Determination of naloxone-3-glucuronide in human plasma and urine by HILIC–MS/MS

• We developed a HILIC/MS/MS method for direct determination of N3G in human plasma and urine.
• HILIC/MS/MS offers significant advantages including simple sample pretreatment without hydrolysis and enhanced sensitivity.
• HILIC/MS/MS has been successfully applied to a pharmacokinetic study of N3G.
• We report the excretion fraction of N3G in urine following oral administration of naloxone.

A hydrophilic interaction chromatography–tandem mass spectrometric (HILIC–MS/MS) method was developed for the direct determination of naloxone-3-glucuronide (N3G) in human plasma and urine. After a straightforward sample preparation by protein precipitation, N3G was analyzed directly without the need for hydrolysis. Chromatographic separation was performed on a HILIC column. The mobile phase was composed of acetonitrile–10 mmol/L ammonium formate (86:14, v/v), with a flow rate of 0.4 mL/min. The detection was performed on a triple quadrupole tandem mass spectrometer by multiple reaction monitoring (MRM) mode via positive electrospray ionisation (ESI+) source. The linear calibration range was 0.5 to 200 ng/mL in plasma and 10 to 5000 ng/mL in urine (r2 > 0.99). The intra- and inter-day precision (relative standard deviation, RSD) values were below 15% and the accuracies (relative error, RE) were −7.1% to 2.8% in plasma and −1.3% to 10.3% in urine at three quality control levels. In human subjects receiving 100 mg tilidine and 8 mg naloxone, mean AUC0–24 of N3G was 160.93 ± 52.77 ng/mL h and mean Cmax was 75.33 ± 25.27 ng/mL. In 24-h urine samples, 8.0% of the dose was excreted in the form of N3G in urine. These results demonstrated a new method suitable for in vivo pharmacokinetic studies of N3G.