Sakae Higashidate

Determination of femtomole concentrations of catecholamines by high-performance liquid chromatography with peroxyoxalate chemiluminescence detection

A highly sensitive method for determination of the plasma catecholamines, norepinephrine (NE), epinephrine (E) and dopamine (DA) is described. The method consists of the extraction of the catecholamines, using 3,4-dihydroxybenzylamine as internal standard, from plasma with alumina (5 mg), followed by a reversed-phase column separation, on-column fluorogenic derivatization with ethylenediamine (ED) and post-column peroxyoxalate chemiluminescent reaction detection utilizing bis[4-nitro-2-(3,6,9-trioxadecyl-oxycarbonyl)phenyl] oxalate (TDPO) and hydrogen peroxide. In order to optimize the reaction conditions for high-performance liquid chromatography to obtain highly sensitive detection, the effects of changing reagent compositions on the chemiluminescence yield were investigated. The following are the optimized conditions. Eluent, a mixture of 50 mmol l-1 potassium acetate (pH 3.20)-50 mmol l-1 potassium phosphate (pH 3.20)-acetonitrile (90.15 + 4.85 + 3 v/v/v) containing 1 mmol l-1 sodium hexanesulfonate (40 degrees C) and flow rate, 0.5 ml min-1. Fluorogenic reagent solution, 105 mmol l-1 ED and 175 mmol l-1 imidazole in acetonitrile-ethanol (90 + 10 v/v) and flow rate, 0.25 ml min-1. Reaction coil (15 m x 0.5 mm i.d.) heated at 80 degrees C. Chemiluminogenic reagent solution, 0.25 mmol l-1 TDPO, 150 mmol l-1 hydrogen peroxide and 110 mmol l-1 trifluoroacetic acid in dioxane-ethyl acetate (50:50 v/v) and flow rate, 1.4 ml min-1. The detection limits for all the catecholamines were 1 fmol (signal-to-noise ratio at 2). The standard deviations of the method for the determination of NE, E and DA added to rat plasma (2.5 nM) were 3, 3 and 4%, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Sensitive detection of oxo-steroids and oxo-bile acids by liquid chromatography with peroxyoxalate chemiluminescence detection

Abstract Oxo-steroids and oxo-bile acid ethyl esters were derivatized with 5- N,N -dimethylamino-naphthalene-1-sulphonohydrazide (DNS-hydrazine) to DNS-hydrazone in the presence of hydrochloric acid in ethanol or trifluoroacetic acid in benzene, purified by high-performance gel-permeation chromatography, separated on an ODS column with an eluent containing tetrahydrofuran and detected via a peroxyoxalate chemiluminescence reaction using bis[4-nitro-2-(3,6,9-trioxadecyloxycarbonyl)phenyl] oxalate (TDPO). Sensitive detection (femtomole level) of each oxo-steroid which appeared as a single peak was achieved. The procedure for the isolation of oxo-bile acids developed for GC-MS allowed the detection by this system of an unusual oxo-bile acid, 7α-hydroxy-3-oxo-5β-cholanic acid, at the nanomole l −1 level in urine from a patient with cholestatic liver disease.

Sensitive assay system for bile acids and steroids having hydroxyl groups utilizing high-performance liquid chromatography with peroxyoxalate chemiluminescence detection.

3 alpha- or 3 beta-hydroxysteroids, such as bile acids (free and glycine and taurine conjugates), 3 beta-hydoxy-5-cholenic acid, pregnanediol, 5-pregnene-3 beta, 20 beta-diol and 5-pregnene-3-beta,20 alpha-diol, were converted to 3-oxosteroids by enzymatic reaction using immobilized hydroxysteroid dehydrogenase, derivatized with dansylhydrazine to the corresponding dansyl hydrazones and purified by gel permeation chromatography. The dansyl hydrazones were chromatographed on a C18 column with a tetrahydrofuran-containing eluent and detected at the level of a few femtomoles by a peroxyoxalate chemiluminescence post-column reaction using bis[4-nitro-2-(3,6,9-trioxadecyloxycarbonyl)phenyl] oxalate as a chemilumigenic reagent. The dansyl hydrazones of chenodeoxycholic acid and deoxycholic acid (free and glycine and taurine conjugates) in particular, which coeluted under the chromatographic conditions above, were separated using an eluent including acetonitrile and 2,6-di-O-methyl-beta-cyclodextrin and detected in the same way.

Effect of tetrahydrofuran on reversed-phase liquid chromatographic separation of dansylhydrazones of oxo-steroids

The oxo-steroids corticosterone, testosterone and progresterone were converted with 5-N,N-dimethylaminonaphthalene- 1 -sulphonohydrazide (DNS-hydrazine) into DNS-hydrazones, which were separated by reversed-phase liquid chromatography and detected fluorimetrically. The anti and syn conformers of the hydrazones were recognized on a C18 column with eluents consisting of imidazole buffer (nitrate, pH 6.0) containing acetonitrile, methanol, acetone or dioxane, and thus two peaks were observed for each oxo-steroid derivative. On the other hand, a single peak was obtained when an eluent containing tetrahydrofuran was used, affording highly sensitive detection of oxo-steroids. A further improvement in the detection of oxo-steroids down to subpicomole level was achieved by adding a gel permeation chromatographic clean-up procedure to remove the excess reagent before the analysis by reversed-phase liquid chromatography.

New High-Speed Fully Automated Guanidino Compound Analyzer

The introduction of dialysis has made rapid progress in the field of research and clinical treatment of renal failure, and has contributed very much to physiological elucidation of uremia. In uremia, concentrations of serum creatinine, BUN (Blood urea nitrogen), uric acid and guanidino compounds increase, and abnormalities of water, electrolytes, and acid-base equilibrium are seen. In addition, there are still many unknown substances and unknown factors, which play an important role in this disease. These substances are generally called uremic toxins. It is essential to investigate uremic toxins for the elucidation of uremia and the establishment of proper dialysis. Many compounds from low molecular weight to middle molecular weight have been investigated as suspicious candidates for the uremic toxin. Among these compounds, guanidines such as methylguanidine (MG), guanidinosuccinic acid (GSA) and guanidine (G) have been suggested. These guanidino compounds are also considered as causes for abnormalities in brain metabolism.