Decades of chromatography research done with DryLab®.

Journal of Pharmaceutical and Biomedical Analysis · 2017

Renewal of an old European Pharmacopoeia method for Terazosin using modeling with mass spectrometric peak tracking

Róbert Kormány, Imre Molnár, Jenő Fekete

An older method for terazosin was reworked in order to reduce the analysis time from 90min (2×45min) to below 5min. The method in European Pharmacopoeia (Ph.Eur.) investigates the specified impurities separately. The reason of the different methods is that the retention of two impurities is not adequate in reversed phase, not even with 100% water. Therefore ion-pair-chromatography has to be applied and since that two impurities absorb at low UV-wavelength they had to be analyzed by different method than the other specified impurities. In our new method we could improve the retention with pH elevation using a new type of stationary phases available for high pH applications. Also a detection wavelength could be selected that is appropriate for the detection and quantification of all impurities. The method development is the bottleneck of liquid chromatography even today, when more and more fast chromatographic systems are used. Expert knowledge with intelligent programs is available to reduce the time of method development and offer extra information about the robustness of the separation. Design of Experiments (DoE) for simultaneous optimization of gradient time (tG), temperature (T) and ternary eluent composition (tC) requires 12 experiments. A good alternative way to identify a certain peak in different chromatograms is the molecular mass of the compound, due to its high specificity. Liquid Chromatography–Mass Spectrometry (LC–MS) is now a routine technique and increasingly available in laboratories. In our experiment for the resolution- and retention modeling the DryLab4 method development software (Version 4.2) was used. In recent versions of the software the use of (m/z)-MS-data is possible along the UV-peak-area-tracking technology. The modelled and measured chromatograms showed excellent correlations. The average retention time deviations were ca. 0.5s and there was no difference between the predicted and measured Rs,crit −values.

Journal of Chromatography A · 2013

Intergroup cross-comparison for the evaluation of data-interchangeability from various chromatographic tests

Chamseddin Chamseddin, Imre Molnár, Thomas Jira

The recently dramatic increase in the available choices of reversed-phase columns could be an advantage of this mode of separation. However, due to the insufficiency of available information in terms of the exact functionality of these phases and the similarities and differences between these newly introduced and conventional reversed-phase columns, it is now somehow problematic to determine which could be the best column for a given analytical problem. There is no single column that will give us a good separation for all applications. As a result, there have been several attempts to develop testing strategies to characterize column chemistries. In this study three of the most widely used and acceptable approaches for the characterization of reversed-phase columns, which are Tanaka, United States Pharmacopeia (USP), and Snyder–Dolan, are systemically applied to investigate the chromatographic properties of calixarene- and resorcinarene-bonded stationary phases, polar-embedded and polar-endcapped stationary phases, phenyl and ether-linked phenyl with the presence of conventional alkyl-bonded phases (octyl- and octadecylsilane). Although all column classification systems aim to evaluate “more or less” the same characteristics, each system uses different test mixtures in different chromatographic conditions. It is therefore very important to evaluate the similarities and differences in the resulted “column parameters” and the possible interchangeability of them. The results of this comparative study show that the used parameters of Tanaka and of Snyder–Dolan have in many cases a good to very good correlation. The USP approach, which is based on single run, is related to Tanaka and Snyder–Dolan only in terms of hydrophobic characters, and no relation could establish in the other parameters. The hydrophobic-subtraction model could be extended to describe the ligand–solute interactions of calixarene- and resorcinarene-bonded stationary phases, which are belonging to reversed phase material. However they show, depending on the analytes, some additional interactions, since their steric, polar and ionic properties are different compared to those of conventional alkyl-bonded phases.

Journal of Pharmaceutical and Biomedical Analysis · 2014

In silico robustness testing of a compendial HPLC purity method by using of a multidimensional design space build by chromatography modeling—Case study pramipexole

Alexander H. Schmidt, Mijo Stanic, Imre Molnár

Purity testing of the active pharmaceutical ingredient (API) pramipexole is performed using an official (compendial) and harmonized method published in the European Pharmacopeia (E.P.) and United States Pharmacopeia (USP). According to this monograph the successful chromatographic separation of the API from impurities is achieved on a C18 column with gradient elution of an ion pairing buffer of pH 3.0 (mobile phase A) and acetonitrile (mobile phase B). Although not recommended in general, compendial methods are often adapted for purity testing of generic formulations. In this paper a novel approach to evaluate method robustness of an adapted method – prior of full method validation – is described. Based on Quality-by-Design (QbD) principles, a small number of experiments are performed, which after entering them into a chromatography modeling software allow to visualize a multidimensional “Design Space”, a region, in which changes in method parameters will not significantly affect the results as defined in the ICH guideline Q8(R2) leading to a more flexible method handling in routine analysis. For two different recommended C18 columns a multidimensional Design Space (Method Operating Design Region, MODR) was constructed to study the robustness of the adapted method with a newly developed Robustness Module. In a full factorial design the following six parameters were varied at three levels (low, nominal, high): gradient time, temperature, pH of the aqueous eluent (A), flow rate, start- and end concentration of the organic mobile phase component (eluent B). The resulting 36=729 experiments were performed in silico from the previously constructed models for Design Space in less than 1min and showed that the required resolution of 2.0 could not be reached in all experiments for the two columns which were recommended by the E.P. (failure rate 25% and 16%, respectively). However, by adjusting the gradient time, we were able to fulfill the requirements with a failure rate of zero. For the aqueous eluent a separate “Eluent Design Space” study was performed, which allows the construction of ionic strength vs. ion pairing concentration models to identify the optimum combination of the concentrations for the buffer and the ion-pairing reagent.

Journal of Pharmaceutical and Biomedical Analysis · 2013

Using an innovative Quality-by-Design approach for development of a stability indicating UHPLC method for ebastine in the API and pharmaceutical formulations

Alexander H. Schmidt, Imre Molnár

A stability-indicating ultra high performance liquid chromatographic (UHPLC) method has been developed for purity testing of ebastine and its pharmaceutical formulations. Successful chromatographic separation of the API from impurities was achieved on a Waters Acquity UPLC BEH C18, 50mm×2.1mm, 1.7μm particle size column with gradient elution of 10mM acetate buffer pH 6.2 and a mixture of acetonitrile/2-propanol (1:1) as the mobile phase. Incorporating Quality by Design (QbD) principles to the method development approach by using the chromatography modeling software DryLab®4 allows the visualization of a “Design Space”, a region in which changes to method parameters will not significantly affect the results as defined in the ICH guideline Q8 (R2). A verification study demonstrated that the established model for Design Space is accurate with a relative error of prediction of only 0.6%. The method was fully validated for specificity, linearity, accuracy and precision, and robustness in compliance to the ICH guideline Q2 (R1). The method was found to be linear in the concentration range from the quantification limit (LOQ) to 125% of the specification limit for ebastine and each of the impurities with correlation coefficients of not less than 0.999. The recovery rate was between 98.15 and 100.30% for each impurity. The repeatability and intermediate precision (RSD) were less than 3.2% for ebastine and each of the impurities. The robustness of the developed method was studied by varying the six parameters: gradient time, temperature, ternary composition of the eluent, flow rate and start and end concentration of the gradient at 3 levels (+1, 0, −1). The resulting 729 experiments were performed in silico from the previously constructed model for Design Space and showed that the required resolution of 2.0 can be reached in all experiments. To prove the stability-indicating performance of the method, forced degradation (acid and base hydrolysis, oxidation, photolytic and thermal stress conditions) of ebastine was carried out. Baseline separation could be achieved for all peaks of the impurities, the degradation products and the API. Total run time was only 4min, which is an impressive 40-fold increase in productivity in comparison to the method published in the Ph. Eur. monograph and allowed purity testing of more than 360 samples per day.

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.

Chromatography Today · 2010

3-Dimensional Retention Modelling of Gradient Time, Ternary Solvent-Strength and Temperature of the Reversed-phase Gradient Liquid Chromatography of a Complex Mixture of 22 Basic and Neutral Analytes using DryLab® 2010

Melvin R. Euerby, Gesa Schad, Hans-Jürgen Rieger, Imre Molnár

The present paper describes a multi-factorial optimization of three critical HPLC method parameters, i.e. gradient time (tG), temperature (T), and ternary composition (B1:B2) based on twelve experiments for the separation of twenty-two pharmaceutically relevant analytes. Examining the effect of these experimental variables on critical resolution and selectivity was carried out in such a way as to systematically vary all three factors simultaneously. The basic element is a gradient time–temperature (tG –T) plane, which is repeated at three different ternary compositions of eluent B between methanol and acetonitrile. The so-defined volume enables the investigation of the critical resolution for a part of the Design Space of a given sample. Multi-dimensional robust regions were successfully defined, graphically depicted and verified. The paper highlights the applicability of this approach for the rapid development of high quality robust LC methodologies.

Journal of Chromatography A · 2025

Extended multidimensional design space studies: Comparing volatile and non-volatile buffer systems in UPLC

Arnold Zöldhegyi, Barnabás Soós, Krisztián Horváth, Imre Molnár, Róbert Kormány

Journal of Chromatography · 1977

Separation of amino acids and peptides on non-polar stationary phases by high-performance liquid chromatography

Imre Molnár, Csaba Horváth

Microparticulate non-polar stationary phases, such as octadecyl-silica offer a rapid and efficient means for the separation of peptides and amino acids by high-performance liquid chromatography. Retention is attributed to hydrophobic interaction between the solutes and the hydrocarbonaceous functions covalently bound to the stationary phase surface. Consequently the species are eluted in the order of increasing hydrophobicity. Various peptide mixtures were analyzed by using gradient elution with increasing acetonitrile concentration in the eluent and monitoring the column effluent at 200 or 210 nm with an UV detector. The separation of angiotensins and enzymic digest of polypeptides illustrates the speed of the method which can be used to assay the purity of peptide hormones such as α-melanotropin and gramicidin or to analyze the composition of reaction mixtures involving peptides. The efficiency of the method is superior to that obtained on the conventionally used ion-exchanger columns, except for hydrophilic amino acids and peptides that are poorly retarded. Nevertheless, with a suitable ionic surfactant in the mobile phase, non-polar stationary phases can be used for the separation of these species as well.

Journal of Chromatography A · 2026

Addressing method transfer and robustness challenges using design space modeling

Arnold Zöldhegyi, Krisztián Horváth, Imre Molnár, Róbert Kormány

In high-performance liquid chromatography (HPLC), robust and easily transferable analytical methods are essential to ensure consistent performance throughout the entire lifecycle of an analytical procedure, including unforeseen future applications. Nevertheless, robustness-, and ruggedness-related issues are frequently reported in industrial practice, particularly during method transfer between laboratories. These challenges often arise from incomplete method understanding and insufficient consideration of HPLC system-related effects, such as dwell volume, extra-column contributions and other, at first less evident, instrument-to-instrument variations in solvent delivery, mixing behavior and column thermal control. In this context, modeling approaches aligned with Analytical Quality by Design (AQbD) principles provide valuable support for both proactive method development and reactive troubleshooting. In the current work, we used DryLab modeling Design Spaces (DSs) to create digitized modeling fingerprints of various instruments, enabling extensive in silico investigations, including in-depth assessment of method transferability and evaluation of subtle, instrument-dependent robustness characteristics.

Journal of Chromatography A · 2026

Overcoming challenges in LC separation of oxysterols through guided design space modelling: A comparison of three stationary phases

Andrea Castellaneta, Ilario Losito, Claudio Crisau, Arnold Zöldhegyi, Zuzana Hrušovská, Hans-Jürgen Rieger, Imre Molnár, Tommaso R. I. Cataldi

Liquid chromatography–mass spectrometry (LC–MS) is an attractive alternative to GC–MS for oxysterol (OS) analysis, as it eliminates the need for chemical derivatization. However, in-source fragmentation during electrospray ionization (ESI) or atmospheric pressure chemical ionization (APCI), usually adopted for LC-MS analysis of OS, may lead to the generation of isomeric ions from non-isomeric precursors. This prevents a complete unambiguous identification along the MS dimension and makes chromatographic separation a fundamental step. In this work, analytical design space modelling empowered by a software-based modelling tool (DryLab®) was employed to optimize the reversed-phase LC separations of ten clinically relevant OS, including isomeric ring-oxidized and side chain-oxidized sterols. Three stationary phases, octadecyl (C18), cyanopropyl (ES-CN), and pentafluorophenyl (F5), were compared in terms of separation efficacy, using HPLC columns of identical geometry (150 × 2.1 mm, 2.7 μm) packed with core–shell particles. Gradient steepness, column temperature, and composition of the organic modifier (mixture of methanol and acetonitrile) in the mobile phase were selected as critical method parameters for successive construction of the design spaces on the selected stationary phases. All the resulting optimized methods resolved positional isomers and diastereomers, excepting 25(R/S)-26-hydroxycholesterol epimers. The C18 stationary phase (at 52 °C) offered high selectivity but demanded the longest run times (ca. 80 min). ES-CN (at 50 °C) and F5 (at 25 °C) stationary phases enabled faster effective runs (< 45 min), with ES-CN also reducing retention/chemical artifacts that are presumably triggered by π-π binding interactions with the stationary phase.

LCGC Supplements · 2013

Application of Quality by Design Principles to a Pharmaceutical Sample Using UHPLC Method Development with Modeling Technologies

Hans-Jürgen Rieger, Róbert Kormány, Imre Molnár

A new type of method development that uses modeling to find the "best" separation for high performance liquid chromatography (HPLC) was investigated and principles of Quality by Design (QbD) were followed when planning the investigation. The process delivered precise results and the method was able be transferred to a routine quality control (QC) laboratory.

Journal of Chromatography A · 1994

Separation of arachidonic acid metabolites by on-line extraction and reversed-phase high-performance liquid chromatography optimized by computer simulation

H. Fritsch, Imre Molnár, M. Wurl

A complex mixture of arachidonic acid metabolites was separated by reversed-phase HPLC using a multi-step gradient, which was modelled by computer-assisted HPLC method development. The metabolites were extracted on-line on a precolumn connected to the analytical column in the same HPLC system. The predictions of the resolution and also the retention times calculated by computer simulation were very accurate when compared with the corresponding experimental run (maximum deviation 0.86%). An appropriate HPLC method and additionally an on-line extraction procedure could be developed with just three experimental HPLC runs. This method could be useful for evaluating the concentrations of arachidonic acid metabolites involved in inflammatory diseases.

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.

Magazine Article · 2021

Comparing Multivariate Eluent Design Spaces for Systematic Characterization of (U)HPLC Columns | LCGC International

Arnold Zöldhegyi, Hans-Jürgen Rieger, Imre Molnár

Presenting a novel chromatographic modelling method to establish QbD-compliant comparative testing of eluent design spaces.

Journal of Chromatography · 1982

High-performance liquid chromatography of proteins: analytical applications

K. Buchholz, B. Gödelmann, Imre Molnár

Analysis of proteins in solution by high-performance liquid chromatography is presented with respect to structural changes in solution, adsorption processes and differentation concerning specific activities. Trypsin and cellulases were taken as examples.

Journal of Chromatography A · 2002

Advanced high-performance liquid chromatography method development: Discovering unexpected choices in chromatography

Imre Molnár, Hans-Jürgen Rieger

The influence of some important experimental parameters on the resolution of compounds as well as the validity of widely used rules of thumb and of common expectations about how to improve resolution is discussed. It will be shown, on the basis of selected examples, that the general expectations about how the experimental parameters have to be adjusted for better resolution does not cover all chances for resolution improvement. The tool for understanding the method and to discover all chances for increasing selectivity is the resolution map of a method.

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.

Journal of Chromatography A · 2012

Quality by Design: Multidimensional exploration of the design space in high performance liquid chromatography method development for better robustness before validation

Kate E. Monks, Imre Molnár, Hans-Jürgen Rieger, Botond Bogáti, E. Szabó

Robust HPLC separations lead to fewer analysis failures and better method transfer as well as providing an assurance of quality. This work presents the systematic development of an optimal, robust, fast UHPLC method for the simultaneous assay of two APIs of an eye drop sample and their impurities, in accordance with Quality by Design principles. Chromatography software is employed to effectively generate design spaces (Method Operable Design Regions), which are subsequently employed to determine the final method conditions and to evaluate robustness prior to validation.

Journal of Chromatography A · 2002

Two-dimensional optimization using different pairs of variables for the reversed-phase high-performance liquid chromatographic separation of a mixture of acidic compounds

Tom H. Jupille, John W. Dolan, Lloyd R. Snyder, Imre Molnár

Computer-facilitated method development has been extended for the simultaneous optimization of any two variables in separations by HPLC and other chromatographic procedures (gas chromatography, supercritical fluid chromatography, capillary electrophoresis, etc.). The application of this approach to HPLC method development is illustrated by the reversed-phase separation of a nine-component mixture of organic acids. Two of four variables (temperature, solvent strength (%B), pH and buffer concentration) were separately optimized in terms of selectivity, and the results are compared in terms of which variables and other conditions are most effective in providing maximum resolution for samples that contain ionizable compounds.

Journal of Chromatography A · 2022

Analytical quality by design-compliant retention modeling for exploring column interchangeabilities in separating ezetimibe and its related substances

Elek Ferencz, Arnold Zöldhegyi, Éva-Katalin Kelemen, Mona Obreja, Melinda Urkon, Emese Sipos, Gergő Tóth, Imre Molnár, Zoltán-István Szabó

There are several potential advantages of using experimental design-based retention modeling for chromatographic method development. Most importantly, through the model-delivered systematic understanding (Design Spaces), users can benefit from increased method consistency, flexibility and robustness that can efficiently be achieved at lesser amount of development time. As a result, modeling tools have always been great supplementary assets and welcomed by both the pharmaceutical industry and the regulatory authorities. Most recently published chapters of ICH however – Q2(R2) and Q14 (both currently drafts) – evidence a further paradigm shift, specifying the elements of model-based development strategies in the so-called “enhanced approach”. The main aim of this study was to investigate the impact of stationary phase chemistries on chromatographic method performance in the application example of ezetimibe and its related substances. A commercial modeling software package (DryLab®) was used to outline three-dimensional experimental design frameworks and acquire model Design Spaces (DSs) of 9 tested columns. This was done by performing 12 input calibration experiments per column, systematically changing critical method parameters (CMPs) as variables such as the gradient time (tG), temperature (T) and the ternary composition (tC) of the mobile phase. The constructed models allowed studying retention behaviors of selected analytes within each separation systems. In the first part of our work, we performed single optimizations for all nine stationary phases with substantially different surface modifications based on their highest achievable critical resolution values. For these optimum points in silico robustness testing was performed, clearly showing a change of CMPs, depending on the column, and specified optimum setpoint. In the second part of our work, we simultaneously compared the three-dimensional virtual separation models to identify all method parameter combinations that could provide at least baseline separation (Rs, crit.>1.50). These overlapping areas between the models described a common method operational design region (MODR) where columns were considered completely interchangeable – in terms of their baseline resolving capability – regardless of their exact physicochemical properties. A final optimized, column-independent working point within the common MODR was selected for verification. Indeed, experimental chromatograms showed excellent agreement with the model; all columns in the common condition were able to yield critical resolution values higher than 2.0, only their retentivity (elution window of peaks) was found different in some cases. Our results underline that a profound understanding of the separation process is of utmost importance andthat in some cases, adequate selectivity is achievable on various stationary phases.