Simultaneous determination of fentanyl and midazolam using high-performance liquid chromatography with ultraviolet detection.

When measuring fentanyl and midazolam simultaneously in the same plasma sample with standard high-performance liquid chromatography-ultraviolet (HPLC-UV) detection, overlap of the fentanyl peak by the midazolam peak occurs, which makes fentanyl determination impossible. We tested the hypothesis that by acidifying the methanol mobile phase with 0.02% perchloric acid, 70%, it would be possible to separate both peaks. The UV detector was set at 200 nm. Calibration curves for fentanyl (range 0-2000 pg/ml) and midazolam (range 0-400 ng/ml) were linear (r>0.99). The detection limits were 200 pg/ml (fentanyl) and 10 ng/ml (midazolam). Precision and accuracy for intra- and inter-assay variability as well as in-line validation with quality control samples (QCS) were acceptable (<15 and 20%, respectively), except for fentanyl QCS of 200 pg/ml (17.8% precision). Although less sensitive than gas chromatography-mass spectrometry (GC-MS), reliable measurements of fentanyl, simultaneously with midazolam, can be performed with this HPLC-UV system.