Rapid indirect separation of glutamine enantiomers by micellar electrokinetic chromatography. Analysis of dietary supplements.

Glutamine is the most abundant free proteinogenic α-amino acid. It is naturally produced in the organism and acts as a precursor for the synthesis of different biologically important molecules (such as proteins or nucleotides). However, under stressful conditions, the organism is unable to produce it in enough amounts to function properly. Thus, glutamine (Gln)-based supplements have become increasingly popular over the last decade. Since legal regulations establish that amino acid-based dietary supplements must contain only the L-enantiomer and not the racemate, adequate chiral methodologies are required to achieve their quality control. In this work, an analytical methodology based on the use of micellar electrokinetic chromatography is proposed for the rapid enantiomeric determination of DL-Gln in dietary supplements. Using (+)-1-(9-fluorenyl)-ethyl chloroformate as a derivatizing agent and ammonium perfluorooctanoate as separation medium, the Gln diastereoisomers formed under optimal conditions were separated in 8 min with a resolution of 2.8. The analytical characteristics of the method were evaluated in terms of linearity, precision, accuracy, and limits of detection/quantitation, and they were found appropriate for the analysis of L-Gln-based dietary supplements.

Rapid enantiomeric separation of indacaterol by electrokinetic chromatography.

The first chiral methodology enabling the separation of indacaterol enantiomers was developed in this work by cyclodextrin-electrokinetic chromatography. Indacaterol (IND) is a chiral drug marketed as a pure enantiomer. Then, the separation and quantification of each enantiomer is of great importance for the quality control of pharmaceutical formulations. After selecting the most suitable chiral selector and background electrolyte, two Box-Behnken designs were achieved to optimize the electrophoretic conditions using two different approaches to shorten analysis times: i) decreasing the capillary length, or ii) performing a short-end injection. Indacaterol enantiomers were separated in less than 5 min with a resolution value of 3.6 under the optimal separation conditions: 0.7% (m/v) carboxymethyl-α-cyclodextrin in 50 mM sodium formate buffer (pH 4.0) and using a short-end injection. Then, the analytical characteristics of the method were evaluated and LODs of 0.05 mg/L for S-IND and 0.04 mg/L for R-IND were achieved. Also, the method allowed the detection of a 0.1% enantiomeric impurity (S-IND) in the R-IND-based pharmaceutical formulations. The developed method was applied to the analysis of two pharmaceutical formulations. Percentages of 97 ± 3% and 103 ± 6% of R-IND with respect to the labeled amounts were found.

Enantiomeric analysis of drugs in water samples by using liquid-liquid microextraction and nano-liquid chromatography.

The nano-LC technique is increasingly used for both fast studies on enantiomeric analysis and test beds of novel stationary phases due to the small volumes involved and the short conditioning and analysis times. In this study, the enantioseparation of 10 drugs from different families was carried out by nano-LC, utilizing silica with immobilized amylose tris(3-chloro-5-methylphenylcarbamate) column. The effect on chiral separation caused by the addition of different salts to the mobile phase was evaluated. To simultaneously separate as many enantiomers as possible, the effect of buffer concentration in the mobile phase was studied, and, to increase the sensitivity, a liquid-liquid microextraction based on the use of isoamyl acetate as sustainable extraction solvent was applied to pre-concentrate four chiral drugs from tap and environmental waters, achieving satisfactory recoveries (>70%).

Synthesis and characterization of carnitine-based ionic liquids and their evaluation as additives in cyclodextrin-electrokinetic chromatography for the chiral separation of thiol amino acids.

In this study, new chiral ionic liquids based on the non-protein amino acid L-carnitine as cationic chiral counterpart and several anions (bis(trifluoromethane)sulfonimide (NTf2-), L-lactate- or Cl-) as counterions were synthesized. Moreover, three different salts based on L-carnitine were also synthesized and the other three were commercially acquired and used for comparison. The synthesized ionic liquids and salts were characterized by nuclear magnetic resonance, fourier transform infrared spectroscopy, high-performance liquid chromatography-mass spectrometry, and elemental analysis. Subsequently, they were used as additives to establish a γ-CD-based dual system for the enantiomeric separation of cysteine and homocysteine (previously derivatized with fluorenylmethoxycarbonyl chloride) by capillary electrokinetic chromatography. The effect of the nature of the anionic counterions and the presence of different substituents on the L-carnitine molecule on the chiral separation of both amino acids was investigated. The enantioseparations obtained with each dual system studied were compared in terms of the enantiomer effective mobilities (µeff) and the effective electrophoretic selectivity (αeff). Practically, all the dual systems evaluated exhibited substantially improved enantioseparations for the two amino acids compared with the single γ-CD system.

Chiral Micellar Electrokinetic Chromatography.

The potential of Micellar Electrokinetic Chromatography to achieve enantiomeric separations is reviewed in this article. The separation principles and the most frequently employed separation strategies to achieve chiral separations by Micellar Electrokinetic Chromatography are described. The use of chiral micellar systems alone or combined with other micellar systems or chiral selectors, as well as of mixtures of achiral micellar systems with chiral selectors is discussed together with the effect of different additives present in the separation medium. Indirect methods based on the derivatization of analytes with chiral derivatizing reagents and the use of achiral micelles are also considered. Preconcentration techniques employed to improve sensitivity and the main approaches developed to facilitate the coupling with Mass Spectrometry are included. The most recent and relevant methodologies developed by chiral Micellar Electrokinetic Chromatography and their applications in different fields are presented.

Enantiomeric determination of econazole and sulconazole by electrokinetic chromatography using hydroxypropyl-ß-cyclodextrin combined with ionic liquids based on L-lysine and L-glutamic acid.

Two analytical methodologies based on the combined use of hydroxypropyl-ß-cyclodextrin and two different amino acid-based chiral ionic liquids (tetrabutylammonium-L-lysine or tetrabutylammonium-L-glutamic acid) in electrokinetic chromatography were developed in this work to perform the enantioselective determination of econazole and sulconazole in pharmaceutical formulations. The influence of different experimental variables such as buffer concentration, applied voltage, nature and concentration of the ionic liquid, temperature and injection time, on the enantiomeric separation was investigated. The combination of hydroxypropyl-ß-cyclodextrin and tetrabutylammonium-L-lysine under the optimized conditions enabled to achieve the enantiomeric determination of both drugs with high enantiomeric resolution (3.5 for econazole and 2.4 for sulconazole). The analytical characteristics of the developed methodologies were evaluated in terms of linearity, precision, LOD, LOQ and recovery showing good performance for the determination of both drugs which were successfully quantitated in pharmaceutical formulations. This work reports the first analytical methodology enabling the enantiomeric determination of sulconazole in pharmaceutical formulations.

Amino acid chiral ionic liquids combined with hydroxypropyl-ß-cyclodextrin for drug enantioseparation by capillary electrophoresis.

Four amino acid chiral ionic liquids were evaluated in dual systems with hydroxypropyl-ß-cyclodextrin to investigate the enantioseparation by CE of a group of seven drugs as model compounds (duloxetine, verapamil, terbutaline, econazole, sulconazole, metoprolol, and nadolol). The use of two of these chiral ionic liquids (tetramethylammonium L-Lysine ([TMA][L-Lys]) and tetramethylammonium L-glutamic acid ([TMA][L-Glu])) as modifiers in CE is reported for the first time in this work whereas tetrabutylammonium L-lysine ([TBA][L-Lys]) and tetrabutylammonium L-glutamic acid ([TBA][L-Glu]) were employed previously in CE although very scarcely. The effect of the nature and the concentration of each ionic liquid added to the separation buffer containing the neutral cyclodextrin on the enantiomeric resolution and the migration time obtained for each drug, was investigated. A synergistic effect was observed when combining each chiral ionic liquid with hydroxypropyl-ß-cyclodextrin in the case of the five compounds for which the cyclodextrin showed enantiomeric discrimination power when used as sole chiral selector (duloxetine, verapamil, terbutaline, econazole, sulconazole). Buffer concentration and pH, temperature and separation voltage were varied in order to optimize the enantiomeric separation of these five compounds using dual systems giving rise to resolutions ranging from 1.1 to 6.6.

Advances in the Determination of Nonprotein Amino Acids in Foods and Biological Samples by Capillary Electrophoresis.

There are hundreds of nonprotein amino acids whose importance in food and biological matrices is still unknown. Many of these compounds can be found in food as products formed during the processing, as metabolic intermediates or because they are added to increase functional and nutritional properties of food. Moreover, this kind of amino acids have also demonstrated to play relevant roles in the pharmaceutical and clinical fields since they may be used therapeutically in the treatment of some pathologies and their levels may be related with some diseases. These facts imply that the analysis of nonprotein amino acids can be useful to obtain relevant information in the food and biological fields. This article reviews the most recent advances in the development of analytical methodologies employing capillary electrophoresis for the achiral and chiral analysis of nonprotein amino acids in food and biological samples. With this aim, the most relevant information concerning the separation and detection of these compounds by capillary electrophoresis is discussed and detailed experimental conditions under which their determination was achieved in food and biological samples are given covering the period of time from 2015 to 2018.