Precision RSD Research Articles

Analytical method development and validation for Estimation of Calcium Dobesilate and Docusate Sodium in Pharmaceutical dosage form with QbD approach

Analytical method development and validation for estimation of Calcium Dobesilate and Docusate Sodium in Pharmaceutical dosage form with QbD approach. Combination of Calcium Dobesilate and Docusate Sodium helps for the treatment of Haemorrhoid. There is no any UV spectroscopy method of this combination due to less absorbance of the Docusate Sodium in various solvent. A specific, precise and accurate RP-HPLC method has been developed and validated for estimation of Calcium Dobesilate and Docusate Sodium in Capsule dosage form. Apply QbD approach by using statistical tool Design of experiment with Box-Behnken design for the optimization of the screening method of RP-HPLC method. Calcium Dobesilate and Docusate Sodium were estimated on Thermoscientificsyncronis C18 (250mm X 4.6mm), 5μm column using Acetonitrile: 0.01M Tetrabutylammonium dihydrogen phosphate pH-4.8(66:34, v/v) as mobile phase with flow rate 1.0 ml/min and detection was carried out at 214 nm. The retention time of Calcium Dobesilate and Docusate Sodium were found to be 2.943 min and 4.190 min respectively. The linearity and range was found to be 50-450 μg/mL for Calcium Dobesilate and 10-90μg/mL for Docusate Sodium. % RSD of precision was found to be less than 2%. %RSD for Robustness parameters was found to be less than 2%. % Recovery of Calcium Dobesilate and Docusate Sodium at different levels were found in the range of 99.451% - 100.082% and 98.071% - 101.187% respectively. So, the developed method was precise, robust and accurate. The assay value for Calcium Dobesilate and Docusate Sodium was found to be 99.772% - 99.874% and 98.159% - 98.736% respectively.

Method Development and Validation of Canagliflozin by using RP-HPLC in Pure and Tablet Dosage Form

A new method was established for simultaneous estimation of Canagliflozin by RP-HPLC method. The chromatographic conditions were successfully developed for the separation of Canagliflozin by using INERTSIL column, C18 (150x4.6)5µm column, flow rate was 1ml/min, mobile phase ratio was Water: Acetonitrile (70:30), detection wavelength was 264nm. The instrument used was Hitachi HPLC Auto Sampler, Separation module 1575. The analytical method was validated according to ICH guidelines (ICH, Q2 (R1)). The linearity study for Canagliflozin was found in concentration range of 1μg-5μg and 100μg-500μg and correlation coefficient (r2) was found to be 0.999 and 0.999, %mean recovery was found to be 100% and 100.5%, %RSD for repeatability was 0.2 and 0.4, %RSD for intermediate precision was 0.5 and 0.1 respectively.

RP-HPLC METHOD FOR SIMULTANEOUS ESTIMATION OF METFORMIN HCL AND CANAGLIFLOZIN IN PHARMACEUTICAL FORMULATION

A simple, specific, accurate, precise and stability-indicating RP-HPLC method was developed and validated for the simultaneous estimation of metformin HCl and canagliflozin in pharmaceutical formulation. The method was developed using the Altima C8 column (150 ×4.6 mm, 0.5 μm) with a mobile phase consisting of acetonitrile and 0.1 % orthophosphoric acid in water (62:38 %V/V) with a flow rate of 1 mL/min. Detection was carried out at 254 nm using a PDA detector. The retention time for metformin HCl and canagliflozin was found to be 2.282 and 3.339 min, respectively. The proposed method was validated for linearity, range, accuracy, precision, robustness, LOD and LOQ. Linearity was observed over a concentration range 15-225 μg/mL for metformin HCl (r2 =0.9995) and 5-40 μg/mL for canagliflozin (r2 =0.9988). The % RSD for intraday and interday precision was found to be 0.13 and 0.20 for metformin HCl and 0.18 and 0.20 for canagliflozin. The LOD and LOQ were found to be 0.16 μg/mL and 0.54 μg/mL for metformin HCl and LOD and LOQ were found to be 0.05 and 0.21 μg/mL for canagliflozin. Metformin HCl and canagliflozin were subjected to stress conditions of degradation including acidic, alkaline, oxidative, thermal and photolysis.

A new simple RP-HPLC Method development, Validation and Forced degradation studies of Bilastine

A new simple, rapid, accurate and precise method for estimation of Bilastine in pharmaceutical dosage form by reverse phase liquid chromatography. The developed method employed mobile phase was Acetonitrile and Ammonium acetate pH 5.0 adjusted with glacial acetic acid with 85:15% v/v and flow rate 1.0ml/min. Method was developed using column C18 Water (150 × 4.6mm, 5µm) and detection wavelength was 215nm. The retention time was found to be 2.519 min. the proposed method was successfully applied to the determination of Bilastine in dosage form. High linearity of developed method was confirmed over concentration range of 25- 150 µg/ml and co-relation co-efficient is 0.996. The percentage RSD for precision and accuracy of the method was found to be less than 2%. The recovery was in the range of 99 – 102% and limit of detection was found to be 0.45µg/ml and limit of quantification was found to be 1.20µg/ml. Bilastine was found to degrade under acid and oxidation conditions. There was no interference of excipient and degradation product in retention time so method was specific. Analytical parameter such as precision, accuracy, limit of detection, limit of quantification and robustness were determined according to international Conference on Harmonization (ICH) guidelines.

Method Development and Validation to Estimate Sofosbuvir in Marketed preparation by UV-Spectroscopy and HPLC along with force Degradation Study

Simple, swift, selective and accurate UV and HPLC methods were developed and validated for estimation of sofosbuvir in bulk and marketed preparation. In the UV spectroscopy method mobile phase used was methanol in 70:30 ratio with a detection wavelength of 260nm and the assay value obtained was 99.36%. The method was validated as stated by ICH in Q2 R1 guidelines in which linearity was detected from 06-30µg/ml range with regression value of 0.999. In the accuracy, precision and robustness studies RSD were below 2%. In HPLC method, Cosmosil C18 (250mm×4.6ID, Particle size: 5µ) column was utilized with methanol: water (70:30) as mobile phase, 0.9ml/min of flow rate, 260nm detection wavelength for estimation of sofosbuvir. Assay value obtained using this optimized parameters was 99.77% with the time of retention of around 4.3 minutes. HPLC method was also validated as stated by ICH in Q2 R1 guidelines in which linearity was noticed to be in the span of 10-50µg/ml with 0.999 of regression coefficient. LOD and LOQ values of the optimized method were 0.5764 and 1.7468µg/ml. In the accuracy, precision, robustness studies the value of RSD was under 2%. The optimized HPLC method was also utilized for the force degradation study, in which it was found that sofosbuvir is susceptible to oxidative, acid, alkaline, photolytic and thermal degradation. From this study it can be concluded that the developed methods can be employed in the routine analysis for sofosbuvir estimation in bulk and marketed preparation and also to determine degradation of drug.

UV Spectrophotometric Method Development and Validation of Luliconazole in Bulk and Formulation

Objective: A new, simple, economical, sensitive, precise and reproducible UV visible spectrophotometric method was developed for the estimation of luliconazole in pure form and pharmaceutical formulation as per ICH guidelines. Method: A UV spectrophotometric method has been developed using methanol and water as solvent to determine the luliconazole in bulk and pharmaceutical dosage formulation. The λmax of luliconazole in methanol and water was found to be 297nm. Results: The drug was proved linear in the range of 3-15µg/ml and exhibited good correlation coefficient (R2= 0.9993) and excellent mean recovery (98-99%). The % RSD for intra-day and inter-day precision was found to be 1.051288 and 1.138658 respectively. The LOD and LOQ of Luliconazole was found to be 1.1168µg/ml and 3.3845µg/ml respectively. This method was successfully applied to luliconazole content in marketed brands and results were in good agreement with the label claims. Conclusion: The method was validated for linearity, precision, repeatability and reproducibility. The obtained results proved that the method can be employed for the routine analysis of luliconazole in bulks as well as in commercial formulations.

Implementing Analytical Quality by Design (AQbD) Approach for Simultaneous Estimation of Tadalafil and Macitentan by RP-HPLC Method

The principle motive behind this work is to develop robust, low cost, and regulatory flexible method for Tadalafil and Macitentan by RP-HPLC using Analytical Quality by Design principles. Failure Mode and Effects Analysis was performed and the risk priority number was calculated, from this analysis independent variables were selected. Optimization of chromatographic condition was performed using Central Composite Design. The separation was achieved using phenomex gemini C18 (150 mm, 4.6 mm, 5μm) column using mobile phase as methanol: 10 mM ammonium formate (74.1 : 25.9 % v/v) (pH: 6.8) at a flow rate 0.94 ml/ min with detection wavelength at 260 nm and 40°C column oven temperature. This method was validated according to ICH Q2 (R1) guideline. Analytical Quality by Design method was numerically optimized with a desirability of 1.0. ANOVA result shows that model was significant for all responses. The method was found to be linear in the range of 0.1-25 μg/ml for Macitentan and 0.4-100 μg/ml for Tadalafil. Percentage recovery was found in the range of 98.6-101.7 % for Macitentan and 98.8-101.8 % for Tadalafil. The RSD of precision and repeatability was found beneath 1 %. AQbD driven method can be highly suitable for analysis of Tadalafil and Macitentan without interference.

A Highly Sensitive LC–MS/MS Method Development and Validation of Fedratinib in Human Plasma and Pharmacokinetic Evaluation in Healthy Rabbits

Background: A simple and sensitive quantitation analytical technique by liquid chromatography–tandem mass spectrometry (LC-MS/MS) is essential for fedratinib in biological media with kinetic study in healthy rabbits. Objective: The main objectives of the present research work are to LC-MS/MS method development and validate procedure for the quantitation of fedratinib and its application to kinetic study in rabbits. Methods: Separation of processed samples were employed on zorbax SB C18 column (50mm×4.6 mm) 3.5µm with a movable phase of methanol, acetonitrile and 0.1% formic acid in the ratio of 30:60:10. The movable phase was monitored through column at 0.8 ml/min flow rate. The drug and ibrutinib internal standard (IS) were evaluated by monitoring the transitions of m/z -525.260/57.07 and 441.2/55.01 for fedratinib and IS respectively in multiple reaction monitoring mode. Results: The linear equation and coefficient of correlation (R2) results were y =0.00348x+0.00245 and 0.9984, respectively. Intra and inter-day precision RSD findings of the developed technique were found in the range of 2.4 - 5.3% for the quality control (QC)-samples (252.56, 1804.0 and 2706 ng/ml). The proposed method was subjected to pharmacokinetic study in healthy rabbits and the kinetic study, fedratinib showed mean AUClast 13190±18.1 hr*ng/ml and Cmax was found to be 3550±4.31 ng/ml in healthy rabbits. Conclusion: The validated method can be applicable for the pharmacokinetic and toxicokinetic studies in the clinical and forensic analysis of fedratinib in different kinds of biological matrices successfully.

Development and Validation of a UV Spectrophotometric Method for the Determination of Dioscin in Tribulmon Tablets

Objective: Develop and validate a novel, sensitive, precise and straightforward UV spectrophotometric method to determine the amount of dioscin in tribulmon tablets. Methods: tribulmon tablets were prepared in the laboratory from medicinal plants Tribulus Terrestris L, native to Mongolia. Extract solutions were prepared from a single batch of tribulmon tablets, and the amount of dioscin was determined by UV spectrophotometric method using a calibration curve of known solutions of the dioscin. A chromogenic reaction was used to determine the amount of dioscin in the tablets. The maximum absorption was 201 nm. Validation parameters were evaluated following ICH guidelines. Results: The correlation coefficient of linearity for the UV spectrophotometric method was 0.9996. The standard working solution was linear from 3.2 to 18.9 μg/mL. The Limit of Detection (LOD) and Limit of Quantitation (LOQ) were 1.9 μg/ mL and 5.9 μg/mL, respectively. The recovery test accuracy was ranged from 97.9 to 100%. The %RSD for repeatability and intra-day precision was 0.45%, 0.13–0.50%, respectively. The %RSD for robustness was 0.26% to 0.40%, respectively. Conclusion: Based on the results of the proposed UV spectrophotometric method, the method we developed to determine dioscin content in tribulmon tablets from Tribulus Terrestris L is sensitive, precise, stable and straightforward. The test’s validation parameters comply with ICH requirements.

Validated Stability Indicating Method for Determination of Indapamide in Pharmaceutical Formulation

A stability indicating method for Indapamide in an oral antihypertensive tablet and bulk dosage forms was developed and validated by reverse phase HPLC (RP-HPLC). The proposed RP-HPLC Shisheido Capcell Pak C18 column was used and column dimension was 250mm × 4.6mm i.d., 5μm partical size at ambient temperature. The mobile phase Methanol and Phosphate Buffer pH 5.8 (60:40) was run in isocratic mode at a flow rate of 1.0ml/minute. The wavelength of Indapamide was selected at 241nm. Indapamide was found as single peak in the chromatogram at 6.58 min. The different chromatographic conditions were applied to determine the drug in pharmaceutical dosage form. The linearity of the proposed method was found in a range of 0.2-1.2μg/ml with coefficient of correlation (r2) value 0.999, slope 8360 and intercept -77.78. The percent RSD of intraday precision and interday precision were found in range of 0.498 - 1.661 and 0.882 - 1.623 respectively. The average accuracy of the method at three different levels of 50, 100 and 150 % were found to be 100.67, 99.50 and 100.65, respectively. The LOQ and LOD expressed as signal to noise ratio (S/N) were found to be 0.03 and 0.08μg/mL with good accuracy and precision. Indapamide was exposed to different stress conditions like alkaline, acidic, thermal, oxidative and photolytic. The stressed samples were analyzed by the proposed method. The proposed methods can be used for routine analysis stability testing and assay of Indapamide in quality control laboratories.

Multivariate optimization and evaluation of quaternary mixture in bulk and co-formulated dosage forms by central composite design

BackgroundThe current study describes the use of central composite design for multivariate optimization of resolution and retention time, taking into account different critical method parameters like organic phase, pH, flow rate, and wavelength for risk assessment. The chromatographic method for the assay of the most effective anti-viral regimen (EPCLUSA, DARVONI, and HARVONI) was developed. An experimental design was presented by sequential investigation of four independent parameters. The method was developed using XTERRA C18 (250 mm × 4.6 mm, 5 μm particle size) column in isocratic mode using potassium dihydrogen phosphate buffer (pH adjusted to 5) and acetonitrile (50:50 % v/v) as mobile phase at a flow rate of 1.0 ml/min and UV detection wavelength of 260 nm.ResultsThe separation of four drugs with fine resolution and preferable retention times was achieved. Retention times of four drugs were found to be 2.96, 3.91, 7.15, and 11.94 min for daclatasvir, sofosbuvir, velpatasvir, and ledipasvir, respectively. The percentage accuracy of labelled claim was in the range of 99–102%, and the pooled %RSD for repeatability, precision, and accuracy was less than 2%.ConclusionThe suggested method was applied for quantification and identification of studied drugs in tablets; the results agreed with the label claim and were validated according to the ICH guidelines. The optimized method can be used for pharmacokinetic and quality control studies.

Stability Indicating RP-HPLC Method Development and Validation for Estimation of Diroximel Fumarate in bulk and its dosage forms

Aim and objective: The aim of present research work is to develop validated RP-HPLC stability indicating method for the quantification of Diroximel Fumarate in bulk and its pharmaceutical dosage forms. Materials and methods: Chromatographic method was carried on C18 column (Waters 250mm x 4.6mm, 5m). Mobile phase was prepared by mixing water: Acetonitrile: 85% OPA: in the ratio of 70:30. The flow rate was 0.1 mL/min and the injection volume was 20μL. The absorbance maxima of Diroximel Fumarate was measured at 215nm. The retention time was found to be 2.390 min. Result: The method was proved to be specific and linear in the range of 50-150μg/mL with correlation coefficient of 0.999. The % RSD for precision was found to be less than 2% and the mean percentage recovery was 100.15%. All the validation parameters were statistically validated according to ICH guidelines and were found to be within acceptance criteria. Conclusion: The developed method was simple, specific, precise, accurate and robust. The described HPLC method can be successfully employed for the analysis of Diroximel Fumarate.

Method Development and Validation of Evening Primrose oil and its Pharmaceutical dosage form by Simple UV- Spectrophotometric Method

In the present work, simple and sensitive UV spectroscopic method has been developed for the quantitative estimation of Evening Primrose Oil in efem capsule. Cyclohexane was used as solvent. In the range of 100-500μg/ml, the linearity of Evening Primrose Oil shows a correlation co-efficient of 0.999. The UV detection wavelength was 233nm. The percentage RSD for precision and accuracy of the method was found to be less than 2%(LOQ) of 23μg/ml and (LOD) of 7.5μg/ml. The method was validated as per the ICH guidelines. The method was successfully applied for routine analysis of Evening Primrose Oil and in formulation dosage form.

Determination α-amylase inhibitor activity of methanol extract of coffee leaves using UV-Vis spectrophotometric method and validation

Diabetes mellitus (DM) is a chronic condition caused by the decrease of insulin. Some studies have shown that coffee leaves act as antidiabetic agents. DM can be characterized by high blood glucose levels. To maintain blood glucose levels in normal conditions can be through the inhibition of carbohydrate catabolism such as inhibition of α-amylase. Assay of α-amylase inhibition of coffee leaves extracts in vitro using UV-Vis Spectrophotometric method has been developed to obtain optimum condition. The optimum condition of measurement wavelength, incubation time, substrate concentration, enzyme concentration, and DNS concentration was 540 nm, 15 min, 0.5 mg/mL, 0.5 U/mL, and 4.38 ppm respectively. The inhibitor concentration of acarbose and extract was 25 ppm and 500 ppm. Evaluation of the validation method showed linear result r = 0.9979 (acarbose); r = 0.997 (extract). Detection limit and quantitation limits of acarbose and extract were 4.7377 ppm; 14.2133 ppm and 108.3539 ppm; 325.0618 ppm. RSD (%) of repeatability precision and intermediate precision test for day 1, day 2, and day 3 were 4.899; 4.899; 6.502 and 4.566 for acarbose and 0.394; 0.394; 0.377 and 0.238 for the extract. The accuracy test showed that the IC50 profile decreased as the number of acarbose additions increased. The IC50 of arabica coffee leaves was 286.804 μg/mL.

Determination of sarecycline by UPLC-MS/MS and its application to pharmacokinetic study in rats

AbstractSarecycline is a narrow-spectrum antibiotic for the treatment of acne, which is a chronic inflammatory disease of the hair follicle sebaceous glands. In the study, UPLC-MS/MS was used to establish a rapid and accurate analytical method. The sarecycline was determined with poziotinib as internal standard (IS) in rat plasma. An ACQUITY UPLC HSS T3 column (2.1 × 100 mm, 1.8 μm) could performe chromatographic separation with the mobile phase (methanol: water of 0.1% formic acid) with gradient elution. The ions of target fragment were m/z 488.19→410.14 for sarecycline and m/z 492.06→354.55 for poziotinib, which could quantify the electrospray ionization of positive multiple reaction monitoring (MRM) mode. The linear calibration curve of the concentration range was 1–1,000 ng/mL for sarecycline with a lower limit of quantification (LLOQ) of 1 ng/mL. The mean recovery was between 82.46 and 95.85% for sarecycline and poziotinib in rat plasma. RSD for precision of inter-day and intra-day were between 3.24 and 13.36%, and the accuracy ranged from 105.26 to 109.75%. The developed and validated method was perfectly used in the pharmacokinetic study and bioavailability of sarecycline after intravenous and oral administration in rats.

Analytical Method Development and Validation for Determination of Selpercatinib by Using RP-HPLC

A novel, specific and precise RP-HPLC method has been developed and validated for the quantification of selpercatinib in pure and its pharmaceutical dosage form. Selpercatinib is a kinase interrupter with increased selectivity for rearranged during transfection (RET) tyrosine kinase receptors (RTKs) above the additional RTK classes. It is used for the treatment of RET fusion-positive non-small cell lung carcinoma (NSCLC). The segregation was accomplished on the Zorbax C18 column (150 x 4.6 mm) with a 5 µ particle size. 0.1% orthophosphoric acid and Acetonitrile (ACN) (60:40 v/v) was used as an optimized mobile phase at a flow rate of 1ml/min. The wavelength selected was 220.0nm. The retention time for selpercatinib was 2.653 min. The linearity of selpercatinib was detected to be 5- 30μg/ml. Linearity equations obtained for selpercatinib was y = 18428x + 2196.2 with correlation coefficient 0.999. The % RSD for precision was found to be less than 2%. %Recovery was obtained as 99.74% for selpercatinib. The LOD and LOQ for selpercatinib were obtained as 0.02 µg/ml and 0.05 µg/ml.

Method Development and Validation for Estimation of TinofovirDisproxil Fumarate in Pharmaceutical Preparation by UV-Spectroscopy and HPLC With Force Degradation Study

Tinofovir Disoproxil Fumarate (TDF) is an acyclic nucleotide diester analog of adenosine monophosphate from the antiviral category utilized in the AIDS and hepatitis B treatment. In the present study, two analytical methods, i.e. UV and HPLC were developed for the TDF’s estimation in pharmaceutical preparation. In the UV method, the mobile phase used was Methanol and water in 60:40 ratio for estimation of the drug at 260 nm and an assay of the (TDF) obtained was 99.53%. The method’s validation was carried out as per ICH Q2 R1 guidelines in which linearity was detected from 10-50 µg/ml range with a regression value of 0.999 Percent RSD value of accuracy, precision and robustness were below 2. In the HPLC method estimation of (TDF) was evaluated on Cosmosil C-18 (250mm×4.6ID, Particle size: 5 µ) column utilizing Methanol: Water (60:40), 0.9 ml/min of flow rate, detection wavelength was 260 nm and the time of retention observed was around 4.63 minutes with the assay value 99.07%. HPLC method was also validated according to ICH guidelines, where linearity was detected in the 10-50 μg/ml of range with the regression coefficient value 0.999. The % RSD of precision, accuracy, and robustness was below 2%. The study of forced degradation was also performed by the HPLC method utilizing methanol: water (60:40) at 260nm. From this study, it can be concluded that the developed methods for estimation of (TDF) drug in pharmaceutical preparation are simple, accurate, precise, and can be utilized in the routine analysis for quantification of the drug in a dosage form.

Low‐level determination of 4‐chlorobutyl‐(S)‐[4‐chloro‐2‐(4‐cyclopropyl‐1,1,1‐trifluoro‐2‐hydroxy‐but‐3yn‐2‐yl)phenyl] carbamate (4‐CTHC) in efavirenz drug substance by LC–MS

A rapid, simple, sensitive, and selective liquid chromatography-tandem mass spectrometry (LC-MS) test method was developed and validated for the trace level determination of 4-chlorobutyl-(S)-[4-chloro-2-(4-cyclopropyl-1,1,1-trifluoro-2-hydroxy-but-3yn-2-yl)phenyl] carbamate (4-CTHC). 4-CTHC is a potential genotoxic impurity in efavirenz drug substance and the acceptable level is 2.5 μg ml-1 with respect to analyte concentration according to ICH M7(R1) Multidisciplinary Guidelines M7(R1). The LC-MS/MS analysis of 4-CTHC impurity was carried out on a Kinetex C18 (150 × 4.6 mm, 5.0 μm) column. In this test procedure, the mobile phase was prepared with buffer (0.1% formic acid in water) and acetonitrile in the ratio of 1:3 (v/v). The method set flow rate was 0.4 ml min-1 . The method was developed with a short run time of <10 min. Selective ion monitoring acquisition mode and negative polarity electrospray ionization mode were used as an MS method to quantify genotoxic impurities at 422.25 Da. The method showed linearity in a range of 0.64-3.71 μg ml-1 with a correlation coefficient of 0.9992. The RSD for intra-day and inter-day precision was found to be <5%. The method's accuracy was in the range of 106.5-112.4% for the genotoxic impurity of 4-CTHC. The procedure was validated as per the current ICH Q2 (R1) guidelines and proved suitable for stability testing in the quality control laboratory for pharmaceutical preparations.

HPLC method validation for quantification of tetrahydrocurcumin in bulk drug and formulation

BackgroundTetrahydrocurcumin (THC), the active metabolite of curcumin, is gaining popularity amongst scientist due to its wide spectrum of pharmacological activities, better stability and colourless nature. The objective of this study was to develop a sensitive, cost-effective RP-HPLC method for the estimation of THC in bulk drug substance and formulation.ResultsEfficient chromatographic separation was achieved on Hypersil BDS, C18 column, 250 mm × 4.6 mm, 5 μm column by isocratic elution with mobile phase comprising of acetonitrile: methanol: water (40:23:37% V/V); adjusted to a pH of 3.0 ± 0.05. The flow rate of the mobile phase was 1.0 ml/min with a column temperature of 25 °C. UV detector was used for the analysis and detection was carried out at 280 nm. The developed method was validated according to ICH guidelines with respect to system suitability, linearity, accuracy, precision and robustness. The theoretical plates were found to be more than 5800. The method showed linearity over the range of 4 to 60 μg/ml with R2 = 0.9998. The accuracy of the method in terms of recovery study was 98.23-99.99%. The %RSD for intra-day and inter-day precision were 0.272 and 0.275, respectively. The method was found to be robust with respect to change in wavelength, flow rate and column temperature.ConclusionThe analytical method was found satisfactory on validation as per ICH guidelines. Hence, it can be routinely used for quantification of THC in bulk drug and formulation.

Interest of flow injection spectrophotometry as an orthogonal method for analyzing biomolecule aggregates: Application to stressed monoclonal antibody study

This study aimed to explore the suitability of flow injection spectrophotometry (FIS) to analyze three degraded therapeutic monoclonal antibodies (bevacizumab, nivolumab, and rituximab). For this purpose, aggregates were generated with stirring, freeze-thaw, and heat stresses. The intact and stressed mab samples were filtered with 0.22 µm hydrophilic filters and analyzed by size exclusion chromatography (SEC), cation-exchange chromatography (CEX), and FIS. In terms of quantitative and qualitative analysis, protein loss and structural changes were assessed. Various aggregates profiles were obtained according to the mabs and the stresses. FIS allowed performing very satisfactory quantifications for each mab with intermediate precision RSD < 3.0 % and recovery between 97.9 and 102.0 %. From the protein loss measurements, it appears that SEC underestimates the mab aggregate proportions up to two times less as compared with FIS since the latter avoids any non-specific interactions (electrostatic or hydrophobic interactions). Using second derivative spectroscopy and multivariate data analysis, we noticed apparent structural differences, located in the regions 245–265 nm for rituximab and nivolumab and 280–300 nm for bevacizumab, depending on the stress. The FIS complementarity with the other techniques used in this study allowed us to demonstrate that the three mabs behave differently for a given stress condition. While extreme mechanical stress formed large aggregates irrespective of the mabs, rituximab showed to be less stable and more sensitive than the two other mabs under freeze-thaw and heat stresses, generating large aggregates (>200 nm) and partial unfolding. Nivolumab tends to form small aggregates less than 50 nm when heated and freeze-thawed. Moreover, freeze-thaw seems to generate native IgG-1 aggregates with rituximab. Similarly, bevacizumab showed to form these IgG-1 aggregates and was resistant to freeze-thaw, likely thanks to trehalose cryoprotectant from its formulation. Finally, FIS associated with multivariate analysis can provide rich information in one single run and appears to be a fast, simple, and reliable method to set complementary and orthogonal approaches for protein aggregates monitoring.