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)

A fully automated HPLC method for the determination of catecholamines in biological samples utilizing ethylenediamine condensation and peroxyoxalate chemiluminescence detection.

A fully automated in-line extraction reversed-phase high-performance liquid chromatography (HPLC) method with chemiluminescence detection was developed for the analysis of human and rat plasma catecholamines (CAs), norepinephrine (NE), epinephrine (E) and dopamine (DA). N-Methyldopamine (N-MeDA) was used as an internal standard. The method involves collection of plasma samples, which are first diluted with a sample dilution buffer containing N-MeDA, and in-line extraction of CAs using a carboxylic acid small resin precolumn (SERUMOUT-CEX). This pre-extraction process was coupled with an HPLC system including reversed-phase mode separation on an analytical column (TSK gel ODS-80Ts), fluorogenic derivatization with ethylenediamine (ED) and finally postcolumn peroxyoxalate chemiluminescence reaction detection using bis [4-nitro-2-(3,6,9-trioxadecyloxycarbonyl)phenyl]oxalate (TDPO) and hydrogen peroxide. The optimized mobile phase compositions, flow rates, operation timing for the adsorption and desorption of CAs in the precolumn, the separation in the analytical column and the optimum fluorogenic and chemiluminogenic reaction conditions were investigated. The detection limit for all the CAs was about 1 fmol (signal-to-noise ratio is 2). Excellent linearity of the calibration curves for CAs was observed in the range from 5 to 500 fmol for each CA using the internal standard. The relative standard deviations of the method for determining NE (183 fmol), E (23.6 fmol) and DA (6.1 fmol) in 50 microL of human plasma (n = 3) were 2.8, 2.7 and 3.1%, respectively, for the within-day assay and 5.0, 3.8 and 4.0%, respectively, for the between-day assay. The method was applicable to the determination of CAs in 25-50 microL of human or rat plasma.

Analysis of 3-sulfated and nonsulfated bile acids by one-step solvolysis and high performance liquid chromatography.

A facile solvolysis procedure of 3-sulfated bile acid was devised using trifluoroacetic acid, tetrahydrofuran, and methanol. The sulfate esters were completely solvolyzed within only 2 hr by the present method. The clinical utility of the solvolysis procedure and high performance liquid chromatography using immobilized 3 alpha-hydroxysteroid dehydrogenase was demonstrated in the analysis of bile acids in serum of patients with obstructive jaundice. The quantities of 3-sulfated bile acids were calculated from the difference in the amount of bile acids before and after solvolysis. A significantly large proportion of 3-sulfated glycochenodeoxycholic acid, i.e., 21.9 to 31.3% of total glycochenodeoxycholic acid, was found in the serum of patients with obstructive jaundice. Thus, the present method permits simultaneous quantitation of 3-sulfated as well as nonsulfated bile acids in biological samples.

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.

Enantiomer resolution of D- and L-alpha-amino acid derivatives by supercritical fluid chromatography on novel chiral diamide phases with carbon dioxide.

The rapid resolution of racemic N-4-nitrobenzoylamino acid isopropyl esters was accomplished without the loss of enantioselectivity by supercritical fluid chromatography (SFC) on novel chiral valine-diamide phases with carbon dioxide and a polar methanol modifier. In each stationary phase, a chiral moiety was anchored to the silica gel surface by a long decamethylene spacer. The enantioselectivity in SFC was comparable to that in liquid chromatography using 2-propanol-n-hexane. The time required for analysis was less than 5 min, and the range of enantiomer resolution (Rs) was 10.8-1.25. On using 2-propanol in place of methanol the separation was improved, but was accompanied by a decrease in column efficiency. The end-capping effect of the remaining surface silanols on enantiomer resolution is discussed.

Relation between blood pressure and plasma catecholamine concentration after administration of calcium antagonists to rats.

Three types of calcium antagonists, diltiazem, verapamil and nicardipine, were separately infused into Sprague-Dawley (SD) rats (under pentobarbital anesthesia n = 5) through the left femoral vein at four different flow rates. Mean arterial blood pressure, heart rate and the concentration of plasma catecholamines (CAs), epinephrine (E), norepinephrine (NE) and dopamine (DA), were measured for each calcium antagonist, and the correlations between them were studied. Blood samples were collected within the infusion from common jugular vein. Plasma concentrations of CAs were determined by a HPLC-ethylenediamine condensation reaction-peroxyoxalate chemiluminescence detection system (HPLC-ED-PO-CL). The plasma concentration of CAs increased corresponding to the blood pressure reduction. The reduction induced by each calcium antagonist correlated with the logarithm of plasma NE concentration. The relation was expressed as Y = -alpha log X+m (Y, blood pressure; X, concentration of plasma NE; alpha, slope; and m, intercept). The correlation coefficients (rs) were -0.950 (diltiazem), -0.975 (verapamil) and -0.978 (nicardipine) (versus -0.734 for control). The alpha for nicardipine (108.4) was greater than those of diltiazem (85.4) and verapamil (80.8) (versus 31.0 for control), meaning that blood pressure reduction was greater in the case of nicardipine than diltiazem and verapamil, with an identical increment of plasma NE concentration. These data indicate that the contribution of the sympathetic nervous system to maintaining blood pressure reduced by nicardipine is less than that observed following the infusion of diltiazem and verapamil. Similar good inverse correlations between blood pressure and the logarithm of plasma concentration of E were observed with the three drugs infused (r = -0.928, -0.966, and -0.948 for diltiazem, verapmil and nicardipine, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

Relations between blood pressure and plasma norepinephrine concentrations after administration of diltiazem to rats: HPLC-peroxyoxalate chemiluminescence determination on an individual basis.

A calcium antagonist, diltiazem, was infused continuously into Sprague-Dawley rats through the left femoral vein at four different flow rates. The mean arterial blood pressure and concentrations of plasma norepinephrine (NE) were measured in each single rat (n = 5) and the correlations between them were studied. Blood (150 microL) was collected 13 times during the infusion. Plasma NE was determined by HPLC-ethylenediamine condensation reaction-peroxyoxalate chemiluminescence detection system (HPLC-ED-PO-CL). In four cases from 5 rats, the blood pressure reduction caused by diltiazem was inversely correlated to logarithm of plasma NE concentration. The relation was expressed as Y = -alogX+m. The coefficients of correlation were -0.9506, -0.9293, -0.9341 and -0.8675, respectively. The correlation for the last rat was worse (r = -0.0799). The good correlation would imply that the sympathetic nervous system released NE to maintain blood pressure up to the normal level, responding to the sympathetic nervous system released NE to maintain blood pressure up to the normal level, responding to the blood pressure reduction caused by diltiazem. The present experiment proved the feasibility of the determination method of NE utilizing HPLC-ED-PO-CL detection in applying to the individual rats.

Sensitive determination of (-)-isoproterenol and (-)-(R)-1-(3,4- dihydroxyphenyl)-2-[(3,4-dimethoxyphenethyl)amino] ethanol (T-0509), a cardiotonic agent, in rat plasma utilizing a fully automated catecholamine analyser.

(-)-Isoproterenol (ISO), used as bronchodilator, and (-)-(R)-1-(3,4-dihydroxyphenyl)-2-[(3,4-dimethoxyphenethyl)amino]ethanol (T-0509), a new cardiotonic agent, were determined in rat plasma (only 25 microL) using a fully automated catecholamine (CA) analyser that includes in-line pre-extraction of CAs, coupled with an HPLC-ethylenediamine condensation reaction and a peroxyoxalate chemiluminescence detection system (HPLC-ED-PO-CL). The chromatographic conditions were: precolumn, SERUMOUT-CEX; buffer for delivering CAs in the precolumn, 10 mM potassium phosphate buffer (pH 7.5)/ethanol (92 + 8, v/v) (1 mL/min); precolumn clean-up solution, 4% phosphoric acid/acetonitrile (50 + 50, v/v) (1 mL/min); analytical column, TSKgel ODS-80Ts, 250 x 4.6 mm i.d.; eluent, 75 mM potassium acetate buffer (pH 3.2)/50 mM potassium phosphate buffer (pH 3.2)/acetonitrile (83.6 + 4.4 + 12, v/v/v for ISO and 76 + 4 + 20 v/v/v for T-0509) (1 mL/min); fluorogenic reagent solution, 105 mM ED and 175 mM imidazole in acetonitrile/ethanol (90 + 10, v/v) (0.25 mL/min); chemiluminogenic reaction solution, 0.25 mM TDPO, 150 mM hydrogen peroxide and 110 mM TFA in dioxane/ethyl acetate (50 + 50, v/v) (1.4 mL/min). Colterol, (+/-)-1-(3,4-dihydroxyphenyl)-2-[(1,1-dimethyl)ethylamino]ethanol, (+/-)-N-t-butylnorepinephrine was used as an internal standard (IS) for ISO, and (+/-)-1-(3,4-dihydroxyphenyl)-2-[(3,4,5- trimethoxyphenethyl)amino]ethanol, (+/-)-(3,4,5-trimethoxyphenethyl- aminomethyl)-3,4-dihydroxybenzylalcohol (T-1583) for T-0509. The detection limits for ISO and T-0509 were 1.3 and 0.9 fmol on injection, respectively.