Decades of chromatography research done with DryLab®.

Journal of Chromatography A · 2012

A general strategy for performing temperature-programming in high performance liquid chromatography—Further improvements in the accuracy of retention time predictions of segmented temperature gradients

Steffen Wiese, Thorsten Teutenberg, Torsten C. Schmidt

In the present work it is shown that the linear elution strength (LES) model which was adapted from temperature-programming gas chromatography (GC) can also be employed for systematic method development in high-temperature liquid chromatography (HT-HPLC). The ability to predict isothermal retention times based on temperature-gradient as well as isothermal input data was investigated. For a small temperature interval of ΔT=40 °C, both approaches result in very similar predictions. Average relative errors of predicted retention times of 2.7% and 1.9% were observed for simulations based on isothermal and temperature-gradient measurements, respectively. Concurrently, it was investigated whether the accuracy of retention time predictions of segmented temperature gradients can be further improved by temperature dependent calculation of the parameter ST of the LES relationship. It was found that the accuracy of retention time predictions of multi-step temperature gradients can be improved to around 1.5%, if ST was also calculated temperature dependent. The adjusted experimental design making use of four temperature-gradient measurements was applied for systematic method development of selected food additives by high-temperature liquid chromatography. Method development was performed within a temperature interval from 40°C to 180°C using water as mobile phase. Two separation methods were established where selected food additives were baseline separated. In addition, a good agreement between simulation and experiment was observed, because an average relative error of predicted retention times of complex segmented temperature gradients less than 5% was observed. Finally, a schedule of recommendations to assist the practitioner during systematic method development in high-temperature liquid chromatography was established.

Journal of Pharmaceutical and Biomedical Analysis · 2012

Analysis of recombinant monoclonal antibodies by RPLC: Toward a generic method development approach

Szabolcs Fekete, Serge Rudaz, Jenő Fekete, Davy Guillarme

Monoclonal antibodies (mAbs) are an emerging class of therapeutic agents that have recently gained importance. To attain acceptable kinetic performance with mAbs in reversed phase liquid chromatography, there is a need to work with the last generation of wide-pore sub-2μm fully porous or core–shell particles stationary phases. In addition, temperature in the range 60–90°C was found to be mandatory to limit adsorption phenomenon of mAbs and their fragments. A generic method development strategy was proposed to account for the selectivity, efficiency, recovery, and the possible thermal degradation. This study also demonstrated that the gradient steepness and temperature cannot be optimized using van’t Hoff type linear models. Similarly, the common linear solvent strength model also generated some error in predicting the retention times. In contrast, when quadratic models were employed, the prediction accuracy of retention times was found to be excellent (relative error between 0.5 and 1%) using a reasonable number of experiments (9 or 6 experiments for optimization of gradient time and temperature, which requires between 6 and 8h). Two separations of mAbs fragments were performed to demonstrate the reliability of the quadratic approach.

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.

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 Pharmaceutical Sciences · 2011

Development of Quality-By-Design Analytical Methods

Frederick G. Vogt, Alireza S. Kord

Quality-by-design (QbD) is a systematic approach to drug development, which begins with predefined objectives, and uses science and risk management approaches to gain product and process understanding and ultimately process control. The concept of QbD can be extended to analytical methods. QbD mandates the definition of a goal for the method, and emphasizes thorough evaluation and scouting of alternative methods in a systematic way to obtain optimal method performance. Candidate methods are then carefully assessed in a structured manner for risks, and are challenged to determine if robustness and ruggedness criteria are satisfied. As a result of these studies, the method performance can be understood and improved if necessary, and a control strategy can be defined to manage risk and ensure the method performs as desired when validated and deployed. In this review, the current state of analytical QbD in the industry is detailed with examples of the application of analytical QbD principles to a range of analytical methods, including high-performance liquid chromatography, Karl Fischer titration for moisture content, vibrational spectroscopy for chemical identification, quantitative color measurement, and trace analysis for genotoxic impurities.

Journal of Pharmaceutical and Biomedical Analysis · 2011

Baclofen ester and carbamate prodrug candidates: A simultaneous chromatographic assay, resolution optimized with DryLab®

Rasha Hanafi, Salwa Mosad, Khaled Abouzid, Raimund Nieß, Hilde Spahn-Langguth

Baclofen exhibits insufficient CNS-availability when dosed systemically. Hence, prodrug candidates (methyl, ethyl, 1-propyl, 2-propyl and butyl 4-(tert-butoxycarbonyl amino)-3-(4-chlorophenyl) butanoate) were synthesized aiming at CNS-levels appropriate for the treatment of spastic disorders. The characterization of some biopharmaceutically highly relevant physicochemical properties (LogP and aqueous solubility) and the evaluation of biophase levels represent one important component of the project. The overall research aim was to generate an HPLC optimized method using DryLab®, a simulation software for the optimization of a RP-HPLC method, which was optimized using a simulation software (DryLab®), for the simultaneous determination of baclofen and ten synthesized prodrug candidates. The chromatographic resolution predicted and obtained via the simulation is Rs >1.5 for all baclofen derivatives, as well as, with parent baclofen. The method was used to assay the prodrugs and determine their purities, solubility and lipophilicity parameters. The designed analytical method also permits the tracking of the new prodrug candidates’ hydrolysis in vitro and in vivo. The determined physicochemical properties indicate for some of the compounds that they might be suitable for CNS-targeting which was exemplified by the detection of significant baclofen levels in rat brain tissues following an i.p. dose of ethyl carbamate (vs. ethyl ester, for which only traces of baclofen were detected).

Journal of Pharmaceutical and Biomedical Analysis · 2011

X-ray powder diffractometry and liquid chromatography studies of sibutramine and its analogues content in herbal dietary supplements

K. Stypułkowska, A. Błażewicz, J. Maurin, K. Sarna, Z. Fijałek

The contemporary societies of the developed countries are prone to use traditional far-east medicines as remedies for all diseases. Some of them, such as obesity, might be classified as civilization diseases. Combating the problem, people try not only several miraculous diets but also herbal infusions (teas) and variety of “herbal” preparations. All these believing that such treatment is healthy and harmless as far as it is “natural”. Leaving out of the way the question if herbal medicines can be taken safely without doctors’ control the query arises if the common preparations are strictly natural and herbal. Here we report examples of quality studies of such medicines using both X-ray powder diffraction (XRPD) and liquid chromatography (LC) with various types of detection: ultraviolet (UV), coulometric electrode array (CEAD) and time-of-flight mass spectrometry (TOF-MS). Especially the XRPD assisted with an optical microscopy seems to be useful as a fast screening method of general sample composition of such preparations. First of all it can discriminate between capsules containing pure herbal materials and those with some chemical.

Analytica Chimica Acta · 2011

Combined use of algorithms for peak picking, peak tracking and retention modelling to optimize the chromatographic conditions for liquid chromatography–mass spectrometry analysis of fluocinolone acetonide and its degradation products

Mattias J. Fredriksson, Patrik Petersson, Bengt-Olof Axelsson, Dan Bylund

A strategy for rapid optimization of liquid chromatography column temperature and gradient shape is presented. The optimization as such is based on the well established retention and peak width models implemented in software like e.g. DryLab and LC simulator. The novel part of the strategy is a highly automated processing algorithm for detection and tracking of chromatographic peaks in noisy liquid chromatography–mass spectrometry (LC–MS) data. The strategy is presented and visualized by the optimization of the separation of two degradants present in ultraviolet (UV) exposed fluocinolone acetonide. It should be stressed, however, that it can be utilized for LC–MS analysis of any sample and application where several runs are conducted on the same sample. In the application presented, 30 components that were difficult or impossible to detect in the UV data could be automatically detected and tracked in the MS data by using the proposed strategy. The number of correctly tracked components was above 95%. Using the parameters from the reconstructed data sets to the model gave good agreement between predicted and observed retention times at optimal conditions. The area of the smallest tracked component was estimated to 0.08% compared to the main component, a level relevant for the characterization of impurities in the pharmaceutical industry.

The Column · 2011

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

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

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

Analytica Chimica Acta · 2011

A stepwise strategy employing automated screening and DryLab modeling for the development of robust methods for challenging high performance liquid chromatography separations: A case study

K. Jayaraman, A. J. Alexander, Y. Hu, F. P. Tomasella

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.

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.

American Laboratory · 2010

Strategy for Improving HPLC Retention Time Prediction Accuracy Using Chromatography Simulation Software

A. Makarov, Zhi Chen, F. Chan, R. Bhashyam, R. Lobrutto, R. Vegesna

The basis for Quality by Design of chromatographic methods can be broadly categorized into two areas: knowledge space and design space. The knowledge space encompasses all considerations made, all experiments conducted, and all knowledge gained in the development of a method (i.e., column screening or pH screening experiments). The knowledge space forms the basis for delineating a design space within which one can modify the chromatographic factors (i.e., gradient slope, temperature, and buffer concentration, all at a defined pH) without significantly impacting the final quality of the method and making sure all of the chromatographic figures of merit can still be met (resolution of critical pairs, tailing factor, selectivity, etc.).

Wiley · 2010

Equipment

Lloyd R. Snyder, J. J. Kirkland, John W. Dolan

LaborPraxis · 2010

Computergestützte Methodenentwicklung in der HPLC

Steffen Wiese, Thorsten Teutenberg, Torsten C. Schmidt, Hans-Jürgen Rieger, Imre Molnár

In den letzten Jahren wurde eine Reihe von Verfahren entwickelt, für die eine Temperaturgradientenelution in der Flüssigchromatographie unumgänglich ist. Lesen Sie, wie bereits existierende Retentionsmodelle angepasst werden können, um HPLC-Methoden mittels Temperaturgradientenelution unter isokratischen Bedingungen zu entwickeln.

Journal of Chromatography A · 2010

Aspects of the “Design Space” in high pressure liquid chromatography method development

I. Molnár, H. -J. Rieger, K. E. Monks

The present paper describes a multifactorial optimization of 4 critical HPLC method parameters, i.e. gradient time (tG), temperature (T), pH and ternary composition (B1:B2) based on 36 experiments. The effect of these experimental variables on critical resolution and selectivity was carried out in such a way as to systematically vary all four factors simultaneously. The basic element is a gradient time–temperature (tG–T) plane, which is repeated at three different pH's of the eluent A and 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. Further improvement of the analysis time, with conservation of the previously optimized selectivity, was possible by reducing the gradient time and increasing the flow rate. Multidimensional robust regions were successfully defined and graphically depicted.

Journal of Pharmaceutical and Biomedical Analysis · 2009

Validated UPLC method for the fast and sensitive determination of steroid residues in support of cleaning validation in formulation area

Szabolcs Fekete, Jenő Fekete, Katalin Ganzler

An ultra performance liquid chromatographic (UPLC) method was developed for simultaneous determination of seven steroid (dienogest, finasteride, gestodene, levonorgestrel, estradiol, ethinylestradiol, and norethisterone acetate) active pharmaceutical ingredient (API) residues. A new, generic method is presented, with which it is possible to verify the cleaning process of a steroid producing equipment line used for the production of various pharmaceuticals. The UPLC method was validated using an UPLC™ BEH C18 column with a particle size of 1.7μm (50mm×2.1mm) and acetonitrile–water (48:52, v/v) as mobile phase at a flow rate of 0.55ml/min. Method development and method validation for cleaning control analysis are described. The rapid UPLC method is suitable for cleaning control assays within good manufacturing practices (GMP) of the pharmaceutical industry.

Journal of Chromatography A · 2009

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

Szabolcs Fekete, Jenő Fekete, Imre Molnár, Katalin Ganzler

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

Journal of Pharmaceutical 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 Pharmaceutical and Biomedical Analysis · 2009

Optimization of separation and determination of moxifloxacin and its related substances by RP-HPLC

Predrag Djurdjevic, Andrija Ciric, Aleksandra Djurdjevic, Milena Jelikic Stankov

A RP-HPLC method for the separation and determination of impurities of moxifloxacin, in its pharmaceutical forms as well as moxifloxacin degradation products, was developed with the aid of DryLab® software and chemometric (response surface) approach. The separation of four synthesis-related impurities was achieved on a Waters C18 XTerra column using a mobile phase of (water+triethylamine (2%, v/v)): acetonitrile=90:10 (v/v%); the pH of water phase being adjusted with phosphoric acid to 6.0. Flow rate of the mobile phase was 1.5ml/min and UV detection at 290nm was employed. The column was thermostated at 45°C. The resolution between the two least resolved impurity peaks was in average, Rs,min>1.5. Method validation parameters indicate linear dynamic range 0.2–2.0μg/ml with LOQ ca. 0.20μg/ml and LOD ca. 0.05μg/ml for all analytes. The method was applied for the impurities determination in drug tablets and infusion (Avelox®, Bayer AG) and for degradation products determination in a stability study of moxifloxacin. The impurity content in the tablets and infusion was quantified as 0.1% of total drug. Two degradation products were noted under hydrolytic conditions. The method can also be used for rapid and accurate quantification of moxifloxacin hydrochloride in its tablets during stability testing.