"There are many differing opinions regarding the optimal methods for forensic hair analysis, especially concerning pretreatment and extraction parameters. Extraction methods include enzymatic, acid/base, and solvent techniques, each with their own advantages and disadvantages. The Society of Hair Testing (SoHT) suggests that any extraction technique can be utilized with the use of adequate control samples. This lack of consensus regarding best practice extraction methods contributes to possible bias in hair testing across multiple laboratories. Previous research in this laboratory has focused on pretreatment parameters, such as hair particle size, extraction solvent to sample size ratio, and extraction time. However, published work has not assessed the best extraction techniques for different drugs and metabolites of interest.

Authentic hair reference material (HRM) containing the drugs of interest was obtained from a commercial source. Samples of 20 mg each were weighed into steel milling jars. The hair samples were pulverized into a powder using a Retsch MM200 ball mill with chrome-steel milling beads at 3,800 rpm for 30 s. The samples were extracted using one of three techniques: a 2-h incubation in 12.5 µL/mg hair of methanol:acetonitrile:2 mM ammonium formate (25:25:50, v/v/v) at 37ºC (solvent swelling technique), a 2-h incubation in 12.5 µL/mg hair of 1 M NaOH at 37ºC (base technique), or a 2-h incubation in 12.5 µL/mg hair of aqueous 12 mg/mL dithiothreitol and 2 mg/mL proteinase K at 37ºC (enzymatic degradation technique). The samples were then transferred into Eppendorf tubes and centrifuged for 30 min. Post-centrifugation, the eluent was subjected to solid phase extraction using an Agilent Bond Elut LRC mixed mode C8 and strong cation-exchange (SCX) cartridge. An Agilent 1290/6460 LC-QqQ-MS was used for analysis.

Recovery of each drug by each extraction technique was evaluated to determine which methods were most effective. Those with the highest extraction recovery included solvent swelling (for diazepam, nordiazepam, cocaine, p-hydroxycocaine, oxycodone, and 6-acetylmorphine), base (for fentanyl), and enzymatic degradation (for methamphetamine, cocaethylene, norcocaine, and morphine). In conclusion, the most effective extraction technique for removing drugs/metabolites from authentic HRM varies based on the physicochemical properties of the drugs of interest."