Validation of a New Recombinant Antibody Fragment (rFab)-Based Homogeneous Enzyme Immunoassay for the Highly Specific Detection of 6-Acetylmorphine in Urine

J Anal Toxicol. 2015 Nov-Dec;39(9):726-33. doi: 10.1093/jat/bkv090. Epub 2015 Aug 26.


The detection of 6-acetylmorphine (6-AM) in urine by immunoassay methods is challenging due to its short half-life and its similarity in structure to many commonly abused opiates that are often present at very high concentrations in urine. Current 6-AM homogeneous enzyme immunoassays use lyophilized reagents because of the instability of 6-AM in water or lack of the required specificity due to high cross-reactivity with morphine. A new 6-AM rFab-based homogeneous enzyme immunoassay (HEIA) has been developed with highly improved specificity. Using a cutoff concentration of 10 ng/mL, morphine or morphine glucuronides did not produce a positive signal up to 300,000 or 1,000,000 ng/mL, respectively. Assay imprecision (n = 80) was less than 1.5% using four replicates per day for 20 days over the range 0-20 ng/mL. Cross-reactivity with structurally related or non-related compounds was assessed at concentrations up to 1,000,000 ng/mL. Interferences from endogenous compounds at ±25% cutoff were also performed at the concentrations ranging from 100,000 to 500,000 ng/mL. The effect of varied pH values on assay performance at ±25% cutoff was investigated; no false-positive or false-negative results were observed between pH 4 and -11. Based on the analysis of 149 authentic urine samples, the accuracy of the 6-AM HEIA compared with LC-MS-MS was 100%. These results demonstrated that rFab can be suitable for traditional HEIA with desired detection sensitivity and stability.

Publication types

  • Validation Study

MeSH terms

  • Analgesics, Opioid / urine
  • Chromatography, Liquid
  • Half-Life
  • Humans
  • Hydrogen-Ion Concentration
  • Immunoenzyme Techniques*
  • Immunoglobulin Fragments / chemistry*
  • Limit of Detection
  • Morphine / urine
  • Morphine Derivatives / urine*
  • Sensitivity and Specificity
  • Specimen Handling
  • Substance Abuse Detection
  • Tandem Mass Spectrometry


  • Analgesics, Opioid
  • Immunoglobulin Fragments
  • Morphine Derivatives
  • Morphine
  • 6-O-monoacetylmorphine