Simultaneous HPLC Determination of Acetaminophen and Four of Its Major Metabolites in Small-Volume Biologic Fluids
| Autor: | Yumiko Honda, Jin Feng Qi, Satoru Mineshita |
|---|---|
| Rok vydání: | 2002 |
| Předmět: | |
| Zdroj: | Rinsho yakuri/Japanese Journal of Clinical Pharmacology and Therapeutics. 33:263-275 |
| ISSN: | 1882-8272 0388-1601 |
| DOI: | 10.3999/jscpt.33.6_263 |
| Popis: | An HPLC method for quantifying acetaminophen (APAP) and four of its major metabolites: glucuronide, cysteine, sulfate, and mercapturate conjugates in biologic fluids has been developed. Proteinous samples (25μl) were deproteinized with ZnSO4-saturated acetonitrile, and urine (100μl) was extracted under alkaline conditions with chloroform-isopropanol (95: 5, v/v) followed by N2 evaporation. The separations were resolved on a 250mm×4.6mm ID, Phenomenex-ODS reverse-phase column with a simple isocratic mobile phase composed of 0.73 M acetic acid in water (pH 2.44) methanol (90: 10, v/v, plus 15μl/L n-octylamine for serum/plasma; 80: 20, v/v, for saliva and 92: 8, v/v, plus 30μl/L n-octylamine for urine). All compounds were simultaneously detected at a wavelength of 260 nm using the structural analog of APAP, 3-acetamidophenol, as an internal standard. Analysis was achieved within 9 min (for serum/plasma) and 4.5 min (for saliva) or 13.8 min (for urine) per run at a flow rate of 1.5 ml/min. The limits of detection ranged between 6 and 16 ng/ml, while limits of quantitation were 11-103 ng/ml for the compounds. The relationship between peak areas and concentration was linear over the range of 0.088 and 70.00μg/ml for APAP, 0.074 and 14.75μg/ml for APAP cysteine, 0.123 and 197.30μg/ml for APAP glucuronide, 0.205 and 390.21μg/ml for APAP sulfate, and 0.124 and 12.40μg/ml for APAP mercapturate. The resolution chromatograms showed no interfering peaks from endogenous components of biologic fluids. Accuracy of the method ranged from -9.0 to 11.1%, and intra-day and inter-day coefficient of variation ranged from 0.2 to 9.3% and 0.1 to 5.7% for all compounds. Extraction efficiencies of urine samples were between 90.1% and 111.6%. Applicability of the method was examined by a pharmacokinetic study in men and male rats that received one dose of 10 mg/kg of APAP. |
| Databáze: | OpenAIRE |
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