ICH Q2 Guidelines Research Articles

Anion exchange-HPLC method for evaluating the encapsulation efficiency of mRNA-loaded lipid nanoparticles using analytical quality by design

Lipid nanoparticles (LNPs) are emerging nucleic acid delivery systems in the development of mRNA therapeutics such as the severe acute respiratory syndrome coronavirus 2 vaccines. However, a suitable analytical method for evaluating the encapsulation efficiency (EE) of the LNPs is required to ensure drug efficacy, as current analytical methods exhibit throughput issues and require long analysis times. Hence, we developed and validated an anion-exchange HPLC method using Analytical Quality by Design. Three critical method parameters (CMPs) were identified using risk assessment and Design of Experiments: column temperature, flow rate, and sodium perchlorate concentration. The CMPs were optimized using Face-Centered Central Composite Design. The discriminating power of the optimized HPLC method and RiboGreen assay was comparable. The main advantage of this method is that LNPs can be directly injected into the HPLC system without bursting the LNPs loaded with encapsulated poly(A). The optimized HPLC method was validated as robust, high-throughput, and sufficiently sensitive according to the ICH Q2 guidelines. We believe our findings could promote efficient LNPs-based drug development.

ESTIMATION OF RESVERATROL AND GLABRIDIN IN GEL FORMULATION BY UV SPECTROSCOPY

Acne and hyperpigmentation are the most common problems in adolescents globally. Resveratrol and glabridin are proven to be effective against acne and hyperpigmentation. We developed a gel comprising these two actives for acne and hyperpigmentation treatment. The purpose was to establish a reliable UV method of analysis for quantification of resveratrol and glabridin, with high precision and accuracy. UV spectral analysis indicated 306nm and 281nm to be the λmax for resveratrol and glabridin, respectively, in methanol. The linearity studies conducted as per ICH guidelines Q2 R1 resulted in a correlation coefficient (r2 ) of 0.99. This method was used to assay the developed gel for resveratrol and glabridin content and demonstrated the suitability of this UV spectroscopic method for simultaneous quantification. It can be applied for other delivery systems with slight modification.

Determination of Mesalamine in Bulk and Suppository Dosage Forms through the Development and Validation of Stability- indicating RP-HPLC Method

Background: In the current study, a simple and cost-effective stability-indicating RP-HPLC method was developed and validated to estimate the mesalamine from both bulk and pharmaceutical dosage forms. Methods: An isocratic HPLC method using a reverse phase HiQSilC18 column (250 x 4.6 mm, 5μm) and a mobile phase methanol: ammonium acetate buffer (90:10 v/v) were employed as the mobile phase with a flow rate of 1 mL/min at 25°C. Detection was carried out at 305 nm, and the injection volume was 20μl. The developed method was validated as per ICH Q2 guidelines. Mesalamine has been subjected to various stress testing conditions, such as hydrolysis of acid and base, thermal degradation, oxidation, and photolysis. Also, methods have been validated with regard to linearity, accuracy, precision, and robustness. Results: The RT of mesalamine was determined to be 3.550 min ± 0.024 minutes, providing a reliable marker for its identification. The method was found to be linear between 5-30 μg/mL concentration with (R²) of 0.994. This demonstrated the method's ability to measure varying concentrations of mesalamine accurately. Additionally, the percentage recovery of mesalamine was approximately 100%, confirming the accuracy of the developed method. The parameters for system suitability have also been found to be within acceptable limits. Force degradation studies reinforced the method's selectivity and sensitivity in detecting mesalamine under various degradation scenarios. Notably, mesalamine significantly degraded in an acidic environment. Conclusion: In conclusion, our proposed RP-HPLC method provides a sensitive, accurate, and precise means of analyzing mesalamine in both bulk and pharmaceutical dosage forms.

Simultaneous Estimation and Stability Indicating Method of Vildagliptin And Dapagliflozin by RP-HPLC in Pharmaceutical Dosage Form

Vildagliptin and Dapagliflozin drugs are used for the management of diabetes.The primary objective of the proposed research was to develop and validate analytical methods for simultaneous quantification of vildagliptin and dapagliflozin in pharmaceutical dosage form. Both the drugs were subjected to force degradation to ensure stability of pharmaceutical products. The HPLC method development was carried out on C18 column (250mm*4.6mm,5µm particle size). The chromatographic separation was achieved by isocratic mode with a mixture of Methanol: 0.01% Trifluoro acetic acid (pH-2.78) in the ratio of 95:05 % v/v as mobile phase. Flow rate was kept at 0.8 ml/min and selected wavelength for estimation was 210 nm. Stress testing with acid, alkaline, oxidative and thermal degradation was performed. The retention time was found to be 2.282 min and 4.070 min for vildagliptin and dapagliflozin, respectively. The drugs response found to be linear with correlation coefficient value was nearly 0.999 for vildagliptin and dapagliflozin with linearity range of 10 – 60 µg/ml. The recoveries from studies were found to be 99.78% and 100.17% for vildagliptin and dapagliflozin respectively. LOD and LOQ were found to be lower, hence the method proved sensitivity. Method was validated as per ICH guideline Q2(R1). Drugs had shown more degradation in oxidative condition, 10.62% and 13.52% for vildagliptin and dapagliflozin, respectively. The proposed method would be successfully applied for the stability indicating, impurity separation, qualitative and quantitative estimation of vildagliptin and dapagliflozin in pharmaceutical dosage form and in process quality control analysis.

Comprehensive analysis of vitamin D3 impurities in oily drug products using supercritical fluid chromatography-mass spectrometry.

Vitamin D3, an essential micronutrient, often requires supplementation via medicines or food supplements, which necessitate quality control (QC). This study presents the development of a method for detecting and quantifying seven impurities of vitamin D3 in oily drug products using supercritical fluid chromatography-mass spectrometry (SFC-MS). Targeted impurities include two esters of vitamin D3 and five non-esters including four that are isobaric to vitamin D3. Firstly, a screening study highlighted the Torus 1-AA column and acetonitrile modifier as adequate for the separation, followed by optimization of the SFC conditions. Secondly, make-up solvent composition and MS settings were optimized to reach high sensitivity. For both the separation and MS response, the screening design of experiments proved useful. Lastly, a fast saponification and liquid-liquid extraction method was developed, enabling efficient sample cleanup and impurities recovery from the complex oily matrix. The SFC-MS method suitability was assessed in two validation studies. The first study employed the ICH Q2 guideline for impurity limit test to demonstrate method specificity and establish a limit of detection (LOD) and a limit of quantification (LOQ) at 0.2% and 0.5%, respectively, for ester impurities. The second study conducted a comprehensive quantitative assessment for three non-ester impurities using a total error approach, determining method validity through accuracy profiles. The validated method exhibited reliable performance across impurity concentrations from 0.1% to 2.0%, with estimated LODs ranging from 2 to 7ng/mL. This study further promotes SFC-MS as a valuable, versatile, and green tool for routine pharmaceutical QC.

ESTABLISHMENT AND AUTHENTICATION OF A UV SPECTROPHOTOMETRIC APPROACH FOR MEASURING EMBELIN CONTENT IN BULK AND FORMULATED SAMPLE

A UV-spectrophotometric method for the estimation of embelin isolated from Embelia ribes berries as per ICH guidelines Q2 (R1) was developed. It is simple, quick, accurate, and affordable. The wavelength of embelin was found to be 294.3 nm; and was linear in the concentration range 2-12 μg mL-1 with 0.997 correlation coefficient. The method was applied to pharmaceutical formulation and the drug estimated was found to be 97.99 % and was in good agreement with the label claim. The accuracy of the method was performed at three different levels, between 80 to 120 %; with % recovery obtained 98.54 - 99.98 %. The low values of % RSD indicate that the method is accurate and reproducible. Interday, intraday variations, and repeatability were studied as precision parameters. A lower % RSD value than 2 indicates the developed method is precise. Ruggedness of the proposed method was studied with the aid of two analysts.

A Robust HPLC Approach for Quantitation of Camptothecin in Mesoporous Silica Nanoparticles Matrix and in the Presence of Its Degradation Products.

Camptothecin is a potent anticancer drug used for the treatment of various cancers. The goal of this research investigation was to develop and validate a new stability-indicating HPLC technique for the quantitative assessment of camptothecin in in-house developed mesoporous silica nanoparticles, a novel nanoformulation matrix for the treatment of cancer. The Waters Inertsil® HPLC column (C18) was used for the chromatographic separation, with a flow rate of 1 mL/min, a column oven temperature of 40°C, an injection volume of 10 µL, a detection wavelength of 216 nm, and a 10 min runtime overall. An isocratic blend of phosphate buffer (10 mM, pH7.0) and acetonitrile (60:40, v/v) served as the mobile phase. Various stress conditions including acid, alkali, oxidative, photolytic, thermal, and humidity environments were tested for the quantitative estimation of the camptothecin through the proposed method. The results demonstrated that the proposed method is specific (peak purity ≥0.999), accurate (99.69-100.64% w/w), precise (RSD, % <2.0), and sensitive (LOD-0.17 µg and LOQ-0.56 µg) in accordance with ICH guideline Q2 (R1). Any unidentified degradation products did not interfere with the drug's estimation. Furthermore, the current method of analysis has eliminated any excipient interference from the matrix effect caused by the numerous excipients of the formulation matrix. To quantify camptothecin for routine assay purposes, this research work offers a novel and straightforward HPLC methodology with optimized chromatographic parameters, contributing to the research and development community while ensuring an appropriate and efficient use of the drug through a variety of nanoformulation for cancer treatment. The stability-indicating HPLC method was found to be specific and suitable for routine analysis of camptothecin. The absence of any interference from excipients was confirmed by forced degradation studies.

Spectrofluorimetric determination of butylated hydroxytoluene and butylated hydroxyanisole in their combined formulation: application to butylated hydroxyanisole residual analysis in milk and butter

Butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA) are two antioxidants that have been extensively used in many applications. Both are well known for their debatable health risks due to their multiple intake sources. Therefore, conservative limits are set for them in different regulations adapted to the matrices in which they exist. Here we present a simple spectrofluorimetric method for the determination of BHT and BHA based on their native fluorescence and synchronous scanning mode. The type of solvent and the interval between emission and excitation wavelengths were carefully optimized. Under the optimized conditions, good linearities were obtained between the emission intensity and the corresponding concentrations of BHT and BHA over the range of 3–18 µg/mL and 0.1–7 µg/mL, respectively with a good correlation coefficient (r > 0.99). The limits of detection were 0.9 and 0.02 µg/mL, and the quantification limits were 3 and 0.05 µg/mL for BHT and BHA, respectively. The suggested procedure was validated according to ICH guidelines Q2 (R1). Furthermore, the method’s greenness was assessed by three different methods, and it proved to be eco-reasonable. The method was successfully applied to the determination of BHT and BHA in pharmaceutical formulations. We also applied the suggested method for monitoring the residual BHA in conventional, powdered milk and butter, with good recovery in spiked samples.

Development and Validation of RP-HPLC Method for Routine Analysis of Osimertinib

Osimertinib is a newly approved treatment line for the non-small cell lung carcinoma actively caused by mutation in EGFR and T790M. The present study was targeted to originate a validated method for the qualification as well as quantification of osimertinib through RP-HPLC in bulk and its pharmaceutical formulation. The mobile phase, 65% triethylamine buffer (pH 2.5) and 35% acetonitrile (v/v) was run through a prontosil column (C18, 4.6×150 mm i.d., 3.5 μm particle size) at a flow rate of 0.7 ml/min. A PDA plus detector was used to scan the absorbance over 210 to 360 nm. Validation study performed following the ICH guidelines Q2 (R1) was found the developed method to be specific, precise, accurate, linear over the range of 64–96 μg/ml with R2 &gt; 0.999, robust and rugged. The % recovery of osimertinib at different levels ranged between 99.32% and 100.37% from tablet formulation (Osicent). The method holds promise for analysis of osimertinib in bulk, pharmaceutical formulations and for further research. Bangladesh Pharmaceutical Journal 27(1): 85-91, 2024 (January)

QbD-Based Stability-Indicating RP-HPLC Method Development and Validation for the Estimation of Favipiravir-An Eco-Friendly Approach.

Analytical quality by design (AQbD) affords a systematic scaffolding to triumph a continuously validated, robust assay as well as life cycle management. The resuscitative repurposed drug favipiravir, an oral drug approved for reemerging pandemic influenza in Japan in 2014, is used for the treatment of life-threatening pathogens such as Ebola, Lassa virus, and currently COVID-19. Favipiravir is gaining a great deal of medical importance due to its pharmaceutical applications. To develop and validate a risk-based stability-indicating RP-HPLC method employing an AQbD approach using Central Composite Design (Design Expert Software 13.0) for the estimation of favipiravir. The Quality Target Product Profile optimized were flow rate and mobile phase composition, thus assessing the critical analytical attributes (retention time, tailing factor, and number of theoretical plates) as the constraints of method robustness. The proposed technique was optimized with a C18 (150 × 4.6 mm, 5 µm) column and 0.1% orthophosphoric acid buffer-acetonitrile (50:50, v/v) as the mobile phase at a flow rate of 1 mL/min using diode-array detector (230 nm) eluted favipiravir at 2.3 min. The optimized method validated as per ICH guideline Q2 (R1) was found to be eco-friendly, simple, precise (RSD 0.0051-1.2%), accurate (99.86-100.22%), linear (25-150 µg/mL), rugged (RSD 0.70%), and robust (RSD 0.6-1.6%) with a limit of detection and limit of quantitation of 1.140 µg/mL and 4.424 µg/mL, respectively. Forced degradation studies (acidic, alkaline, thermal, photolytic, and oxidative conditions) revealed the suitability of the AQbD method for the analysis of favipiravir in tablet formulation.The developed and validated AQbD method is less time consuming and can be used in the industry for routine quality control/analysis of bulk drug and marketed Favipiravir products. A robust Design of Experiment enhanced stability-indicating analytical method was developed and validated for the estimation of favipiravir. Furthermore, the contemporary method would aid in extending the analysis of favipiravir in other formulations.

Development of chromatographic methods to determine multivitamins formulation depending on their solubility and polarity: comparative study using three greenness assessment tools

High performance liquid chromatography is one of the techniques of choice for the separation and quantitative determination of drugs in mixture form. Ipriflavone, ascorbic acid, pyridoxine, vitamin D3, and lysine are formulated together as an adjuvant combination in osteoporosis. In this work, we developed and validated two complementary high performance liquid chromatographic methods to determine the five compounds in their pharmaceutical dosage form. The first method (method A) was capable of determining ipriflavone, ascorbic acid, pyridoxine, and vitamin D3 in their bulk and combined pharmaceutical formulation. The method is based on Liquid Chromatographic separation with UV detection at 254 nm using Agilent Eclipse XDB-C18 column with a mobile phase consisting of 25 mM ammonium acetate buffer (pH 4.2): methanol in gradient mode. Due to the high polarity of lysine, it was difficult to achieve satisfactory retention on reversed phase columns. So, we separated it on a strong cation exchange column (Exsil 100 SCX) without derivatization with a mobile phase consisting of 10 mM sodium dihydrogen phosphate and 200 mM sodium chloride (pH 6) with UV detection at 210 nm (method B). Validation of the proposed methods was performed according to ICH guidelines Q2(R1). The proposed methods proved to be valid for selective analysis of the stated drugs in their bulk and combined pharmaceutical formulation. Greenness assessment of the developed methods was evaluated using three assessment tools: ESA, GAPI and the most recently developed tool AGREE, showing a satisfactory comprehensive guide of the greenness of the developed methods.

Stability Indicating Reverse Phase Ultra Performance Liquid Chromatography Method Development and Validation for Amiloride and Hydrochlorothiazide Tablet in the Presence of Degradation Products and Application to Green Analytical Principles

Amiloride (AML) and hydrochlorothiazide (HCTZ) combination is used alone or with other medicines to treat high blood pressure (hypertension). The present article provides the development and validation of an reverse phase ultra performance liquid chromatography (RP-UPLC) approach for the quantification of AML, HCTZ, and related impurities in pharmaceutical dosage forms. The analytical column used for the separation of impurities is the Waters X bridge C18 3.5 μm (50 mm X 4.6 mm). Used 0.1% formic acid and ethanol as MP in an isocratic mode with 0.3 mL/min as flow rate and 3 μL as injection volume, at 254 nm as detection in UV and 12 minutes as total run time. The samples were prepared specifically to undergo forced degradation, which involved subjecting them to hydrolysis, oxidation, thermal, and photolytic conditions. The technique underwent validation by the principles set forth by the ICH Q2 guidelines. The validation confirmed that the method possesses specificity, linearity, ruggedness, robustness and accuracy. The methodology employed exhibited a linear relationship extending from 10 to 150% for all impurities. The recovery analysis was conducted throughout a range of concentrations, starting from 10 to 150% concentration. The average recovery value was determined to be within acceptable limits. The evidence of degradation and the findings of the verified study suggest that the nature of the subject under investigation is stable. Hence, this approach might be employed within the domains of pharmaceutical research and development and quality control departments. Green analytical chemistry tools are used to assess the method’s greenness and calculated using GAPI, AGREE and ecological-scale and found excellent green of &gt;75%.

Development of Reverse Phase HPLC Method and Validation: Sofosbuvir and Velpatasvir Quantification in Bulk and Tablets

A new reverse-phase high-performance liquid chromatography (RP-HPLC) method for measuring the amount of sofosbuvir and velpatasvir in active pharmaceutical ingredients (API) and tablet forms has been developed and the method was also validated. Discovery column C18 150 mm x 4.6 mm, pore size of 5 μm was used for the analysis of drugs. Elution of drugs was achieved with 0.1% OPA Buffer: Acetonitrile mixed in the proportion of 40:60 v/v was used as mobile phase, maintaining 1.0 mL/min as flow rate, column temperature maintained at 30°C and uv detector wavelength was fixed at 272 nm. Sofosbuvir and velpatasvir were eluted at retention time (RT) of 2.183 and 3.038 minutes, respectively. Linearity parameter was performed in the levels from 25 to 150% and the square of correlation coefficient (r2) was found to be 0.9992. The %recovery obtained for sofosbuvir was 99.6 and for velpatasvir was 99.4. In precision %RSD for sofosbuvir was found to be 0.4 and for velpatasvir was 0.9. Validation was performed for the developed method and all of the validated parameter results obtained was within the ICH Q2 guidelines acceptance range. So, the developed RP-HPLC method can be used in pharmaceutical industry for quantification of sofosbuvir as well as velpatasvir at once in API and tablet dosage form.

Stability Indicating RP-HPLC Method Development and Validation for Simultaneous Estimation of Dapagliflozin Propanediol Monohydrate and Teneligliptin Hydrobromide Hydrate in Synthetic Mixture

For the quantitative measurement of Dapagliflozin propanediol monohydrate and Teneligliptin hydrobromide hydrate in synthetic mixture form in the presence of its degradants, precise, accurate, robust, cost-effective, and isocratic stability indicating RP-HPLC method was developed and validated. The mobile phase comprises [Methanol: 20 mM Ammonium formate (70:30 v/v)] at a flow rate of 1.0 ml/min, injection volume of 20 µl, and UV detection at 225 nm. Separation was accomplished using Gemini, C18 column. Teneligliptin hydrobromide hydrate and Dapagliflozin propanediol monohydrate were eluted with retention times of 6.65 minutes and 4.20 minutes, respectively. This procedure was approved following ICH guideline Q2 (R1). The calibration plots for Dapagliflozin propanediol monohydrate and Teneligliptin hydrobromide hydrate have correlation coefficients of 0.9995 and 0.9996 over the concentration ranges of 5-100 µg/ml and 10-200 µg/ml respectively. For Dapagliflozin propanediol monohydrate and Teneligliptin hydrobromide hydrate, accuracy ranged from 99.81-100.78% and 99.13-100.69%, respectively. For Dapagliflozin propanediol monohydrate and Teneligliptin hydrobromide hydrate, the LOD was found to be 0.947 µg/ml and 1.355 µg/ml. In contrast, the LOQ was 2.869 µg/ml and 4.107 µg/ml, respectively. The findings demonstrated the applicability of the devised approach for routine analysis of Teneligliptin hydrobromide hydrate and Dapagliflozin propanediol monohydrate in a synthetic mixture form with its degradants.

Continuous wavelet transform for solving the problem of minor components in quantitation of pharmaceuticals: a case study on the mixture of ibuprofen and phenylephrine with its degradation products

The presence of minor components represents a challenging problem in spectrophotometric analysis of pharmaceuticals. If one component has a low absorptivity or present in a low concentration compared to the other components, this will hinder its quantitation by spectrophotometric methods. Continuous Wavelet Transform (CWT) as a signal processing technique was utilized to figure out a solution to such a problem. A comparative study was established between traditional derivative spectrophotometry (Numerical Differentiation, ND) and CWT to indicate the advantages and limitations of each technique and possibility of solving the problem of minor components. A mixture of ibuprofen (IBU) and phenylephrine (PHE) with its degradation products forming a ternary mixture was used for comparing the two techniques. The two techniques were applied on raw spectral data and on ratio spectra data resulting in four methods, namely ND, CWT, Derivative Ratio-Zero Crossing (DRZC) and Continuous Wavelet Transform Ratio-Zero Crossing (CWTR-ZC) methods. By comparing the results in laboratory prepared mixtures, CWT technique showed advantages in analysis of mixtures with minor components than ND. The proposed methods were validated according to the ICH guideline Q2(R1), where their linearity was established with correlation coefficient ranging from 0.9995 to 0.9999. The linearity was in the range 3–40 μg/mL for PHE in all methods, while for IBU it was 20–180 and 30–180 μg/mL in CWT and ND methods, respectively. The CWT methods were applied for quantitative determination of the drugs in their dosage form showing the ability of the methods to quantitate minor components in pharmaceutical formulations.

Quantification of Oligonucleotides Using Tandem Mass Spectrometry with Isobaric Internal Standards.

In recent years, oligonucleotides have become more important in research, drug approvals and medical therapies. Due to this growing interest in pharmaceutical applications, it is essential to develop reliable analytical methods for this substance class. In this work, we present a quantification method using liquid chromatography coupled with tandem mass spectrometry by applying an isobaric oligonucleotide standard. In addition to a proof of principle, we perform a method qualification to assess its readiness for validation according to ICH Q2 guidelines. In addition to good linearity, sensitivity, accuracy and recovery, the method showed no significant matrix effects. Furthermore, we demonstrated the application of the method by applying the quantification in a biological matrix, as well as an exemplary degradation of an oligonucleotide in bovine plasma.

Green HPLC Method for Simultaneous Analysis of Three Natural Antioxidants by Analytical Quality by Design.

Glutathione, silybin, and curcumin are well-known potential antioxidants that are recommended as adjuvant therapy in cancer treatment. Based on the principles of Analytical Quality by Design (AQbD) and green analytical chemistry, a simple, robust, and environmentally benign HPLC method for the simultaneous estimation of glutathione, silybin, and curcumin in bulk and formulation was performed. Elution was achieved by an Agilent Eclipse XDB C18 (150 mm × 4.6 mm id, 3.5 μm) column using a gradient mobile phase composed of ethanol-water pH 6.7 (with 0.1%, v/v orthophosphoric acid) and 1.07 mL/min flow rate with PDA detection at 215 nm. Critical method variables were identified by risk assessment using an Ishikawa diagram, and multivariate optimization of the experimental conditions for the HPLC technique was accomplished by central composite design using design of experiments (DoE) software. The separation was achieved within 15 min, where the retention time of glutathione, silybin, and curcumin were 3.3, 4.9, and 7.3 min, respectively. The standard curve was linear in the range of 3.75-26.25 µg/mL for glutathione, 62.50-437.50 µg/mL for silybin, and 12.5-87.50 µg/mL for curcumin. The developed method was validated as per ICH guidelines Q2 (R1), and all the parameters are within specified limits. The proposed method is simple, precise, and robust, which can be employed for routine analysis and also concluded to be a greener approach according to AGREE, Green Analytical Procedure Index, and analytical eco-scale tools. The chosen antioxidants were evaluated for the very first time simultaneously using the chromatographic technique in bulk and dosage forms employing green solvents. The peak purity of all three compounds was studied using a PDA detector. Wastage was reduced in terms of time, cost, and solvents by employing AQbD elements and tools. Complete application of environmentally sustainable safe solvents were employed.

Characterization of Forced Degradants of Tegafur, Gimeracil, and Oteracil Potassium by Liquid Chromatographic-Electrospray Ionization-Mass Spectrometry and Simultaneous Estimation of Triple Combination in Drug Substance and Finished Pharmaceutical Dosage Form.

Tegafur, gimeracil, and oteracil potassium are widely used pharmaceuticals to treat lung cancers of the gastrointestinal tract, such as those of the oral cavity, esophagus, colon and rectum, and pancreas, as well as non-small cell lung cancers. The literature review revealed that no study has yet offered a completely stability-demonstrating, validated liquid chromatography-mass spectrometric approach for the concurrent estimation of tegafur, gimeracil, and oteracil potassium, along with all known degradation products. The simultaneous detection of tegafur, gimeracil, and oteracil potassium and their forced degradation product characterization necessitated the invention of a simpler, faster, and less expensive method. Therefore, this study aimed to follow the ICH method validation standards to develop and validate a fast, easy, and rugged liquid chromatography-mass spectrometry (LC-MS) technique for the concurrent estimation of tegafur, gimeracil, and oteracil potassium in the drug substance and the finished dosage form. Tegafur, gimeracil, and oteracil potassium were examined on the Waters HPLC Alliance system, coupled to the SCIEX QTRAP 5500 mass spectrometer, and endowed with an interface capable of carrying electrospray ionization. The tegafur, gimeracil, and oteracil peaks eluted at retention times of 2.338 min, 3.756 min, and 5.338 min, respectively. The limit of detection values of tegafur, gimeracil, and oteracil were detected to be 0.6, 0.174, and 0.474 μg/mL, respectively. The results for the quantification limit were calculated at 2.0, 0.58, and 1.58 µg/mL concentrations, respectively. Tegafur, gimeracil, and oteracil had linear ranges of 50-300 µg/ml, 14.5-87 µg/ml, and 39.5-237 µg/ml, with regression coefficients of 0.99956, 0.99986, and 0.999479, respectively. The accuracy values of tegafur, gimeracil, and oteracil in the ranges of 50%, 100%, and 150% were determined at 99.9%, 99.9%, and 99.4%, respectively. The RSD for the six replicates was less than 2% for precision. According to the ICH Q2 guidelines, this approach was effectively evaluated with LC-MS to validate the chemical structures of the freshly created tegafur, gimeracil, and oteracil degradation products. An accurate and sensitive LC-MS technique was developed and validated for the concurrent quantification of tegafur, gimeracil, and oteracil potassium in the drug material and the medicinal dosage form.

Characterization, quantification and a multi-computational in silico toxicity assessment of impurity (feruloyl methane) in synthetic curcumin using RP-HPLC-UV technique

Feruloyl Methane (FM) is a common impurity in Synthetic Curcumin (SC) that affects its purity and potency. The identification and quantification of FM is crucial to ensure the quality and safety of SC based drugs. The current study aims to develop and validate a simple, rapid and cost-effective analytical technique for the precise and accurate quantification of FM in SC using RP-HPLC with a UV-Vis detector (Ultraviolet/Visible) and assessment of its toxicity by multi-computational methods. The developed HPLC method with a UV-Vis detector enabled accurate identification and quantification of FM in SC. The optimized method was validated in accordance with ICH guidelines Q2(R1) and all parameters were found to be within the standard acceptance range. The ideal run time was determined to be 10 min and the impurity eluting at a retention time of 2.65 min was characterized using spectral techniques viz., mass spectrometry, FTIR and 1 H NMR, confirming the presence of FM. The amount of FM in SC was estimated to be 8.26 µg/kg. In addition, toxicity assessments using in silico tools such as ProTox- II, ADMETlab 2.0 and PASS Online indicated that the presence of FM in SC is not safe for human consumption. In conclusion, the developed method is not only capable of quantifying FM but also aids in distinguishing Natural Curcumin (NC) adulterated with SC and can be applied to a wide range of fields such as natural drug analysis, food analysis and toxicity prediction.

RP-HPLC Method Development for Simultaneous Estimation of Brimonidine Tartrate and Timolol Maleate in Ophthalmic Preparation

Introduction: A simple, precise, rapid, and reproducible RP-HPLC method is developed for the simultaneous estimation of brimonidine tartrate and timolol maleate present in ophthalmic dosage forms.&#x0D; Method: Gradient RP-HPLC is carried out at 35℃ on column C18 (25 cm X 4.6 mm, 5 µm) with mobile phase a ACN and buffer (25 ml ACN &amp; 475 ml buffer) and mobile phase B water and ACN (37.5 ml water &amp; 212.5 ml ACN) at a flow rate of 1.2ml/min. detection is carried out using DAD detector at 248 nm. Parameter such as linearity, accuracy, precision, specificity, LOD, LOQ and robustness are studied as per ICH guidelines Q2 (R1). &#x0D; Results: The linearity range of Brimonidine tartrate and Timolol maleate are 10-50 µg/ml and 4-20 µg/ml. The accuracy for both Brimonidine Tartrate and Timolol Maleate are in range of 100-101%. The RSD for Brimonidine Tartrate and Timolol Maleate are less than 2 for precision, specificity, robustness respectively. &#x0D; Conclusion: This RP-HPLC method can be highly suitable for analysis of brimonidine tartrate and timolol maleate without interference in combined dosage form.