Phase High-performance Liquid Chromatography Method Research Articles

Bioanalytical method development and validation for the quantification of raloxifene hydrochloride from mice plasma by RP-HPLC

Raloxifene hydrochloride (RLX) belongs to the Selective estrogen receptor modulator (SERM) class of drugs, exhibiting diverse affinity to estrogen receptors. The drug is known for its anti-cancer and anti-osteoporosis activity. The current study employs a Reverse Phase High-Performance Liquid Chromatography (RP-HPLC) method that is sensitive and time-efficient for quantitative evaluation of RLX from mice plasma, using tamoxifen citrate as an internal standard (IS). The protein precipitation technique was used to extract the RLX and IS. RLX was separated using a C8 column with an isocratic elution of a mobile phase consisting of acetonitrile and Milli-Q water (pH 3) at a 1 mL/min flow rate. The ultraviolet (UV) detection of the analyte and IS were carried out at 287 nm. The linearity plot was constructed in the range of 100–3200 ng/mL with a correlation coefficient of r2 ≥ 0.9996. The inter-day and intra-day accuracy and precision were found to be within acceptable limits. Stability studies revealed that the analyte remained stable. The validated RP-HPLC method was successfully applied to quantify RLX after oral administration in mice, and the pharmacokinetic (PK) parameters were established. The present RP-HPLC method could be a beneficial tool for the fast and reliable estimation of future PK interactions, PK analysis of formulations, and therapeutic drug monitoring.

Analytical Method Development and Optimization on High Performance Liquid Chromatography for Related Substances Test of Gliclazide Drug as Active Pharmaceutical Ingredient

Diabetes mellitus, particularly Type 2 Diabetes Mellitus (T2DM), presents a significant global health challenge, necessitating continuous advancements in therapeutic strategies. Gliclazide, an oral selective inhibitor of sodium glucose co-transporter-2 (SGLT2), has emerged as a promising agent for managing T2DM by facilitating urinary glucose excretion and reducing plasma glucose levels. However, ensuring the quality and efficacy of pharmaceutical formulations remains crucial for regulatory approval and clinical effectiveness. In this study, we present the development of an improved stability-indicating Reverse Phase-High Performance Liquid Chromatography (RP-HPLC) method for the precise quantification of Gliclazide. The novel analytical method employs 0.1% Potassium Phosphate in water 0.1% Potassium Phosphate in methanol (MeOH) as the mobile phase, demonstrating enhanced sensitivity, cost-effectiveness, and reduced reliance on organic chemicals. Through rigorous validation following International Conference on Harmonisation (ICH) guidelines, the developed method exhibited superior performance in terms of retention times compared to existing methodologies. The proposed analytical approach holds promise for routine analysis of Gliclazide in both bulk and tablet formulations, offering a valuable tool for pharmaceutical quality control and clinical research in the management of T2DM.

A Validated Chiral Chromatography Method for Enantiomeric Separation of Pomalidomide in Human Plasma.

In the present work, new chiral stationary phase high-performance liquid chromatography (CSP-HPLC) method was established and validated for the quantification of pomalidomide (PMD) enantiomers in human plasma. The chromatographic enantiomeric separation was achieved on a Daicel-CSP, Chiralpack IA 4.6 × 250mm, 5μm; because of its advantages of high degree of retention, high resolution capacity, better reproducibility, ability to produce lower back pressure and low degree of tailing. The mobile phase was maintained as methanol: glacial acetic acid (499.50ml:50μL). Ultraviolet wavelength for detection was 220nm. PMD enantiomer-I and enantiomer-II were separated at 8.83 and 15.34min, respectively. Limit of detection and limit of quantification for each enantiomer and the calibration curve of standard PMD was linear in range between 10-5,000ngmL-1. The method was validated according to The International Council for Harmonisation of Technical Requirements of Pharmaceuticals for Human Use (ICH(Q2R1)) specific guidelines. We found no interference peak with PMD chromatogram obtained. This is a simple, reliable and specific method for detection and quantification of enantiomer of PMD in human plasma sample.

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.

Optimization of a Validated Liquid Chromatographic Method with Chemometric Approach for Estimation of Eugenol from Seed Powder Extract and Perform Anti-oxidant Activity

Background:: Developed and validated a new reverse phase high-performance liquid chromatographic (RP-HPLC) method, and it is prompt, precise, sensitive and robust for the esti-mation of eugenol in seed powder extract of Myristica fragrans. Objective:: The chemometric approach was utilized to obtain a rugged and definitive chromato-graphic method for the purpose. Methods:: Method variables such as acetonitrile (%) and flow rate were investigated for robust-ness and optimization by using a face-centered cubic design (FCCD). The Design Expert 12.0.1.0 software has been employed for this optimization. Further, the effects of factors were monitored on the concentrations of eugenol recovered from seed powder extract. Chromatograms have been developed by using an optimized mobile phase mixture containing methanol-water-acetonitrile (10:40:50, v/v/v) and Symmetry® C18 column (5 μm, 3.9 ×150 mm). The mobile phase was de-rived at a flow rate of 1 mL/min, and estimation of eugenol was performed at ʎmax 272 nm. Results:: Validation of the method has been carried out to reveal its selectivity, linearity, preci-sion, accuracy, LOD, and LOQ. Linear calibration plot for eugenol was held over the concentra-tion across 6.25 and 100 μg/mL (R2 = 0.999). The coefficient of variation was less than 1%, and accurate recovery of eugenol was observed between 96.80 and 99.56 %. The LOD and LOQ have been established to be 1.97 and 6.25 μg/mL, respectively. Intraday and Inter-day coefficients of variation have 1.81-1.91 and 1.92-1.57, respectively. Antioxidant activity (AA) by DPPH assay of seed powder extract in five different solvents was performed, and % AA activity was calculat-ed against ascorbic acid. Conclusion:: The validated method has been found to be highly robust and will be applied for the analysis of eugenol in formulation. The highest % AA has been reported in hexane solvent.

Ecofriendly bioanalytical validated RP-HPLC method for simultaneous determination of COVID-19 co-prescribed drugs employing quality by design and green chemistry

Molnupiravir, dexamethasone, ertapenem, and remdesivir are among the most frequently co-prescribed drugs used for COVID-19 virus treatment. For the first time, a simple isocratic reversed phase-high performance liquid chromatographic (RP-HPLC) method employing a photodiode array detector has been developed and validated for their simultaneous determination in spiked human plasma using rivaroxaban as an internal standard by the aid of quality by design and in accordance with the criteria of green chemistry. The separation was carried out utilizing a BDS Hypersil ® C18 column (300 × 4.6 mm), 5 μm particle size as the stationary phase using mobile phase consisted of methanol: acidified water; adjusted at pH 4.0 with 0.1 M orthophosphoric acid; with ratio (70:30, v:v) delivered at flow rate 0.6 mL/min, temperature adjusted at 20 °C, photodiode array detector maintained at 240 nm. The method was validated according to bioanalytical FDA guidelines with respect to linearity, accuracy, precision, selectivity, recovery, and stability. The method was found to be linear in the range of 4.00–120.00 μg/mL for molnupiravir, 10.00–140.00 μg/mL for dexamethasone, 10.00–160.00 μg/mL for ertapenem and 4.00–120.00 μg/mL for remdesivir. It is worth mentioning that the previously mentioned ranges covered the Cmax of the drugs. Finally, the green analytical score has been evaluated, and it was found to be an excellent green analytical method based on analytical eco-scale system penalty points. Moreover, it was assessed by analytical GREEnness metric approach and green analytical procedure index tool.

Validation of an Ion-Pair Reverse Phase High-Performance Liquid Chromatography Method for the Detection of Major Components and Related Substances in Diquafosol Sodium Eye Drops.

A simple, feasible, isocratic elution, and stable reversed-phase high performance liquid chromatography method was established and verified. The chromatographic conditions are as follows: EF-C18H, 4.6 × 250 mm, 5 μm column; column temperature 30 °C; for the mobile phase 27.2 g of KH2PO4 and 8.5 g of tetrabutylammonium hydrogen sulfate were taken, 2500 mL of water was added to dissolve, and the pH was adjusted to 6.7 with phosphoric acid:methanol solution with a ratio of 84:16 (V:V). The flow rate was 1.0 mL/min; the injection volume was 10 μL; and the wavelength was 262 nm. According to the current ICH guidelines, the developed method was verified, and the system suitability, specificity, LOD, LOQ, linearity, range, accuracy, repeatability, durability, and solution stability of the proposed method were verified. The validation results demonstrated that the LOQ for the method was 0.05% and the LOD was 0.02%. The content was detected within the concentration range of 300 to 900 μg/mL. The relationship between concentration and measurement was linear, with an r2 of >0.999. The concentration of impurities ranged from 0.3 to 4.5 μg/mL. A good linear correlation was observed within the range of g/mL, with a coefficient of determination r2 greater than 0.999. The accuracy and repeatability met the specified criteria.

An Eco-Friendly RP-HPLC Method Development and Validation for Quantification of Favipiravir in Bulk and Tablet Dosage Form Followed by Forced Degradation Study.

In this work, an eco-friendly simple, precise reverse phase high-performance liquid chromatography (HPLC) method has been developed and validated for Favipiravir in bulk and tablet dosage form followed by its force degradation study. The proposed method was validated to obtain official requirements including stability, accuracy, precision, linearity, robustness and selectivity as per International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) Guidelines. The estimation was developed on C (18) column reversed-phase using the mobile phase composition as methanol:water (10:90v/v). The flow rate was set as 1ml/min, and the maximum absorption was observed at 323nm using Shimadzu Photo Diode Array detector. The Favipiravir, drug showed a precise and good linearity at the concentration ranges of 10-50μg/mL. The Revearse Phase High Perforance Liquid Chromatography assay showed the highest purity ranging from 99.90 to 100.02% for Favipiravir, tablet dosage form, and 100.15% was the mean percentage purity. The percent recovery was found within the acceptance limit of (98.6-100.0%). Intra- and inter-day precision studies of the method were less than the maximum allowable limit percentage of relative standard deviation ≤ 2.0. The Favipiravir retention time was found to be 5.00min. To examine the stability of the drug, various forced degradation studies were conducted on Favipiravir Active Pharmaceutical Ingredient. The developed method was validated according to the ICH guidelines. A very quick, cost-effective, precise and accurate HPLC method for the determination of Favipiravir has been developed and validated in compliance with ICH guidance Q2.

A Validated RP-HPLC Method for Simultaneous Quantification of Abacavir and Zidovudine and their Impurities in Combined Dosage Forms

Develop a simple, accurate, and precise method for simultaneous analysis of Abacavir and Zidovudine, along with their related impurities, in combined pharmaceutical dosage forms. A reverse- phase high-performance liquid chromatography (RP-HPLC) method was developed using an LC-20 AT C18 column and a Water: Methanol (80:20) mobile phase. Detection was carried out at 240 nm. The method successfully separated Abacavir, Zidovudine, and their respective impurities with distinct retention times. Validation demonstrated good linearity, accuracy, and precision for both drugs and their impurities. Limits of detection (LOD) and quantification (LOQ) were determined and found to be suitable for analyzing impurities in commercial dosage forms. The developed RP-HPLC method offers a simple, accurate, and reliable tool for simultaneous quantification of Abacavir, Zidovudine, and their impurities in combined dosage forms, thereby contributing to quality control and ensuring drug safety. Keywords Abacavir, Zidovudine, Related Substance RP-HPLC Method, ICH Q2 (R1) guidelines

A SIMPLE RP-HPLC METHOD DEVELOPMENT AND VERIFICATION OF THE DISSOLUTION OF BROMELAIN-A COMPLEX MIXTURE OF PROTEOLYTIC ENZYMES, IN DELAYED-RELEASE TABLETS

Objective: To develop a simple, accurate, precise and linear Reverse Phase High-Performance Liquid Chromatographic (RP-HPLC) method and verify for the quantitative estimation (Dissolution) of Bromelain in delayed-release tablets. Methods: The optimized RP-HPLC method for both acid and buffer stage dissolutions of delayed-release tablets uses Zorbax 300 SB-C8 column (150 mm X 4.6 mm; 3.5μ), a mobile phase-A of 0.1% trifluoroacetic acid in water and a mobile phase-B of 0.1% Trifluoroacetic acid in Acetonitrile in the gradient proportion, flow rate of 1.0 ml/min, injection volume of 25 µl, detection wavelength of 280 nm using a UV/PDA detector, column temperature of 40 °C, sample tray/compartment temperature of 5 °C and a run time of 20 min. Results: The developed method gave Bromelain eluting at about 6 min. Bromelain exhibited linearity in the range 53.4-800.6 μg/ml (r2=0.99992). The precision is exemplified by relative standard deviation of 1.3 and 2.3% for acid and buffer stages, respectively. Percentage recovery of the drug was found to be between 90.0 and 110.0 during accuracy studies. Conclusion: A simple, accurate, precise, and linear RP-HPLC method was developed and verified for the quantitative estimation (Dissolution) of Bromelain in tablets and hence this method can be explored for the analysis of Bromelain in tablets in various pharmaceutical industries.

A Validated RP-HPLC Method for Simultaneous Estimation of Benidipine and Telmisartan in Bulk and Tablet Dosage Form

Background: In this work, a novel, simple and accurate reverse phase high-performance liquid chromatographic method with UV detection is presented for the simultaneous estimation of Benidipineand Telmisartan in API and marketed formulations. Objective: To develop and validate this method in accordance to regulatory guidelines i.e. ICH Q2B-R1 for analytical parameters. Methods: In RP-HPLC the separation was achieved using a Xbridge C18 (250x 4.6 i.d; 5µm) column with isocratic elution using a mobile phase consisting of methanol: acetonitrile (80:20v/v/ with 0.1% v/v TEA) adjusted to pH 5.2 with glacial acetic acid at 1mL/min flow for 10min. Benidipine and Telmisartan, were detected at dual wavelength at 237 and 297nm with retention time (Rt) 6.891 and 8.997 minutes respectively. Results: Our new methods (RP-HPLC) were found to (r2>0.999) for both Benidipine and Telmisartan. The developed methods are highly selective, precise and reproducible with %RSD < 2%, Moreover the methods are accurate with recovery more than 99% in both methods. Conclusion: The RP-HPLC method results are efficient, simple and easy. These highly sensitive methods were successfully applied for assay determination of both Benidipine and Telmisartan at fixed dose combination.

Extraction of phenolic and flavonoid compounds and evaluation of their antioxidant activity in saffron anthers (Crocus sativus L.)

Saffron (Crocus sativus L.) is the most expensive spice in the world and rich in a variety of bioactive compounds including phenolic acids, flavonoids, and vitamins. Saffron is used in food, cosmetics, perfumery, and dye industries due to its color, taste, aroma, and medicinal properties. The extraction of bioactive compounds imposes a constant search for economically and environmentally viable extraction strategies for higher yields including the solvent type. The research was carried out to evaluate total phenolic content (TPC), total flavonoid content (TFC), and antioxidant activity of C. sativus L. anthers using different extraction solvents including ethanol, methanol, and distilled water. The results showed that ethanol was the most effective extraction solvent type exhibiting the highest TPC (7.29 mg GAE g-1 DW), TFC (3.77 mg QE g-1 DW), and antioxidant activity of flavonoids (88%) and ascorbic acid (76.07%). Flavonoids proved to be stronger antioxidants than ascorbic acid, irrespective of solvent type, however the ethanolic extracts showed significantly higher antioxidant activity. A Reverse Phase High-Performance Liquid Chromatography method revealed that gallic acid (3.1 mg g-1), syringic acid (0.2 mg g-1), and vanillic acid (0.13 mg g-1) were the main phenolic compounds detected in the dried anther ethanolic extracts, while quercetin (2.13 mg g-1), pyrogallol (1.73 mg g-1), kaempferol (1.2 mg g-1), rutin (0.2 mg g-1), and tricin (0.1 mg g-1) the main flavonoid compounds. Thus, apart from the spice, constituted by the red stigmas, the production of other flower parts such as the anthers, so far considered agricultural waste can constitute a rich source of bioactive compounds of high antioxidant potential by setting quality standards for new products’ development and ensuring better valorization of saffron’s bioresidues in Mashhad region of Iran.

A Novel, Economical RP-HPLC Method for Quantification of Cefixime in Bulk and Pharmaceutical Solid Dosage form

A new, precise, economical, and linear gradient reverse phase high-performance liquid chromatography (RP-HPLC) method has been developed and validated for assay determination in Cefixime. The chromatographic separation was achieved with Hypersil C18(250*4.6)mmand 3 µm particle size column. The flow rate was 1.0 mL/min and eluents were detected at 220 nm using PDA detector. The retention time was found to be 7.3 min.The percentage recoveries for molecules were found to be in the range of 99–101%. The calibration curve demonstrated good linearity in the range of 20–100 gm/ml. for Cefixime. The approach has been validated in accordance with the International Conference on Harmonization's regulatory criteria. The evaluated parameters are precision, linearity, detection limit, quantification limit, specificity, accuracy, and robustness.The technique may be applied to stability investigations as well as routine analysis to identify and quantify Cefixime in pharmaceutical dosage form .
 Keywords: Cefixim. Reversed Phase High Performance Liquid Chromatography. Assay substances. Methanol

Evaluation of Quercetin's Bioenhancing Effect on Oral Pharmacokinetics of Rosuvastatin in Wistar Rats Using RP-HPLC Method

Background: The absolute oral bioavailability of rosuvastatin (RST), a secondgeneration statin, is low i.e. 20% and only 10% is recovered as metabolite N-desmethy l rosuvistatin. Since it is a hydrophilic statin, RST relies on the organic anion transporting polypeptide- 1B1 (OATP-1B1), as the key mechanism for active transport into hepatocytes. Quercetin (QUE) being a bio enhancer and inhibitor of OATP1B1 can augment the bioavailability and pharmacokinetics of RST. Objective: The present study includes the development of a simple and validated bioanalytical Reverse Phase High-Performance Liquid Chromatography (RP-HPLC) method for the estimation of RST and to study the effect of co-administration of QUE as a bio enhancer on its bioavailability. Methodology: An analytical column of Kromasil 100, C18 (250 mm × 4.6 mm, 5 μm), was used for chromatographic separationand acetonitrile (ACN): acetic acid buffer pH 3.0 adjusted with glacial acetic acid (55:45 Vol. %) as mobile phase with flow rate 1.0 ml/min monitored at 242 nm. The ACN: methanol (50:50 Vol. %) was employed as the final solvent for extraction. The developed method has been successfully applied in a study on the pharmacokinetics of the drug RST in rats after co-administration of QUE, which was carried out using non-compartmental analysis in order to estimate the blood concentration of the drug. Results: The pharmacokinetics of RST was found to be altered significantly (highest concentration of RST in the blood (Cmax) = 67.3 ng/ml to 122.2 ng/ml) (p <0.001), area under curve (AUC)0-t (p <0.0001) and AUC0-inf (p =0.0005) when co-administered with QUE at 120 min (tmax). Conclusion: The results are in accordance with the fact that QUE increases plasma levels in rats through herb-drug interactions.

Comprehensive Physicochemical Characterization, In Vitro Membrane Permeation, and In Vitro Human Skin Cell Culture of a Novel TOPK Inhibitor, HI-TOPK-032.

Nonmelanoma skin cancers (NMSC) are the most common skin cancers, and about 5.4 million people are diagnosed each year in the United States. A newly developed T-lymphokine-activated killer cell-originated protein kinase (TOPK) inhibitor, HI-TOPK-032, is effective in suppressing colon cancer cell growth, inducing the apoptosis of colon cancer cells and ultraviolet (UV) light-induced squamous cell carcinoma (SCC). This study aimed to investigate the physicochemical properties, permeation behavior, and cytotoxicity potential of HI-TOPK-032 prior to the development of a suitable topical formulation for targeted skin drug delivery. Techniques such as scanning electron microscopy (SEM), energy-dispersive X-ray (EDX) spectroscopy, differential scanning calorimetry (DSC), hot-stage microscopy (HSM), X-ray powder diffraction (XRPD), Karl Fisher (KF) coulometric titration, Raman spectrometry, confocal Raman microscopy (CRM), attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR), and Fourier transform infrared microscopy were used to characterize HI-TOPK-032. The dose effect of HI-TOPK-032 on in vitro cell viability was evaluated using a 2D cell culture of the human skin keratinocyte cell line (HaCaT) and primary normal human epidermal keratinocytes (NHEKs). Transepithelial electrical resistance (TEER) at the air-liquid interface as a function of dose and time was measured on the HaCAT human skin cell line. The membrane permeation behavior of HI-TOPK-032 was tested using the Strat-M® synthetic biomimetic membrane with an in vitro Franz cell diffusion system. The physicochemical evaluation results confirmed the amorphous nature of the drug and the homogeneity of the sample with all characteristic chemical peaks. The in vitro cell viability assay results confirmed 100% cell viability up to 10 µM of HI-TOPK-032. Further, a rapid, specific, precise, and validated reverse phase-high performance liquid chromatography (RP-HPLC) method for the quantitative estimation of HI-TOPK-032 was developed. This is the first systematic and comprehensive characterization of HI-TOPK-032 and a report of these findings.

Validation and development of RP-HPLC method for quantification of glibenclamide in rat plasma and its application to pharmacokinetic studies in wistar rats

Herein, we report a novel, simple, specific, accurate and cost-friendly validated reverse phase-high performance liquid chromatographic (RP-HPLC) method for the quantification of second generation sulphonylurea based antidiabetic drug, glibenclamide (GLB) in rat plasma and its application to calculate pharmacokinetic parameters in wistar rats. The internal standard used was flufenamic acid. The chromatographic separation was conducted on C18 column (250 mm × 4.6 mm x 5 μm, Agilent-Zorbax, SB) using isocratic elution with mobile phase containing Acetonitrile: Water (1:1; v/v) pH adjusted to 4.0 with 0.03 % glacial acetic acid and detected by photo-diode array as detector. Calibration curves made in the rat plasma were linear in the range of 50–1200 ng/ml with r2 = 0.998. The LLOQ was 40 ng/ml. This method was effectively applied for pharmacokinetic studies of Glibenclamide following administration through oral route as solid dispersion formulation to Wistar rats. Several methods are available in the literature which can be employed for the quantification of Glibenclamide but such methods are tedious, provide lower sensitivity, less simultaneous resolution and are time-consuming. Therefore the present methods suits best for the quantification of Glibenclamide from Wistar rats.

Development and validation of analytical procedure for analysis of Amoxiciline, Metronidazole and Omeprazole, used as anti- Helicobacter pylori agents alone and in mixture

Background: The contemporary treatment of ulcerogenic diseases and gastroesophageal reflux disease is related usually to application of a combination of imidazole-based antibacterial, antibiotic and proton pump inhibitor. In the current study, the three most common representatives Amoxicilline (AMO), Metronidazole (MET) and Omeprazole (OME), respectively, are subjected to analysis through classical analytical procedure, providing high level accuracy, sensitivity and good separation abilities. As such a UV/VIS method was applied as a well known identification and quantitation technique for analyses in various samples. Furthermore, this technique is known to be a good detection method in combination with chromatographic systems. Purpose: A simple, specific, accurate and precise reverse phase-high performance liquid chromatographic method has been developed for the simultaneous determination of Amoxicillin Trihydrate (AMO), Metronidazole (MET) and Omeprazole (OME) in synthetic mixture. Materials and methods: Some important parameters like pH of the mobile phase, concentration of the acid or buffer solution, percentage and type of the organic modifier, etc. were tested for a good chromatographic separation. The sample was analyzed using a mobile phase of Acetonitrile: Phosphate buffer (pH=7.6±0.1) (40:60 v/v). The flow rate was 1.0 mL/ min with detection at 280 nm. Results: The retention time for AMO, MET and OME was found to be 1.67, 2.86 and 5.99 min respectively, and the recoveries in the synthetic mixture were between 98 and 102%. The validated method was linear over the concentration range of 25 to 200 μg/mL for AMO, 12.5 to 100 μg/mL for MET and 5–40 µg/mL for OME, with a correlation coefficient > 0.999. Conclusion: The developed method has been validated in accordance with the International Conference on Harmonization (ICH) guidelines and showed excellent linearity, accuracy, precision, specificity, robustness, as well as system suitability results within the acceptance criteria.

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

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

Antioxidant activity and reduction potential of dietary herbs on acrylamide formation in asparagine-glucose reaction model

ABSTRACT Natural ingredients are increasingly being used to prevent the formation of acrylamide during food processing. This study investigated the antioxidant activities of Ocimum basilicum (basil), Lippia adoensis var. adoensis (kesse), Lippia adoensis var. koseret (koseret), Rosmarinus officinalis (rosemary), and Thymus schimperi (tosign) extracts and their acrylamide reduction effect in the asparagine-glucose model system. Folin-Ciocalteu and aluminum chloride methods were used to calculate the total phenolic content (TPC) and total flavonoid content (TFC). Antioxidant activities were determined using 2,2-Diphenyl-1-Picryl-Hydrazyl (DPPH) scavenging and phosphomolybdenum assay methods. Acrylamide contents of the samples were determined by a reversed phase high-performance liquid chromatography (RP-HPLC-DAD) method. Kesse had the highest concentrations of TPC (30.20 ± 1.23 mg gallic acid equivalent per gram of herb extract), TFC (15.87 ± 0.55 mg catechin equivalents per gram), and the strongest DPPH scavenging activity (IC50 = 108 μg/mL). The amount of acrylamide formed during the frying process was decreased by a factor of 26.8% to 65.81% with the addition of dietary herb extracts to the asparagine-glucose reaction model. The amount of acrylamide formed from an asparagine-glucose reaction model containing herb extracts was negatively correlated with the total phenolic and total flavonoid content with, R2 values of 0.920 and 0.930. This study suggests a potential application of these dietary herb extracts as a natural antioxidant for lowering acrylamide formation during the Maillard reaction in fried food products.

Development and validation of RP-HPLC method for estimation of anti- hypothyroidism drug in capsule dosage form

A simple, accurate, sensitive and precise Reverse Phase-High Performance Liquid Chromatographic assay method for estimation of Levothyroxine sodium in dosage form was successfully developed. The chromatographic separation was performed on Welch Ultisil XB-CN, 150 x 4.6 mm, 5µm. column. The mobile phase consists of 0.05% OPA Buffer solution, and Acetonitrile in the ratio of 72:28, v/v. Mobile phase was delivered at a flow rate of 1.0 ml/min. Samples were injected 50 μL the column temperature was kept at 40 0C and sample temperature 150C. The wavelength 221 nm were selected for the evaluation of the chromatogram. The retention time of the drug was found to be 3.7 min. The developed method was found to be linear in a concentration range of 5-15 μg/ml of the drug (r2= 0.999). The low value of % RSD indicates reproducibility of the method. Thus this method can be used for routine analysis of pomalidomide in formulation and to check the stability of bulk samples.