A novel simultaneous high performance liquid chromatography-PDA method for the determination of Tenofovir AF, Darunavir, Emtricitabine and Cobicistat in bulk and its application to marketed formulation

Background The present research article involves the simultaneous determination of Tenofovir alafenamide, Darunavir, Emtricitabine and Cobicistat in bulk as well as in tablet dosage form using high performance liquid chromatography. Result The separation was performed using DIKMA Spursil, C18, ODS, (4.6 × 150 mm × 5 µm) analytical column using the mobile phase acetonitrile and 0.1% Orthophosphoric acid in the volume ratio of 70:30 at pH 3. The eluents were detected using PDA detector at 254.0 nm. After optimization subsequent validation study of different parameters was performed by utilizing the optimised condition as per the ICH guidelines. Under this optimised conditions Tenofovir alafenamide, Darunavir, Emtricitabine and Cobicistat were eluted at 2.287 min, 2.507 min, 4.062 min, 6.011 min respectively. Percentage assay was found 99.21% for Tenofovir alafenamide, 99.80% for Darunavir, 99.80% for Emtricitabine and 99.84% for Cobicistat. Tenofovir alafenamide was found linear in the range of 2.0–10.0 µg/mL, Darunavir (160.0–800.0 µg/mL), Emtricitabine (40.0–200.0 µg/mL) and for cobicistat (30.0–150.0 µg/mL). The corelation coefficient was found 0.999 for all the APIs. The detection limit was found 0.14 µg/mL for Tenofovir alafenamide, 2.14 µg/mL for Darunavir, 0.6 µg/mL for Emtricitabine and 7.32 µg/mL for cobicistat. In the LOQ study the quantitation limit was found 0.47 µg/mL for Tenofovir alafenamide, 7.12 µg/mL for Darunavir, 2.10 µg/mL, for Emtricitabine and 24.42 µg/mL for cobicistat. Conclusion All the studied API’s has been highly resolute utilizing the optimised condition and found extremely suitable for the determination of all of them simultaneously in marketed dosage form as well as in the bulk form.

Development of Validated RP-HPLC Method for Estimation of Empagliflozin and Metformin in Combined Formulation

Aim: The aim of the present study include development of validated RP-HPLC method for estimation of Empagliflozin and Metformin in combined dosage form by using LC-MS compatible volatile mobile phase. Methodology: Appropriate separation of drugs was achieved using C18 column as a stationary phase and Acetonitrile: Water (50: 50, v/v) at a flow rate 1mL/min as mobile phase. Detection was done at 230 nm. Results: The Rt of Metformin and Empagliflozin was found to be 2.20 ± 0.02 min and 3.64 ± 0.02 min respectively. When the marketed formulation was analyzed by the developed method, the % drug contents were found to be 98.57 ± 1.28 and 99.86 ± 1.02 %w /w for Empagliflozin and Metformin, respectively. The method was found to be linear in a range of 11.25 – 56.25 μg/mL for Empagliflozin and 85 – 425 μg/mL for Metformin. Detection limit and quantitation limit were found to be 0.30 and 0.92 μg/mL for Empagliflozin and 1.12 and 3.36 μg/mL for Metformin, respectively. The accuracy and precision results were found to be near 100 % w/w for both the drugs. The method was also found to be robust and specific. Conclusion: The developed RP-HPLC method was found to be linear, sensitive, accurate, precise, specific and robust for the analysis of Empagliflozin and Metformin in combined dosage form.

Validation Of Spectrophotometry-Visble Method On The Determination Of Borax Levels In Meatballs

Borax, in illictic additive substance, is added on certain food product as a e preservative and rubbery. Therefore, the determination of borax in the food product such as meatball is very impotant in view of meatball is a food product often consumed by community. Vis- Spectrophotometric method with curcumine 0.125% as a reagent and glacial acetic acid-sulphuric acid has been used for determination of borax in this research. Curcumin reagent was selected because sensitivity of the method and the reproducibility of the results are affected by quality of the reagent other than rigorous observance of the reaction conditions (temperature, time, reagent quantities). Glacial acetic acid- sulphuric acid was used to create acid condition, so that curcumin and boron form a violetred 2:1 complex called rosocyanin. The optimum result was obtained when 1/. ml solution of 0.125% curcuumin and 1.0 ml concentrated sulphuric acid were added and the absorbance was measured after 70 minutes at 547 nm. The results showed linear regression y = 1.3127x – 0.0994, r = 0.9690 > r table (n = 5) is 0.878 and p = 0.007 (p< 0.01) and Vxo is was 15,53%. The detection limit and quantitation limit were 9.7.10-4 ppm and 2.94. 10-3 ppm respectively. The recovery and coefficient variation were 47.56%±3,92%. Determination of borax in three meatball samples which were taken from a location in Surabaya showed that the sample contained borax with concentration of 0.0205; 0.0151; 0.0210 (% w/w) respectively.

Validation of the Developed Zero-Order Infrared Spectrophotometry Method for Qualitative and Quantitative Analyses of Tranexamic Acid in Marketed Tablets

(1) Background: The functional groups present in tranexamic acid allow direct infrared detection analysis. This study aimed to develop, apply, and validate an infrared spectrophotometry method used for qualitative and quantitative analyses of tranexamic acid in marketed tablets. (2) Methods: This was a descriptive observational study that consisted of several stages: determining the specific wavenumber for analysis, obtaining a simple linear regression equation, analyzing tranexamic acid both qualitatively and quantitatively, and validating the developed method for routine analysis. (3) Results: The peak analysis obtained a range of baseline wavenumbers from 1679.17 to 1295.25 cm−1. The regression equation obtained was Y = 310.8527 × X + 0.9718, and the coefficient of determination (R2) obtained was 0.9994. The tranexamic acids in marketed tablets overall have a similarity index value of more than 0.90 and overall have levels ranging between 97.0% and 103.0%. The infrared spectrophotometry method that was successfully developed, applied, and validated for qualitative and quantitative analyses of tranexamic acid in marketed tablets meets the requirements both qualitatively and quantitatively of the tablet monograph. (4) Conclusions: The infrared spectrophotometry method has been validated and meets the requirements for accuracy, precision, detection limit, quantitation limit, linearity, range, and specificity.

QUANTIFICATION OF BILASTINE AND MONTELUKAST COMBINATION IN FORMULATIONS UTILIZING LIQUID CHROMATOGRAPHY: STABILITY STUDIES

Objective: We have developed a “stability-indicating RP-HPLC” procedure for the Bilastine (BLS) and montelukast (MTL) analysis of tablets. Methods: The quantification of BLS and MTL combination was implemented utilising a Waters column (C18, 5 μm, 250 mm and 4.6 mm). Isocratic mobile phase had 60% volume KH2PO4 of 0.1M strength with pH 4.2 units and 40% volume methanol at a flow with 1.0 ml/min speed. UV detection at 232 nm was done to examine BLS and MTL. Stability experiments of BLS and MTL under distinctive environments of stress were also performed. Results: The BLS and MTL were eluted at 1.810 min and 2.551 min, respectively. The responses were found to be linear for the concentration ranges of 10-30 µg/ml (BLS) and 5-15 µg/ml (MTL). Percent comparative standard deviance for precision was 0.331% (BLS) and 0.486% (MTL). Percent assay for accuracy was 98.96% (BLS) and 99.00% (MTL). The detection limit and quantitation limit measures for BLS were 0.018 µg/ml and 0.059 µg/ml, respectively, while for MTL it was 0.024 µg/ml and 0.081 µg/ml, respectively. Robustness studies authorized that the method is robust with percent comparative standard deviance of a highest 1.950%. Conclusion: The developed “stability-indicating RP-HPLC” procedure for the BLS and MTL analysis is simple, sensitive, precise, specific and robust, making it appropriate to the assessment of BLS and MTL in a tablet formulation.

New stability indicating RP-HPLC-PDA method for determination of mifepristone in bulk and tablet formulation

Background Mifepristone is progestational and glucocorticoid hormone antagonist. The objective of this study is to develop simple and economical stability indicating RP-HPLC method for the determination of mifepristone in bulk and tablet formulation. Result The chromatographic separation was achieved on Qualisil BDS C8 column with mobile phase containing of mixture of Buffer (Potassium dihydrogen ortho phosphate, pH to 3.0 with ortho phosphoric acid) and Organic Solvent (Acetonitrile) 60:40 v/v pumped at flow rate 0.6 mL min−1. The detection of elute was performed using PDA detector at 305 nm. Mifepristone was eluted at 8.67 min. According to international conference on harmonization Q2(R1) guideline, method was validated and shows satisfactory results for accuracy, precision, linearity, ruggedness, robustness, detection limit, quantitation limit. The method indicated to be linear in the series of concentration 3–18 µg mL−1, and correlation coefficient was 0.9997. In acidic, basic, oxidative, thermal, photolytic forced degradation conditions, the peak of degradation product was clearly and well separated from drug peak without any interference in quantitative analysis. This represents stability indicating nature of established method. Conclusion The established RP-HPLC method is simple, accurate, specific, precise, robust, rugged, sensitive, and economical in nature which can be utilized for routine analysis of mifepristone in bulk and pharmaceutical formulation.

A Validated Stability Indicating RP-HPLC Method for Quantification of Cilnidipine in Bulk and in Tablet Dosage Form

A stability indicating RP-HPLC method has been developed for quantification of Cilnidipine in bulk and in tablet dosage form. The chromatographic analysis was accomplished at ambient temperature on Xttera RP18 (100 x 4.6 mm, 3.5 µm) column and 1 mL/min flow rate by using Eluent composed of 10 mM phosphate buffer pH 2.6 with Acetonitrile (300:700, v/v). The UV detection at the wavelength of 240 nm was carried out using 20 µL injection volume. The Cilnidipine retention time was found to be 3.029 min. The method in the range of 40.0573 – 120.1719 µg/mL was found to be linear (R2 = 0.999) with a detection limit and quantitation limit of 1.2038 and 3.6478 μg/mL, respectively. The mean recovery % over the three tested levels of 50, 100, and 150% were found to be 98.74, 99.60, and 98.23%, respectively. The mean % assay of 99.29 for method repeatability and 98.82 for intermediate precision were found with % RSD of 0.68 and 0.31, respectively. Cilnidipine drug substance and their product exposed to acid, alkali, oxidative, thermal, photolytic, and humidity stress conditions. The acid, alkali, and photolytic induced stress studies signifying the formation of a variety of degradants and their peaks were well resolved from that of active analyte peak. Hence, it is recommended that the Cilnidipine drug substance, as well as drug product, should be store in a tightly closed container protected from light. The method as per ICH guidelines was validated for specificity, linearity, detection limit, quantitation limit, precision, accuracy, robustness, solution stability, and can be effectively used for routine analysis.

A Multicentre, Efficacy and Safety Study of Methotrexate to Increase Response Rates in Patients With Uncontrolled Gout Receiving Pegloticase (MIRROR): 12-Month Efficacy, Safety, Immunogenicity, and Pharmacokinetic Findings of an Open-label Study

Background: Publications suggest immunomodulation co-therapy improves responder rates in uncontrolled/refractory gout patients undergoing pegloticase treatment. The MIRROR open-label trial showed a 6-month pegloticase+methotrexate co-therapy responder rate of 79%, compared to an established 42% pegloticase monotherapy responder rate. Longer-term efficacy/safety data are presented here.Methods: Uncontrolled gout patients (serum urate [SU]≥6 mg/dL and SU≥6 mg/dL despite urate-lowering therapy [ULT], ULT intolerance, or functionally-limiting tophi) were included. Patients with immunocompromised status, G6PD deficiency, severe kidney disease, or methotrexate contraindication were excluded. Oral methotrexate (15 mg/week) and folic acid (1 mg/day) were administered 4-weeks before and during pegloticase therapy. Twelve-month responder rate (SU<6 mg/dL for ≥80% during Month 12), 52-week change from baseline in SU, and extended safety were examined. Efficacy analyses were performed for patients receiving ≥1 pegloticase infusion. PK/anti-drug antibodies (ADAs) were examined and related to efficacy/safety findings. Results: Fourteen patients were included (all male, 49.3 ± 8.7 years, 13.8 ± 7.4 year gout history, pre-therapy SU: 9.2 ± 2.5 mg/dL). Three patients were non-responders and discontinued study treatment before 24-weeks, one patient exited the study per-protocol at 24-weeks (enrolled prior to treatment extension amendment), and 10 remained in study through Week 52. Of the 10, 8 completed 52-weeks of pegloticase+methotrexate and were 12-month responders. The remaining two discontinued pegloticase+methotrexate at Week 24 (met treatment goals) and stayed in study under observation (allopurinol prescribed at physicians’ discretion); one remained a responder at 12-months. At 52-weeks, change from baseline in SU was -8.2 ± 4.1 mg/dL (SU: 1.1 ± 2.4 mg/dL, n=10). Gout flares were common early in treatment but progressively decreased while on therapy (Weeks 1-12: 13/14 [92.9%], Weeks 36-52: 2/8 [25.0%]). One patient recovered from sepsis (serious AE). Two non-responders developed high ADA titres; fewer patients had trough concentrations (Cmin) below quantitation limit (BQL) and median Cmin was higher (1.03 mg/mL vs. BQL) than in pegloticase monotherapy trials.Conclusions: Methotrexate+pegloticase co-therapy was well-tolerated over 12-months, with sustained SU lowering, progressive gout flare reduction, and no new safety concerns. Antibody/PK findings suggest methotrexate attenuates ADA formation, coincident with higher treatment response rates.Trial registration: ClinicalTrials.gov: NCT03635957, registered 17 August 2018, https://clinicaltrials.gov/ct2/show/NCT03635957

Development of Microchip Isotachophoresis Coupled with Ion Mobility Spectrometry and Evaluation of Its Potential for the Analysis of Food, Biological and Pharmaceutical Samples

An online coupling of microchip isotachophoresis (µITP) with ion mobility spectrometry (IMS) using thermal evaporation interface is reported for the first time. This combination integrates preconcentration power of the µITP followed by unambiguous identification of trace compounds in complex samples by IMS. Short-chain carboxylic acids, chosen as model analytes, were first separated by the µITP in a discontinuous electrolyte system at pH 5–6, and subsequently evaporated at 130 °C during their transfer to the IMS analyzer. Various parameters, affecting the transfer of the separated sample components through the evaporation system, were optimized to minimize dispersion and loss of the analytes as well as to improve sensitivity. The following analytical attributes were obtained for carboxylic acids in the standard solutions: 0.1–0.3 mg L−1 detection limits, 0.4–0.9 mg L−1 quantitation limits, linear calibration range from the quantitation limit to 75 mg L−1, 0.2–0.3% RSD of the IMS response and 98–102% accuracy. The analytical potential of the developed µITP-IMS combination was demonstrated on the analysis of various food, pharmaceutical and biological samples, in which the studied acids are naturally present. These include: apple vinegar, wine, fish sauce, saliva and ear drops. In the real samples, 0.3–0.6% RSD of the IMS response and 93–109% accuracy were obtained.

Development of Differential Pulse Anodic Stripping Voltammetry Technique for Cadmium(II) Detection and Its Application in Water Spinach

Cadmium is a toxic pollutant that is harmful to the environment and humans. The purpose of this research was to develop a method for cadmium(II) detection using differential pulse anodic stripping voltammetry (DPASV) using a glassy carbon electrode. The developed method was then applied for cadmium detection in the vegetable samples which is water spinach. The developed method was optimized in several parameters such as potential window, deposition potential, deposition time, and scan rate. The developed method for cadmium(II) detection was also investigated in its analytical performance includes linearity, precision, detection limit, and quantitation limit. The optimum conditions for cadmium(II) detection in 0.1 M KCl using DPASV technique obtained such as potential window from -1200 to -100 mV, deposition potential of -1100 mV (vs Ag/AgCl), and deposition time of 360 s. It was obtained good linearity for cadmium(II) detection using the DPASV technique with an R2 of 0.996. The precision was expressed as %SBR with 0.66%. The detection and quantitation limits for cadmium(II) detection were 0.4206 µM~0.0771 ppm and 0.5525 µM~0.1013 ppm, respectively. The developed method was then applied for cadmium(II) measurement in the water spinach sample and the obtained cadmium(II) concentration in water spinach was 0.2399 mg/Kg.