Development of New Monolithic Polysaccharide-Based Columns for HPLC Enantiomeric Separation

Chondroitin sulfate A and maltodextrin have been studied as potential chiral selectors to provide new polysaccharide-based chiral stationary phases. A capillary electrophoresis method has been involved to figure out the enantiomers recognition behaviour in the presence of chiral selector. Chondroitin sulfate A 0.3% w/v and maltodextrin 10% w/v which found to be the optimal values in CE were immobilized onto monolithic silica epoxy and monolithic silica amine columns by Schiff base reaction respectively. The immobilized chondroitin sulfate A-based and maltodextrin-based CSPs have shown intial enantiomeric separation of amlodipine and verapamil as model compound of by HPLC

Label-free microscale thermophoresis for the study of lactoferrin-drug interaction

In this study, a microscale thermophoretic (MST) method was developed to investigate lactoferrin-drug interaction using label-free system which depends on the intrinsic fluorescence of one interacting partner. The interaction between lactoferrin and the iron chelator deferiprone was first investigated to evaluate the excretion of deferiprone into mother milk during lactation. Furthermore, the interaction between lactoferrin and amphotericin B was investigated for possible explanation of the observed synergistic effect as antifungal effect. The experiments were performed on Monolith NT.115 LabelFree® (NanoTemper Technologies, Munich, Germany). Different concentrations of the intended drugs in the range of 0.007- 250 μM were titrated against 120 nM fixed concentration of lactoferrin which was dissolved in 0.1 M tris buffer at pH 7.4. The measurements were performed using standard capillaries at 20% excitation power and medium MST power. The results indicated a significant interaction between lactoferrin and deferiprone however, no significant interaction between lactoferrin and amphotericin B was observed. The data were analyzed using NT Analysis software which was provided by NanoTemper Technologies. The estimated binding constant for lactoferrin-deferiprone interaction was (8.9 x 10 -6 ± 1.6 SD) which is reported for the first time. This significant binding between lactoferrin and deferiprone may potentiates the drug secretion into mother milk. The technique shows fast and simple approach to study protein-drug interaction without complicated labelling procedure

Immobilization of Chondroitin Sulfate A into Monolithic Epoxy Column for Chiral Separation

Chondroitin sulfate A was successfully immobilized into epoxy monolithic column at a concentration of 3% (w/v) in the presence of ethylene diamine. The epoxy group of monolithic column was first converted to aldehyde group by successive hydrolization and oxidation. A Schiff base reaction at pH 8.0 was used to attach the diamine-spacer to aldehyde group. The chondroitin sulfate A was introduced into the monolithic column by circulating the solution at a flow rate of 0.1 mL/min for 24 hours. The chondroitin sulfate A-immobilized epoxy column was evaluated for chiral separation of verapamil enantiomers under optimized HPLC conditions at a wavelength of 230 nm. As a mobile phase, 20 mM Na2HPO4 (pH 2.9) was used. A resolution (Rs) of about 1.5 was achieved for the separation of verapamil enantiomers. A good repeatability of the retention time at two concentration levels (n=8) with RSD < 1% was obtained. The linear responses of verapamil enantiomers were in the range of 1.0-3.0 ppm with R2 of about 0.994

Greener and Whiter Analytical Chemistry Using Cyrene as a More Sustainable and Eco-Friendlier Mobile Phase Constituent in Chromatography

Cyrene (dihydrolevoglucosenone) was evaluated for the first time as a potential sustainable mobile phase solvent in reversed-phase chromatography. As a benign biodegradable solvent, Cyrene is an attractive replacement to classical non-green organic chromatographic solvents such as acetonitrile and a modifier, co-eluent to known green solvents such as ethanol. Compared to ethanol, Cyrene is less toxic, non-flammable, biobased, biodegradable, and a cheaper solvent. A fire safety spider chart was generated to compare the properties of Cyrene to ethanol and show its superiority as a greener solvent. Cyrene’s behavior, advantages, and drawbacks in reversed-phase chromatography, including the cut-off value of 350 nm, elution power, selectivity, and effect on the column, were investigated using a model drug mixture of moxifloxacin and metronidazole. A monolithic C18 (100 × 4.6 mm) column was used as a stationary phase. Different ratios of Cyrene: ethanol with an aqueous portion of sodium acetate buffer mobile phases were tested. A mobile phase consisting of Cyrene: ethanol: 0.1 M sodium acetate buffer pH 4.25 (8:13:79, v/v/v) was selected as the most suitable mobile phase system for separating and simultaneously determining metronidazole and moxifloxacin. The greenness and whiteness of the method were evaluated using the qualitative green assessment tool AGREE and the white analytical chemistry assessment tool RGB12. Further potentials of Cyrene as a solvent or modifier in normal phase chromatography, liquid chromatography–mass spectrometry, and supercritical fluid chromatography are discussed

Thermophoresis for characterizing biomolecular interaction

The study of biomolecular interactions is crucial to get more insight into the biological system. The interactions of protein-protein, protein-nucleic acids, protein-sugars, nucleic acid-nucleic acids and proteinsmall molecules are supporting therapeutics and technological developments. Recently, the development in a large number of analytical techniques for characterizing biomolecular interactions reflect the promising research investments in this field. In this review, microscale thermophoresis technology (MST) is presented as an analytical technique for characterizing biomolecular interactions. Recent years have seen much progress and several applications established. MST is a powerful technique in quantitation of binding events based on the movement of molecules in microscopic temperature gradient. Simplicity, free solutions analysis, low sample volume, short analysis time, and immobilization free are the MST advantages over other competitive techniques. A wide range of studies in biomolecular interactions have been successfully carried out using MST, which tend to the versatility of the technique to use in screening binding events in order to save time, cost and obtained high data qualit

Comparing monolithic and fused core HPLC columns for fast chromatographic analysis of fat soluble vitamins

HPLC stationary phases of monolithic and fused core type can be used to achieve fast chromatographic separation as an alternative to UPLC. In this study, monolithic and fused core stationary phases are compared for fast separation of four fat-soluble vitamins. Three new methods on the first and second generation monolithic silica RP-18e columns and a fused core pentafluoro-phenyl propyl column were developed. Application of three fused core columns offered comparable separations of retinyl palmitate, DL-α-tocopheryl acetate, cholecalciferol and menadione in terms of elution speed and separation efficiency. Separation was achieved in approx. 5 min with good resolution (Rs >5) and precision (RSD ≤ 0.6 %). Monolithic columns showed, however, a higher number of theoretical plates, better precision and lower column backpressure than the fused core column. The three developed methods were successfully applied to separate and quantitate fat-soluble vitamins in commercial products

Validated Capillary Zone Electrophoresis Method for Impurity Profiling and Determination of NiII(3-OMe-Salophene)

A capillary zone electrophoresis method was developed for the determination of NiII(3-OMe-salophene), a substance with anticancer activity in vitro. A fused silica capillary (56 cm × 100 µm) was used for this purpose. The method was optimized in terms of parameters affecting the electrophoretic conditions in order to optimize separation efficiency and total time of migration. The analysis was best performed using an operating buffer of 50 mM borate, adjusted to pH 9.3, mixed with acetonitrile (50%, v/v) as organic modifier. Injections were performed hydrodynamically by applying a pressure of 50 mbar for 8 s, and a 30 kV separation voltage was selected at 25 °C. Detection was carried out at 250 nm using diode array detector (DAD). The method allowed the separation of NiII(3-OMe-salophene) from four other structurally related impurities in a total migration time (tm) of 8 min. Peak identification was achieved using the standard reference of individual impurities. The purity of the migrated NiII(3-OMe-salophene) was confirmed by Ultra-violet (UV) scan overlay depending on DAD. The linear ranges for the determination of NiII(3-OMe-salophene) was 400–20,000 ng mL−1 with limit of detection (LOD) of 120 ng mL−1. Acceptable intra-day and inter-day precisions were achieved (%relative standard deviation (RSD) results were less than 0.76% and 0.30%, respectively). The proposed method was assessed for greenness and compared to reported methodologies to prove superiority

Investigation of the enantioselective interaction between selected drug enantiomers and human serum albumin by mobility shift-affinity capillary electrophoresis

Mobility shift-affinity capillary electrophoresis was employed for enantioseparation and simultaneous binding constant determination. Human serum albumin was used as a chiral selector in the background electrolyte composed of 20 mM phosphate buffer, pH 7.4. The applied setup supports a high mobility shift since albumin and the drug-albumin complex hold negative net charges, while model compounds of amlodipine and verapamil are positively charged. In order to have an accurate effective mobility determination, the Haarhoff-van der Linde function was utilized. Subsequently, the association constant was determined by nonlinear regression analysis of the dependence of effective mobilities on the total protein concentration. Differences in the apparent binding status between the enantiomers lead to mobility shifts of different extends (α). This resulted in enantioresolutions of Rs = 1.05–3.63 for both drug models. R-(+)-Verapamil (KA 1844 M−1) proved to bind stronger to human serum albumin compared to S-(−)-verapamil (KA 6.6 M−1). The association constant of S-(−)-amlodipine (KA 25 073 M−1) was found to be slightly higher compared to its antipode (KA 22 620 M−1) when applying the racemic mixture. The low measurement uncertainty of this approach was demonstrated by the close agreement of the association constant of the enantiopure S-(−)-form (KA 25 101 M−1)

Protein-Based Affinity Capillary Electrophoresis for Enantioseparation of Calcium Channel Blockers

The development of separation methods for chiral compounds has been an interesting field in pharmaceutical research, particularly the use of proteins as the binding agent. ln this study, the enantioselectivity of calcium channel blockers was investigated using affinity capillary electrophoresis, with human serum as a chiral selector. For this purpose, positively charged of racemic drugs, amlodipine and verapamil were selected as chiral drug models. As the sample was introduced in the capillary electrophoresis system, enantiomer bound to human serum albumin in different extents. Baseline separation was achieved in bare-fused silica capillary with a 20 mM phosphate buffer pH 7 .4, at 15 kV applied voltage and 25 oC. The difference in apparent mobility shifts of enantiomers corresponds to the resolution value of 1.0-3.9. Association constant of the enantiomers was determined using nonlinear regression. R- (+)-verapamil performed to be bound stronger to serum albumin compared with S-(-)-verapamil. The Kn value of S- (-)-amlodipine in the racemic mixture was found to be higher than its antipode. ln addition to this, a close agreement with the S-(-)- enantiomer was achieved for amlodipine

Simultaneous Enantiomeric Separation and Binding Constants Determination by Affinity Capillary Electrophoresis using Human Serum Albumin

The binding of chiral drugs to plasma proteins is potentially enantioselective and frequently exhibits different pharmacological activities. Serum albumin is one of the most important proteins in human plasma and is commonly used as protein ligand in chiral capillary electrophoresis. Furthermore, human serum albumin has been reported as a stereoselective agent for studying enantioselective interactions. The estimation of binding constant highly contributes to enantiomeric-drugs development and quality control. In this study, mobility-shift affinity capillary electrophoresis was employed for the simultaneous enantiomeric separation and determination of binding constant. The enantiomeric separation was conducted at physiological pH of phosphate buffer containing human serum albumin as background electrolyte. Accordingly, a net negative charge of protein- ligand was obtained. Therefore, positively charged racemic drugs of amlodipine and verapamil were selected as chiral model compounds. Electrophoretic parameters of resolution and effective mobility were evaluated. Subsequently, association constant was determined through nonlinear regression of effective mobilities and total ligand concentrations. In the particular separation conditions, human serum albumin in the range of 30-110 μM displayed stereoselectivity to verapamil and amlodipine. As the sample was introduced into the capillary electrophoresis system, enantiomers bound the human serum albumin to different extents. The difference in apparent mobility shifts between enantiomers corresponds to the resolution of about 1.05-3.93. Moreover, R-(+)-verapamil proved to be bound stronger to human serum albumin compared with S-(-)- verapamil. The association constant of S-(-)-amlodipine was found to be higher compared to its antipode when applying the racemic mixture. In addition to this, a close agreement with the S-(-)-enantiomer was achieved