A METHOD FOR THE PREPARATION OF CALIBRATION CURVES FOR ACETAMINOPHEN GLUCURONIDE AND ACETAMINOPHEN SULFATE IN RABBIT URINE WITHOUT USE OF AUTHENTIC COMPOUNDS IN HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

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A method for the preparation of calibration curves for acetaminophen glucuronide (NAPAG) and acetaminophen sulfate (NAPAS) in rabbit urine without use of authentic compounds in high-performance liquid chromatography was examined. Rabbits were dosed intravenously with acetaminophen (NAPA, 30 mg/kg). Urine was collected and diluted. A plot of the peak area ratio of NAPAG to internal standard against NAPA concentration after the hydrolysis of diluted urine with β-glucuronidase was linear and passed through the origin. A linear tendency was also observed in the plot of the peak area ratio of NAPAS to internal standard against NAPA concentration calculated by the difference between the peak area ratio of NAPA after the hydrolysis with β-glucuronidase and that with β-glucuronidase/arylsulfatase. Thus, once the calibration curve has been prepared following the enzyme hydrolysis of NAPAG and NAPAS, then the concentration of NAPAG and NAPAS in the sample solution can be calculated from the peak of NAPAG and NAPAS, respectively. The method is simple, and has the advantage that pure standards of the individual NAPA metabolites are not required.
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