Decades of chromatography research done with DryLab®.

Chemia Analityczna · 1996

Computer Optimization for RP-HPLC Separation of Some Nucleosides

T. H. Dzido, A. Sory

The DrylabG software (LC Resources, Lafayette, CA, USA) was applied for optimization of HPLC resolution of some nucleosides in the reversed-phase systems. Two preliminary runs based on linear gradient range of acetonitrile (ACN) from 0% (pure buffer) to 20% and of methanol (MeOH) from 0% to 50% are shown to be satisfactory for optimization of the resolution. A good agreement between simulated and experimental chromatograms was observed.

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.

Journal of Chromatography B: Biomedical Sciences and Applications · 1997

Computerized optimization of the high-performance liquid chromatographic enantioseparation of a mixture of 4-dinitrophenyl amino acids on a quinine carbamate-type chiral stationary phase using drylab

Michael Lämmerhofer, Paolo Di Eugenio, Imre Molnár, Wolfgang Lindner

A method is proposed for the sensitive chiral analysis of amino acid enantiomers by high-performance liquid chromatography (HPLC). Thus the enantiomers of a mixture of seven racemic amino acids were resolved as their DNP derivatives from each other and from the peak of the hydrolyzed reagent, employing a quinine carbamate-based chiral anion exchange-type chiral stationary phase (CSP) and aqueous buffered mobile phases. However, the initial isocratic chromatogram yielded many peak overlaps although the corresponding enantiomers were well resolved. Therefore, the separation of the complex mixture had to be optimized; we utilized the commercial computer method development software drylab. First, the influence of the manifold mobile phase parameters and chromatographic conditions (pH, type and content of organic modifier, buffer concentration, temperature, type of co-ion, etc.) on retention and resolution was studied by isocratic elution. Furthermore, with such optimized conditions linear and multi-segmented organic modifier and buffer salt gradients, respectively, were simulated with the computer program and experimentally verified. Average errors of prediction of retention times lay between 2 and 8%. Finally, a highly improved HPLC gradient method resulted in almost all components being baseline separated and equally spaced and accelerated by a factor of more than 3 compared to the initial run.

Journal of Pharmaceutical and Biomedical Analysis · 2020

Updating the European Pharmacopoeia impurity profiling method for terazosin and suggesting alternative columns

Dániel Enesei, Imre Kapui, Szabolcs Fekete, Róbert Kormány

This work was motivated by the demand of European Directorate for the Quality of Medicines and HealthCare (EDQM). A new liquid chromatographic (LC) method was developed for terazosin impurity profiling to replace the old European Pharmacopoeia (Ph. Eur.) method. This new method is published as part of the new Ph. Eur. monograph proposal of terazosin in Pharmeuropa issue 32.2. The aim of the method renewal was to cut the analysis time from 90 min (2 × 45 min) down to below 20 min. The Ph. Eur. monograph method is based on two different chromatographic separations to analyze the specified impurities of terazosin. The reason for the two methods is that two of the impurities are not sufficiently retained in reversed phase (RP) conditions, not even with 100% water as eluent. Therefore, next to RP, an ion-pair (IP) chromatographic method has to be applied to analyze those two impurities. With our new proposed method it was possible to appropriately increase the retention of the two critical compounds using alternative stationary phases (instead of a C18 phase which is suggested by the Ph. Eur. method). Applying a pentafluoro-phenyl (PFP) stationary phase, it was feasible to separate and adequately retain all the impurities. The detection wavelength was also changed compared to the Ph. Eur. method and is now appropriate for the detection and quantification of all impurities using perchloric acid in the mobile phase at low pH. Another goal of the present study was to develop a generic workflow and to evaluate the chromatographic resolution in a wide range of method variables and suggest some replacement columns for terazosin impurity profiling. Retention modeling was applied to study the chromatographic behavior of the compounds of interest and visualize resolution for the different columns, where a given criterion is fulfilled. A zone (set of chromatographic conditions) of a robust space could be then quickly identified by the overlay of the individual response surfaces (resolution maps). It was also demonstrated that two columns from different providers (Kinetex F5 and SpeedCore PFP) can be used as replacement columns, providing sufficient resolution at the same working point and a high degree of robustness.

Chromatographia · 2011

From Csaba Horváth to Quality by Design: Visualizing Design Space in Selectivity Exploration of HPLC Separations

Imre Molnár, K.E. Monks

The present paper starts by taking a look back at some of the pioneering work in high pressure liquid chromatography (HPLC) that went on in Csaba Horváth’s laboratory in 1970s, through the eyes of I. Molnár. It then goes on to describe a very modern approach to HPLC method development within the Quality by Design framework: the multifactorial optimization of three critical HPLC method parameters, i.e. gradient time (tG), temperature (T), and ternary composition (B1:B2) based on 12 experiments. 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.

Journal of Chromatography A · 2020

Simultaneous determination of loading capacity and selectivity in preparative off-line two-dimensional separation: An application for purification of corilagin from Pomegranate flower extracts

Guliqire Adili, Guangying Sun, Munire Abuduaini, Yongxin Zhao, Rahima Abdulla, Haji Akber Aisa

This work describes the development of capacity orthogonal chromatography (COC), a new technique for simultaneously determining the loading capacity and orthogonality during the construction of two-dimensional (2D) separations. Three steps were required for the construction of a COC based on the correlation between the selectivity factor (α) and both orthogonality and loading capacity. (1) α values of the impurities-target compound were used to normalize the retention of the impurities around the target compound. (2) α values were input into four quadrants of a coordinate system to identify correlations between orthogonality and loading capacity. α values of the impurities must be greater in the first dimension than the second dimension, with iterated analyses performed until an αmax is obtained for the two purification methods. (3) Touch-peak separation using the first-dimensional αmax was performed and the target compound was collected. The co-eluted impurities are further separated in the second dimension. To test the efficiency of this technique, a COC using two methods on a standard C18 column was developed to purify corilagin from pomegranate flower extract. Despite its low abundance, 288 mg of corilagin was obtained by COC and further purified by LH-20 gel chromatography to obtain the compound with an 80.0% recovery and 98.4% purity. Compared to COC, the purity of corilagin independently obtained using the same purification methods and identical loading capacity was poor (60.1% and 61.6%). These results indicate that COC is a useful tool for extending loading capacity in the development of preparative 2D separations.

Journal of Pharmaceutical and Biomedical Analysis · 2014

Importance of instrumentation for fast liquid chromatography in pharmaceutical analysis

Szabolcs Fekete, Isabelle Kohler, Serge Rudaz, Davy Guillarme

In the last decade, an important technical evolution has occurred in pharmaceutical analysis with numerous innovative supports and advanced instruments that have been proposed to achieve fast or ultra-fast separations in LC with an excellent sensitivity and ease of operation. Among the proposed strategies to increase the throughput, the use of short narrow-bore columns packed with sub-3μm core–shell and porous sub-2μm particles have emerged as the gold standards. Nevertheless, to take the full benefits of these modern supports, a suitable chromatographic system has to be employed. This review summarizes the instrumental needs and challenges in terms of extra-column variance, dwell volume, maximum system pressure, detector data acquisition rate, and injection cycle time. In addition, because of their reasonable pressure drop, the use of columns packed with sub-3μm core–shell particles on a conventional LC instrument is discussed in detail. A methodology is proposed to check the compatibility between stationary phase and instrument, and some solutions are proposed to improve the performance of standard instruments. Finally, because the column technology is evolving faster than instrumentation, it is nowadays possible to purchase short, narrow-bore columns packed with 1.3μm core–shell particles. Micro columns (1mm I.D.) packed with 1.7–1.9μm porous particles are also available from several providers, which limit frictional heating effects and reduce solvent and sample consumption. However, it remains difficult to find instruments compatible with such column geometries and a severe loss of performance may be observed due to the system itself.

Journal of Pharmaceutical and Biomedical Analysis · 2016

Practical method development for the separation of monoclonal antibodies and antibody-drug-conjugate species in hydrophobic interaction chromatography, part 1: optimization of the mobile phase

Marta Rodriguez-Aller, Davy Guillarme, Alain Beck, Szabolcs Fekete

The goal of this work is to provide some recommendations for method development in HIC using monoclonal antibodies (mAbs) and antibody-drug conjugates (ADCs) as model drug candidates. The effects of gradient steepness, mobile phase pH, salt concentration and type, as well as organic modifier were evaluated for tuning selectivity and retention in HIC. Except the nature of the stationary phase, which was not discussed in this study, the most important parameter for modifying selectivity was the gradient steepness. The addition of organic solvent (up to 15% isopropanol) in the mobile phase was also found to be useful for mAbs analysis, since it could provide some changes in elution order, in some cases. On the contrary, isopropanol was not beneficial with ADCs, since the most hydrophobic DAR species (DAR6 and DAR8) cannot be eluted from the stationary phase under these conditions. This study also illustrates the possibility to perform HIC method development using optimization software, such as Drylab. The optimum conditions suggested by the software were tested using therapeutic mAbs and commercial cysteine linked ADC (brentuximab-vedotin) and the average retention time errors between predicted and experimental retention times were ∼1%.

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.

Journal of Pharmaceutical and Biomedical Analysis · 2013

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

Alexander H. Schmidt, Imre Molnár

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

Analytica Chimica Acta · 2021

Enantioselective multiple heart cutting online two-dimensional liquid chromatography-mass spectrometry of all proteinogenic amino acids with second dimension chiral separations in one-minute time scales on a chiral tandem column

Ryan Karongo, Min Ge, Christian Geibel, Jeannie Horak, Michael Lämmerhofer

In this work, we present a unique, robust and fully automated analytical platform technology for the enantioselective amino acid analysis using a multiple heart cutting RPLC-enantio/stereoselective HPLC-ESI-QTOF-MS method. This 2D-LC method allows the full enantioselective separation of 20 proteinogenic AAs plus 5 isobaric analogues, namely allo-Threonine (aThr), homoserine (Hse), allo-isoleucine (aIle), tert-Leucine (Tle) and Norleucine (Nle), after pre-column derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC; AccQ). This N-terminal AA-derivatization method introduces on the one hand beneficial chromatographic properties for 1D RP-LC (stronger retention) and 2D chiral separation (better chiral recognition), and on the other hand favorable detection properties with its chromophoric, fluorophoric, and easily ionizable quinoline mass tag. The entire separation occurs within a total 2DLC run time of 45 min, which includes the 1D-RP run and the 68 s 2D chiral separations of 30 heart-cuts (from the 1D-RP-run) on a chiral quinine carbamate (core-shell QNAX/fully porous ZWIX) tandem column. This relatively short overall run time was only possible by utilizing the highly efficient “smart peak parking” algorithm for the heart cuts and the resulting optimized analysis order thereof. 1D retention time precisions of <0.21% RSD were a requirement for the time-based sampling mode and finally led to a robust, fully automated enantioselective amino acid analysis platform. This achiral-chiral 2DLC method was applied for the amino acid stereoconfiguration assignment of three peptides (aureobasidin A, a lipopeptide research sample, and octreotide) using an L-[u-13C15N] labelled internal AA standard mix spiked to each sample. The isotopically labelled L-AA standard allowed an easy and straightforward identification and configuration assignment, as well as the relative quantification of amino acids within the investigated peptides, allowing the direct determination of the number of respective amino acids and their chirality within a peptide.

Journal of Chromatography · 1989

Peak tracking in high-performance liquid chromatography based on normalized band areas: A ribosomal protein sample as an example

I. Molnár, R. Boysen, P. Jekow

Complex chromatograms that result from reversed-pahse gradient elution often exhibit changes in band order when the gradient steepness is changed. This complicates the interpretation of the resulting separation, and prevents the application of computer simulation for method development. A simple procedure based on normalized band areas was used to match bands between runs where the gradient steepness has been changed. In one example involving a Thermus aquaticus ribosomal protein sample, it was possible to find an additional band that was not apparent in tw initial experimental runs with different gradient slopes.

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).

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.

Liquid Chromatography (Second Edition) · 2017

Chapter 14 - Method development in liquid chromatography

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

A general approach to method development based on the concept of quality by design is described. The workflow includes problem definition, mode selection, selectivity optimization, system optimization, and method validation. Computer-assisted methods can support some of these decision steps minimizing the time required to build a robust method. The approach used is generally applicable, but practical illustrations will largely be restricted to isocratic and gradient elution methods in reversed-phase liquid chromatography. Critical parameters are identified as column selection, identification of the mobile-phase composition, gradient time, temperature, pH (for ionizable compounds), and buffer concentration. We focus on how to select these variables and their influence on the quality criteria for defining method suitability.

Journal of Chromatography A · 2020

Improving selectivity and performing online on-column fractioning in liquid chromatography for the separation of therapeutic biopharmaceutical products

Szabolcs Fekete, Harald Ritchie, Jason Lawhorn, Jean-Luc Veuthey, Davy Guillarme

A novel column-coupling approach is suggested to improve both the selectivity and efficiency of protein separations in liquid chromatography. Protein separations often suffer from limited selectivity or not appropriate resolving power. For a new biopharmaceutical product, the identification of the main and minor variant species is required. For that purpose, often offline collection fractioning is applied which is time consuming and regularly dilute the samples to an unacceptable extent. By serially coupling columns in the order of their increasing retentivity and applying “multi-isocratic” elution mode, indeed any (arbitrary) selectivity can be attained. Moreover, if a protein peak is trapped at the inlet of a later column segment – of a coupled system -, its band will be refocused and elute in unprecedented sharp peak. Furthermore, it becomes possible to perform online on-column fractioning of protein species within a very short analysis time (∼ 1 min) and without sample dilution. Two-, three- or multiple column systems can be developed and applied for complex sample separations (such as antibody mixtures). This new methodology can be particularly useful to improve the analysis (and therefore, safety and quality) of therapeutic mAbs and related products and offers benefits compared to offline fractionating. It is also demonstrated in this proof of concept study, that methyl (C1) modified RP phase has a great potential for protein separations despite it is not commercially available in state-of-the-art wide pore superficially porous particle format..

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 · 2017

A workflow for column interchangeability in liquid chromatography using modeling software and quality-by-design principles

Róbert Kormány, Katalin Tamás, Davy Guillarme, Szabolcs Fekete

The goal of the present study was to develop a generic workflow to evaluate the chromatographic resolution in a large design space and easily find some replacement column for the method. To attain this objective from a limited number of initial experiments, modern LC modeling software (Drylab) was employed to study the behaviour of the compounds and visually compare the parts of design spaces obtained with different columns, where a given criterion of critical resolution is fullfilled. A zone of robust space can then easily be found by overlapping design spaces. By using 50×2.1mm columns packed with sub–2μm fully porous particles (UHPLC), the resolution in the entire design space can be modeled on the basis of only 2–3h experimental work per column. To demonstrate the applicability of the developed procedure, amlodipine and its related pharmacopeia impurities were selected as a case study. It was demonstrated that two columns from different providers (Waters Acquity HSS C18, Thermo Hypersil Gold C18) can be interchanged, providing a sufficient resolution at the same working point and a high degree of robustness around this condition.

John Wiley · 2007

High-performance gradient elution: the practical application of the linear-solvent-strength model

Lloyd R. Snyder, John W. Dolan

Journal of Pharmaceutical and Biomedical Analysis · 2014

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

Alexander H. Schmidt, Mijo Stanic, Imre Molnár

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