BACKGROUND

Xylazine is an α-2 adrenoreceptor agonist used as a sedative/analgesic in veterinary medicine. Xylazine is known to be present within the street supply of opiates in urban Philadelphia. Medical staff at our hospital asked if we could test for xylazine in fentanyl screen-positive urine samples. We developed an LC-MS/MS assay for this purpose, and determined prevalence of xylazine among fentanyl screen-positive urine samples at our hospital.

METHODS

The LC-MS/MS assay utilized d5-norfentanyl as internal standard (IS). One hundred microliter samples were extracted with 200 µl of MeOH/IS. LC was performed using a Phenomenex Kinetix C18 column (100 A, 5 µm, 50 × 4.6 mm) at 40 °C. Time-variable mobile phases (A = H2O, 0.1% formic acid; B = MeOH, 0.1% formic acid) were used at a fixed flow rate of 0.5 ml/min. MS/MS used positive electrospray ionization, monitoring m/z transitions of 221 > 164 for xylazine (primary), 221 > 90 for xylazine (qualifier), and 238 > 84 for d5-norfentanyl (IS). Retention time was 3.9 min for both xylazine and IS.

RESULTS

Calibration curve was linear (0-500 ng/ml; r > 0.99). Inter-assay CVs (n = 20) were 5.2% (18 ng/ml) and 6.6% (95 ng/ml). Lower limit of detection was set at 10 ng/ml (CV = 15%). Among 81 urine samples that were screen-positive for fentanyl (Ark Diagnostics immunoassay), 63 (78%) were positive for xylazine (>10 ng/ml).

CONCLUSIONS

By LC-MS/MS, there was high prevalence (78%) of xylazine in fentanyl screen-positive urine samples submitted to the laboratory. Because α-2 adrenoreceptor agonists may be used in treatment of opioid addiction, knowledge of xylazine exposure may be clinically useful to guide patient management.