Sensitive Determination of Fentanyl in Low-Volume Serum Samples by LC-MS/MS

Fentanyl is a widely used drug in the management of pain. Present LC-MS/MS methods for analysis of fentanyl require a large volume of serum, but yet the sensitivity was at about 50 pg/mL. Here, we report a modified liquid-liquid extraction method for the analysis of fentanyl in serum. The method is very sensitive with a LLOQ of 5 pg/mL while using only 0.175 mL of serum for analysis. The separation was performed on a Zorbax XDB-C18 column (4.6 × 50 mm, 1.8 μm, 600 bar) using a mobile phase of water: acetonitrile (70:30 v/v) with 0.1% formic acid that was pumped isocratically at a flow rate of 0.5 mL per minute. The calibration curve was found to be linear over a range of 5–10,000 pg/mL. The inter-day and intra-day accuracy and precision were tested using low (20 pg/mL), medium (1000 pg/mL), and high (5000 pg/mL) quality control samples of fentanyl prepared in blank human serum and were within ± 15% of the nominal value. Fentanyl was also found to be stable in various storage and sample preparation conditions, including short-term bench-top storage (for 5 h), freeze-thaw cycling (three cycles), long-term frozen condition (4.5 months at - 70°C), and post-preparative storage (for 48 h).