C18 Column Research Articles

Kinetics and Thermodynamics of Lactose Mutarotation through Chromatography.

The mutarotation kinetics and thermodynamics of the reaction α-lactose ⇌ β-lactose have been measured in dilute solutions using liquid chromatography without any derivatization step, using a C18 column and pure water as the mobile phase. The effect of temperature (0.5-45 °C) of the starting powder composition (α-lactose-rich or β-lactose-rich powder) and of the solvent composition (water with up to 35% weight fraction of seven organic solvents) has been experimentally investigated. Increasing the temperature leads to faster kinetics, following an Arrhenius model, and to slightly decreasing concentration-based equilibrium ratio. Conversely, increasing the weight fraction of organic solvent at 25 °C resulted in slower kinetics and smaller concentration-based equilibrium ratio. The starting powder composition is shown not to influence the kinetics or thermodynamics of the process. The corresponding parameter estimation problem is thoroughly discussed, taking into account the small difference in response factors of the lactose diastereomers.

Detection of aflatoxin in raw and pasteurized milk by high-performance liquid chromatography (HPLC) in central Ethiopia

Aflatoxins are a group of structurally related mycotoxins produced by the Aspergillus flavus, Aspergillus parasiticus, and Aspergillus nomius species as secondary metabolites. Well-known forms of Aflatoxin are AFB1, AFB2, AFG1, AFG2, AFM1, and AFM2. aflatoxin B1 is the most prevalent one. It can be metabolized in the liver and excreted as aflatoxin M1 in milk. Both forms have mutagenic and carcinogenic effects. In Ethiopia, there is limited information on the occurrence and contamination level of aflatoxin in raw and pasteurized milk. The present study was conducted in the Addis Ababa milk shed area to detect and quantify the level of Aflatoxin M1 in raw and pasteurized milk. A cross-sectional study was conducted from October 2020 to May 2021 on a total of 114 cow milk samples consisting of 60 raw milk and 54 pasteurized milk samples with the aim of detecting and quantifying the amount of aflatoxin M1 in raw and pasteurized milk in central Ethiopia by high-performance liquid chromatography (HPLC) using C18 column with fluorescence detector. AFM1 was detected in 79 (69.3%) of the 114 tested milk samples. The maximum and mean concentrations were 0.893 µg/L and 0.0465 µg/L, respectively. 25.4% of them contain AFM1 above the maximum limit of EU (0.05 µg/L), and 1.8% contained above the maximum limit of CES278 and FDA (0.5 µg/L). 26.7% of the samples from Sebata and 6.7% from Sululta contain above the maximum limit of EU (0.05 µg/L). Higher contamination of AFM1 was detected in pasteurized milk (96.3%) than in raw milk (16.7%). 35.2% of pasteurized and 16% of raw milk contained AFM1 above the maximum limit of EU0.05 µg /L. The study results showed a significant difference in AFM1 occurrence with feed type, storage time, and milk type. The current AFM1 concentration level in raw and pasteurized milk was not safe for human consumption in the study areas. Due to its heat resistance, AFM1 is found in pasteurized milk and has great health effects. Creating awareness of feed management for feed producers and farmers and developing risk mitigation methods are crucial in order to reduce public health threats.

In vitro dissolution equivalence of Jordanian sildenafil generics via validated, stability-indicating HPLC method.

This study was conducted to compare dissolution profiles of four Jordanian registered sildenafil (SDF) products to the originator. Dissolution samples were analyzed utilizing a validated and stability-indicating HPLC method in human plasma. Validation was performed forspecificity, linearity, limit of detection, lower limit of quantification, precision, trueness and stability. SDF was extracted from plasma samples using liquid-liquid extraction. The analysis wasperformed utilizing isocratic elution on C18 column with 1.0ml/min flow rate. The regression value was ∼0.999 over 3days with drug recovery between86.6 to 89.8%with 10ng/ml lower limit of quantitation. This method displayed a good selectivity of SDF with improved stability under various conditions. The method was used for SDF quantification in dissolution medium. Similarity factors for local products varied according to the used mediums, but all SDF local products passed the dissolution in vitro test since all of them showed a released of >85% after 60min at the dissolution mediums.

An LC-MS/MS method for determination of the bromodomain inhibitor ZEN-3694 and its metabolite ZEN-3791 in human plasma.

We have developed and validated a novel LC-MS/MS method for the simultaneous quantification of ZEN-3694 and its active metabolite ZEN-3791 in human plasma afterprotein precipitation. Stable isotope-labeled versions were used as internal standards. Chromatographic separation was achieved on a Kinetex C18 column using 0.1% formic acid in H2O and 0.1% formic acid in MeOH as mobile phases. Detection was performed via positive electrospray ionization mode with multiple reaction monitoring. The assay exhibited linearity in the concentration range of 5-5000ng/ml for both analytes. Intra- and inter-assay precision and accuracy were within ±11%. ZEN-3694 and ZEN-3791 recoveries were between 93 and 105%. This LC-MS/MS assay is an essential tool to study ZEN-3694 in an ongoing clinical trial (NCT04840589).

Simultaneous HPLC determination of remdesivir and dexamethasone in the presence of metformin, sitagliptin, and glimepiride in a synthetic mixture and spiked human plasma

The COVID-19 pandemic has raised many questions regarding the control and therapy of type 2 diabetes and the higher risk of severe disease progression. One of the therapeutic regimens used in moderate and severe cases of COVID-19, endorsed by the World Health Organization, involves the administration of an antiviral medicinal product and a corticosteroid. The present study describes the development of a liquid chromatographic method for the simultaneous separation and quantification of Remdesivir and Dexamethasone in the presence of Metformin, Sitagliptin, and Glimepiride in a synthetic mixture. The developed method also allows determination of Remdesivir, Dexamethasone, and Glimepiride in spiked plasma samples, using Sitagliptin as internal standard. A mixture of acetonitrile and potassium dihydrogen phosphate buffer (pH 3) in a ratio of 45:55 v/v was used as а mobile phase on a C18 column. The recovery percentages from plasma ranged from 85.1 to 108.5%. The developed method can serve in routine quality control and clinical laboratory practice.

Mechanistic studies to understand peak tailing due to sulfinic acid- and carboxylic acid-silanophilic interactions in reversed-phase liquid chromatography

Silanophilic interactions are a primary contributor to peak tailing of acidic pharmaceutical compounds, thus a thorough understanding is especially important for reversed-phase liquid chromatography (RPLC) method development. Herein, a sulfinic acid compound that exhibited severe peak tailing in RPLC with acidic mobile phases was carefully studied. Results indicated that the neutral protonated form of the sulfinic acid is involved in the strong interaction that leads to peak tailing, but that tailing can be mitigated with a blocking effect achieved through use of acetic acid modifier in the mobile phase. Peak tailing was also observed with other structurally-similar sulfinic acids and carboxylic acids but was, in general, less severe with the latter. The Hydrophobic Subtraction Model (HSM) was applied to six commercial C18 columns that exhibited different tailing behaviors for the sulfinic acid compound in attempts to identify key sites of interaction within the stationary phase. A combination of heated acid column wash experiments and density functional theory (DFT) calculations indicate that the differential interactions of the acids with vicinal silanol pairs in the stationary phase play a major role in peak tailing.

Comprehensive Characterization of Triterpene Saponins in Rhizoma Panacis Japonici by Offline Two-Dimensional Liquid Chromatography Coupled to Quadrupole Time-of-Flight Mass Spectrometry.

Rhizoma Panacis Japonici (RPJ) is an ancient herbal medicine from China that has long been employed for its medicinal benefits in relieving arthritis physical debility and diverse afflictions. The primary bioactive constituents found in RPJ are triterpene saponins, which exhibit numerous pharmacological actions, including anti-inflammatory, antioxidant, and immunomodulating effects. The present study established a straightforward and effective approach for characterizing triterpene saponins in RPJ. An offline HILIC × RP LC/QTOF-MS method was developed, along with a self-constructed in-house database containing 612 saponins reported in the Panax genus and 228 predicted metabolites. The approach achieved good chromatographic performance in isolating triterpene saponins of RPJ, with the HILIC column as the first dimension (1D) and the BEH C18 column as the second dimension (2D). The developed two-dimensional liquid chromatography system exhibited an orthogonality of 0.61 and a peak capacity of 1249. Detection was performed using a QTOF mass spectrometer in a data-independent manner (MSE) in a negative ion mode. Using the in-house database, the collected MS data were processed by an automatic workflow on UNIFI 1.8.2 software, which included data correction, matching of precursor and product ions, and peak annotation. In this study, 307 saponins were characterized from RPJ and 76 saponins were identified for the first time in Panax japonicus. This research not only enhances our understanding of the chemical characteristics of RPJ but also offers a simple and efficient method for analyzing the complex composition of herbal medicine.

Development of an easy-to-set-up multiple heart-cutting achiral-chiral LC-LC method for the analysis of branched-chain amino acids in commercial tablets.

In this paper, the development and application of a multiple heart-cutting achiral-chiral LC-LC method (mLC-LC) for the analysis of dansylated (Dns) branched-chain amino acids in commercial tablets are described. In the first dimension, a Waters Xbridge RP C18 achiral column was used under gradient conditions with buffered aqueous solution and acetonitrile. The elution order Dns-valine (Dns-Val)<Dns-isoleucine (Dns-Ile)<Dns-leucine (Dns-Leu) turned out with full resolution between adjacent peaks: 7.25 and 1.50 for the Val/Ile and the Ile/Leu pairs, respectively. A "research" validation study was performed, revealing high accuracy (Recovery%) and precision (RSD%) using two external set solutions, respectively, in the range 93.7%-104.1% and 0.4%-3.2%. The C18 column was connected via a two-position six-port switching valve to the quinidine-based Chiralpak quinidine-anion-exchange chiral column. A water/acetonitrile, 30/70 (v/v) with 50mM ammonium acetate (apparent pH of 5.5) eluent allowed getting the three enantiomers' pairs resolved: RS equal to 4.3 for Dns-Val and Dns-Ile, and 1.7 for Dns-Leu. The application of the mLC-LC method confirmed that the content of Val, Ile, and Leu in the tablets was compliant with that labeled by the producer. Only l-enantiomers were found in the food supplement, as confirmed by LC-MS/MS analysis.

Dispersive Solid-Phase Extraction and Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry-A Rapid and Accurate Method for Detecting 10 Macrolide Residues in Aquatic Products.

The amount of macrolide (MAL) residues in aquatic products, including oleandomycin (OLD), erythromycin (ERM), clarithromycin (CLA), azithromycin (AZI), kitasamycin (KIT), josamycin (JOS), spiramycin (SPI), tilmicosin (TIL), tylosin (TYL), and roxithromycin (ROX), was determined using solid-phase extraction and ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The residues were extracted with 1% ammonia acetonitrile solution and purified by neutral alumina adsorption. Chromatographic separation was completed on an ACQUITY UPLC BEH C18 column with acetonitrile-0.1% formic acid aqueous solution as the mobile phase, and mass spectrometry detection was performed by multiple reaction monitoring scanning with the positive mode in an electrospray ion source (ESI+). Five isotopically labeled compounds were used as internal standards for quality control purposes. The findings indicated that across the mass concentration span of 1.0-100 μg/L, there was a strong linear correlation (R2 > 0.99) between the concentration and instrumental response for the 10 MALs. The limit of detection of UPLC-MS/MS was 0.25-0.50 μg/kg, and the limit of quantitation was 0.5-1.0 μg/kg. The added recovery of blank matrix samples at standard gradient levels (1.0, 5.0, and 50.0 μg/kg) was 83.1-116.6%, and the intra-day precision and inter-day precisions were 3.7 and 13.8%, respectively. The method is simple and fast, with high accuracy and good repeatability, in line with the requirements for accurate qualitative and quantitative analysis of the residues for 10 MALs in aquatic products.

Molecular Docking and ADMET Analysis Strategy-Based Stability Indicating RP-HPLC-PDA Method Development and Validation of Toremifene.

The purpose of this research is to develop an analytical method and validate it according to ICH guidelines for the estimation of Toremifene by RP-HPLC-PDA with molecular docking and ADMET analysis. From molecular docking, it came to know the receptor affinity specifically to estrogen receptors (ERα and ERβ), which are responsible for cancer therapy. ADMET analyses secure its therapeutic potential as well safety of the drug. An isocratic method has developed by RP-HPLC-PDA (AGILENT 1100) with symmetry of 100 mm x 4.6 mm x 5 μm particle size C18 column and optimise mobile phase is methanol: 0.1% OPA (orthophosphoric acid) water ratio of 43:57% v/v. Under different conditions like acidic, alkaline, oxidative, and neutral environments, toremifene was tested for degradation. The developed method is validated in accordance with ICH guidelines. A calibration curve with an r2 value of 0.9987 has been prepared across the range of 10 to 50 μg/ml with five standard dilutions. The retention time of the drug is 5.575 minutes. The validation results are system suitability (%RSD-0.76), inter-day precision (%RSD 0.14-0.29), intraday precision (%RSD 0.08-0.34), accuracy (%RSD 0.16-0.96), and robustness (%RSD 0.16-0.35). In different intended conditions, four peaks are in 1 N HCl, two peaks in 1 N NaOH, three peaks in 10% H2O2 (1hr), and one peak in neutral. Toremifene, a Selective Estrogen Receptor Modulator (SERM), Drug pharmacokinetic properties and receptor binding affinity results are helpful in designing the analytical method. Developing the RP-HPLC-PDA method is found to be novel, simple and precise. It could be used for testing toremifene in bulk and pharmaceutical tablet dosage forms in quality control, as well as stability tests.

Simultaneous determination of four non paraben preservatives (benzyl alcohol, phenoxyethanol, sodium benzoate, and salicylic acid) in shampoos by an isocratic HPLC method

Salicylic acid (SA), sodium benzoate (SB), phenoxyethanol (PO), and benzyl alcohol (BA) are “non paraben” preservatives commonly used in cosmetic products like shampoos, and often in combination of at least two compounds to assure their antimicrobial effectiveness at low concentrations. In this study, an HPLC-DAD method was developed and validated for simultaneous determination of SA, SB, PO, and BA in commercial shampoos. The method was carried out using an YMC Triart C18 (250 × 4.6 mm, 5 µm) column with a mixture of methanol and 0.02 M solution of phosphate buffer pH 2.7 (35 : 65, v/v) as mobile phase. The flow rate of mobile phase was maintained at 1.0 mL per minute, the injection volume was 20 µL and the UV detection was done at 208 nm for BA and at 230 nm for SA, SB and PO. The method was fully validated according to requirements on analytical method performance of AOAC International and validation results confirmed its reliability and suitability for intended application. The method was employed to determine the four “non paraben” preservatives in 18 commercial shampoos, finding the actual content of these substances in all samples conformed to their regulatory limits.

Targeted ultra performance liquid chromatography tandem mass spectrometry procedures for the diagnosis of inborn errors of metabolism: validation through ERNDIM external quality assessment schemes.

Early diagnosis of inborn errors of metabolism (IEM) is crucial to ensure early detection of conditions which are treatable. This study reports on targeted metabolomic procedures for the diagnosis of IEM of amino acids, acylcarnitines, creatine/guanidinoacetate, purines/pyrimidines and oligosaccharides, and describes its validation through external quality assessment schemes (EQA). Analysis was performed on a Waters ACQUITY UPLC H-class system coupled to a Waters Xevo triple-quadrupole (TQD) mass spectrometer, operating in both positive and negative electrospray ionization mode. Chromatographic separation was performed on a CORTECS C18 column (2.1×150, 1.6 µm). Data were collected by multiple reaction monitoring. The internal and EQA results were generally adequate, with a few exceptions. We calculated the relative measurement error (RME) and only a few metabolites displayed a RME higher than 30 % (asparagine and some acylcarnitine species). For oligosaccharides, semi-quantitative analysis of an educational panel clearly identified the 8 different diseases included. Overall, we have validated our analytical system through an external quality control assessment. This validation will contribute to harmonization between laboratories, thus improving identification and management of patients with IEM.

A new validated high‐performance liquid chromatography method for the determination of regorafenib in rat plasma: Application for pharmacokinetic study

AbstractThis study reports a rapid and sensitive reversed‐phase high‐performance liquid chromatography method with ultraviolet detection for the determination of regorafenib (REG) in rat plasma. For the extraction of REG from plasma and the precipitation of plasma proteins, a rapid one‐step precipitation method was performed with methanol. The separation was performed in a methanol‐water mixture (80:20, v:v) mobile phase system with a C18 column and monitored at a wavelength of 258 nm. Nilotinib was used as an internal standard. The linear dynamic range of REG was determined between 5 and 500 ng/mL in standard solution and plasma. The limit of detection of REG from the standard solution was found to be 1.23 ng/mL and the limit of quantitation was 3.73 ng/mL. The limit of detection of REG from plasma was found to be 1.55 ng/mL and the limit of quantitation was 4.70 ng/mL. Regorafenib was administered at 5 mg/kg to 10 healthy male Sprague Dawley rats, and pharmacokinetic results were evaluated. It exhibited a high recovery of 93.82% in plasma. Due to ease of sample preparation, high susceptibility, specificity, and certainty, the described method can be used successfully in the quantification of REG in in‐vivo plasma and in routine analyses in clinical laboratories.

Comparative Pharmacokinetics Research of 13 Bioactive Components of Jieyu Pills in Control and Attention Deficit Hyperactivity Disorder Model Rats Based on UPLC-Orbitrap Fusion MS.

Jieyu Pills (JYPs), a Chinese medicine consisting of 10 herbal elements, have displayed promising clinical effectiveness and low by-effects in the treatment of depression. Prior investigations mostly focused on elucidating the mechanism and therapeutic efficacy of JYPs. In our earlier study, we provided an analysis of the chemical composition, serum pharmacochemistry, and concentrations of the main bioactive chemicals found in JYPs. However, our precise understanding of the pharmacokinetics and metabolism remained vague. This study involved a comprehensive and meticulous examination of the pharmacokinetics of 13 bioactive compounds in JYPs. Using UPLC-Orbitrap Fusion MS, we analyzed the metabolic characteristics and established the pharmacokinetic parameters in both control rats and model rats with attention deficit hyperactivity disorder (ADHD) following oral administration of the drug. Before analysis, plasma samples that were collected at different time intervals after the administration underwent methanol pre-treatment with Puerarin used as the internal standard (IS) solution. Subsequently, the sample was chromatographed on a C18 column employing gradient elution. The mobile phase consisted of methanol solution containing 0.1% formic acid in water. The electrospray ionization source (ESI) was utilized for ionization, whereas the scanning mode employed was selected ion monitoring (SIM). The UPLC-Orbitrap Fusion MS method was subjected to a comprehensive validation process to assess its performance. The method demonstrated excellent linearity (r ≥ 0.9944), precise measurements (RSD < 8.78%), accurate results (RE: -7.88% to 8.98%), and appropriate extraction recoveries (87.83-102.23%). Additionally, the method exhibited minimal matrix effects (87.58-101.08%) and satisfactory stability (RSD: 1.52-12.42%). These results demonstrated adherence to the criteria for evaluating and determining biological material. The 13 bioactive compounds exhibited unique pharmacokinetic patterns in vivo. In control rats, all bioactive compounds except Ferulic acid exhibited linear pharmacokinetics within the dose ranges. In the ADHD model, the absorption rate and amount of most of the components were both observed to have increased. Essentially, this work is an important reference for examining the metabolism of JYPs and providing guidelines for clinical therapy.

Validation of RP-HPLC Method for the Estimation of Timolol Maleate in Fast Dissolving Tablets

A simple, precise and reproducible validation method was develop for Timolol Maleate as per ICH (International Council for Harmonization) guidelines Q2R(1) and subsequently stability indicating method as per ICH guidelines Q1A(R2). Chromatographic evaluation was carried out on C18 column of Agilent (25 cm x 4.6 mm), (i.d. 5 µm.) with a mobile phase consist of Methanol : Water (20:80 v/v). Flowrate was maintained to 0.8ml/min. The stability indicating data revealed that Timolol Maleate was found to be unstable alkaline condition. The validated method was applied various validation parameters like Linearity(calibrationcurve),Precision, Accuracy,Robustness,LOD and LOQ were found to be 0.9μg/ml and 2μg/ml. The validated method can be used for estimation of Timolol Maleate in prepared Fast DissolvingTablets (FDTs) by using Croscar mellose Sodium(CCS) as a superdisintegrant a calculated their optical parameters.

Identification and characterization of impurities in an insecticide, commercial chlorantraniliprole using liquid chromatography-tandem mass spectrometry.

Ensuring the global safety and effectiveness of agrochemicals has become imperative. An in-depth understanding of impurity profiles of products is crucial, especially for high-demand agrochemicals, where impurities may be more toxic and persistent than original agrochemicals. This study focuses on the detection and identification of impurities in a commercial chlorantraniliprole (CAP), an anthranilic diamide class broad-spectrum insecticide. Commercial CAP was collected from an agrochemical supplier in India and was analyzed using a high-performance liquid chromatography-photodiode array (HPLC-PDA) (Agilent 1260; wavelength, 220 nm) with a Zorbax RP SB-C18 (250 × 4.6mm, 5μm) column and liquid chromatography-mass spectrometry (LC-MS) (Agilent 6545 quadrupole time of flight (Q-TOF)) techniques to identify the impurities. The impurities were isolated by preparative HPLC using a Zorbax-DB C18 (250 × 9.4mm, 5μm) column. liquid chromatography- tandem mass spectrometry (LC-MS/MS) experiments (Q-TOF) were performed on CAP and its impurities to obtain their structural data. HPLC-PDA analysis of CAP showed four major impurities (IM-1 to IM-4) ranging from 0.76% to 4.1%. The positive ion electrospray ionization (ESI) mass spectra of CAP and its impurities showed dominant [M + H]+ ions in addition to [M + Na]+ , [M + K]+ , and [2M + Na]+ ions. High-resolution mass spectrometry (HRMS) data provided the elemental composition of the compounds, and isotopic distribution patterns revealed the number of Cl and/or Br atoms present in them. The structures of impurities were proposed based on the LC-MS/MS) data and further confirmed by nuclear magnetic resonance (NMR) data on isolated impurities/synthesis. The quality and impurities of CAP, a popular insecticide, must be assessed and described for its efficacy and safety. In this study, four impurities of CAP were detected using HPLC and successfully characterized using LC-HRMS, LC-MS/MS, and NMR data. The method is useful for verifying the purity of CAP as well as helping in the identification of its possible impurities.

Cyclic Voltammetric Behaviour and High-Performance Liquid Chromatography Amperometric Determination of Levamisole

The electrochemical oxidation of levamisole, a glassy carbon electrode, was investigated over the pH range 2.0–10.0. Cyclic voltammetric investigations showed a single oxidation process was recorded, with a peak potential (Ep) shown to be pH-dependent in the range 5.0–8.0; between pH 2.0 and pH 5.0, and above pH 8.0, the Ep was found to be independent of pH, indicating apparent pKa values of 5.0 and 8.0. Peak currents were found to increase with increasing pH values. This voltammetric oxidation process was found to be consistent with a two-electron, two-proton oxidation to the corresponding sulfoxide. Based on these findings, the development of a of method based on the high-performance liquid chromatography separation of levamisole, with electrochemical detection being used for its determination, was explored. The chromatographic conditions required for the separation of levamisole were first investigated and optimized using UV detection. The conditions were identified as a 150 mm × 4.6 mm, 5 µm C18 column with a mobile phase consisting of 50% methanol, and 50%, 50 mM, pH 8.0 phosphate buffer. The technique of hydrodynamic voltammetry was applied to optimize the applied potential required for the determination of levamisole, identified as +2.3 V versus a stainless-steel pseudo-reference counter-electrode. Under the optimized conditions, levamisole exhibited a linear response of 1.00–20 mg/L (R2 = 0.999), with a detection limit of 0.27 mg/L. The possibility of determining levamisole in artificial urine was shown to be possible via simple dilution in the mobile phase. Mean recoveries of 99.7%, and 94.6%, with associated coefficients of variation of 8.2% and 10.2%, respectively, were obtained for 1.25 µg/mL (n = 5) and 2.50 µg/mL (n = 5).

Simultaneous monitoring of seven antiepileptic drugs by dried blood spot and dried plasma spot sampling: method validation and clinical application of a LC–MS/MS-based technique

Alternative blood sampling strategy can enhance the application of therapeutic drug monitoring (TDM), then improve precision therapy and medication compliance. In developing nations, alternative sampling strategy that allows self-sampling and room temperature transport is especially important. This study validates the use of dried blood spot (DBS) and dried plasma spot (DPS) sampling along with liquid chromatography–tandem mass spectrometry (LC–MS/MS) for analyzing seven common antiepileptic drugs (AEDs) (phenytoin, lamotrigine, levetiracetam, topiramate, carbamazepine, oxcarbazepine and its active metabolite 10,11-dihydro-10-hydroxy carbamazepine) and evaluates their applicability to clinical practice. Following simple protein precipitation with acetonitrile, the AEDs were separated on a C18 column by gradient elution with a mobile phase consisting of acetonitrile–water–0.1% formic acid at a flow rate of 0.65 mL/min. The method provided linear analysis over the tested concentration ranges, with a total run time of 7 min. Intra- and inter-assay precision for all quality controls were ≤12% with accuracies of 85.9%–113%. The average extraction efficiencies were 69.0%–92.4% for DBS and 65.9%–96.5% for DPS, and no significant matrix effects were observed. The AEDs were stable in all samples for seven days at room temprature and 40°C. There was good correlation between the dry and wet plasma concentrations with greater accuracy for DPS compared to DBS indicating that alternative sampling strategy using DBS and DPS are suitable for monitoring the concentrations of AEDs with satisfied performance and logistical advantages.

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.

A RELIABLE RP-UPLC-TUV METHOD FOR SIMULTANEOUS ESTIMATION OF CLARITHROMYCIN, AMOXICILLIN, AND VONOPRAZAN IN CO-PACKED PHARMACEUTICAL DOSAGE FORMS: METHOD DEVELOPMENT AND VALIDATION WITH STABILITY INDICATING PROPERTIES

Objective: The study aims to develop a reliable RP-UPLC-TUV method for simultaneous estimation of Clarithromycin, Amoxicillin, and Vonoprazan in bulk and combined dosage. Methods: A simple, specific, and reliable method for determining Clarithromycin, Amoxicillin, and Vonoprazan has been developed using the RP-UPLC method. In order to successfully separate Clarithromycin, Amoxicillin, and Vonoprazan, 1.0 µl of a 100 % level solution was injected into a Hibar C18 (100 x 2.1 mm and 2 µm) column. The mobile phase consisted of Ammonium Acetate and Acetonitrile in equal volumes, and the flow rate was kept at 0.3 ml/min while the detection wavelength was set to 210 nm. Both the column and the injection port were kept at a temperature of 30 °C at all times. Results: The retention time (RT) of Clarithromycin, Amoxicillin, and Vonoprazan was observed at 1.24 min, 0.97 min and 1.66 min, correspondingly with accepted system suitability. The linear responses were observed for Clarithromycin, Amoxicillin, and Vonoprazan in the range of 25 to 150 µg/ml, 25 to 150 µg/ml and 1 to 6 µg/ml, respectively. The LOD and LOQ values were calculated to 0.07 µg/ml and 0.22 µg/ml for Clarithromycin, 0.81 µg/ml and 2.45 µg/ml for Amoxicillin and 0.03 µg/ml and 0.09 µg/ml for Vonoprazan. The % RSD values of both precision were assessed in the range of 0.8-1.4. The mean recovery of Clarithromycin, Amoxicillin, and Vonoprazan was in the range of 99.66 %-100.88 %. The statistical analysis of the validation parameters confirmed that the approach was reliable in terms of its accuracy, sensitivity, and precision while also exhibiting a high degree of sensitivity. The study of analytes in a variety of stressful situations guarantees the stability of the substances, ensuring that they represent the method's stability indication. Conclusion: The newly established technique is quite effective in separating Clarithromycin, Amoxicillin, and Vonoprazan from one another. Also separated with excellent resolution were the degradation products that were formed as a result of the stress conditions. The study concluded that the developed method has considerable adoption in the pharmaceutical sector.