RP-LC Method Research Articles

Use of green RPLC methods as an alternative to conventional RPLC methods in the determination of the pK a values of some H 2 receptor antagonists

Use of green RPLC methods as an alternative to conventional RPLC methods in the determination of the <i> pK _a </i> values of some H ₂ receptor antagonists

Switch from The Traditional RPLC Method to the Green RPLC Methods in The Determination of The Ionization Constant (pKa) of Favipiravir

AbstractIn this study, the ionization constant (pKa) of favipiravir was determined using both green and the traditional RPLC methods. To determine these values, acetonitrile‐water and methanol‐water mixtures were used in the traditional RPLC methods, while ethanol‐water and micellar solvent‐free mixtures were used in the green RPLC methods. The pKa value of favipiravir was calculated by evaluating the chromatographic data obtained in the investigated hydro‐organic and micellar solvent‐free mixtures using two different calculation methods. In the determination of their pKa values in water from the pKa values of the investigated compounds determined in hydro‐organic mixtures, the relationship between some macroscopic values of acetonitrile, methanol, ethanol and the values was used, while the values of the compounds could be determined directly in micellar solvent‐free mobile phases. It was found that the directly and indirectly calculated values were quite compatible with each other. Using the value calculated for favipiravir, the ionization percentages at different pH values (1–12) were calculated by the Henderson‐Hasselbalch equation. The environmental impact of the RPLC methods used in this study was assessed using the Green Metrics tools GAPI and AGREE.

GREEN HPLC DETERMINATION OF PHENYTOIN AND METHOD VALIDATION

Objective: In this study, the chromatographic behavior of the antiepileptic drug phenytoin was determined by the green HPLC method. The optimization of the developed method was based on the capacity factor values of phenytoin in varying water-ethanol binary mixtures and the ethanol concentration in the mobile phase where the compound was analyzed. Material and Method: Ethanol-water binary mixtures containing 35%, 40%, and 45% (v/v) ethanol were used in the optimization for the determination performed by the RPLC method. Retention times of the compound were determined with the Zorbax SB-CN (150x4.6 mm, 3.5 µm ID) column. Analyzes were performed at a constant flow rate (0.3 ml/min) and column temperature (37°C). The optimum condition for quantitative analysis was determined as an ethanol-water binary mixture containing 40% (v/v) ethanol with a pH of 6.5. Result and Discussion: In this study, the hydrophobicity of phenytoin was calculated using the logk-φ relationship. The optimum condition was determined using the obtained chromatographic data, and the quantitative determination of phenytoin in the commercial tablet formulation was made by the internal standard method. Under these conditions, excellent linearity (r&amp;gt;0.99) was obtained in the concentration range of 0.8-2.8 μg/ml. The detection limit of the developed method is 0.021 μg/ml; the limit of quantitation was calculated as 0.064 μg/ml. The recovery value of the method was determined as 99.61%. It was concluded that the parameters of precision, accuracy, and method robustness were appropriate for the validation procedures.

Determination of ionization/protonation constant values of some guanine derivative drugs by traditional/ green RPLC and UV–Vis spectrophotometric methods

In this study, the greenreverse-phase liquid chromatography (RPLC) method was used in addition to the traditional RPLC method to determine the ionization/protonation constants (pKa) of the guanine derivatives ganciclovir, valacyclovir, and valganciclovir. The determination of these values was carried out in binary acetonitrile–water mixtures (for the traditional RPLC method), ethanol–water binary mixtures (for the green RPLC method), and in aqueous solution of a micelle-forming surfactant (for pure micellar chromatography). The pKa values sspKa of the compounds were calculated using the linear solvation energy relationship (LSER) method in the studied hydro-organic and micellar solvent-free mixtures. Moreover, the UV–Vis spectrophotometric method was used to compare the sspKa values of ganciclovir, valaciclovir, and valganciclovir obtained in a acetonitrile–water medium using another method. The pKa values ​​of the examined compounds in water were calculated using the linear relationship between the sspKa values ​​determined in hydroorganic mixtures and some macroscopic values ​​of acetonitrile and ethanol. Additionally, wwpKa values ​​were determined in the micellar solvent-free mobile phase. Subsequently, the wwpKa values calculated by these direct and indirect methods were compared and the agreement between them was excellent. By using the wwpKa value calculated for each compound, the ionization percentages were calculated at different pH (1–12) values with the Henderson-Hasselbalch equation. The environmental impact of the RPLC methods used in this study was assessed using the green metrics tools GAPI and AGREE. The result of the investigation was that the environmental suitability of the green methods is satisfactory.

Exploring the chemical subspace of RPLC: A data driven approach

BackgroundThe chemical space is comprised of a vast number of possible structures, of which an unknown portion comprises the human and environmental exposome. Such samples are frequently analyzed using non-targeted analysis via liquid chromatography (LC) coupled to high-resolution mass spectrometry often employing a reversed phase (RP) column. However, prior to analysis, the contents of these samples are unknown and could be comprised of thousands of known and unknown chemical constituents. Moreover, it is unknown which part of the chemical space is sufficiently retained and eluted using RPLC. ResultsWe present a generic framework that uses a data driven approach to predict whether molecules fall ‘inside’, ‘maybe’ inside, or ‘outside’ of the RPLC subspace. Firstly, three retention index random forest (RF) regression models were constructed that showed that molecular fingerprints are able to predict RPLC retention behavior. Secondly, these models were used to set up the dataset for building an RPLC RF classification model. The RPLC classification model was able to correctly predict whether a chemical belonged to the RPLC subspace with an accuracy of 92% for the testing set. Finally, applying this model to the 91 737 small molecules (i.e., ≤1 000 Da) in NORMAN SusDat showed that 19.1% fall ‘outside’ of the RPLC subspace. Significance and noveltyThe RPLC chemical space model provides a major step towards mapping the chemical space and is able to assess whether chemicals can potentially be measured with an RPLC method (i.e., not every RPLC method) or if a different selectivity should be considered. Moreover, knowing which chemicals are outside of the RPLC subspace can assist in reducing potential candidates for library searching and avoid screening for chemicals that will not be present in RPLC data.

Comparison of Classical and Green RPLC Methods in the Determination of pKa Values of Some Piperazine Group Antihistamines.

In this study, protonation constant values and liquid chromatographic behaviors of hydrophobic cyclizine, chlorcyclizine, hydroxyzine, cinnarizine, cetirizine, meclizine, and buclizine in some water-organic solvent binary mixtures were examined for the first time using classical and green reverse phase liquid chromatography methods. In the isocratic study, the relationship of the retention time and mobile phase pH in water-organic solvent binary mixtures containing acetonitrile (45, 50, 55, 60, 65%, v/v), methanol (60, 65, 70, 75%, v/v) and ethanol (45, 50, 55, 56, 59, 60, 62, 65%, v/v) were determined at 37 °C. In the study, XBridge C18 and Gemini NX C18 columns suitable for the chemical properties of basic compounds were used. The obtained liquid chromatographic data were analyzed using the linear solvation energy relationship methodology and the SOLVER program. The aqueous protonation constant values of the investigated compounds were calculated using the linear relationship between the protonation constant data calculated in studied binary mixtures and some macroscopic constant values of the solvents used. The greenness of methods developed using three different solvents was evaluated with the Analytical Greenness Metric Approach, the Green Analytical Procedures Index, and the Green Solvent Selection Tool approaches.

Label-Free Composition Analysis of Supramolecular Polymer-Nanoparticle Hydrogels by Reversed-Phase Liquid Chromatography Coupled with a Charged Aerosol Detector.

Supramolecular hydrogels formed through polymer-nanoparticle interactions are promising biocompatible materials for translational medicines. This class of hydrogels exhibits shear-thinning behavior and rapid recovery of mechanical properties, providing desirable attributes for formulating sprayable and injectable therapeutics. Characterization of hydrogel composition and loading of encapsulated drugs is critical to achieving the desired rheological behavior as well as tunable in vitro and in vivo payload release kinetics. However, quantitation of hydrogel composition is challenging due to material complexity, heterogeneity, high molecular weight, and the lack of chromophores. Here, we present a label-free approach to simultaneously determine hydrogel polymeric components and encapsulated payloads by coupling a reversed phase liquid chromatographic method with a charged aerosol detector (RPLC-CAD). The hydrogel studied consists of modified hydroxypropylmethylcellulose, self-assembled PEG-b-PLA nanoparticles, and a therapeutic compound, bimatoprost. The three components were resolved and quantitated using the RPLC-CAD method with a C4 stationary phase. The method demonstrated robust performance, applicability to alternative cargos (i.e., proteins) and was suitable for composition analysis as well as for evaluating in vitro release of cargos from the hydrogel. Moreover, this method can be used to monitor polymer degradation and material stability, which can be further elucidated by coupling the RPLC method with (1) a multi-angle light scattering detector (RPLC-MALS) or (2) high resolution mass spectrometry (RPLC-MS) and a Fourier-transform based deconvolution algorithm. We envision that this analytical strategy could be generalized to characterize critical quality attributes of other classes of supramolecular hydrogels, establish structure-property relationships, and provide rational design guidance in hydrogel drug product development.

Determination of protonation constant value of thiabendazole in ethanol-water binary mixtures by green RPLC method

Determination of protonation constant value of thiabendazole in ethanol-water binary mixtures by green RPLC method

Evaluation of the RP-LC method for determining the pKa of abiraterone and its assay

AbstractA straightforward, dependable, and quick RP-LC method for the analysis of abiraterone acetate in its dose form and human urine has been devised. With DAD detection, sensitivity was reported to be high. The LOD and LOQ of the procedure were deemed adequate. The suggested approach was exhaustively validated in accordance with ICH requirements, and the findings demonstrated that it was exact, accurate, selective, and sensitive for the analysis of this pharmaceutical. The chromatographic separation was realized using a X-Terra RP-18 (150 × 4.60 mm i.d. × 5 μm) column and a UV detector set at 255 and 267 nm. In addition, pKa values were calculated based on the relationship between the retention factor and the pH of the mobile phase. The influence of the composition of the mobile phase on the ionization constant was investigated by measuring the pKa at various acetonitrile–water combinations ranging from 50 to 70% (v/v).

A column screening study: Validation of a RP-LC method for determination of teriflunomide with pentafluorophenyl stationary phase

The research focuses on column screening in order to develop a novel validation method to separate Teriflunomide significantly using comparative and equivalent column selection studies. This gives stationary phase efficiency relative to height equivalent to a theoretical plate (HETP) (>16000) and symmetry factor (<2.0%). After extensive column chemistry and technique development, the advanced Pentafluorophenyl (PFP) column separates halogenated drugs better. Teriflunomide is fluorinated drug, hence PFP is optimal for analysis. For statistical data comparison, the same chromatographic method was developed and transferred to another YMC Pack Pro C18 column that gives almost the same results as PFP column in terms of carbon load, pore size, and surface area. The refined and validated method yielded equivalent results with two stationary phases using a single analysis procedure. Column screening, technique creation, transfer, and validation with comparison studies with correlation coefficients over 0.999 are unique. The statistical data from both columns meets ICH requirements. The sensitive, reliable, robust, and reproducible approach is easy to apply in regular laboratory analysis. Data analysis showed that the validation study with PFP is better (LoD - 0.0015 ppm) for column ageing due to smaller injection volume and amount loaded in the column, shorter run time, and cheaper analysis cost. This research helps with column selection and method transfer with diverse column specifications. With less than 2.0% of RSD, the column screening analytical method validation is linear, precise, accurate, rugged, and efficient.

Liquid Chromatography Methods for Analysis of mRNA Poly(A) Tail Length and Heterogeneity.

Messenger RNA (mRNA) is a new class of therapeutic compounds. The current advances in mRNA technology require the development of efficient analytical methods. In this work, we describe the development of several methods for measurement of mRNA poly(A) tail length and heterogeneity. Poly(A) tail was first cleaved from mRNA with the RNase T1 enzyme. The average length of a liberated poly(A) tail was analyzed with the size exclusion chromatography method. Size heterogeneity of the poly(A) tail was estimated with high-resolution ion-pair reversed phase liquid chromatography (IP RP LC). The IP RP LC method provides resolution of poly(A) tail oligonucleotide variants up to 150 nucleotide long. Both methods use a robust ultraviolet detection suitable for mRNA analysis in quality control laboratories. The results were confirmed by the LC-mass spectrometry (LC MS) analysis of the same mRNA sample. The poly(A) tail length and heterogeneity results were in good agreement.

Development and Validation of the RPLC Method for the Determination of Valaciclovir in Human Urine

Sunulan çalışmada, herpes ve megalovirüslerin sebep olduğu enfeksiyonların tedavisinde tercihli olarak kullanılan ilaçlardan valasiklovirin katkılandırılmış idrarda ters faz sıvı kromatografik yöntemle tayini için yöntem geliştirilmiştir. Yöntem geliştirmede, valasiklovirin sistematik olarak pH-kapasite faktörü ilişkisini değerlendirilip, mobil faz optimizasyonu gerçekleştirilmiştir. İnceleme sonunda, optimum ayırma koşulu %4 asetonitril-su (h/h), pH 5,0 ve 37oC kolon sıcaklığı olarak belirlenmiştir. Sonrasında, belirlenen optimum koşulun Uluslararası Harmonizasyon Topluluğu (ICH) yönergelerine göre validasyonu gerçekleştirilmiştir. Geliştirilen yöntem 2-12 μg/mL derişim aralığında muhteşem doğrusallık göstermiştir ve idrar ortamında gerçekleştirilen geri kazanma sonuçları (%) valasiklovirin iki farklı derişimi için 100,147±0,800 ve 100,208±0,604 olarak bulunmuştur. Ayrıca, geliştirilen yönteme sağlamlık testleri de uygulanmıştır ve elde edilen sonuçlar t-testiyle değerlendirilmiştir. Elde edilen validasyon ve sağlamlık testleri sonucunda, geliştirilen yöntemin hassas, tekrar edilebilir, doğru, kesin ve sağlam olduğu bulunmuştur. Bundan dolayı, geliştirilen yöntemin rutin analizler için uygun olduğu belirlenmiştir.

Implementing reversed-phase and hydrophilic interaction liquid chromatography into the characterization of DTPA-ramucirumab conjugate before radiolabeling

Radioimmunoconjugates represent a promising class of therapeutics and diagnostics. The characterization of intermediate chelator-antibody products, i.e., without the radionuclide, is frequently omitted, bringing significant uncertainty in the radioimmunoconjugate preparation. In the present study, we explored the utility of reversed-phase (RPLC) and hydrophilic interaction (HILIC) liquid chromatography with UV detection to characterize ramucirumab stochastically conjugated with p-SCN-Bn-CHX-A"-DTPA chelator (shortly DTPA). The conjugation was well reflected in RPLC chromatograms, while chromatograms from HILIC were significantly less informative. RPLC analyses at the intact level confirmed that the conjugation resulted in a heterogeneous mixture of modified ramucirumab. Moreover, the RPLC of DTPA-ramucirumab confirmed heterogeneous conjugation of all subunits. The peptide mapping did not reveal substantial changes after the conjugation, indicating that most parts of ramucirumab molecules remained unmodified and that the DTPA chelator was bound to various sites. Eventually, the RPLC method for analysis of intact ramucirumab was successfully applied to online monitoring of conjugation reaction in 1 h intervals for a total of 24 h synthesis, which readily reflected the structural changes of ramucirumab in the form of retention time shift by 0.21 min and increase in peak width by 0.22 min. The results were obtained in real-time, practically under 10 min per monitoring cycle. To the best of our knowledge, our study represents the first evaluation of RPLC and HILIC to assess the quality of intermediates during the on-site preparation of radioimmunoconjugates prior to radiolabeling.

Optimization of Mass Spectrometric Parameters in Data Dependent Acquisition for Untargeted Metabolomics on the Basis of Putative Assignments.

Optimization of mass spectrometric parameters for a data dependent acquisition (DDA) experiment is essential to increase the MS/MS coverage and hence increase metabolite identifications in untargeted metabolomics. We explored the influence of mass spectrometric parameters including mass resolution, radio frequency (RF) level, signal intensity threshold, number of MS/MS events, cycle time, collision energy, maximum ion injection time (MIT), dynamic exclusion, and automatic gain control (AGC) target value on metabolite annotations on an Exploris 480-Orbitrap mass spectrometer. Optimal annotation results were obtained by performing ten data dependent MS/MS scans with a mass isolation window of 2.0 m/z and a minimum signal intensity threshold of 1 × 104 at a mass resolution of 180,000 for MS and 30,000 for MS/MS, while maintaining the RF level at 70%. Furthermore, combining an AGC target value of 5 × 106 and MIT of 100 ms for MS and an AGC target value of 1 × 105 and an MIT of 50 ms for MS/MS scans provided an improved number of annotated metabolites. A 10 s exclusion duration and a two stepped collision energy were optimal for higher spectral quality. These findings confirm that MS parameters do influence metabolomics results, and propose strategies for increasing metabolite coverage in untargeted metabolomics. A limitation of this work is that our parameters were only optimized for one RPLC method on single matrix and may be different for other protocols. Additionally, no metabolites were identified at level 1 confidence. The results presented here are based on metabolite annotations and need to be validated with authentic standards.

Monitoring of Tetracycline Group Antibiotic Residues in Various Food Products of Animal Origin in the Turkish Market

The aim of this study was to detect the presence of antibiotic residues in foods of animal origin, including 42 pieces of chicken gizzard and 46 pieces of bovine kidney and 102 chicken eggs belonging to various brands. These samples were gathered from December 2020 to April 2021 in the Aegean province of Turkey. A sensitive, simple, rapid, experimentally convenient and cost effective RP-LC method with high recovery output was developed. The method was thoroughly validated for the optimized parameters and produced satisfactory results. The analysis of bovine offal by the developed RP-LC method showed the presence of oxytetracycline, tetracycline, and chlortetracycline residues in 14 (30.43%) kidney samples. Chlortetracycline was detected in 7 (16.67%) chicken gizzard samples. In addition, the analysis of chicken eggs revealed the presence of oxytetracycline and tetracycline residues in nine egg samples (8.82%). Since, the amount of antibiotic residues in these samples was below the detection limit, quantification could not be carried out. Only one (0.98%) of the 102 egg samples exceeded the MRL (267.1 mg/kg) for oxytetracycline concentration. According to the study's overall findings, it is recommended that tetracycline antibiotics should be regularly checked in a variety of foods made from animals because they were found in 32 out of 190 analysed samples. Tetracycline residues may pose dangers to human health, so it's important to conduct further research and more information should be given for both producers and consumers.

RP-LC Method Development and Validation for Dasatinib Forced Degradation Study: Isolation and Structural Characterization by NMR and HRMS.

A reverse phase high-performance liquid chromatography (HPLC) method has been developed for the quantification of a typical drug Dasatinib (DST) and its related impurities in pharmaceuticals. Kinetex C18 (4.6 × 150mm, 5μm) column was used in the chromatographic separations, using buffer (1.36g of KH2PO4 in 1000mL of water, pH = 7.8; adjusted with diluted KOH solution) with solvent as acetonitrile and mode of elution as the gradient. The flow rate is 0.9mL/min, column oven temperature as 45°C and the overall gradient run time as 65min. The developed method was found to produce symmetric and good separation between the process-related and degradation impurities. Method optimization is achieved with photodiode array at 305nm over the concentration range of 0.5mg/mL and degradation studies were carried out under acidic, alkaline, oxidative, photolytic and thermal conditions to demonstrate the stability indicating capability of the method. Two major impurities were found in forced degradation studies in the HPLC analysis, the unknown, acid degradants were enriched and isolated by preparative HPLC, then characterized through high-resolution mass spectrometry, nuclear magnetic resonance spectroscopy and Fourier transform infrared spectroscopy. The unknown acid degradation impurity was showing Exact Mass of 521.11, molecular formula C22H25Cl2N7O2S and its chemical name as 2-(5-chloro-6-(4-(2-hydroxyethyl) piperazin-1-yl)-2-methylpyrimidin-4-ylamino)-N-(2-chloro-6-methylphenyl) thiazole-5-carboxamide. Another impurity (oxidative degradant) found as known DST N-oxide Impurity-L and its chemical name as 4-(6-((5-((2-chloro-6-methylphenyl) carbamoyl) thiazol-2-yl) amino)-2-methylpyrimidin-4-yl)-1-(2-hydroxyethyl) piperazine 1-oxide. The analytical HPLC method was further validated as per ICH guidelines.

Pravastatinin Su ve Su-Asetonitril İkili Karışımlarında İyonlaşma Sabiti Değerinin RPLC Metoduyla Belirlenmesi

Ters faz sıvı kromatografi (RPLC) yöntemi asidik ve bazik fonksiyonel grup içeren bileşiklerin iyonlaşma/protonasyon (pKa) sabitlerinin tayini için tercih edilmektedir. pKa değeri, ilaçların absorpsiyon, dağılım, metabolizma, atılım, toksitite (ADMET) özelliklerini tahmin etmeye yarayan önemli bir parametredir. Sunulan bu çalışmada, pravastatinin su-asetonitril ikili karışımında pKa değerinin tayini için RPLC metot geliştirilmiş ve asetonitrilin mobil fazdaki miktarının değişimiyle pravastatinin kromatografik davranışı da belirlenmiştir. Çalışma Nücleosil C4 (250×4,6 mm, 5 µm) kolonda, 37°C’de ve 1 mL/dakika sabit akış hızında gerçekleştirilmiştir. Elde edilen alıkonma verileriyle doğrusal olmayan model kullanılarak bileşiğin pKa değeri belirlenmiştir. Hidrofilik özellikte olan pravastatinin su ortamındaki pKa değeri farklı organik modifiyer-su karışımlarında tayin edilen mobil faz pKa değerleri ve asetonitrilin makroskopik sabitleri (mol kesri, dielektrik sabiti, vs.), çözücü-çözünen arasındaki spesifik solvasyon etkileşim parametreleri kullanılarak hesaplanmıştır. Hesaplanan bu önemli fizikokimyasal parametrenin literatür verileriyle uyumlu olduğu görülmüştür.

Liquid chromatography-mass spectrometry metabolite library for metabolomics: Evaluating column suitability using a scoring approach

Untargeted metabolomic studies require an extensive set of analyte (metabolic) information to be obtained from each analyzed sample. Thus, highly selective, and efficient analytical methodologies together with reversed-phase (RP) or hydrophilic interaction liquid chromatography (HILIC) are usually applied in these approaches. Here, we present a performance comparison of five different chromatographic columns (C18, C8, RP Amide, zicHILIC, OH5 HILIC phases) to evaluate their sufficiency of analysis for a large analyte library, consisting of 817 authentic standards. By taking into account experimental chromatographic parameters (i.e. retention time, peak tailing and asymmetry, FWHM, signal-to-noise ratio and peak area and intensity), the proposed column scoring approach provides a simple criterion that may assist analysis in the select of a stationary phase for those metabolites of interest. RPLC methods offered better results regarding metabolic library coverage, while the zicHILIC stationary phase delivered a bigger number of properly eluted compounds. This study demonstrates the importance of choosing the most suitable configuration for the analysis of different metabolic classes.

Development and validation of RPLC method for the simultaneous analysis of ACE inhibitors in tablet formulations

Hypertension is a disease that can progress for many years without symptoms or complications. This disease can cause death and permanent damage to vital organs such as the kidney, heart, and brain. However, when hypertension is treated effectively, these risk factors may decrease significantly. Although many antihypertensive drugs are used in the treatment of hypertension, ACE inhibitors are a new class among these drugs, and their separation and quantitative determinations are important for the success of preclinical, biopharmaceutical, and clinical studies. For this reason, in this study, the simultaneous determination of cilazapril, benazepril, and quinapril from ACE inhibitors, the validation of the developed method, and the quantitative determination of commercial drug dosage forms are aimed. To obtain the optimum liquid chromatographic separation condition, a systematic approach based on the relationship between mobile phase pH and retention time was used. The mobile phase organic modified concentration and column temperature parameters, which are known to affect the retention times of the compounds, were kept constant. When deciding on these parameters, few experiments were carried out.

Estimation of Trometamol content in Gadobutrol solution for intravenous administration by using RP-LC with Refractive Index DetectorV

Extremely responsive technique designed for the willpower of trometamol for instance tris(hydroxymethyl) aminomethane in gadobutrol by RP-LC technique. Quantification of trometamol content in gadobutrol samples by HPLC with refractive index detector (RID). Trometamol was UV inactive compound. Trometamol was determined by RP-LC method using Inertsil NH2 (250x4.6mm, 5µm) column as motionless segment. Column temperature maintained 30°C, inoculation quantity 10µL, flow velocity was 0.3 ml/min, sample cooler temperature 25°C. The mixture of phosphate buffer and cyanomethane in the ratio of 990:10 (volume/volume) was utilized as movable segment. The technique legalization erstwhile conceded as indicated by ICH guidelines.