantification of Capecitabine in Tablets and Forced Degradation Samples

A simple, rapid accurate and forced degradation indicating RP-HPLC method was developed for the determination of Capecitabine in pure and tablet dosage form. The mobile phase used contains 0.1% Acetic acid, Methanol and Acetonitrile in the ratio of 35:60:5 v/v. The linearity range is found over the concentration range of 50-150µg/ml. The Inertsil ODS, C18, 3V, 250 x 4.6 mm, 5µm with UV detection 250 nm is used. The RT is found to be 6.4 mins. The method was statistically validated for accuracy, linearity, precision, robustness, specificity and range. Quantitative and recovery studies of the dosage form were also carried out and analyzed; the % RSD from recovery studies was found to be within limits. The specificity of the method was ascertained by forced degradation studies by acid, alkali hydrolysis and oxidation and about 35% of drug is degraded in acidic medium. Due to simplicity, rapidity and accuracy of the method, the method will be useful for routine analysis and checking purity of Capecitabine tablet. The method further can be investigated for pharmacokinetic and biopharmaceutical analysis.

Forced Degradation Study of Monoclonal Antibody Using Two- Dimensional Liquid Chromatography

Monoclonal antibody (mAb) products are extraordinarily heterogeneous due to the presence of a variety of enzymatic and chemical modifications, such as deamidation, isomerization, oxidation, glycosylation, glycation, terminal cyclization, aggregation, and fragmentation. Forced degradation study is a common practice to assess the potential modifications and degradation pathways of mAbs upon extreme conditions, including light exposure and extreme local pH. The degraded samples are subject to characterization using a wide array of analyses, including ion exchange chromatography (IEC) for charge variants and size exclusion chromatography (SEC) for size variants. In this proof-of-concept study, a two-dimensional liquid chromatography (2D-LC) approach was successfully applied to mAb forced degradation samples for evaluation of size distribution in the IEC charge profile. The charge variant peaks in first-dimension (1D) IEC were fractioned by a heart-cutting schedule, and the integrated peak parking feature accommodated later-eluting 1D cuts when the previous ones were still under second-dimension (2D) analysis. Thus, for a singly IEC injection, all IEC fractions were acquired by the 2D for SEC analysis. This study demonstrated that 2D SEC was compatible with 1D IEC for 2D-LC analysis. The method sensitivity was sufficient to determine the aggregates and fragments in each individual IEC cut. The calculated total aggregates and fragments in the stressed mAb-1 samples were comparable to those detected by one dimensional SEC. Most importantly, the 2D IEC-SEC approach was capable of bridging the mAb charge and size variants in a real-time and efficient manner. It was observed that aggregates were enriched in the most basic region in the IEC charge profile, especially in the highly degraded samples. This is the first IEC-SEC 2D-LC application for intact antibody analysis as per the authors’ knowledge.

Forced degradation of nepafenac: Development and validation of stability indicating UHPLC method

This paper presents stability study of the nonsteroidal anti-inflammatory drug (NSAID) nepafenac. In order to investigate stability of nepafenac, it was subjected to forced degradation under different stress conditions: acid and base hydrolysis, oxidation, humidity, heat and light. A novel stability indicating reverse phase ultra high performance liquid chromatographic (UHPLC) method coupled to ultraviolet detector has been developed to separate nepafenac and all related compounds (2-aminobenzophenone, Cl-thionepafenac, thionepafenac, Cl-nepafenac, hydroxy-nepafenac, and cyclic-nepafenac). Efficient chromatographic separation was achieved on a Waters Acquity BEH C18 stationary phase with a gradient elution. Quantification was carried out at 235nm at a flow rate of 0.6mL/min−1. The resolution between nepafenac and six potential impurities is found to be greater than 2.0. The developed method was validated with respect to specificity, LOD, LOQ, linearity, precision, accuracy and robustness. The r2 values for nepafenac and six potential impurities were all greater than 0.999. The developed method is capable to detect impurities of nepafenac at a level of 0.005% with respect to test concentration of 1.0mg/mL. Significant degradation is observed in acid, base and oxidative degradation conditions and degradation products (DPs) were identified using mass spectrometry analysis; two of them were found to be a known process related impurities (hydroxy- and cyclic-nepafenac) whereas four degradation products were identified as new degradation impurities. The forced degradation samples were assayed against a qualified reference standard and the mass balance was found to be close to 99.5%.

Liquid chromatography and liquid chromatography-mass spectrometry analysis of donepezil degradation products

This study describes the investigation of degradation products of donepezil (DP) using stability indicating RP-HPLC method for determination of donepezil, which is a centrally acting reversible acetylcholinesterase inhibitor. In order to investigate the stability of drug and formed degradation products, a forced degradation study of drug sample and finished product under different forced degradation conditions has been conducted. Donepezil hydrochloride and donepezil tablets were subjected to stress degradation conditions recommended by International Conference on Harmonization (ICH). Donepezil hydrochloride solutions were subjected to acid and alkali hydrolysis, chemical oxidation and thermal degradation. Significant degradation was observed under alkali hydrolysis and oxidative degradation conditions. Additional degradation products were observed under the conditions of oxidative degradation. The degradation products observed during forced degradation studies were monitored using the high performance liquid chromatography (HPLC) method developed. The parent method was modified in order to obtain LC-MS compatible method which was used to identify the degradation products from forced degradation samples using high resolution mass spectrometry. The mass spectrum provided the precise mass from which derived molecular formula of drug substance and degradation products formed and proved the specificity of the method unambiguously.

Validated Stability-Indicating RP-HPLC Method for the Estimation of Degradation Behaviour of Organic Peroxide and Third-Generation Synthetic Retinoids in Topical Pharmaceutical Dosage Formulation.

The objective of the current study was to establish a validated stability-indicating, high-performance liquid chromatographic method to determine the purity of benzoyl peroxide (BPO) and adapalene (ADP) in the presence of its impurities, forced degradation products, and placebo in pharmaceutical dosage forms. The desired chromatographic separation was achieved on the Kinetex™ C18 (250 × 4.6 mm, 5 µm) column using gradient elution at 272 nm detection wavelength. The optimized mobile phase consisted of solvent A (mixture of 0.1% v/v glacial acetic acid in water and acetonitrile in the ratio of 80:20 v/v, respectively) and solvent B (mixture of acetonitrile: tetrahydrofuran: methanol in the ratio of 50:30:20 v/v/v, respectively). The stability-indicating capability of the developed method was established by analysing forced degradation samples in which the spectral purity of BPO and ADP along with separation of all degradation products from the analyte peaks was achieved. The developed method was validated as per ICH guidelines with respect to specificity, linearity, limit of detection, limit of quantification, accuracy, precision, and robustness.

Quality by Design-Based Development of a Stability-Indicating RP-HPLC Method for the Simultaneous Determination of Methylparaben, Propylparaben, Diethylamino Hydroxybenzoyl Hexyl Benzoate, and Octinoxate in Topical Pharmaceutical Formulation.

A stability-indicating RP-HPLC method has been developed and validated for the simultaneous determination of methylparaben (MP), propylparaben (PP), diethylamino hydroxybenzoyl hexyl benzoate (DAHHB), and octinoxate (OCT) in topical pharmaceutical formulation. The desired chromatographic separation was achieved on the KinetexTM C18 (250 × 4.6 mm, 5 μm) column using gradient elution at 257 nm detection wavelength. The optimized mobile phase consisted of a buffer : acetonitrile : tetrahydrofuran (60 : 30 : 10, v/v/v) as solvent A and acetonitrile : tetrahydrofuran (70 : 30, v/v) as solvent B. The method showed linearity over the range of 0.19–148.4 μg/mL, 0.23–15.3 μg/mL, 1.97–600.5 μg/mL, and 1.85–451.5 μg/mL for MP, PP, DAHHB, and OCT, respectively. Recovery for all the components was found to be in the range of 98–102%. The stability-indicating capability of the developed method was established by analysing the forced degradation samples in which the spectral purity of MP, PP, DAHHB, and OCT, along with the separation of the degradation products from the analyte peaks, was achieved. The proposed method was successfully applied for the quantitative determination of MP, PP, DAHHB, and OCT in the lotion sample. The design expert with ANOVA software with the linear model was applied and a 24 full factorial design was employed to estimate the model coefficients and also to check the robustness of the method. Results of the two-level full factorial design, 24 with 20 runs including four centrepoint analysis based on the variance analysis (ANOVA), demonstrated that all four factors, as well as the interactions of resolution between DAHHB and OCT are statistically significant.

Development and Validation of a Novel Stability Indicating RP-UPLC Method for Simultaneous Estimation of Methylparaben, Mometasone Furoate and Eberconazole Nitrate in Combined Topical Pharmaceutical Dosage Form

A novel, rapid, simple, stability indicating RP-UPLC method development and validation for simultaneous estimation of methylparaben (MP), mometasone furoate (MF) and eberconazole nitrate (EN) in topical pharmaceutical dosage form. Isocratic chromatographic separation was achieved on Acquity BEH C18, column using mobile phase consist of buffer (0.1%v/v triethylamine in water pH 6.0 by Glacial acetic acid), acetonitrile in the ratio (40:60, v/v) at flow rate 0.4 mL/min and detection was monitored at 235 nm by using photo-diode array detector. The method shows excellent linearity over the range of 2.75-152.7 µg/mL, 4.45-60.7 µg/mL and 9.10-606.6 µg/mL for methylparaben, mometasone furoate and eberconazole nitrate respectively. Recovery was found for all the components to be in the range of 98.4-101.7%. Stability indicating capability of developed method was established by analysing forced degradation samples in which spectral purity of methylparaben, mometasone furoate and eberconazole nitrate along with separation of degradation products from analytes peak. The proposed method was successfully applied for the quantitative estimation of MP, MF and EN in cream sample.

Stability of dexmedetomidine in polyvinyl chloride bags containing 0.9% sodium chloride injection

The stability of dexmedetomidine in polyvinyl chloride (PVC) bags containing 0.9% sodium chloride injection was studied. Dexmedetomidine solutions (4, 8, 12, and 20 μg/mL; n = 6 for each) were prepared by removing 2, 4, 6, and 10 mL of 0.9% sodium chloride injection, respectively, from 50-mL PVC bags and injecting 2, 4, 6, and 10 mL of dexmedetomidine 100 μg/mL, respectively. To ensure a homogeneous mixture, the contents of each bag was manually mixed initially and before each sample was removed. All compounding was conducted by a single pharmacist using aseptic technique in a horizontal-laminar-airflow hood at 25 °C. Forced-degradation studies were conducted at 70 ± 1 °C. Stability samples were analyzed using high-performance liquid chromatography electrospray ionization-tandem mass spectrometry (LC/MS-MS) and high-performance liquid chromatography-ultraviolet-light (HPLC/UV) absorbance. Forced-degradation samples were monitored using LC/MS-MS, HPLC/UV, and gas chromatography-MS. Dexmedetomidine solutions were very stable at 23 ± 2 °C at all four concentrations over the 48-hour testing period. As determined via LC/MS-MS and HPLC/UV methods, over 97% of the initial concentration of dexmedetomidine remained after 48 hours. Extensive HPLC/UV active degradation products could be observed in basic conditions; only minor UV active degradation products were observed in acidic, oxidative, and photochemical conditions. Dexmedetomidine hydrochloride 4, 8, 12, and 20 μg/mL stored in PVC bags at 23 ± 2 °C was stable for 48 hours, despite a slight decrease in solution pH seen with increasing dexmedetomidine concentrations.

Development and validation of stability-indicating UPLC method for the determination of lafutidine and its impurities in bulk and pharmaceutical dosage form

Abstract Background A simple and rapid stability-indicating, reversed phase ultra-performance liquid chromatography (UPLC) method was developed for the quantitative determination of lafutidine and its four potential impurities. Results Separation was achieved on Acquity BEH-shield RP18 UPLC column (3.0 mm × 100 mm, 1.7 μm) under the gradient mode of elution by using mobile phase A (0.02M diammonium hydrogen phosphate/acetonitrile, 80:20 v/v) and mobile phase B (0.02M diammonium hydrogen phosphate/acetonitrile, 30:70 v/v). The flow rate was maintained at 0.5 mL min−1. UV detection was carried out at 276 nm. Conclusions Stability-indicating capability of the developed method is established by analyzing forced degradation samples in which the spectral purity of lafutidine is ascertained along with the separation of degradation products from analyte peak. The developed UPLC method is validated as per International Conference on Harmonization guidelines with respect to system suitability, specificity, precision, sensitivity, accuracy, linearity, and robustness.

Development and Validation of a Stability-Indicating RP-HPLC Method for Simultaneous Determination of Phenoxyethanol, Methylparaben, Propylparaben, Mometasone Furoate, and Tazarotene in Topical Pharmaceutical Dosage Formulation

A stability-indicating RP-HPLC method has been developed and validated for the simultaneous determination of phenoxyethanol (PE), methylparaben (MP), propylparaben (PP), mometasone furoate (MF), and tazarotene (TA) in topical pharmaceutical dosage formulation. The desired chromatographic separation was achieved on the Waters X-Bridge™ C18 (50×4.6mm, 3.5μ) column using gradient elution at 256 nm detection wavelength. The optimized mobile phase consisted of 0.1%v/v orthophosphoric acid in water as solvent-A and acetonitrile as solvent-B. The method showed linearity over the range of 5.88–61.76 μg/mL, 0.18–62.36 μg/mL, 0.17–6.26 μg/mL, 0.47–31.22 μg/mL, and 0.44–30.45 μg/mL for PE, MP, PP, MF, and TA, respectively. The recovery for all of the components was in the range of 98–102%. The stability-indicating capability of the developed method was established by analysing the forced degradation samples, in which the spectral purity of PE, MP, PP, MF, and TA along with the separation of degradation products from the analyte peaks was achieved. The proposed method was successfully applied for the quantitative determination of PE, MP, PP, MF, and TA in a cream sample.

HPLC Stability Indicating Assay Method for Metformin Hydrochloride in Bulk Drug and Tablets and Cytotoxicity of Degradation Products

The present work reports a stability-indicating reversed phase ion pair liquid chromatography method for quantitative determination of metformin hydrochloride (MTF) (drug and tablets) and assessment of the cytotoxicity of degraded MTF. Chromatographic separation was performed on a C18 (Phenomenex® Luna), 5.0 μm (250 mm x 4.6 mm) column using isocratic elution. The optimized mobile phase consisted of 10 mmol L-1 heptane sulphonic acid (pH 3.0), acetonitrile and methanol (75:8:17 v/v), flow 1.0 mL.min-1, at 30 ºC. The eluted compounds were monitored at 210 nm wavelength using a DAD detector. The stability indicating capability of the developed method was established by analyzing forced degradation samples (acid, alkali, neutral, oxidative and photolytic condition) in which the spectral purity of MTF was ascertained, along with the separation of degradation products from the analyte peak. The cytotoxicity of MTF and the degraded drug was analyzed in the L929 cell test by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The 500 mg of commercial MTF tablets (Reference, Generic and Similar drugs) showed a similar chromatographic profile, but lower intensity degradation in the same conditions, in relation to the bulk drug in almost forced conditions. Neither the degraded drug samples, nor the tablet samples degraded in acid conditions, showed any cytotoxicity. The developed stability-indicating ion-pair HPLC method was fully validated according to the International Conference on Harmonization (ICH) guidelines. The degraded sample of MTF showed no affect on cell viability, compared with the non-degraded drug. Keywords: Bulk Drug, Cytotoxicity, Degradation Products, HPLC-UV, Ion-pair liquid chromatography, Metformin hydrochloride, Method validation, MTT assay, Stability-indicating assay method, Tablets

Development of a Stability-Indicating RP-LC Method for the Separation of a Critical Pair of Impurities and their Degradants in Zafirlukast

A high-throughput reverse-phase liquid chromatographic (RP-LC) method is developed for the quantification of zafirlukast and its related impurities in drug substance. The separation of known impurities is accomplished using a short (50 mm) LC column with sub-2-µm particle size in a relatively short run-time. A linear gradient elution involves ammonium formate and acetonitrile as mobile phase. The critical impurity pair is the meta and para isomers of zafirlukast, which are known to be potential impurities of zafirlukast, whose resolution is sensitive to pH. The stability-indicating capability of the developed method is demonstrated using forced degradation samples from stress conditions such as hydrolysis, oxidation, thermal and photolytic degradation. The developed RP-LC method is validated in accordance with International Conference on Harmonization requirements. The results from the validation study indicate that this RP-LC method can be used for the determination of synthetic and degradation impurities in regular quality control analysis for the drug substance.