DL-amino Acids Research Articles

Sustainable solutions for direct TLC enantioseparation with in-home thought-out, prepared/modified chiral stationary phases.

TLC is used globally, yet less attention has been paid to TLC (in enantioseparation) despite its advantages. The present paper describes/reviews successfully practiced direct approaches of 'chiral additive in achiral stationary phase' (as an application of in-home thought out, prepared, tested, and modified chiral stationary phase), 'pre-mixing of chiral reagent with the enantiomeric mixture' (an approach using both achiral phases during chromatographic separation) and 'chiral additive in mobile phase', and chiral ligand exchange for enantioseparation of DL-amino acids, their derivatives, and some active pharmaceutical ingredients. It provided efficient enantioseparation, quantitative determination, and isolation of native forms via in-situ formation of non-covalent diastereomeric pair. The mechanism of enantioseparation in these approaches has been discussed along with the isolation and establishment of the structure of diastereomers. This may help chemists gain useful insights into fields outside their specialization and the experts get brief accounts of recent key developments, providing solutions for sustainable development of less expensive methods for control of enantiomeric purity and isolation of native enantiomers.

Simultaneous determination of free DL-amino acids in human hair with a novel DBD-M-Pro derivatization by UHPLC-HRMS: An application in diabetes patients

Human hair is a non-invasive biological sample that is easy to collect and store and can reflect long-term body health. However, the correlation between DL-amino acids and metabolic diseases in hair samples has not been studied. Therefore, we propose a novel UHPLC-HRMS method for analyzing seven free chiral amino acids (DL-Thr, DL-Glu, DL-Ala, DL-Val, DL-Pro, DL-Leu, and DL-Phe) simultaneously in hair samples by derivatization of chiral probe 4-(N,N-dmethylaminosulfonyl)-2,1,3-benzoxadiazole-trans-2-methyl-L-proline (DBD-M-Pro) labeled with targeted amino functional groups. Gradient elution was carried out using an ACQUITYTM BEH C18 (100×2.1 mm,1.7 μm) column with a mobile phase of 0.15 % formic acid (FA) in 10 mM ammonium acetate (CH3-COONH4) and 0.2 % FA in acetonitrile. The labelled DL-amino acid diastereoisomers could be completely separated, with a resolution (Rs) of 1.59–11.44. These amino acids show a strong linear correlation within the range of 3.1–99.2 pmol (R2 ≥ 0.9990). Intraday and interday precision was 1.87 %–14.87 %. The average recovery was 96.12 %–105.33 %. The limit of detection (LOD) ranged from 0.29 to 2.11 pmol. We then employed the method to determine the concentration of free chiral amino acids in hair samples from 30 healthy volunteers (HVs) and 30 diabetes patients (DPs). Male diabetes patients had significantly higher levels of L-Thr, L-Val, L-Leu (p < 0.05), and D-Ala (p < 0.01) in their hair samples than male healthy volunteers and female diabetes patients had significantly higher levels of D-Ala (p < 0.05) in their hair samples than female healthy volunteers. This is the first study to confirm the feasibility of using free DL-amino acids in human hair as potential biomarkers for diabetes.

Urinary D-amino acid profiles in cats with chronic kidney disease.

Chronic kidney disease (CKD) is highly prevalent in domestic cats. This study aimed to compare urinary D-amino acid levels between control and CKD-afflicted cats as a novel noninvasive method for assessing CKD. Cats were divided into control and CKD stage II groups in accordance with the International Renal Interest Society guidelines. The urinary DL-amino acid levels of the cats were analyzed using chiral tandem liquid chromatography-tandem mass spectrometry, and their medical records were investigated. The CKD group had considerably lower urinary D-amino acid concentrations and enantiomeric ratios than the control group. The total urinary D-amino acid contents significantly correlated with blood parameters (creatinine and urea nitrogen). These findings may contribute towards the detection of CKD stage II in domestic cats.

Simultaneous analysis of DL-Amino acids in foods and beverages using a highly sensitive chiral resolution labeling reagent

Amino acids with various functions are abundant in living organisms and foods. Recent advances in analytical technology show that trace amounts of D-amino acids exist in living organisms and foods. In addition, studies show that these amino acids are involved in various physiological functions that differ from those of L-amino acids. Thus, a technique for analyzing DL-amino acids is required. However, the simultaneous separation and highly sensitive detection of DL-amino acids are complicated; therefore, highly sensitive analytical methods that can rapidly separate and identify compounds are required. We previously developed our original chiral resolution labeling reagents for the separation and highly sensitive detection of DL-amino acids. Here, we developed a simple method for the rapid separation and highly sensitive detection of DL-amino acids in various foods and beverages by liquid chromatography–mass spectrometry (LC–MS) using an octadecyl (C18) column after labeling with 1-fluoro-2,4-dinitrophenyl-5-D-leucine-N,N-dimethylethylenediamineamide (D-FDLDA; enantiomeric excess > 99.9 %). In addition, we synthesized a stable isotope (13C6)-labeled D-FDLDA (13C6-D-FDLDA) and established an analytical method that can accurately identify the peak of each DL-amino acid. MS sensitivity of DL-amino acids labeled with our labeling reagent was higher than that of conventional labeling reagents (Marfey’s reagents). The labeling reagent was neither desorbed from each DL-amino acid nor degraded for at least 1 week at 4 °C. Furthermore, we determined the DL-amino acid contents in foods and beverages using the proposed method, and differences in the total amino acid content and D/L ratio in each food and beverage were observed.

Solubility of Four DL-Amino Acids at Nine Equidistant Temperatures in Aqueous Mixtures of Dipolar Aprotic N,N-Dimethylformamide and the Solution Thermodynamics

Solubility of Four DL-Amino Acids at Nine Equidistant Temperatures in Aqueous Mixtures of Dipolar Aprotic N,N-Dimethylformamide and the Solution Thermodynamics

Separation and Identification of Isoleucine Enantiomers and Diastereomers Using an Original Chiral Resolution Labeling Reagent.

D-Amino acids, which are present in small amounts in living organisms, are responsible for a variety of physiological functions. Some bioactive/biomolecular peptides also contain D-amino acids in their sequences; such peptides express different functions than peptides composed only of L-form amino acids. Among the 20 amino acids that make up proteins, threonine (Thr) and isoleucine (Ile) have two chiral carbons and thus have two enantiomers and diastereomers. These stereoisomers have been previously analyzed through HPLC using chiral columns or chiral resolution labeling reagents. However, the separation and identification of these stereoisomers are highly laborious and complicated. Herein, we propose an analytical method for the separation and identification of Ile stereoisomers through LC-MS using our original chiral resolution labeling reagent, 1-fluoro-2,4-dinitrophenyl-5-L-valine-N,N-dimethylethylenediamine-amide (L-FDVDA) and a PBr column packed with pentabromobenzyl-modified silica gel. Twenty DL-amino acids including Thr stereoisomers (41 amino acids including glycine) were separated and identified using C18 column. Ile stereoisomers could be separated using not a C18 column but a PBr column. Additionally, we showed that peptides containing Thr and Ile stereoisomers can be accurately detected through labeling with L-FDVDA.

Simultaneous quantification of five DL-amino acids in beer by UHPLC–MS/MS using a novel chiral mass spectrometry probe

Simultaneous quantification of five DL-amino acids in beer by UHPLC–MS/MS using a novel chiral mass spectrometry probe

Development of a diphenyl sulfide structure derivatization reagent for amino acid enantiomers analysis: Application of dynamic monitoring in human urine after drinking wine

We developed a novel chiral mass spectrometry derivatization reagent (S)-(3-(4-carboxythiazolidin-3-yl)-3-oxopropyl) diphenylsulfonium (CTOD) with a positively charged sulfur-containing structure for high-sensitivity detection of the chiral resolution of amino acid enantiomers. CTOD reacted with DL-amino acids at 60oC for 60 min to generate the corresponding diastereomers, fifteen chiral amino acid-derived products were separated. Resolution (Rs) values were of the range 1.54-4.36, except Asn 1.07, achieving good separation. A highly sensitive and selective UHPLC-MS/MS method for the simultaneous determination and chiral separation of five chiral amino acids (Pro, Ala, Glu, Asp, and Phe) based on CTOD derivatization was established and applied to the detection of chiral amino acids in different wines. The diastereomeric resolution of the five amino acids was 1.71–5.42, and an excellent linear relationship was obtained in the range of 0.25–500 pmol (R2 ≥0.9993). The detection limit was 0.05–0.25 pmol. The intra- and inter-day precisions were 0.51–5.76% and 0.78–5.18%, respectively, and the average recovery was 90.03–99.99%. In addition, the metabolic concentration of chiral amino acids was monitored after drinking red wine and white wine, and the fitting curve of metabolic concentration was drawn.

Highly sensitive novel fluorescent chiral probe possessing (S)-2-methylproline structures for the determination of chiral amino compounds by ultra-performance liquid chromatography with fluorescence: An application in the saliva of healthy volunteer

We developed a novel fluorescent chiral probe, DBD-trans-2-methyl-L-proline (DBD-M-Pro), which can be used to target recognition of amino functional groups using chiral resolution. To investigate the chiral resolution efficiency, 20 chiral amino enantiomers (19 DL-amino acids and phenylethylamine) were labeled using ultra-performance liquid chromatography (UPLC) with a fluorescence (FL) system. Diastereomers were formed by the reactions of DBD-M-Pro with enantiomers of amino functional groups at 60 °C for 60 min and detected on a BEH C18 column (100 × 2.1 mm, 1.7 μm). Gradient elution of 10 mM ammonium acetate with 0.05% formic acid (FA) aqueous solution and 0.1% FA acetonitrile or 0.1% FA methanol solution was performed at an excitation wavelength (Ex) 460 nm and emission wavelength (Em) 550 nm. Each resulting derivative of D- and L- type was effectively separated. The results showed that the resolution (Rs) of 17 amino acids and phenylethylamine (PEA) in the range of 1.59–24.11, except for histidine (His) (Rs = 1.32) and serine (Ser) (Rs = 1.47), achieved completely separation. The DBD-M-Pro chiral probe has a robust chiral selectivity for D-amino acids. Furthermore, a new method for the simultaneous determination of six DL-amino acids (Pro, Val, Trp, Phe, Leu, Lys) in human saliva was developed. The proposed method showed resolution values of 1.78–16.38, and an excellent linear relationship was obtained in the range of 2.5–500 pmol (R2 ≥ 0.9990). The limit of detection (S/N = 3) ranged from 0.5 to 3.75 pmol. The intra-day and inter-day coefficient of variation (CV) were within the range of 1.75–11.73%. The average addition recoveries in saliva ranged from 95.99 to 106.97%. The methodology was used to determine the content of DL-amino acids and the D/L-amino acid ratio in the saliva of 40 healthy volunteers (15 males and 25 females), as well as evaluating the differences between men and women. Our study suggests that the DBD-M-Pro chiral probe could be an effective tool for screening potential D-amino acid biomarkers in disease.

Rapid enantioselective amino acid analysis by ultra-high performance liquid chromatography-mass spectrometry combining 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate derivatization with core-shell quinine carbamate anion exchanger separation

Amino acid analysis (AAA) is of central importance for the characterization of the amino acid composition of proteins, peptides, pharmaceutical formulations, dietary supplements, in bioanalysis and metabolomics. Common methodologies are based on achiral assays and hence do not consider distinction of D and L-amino acid enantiomers. This may be misleading in many instances, because therapeutic peptides and natural peptides synthesized by non-ribosomal peptide synthetases (NRPSs) frequently contain D-amino acids to achieve proteolytic stability. Furthermore, stereochemical integrity control of peptides made from L-amino acids also need stereoselective assays, and racemization in natural products (proteins, peptides, amino acids) have shown to be useful biomarkers of disease and require assays, which can provide information about the individual stereoisomers. Hence, rapid enantioselective amino acid analysis (ReAAA) is of utmost importance. However, currently employed enantioselective assays are often only focusing on a limited number of amino acids or require long analysis times making them incompatible for high-throughput sample analysis. Here, we present an ReAAA assay, which is based on a fast UHPLC enantiomer separation using a short QN-AX core-shell particle column (2.7 µm) coupled to electrospray ionization quadrupole time of flight mass spectrometric (ESI-QTOF-MS) detection. Amino acid samples are mixed with a [u-13C15N]-labelled L- or DL-amino acid standard mixture, precolumn derivatized with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) reagent and subjected to UHPLC-ESI-MS analysis. A number of chromatographic variables have been optimized (buffer additives and concentration, water content, flow rate and temperature) to achieve the goal of simultaneous amino acid enantiomer separation of all proteinogenic amino acids within the shortest possible analysis time. A mobile phase consisting of 50 mM NH4FA, 50 mM FA and 0.5% H2O in MeOH combined with a flow rate of 1 mL/min and a column temperature of 30 °C has been identified as optimal. With these conditions, all of the proteinogenic DL-enantiomers (except for Arg) can be separated in less than 2.5 min. If D-Arg and distinction between D-Leu/D-Ile is of concern, a second-tier method can accomplish this goal. The method can be very useful for ReAAA when high sample throughput is a major demand. Its utility was demonstrated by ReAAA of a lipopeptide hydrolysate which contained several D-amino acids and of an amino acid supplement in which minor D-amino acid impurities could be detected.

Solvent Effect on the Preparation of Ionic Cocrystals of dl-Amino Acids with Lithium Chloride: Conglomerate versus Racemate Formation

The hydrophobic dl-amino acids alanine, valine, leucine, and isoleucine have been cocrystallized with LiCl via solid-state and solution methods, and the effect of preparation conditions and solvent choice on the racemic versus conglomerate formation has been investigated. For the sake of comparison, enantiopure l-amino acids have also been reacted with LiCl in the same experimental conditions. With dl-alanine only, a racemic ionic cocrystal of formula dl-alanine·LiCl·H2O is obtained, irrespective of the preparation conditions, while the amino acids dl-valine and dl-leucine undergo spontaneous chiral resolution when MeOH is used in ball milling conditions, yielding monohydrated conglomerates, which at ambient conditions convert over time into the racemic ionic cocrystals dl-Val·LiCl·H2O and dl-Leu·LiCl·1.5H2O; these racemic ionic cocrystals (ICCs) are otherwise obtained in a single step if water is employed instead of MeOH, both in ball milling and solution conditions. dl-Isoleucine behaves differently, and product characterization is complicated by the presence of dl-alloisoleucine (dl-aIle) in the commercial starting material; solution crystallization in the presence of excess LiCl, however, unexpectedly results in the formation of the alloisoleucine conglomerate d-aIle·LiCl·H2O and l-aIle·LiCl·H2O, together with unreacted dl-isoleucine. Solid-state syntheses of the ionic cocrystals proceed in most cases via formation of intermediate metastable polymorphs; phase identification and structural characterization for all ICCs have been conducted via single crystal and/or powder X-ray diffraction.

Simultaneous Determination of Free DL-Amino Acids in Natto with Novel Fluorescent Derivatization by UPLC-FL

A new simple and sensitive method for the simultaneous detection of free DL-amino acids (DL-AA) in natto was established by UPLC-fluorescence (FL) using the newly developed fluorescence derivatization reagent DBD-trans-2-methyl-L-proline (DBD-M-Pro) by precolumn derivatization. DBD-M-Pro reacts with DL-amino acids at 60 °C for 60 min. Using BEH C18 (2.1 mm × 100 mm, 1.7 μm) column gradient analysis for 20 min, the resolution of diastereomers after the derivatization of four DL-amino acids was 1.65–3.50. The detection limit for amino acids was 0.25–2.5 pmol. The average recovery (%) of DL-amino acids was 86.65–118.70%. The method was applied to the detection of chiral amino acids in natto beans and mucus, and four chiral amino acids, DL-Gln, DL-Asp, DL-Glu, and DL-Ala, were simultaneously detected. At the same time, the DL-amino acid content and D/L ratio of three different manufacturers of natto were compared quantitatively.

Preparation of Vortex Porous Graphene Chiral Membrane for Enantioselective Separation.

Chiral materials are usually the key to the separation of chiral membranes. In this work, we propose a new strategy that chiral porous graphene membrane can be fabricated from nonchiral porous graphene by mechanical stirring to induce vortex structure. Porous graphene with controlled, nanosized pores was synthesized by a newly designed, one-pot process directly from graphite as opposed to graphene oxide. Then porous graphene was immobilized on ultrafiltration membrane through filtering while stirring to form porous graphene membrane, which was applied for enantioselective separation toward DL-amino acids: for example, the separation factor of l-/d-phenylalanine reached 4.76. Interestingly, we first observed that the front and back sides of the porous graphene membrane exhibited opposite optical activities.

Solvation Thermodynamics of Amino Acids with Hydrophobic and Hydrophilic Side Chains in Aqueous Mixtures of Protic Ethylene Glycol

The zwitterionic as well as solvophilic and solvophobic solvation behavior of DL-tyrosine, DL-leucine, DL-isoleucine and DL-threonine in aqueous and aqueous ethylene glycol systems have been studied at 298.15 K. For this study the solubilities of these amino acids in different solvent mixtures were measured at nine equidistant temperatures in the range of 278.15–318.15 K under 0.1 MPa pressure using the ‘formol titrimetric’ method. The order of experimentally found solubilities is DL-Thr > DL-Ile > DL-Leu > DL-Tyr. The standard transfer Gibbs energies, $$\Delta G_{{\text{t}}}^{{0}} (i)$$ , and entropies, $$T\Delta S_{{\text{t}}}^{{0}} (i)$$ of transfer were computed using the solubility data and are discussed. The observed $$\Delta G_{{\text{t}}}^{{0}} (i)$$ and $$T\Delta S_{{\text{t}}}^{{0}} (i)$$ versus composition profiles are in confusing order because of various interaction effects. The chemical effects of the transfer Gibbs energies $$\Delta G_{{\text{t,ch}}}^{{0}} (i)$$ and entropies of transfer values $$T\Delta S_{{\text{t,ch}}}^{{0}} (i)$$ have been obtained after removal of the cavity forming effect, dipole–dipole and dipole-induced-dipole interaction effects. The chemical contribution of these DL-amino acids are guided by the united effects of gradual change in the dispersion interaction, basicity and acidity, hydrogen bonding effects, solvophobic and solvophilic solvation of aqueous ethylene glycol as compared to those in the reference solvent, pure water. The solvophilic and solvophobic solvation effects of the transfer Gibbs energies, $$\Delta G_{{\text{t,H1HbH}}}^{{0}} (i)$$ , and entropies of transfer, $$T\Delta S_{{\text{t,H1HbH}}}^{{0}} (i)$$ , for the amino acids have been obtained after subtraction of the dispersion effect from the chemical transfer energies, which are guided by the side chains with amino acids.

Preparation and evaluation of an ethylenediamine dicarboxyethyl diamido-bridged bis(β-cyclodextrin)-bonded chiral stationary phase for high performance liquid chromatography

An ethylenediamine dicarboxyethyl diacetamido-bridged bis(β-cyclodextrin) was firstly synthesized through the reaction of 6-deoxy-6-amino-β-cyclodextrin (NH2-CD) with ethylenediaminetetraacetic dianhydride. Then it was bonded onto the surface of silica gel SBA-15 to obtain an ethylenediamine dicarboxyethyl diacetamido-bridged bis(β-CD)-bonded chiral stationary phase (EBCDP). The structures of the bridged bis(β-CD) and EBCDP were characterized by infrared spectroscopy, mass spectrometry, elemental analysis and thermogravimetric analysis, accordingly. The chiral chromatographic performances of EBCDP were systematically evaluated by separating 28 racemic analytes in the reversed-phase or polar organic mode, including eight flavanones, eight bolckers, five dansyl-amino acids, three DL-amino acids and four other common drugs. As a result, the relatively high enantioselectivity of EBCDP was observed in comparison with a native β-CD-CSP (CDSP). All selected analytes were separated on EBCDP, of which 20 analytes had resolutions up to baseline, 2′-hydroxyflavanone and arotinolol had resolutions up to 4.35 and 2.05 in about 30 min, respectively, whereas CDSP only separated 11 analytes with low resolutions (0.55~1.69). Moreover, EBCDP was able to utilize the complexation of the bridging linker (ethylenediamine dicarboxyethyl diamide group, EDTA-based) to realize direct separations of DL-amino acids with a mobile phase containing copper ion (Cu2+), which was similar to the chiral ligand exchange chromatography. Unlike the native cyclodextrin with small cavity (~242 Å3), the bridged bis(β-CD) combined two β-CD units with a bridging linker, having a well-organized “pseudo-cavity” as an organic whole to encapsulate more analytes, which made EBCDP have broad-spectrum applications in chiral separations.

Co-crystallization of racemic amino acids with ZnCl2: an investigation of chiral selectivity upon coordination to the metal centre

A preference for racemic vs. meso-crystal formation has been observed upon the cocrystallization of dl-amino acids with ZnCl2, as a result of homo vs. heterochiral competition.

Evaluation of chiral separation efficiency of a novel OTPTHE derivatization reagent: Applications to liquid-chromatographic determination of DL-serine in human plasma.

A novel chiral derivatization reagent, the N-[1-oxo-5-(triphenylphosphonium)pentyl]- (R)-1,3-thiazolidinyl-4-N-hydroxysuccinimide ester bromide salt (OTPTHE), was developed for the separation and selective detection of chiral DL-amino acids by RP-HPLC analysis. The OTPTHE reacted with DL-amino acids at 60°C maintained for 30 minutes in the presence of 100 mM borate buffer (pH 9.5). The separability of the diastereomeric derivatives was evaluated in terms of the resolution value (Rs) using 13 kinds of DL-amino acids, which were completely separated by reversed-phase chromatography using C18 column at 254 nm. The Rs of the DL-amino acids varied from 1.62 to 2.51. As for the application of the DL-amino acids, the determination of DL-Ser in the human plasma of healthy volunteers was performed based on our developed method. It was shown that linear calibrations were available with high coefficients of correlation (r2 > 0.9997). The limit of detection (S/N = 3) of the DL-Ser enantiomers was 5.0 pmol; the relative standard deviations of the intraday and interday variations were below 4.56%; the accuracy ranged between 95.40%-110.06% and 95.45%-109.80%, respectively; the mean recoveries (%) of the DL-Ser spiked in the human plasma were 99.49%-103.74%. The amounts of DL-Ser in the human plasma of healthy volunteers were determined.

Stereoselective separation of underivatized and 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate derivatized amino acids using zwitterionic quinine and quinidine type stationary phases by liquid chromatography–High resolution mass spectrometry

Amino acids play an important role in cellular processes and are building blocks for peptides and proteins, which take part in regulatory processes within each organism. Hence a large variety of biotechnologically or synthetically produced therapeutic drugs are peptides and proteins. Due to the chiral nature of amino acids and the large variety of common, uncommon and newly synthesized amino acid type compounds, stereoselective separation tools combined with mass spectrometric detection are important in research as well as purity control of therapeutics in industry. Since structural isomers and epimers of common amino acids are isobaric to each other, stereoselective separation is key to their identification. For this purpose zwitterionic quinine and quinidine type chiral stationary phases Chiralpak ZWIX(+) and Chiralpak ZWIX(-) were investigated for their separation performance for underivatized and 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC; AccQ) derivatized proteinogenic amino acids, uncommon amino acids and their isobaric analogs such as allo-threonine, homoserine, allo-isoleucine and homocysteine by HPLC-ESI-QTOF-MS. Cystine and homocystine were reduced with dithiothreitol and S-alkylated with iodoacetic acid and iodoacetamide. In general, derivatization with AQC and thiol alkylation increased the detection sensitivity and resolution of acidic, basic and polar amino acids significantly (e.g. separation factor of Asp increased from 1.00 to 2.29 for Asp-AQC).In addition, throughout this study a u-13C15N-L-amino acid metabolomics mixture was added to the DL-amino acid test solution and used as a co-eluting peak assignment standard to identify the corresponding u-12C14N-L-amino acid peak and hence determine the elution order of the enantiomer pairs for complex mixtures within a single run, employing the same separation conditions for underivatized and AQC-derivatized amino acids and their isobaric analogs.

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.

DL-Amino Acid Analysis Based on Labeling with Light and Heavy Isotopic Reagents Followed by UPLC-ESI-MS/MS.

L-Pyroglutamic acid succinimidyl ester (L-PGA-OSu) and its isotopic variant (L-PGA[d5]-OSu) were synthesized and used as the chiral labeling reagents for the enantioseparation of amino acids by reversed-phase UPLC-ESI-MS/MS. The enantiomers of amino acids were labeled with the reagents at 60°C for 10min in an alkaline medium. The resulting diastereomers were well separated by the reversed-phase chromatography using an ODS column, packed with small particles (1.7μm) (Rs=1.95-8.05). A highly sensitive detection at a low-fmol level (0.5-3.2fmol) was obtained from the selected reaction monitoring (SRM) chromatograms. An isotope labeling strategy using light and heavy variants for the differential analysis of the DL-amino acids in different sample groups is also presented in this paper. The ratios of D/L-alanine in different yogurt products were successfully determined by the proposed method. The D/L ratios were almost comparable to those obtained from only using light reagent (i.e., L-PGA-OSu). Therefore, the proposed strategy seems to be useful for the differential analysis of DL-amino acids, not only in food products but also in biological samples.