HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHIC METHOD FOR THE QUANTITATIVE-DETERMINATION OF BUTORPHANOL, HYDROXYBUTORPHANOL, AND NORBUTORPHANOL INHUMAN URINE USING FLUORESCENCE DETECTION

A sensitive, quantitative reversed-phase high-performance liquid chrom atographic method has been established for the simultaneous determinat ion of butorphanol, a synthetic opioid, and its metabolites, hydroxybu torphanol and norbutorphanol, in human urine samples. The method invol ved extraction of butorphanol, hydroxybutorphanol, and norbutorphanol from urine (1.0 ml), buffered with 0.1 ml of 1.0 M ammonium acetate (p H 6.0), onto 1-ml Cyano Bond Elut columns. The eluent was evaporated u nder nitrogen and low heat, and reconstituted with the HPLC mobile pha se, acetonitrile-methanol-water (20:10:70, v/v/v), containing 10 mM am monium acetate and 10 mM TMAH (pH 5.0). The samples were chromatograph ed on a reversed-phase octyl 5-mum column. The analysis was accomplish ed by detection of the fluorescence of the three analytes, at excitati on and emission wavelengths of 200 nm and_325 nm, respectively. The re tention times for hydroxybutorphanol, norbutorphanol, the internal sta ndard, and butorphanol were 5.5, 9.0, 13.0, and 23.4 min respectively. The validated quantitation range of the method was 1-100 ng/ml for bu torphanol and hydroxybutorphanol, and 2-200 ng/ml for norbutorphanol i n urine. The observed recoveries for butorphanol, hydroxybutorphanol, and norbutorphanol were 93%, 72%, and 50%, respectively. Standard curv e correlation coefficients of 0.995 or greater were obtained during va lidation experiments and analysis of study samples. The method was app lied on study samples from a clinical study of butorphanol, providing a pharmacokinetic profiling of butorphanol.