Methanol Buffer Research Articles

Development of simple stability indicating RP-HPLC method to estimate ferulic acid in niosomes and marketed products using quality by design approach

Ferulic acid found in Feru-guard which is obtained from rice bran. This compound occurs in a wide range of plant sources including pineapple, various grasses, rice, leaves, beans, coffee beans, oats, grains, vegetables, flowers, fruits, peanuts and nuts. Particularly, it received a lot of attention due to its potential therapeutic uses in different pathologies, including metabolic, viral infections, cancer, cardiovascular, skin disorders, diabetes, hepatotoxicity and neurodegeneration. The article describes the methodical development of a simple, fast, highly sensitive, durable, efficient and economically viable reversed-phase high-performance liquid chromatography (RP-HPLC) technology for measuring ferulic acid. The analysis was Performed using the Central composite design (CCD), which offers the advantage of employing a reduced number of design points and experimental runs. Employing the response surface methodology via central composite design (CCD), the study thoroughly assessed the influence of these factors. The proposed analytical method underwent comprehensive validation, ensuring its reliability as per the guidelines outlined in ICH Q2A and ICH Q2B.The preferred method employed a mobile phase consisting of Methanol and OPA buffer (50:50 % v/v, pH maintain to 4.2 with 0.1 % orthophosphoric acid), operating at a flow rate of 1 mL min⁻1,with a Chemsil ODS column, the wavelength of detection was set at 320 nm. According to the novel analytical method, Ferulic Acid exhibited a peak retention time of approximately 6.13 min. The method demonstrated linearity range of 0.5–16 μg/ml. The developed approach proved to be robust, precise and accurate, with a recovery rate of 98 %. It achieved % relative standard deviation (RSD) of below 2 %,The current stable RP-HPLC method was effectively employed for the characterization of niosomes loaded with Ferulic Acid. Furthermore, degradation studies were conducted under various conditions to elucidate the degradation pathways and resultant products.

DoE-Aided Optimization of RP-HPLC Method for Simultaneous Estimation of Amoxicillin and Tinidazole Loaded Mucoadhesive GRDDS Formulation for the Treatment of H. pylori

Helicobacter pylori (H. pylori) infection is one of the primary risk factors of peptic ulcer disease worldwide. Treatment of H. pylori with the conventional dosage form is often challenging due to the ineffective reach of the antibiotics to the inner layers of gastric mucosa, where the organism resides. This study developed an eco-friendly, stability-indicating RP-HPLC method to simultaneously estimate amoxicillin and tinidazole from mucoadhesive formulation targeting H. pylori infection. The mucoadhesive GRDDS formulation of antibiotics was developed with a goal of improving bioavailability at the gastric mucosa. The multivariate Box–Behnken design (BBD) was utilized to optimize chromatographic parameters. Independent variable such as ratio of mobile phase, flow rate, pH and injections volume were optimized using DoE, and analyzed using perturbation plots. A desirability of 0.981 was achieved for the optimized variables. The optimized method utilized methanol and phosphate buffer (25:75) at pH 6.3 as the mobile phase in an isocratic elution mode on a Luna ODS C18 column kept at 25 °C as the stationary phase. The method was linear from 0.25 to 20 µg/mL, for both the drugs with R2 values of 0.9993 and 0.9997 for amoxicillin and tinidazole, respectively. This validated RP-HPLC technique demonstrated selectivity in the presence of possible degradation products and excipients present in the mucoadhesive GRDDS beads. The method was used for the determination of entrapment efficiency and in vitro release profile for tinidazole and amoxicillin in the mucoadhesive GRDDS formulation.Graphical

An eco-friendly liquid chromatographic analysis of the triple therapy protocol of amoxicillin, metronidazole and vonoprazan for H. Pylori eradication: application to combined dosage forms and simulated gastric fluid

Helicobacter pylori has a big sway when peptic ulcers are concerned. For its eradication, different protocols have been approved. Among which, the tripartite therapy protocol which embraces vonoprazan as potassium competitive acid blocker in combination with amoxicillin and metronidazole as antibiotics. An environmentally benign HPLC method is addressed in order to simultaneously determine amoxicillin (AMX), metronidazole (MET) and vonoprazan (VPZ) in bulk powder and combined tablet mixture. Full separation of AMX, MET and VPZ is accomplished using C8 column, and a gradient mobile phase system, composed of methanol and phosphate buffer of a pH value of 5. Fine linearity in the concentration ranges 50–600 µg mL−1 amoxicillin, 50–400 µg mL−1 metronidazole and 10–100 µg mL−1 vonoprazan was denoted by the high correlation coefficient (0.9999). The method accuracy and precision are confirmed upon analyzing AMX, MET and VPZ triple therapy not only in their synthetic mixtures and combined tablet mixtures but also in their combined tablet mixtures in simulated gastric fluid. AMX, MET and VPZ triple therapy could be routinely analyzed in QC labs, in case of being co-formulated, using the presented method. Three different assessment tools were adopted revealing the benign environmental impact of presented method.

Development and Validation of Stability Indicting HPLC Method for the Separation and Simultaneous Analysis of Timolol, Dorzolamide and Latanoprost Inophthalmic formulations

The present work is intended to establish a simple, precise and sensitive stability indicating HPLC method for the separation and simultaneous quantification of timolol, dorzolamide and latanoprost in pharmaceutical formulations. The separation of analytes was achieved on Spherisorb ods2 C18 (250mm × 4.6mm; 5µ)as stationary phase, methanol, acetonitrile and phosphate buffer (pH 5.2) in 55:45:05 (v/v) as mobile phase at 1.0 mL/min and UV detection at 239nm. In this condition, well resolved, retained peaks were identified at 3.45 min fortimolol, 2.66min for dorzolamideand 5.43min for latanoprost. The method reports 0.313µg/mL, 1.25µg/mL and 0.003µg/mL for timolol, dorzolamide and latanoprost respectivelyas LOD that proves that the method have enough sensitivity levels for the detectionanalytes in samples. The method passes all the validation parameters as per the guidelines proved that the method was valid. The method can shows very less % degradation in various stress studies such as acidic, base, peroxide, thermal and UV light conditions and can effectively separate various stress degradation compounds and confirms the stability indicating nature of the method. The method applicability was assessed by analysing the drug content in ophthalmic drops and reports the % assay of be 98.48, 99.37 and 98.32% for timolol, dorzolamide and latanoprost respectively. Based on the results, it can be concluded that the method can adequately suitable for the separation and quantification of timolol, dorzolamide and latanoprost and hence can be applicable for the routine analysis of timolol, dorzolamide and latanoprostin single or any combined ophthalmic formulations.

METHOD DEVELOPMENT AND VALIDATION OF RAPID ISOCRATIC RP-HPLC METHOD FOR SIMULTANEOUS ESTIMATION OF PARACETAMOL, CAFFEINE, AND PROPYPHENAZONE IN PHARMACEUTICAL FORMULATION

Objective: The objective is to develop a novel, rapid, simple, precise, accurate, and reproducible RP-HPLC method for simultaneous estimation of paracetamol, caffeine, and propyphenazone in bulk and a pharmaceutical dosage form. Method: Optimized chromatographic conditions were used for isocratic elution with Shimadzu C18 (4.6×250 mm, 5 μm), methanol, and 20mM phosphate buffer (60:40, v/v, pH 2.5) as mobile phase, flow rate 1.2 mL/min, and UV detector at λ max 272 nm. The method was validated for specificity, precision, linearity, accuracy, robustness, and solution stability as per the International Council for Harmonization (ICH) guidelines. Result: It was discovered that the retention time for paracetamol, caffeine, and propyphenazone was found to be 2.6 min, 3.0 min, and 7.5 min. The method proved to be rapid, simple, linear (R2>0.999), precise {relative standard deviation (RSD<2.0%)}, accurate (recovery 98–102%), sensitive, and robust. Conclusion: The proposed novel isocratic RP-HPLC method was found to be rapid (short run time of 10 min), highly selective, accurate, and sensitive. The method has been successfully applied to the simultaneous analysis of paracetamol, caffeine, and propyphenazone in a pharmaceutical formulation.

Validated Stability Indicating RP-HPLC Method for the Quantification of Process Related Impurities of Solifenacin and Mirabegron in Pharmaceutical Formulations

To assess solifenacin (SOL) and mirabegron (MER) simultaneously, a verified reverse-phase high-performance liquid chromatography (RP-HPLC) method has been developed to indicate stability. The method was thoroughly evaluated and found to meet satisfactory criteria for precision, linearity, accuracy, limits on detection, and robustness, limits on quantitation. The quantitation wavelength of 231 nm was determined. Linearity was successfully demonstrated across concentration ranges of 5 to 25 μg/mL of solifenacin and 50 to 250 μg/mL of mirabegron. RPHPLC separations was conducted employing a Phenomenex L. C18 column measuring 250 x 4.6 mm and containing particles as small as 5 μm. The methanol and phosphate buffer (pH 7) were combined in a volumetric ratio of 25:75 to create the mobile phase. The separation is accomplished at a 0.7 mL per minute flow rate. Time spent in retention for mirabegron and solifenacin had been established at 5.521 and 9.161 minutes, respectively. Forced degradation studies validated the stability-indicating character of the approach, which included hydrolysis under acidic and basic conditions, exposure to H2O2, thermal degradation, and photodegradation. Mirabegron and solifenacin exhibited 10 to 20% degradation under the specified conditions. Importantly, the process evaluated the two prescription drugs in detail with all degradation products generated during the forced degradation experiments. This developed method is characterized as straightforward, specific, and cost-effective, making them suitable of the simultaneous estimate of mirabegron with solifenacin in tabs dose forms.

A Green UPLC Method for the Simultaneous determination of Tenofovir and Amoxicillin in Biological Fluids and Dosage Forms

This paper elucidates the process of developing and validating the UPLC technique, aiming to enable the concurrent measurement of tenofovir and amoxicillin. This UPLC method is accurate and sensitive. The experimental setup involved utilizing a Kinetex 1.7μ C18 100A (2.1-mm × 50-mm) column maintained at a temperature of 30°C. The mobile phase employed a combination of methanol and phosphate buffer (0.1% orthophosphoric acid in water, pH 3.5) in a ratio of 30:70 (v/v). The flow rate was set at 0.2mL/min, and the detection wavelength for analysis was 230nm. All these conditions resulted in optimum chromatographic separation. This developed method of UPLC took less than 4 minutes to separate this mixture. The detection limits for two medications were determined to be 0.45µg/mL for tenofovir and 0.26µg/mL for amoxicillin. The calibration plots of both substances exhibited satisfactory linearity within the concentration range of 1.0 - 2.0 µg/mL. Remarkably, the method demonstrated excellent performance with high percentage recoveries ranging from 96.8% to 102.92% and low percentage relative standard deviation (%RSD) values below 2%. Consequently, the proposed methodology proved to be highly effective for detecting the targeted drugs in their respective dosage forms.

Challenges and Strategies to develop RP-HPLC Method of L-Arginine with Polyphenolic compounds

While developing a method of RP-HPLC for a new compound or combination of drugs simultaneously, many challenges are faced by the researcher pertaining to effective elution and sufficient resolution of a mixture of compounds. Many factors come into the picture, which reflects the ultimate elution of chemical compounds. Factors like the physicochemical properties of chemical compounds, the chemical nature of the mobile phase, instrumental factors, and experimental conditions play crucial roles in RP-HPLC method development. This research article discusses the challenges faced while developing a method of L-Arg along with polyphenolic compounds. Both the compounds have contrasting solubility profiles as L-Arg is soluble in water but PIC is soluble in organic solvents. Strategies were used to develop RP-HPLC of this combination with mobile phases like acetonitrile, orthophosphoric acid, methanol, and potassium dihydrogen phosphate buffer of pH 2.6. The present article provides an insightful approach to develop a new RP-HPLC method for a combination of compounds having related physicochemical characteristics.

A cheap and straightforward method for the selective isolation of histidine-derived natural products using nickel(II) phosphate.

Nickel-based resins have long been used in biochemical studies for the purification of His-tagged proteins, but their potential in the isolation of histidine-derived small molecules has not been investigated to date. Many agriculturally-important mycotoxins incorporate histidine residues, as do natural products from both plants and bacteria. Here, a highly-selective solid-phase extraction method is described for the purification of histidine-derived natural products using the insoluble nickel salt Ni3(PO4)2. This led to the highly-selective binding and elution of two natural products, meleagrin and gartryprostatin C, from two fungal strains, Penicillium chrysogenum and Aspergillus sclerotiorum, respectively. A simple protocol involving binding, washing with water and methanol, and elution with methanolic acetate buffer, gave 65-75% recovery of both compounds directly from crude extracts of the two fungi. The procedure can easily be used as part of a multi-step isolation process in combination with HPLC to yield the purified alkaloids.

Spectroscopic method to estimate 3-Acetyl-11-keto-β-boswellic acid in complex lipid nanocarriers and skin matrices

3-Acetyl-11-keto-β-boswellic acid (AKBA) is a phytoconstituent derived from Boswellia serrata extract. It shows anti-rheumatic activity by selectively and non-competitively inhibiting the 5-Lipoxygenase (5-LOX) enzyme. AKBA is utilized for complex nanocarriers for its effective topical delivery at the arthritic site. There is a demand to develop a rapid, sensitive, and economical UV–Visible spectroscopic method for AKBA, which can be utilized in nanoformulation designing and day-to-day analysis of in vitro and ex vivo samples. In this research work, an analytical method was developed for the estimation of AKBA in two solvent systems, methanol and phosphate buffer saline pH 7.4, using a UV–Visible spectrophotometer. Further, the developed method was validated for linearity, range, limit of detection, limit of quantification, accuracy, precision, solution stability, and specificity as per regulatory guidelines. The method exhibited a linearity range of 10–50 µg/mL in both media with the obtained value of the high correlation coefficient (>0.9995). Interday and intraday precision samples showed less than 2 % relative standard deviation (% RSD) that exhibited the reliability of the developed method. The accuracy study reflected the % recovery between 99 and 101 % in both media. Further, the developed method was explored to quantify AKBA in formulated lipid nanocarriers (LNCs) formulation. The methanol method was effectively used to estimate the entrapment efficiency in LNCs and skin retention of AKBA. For the quantification of AKBA in in vitro release and ex vivo permeation samples, the phosphate buffer saline pH 7.4 method was utilized. Results indicated that the method could quantify the AKBA in the presence of different sample matrices with reproducibility in nature. In conclusion, a sensitive, simple, economical, precise, and accurate UV–Visible spectroscopic method was developed and validated to quantify AKBA.

Development and evaluation of a protease inhibitor antiretroviral drug-loaded carbon nanotube delivery system for enhanced efficacy in HIV treatment

The primary objective of this study was to enhance the effectiveness of the protease inhibitor antiretroviral drug by designing a novel delivery system using carboxylated multiwalled carbon nanotubes (COOH-MWCNTs). To achieve this, Fosamprenavir calcium (FPV), a prodrug of amprenavir known for inhibiting the proteolytic cleavage of immature virions, was selected as the protease inhibitor antiretroviral drug, and loaded onto COOH-MWCNTs using a direct loading method. The structural specificity of the drug-loaded MWCNTs, the percent entrapment efficiency, and in vitro drug release were rigorously evaluated for the developed formulation, referred to as FPV-MWCNT. Fourier transform infrared (FTIR) spectroscopy, Field emission scanning electron microscopy (FE-SEM), Raman spectroscopy, and atomic force microscopy (AFM) techniques were employed to confirm the structural integrity and specificity of the FPV-MWCNT formulation. The results demonstrated a remarkable entrapment efficiency of 79.57 ± 0.4 %, indicating the successful loading of FPV onto COOH-MWCNTs. FE-SEM and AFM analyses further confirmed the well-dispersed and elongated structure of the FPV-MWCNT formulation, without any signs of fracture, ensuring the stability and integrity of the drug delivery system. Moreover, particle size analysis revealed an average size of 290.1 nm, firmly establishing the nanoscale range of the formulation, with a zeta potential of 0.230 mV, signifying the system's colloidal stability. In vitro drug release studies conducted in methanolic phosphate buffer saline (PBS) at pH 7.4 and methanolic acetate buffer at pH 5 demonstrated sustained drug release from the FPV-MWCNT formulation. Over a period of 96 h, the formulation exhibited a cumulative drug release of 91.43 ± 2.3 %, showcasing the controlled and sustained release profile. Furthermore, hemolysis studies indicated a notable reduction in the toxicity of both FPV and MWCNT upon conjugation, although the percent hemolysis increased with higher concentrations, suggesting the need for careful consideration of dosage levels.In conclusion, the findings from this study underscore the potential of the FPV-MWCNT formulation as an effective and promising drug-conjugated system for delivering antiretroviral drugs. The successful encapsulation, sustained drug release, and reduced toxicity make FPV-MWCNT a compelling candidate for enhancing the therapeutic efficacy of protease inhibitor antiretroviral drugs in the treatment of HIV. The developed delivery system holds great promise for future advancements in HIV treatment and paves the way for further research and development in the field of drug delivery utilizing carbon nanotube-based systems.

A novel eco-friendly HPLC method with dual detection modes for versatile quantification of dutasteride and silodosin in pharmaceutical formulation, dissolution testing and spiked human plasma

With the increased production of new fixed combinations and cheaper generic drugs, the need for a single, sensitive and reliable analytical method is hugely evoked to cover the versatile and prerequisite tests of in-vitro and in-vivo bioequivalence studies. A new fixed dose combination, containing dutasteride (DUT) and silodosin (SLD), has been recently launched for relieving moderate and severe symptoms of benign prostatic hyperplasia. Consequently, this concurrent study was dedicated to present, for the first time, eco-friendly high-performance liquid chromatographic (HPLC) methods coupled with dual-mode detection, for the simultaneous quantification of both drugs in versatile real samples. The optimized chromatographic separation was achieved on a Waters XBridge® (150×4.6 mm, 5 μm) C18 analytical column with a mobile phase composed of methanol & phosphate buffer pH 6.0 at ratio (77: 23, v/v) and pumped at 1.0 mL/min flow rate. In the first HPLC method, drug quantification was performed at 245 nm achieving linearity range of 3–120 µg/mL, while the second one made good use of the unique features of both drugs in possessing native fluorescence for more sensitive linearity ranges (50–1000 ng/mL for DUT & 80–2500 ng/mL for SLD). Both methods were validated and successfully applied for assay dosage forms as well as testing content uniformity. In addition, the capability of the proposed HPLC-fluorometric method in detecting drugs in nano-gram scale paved the way not only for studying their dissolution behavior in various dissolution media but also analyzing spiked human plasma samples extracted by liquid-liquid extraction. Finally, sustainability of the proposed methods was clearly declared, in comparison to the official ones, via environmental, health and safety (EHS) tool and analytical greenness (AGREE) software.

ANALYTICAL UV SPECTROSCOPY METHOD DEVELOPMENT AND VALIDATION STUDIES FOR SIMULTANEOUS ESTIMATION OF METFORMIN HCL AND QUERCETIN

In the current investigation, we have designed and assessed a simple and swift analytical approach employing UV spectroscopy for the simultaneous quantification of the analytes metformin and quercetin with excellent precision and accuracy. The wavelengths of interest are the wavelengths at which both the drugs show maximum absorbance: 233 nm for metformin and 256 nm for quercetin. Linearity study, conducted in methanol and phosphate buffer, yielded a correlation coefficient (r2 ) of 0.99. The validation study for the developed method was conducted in accordance with ICH Q2 R1 guidelines. The percent recovery was 95% to 105%, and the percent relative standard deviation was <2, demonstrating the accuracy and precision of this method. This method can be applied to analysis of the two compounds in fixed dose formulations using simple UV spectroscopy.

Green chromatographic methods for determination of co-formulated lidocaine hydrochloride and miconazole nitrate along with an endocrine disruptor preservative and potential impurity

Recently, green analytical chemistry (GAC) is a key issue towards the idea of sustainability, the analytical community is focused on developing analytical methods that incorporate green chemistry principles to minimize adverse impacts on the environment and humans. Herein, we present 2 sustainable, selective, and validated chromatographic methods. Initially, lidocaine hydrochloride (LDC) and miconazole nitrate (MIC) with two preservatives; methyl paraben (MTP) and saccharin sodium (SAC) were chromatographed via TLC–densitometric method which employed ethyl acetate: methanol: formic acid (9:1:0.1, by volume) as the mobile phase with UV detection at 220.0 nm, good correlation was obtained in the range of 0.3–3.0 µg/band for MIC and LDC. Following that, RP-HPLC was successfully applied for separating quinary mixture of LDC, MIC, MTP, SAC along with LDC impurity; dimethyl aniline (DMA) using C18 column, and a gradient green mobile phase composed of methanol and phosphate buffer (pH 6.0) in different ratios with a flow rate 1.5 mL/min and UV detection at 210.0 nm, linearity ranges from 1.00 to 100.00 µg/mL for MIC, 2.00–100.00 µg/mL for LDC and 1.00–-20.00 µg/mL for MTP and DMA. No records to date regarding the determination of the two drugs, besides MTP and DMA. The proposed methods were validated according to the ICH guidelines and applied successfully to the analysis of the compounds. The methods' results were statistically compared to those obtained by applying the reported one, indicating no significant difference regarding both accuracy and precision. The methods' greenness profiles have been assessed and compared with those of the reported method using different assessment tools.Graphical

Molecularly imprinted polymer membranes based on single template for recognition and extraction of carbamazepine and its structurally analogues

In this work, a novel molecularly imprinted polymer membrane (MIPM) using oxcarbazepine (OXC) as the single template and polyvinylidene difluoride membrane as the subtract was fabricated for recognition and extraction of carbamazepine (CBZ) and its analogues including OXC, carbamazepine-10,11-epoxide (CBZE), and monohydroxy derivative 10, 11-dihydro-10-hydroxy carbamazepine (MHD). The prepared MIPMs were characterized by scanning electron microscope, Fourier transform infrared spectroscopy, Brunaner-emmet-teller measurements and X-ray photoelectron spectroscopy, showing the successful construction of uniform and porous polymers on the surface of membranes. A series of adsorption affinity tests were investigated, indicating the prepared materials had specific recognition capacity and excellent stability as novel sorbents. The maximum static adsorption capacities of MIPMs towards OXC, CBZ, MHD or CBZE was 4.246, 3.621, 2.316, or 4.877 mg·g−1, respectively. The MIPMs showed reusability and reproducibility. The usability of MIPMs was evaluated by combining MIPMs extraction and high performance liquid chromatography detection for the determination of CBZ, CBZE, OXC and MHD in different matrices including methanol, normal saline, phosphate buffer solution, and plasma. The mean recovery of four target compounds in methanol, normal saline and phosphate buffer solution was more than 75 %, while the mean recovery of CBZ and CBZE in plasma was more than 80 %. The results demonstrated that the proposed MIPMs is very suitable for the determination of OXC, CBZ, MHD and/or CBZE in different matrices.

Development and Validation of Chemometric Assisted RP-HPLC Method for Simultaneous Estimation of Perindopril Erbumine, Indapamide, and Amlodipine Besylate in Bulk and Pharmaceutical Formulation

This research paper illustrates a latterly developed, optimized and validated gradient RP-HPLC approach for simultaneous analysis ofIndapamide, Perindopril erbumine and Amlodipine besylate in bulk and pharmaceutical formulation with the assistance of quality by design. Qualityis predicated on desired and predetermined specifications. Understanding various factors, dependent variables, and their interconnection effectsby a desired set of experiments on the responses to be analyzed is an important component of QbD. Several operating conditions of variousprocesses optimization, chromatographic separation performance improvement, and high extraction efficiency were attained by using QbD. Thepowerful chromatographic conditions were done using the HypersilC18 column (250mm × 4.6mm, 5μm particle Size). The UV detector wasadjusted to 215nm. Design of experiments (DoE) was applied for multivariate optimization of the experimental conditions of the RP-HPLCmethod. Three independent factors, mobile phase composition, phosphate buffer strength, and flow rate, were used to design mathematicalmodels. Central composite design (CCD) was used to examine the response surface methodology and fully examine the results of theseindependent factors. The desirability function was used to optimize the retention time and resolution of the analytes simultaneously. Theimproved and anticipated data from the contour diagram consisted of methanol and phosphate buffer (pH 2.5, strength 0.05M) in the ratio of65:35, respectively, at a flow rate of 1.1 ml/min. Using these optimum conditions, baseline separation of both drugs with good resolution and runtime of less than 5.0 min was achieved. The novelty of the developed method was time-consuming, cost-effective, and sensitive. The optimizedassay conditions were validated according to ICH guidelines. Under the optimized state, the linearity ranges were found to be 10-40 μg/mL, 32–128 μg/mL, and 40-160 μg/mL for Indapamide, Perindopril erbumine, and amlodipine besylate, respectively, with correlation coefficients (R2) of0.999. The mean accuracy studied ranged from 99.18 to 99.58%. The percentage coefficient variation value for the precision study was lower than1%. The proposed method showed good precision and repeatability. Hence the developed RP-HPLC method using quality by design can be usedas a routine quality control analysis of indapamide, perindopril erbumine, and amlodipine besylate.

Box–Behnken design-assisted optimization of RP-HPLC method for the estimation of evogliptin tartrate by analytical quality by design

BackgroundA quality by design approach can potentially lead to a more robust/rugged method development due to emphasis on the risk assessment and management. By carefully understanding the step-by-step procedure for analytical QbD-based optimization parameters, such as analytical target profile and critical quality attributes (CQAs), was assessed. The present study describes the simple, rapid, sensitive and cost-effective RP-HPLC method development and validation for the estimation of evogliptin tartrate in pharmaceutical dosage form.ResultsThe factor screening studies were performed using Box–Behnken design by three key components of the RP-HPLC method (mobile phase, pH and flow rate). The chromatographic conditions were optimized with the Design Expert software trial version 13.0. The optimal chromatographic separation was achieved having water C18 column (250 mm × 4.6 mm, 5 μ) and using mobile phase as a methanol and phosphate buffer (pH 4.5) 60:40% v/v with a flow rate 1.0 ml/min and UV detection at 267 nm. The Box–Behnken experimental design describes the interrelationship of mobile phase, pH and flow rate at three different levels, and responses of retention time and tailing factor were observed with response surface plot and statistical data. The developed method was validated as per recommended ICH guidelines which revealed the high degree of linear, precise, accurate, sensitive and robust method over the existing RP-HPLC method for evogliptin tartrate.ConclusionThe developed QbD-based method helped in generating a design space and operating space with knowledge of all method performance characteristics, and RP-HPLC method takes less time and can be used in the industry for routine quality control of bulk and marketed formulation of evogliptin tartrate.

One-pot synthesis of a poly(styrene-acrylic acid) copolymer-modified silica stationary phase and its applications in mixed-mode liquid chromatography

As modified ligands with a wide range of sources, abundant functional groups, and good biocompatibility, polymers have been widely used in the development of silica-based chromatographic stationary phases. In this study, a poly(styrene-acrylic acid) copolymer-modified silica stationary phase (SiO2@P(St-b-AA)) was prepared via one-pot free-radical polymerization. In this stationary phase, styrene and acrylic acid were used as functional repeating units for polymerization and vinyltrimethoxylsilane (VTMS) was used as a silane coupling agent to link the copolymer and silica. Various characterization methods, such as Fourier transform infrared (FT-IR) spectroscopy, thermogravimetric analysis (TGA), scanning electron microscopy (SEM), N2 adsorption-desorption analysis, and Zeta potential analysis, confirmed the successful preparation of the SiO2@P(St-b-AA) stationary phase, which had a well-maintained uniform spherical and mesoporous structure. The retention mechanisms and separation performance of the SiO2@P(St-b-AA) stationary phase in multiple separation modes were then evaluated. Hydrophobic and hydrophilic analytes as well as ionic compounds were selected as probes for different separation modes, and changes in the retention of the analytes under various chromatographic conditions, including different methanol or acetonitrile contents and buffer pH values, were investigated. In reversed-phase liquid chromatography (RPLC) mode, the retention factors of alkyl benzenes and polycyclic aromatic hydrocarbons (PAHs) on the stationary phase decreased with increasing methanol content in the mobile phase. This finding could be attributed to the hydrophobic and π-π interactions between the benzene ring and analytes. The retention changes of alkyl benzenes and PAHs revealed that the SiO2@P(St-b-AA) stationary phase, similar to the C18 stationary phase, exhibited a typical reversed-phase retention behavior. In hydrophilic interaction liquid chromatography (HILIC) mode, as the acetonitrile content increased, the retention factors of hydrophilic analytes gradually increased, and a typical hydrophilic interaction retention mechanism was inferred. In addition to hydrophilic interaction, the stationary phase also demonstrated hydrogen-bonding and electrostatic interactions with the analytes. Compared with the C18 and Amide stationary phases prepared by our groups, the SiO2@P(St-b-AA) stationary phase exhibited excellent separation performance for the model analytes in the RPLC and HILIC modes. Owing to the presence of charged carboxylic acid groups in the SiO2@P(St-b-AA) stationary phase, exploring its retention mechanism in ionic exchange chromatography (IEC) mode is of great importance. The effect of the mobile phase pH on the retention time of organic bases and acids was further studied to explore the electrostatic interaction between the stationary phase and charged analytes. The results revealed that the stationary phase has weak cation exchange ability toward organic bases and electrostatically repels organic acids. Moreover, the retention of organic bases and acids on the stationary phase was influenced by the analyte structure and mobile phase. Thus, the SiO2@P(St-b-AA) stationary phase could provide multiple interactions, as demonstrated by the separation modes described above. The SiO2@P(St-b-AA) stationary phase showed excellent performance and reproducibility in the separation of mixed samples with different polar components, indicating that it has promising application potential in mixed-mode liquid chromatography. Further investigation of the proposed method confirmed its repeatability and stability. In summary, this study not only described a novel stationary phase that could be used in RPLC, HILIC, and IEC modes but also presented a facile "one-pot" preparation approach that could provide a new route for the development of novel polymer-modified silica stationary phases.

UV Spectrophotometric and UPLC assay methods for the determination of Palbociclib in bulk and Tablet Dosage Form

The goal of this study was to create a sensitive and reliable UV-spectrophotometric and reverse phase liquid chromatographic method for determining Palbociclib in bulk and pharmaceutical dosage forms. The current UV approach uses methanol and phosphate buffer as a solvent to evaluate absorption at a maximum wavelength of 220nm. Waters Acquity BEH C18 (2.1 x 50mm, 1.7µm) UPLC column with Empower 2 was used in the UPLC procedure. The mobile phase was made up of 50:50 (v/v) methanol and phosphate buffer.The drug obeyed Beer Lambert's law in the UV technique in the concentration range of 1-70µg/ml with a regression coefficient of 0.999. The linearity level in the UPLC technique was 1-100µg/ml. In the validation investigation, the developed UV and UPLC procedures were successful. For the UPLC and UV methods, the percentage purity of the marketed dosage form of Palbociclib was found to be 98.76 percent and 98.84 percent, respectively. The percent RSD of all validation parameters investigated, as suggested by the ICH recommendations, was determined to be less than 2 in both techniques. The two methods established for determining Palbociclib in bulk and tablet doses were found to be accurate, simple, and validated.

Optimized Suspension Trapping Method for Phosphoproteomics Sample Preparation.

A successful mass spectrometry-based phosphoproteomics analysis relies on effective sample preparation strategies. Suspension trapping (S-Trap) is a novel, rapid, and universal method of sample preparation that is increasingly applied in bottom-up proteomics studies. However, the performance of the S-Trap protocol for phosphoproteomics studies is unclear. In the existing S-Trap protocol, the addition of phosphoric acid (PA) and methanol buffer creates a fine protein suspension to capture proteins on a filter and is a critical step for subsequent protein digestion. Herein, we demonstrate that this addition of PA is detrimental to downstream phosphopeptide enrichment, rendering the standard S-Trap protocol suboptimal for phosphoproteomics. In this study, the performance of the S-Trap digestion for proteomics and phosphoproteomics is systematically evaluated in large-scale and small-scale samples. The results of this comparative analysis show that an optimized S-Trap approach, where trifluoroacetic acid is substituted for PA, is a simple and effective method to prepare samples for phosphoproteomics. Our optimized S-Trap protocol is applied to extracellular vesicles to demonstrate superior sample preparation workflow for low-abundance, membrane-rich samples.