Original article Liquid chromatography–mass spectrometric analysis of buprenorphine and its N-dealkylated metabolite norbuprenorphine in rat brain tissue and plasma Hongfei Yue a , Michael R. Borenstein b , Susan A. Jansen a , Robert B. Raffa b, * a Department of Chemistry, Temple University, Philadelphia, PA, USA b School of Pharmacy, Temple University, 3307 N. Broad Street, Philadelphia, PA 19140, USA Received 11 March 2005; accepted 4 April 2005 Abstract Introduction: A specific, accurate, and reproducible liquid chromatography – mass spectrometric (LC/MS) method was developed and validated that allows simultaneous measurement of the centrally acting analgesic buprenorphine and its major metabolite, norbuprenorphine, in rat brain and plasma samples. Methods: A 96-well plate solid phase extraction (SPE) procedure was developed for buprenorphine and norbuprenorphine using mixed-mode cation-exchange reversed-phase sorbent. An LC method using a C8 column with isocratic mobile phase (80:20 water/acetonitrile with 20 mM ammonium acetate and 0.1% acetic acid) was developed for reproducible and selective separation. A quadrupole mass spectrometer with atmospheric electrospray ionization source under positive ion mode was used for detection. d 4 -Buprenorphine and d 3 -norbuprenorphine were used as internal standards. Results: The calibration curves for buprenorphine and norbuprenorphine in plasma and brain tissue were linear within the range of 7 to 8333 ng/ml (plasma) and 5 to 5000 ng/g (brain). The lower limit of quantification for both buprenorphine and norbuprenorphine from brain tissue was 5 ng/g, and from plasma was 7 ng/ml. Assay accuracy and precision of back-calculated standards were within T 15%. Discussion: This method will be useful for investigation of buprenorphine’s mechanism of action and clinical profile. D 2005 Elsevier Inc. All rights reserved. Keywords: Buprenorphine; Norbuprenorphine; LC/MS; Methods; Plasma; Brain; Rat 1. Introduction Buprenorphine, [5a,7a (S )]-17-(Cyclopropylmethyl-a- (1,1-dimethylethyl)-4,5-epoxy-18,19-dihydro-3-hydroxy-6- methoxy- a-methyl-6,14-ethenomorphinan-7-methanol (C 29 H 41 NO 4 , mol. wt. 467.64), is a centrally acting opioid clinically important for its use as an analgesic and for the treatment of opioid abuse and dependence (Cowan & Lewis, 1995). The mechanism of action of buprenorphine has not been fully elucidated, but it has been speculated that buprenorphine’s N-dealkylated metabolite (norbuprenor- phine) (Fig. 1) contributes to the pharmacologic profile of the parent drug (Huang, Kehner, Cowan, & Liu-Chen, 2001). However, this possibility is predicated on sufficient metabolic conversion of buprenorphine to norbuprenorphine and passage of norbuprenorphine across the blood –brain barrier. The purpose of the present study was to develop a specific measure of the plasma and brain levels of buprenorphine and norbuprenorphine in samples from rats administered antinociceptive (analgesic) doses of buprenor- phine intravenously. Various methods have been previously used to determine buprenorphine in biological matrices, including immuno- assay (Cirimele, Etienne, Villain, Ludes, & Kintz, 2004; Cirimele, Kintz, Lohner, & Ludes, 2003; Debrabandere, Van Boven, & Daenens, 1995), HPLC with UV (Lagrange, Pehourcq, Baumevieille, & Begaud, 1998), fluorescence (Ho, Wang, Ho, & Hu, 1991), or electrochemical (Debra- 1056-8719/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.vascn.2005.04.012 * Corresponding author. Tel.: +1 215 707 4976; fax: +1 215 707 5228. E-mail address: robert.raffa@temple.edu (R.B. Raffa). Journal of Pharmacological and Toxicological Methods 52 (2005) 314 – 322 www.elsevier.com/locate/jpharmtox