Decades of chromatography research done with DryLab®.

Software-assisted method development in high performance liquid chromatography · 2019

Modeling of HPLC Methods Using QbD Principles in HPLC

Szabolcs Fekete, Imre Molnár, Imre Molnár, Hans-Jürgen Rieger, Róbert Kormány

HPLC method modeling is becoming a powerful tool to be used in the communication about method quality in HPLC between different labs, different companies, and between companies and regulatory agencies. The understanding of simple rules of peak movements will facilitate the development of new drugs, which are badly needed for smaller patient populations. The new features of HPLC modeling software, such as 3D resolution map, the modeled robustness testing, a practicable method transfer, or a method knowledge management offer a closed loop of all information about the birth and practical use of a method, and it further suggests the use of such software solutions in regulated laboratories to make analyst's life easier-especially in the pharmaceutical industry.

The Column · 2012

Practical Application of Quality by Design Principles to The Development of an HPLC Assay Method for an API and Impurities

Kate E. Monks, Imre Molnár, Hans-Jürgen Rieger

A method for the assay of an active pharmaceutical ingredient (API) and five known and unknown impurities was developed in accordance with Quality by Design (QbD) principles. Highly influential separation parameters were simultaneously and systematically studied so that the quality of the HPLC method could be understood, controlled and ensured. Consequently, during routine analysis out of specification (OOS) results can be corrected more effectively.

LC GC Europe · 2002

Optimizing Multilinear Gradients in HPLC

Tom H. Jupille, Lloyd R. Snyder, Imre Molnár

Multilinear gradients have not been widely used in general purpose high performance liquid chromatography, in part because of experimental inconvenience in method development. Even with the use of computer modelling, identifying an optimum set of breakpoints has primarily been done by trial and error. Combining a spreadsheet with controllable chromatography modelling software has allowed us to implement a systematic approach to bi- and tri-linear gradient optimization. Application of this approach to 10 randomly generated sample sets suggests that the improvement in critical resolution that can be obtained is considerably smaller than that obtained through the use of a second selectivity variable. Because no additional experimental time (and only limited computational time) is required once an optimum linear gradient has been modelled, however, any improvement thus gained is essentially "free".

World Scientific Publishing Europe Ltd · 2019

Software-assisted method development in high performance liquid chromatography

Imre Molnár, Szabolcs Fekete

"This handbook gives a general overview of the possibilities in recent developments in chromatographic retention modeling. As a result of the latest developments in modeling software, several new features are now accessible, opening a new level in HPLC method development. Many of these current possibilities in software assisted liquid chromatographic method modeling for analytical purposes are presented. Several modes of chromatography, including Reversed-Phase Liquid Chromatography (RPLC), Ion Exchange Chromatography (IEX), Hydrophobic Interaction Chromatography (HIC), and Hydrophilic Interaction Liquid Chromatography (HILIC) are explained in detail. For all these chromatographic modes, the most important variables for tuning retention and selectivity are exposed. Beside the industrial and practical benefits of retention modeling, the possibilities in teaching and education are also illustrated. Finally, numerous representative industrial examples are shown, to highlight the benefits, time and cost savings offered by state-of-the-art software assisted HPLC method development."--Publisher's website

Peptides: Proceedings of the 5th American Peptide Symposium · 1977

Rapid Analysis of Peptide Mixtures by High Performance Liquid Chromatography With Nonpolar Stationary Phases

Murray Goodman, Johannes Meienhofer, Imre Molnár, Csaba Horváth

Journal of Pharmaceutical and Biomedical Analysis · 2015

Establishing column batch repeatability according to Quality by Design (QbD) principles using modeling software

Norbert Rácz, Róbert Kormány, Jenő Fekete, Imre Molnár

Column technology needs further improvement even today. To get information of batch-to-batch repeatability, intelligent modeling software was applied. Twelve columns from the same production process, but from different batches were compared in this work. In this paper, the retention parameters of these columns with real life sample solutes were studied. The following parameters were selected for measurements: gradient time, temperature and pH. Based on calculated results, batch-to-batch repeatability of BEH columns was evaluated. Two parallel measurements on two columns from the same batch were performed to obtain information about the quality of packing. Calculating the average of individual working points at the highest critical resolution (Rs,crit) it was found that the robustness, calculated with a newly released robustness module, had a success rate >98% among the predicted 36=729 experiments for all 12 columns. With the help of retention modeling all substances could be separated independently from the batch and/or packing, using the same conditions, having high robustness of the experiments.

The Column · 2014

UHPLC Method Development and Modelling in the Framework of Quality by Design

Imre Molnár, Hans-Jürgen Rieger, Alexander Schmidt, Jenő Fekete, Róbert Kormány

The goals in ultrahigh-pressure liquid chromatography (UHPLC) method development are to first find the best separation, second find the best column, and third find the most robust method in a multifactorial Design Space. Trial and error methods are not sufficient anymore and solid science based on Quality by Design (QbD) principles is required.

Journal of Pharmaceutical and Biomedical Analysis · 2013

Exploring better column selectivity choices in ultra-high performance liquid chromatography using Quality by Design principles

Róbert Kormány, Imre Molnár, Hans-Jürgen Rieger

An older method for amlodipine was reworked with the goal to reduce the analysis time of 60min below 6min. To select the best column for short and robust analysis, 9 different UHPLC column chemistries were investigated using 3-dimensional resolution spaces based on 12 experiments using modelling software. The main variables used were gradient time (tG), temperature (T) and the pH of eluent A. The best critical resolution was calculated and located in a 3-dimensional space in an automated fashion and the corresponding best experiments were carried out. The work (9×12=108 runs) was finished with an UHPLC instrument in less than 24h. The comparison between predictions and real experiments showed an excellent correlation with differences typically less than 0.04min (<3s) in average, although the set points were located at quite different conditions on gradient times, pH's and temperatures for the individual columns. All columns could perform the required baseline separation at their individual best working points with satisfactory results.

The Column · 2011

Experimental Combination of Method Development Strategies in a Working Environment of Different Instrumental Set-ups

Imre Molnár, Kate E. Monks, Björn-Thoralf Erxleben

Experimental Combination of Method Development Strategies in a Working Environment of Different Instrumental Set-ups

Journal of Chromatography B: Biomedical Sciences and Applications · 1977

Rapid separation of urinary acids by high-performance liquid chromatography

Imre Molnár, Csaba Horváth

Over a hundred acidic urinary constituents were separated within 30 min by using 5-μm octadecyl-silica columns and gradient elution with increasing acetonitrile concentration in dilute aqueous phosphoric acid solution at 70°. The column effluent was monitored with a UV detector at 280 nm or with a fluorescence detector at 260 nm excitation and 340 nm emission wavelengths. The high sensitivity and speed of analysis, the excellent reproducibility and adequate resolution obtained suggest that this technique may be useful to obtain metabolic profiles in routine clinical work.

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.

Journal of Chromatography A · 2006

Retention modelling in ternary solvent-strength gradient elution reversed-phase chromatography using 30mm columns

Melvin R. Euerby, Federico Scannapieco, Hans-Jürgen Rieger, Imre Molnár

An optimization strategy for ternary solvent-strength gradient elution RP chromatography is described in which a two-dimensional model of gradient time (2 levels) against ternary proportions of organic modifiers (4 levels) was constructed. From the resolution surface the optimum ratio of organic modifiers could be selected. Excellent retention time and acceptable peak width and resolution simulations were obtained. The separation could be further optimized from the same input data by using a standard one-dimensional model in order to optimize for gradient slope, duration and shape. Excellent retention time and acceptable peak width and resolution simulations were obtained (<1, 2 and 6% error, respectively).

Journal of Chromatography · 1976

Solvophobic interactions in liquid chromatography with nonpolar stationary phases

Csaba Horváth, Wayne Melander, Imre Molnár

Solute interaction with nonpolar stationary phases in liquid chromatography is examined on the basis of the solvophobic theory. The chromatographic process is viewed as a reversible association of the solute with the hydrocarbonaceous ligands of bonded phases. A detailed analysis of the effect of the solvent on this process yields an expression for the capacity factor with essentially no adjustable constants. The theory satisfactorily accounts for the factors affecting solute retention under a wide range of experimental conditions. It makes possible the characterization of the solvophobic (eluent) strength of mixed solvents having different composition and the evaluation of the various solvophobic forces representing incremental values of the logarithm of the capacity factor. The wide applicability of nonpolar stationary phases (reversed phases) in liquid chromatography is demonstrated by the rapid separation of biogenic acids and bases on octadecylsilica columns with neat aqueous elements.

Chromatographia · 2014

Robust UHPLC Separation Method Development for Multi-API Product Containing Amlodipine and Bisoprolol: The Impact of Column Selection

Róbert Kormány, Imre Molnár, Jenő Fekete, Davy Guillarme, Szabolcs Fekete

This paper describes a new and fast ultra-high pressure liquid chromatographic separation of amlodipine and bisoprolol and all their closely related compounds, for impurity profiling purposes. Computer-assisted method development was applied and the impact of several state-of-the-art stationary phase column chemistries (50 × 2.1 mm, sub-2 μm, and core–shell type materials) on the achievable selectivity and resolution was investigated. The work was performed according to quality by design principles using design of experiment with three experimental factors; namely the gradient time (tG), temperature (T), and mobile phase pH. Thanks to modeling software, it was proved that the separation of all compounds was feasible on numerous column chemistries within <10 min, by proper adjustments of variables. It was also demonstrated that the reliability of predictions was good, as the predicted retention times and resolutions were in good agreement with the experimental ones. The final, optimized method separates 16 peaks related to amlodipine and bisoprolol within 7 min, ensuring baseline resolution between all peak-pairs.

Analytical Chemistry · 1977

Enhancement of retention by ion-pair formation in liquid chromatography with nonpolar stationary phases

Csaba Horváth, Wayne Melander, Imre Molnár, Petra Molnár

In Ion-pair reversed-phase chromatography, the retention of ionized analytes on a nonpolar bonded stationary phase is enhanced by the presence of a "hydrophobic" counterlon (hetaeron) in the mobile phase. Either ion-pair formation in the mobile phase with relatively strong retention of the complex or the conversion of the stationary phase into an ion-exchanger may explain the phenomenon. Analysis of the pertinent equilibria shows that the observed hyperbolic or parabolic dependence of the capacity factors on the hetaeron concentration cannot shed light on the mechanism. The experimental data obtained for the retention of catecholamlnes by using C4-C10 alkyl sulfates and other similar hetaerons in a wide concentration range, however, could be mechanistically interpreted from the chain length dependence of the parameters for the relationship between the capacity factors and hetaeron concentration. Although the results clearly demonstrate that in the system investigated, ion-pair formation governs retention, ion-exchange mechanism can be operative under certain conditions. Changes in retention upon addition of salt to the eluent are treated both theoretically and experimentally. The effect of organic solvents on the behavior of the chromatographlc system is discussed in view of the proposed theory.

Journal of Chromatography A · 2009

Rapid high performance liquid chromatography method development with high prediction accuracy, using 5cm long narrow bore columns packed with sub-2μm particles and Design Space computer modeling

Szabolcs Fekete, Jenő Fekete, Imre Molnár, Katalin Ganzler

Many different strategies of reversed phase high performance liquid chromatographic (RP-HPLC) method development are used today. This paper describes a strategy for the systematic development of ultrahigh-pressure liquid chromatographic (UHPLC or UPLC) methods using 5cm×2.1mm columns packed with sub-2μm particles and computer simulation (DryLab® package). Data for the accuracy of computer modeling in the Design Space under ultrahigh-pressure conditions are reported. An acceptable accuracy for these predictions of the computer models is presented. This work illustrates a method development strategy, focusing on time reduction up to a factor 3–5, compared to the conventional HPLC method development and exhibits parts of the Design Space elaboration as requested by the FDA and ICH Q8R1. Furthermore this paper demonstrates the accuracy of retention time prediction at elevated pressure (enhanced flow-rate) and shows that the computer-assisted simulation can be applied with sufficient precision for UHPLC applications (p>400bar). Examples of fast and effective method development in pharmaceutical analysis, both for gradient and isocratic separations are presented.

Journal of Pharmaceutical and Biomedical Analysis · 2017

Separation of antibody drug conjugate species by RPLC: A generic method development approach

Szabolcs Fekete, Imre Molnár, Davy Guillarme

This study reports the use of modelling software for the successful method development of IgG1 cysteine conjugated antibody drug conjugate (ADC) in RPLC. The goal of such a method is to be able to calculate the average drug to antibody ratio (DAR) of and ADC product. A generic method development strategy was proposed including the optimization of mobile phase temperature, gradient profile and mobile phase ternary composition. For the first time, a 3D retention modelling was presented for large therapeutic protein. Based on a limited number of preliminary experiments, a fast and efficient separation of the DAR species of a commercial ADC sample, namely brentuximab vedotin, was achieved. The prediction offered by the retention model was found to be highly reliable, with an average error of retention time prediction always lower than 0.5% using a 2D or 3D retention models. For routine purpose, four to six initial experiments were required to build the 2D retention models, while 12 experiments were recommended to create the 3D model. At the end, RPLC can therefore be considered as a good method for estimating the average DAR of an ADC, based on the observed peak area ratios of RPLC chromatogram of the reduced ADC sample.

Journal of Pharmaceutical and Biomedical Analysis · 2018

Implementation of a generic liquid chromatographic method development workflow: Application to the analysis of phytocannabinoids and Cannabis sativa extracts

Szabolcs Fekete, Vida Sadat-Noorbakhsh, Cedric Schelling, Imre Molnár, Davy Guillarme, Serge Rudaz, Jean-Luc Veuthey

A generic liquid chromatographic method development workflow was developed and successfully applied to the analysis of phytocannabinoids and Cannabis sativa extracts. Our method development procedure consists in four steps: i)The screening of primary parameters (i.e. stationary phase nature, organic modifier nature and approximate mobile phase pH) was carried out with a generic gradient on a short narrow bore column, using a system able to accommodate numerous solvents/buffers and columns. Instead of complete peak tracking, the number of peaks which can be separated was considered as a response at this level, to save time.ii)The optimization of secondary parameters (i.e. gradient conditions, mobile phase temperature and pH within a narrow range) requires only 12 initial experiments and the use of HPLC modeling software for data treatment. It allows to find out the best retention and selectivity for the selected compounds. Peak tracking was performed with a single quadruple mass detector in single ion recording mode, and UV detection (in a broad wavelength range).iii)The refinement step allows to further adjust column efficiency, by tuning column length and mobile phase flow rate. This can also be done virtually using HPLC modeling software.iv)The robustness testing step was also evaluated from a virtual experimental design. Success rate and regression coefficients were estimated in about 1 min, without the need to perform any real experiment. At the end, this method development workflow was performed in less than 4 days and minimizes the costs of the method development in liquid chromatography.

LC GC Europe · 2001

Unexpected Results in Chromatography

Imre Molnár

Unusual experiments can provide surprisingly good analytical solutions. When developing Chromatographic methods, analysts must use a combination of experience and instinct to choose initial starting conditions. This is often followed by a period of trial-and-error optimization until the desired methodology is achieved. This article illustrates how the process of Chromatographic method development can be improved using computer modelling and simulation.

Journal of Pharmaceutical and Biomedical Analysis · 2018

Simultaneous optimization of mobile phase composition and pH using retention modeling and experimental design

Norbert Rácz, Imre Molnár, Arnold Zöldhegyi, Hans-Jürgen Rieger, Róbert Kormány

Chromatographic methods are progressing continuously. Increasing sample complexity and safety expectations lead to higher regulatory demands, hence challenges in liquid chromatography analysis are rising, even today, when faster and faster chromatographic systems are extensively employed and become widely accessible for successful method development. The goal of this study was to investigate the impact of mobile phase influences as important factors of selectivity tuning in method development. This would mitigate mobile phase-related robustness issues throughout the method’s lifecycle. To discover and understand these effects, a new module of chromatographic modeling software DryLab (ver. 4.3.4. beta) was introduced and a special experimental design (DoE) was tested, allowing the simultaneous optimization of solvent-dependent parameters, such as gradient time (tG), ternary eluent composition (tC) and pH, requiring 18 input experiments (2 × 3 × 3 = 18). Additionally, the model creation, using a UPLC system and a narrow bore column (50 × 2.1 mm), the entire experimental work could be finished in 2–3 hours. To demonstrate the applicability of this new design, amlodipine and its related pharmacopoeia impurities (A–H) were subjected to be used in a case study. Predicted vs. Experimental (or Verification) runs showed excellent agreement, average retention time deviations were typically less than 1 s. Modelled robustness testing was also performed, elucidating all important mobile phase and instrument parameters that could influence a method’s lifetime performance. Furthermore, as the in silico robustness testing is the least time consuming part of the method development process, it can be used extensively to evaluate robustness even at the very early part in stage 1 of the Method Life Cycle (MLC).