Literature | MOLNÁR-INSTITUTE

Journal of Pharmaceutical and Biomedical Analysis · 2018

Automated UHPLC separation of 10 pharmaceutical compounds using software-modeling

Arnold Zöldhegyi, Hans-Jürgen Rieger, Imre Molnár, Liliya Fekhretdinova

Human mistakes are still one of the main reasons of underlying regulatory affairs that in a compliance with FDA’s Data Integrity and Analytical Quality by Design (AQbD) must be eliminated. To develop smooth, fast and robust methods that are free of human failures, a state-of-the-art automation was presented. For the scope of this study, a commercial software (DryLab) and a model mixture of 10 drugs were subjected to testing. Following AQbD-principles, the best available working point was selected and conformational experimental runs, i.e. the six worst cases of the conducted robustness calculation, were performed. Simulated results were found to be in excellent agreement with the experimental ones, proving the usefulness and effectiveness of an automated, software-assisted analytical method development.

Journal of Pharmaceutical and Biomedical Analysis · 2018

An overview of experimental designs in HPLC method development and validation

Prafulla Kumar Sahu, Nageswara Rao Ramisetti, Teresa Cecchi, Suryakanta Swain, Chandra Sekhar Patro, Jagadeesh Panda

Chemometric approaches have been increasingly viewed as precious complements to high performance liquid chromatographic practices, since a large number of variables can be simultaneously controlled to achieve the desired separations. Moreover, their applications may efficiently identify and optimize the significant factors to accomplish competent results through limited experimental trials. The present manuscript discusses usefulness of various chemometric approaches in high and ultra performance liquid chromatography for (i) methods development from dissolution studies and sample preparation to detection, considering the progressive substitution of traditional detectors with tandem mass spectrometry instruments and the importance of stability indicating assays (ii) method validation through screening and optimization designs. Choice of appropriate types of experimental designs so as to either screen the most influential factors or optimize the selected factors’ combination and the mathematical models in chemometry have been briefly recalled and the advantages of chemometric approaches have been emphasized. The evolution of the design of experiments to the Quality by Design paradigm for method development has been reviewed and the Six Sigma practice as a quality indicator in chromatography has been explained. Chemometric applications and various strategies in chromatographic separations have been described.

Journal of Pharmaceutical and Biomedical Analysis · 2014

Reliability of computer-assisted method transfer between several column dimensions packed with 1.3–5μm core–shell particles and between various instruments

Róbert Kormány, Jenő Fekete, Davy Guillarme, Szabolcs Fekete

In this contribution, the possibility to automatically transfer RPLC methods between different column dimensions and instruments was evaluated using commercial modelling software. The method transfer reliability was tested with loratadine and its 7 related pharmacopeial impurities. In this study, state-of-the-art columns packed with superficially porous particles of 5, 2.6, 1.7 and 1.3μm particles were exclusively employed. A fast baseline separation of loratadine and related impurities (Rs,min=2.49) was achieved under the best analytical conditions (i.e. column of 50mm×2.1mm, 1.3μm, 10–90% ACN in 5min, T=40°C, pH=3, F=0.5ml/min). This optimal method was successfully tested on columns packed with other particle sizes, namely 1.7 and 2.6μm, to reduce pressure drop. The selectivities and retentions remained identical, while the peak widths were logically wider, leading to a reduction of peak capacity from 203 to 181 and 159 on the 1.3, 1.7 and 2.6μm particles, respectively. On the minimum, the resolution was equal to 1.54 on the 50mm×2.1mm, 2.6μm stationary phase. Next to this, the method was transferred to columns of different lengths, inner diameters and particle sizes (100mm×3mm, 2.6μm or 150mm×4.6mm, 5μm). These columns were used on other LC instruments possessing larger dwell volumes. The modelling software employed for developing the original method was able to calculate the new gradient conditions to be used. The accuracy of prediction was excellent, as the average retention time errors between predicted and observed chromatograms were −0.11% and 0.45% when transferring the method to 100mm×3mm and 150mm×4.6mm columns, respectively. This work proves the usefulness and validity of HPLC modelling software for transferring methods between different instruments, column dimensions and/or flow rates.

Journal of Chromatography · 1990

High-performance liquid chromatographic computer simulation based on a restricted multi-parameter approach: II. Applications

L. R. Snyder, J. W. Dolan, D. C. Lommen

A computer program (DryLab® MP) is described that allows restricted multi-parameter mapping for any number (or kind) of separation variables, based on only a few experiments. Multi-parameter computer simulation can be used to develop an high-performance liquid chromatographic method from the beginning, or it can be used to enhance a method developed by other means; e.g., by trial-and-error, single-parameter mapping, etc. The software can also be used to evaluate (and improve) method ruggedness. Finally, various problems (column-to-column variability, change of retention with ambient temperature fluctuations, experimental errors, etc.) that are commonly encountered during routine operation can be handled in the same general way. Examples of these various applications are given.

Journal of Chromatography · 1992

Application of the gradient elution technique: Demonstration with a special test mixture and the DryLab G/plus method development software

R. Däppen, I. Molnár

A mixture of ten compounds with an overlapping peak pair was analysed with a 90-min elution gradient. To improve the separation, two reversed-phase gradients differing by a factor of 3 in their run times, were applied. Contrary to expectation, two peak pairs were less well separated in the gradient run with the lower slope. The relative resolution map provided a rapid solution to the problem: a gradient with 16-min run time gave the best separation of the mixture. The simulated chromatogram was verified experimentally. The differences between the predicted and experimental retention times averaged 0.03 min. Further improvement was obtained using a segmented gradient, which adequately separately all peaks in only 9 min.

Journal of Chromatography A · 2019

Is hydrophobic interaction chromatography the most suitable technique to characterize site-specific antibody-drug conjugates?

Valentina D’Atri, Reinhard Pell, Adrian Clarke, Davy Guillarme, Szabolcs Fekete

Antibody drug conjugates (ADCs) belong to the fastest growing class of therapeutic agents for cancer therapy. In preclinical and clinical studies, there is a significant number of site-specific ADCs (also known as third generation ADCs), which are more homogeneous than their previous generations. These new ADC formats, in which the inter-chain disulphide bridges (hinge cysteines) are not reduced, also need to be deeply characterized. In particular, various quality attributes (QAs) have to be determined, such as free antibody level, average drug to antibody ratio (DAR) and drug distribution. In this contribution, a non-commercial site-specific conjugated ADC has been analyzed by RPLC. Our results demonstrated that RPLC has a huge potential to determine QAs and can replace the historically used HIC methods as RPLC provides better separation quality for such type of ADCs. Site-specific ADCs can be analyzed in RPLC at intact level without the need for sample preparation. A further advantage of RPLC is that it enables the direct coupling to MS and thus allows the fine identification of all eluting species.

Journal of Chromatography A · 1996

High-performance liquid chromatographic separation of the impurities in a pharmaceutical raw material with the aid of computer simulation

H. W. Bilke, Imre Molnár, Ch. Gernet

A complex pharmaceutical raw material was characterized by means of reversed-phase gradient elution. By varying gradient steepness and mobile-phase pH, it was possible to optimize band spacing so as to separate 16 impurities or degradation products from the drug substance. Computer simulation was useful in interpreting these complex chromatograms and determining the maximum number of peaks that could be separated in this way. A marginal separation of all 17 sample components could be obtained, but the resulting method was quite pH-sensitive and therefore not very rugged. As an alternative, a rugged method was developed that separates the drug substance from all other sample components. The present study also describes how present computer simulation software for isocratic separation can be used to predict resolution for gradient elution runs as a function of pH.

Journal of Chromatography A · 2014

Implementation of gradients of organic solvent in micellar liquid chromatography using DryLab®: Separation of basic compounds in urine samples

J. Rodenas-Montano, C. Ortiz-Bolsico, M. J. Ruiz-Angel, M. C. García-Alvarez-Coque

In micellar liquid chromatography (MLC), chromatographic peaks are more evenly distributed compared to conventional reversed-phase liquid chromatography (RPLC). This is the reason that most procedures are implemented using isocratic elution. However, gradient elution may be still useful in MLC to analyse mixtures of compounds within a wide range of polarities, decreasing the analysis time. Also, it benefits the determination of moderately to low polar compounds in physiological fluids performing direct injection: an initial micellar eluent with a low organic solvent content, or a pure micellar (without surfactant) solution, will provide better protection of the column against the proteins in the physiological fluid, and once the proteins are swept away, the elution strength can be increased using a positive linear gradient of organic solvent to reduce the analysis time. This work aims to encourage analysts to implement gradients of organic solvent in MLC, which is rather simple and allows rapid analytical procedures without pre-treatment or the need of re-equilibration. The implementation of gradient elution is illustrated through the separation of eight basic compounds (β-blockers) in urine samples directly injected into the chromatograph, the most hydrophobic showing large retention in both conventional RPLC and MLC. The use of the DryLab® software to optimise gradients of organic solvent with eluents containing a fixed amount of surfactant above the critical micellar concentration is shown to provide satisfactory predictions, and can facilitate greatly the implementation of gradient protocols.

Journal of Pharmaceutical and Biomedical Analysis · 2015

Development of a purity control strategy for pemetrexed disodium and validation of associated analytical methodology

Anne Warner, Irina Piraner, Heather Weimer, Kevin White

Stability-indicating reversed phase HPLC methods have been developed and validated for the determination of 13 potential process and degradation impurities in pemetrexed disodium drug substance (DS) and pemetrexed for injection drug product (DP). This paper describes the development of HPLC-UV impurity methods for drug substance and drug product. Relative response factors (RRF) have been determined using HPLC-UV in tandem with CAD or by NMR detection. Conditions for the generation of system suitability solutions are described and assure adequate chromatographic resolution and peak identification without the need for impurity reference standards. The methods were fully validated and demonstrated to have acceptable specificity, linearity, accuracy, repeatability, intermediate precision, detection/quantitation limit, and robustness.

Analytical and Bioanalytical Chemistry · 2011

Determination of stilbene derivatives in Burgundy red wines by ultra-high-pressure liquid chromatography

Lemia Boutegrabet, Agnes Fekete, Norbert Hertkorn, Yorgos Papastamoulis, Pierre Waffo-Téguo, Jean Michel Mérillon, Philippe Jeandet, Régis D. Gougeon, Philippe Schmitt-Kopplin

The polyphenols, for example stilbenes and flavonoids, are an important family of compounds present in grapes and wines. Several studies have shown that stilbenes are antioxidants and cancer-preventing agents. For the first time, eight natural stilbenes (trans-resveratrol, trans-piceid, cis-piceid, trans-astringin, trans-piceatannol, (+)-trans-ε-viniferin, pallidol, and hopeaphenol), isolated and purified from Vitis vinifera, were simultaneously analysed by ultra-high-pressure liquid chromatography coupled with photodiode-array detection. Separation of the stilbenes by UHPLC was optimized with the assistance of “Quality-by-Design” commercial software. Four different reversed-phase columns packed with 1.5–1.7-μm particles were tested and compared for their retention behaviour and separation efficiency. On the basis of the performance characteristics determined, the VisionHT C18 HL column was selected for the stilbenes studied, because resolution of the critical pair was 1.5 with a peak width of 2–4 s. The optimized method resulted in highly repeatable retention times (RSD 0.03–0.07%), peak areas (RSD 3–6%), and linear ranges were between 0.005 and 50 mg L−1 for most of the compounds. All stilbenes, except trans-astringin, trans-piceatannol, and pallidol were identified and quantified in Burgundy red wines at different concentrations after direct injection of the wines.

Journal of Chromatography · 1989

Drylab® computer simulation for high-performance liquid chromatographic method development: I. Isocratic elution

L. R. Snyder, J. W. Dolan, D. C. Lommen

Computer simulation (DryLab software) as a means of facilitating the development of isocratic high-performance liquid chromatographic methods is reviewed. The various features of computer simulation are discussed and several examples of its application are presented.

Chromatographia · 2012

Enhancing the Quality of Separation in One-Dimensional Peptide Mapping Using Mathematical Transformation

Róbert Berky, Szabolcs Fekete, Jenő Fekete

In this study, some practical examples are presented that show the quality of separations using very efficient columns packed with the latest generation of core shell sub-3 μm and fully porous sub-2 μm particles in one-dimensional peptide separations. This paper shows an approach for the analysis of proteins, such as high-resolution separations, and a data transformation process to improve peak recognition and analysis. Applying power functions on raw chromatographic data can be a neat tool in the field of biosimilar analysis, especially in comparability studies regarding the quality (primary structure) of proteins. Based on the results presented here, it can be stated that the use of power functions is beneficial for the comparison of chromatograms when peak areas are considered but has no effect when using peak heights. In this study, the new Acquity CSH columns (C18 and phenyl-hexyl) and the core–shell type wide pore Ascentis Express Peptide ES C18 material were applied with great success in peptide mapping. Finally, using phenyl-hexyl stationary phase in peptide separation seems to be a good alternative to the generally applied C18 or C4 phases.

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.

Journal of Pharmaceutical and Biomedical Analysis · 2024

Updating the European Pharmacopoeia impurity profiling method for cetirizine and suggesting alternative column, using design space comparison

Róbert Kormány, Barnabás Soós, Krisztián Horváth

The goal of the present study was to develop a generic workflow to evaluate the chromatographic resolution in a design space and find replacement column for the new method. To attain this objective, a limited number of initial experiments have been performed, and a modeling tool was employed to study and compare design spaces obtained with different columns. By overlaying the different individual resolution maps (design spaces), it is possible to quickly identify a robust zone where the different columns meet a given resolution criterion. This new feature of the modeling tool is very useful for finding alternative columns for a given separation, rather than the usual column tests. It was also demonstrated that two different columns can be used as equivalents (replacement columns), providing sufficient resolution at the same working point and with a high degree of robustness.

TrAC Trends in Analytical Chemistry · 1987

High-performance liquid chromatographic method-development using computer simulation

L. R. Snyder, J. W. Dolan, M. A. Quarry

Computer-assisted method-development schemes for high-performance liquid chromatography have taken another step forward with the introduction of 'computer simulation'. Software for the application of this technique is described and illustrated.

Journal of Chromatography · 1989

Drylab® computer simulation for high-performance liquid chromatographic method development: II. Gradient Elution

J. W. Dolan, D. C. Lommen, L. R. Snyder

Computer simulation (DryLab software) as an aid for the development of gradient high-performance liquid chromatographic methods is reviewed. Several examples of its application are presented and the accuracy of such predictions is discussed.

Journal of Chromatography A · 2017

Optimization of non-linear gradient in hydrophobic interaction chromatography for the analytical characterization of antibody-drug conjugates

Balázs Bobály, Giuseppe Marco Randazzo, Serge Rudaz, Davy Guillarme, Szabolcs Fekete

The goal of this work was to evaluate the potential of non-linear gradients in hydrophobic interaction chromatography (HIC), to improve the separation between the different homologous species (drug-to-antibody, DAR) of commercial antibody-drug conjugates (ADC). The selectivities between Brentuximab Vedotin species were measured using three different gradient profiles, namely linear, power function based and logarithmic ones. The logarithmic gradient provides the most equidistant retention distribution for the DAR species and offers the best overall separation of cysteine linked ADC in HIC. Another important advantage of the logarithmic gradient, is its peak focusing effect for the DAR0 species, which is particularly useful to improve the quantitation limit of DAR0. Finally, the logarithmic behavior of DAR species of ADC in HIC was modelled using two different approaches, based on i) the linear solvent strength theory (LSS) and two scouting linear gradients and ii) a new derived equation and two logarithmic scouting gradients. In both cases, the retention predictions were excellent and systematically below 3% compared to the experimental values.

Journal of Chromatography B · 2021

Integrated analytical workflow for chromatographic profiling and metabolite annotation of a cytotoxic Phorbas amaranthus extract

Bruno S. do Amaral, Fernanda B. da Silva, Gabriel Mazzi Leme, Letícia S. S. Schmitz, Paula C. Jimenez, Roberto Carlos Campos Martins, Quezia B. Cass, Alessandra L. Valverde

Phorbas is a widely studied genus of marine sponge and produce structurally rich cytotoxic metabolites. Still, only few studies have assessed metabolites present in Brazilian species. To circumvent redundancy, in this work, we applied and herein report the use of a scouting liquid chromatographic system associate to the design of experiment produced by the DryLab® software to obtain a fast and efficient chromatographic separation of the active hexane fraction, further enabling untargeted high-resolution mass spectrometry (HRMS) data. To this end, a crude hydroalcoholic extract of the sponge Phorbas amaranthus collected in Brazilian coast was prepared and partitioned. The cytotoxicity of the crude extract and the fractions was evaluated using tumor cell culture models. Fragmentation pathways assembled from HRMS data allowed the annotation of 18 known Phorbas metabolites, while 17 metabolites were inferred based on Global Natural Product Social Molecular Networking (GNPS), matching with a further 29 metabolites annotated through molecular subnetwork. The workflow employed demonstrates that chromatographic method development can be accelerated by the use of automated scouting systems and DryLab®, which is useful for profiling natural product libraries, as well as data curation by molecular clusters and should be incorporated to the tools of natural product chemists.

Journal of Chromatography A · 2003

Computer-assisted method development and optimization in high-performance liquid chromatography

T. H. Hoang, D. Cuerrier, S. McClintock, M. Di Maso

This paper reports the use of DryLab, a computer simulation software package, to assist in the development and optimization of a reversed-phase high-performance liquid chromatographic (HPLC) method for the separation of a model drug candidate and its degradation products. Prior to the optimization process, columns with various bonded phases are evaluated for their chromatographic performance using the sample of interest. Simultaneous optimization of two separation variables and the use of resolution maps to predict the optimal conditions are illustrated. Options to optimize column conditions (column length and flow-rate) to further reduce run time are briefly discussed. The accuracy of DryLab-predicted retention times and resolution is compared with experimental values. The DryLab software used in this study provided satisfactory predictions for the selected model, with average errors of less than 3.5 and 11.8% for retention time and resolution, respectively.

HPLC 2023 · 2023

Software-supported HPLC Method Development for Separating Four Regioisomers of a Sugammadex-related Impurity

Dominika Herr, Arnold Zöldhegyi, Péter Soma Szakály, Erzsébet Varga

This research focuses on a simple, relatively inexpensive reversed-phase high-pressure liquid chromatography (RP-HPLC) application in combination with UV-detection aiming for the separation of four relevant regioisomers (marked as AB, AC, AD, AE) of Di-OH-SGM (Hexakis(6-deoxy-6-(2-carboxyethyl)thio)-gamma- cyclodextrin). The presence of these regioisomers in the synthesized Di-OH-SGM reference material has already been proven by MS and NMR spectroscopy. Thus, the main goal of our project was to baseline-resolve these regioisomers—as this is required for the qualification of the synthetized Di-OH-SGM material. Although our preliminary results on the selected C18 stationary phase provided a promising initial separation, only Rs,crit. = 0.9 of peaks could be realized. Hence, we decided to apply a systematic modeling approach (DryLab) to contextualize data and to try optimizing critical method parameters. We found that by lowering column temperature, better separation but still no baseline separation of peaks was possible—while the modeling software revealed some unique features of these molecules. The poster will present the current results of our software-supported development process, underlining the key benefits of modeling approaches in better understanding the complex nature and chromatographic behavior of regioisomers of Di-OH-SGM.

Decades of chromatography research done with DryLab®.