Kazutake Shimada

New derivatization reagents for the resolution of amino acid enantiomers by high-performance liquid chromatography

Abstract A new derivatization procedure has been developed for converting enantiomeric amines into diastereomers for resolution by high-performance liquid chromatography. Two chiral reagents, (−)- a -methoxy- a -methyl-1-naphthaleneacetic acid and (−)- a -methoxy- a -methyl-2-naphthaleneacetic acid, were prepared and optically resolved by fractional crystallization of their (+)- a -methylbenzylamine salts. The diastereomeric amides formed from amino acid methyl esters and (−)- a -methoxy- a -mthyl-1-naphthaleneacetic acid by the N,N′-dicyclohexylcarbodiimide method were efficiently resolved on a normal-phase column and responded with satisfactory sensitivity in the ultraviolet detector.

Studies on steroids. CXXVIII. Separation and determination of bile acids by high-performance liquid chromatography.

A method is described for the simultaneous determination of major bile acids by high-performance liquid chromatography without prior hydrolysis. A mixture of bile acids is divided into the free, glyco- and tauro-conjugate groups by thin-layer chromatography. Separation of each group into cholate, ursodeoxycholate, chenodeoxycholate, deoxycholate and lithocholate is attained in two stages on a muBondapak C18 column; first, 0.3% ammonium carbonate-acetonitrile (9:4) is used as a mobile phase for the separation of the last three compounds. Subsequently cholate and ursodeoxycholate are resolved by chromatography in 0.3% ammonium carbonate-acetonitrile (11:4).

Determination of captopril in human blood by high-performance liquid chromatography with electrochemical detection

A new method for quantitation of captopril in human blood is described. Captopril was derivatized with N-(4-dimethylaminophenyl)maleimide into the electrochemically active adduct. The derivative was separated and determined by high-performance liquid chromatography with an electrochemical detector on a reversed-phase column. The proposed method was satisfactory for determination of captopril in whole blood with respect to accuracy and precision. The detection limit of captopril thereby obtained was 10 ng/ml. The blood levels of captopril in patients orally given an official dose were measured by the present method.

Sensitive derivatization reagents for thiol compounds in high-performance liquid chromatography with electrochemical detection

Three N-substituted maleimides were tested as derivatizing reagents; N-(4-anilinophenyl) maleimide (APM) was the most favorable in terms of sensitivity and reactivity. N-Acetyl-l-cysrteine, glutathione, l-cysteine and d-penicillamine were readily converted into the adducts with APM. Picogram levels of these thiol compounds were separated and quantified.

New derivatization of amines for high-performance liquid chromatography with electrochemical detection

Abstract N-(4-Anilinophenyl)isomaleimide (APIM) and N-(4-anilinophenyl)isophthalimide (APIP) were prepared and evaluated for precolumn derivatization of amines in liquid chromatography with electrochemical detection. Phenethylamine and piperidine were taken as the model compounds for primary and secondary amines, respectively. Derivatization of amines with these reagents was complete within 20 min at room temperature in acetonitrile—0.05 M borate buffer (pH 9.0) (1:1). The derivatives formed with APIM were more responsive to an electrochemical detector than those formed with APIP. The detection limit for the phenethylamine—APIM adduct was ca. 0.1 pmol. The electrochemical response was found to be linear in the range 0.1–10 ng of phenethylamine.

New ferrocene reagents for derivatization of alcohols in high-performance liquid chromatography with electrochemical detection

Abstract New derivatization reagents possessing ferrocene as an electrophore have been developed for high-performance liquid chromatography with electrochemical detection. Among several reagents examined, ferrocenoyl azide and 3-ferrocenylpropionl azide proved to be satisfactory for use in derivatization of alocholic hydroxyl compounds with respect to reactivity and sensitivity. Hydroxysteroids were readily condensed with these car☐ylic acid azides under mild conditions to provide urethanes, exhibiting maximum sensitivity at +0.4 V vs . a silver—silver chloride reference electrode with a detection limit of 0.5 pmol. This derivatization method was found to be applicable to the characterization of the products formed from digoxigenin by in vitro bioconversion.

Derivatization of Ketosteroids for High-Performance Liquid Chromatography with Electrochemical Detection

Abstract A highly sensitive and selective derivatization of ketosteroids for use in high-performance liquid chromatography with electrochemical detection (HPLC/EC) is described. When the detector response for three phenylhydrazone derivatives was compared with one another using dehydroepiandrosterone, the p-nitrophenylhydrazone showed the highest sensitivity with a detection limit of 200 pg. Dehydroepiandrosterone and other principal 17-ketosteroids in human blood were quantitatively derivatized into p-nitrophenylhydrazones and efficiently separated by HPLC/EC on a μBondapak C1 8 column using 0.5%. NH4H2PO4/methanol (2:7, v/v).

Determination of fatty acids by high-performance liquid chromatography with electrochemical detection using a ferrocene derivatization reagent

Abstract New derivatization method using ferrocene reagents has been developed for the determination of fatty acids by high-performance liquid chromatography with electrochemical detection. Condensation of fatty acids with 3-bromoacetyl-1, 1-dimethyl-ferrocene was effected in the presence of 18-crown-6 and potassium fluoride. The resulting esters showed the satisfactory sensitivity at +0.60 V vs. an Ag/AgCl reference electrode with a detection limit of 0.5 pmole. Also the high selectivity was obtained by using a twin electrode electrochemical detector. The proposed derivatization method was found applicable to the determination of fatty acids in human serum.

Sensitive ferrocene reagents for derivatization of thiol compounds in high-performance liquid chromatography with dual-electrode coulometric dectection

Three N-substituted maleimides possessing ferrocene as an electrophore were prepared and evaluated for pre-column derivatization of thiol compounds in high-performance liquid chromatography with dual-electrode coulometric detection. The utility of these reagents was investigated by employing N-acetyl-L-cysteine as a model compound. Among the three, N-(ferrocenyl) maleimide was the most favourable reagent with respect to reactivity, stability and electrochemical properties. The dual-electrode coulometric detection of the adduct showed high selectivity and sensitivity with a detection limit of 0.06 pmol. The proposed method is applicable to the determination of glutathione in biological specimens.

Studies on steroids : CLXV. Determination of isomeric catechol estrogens in pregnancy urine by high-performance liquid chromatography with electrochemical detection

A method for the determination of 2- and 4- hydroxylated estrone and estradiol in pregnancy urine by high-performance liquid chromatography with electrochemical detection (HPLC-ECD) is described. The urine catechol estrogens were deconjugated, purified by adsorption on alumina, and subjected to HPLC-ECD. Two pairs of isomeric catechol estrogens were distinctly separated on a microBondapak c18 column with acetonitrile-0.5% ammonium dihydrogen phosphate (pH 3.0). The amounts of these four compounds were satisfactorily determined with a quantitation limit of 1 ng using 4-hydroxy-16 oxoestradiol 17 acetate as an internal standard. The validity of the present methods for the determination of urine catechol estrogens was verified by the recovery test.