On-line desalting and determination of morphine, morphine-3-glucuronide and morphine-6-glucuronide in microdialysis and plasma samples using column switching and liquid chromatography/tandem mass spectrometry.
A sensitive and reproducible method for the determination of morphine and the metabolites morphine-3-glucuronide (M3G) and morphine-6-glucuronide (M6G) was developed. The method was validated for perfusion fluid used in microdialysis as well as for sheep and human plasma. A C18 guard column was used to desalt the samples before analytical separation on a ZIC HILIC (hydrophilic interaction chromatography) column and detection with tandem mass spectrometry (MS/MS). The mobile phases were 0.05% trifluoroacetic acid (TFA) for desalting and acetonitrile/5 mM ammonium acetate (70:30) for separation. Microdialysis samples (5 microL) were directly injected onto the system. The lower limits of quantification (LLOQ) for morphine, M3G and M6G were 0.50, 0.22 and 0.55 ng/mL, respectively, and the method was linear from LLOQ to 200 ng/mL. For plasma, a volume of 100 microL was precipitated with acetonitrile containing internal standards (deuterated morphine and metabolites). The supernatant was evaporated and reconstituted in 0.05% TFA before the desalting process. The LLOQs for sheep plasma were 2.0 and 3.1 ng/mL and the ranges were 2.0-2000 and 3.1-3100 ng/mL for morphine and M3G, respectively. For human plasma, the LLOQs were 0.78, 1.49 and 0.53 ng/mL and the ranges were 0.78-500, 1.49-1000 and 0.53-500 ng/mL for morphine, M3G and M6G, respectively.
Bengtsson
Jörgen
J
Division of Pharmacokinetics and Drug Therapy, Department of Pharmaceutical Biosciences, Box 591, SE-751 24 Uppsala, Sweden. Jorgen.Bengtsson@farmbio.uu.se
Jansson
Britt
B
Hammarlund-Udenaes
Margareta
M
D000697:Central Nervous System Stimulants / Q000097:blood*; D002851:Chromatography, High Pressure Liquid; D006801:Humans; D017551:Microdialysis / Q000379:methods*; D009020:Morphine / Q000097:blood*; D009022:Morphine Derivatives / Q000097:blood*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
D000697:Central Nervous System Stimulants; D009022:Morphine Derivatives; C035349:morphine-6-glucuronide; D009020:Morphine; C018108:morphine-3-glucuronide