Separation Of Amino Acid Enantiomers Research Articles

Automated sample preparation with 6-Aminoquinolyl-N-hydroxysuccinimidyl carbamate and iodoacetamide derivatization reagents for enantioselective liquid chromatography tandem mass spectrometry amino acid analysis

Enantioselective amino acid analysis is gaining increasing importance in pharmaceutical, biomedical and food sciences. While there are many methods available for enantiomer separation of amino acids, the simultaneous analysis of all chiral proteinogenic amino acids by a single method with one column and a single condition is still challenging. Herein, we report an enantioselective high-performance liquid chromatography-tandem mass spectrometry (LC-MS) assay using Chiralpak QN-AX as chiral column. With 6-aminoquinolyl-N-hydrosysuccinimidyl carbamate (AQC) as derivatization reagent, efficient enantioselective separation of D- and L-amino acids using HPLC has become possible. Thiol-containing amino acids like Cys are alkylated prior to AQC-labelling. A protocol for automated sample preparation including both derivatization step and calibrator preparation is presented. For compensating matrix effects, u-13C15N-labelled internal standards (IS) were employed. The method was validated and applied to the enantioselective analysis of amino acids in a bacterial fermentation broth.

Ultrafast simultaneous chiral analysis of native amino acid enantiomers using supercritical fluid chromatography/tandem mass spectrometry

In the chiral separation of amino acids, liquid chromatography has been mainly used because of the physicochemical properties of the analytes. To date, only few reports of the use of supercritical fluid chromatography (SFC) for the analysis of chiral amino acids exist, and there is much room for improvement in terms of the number of measurable amino acids, peak shape, and analysis time. In this study, we developed a novel method for the chiral analysis of native amino acids using a system combining SFC and tandem mass spectrometry. Specifically, the separation of amino acid enantiomers was investigated using a CROWNPAK CR-I(+) column with a chiral stationary phase of optically active crown ether. Methanol/water mobile phase with trifluoroacetic acid as a modifier based on supercritical carbon dioxide (CO2) was used. At a low modifier concentration of 30% for the separation of hydrophilic compounds, 18 proteinogenic amino acid enantiomers except glycine and proline were successfully separated with resolution (Rs) = 1.96–33.62 within 6.5 min. In attempt to shorten the analysis time, the flow rate was increased; using a CO2/modifier ratio of 60/40 at a flow rate of 3 mL/min, ultrafast chromatography of 17 amino acid enantiomers, except histidine, was achieved with retention time ≤ 1 min and resolution ≥ 1.5. The developed ultrafast chiral separation method was verified by analyzing a commercially available black vinegar, which detected eight kinds of d-amino acids. The present method has thus confirmed to be successful and practical in terms of both analyte coverage and throughput.

Powerful Steroid-Based Chiral Selector for High-Throughput Enantiomeric Separation of α-Amino Acids Utilizing Ion Mobility-Mass Spectrometry.

Stereospecific recognition of amino acids (AAs) plays a crucial role in chiral biomarker-based diagnosis and prognosis. Separation of AA enantiomers is a long and tedious task due to the requirement of AA derivatization prior to the chromatographic or electrophoretic steps which are also time-consuming. Here, a mass-tagged chiral selector named [d0]/[d5]-estradiol-3-benzoate-17β-chloroformate ([d0]/[d5]-17β-EBC) with high reactivity and good enantiomeric resolution in regard to AAs was developed. After a quick and easy chemical derivatization step of AAs using 17β-EBC as the single chiral selector before ion mobility-mass spectrometry analysis, good enantiomer separation was achieved for 19 chiral proteinogenic AAs in a single analytical run (∼2 s). A linear calibration curve of enantiomeric excess was also established using [d0]/[d5]-17β-EBC. It was demonstrated to be capable of determining enantiomeric ratios down to 0.5% in the nanomolar range. 17β-EBC was successfully applied to investigate the absolute configuration of AAs among peptide drugs and detect trace levels of d-AAs in complex biological samples. These results indicated that [d0]/[d5]-17β-EBC may contribute to entail a valuable step forward in peptide drug quality control and discovering chiral disease biomarkers.

Application of achiral-chiral two-dimensional HPLC for separation of phenylalanine and tryptophan enantiomers in dietary supplement.

This study developed a two-dimensional heart-cutting LC method for the separation of amino acid enantiomers. Two approaches for achiral separation of amino acids, phenylalanine and tryptophan, were selected. Amino acids were separated on C18 or hydrophilic interaction liquid chromatography (HILIC) columns in first dimension after their enantiomer separation on a teicoplanin chiral column in second dimension. Mobile phases for both separation systems were optimized by testing different types of organic modifiers, concentrations of ion-pair agent (sodium 1-octanesulfonate), and ionic modifier (ammonium acetate). The resolution of enantiomers higher than 1.5 for both amino acids was achieved using a C18-teicoplanin coupled column separation system with mobile phases methanol/2 mM sodium 1-octanesulfonate (10:90 and 75:25, step gradient between achiral and chiral columns, respectively). The lower resolution of amino acid enantiomers (RS ˃ 0.9), but higher column efficiency, was achieved on a HILIC-teicoplanin separation system with mobile phases acetonitrile/50 mM ammonium acetate (90:10 and 80:20, step gradient between achiral and chiral columns, respectively). The developed heart-cutting 2D-LC methods were validated in terms of linearity, limit of detection, limit of quantification, precision, and accuracy. The results suggested that the developed methods were applicable for the simultaneous determination of amino acid enantiomers in the dietary supplement. The sample contained l-phenylalanine and l-tryptophan.

Fast enantiomeric separation of amino acids using liquid chromatography/mass spectrometry on a chiral crown ether stationary phase

Fast enantiomeric separation of amino acids was studied by liquid chromatography/mass spectrometry (LC/MS) on a chiral crown ether stationary phase. A chiral crown ether bonded silica column (3mm internal diameter (i.d.), 5cm long) packed with 3μm particles was employed instead of a 15cm column packed with 5μm particles used in our previous study. In addition, the extra-column variance, becoming more serious for smaller columns, was reduced by replacing 0.127mm i.d. post-column tubes with shorter, smaller-diameter (0.0635mm i.d.) tubes. The results demonstrated the benefits of using shorter columns packed with smaller particles and the reduction of the extra-column band broadening for fast enantiomeric separation. Finally, the enantiomeric separation of 18 pairs of proteinogenic amino acids was achieved within 2min with a resolution (Rs) > 1.5 for each pair using an isocratic mobile phase of acetonitrile/water/trifluoroacetic acid (ACN/W/TFA)= 96/4/0.5, and a flow rate 1.2mL/min at 30°C. This is the highest throughput method for simultaneous chiral separation of all proteinogenic amino acids except proline to date.

TLC-based enantiomeric separation of amino acids onto β-CD-incorporated glutaraldehyde-crosslinked PVA electrospun fiber stationary phase

Simple and economical methods for chiral separations are always needed in synthesis and drug development and as biomarkers, besides many other useful applications. Cyclodextrins (CDs) are chiral host molecules and have been used to separate a number of chiral analytes. In this study, we have successfully prepared electrospun films of β-CD incorporated into polyvinyl alcohol (PVA) through glutaraldehyde (GA) crosslinking. These films of β-CD-PVA-GA electrospun fibers are characterized by Fourier transform infrared (FTIR) and scanning electron microscopy (SEM), which were subsequently used for thin-layer chromatography (TLC)-based enantiomeric separation of histidine and serine pairs. Amino acids were detected by spraying the chromatograms with the ninhydrin solution. Among various solvent systems employed, it was found that the separation of serine enantiomers with a resolution of 1.6 was possible with the mobile phase ethanol–butanol–ethyl acetate–water–acetone (4:5:5:0.5:1.5,v/v), and histidine enantiomers with a resolution of 1.4 were possible with the mobile phase ethanol–butanol–ethyl acetate–water–acetone (4:5:4.5:0.5:1.5,v/v). This proves that the prepared stationary phase is efficient in enatioresolution of selected amino acid pairs and can be further examined for physiological samples.

Chiral Discrimination of DL-Amino Acids by Trapped Ion Mobility Spectrometry after Derivatization with (+)-1-(9-Fluorenyl)ethyl Chloroformate.

A novel analytical method based on hybrid trapped ion mobility spectrometry-time-of-flight mass spectrometry (TIMS-TOFMS) has been developed to achieve fast enantiomeric separation of amino acids (AAs). Resolution of chiral AAs was achieved by forming diastereomers through derivatization with the chiral agent (+)-1-(9-fluorenyl)ethyl chloroformate (FLEC), avoiding the use of reference compounds. Electrospray ionization (ESI) in positive mode yielded sodiated FLEC-AAs ions of which the diastereomers could be separated by TIMS. The effect of other alkali metal ions (such as Li and K) on the enantioselectivity was studied, but chiral discrimination was only observed for Na. TIMS conditions, including voltage ramp, ramp time, and accumulation time were optimized for each AA, and collision cross sections (CCSs) were determined for all diastereomers. The migration order of the DL enantiomers was found to be dependent on the structure of the AA. The resulting TIMS resolution (K0/ΔK0) for the FLEC-AA diastereomers on average was 115, requiring a mobility (K0) difference of about 0.009 cm2/(V s) to achieve 50%-valley separation. From the 21 AAs studied, enantiomer separation was achieved for 17 AAs with mobility differences ranging from 0.009 for lysine up to 0.061 cm2/(V s) for asparagine. Moreover, the presented methodology provided mutual separation of various AAs, allowing chiral analysis of multiple AAs simultaneously which may be challenging with previous enantioselective IMS approaches. It appeared possible to fully resolve all studied DL-AAs using three distinct TIMS methods, resulting in a total MS run time of about 3 min (1 min per method) and a total analysis time (including derivatization) of less than 15 min. The method demonstrated capable to determine enantiomeric ratios down to 2.5% with detection limits for the D enantiomers in the nanomolar range. This new TIMS-based methodology opens up possibilities for easy and fast analysis of AA enantiomers.

Chiral separation and determination of amino acid enantiomers in fruit juice by open-tubular nano liquid chromatography.

A novel chiral porous-layer stationary phase was developed for use in open-tubular nano liquid chromatography. The stationary phase was prepared by an in-situ polymerization of 3-chloro-2-hydroxypropylmethacrylate (HPMA-Cl) and ethylene dimethacrylate (EDMA). The reactive chloro groups at the surface of the porous stationary phase were reacted with β-Cyclodextrin (β-CD). The resulting morphology was characterized by using scanning electron microscopy (SEM) and Fourier-transform infrared spectroscopy (FT-IR). The chromatographic performance of the column was evaluated by hydrophilic interaction chromatography (HILIC). Amino acids were used as test solutes. The running buffer conditions for the enantioseparation were found to be 85% acetonitrile (ACN):10%MeOH: 5% H2 O at 0.1% v/v trifluoro acetic acid (TFA) (flow rate: 800nL/min). The enantioseparation provided high theoretical plate numbers up to 26000 platesm-1 . A good retention capacity within satisfactory retention times was also achieved. Real sample applicability of this column to the separation of amino acid enantiomers in fruit juice sample was demonstrated.

Separation and detection of free d- and l-amino acids in tea by off-line two-dimensional liquid chromatography

An off-line 2D-HPLC method was employed to achieve enantiomeric separation of amino acids in tea samples. 16 free amino acids in longjing, black, oolong, and pu-erh teas were separated and collected after FMOC-Cl derivatization in the first dimension, and the fractions were collected and concentrated and then separated on a chiral column.

Separation of amino acid enantiomers by a capillary modified with a metal–organic framework

Covalent bonding of homochiral metal–organic framework in capillaries for amino acid enantiomer separation by capillary electrochromatography.

Specific cooperative effect for the enantiomeric separation of amino acids using aqueous two-phase systems with task-specific ionic liquids

Aqueous two-phase systems (ATPS) based on hydrophilic ionic liquid (IL) and inorganic salt solution were designed and prepared for the enantiomeric separation of racemic amino acids. Two different kinds of hydrophilic ionic liquids (IL-1 and IL-2) containing functional groups were synthesized to separate racemic amino acids. Preliminary experiments showed that d-enantiomer of amino acids cooperatively interacted with ILs, which pushed d-enantiomer to remain in the bottom IL-rich phase. By contrast, l-enantiomer was transferred into the top Na2SO4-rich phase. The enantioselectivity of IL-1 was better than that of IL-2 because of their different intermolecular interactions. Various factors influencing separation efficiency were also systematically investigated including extraction time, IL volume and temperature. Furthermore, the mechanism was studied by 1H NMR and DFT calculations, which showed that the hydrogen bond between the carboxylate and amide groups and the resonance-assisted hydrogen bond between amino and hydroxyl groups conditioned the movement between the residues and ILs. Finally, IL-1 was validated with other general amino acids by the same procedures based on ATPS.

Model-based identification of optimal operating conditions for amino acid simulated moving bed enantioseparation using a macrocyclic glycopeptide stationary phase

Teicoplanin aglycone columns allow efficient separation of amino acid enantiomers in aqueous mobile phases and enable robust and predictable simulated moving bed (SMB) separation of racemic methionine despite a dependency of the adsorption behavior on the column history (memory effect). In this work we systematically investigated the influence of the mobile phase (methanol content) and temperature on SMB performance using a model-based optimization approach that accounts for methionine solubility, adsorption behavior and back pressure. Adsorption isotherms became more favorable with increasing methanol content but methionine solubility was decreased and back pressure increased. Numerical optimization suggested a moderate methanol content (25–35%) for most efficient operation. Higher temperature had a positive effect on specific productivity and desorbent requirement due to higher methionine solubility, lower back pressure and virtually invariant selectivity at high loadings of racemic methionine. However, process robustness (defined as a difference in flow rate ratios) decreased strongly with increasing temperature to the extent that any significant increase in temperature over 32°C will likely result in operating points that cannot be realized technically even with the lab-scale piston pump SMB system employed in this study.

Potential of vancomycin for the enantiomeric resolution of FMOC‐amino acids by capillary electrophoresis‐ion‐trap‐mass spectrometry

The potential of the antibiotic vancomycin (VC) as chiral selector for the enantiomeric separation of amino acids by CE-ESI-MS/MS² was investigated for the first time in this work. Derivatization of amino acids with FMOC-Cl was carried out to enable their interaction with VC as well as the formation of precursor ions with larger m/z which were employed in MS² experiments. The partial filling of a coated capillary was employed to avoid the loss in MS sensitivity originated by the introduction of VC in the ionization source. Under optimized conditions, the simultaneous enantiomeric separation and unequivocal identification of 17 amino acids (two of them being nonprotein amino acids) took place in about 20 min with LODs in the micromolar range.

Preparation, characterization and application of β-cyclodextrin-glutaraldehyde crosslinked membrane for the enantiomeric separation of amino acids

The polysulfone membrane (PS) and β-cyclodextrin glutaraldehyde crosslinked polysulfone membrane (β-CDXM) were prepared by phase inversion technique from the solutions prepared by solubilizing polysulfone polymer, β-cyclodextrin in N, N-dimethyl formamide (DMF) and adding glutaraldehyde. The membranes were extensively characterized by various techniques including attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR), X-ray diffraction (XRD), thermo-gravimetric analysis (TGA), scanning electron microscopy (SEM) and atomic force microscopy (AFM) to correlate membrane characteristics with their performance. ATR-FTIR and XRD studies indicated strong hydrogen bonding interactions between polysulfone and glutaraldehyde crosslinked (XβCD) network. TGA indicates a different range of thermal degradation due to change in enthalpy and presence of crosslinked structure. The membranes were tested by performing separation of enantiomers from racemic phenylalanine (Phe) and racemic tryptophan (Trp) in reverse osmosis. The results of experiments indicated that incorporation of β-cyclodextrin in polysulfone polymer had provided enantiomer discriminating capability to the membrane therefore β-CDXM had exhibited enantioselectivity for D-Phe and D-Trp. The observation confirmed that chiral environment is essential for enantiomer separation. The maximum enantioselectivity (>81%) was observed for the D-Phe.

Separation of tryptophan enantiomers with polypyrrole electrode column by potential-induced technique

Polypyrrole (PPy), synthesized by chemical oxidation in the presence of l-tryptophan ( l-Trp) as template molecules, was packed into a porous ceramic column as the conductive stationary phase to fabricate an electrode column which can be regarded as a working electrode of a three-electrode system from an electrochemical point of view. The electric potential of this column can be easily controlled by a potentiostat. l-Trp anions were ejected from the PPy backbone after overoxidation of PPy, and thus cavities complementary to l-Trp were left in the stationary phase. When the solution containing l- or d-Trp was pumped through the column packed with imprinted overoxidized polypyrrole (oPPy), a negative potential was applied on the electrode column for re-binding Trp enantiomers. The novel electrode column offers an excellent chiral selectivity for l-Trp over d-Trp as the result of stereochemistry, and it also displays satisfactory selectivity for tyrosine (Tyr) enantiomers which is analogous to Trp in structure. The advantage of this method proposed over previously reported is that the separation of amino acids enantiomers can be performed in a high efficiency by controlling the potential applied on the electrode column.

ChemInform Abstract: α-Methoxy-α-trifluoromethylpropionic Acid (MTPr). A New Chiral Derivatizing Reagent for GC Separation of Enantiomeric Amino Acids.

A title homochiral acid has been synthesized and shown to be an efficient chiral derivatizing reagent for capillary gas chromatographic separation of enantiomeric amino acids at considerably low column temperature.

Investigation on chiral capillary columns and their application in separation of enantiomers

Six chiral diamide stationary phases (CSPs), namely N-(3-carbobenzoxypropionyl)-L-Val-tert-butylamide (CSP-1), N-undecenoyl-L-Val-S-α-phenylethylamide (CSP-2), N-undecenoyl-L-Val-R-α-phenylethylamide (CSP-3), OV-225-L-Val-tert-butylamide (CSP-4), XE-60-L-Val-tert-butylamide (CSP-5) and polycyanoethyl vinyl siloxane-L-Val-tert-butylamide (CSP-6), were investigated and CSP-6 was crosslinked within narrow bore (70 μm) fused silica capillary columns. The separation of amino acid enantiomers on this narrow bore column by gas chromatography (GC) is illustrated.

Chiral ligand‐exchange CE assays for separation of amino acid enantiomers and determination of enzyme kinetic constant

This paper deals with studies on the use of Zn(II)-L-ornithine complex as a chiral selecting system for the enantioseparation and UV detection of amino acids (AAs) by using the principle of ligand-exchange CE. Successful enantioseparation of three pairs of label-free aromatic AAs and four pairs of labeled AA enantiomers have been achieved with a buffer of 100.0 mM boric acid, 5.0 mM ammonium acetate, 3.0 mM ZnSO4 and 6.0 mM L-Orn at pH 8.2. This new method was shown to be applicable to the quantitative analysis of D- and L-aromatic AAs, with a linear range between 12.5 and 800.0 microg/mL, and a correlation coefficient above 0.99. Thus this assay, which is facile and relatively rapid, allows us to measure the enzyme catalytic activity in the incubation of D,L-AAs with D-AA oxidase. Using this new method, we can determine the enzyme kinetic constant, lending insight into potential enzyme mechanism.

Capillary electrophoresis and column chromatography in biomedical chiral amino acid analysis

Free amino acids are typically quantified as the sum of their enantiomers, because in terrestrial organisms they mainly exist in the left-handed form. However, with increasing understanding of the biological significance of right-handed amino acids interest in enantioselective quantification of amino acids has steadily increased. Initially, electrophoretic and chromatographic methods using chiral (pseudo)-stationary phases or chiral eluents were applied to the separation of amino acid enantiomers. Later, derivatization of amino acids prior to chromatography with chiral reagents gained in popularity, because the diastereomers formed can be resolved on conventional reversed-phase columns. Novel multi-interaction chiral columns turned attention back to direct chiral chromatographic methods. Hyphenation to mass spectrometry has increasingly replaced optical detection because of superior selectivity, although this has not obviated the need for baseline resolution of amino acid enantiomers. Despite the progress made, enantioselective separation and quantification of amino acids remains an analytical challenge owing to frequently incomplete resolution of all naturally occurring enantiomers and insufficient sensitivity for the determination of the trace amounts of D-amino acids typically found in biological fluids and tissues.

Descriptive structure–separation relationship studies in chiral ligand‐exchange chromatography

A new approach to model and interpret the separation of amino acid enantiomers is described. The descriptive structure-separation relationship (DSSR) study of a set of amino acid enantiomer couples analyzed with two chiral selectors S-benzyl-(R)-cysteine ((R)-SBC) and S-trityl-(R)-cysteine ((R)-STC) has been performed with the aim to highlight the molecular properties of the analyte affecting the enantiodiscrimination process. Although with some exceptions, the classification models obtained with (R)-STC indicate that the partial positive surface area (PPSA-1) is the suitable descriptor to account for the separation of both (R)- and (S)-enantiomers. On the other hand, two different descriptors characterize the separation process with (R)-SBC. While fractional negatively charged partial surface area (FNSA-3) results the discriminating descriptor for (R)-enantiomers, the relative polar surface area (RPSA) is able to best discriminate among (S)-enantiomers.