Rikizo Horikawa

Resolution of DL-Valine by Countercurrent Solvent Extraction with Continuous Sample Feeding

Abstract DL-Valine was resolved completely by a solvent extraction system with a continuous sample feeding process called continuous countercurrent fractional extraction (CFE). The two-phase system contained n-butanol and water with a copper(II) complex of N-n-dodecyl-L-hydroxproline as a resolving reagent. The upper (alcohol-rich) phase and the lower (water-rich) phase proceeded in the segmented extraction columns countercurrently; the D-isomer was recovered in the upper phase, while the L-isomer was recovered in the lower phase. The optical purity of the enantiomers obtained was consistently 99.5% or higher. The D-isomer extracted in the upper phase was recovered completely by using a backextraction column in series after the extraction column, allowing the upper phase to be used repeatedly for the resolution. The efficiency of the CFE system was estimated at more than 24 theoretical stages.

Spectrophotometric Determination of Carboxylic acids with 2-Mitrophenylhydrazine in Aqueous Solution

Abstract Carboxylic acids were determined spectrophotometrically by converting them into 2-nitrophenylhydrazides. The coupling reaction of carboxylic acids with 2-nitrophenylhydrazine was effected by a water-soluble carbociimide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, and proceeded efficiently in aqueous solution buffered at pH 4.5 with pyridine and hydrochloric acid. The yields of hydrazides from acetic and benzoic acids were nearly quantitative. The reaction mixture was then made strongly alkaline, and after heating to reduce the blank absorbance, the absorbance of the hydrazide was measured at around 540 nm. Calibration curves of various carboxylic acids were linear between 0.02 and 0.5 μmol through the origin.

Spectrophotometry Determination of Carboxylic Acids by Ferric Hydroxamate Formation with Water-Soluble Carbodiimide

Abstract Carboxylic acids in aqueous solution were determined spectrophotometrically as ferric hydroxamates. A water-soluble carbodiimide, l-cyclohexyl-3-(2-morpho-linyl-(4)-ethyl)carbodiimide metho-p-toluenesulfonate, was used as a cuopling agent to form hydroxamic acids. The coupling reaction was carried out at 37°C for 30 min in a solution buffered at pH 5.3 with pyridine and hydrochloric acid. Calibration curves of benzoic, acetic, citric, and a-ketoglutaric acids were linear between 0.25 and 8 μmol.

Flow injection determination of drugs by specific detection of carboxylic acids

The spectrophotometric determination of various drugs having a carboxyl function is described. The method involves flow injection and specific detection of carboxylic acids based on the formation of 2-nitrophenyl-hydrazide derivatives mediated by a water-soluble carbodiimide. Using this system, the linear calibration range spanned three orders of magnitude from 125 pmol to 200 nmol and both hydrophilic and hydrophobic drugs could be determined easily at a sampling rate of 100 per hour. A number of drug preparations were also analysed with a coefficient of variation of less than 1%(n= 10).

Rapid determination of glycine- and taurine-conjugated bile acids in human bile by high-performance liquid chromatography

With use of an anion-exchange packing, TSK Gel IEX 540 DEAE, for high-performance liquid column chromatography, glycine- and taurine-conjugated bile acids were separated in 10 min and detected with a differential refractometer. Human bile could be analyzed after a simple pretreatment. The purity of the peaks of glycine- and taurine-conjugated bile acids in human bile was confirmed by enzymatic determinations using 3 alpha-hydroxysteroid:NAD oxidoreductase. The molar ratios of the two forms of the conjugates (glycine/taurine ratios) in bile from normal subjects and from patients suffering from various hepatobiliary diseases were measured.

Fluorometric determination of Δ4-3-ketosteroids using aluminum salt and isonicotinylhydrazine

Abstract We have developed a new method for the fluorometric determination of Δ 4 -3-ketosteroids. In a methanolic solution of aluminum salt, Δ 4 -3-ketosteroids reacted with isonicotinylhydrazine (INH), and the resulting hydrazone fluoresced through complex formation with the aluminum ion in the solution. As little as 0.25 nmol of cortisol can be determined by this method.

Mechanized assay of serum cholinesterase by specific colorimetric detection of released acid

An automated assay method has been developed for the measurement of serum cholinesterase activity. The samples were prepared by an automated liquid handling unit and incubated for 9.7 min at 30 degrees C, followed by automatic injection into a colorimetric flow injection determination system for acetic acid liberated from acetylcholine by cholinesterase catalytic activity. The coloration reaction employed is based upon the formation of 2-nitrophenylhydrazide utilizing a water-soluble carbodiimide and has a high selectivity for carboxylic acids. The coefficients of variation of the proposed method were 2.4% for within-run analysis (n = 14) and 2.6% for between-run analysis (n = 6). Sera of 55 hospitalized patients were analyzed and the values obtained correlated well with an automated differential pH method (gamma = 0.989).

Determination of catecholamines by radioenzymatic assay using ion-pair liquid chromatography

A simple radioenzymatic method for the simultaneous determination of norepinephrine, epinephrine and dopamine in human serum was developed. catecholamines were converted to normetanephrine (NMN), metanephrine (MN), 3-methoxytyramine (3-MT) and their 4-O-methyl isomers by catechol O-methyl transferase with S-adenosylmethionine-(3H-methyl) ([3H]SAM). After addition of unlabelled NMN, MN and 3-MT as carriers, the methyl derivatives were isolated from the reaction mixture by passing through a small boric acid gel column and separated from each other by ion-pair liquid chromatography. Interference by tailing of radioactivity of [3H] SAM and its degradation products eluted earlier was minimized by the use of a precolumn and a branched flow path.