Highlights:

• Software assisted, automated approach was used for LC method development
• Ultra-short protein columns enabled 1–2 min analysis time
• Complete method development for four chromatographic modes required only 2 days
• Conventional chromatographic techniques can quickly be transferred to short columns

Highlights

• Computer-aided chromatographic method development for the separation of ezetimibe and its related substances
• Design space comparison - new approach for testing column interchangeability
• Mechanistic retention modeling and a 3D experimental design based approach via DryLab® software
• Baseline separation of the analytes achieved on nine different stationary phases with various chemistries
• Multivariate in silico robustness testing performed to identify the critical method parameters and to set up control strategy for routine applications
• General specification with suitable working point yielding similar results on six stationary phases
• Interchangeability of the columns proved in early phase method development

Highlights

• Therapeutic protein complexes separated at superheated conditions.
• Complex dissociation is complete at superheated conditions.
• On-column degradation can be limited by fast gradients and ultrashort columns.

Highlights

• New stationary phases with low hydrophobicity were tested for protein separations
• The new ES-LH stationary phase showed excellent protein recovery
• The ES-LH and C1 materials were found to be less retentive than commercial phases
• The ES-LH phase showed novel selectivity due to its unique surface chemistry

In-process control (IPC) is an important task during chemical syntheses in pharmaceutical industry. Despite the fact that each chemical reaction is unique, the most common analytical technique used for IPC analysis is high performance liquid chromatography (HPLC). Today, the so-called “Quality by Design” (QbD) principle is often being applied rather than “Trial and Error” approach for HPLC method development. The QbD approach requires only for a very few experimental measurements to find the appropriate stationary phase and optimal chromatographic conditions such as the composition of mobile phase, gradient steepness or time (tG), temperature (T), and mobile phase pH. In this study, the applicability of a multifactorial liquid chromatographic optimization software was studied in an extended knowledge space. Using state-of-the-art ultra-high performance liquid chromatography (UHPLC), the analysis time can significantly be shortened. By using UHPLC, it is possible to analyse the composition of the reaction mixture within few minutes. In this work, a mixture of route of synthesis of apixaban was analysed on short narrow bore column (50 × 2.1 mm, packed with sub-2 µm particles) resulting in short analysis time. The aim of the study was to cover a relatively narrow range of method parameters (tG
, T, pH) in order to find a robust working point (zone). The results of the virtual (modeled) robustness testing were systematically compared to experimental measurements and Design of Experiments (DoE) based predictions.

Highlights:

• Quality-by-design based liquid chromatographic method for new chemical entity
• Exploration of design space using 2D and 3D chromatographic retention modeling
• Establishment of a methodology for achieving analytical method control space
• Demonstration of dependence of method control space on control strategy
• Use of DryLab^® to evaluate method robustness with acceptable accuracy

Highlights

In the present work, we describe the fundamental and practical advantages of a new strategy to improve the resolution of very closely eluting peaks within therapeutic protein samples.

This approach involves the use of multiple isocratic steps, together with the addition of a steep negative gradient segment (with a decrease in mobile phase strength) to "park" a slightly more retained peak somewhere along the column (at a given migration distance), while a slightly less retained compound can be eluted.

First, some model calculations were performed to highlight the potential of this innovative approach. For this purpose, the retention parameters (logk₀ and S) for two case studies were considered, namely the analysis of a mixture of two therapeutic mAbs (simple to resolve sample) and separation of a therapeutic mAb from its main variant (challenging to resolve sample). The results confirm that the insertion of a negative segment into a multi-isocratic elution program can be a good tool to improve selectivity between critical peak pairs. However, it is also important to keep in mind that this approach only works with large solutes, which more or less follow an "on-off" type elution behavior.

Two real applications were successfully developed to illustrate the practical advantage of this new approach, including the separation of a therapeutic mAb from its main variant possessing very close elution behavior, and the separation of a carrier protein from an intact mAb as might be encountered in a quantitative bioanalysis assay. These two examples demonstrate that improved selectivity can be achieved for protein RPLC through the inclusion of a negative gradient slope that selectively bifurcates the elution of two or more peaks of interest.

Highlights:

• Phorbas amaranthus extract as natural products library
• DryLab® software for Design of Experiments in association with scouting systems
• Sixty-four metabolites chemically characterized by LC-HRMS
• New metabolites inferred based on Global Natural Product Social Molecular Networking (GNPS)

Highlights:

• • New superficially porous material for analyzing polycyclic aromatic hydrocarbons.
  • In silico modeling aided development of material use conditions.
  • Comparison between superficially porous and fully porous materials.
  • Good resolution for robust sub-minute separation of 16-component mix.

Highlights:

• DryLab Assisted modeling of secondary method factors.
• Timely and accurate analytics needed for recombinant protein production.
• Chromatographic modeling accelerated method development using an AQbD approach.
• Robustness of Method Operable Design Region tested in-silico and experimentally.
• Developed RPLC-Method is superior to current analytical techniques.

Highlights:

• A new (U)HPLC method was developed for terazosin impurity profiling with DryLab to update the Ph. Eur. Method.
• A generic workflow has been proposed to suggest replacement columns for a given separation.
• Column comparison was based on the visualization and overlay of design spaces.
• The new method was validated in accordance with international guidelines.

This study reports the use of retention modeling software for the successful method development of 24 injectable antineoplastic agents. Firstly, a generic screening of several stationary and mobile phases (using various organic modifiers and pH) was achieved. Then, an optimization procedure of mobile phase temperature, gradient profile and mobile phase binary composition was conducted through only 28 real experiments using retention modeling software for data treatment. Finally, the optimized separation was achieved with a mobile phase consisting in 10 mM acetic acid at pH 5.1 (A) and acetonitrile (B). A Waters CORTECS^® T3 column (100 × 2.1 mm, 1.6 μm) operated at 25 °C with a gradient time of 17.5 min (0-51%B) at a flow rate of 0.4 mL/min was used. The prediction offered by the retention model was found to be highly reliable, with an average error lower than 1%. A robustness testing step was also assessed from a virtual experimental design. Success rate and regression coefficient were evaluated without the need to perform any real experiment. The developed LC-MS method was successfully applied to the analysis of pharmaceutical formulations and wiping samples from working environment.

Highlights:

• ADC consists of a recombinant mAb covalently linked to a cytotoxic molecule.
• Hydrophobic interaction chromatography represents the gold standard for ADC analysis.
• New site-specific ADC formats are more homogeneous than their previous generations.
• RPLC is proposed as alternative method to HIC for site-specific ADC characterization.
• Retention and resolution modeling was performed with DryLab®4 software.

Highlights:

• Alternative buffer systems are suggested for CEX separation of mAbs.
• Benefits of salt mediated pH gradient are discussed.
• The combination of a “true” pH- and salt gradients extends the possibilities of method development.
• Retention modeling and method optimization were performed by DryLab®4
• Complete method development is feasible within 6 h.

Highlights:

• Alternative to gas chromatographic methods for fatty acid identification developed.
• DoE optimized methods for peak separation of fatty acids and 18:1 isomers.
• The DoE software Drylab was used to calculate optimized and robust methods for peak separation with a full factorial design.
• Separation of cis/trans and structural fatty acid isomers using derivatization.
• Indirect polysorbate 80 quantification (0.05–5.83 μg/mL) achieved using oleic acid.

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 (t_G), ternary eluent composition (t_C) 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).

Highlights:

• Proper correlation between logD and relative retention in LC.
• logD calculation is a good basis for starting method screening.
• Drylab software modeling tool to comply analytical QbD requirements in regards to lifecycle principles.
• In silico generation of extended knowledge space possible.
• Extreme high robustness of resulting HPLC method with improved reliability and understanding.

A generic liquid chromatographic method development workflow was developed and successfully applied to the analysis of phytocannabinoids and Cannabis sativaextracts. 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.

Drug impurity and degradation profiling mean the detection, structure elucidation and quantitative determination of impurities and degradation products in bulk drug materials and pharmaceutical formulations. This is today one of the most important fields of activities in pharmaceutical analysis. The reason for this is that unidentified, potentially toxicimpurities are health hazards, and in order to increase the safety of drug therapy, impurities should be identified and determined by selective methods.

The aim of this review is to characterise the state-of-art in the field of impurity and degradation profiling of drugs based on papers published in the last decade. The separation and determination of impurities and degradants with a known structure are discussed, but emphasis is placed on the structure elucidation and determination of new (unknown) impurities and degradation products by off-line and on-line chromatographic-spectroscopic methods. The analytical aspects of enantiomeric purity of chiral drugs are also discussed.

Highlights:

• A wide-pore silica-based SPP material with a high coverage phenyl bonding was evaluated.
• Kinetic performance, recovery and selectivity were systematically studied.
• Optimal conditions were determined using Drylab 4 software.
• The possibility to replace TFA with FA was studied.
• The new column was applied for the separation of mAb sub-units and ADC species.

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

Hydrophilic interaction liquid chromatography (HILIC) is a well-established technique for the separation and analysis of small polar compounds. A recently introduced widepore stationary phase expanded HILIC applications to larger molecules, such as therapeutic proteins. In this paper, we present some generic HILIC conditions adapted for a wide range of FDA and EMA approved recombinant monoclonal antibody (mAb) species and for an antibody-drug conjugate (ADC). Seven approved mAbs possessing various isoelectric point (pI) and hydrophobicity as well as a cysteine conjugated ADC were used in this study. Samples were digested by IdeS enzyme and digests were further fragmented by chemical reduction. The resulting fragments were separated by HILIC. The main benefit of HILIC was the separation of polar variants (glycovariants) in a reasonable analysis time at the protein level, which is not feasible with other chromatographic modes. Three samples were selected and chromatographic conditions were further optimized to maximize resolution. A commercial software was used to build up retention models. Experimental and predicted chromatograms showed good agreement and the average error of retention time prediction was less than 2%. Recovery of various species and sample stability under the applied conditions were also discussed.

Highlights:

• We developed UHPLC method strategy for investigation of column interchangeability using modeling software.
• The selected four stationary phases have some obvious differences in terms of surface area, coverage, carbon load and endcapping, but three of them could provide baseline separation in the same design space.
• At the end, by using DryLab LC modeling software, it was found that two of the four columns share the same working point and are robust around this condition.
• The predicted results were in good agreement with the experimental ones.

Abstract

In this era of competition quality has been given prime magnitude; failure to meet such quality allied goals produces massive shift of company in share of market. In this context pharmaceutical industry is utmost regulated industry as it is governed by authoritative regulatory bodies. “Quality could be planned and most of quality deficit arises in the way process is planned and developed”, this thought of well known quality expert Joseph Moses Juran gives foundation to the concept of quality by design (QbD). USFDA launched a pilot programme in 2005 to permit participating firms a prospect to submit chemistry, manufacturing, and controls (CMC) of NDA information representing application of QbD. Now USFDA is accelerating QbD drive by making warning to generic manufacturers from January 2013. QbD has its perspectives to contribute the drug design, development, and manufacture of high-quality drug products. In the present review basic consideration of the QbD approach, its historical background, and regulatory needs are discussed. In detail explanation of elements of QbD i.e. method intent, design of experiment, and risk assessment is given. Application of QbD to pharmaceutical and biopharmaceutical processes, development and unit operation associated with it are briefly mentioned. Detail account of QbD to analytical technique is explained thoroughly by referencing examples. Commerially available chromatography optimization software, DryLab was used to perform computer simulations.

Highlights:

• New trends in chromatographic method development.
• Recent developments in chromatographic screening, modeling and knowledge management.
• Evolution of strategies for pharmaceutical method development in SFC and HPLC.
• DryLab was the first software for the modeling and simulation of reversed phase HPLC separations.
• The future outlook for improved method development algorithms and intelligent systems.

The aim of this article is to illustrate the current status of computer-assisted method development and retention modelling. This study focuses on the successful method development of typical small pharmaceutical compounds (impurity profiling) and large therapeutic proteins. By choosing appropriate initial conditions, the method development can be performed in less than one day. However, for small molecules possessing different physicochemical properties, the conditions can be multifarious, while for biopharmaceuticals (for example, monoclonal antibodies [mAbs], antibody–drug conjugates [ADCs]), a generic method can easily be developed. In addition to retention modelling and optimization, the potential of simulated robustness testing is also demonstrated. Depending on the applied retention model, the impact of any change among six experimental parameters (t_G, T, pH, ternary composition, flow rate, and initial- and final mobile phase compositions) on the separation can be assessed using a 2⁶ or 3⁶ type virtual factorial design. No additional experiments are required to perform the robustness evaluation. Finally, virtual method transfer between different chromatographic systems is demonstrated.

This study reports the use of modelling software for the successful method development of IgG1 cysteine conjugated antibody drug conjugate (ADC) in RPLC. The goal of such a method is to be able to calculate the average drug to antibody ratio (DAR) of and ADC product. A generic method development strategy was proposed including the optimization of mobile phase temperature, gradient profile and mobile phase ternary composition. For the first time, a 3D retention modelling was presented for large therapeutic protein. Based on a limited number of preliminary experiments, a fast and efficient separation of the DAR species of a commercial ADC sample, namely brentuximab vedotin, was achieved. The prediction offered by the retention model was found to be highly reliable, with an average error of retention time prediction always lower than 0.5% using a 2D or 3D retention models. For routine purpose, four to six initial experiments were required to build the 2D retention models, while 12 experiments were recommended to create the 3D model. At the end, RPLC can therefore be considered as a good method for estimating the average DAR of an ADC, based on the observed peak area ratios of RPLC chromatogram of the reduced ADC sample.

HPLC method modeling is becoming a powerful tool to be used in the communication about method quality in HPLC between different labs, different companies, and between companies and regulatory agencies. The understanding of simple rules of peak movements will facilitate the development of new drugs, which are badly needed for smaller patient populations. The new features of HPLC modeling software, such as 3D resolution map, the modeled robustness testing, a practicable method transfer, or a method knowledge management offer a closed loop of all information about the birth and practical use of a method, and it further suggests the use of such software solutions in regulated laboratories to make analyst's life easier-especially in the pharmaceutical industry.

Modeling of HPLC methods using QbD principles in HPLC.

• Emphasizes the integration of chromatographic methods and sample preparation.
• Explains how liquid chromatography is used in different industrial sectors.
• Covers the most interesting and valuable applications in different fields, e.g., proteomic, metabolomics, foodomics, pollutants and contaminants, and drug analysis (forensic, toxicological, pharmaceutical, biomedical).
• Includes references and tables with commonly used data to facilitate research, practical work, comparison of results, and decision-making.

In the pharmaceutical industry, the analysis of atropisomers is of considerable interest from a scientific and regulatory perspective. The compound of interest contains two stereogenic axes due to the hindered rotation around the single bonds connecting the aryl groups, which results in four potential configurational isomers (atropisomers). The separation of the four atropisomers is achieved on a derivatized β-cyclodextrin bonded stationary phase. Further investigation shows that low temperature conditions, including sample preparation (−70 °C), sample storage (−70 °C), and chromatographic separation (6 °C), were critical to preventing interconversion. LC-UV-Laser Polarimetric analysis identified peak 1/2 as a pair of enantiomers and peak 3/4 as another. Thermodynamic analysis of the retention data indicated that the separation of the pairs of enantiomers is primarily enthalpy controlled as indicated by the positive slope of the van’t Huff plot. The difference in absolute Δ (Δ H), ranged from 2.20 kJ/mol to 2.42 kJ/mol.

This review summarizes the use of computer assisted liquid chromatographic method development for the analytical characterization of protein biopharmaceuticals. Several modes of chromatography including reversed-phase liquid chromatography (RPLC), ion exchange chromatography (IEX), hydrophobic inter- action chromatography (HIC) and some perspectives are discussed. For all these chromatographic modes, the most important variables for tuning retention and selectivity are exposed. Then, the retention models that were applied in the literature in RPLC, IEX and HIC are described and critically discussed. Finally, some representative examples of separation of therapeutic proteins and mAbs are shown, to illustrate the possibilities offered by the retention modeling approach. At the end, the reliability of the models was excellent, whatever the chromatographic mode, and the retention time prediction errors were systematically below 2%. In addition, a significant amount of time can be saved during method development and robustness testing.

A platform analytical quality by design approach for methods development is presented in this paper. This approach is not limited just to method development following the same logical Analytical quality by design (AQbD) process, it is also exploited across a range of applications in method development with commonality in equipment and procedures. As demonstrated by the development process of 3 methods, the systematic approach strategy offers a thorough understanding of the method scientific strength. The knowledge gained from the UHPLC-UV peptide mapping method can be easily transferred to the UHPLC–MS oxidation method and the UHPLC-UV C-terminal heterogeneity methods of the same protein.

Using the DryLab simulation, the cost saving was tremendous. It shortened the method development time from the typical 1-3 months to about 1 week. Importantly, the unknown factors in chromatography became more predictable. The direct cost saving involved labor, consumable, and instrument time. Even more significant were the lateral benefits, as the productivity of scientists could be increased by multiple factors.

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.

Highlights:

• Pemetrexed sodium purity methods were developed and validated.
• Information and structure of 13 main impurities for pemetrexed sodium are provided.
• Conditions for the system suitability solutions in situ preparation are described.
• DryLab software tool was used to provide efficiency.
• The system suitability assures the main impurities resolution and identification.
• Relative response factors were determined using HPLC-UV in tandem with CAD or in combination with NMR.

Twelve columns of same brand name but different batches were compared. 3D design of experiment modeled by DryLab was used to compare column selectivity and performance. 3D resolution space allows establishing better method robustness before validation. A multivariable design space (MODR) allows flexible routine work with columns. This procedure allows easy column replacement to retain method robustness.

Highlights:

• Columns for two-dimensional chromatography were compared to maximise orthogonality.
• Simulation software was employed to rapidly optimise 2D-HPLC operating conditions.
• 2 Optimisation protocols were compared to find the best 2D-HPLC column combination.
• Both protocols were proven to be effective at predicting the final orthogonality.
• The operating conditions were adjusted with DryLab and the simulated chromatograms were compared to the real runs.

Gas phase emission samples of carbonyl compounds (CCs) were collected from a research ship diesel engine at Rostock University, Germany. The ship engine was operated using two different types of fuels, heavy fuel oil (HFO) and diesel fuel (DF). Sampling of CCs was performed from diluted exhaust using cartridges and impingers. Both sampling methods involved the derivatization of CCs with 2,4-Dinitrophenylhydrazine (DNPH). The CCs-hydrazone derivatives were analyzed by two analytical techniques: High Performance Liquid Chromatography–Diode Array Detector (HPLC–DAD) and Gas Chromatography–Selective Ion Monitoring–Mass Spectrometry (GC–SIM–MS). Analysis of DNPH cartridges by GC–SIM–MS method has resulted in the identification of 19 CCs in both fuel operations. These CCs include ten aliphatic aldehydes (formaldehyde, acetaldehyde, propanal, isobutanal, butanal, isopentanal, pentanal, hexanal, octanal, nonanal), three unsaturated aldehydes (acrolein, methacrolein, crotonaldehyde), three aromatic aldehyde (benzaldehyde, p-tolualdehyde, m,o-molualdehyde), two ketones (acetone, butanone) and one heterocyclic aldehyde (furfural). In general, all CCs under investigation were detected with higher emission factors in HFO than DF. The total carbonyl emission factor was determined and found to be 6700 and 2300 μg kWh−1 for the operation with HFO and DF respectively. Formaldehyde and acetaldehyde were found to be the dominant carbonyls in the gas phase of ship engine emission. Formaldehyde emissions factor varied from 3870 μg kWh−1 in HFO operation to 1540 μg kWh−1 in DF operation, which is 4–30 times higher than those of other carbonyls. Emission profile contribution of CCs showed also a different pattern between HFO and DF operation. The contribution of formaldehyde was found to be 58% of the emission profile of HFO and about 67% of the emission profile of DF. Acetaldehyde showed opposite behavior with higher contribution of 16% in HFO compared to 11% for DF. Heavier carbonyls (more than two carbon atoms) showed also more contribution in the emission profile of the HFO fuel (26%) than in DF (22%).

Highlights:

• DryLab employs to find the optimum time of a simple (or segmented) gradient, yielding best resolution of a particular sample.
• Short silica-based monolithic columns can be used with fast 1 min- gradients up to 5 mL/min.
• Isocratic retention parameters were used for prediction and modeling gradient chromatograms.
• The gradient range was optimized for maximum peak capacities within a fixed gradient time.

Highlights:

• The study of theoretical principles of acid–base equilibrium has been reviewed.
• The proton transfer process is often present in the analytical separation practice.
• The influence of temperature on secondary chemical equilibria is examined.
• The focus is laid on liquid chromatography and capillary electrophoresis.
• Temperature can be a useful variable to modify selectivity under predictable basis.

A new and fast ultra-high pressure liquid chromatographic separation of amlodipine and bisoprolol and all their closely related compounds is described for impurity profiling purposes. Computer-assisted method development with DryLab was applied and the impact of several state-of- the-art stationary phase column chemistries (50 × 2.1 mm, sub-2 μm, and core–shell type materials) on the achievable selectivity and resolution was investigated. The work was performed according to QbD-principles using design of experiment with three experimental factors: gradient time (tG), temperature (T), and mobile phase pH. DryLab proves that the separation of all compounds was feasible on numerous column chemistries within <10 min, by proper adjustments of variables. It was also demonstrated that the reliability of predictions was good, as the predicted retention times and resolutions were in good agreement with the experimental ones. The final, optimized method separates 16 peaks related to amlodipine and bisoprolol within 7 min, ensuring baseline separation between all peak-pairs.

In-silico optimised two-dimensional high performance liquid chromatographic (2D-HPLC) separations of a model methamphetamine seizure sample are described, where an excellentmatch between simulated and real separations was observed. DryLab optimisation reduced 2D-HPLC development time significantly. Targeted separation of model compounds was completed with significantly reduced method development time. This separation was completed in the heart-cutting mode of 2D-HPLC where C18 columns were used in both dimensions taking advantage of the selectivity difference of methanol and acetonitrile as the mobile phases. This method development protocol is most significant when optimising the separation of chemically similar chemical compounds as it eliminates potentially hours of trial and error injections to identify the optimised experimental conditions. After only four screening injections the gradient profile for both 2D-HPLC dimensions could be optimised via simulations, ensuring the baseline resolution of diastereomers (ephedrine and pseudoephedrine) in 9.7 min. Depending on which diastereomer is present the potential synthetic pathway can be categorised.

This study describes the development of liquid chromatographic methods for the simultaneous separation of some of the most popular local anesthetics in pharmaceutical preparations and medical praxis (benzocaine, bupivacaine, chloroprocaine lidocaine, oxybuprocaine, prilocaine, procaine, propipocaine and tetracaine) based on a systematic approach using experimental design methodology in which one or more factors are changed at the same time. The strategy employs a chromatography modeling software for the simultaneous optimization of critical chromatographic parameters, which are gradient time tG, temperature T and the ternary composition of the organic eluent B.

DryLab is one of the most established software for chromatography modeling, which allows for modeling of chromatographic separations based on input data from two or more experimental runs. The use of DryLab for HPLC modeling to facilitate methods development was well documented in the last 27 years. In this time a continuous development occurred to the software which enabled it to cope more with the ongoing technological progress. On the other hand, a number of published studies exist that deal with the use of DryLab in different chromatographic modes and wide application ranges. DryLab is applied to solve different analytical problems in pharmaceutical analysis, which deal mostly with the separation of active pharmaceutical ingredients (APIs) in the presence of their impurities and/or their degradation products. In the field of phytochemical analysis many applications on complex plant extracts are also available. Moreover, DryLab has been successfully applied to optimize the separation of different groups of environmental pollutants, peptides and proteins and metabolites.

Highlights:

• Implementation of organic solvent gradients in micellar liquid chromatography, MLC.
• Screening of β-blockers in urine samples with direct injection in gradient MLC.
• 1-Propanol gradients allows the elution of β-blockers of diverse polarity.
• Gradient elution starting with pure micellar solution benefits direct injection.
• Optimisation of organic solvent gradients in MLC can be performed with DryLab®.

Quality by design (QbD) is an approach to product development where a comprehensive understanding of the effect of various inputs (e.g. process parameters, materials) on the final product (active pharmaceutical ingredient or drug product), leads to a process or product design that ensures reliable manufacture of a high-quality product. Analogous to QbD for product, QbD may also be applied to analytical methods, by defining the required analytical performance and systematically relating how the technique and method parameters relate to analytical performance. Achieving the appropriate level of method understanding typically involves a multifactorial approach. The factors to be studied are identified via a systematic risk analysis, which allows truly impactful parameters to be further investigated in studies applying statistical design of experiments (DOE) or mechanistic models to develop a multidimensional region of experimental space in which the effects of key factors are understood and documented. An operating region can then be defined where there is high confidence that the method will operate reliably. Data generated during use of the method can be monitored over time to ensure continued adequate performance of the analysis. 

Highlights:

• A suitable chromatographic system has to be employed to take the full benefits of modern LC columns.
• Extra-column band broadening and gradient delay volume have to be minimized.
• The upper pressure limit of UHPLC systems is less critical in the case of fast-LC separations.
• Acquisition rate is sufficient with spectroscopic detectors but much more critical with MS devices.
• During the last few years, column technology has evolved faster than instrumentation.

The goals in ultrahigh-pressure liquid chromatography (UHPLC) method development are to first find the best separation, second find the best column, and third find the most robust method in a multifactorial Design Space. Trial and error methods are not sufficient anymore and solid science based on Quality by Design (QbD) principles is required.

The goal of this study was to evaluate the accuracy of simulated robustness testing using commercial mod- elling software (DryLab) and state-of-the-art stationary phases. For this purpose, a mixture of amlodipine and its seven related impurities was analyzed on short narrow bore columns (50 × 2.1 mm, packed with sub-2  m particles) providing short analysis times. The performance of commercial modelling software for robustness testing was systematically compared to experimental measurements and DoE based pre- dictions. We have demonstrated that the reliability of predictions was good, since the predicted retention times and resolutions were in good agreement with the experimental ones at the edges of the design space. In average, the retention time relative errors were <1.0%, while the predicted critical resolution errors were comprised between 6.9 and 17.2%. Because the simulated robustness testing requires signif- icantly less experimental work than the DoE based predictions, we think that robustness could now be investigated in the early stage of method development.

Moreover, the column interchangeability, which is also an important part of robustness testing, was investigated considering five different C8 and C18 columns packed with sub-2  m particles. Again, thanks to modelling software, we proved that the separation was feasible on all columns within the same analysis time (less than 4 min), by proper adjustments of variables.

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

Highlights:

• Innovative QbD-based approach for robustness testing of a compendial HPLC method.
• A Design Space was constructed to study the robustness in silico.
• Required peak resolution of 2.0 could not be reached in all experiments.
• After adjusting the gradient time the requirements were fulfilled.
• A separate “Eluent Design Space” study was performed.

The aim of this study was to apply quality-by-design principles to build in a more scientific and risk-based multifactorial strategy in the development of an ultrahigh-pressure liquid chromatography (UHPLC) method for omeprazole and its related impurities.

The work presents a quality-by-design–based method development strategy for a method that tests the purity of omeprazole. The scientific and risk-based multifactorial method development strategy uses visual chro- matographic modeling as a fast and easy-to-use development tool. To speed up the method development process, all experiments are performed on a UHPLC system. The final method is successfully transferred to HPLC conditions. Predicted and experimental retention times are verified to confirm accuracy of the model.

The International Conference on Harmonisation (ICH) guideline for the Validation of Analytical Procedures (ICHQ2(R1)) currently covers validation procedures for the four most common analytical tests: identification tests, quantitative tests for impurities, limit tests for the control of impurities and quantitative tests for the active moiety(ies) in APIs (active pharmaceutical ingredients) or drug products. The key underlying concepts and strategies are equally applicable to other analytical methodologies; e.g. particle size analysis, dissolution, etc. Typical validation parameters covered in the guideline include accuracy, precision, specificity, detection limits (DL / LOD) and quantitation limits (QL / LOQ), linearity, range and robustness.

Highlights:

• Test parameters of Tanaka and of Snyder showed surprisingly a high correlation.
• USP test is related to Tanaka and Snyder only in terms of hydrophobic characters.
• Some differences in the correlation are shown between aromatic and alkyl phases.
• The hydrophobic-subtraction model is extended to describe calixarene-bonded phases.

This paper presents new reversed phase liquid chro-matographic 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. Elution of sulfonamides was carried out under acidic, neutral, and basic conditions using methanol. 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 with DryLab4. 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.

A new type of method development that uses modeling by DryLab4 to find the "best" separation for high performance liquid chromatography (HPLC) was investigated. Principles of Quality by Design (QbD) were followed when planning the investigation. A simple Design of Experiments (DoE) with only three measured factors — gradient time, pH, and temperature — was used with different columns. The basic experiments were saved in an electronic file with the peak tracking results. After calculating a Design Space, the best prediction was compared with a confirmation run. The process delivered precise results and the method was able be transferred to a routine quality control (QC) laboratory.

Highlights:

• The influence of changing %B (or tG), °C, and solvent type on selectivity is reexamined.
• These changes can be as effective as changing pH or column type when adjusting selectivity.
• These changes can mimic changes in pH, reducing or eliminating the need to optimize pH.
• Such continuous changes can be more effective than a step-wise change in column type

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 DryLab4 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) for DryLab4 modeling 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. With the support of DryLab4 all columns could perform the required baseline separation at their individual best working points with satisfactory results.

An HPLC-UV method to determine compounds having a hydroxyl functional group in plant essential oils is developed. Separation conditions were optimized using the DryLab® method development software, a large sensitivity enhancement was obtained.

In this work, DryLab is used to accurately determine the design space for critical resolution in the analysis of water-soluble vitamins by HPLC. The multifactorial optimization of three measured parameters (gradient time, temperature, ternary eluent composition (B1/B2)) and seven calculated ones (flow rate, column length, column internal diameter, dwell volume, extracolumn volume, %B [start], and %B [end]) are illustrated. DryLab is used to examine multifactorial effects of these 3+7 parameters on critical resolution and selectivity. Multidimensional robust regions of high critical Rs were defined and graphically verified. The optimum method was selected based on the best resolution separation in the shortest run time. Predicted retention times of all peaks were found to be in excellent match with the virtual ones.

Highlights:

• Impurity profiling of a triple combination anti-HIV drug by SFC and RPLC is reported.
• Method development strategies and figures of merit with UV detection are discussed.
• A high degree of orthogonality was achieved with SFC.
• SFC also gave a more uniform distribution of components across the separation space.
• Potential impurities/degradation products were quantitated at the ≤0.1 area% level.

Highlights:

• Comparative study between column selectivity in RP-LC obtained for a real biomedical separation was reported.
• We performed classification of 24 RP-LC columns based on the QSRR approach.
• The reliability of the theoretical QSRR-model results was examined against column test performance based on the LC analysis of moclobemide and its two metabolites using two chemometric techniques.
• Principal component analysis (PCA) and hierarchical clustering analysis (HCA) were applied.

Free amino acids are closely related to the savory taste and beneficial effects of tea, and high-performance liquid chromatography (HPLC) is the most widespread analytical approach for simultaneous determination of free amino acids in tea. However, the reported HPLC methods have drawbacks such as long run times, low resolution, or poor efficiency. In this study, a special amino acid analysis column was used to separate and verify 21 amino acids including l-theanine, the predominant amino acid in tea. The mobile phases, including the sodium acetate and acetic acid concentration in buffer B, and the pH and concentration of sodium acetate in buffer A were optimized. The elution gradients were optimized using DryLab software. In this way, an online o-phthaldialdehyde precolumn derivatization HPLC-fluorescence detection method was developed for simultaneous determination of 21 amino acids. Comparison to other HPLC methods for simultaneous determination of free amino acids in tea showed that our method is easy (automated derivatization), quick (30 min), inexpensive, and green (using a minimum of solution). It has good resolution (≥1.8) and high selectivity (interpark time ≥ 0.5 min). Free amino acids in six tea samples were analyzed. This work provides an HPLC method to simultaneously measure 21 amino acids in tea and potential in other food products.

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, 1.7 µm 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 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 runtime was only 4 min,which is an impressive 40-fold increase in productivity in comparison to themethod published in the Ph. Eur.monograph and allowed purity testing of more than 360 samples per day.

This article discusses the development of chromatography modelling of the last 30 years from the first software package for calculating resolution and capacity factors to the visual modelling of chromatograms for testing peak movements with altering elution conditions. Different approaches are discussed, such as retention modelling based on measurements, others based on molecular structure or on statistical considerations. The state-of-the-art will be demonstrated using DryLab with a few applications of industrial importance.

This article aims to investigate the predictability of chromatographic behavior of enantiomers using DryLab to predict the effect of changing various chromatographic parameters on resolution in the reversed phase mode. Three different types of chiral stationary phases were tested for predictability.High rates of accuracy allow for the conclusion that Chirobiotic V reversed phase retention mechanism follows the solvophobic theory.

In the pharmaceutical field, there is considerable interest in the use of peptides and proteins for therapeutic purposes. There are various ways to characterize such complex samples, but during the last few years, a significant number of technological developments have been brought to the field of RPLC and RPLC-MS. Thus, the present review focuses first on the basics of RPLC for peptides and proteins, including the inherent problems, some possible solutions and some directions for developing a new RPLC method that is dedicated to biomolecules. Then the latest advances in RPLC, such as wide-pore core-shell particles, fully porous sub-2 μm particles, organic monoliths, porous layer open tubular columns and elevated temperature, are described and critically discussed in terms of both kinetic efficiency and selectivity using DryLab. Numerous applications with real samples are presented that confirm the relevance of these different strategies. Finally, one of the key advantages of RPLC for peptides and proteins over other historical approaches is its inherent compatibility with MS using both MALDI and ESI sources.

The validity of the extended Tanaka column characterization procedure against the retention behavior of 101 analytes of widely differing properties chromatographed on five differing stationary phase chemistries has been established using a chemometric technique called principal component analysis (PCA). It was concluded that the simple and conveniently determined column characterization parameters covered the same space in the PCA loading plot as the retention times for the 101 differing analytes. This confirms that the ten column characterization parameters of the extended Tanaka protocol encode the same information as the retention times of the 101 analytes. Significant selectivity differences were observed between stationary phases and the mobile-phase modifiers – MeOH and MeCN. PCA contribution plots served as a convenient way to highlight specific selectivity differences between stationary phases. logD values exhibited a poor correlation with retention indicating that retention in RP-LC is not solely dictated by the analytes hydrophobicity. The use of MeOH was found to generate greater selectivity differences with the five stationary phases than when MeCN is used.

The application of factor analysis (FA) method in classification of the antitumor acridinones based on high-performance liquid chromatography (HPLC) retention data and calculated parameters of lipophilicity as well as some nonempirical structural parameters was studied. First, a group of 19 acridinone (imidazoacridinone and triazoloacridinone) derivatives was chromatographed in six RP-HPLC systems, and the values of their HPLC retention data as retention times determined in both 10 min and 30 min gradient times were obtained as well as log kw (retention factor log k extrapolated to 0% organic modifier) parameters using DryLab 4 program were calculated. 

A new, simple, rapid, sensitive and economical spectrophotometric method has been developed and validated for estimation of lercanidipine in pure and its pharmaceutical formulation like tablet. During the development of formulations containing lercanidipine in its solid dosage form, analytical methods will serve as assay method for quantitation of the lercanidipine during product developmental stages. The present work consist of estimation of lercanidipine by difference spectrophotometry which is based on shifting of λ max by changing the pH of the solution by adding 0.1M HCl and 0.1M NaOH the absorption maximum was obtained. Linearity of the response was demonstrated for the drug for a range fulfilling Beer’s law, which is 5-25 μg/ml. The absorption maxima of lercanidipine were obtained at 260 nm in 0.1M NaOH, and 240 nm in 0.1M HCl. The results of analysis have been validated statistically and by recovery studies. The method was extended to pharmaceutical formulation and there were no interferences from any excipients and diluents. The full analytical validation was performed according to International Conference on Harmonization Q2R1 guidelines for validation of analytical procedures, supported by DryLab-models. 

Following administration of the acidic drug tolmetin (TOL) anaphylactic reactions occurred, which have been hypothesized to be related to the formation of reactive acyl glucuronides. Recently, glutathione adducts have been detected upon incubation of TOL with human liver microsomal preparations, which proved that oxidative activation might also be a pathway of formation of reactive—possibly toxic—glutathione metabolites of TOL. The aim of this work was to develop a new and robust HPLC method to investigate the in vivo effect of 2 coadministered drugs/nutritional supplements on the kinetics of TOL in rats (cimetidine, CIM) known to be a potent inhibitor of CYP3A4, an enzyme that catalyzes the oxidative metabolism and Quercetin, and QUE which induces UGT1A6, an enzyme involved in glucuronidation of acidic drugs. DryLab, a computer modeling software package, was used to assist in the development and optimization of the HPLC method used for separation of TOL and the two potential kinetic modulators together with three potential internal standards (zomepirac, carvedilol and fexofenadine). The method was validated in biological samples obtained from rats. 

Robust HPLC separations lead to fewer analysis failures and better method transfer as well as providing an assurance of quality. This work presents the systematic development of an optimal, robust, fast UHPLC method for the simultaneous assay of two APIs of an eye drop sample and their impurities, in accordance with Quality by Design principles. DryLab chromatography modeling software is employed to effectively generate design spaces (Method Operable Design Regions, MODR's), which are subsequently employed to determine the final method conditions and to evaluate robustness prior to validation.

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

The retention behavior of a range of statistical poly(styrene/ethylacrylate) copolymers is investigated, in order to determine the possibility to predict retention volumes of these copolymers based on a suitable chromatographic retention model using the DryLab software. It was found that the composition of elution in gradient chromatography of the copolymers is closely related to the eluent composition at which, in isocratic chromatography, the transition from elution in adsorption to exclusion mode occurs. For homopolymers this transition takes place at a critical eluent composition at which the molar mass dependence of elution volume vanishes. Thus, similar critical eluent compositions can be defined for statistical copolymers. The existence of a critical eluent composition is further supported by the narrower peak width, indicating that the broad molar mass distribution of the samples does not contribute to the retention volume. It is shown that the existing retention model for homopolymers allows for correct quantitative predictions of retention volumes based on only three appropriate initial experiments. The selection of these initial experiments involves a gradient run and two isocratic experiments, one at the composition of elution calculated from first gradient run and second at a slightly higher eluent strength.

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 k_w) and log P data calculated using some commonly available software, are discussed.

In the present work it is shown that the linear elution strength (LES) model can be employed to predict retention times for segmented-temperature gradients based on temperature-gradient input data in liquid chromatography (LC) with high accuracy using DryLab-software. In order to predict retention times for temperature gradients with different start temperatures in LC, another relationship is required to describe the influence of temperature on retention. It could be shown that a plot of lnk vs. T yields more reliable isothermal/isocratic retention time predictions than a plot of lnk vs. 1/T which is usually employed. Retention times can be predicted with a maximal relative error of 5.5%(average relative error: 2.9%). As an example, the systematic method development for an isothermal as well as a temperature gradient separation of selected sulfonamides by means of the adapted LES model is demonstrated using a pure water mobile phase. 

DryLab^® was used with excellent results with superficially porous particles (Fused-Core^®) which were designed with 160 Å pores. These particles show superior kinetics (lower resistance to mass transfer), allowing fast separations of peptides and small proteins (molecular weights of 15,000). The high efficiency and relatively low back pressure of these 2.7 μm Fused-Core particles has been maintained so that separations can be performed with conventional HPLC instruments.11 synthetic peptides (50 ng each) Fig. 12 show an excellent comparison of DryLab^® predicted chromatogram to experimental ones. Chromatographic optimizations were conducted using DryLab^® from the Molnár-Institute for Applied Chromatography. Longer columns can be used for higher resolution of complex mixtures of peptides, such as proteolytic digests. Highly reproducible separations of peptides at elevated temperatures with low pH mobile phases are maintained as a result of a stable bonded stationary phase. The utility of such highly stable materials is exemplified by separations of problematic amyloid peptides at low pH (TFA mobile phase) at an operational temperature of 100 °C.

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 latest generation of wide-pore sub-2 µm fully porous or core-shell particles stationary phases and the DryLab selectivity modeling software. 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 using DryLab.  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 8 h). Two separations of mAbs fragments were performed to demonstrate the reliability of the quadratic approach.

A method for the assay of an active pharmaceutical ingredient (API) and five known and unknown impurities was developed in accordance with Quality by Design (QbD) principles and using DryLab as a modeling software. Highly influential separation parameters were simultaneously and systematically studied so that the quality of the HPLC method could be understood, controlled and ensured. Consequently, during routine analysis out of specification (OOS) results can be corrected more effectively.

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 polyphenols, for example stilbenes and flavonoids, are an important family of compounds present in grapes and wine. For the first time, eight natural stilbenes (trans-resveratrol, trans-piceid, cis-piceid, trans-astringin, trans-piceatannol, (+)-trans-Ɛ-viniferin, pallidol, and hopeaphenol), isolated and purified from Vitis vinifera, were simultaneously analysed by UHPLC coupled with photodiode-array detection. Separation of the stilbenes was optimized with the assistance of Quality-by-Design commercial software DryLab. Four different reversed-phase columns packed with 1.5-1.7 µm particles were tested and compared for their retention behaviour and separation efficiency. 

The optimization of chromatographic separation was achieved using DryLab Software (Molnár-Institute, Berlin, Germany). Specifically, the experiments were carried out in a systematic way. 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. 

A stepwise method development strategy has been employed to develop a robust HPLC method to resolve several closely eluting structurally related impurities in an active pharmaceutical ingredient (API). This strategy consisted of automated column screening, optimization of the most critical chromatographic parameters, DryLab modeling, and experimental verification of optimized separation conditions. DryLab was used to predict an optimized gradient profile and separation temperature and these predictions were verified experimentally. A discussion of the accuracy of these predictions is presented. The robustness of the method was verified and the ability of DryLab to predict, with reasonable accuracy, the outcome of such robustness studies was also examined. Once the robustness was established by the DryLab predictions the remainder of the subsequent verification by experiment becomes a simple reiterative exercise. It is critical to adopt a rational strategy, as demonstrated here, to evaluate the interplay of these factors, thereby greatly enhancing method development efficiency.

The current paper presents a novel approach to applying Quality by Design (QbD) principles to the development of high pressure reversed phase liquid chromatography (HPLC) methods using DryLab. 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.

This paper describes systematic method development strategies used with different instrumental set-ups according to Quality by Design principles for a sample of toxicological interest. Experiments for DryLab modeling were run in an automated fashion on three Shimadzu instruments, and the accuracy of all models was confirmed to be excellent. Generated models were employed to reduce the original method time of 45 minutes to just over 3 minutes. Additionally, DryLab models were shown to successfully emulate experimental changes typically encountered during a method transfer process (dwell volume, extra column volume, flow rate, column dimensions).

This paper builds a bridge between the pioneering work of Csaba Horváth at the dawn of high pressure liquid chromatography during the 1970s and the present state of the art technologies in the field more than four decades later. In recognition of a lifetime of achievement, a small piece in Csaba Horváth's large work together with Imre Molnár is specifically honored. The work emphasizes the importance of understanding retention phenomena, which started with Csaba Horváth’s Solvophobic Theory and nowadays is the base of the ever-growing Quality by Design movement. It is shown how the latest advances in modeling software can be used to gain insight into retention behavior and how this knowledge can be applied to the development of robust reversed phase liquid chromatography methods.

A multifactorial optimization of three critical HPLC parameters is described. Gradient time (tG), temperature (T), and ternary composition (B1:B2) are optimized for the separation of 22 pharmaceutically relevant analytes based on 12 experimental runs. The effects of these variables on critical resolution and selectivity were examined as all three factors were varied simultaneously. Robust conditions for separation were defined using the Design Space and then verified. This paper demonstrates the applicability of this approach to rapid development of high-quality LC methods using DryLab.

This paper describes the use of "knowledge space" and "design space" for Quality by Design of chromatographic methods using DryLab. The authors look specifically at the selection of the proper range of gradient steepness for experimental input data and its effect on predicted results. They demonstrate that the interpolated simulation as well as the extrapolated simulation results, when the initial slopes were close in range to the target slope, provided good predictions with overall percent error difference of experimental retention times of less than 1%.

This paper describes a multifactorial optimization of four 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 pHs 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.

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 astructured 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 colormeasurement, and trace analysis for genotoxic impurities.

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. Two gradients—two column temperature basic model runs were carried out and DryLab was used to predict the optimal solvent ratio, which would give sufficient resolution (Rs > 1.5) between the compounds and peaks originated from sampling matrix. The 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 1.7m (50mm×2.1mm) and acetonitrile–water (48:52, v/v) as mobile phase at a flow rate of 0.55 ml/min. Method development and method validation for cleaning control analysis are described. The rapid UPLCmethod is suitable for cleaning control assays within good manufacturing practices (GMP) of the pharmaceutical industry.

In the present study a computer simulation software DryLab and a chemometric (response surface) approach were used in developing and optimizing a reverse-phase HPLC separation of moxifloxacin and its related impurities and degradation products of moxifloxacin. 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. The column was thermostated at 45 °C. The resolution between the two least resolved impurity peaks was in average, R_s,min > 1.5. Method validation parameters indicate linear dynamic range 0.2–2.0 μg/ml with LOQ ca. 0.20 μg/ml and LOD ca. 0.05 μg/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. 

A comprehensive automated strategy for the development of a RPLC-UV-method for a basic drug candidate (pKa 7.7), its impurities, and degradants was demonstrated by using multidimensional screening and analysis (MeDuSA) and DryLab computer optimization software. DryLab software was used to model the effect of temperature, pH, and ionic strength of the buffer on resolution and peak shape to determine the design space of the method and establish the optimal operating conditions. In silico optimization supported by DryLab enabled the determination of the optimum conditions without carrying out trial-and-error simulations. Excellent agreement between DryLab simulation and experimental results was obtained. As a result, decreased run time (from 35 to 14 min compared to the original method) was developed.

The paper describes a strategy for the systematic development of ultra high pressure liquid chromatographic (UHPLC or UPLC) methods using 5 cm × 2.1mm columns packed with sub-2µm particles and computer simulation with the DryLab® package. Data for the accuracy of computer modeling in the Design Space under UPLC conditions are reported. An acceptable accuracy for these predictions of the computer models is presented. The 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 > 400 bar). Examples of fast and effective method development in pharmaceutical analysis, both for gradient and isocratic separations are presented.

The main objective in all optimization procedures is to define the most appropriate conditions for rapid, sensitive, precise, and reproducible analysis, as economically as possible. Experimental design and DryLab optimization software have been used to optimize a liquid chromatographic method for separation of lercanidipine and its three impurities. In both methods of optimization the acetonitrile content and pH of the mobile phase were factors extracted for analysis, resolution of a critical pair was output in both cases. Data obtained from both optimization methods were compared and appropriate conclusions were extracted with the objective of gaining a complete view of chromatographic behavior. Detailed description was obtained by use of a three-dimensional graph and DryLab maps.

A stepwise method development strategy was employed in developing a robust HPLC method to resolve several closely eluting process impurities associated with a novel diabetes compound. The strategy consisted of rapid column screening, optimization of mobile phase compositions and separation temperature, DryLab modelling, and experimental verification of optimized separation conditions. The column evaluation process involved screening of a series of 20 columns varying in bonding chemistry using four sets of mobile phases composed of water, ACN and/or MeOH at three different pH’s. The screening process resulted in identifying two promising columns: XBridge Shield RP18 and SunFire C18. The effects of organic modifiers and separation temperatures were then evaluated to narrow down the chromatographic separation parameters. DryLab® was used to predict optimized gradient profile and separation temperature. Finally, the DryLab® predictions were verified experimentally. The study demonstrates that factors such as stationary phase composition, organic modifiers, pH and separation temperature have profound and often complex effects on chromatographic conditions. Therefore, it is critical to adopt a rational strategy as demonstrated here to evaluate the interplay of these factors, there by greatly enhancing method development efficiency.

The development of gradient methods in HPLC is a difficult task. The transfer of the methods requires deep understanding the process in the column and the factors, which are required for a safe operation in the routine lab. The talk will discuss the aspects, how to make reliable methods using computerized design in the development.

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.

An optimization strategy for ternary solvent-strength gradient elution RP chromatography is described in which a 2-dimensional model of gradient time (2 levels) against ternary proportions of organic modifiers (4 levels) was constructed. Modelling was performed using Drylab. From the resolution surface the optimum ratio of organic modifiers could be selected. Excellent retention time and acceptable peak width and resolution simulations were obtained. The separation could be further optimized from the same input data by using a standard one-dimensional model in order to optimize for gradient slope, duration and shape. Excellent retention time and acceptable peak width and resolution simulations were obtained (< 1, 2 and 6% error respectively).

The suitability of three different retention models to predict the retention times of poly(ethylene glycol)s (PEGs) in gradient and isocratic chromatography was investigated. The models investigated were the linear (LSSM) and the quadratic solvent strength model (QSSM). In addition, a model describing the retention behaviour of polymers was extended to account for gradient elution (PM). It was found that all models are suited to properly predict gradient retention volumes provided the extraction of the analyte specific parameters is performed from gradient experiments as well. The LSSM and QSSM on principle cannot describe retention behaviour under critical or SEC conditions. Since the PM is designed to cover all three modes of polymer chromatography, it is therefore superior to the other models. However, the determination of the analyte specific parameters, which are needed to calibrate the retention behaviour, strongly depend on the suitable selection of initial experiments. A useful strategy for a purposeful selection of these calibration experiments is proposed.

Attempts to theoretically address the problems involved in transferring linear gradient elution methods have been somewhat ad hoc due to the simplifying assumptions usually made in conventional gradient elution theory. Until now, all equations based on the k* parameter of linear gradient elution theory used as the basis for predicting the separation selectivity have not explicitly included the effect of the dwell volume (VD). Using an exact equation for predicting k*, that is, one which fully accounts in an a priori fashion for VD, we find a set of simple yet exact equations which unequivocally must be satisfied to transfer an optimized linear gradient elution method from one system (column or instrument or both) to another. 

This paper is written to honor Csaba Horváth and to remember his work on Reversed Phase Chromatography, (RPC) a theoretical fundament of the mechanism of retention on nonpolar stationary phases, called the "Solvophobic Theory" from the subjective point of view of the author. The paper is trying to compile a few stations in the development of this important theory, which is valid more than ever and look out for its consequences in developing robust methods for routine work, especially in the daily applications of RPC in industrial settings worldwide. It was Horváth, who laid the fundaments of this valuable technique, which makes the application of RPC to a still growing use in scientific research and in pharmaceutical and chemical production. Although the Solvophobic Theory of RPC was reflecting only a part of Horváth's scientific work, the impact of RPC in life science is tremendous and the technique RPC is today one of the most popular, most widely used tools in analytical chemistry and will remain for long time in use due to its stability and to its robustness.

The separation of anionic, cationic, and neutral drugs in microemulsion electrokinetic chromatography (MEEKC) was studied. The concentration of sodium dodecyl sulfate (SDS, surfactant) and 2-propanol (organic solvent) was varied in a three-level full factorial design. 29 different model substances were chosen with different hydrophobicities and charges (neutral, positive, and negative).

DryLab is used for the optimization of a model drug candidate and its degradation products. Accuracy of DryLab predicted retention times and resolution is compared with experimental values.

Multi-linear gradients have not been widely used in general-purpose HPLC in part because of experimental inconvenience in method development. Even with the use of computer modeling, identifying an optimum set of breakpoints has been done primarily by trial and error. Combining a spreadsheet with controllable chromatography modeling software has allowed to implement a systematic approach to bi- and tri-linear gradient optimization.

The development of the DryLab Software is a special achievement in analytical HPLC, which took place in the last 16 years. This paper tries to collect historical building stones and fundaments, which were laid down by the eminent work of Lloyd Snyder, John Dolan, Tom Jupille and many other enthusiastic scientist, from which DryLab has been put together to its state, where it is today. DryLab, being always a subject to changes, according to the needs of the user, never stopped to go on. Under the force of an ever changing science market, the development team of DryLab had to consider not just scientific improvements, but also new technological achievements, such as the introduction of Windows 1.0 and 3.1, later Windows NT and Windows 2000. The recent availability of new 32 bit program-ming tools allowed to carry out calculations of chromatograms much faster, to be able to show peak movements, which result of slight changes - for example - in eluent pH. DryLab is a great success of an interdisciplinary and intercontinental cooperation of many scientists.

For ionizable compounds such as organic acids, best results were obtained recently with simultaneous optimization of %B and pH, regardless of ionic strength or temperature. Changes in the pH of eluent A, adjusted to bracket the pK-values of acids (works also for bases and zwitterions), help to understand changes in critical resolution values due to shifts in peak positions.

A new strategy for neutral compounds, contained in many phytopharmaceuticals, was presented at HPLC 2001 in Maastricht by Molnár and Schmidt. The systematic work with kava pyrones and three different organic modifiers, methanol, acetonitrile and 2-propanol, by simultanously changing gradient slope versus temperature or gradient slope versus pH reveals the true composition of such mixtures.

The analytical chemist is interested to learn more about the influence of the experimental parameters on the resolution, but can often only rely on experiments, he was able to carry out in a given time in a project. There are however often as many chances to improve resolution in the "unexpected" direction as by varying them in the "expected" way. The tools for understanding the method and discover all chances for improved selectivity are the different resolution maps.

DryLab is used in a study in which the linear solvent strength model of gradient elution is applied to estimate parameters of lipophilicity and acidity of a series of drugs and model chemicals.

Drylab G software has been used to optimize the reversed-phase HPLC separation of taxoids and co-extracted substances from yew. Two preliminary runs based on a linear gradient from 5 to 100% acetonitrile (ACN) in 20 or 60 min were shown to be satisfactory for optimization of resolution. The optimization experiments were performed on a purified extract fortified with taxoid standards. The identity and purity of the peaks were verified by use of photodiode-array detection. Agreement between simulated and experimental data was good. The optimized chromatographic system can be used for quantitative analysis of paclitaxel, cephalomannine, and 10-deacetylbaccatin in yew extracts.

Reversed-phase columns are widely used in assays based on high-performance liquid chromatography (HPLC). When such assays are repeated over time, it is often necessary to replace the column. In such cases, the selectivity of columns from different production batches may prove sufficiently variable to result in a failed separation. It is possible to compensate for differences in column selectivity by making small changes (adjustments) in separation conditions. The present paper describes an efficient procedure for choosing adjusted conditions and discusses its general applicability.

Two different stationary phases, carbon coated ZrO2 and C18 modified silica were compared. They show very different selectivity. The DryLab software was used to evaluate the different selectivities using a sample of triazine herbicides.

DryLab-Software was used to determine the optimum column temperature and mobile phase pH for the separationof mixtures of roxithromicin and related compounds.

Unusual experiments can provide surprisingly good analytical solutions. When developing chromatographic methods, analysts use in most cases a combination of experience and instinct to choose initial starting conditions. This is often followed by a period of trial-and-error optimization, until the desired method is achieved. The article illustrates, how the process of chromatographic method development can be improved using computer modelling and simulation.

A computer assisted method for the scale up of a column liquid chromatographic separation from the analytical to preparative scale is successfully proposed to make the up-scaling process easier and faster. The test sample consisting of six phenolic compounds was chromatographed on an analytical HPLC column using two different gradient runs. Based on the chromatographic data achieved from these initial analyses, elution behaviour of compounds could be simulated for alternative conditions using the DryLab program. A simulative replacement of the analytical column with a preparative scale medium pressure LC (MPLC) column allowed the determination of gradient profile to allow sufficient separation in the preparative scale. A test run carried out in suggested simulated conditions matched well with expected elution times. Furthermore, this upscaling procedure was successfully applied to a plant extract.

DryLab is used to optimize mobile phase and temperature after evaluating chromatograms of gradient elution separations performed automatically by column switching. The automated procedure was applied to more than three dozen substances (steroidal intermediates) with a time savings of more than a third.

DryLab is used in method development with the goal of enhancing separations through the exclusive use of gradient time and column temperature. The resulting method is simple, fast, demonstrates excellent transferability and is ideal for the quantitative analysis of pigments in dilute natural water samples.

Rational selection of optimized experimental conditions for chromatographic separation of analytes is realized nowadays by means of specialized method development software. Two such programs, DryLab (LC Resources, USA, in Europe: Molnár-Institut, Berlin) and ChromSword (Merck, Darmstadt), were compared in a few aspects in this paper. The aim was to make a comparison of the quality of the software packages in the separation of neutral compounds, performed isocratically in RP-HPLC systems. A discussion of the differences in predicted and experimental chromatographic retention parameters is reported. The conclusion reached is, that the two programs provide good predictions of retention data, when predictions are based on %B changes using two initial experimental runs. An additional option of ChromSword, employing the quantitative structure-retention relationship (QSRR), did not to provide precise predictions of the separation, because molecular structural data were used as inputs. Predictions based on molecular structure were unaccurate. The comparison of the performance was only done with ca. 5% of the funtional capabilities, which DryLab was offering. Gradient data were not compared at all.

Excellent description of the mathematical background and practical aspects of HPLC gradient elution technique as a fundamental of the DryLab-software .

Development of a method for a high performance liquid chromatography (HPLC) separation can be a major undertaking. Before the separation can be made, the sample must be in a suitable form to inject, and pretreatment steps are often required to remove major interferences or materials that might shorten the column life. After conditions for adequate separation are determined, some level of method validation is usually performed. Sample pretreatment and method validation are beyond the scope of the present discussion, which concentrates on achieving separation. This article describes only the major steps that are required for most samples. For additional information, the reader is urged to consult the reference by Snyder et al. (see Further Reading) which covers HPLC method development in detail. Additional method development information can be found in the other monographs listed.

Software is described that allows the rapid development of separations by means of isocratic reversed-phase liquid chromatography (RP-LC) based on the optimization of column temperature (T) and mobile phase strength (%B). For a given sample, four initial experiments are carried out at two different temperatures, using either isocratic or (better) gradient elution. If isocratic experiments are chosen for computer simulation, it is necessary to select appropriate values of %B for these initial runs. Literature data for solute retention as a function of T are reviewed as a basis for estimating values of %B at the two values of T selected. The paper describes use of a newly introduced version of DryLab to optimize reversed-phase isocratic separations by varying temperature and %B.

The choice of T and t_G as variables for controlling selectivity and resolution during reversed-phase liquid chromatography (RPLC) method development can be used to minimize problems caused by column batch-to-batch irreproducibility. When a new column fails to provide adequate separation of the sample, altered values of T and t_G can be predicted that will restore the separation obtained with the previous column. Alternatively, columns from different manufacturers can be tested during method development, in order to find a common set of conditions (T and t_G) that provide acceptable separation with two or more of these columns. In this way, any of several columns from different sources become usable for the method. Examples are shown of these different computer-assisted procedures for minimizing problems due to column variability.

The difficulty in separating two compounds generally increases as the molecular structures of the two compounds become more similar. Isomers represent a "worst case" scenario, which can serve as a test of the efficacy of a given method development approach. We have advocated the use of DryLab for method development, with particular stress on simultaneous changes in temperature T and either isocratic %B or gradient time tG for the purpose of optimizing selectivity and band spacing. The application of the latter procedure to 137 different isomer pairs resulted in the separation of 90% of these pairs with a resolution of at least Rs = 1.0. It is concluded that optimizing temperature and gradient time is a good first step in method development.

When separations by reversed-phase liquid chromatography (RP-LC) are carried out at temperatures other than ambient, resulting retention times and bandwidths can depend on the equipment used. As a result, an RP-LC separation that is adequate when carried out on one LC system may prove inadequate when the separation is repeated on a second system. In the present study, various temperature-related problems that can result in a failure of method transfer for non-ambient RP-LC methods were examined using DryLab. Means for correcting for such effects, and thereby ensuring method transferability, are described. Using temperature to optimize HPLC separation, care must be taken to ensure that the column is at the correct temperature. An experimental study is described that leads to simple rules for ensuring good method transfer for methods run at temperatures > ambient.

Optimization of the HPLC separation conditions for the determination of albendazol and its main metabolites by gradient elution using a 2-dimensional tG-T-DryLab-model is demonstrated. The optimal separation of the compounds of interest from endogenous plasma constituens was obtained by simultaneously optimizing gradient range, temperature and gradient time. DryLab sufficiently resolved the peaks of interest from the endogenous plasma components. The results show excellent comparisons of the DryLab models with the real experiments.

Different C18 columns were used with DryLab for the optimization of temperature and gradient steepness for the separation of impurities from a pharmaceutical product. For this application, each of nine different columns gave similar results (a resolution Rs equal to 2.1-2.7), while a column with an embedded polar group gave somewhat better separation (Rs = 3.2).

Four experimental runs where temperature T and gradient time t_G are varied allow the computer-prediction of reversed-phase liquid chromatographic (RPLC) separation for different combinations of temperature and gradient time. This in turn can provide significant changes in selectivity and a resulting optimization of separation. If this procedure is repeated for different columns, additional control over selectivity and resolution becomes possible. The simultaneous variation of T and t_G for columns from different sources was studied for two samples, as a means of evaluating the general advantage of this approach for RPLC method development. Changes in relative retention with T were found to be approximately constant for different values of t_G and for different RPLC columns; similarly, changes in relative retention with tG were roughly independent of changes in temperature or the column. The latter relationships can be useful in matching ("tracking") peaks between runs during method development based on the present approach, as well as for other applications discussed in here and in Part II.

The separation of samples that contain more than 15 to 20 analytes (n>15–20) is typically difficult and usually requires gradient elution. We have examined the reversed-phase liquid chromatographic separation of 24 samples with 8≤n≤48 as a function of temperature T and gradient time tG. The required peak capacity was determined for each sample, after selecting T and tG for optimum selectivity and maximum sample resolution. Comparison of these results with estimates of the maximum possible peak capacity in reversed-phase gradient elution was used to quantify the maximum value of n for some required sample resolution (when T and tG have been optimized). These results were also compared with literature studies of similar isocratic separations as a function of ternary-solvent mobile phase composition, where the proportions of methanol (MeOH), tetrahydrofuran (THF) and water were varied simultaneously. This in turn provides information on the relative effectiveness of these two different method development procedures (optimization of T and tG vs. % MeOH and % THF) for changing selectivity and achieving maximum resolution.

By optimizing column temperature T and gradient time tG, complex samples can often be separated by means of reversed-phase high-performance liquid chromatography (RP-LC). Conclusions reached in Part I suggest that the complete separation of such samples will be difficult, however, when more than 15–20 components are present in the sample. An alternative approach is to carry out two separations with different conditions (T, tG) in each run. The combination of results from these two runs then allows the total analysis of the sample, providing that every sample component is adequately resolved in one run or the other. Examples of this approach, carried out by means of computer simulation, are shown here for several samples of varying complexity. Also considered is the ability of a single separation where T and tG are optimized to enable the separation and analysis of one or more individual sample components from complex mixtures (e.g., drugs in animal plasma), including the resolution of isomeric compounds from each other.

Previous studies have shown that four experimental runs, where both temperature T and gradient time tG are varied, can be used for the reliable prediction of separation as a function of these two variables (two-dimensional optimization). Computer simulation (e.g., DryLab) can then be used to predict “optimized” conditions for maximum sample resolution using either isocratic or gradient elution. Samples that contain a large number of components (e.g., n>15–20) present a greater challenge. Resolution for these more complex samples is often quite sensitive to small changes in T or tG, in turn requiring greater accuracy in predictions that result from computer simulation. In the present study of several samples, we have examined computer simulation errors that can arise from inexact expressions for retention time as a function of T, tG or isocratic %B. Resulting conclusions are applicable to both complex and simpler samples, in either one- or two-dimensional optimization. Means to anticipate and minimize the impact of these predictive errors are examined.

Three ion chromatography (IC) retention models, namely the linear solvent strength model (LSSM), empirical end points model (EEPM) and three-point curve fitting using DryLab from LC Resources were evaluated in terms of their ability to predict retention factors for inorganic anions separated on a Dionex AS11 column using electrolytically generated hydroxide eluents. Extensive experimental retention data were gathered for 21 anions (fluoride, acetate, formate, bromate, chloride, nitrite, methanesulfonate, bromide, chlorate, nitrate, iodide, thiocyanate, succinate, sulfate, tartrate, oxalate, tungstate, phthalate, chromate, thiosulfate and phosphate) using hydroxide eluents of varying concentration. Although the purely theoretical LSSM was found to give adequate performance, the EEPM (in which a linear relationship is assumed between the logarithm of retention factor and the logarithm of eluent strength, but the slope is determined empirically) and DryLab performed better, with DryLab giving the best accuracy and precision of the three models. The EEPM and DryLab were also shown to have advantages in terms of their low knowledge requirements and ease of solution. Compared with IC using dual eluent species, the retention behaviour in IC using single eluent species was found to be easier to model by both theoretical and empirical approaches.

The optimized reversed-phase HPLC separation of 14 different samples is reported, based on simultaneous changes in temperature and gradient steepness. Four experimental runs are required for each sample, following which preferred conditions can be predicted using computer simulation software (DryLab). The overall accuracy and effectiveness of this method development approach is discussed, with particular attention to the use of resolution maps provided by the software. These maps are useful for maximizing resolution for the total sample, for optimizing the separation of a smaller number of selected sample compounds, and as an initial step in the separation of more demanding samples.

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.

In gradient elution separations, it may be required to change either column length (to increase resolution or shorten run time) or column diameter (for an increase in sensitivity or for preparative separations). In either of these changes of column dimensions, it is usually desired to maintain the same relative band spacing (selectivity), so as to increase resolution in proportion to (column plate number)1/2 when increasing column length, or to maintain constant resolution when changing column diameter. Changes in band spacing as a result of change in column size are of special concern when developing procedures for preparative chromatography under gradient conditions.

A method is proposed for the sensitive chiral analysis of amino acid enantiomers by high-performance liquid chromatography (HPLC) using computer modeling with DryLab. 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. 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.

When carrying out HPLC method development, it is often necessary to vary the relative retention of the sample (values of alpha) 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 alpha 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 (alpha). Various practical problems which must be balanced against this ability of a variable to change value of alpha are also discussed. The selection of any two variables for their simultaneous use in controlling alpha is also examined.

It has been shown previously that computer simulation based on two initial experiments can predict separation in reversed-phase gradient elution as a function of gradient conditions (gradient steepness, gradient range and gradient shape) and column conditions (column length, flow-rate and particle size). The present study extends this capability for changes in temperature. Four initial experiments (two different gradient times, two different temperatures) provide input data that allow predictions of separation as a function of temperature as well as gradient and column conditions. A semi-empirical relationship, tR=a+bT, is able to relate gradient retention time tR to column temperature T (other conditions constant). The accuracy of this approach has been evaluated for 102 solutes and a variety of experimental conditions, including the use of five different HPLC instruments (four different models).

A change in temperature (T) or gradient steepness (b) can result in changes in reversed-phase selectivity (α). The magnitude of these changes in α will vary with other separation conditions (column, pH, etc.) and with sample type. In this paper, selectivity changes as a function of T and b are discussed and a simple treatment that allows changes in selectivity to be compared quantitatively for different samples and HPLC conditions is developed. Following papers in this series will apply this theory to arrive at conclusions concerning the use of temperature and gradient steepness in HPLC method development. The present treatment assumes that gradient-steepness selectivity (measured by the parameter S) does not change significantly with temperature. Data for a wide range of compound types and conditions are provided in support of this assumption.

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.

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.

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.

A single gradient elution run with water (A) to acetonitrile (B) as mobile phase can be used to estimate preferred conditions for subsequent method development experiments based on RP-HPLC. 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. 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. 

Initial method development experiments for both neutral and ionic samples are best carried out with reversed-phase high-performance liquid chromatography (HPLC) using acetonitrile-methanol-buffer mobile phases. The preceding paper (Part I) suggests the use of an acetonitrile-buffer gradient to start method development. In this paper, optimum conditions for this first separation are discussed. Sequential method development experiments plus computer simulations are then used to obtain a final HPLC method. In this connection, we have examined how many experiments are required for reliable predictions of ternary-solvent retention. Three experiments are sufficient to predict isoeluotropic retention for methanol-acetonitrile-buffer ternaries where solvent strength does not vary, but five experiments are required for ternaries that contain tetrahydrofuran.

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

Isocratic optimization of coumarin, potassium sorbate, ascorbic acid, sodium benzoate, vanillin, ethylvanillin, methylparaben, ethylparaben, sodium saccharin

Computer simulation by DryLab G/plus has proved to be an invaluable tool in the rapid development of analytical HPLC methods for antibiotics. A glycopeptide antibiotic under study at this Research Center was taken into consideration as a case study. Retention data from two preliminary experiments have allowed us to perform several simulations which greatly shortened the time normally required for the identification of the optimum gradient conditions. The “key steps” in the simulation process have been experimentally verified and a more than satisfactory agreement between calculated and experimental retention times was consistently found.

A complex mixture of arachidonic acid metabolites was separated by reversed-phase HPLC using a multi-step gradient, which was modelled by computer-assisted HPLC method development. The metabolites were extracted on-line on a precolumn connected to the analytical column in the same HPLC system. The predictions of the resolution and also the retention times calculated by computer simulation were very accurate when compared with the corresponding experimental run (maximum deviation 0.86%). An appropriate HPLC method and additionally an on-line extraction procedure could be developed with just three experimental HPLC runs. This method could be useful for evaluating the concentrations of arachidonic acid metabolites involved in inflammatory diseases.

Peptide and protein samples are often complex mixtures that contain a number of individual compounds. The initial HPLC separation of such samples typically results in the poor resolution of one or more band pairs. Various means have been suggested tor varying separation selectivity so as to minimize this problem. In this study of a tryptic digest of recombinant human growth hormone, the simultaneous variation of temperature and gradient steepness was found to be a convenient and effective means of varying selectivity and optimizing the separation. The use of computer simulation greatly facilitated this investigation.

Changes in band spacing as a function of temperature and/or gradient steepness were investigated for four peptide or protein samples. Reversed-phase HPLC in a gradient mode was used to separate tryptic digests of tissue plasminogen activator and calmodulin. Additionally, a synthetic peptide mixture and a storage protein sample from wheat were studied. Simultaneous changes in gradient steepness and temperature were found to provide considerable control over band spacing and sample resolution.

The effects of temperature and gradient steepness on selectivity in these systems appear to be complementary. Simultaneous optimization of both temperature and gradient steepness thus represents a powerful and convenient means of controlling band spacing and separation. Because of the complexity of these sample chromatograms, computer simulation proved to be a useful tool in both interpreting these experiments and in optimizing final separations.

DryLab G/plus and DryLab I/plus (LC Resources) are shown to be effective aids in the development and optimization of gradient and isocratic HPLC conditions for the assay of drug substances and related compounds. Data obtained after two experimental runs in the laboratory are entered into the appropriate program where HPLC conditions can be altered (e.g. flow-rate, column dimensions, mobile phase composition, gradient steepness and shape, etc.) to arrive at optimum separation conditions with less analyst time required. The computer simulations from DryLab G/plus are shown to be suitably accurate under “real life” conditions in the development of gradient purity methods for two drug substances (Zalospirone and WY-47 384) and two synthetic intermediates (cyclooctatetraene and 2-methylcarboxybenzaldehyde). Moreover, DryLab I/plus was shown to be accurate in predicting isocratic retention for the separation of impurities in cyclooctatetraene, both in scaling down to small columns for speed and scaling up to a semi-preparative separation for isolation of impurities.

The pharmaceutically important conjugated estrogens of the type excreted by pregnant mares were baseline-resolved by gas chromatography (GC) on a SE-30 fused-silica open tubular column after acid hydrolysis and conversion to their tert.-butyldimethylsilyl derivatives. The temperature-programmed conditions were optimized with the aid of DryLab GC software with excellent agreement between the predicted and experimental results. The composition of conjugated estrogens in Premarin tablets is described as an application of the method.

The separation by reversed-phase high-performance liquid chromatography of Rp and Sp diastereomers of phosphate-methylated DNA and RNA dinucleotides was studied with respect to pH, organic modifier type and concentration and reversed-phase packing material. Drylab G was used to deduce optimum conditions. On the basis of the observed discrepancies between the computer predictions and experimental results, the gradient operation procedure with volatile buffers was improved. By repetitive chromatography on a 250 × 22 mm I.D. reversed-phase column, fourteen diastereomeric pairs were obtained in at least 97% purity and 60% yield, in amounts of 10–100 mg.

Description of the general principles underlying DryLab I/mp.

Computer simulation was used to optimize the separation of a tryptic digest of recombinant human growth hormone using reversed-phase high-performance liquid chromatography in a gradient mode. DryLab G/plus software modelled the retention behavior of the complex tryptic digest mixture as a function of gradient conditions, based on data from two experimental gradient runs. The theoretical optimum separation conditions were rapidly obtained and reproduced experimentally. Resolution did not simply increase as gradient steepness was decreased, rather, an intermediate gradient time provided maximum sample resolution. The simulation results also indicate that the method is reasonably rugged, with little change in the separation expected for different high-performance liquid chromatography systems, and changes in the separation can be compensated by a change in the gradient steepness. Computer simulation can also be useful to quickly reoptimize conditions for a new column, if it fails to provide the same separation.

Computer simulation depicting column temperature—solute retention relationships in gas chromatography is a potentially effective instructional tool and can reinforce theoretical concepts presented in the classroom. The role of computer simulation of gas chromatographic separations in an academic laboratory is described for simple mixtures of alcohols and hydrocarbons for which retention data and chromatograms are accurately predicted. The technique is also illustrated for a more complex sample, peppermint oil, where computer-generated chromatographic data compare favorably with the corresponding experimental data. The approach can be incorporated into presently conducted experiments in the analytical or organic chemistry laboratory and is applicable for both isothermal and temperature-programmed separations.

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

The method described in this paper was developed with the help of the gradient elution technique. With known dwell volume, gradient elution could be used in routine work without problems. Computer supported HPLC-method development allows the control of a large number of experiments on the screen and verification only of the experimentally promising ones. The method has been developed in 3 days and is approved in practice. With the method, 5 by-products of the synthesis of Atenolol could be identified and quantitatively determined.

Summary of symposium on computer-assisted method development from the 1991 Pittsburgh Conference.

Application of DryLab GC to wide range of environmental, pharmaceutical, and process samples.

An experimental study was carried out for the separation of a series of homologous 2-ketoalkanes by reversed-phase gradient elution. Both linear and non-linear gradients were used. Computer simulation was applied to these same separations, and comparisons were made between experimental and predicted results (values of retention time, retention time difference and bandwidth). Errors in the predicted separation were generally small and similar for both linear and non-linear gradients. Somewhat larger errors (retention time differences) were found in non-linear gradient separations for bands eluting after a change in gradient steepness. This can be attributed to the dispersion (rounding) of the gradient as it passes through the high-performance liquid chromatographic equipment. Computer simulation was demonstrated to provide predictions that are adequate for the purposes of developing an optimized gradient separation.

N,N′-1,2-Ethylenediylbis-l-cysteine dietyl ester (ECD) is a chiral compound and is a key component of Neurolite, a brain imaging agent. In order for the material to be used in the manufacture of Neurolite, it must meet optical rotation and other purity specifications. The optical rotation of ECD is affected by the presence of oxidation-related impurities of the parent material. Prior to this work, the optical rotation was used as a gross indication of these impurities. During product development, information regarding the impurity profile became necessary in order to understand and monitor ECD degradation. A gradient elution high-performance liquid chromatographic method compatible with liquid chromatography—mass spectrometry was developed and optimized using Drylab G software. System suitability of the method was assessed by adding l-methionine ethyl ester and acetophenone to the ECD standard as resolution markers. Comparison of the resolution between each marker and ECD with previous measures of resolution ensures sufficient zone capacity to resolve all potential impurities.

The use of computer simulation software for high-performance liquid chromatographic (HPLC) method development is considered. In particular, gradient elution data entered into DryLab G/plus are used to predict isocratic retention times. The motivation was to establish whether data generated in a research-grade, gradient environment might be used to simulate accurately isocratic HPLC conditions applicable to a process monitoring operation. Good agreement between experimentally obtained and computer-predicted retention times for a homologous series of alkylketones was found for the conversion from gradient to isocratic elution conditions.

DryLab G software was used for the optimization of gradient elution programs in reversed-phase high-performance liquid chromatography applied to the chromatographic analysis of flavonoids present in marsh-mallow (Althaea officinalis). The optimization experiments were carried out for a mixture of eight standard solutes (isolated previously from the plant) and then applied to an extract from the flowers of marsh-mallow. Computer simulation experiments allowed convenient analytical conditions to be chosen. The use of two modifiers, methanol and acetonitrile, made the identification of separated components more certain. Good agreement between simulated and experimental chromatograms was obtained.

Rapid development of robust and reliable high-performance liquid chromatographic methods for routine quality control of Ginkgo biloba is possible with computer simulation. The goal is to reduce method development time and to increase transparency of the complex composition of plant extracts. With only two basic experiments and a peak tracking process based on the total area ratio compared to the individual peak-area ratios a robust method with more than 50 simulated experiments was completed in 8 h. The best method has been verified experimentally. The correlation between the best simulated run and the final experiment was satisfactory.

The use of computer simulation for developing optimized gas chromatographic (GC) separations is illustrated for several samples. Resolution maps (plots of Rsvs. heating rate r) for different starting temperatures provide a means for the rapid exploration of separation as a function of the temperature program in the case of programmed-temperature GC. Isothermal separations are easily developed by trial and error, because of the speed of computer simulation. More complex samples may require multi-ramp temperature programs, t

Computer-simulation with commercially available software (DryLab GC) allows the prediction of isothermal or temperature-programmed gas chromatographic (GC) separation as a function of experimental conditions. In either case, two experimental runs are carried out initially, using a linear temperature program (heating rate different, all other conditions the same). Data from these two runs are entered into the computer, and separation can then be predicted for other conditions: different temperatures in the case of isothermal runs, or any kind of temperature program for programmed runs.

The reliability of resulting predictions was evaluated in the present study for several samples and a wide ranger in separation conditions. Retention time predictions were usually accurate within a few percent, and sample resolution was predicted within about ± 10%. The use of computer simulation should be a considerable help for the rapid development of superior GC methods.

Several different samples and three stationary phases of varying polarity have been examined for changes in band spacing as a function of temperature. The results of these studies have been expressed as a relative variability in the temperature coefficients of retention (S) for adjacent bands. A theoretical analysis suggests that useful changes in band spacing vs. temperature (or heating rate) can be expected when the difference in S values (ΔS) for two bands is larger than 1 to 2%. The samples studied exhibited variations in average values of ΔS of 0.6–6%. This suggests that optimizing the (isothermal) temperature or (programmed) heating rate of a gas chromatographic separation will often be advantageous.

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

Examples of DryLab use in pharmaceutical method development.

Description of commercial program and application to a complex sample (spearmint oil).

A review of the use of gradient elution and computer simulation (DryLab G/plus) for the separation of large biomolecules.

Application of DryLab G/plus to the separation of the 30S ribosomal proteins.

If the dependence of retention on temperature is specified for the various components of a sample in isothermal gas chromatography (GC), it is possible to predict retention, bandwidth, and resolution for programmed-temperature GC separations as a function of experimental conditions. The use of a linear-elution-strength (LES) approximation for isothermal retention allows these predictions to be carried out more easily and conveniently, in turn facilitating rapid simulations with a personal computer. This approach to GC method development appears promising, especially if segmented-temperature programs are used. The LES approximation also provides added insight into how different factors affect separation in programmed-temperature GC.

A computer program (DryLab MP®) is described for the simulation of HPLC separations where two or more variables (e.g., temperature and pH) are changed simultaneously. It is assumed that preliminary method development has resulted in a mobile phase of appropriate strength, such that 1 < k′ < 20 for all bands in the chromatogram. If it is desired to simulate separation as a function of changes in the values of n variables, then one or two additional runs are carried out for each variable —changing only that variable. Retention times for each of the latter runs are entered into the computer, and predictions of separation as a function of all conditions are now possible. Because simultaneous changes in two or more variables can lead to significant interaction effects and less accurate predictions, the software evaluates each simulation for possible errors. Allowed conditions must be capable of predicting values of α with an accuracy better than ± 2% (1 S.D.). The present computer program has a number of possible applications during and following method development, as discussed in the following paper (Part II).

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

Computer simulation was used to optimize high-performance liquid chromatography of phenol and its chloro and nitro derivatives. On the basis of two linear gradient runs of different steepness (RP-18—water + methanol + 1% acetic acid), several simulated gradient runs allowed the optimum gradient programme and flow-rate to be chosen so that the time of analysis could be considerably shortened. Good agreement between simulated and experimental chromatograms was obtained in spite of changes in experimental conditions.

Brief review of currently available expert system and simulation software approaches.

Use of DryLab programs as the core of a systematic method development strategy.

Requirements for new pharmaceutical products and their impact on applications of high-performance liquid chromatography (HPLC) are discussed. The strengths and weaknesses of HPLC in this context are evaluated and compared with current trends and expectation in separation science.

Computer-aided method development techiques have been commercially available for a number of years. Each of these procedures has strengths and weaknesses. In the past, hidden difficulties in the practical application of computeraided method development have discouraged their widespread use. This paper proposes the complementary use of these techniques so that their strengths are maximized while their weaknesses are compensated for. The result is a method development strategy that obtains simple solutions for simple separation problems and reserves the more complex solutions for difficult separations.

Isocratic method for the separation of nitrobenzene, 2,6-dinitrotoluene, benzene, 2-nitrotoluene, 3-nitrotoluene, 4-nitrotoluene, 2-nitro-1,3-xylene, 4-nitro-1,2-xylene. The most robust Separation is available at (60:40)(MeOH:Water) on a 250x4.6mm, 5 µm C18 column at 2 ml/min flowrate.

Summary

The ribosomal 50S and 30S subunit proteins (r-proteins) of Thermus aquaticus have, for the first time, been characterized by size exclusion chromatography (SEC) and by reversed phase high performance liquid chromatography (RPC). To ensure that the best resolution in the RPC was obtained. the elution conditions, such as gradient time, flow rate, temperature, ionic strength of the eluent and the type of stationary phase were optimized. Correlation between experimentally found retention times and those predicted by DryLab G was better than 0.7% over 30 peaks. Protein fractions from RPC runs were desalted and processed by gel electrophoresis so that the ribosomal proteins could be identified by their position on SDS-polyacrylamide gels. The enhanced speed and quality of separation which has been achieved in this study is expected to bring advantages in experimental work with ribosomal proteins as well as with other biopolymers. In our case the high resolution technique provides a basis for the preparation of a collection of individual ribosomal protein components for future rRNA-protein interaction studies.

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.

The separation of seven o-phthalaldehyde-derivatized amino acids by reversed-phase gradient elution was studied as a function of gradient time and mobile phase flow-rate. The resulting separations were compared with those from computer simulation (Drylab G). Predictions of retention time via computer simulation were found to be quite accurate, being about ±0.7% for retention time and about ±7% for retention-time differences (resolution). Predictions of band width were accurate within about ±15% for all but the steepest gradients (b > 1.0). Consequently, the ability of computer simulation to predict chromatograms reliably as a function of gradient conditions and flow-rate was confirmed for a sample that is representative of “real life”. For very steep gradients (b > 1.0), significant errors in band width were observed. The source of these errors could arise from various effects which are discussed.

Using DryLab G to identify a "hidden" band in a protein separation. 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.

The herbicide fluroxypyr 1-methylheptyl ester was separated from its acid form (fluroxypyr) and two soil metabolites (a pyridinol and a methoxypyridine) by high-performance liquid chromatography (HPLC) with the acid of Drylab G. A difference in retention time of at least 6 min between each compound was achieved, to allow complete separation of radiolabeled components during collection of 1-min fractions. The total elution time was 31 min. Actual HPLC retention times differed from DryLab G predictions by 0.5 min or less.

High-performance liquid chromatographic (HPLC) methods for analyzing new drugs and their synthetic intermediates are needed as the synthesis is optimized and scaled up from making milligram amounts for initial evaluation of biological activity to producing kilogram amounts of the drug for thorough testing purposes. The most efficient solution is a single HPLC method that can be used for each step of the synthesis. A practical approach for the development of a single HPLC method is the use of computer-assisted method development to maximize the resolution within a reasonable analysis time. The computer program DryLab I was used in the development of an HPLC assay for the synthetic intermediates of a leukotriene inhibitor. The use of DryLab I with binary mixtures of organic solvents in the organic portion of reversed-phase HPLC systems is reported. With the retention data from two initial analyses, resolution can be optimized as a function of solvent strength.

Using DryLab G to identify a "hidden" band in a protein separation. 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.

A method for prediction ion-exchange isocratic capacity factors from two initial gradient runs is developed. This does not assume so-called linear solvent strength (LSS) conditions, which cause significant errors in k'(C) vs. C relationships in ion-exchange chromatography. The errors associated with this approach and the LSS model are examined. The present approach allows a more accurate prediction of isocratic capacity factors for ion-exchange chromatography. Experimental application of the method to a variety of compounds, including peptides, polynucleotides and polysaccharides, separated by ion-exchange chromatography is described.

Optimization of high-performance liquid chromatography for application to a cough medication (Tussagesic) and its decomposition and byproducts was performed. Special emphasis was placed on the optimization of all parameters that relate to the chemical selectivity of the separation process itself and on the final proof of the ruggedness of the optimized system. All analytes can be reliably determined and the response surface can be represented graphically. This provides a means for improved transfer of methods between laboratories and for efficient system documentation.

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

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

Computer simulations can be used to develop high-performance liquid chromatographic gradient elution methods. However, the usefulness of this approach depends on the accuracy of the resulting predictions. Possible sources of error in computer simulation for the prediction of separation based on gradient elution have been investigated. This has in turn led to recommendations for minimizing such errors. With suitable precautions it appears possible to make adequately reliable predictions of separation by gradient elution. Several examples with protein mixtures as samples are reported.

The effect of the gradient on high-performance liquid chromatographic separations has been examined from a theoretical standpoint, using computer simulations to visualize the effects of different variables. Samples to be separated by gradient elution can be classified according to their separation characteristics into three groups (referred to here as cases I, II and III). Each of these sample types responds differently to a change in gradient conditions.

Case III samples exhibit changes in band spacing when the gradient conditions are varied, and gradients composed of multiple linear segments are especially useful for controlling band spacing and resolution for such samples. The effect of different gradient conditions (starting %B, gradient steepness and gradient shape) on the separation of case III samples is examined in detail.

Application of principles of DryLab G.

The commercially available computer program, DryLab, for optimization of separations by high-performance liquid chromatography (HPLC) using binary solvent mixtures is used to improve an HPLC method for separation of the bitter principle, limonin, in grapefruit and navel orange juices. Best conditions for separation of limonin in a reasonable time are 30 to 32% acetonitrile in water at 0.9 mL/min using a 5-μm C18 column 10 cm long. These conditions are used to analyze grapefruit and navel orange juice samples, and these HPLC results are compared with values determined by enzyme immunoassay or thin-layer chromatography (TLC) on the same samples.

Application of DryLab G method development strategy to a real sample.

Recommendations are presented for an efficient approach to the design of optimized gradients for complex samples using computer simulation. Examples based on the separation of polyaromatic hydrocarbon and ribosomal protein mixtures are shown.

Solvent-strength selectivity refers to the variation of band spacing by changing the %-organic in the mobile phase (ion-pair or reversed-phase HPLC). A review of the literature has been combined with new experimental data to illustrate the general potential of this approach for HPLC optimization. It appears that most samples exhibit significant changes in band spacing method development based on solvent-strength optimization plus computer simulation (DryLab software) are given for illustration. For relatively simple mixtures (10 or fewer components), it appears that solvent-strength optimization compares favorably with other methods such as mapping the organic-solvent selectivity of methanol, acetonitrile, tetrahydrofuran, and water.

A personal-computer program (DryLab G) is described for the simulation of reversed-phase gradient-elution separations. After two experimental gradient runs are carried out initially, this program allows the user to develop a final separation by varying gradient conditions (gradient time, initial and final %-organic in the mobile phase, gradient shape), column dimensions, flowrate and particle size. This approach takes advantage of “solvent-strength selectivity”, as reported recently [28] for isocratic separations. Method development using this procedure can result in better separations with much less effort. Examples of its validation and application are presented.

A general model for describing gradient elution separations of peptides and proteins by reversed-phase high-performance liquid chromatography (HPLC) has been presented previously. This model has now been modified so that it can be applied to any of the four HPLC methods used for separating biological macromolecules: reversed-phase, ion-exchange, hydrophobic-interaction and size-exclusion chromatography, carried out in either an isocratic or gradient elution mode. The role of sample molecule structure and the particular column used has been further studied, so that previous empirical parameters for different column/sample choices can now be estimated from three physical properties of the sample and the column: (a) sample molecular weight, (b) native vs. denatured sample, (c) column packing pore diameter. This eliminates much of the empiricism of our preceding model, and minimizes the number of experimental runs now required in order to apply the model in practice

Application of DryLab 4-5 (the first DryLab program to model retention effects) to the problem of column-to-column reproducibility, adjusting conditions to minimize retention differences.

Computer simulation uses two experimental HPLC runs to allow prediction of sample retention as a function of mobile phase composition or gradient conditions. The general approach is rigorous, but it is assumed that reversed-phase retention obeys the relationship log k' = log kw - S 0. Small deviations in this relationship can lead to error in predicted retention times. We have studied this error empirically for several different reversed-phase systems. This provides a basis for partially correcting these errors, and suggests recommendations for avoiding significant errors during computer simulation.Further work on improving the accuracy of DryLab I and DryLab G predictions, verification of this approach. 

Column plate numbers, N, were measured for 12 different proteins as a function of mobile phase flow-rate in two gel filtration systems (either denaturing or non-denaturing conditions). These data were used to extend a previous model that predicts bandwidths in reversed-phase and ion-exchange chromatography. Restriction of diffusion of large molecules within column packing pores is now defined more precisely, with a single relationship describing this effect for both reversed-phase and size-exclusion chromatography (SEC) (and presumably other high-performance liquid chromatography systems). Separations by gel filtration (SEC) are now included in our general model. A total of 17 flow-rate studies were carried out, involving different proteins, columns and/or mobile phase conditions (denaturing or non-denaturing). Comparisons of plate numbers predicted by the model with experimental values were satisfactory in 15 out of 17 cases. The remaining two cases appear to represent "non-well-behaved" systems, where experimental bandwidths were higher than predicted values by more than 20%. Initial attempts at understanding the origin of these non-ideal effects are described.

First description of DryLab 1 (the ancestor of the column optimization portion of the present DryLab for Windows) as applied to a steroid sample.

First description of DryLab 4-5 (the ancestor of the binary isocratic reversed phase module of DryLab for Windows) as applied to a mixture of nitro-aromatic compounds.

A general model has recently been proposed for the separation of peptides and proteins using reverse-phase gradient elution liquid chromatography. One application of this model suggests that flow rate, gradient time, or column configuration can be varied for band spacing control in the separation of enzymatic digests of proteins. Here a systematic procedure is described that uses repeated separations with different flow rates to maximize the separation of individual peaks within the chromatogram. From these initial separations it is possible to choose an optimum flow rate for the separation of a given sample. It is important in this approach to identify which bands in the various separations correspond to the same peptide. Various peak-tracking procedures are discussed and illustrated.

Summary of the basic model that underlies DryLab G applied to large molecules.

Application of the model described in J. Chromatogr., 327, 27 (1985) to the separation of protein/peptide mixtures by reversed-phase gradient elution.

A previously reported model describes retention and band width as functions of experimental conditions, for the reversed-phase gradient elution separation of peptides and proteins. The model begins by relating separation in gradient elution to corresponding separations by isocratic elution (same high-performance liquid chromatographic system). The use of certain semi-empirical relationships then allows band width to be predicted for samples for which isocratic data are unavailable. This in turn allows the prediction of band width, peak capacity and relative peak height (or peak volume) as a function of experimental conditions such as column dimensions, gradient conditions, mobile phase flow-rate, sample molecular weight, etc. The next paper [J. Chromatogr., 327 (1985) 93] demonstrates the practical value of this approach for optimizing the separation of peptide and protein mixtures.In the present paper it is first shown that isocratic data for desamido-insulin (6000 daltons) allow the accurate prediction of band widths as a function of experimental conditions in related gradient separations. It is further shown that the present model accurately predicts how band widths vary with experimental conditions for peptides and proteins having molecular weights in the range 600–80 000 daltons.

The presently accepted theory for gradient separations of small molecules has been used to develop a predictive model for peptides and proteins as samples, using reversed-phase high-performance liquid chromatography. Given the experimental conditions (gradient time, flow-rate, temperature, etc.), the molecular weight of the sample, and certain column characteristics (Knox parameters, column dimensions, particle diameter, etc.), it is possible to calculate the overall results of a given separation by gradient elution: peak capacity or average resolution, peak volume or relative peak height, etc. This information can in turn facilitate the optimized separation of any sample. The present model assumes that isocratic and gradient retention are interrelated for peptide molecules, in the same fashion as for small molecules. This assumption has been verified for various peptides and proteins and further used to gain new insight into the control of retention and band-spacing in gradient elution.

Equipment for high-performance liquid chromatographic (HPLC) gradient elution generally distorts the gradient selected by the operator, which in turn affects the retention of solutes separated by gradient elution. A theoretical analysis describes these gradient distortions as a function of equipment design and operating conditions. Comparisons of theory with experimental data show generally good agreement. As a result, it is now possible to select gradient conditions for minimal gradient distortion, or to correct for the effect of gradient distortion on solute retention. This will be shown in later papers to allow the use of gradient elution in new ways for more efficient method development and optimization of separation by HPLC.An analysis of instrumental factors that can limit the accuracy of gradient retention data.

Under “ideal” conditions it is possible to model retention in gradient elution so as to be able to calculate retention times, tG, as a function of isocratic retention in corresponding liquid chromatographic systems. In this paper we consider various “non-ideal” processes that lead to errors in calculated values of tG. 

An HPLC-method for the measurement of blood Cyclosporin A levels (CyA) of renal allograft transplanted patients within 9 min is described. After a simple protein precipitation of the blood the supernatant is transferred to an HPLC-system. The short time of analysis is obtained by a step gradient elution technique and a precolumn separation of the fractions of interest followed by a backflush regeneration step of the precolumn. The analysis of the fraction of interest takes place on a column with high resolution power as long as the precolumn is regenerated. CyA is monitored by UV-absorption at 206 nm. Detection of 20 to 2000 ng/ml Cya allows the use of the method for patient monitoring and for research purposes.

Preliminary model for predicting large-molecule separations by gradient elution, particularly for reversed-phase LC

Reduced plate height (h) vs. reduced velocity (v) plots have been measured over a wide range of v for 36 high-performance liquid chromatographic systems. Column type was varied over wide limits and solute capacity factor (k′) values were changed over the range 0.6–22. Resulting data can be accurately described by the Knox equation h = Av1/3 + B/v + Cv, where A is roughly constant (A = 0.5–0.8) for all columns studied, but values of B and C are strongly dependent on column type and solute k′ values. Reduced plate height (h) vs. reduced velocity (v) plots have been measured over a wide range of v for 36 high-performance liquid chromatographic systems. Resulting data can be accurately described by the Knox equation h = Av1/3 + B/v + Cv, where A is roughly constant (A = 0.5–0.8) for all columns studied, but values of B and C are strongly dependent on column type and solute k′ values. 

Analysis of proteins in solution by high-performance liquid chromatography is presented with respect to structural changes in solution, adsorption processes and differentation concerning specific activities. Trypsin and cellulases were taken as examples.

Development of the basic theory relating gradient and isocratic separations, essential to later work on DryLab I and DryLab G.

Charcoal, a nonpolar sorbent, had been widely used for the separation of peptides1 before the advent of ion-exchange chromatography. Recent developments in high performance liquid chromatography revived the interest in the use of nonpolar stationary phases for the separation of biological substances by “reversed phase” chromatography, which employs columns packed with 5 or 10µm porous silica particles having hydrocarbonaceous functions covalently bound to the surface.

This report illustrates the potential of this type of chromatography for the rapid analysis of minute quantities of peptide mixtures. The results suggest that octadecyl-silica columns can be used for fast separation of a wide variety of peptides. By monitoring the column effluent with a UV-detector at 200 nm, the sample components can be analyzed at the subnanomole level without the formation of UV absorbing or fluorescent derivatives.

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" counterion (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.

The effect of solute ionization on the retention of weak acids, bases, and ampholytes on octadecylsilica was investigated both theoretically and experimentally. The retention was attributed to a reversible association of the stationary phase. A phenomenological treatment of the corresponding equilibria was developed for various types of ionogenic substances. The energetics of the association process was analyzed in a rigorous fashion in the light of the solvophobic theory and a semi-empirical extension of the Debye-Hückel theory to high ionic strength. The predicted effect of solute ionization on the capacity factors was substantiated by experimental data. The observed dependence of the capacity factors on the ionic strength of the eluent and the hydrophobic surface of the solute molecules showed good agreement with the theory. The advantages of the technique in the separation of biological substances are illustrated.

Microparticulate non-polar stationary phases, such as octadecyl-silica offer a rapid and efficient means for the separation of peptides and amino acids by high-performance liquid chromatography. Retention is attributed to hydrophobic interaction between the solutes and the hydrocarbonaceous functions covalently bound to the stationary phase surface. Consequently the species are eluted in the order of increasing hydrophobicity. Various peptide mixtures were analyzed by using gradient elution with increasing acetonitrile concentration in the eluent and monitoring the column effluent at 200 or 210 nm with an UV detector. The separation of angiotensins and enzymic digest of polypeptides illustrates the speed of the method which can be used to assay the purity of peptide hormones such as α-melanotropin and gramicidin or to analyze the composition of reaction mixtures involving peptides. The efficiency of the method is superior to that obtained on the conventionally used ion-exchanger columns, except for hydrophilic amino acids and peptides that are poorly retarded. Nevertheless, with a suitable ionic surfactant in the mobile phase, non-polar stationary phases can be used for the separation of these species as well.

0ver a hundred acidic urinary constituents were separated within 30 min by using 5-µm octadecylsilica columns and gradient elution with increasing acetonitrile concentration in dilute aqueous phosphoric acid solution at 70°. The column effluent was monitored with a UV detector at 280 nm or with a fluorescence detector at 260 nm excitation and 340 nm emission wavelengths. The high sensitivity and speed of analysis, the excellent reproducibility and adequate resolution obtained suggest that this technique may be useful to obtain metabolic profiles in routine clinical work.