Chiral Micellar Electrokinetic Chromatography Research Articles

Bile Salts in Chiral Micellar Electrokinetic Chromatography: 2000-2020.

Bile salts are naturally occurring chiral surfactants that are able to solubilize hydrophobic compounds. Because of this ability, bile salts were exploited as chiral selectors added to the background solution (BGS) in the chiral micellar electrokinetic chromatography (MEKC) of various small molecules. In this review, we aimed to examine the developments in research on chiral MEKC using bile salts as chiral selectors over the past 20 years. The review begins with a discussion of the aggregation of bile salts in chiral recognition and separation, followed by the use of single bile salts and bile salts with other chiral selectors (i.e., cyclodextrins, proteins and single-stranded DNA aptamers). Advanced techniques such as partial-filling MEKC, stacking and single-drop microextraction were considered. Potential applications to real samples, including enantiomeric impurity analysis, were also discussed.

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.

Carbohydrate-Based Polymeric Surfactants for Chiral Micellar Electrokinetic Chromatography (CMEKC) Coupled to Mass Spectrometry.

Polymeric surfactants (molecular micelles, MoMs) with a variety of chiral head groups and chain lengths may be the most promising chiral selectors when used for sensitive detection of chiral compounds in micellar electrokinetic chromatography-mass spectrometry (MEKC-MS). Various carbohydrate-based MS compatible surfactants with phosphate and sulfate head groups have been recently synthesized in our laboratory for its application in CMEKC-MS. In this chapter, we illustrate that the synthesized glucopyranoside-based MoMs are fully compatible with electrospray ionization MS and can be successfully used as a chiral selector for high-throughput screening of multiple chiral compounds using MRM mode in CMEKC-MS/MS experiments. This chapter describes in detail synthesis and utility of α- and β-glucopyranoside-based polymeric surfactant with two different chain lengths and head groups. The presented examples optimize the effect of appropriate millimolar concentration of monomer sugar surfactants required for polymerization as it affects the separations of acidic and basic compounds. Under the optimized concentration of the monomer needed for polymerization (i.e., an equivalent monomer concentration of MoMs), the superiority of MEKC-MS over MEKC-UV is evident. Structurally similar basic drugs with the difference in hydrophobicity are first tested in MEKC-MS to find the optimum head group and optimum chain length with the aim for developing a widely applicable polymeric glucopyranoside-based surfactant. The partial enantioresolution of several structurally similar basic compounds is significantly improved when switching from one head group to another head group of the glucopyranoside MoMs. Thus, complementary separations using poly-N-β-D-SUGP versus poly-N-β-D-SUGS were seen. This phenomenon also exists when comparing the MoMs, which differ in an anomeric configuration such as poly-N-α-D-SUGP and poly-N-β-D-SUGP.

Chiral micellar electrokinetic chromatographic separation for determination of L- and D-primary amines released from murine islets of Langerhans.

D-amino acids have been located in various tissues including the endocrine portion of the pancreas, the islets of Langerhans. D-Serine (D-Ser), is of particular interest since it is an agonist for the ionotropic N-methyl-D-aspartate receptors. To examine the potential release of D-Ser and other D-amino acids from islets, a chiral micellar electrokinetic chromatography method was developed by derivatizing primary amines with 2,3-naphthalenedicarboxaldehyde and to achieve resolution of the enantiomers, two surfactants were used in the separation, sodium dodecyl sulfate and sodium deoxycholate. With the optimized conditions, 7 of 13 enantiomeric pairs that were tested had greater than baseline resolution, while the resolution of numerous other L-amino acids and small molecules were maintained. For the 17 compounds that were fully resolved, limits of detection were less than 10 nM. The resulting optimized separation method produced high efficiency peaks, with an average of 300,000 theoretical plates per peak and a peak capacity of 120. The method was used to examine the release of small molecules from groups of 50 murine islets of Langerhans. A peak was detected from islets incubated with 20 mM glucose that co-migrated with a D-Ser standard, although its level was below the quantifiable limit.

Effect of low concentration sodium dodecyl sulfate on the electromigration of palonosetron hydrochloride stereoisomers in micellar electrokinetic chromatography

The effect of low concentrations of sodium dodecyl sulfate (SDS) on the separation of palonosetron hydrochloride (PALO) stereoisomers by micellar electrokinetic chromatography (MEKC) has been investigated. It was found that the addition of SDS prolongs the migration time and the migration order of four stereoisomers changes regularly with the SDS concentration. Good separations for all the four stereoisomers were achieved at appropriate SDS concentration. The effect of SDS on the electromigration (mobilities) of PALO stereoisomers has been studied, in order to explain its effect on the separation by MEKC. It was found that low concentrations of SDS added into the separation media forms negatively charged complexes with PALO stereoisomers and hence reverses their electromigration direction. Furthermore, the migration order between two enantiomeric pairs is also reversed because the enantiomeric pair with a bigger positive mobility than that of another pair turns to have a bigger negative mobility when bound with SDS. Based on these results, the effect of SDS on the MEKC separation of PALO stereoisomers was elucidated reasonably. The performance of the developed chiral MEKC method was validated by the analysis of a real sample.

Development of a chiral micellar electrokinetic chromatography–tandem mass spectrometry assay for simultaneous analysis of warfarin and hydroxywarfarin metabolites: Application to the analysis of patients serum samples

The enantioseparation of warfarin (WAR) along with the five positional and optical isomers is challenging because of the difficulty to simultaneously separate and quantitate these chiral compounds. Currently, no effective chiral CE–MS methods exist for the simultaneous enantioseparation of WAR and all its hydroxylated metabolites in a single run. Polymeric surfactants (aka. molecular micelles) are particularly compatible with micellar electrokinetic chromatography–mass spectrometry (MEKC–MS) because they have a wider elution window for enantioseparation and do not interfere with the MS detection of chiral drugs. Using polysodium N-undecenoyl-l,l-leucylvalinate (poly-l,l-SULV) as a chiral pseudophase in MEKC–MS baseline separation of WAR, its five metabolites along with the internal standard was obtained in 45min. This is in comparison to 100min required for separation of the same mixture with packed column CEC–MS using a vancomycin chiral stationary phase. Serum samples were extracted with mixed-mode anion-exchange (MAX) cartridge with recoveries of greater than 85.2% for all WAR and hydroxywarfarin (OH-WAR) metabolites. Utilizing the tandem MS and multiple reaction monitoring mode, the MEKC–MS/MS method was used to simultaneously generate calibration curves over a concentration range from 2 to 5000ng/mL for R- and S-warfarin, 5 to 1000ng/mL for R- and S-6-, 7-, 8- and 10-OH-WAR and 10 to 1000ng/mL for R and S-4′-OH-WAR. For the first time, the limits of detection and quantitation for most WAR metabolites by MEKC–MS/MS were found to be at levels of 2 and 5ng/mL, respectively. The method was successfully applied for the first time to analyze WAR and its metabolites in plasma samples of 55 patients undergoing WAR therapy, demonstrating the potential of chiral MEKC–MS/MS method to accurately quantitate with high sensitivity.

Analysis of chiral amino acids in cerebrospinal fluid samples linked to different stages of Alzheimer disease

Chiral micellar electrokinetic chromatography with laser-induced fluorescence detection (chiral-MEKC-LIF) was used to investigate D- and L-amino acid contents in cerebrospinal fluid (CSF) samples related to different Alzheimer disease (AD) stages. CSF samples were taken from (i) control subjects (S1 pool), (ii) subjects showing a mild cognitive impairment who remained stable (S2 pool), (iii) subjects showing an mild cognitive impairment that progressed to AD (S3 pool) and (iv) subjects diagnosed with AD (S4 pool). The optimized procedure only needed 10 μL of CSF and it included sample cleaning, derivatization with FITC and chiral-MEKC-LIF separation. Eighteen standard amino acids were baseline separated with efficiencies up to 703,000 plates/m, high sensitivity (LODs in the nM range) and good resolution (values ranging from 2.6 to 9.5). Using this method, L-Arg, L-Leu, L-Gln, γ-aminobutyric acid, L-Ser, D-Ser, L-Ala, Gly, L-Lys, L-Glu and L-Asp were detected in all the CSF samples. S3 and S4 samples (i.e. AD subjects) showed significant lower amounts of L-Arg L-Lys, L-Glu and L-Asp compared to the non-AD S1 and S2 samples, showing in the S4 group the lowest amounts of L-Arg L-Lys, L-Glu and L-Asp. Moreover, γ-aminobutyric acid was significantly higher in AD subjects with the highest amount also found for S4. No significant differences were observed for the rest of amino acids including D-Ser. Based on the obtained chiral-MEKC-LIF data, it was possible to correctly classify all the samples into the four groups. These results demonstrate that the use of enantioselective procedures as the one developed in this work can provide some new light on the investigations of AD, including the discovery of new biomarkers related to different stages of AD.

Chiral analysis of amino acids from conventional and transgenic yeasts

Autolysis of Saccharomyces cerevisiae yeast is the main source of molecules that contribute to the quality of sparkling wines made by the traditional method. In this work, a genetically modified yeast (LS11) is compared to its isogenic wild type strain (BY4741) after autolysis. Chiral micellar electrokinetic chromatography with laser-induced fluorescence detection (chiral-MEKC–LIF) is used to identify and quantify the main d- and l-amino acids from both strains after accelerated autolysis. The procedure includes amino acids extraction, derivatization with FITC and chiral-MEKC–LIF separation in a background electrolyte composed of 100 mM sodium tetraborate, 30 mM SDS, 20 mM β-CD at pH 10.0. The d- and l-forms of Arg, Asn, Ala, Glu and Asp, corresponding to the major amino acids found in these samples, are separated in less than 30 min with efficiencies up to 800,000 plates/m and high sensitivity (i.e., LODs as low as 40 nM were obtained for d-Arg for a signal to noise ratio of three). From these results it is corroborated that the genetic modification brings a faster autolysis of the yeast, releasing a higher amount of l-amino acids to the medium in a short time. Interestingly, the pattern of release of d-amino acids was also different between the transgenic and the conventional yeast strains.

Analysis of Chiral Amino Acids in Conventional and Transgenic Maize

In this work, a new chiral micellar electrokinetic chromatography with laser-induced fluorescence detection (chiral-MEKC-LIF) method is proposed to identify and quantify D- and L-amino acids in three lines of transgenic maize and their corresponding nontransgenic parental lines grown under identical conditions. The optimized procedure includes amino acids extraction, derivatization with FITC and chiral-MEKC-LIF separation in a background electrolyte composed of 100 mM sodium tetraborate, 80 mM SDS, and 20 mM beta-CD at pH 10.0. The D- and L-forms of Arg, Ser, Ala, Glu, and Asp, corresponding to the majority amino acids usually found in maize, are separated in less than 25 min with efficiencies up to 890,000 plates/m and high sensitivity (i.e., LODs as low as 160 nM were obtained for D-Arg for a signal-to-noise ratio of three), allowing the detection of 1% D-Arg in the presence of 99% of its opposite enantiomer. Using this method, different D-amino acids are detected in all investigated maize samples providing the reproducible quantification of the D-enantiomeric excess (% d-aa) for each amino acid calculated as % D-aa = 100D-aa/(D-aa + L-aa). Thus, significant differences were observed among the % d-aa values for the different conventional varieties (Aristis, Tietar, and PR33P66 maize) as could be expected from their natural variability. More interestingly, comparing each conventional maize with its corresponding transgenic line, very similar % D-aa values were obtained for one of the studied maize couples (Tietar vs Tietar-Bt) what could be presented as a new proof of their substantial equivalence. However, significant differences in the % d-aa values were observed for the other lines of maize studied. It is concluded that enantioselective procedures can open new perspectives in the study of transgenic organisms in order to corroborate (or not) the equivalence with their conventional counterparts.

Separation and determination of warfarin enantiomers in human plasma using a novel polymeric surfactant for micellar electrokinetic chromatography–mass spectrometry

Warfarin is a widely used oral anticoagulant which is mostly administrated as a racemic mixture containing equal amount of R- and S-enantiomers. The two enantiomers are shown to exhibit significant differences in pharmacokinetics and pharmacodynamics. In this study, a new chiral micellar electrokinetic chromatography–mass spectrometry (MEKC–MS) method has been developed using a polymeric chiral surfactant, polysodium N-undecenoyl- l,l-leucyl-valinate (poly- l,l-SULV), as a pseudostationary phase for the chiral separation of (±)-warfarin (WAR) and (±)-coumachlor (COU, internal standard). Under optimum MEKC–MS conditions, the enantio-separation of both (±)-WAR and (±)-COU was achieved within 23 min. Calibration curves were linear ( R = 0.995 for ( R)-WAR and R = 0.989 for ( S)-WAR) over the concentration range 0.25–5.0 μg/mL. The MS detection was found to be superior over the commonly used UV detection in terms of selectivity and sensitivity with LOD as low as 0.1 μg/mL in human plasma. The method was successfully applied to determine WAR enantiomeric ratio in patients’ plasma undergoing warfarin therapy.

Chiral micellar electrokinetic chromatography.

Chiral micellar electrokinetic chromatography.

Micellar electrokinetic chromatography-mass spectrometry using a polymerized chiral surfactant.

The coupling of chiral micellar electrokinetic chromatography (CMEKC) to mass spectrometry (MS) using conventional surfactant [above the critical micelle concentration (cmc)] is very challenging. Preliminary investigation in this laboratory indicates that the use of a chiral polymeric surfactant provides one possible solution to this difficult coupling. This is because of many positive attributes of micelle polymers which include zero cmc, lower surface activity, low volatility, high electrophoretic mobility, and function as a suitable separation medium even at lower concentrations of pseudophases. In this work, the feasibility of using poly(sodium N-undecanoyl-L-valinate (poly-L-SUV) in CMEKC-MS is demonstrated. After CMEKC separation, enantiomers of 1,1'-binaphthol (BOH) were detected using electrospray ionization mass spectrometry (ESI-MS) by selected ion monitoring (SIM) in the negative ion mode. Although in the SIM mode ESI-MS parameters (nebulizer pressure, drying gas flow rate, drying gas temperature, and sheath liquid flow rate) affected only the signal-to-noise ratio of (+/-)BOH, two of the ESI-MS parameters (nebulizer pressure, sheath flow rate) were found to have a significant impact on chiral resolution of (+/-)BOH. At the optimum ESI-MS conditions, the enantioseparation of (+/-)BOH was successfully accomplished by varying the buffer pH, concentration of the volatile background electrolyte, and poly-L-SUV.