On the use of solid phase ion exchangers for isolation of amino acids from liquid samples and their enantioselective gas chromatographic analysis

This work focuses on the development of a suitable working procedure for preconcentration of amino acids enantiomers from water samples using a solid phase extraction. The three types of ion exchangers with various capabilities have been used. The effect of experimental conditions in SPE procedure employing strong anion exchange (SAX), weak (WCX) and strong cation exchange (SCX) cartridges (such as sample volume, pH, origin of elution solvent and its volume) on effective preconcentration of the model set of amino acids has been studied in detail. The enantiomers of isolated and preconcentrated amino acids have been analysed by GC on three capillary columns coated with chiral selectors. The different amino acids derivatives have been investigated in order to achieve optimal resolution of biogenic amino acids and their enantiomers. The best separation of amino acid enantiomers has been obtained on a Chirasil-L-Val column analysing their N-TFA methyl esters. It has been shown that SCX-SPE cartridge with sulfonic groups attached on silicagel support is most suitable for isolation and preconcentration of amino acids from water samples. For this sample treatment procedure, the overall recovery of extraction process has been calculated as an average value from three measurements. It has been found, that recoveries are practically identical for both enantiomers of a particular amino acid and varies in the range 75-99% depending on the type of amino acid. The effectivity of this sample preparation and GC method has been verified by preconcentration of amino acids from orange juice fortified by racemic mixture of some selected amino acids.     

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. Chiral GC-MS analysis of heptafluorobutanol/pentafluoropropionanhydride amino acid derivatives on an Rt-gDEXsa column     

Isolation of the 9-fluorenylmethyl chloroformate derivative in the determination of sulphamethazine

Derivatisation of amine-containing analytes with 9-fluorenylmethyl chloroformate (FMOC) to form fluorescent adducts requires a large excess of FMOC. This excess hydrolyses to form FMOC-OH, which is also fluorescent. Solvent extraction has been investigated as a means of isolating the sulphamethazine (SMZ) adduct (FMOC-SMZ) from the hydrolysis product in order to perform rapid spectrophotometric or spectrofluorimetric assays. However, even under the most favourable pH conditions possible, FMOC-OH was not totally removed. Attempts to enhance the separation by reaction of FMOC-OH with 1-ethoxy-4-dichloro-S-triazinylnaphthalene (EDTN) or by acetylation were also unsuccessful. On the other hand, reaction of FMOC with mixed substrates, followed by two pentane extractions to remove the excess FMOC and direct injection into an HPLC provides the desired separations on a reversed phase column (RPLC) with methanol-modified, (pH 3.5) phosphate buffers. FMOC-SMZ is readily separated from FMOC-OH under all elution conditions, from the FMOC-amino acids (under gradient conditions or isocratically up to 75% methanol), and from other FMOC-sulphonamides and FMOC-dihydrofolate reductase inhibitors (isocratically up to 70% methanol). Hence conversion to the FMOC derivatives permits SMZ to be separated from all of the potential interferants tested by isocratic elution with 70% methanol in RPLC. Analysis for the amino acid derivatives of FMOC may be done without interference from SMZ in samples.     

Determination of S-carboxymethyl-L-cysteine and some of its metabolites in urine and serum by high-performance liquid chromatography using fluorescent pre-column labelling.

Pre-column labelling techniques are described for the determination of S-carboxymethyl-L-cysteine (CMC) and its metabolites in urine and plasma samples by high-performance liquid chromatography (HPLC) without prior extraction. All substances containing an amino group were converted into fluorescent fluorenylmethyl derivatives with 9-fluorenylmethyloxycarbonyl chloride (FMOC). Deaminated or N-acetylated carbocysteine metabolites were coupled with 1-pyrenyldiazomethane (PDAM) to give fluorescent PDAM esters. Similar results were obtained with the two commercially available and stable diazomethane derivatives PDAM and 9-anthryldiazomethane (ADAM). Following double derivatization with PDAM and FMOC, in a single chromatographic run with two fluorescence detectors connected in series, amines and amino(carboxylic) acids could be detected by their FMOC residues and, simultaneously, carboxylic acids were detected as fluorescent PDAM esters. The (R) and (S) enantiomers of the sulphoxides of CMC, of methylcysteine and of N-acetyl CMC were separated, although the reversed-phase HPLC system did not contain a chiral additive or stationary phase designed for the separation of enantiomers. The methods do not include liquid extraction steps and can therefore be performed either manually or automatically using an HPLC autosampler. These methods were used for the investigation of a disputed pharmacogenetic polymorphism of S-oxidation of CMC in humans, which until now has most often been studied using paper chromatography. The described techniques were applied to the determination of CMC and its metabolites in human urine and plasma samples.     

Liquid chromatographic enantiomer resolution of N-fluorenylmethoxycarbonyl alpha-amino acids and their ester derivatives on polysaccharide-derived chiral stationary phases

The liquid chromatographic enantiomer separation of N-fluorenylmethoxycarbonyl (FMOC) protected alpha-amino acids and their ethyl ester derivatives was performed on polysaccharide-derived chiral stationary phases, Chiralcel OD, Chiralpak AD, and Chiralpak AS. In general, Chiralcel OD and Chiralpak AD showed good performance for resolution of N-FMOC alpha-amino acids and their ethyl esters, respectively. All investigated N-FMOC alpha-amino acid enantiomers were baseline separated on Chiralcel OD or Chiralpak AD, whereas N-FMOC alpha-amino acid ethyl ester enantiomers were baseline resolved (alpha = 1.15-3.03) on Chiralpak AD, except for two analytes. The L-enantiomers of all examined FMOC alpha-amino acid ethyl ester derivatives are preferentially retained on Chiralpak AD, while the elution orders of the other enantiomer separations are not consistent.     

Separation of β-blocker and amino acid enantiomers on a mixed chiral sorbent modified with macrocyclic antibiotics eremomycin and vancomycin

A mixed chiral sorbent based on silica with immobilized macrocyclic antibiotics eremomycin and vancomycin was synthesized. A possibility of the separation of enantiomers of β-blockers (metoprolol, pindolol, alprenolol, oxprenolol, labetalol, and atenolol) and amino acids (tryptophan, phenylalanine, DOPA, methionine, and acetyl glutamic acid) on this chiral sorbent by HPLC was studied. The influence of the composition of the mobile phase (pH of buffer solution, its concentration, content of organic modifier, and its nature) on the retention times of β-blocker and amino acid enantiomers, selectivity, and resolution of peaks was studied. It was shown that the mixed chiral sorbent has enantioselectivity to both classes of compounds, while silica modified with vancomycin has no ability to the separation of enantiomers of non-derivatized amino acids, and silica modified with eremomycin has no ability to the separation of β-blocker enantiomers. High values of resolution for amino acids (max Rs > 4) and β-blockers (max Rs > 1) were obtained.     

An evaluation of the structural requirements for the separation of propranolol enantiomers on Pirkle phases following achiral derivatisation

Summary The chromatographic performance of a series of isocyanate and isothiocyanate derivatives of (±)-propranolol has been investigated on three Pirkle-type chiral stationary phases (CSP's), 3,5-dinitrobenzoylphenylglycine (PHE), 3,5-dinitrobenzoylleucine (LEU) and phenethylpropylurea (PEPU). The reaction yield is independent of the substituent group but is affected by choice of solvent. The urea or thiourea derivative group formed is responsible for any separation observed on the amino acid based phases, the retention being related to structure. The structure-retention relationship is not apparent for the -basic PEPU phase, moreover resolution is not simply related to retention for all three phases. Inversion of elution order of the enantiomers is observed with certain derivatives on the LEU column. The naphthyl and nitrophenyl derivatives generally exhibit enhanced retention, with the naphthylurea derivative generally giving the best resolution on these phases.     

Determination of Amino Acid Enantiomers in Pharmaceuticals by Reversed-Phase High-Performance Liquid Chromatography

Precolumn derivatization with the reagent o-phthalic aldehyde/N-acetyl-L-cysteine (OPA/NAC) was used for the determination of amino acid enantiomers by reversed-phase high-performance liquid chromatography. The influence of the composition and pH of the eluent on the separation of the resulting derivatives was studied with the example of four amino acids. It was found that the highest selectivity and efficiency of the separation of OPA/NAC derivatives of amino acids is attained with the use of the eluent methanol–0.01 M Na₂HPO₄ (pH 6.0). The optimum composition of the mobile phase and conditions of the gradient elution were selected for the separation of a mixture of 20 amino acid derivatives. A procedure was developed for the determination of amino acid enantiomers in parenteral nutrition preparations. The procedure was used for the determination of D-isomers of arginine, alanine, methionine, phenylalanine, and leucine in the preparation Polyamine.     

Direct high-performance liquid chromatographic enantioseparation of free α-, β- and γ-aminophosphonic acids employing cinchona-based chiral zwitterionic ion exchangers

We report a chiral high-performance liquid chromatographic enantioseparation method for free α-aminophosphonic, β-aminophosphonic, and γ-aminophosphonic acids, aminohydroxyphosphonic acids, and aromatic aminophosphinic acids with different substitution patterns. Enantioseparation of these synthons was achieved by means of high-performance liquid chromatography on CHIRALPAK ZWIX(+) and ZWIX(-) (cinchona-based chiral zwitterionic ion exchangers) under polar organic chromatographic elution conditions. Mobile phase characteristics such as acid-to-base ratio, type of counterion, and solvent composition were systematically varied in order to investigate their effect on the separation performance and to achieve optimal separation conditions for the set of analytes. Under the optimized conditions, 32 of 37 racemic aminophosphonic acids studied reached baseline separation when we employed a single generic mass-spectrometry-compatible mobile phase, with reversal of the elution order when we used (+) and (-) versions of the chiral stationary phase.

Recent advances in on-line concentration and separation of amino acids using capillary electrophoresis

This article highlights recent methodological developments in the on-line concentration and separation of amino acids and their enantiomers using capillary electrophoresis. Sections are dedicated to recent contributions to on-line concentration strategies such as field-amplified sample stacking, large-volume sample stacking, dynamic pH junction, transient isotachophoresis, sweeping, and the combination of two methods. The main applications, advantages, and limitations of these procedures in the biological, food, and pharmaceutical fields are addressed. Comprehensive tables listing on-line techniques for the concentration and separation of amino acids and their enantiomers, categorized by the stacking strategies used, background electrolytes, sample matrix, limit of detection, and enhancement factor, are provided.

Zwitterionic chiral stationary phases based on cinchona and chiral sulfonic acids for the direct stereoselective separation of amino acids and other amphoteric compounds

An extensive series of free amino acids and analogs were directly resolved into enantiomers (and stereoisomers where appropriate) by HPLC on zwitterionic chiral stationary phases (Chiralpak ZWIX(+) and Chiralpak ZWIX(?)). The interaction and chiral recognition mechanisms were based on the synergistic double ion‐paring process between the analyte and the chiral selectors. The chiral separation and elution order were found to be predictable for primary α‐amino acids with apolar aliphatic side chains. A systematic investigation was undertaken to gain an insight into the influence of the structural features on the enantiorecognition. The presence of polar and/or aromatic groups in the analyte structure is believed to tune the double ion‐paring equilibrium by the involvement of the secondary interaction forces such as hydrogen bonding, Van der Waals forces and π–π stacking in concert with steric parameters. The ZWIX chiral columns were able to separate enantiomers and stereoisomers of various amphoteric compounds with no need for precolumn derivatization. Column switching between ZWIX(+) and ZWIX(?) is believed to be an instrumental tool to reverse or control the enantiomers elution order, due to the complementarity of the applied chiral selectors.     

Simple and low-cost high-performance liquid chromatographic method for determination of D- and L-amino acids

In this article, a simple and low-cost method for the analysis of amino acid enantiomers by using high-performance liquid chromatography (HPLC) is described. In this method, the amino acids are modified to diastereomers in order to be separated into enantiomers on a usual C(18) reversed-phase column. Methanol instead of acetonitrile is used as an elution solvent; the results of HPLC with methanol elution are comparable with those of HPLC with acetonitrile elution. Sub-nanomolar sensitivity is attained by measuring the absorbance at 340 nm in analysis of 15 amino-acid enantiomers.     

Assessment and application of Marfey’s reagent and analogs in enantioseparation: a decade’s perspective

Of the various methods available for high‐performance liquid chromatography separation of enantiomers (of e.g. amino acids and amino group containing compounds) by the pre‐column derivatization approach, use of Marfey’s reagent has been most successful with continued application since its introduction in 1984. The reagent is prepared from difluoro dinitro benzene by nucleophilic substitution of one of its F atoms by l‐ alanine amide. There is flexibility to prepare several chiral variants (by substituting the F atom with different chiral auxiliaries) and to tailor the hydrophobicity and resolution, ultimately, of the diastereomeric derivatives. The present paper assesses and reviews applications of Marfey’s reagent and its chiral variants (i.e. other FDNP reagents) for enantioseparation of certain amino group containing drugs/amino acids, and to provide some case studies on enantiomeric separations that are important for the pharmaceutical industry. Various explanations for separation mechanism and elution order using FDNP reagents are included and the question of the configuration of the corresponding enantiomer using an indirect approach has also been addressed.     

Direct separation of amino enantiomers by high performance ligand exchange chromatography on chemically bonded chiral phases

Summary Various chiral chemically bonded stationary phases for the separation of amino acid enantiomers by high performance ligand exchange chromatography are described. The phases were synthesized by treating silica gel with 3-glycidoxypropyltrimethoxysilane and bonding L-amino acids such as L-azetidine carboxylic acid, L-pipecolic acid or L-phenylalanine to the product. The best results were obtained with L-pipecolic acid as a fixed ligand. Nearly all common amino acid enantiomers could be separated.Presented at the 14th International Symposium on Chromatography London, September, 1982     

Liquid‐Chromatography Quantitative Analysis of 20 Amino Acids after Derivatization with FMOC‐Cl and Its Application to Different Origin Radix isatidis

We developed a simple, rapid and reliable method for determination of 20 common amino acids based on derivatization with 9‐fluorenylmethyl chloroformate (FMOC‐Cl) and RP‐LC/UV, this method was first introduced into quantitative analysis of amino acids. The amino groups of amino acids were trapped with FMOC‐Cl to form amino acid‐FMOC‐Cl adducts which can be suitable for LC‐UV. Chromatographic separation was performed on a C₁₈ column with a mobile phase gradient consisting of acetonitrile and sodium acetate solution. This method was shown to be sensitive for 20 common amino acids. In the intra‐day precisions assay, the range of RSDs was 3.21‐7.67% with accuracies of 92.34‐102.51%; for the inter‐day precisions assay, the range of RSDs was 5.82‐9.19% with accuracies of 90.25‐100.63%. The results also indicated that solutions of amino acids‐FMOC‐Cl can be kept at room temperature for at least 24 h without showing significant losses in the quantified values. The validated method was successfully applied to the determination of major four kinds of amino acids in R. isatidis samples (Arg, Pro, Met and Val). The total content of amino acids in different origin R. isatidis was 13.32‐19.16 mg/g. The differences between R. isatidis samples were large using HCA.     

Enantiomer separation and indirect chromatographic absolute configuration prediction of chiral pirinixic acid derivatives: Limitations of polysaccharide-type chiral stationary phases in comparison to chiral anion-exchangers

Chiral α-arylthiocarboxylic acids with different substitution patterns, representing new pirinixic acid derivatives with dual PPARα/γ agonistic activities, have been separated into enantiomers on tert-butylcarbamoylquinine and quinidine based chiral anion-exchangers and amylose tris(3,5-dimethylphenylcarbamate) coated silica on analytical and preparative scale. Absolute configurations of individual enantiomers were assigned chromatographically via elution orders on the chiral anion-exchangers and were confirmed by stereoselective syntheses via Ewans auxiliaries that have lead to enantiomeric products with known absolute configurations. The results of both methods were in full agreement. Moreover, the receptor stereoselectivity in PPARα transactivation activities was consistent within the test set of structurally related compounds. Limited correlation (between elution order and substitution) was observed within the set of α-arylthiocarboxylic acids on the amylose tris(3,5-dimethylphenylcarbamate) based chiral stationary phase (CSP), in particular the elution order changed with remote substitution. This clearly demonstrates the risks of chromatographic absolute configuration assignments by prediction from one structural analog to another one, especially with CSPs such as polysaccharide CSPs that are recognized for their broad applicability due to multiple binding and chiral recognition modes. It is therefore of utmost importance that such chromatographic absolute configuration predictions by extrapolation to structural analogs are combined with orthogonal methods for verification of the results.     

Post-column labeling techniques in amino acid analysis by liquid chromatography

Amino acid analysis (AAA) has always presented an analytical challenge in terms of sample preparation, separation, and detection. Because of the vast number of amino acids, various separation methods have been applied taking into consideration the large differences in their chemical structures, which span from nonpolar to highly polar side chains. Numerous separation methods have been developed in the past 60 years, and impressive achievements have been made in the fields of separation, derivatization, and detection of amino acids (AAs). Among the separation methods, liquid chromatography (LC) prevailed in the AAA field using either pre-column or post-column labeling techniques in order to improve either separation of AAs or selectivity and sensitivity of AAA. Of the two approaches, the post-column technique is a more rugged and reproducible method and provides excellent AAs separation relatively free from interferences. This review considers current separations combined with post-column labeling techniques for AAA, comparison with the pre-column methods, and the strategies used to develop effective post-column methodology. The focus of the article is on LC methods coupled with post-column labeling techniques and studying the reactions to achieve optimum post-column derivatization (PCD) conditions in order to increase sensitivity and selectivity using various types of detectors (UV–Vis, fluorescence, electrochemical etc.) and illustrating the versatility of the PCD methods for practical analysis.

Enantiomeric separation of D/L-carnitine using HPLC and CZE after derivatization

Summary Carnitine is an essential component in tissues of animals, higher plants and many microorganisms. Whereas the L-carnitine enantiomer plays an important role in the metabolism of long chain fatty acids, D-carnitine has a considerable toxic influence on biochemical processes. The analytical separation of D-and L-carnitine depends upon derivatization with UV-or fluorescently active substances, e.g. FMOC and (+)/(–)-FLEC. The separation of diastereomeric (+)- and (–)-FLEC carnitine esters was performed successfully with capillary zone electrophoresis (CZE) and HPLC, after optimization of the derivatization process and of the composition and pH of the buffer, using UV- and fluorescence detection. With HPLC separation a detection limit of the carnitine esters of 5 mol/l when using fluorescence detection was achieved. With both separation systems baseline resolution and short analysis times could be obtained. The enantiomeric FMOC derivatives could be separated using the electrophoretic system and acidic buffers with high concentrations of an osmotic flow modifier together with -cyclodextrine as chiral selector. The applicability of the optimized separation conditions are demonstrated in the analysis of agar culture medium inoculated withPseudomonas putida and of pharmaceutical formulations. In all samples very low amounts of D- or L-carnitine could be determined in the presence of the other enantiomeric form. Problems caused by the impurity of the carnitine standards or the derivatization agent (+)/(–)-FLEC are discussed.     

Retention behaviour of binaphthyl compounds in enantiomeric separation by microcolumn liquid chromatography with micellar bile-salt mobile phases

Summary Optical isomers of substituted binaphthyl, compounds such as 2,2-dihydroxy-1,1-dinaphthyl and 1,1-binaphthyl-2,2-diyl hydrogenphosphate are, separated by HPLC using micellar bile-salt mobile phases. Operating conditions which affect the optical resolution of these enantiomers are examined. The largest separation factor achieved for these enantiomers is 2.74. The elution order of the latter enantiomers changes with mobile phase composition.     

Derivatives for separation of amino acid enantiomers

Summary An optimum gas chromatographic separation of all protein amino acids in one run on capillaries coated with Chirasil-Val is difficult to achieve. Overlap of enantiomers of different amino acids may occur because the relative retention times depend upon the overall polarity of the stationary phase, the film thickness and the actual temperature programm. Employment of different derivatives formed by esterification with isopropanol, n-propanol, isobutanol and n-butanol and by acylation with trifluoroacetic, pentafluoropropionic and heptafluorobutyric anhydrides yields patterns of relative elution of all amino acid enantiomers. Thus, even critical pairs of amino acid enantiomers can be separated or shifted in their relative retention times. All amino acid enantiomers can be separated and quantitatively estimated.