With the rise in the use and misuse of prescription opioids, there is an increasing need for the confirmed dentification of opioid analgesics in toxicology laboratories. The goals of this study were to (i) systematically evaluate the hydrolysis efficiency of four β-glucuronidase enzymes under optimizedcondition; (ii) evaluate compound recovery, matrix effects and precision of three protein precipitation plates and (iii) develop and validate a qualitative liquid-chromatography mass spectrometry (LC–MS/MS) assay to identify 13 opioids in urine. A recombinant β-glucuronidase exhibited the best overall hydrolysis efficiency for seven opioid glucuronide conjugates compared with β-glucuronidase from red abalone, Escherichia coli and Patella vulgata. One of the protein precipitation plates tested exhibited overall better recovery of the opioids and lower ion suppression compared with the other two plates. An ESI positive mode LC–MS/MS assay for qualitative opioid analysis was developed and validated. Linearity, LOD, precision, matrix effect, recovery, carryover and interference of the method were
evaluated. Sixty-two patient samples were analyzed by both a legacy GC–MS opioid method and the LC–MS/MSmethod, and 22 sampleswere analyzed by the LC–MS/MS and an LC–MS/MS reference method. The results of the comparisons showed good concordance. Overall, we described an efficient sample preparation procedure for a sensitive qualitative opioid confirmation assay in urine.