Literature | MOLNÁR-INSTITUTE

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.

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 Liquid Chromatography & Related Technologies · 2013

Analysis of Sulfonamide Residues in Real Honey Samples Using Liquid Chromatography with Fluorescence and Tandem Mass Spectrometry Detection

Ádám Tölgyesi, Róbert Berky, Katalin Békési, Szabolcs Fekete, Jenő Fekete, Virender K. Sharma

This paper presents new reversed phase liquid chromatographic methods (HPLC-FLD and LC-MS/MS) for the quantification of sulfonamides in spiked and incurred honey samples. The sample preparation was optimized using Oasis HLB (hydrophilic–lipophilic balance) solid-phase extraction (SPE) cartridge. Elutions of sulfonamides were carried out under acidic, neutral, and basic conditions using methanol. Recoveries under acid condition were in the range from 66.8–90%, which were approximately 10% higher than those obtained under other conditions. The sample clean-up was also tested using Strata-XL cartridges. The HPLC-FLD separation was performed using a Varian C18 column and a ternary (methanol-acetonitrile-phosphate buffer, pH 5) mobile phase resulting good selectivity for the determination. The robustness of the ternary gradient method was evaluated by computer simulation (DryLab). LC-MS/MS separation was carried out on a Kinetex XB core-shell type HPLC column that enabled a low limit of detection (0.01–0.5 µg/kg) and faster separation (6 min). The developed methods were validated in accordance with the European Union Commission Decision 2002/657/EC and were applied successfully for more than four hundred honey samples (under a national monitoring program). The concentrations of sulfadimethoxine, sulfachloropyridazine, and trimethoprim residues in samples were found in a concentration range from 0.03 up to 686 µg/kg.

Journal of Pharmaceutical and Biomedical Analysis · 2020

Development of a generic reversed-phase liquid chromatography method for protein quantification using analytical quality-by-design principles

Julian Kopp, Florian B. Zauner, Andreas Pell, Johanna Hausjell, Diana Humer, Julian Ebner, Christoph Herwig, Oliver Spadiut, Christoph Slouka, Reinhard Pell

Biopharmaceutical drug substances are generally produced using fermentation technology and are subsequently purified in the following downstream process. For the determination of critical quality attributes (CQAs), such as target protein titer and purity, monitoring tools are required before quality control analysis. We herein present a novel reversed phase liquid chromatography method (RPLC), which enables facile and robust protein quantification during upstream and downstream processing of intracellularly produced proteins in E. coli. The overall goal was to develop a fast, robust and mass spectrometry compatible method which can baseline resolve and quantify each protein of interest. Method development consisted of three steps, oriented on an Analytical Quality by Design (AQbD) workflow: (i) the stationary phase as primary parameter was chosen based on state-of-the art technology thus minimizing protein on-column adsorption and providing high efficiency, (ii) secondary parameters (i.e. gradient conditions and column temperature) were optimized applying chromatographic modeling, and (iii) the established Method Operable Design Region (MODR) was challenged and confirmed during robustness testing, performed in-silico and experimentally by a Design of experiment (DoE) based approach. Finally, we validated the RPLC method for pivotal validation parameters (i.e. linearity, limit of quantification, and repeatability) and compared it for protein quantification against a well-established analytical methodology. The outcome of this study shows (i) a protocol for RPLC development using an AQbD principle for new method generation and (ii) a highly versatile RPLC method, suited for quick and straightforward recombinant protein titer measurement being applicable for the detection of a broad range of proteins.

Food Chemistry · 2018

Identification of cis/trans isomers of menaquinone-7 in food as exemplified by dietary supplements

Arkadiusz Szterk, Adam Zmysłowski, Katarzyna Bus

For the first time, the cis/trans isomer content of menaquinone-7 in food products has been identified and marked. A novel method of marking isomers of vitamin K2MK-7 in dietary supplements was developed and validated. Five different isomers of cis/trans vitamin K2MK-7 were identified. Identification of cis/trans isomers was performed by HRMS-QTOF, whereas their quantities were determined by using CAD and DAD detectors. In the majority of cases, the content of biologically active all trans vitamin K2MK-7 was below its declared content. The content of all trans K2MK-7 was in the range between 5.5 and 49 µg in pills. In one of the studied supplements, this vitamin was not found, regardless of the claim on the product label. The content of cis/trans isomers in certain dietary supplements exceeded the content of all trans K2MK-7 by up to 3.7 times.

Journal of Chromatography A · 2006

Application of a column selection system and DryLab software for high-performance liquid chromatography method development

Ryan M. Krisko, Kieran McLaughlin, Michael J. Koenigbauer, Craig E. Lunte

This paper describes a strategy for the development of chromatographic methods for drug candidates based upon the use of simple MS compatible mobile phases and optimization of the chromatographic selectivity through variations of the stationary phase and mobile phase pH. The strategy employs an automated column selection system and a series of HPLC columns, varying in hydrophobicity and silanol activity, in combination with DryLab software to develop chromatographic methods for the separation of mixtures of bupivacaine and its metabolites; acidic, basic, and neutral compounds; and atenolol, nitrendipine, and their degradation products.

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 · 1998

Simultaneous variation of temperature and gradient steepness for reversed-phase high-performance liquid chromatography method development: II. The use of further changes in conditions

J. W Dolan, L. R Snyder, D. L Saunders, L Van Heukelem

The preceding paper (Part I) suggests that simply optimizing temperature and gradient steepness will often provide an adequate reversed-phase HPLC separation. In some cases, however, this procedure will prove unsuccessful, and then further method-development experiments (involving change in other separation conditions) will be required. One strategy is to change a variable other than temperature or gradient steepness, followed by re-optimization of the latter two variables. The present paper examines the application of this approach with the aid of computer simulation to several samples.

Journal of Pharmaceutical and Biomedical Analysis · 2021

Computer-assisted UHPLC method development and optimization for the determination of albendazole and its related substances

Elek Ferencz, Éva-Katalin Kelemen, Mona Obreja, Emese Sipos, Sára Vida, Melinda Urkon, Zoltán-István Szabó

Computer-aided ultrahigh performance liquid chromatographic (UHPLC) method development and optimization was undertaken in order to replace an underperforming European Pharmacopoeia method for the determination of albendazole and its related substances. In the preliminary screening, a temperature-gradient time bidimensional model was chosen to aid selection of the proper stationary phase. Hereinafter temperature-gradient time-ternary composition and temperature-gradient time-pH tridimensional models were applied for the optimization of critical method parameters. The simulation and in silico robustness testing were realized using DryLab modeling software. The final method was validated for quantification of impurities and assay of the active substance according to the current ICH guidance. The validated methods were tested on a real, commercial tablet formulation. The experimental design-based and software-assisted method development proved to be a fast and reliable way of replacing a method with inadequate selectivity and long runtime with a robust UHPLC-based method, which offers baseline separation for all monitored impurities in 10 min. Results confirm that software-based chromatographic modelling can not only speed up the analytical method development process, but also improve the reliability of the developed method.

LC-GC Europe · 2001

Computer-Assisted Optimization of Reversed-Phase HPLC Isocratic Separations of Neutral Compounds

Tomasz Baczek, Roman Kaliszan, H.A. Claessens, M.A. Straten

Rational selection of optimized experimental conditions for chromatographic separation of analytes is realized nowadays by means of specialized computer programs. Two such programs, DryLab (LC Resources, Walnut Creek, California, USA) and ChromSword (Merck KCaA, Darmstadt, Germany), were compared in this research. The aim of the study was to compare the optimization of chromatographic separations of neutral compounds performed isocratically in reversed-phase high performance liquid chromatography (HPLC) systems. A detailed description and discussion of the differences in predicted and experimental chromatographic retention parameters is reported. The conclusion reached is that the two programs provide identical predictions of retention data, when predictions are based on two initial experimental chromatographic measurements. An additional option of ChromSword, employing the quantitative structure-retention relationships (QSRR), appears to provide satisfactory predictions of the separation when molecular structural data along with retention data from a single initial experimental run are used as inputs. Predictions based on molecular structure alone are not very accurate and are not likely to provide useful separation information.

Journal of Chromatography A · 2014

Comparison of liquid chromatography and supercritical fluid chromatography coupled to compact single quadrupole mass spectrometer for targeted in vitro metabolism assay

Dany Spaggiari, Florence Mehl, Vincent Desfontaine, Alexandre Grand-Guillaume Perrenoud, Szabolcs Fekete, Serge Rudaz, Davy Guillarme

The goal of this study was to evaluate the combination of powerful chromatographic methods and compact single quadrupole MS device for simple in vitro cytochrome P450 (CYP) inhibition assay, instead of more expensive triple quadrupole MS/MS detectors. For this purpose, two modern chromatographic approaches (ultra-high pressure liquid chromatography (UHPLC) and ultra-high performance supercritical fluid chromatography (UHPSFC)) were tested in combination with simple MS detector. To show the applicability for an in vitro CYP-mediated metabolism assay using the cocktail approach, a method was first developed in UHPLC–MS to separate a mixture of 8 probe substrates and 8 CYP-specific metabolites. A screening procedure was initially applied to determine the best combination of a column, an organic modifier and a mobile-phase pH, followed by fine tuning of the conditions (i.e., gradient profile, temperature and pH) using HPLC modelling software. A similar sequential method development procedure was also evaluated for UHPSFC–MS. For method development, where peak tracking is necessary, the use of single quadrupole MS was found to be extremely valuable for following the investigated analytes. Ultimately, a baseline separation of the 16 compounds was achieved in both UHPLC–MS and UHPSFC–MS with an analysis time of less than 7min. In a second series of experiments, sensitivity was evaluated, and LOQ values were between 2 and 100ng/mL in UHPLC–MS, while they ranged from 2 to 200ng/mL in UHPSFC–MS. Based on the concentrations employed for the current in vitro phase I metabolism assay, these LOQ values were appropriate for this type of application. Finally, the two analytical methods were applied to in vitro CYP-dependent metabolism testing. Two well-known phytochemical inhibitors, yohimbine and resveratrol, were investigated, and reliable conclusions were drawn from these experiments with both UHPLC–MS and UHPSFC–MS. At the end, the proposed strategy of optimized chromatography combined with simple MS device has been shown to potentially compete with the widely used combination of generic chromatography and highly selective MS/MS device for simple in vitro CYP inhibition assays. In addition, our analytical method may be easier to use in a routine environment; the instrument cost is significantly reduced and the two developed methods fit for purpose.

Journal of Pharmaceutical and Biomedical Analysis · 2016

Practical method development for the separation of monoclonal antibodies and antibody-drug-conjugate species in hydrophobic interaction chromatoraphy, part 2: Optimization of the phase system

Alessandra Cusumano, Davy Guillarme, Alain Beck, Szabolcs Fekete

The goal of this second part was (i) to evaluate the performance of commercially available HIC columns and (ii) to develop a fast and automated “phase system” (i.e. stationary phase and salt type) optimization procedure for the analytical characterization of protein biopharmaceuticals. For this purpose, various therapeutic mAbs (denosumab, palivizumab, pertuzumab, rituximab and bevacizumab) and a cysteine linked ADC (brentuximab-vedotin) were selected as model substances. Several HIC column chemistries (butyl, ether and alkylamide) from different providers were evaluated in four different buffer systems (sodium acetate, sodium chloride, ammonium acetate and ammonium sulfate). As stationary phases, the historical TSK gel Butyl NPR phase and the brand new Thermo MAbPac HIC-10 were found to be the most versatile ones in terms of hydrophobicity, peak capacity and achievable selectivity. As salt types, ammonium sulfate and sodium acetate were found to be particularly well adapted for the analytical characterization of mAbs and ADCs, but it is important to keep in mind that a concentration 2 to 3-times higher of sodium acetate versus ammonium sulfate is required to achieve a similar retention in HIC. After selection of the most appropriate phase systems, the optimization of the separation can be carried out by computer assisted retention modeling in a high throughput manner.

Journal of Chromatography A · 1996

Initial experiments in high-performance liquid chromatographic method development I. Use of a starting gradient run

L. R. Snyder, J. W. Dolan

A single gradient elution run with acetonitrile (B)-water (A) as mobile phase can be used to estimate preferred conditions for subsequent method development experiments based on reversed-phase high-performance liquid chromatography. For a broad range of sample types,that includes both very hydrophilic and hydrophobic compounds, it was found that isocratic retention is given by log k ≈ log kw −4.2ϕ, where ϕ = 0.01 %B. An initial gradient run allows values of log kw to be estimated for each compound in the sample, which then permits compound retention to be approximated as a function of either isocratic or gradient experimental conditions. The use of an initial gradient run in this way provides a rational basis for the subsequent development of a final HPLC method. Predictions based on this initial run can be used to (a) select between isocratic or gradient elution for further experiments and the final method, (b) choose a value of %B to achieve a desired value of k for the initial or final band in isocratic separation and/or (c) choose values of %B for the initial and final mobile phase in gradient elution. The present approach is based on a wide range of sample types and different reversed-phase columns; for this reason it is expected to be reasonably general and accurate. Errors in subsequent predictions based on an initial gradient run can also be estimated as a function of experimental conditions, allowing the selection of conditions for minimum error.

Journal of Chromatography A · 2015

Possibilities of retention prediction in fast gradient liquid chromatography. Part 3: Short silica monolithic columns

Pavel Jandera, Tomáš Hájek

We studied possibilities of prediction of the gradient elution data for alkylbenzenes, flavones and phenolic acids on two short octadecyl silica gel monolithic columns, namely a Chromolith Flash C18, 25×4.6mm, and a “new generation” Chromolith High Resolution C18, 50×4.6mm, in fast 1−2min gradients. With fixed short gradient times and varying gradient ranges of acetonitrile concentration in water, high flow rates of the mobile phase (3−5mL/min) could be used. The gradient elution data were predicted from four gradient models based on two-parameter and three-parameter isocratic retention equations. Various gradient retention models can be used for prediction of chromatograms and optimization of separation within a fixed gradient time. A two-parameter log−log model introduced in 1974 and a three-parameter model introduced in 1980 provided slightly more accurate prediction than the Linear Solvent Strength (LSS) semi-logarithmic two-parameter model, most frequently used in reversed-phase LC. A three-parameter model introduced in 1978 provided slightly improved accuracy of prediction of gradient data with respect to two-parameter models, in contrast to another, more recent three-parameter empirical model introduced in 2010 (which failed for gradients starting at a non-zero concentration of acetonitrile). Both a longer (5cm) and more efficient Chromolith HR column and a shorter (2.5cm) slightly less efficient Chromolith Flash column provide useful separations in fast gradients (1−2min) at high flow rates (3.5−5mL/min), especially in second dimension of two-dimensional LC×LC, in combination with HILIC separation on monolithic microcolumn in D1.

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 Pharmaceutical and Biomedical Analysis · 2013

Classification of LC columns based on the QSRR method and selectivity toward moclobemide and its metabolites

Alina Plenis, Ilona Olędzka, Tomasz Bączek

This paper focuses on a comparative study of the column classification system based on the quantitative structure-retention relationships (QSRR method) and column performance in real biomedical analysis. The assay was carried out for the LC separation of moclobemide and its metabolites in human plasma, using a set of 24 stationary phases. The QSRR models established for the studied stationary phases were compared with the column test performance results under two chemometric techniques – the principal component analysis (PCA) and the hierarchical clustering analysis (HCA). The study confirmed that the stationary phase classes found closely related by the QSRR approach yielded comparable separation for moclobemide and its metabolites. Therefore, the QSRR method could be considered supportive in the selection of a suitable column for the biomedical analysis offering the selection of similar or dissimilar columns with a relatively higher certainty.

Analytica Chimica Acta · 2025

A novel 3D-printed sample preparation method for benzodiazepine quantification in human serum

Dagmara Kroll, Szymon Ulenberg, Paweł Georgiev, Bartosz Marciniak, Gert Desmet, Tomasz Bączek, Mariusz Belka

Background Benzodiazepine abuse remains a significant public health concern. Current sample preparation methods for benzodiazepine analysis from human serum often involve complex procedures that require large sample volumes and extensive organic solvent use. To address these limitations, this study presents a novel and efficient sample preparation method utilizing 3D-printed sorbent devices. Results The 3D-printed devices, fabricated from a thermoplastic composite incorporating C18-modified silica, demonstrated exceptional performance in extracting benzodiazepines from human serum. The method was optimized and validated according to ICH guidelines, ensuring its reliability for quantitative benzodiazepine analysis. Notably, the method required minimal sample and solvent volumes, eliminating the need for protein precipitation, evaporation, and reconstitution. Significance This novel sample preparation approach offers significant advantages over traditional methods, providing a more efficient and environmentally friendly solution for benzodiazepine analysis. The versatility of 3D printing allows for the customization of sorbent devices for various analytes and matrices, expanding the potential applications of this method. Coupled with a rapid and robust LC-MS method optimized with DryLab, this approach presents a valuable and sensitive tool for benzodiazepine monitoring in clinical and toxicological settings.

Decades of chromatography research done with DryLab®.