Literature | MOLNÁR-INSTITUTE

Journal of Chromatography · 1989

Computer-aided optimization of high-performance liquid chromatography in the pharmaceutical industry

E. P. Lankmayr, W. Wegscheider, J. C. Gfeller, N. M. Djordjevic, B. Schreiber

Optimization of high-performance liquid chromatography for application to a cough medication (Tussagesic) and its decomposition and byproducts was performed. Special emphasis was placed on the optimization of all parameters that relate to the chemical selectivity of the separation process itself and on the final proof of the ruggedness of the optimized system. All analytes can be reliably determined and the response surface can be represented graphically. This provides a means for improved transfer of methods between laboratories and for efficient system documentation.

Journal of Pharmaceutical and Biomedical Analysis · 2016

A platform analytical quality by design (AQbD) approach for multiple UHPLC-UV and UHPLC–MS methods development for protein analysis

Jianmei Kochling, Wei Wu, Yimin Hua, Qian Guan, Juan Castaneda-Merced

A platform analytical quality by design approach for methods development is presented in this paper. This approach is not limited just to method development following the same logical Analytical quality by design (AQbD) process, it is also exploited across a range of applications in methods development with commonality in equipment and procedures. As demonstrated by the development process of 3 methods, the systematic approach strategy offers a thorough understanding of the method scientific strength. The knowledge gained from the UHPLC-UV peptide mapping method can be easily transferred to the UHPLC–MS oxidation method and the UHPLC-UV C-terminal heterogeneity methods of the same protein. In addition, the platform AQbD method development strategy ensures method robustness is built in during development. In early phases, a good method can generate reliable data for product development allowing confident decision making. Methods generated following the AQbD approach have great potential for avoiding extensive post-approval analytical method change. While in the commercial phase, high quality data ensures timely data release, reduced regulatory risk, and lowered lab operational cost. Moreover, large, reliable database and knowledge gained during AQbD method development provide strong justifications during regulatory filling for the selection of important parameters or parameter change needs for method validation, and help to justify for removal of unnecessary tests used for product specifications.

Science of The Total Environment · 2020

High-performance liquid chromatography (HPLC)-fluorescence method for determination of bisphenol A diglycidyl ether (BADGE) and its derivatives in canned foods

Mingming Guo, Mingfeng He, Jianjun Zhong, Qiao He, Balarabe B. Ismail, Guoying Chen, Donghong Liu

Bisphenol A diglycidyl ether (BADGE) is used as a raw material for the production of epoxy resins and PVC organosols, which are commonly applied as inner coatings for food cans. BADGE and its derivatives can migrate from coatings to foodstuffs during processing and storage thereby creating adverse health issues. In this work, a method based on high-performance liquid chromatography (HPLC)-fluorescence detection (FLD) method was developed for the rapid determination of BADGE and its five derivatives in canned foods. Modeling software DryLab® was applied for the optimization of separation conditions. An adequate separation was achieved in 5 min including equilibration time, using a core–shell particle column; such application has not been reported so far. Also, the results showed that LOD varied from 0.01 to 0.20 ng/g, while LOQ varied from 0.03 to 0.66 ng/g, and RSD was found to be <8.64%. The analytical recoveries ranged from 70.46 to 103.44%. Excellent validation data revealed that this method is suitable for the investigation of can coating-to-food migration of BADGE and its derivatives. The HPLC-FLD method is rapid, inexpensive and highly efficient, which could be applicable for safety inspection of food contact materials involving BADGE and its derivatives.

Journal of Chromatography A · 1999

Reversed-phase liquid chromatographic separation of complex samples by optimizing temperature and gradient time: I. Peak capacity limitations

J. W. Dolan, L. R. Snyder, N. M. Djordjevic, D. W. Hill, T. J. Waeghe

The separation of samples that contain more than 15 to 20 analytes (n>15–20) is typically difficult and usually requires gradient elution. We have examined the reversed-phase liquid chromatographic separation of 24 samples with 8≤n≤48 as a function of temperature T and gradient time tG. The required peak capacity was determined for each sample, after selecting T and tG for optimum selectivity and maximum sample resolution. Comparison of these results with estimates of the maximum possible peak capacity in reversed-phase gradient elution was used to quantify the maximum value of n for some required sample resolution (when T and tG have been optimized). These results were also compared with literature studies of similar isocratic separations as a function of ternary-solvent mobile phase composition, where the proportions of methanol (MeOH), tetrahydrofuran (THF) and water were varied simultaneously. This in turn provides information on the relative effectiveness of these two different method development procedures (optimization of T and tG vs. % MeOH and % THF) for changing selectivity and achieving maximum resolution.

Journal of Pharmaceutical and Biomedical Analysis · 2016

Hydrophobic interaction chromatography for the characterization of monoclonal antibodies and related products

Szabolcs Fekete, Jean-Luc Veuthey, Alain Beck, Davy Guillarme

Hydrophobic interaction chromatography (HIC) is a historical strategy used for the analytical purification and characterization of proteins. Similarly to what can be done in reversed-phase liquid chromatography (RPLC), HIC is able to separate protein species based on their hydrophobicity, but using different conditions. Compared to RPLC, the main benefit of HIC is its ability to perform separations under non denaturing conditions (i.e. physiological pH conditions, ambient mobile phase temperature and no need for organic solvents) and so an orthogonal method. The goal of this review is to provide a general overview of theoretical and practical aspects of modern HIC applied for the characterization of therapeutic protein biopharmaceuticals including monoclonal antibodies (mAbs), antibody drug conjugates (ADCs) and bispecific antibodies (bsAbs). Therefore, method development approaches, state-of-the-art column technology, applications and future perspectives are described and critically discussed.

Journal of Chromatography A · 2000

Control of column temperature in reversed-phase liquid chromatography

R. G Wolcott, John W. Dolan, Lloyd R. Snyder, Stephen R Bakalyar, Megan A Arnold, Jon A Nichols

When separations by reversed-phase liquid chromatography (RP-LC) are carried out at temperatures other than ambient, resulting retention times and bandwidths can depend on the equipment used. As a result, an RP-LC separation that is adequate when carried out on one LC system may prove inadequate when the separation is repeated on a second system. In the present study, various temperature-related problems which can result in a failure of method transfer for non-ambient RP-LC methods were examined. Means for correcting for such effects and thereby ensuring method transferability are described.

Acta Pharmaceutica Hungarica · 2021

Computer-Assisted Approach for the Development of RP-HPLC Methods for the Separation and Quantification of Bioactive Plant Secondary Metabolites

Mohammed Séghir Daas, Massinissa Faci, Isabella Nicoletti, Malika Douzane, Danilo Corradini

This communication describes the development of computer-assisted RP-HPLC methods for the separation, identification and quantification of phenolic compounds, which are a large class of plant secondary metabolites comprising a great number of heterogeneous structures that range from simple molecules to highly Polymerized compounds. The study has been conducted by a Design of Experiments (DoE) approach that allows the simultaneous optimization of gradient time (tG), column temperature (T), and binary eluent composition on the basis of retention times and peak areas of the analytes of interest, obtained in twelve different experiments. These experiments consist in the linear gradient separations of the investigated compounds performed at two different gradient times and column temperatures, using either the aqueous component of the mobile phase at three different pH values or a combination of two organic solvents at three different volume ratios as the gradient former. The RP-HPLC methods developed by the computer-assisted approach described in this paper were used for the separation and quantification of phenolic compounds occurring in fruits of Olea europaea, extra virgin olive oil (EVOO), and olive mill wastewater (OMWW).

Journal of Chromatography A · 2002

Computerized design of separation strategies by reversed-phase liquid chromatography: development of DryLab software

Imre Molnár

The development of DryLab software is a special achievement in analytical HPLC which took place in the last 16 years. This paper tries to collect some of the historical mile stones and concepts. DryLab, being always subject to change according to the needs of the user, never stopped being developed. Under the influence of an ever changing science market, the DryLab development team had to consider not just scientific improvements, but also new technological achievements, such as the introduction of Windows 1.0 and 3.1, and later Windows NT and 2000. The recent availability of new 32-bit programming tools allowed calculations of chromatograms to be completed more quickly so as to show peak movements which result for example from slight changes in eluent pH. DryLab is a great success of interdisciplinary and intercontinental cooperation by many scientists.

Specification of Drug Substances and Products · 2014

Application of Quality by Design (QbD) to the Development and Validation of Analytical Methods

David K. Lloyd, James Bergum

Quality by design (QbD) is an approach to product development where a comprehensive understanding of the effect of various inputs (e.g. process parameters, materials) on the final product (active pharmaceutical ingredient or drug product), leads to a process or product design that ensures reliable manufacture of a high-quality product. Analogous to QbD for product, QbD may also be applied to analytical methods, by defining the required analytical performance and systematically relating how the technique and method parameters relate to analytical performance. Achieving the appropriate level of method understanding typically involves a multifactorial approach. The factors to be studied are identified via a systematic risk analysis, which allows truly impactful parameters to be further investigated in studies applying statistical design of experiments (DOE) or mechanistic models to develop a multidimensional region of experimental space in which the effects of key factors are understood and documented. An operating region can then be defined where there is high confidence that the method will operate reliably. Data generated during use of the method can be monitored over time to ensure continued adequate performance of the analysis.

Liquid Chromatography (Second Edition) · 2017

Chapter 13 - Solvent selection in liquid chromatography

Guillermo Ramis-Ramos, María Celia García-Álvarez-Coque, José Antonio Navarro-Huerta, Salvatore Fanali, Paul R. Haddad, Colin F. Poole, Marja-Liisa Riekkola

Many solvents and additives are used to prepare mobile phases in liquid chromatography (LC). Also, mixtures of solvents at different ratios are used to modify the mobile-phase properties. This can make solvent selection for method development a puzzling task, unless suitable guidelines are followed. This chapter summarizes the most common strategies used by skilled chromatographers in reversed-phase, normal-phase, and hydrophilic interaction LC. These are based on considerations about the global polarity of solutes, stationary phase, and mobile phase, which determine the elution strength, and on the particular profile of the contributions of intermolecular interactions to the global polarity, which determines the selectivity. The optimization of selectivity using isoeluotropic mixtures on the rational basis provided by solvent-selectivity profiles and by systematic trial-and-error procedures in the isocratic and gradient elution modes is described. The modification of the selectivity by using combinations of solvents is discussed. Additional considerations for mobile-phase selection, including computer-assisted interpretive optimization, sustainability criteria, and use of high temperature, are also commented.

Journal of Chromatography A · 2021

New wide-pore superficially porous stationary phases with low hydrophobicity applied for the analysis of monoclonal antibodies

Szabolcs Fekete, Amarande Murisier, Alain Beck, Jason Lawhorn, Harry Ritchie, Barry Boyes, Davy Guillarme

The article describes the development of new stationary phases for the analysis of proteins in reversed phase liquid chromatography (RPLC). The goal was to have columns offering high recovery at low temperature, low hydrophobicity and novel selectivity. For this purpose, three different ligands bound onto the surface of superficially porous silica-based particles were compared, including trimethyl-silane (C1), ethyl-dimethyl-silane (C2) and N-(trifluoroacetomidyl)-propyl-diisopropylsilane (ES-LH). These three phases were compared with two commercial RPLC phases. In terms of protein recovery, the new ES-LH stationary phase clearly outperforms the other phases for any type of biopharmaceutical sample, and can already be successfully used at a temperature of only 60°C. In terms of retention, the new ES-LH and C1 materials were the less retentive ones, requiring lower organic solvent in the mobile phase. However, it is important to mention that the stability of C1 phase was critical under acidic, high temperature conditions. Finally, some differences were observed in terms of selectivity, particularly for the ES-LH column. Besides the chemical nature of the stationary phase, it was found that the nature of organic modifier also plays a key role in selectivity.

Journal of Chromatography A · 2022

Kinetic performance comparison of superficially porous, fully porous and monolithic reversed-phase columns by gradient kinetic plots for the separation of protein biopharmaceuticals

Simon Jaag, Chunmei Wen, Benjamin Peters, Michael Lämmerhofer

To find the best performing column for the analysis of protein-based biopharmaceuticals is a significant challenge as meanwhile numerous modern columns with distinct stationary phase morphologies are available for reversed-phase liquid chromatography. Especially when besides morphology also several other column factors are different, it is hard to decide about the best performing column a priori. To cope with this problem, in the present work 13 different reversed-phase columns dedicated for protein separations were systematically tested by the gradient kinetic plot method. A comprehensive comparison of columns with different morphologies (monolithic, fully porous and superficially porous particle columns), particle sizes and pore diameters as well as column length was performed. Specific consideration was also given to various monolithic columns which recently shifted a bit out of the prime focus in the scientific literature. The test proteins ranged from small proteins starting from 12 kDa, to medium sized proteins (antibody subunits obtained after IdeS-digestion and disulphide reduction) and an intact antibody. The small proteins cytochrome c, lysozyme and β-lactoglobulin could be analysed with similar performance by the best columns of all three column morphologies while for the antibody fragments specific fully porous and superficially porous particle columns were superior. A 450 Å 3,5 µm superficially porous particle column showed the best performance for the intact antibody while a 1.7 µm fully porous particle column with 300 Å showed equivalent performance to the best superficially porous column with thin shell and 400 Å pore size for proteins between 12 and 25 kDa. While the majority of the columns had C4 bonding chemistry, the silica monolith with C18 bonding and 300 Å mesopore size approximated the best performing particle columns and outperformed a C4 300 Å wide-pore monolith. The current work can support the preferred choice for the most suitable reversed-phase column for protein separations.

Chromatography Today · 2013

Chromatography Modelling in High Performance Liquid Chromatography Method Development

Imre Molnár, Róbert Kormány

This article discusses the development of chromatography modelling of the last 30 years from the first software package for calculating resolution and capacity factors to the visual modelling of chromatograms for testing peak movements with altering elution conditions. Different approaches are discussed, such as retention modelling based on measurements, others based on molecular structure or on statistical considerations. The state-of-the-art will be demonstrated with a few applications of industrial importance.

Journal of Chromatography A · 2002

Lipophilicity and pKa estimates from gradient high-performance liquid chromatography

Roman Kaliszan, Piotr Haber, Tomasz Bączek, Danuta Siluk, Klara Valko

The linear-solvent strength (LSS) model of gradient elution has been applied to estimate parameters of lipophilicity and acidity of a series of drugs and model chemicals. Apparent pKa values and log kw values for individual analytes were determined in 2–3 gradient runs. The first experiment (or first two experiments) uses a wide-range organic modifier gradient with pH chosen for suppressed ionization of the analyte. The result of this experiment allows an estimate of contents of organic modifier of the mobile phase (%B) providing the required retention coefficient, k, for the non-ionized analyte. The following experiment is carried out with the latter %B and a pH-gradient of the aqueous component of the eluent that is sufficient to overlap the possible pKa-value of the analyte. The initial pH of the buffer used to make the mobile phase is selected to insure that the analyte is in non-ionized form. The resulting retention time allows an estimate of pKa in a solvent of the selected %B. At the same time, estimates of log kw can also be obtained. The log kw parameter obtained from gradient HPLC by the approach proposed correlated well with the corresponding value obtained by standard procedure of extrapolation of retention data determined in a series of isocratic measurements. Correlation between log kw and the reference parameter of lipophilicity, log P, was very good for a series of test analytes and satisfactory for a structurally diverse series of drugs. The approach supported with specific detection procedures can be recommended for fast screening of lipophilicity of individual components of complex mixtures like those produced by combinatorial chemistry. The values of pKa obtained in a study were found to correlate with the literature pKa data determined in water for a set of aniline derivatives studied. In case of a series of drugs the correlation was less than moderate if the general procedure of pKa determination was applied.

Chromatographia · 2005

Searching for Robust HPLC Methods – Csaba Horváth and the Solvophobic Theory

Imre Molnár

This paper is written in remembrance of the work of Csaba Horváth on Reversed Phase Chromatography, (RPC) and the fundamental theory of the mechanism of retention on non-polar stationary phases, the ``Solvophobic Theory''. The paper discusses some steps in the development of this important theory and examines its consequences in developing robust methods for routine RPC. Reliable product quality requires the understanding of selectivity changes, which in RPC govern the development of robust and reliable methods involving continuous changes of liquid chromatographic parameters in aqueous eluents. The application of RPC is still growing in scientific research and in pharmaceutical and chemical production. The impact of the Solvophobic Theory in life sciences has been enormous but it was only a part of Horváth's scientific work. RPC is today one of the most popular, most widely used tools in analytical chemistry and will remain so for many years due to its stability and to its robustness.

Journal of Chromatography · 1983

High-performance liquid chromatographic column efficiency as a function of particle composition and geometry and capacity factor

R. W. Stout, J. J. DeStefano, Lloyd R. Snyder

Reduced plate height (h) vs. reduced velocity (v) plots have been measured over a wide range of v for 36 high-performance liquid chromatographic systems. Column type was varied over wide limits and solute capacity factor (k′) values were changed over the range 0.6–22. Resulting data can be accurately described by the Knox equation h = Av1/3 + B/v + Cv, where A is roughly constant (A = 0.5–0.8) for all columns studied, but values of B and C are strongly dependent on column type and solute k′ values. These observations can be rationalized by a quantitative model that recognizes two effects: (a) surface diffusion of solute molecules in the stationary phase (along the pore wall) and (b) restricted diffusion of small solute molecules within particles having narrow pores and long alkyl chains bonded to their surface.

Journal of Pharmaceutical and Biomedical Analysis · 2015

Method development for the separation of monoclonal antibody charge variants in cation exchange chromatography, Part I: Salt gradient approach

Szabolcs Fekete, Alain Beck, Jenő Fekete, Davy Guillarme

Ion exchange chromatography (IEX) is a historical technique widely used for the detailed characterization of therapeutic proteins and can be considered as a reference and powerful technique for the qualitative and quantitative evaluation of charge variants. When applying salt gradient IEX approach for monoclonal antibodies (mAbs) characterization, this approach is described as time-consuming to develop and product-specific. The goal of this study was to tackle these two bottle-necks. By modeling the retention of several commercial mAbs and their variants in IEX, we proved that the mobile phase temperature was not relevant for tuning selectivity, while optimal salt gradient program can be easily found based on only two initial gradients of different slopes. Last but not least, the dependence of retention vs. pH being polynomial, three initial runs at different pH were required to optimize mobile phase pH. Finally, only 9h of initial experiments were necessary to simultaneously optimize salt gradient profile and pH in IEX. The data can then be treated with commercial modeling software to find out the optimal conditions to be used, and accuracy of retention times prediction was excellent (less than 1% variation between predicted and experimental values). Second, we also proved that generic IEX conditions can be applied for the characterization of mAbs possessing a wide range of pI, from 6.7 to 9.1. For this purpose, a strong cation exchange column has to be employed at a pH below 6 and using a proportion of NaCl up to 0.2M. Under these conditions, all the mAbs were properly eluted from the column. Therefore, salt gradient CEX can be considered as a generic multi-product approach.

Journal of Chromatography A · 2000

Reversed-phase separation of achiral isomers by varying temperature and either gradient time or solvent strength

Lloyd R. Snyder, John W. Dolan

The separation of several isomer pairs of widely varied structure was studied as a function of changes in temperature, gradient time, mobile phase pH, column type (“monomeric” vs. “polymeric” C18-silica) and organic solvent (methanol vs. acetonitrile). General conclusions are drawn which may prove useful in future attempts at the separation of these and other achiral isomers.

Journal of Chromatography B: Biomedical Sciences and Applications · 1978

Catecholamines and related compounds: Effect of substituents on retention in reversed-phase chromatography

Imre Molnár, Csaba Horváth

The capacity factors of 32 compounds were measured on octadecyl-silica columns by using neat aqueous phosphate buffer, pH 2.1, as the eluent. The tabulated results allow the estimation of the effect of various substituents on the retention of catecholamines and related compounds under similar chromatographic conditions.

Analytical Chemistry · 1977

Liquid chromatography of ionogenic substances with nonpolar stationary phases

Csaba Horváth, Wayne Melander, Imre Molnár

The effect of solute ionization on the retention of weak acids, bases, and ampholytes on octadecylsilica was investigated both theoretically and experimentally. The retention was attributed to a reversible association of the solutes with the hydrocarbonaceous ligand of the stationary phase. A phenomenological treatment of the corresponding equilibria was developed for various types of ionogenic substances. The energetics of the association process was analyzed in a rigorous fashion in the light of the solvophobic theory and a semi-empirical extension of the Debye-Hiickel theory to high ionic strength. The predicted effect of solute ionization on the capacity factors was substantiated by experimental data. The observed dependence of the capacity factors on the ionic strength of the eluent and the hydrophobic surface of the solute molecules showed good agreement with the theory. The advantages of the technique in the separation of biological substances are illustrated.

Decades of chromatography research done with DryLab®.