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Abstract:

INTRODUCTION Fentanyl, a synthetic narcotic analgesic with high potency and its major metabolite norfentanyl often occur in low doses in biological samples. AIM A highly sensitive UFLC-MS/MS method for determination of fentanyl and norfentanyl in urine and blood was developed and fully validated. METHODS Blood (1ml) and urine samples (2ml) were extracted on mixed-mode cation exchange Bond Elut Plexa PCX SPE cartridges followed by UFLC-MS/MS analysis (Shimadzu Prominence UFLC coupled to 3200 QTRAP, Applied Biosystems). Different sources (APCI vs. ESI), columns (with varying internal diameter and/or particle size) and mobile phases (low pH vs. high pH; acetonitrile vs. methanol) were evaluated. The final method was fully validated. RESULTS In order to obtain a highly sensitive method, several parameters were optimised. In the SPE protocol, an additional alkaline wash step was added in order to decrease matrix effects. Ionization of fentanyl and norfentanyl with ESI was more efficient than APCI. The use of a mobile phase of high pH resulted in higher ESI signals than the conventional low pH mobile phases. A comparison of columns with different internal diameter and/or smaller particles showed optimal resolution and sensitivity when an Acquity C18 column (1.7 µm, 2.1 mm x 50 mm) was used. The final method of preference lasted 6 minutes and gradient elution with 10 mM ammonium bicarbonate (pH 9) and methanol was performed. Deuterium labeled internal standards were used, but with careful evaluation of their stability since deuterium exchange was observed. The method was selective and stable for fentanyl and norfentanyl. No matrix effects and optimal extraction efficiency was seen because of the use of appropriate internal standards. The method was linear from 0.0025 to 5 ng/ml for fentanyl in urine, 0.005 to 10 ng/ml for norfentanyl in urine and 0.01 to 20 ng/ml for both fentanyl and norfentanyl in blood. Accuracy, repeatability and intermediate precision ranged between -15% and 15%. With limits of detection of 0.25 pg/ml for fentanyl and 2.5 pg/ml for norfentanyl in urine and 5 pg/ml for fentanyl and norfentanyl in whole blood this UFLC-MS/MS method has clearly superior sensitivity. CONCLUSION Considering the high sensitivity, this UFLC-MS/MS method is highly appropriate for the analysis of fentanyl and norfentanyl in forensic urine and blood samples.