Development and validation of a sensitive ultra performance liquid chromatography tandem mass spectrometry method for the analysis of fentanyl and its major metabolite norfentanyl in urine and whole blood in forensic context

• Published in: Journal of chromatography. B, Analytical technologies in the biomedical and life sciences Vol. 878; no. 22; pp. 1987 - 1996
• Main Authors: Verplaetse, Ruth, Tytgat, Jan
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 15.07.2010; Elsevier
• Subjects: Analysis; Analytical, structural and metabolic biochemistry; Biological and medical sciences; Chromatography, High Pressure Liquid - methods; Fentanyl; Fentanyl - analogs & derivatives; Fentanyl - blood; Fentanyl - metabolism; Fentanyl - urine; Forensic Sciences; Fundamental and applied biological sciences. Psychology; General pharmacology; High pH mobile phase; Humans; LC–MS/MS; Medical sciences; Norfentanyl; Pharmacology. Drug treatments; Post-mortem samples; Tandem Mass Spectrometry - methods; UFLC or UPLC; UFLC or UPLC; Fentanyl; High pH mobile phase; Norfentanyl; LC–MS/MS; Post-mortem samples; Urine; Validation; Ultra performance liquid chromatography; Biological fluid; Agonist; Metabolite; μ Opioid receptor; Postmortem; HPLC chromatography; Opiates; Narcotic analgesic; Mobile phase; Whole blood; LC-MS/MS; Analysis; Mass spectrometry MS/MS; Development; pH; Phenylpiperidine derivatives
• ISSN: 1570-0232; 1873-376X
• DOI: 10.1016/j.jchromb.2010.05.033

Fentanyl and its major metabolite norfentanyl often occur in low doses in biological samples. Therefore, a highly sensitive liquid chromatography tandem mass spectrometry (LC–MS/MS) method has been developed and fully validated. Sample preparation was performed on a mixed-mode cation exchange solid phase extraction (SPE) cartridge with an additional alkaline wash step to decrease matrix effects and thus increase sensitivity. Ionization of fentanyl and norfentanyl with electrospray ionization (ESI) was more efficient than atmospheric pressure chemical ionization (APCI). The use of a mobile phase of high pH resulted in higher ESI signals than the conventional low pH mobile phases. In the final method, gradient elution with 10 mM ammonium bicarbonate (pH 9) and methanol was performed. A comparison of columns with different internal diameter and/or smaller particles showed optimal resolution and sensitivity when an Acquity C18 column (1.7 μm, 2.1 mm × 50 mm) was used. Deuterium labeled internal standards were used, but with careful evaluation of their stability since loss of deuteriums was seen. With limits of detection of 0.25 pg/ml for fentanyl and 2.5 pg/ml for norfentanyl in urine and 5 pg/ml for fentanyl and norfentanyl in whole blood the presented method is highly appropriate for the analysis of fentanyl and norfentanyl in forensic urine and blood samples.