Decades of chromatography research done with DryLab®.

SEPARATION SCIENCE PLUS · 2024

Retention modeling of therapeutic peptides in sub-/supercritical fluid chromatography

Jonas Neumann, Sebastian Schmidtsdorff, Alexander H. Schmidt, Maria K. Parr

Chromatographic modeling software packages are valuable tools during method optimization steps. These are well established for reversed-phase applications utilizing retention time (RT) prediction to optimize separations and receive robust methods, which is of high interest for the analysis of pharmaceuticals. In contrast to liquid chromatography, the knowledge of RT prediction in supercritical fluid chromatography is limited to a manageable number of studies. This study uses the software DryLab to predict the RTs of the peptides bacitracin (Bac), colistin, tyrothricin (Tyro), and insulin analogs. Gradient time, column temperature, and the ternary composition (terC) of carbon dioxide, methanol (MeOH), and acetonitrile (ACN) in the gradient elution are varied in a feasibility approach using a neutral (Viridis BEH) and an amino-derivatized aromatic (Torus 2-PIC) stationary phase. In the second part, chromatographic optimization is performed in silico through gradient adjustments to optimize the separation of the fingerprint of Bac. The final gradient method utilizes a Viridis BEH column (100 × 3.0 mm, 1.7 μm), carbon dioxide, and a modifier consisting of ACN/MeOH/water/methanesulfonic acid (60:40:2:0.1, v:v:v:v). In addition, changes in the retention order of Tyro compounds with the proportion of the terC in combination with a Torus Diol column are investigated.

Journal of Pharmaceutical and Biomedical Analysis · 2016

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

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

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

Journal of Chromatography A · 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.

Journal of Chromatography A · 2021

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

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

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

Journal of Chromatography A · 2024

Application of chiral stationary phases for the separation of vitamin A acetate isomers

Nicole Schräder, Wan Xia Zhu, Alexander Jaekel, Mo Legelli, Daniel Meyer, Kevin Streckel, Michaela Wirtz, Stefan Lamotte

The separation of vitamin A acetate isomers is essential for quality assurance of e.g. nutrition supplements, cosmetics, and pharmaceutical ingredients. High performance liquid chromatography (HPLC) is currently the most suitable analytical method for tackling this challenging separation task. However, the existing methods based on normal phase chromatography (NPC) are poorly reproducible due to the typical disadvantages of NPC, such as long equilibration times and fluctuation in retention factors. A new reversed phase method developed in our labs allows the separation of the isomers applying a chiral stationary phase (CSP). This phase consists of an immobilized polysaccharide which can be used in every chromatographic mode. However, they are not typically used in reversed phase mode. Through the screening of various stationary phases with different polysaccharide based chiral selectors, the choice of the ideal stationary phase could be confirmed, allowing to draw conclusions about the retention mechanism. The CSP Chiralpak IG-3 was found to be the most suitable among the examined. Regarding the separation mechanism, the spatial helical structure of the polysaccharide derivatives was confirmed to be of particular significance. In addition to the stationary phase, the mobile phase was tested for optimization regarding composition, gradient parameters as well as temperature using chromatographic method optimization software for the sake of method robustness.

Journal of Pharmaceutical and Biomedical Analysis · 2020

A validated UHPLC-MS method for tryptophan metabolites: Application in the diagnosis of multiple sclerosis

Ferenc Tömösi, Gábor Kecskeméti, Edina Katalin Cseh, Elza Szabó, Cecília Rajda, Róbert Kormány, Zoltán Szabó, László Vécsei, Tamás Janáky

The simultaneous quantitative estimation of tryptophan (TRP) and its metabolites represents a great challenge because of their diverse chemical properties, e.g., presence of acidic, basic, and nonpolar functional groups and their immensely different concentrations in biological matrices. A short ultra high-performance liquid chromatography (UHPLC)–tandem mass spectrometry (MS/MS) method was validated for targeted analysis of TRP and its 11 most important metabolites derived via both kynurenine (KYN) and serotonin (SERO) pathways in human serum and cerebrospinal fluid (CSF): SERO, KYN, 3-hydroxyanthranilic acid, 5-hydroxyindoleacetic acid, anthranilic acid, kynurenic acid (KYNA), 3-hydroxykynurenine (3-HK), xanthurenic acid, melatonin, picolinic acid (PICA), and quinolinic acid (QUIN). After selecting the “best” reversed-phase column and organic modifier, DryLab®4 was used to optimize the gradient time and temperature in chromatographic separation. To achieve absolute quantification, deuterium-labeled internal standards were used. Among all compounds, 3 were analyzed in derivatized (butyl ester) forms (3-HK, PICA, and QUIN) and the remaining 9 in underivatized forms. Validation was performed in accordance with the ICH and FDA guidelines to determine the intraday and interday precision, accuracy, sensitivity, and recovery. To demonstrate the applicability of the developed UHPLC–MS/MS method, the aforementioned metabolites were analyzed in serum and CSF samples from patients with multiple sclerosis (multiple sclerosis group) and those with symptomatic or noninflammatory neurological diseases (control group). The concentration of QUIN dramatically increased, whereas that of KYNA slightly decreased in the multiple sclerosis group, resulting in a significantly increased QUIN/KYNA ratio and significantly decreased PICA/QUIN ratio.

Journal of Chromatography A · 1996

Combined use of temperature and solvent strength in reversed-phase gradient elution III. Selectivity for ionizable samples as a function of sample type and pH

P. L. Zhu, John W. Dolan, Lloyd R. Snyder, D. W. Hill, L. Van Heukelem, T. J. Waeghe

The ability of temperature and gradient steepness to change band spacing has been investigated for several ionizable samples that include 8 substituted benzoic acids, 9 substituted anilines, 22 basic drugs, 9 structurally-related herbicide impurities, 7 chlorophylls and 72 peptides and proteins. Mobile phase pH was also varied to determine the effect of sample ionization on temperature and gradient-steepness selectivity.

Journal of Chromatography B: Biomedical Sciences and Applications · 1997

Changing reversed-phase high performance liquid chromatography selectivity Which variables should be tried first?

L. R. Snyder

When carrying out HPLC method development, it is often necessary to vary the relative retention of the sample (values of α) by changing some experimental variable, e.g., solvent type, pH, etc. The choice of which variable will be most suitable for a change in selectivity depends on two conflicting goals: (a) the attainment of maximum changes in α for the better control of resolution and (b) the avoidance of practical problems associated with the use of a given variable to optimize selectivity. This study provides a quantitative evaluation of different variables for their effect on selectivity (α). Various practical problems which must be balanced against this ability of a variable to change values of α are also discussed. The selection of any two variables for their simultaneous use in controlling α is also examined.

Journal of Pharmaceutical and Biomedical Analysis · 2011

Expanding the term “Design Space” in high performance liquid chromatography (I)

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

The current article presents a novel approach to applying Quality by Design (QbD) principles to the development of high pressure reversed phase liquid chromatography (HPLC) methods. Four common critical parameters in HPLC – gradient time, temperature, pH of the aqueous eluent, and stationary phase – are evaluated within the Quality by Design framework by the means of computer modeling software and a column database, to a satisfactory degree. This work proposes the establishment of two mutually complimentary Design Spaces to fully depict a chromatographic method; one Column Design Space (CDS) and one Eluent Design Space (EDS) to describe the influence of the stationary phase and of the mobile phase on the separation selectivity, respectively. The merge of both Design Spaces into one is founded on the continuous nature of the mobile phase influence on retention and the great variety of the stationary phases available.

Journal of Pharmaceutical Innovation · 2009

Application of Multidimensional Screening and Analysis and Computer Simulation Software for Rapid HPLC Method Development

Claudia C. Corredor, John A. Castoro, Joel Young

Journal of Chromatography A · 2018

Structure assisted impurity profiling for rapid method development in liquid chromatography

Sebastian Schmidtsdorff, Alexander H. Schmidt, Maria Kristina Parr

The use of trial-and-error principles is a frequently used technique in method development. This may lead to the fact that analytical methods are used routinely without developers and users having gained extensive and well-founded knowledge about the robustness of their analytical methods and the influence of critical key parameters. This very often leads to unnecessary problems for analysts. A simple way in reverse phase chromatography to simulate the effects of pH value changes on the separation and retention of substances is the pH-dependent calculation of the logD value. With this tool, model substances were used to show how the time requirement for method screening can be considerably reduced in silico and, in addition, extended knowledge about the separation mechanics can be generated. Based on this knowledge, a new method for the purity analysis of carbamazepine was developed within a very short period of time, which improves the performance of the official Ph.Eur. monograph by far. Furthermore, the extremely high robustness of the new method was demonstrated. Using the logD based approach, Quality-by-Design is applied in method development and kept pace with the increasing requirements of regulatory authorities in the pharmaceutical industry.

Journal of Chromatography A · 2020

Rapid analysis of polycyclic aromatic hydrocarbons

Justin M. Godinho, Jason Lawhorn, Barry E. Boyes

Polycyclic aromatic hydrocarbons are a continuing environmental and health concern. The analytical methods developed to analyze this class of compounds have relied on reversed phase liquid chromatography and are often on the order of tens of minutes. Reduction in analysis times through the application of sub-2 μm fully porous and superficially porous support materials can increase the throughput of these LC separations. Herein, we demonstrate similar selectivity between a fully porous 1.8 μm and a 2.7 μm superficially porous material. Separations were individually developed with in silico modeling for a given flow rate determined by the fully porous column's backpressure requirements. Since the 2.7 μm superficially porous materials inherently require less backpressure to achieve similar levels of efficiency as the 1.8 μm fully porous materials, a marked increase in throughput is possible with elevated flow rates. Good resolution for a standard 16-component sample mixture is demonstrated in a sub-minute separation.

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 · 1991

High-performance liquid chromatography retention index and detection of nitrated polycyclic aromatic hydrocarbons

Tyng-Yun Liu, Albert Robbat

Based on reversed-phase isocratic experiments and gradient optimization modeling, acetonitrile was found to provide optimum separation of nitrated polycyclic aromatic hydrocarbons (nitro-PAHs). A 31-min linear gradient between 24% and 80% acetonitrile in water at 35°C and 0.5 ml/min flow-rate was established. Nitro-PAH retention indices, I, were measured under these conditions. It was found that retention index values varied with changing column temperature and/or mobile phase compositions. Diode-array, fluorescence (FD) and chemiluminescence (CD) detection were studied for nitro-PAHs. Diode-array detection responded linearly with detection limits between 2 and 12 ng/compound injected. In addition, dual-wavelength UV absorbance ratio (A230/A254, A330/A254 and A230/A330) measurements at these wavelength pairs were reported. Fluorescence and chemiluminescence provided increased selectivity and sensitivity. Four orders of magnitude linear range were found for both detection methods with detection limits between 10 and 15 pg and 50 pg (on an NO2/compound mole basis), respectively.

Journal of Chromatography A · 2021

A methodology employing retention modeling for achieving control space in liquid chromatography method development using quality by design approach

Karthik Jayaraman, Ashok Kumar Rajendran, Gandhi Santosh Kumar, Hemant Bhutani

This study reports the application of retention modeling and quality by design practices for reverse-phase liquid chromatographic method development of a new chemical entity. Prior to the retention modeling, preliminary screening experiments were performed for the selection of stationary phase, organic modifiers, and method parameters. Based on the results of preliminary method conditions, tG-T (gradient time - temperature) 2-D modeling with 4 input runs, and tG-T-tc (gradient time-temperature-ternary composition) 3-D modeling with 12 input runs were designed to build a model for achieving the optimized separation. Modeling of reverse phase separations was based on the measurement of both retention times and peak areas. A design space with appropriate input variables and control strategy was established prior to optimization and robustness evaluation following the quality by design framework. DryLabⓇ was used to predict the optimized gradient profile and separation temperature. The robustness evaluation was carried out using the multiple factors at a time approach and the control space was established. The interdependence of control space and the control strategy was demonstrated by evaluating method robustness using two levels of system suitability criteria. The predictive accuracy of the retention modeling was established through experimental verification of the in-silico predictions. The quality by design based method development approach demonstrated the in-silico optimization as an integral component of reverse-phase chromatographic method development to evaluate the interplay of factors such as organic modifiers, separation temperature and gradient time, which greatly integrated and enhanced method robustness during method development.

Journal of Chromatography · 1989

Predicting reversed-phase gradient elution separations by computer simulation: A comparison of two different programs

Jack Schmidt

Two computer programs for developing and improving high-performance liquid chromatographic methods, DryLab G and LCSIM, have recently been described. The accuracies of these two programs were examined using experimental (o-phthalaldehyde-derivatized amino acids) and synthetic data. DryLab G, which uses gradient data for input, correctly predicted retention times for various gradient and isocratic separations. Predicted retention times for the simulation of certain isocratic conditions are susceptible to errors in the measured dwell volume, but the prediction of resolution is not seriously affected. LCSIM uses isocratic data for input, and predicted gradient retention times are affected by the accuracy of the measured dwell volume. The resolution of closely eluting analytes was usually predicted within a small fraction of the peak width, i.e., with negligible errors. Examples are given for some unique features of the LCSIM program: solvent switching and mixing, optional descending gradients, graphics display of the separation process and an iterative fit of the separation parameters to the retention characteristics of a new column (as measured from a single run).

Journal of Chromatography A · 1996

Combined use of temperature and solvent strength in reversed-phase gradient elution IV. Selectivity for neutral (non-ionized) samples as a function of sample type and other separation conditions

P. L. Zhu, John W. Dolan, Lloyd R. Snyder, N. M. Djordjevic, D. W. Hill, J. -T. Lin, L. C. Sander, L. Van Heukelem

The separation of nine un-ionized samples was studied as a function of temperature (T) and gradient steepness (b). Selectivity values Δlog α∗ were obtained for 160 compounds, ranging from nonpolar hydrocarbons to very polar drugs. Selectivity varied markedly with sample type: nonpolar compounds such as aromatic hydrocarbons and fatty acid methyl esters generally showed only modest changes in band spacing as temperature or gradient steepness was varied. More polar samples exhibited larger changes in α (Δlog α∗) when temperature and/or gradient steepness wee changed, but the largest values of Δlog α∗ for these non-ionized samples are less than the average value of Δlog α∗ for the ionized samples of Part III [1]. Poly-functional silane (“polymeric”) columns exhibit slightly increased b- and/or T-selectivity for some samples.

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.

Open Chemistry · 2012

Determination of lipophilicity for antitumor acridinone derivatives supported by gradient high-performance liquid chromatography method

Marcin Koba, Mariusz Belka, Tomasz Ciesielski, Tomasz Bączek

The lipophilicity values of selected acridinone (imidazoacridinone and triazoloacridinone) derivatives were measured by gradient reversed-phase high-performance liquid chromatography (RP-HPLC) using a C18 stationary phase with a water/acetonitrile mixture as a mobile phase. The retention times obtained served as input data and appropriate log kw values (i.e., the retention factor log kw extrapolated to 0% organic modifier) as an alternative to log P were calculated using the DryLab program. The relationships between the lipophilicity (log kw) and the chemical structure of the studied compounds, as well as correlation between experimentally determined lipophilicities (log kw) and log P data calculated using some commonly available software, are discussed.

Liquid Chromatography · 2017

Chapter 1 - Milestones in the development of liquid chromatography

Lloyd R. Snyder, John W. Dolan, Salvatore Fanali, Paul R. Haddad, Colin F. Poole, Marja-Liisa Riekkola

The evolution of high-performance liquid chromatography is reviewed with an emphasis on the innovations that occurred in the technique in response to the sample needs. The general themes are the development and/or applications of the basic theory as catalyst for change, invention of new chromatographic modes, evolution of column technology, and the development and improvement of instrumentation. Steady progress in these areas rather than sudden change is responsible for the column chemistries, particle technology, instrumentation, and data handling tools available today.