Literature | MOLNÁR-INSTITUTE

Journal of Chromatography · 1977

Separation of amino acids and peptides on non-polar stationary phases by high-performance liquid chromatography

Imre Molnár, Csaba Horváth

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.

Journal of Chromatography · 1989

Computer-aided optimization of high-performance liquid chromatography in the pharmaceutical industry

E. P. Lankmayr, W. Wegscheider, J. C. Gfeller, N. M. Djordjevic, B. Schreiber

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.

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).

Fresenius' Journal of Analytical Chemistry · 2001

Automatization for development of HPLC methods

M. Pfeffer, H. Windt

Within the frame of inprocess analytics of the synthesis of pharmaceutical drugs a lot of HPLC methods are required for checking the quality of intermediates and drug substances. The methods have to be developed in terms of optimal selectivity and low limit of detection, minimum running time and chromatographic robustness. The goal was to shorten the method development process. Therefore, the screening of stationary phases was automated by means of switching modules equipped with 12 HPLC columns. Mobile phase and temperature could be optimized by using Drylab after evaluating chromatograms of gradient elutions performed automatically. The column switching module was applied for more than three dozens of substances, e.g. steroidal intermediates. Resolution (especially of isomeres), peak shape and number of peaks turned out to be the criteria for selection of the appropriate stationary phase. On the basis of the "best" column the composition of the "best" eluent was usually defined rapidly and with less effort. This approach leads to savings in manpower by more than one third. Overnight, impurity profiles of the intermediates were obtained yielding robust HPLC methods with high selectivity and minimized elution time.

Chromatographia · 2001

Isocratic liquid chromatographic method for the analysis of roxithromycin and structurally related substances in bulk samples

H. K. Chepkwony, F. N. Kamau, E. Rodriguez, E. Roets, J. Hoogmartens

A selective, repeatable, and robust isocratic liquid chromatographic method has been developed for the analysis of bulk samples of roxithromycin, a semi-synthetic macrohde antibiotic. The method involves the use of XTerra RP18 as stationary phase and acetonitrile-2.0 M (NH4)2HPO+ pH 6.4-water, 25:30:45, (v/v) as mobile phase, delivered at a flow rate of 1.0mL min 1. Detection was by UVabsorbance at 215 nm. Sufficient separation of roxithromycin from its homolog containing one more methyleneoxy group (roxithromycin G) and from other related substances was achieved. The robustnessof the method was evaluated by means of a full-factorial experimental design.

Journal of Chromatography · 1993

Use of computer simulations in the development of gradient and isocratic high-performance liquid chromatography methods for analysis of drug compounds and synthetic intermediates

Loren Wrisley

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.

Advanced Materials Technologies · 2020

A Novel Approach to Glycan Method Development for Biotherapeutics using Superficially Porous Particle Technology

Joseph L. Glajch, Andrew Harron, Benjamin P. Libert

In this white paper, Advanced Materials Technology presents a novel method for separating and identifying glycans. Glycans are instrumental in cellular signaling, therefore a specific protein’s glycosylation can influence the effectiveness of the biotherapeutic. Given the importance of glycosylation to the overall performance of a glycoprotein, there is a need to completely characterize and then monitor these glycans for both research and manufacturing purposes.

Journal of Chromatography A · 1999

Critical comparison of retention models for optimisation of the separation of anions in ion chromatography: III. Anion chromatography using hydroxide eluents on a Dionex AS11 stationary phase

John E. Madden, Nebojsa Avdalovic, Peter E. Jackson, Paul R. Haddad

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.

Journal of Pharmaceutical and Biomedical Analysis · 2019

Tuning selectivity in cation-exchange chromatography applied for monoclonal antibody separations, part 1: Alternative mobile phases and fine tuning of the separation

Evelin Farsang, Amarande Murisier, Krisztián Horváth, Alain Beck, Róbert Kormány, Davy Guillarme, Szabolcs Fekete

Cation exchange chromatography (CEX) of therapeutic monoclonal antibodies is generally performed with either salt gradient (MES buffer + NaCl) or using commercial pH gradient buffer. The goal of this study was to find out some alternative buffer systems for CEX separation of mAbs, which may offer alternative selectivity, while maintaining similar peak shapes. Among the new buffers that were tested, (N-morpholino)ethanesulfonic acid (MES) / 1,3-diamino-2-propanol (DAP), and citric acid / 2-(cyclohexylamino)ethanesulfonic acid (CHES) systems were particularly promising, especially when combining them with a moderate salt gradient of NaCl. This two buffer system provides an equivalent or slightly better separation than the standard, mobile phases for therapeutic mAbs. It was also demonstrated that working with salt-mediated pH gradients, allows to extend the possibilities in method development, since the concentration of salt in the mobile phase has a significant impact on selectivity. Using HPLC modeling software (Drylab), it was possible to successfully develop CEX methods for authentic mAb samples within only 6 h, by optimizing the gradient steepness and salt concentration in the B eluent.

Acta Pharmaceutica Hungarica · 2021

Computer-Assisted Approach for the Development of RP-HPLC Methods for the Separation and Quantification of Bioactive Plant Secondary Metabolites

Mohammed Séghir Daas, Massinissa Faci, Isabella Nicoletti, Malika Douzane, Danilo Corradini

This communication describes the development of computer-assisted RP-HPLC methods for the separation, identification and quantification of phenolic compounds, which are a large class of plant secondary metabolites comprising a great number of heterogeneous structures that range from simple molecules to highly Polymerized compounds. The study has been conducted by a Design of Experiments (DoE) approach that allows the simultaneous optimization of gradient time (tG), column temperature (T), and binary eluent composition on the basis of retention times and peak areas of the analytes of interest, obtained in twelve different experiments. These experiments consist in the linear gradient separations of the investigated compounds performed at two different gradient times and column temperatures, using either the aqueous component of the mobile phase at three different pH values or a combination of two organic solvents at three different volume ratios as the gradient former. The RP-HPLC methods developed by the computer-assisted approach described in this paper were used for the separation and quantification of phenolic compounds occurring in fruits of Olea europaea, extra virgin olive oil (EVOO), and olive mill wastewater (OMWW).

Journal of Pharmaceutical Analysis · 2017

Separation of atropisomers by chiral liquid chromatography and thermodynamic analysis of separation mechanism

Ling Zhang, Yue Hu, Elizabeth Galella, Frank P. Tomasella, William P. Fish

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 2.20kJ/mol to 2.42kJ/mol.

European Food Research and Technology · 2026

QbD-based chromatographic phenolic profiling of lentisk and apricot kernel oils with defined chemical composition and antioxidant properties

Kamelia Zane, Anis Chikhoune, Mikhail Shashkov, Alexander Vasilyevich Piligaev, Enrique Sentandreu, Ahmed Benchabane, Isabella Nicoletti, Susana Maria Cardoso, Danilo Corradini

Lentisk (L) and apricot kernel (AKO) oils were characterized according to their phenolic, non-polar and antioxidant profiles. A quality by design (QbD) principles approach optimized phenolic analysis of samples through liquid chromatography coupled to diode array and mass spectrometry (LC-DAD-MS) analyses, firstly reporting catechol in L and vanillin and p-hydroxybenzoic acid in AKO oils. Non-polar fractions of L and AKO was featured by LC-DAD-MS analysis, determining 20 and 14 acylglycerols, respectively, most of them di- and tri-unsaturated acylglycerols. Moreover, analysis of fatty acids, tocopherols and phytosterols by GC-MS research unveiled high contents of unsaturated fatty acids (67% and 89% total content in L and AKO, respectively), mainly oleic, and relevant amounts of tocopherols (384 vs. 471 mg/kg) and physterols (1852 vs. 3511 mg/kg) in L and AKO samples, respectively. Spectrophotometric assays determined low carotenoid (2.18 vs. 0.18 mg luteolin/kg) and chlorophyll (1.51 vs. 0.08 mg pheophytine/kg) contents in L and AKO samples, but demonstrated their high antioxidant/antiradical capacity values. High quality nutritional value of lentisk and apricot kernel oils together to their relevant antioxidant/antiradical properties may favor their use as source of natural bioactive compounds by food and pharmaceutical industries, favoring circular economy through the reuse of agricultural by-products.

Journal of Chromatography A · 1998

Maintaining fixed band spacing when changing column dimensions in gradient elution

J. W Dolan, L. R Snyder

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. A general rule for avoiding changes in band spacing in these situations is to maintain the quantity [(gradient time)×(flow-rate)/(column volume)] constant, while holding the initial and final gradient mobile phase compositions (%B) fixed. This rule is only valid as long as the equipment hold-up volume (dwell volume) is negligible, or if all sample components are strongly retained at the start of the gradient. When neither of the latter conditions apply, then significant changes in band spacing may result when changing column size. Rules are presented for recognizing this potential problem for a given sample/HPLC-equipment combination, and adjustments in separation conditions that can avoid this problem are discussed. 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.

Toxicology and Applied Pharmacology · 2016

An extensive cocktail approach for rapid risk assessment of in vitro CYP450 direct reversible inhibition by xenobiotic exposure

Dany Spaggiari, Youssef Daali, Serge Rudaz

Acute exposure to environmental factors strongly affects the metabolic activity of cytochrome P450 (P450). As a consequence, the risk of interaction could be increased, modifying the clinical outcomes of a medication. Because toxic agents cannot be administered to humans for ethical reasons, in vitro approaches are therefore essential to evaluate their impact on P450 activities. In this work, an extensive cocktail mixture was developed and validated for in vitro P450 inhibition studies using human liver microsomes (HLM). The cocktail comprised eleven P450-specific probe substrates to simultaneously assess the activities of the following isoforms: 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 2J2 and subfamily 3A. The high selectivity and sensitivity of the developed UHPLC-MS/MS method were critical for the success of this methodology, whose main advantages are: (i) the use of eleven probe substrates with minimized interactions, (ii) a low HLM concentration, (iii) fast incubation (5min) and (iv) the use of metabolic ratios as microsomal P450 activities markers. This cocktail approach was successfully validated by comparing the obtained IC50 values for model inhibitors with those generated with the conventional single probe methods. Accordingly, reliable inhibition values could be generated 10-fold faster using a 10-fold smaller amount of HLM compared to individual assays. This approach was applied to assess the P450 inhibition potential of widespread insecticides, namely, chlorpyrifos, fenitrothion, methylparathion and profenofos. In all cases, P450 2B6 was the most affected with IC50 values in the nanomolar range. For the first time, mixtures of these four insecticides incubated at low concentrations showed a cumulative inhibitory in vitro effect on P450 2B6.

Journal of Pharmaceutical and Biomedical Analysis · 2009

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

Szabolcs Fekete, Jenő Fekete, Katalin Ganzler

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

LaborPraxis · 2010

Computergestützte Methodenentwicklung in der HPLC

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

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

Journal of Chromatography A · 2019

Development of a design of experiments optimized method for quantification of polysorbate 80 based on oleic acid using UHPLC-MS

Julia Puschmann, Dirk-H. Evers, Christel C. Müller-Goymann, Michael E. Herbig

The aim was to develop a straightforward UHPLC-MS quantification method for polysorbate 80 using oleic acid as surrogate marker, which was the commonest substance within the emulsifier. However, hydrolysis of polysorbate 80 and subsequent analysis of fatty acids revealed a co-elution of oleic acid and an isomer while all the other fatty acids were successfully separated by varying retention times and mass-to-charge ratios. For identification and separation of the isomer a derivatization method was evaluated. Oxidation to the corresponding dihydroxystearic acids with potassium permanganate resulted in peak separation of cis/trans and structural isomers of the 18:1 fatty acids. Hydrolyzed and derivatized polysorbate 80 was quantified indirectly in the range of 0.046–5.83 μg/mL (R2 > 0.997) with a limit of detection of 11.4 ng/mL. Quantification of polysorbate 80 using oleic acid as a surrogate marker showed good reproducibility and linearity. As all isomers of the 18:1 fatty acids were successfully separated, the previously co-eluting peak was identified as elaidic acid and was found as a component in the mixture of the emulsifier polysorbate 80. Additionally, cis-vaccenic acid was separated as a second co-eluting isomer. Therefore, derivatization led to successful chromatographical separation of cis/trans and structural 18:1 fatty acid isomers.

Science of The Total Environment · 2020

High-performance liquid chromatography (HPLC)-fluorescence method for determination of bisphenol A diglycidyl ether (BADGE) and its derivatives in canned foods

Mingming Guo, Mingfeng He, Jianjun Zhong, Qiao He, Balarabe B. Ismail, Guoying Chen, Donghong Liu

Bisphenol A diglycidyl ether (BADGE) is used as a raw material for the production of epoxy resins and PVC organosols, which are commonly applied as inner coatings for food cans. BADGE and its derivatives can migrate from coatings to foodstuffs during processing and storage thereby creating adverse health issues. In this work, a method based on high-performance liquid chromatography (HPLC)-fluorescence detection (FLD) method was developed for the rapid determination of BADGE and its five derivatives in canned foods. Modeling software DryLab® was applied for the optimization of separation conditions. An adequate separation was achieved in 5 min including equilibration time, using a core–shell particle column; such application has not been reported so far. Also, the results showed that LOD varied from 0.01 to 0.20 ng/g, while LOQ varied from 0.03 to 0.66 ng/g, and RSD was found to be <8.64%. The analytical recoveries ranged from 70.46 to 103.44%. Excellent validation data revealed that this method is suitable for the investigation of can coating-to-food migration of BADGE and its derivatives. The HPLC-FLD method is rapid, inexpensive and highly efficient, which could be applicable for safety inspection of food contact materials involving BADGE and its derivatives.

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.

Journal of Pharmaceutical and Biomedical Analysis · 2011

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

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

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

Decades of chromatography research done with DryLab®.