Mass Balance in Pharmaceutical Stress Testing: A Review of Principles and Practical Applications.

Stress testing (also known as forced degradation) of pharmaceutical drug substances and products is a critical part of the drug development process, providing insight into the degradation pathways of drug substances and drug products. This information is used to support the development of stability-indicating methods (SIMs) capable of detecting pharmaceutically relevant degradation products that might potentially be observed during manufacturing, long-term storage, distribution, and use. Assessing mass balance of stressed samples is a key aspect of developing SIMs and is a regulatory expectation. However, the approaches to measure, calculate, and interpret mass balance can vary among different pharmaceutical companies. Such disparities also pose difficulties for health authorities when reviewing mass balance assessments, which may result in the potential delay of drug application approvals. The authors have gathered input from 10 pharma companies to map out a practical review of science-based approaches and technical details to assess and interpret mass balance results. Key concepts of mass balance are introduced, various mass balance calculations are demonstrated, and recommendations on how to investigate poor mass balance results are presented using real-world case studies. Herein we provide a single source reference on the topic of mass balance in pharmaceutical forced degradation for small molecule drug substances and drug products in support of regulatory submissions with the goal of facilitating a shared understanding among pharmaceutical scientists and health authorities.

AQbD-guided development and validation of an innovative extraction procedure and stability-indicating RP-HPLC method for quantification of posaconazole in tablet formulation.

OBJECTIVE: The objective of this work is to develop a stability-indicating HPLC method for the quantification of posaconazole (PCZ) in tablet formulation using an Analytical Quality by Design (AQbD) approach. MATERIALS AND METHODS: The development process involved the Design of Experiments (DOE) utilizing distinctive constraints mixture design for mobile phase ratio optimization and a 2-level factorial design for selection of extraction diluent compositions. Key responses measured included % assay and system suitability parameters. Method operable design regions (MODR) were determined, and final optimum conditions were selected. Forced degradation studies were conducted to assess method stability. RESULTS: The optimized HPLC method employed a Zorbax C18 column with a mobile phase consisting of pH 3.5 10mM phosphate buffer, acetonitrile, and methanol in a ratio of 30:53:17% v/v/v. The method demonstrated stability-indicating capabilities, with PCZ degradation observed in acidic and oxidative environments, while remaining stable in alkali. Peak purity analysis from Empower software confirmed the absence of interaction with degradants. Validation according to ICH Q2 (R2) guidelines showed precision, linearity over the range of 0.25 to 376µg/mL, and accuracy demonstrated through recovery studies from 50 to 150%. CONCLUSION: The developed HPLC method utilizing AQbD approach is specific, robust, precise, and accurate for the quantification of PCZ in tablet formulations, thus suitable for routine analysis.

Development and validation of stability-indicating method of etrasimod by HPLC/DAD/MS/MS technique with greenness profiling.

Etrasimod, a novel selective sphingosine-1-phosphate receptor modulator, was recently approved by the U.S. Food and Drug Administration and the European Medicinal Agency for the treatment of moderately to severely active ulcerative colitis in adults. In this research, the forced degradation study as an integral part of drug product and packaging development, which generates data on degradation mechanisms, is published. The development and validation of the stability-indicating method using a superior high-performance liquid chromatography technique coupled with a diode array detector and tandem mass spectrometer was performed to support the forced degradation study and monitor the formation of degradation products. Etrasimod demonstrated good stability under elevated temperature and basic stress conditions, while acidic hydrolysis, oxidative, and photolytic degradation produced eight degradation products. The knowledge of degradation products will be useful in the long-term stability study for establishing the acceptance criteria of etrasimod as a drug substance and dosage form during quality control and stability assessment. The eco-friendliness of the developed forced degradation procedure was evaluated using various greenness appraisal tools. The green metric tools showed that the forced degradation procedure obeys eco-friendly conditions.

Development of a novel quality by design-enabled stability-indicating HPLC method and its validation for the quantification of nirmatrelvir in bulk and pharmaceutical dosage forms.

A systematic and novel quality by design-enabled, rapid, simple, and economic stability-indicating HPLC method for quantifying nirmatrelvir (NMT) was successfully developed and validated. An analytical target profile (ATP) was established, and critical analytical attributes (CAAs) were allocated to meet the ATP requirements. The method used chromatographic separation using a Purosphere column with a 4.6 mm inner diameter × 250 mm (2.5 µm). The analysis occurred at 50°C with a flow rate of 1.2 mL/min and detection at 220 nm. A 10 µL sample was injected, and the mobile phase consisted of two components: mobile phase A, containing 0.1% formic acid in water (20%), and mobile phase B, containing 0.1% formic acid in acetonitrile (80%). The diluent was prepared by mixing acetonitrile and water at a 90:10 v/v ratio. The retention time for the analyte was determined to be 2.78 min. Accuracy exceeded 99%, and the correlation coefficient was greater than 0.999. The validated HPLC method was characterized as precise, accurate, and robust. Significantly, NMT was found to be susceptible to alkaline, acidic, and peroxide conditions during forced degradation testing. The stability-indicating method developed effectively separated the degradation products formed during stress testing, underlining its effectiveness in stability testing and offering accuracy, reliability, and sensitivity in determining NMT.

Development and validation of stability indicating RP-HPLC method for simultaneous estimation of silodosin and mirabegron in synthetic mixture.

The study focuses to validate and develop a precise, simple and accurate stability indicating RP-HPLC method for estimation simultaneously of and silodosin and mirabegron in synthetic mixture. The chromatographic separation was achieved by using Shimpack Solar C18 column (250×4.5mm, 5µm) with acetonitrile: 5mM ammonium acetate in ratio of 90:10% v/v as a mobile phase at a constant flow rate of about 1.2mL/min. The development and validation were carried out at detection wavelength of 229nm. We developed a robust RP-HPLC method, validated for linearity, precision, accuracy, specificity, and system suitability. The method demonstrated excellent linearity with correlation coefficient value r2 was nearly 0.998 with linearity range 8-18µg/mL for Silodosin and 24-54µg/mL for mirabegron. LOD and LOQ were found to be lower; hence, the method is sensitive. Percentage recovery was obtained 99.97% and 99.99% for silodosin and mirabegron, respectively. In case of precision, robustness and repeatability, RSD was found to be less than 2. The validated and developed RP-HPLC method offers an efficient and practical approach for the simultaneous quantification of silodosin and mirabegron in pharmaceutical formulations, making it a valuable tool for quality control and pharmaceutical research.

Separation and simultaneous estimation of enantiomers and Diastereomers of muscarinic receptor antagonist Solifenacin using stability-indicating Normal-phase HPLC technique with chiral stationary phase amylose tris-(3,5-dimethylphenylcarbamate).

The R,S-enantiomer impurity and diastereomer impurities (S,S-isomer and R,R-isomer) of the solifenacin (S,R-enantiomer) were effectively separated and quantified simultaneously utilizing normal-phase high-performance liquid chromatography with a chiral stationary phase consisting of amylose tris (3,5-dimethylphenylcarbamate) coated on silica-gel (Chiralpak, AD-H). The enantiomeric and stereo-selective separation was achieved within a run time of 35 minutes using a mobile phase of 'n-hexane, ethanol, and diethylamine' in an isocratic elution mode with a detection wavelength of 220 nm. The validation attributes assessed were accuracy (which showed excellent recoveries between 97.5% and 100.4%) and linearity (which was proven in the range of 0.081-1.275 µg.mL-1 , with a linear regression of 0.999). The stress testing experiments proved that the developed methodology by the HPLC technique has stability-indicating characteristics, as all closely eluting peak pairs were separated well with a resolution of 2.3 and without any interference. The proposed methodology was highly efficient in separating and simultaneously determining the chiral impurities (enantiomers and diastereomers) of the solifenacin in the release and stability sample analyses of drug substances and tablets in pharmaceutical formulations.

Validated Stability-Indicating RP-HPLC Method for Daclatasvir in Tablets.

Objectives: The current study goal was to create a precise, sensitive, and validated reverse phase-high performance liquid chromatography (RP-HPLC) method for assessing the direct-acting antiviral daclatasvir (DCV) as well as to evaluate the stability of DCV in both drug and tablet formulations. The current investigation was to display stability indicating methods under different stress conditions, including hydrolysis (acidic, basic, and neutral), oxidation, and photolysis. Materials and Methods: All experiments were performed on HPLC Agilent 1100 with a stainless steel Hypersil C18 column having a particle size of 5 µm and a dimension of 4.6 x 250 mm. The mobile phase chosen was acetonitrile: 0.05% o-phosphoric acid (50:50 v/v) in isocratic mode with 0.7 mL/min flow rate and wavelength 315 nm was selected for detection. Results: This method was validated for linearity and range, accuracy, precision, limit of detection, limit of quantification, and robustness in accordance with International Council for Harmonisation (ICH) requirements. The results were satisfactory. It was observed that retention time (tR) was 3.760 ± 0.01 min. In acidic conditions, DCV degradans show tR at 3.863, 4.121, and 4.783 min and tandem mass spectrometry (MS/MS) spectra scans had m/z 339.1, 561.2 fragment ions. In basic condition, DCV degradans show tR at 5.188, 5.469 min and MS/MS spectra scans having m/z 294.1, 339.1, 505.2, 527.2 fragment ions. In oxidation conditions, DCV degradans shows tR at 4.038 min and MS/MS spectra scans having m/z 301.1 and 339.1 fragment ions were observed. Conclusion: All the mass fragments exhibited additional degradation observed for different stress conditions. This will help to identify the structure of the degradant and its pathways. No degradation was observed in neutral and photolytic conditions.

Stability-indicating method development and validation for quantitative estimation of organic impurities of the antidiabetic drug glipizide in drug substance and pharmaceutical dosage form using HPLC.

Glipizide is an antidiabetic drug used for the treatment of type 2 diabetes. A simple, reliable and robust reverse-phase liquid chromatographic method (RP-HPLC) was developed and validated as per International Conference on Harmonization Q2(R1) for estimating the impurities of glipizide in pharmaceutical formulations. The chromatographic separation was carried out on a Phenomenex Luna C18 (2), 250 × 4.6 mm, 5 µm with a binary solvent delivery system [MP-A, a homogenous mixture of water and acetonitrile in a ratio of 90:10 (v/v) and 1 ml of orthophosphoric acid; and MP-B, a homogenous mixture of water and acetonitrile in a ratio of 10:90 (v/v) and 1 ml of orthophosphoric acid] with a detection wavelength of 225 nm, a column temperature of 30°C, a flow rate of 1.5 ml/min, and an injection volume of 20 µl. All process, degradant and unknown impurities were separated well with a resolution >2.2 and were estimated accurately without any interference. The recovered values and regression values were 98.7-100.5% and R2 > 0.9999, respectively. The recovery and linearity studies covered the quantitation limit to 150% of the specification limit. The stability-indicating properties of the developed RP-HPLC method was assessed from the forced degradation studies. The developed method was successfully applied for real-time sample analysis of the glipizide dosage form.

Separation and quantitative estimation of stereo-selective enantiomers of montelukast in pharmaceutical drug substance and tablets dosage forms by using stability-indicating normal phase-HPLC method.

Montelukast sodium (MLS) is a leukotriene receptor antagonist that relieves asthma, bronchospasm, allergic rhinitis, and urticaria. A simple, robust, and stability-indicating normal phase high-performance liquid chromatography method was developed to separate and quantitatively estimate the S-enantiomer of MLS. The chiral separation was achieved using USP L51 packing material along with a mobile phase consisting of a solvent mixture (n-hexane, ethanol, and propionic acid), a flow rate of 1.0 mL/min, a detection wavelength of 284 nm, a column temperature of 30°C and an injection volume of 20 µL. The enantiomers peaks were well separated from the peaks of the placebo, diluting solvent, MLS, and its known impurities with a resolution of more than 2.2 and with no interference. Accuracy and linearity were studied in a range of 0.36-3.597 µg/mL (0.03%-0.30%), with good recoveries between 92.5% and 96.8% and a linear regression coefficient above 0.996. The suggested chiral chromatography method is being considered as an alternative and equivalent method to the United States Pharmacopeia and European Pharmacopeia monographs. The developed method was effectively employed for the study of release and stability samples of MLS. This HPLC method is also capable of separating and estimating the stereo-selective isomers (R- and S-enantiomers) of sulfoxide impurity of MLS in pharmaceutical medicine.

Stability-indicating normal-phase HPLC method development for separation and quantitative estimation of S-enantiomer of lacosamide in pharmaceutical drug substance and tablet dosage form.

Lacosamide (LA) is an antiepileptic medicine that is used to treat tonic-clonic seizures, partial-onset seizures, mental problems, and pain. A simple, effective, and reliable normal-phase liquid chromatographic technique was developed and validated to separate and estimate the enantiomer of (S-enantiomer) LA in pharmaceutical drug substance and drug product. Normal-phase LC was performed using USP L40 packing material (250 × 4.6 mm, 5 µm) and a mobile phase of n-hexane and ethanol at 1.0 ml min-1 . The detection wavelength, column temperature, and injection volume used were 210 nm, 25°C, and 20 µl, respectively. The enantiomers (LA and S-enantiomer) were completely separated using a minimum resolution of 5.8 and accurately quantified without any interference in a 25-min run time. Accuracy study for stereoselective and enantiomeric purity trials was conducted between 10 and 200%, with recovery values ranging from 99.4 to 103.1% and linear regression values >0.997. The stability-indicating characteristics were assessed using forced degradation tests. The proposed normal-phase HPLC technique is an alternate approach to the official monograph methods (USP and Ph.Eur.) of LA, and it was successfully used in the evaluation of release and stability samples for both tablet dosage forms and pharmaceutical substances.

Stability-indicating method development and validation for quantitative estimation of assay and organic impurities of antiviral drug baloxavir marboxil in drug substance and pharmaceutical dosage form using HPLC and LC-MS methods.

Baloxavir marboxil (BXM) is a polymerase acidic endonuclease inhibitor used as an antiviral drug. A simple, reliable, and robust liquid chromatographic method was developed and validated per International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) Q2(R1) for estimating the assay and impurities of BXM in drug substance and pharmaceutical formulations. The chromatographic separation was carried out on C18 (100 × 4.6 mm, 5 µm) with binary solvent delivery system (A:0.1% trifluoroacetic acid in water; B:0.1% trifluoroacetic-acid in acetonitrile) along with detection wavelength of 260 nm, column temperature of 57°C, flow of 1.2 mL/min and injection volume of 10 µL. All five known impurities and unknown impurities were separated well with resolution >1.7 and were estimated accurately without any interference. Recovered values and regression value were 99.5%-101.2% and R2  > 0.999, respectively. The recovery and linearity studies covered from 50% to 150% for assay, and quantitation limit, 120% for five BXM impurities. Stability-indicating property of the HPLC developed method was assessed from the forced degradation studies. The mass spectral data of unknown impurity formed under oxidation stress condition were discussed. The developed method was also successfully utilized for stability sample analysis of drug substance and tablet dosage form.

Green analytical chemistry and experimental design: a combined approach for the analysis of zonisamide.

Green analytical chemistry principles, as well as experimental design, are a combined approach adopted to develop sensitive reproducible stability indicating HPLC method for Zonisamide (ZNS) determination. The optimal conditions for three chromatographic parameters were determined using a central composite design of the response surface. Kromasil C18 column (150 mm × 4.6 mm, 5 µm) was utilized with ethanol, H2O (30:70 v/v) as a mobile phase at a flow rate of 1 mL/min at 35 °C. Good reproducibility and high sensitivity were achieved along (0.5-10 µg/mL) concentration range. In contrast, the TLC-densitometric method was performed on aluminum plates precoated with silica gel 60F254 as a stationary phase and chloroform: methanol: acetic acid (8:1.5:0.5 by volume) as a developing system. Reproducible results were obtained in the range of (2-10 µg/band). The chromatograms of HPLC and TLC were scanned at 280 nm and 240 nm, respectively. The suggested methods have been validated following ICH guidelines, and no statistically significant differences were detected between the results of the current study and the official USP method. It was also found that using experimental design implements the green concept by reducing the environmental impact. Finally, Eco-Scale, GAPI and AGREE were used to assess the environmental impacts of the suggested methods.

An ultra-high-performance chromatography method to study the long term stability of gemcitabine in dose banding conditions.

Gemcitabine is an analogue of cytidine arabinoside, used alone or in combination chemotherapy to treat various type of cancer. The dose-banding of gemcitabine provides the opportunity to anticipate the preparation of this anticancer drug on condition of carrying out stability studies. The aim of this study is to develop and validate a stability-indicating ultra-high-performance Liquid Chromatography (UHPLC) method for measuring the concentration of gemcitabine and to evaluate its stability at standardised rounded doses in polyolefin bags. The UHPLC with photodiode array (PDA) detector method was developed and validated (linearity, precision, accuracy, limits of detection and quantification, robustness and degradation test). Thirty polyolefin bags of gemcitabine (1600 mg/292 ml (n = 10), 1800 mg/297 ml (n = 10) and 2000 mg/303 ml (n = 10)) were prepared under aseptic conditions and stored at 5 ± 3 °C and 23 ± 2 °C for 49 days. Physical stability tests were periodically performed: visual and microscopic inspection and optical densities. The chemical stability was evaluated through pH monitoring and chromatographic assays. The results confirm the stability of Gemcitabine at selected standardised rounded doses of 1600 mg, 1800 mg and 2000 mg in NaCl 0.9% polyolefin bags for at least 49 days at 5 ± 3 °C and 23 ± 2 °C, allowing in-advance preparation.

A Validated Stability-Indicating HPLC-PDA Method for Tolnaftate: Identification, Characterization and In Silico Toxicity Predictions of Major Degradation Products.

Simple and rapid stability indicating High Performance Liquid Chromatography-Photo Diode Array (HPLC-PDA) method was developed and validated for the estimation of tolnaftate in the presence of its forced degradation products. The method employed SunQSil C18 column (250 mm × 4.6 mm, 5 µm) as stationary phase and acetonitrile:water (85:15, v/v) as mobile phase. Retention time of tolnaftate was 6.9 min. Acid and alkali hydrolysis, oxidation, photo degradation and thermal degradation studies were carried out to evaluate the degradation behavior of tolnaftate. The developed and optimized method was validated as per International Conferences on Harmonization (ICH) guidelines. Limit of detection and limit of quantitation were found to be 0.092 and 0.276 µg/mL, respectively. Linearity was observed in a concentration range of 0.276-6 µg/mL with R2 = 0.9936. %Recovery was found to be between 98.28% and 100.71%. The developed and validated Reversed Phase-High Performance Liquid Chromatography (RP-HPLC) method was successfully applied for quantification of tolnaftate in in-house topical solution. Major base and oxidative degradation products were identified and characterized by liquid chromatography-electrospray ionization mass spectrometry. The probable mechanisms for the formation of degradation products were predicted based on the fragmentation pattern of degradation products. The in silico dermal penetration predictions and carcinogenicity of degradation products were evaluated by using QikProp and CarcinoPred-EL functionality.

Stability of Morphine Sulfate-Clonidine and Sufentanil-Clonidine Mixtures.

BACKGROUND: Spinal analgesia is recommended for intractable cancer pain. Morphine-clonidine and sufentanil-clonidine are often used in association in intrathecal drug delivery systems, injected by intraabdominal pumps. To refill these pumps and to limit patient transport, it may be necessary to ship the mixtures in polypropylene syringes to peripheral establishments located near patient homes. The purpose of this study is to determine the stability of morphine-clonidine and sufentanil-clonidine mixtures in polypropylene syringes to ensure the best and safest transport conditions and in implantable pumps for intrathecal use. MATERIALS AND METHODS: The stability study method was conceived according to the International Council for Harmonization guidelines. For polypropylene syringes, four different mixtures of morphine-clonidine and sufentanil-clonidine were assessed over seven days. Two storage temperatures were tested (5 ± 3 °C and 25 ± 2 °C). For implantable pumps, two different mixtures of morphine-clonidine and sufentanil-clonidine were assessed over 28 days and stored at 37 °C. RESULTS: For the morphine-clonidine mixtures in polypropylene syringes, all mixtures remained stable for five days in both storage conditions (5 ± 3 °C and 25 ± 2 °C) because of relative concentrations systematically positioned between 90% and 110% (95% CIs of the mean of three samples). The two mixtures in implantable pumps remained stable for 28 days. For the sufentanil-clonidine mixtures in polypropylene syringes, cold conservation kept all the preparations stable for seven days, whereas a quick degradation was observed after only two days for ambient storage conditions. This result is similar to that with an implantable pump, in which the concentration is <90% on day 7 for low concentration mixtures. No visual modification, no significant pH modification, and no changes in turbidity assays were observed in either study. CONCLUSION: This study shows the stability of the morphine-clonidine mixtures in syringes stored at 5 °C for five days and in implantable pumps stored at 37 °C for 28 days. For the sufentanil-clonidine mixtures, the results show stability in syringes for seven days at 5 °C. Pump results show stability of seven days for low concentrations and 28 days for high concentrations.

Development and validation of a stability-indicating, single HPLC method for sacubitril-valsartan and their stereoisomers and identification of forced degradation products using LC-MS/MS.

The aim of this research work was to develop and validate a stability-indicating, single reversed-phase HPLC method for the separation of five impurities, including enantiomers, diastereomers, and degradation products in sacubitril-valsartan tablets. The method was developed using a Chiralcel OJ-RH column (150 × 4.6 mm, 5 µm) at 45°C with a gradient program of (T/%B) 0.01/25, 10.0/25, 25/38, 37.0/45, 39.0/25, and 45.0/25 at a flow rate of 0.8 ml/min. Mobile phase A consisted of 1 ml of trifluoroacetic acid in 1000 ml of Milli-Q water. Mobile phase B consisted of 1 ml of trifluoroacetic acid in a mixture of acetonitrile and methanol in the ratio of 950:50 (v/v). Sacubitril, valsartan, and their five impurities were monitored at 254 nm. Degradation was not observed when sacubitril-valsartan was subjected to heat, light, hydrolytic, and oxidation conditions. In acid degradation study (1 N HCl/60°C/2 h) impurity 1 (m/z 383.44) was formed, and in base degradation study (0.1 N NaOH/40°C/1 h) impurities 1 and 5 (m/z 265.35) were formed; both impurities were confirmed using LC-MS. The degradation products, enantiomers, and diastereomers were well separated from sacubitril and valsartan, proving the stability-indicating power of the method. The developed method was validated per the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use guidelines. The inter- and intra-day percentage relative standard deviation for sacubitril, valsartan, and their five impurities was less than 5.2%, recovery of the five impurities was between 93 and 105%, and linearity was ≥0.999. The limit of detection was 0.030-0.048 µg/ml, and the limit of quantification was 0.100-0.160 µg/ml.

Novel Eco-Friendly Stability Indicating Capillary Zone Electrophoresis Method for Determination of Aripiprazole in Tablet Dosage form: DoE Directed Optimization, Development and Method Validation.

In this work, a novel environment-friendly stability indicating capillary zone electrophoresis (CZE) method has been developed and validated for assaying the aripiprazole (ARP) in tablet dosage form. The separation of ARP from its degradation products and internal standard was achieved using a fused silica capillary column (30.2 cm x 75 µm ID), a background electrolyte containing 6 mmol L-1 ammonium formate buffer (pH 3) with 5% methanol under a potential of 15 kV and detection at 214 nm. The stability indicating ability of the method was investigated by analyzing ARP after being subjected to acidic, alkaline, thermal, photolytic, and oxidative stress conditions, according to ICH guidelines. Design of experiments was used during forced degradation and method optimization. Oxidation was the main degradation pathway among those evaluated. The drug was separated from its oxidative degradation products in less than 4 min. CZE method was linear between 60 - 140 µg mL-1, R2 = 0.9980, precise (intra-day 0.88% and inter-day 1.30%). The average recovery was 100.93 ± 0.77%. This is the first method in the literature for quantification of ARP in the presence of its related degradation products with high separation efficiency, low operation cost and minimum solvent consumption. This method could be helpful in the routine quality control analysis in the pharmaceutical industries with least harmful effect on the environment. CZE is considered an eco-friendly alternative of conventionally HPLC methods.

Development and validation of novel RP-HPLC method for midostaurin determination using analytical quality by design approach from regulatory perspective and determination of major degradation compounds of midostaurin using LC-MS.

Midostaurin (MTN), designated as an orphan medicinal product, is emerging as an important drug for treating acute myeloid leukemia and advanced systematic mastocytosis. The proposed method was developed and validated to evaluate the related impurities of MTN. The impurities were separated using a YMC Trait C18 ExRS column (150 × 4.6 mm, 3 µm). Mobile phase A consisted of a 10-mM concentration of phosphate buffer adjusted to pH 3.0 with diluted orthophosphoric acid, and mobile phase B consisted of 90% acetonitrile and 10% water. The optimized chromatographic conditions were as follows: flow rate, 0.5 mL min-1 ; injection volume, 10 µL; UV detection, 290 nm; and linear gradient program, up to 65 min. The method was developed using an analytical quality by design approach. A systematic flow chart shows the evaluation, control, and life cycle management method. As part of method evaluation, risk assessment was conducted. The method has been validated per current guidelines of the International Conference on Harmonization. The recovery study and linearity ranges were established from the limit of quantification to 150% optimal concentrations. The recovery was found to be between 95.5 and 102.5%, and linearity (r2 ) was 0.9998-0.9999 for all the identified impurities. The method precision results were achieved below 10% of relative standard deviation. Forced degradation studies were performed under chemical and physical stress conditions. The compound was sensitive to chemical stress conditions. During the study, the analyte degraded and was converted into the identified degradation impurities, and its molecular mass was found using the LC-MS technique.

Stability-indicating liquid chromatography method development for assay and impurity profiling of amitriptyline hydrochloride in tablet dosage form and forced degradation study.

Amitriptyline hydrochloride is an antidepressant drug with sedative effects used to treat the symptoms of anxiety, agitation with depression and schizophrenia with depression. A reversed-phase high-performance liquid chromatography method was developed to separate and quantitatively determine the assay and four organic impurities of amitriptyline in tablet dosage form and bulk drugs using a C18 column in an isocratic elution mode with mobile phase consisting of a mixture of pH 7.5 phosphate buffer and methanol. The pH conditions used in the chromatographic separation are discussed. The stability-indicating characteristics of the proposed method were proved using stress testing [5 m HCl at 80°C/1 h, 5 m NaOH at 80°C/1 h, H2 O (v/w) at 80°C/1 h, 6% H2 O2 (v/v) at 25°C/1 h, dry heat at 105°C/24 h and UV-vis light/4 days] and validated for specificity, detection limit, quantitation limit, linearity, precision, accuracy and robustness. For amitriptyline and its four known organic impurities, the quantitation limits, linearity and recoveries were in the ranges 0.25-3.0 µg/ml (r2 > 0.999) and 87.9-107.6%, respectively. The mass (m/z) spectral data of amitriptyline hydrochloride and its impurity are discussed. The proposed LC method is also suitable for impurity profiling and assay determination of amitriptyline in bulk drugs and pharmaceutical formulations.

Stability-indicating HPLC method development and validation of rivaroxaban impurities and identification of forced degradation products using LC-MS/MS.

The current aim of this study is to develop a simple, sensitive, and robust analytical method that can separate the Rivaroxaban and its related impurities by using HPLC. This new method can separate enantiomers and all the process-related impurities of rivaroxaban. This method can also be used to determine the assay of rivaroxaban in drug substances and drug products. This new method was developed using a Chiralpak IC (250 × 4.6 mm, 5 µm) column at 27°C with a gradient program of 25.0 min at a flow rate of 0.8 mL/min. Mobile phase A consisted of acetonitrile, ethanol, and n-butyl amine in the ratio of 95:5:0.5 (v/v/v), respectively. Mobile phase B consisted of a mixture of Milli-Q water, methanol, and n-butyl amine in the ratio of 50:50:0.5 (v/v/v), respectively. The detector wavelength was 254 nm. Rivaroxaban was subjected to the forced degradation conditions (stress conditions) of hydrolysis, base, acid, thermal, photolysis, and oxidation. The degradation products were well separated from rivaroxaban peak and enantiomer peak, and its potential impurities prove the stability-indicating power of the method. The proposed new stability-indicating method was validated with respect to specificity, precision, accuracy, limit of quantification, limit of detection, linearity robustness, and roundness per International Conference on Harmonization guidelines. The %RSD (relative standard deviation) for six sample preparations for rivaroxaban and impurities less than 10% and the recovery is between 90 and 110%. The current method can be used in quality control labs for analysis of commercial products.