C18 HPLC Research Articles

Development and Validation of a New Eco-Friendly HPLC-PDA Bioanalytical Method for Studying Pharmacokinetics of Seliciclib

Background and Objectives: Seliciclib (SEL) is the first selective, orally bioavailable potential drug containing cyclin-dependent kinase inhibitors. Preclinical studies showed antitumor activity in a broad range of human tumor xenografts, neurodegenerative diseases, renal dysfunctions, viral infections, and chronic inflammatory disorders. To support the pharmacokinetics and aid in therapeutic monitoring of SEL following its administration for therapy, an efficient analytical tool capable of quantifying the concentrations of SEL in blood plasma is needed. In the literature, there is no existing method for quantifying SEL in plasma samples. This study introduces the first HPLC method with a photodiode array (PDA) detector for the quantitation of SEL in plasma. Materials and Methods: The chromatographic resolution of SEL and linifanib as an internal standard (IS) was achieved on Zorbax Eclipse Plus C18 HPLC column (150 mm length × 4.6 mm internal diameter, 5 µm particle size), with a mobile phase composed of acetonitrile–ammonium acetate, pH 5 (50:50, v/v) at a flow rate of 1.0 mL min−1. Both SEL and IS were detected by PDA at 230 nm. The method was validated according to the ICH guidelines for bioanalytical method validation. Results: The method exhibited linearity in concentrations ranging from 50 to 1000 ng mL−1, with a limit of quantitation of 66.1 ng mL−1. All remaining validation parameters satisfied the ICH validation criteria. The environmental sustainability of the method was verified using three extensive tools. The proposed HPLC-PDA method was effectively utilized to study the pharmacokinetics of SEL in rats after a single oral administration of 25 mg/kg. Conclusions: The proposed method stands as a valuable tool for studying SELs for pharmacokinetics in humans. It aids in achieving the targeted therapeutic advantages and safety of treatment with SEL by optimizing the SEL dosage and dosing schedule.

Development of a RP-HPLC method for simultaneous determination of atenolol, metoprolol tartrate and phenol red for in-situ rat intestinal perfusion studies

Single-pass intestinal perfusion (SPIP) method is a widely used experimental model to determine the intestinal permeability of drugs. These studies are performed in the presence of a reference standard (metoprolol, MT) and a zero permeability marker (phenol red, PR). Therefore, it is important to develop a validated method for simultaneous determination of the investigated compound along with MT and PR. The aim of this study was to develop a reversed phase high-performance liquid chromatography (RP-HPLC) method with UV-detection for the simultaneous determination of atenolol (ATN), MT, and PR in the perfusion medium used in SPIP experiments. Separation of compounds were performed using an InertSustain C18 (250 × 4.6 mm, 5 µm) HPLC column at 35 °C. The mobile phase was a mixture of acetonitrile and phosphate buffer (pH 7.0, 12.5 mM) in gradient elution, and was delivered at a flow rate of 1 mL/min. The acetonitrile ratio of the mobile phase increased linearly from 10 to 35 % over 15 min. The injection volume was 20 µL, and ATN, MT and PR were detected at 224 nm. The retention times under optimum HPLC conditions were 5.028 min, 12.401 min, and 13.507 min for ATN, MT and PR, respectively. The developed RP-HPLC method was validated for selectivity, specificity, calibration curve and range, accuracy and precision, carry-over effect, stability, reinjection reproducibility, recovery and robustness. The method was linear for ATN (0.76–50 μg/mL), MT (1.14–50 μg/mL), and PR (0.47–20 μg/mL) with determination coefficients of 0.9999, 0.9994 and 0.9998, respectively. The results obtained for all validation parameters of the developed RP-HPLC method met the required limits of the ICH M10 Guideline.

Development and validation of HPLC-ultraviolet method for quantitative determination of pritelivir in human placental perfusion medium.

A simple and reliable HPLC-ultraviolet (HPLC-UV) method was developed and validated for the quantification of pritelivir in the samples of medium from the experiments utilizing the ex vivo technique of dual perfusion of the human placental lobule. Phenacetin was used as an internal standard (IS) in our HPLC-UV method. Chromatographic separation of pritelivir and phenacetin was achieved on a Waters Symmetry C18 HPLC column (100 × 2.1 mm, 3.5 μm) at ambient temperature (22-25°C). The mobile phase was composed of 50% methanol in deionized water (v/v), the flow rate for isocratic elution was established at 0.25 mL/min, and the detection wavelength for pritelivir and IS was set at 254 nm. Pritelivir and IS were extracted with the protein precipitation method using methanol as a solvent. The calibration curve for pritelivir exhibited linearity (r2 > 0.99) within the concentration range from 0.155 to 6.62 μg/mL. Within- and between-day accuracy ranged from 97% to 110% with relative standard deviation (RSD) values not exceeding 10%. The extraction recovery of pritelivir and IS ranged from 89% to 91% with RSD not exceeding 7%. Pritelivir was stable under the storage and sample handling conditions. This validated HPLC-UV method was utilized to quantify pritelivir in the placental perfusion medium samples, and the resulting concentrations were authenticated with incurred sample reanalysis to confirm the reliability of the method.

Study of the process of tetracycline absorption by soil and its transformation during acid desorption

Introduction. Currently, control over the soil entry of antibiotics used in agriculture is becoming an urgent problem of environmental hygiene. Tetracycline can enter the soil with the excrement of domestic animals and affect biological objects, both microbiological and plant. The chemical form of tetracycline in the soil and the options for its transformation, in particular during sample preparation for analysis, are factors that determine the intensity of this effect and the reliability of analytical control. The lack of information about the transformation of the drug under chemical influence determined the relevance of the research. Materials and methods. The studies were performed using the Stayer chromatographic system with a Shodex IC YS-50 150/4.6 cation column and a Kromasil C18 HPLC column with amperometric and conductometric detection. The objects of research were model soil samples with different tetracycline contents. When conducting research, soddy-podzolic soil from the «Nemchinovka» experimental field (Moscow region) and GSO No. chernozem were used. Results. Solutions obtained after acid-methanol desorption of tetracycline from soil samples were studied using HPLC and IC (Ionochromatographic methods). As a result of desorption, complex forms of substances, including both natural organic substances of the soil and tetracycline components, were established to pass into the aqueous phase. These forms of substances are fixed with a Shodex IC YS-50 cation exchange column and are not fixed by HPLC with a Kromasil C18 column. The possibility of isolating a chromatographic signal corresponding to the tetracycline component using ion chromatography has been demonstrated. Limitations. To create a method for measuring tetracycline in soil, it is necessary to test a larger number of soil species that differ in the content of organic matter to identify in detail the interfering effect of the organic matrix during chromatographic analysis. Conclusion. Research has shown tetracycline chemically to bind to soil matter, forming cationic complexes. After treating the soil with a mixture of concentrated hydrochloric acid and methanol, tetracycline is quantitatively extracted from the soil in combination with natural soil organic matter. These forms of substances are detected by a Shodex IC YS-50 cationic column and are not detected by HPLC with a Kromasil C18 column. Selective analysis of the cationic form containing the tetracycline moiety is possible.

Utilizing central composite design (CCD) in the optimization of new HPLC-PDA method for the assay of daridorexant: Application to the analysis in bulk and tablet dosage forms

In this study, we developed a robust Quality by Design guided reverse phase high-performance liquid chromatographic method for quantifying daridorexant with simplicity, speed, sensitivity, reproducibility, and cost-effectiveness. Utilizing a Waters e2690 alliance HPLC system with Empower 2® software control and a SpursilTM C18 HPLC column, critical method parameters such as flow rate, % aqueous buffer and buffer pH were identified through preliminary trials and risk assessment studies. Optimization via central composite design as part of a Design of Experiments framework, with retention time and tailing factor as critical analytical attributes, resulted in optimized chromatographic conditions: 30:70 (% v/v) phosphate buffer (pH 3) and acetonitrile as mobile phase, flowing at 1.0 mL/min, with detection at 270 nm. The method exhibited linearity within a range of 2.0-10.0 μg/ mL (correlation coefficient: 0.9997) and detection, quantitation limits of 0.089 μg/mL and 0.271 μg/mL, respectively. Validation according to ICH Q2 (R1) guidelines confirmed the method’s robustness, precision, accuracy, sensitivity, and high linearity for daridorexant estimation in both bulk drug and marketed formulations.

Correlation of solvent strength parameter with two molecular descriptors in reversed-phase liquid chromatography

A new hydrophobicity index has been previously developed and reported (Rev. Roum. Chim., 2023, submitted). The new index is based only on the solvent strength parameter (S) that describes the linear dependences (log k = log kw - S) between the logarithm of the retention factor (k) and volume fraction of the organic modifier in mobile phase () in an HPLC separation. The correlations between the new hydrophobicity index and octanol-water partition constant (log Kow) were evaluated and reported in present study for a set of 20 solutes by using seven C18 HPLC columns and two organic modifiers, typically utilized in reversed-phase HPLC applications (acetonitrile and methanol). All the correlations were good, showing values above 0.85. Correlations between the extrapolated values of retention factor to zero percent of the organic modifier in mobile phase, log kw, and log Kow showed also good determination coefficients R2, unlike an older hydrophobicity index based on the ratio between log kw and S, which was generally characterized by lower R2 than 0.8 for acetonitrile and 0.7 for methanol.

Design of Experimental Approach for Development of Rapid High Performance Liquid Chromatographic Process for Simultaneous Estimation of Metoprolol, Telmisartan, and Amlodipine from Formulation: Greenness and Whiteness Evaluation.

The design of an experimental approach, the Box-Behnken design, was implemented to optimize the chromatographic condition to develop a rapid HPLC procedure for quantification of a ternary mixture of metoprolol (MET), telmisartan (TEL), and amlodipine (AML) from the formulation. The perturbation plots, contour, and 3D response surface pictures were developed to study the impact of each variable on the analytes' retention time and the probable interaction between the parameters with fewer chromatographic runs. The optimized HPLC method separated the three analytes within 5 min with excellent selectivity and peak shape on a Zorbax C18 HPLC column using acetonitrile and phosphate buffer (20 mM, pH 5.8) with isocratic elution at a 1.1 mL/min flowrate. A wavelength 230 nm was utilized to monitor the elute. The validation of proposed method demonstrated a wide linearity range of 10-200 µg/mL for MET and TEL and 5-50 µg/mL for AML along with an excellent correlation coefficient. The correctness of the HPLC approach was further confirmed by excellent recovery of the added amount of analytes utilizing the standard addition technique. The recommended HPLC approach was employed safely for quality assurance of the formulation, because the evaluation of the method's greenness and whiteness confirmed the environmentally friendly nature of the approach.

Quantitative Estimation of Mangiferin and Molecular Docking Simulation of Salacia reticulata Formulation

The phytochemical constituents present in herbal products need to be determined in their prescribed strength in order to ensure their efficacy and product quality in formulations. The marker-based standardization of herbal products is a well-accepted concept. In this study Mangiferin is used as marker to estimate the amount of Mangiferin present in the formulation Salacia Lin. Veggie capsules containing 400mg of Salacia reticulata extract to treat type 2 diabetes mellitus. The method was validated as per the international conference on harmonization (ICH) guidelines that is applicable in industry as well as in academia. The method was developed using reverse phase, Analytical column used for the separation of analytes, Phenomenex HPLC C18 (250 X 4.6 mm, 5 μm). The run time was of 7 min. The mobile phase used was acetonitrile and orthophosphoric acid (pH adjusted to 3.5) in the ratio 85:15 at a flow rate of 0.5ml/min, column temperature was maintained at 28°C and a detection wavelength of 257 nm using a photodiode array detector. The optimized method that was developed resulted in the elution of Mangiferin at 4.83min and % recovery was between 97.09 to 101.57. Molecular docking investigation was done using Schrodinger software. The binding affinity of phytoconstituents present in Salacia reticulata to intestinal enzymes alpha amylase was investigated to study their possible inhibitory mechanism. The physicochemical and drug-likeness properties of the phytoconstituents were evaluated. The phytoconstituent salacinol showed highest docking score (- 9.592) with alpha amylase (2QV4 obtained from protein data bank) and Mangiferin showed a score of -8.235 in comparison with standard acarbose ( -10.274). Studies showed phytoconstituents Mangiferin, Salacinol and Kotalanol can be potential inhibitors of 2QV4, and potent drug candidates for T2DM. However, further studies on these phytoconstituents should be carried out by wet lab experiments to prove their effectiveness.

DEVELOPMENT AND VALIDATION OF NOVEL RP-HPLC METHOD FOR THE SIMULTANEOUS ESTIMATION OF SALICYLIC ACID AND BECLOMETHASONE DIPROPIONATE IN A TOPICAL OINTMENT DOSAGE FORM

Objective: The objective of the study is to develop and validate an analytical method to simultaneously estimate salicylic acid (SA) and beclomethasone dipropionate (BD) in a topical ointment formulation. Methods: The simultaneous estimation of SA and BD in a topical ointment formulation was carried out by developing and validating a novel, accurate, and economical isocratic reversed-phase high-performance liquid chromatographic (RP-HPLC) method. Separation was achieved by chromatographic technique using a prontosil HPLC C18 (250×4.6 mm) column with a particle size of 5 μm. The mobile phase utilized for this study includes methanol, acetonitrile, and 0.1% orthophosphoric acid in the ratio 50:35:15 v/v/v, respectively. The flow rate of 1 mL/min and column temperature of 28±2°C were set. The detection of the two drugs was carried out at 235 nm using an ultraviolet detector. It was observed that SA and BD were retained at 3.59 min and 6.00 min, respectively. Results: The RP-HPLC method was found to be linear with excellent correlation between peak areas and concentrations of 30–108 µg/mL for SA and 1–3.6 µg/mL for BD. The observed recovery data was obtained within the acceptance range of 98–102%, which confirmed the accuracy of the developed method. The two drugs, SA and BD, showed good resolution with a short analysis time of 7 min. Conclusion: The method was successfully created and validated in compliance with the recommendations of the International Conference on Harmonization for specificity, precision, linearity, accuracy, and robustness.

High-performance liquid chromatography with fluorescence detection method for measuring colistin in human serum and its application in critically ill patients treated with colistin methanesulfonate sodium

Colistin, in the form of colistin methanesulfonate sodium (CMS), has a narrow therapeutic window, and colistin levels must be monitored during treatment. The aim of this study was to develop and validate a high-performance liquid chromatography-fluorescence detection (HPLC-FLD) method for measuring colistin in human serum. Colistin was extracted from human serum using trichloroacetic acid and methanol for protein precipitation, followed by in-solid phase extraction derivatization with 9-fluorenylmethyl chloroformate. HPLC-FLD system using a reverse-phase HPLC C18 column and a mobile phase consisting of acetonitrile, tetrahydrofuran, and water (80%:4%:16%) were used for the quantification of colistin A, colistin B, and polymyxin B (internal standard) in human serum. The extraction recoveries were between 71% and 103%. Linear calibration curves were obtained for colistin in concentrations from 0.3 to 8.0 μg/mL, with good fit (r2 = 0.9993). The lower limit of quantitation was 0.3 μg/mL. The intra- and inter-day precision of the assay was 0.5–5% and 3.5–9.4%, respectively. The accuracy ranged from 98% to 100%. This validated method was successfully applied to the analysis of serum-receiving CMS in critically ill patients. This method will be useful to assist colistin dosage adjustment based on individual blood colistin levels for optimization of colistin therapy.

Validation of analytical HPLC with post-column injection as a method for rapid and precise quantification of radiochemical yields

Accurate assessment of isolated radiochemical yields (RCYs) is a prerequisite for efficient and reliable optimization of labeling reactions. In practice, radiochemical conversions (RCCs) determined by HPLC analysis of crude reaction mixtures are often used to estimate RCYs. However, incomplete recovery of radioactivity from the stationary phase can lead to significant inaccuracies if RCCs are calculated based on the activity eluted from the column (i.e. the summed integrals of all peaks). Here, we validate a simple and practical method that overcomes problems associated with retention of activity on the column by determination of the total activity in the sample using post-column injection. Post-column injections were carried out using an additional injection valve, which was placed between the outlet of the HPLC column and the inlet of the detectors. 2-[18F]Fluoropyridine ([18F]FPy) and 8-cyclopentyl-3-(3-[18F]fluoropropyl)-1-propylxanthine ([18F]CPFPX) were prepared with radiochemical purities of > 99.8% and mixed with [18F]fluoride at a ratio of 1:1 to simulate reaction mixtures obtained by radiolabeling reactions with an RCC of 50%. The samples were analyzed on three different C18 HPLC columns using neutral and acidic mobile phases. RCCs determined using the summed area of all peaks in the chromatograms were compared with those determined using post-column injection. Additionally, RCCs determined by post-column injection were corrected for activity losses before, during and after radiosyntheses to afford analytical RCYs, which were compared with isolated RCYs. Determination of RCCs based on the summed area of all peaks gave correct results under certain chromatographic conditions, but led to overestimation of the actual RCCs by up to 50% in other cases. In contrast, determination of RCCs using post-column injection provided precise results in all cases, and often significantly reduced analysis time. Moreover, analytical RCYs calculated from RCCs determined by post-column injection showed excellent agreement with isolated RCYs (<3% deviation). In conclusion, HPLC analysis using post-column injection enables reliable determination of RCCs independent of the chromatographic conditions and, together with a simple activity balance, rapid and accurate prediction of isolated RCYs.

Identification and Quantification of 29 Active Substances by HPLC–ESI-MS/MS in Lyophilized Swine Manure Samples

Veterinary drugs are frequently employed to treat and prevent diseases in food-producing animals to improve animal health and to avoid the introduction of microorganisms into the food chain. The analysis of the presence of pharmaceutical residues in animal manure could help to evaluate the legal and illegal practices during food production without harming the animals and to correctly manage manure when it is going to be applied as a fertilizer. This article describes a method for the simultaneous analysis of 29 active substances, mostly antibiotics and antiparasitic agents. Substances were extracted from lyophilized manure with a methanol:McIlvaine solution and analyzed with HPLC-ESI-MS/MS and a C18 HPLC column. The method was validated following European guidelines, the achieved trueness was between 63 and 128% (depending on the analytes), and the linearity was between 100 and 1500 µg/kg. The applicability of the method was demonstrated in 40 manure samples collected from pig farms where tetracycline was quantified in 7.5% of the samples. These results show the viability of this non-invasive method for the control of the legal and illegal administration of pharmaceuticals in food-producing animals.

Determination of clobetasol propionate in cosmetics by high performance liquid chromatography with pre-column fluorescence derivatization of 4-hydrazino-7-nitrobenzo [C] [1,2,5] oxadiazole

In this study, a fluorescent labeling reagent 4-hydrazino-7-nitrobenzo [c] [1,2,5] oxadiazole (NBD-NHNH2) was synthesized and used for fluorescence detection of clobetasol propionate in cosmetics by high performance liquid chromatography. In the presence of acidic catalyst (pH = 6.0), NBD-NHNH2 reacted at 45 °C for 30 min, and clobetasol propionate can be labeled quickly and efficiently. These derivatives have excellent stability and fluorescent properties. The maximum excitation wavelength was 450 nm, and the emission wavelength was 550 nm. The four cosmetic formulations were completely separated on a Waters e2695 HPLC C18 column by gradient elution. Under the optimal conditions, the standard curve showed a good linear response, and the correlation coefficient of r 2 = 0.9999. The LOD and LOQ were 0.01 ng/mL and 0.03 ng/mL, respectively. Therefore, NBD-NHNH2 was successfully applied in the detection of clobetasol propionate illegal added in cosmetics.

Synthesis of 5’-fluorophosphate-modified short-interfering RNAs

We have developed an improved scheme for the synthesis of a mono-fluorinated phosphoramidite for the 5’-modification of nucleic acids using standard phosphoramidite chemistry. We describe the first report of a phosphofluoridate modified siRNA strand and have evaluated C18 HPLC for purification of modified strands from unreacted siRNA strands. Lastly, the biological activity of the high purity siRNA strands, when placed on the sense and/or antisense strand, was evaluated to assess the impact of 5’ phosphofluoridate modifications on siRNA activity.

Green Chromatographic Method for Determination of Active Pharmaceutical Ingredient, Preservative, and Antioxidant in an Injectable Formulation: Robustness by Design Expert.

We report an efficient HPLC method for simultaneous qualitative and quantitative analysis of lincosamide antibiotic injectable formulations containing Clindamycin phosphate (CMN), benzyl alcohol (BA), and ethylenediaminetetraacetic acid (EDTA) as major ingredients. The three components were separated by Phenomenex prodigy C8 (250 mm × 4.6 mm, 5 μm) HPLC column, flow rate 1.1 mL/min, injection volume 30 μL, and column temperature 35 °C, using 0.05 M sodium acetate buffer (pH 4.5) with acetonitrile (ACN) in the ratio of 80:20 (v/v). The detection wavelength was set as 240 nm. The method was validated as per International Conference on Harmonization (ICH) guidelines and was confirmed to be specific, precise, accurate, and linear. Method robustness was executed by utilizing quality in the design of the experiment. Accuracy results were found to be 99.3–100.5% for CMN, 99.3–100.8% for BA, and 99.1–100.3% for EDTA. Precision results were obtained as % relative standard deviation (RSD): 0.6% for CMN, 0.4% for BA, and 0.4% for EDTA. Correlation coefficient (r2) values were obtained as >0.999 for the three components. Analytical solutions are stable for 48 h at benchtop and refrigerator conditions. The greenness of the analytical method was evaluated by the Green Analytical Procedure Index (GAPI), National Environmental Method Index (NEMI), analytical eco-scale, and Analytical Greenness (AGREE) tools to confirm that the method is eco-friendly.

HPLC Quantification and Stability Study of Antilipidemic Polyherbal Formulation

Hyperlipidemia is one of the major risk factor for cardio vascular disorders, which accounts for the one third of total death in the world. In the present study an Antilipidemic polyherbal formulation was prepared and contains four herbal drugs namely Murraya koenigi leaves, Acacia catechu bark, Emblica officinalis fruits and leaves of Gymnema sylvestra. The study involved HPLC marker-based quantification and shelf-life evaluation of a traditional Antilipidemic formulation. The quantification method for the biomarkers present in the polyherbal formulation was developed using reverse phase method by using shim-pack HPLC C18 column (250 X 4.6 mm, 5 ?m). The four phytoconstituents selected for the study are mahanine, quercetin, gallic acid and gymnemic acid respectively. The percentage content of quercetin, gallic acid, gymnemic acid and mahanine in the PHF was found to be 0.82%w/w,10.15%w/w, 2.77%w/w and 14.19%w/w respectively. Shelf life is an important component that is mandatory to be displayed on the label of all medicinal products. This is also applicable to Ayurveda. The shelf life of polyherbal formulations is also assessed in this study using an accelerated stability analysis. At a temperature of 40.2 °C and a relative humidity of 75%, physicochemical parameters were measured. The analysis was repeated at 1, 3, and 6-month intervals, with the average 10% deterioration time calculated and extrapolated to determine the shelf life. It was found that the shelf life of formulated Antilipidemic polyherbal formulation was 2 years 2 months. The polyherbal formulation prepared and evaluated can be effectively used for the treatment of hyperlipidemia and the preparation can be used without any deterioration for a period of 2 years.

Analytical method development and validation of rivaroxaban in bulk and pharmaceutical dosage form by using RP-HPLC

A simple, specific, accurate, and precise reverse phase high performance liquid chromatography (RP-HPLC0 method was developed and validated to analyse Rivaroxaban bulk dosage form. Using Sun Q C18 HPLC column separation was carried out. This was maintained at ambient temperature. During separation mobile phase consist of Acetonitrile : Buffer(sodium acetate buffer) 80:20 was delivered at a flow rate of 1mL/min. Using UV detector analysis was carried out at the wavelength 249 nm. RP-HPLC method was validated by using various parameter like system suitability, linearity, specificity, precision, limit of detection, limit of quantification and robustness. The RP-HPLC method was found to be linear over the concentration range from 5-30 µg/mL (r2 =0.994). Retention time for bulk rivaroxaban was found to be 3.69 min. LOQ of method was 1.331 µg/mL and LOD 0.439 µg/mL. Thus the developed RP-HPLC method was found to be robust which can be applied for the regular analysis of Rivaroxaban in the bulk as well as pharmaceutical dosage form

Analytical method development and validation for estimation of dolutegravir in tablet dosage form

A simple, specific, accurate, and precise reverse phase high performance liquid chromatography method was developed and validated to analyse Dolutegravir Sodium bulk dosage form. Using Princeton C18 HPLC column separation was carried out. This was maintained at 25ºC temperature. Shimadzu LC -20A prominence with LC solution softwarering separation mobile phase consist of Methanol: Water (0.1% OPA) in ratio of 80:20 v/v was delivered at a flow rate of 1mL/min. Using UV detector analysis was carried out at the wavelength 254 nm. RP-HPLC method was validated by using various parameter like system suitability, linearity, specificity, precision, limit of detection, limit of quantification and robustness. The RP-HPLC method was found to be linear over the concentration range from 10-60 µg/mL (r2 =0.9998). Retention time for bulk Dolutegravir Sodium was found to be 4.383 min. LOQ of method was 1.3274 µg/mL and LOD 0.4380 µg/mL. Thus the developed RP-HPLC method was found to be robust which can be applied for the regular analysis of Dolutegravir Sodium in the bulk as well as pharmaceutical dosage form.

Green Liquid Chromatography Method for the Determination of Related Substances Present in Olopatadine HCl Nasal Spray Formulation, Robustness by Design Expert.

Dual therapeutic nature drug mast cell stabilizer and histamine receptor antagonist olopatadine hydrochloride (OPT) nasal spray does not have an official monograph, and no literature is available. Eye drops formulation had the official monograph for impurities, but the determination was done in two methods. A simple and effective green liquid chromatography method to develop and validate for the related substances of OPT nasal spray formulation. A 25 min gradient method was employed to separate impurities and OPT with a 1.0 mL/min flow rate using a Boston green C8 (150 mm × 4.6 mm, 5 µm) HPLC column. The set wavelength and column oven temperatures were 299 nm and 30°C, respectively. pH 3.5 phosphate buffer-acetonitrile in the ratio of (70:30, v/v) as mobile phase A and (50:50, v/v) ratio as mobile phase B. A Quality by Design (QbD) based Design of Experiments (DoE) was employed to evaluate the robustness characteristics of the analytical method validation. The obtained RSD from the precision and intermediate precision was 0.4 to 4.1%. The % recovery of the impurities from LOQ to 150% of specification level was 87.5 to 110.3%. The linear regression curves for the impurities with a correlation coefficient of >0.999 indicate that all peak responses are linear with the concentration. The sample and standard solutions were stable for 24 h at benchtop and refrigerator conditions. All the critical peaks were well separated from the forced degradation studies' diluent, placebo, and generated degradation peaks. The method validation data and QbD based robustness study results indicate that the developed impurities method fits the routine quality control laboratory use. National Environmental Index (NMEI), Green Analytical Procedure Index (GAPI), Analytical Eco-scale and Analytical Greenness (AGREE) tools expressed the method's greenness. The proposed method is QbD utilized and green chemistry assessed impurities determination method for OPT in nasal spray formulation.

Implementation of Quality by Design Approach for Optimization of RP-HPLC Method for Quantification of Abiraterone Acetate in Solid Dispersion in Forced Degradation Studies

Background: Abiraterone acetate is a derivative of steroidal progesterone, used as a first-line therapy for metastatic castration of prostate cancer. Objective: The present study encompasses the design of an experiment approach for developing a simple, reliable, and rapid RP-HPLC method for the estimation of abiraterone acetate. Method: The chromatographic separation was efficiently conducted on a Hypersil Gold C18 (50 x 4.6 mm, 5 µm) HPLC column, using the mobile phase composition of acetonitrile: dibasic potassium phosphate (0.01 mM) in the ratio of 80:20 (%v/v) at pH 6.5 with an isocratic elution mode. Furthermore, the different force degradation study including hydrolysis, oxidation, thermal, and photolytic was performed for abiraterone acetate. Result: The dynamic linearity was established in the concentration range of 0.5-10 µg/mL with r2 of 0.9998. Furthermore, the limit of detection and the limit of quantitation were 0.0978 µg/mL and 0.3260 µg/mL. The degradation of abiraterone acetate was shown in both acidic (54.16 ± 0.247 after 24 hrs) and basic conditions (35.06 ± 0.458 after 24 hrs). Furthermore, the developed method was successfully employed to quantify abiraterone acetate in bulk powder and the solid dispersion did not show any change in the retention time. Conclusion: The developed method was validated according to the ICH Q2 (R1) specification, which was found to be sensitive, accurate, precise, robust, linear, and selective compared to the reported chromatographic method.