Hans Brückner

Liquid chromatographic determination of d- and l-amino acids by derivatization with o-phthaldialdehyde and chiral thiols: Applications with reference to biosciences

Abstract A high-performance liquid chromatographic procedure was developed for the fully automated precolumn derivatization of amino acids by derivatization with o -phthaldialdehyde (OPA) together with the chiral thiols N-isobutyryl- l -cysteine (IBLC) and N-isobutyryl- d -cysteine (IBDC) [(CH 3 ) 2 CHCONHCH(CH 2 SH)COOH]. The derivatization is completed within 2 min at room temperature and the resulting diastereomeric isoindol derivatives exhibit linearity with respect to fluorescence in the range 25–1000 pmol. The derivatives are completely separated within 75 min on an octadecylsilyl stationary phase using a linear gradient generated by sodium acetate buffer (pH 5.95) and methanol-acetonitrile. Fluorescence detection at an excitation wavelength of 230 nm and an emission wavelength of 445 nm makes possible the detection of 1–2 pmol of an amino acid enantiomer. Derivatization with OPA-IBLC permits the separation of a 41-component standard containing seventeen protein l -amino acids and, in addition, glycine, dl -Cysteic acid, the internal standard l - homo -arginine and the non-protein amino acids α-aminoisobutyric acid and dl -isovaline. Replacement of OPA-IBLC with OPA-IBDC (or vice versa ) leads to a reversal in the elution of the derivatives of d - and l -amino acids. The applicability of the method to various fields of biosciences is demonstrated with the detection and determination of d -amino acids in bacteria, microfungi, higher plants, invertebrates and vertebrates, including man, and in the amino acid-containing Murchison meteorite.

High-performance liquid chromatographic separation of dl-amino acids derivatized with chiral variants of Sanger's reagent

Abstract Substitution of one fluorine atom in 1,5-difluoro-2,4-dinitrobenzene by l -alanine amide yields N 2 -(5-fluoro-2,4-dinitrophenyl)- l -alanine amide (FDNP-Ala-NH 2 , “Marfeys reagent”). This reagent (or analogues, termed FDNP reagents, in which Ala is replaced with other chiral α-amino acids (AAs), oligopeptides or amino components) might be considered as a chiral variant of “Sangers reagent” (1-fluoro-2,4-dinitrobenzene). The remaining fluorine atom in FDNP reagents can be substituted by chiral AAs to furnish diastereomeric derivatives which are separable by high-performance liquid chromatography (HPLC). A number of chiral variants of Sangers reagent have been synthesized with the general structures (a) FDNP-Val-NHR (R = H, tert. -butyl, chiral aralkyl, phenyl, p -nitrophenyl), (b) FDNP-Val-OR (R = H, CH 3 , tert. -butyl), (c) FDNP-(Ala) n -NH 2 , n = 1,2, and (d) FDNP-NHR (R = aralkyl and hydroxyalkyl), and their ability to resolve certain dl -AAS as diastereomers in reversed-phase HPLC was investigated. Reagents with the structures (a), (b) and (c), but not (d), made possible the separation of dl -AAs as diastereomers. From the results obtained and from observations of Corey-Pauling-Koltun (CPK) space-filling molecular models of the diastereomers, it is concluded that the AA side-chains and the carboxy and carboxamide substituents in the reagents have an influence on the retention times of diastereomers and that the differences in free energy as a result of the formation of an intramolecular hydrogen bridge in the l-l and non-formation in the d-l diastereomers (first letter refers to the configuration of the amino acid to be analysed) are in particular responsible for large differences in the retention times of certain diastereomers in HPLC.

Gas chromatographic characterization of free d-amino acids in the blood serum of patients with renal disorders and of healthy volunteers

A capillary gas chromatographic method, using the chiral stationary phase Chirasil-L-Val, after treatment and isolation with Dowex 50W X8 cation exchanger and conversion into trifluoroacetyl-1-propyl esters or pentafluoropropionyl-1 (or 2)-propyl esters, has been developed for the determination of the relative amounts of free D-amino acids in the blood serum of eighteen patients with renal failure (continuous ambulatory peritoneal dialysis (CAPD), n = 11; hemodialysis, n = 5; nephrotic syndrome, n = 2) and compared with data obtained from healthy volunteers (n = 5). Significant amounts of D-Ala (0.5-13%) and D-Asx (1.5-7.7%; Asx = Asp + Asn) were found in all serum samples. D-Ser was detected in the serum of all patients with renal disorders and, in addition, D-Pro (0.6-2.5%) was found in the serum of all patients undergoing hemodialysis and with nephrotic syndrome. D-Ser (2.9-3.1%) and D-Pro (0.6-0.9%) were also found in the samples of three volunteers. D-Leu (1.2-1.7%) was present in three patients with CAPD, and D-Glx (0.3-1.3%; Glx = Glu + Gln) was present in eight of eighteen patients with renal malfunction. Linear regression analysis of the relative amounts of D-amino acids and the serum creatinine levels of all donors revealed positive correlation factors for D-Asx (r = 0.748) and D-Ser-(r = 0.667), but not for D-Pro and D-Ala. Remarkably high amounts of D-Ser (12.1 and 19.8%) were found in two hemodialysates investigated. Participation of intestinal bacteria and nutrition are discussed as possible sources of serum D-amino acids. An increase of some D-amino acids in the serum of patients with renal diseases might be explained, in part, by decreased activity of renal D-amino acid oxidase.

Automated enantioseparation of amino acids by derivatization with o-phthaldialdehyde and n-acylated cysteines.

The enantioseparation of standard mixtures composed of protein DL-amino acids was performed by reversed-phase high-performance liquid chromatography of the corresponding diastereomeric isoindolyl derivatives, formed by automated precolumn derivatization with o-phthaldialdehyde (OPA) and a series of N-acyl-L-cysteines(Acyl-Cys). A photodiode-array detector, operating at 338 nm, was used for detection. In order to evaluate systematically the influence of the structures of the acyl group in the chiral thiol reagents, a series of novel N-acyl-L-cysteines was synthesized [acyl = n-butyryl, isobutyryl (i-But), pivaloyl, benzoyl) and the chromatographic behaviour of the diastereomers formed was compared with those of already known reagents, N-acetyl-L-cysteine and N-ter.-butyloxycarbonyl-L-cysteine. All Acyl-Cys derivatives of DL-amino acids were resolved. In particular, i-But-Cys gave the highest resolutions for most of the amino acid enantiomers in comparison with the other Acyl-Cys. Investigation of yoghurt using OPA-acetyl-Cys demonstrated the applicability of the method to a complex food matrix and the occurrence of D-Asp, D-Glu and D-Ala in this dairy product.

Fully automated high-performance liquid chromatographic separation of DL-amino acids derivatized witho-phthaldialdehyde together withN-isobutyryl-cysteine. Application to food samples

SummaryA method is presented that enables the fully automated precolumn derivatization of mixtures of DL-amino acids (DL-AA) witho-phthaldialdehyde together withN-isobutyryl-L(orD)-cysteine. HPLC on a 250 mm×4 mm i.d. column packed with Shandon Hypersil ODS, 5 μm, and a linear gradient formed from 23 mM sodium acetate (pH 6.0) and methanol/acetonitrile (600 ml+50 ml) separates completely an AA standard composed of 17 pairs of DL-AA (including Asn and Gln), Gly and the internal standard L-homo-Arg, within 75 min at a flow rate at 1 ml/min. Applications are shown of the determination of free D-AA isolated from an orange juice concentrate and from soy sauce, and the detection of D-AA in a gelatine total hydrolysate. In the case of these foodstuffs fluorescence detection (excitation at 230 nm, emission at 445 nm) allows the routine detection of 5–10 pmol per AA; and approx. 0.2–1% D-AA, in an excess of L-AA, are quantifiable.

Chromatographic determination of D-amino acids as native constituents of vegetables and fruits

SummaryFree D-amino acids (D-AA) were detected as native constituents in juices of vegetables (cultivars of cabbage, tomato, carrot, garlic) and fruits (organes, clementine, grapefruit, lemon, apples, pear, grapes) using gas chromatography (GC) or high-performance liquid chromatography (LC). For investigation by GC, AA enantiomers were converted into theirN(O)-pentafluoropropionyl 2-propyl esters and resolved on a Chirasil-L-Val capillary column. For determination by LC, precolumn derivatization of AA enantiomers usingo-phthaldialdehyde together with the chiral thiolsN-isobutyryl-L-cysteine orN-isobutyryl-D-cysteine and fluorescence detection of the diastereomeric isoindole derivatives, resolvable on an octadecylsilyl stationary phase, were used. D-Ala (0.6–3.8%) was detected in all freshly pressed plant juices usually in the highest relative amounts. Other D-AA detected were D-Asx (0.1–1.9%), D-Glx (0–1.3%), D-Ser (0–1.7%), D-Arg (0.4–1.2%, in grapes, orange, grapefruit, and clementine) and D-Leu and D-Val (1% in cabbage). Absolute amounts of native D-AA were totally 28–57 μmol L−1 in fruit juices, 14.5 μmol L−1 in a tomato juice and 8.5 μmol L−1 in a carrot juice.

Use of chiral monohalo-s-triazine reagents for the liquid chromatographic resolution of dl-amino acids

Abstract Nucleophilic replacement of one halogen atom (chlorine, fluorine) in the trihalo-s-triazines (2,4,6-trihalo-1,3,5-triazines), cyanuric chloride or cyanuric fluoride by reaction with either methanol, 2-naphthol, 1-methoxynaphthalene, or 4-aminoazobenzene furnished ultraviolet-absorbing, fluorescent of chromogenic dihalo-s-triazines. Substitution of a further halogen atom in these compounds by reaction with l -alanine amide provided chiral monohalo-s-triazines. The remaining halogen atom was substituted by reaction with selected d - or l -amino acids to form diastereomeric derivatives which were separated by reverse-phase (C18) high-performance liquid chromatography by using mixtures of water, acetonitrile and trifluoroacetic acid as eluents. Because of its possibilities for selection among a large number of detection groups in combination with various chiral moieties, the approach is considered to be a general method for the design and construction of tailor-made reagents suitable for precolumn derivatization and indirect liquid chromatographic separation of amino acid enantiomers.

Quantification of D-amino acids in human urine using GC-MS and HPLC.

SummaryThe relative amounts of free D-amino acids (D-AA) in the urine of seven healthy volunteers (age 27 to 49 years) were determined using chiral phase (Chirasil-L-Val) capillary gas chromatography in conjunction with selected ion monitoring mass spectrometry. The absolute amounts of free D-AA were determined by pre-column derivatization of the amino acids witho-phthaldialdehyde andN-isobutyryl-L-cysteine followed by high-performance liquid chromatographic separation and fluorescence detection of the isoindol derivatives formed. The following most abundant D-AA were found (highest and lowest absolute and relative amounts): D-Ser (379.8 — 30.1µMol/L; 56.5 — 19.0%), D-Ala (53.8 — 7.6µMol/L; 19.6 — 5.7%), D-Thr (5.8 — 0.25µMol/L; 3.4 — 1.0%), D-Val (3.7 — 0µMol/L; 4.2 — 0%), and D-Phe (3.5 — 0.35µMol/L; 4.8 — 1.4%).

Liquid chromatographic determination of D-amino acids in cheese and cow milk. Implication of starter cultures, amino acid racemases, and rumen microorganisms on formation, and nutritional considerations

SummaryFree L- and D-amino acids (L-AA, D-AA) were isolated from an Appenzeller cheese, from raw milk, and from an ethanolic extract as well as a total hydrolysate of cows rumen microorganisms, and their relative amounts were determined by reversed-phase high-performance liquid chromatography after derivatization witho-phthaldialdehyde together withN-isobutyryl-L-(or D)-cysteine. D-Ala, D-Asp and D-Glu were found, among other D-AA in all cases and a microbial origin of free D-AA found in cheese and milk was rationalized. From the results, and taking other findings of the occurrence of D-AA in food and beverages into account, the highest intake of D-AA is to be expected from the consumption of ripened cheeses. From the presence of D-amino acid oxidases in human kidney, liver, and brain and from reports on the intravenous administration of racemic AA to humans and their metabolisation it is concluded that intake of free D-AA found in food is no threat for human beings.

Liquid chromatographic separation of amino acid enantiomers on a silica-bonded chiral s-triazine column

Abstract A chiral derivatizing reagent (CDR) was synthesized by consecutive nucleophilic replacement of two chlorine atoms in 2,4,6-trichloro-1,3,5-triazine (cyanuric chloride) by l -valinamide and l -phenylalaninamide, yielding N -[4-((S)-1- carbamoyl -2- methyl-propylamino )-6- chloro -[1,3,5] triazin -2- yl ]- l - phenylalanine . This CDR was used for the derivatization of free dl -amino acids, followed by the liquid chromatographic separation of the diastereomers thus formed, and for the synthesis of a chiral stationary phase (CSP). The CSP was synthesized by substitution of the remaining chlorine atom with aminopropylsilica, yielding {3-[4-(( S )-1-carbamoyl-2-methyl-propylamino)-6-(( S )-1-carbamoyl-2-phenyl-ethylamino)-[1,3,5] triazin-2-ylamino]-propyl}-functionalized silica gel. This new CSP was found to effect, in part very high, resolutions for enantiomers of dansylamino acids when mixtures of acetonitrile and 0.01 M sodium acetate buffer (pH 4) were used as eluents.