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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
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, Division of Pharmacokinetics and Drug Therapy. (Hammarlund-Udenaes)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, Division of Pharmacokinetics and Drug Therapy. (Hammarlund-Udenaes)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, Division of Pharmacokinetics and Drug Therapy. (Hammarlund-Udenaes)
2005 (English)In: Rapid Communications in Mass Spectrometry, ISSN 0951-4198, E-ISSN 1097-0231, Vol. 19, no 15, 2116-2122 p.Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
John Wiley & Sons , 2005. Vol. 19, no 15, 2116-2122 p.
National Category
Pharmaceutical Sciences
Identifiers
URN: urn:nbn:se:uu:diva-103231DOI: 10.1002/rcm.2035PubMedID: 15988726OAI: oai:DiVA.org:uu-103231DiVA: diva2:217792
Available from: 2009-05-15 Created: 2009-05-15 Last updated: 2017-12-13Bibliographically approved
In thesis
1. Developmental Aspects of Drug Transport Across the Blood-Brain Barrier
Open this publication in new window or tab >>Developmental Aspects of Drug Transport Across the Blood-Brain Barrier
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The developmental aspect of drug transport across the blood-brain barrier (BBB) was investigated. Microdialysis was used to study unbound morphine BBB transport at different ages in sheep. An in vitro study was performed to find differentially expressed genes in brain capillary-rich fractions of the brain in rats of different ages. Microdialysis and brain-to-plasma ratios were used to study the contribution of breast cancer resistance protein (Bcrp) to the transport of nitrofurantoin (NTF) across the BBB of rats during development as well as in adult rats and mice.

A method of analysing morphine and its metabolites in plasma and microdialysis samples was developed and validated. The in vivo recovery of deuterated morphine, used as a calibrator in microdialysis experiments, was not affected by the presence of morphine in the tissue. A net influx of morphine was observed in premature lambs and adult sheep, in contrast to the efflux seen in other species. This influx decreased with age, indicating that the morphine transport across the BBB changes with age. In contrast, the transport of the morphine metabolite morphine-3-glucuronide (M3G) did not change with age. Microarray data indicated that several active transporters are differentially expressed with age. Moreover, the mRNA expression levels of Abcg2 (Bcrp) and Slc22a8 (organic anion transporter 3) changed with age when quantified using real-time polymerase chain reaction. In contrast, the expression of Abcb1 (P-glycoprotein) and occludin (a tight junction protein) did not change with age. In rats, the brain distribution of NTF decreased with age due to increased protein binding in plasma. The concentration ratio of unbound NTF across the BBB was low in the adult rat, due to intra-brain metabolism and/or efflux by other transporters. Bcrp did not appear to have a significant contribution in the developing rat or in knock-out mice compared to wild-type controls with regard to NTF BBB transport.

In conclusion, in vitro studies showed that the expression levels of some genes changed with age, presumably affecting subsequent drug distribution to the brain. Further, in vivo studies showed that distribution across the BBB changed with age for morphine but not for M3G or NTF.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2009. 60 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy, ISSN 1651-6192 ; 110
Keyword
Blood-brain barrier, development, active transport, tight junction proteins, microdialysis, recovery, morphine, nitrofurantoin, Bcrp, microarray, real-time PCR, in vitro, in vivo, LC-MS/MS
National Category
Pharmaceutical Sciences
Research subject
Pharmacokinetics and Drug Therapy
Identifiers
urn:nbn:se:uu:diva-108374 (URN)978-91-554-7627-4 (ISBN)
Public defence
2009-11-26, B42, Biomedicinskt Centrum, Husargatan 3, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2009-11-04 Created: 2009-09-17 Last updated: 2011-05-11Bibliographically approved

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Bengtsson, JörgenJansson, BrittHammarlund-Udenaes, Margareta

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