Quantitation of polar analytes using column-switching: Application to oxycodone and three metabolites in human plasma

• Published in: Journal of chromatography. B, Analytical technologies in the biomedical and life sciences Analytical technologies in the biomedical and life sciences, 2010-03, Vol.878 (7), p.637-644
• Main Authors: Wagner, M., Bourgogne, E., Varesio, E., Hopfgartner, G.
• Format: Article
• Language: English
• Subjects: Analysis; Analytical, structural and metabolic biochemistry; Atmospheric pressure; Biological and medical sciences; Cartridges; Chromatography, Liquid - methods; Column-switching; Fundamental and applied biological sciences. Psychology; General pharmacology; Human; Humans; Hydrogen-Ion Concentration; Ionization; Least-Squares Analysis; Liquid chromatography; Mass spectrometry; Medical sciences; Metabolites; Methyl alcohol; Morphinans - blood; Opioid receptor agonist; Oxycodone - blood; Oxymorphone - blood; Perchlorates; Perchloric acid; Pharmacology. Drug treatments; Plasma; Quantitative analysis; Reproducibility of Results; Sensitivity and Specificity
• ISSN: 1570-0232; 1873-376X
• DOI: 10.1016/j.jchromb.2010.01.014

We present herein a sensitive and selective assay for the determination of oxycodone and its main metabolites, oxymorphone, noroxycodone and noroxymorphone in human plasma, using column-switching and liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS). Sample preparation comprised protein precipitation with perchloric acid. After neutralization, the supernatant was injected without any evaporation step onto a polymeric, pH-resistant cartridge (HySphere Resin GP 10–12 μm) for sample clean-up (Prospekt II). The latter operation was achieved by using alkaline conditions to ensure retention of analytes and methanol for matrix interference removal. More than two hundred plasma samples could be analyzed with a single cartridge. Analytes were desorbed in the backflush mode and were separated on a conventional reversed phase column (XTerra MS 4.6 × 50 mm, 3.5 μm), using an acidic mobile phase (i.e. containing 0.1% of formic acid). Mass spectrometric detection was achieved with a 4000 Q TRAP equipped with an atmospheric pressure chemical ionization (APCI) source, in positive ionization mode, operated in the selected reaction monitoring mode (SRM). Starting from a plasma volume of 250 μl, quantification ranges were 25–10,000 pg/ml for OXM and NOXM and 50–10,000 pg/ml for OXC and NOXC. Accuracy was found to be within 98% and 108% and precision better than 7%. Replicate determination of incurred or study samples ensured the method to be reproducible and usable for clinical studies.