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Validation of the Neogen® Fentanyl ELISA Kit for Blood and Urine.

The Neogen® Fentanyl ready-to-use enzyme-linked immunosorbent assay kit was validated following the Scientific Working Group for Forensic Toxicology Standard Practices for Method Validation in Forensic Toxicology Laboratory Guidelines. Two decision points, 0.5 and 1 ng/mL, were successfully validated for whole blood. For urine, two decision points, 1 and 5 ng/mL, were also successfully validated. The validation included the evaluation of sensitivity, precision, specificity, carryover, plate drift, ruggedness/robustness and a case sample evaluation. The empirically determined limit of detection was 0.25 ng/mL for blood and 0.5 ng/mL for urine. Precision was determined at five different concentrations ranging from 0.25 to 1.5 ng/mL with 15 replicates at each level for whole blood and demonstrated a <2.4% coefficient of variation (CV). In urine, the CV was <5.6% at six different concentrations from 0.5 to 7.5 ng/mL with 15 replicates at each level. Cross-reactivity was evaluated for norfentanyl, acetyl fentanyl, 4-anilino-N-phenethylpiperidine, beta-hydroxythiofentanyl, butyryl fentanyl and furanyl fentanyl.