In a presentation given at TIAFT 2018 (P277) by M.F.Fogerty et al., the development and optimization of a method for the following compounds was presented: fentanyl, norfentanyl, furanylfentanyl, butyrylfentanyl, methoxyacetylfentanyl, despropionylfentanyl, tetrahydrofuranylfentanyl, fluoro-isobutyrylfentanyl (FIBF), acrylfentanyl, para-fluorofentanyl, ortho-fluorofentanyl, carfentanil, α-methylfentanyl, β-methylfentanyl, isobutyrylfentanyl, para-methylfentanyl, cyclopentylfentanyl, cyclopropylfentanyl and β-hydroxyfentanyl. UCT’s famous Clean Screen® DAU was used to extract the compounds from whole blood samples.
Columns were conditioned and washed before eluting with ethyl acetate/acetonitrile/ammonium hydroxide. The eluent was evaporated and reconstituted in mobile phase. Analysis was performed using UPLC with Tandem Mass Spectrometry. Chromatographic separation was achieved using a Poroshell EC C-18 column (3.0 mm x 150 mm, 2.7 um). The mobile phases used were 5 mM ammonium formate (pH=3) and 0.1% formic acid in methanol. A fit-for-purpose method validation adapted from SWGTOX was performed by assessing the following criteria over five days: bias, precision, linear dynamic range, interferences and carryover.
The criteria for controls were as follows: average bias ±20%, within and between run precision coefficient of variation less than 15%. Results: Calibration curves were calculated using a quadratic fit with 1/x weighting regression analysis with the resulting range of 0.1-100 ng per mL. All analytes in this method, except for 4-ANPP and carfentanil (criteria expanded to 30%), passed all parameters of the validation listed above. All compounds were stable in matrix, except for norfentanyl, at -20°C and -80°C for 30 days. Norfentanyl was stable for 14 days at -20°C and -80°C. The assay’s limits of detection (LOD) was 6 pg/mL. The method was applied to genuine post-mortem blood samples where 42 cases were found to be positive for cyclopropylfentanyl and methoxyacetylfentanyl. The mean and median concentration for cyclopropylfentanyl (n=32) was 15.2 (±11.9) and 12.3 ng per mL, respectively. Methoxyacetylfentanyl cases (n=14) averaged 17.7 (±11.5) ng per mL, with a median of 15.1 ng per mL. Other fentanyl analogs detected in combination in these cases included acrylfentanyl, FIBF, furanylfentanyl, acetylfentanyl and U-47700.