Yoshio Machida

Enantiomer separation of amino compounds by a novel chiral stationary phase derived from crown ether

Abstract A novel chiral stationary phase (CSP-18C6I) was prepared by immobilizing (+)-18-crown-6 tetracarboxylic acid on 3-aminopropylsilanized silica-gel to separate enantiomers of drugs having a primary amino group. The chiral crown ether was combined with 3-aminopropyl silica gel (0.85 mmol of amine per gram of gel) to provide CSP-18C6I having a chiral selector loading of 0.26 mmol per gram of gel. This CSP-18C6I showed good chiral recognition for thirteen out of eighteen dl -amino acids and seven racemic aminoalcohols using a dilute aqueous solution of perchloric acid as the eluent. Afloqualone (a muscle relaxant), primaquine (an antimalarial), and 1-(1-naphthyl)ethylamine (1-NEA) were resolved on the CSP-18C6I. Furthermore, alanine-β-naphthylamide (Ala-β-NA), which is hydrophobic and did not elute within 60 min by the commercially available CROWNPAK CR(+) with 15% methanol and a column temperature of 40°C, was successfully enantioseparated by the novel CSP-18C6I.

Nuclear magnetic resonance studies for the chiral recognition of (+)-(R)-18-crown-6-tetracarboxylic acid to amino compounds.

Chiral recognition capability of (+)-(R)-18-crown-6-tetracarboxylic acid (18C6H(4)) to various amino compounds containing 16 amino acids, five alkyl amines, seven aminoalcohols and other amino compounds in nuclear magnetic resonance (1H-NMR) analysis was investigated. In general, amino compounds having an aromatic ring were well chiral recognized with 18C6H(4) compared with those having no aromatic ring. Effects of 18C6H(4) concentration and the kind of deuterated solvents (D(2)O, CD(3)OD and CD(3)CN) for measurement on the chiral recognition was investigated in detail. Concentration of 5 equivalent 18C6H(4) to the amino compounds was found to be sufficient for the chiral recognition. On the other hand, an effective deuterated solvent (D(2)O, CD(3)OD or CD(3)CN) for measurement was different in each compound. Distinguishment of 1.0% of the minor enantiomer (D-form) in L-alanine-beta-naphthylamide was found to be possible by 1H-NMR employing 18C6H(4) as a chiral shift reagent.

Nuclear magnetic resonance studies for the chiral recognition of the novel chiral stationary phase derived from 18-crown-6 tetracarboxylic acid

Abstract Enantioselectivities observed in high-performance liquid chromatography (HPLC) with the novel chiral stationary phase (CSP-18C6I) derived from (+)-18-crown-6 tetracarboxylic acid (18C6H4) were investigated by using nuclear magnetic resonance (NMR) spectrometry. The elution orders in CSP-18C6I, that is, the S-enantiomer of 1-(1-naphthyl)ethylamine (1-NEA) and the l -enantiomer (S-form) of alanine-β-naphthylamide (Ala-β-NA) eluted prior to each corresponding enantiomer, were successfully explained on the basis of the apparent binding constants (Ka) of the enantiomers to the CSP moiety which were calculated from 1H-NMR experiments. Detailed HPLC and NMR studies for the chiral recognition of racemic amino compounds with 18C6H4 hosts showed that 1H-NMR spectrometry is a useful technique for the investigation of the chiral recognition mechanism in HPLC. Additionally, it was found 18C6H4 can be recommended as a useful chiral shift reagent for the enantiomeric excess determination by 1H-NMR.

Crystallographic studies for the chiral recognition of the novel chiral stationary phase derived from (+)‐(R)‐18‐crown‐6 tetracarboxylic acid

Chiral discrimination observed in high-performance liquid chromatography (HPLC) with the novel chiral stationary phase (CSP-18C6I) derived from (+)-(R)-18-crown-6 tetracarboxylic acid [(+)-18C6H4] was investigated by X-ray crystallographic analysis of the complex composed of the R-enantiomer of 1-(1-naphthyl)ethylamine (1-NEA) and (+)-18C6H4. Mixtures of 1-NEA (the R- or S-enantiomer) and (+)-18C6H4 were dissolved in methanol-water (1:1) solution and allowed to stand for crystallization. The R-enantiomer crystallized with (+)-18C6H4 as a co-crystal, although the S-enantiomer did not. This result was in good agreement with the enantiomer elution order of 1-NEA in CSP-18C6I. The apparent binding constants (Ka) of the enantiomers to the (+)-18C6H4 obtained from 1H-NMR experiments also supported the above-mentioned result. The X-ray crystal structure of the 1:1 complex of the R-enantiomer and (+)-18C6H4 indicated the four sets of hydrogen bond association between the naphthylethylammonium cation and oxygen of polyether ring or carbonyl group of (+)-18C6H4. Chirality 11:173–178, 1999.

Enantiomeric purity determination of acetyl-l-carnitine by reversed-phase high-performance liquid chromatography using chiral derivatization

An indirect HPLC enantioseparation method for the determination of acetyl-D-carnitine (D-AC) in acetyl-L-carnitine (L-AC) was developed. L-AC was derivatized with a chiral amino compound which has a chromophore for UV detection. Six chiral amino compounds were examined as chiral derivatization reagents. Among them, enantiomers of acetylcarnitine derivatized with L-alanine-beta-naphthylamide (L-Ala-beta-NA) were successfully separated on an ODS column within 10 min with Rs = 1.94 and alpha = 1.10. Quantitation was achieved through UV detection at 254 nm. The derivatization reaction of L-AC with L-Ala-beta-NA was completed in less than 10 min at room temperature (ca. 20 degrees C). Validation data such as linearity, detection limit, and precision are also presented. The detection limit of D-AC in L-AC in this method was below 0.05% (visual evaluation). This method was found to be applicable as a practical quality control method for the enantiomeric purity determination of L-AC.

Enantiomeric separation of diols and β-amino alcohols by chiral stationary phase derived from (R,R)-tartramide

A novel chiral stationary phase (CSP) having a (R,R)-tartramide derivative as a chiral moiety was synthesized. This CSP showed good chiral recognition for 1,2-diols, bi-β-naphthol and β-amino alcohols (β-blockers) without any derivatization. The driving force of enantiomeric separation was assumed to be the dual hydrogen-bonding association and π-π interaction between the solute enantiomers and the chiral moiety of CSP.

Chromatography of crotamiton and its application to the determination of active ingredients in ointments.

Crotamiton, which is a mixture of cis and trans isomers, was investigated by several separation techniques. One of the HPLC modes, in which crotamiton eluted as a single peak, was selected for the determination of five active ingredients (crotamiton, prednisolone, glycyrrhetinic acid, dibucaine and chlorhexidine hydrochloride) in an ointment. The simultaneous determination was performed using isocratic reversed-phase mode within 20 min by employing an octyl (C8) column and a mobile phase containing sodium dodecyl sulfate (SDS) and 2-propanol. The method was successfully applied to quality control and stability testing of the ointment.