m-Hydroxybenzoylecgonine: An Important Contributor to the Immunoreactivity in Assays for Benzoylecgonine in Meconium

Meconium has been reported to be a more suitable specimen than maternal or neonatal urine for detecting fetal exposure to cocaine. In a study comparing various immunoassays with gas chromatography/mass spectrometry (GC/MS), several unexplained discrepancies among the assays were noted. Using methanol extracts of meconium samples, an immunoreactive spot that was more polar than benzoylecgonine was detected by thin-layer chromatography (TLC). An extract of this spot analyzed by GC/MS yielded a fragmentation pattern indicative of an aryl hydroxylated benzoylecgonine. Standards of m-hydroxybenzoylecgonine, o-hydroxybenzoylecgonine, and p-hydroxybenzoylecgonine were synthesized; it was determined that m-hydroxybenzoylecgonine had the same retention time and ion ratios as the TLC immunoreactive spot. Furthermore, m-hydroxybenzoylecgonine proved to be immunoreactive. Ten meconium samples immunoreactive for benzoylecgonine were analyzed by GC/MS. Results before and after hydrolysis with β-glucuronidase (type IX) showed free m-hydroxybenzoylecgonine comprising 59 to 94% of the total m-hydroxybenzoylecgonine and showed total m-hydroxybenzoylecgonine values ranging from 0.2 to 6.3 times as high as benzoylecgonine. Therefore, m-hydroxybenzoylecgonine appears to be a quantitatively important cocaine metabolite in meconium, which is responsible for a significant portion of the discrepancy between benzoylecgonine concentrations in meconium extracts as measured by immunoassay and GC/MS.