A sensitive method for the determination of 3 alpha,5 beta-tetrahydroaldosterone (THALD) in human urine is described. The method uses high-performance liquid chromatography with chemiluminescence detection. Urinary THALD, released by enzyme hydrolysis, is isolated and concentrated using a Sephadex G-25M column and Bond-Elut C1 cartridges, and then oxidized by copper(II) acetate to form the corresponding glyoxal derivative. The glyoxal derivative is converted into the chemiluminescent quinoxaline by reaction with 4,5-diaminophthalhydrazide. The chemiluminescent quinoxaline is separated within 50 min on a reversed-phase column (TSKgel ODS-120T) with isocratic elution, followed by chemiluminescence detection; the chemiluminescence is produced by the reaction of the quinoxaline with hydrogen peroxide in the presence of potassium hexacyanoferrate(III) in alkaline solution. The detection limit for THALD is 0.6 pmol (220 pg) ml-1 in urine [1.5 fmol (0.53 pg) per 20 microliters injection] at a signal-to-noise ratio of 3. This method permits the sensitive and precise determination of THALD in human urine (50 microliters) from normal subjects and a patient with primary aldosteronism.

No abstract

KeywordsHigh-performance liquid hydrazide, plasma chromatography, dexamethasone, chemiluminescence, 4,5-diaminophthalDexamethasone, 9a-fluoro-16a-methyl-1 1f3,17a,21-trihydroxy-l,4-pregnadiene-3,20-dione, is a synthetic corticosteroid which has been widely used for the treatment of various kinds of diseases such as endocrinopathy, rheumatism and nephrotic syndorome owing to its anti-allergic and anti-inflammatory actions. Because dexamethasone has strong efficacy against the diseases, the dose of the drug is much smaller (several mg per day) than those of other synthetic corticosteroids. Therefore, a sensitive method is required for the determination of dexamethasone in plasma after administration for efficient and safety use of the drug.Several methods involving radioimmunoassay (RIA)1-3, gas chromatography-mass spectrometry (GC-MS)4 and liquid chromatography (LC)s-12 have been reported for the determination of dexamethasone in plasma/serum. Although the RIA methods are very sensitive, they require chromatographic purification to avoid crossreaction with other endogeneous corticosteroids. The GC-MS method needs a relatively complicated procedure for sample pretreatment. Spectrophotometric LC5-11 are not very sensitive and thus require a large amount of plasma (2 -3 ml). A fluorometric LC12, which involves precolumn derivatization with 9-anthroylnitrile, is fairly sensitive. However, the method requires solidphase extraction cartridges for the clean-up of plasma and the elimination of the excess reagent before and after the derivatization.We have previously reported a chemiluminometric LC method13 for the determination of 3a,5/3-tetrahydroaldosterone in human urine. The method is based on the conversion of the corticosteroid into the corresponding glyoxal compound by copper(II) acetate oxidation. The glyoxal compound is derivatized with 4,5-diaminophthalhydrazide (DPH), a chemiluminescence derivatization reagent for a-dicarbonyl compounds14, to yield a chemiluminescent quinoxaline. The resulting quinoxaline produces chemiluminescence by reaction with hydrogen peroxide in the presence of potassium hexacyanoferrate(III) in alkaline medium after LC separation.Recently, it was found that dexamethasone generates chemiluminescence by the same derivatization and chemiluminescence reactions as described above. Our present objective is to develop a sensitive and selective LC method involving a simple pretreatment of plasma samples by precolumn Chemiluminescence derivatization with DPH for the quantification of dexamethasone in plasma. Beclomethasone (9a-chloro-16/3-methyl-1 1f3,17a,21-trihydroxy-l,4-pregnadiene-3,20-dione), which has a structure similar to that of dexamethasone, was used as an internal standard for the precise determination of dexamethasone.Dexamethasone concentrations in plasma from a healthy volunteer after oral administration of dexamethasone were measured by the established method.

A simple and sensitive method for the simultaneous determination of 3 alpha, 5 beta-tetrahydroaldosterone (THALD) and cortisol in human urine is described. The method uses high performance liquid chromatography with fluorescence detection. THALD and cortisol, released by enzyme hydrolysis, and fludrocortisone (internal standard) are isolated by a Sephadex G-25M column and a Bond-Elut C18 cartridge, and then oxidized by cupric acetate to form the corresponding glyoxal derivatives. The glyoxal derivatives are converted into the fluorescent quinoxalines by reaction with 1,2-diamino-4,5-methylenedioxybenzene. The quinoxalines are successfully separated on a reversed phase column (L-column ODS) with isocratic elution and monitored fluorimetrically. The detection limits for THALD and cortisol are 0.45 and 1.18 ng/mL urine (0.65 and 2.65 pmol/100 microL injection volume), respectively, at a signal-to-noise ratio of 3 in a 100 microL injection volume. This method permits the precise and sensitive determination of THALD and cortisol in human urine (2 mL).