We established GC-MS/MS and LC-MS/MS analysis methods for nine fentanyl drugs in hair samples. Human hairs were prepared by soaking in a solution of water-dimethyl sulfoxide with target analytes. The drugs were norfentanyl, acetyl fentanyl, -fluorofentanyl, isobutyryl fentanyl, fentanyl, thiofentanyl, 4-fluoroisobutyr fentanyl, ocfentanil, and tetrahydrofuran fentanyl. For a single-factor experiment, a Box-Behnken design-response surface was used to optimize the pretreatment conditions of samples. The prepared samples were quantitatively analyzed by GC-MS/MS and LC-MS/MS. The working curve method was used for quantitative analysis with fentanyl-D5 as the internal standard. The concentrations of the nine fentanyl drugs in the samples were 1.488-6.494 ng mg, RSDs < 5.0%. For GC-MS/MS, the linear range of the nine fentanyl drugs was 0.5-5.0 ng mg, > 0.999. The detection limits were 0.02-0.05 ng mg, and the recovery rates were >86%. For LC-MS/MS, the nine fentanyl drugs had an excellent linear relationship within the concentration range of 3.0-220.0 pg mg, > 0.999. The detection limits were 0.05 pg mg and the recovery rates were >84%. The established methods were used for the detection of fentanyl drugs in human hairs, with high sensitivity, accuracy, and specificity. These two methods can be used for the certification of fentanyl certified reference substances (CRMs). In the experiment, the developed hair CRMs, which will continue to be studied in the future, are expected to be used in forensic drug abuse detection.
