Contact Us

Literature

Decades of chromatography research done with DryLab®.

Browse through a complete list of over 280 publications that have been published using DryLab®.

Try searching for:Imre MolnárCsaba Horváthlarge moleculesion-exchangereverse phase

Showing 81–100 of 282 documents

Atmospheric Environment · 2015

Gas phase carbonyl compounds in ship emissions: Differences between diesel fuel and heavy fuel oil operation

Ahmed A. Reda, J. Schnelle-Kreis, J. Orasche, G. Abbaszade, J. Lintelmann, J. M. Arteaga-Salas, B. Stengel, R. Rabe, H. Harndorf, O. Sippula, T. Streibel, R. Zimmermann

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%).

  • Carbonyl compounds
  • DNPH
  • GC–SIM–MS
  • Heavy fuel oil
  • Ship emission
View PDFRead online

Journal of Chromatography A · 2015

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

Pavel Jandera, Tomáš Hájek

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

  • Fast separations
  • Gradient elution
  • Modeling of chromatography
  • Monolithic columns
  • Two-dimensional chromatography
View PDFRead online

Journal of Pharmaceutical and Biomedical Analysis · 2015

Method development for the separation of monoclonal antibody charge variants in cation exchange chromatography, Part II: pH gradient approach

Szabolcs Fekete, Alain Beck, Jenő Fekete, Davy Guillarme

The cation exhange pH gradient approach was evaluated for the characterization of 10 model monoclonal antibodies including panitumumab, natalizumab, cetuximab, bevacizumab, trastuzumab, rituximab, palivizumab, adalimumab, denosumab and ofatumumab. This work shows that retention and resolution can be modelled in cation exchange pH gradient mode, based on only four initial runs (i.e. two gradient times and two mobile phase temperature). Only 6h were required for a complete method optimization when using a 100mm×4.6mm strong cation exchange column. The accuracy of the predictions was excellent, with an average difference between predicted and experimental retention times of about 1%. The 10 model antibodies were successfully eluted in both pH and salt gradient modes, proving that both modes of elution can be considered as multi-product charge sensitive separation methods. For most of the compounds, the variants were better resolved in the salt gradient mode and the peak capacities were also higher in the salt gradient approach. These observations confirm that pH gradient approach may be of lower interest than salt gradient cation exchange chromatography for antibody characterization.

  • Cetuximab
  • Ion exchange
  • Method development
  • Monoclonal antibody
  • pH gradient
View PDFRead online

Journal of Pharmaceutical and Biomedical Analysis · 2015

Establishing column batch repeatability according to Quality by Design (QbD) principles using modeling software

Norbert Rácz, Róbert Kormány, Jenő Fekete, Imre Molnár

Column technology needs further improvement even today. To get information of batch-to-batch repeatability, intelligent modeling software was applied. Twelve columns from the same production process, but from different batches were compared in this work. In this paper, the retention parameters of these columns with real life sample solutes were studied. The following parameters were selected for measurements: gradient time, temperature and pH. Based on calculated results, batch-to-batch repeatability of BEH columns was evaluated. Two parallel measurements on two columns from the same batch were performed to obtain information about the quality of packing. Calculating the average of individual working points at the highest critical resolution (Rs,crit) it was found that the robustness, calculated with a newly released robustness module, had a success rate >98% among the predicted 36=729 experiments for all 12 columns. With the help of retention modeling all substances could be separated independently from the batch and/or packing, using the same conditions, having high robustness of the experiments.

  • DOE
  • DryLab
  • QbD
  • Robustness
  • UPLC-column comparison
View PDFRead online

Talanta · 2015

Protocols for finding the most orthogonal dimensions for two-dimensional high performance liquid chromatography

Danielle N. Bassanese, Brendan J. Holland, Xavier A. Conlan, Paul S. Francis, Neil W. Barnett, Paul G. Stevenson

The selection of two high performance liquid chromatography (HPLC) columns with vastly different retention mechanisms is vital for performing effective two-dimensional (2D-) HPLC. This paper reports on a systematic method to select a pair of HPLC columns that provide the most different separations for a given sample. This was completed with the aid of a HPLC simulator that predicted retention profiles on the basis of real experimental data, which is difficult when the contents of sample matrices are largely-or completely-unknown. Peaks from the same compounds must first be matched between chromatograms to compare the retention profiles and optimised 2D-HPLC column selection. In this work, two methods of matching peaks between chromatograms were explored and an optimal pair of chromatography columns was selected for 2D-HPLC. First, a series of 17 antioxidants were selected as an analogue for a coffee extract. The predicted orthogonality of the standards was 39%, according to the fractional surface coverage ‘bins’ method, which was close to the actual space utilisation of the standard mixture, 44%. Moreover, the orthogonality for the 2D-HPLC of coffee matched the predicted value of 38%. The second method employed a complex sample matrix of urine to optimise the column selections. Seven peaks were confidently matched between chromatograms by comparing relative peak areas of two detection strategies: UV absorbance and potassium permanganate chemiluminescence. It was found that the optimal combinations had an orthogonality of 35% while the actual value was closer to 30%.

  • Bins method
  • HPLC simulation
  • Orthogonality
  • Potassium permanganate chemiluminescence
  • Stationary phase optimisation
  • Two-dimensional high performance liquid chromatography
View PDFRead online

Journal of Pharmaceutical and Biomedical Analysis · 2015

Method development for the separation of monoclonal antibody charge variants in cation exchange chromatography, Part I: Salt gradient approach

Szabolcs Fekete, Alain Beck, Jenő Fekete, Davy Guillarme

Ion exchange chromatography (IEX) is a historical technique widely used for the detailed characterization of therapeutic proteins and can be considered as a reference and powerful technique for the qualitative and quantitative evaluation of charge variants. When applying salt gradient IEX approach for monoclonal antibodies (mAbs) characterization, this approach is described as time-consuming to develop and product-specific. The goal of this study was to tackle these two bottle-necks. By modeling the retention of several commercial mAbs and their variants in IEX, we proved that the mobile phase temperature was not relevant for tuning selectivity, while optimal salt gradient program can be easily found based on only two initial gradients of different slopes. Last but not least, the dependence of retention vs. pH being polynomial, three initial runs at different pH were required to optimize mobile phase pH. Finally, only 9h of initial experiments were necessary to simultaneously optimize salt gradient profile and pH in IEX. The data can then be treated with commercial modeling software to find out the optimal conditions to be used, and accuracy of retention times prediction was excellent (less than 1% variation between predicted and experimental values). Second, we also proved that generic IEX conditions can be applied for the characterization of mAbs possessing a wide range of pI, from 6.7 to 9.1. For this purpose, a strong cation exchange column has to be employed at a pH below 6 and using a proportion of NaCl up to 0.2M. Under these conditions, all the mAbs were properly eluted from the column. Therefore, salt gradient CEX can be considered as a generic multi-product approach.

  • Cetuximab
  • Ion exchange
  • Method development
  • Monoclonal antibody
  • Salt gradient
View PDFRead online

Specification of Drug Substances and Products · 2014

Application of Quality by Design (QbD) to the Development and Validation of Analytical Methods

David K. Lloyd, James Bergum

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.

View PDF

Journal of Pharmaceutical and Biomedical Analysis · 2014

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

Alexander H. Schmidt, Mijo Stanic, Imre Molnár

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

  • Design Space
  • HPLC method development
  • Pramipexole
  • Quality by Design
  • Robustness in routine quality control
View PDFRead online

LCGC North America · 2014

Quality by Design in Pharmaceutical Analysis Using Computer Simulation with UHPLC

Jenő Fekete, Róbert Kormány, Imre Molnár

In this study, the quality-by-design principle is applied instead of trial-and-error in the development of a liquid chromatography method. With few measurements, the appropriate stationary phase and chromatographic conditions such as the composition of mobile phase, gradient time, temperature, and pH can be determined. A mixture of an active pharmaceutical ingredient and its 13 impurities was analyzed on a short narrow-bore column (50 mm × 2.1 mm, packed with sub-2-µm particles) providing short analysis times. The performance of commercial modeling software for robustness testing was systematically compared to experimental measurements and design-of-experiment–based predictions.

Journal of Pharmaceutical and Biomedical Analysis · 2014

Importance of instrumentation for fast liquid chromatography in pharmaceutical analysis

Szabolcs Fekete, Isabelle Kohler, Serge Rudaz, Davy Guillarme

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

  • Core–shell
  • Extra-column variance
  • Gradient dwell volume
  • Maximum system pressure
  • UHPLC
View PDFRead online

Journal of Pharmaceutical and Biomedical Analysis · 2014

Reliability of simulated robustness testing in fast liquid chromatography, using state-of-the-art column technology, instrumentation and modelling software

Róbert Kormány, Jenő Fekete, Davy Guillarme, Szabolcs Fekete

The goal of this study was to evaluate the accuracy of simulated robustness testing using commercial modelling 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 (50×2.1mm, packed with sub-2μm particles) providing short analysis times. The performance of commercial modelling software for robustness testing was systematically compared to experimental measurements and DoE based predictions. 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 significantly 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-2μm particles. Again, thanks to modelling software, we proved that the separation was feasible on all columns within the same analysis time (less than 4min), by proper adjustments of variables.

  • Column interchangeability
  • DryLab
  • Method development
  • Modelling software
  • Robustness
  • UHPLC
View PDFRead online

Journal of Pharmaceutical and Biomedical Analysis · 2014

Reliability of computer-assisted method transfer between several column dimensions packed with 1.3–5μm core–shell particles and between various instruments

Róbert Kormány, Jenő Fekete, Davy Guillarme, Szabolcs Fekete

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 1.3μm 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 50mm×2.1mm, 1.3μm, 10–90% ACN in 5min, T=40°C, pH=3, F=0.5ml/min). This optimal method was successfully tested on columns packed with other particle sizes, namely 1.7 and 2.6μm, 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 2.6μm particles, respectively. On the minimum, the resolution was equal to 1.54 on the 50mm×2.1mm, 2.6μm stationary phase. Next to this, the method was transferred to columns of different lengths, inner diameters and particle sizes (100mm×3mm, 2.6μm or 150mm×4.6mm, 5μm). 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 100mm×3mm and 150mm×4.6mm columns, respectively. This work proves the usefulness and validity of HPLC modelling software for transferring methods between different instruments, column dimensions and/or flow rates.

  • Core–shell particles
  • DryLab
  • Method development
  • Method transfer
  • Modelling software
View PDFRead online

Journal of Chromatography A · 2014

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

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

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

  • Cocktail approach
  • Compact single quadrupole
  • MS-supported automated method development
  • UHPLC–MS
  • UHPSFC–MS
  • metabolism
View PDFRead online

The Column · 2014

UHPLC Method Development and Modelling in the Framework of Quality by Design

Imre Molnár, Hans-Jürgen Rieger, Alexander Schmidt, Jenő Fekete, Róbert Kormány

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.

View PDF

Forensic Science International · 2014

DryLab® optimised two-dimensional high performance liquid chromatography for differentiation of ephedrine and pseudoephedrine based methamphetamine samples

Luke M. Andrighetto, Paul G. Stevenson, James R. Pearson, Luke C. Henderson, Xavier A. Conlan

In-silico optimised two-dimensional high performance liquid chromatographic (2D-HPLC) separations of a model methamphetamine seizure sample are described, where an excellent match between simulated and real separations was observed. 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 9.7min. Depending on which diastereomer is present the potential synthetic pathway can be categorised.

  • Drylab
  • Ephedrine
  • Methamphetamine
  • Multidimensional high performance liquid chromatography
  • Pseudoephedrine
View PDFRead online

Chromatographia · 2014

Robust UHPLC Separation Method Development for Multi-API Product Containing Amlodipine and Bisoprolol: The Impact of Column Selection

Róbert Kormány, Imre Molnár, Jenő Fekete, Davy Guillarme, Szabolcs Fekete

This paper describes a new and fast ultra-high pressure liquid chromatographic separation of amlodipine and bisoprolol and all their closely related compounds, for impurity profiling purposes. Computer-assisted method development 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 quality by design principles using design of experiment with three experimental factors; namely the gradient time (tG), temperature (T), and mobile phase pH. Thanks to modeling software, it was proved 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 resolution between all peak-pairs.

  • Amlodipine
  • Bisoprolol
  • DryLab
  • Method development
  • Quality by design (QbD)
  • UHPLC
View PDFRead online

Analytical Methods · 2014

Multifactorial design principles applied for the simultaneous separation of local anesthetics using chromatography modeling software

Chamseddin Chamseddin, Thomas Jira

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. Three different stationary phases were investigated, which are: Kromasil C18, Prontosil C18-AQ and Luna Phenyl-Hexyl. To build the design space for each column, 12 initial experiments were carried out by systematical variation of the selected critical parameters simultaneously. The chromatographic conditions of these initial runs are based on two different gradient times (tG = 20 and 60 min, linear gradient system from 10–90% organic modifier B) each at two different temperatures (T = 25 and 40 °C) repeated at three different ternary composition of the eluent B (a) 100% acetonitrile, (b) acetonitrile–methanol (50 : 50) (v/v), and (c) 100% methanol. In all experiments the pH value of the eluent A (20 mM phosphate buffer) was kept at 3.0. The mixture of local anesthetics could be well separated on all three stationary phases. Although not demonstrated in this paper, this method should be suitable for the analysis of LAs in pharmaceutical preparations or to detect them in some illegal cosmetics. The results showed that the selectivity and the elution order were similar on Kromasil C18 and Prontosil C18-AQ. On the other hand, a unique selectivity is resulted on Luna Phenyl-Hexyl, which shows, depending on the analytes, some additional interactions, since the separation mechanism on this column is influenced by its different steric and polar properties compared to the separation mechanism of alkyl-bonded phases. The predictions and real experiments were strongly correlated with an average absolute error (ΔtR) of 0.13 min (<8 s).

View PDFRead online

Journal of Chromatography A · 2014

Implementation of gradients of organic solvent in micellar liquid chromatography using DryLab®: Separation of basic compounds in urine samples

J. Rodenas-Montano, C. Ortiz-Bolsico, M. J. Ruiz-Angel, M. C. García-Alvarez-Coque

In micellar liquid chromatography (MLC), chromatographic peaks are more evenly distributed compared to conventional reversed-phase liquid chromatography (RPLC). This is the reason that most procedures are implemented using isocratic elution. However, gradient elution may be still useful in MLC to analyse mixtures of compounds within a wide range of polarities, decreasing the analysis time. Also, it benefits the determination of moderately to low polar compounds in physiological fluids performing direct injection: an initial micellar eluent with a low organic solvent content, or a pure micellar (without surfactant) solution, will provide better protection of the column against the proteins in the physiological fluid, and once the proteins are swept away, the elution strength can be increased using a positive linear gradient of organic solvent to reduce the analysis time. This work aims to encourage analysts to implement gradients of organic solvent in MLC, which is rather simple and allows rapid analytical procedures without pre-treatment or the need of re-equilibration. The implementation of gradient elution is illustrated through the separation of eight basic compounds (β-blockers) in urine samples directly injected into the chromatograph, the most hydrophobic showing large retention in both conventional RPLC and MLC. The use of the DryLab® software to optimise gradients of organic solvent with eluents containing a fixed amount of surfactant above the critical micellar concentration is shown to provide satisfactory predictions, and can facilitate greatly the implementation of gradient protocols.

  • 1-Propanol
  • Basic compounds
  • Gradient elution
  • Micellar liquid chromatography
  • Sodium dodecyl sulphate
  • Urine samples
View PDFRead online

LCGC North America · 2014

Rapid UHPLC Method Development for Omeprazole Analysis in a Quality-by-Design Framework and Transfer to HPLC Using Chromatographic Modeling

Mijo Stanic, Alexander Schmidt

Quality-by-design principles are applied to build in a more scientific and risk-based multifactorial strategy in the development of a UHPLC method for analyzing a drug and its related impurities. 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.

View PDF

Chromatography Today · 2013

Chromatography Modelling in High Performance Liquid Chromatography Method Development

Imre Molnár, Róbert Kormány

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 with a few applications of industrial importance.

View PDF