Expanding fentanyl analog testing to include brain specimens: method validation and homogenate stability assessment

The Harris County Institute of Forensic Sciences recently added brain to its fentanyl analog testing method for 14 analogs (fluoroisobutyryl fentanyl, acetyl fentanyl, acryl fentanyl, alfentanil, butyryl fentanyl, carfentanil, fentanyl, para-fluorofentanyl, furanyl fentanyl, methoxyacetyl fentanyl, norcarfentanil, norfentanyl, sufentanil, and valeryl fentanyl) and 3 U-series drugs (U-47700, U-48800, and U-49900). Brain is a protected and isolated organ with lower metabolic activity than other tissues, which can assist in interpreting results and preserving parent drug. Limited publications testing brain samples for fentanyl and fentanyl analogs exist and none describe homogenate stability for these analytes. Validation of the solid phase extraction and liquid chromatography tandem mass spectrometry method followed the ASB 036 Standard Practices for Method Validation in Forensic Toxicology and included limit of detection, limit of quantification, calibration model, bias and precision, ionization suppression/enhancement, interferences, carryover, processed sample stability, and dilution integrity. Carfentanil, fentanyl, furanyl fentanyl and methoxyacetyl fentanyl met quantitative bias and precision acceptance criteria in brain. To assess homogenate stability, brain homogenates (both unpreserved and preserved with 1% sodium fluoride) were fortified with 50 ng/mL of analyte, stored at room temperature (∼20°C), refrigerated (2-8°C), or frozen (∼-20°C), and analyzed in triplicate over a 90-day period. Analytes were considered stable if analyte/internal standard response ratio was within ± 20% of Day 0 and chromatographic peaks met qualitative acceptance criteria. Frozen brain homogenates could be stored for up to 90 days and withstood three freeze/thaw cycles for acetyl fentanyl, alfentanil, fentanyl, para-fluorofentanyl, FIBF, methoxyacetyl fentanyl, and norfentanyl. Brain homogenate stability was improved when frozen and was not impacted by the addition of 1% sodium fluoride. The study herein provides insight into the feasibility of testing brain for fentanyl analogs and their stability under various storage conditions, contributing valuable data to the limited literature on brain toxicology testing.