Received: 5 August 2010 Revised: 4 October 2010 Accepted: 5 October 2010 Published online in Wiley Online Library: 00 Month 2010 Energy-dependent collision-induced dissociation study of buprenorphine and its synthetic precursors Bernadett Biri 1 , Jo ´ zsef Kalma ´r 2 , Lajos Nagy 1 , Attila Sipos 3 , Miklo ´ s Zsuga 1 and Sa ´ndor Ke ´ki 1 * 1 Department of Applied Chemistry, University of Debrecen, H-4032 Debrecen, Egyetem te ´r 1, Hungary 2 Department of Inorganic and Analytical Chemistry, University of Debrecen, H-4032 Debrecen, Egyetem te ´r 1, Hungary 3 Department of Pharmaceutical Chemistry, University of Debrecen and Research Group for Chemistry of Antibiotics of the Hungarian Academy of Sciences, H-4032 Debrecen, Egyetem te ´r 1, Hungary The collision-induced dissociation (CID) of protonated buprenorphine ([MRH] R ) and four related compounds was studied by electrospray quadrupole/time-of-flight mass spectrometry (ESI-QTOF MS). The fragmentation pathways were investigated by using energy-dependent CID and pseudo-MS 3 (in-source CID combined with tandem mass spectrometry (MS/MS)) methods. The first steps of the fragmentation are the parallel losses of the substituents from the non-aromatic ring moieties. Depending on the applied collision energies, a large number of further fragment ions arising from the cross-ring cleavages of the core-ring structure were observed. Based on the experimental results, a generalized fragmentation scheme was developed for the five buprenorphine derivatives highlighting the differences for the alternatively substituted compounds. The collision-energy-dependent fragmen- tation profile of buprenorphine is visualized in a two-dimensional plot to aid its fingerprint identification. Copyright ß 2010 John Wiley & Sons, Ltd. Since the fundamental work of Bentley and co-workers, [1] leading to the synthesis of the pharmaceutically important opioid-agonist ethorphine, [2] agonist-antagonist buprenor- phine and antagonist diprenorphine, [3] a variety of etheno- morphinans have been prepared. [4] The opioid analgesic buprenorphine was approved for the treatment of opioid dependence (Suboxone 1 with naltrexone), [5,6] but it was originally introduced as an analgesic agent (Bupren 1 ) with lower abuse potential than the traditional opioid analgesics, such as morphine. [7] The fundamental toxicological importance of buprenor- phine facilitated the development and validation of several chromatographic methods with mass spectrometric detection for the quantification of this compound and its metabolites, among other opioids in various human samples. [9–17] After adequate sample preparation, high-performance liquid chromatography (HPLC) coupled with electrospray ionization mass spectrometry (ESI-MS) [10,12–16] is often the method of choice. For the discrimination of buprenorphine from related compounds tandem mass spectrometry (MS/ MS) [10–16] is frequently utilized. Considering that no paper has been published on the detailed fragmentation properties of this compound, the aim of the present study was to establish an extensive ESI-MS/MS database on buprenor- phine and four closely related compounds as its synthetic precursors. Emphasis is placed on the alternated fragmenta- tion stability of the differently substituted precursors. The database is supposed to provide support for the MS/MS and fingerprint identification [18,19] of buprenorphine in combined analytical methods. The fragmentation of bupre- norphine was studied by ESI-MS/MS. For further clarifica- tion of the fragmentation scheme pseudo-MS 3 measurements (in-source dissociation combined with tandem mass spec- trometry) of selected fragment ions [20,21] and energy- dependent collision-induced dissociation (ED-CID) [22,23] experiments were performed. The proposed mechanism of fragmentation is discussed in the view of previous studies published on the fragmentation of numerous morphinans: morphine, [24–27] codeine, [24–27] thebaine, [24,25,27] papaver- ine, [27,28] and other related compounds. [24,25,27–29] EXPERIMENTAL Chemicals The chemicals used for the synthesis of the studied com- pounds were Analytical grade, others for the MS measure- ments were HPLC grade and all were purchased from Aldrich (Germany) and used without further purification. Synthesis of buprenorphine and its derivatives The structures of the compounds investigated are shown in Scheme 1. The synthesis of these ethenomorphinans is based on the Diels-Alder addition of methylvinyl ketone to the natural morphinan alkaloid thebaine. [1,8] The result- ing thevinone was catalytically hydrogenated to saturate the C(18)–C(19) double bond and then transformed into the appropriate tertiary alcohols, so called thevinols, Rapid Commun. Mass Spectrom. 2011, 25, 41–49 (wileyonlinelibrary.com) DOI: 10.1002/rcm.4821 Research Article * Correspondence to: S. Ke ´ki, Department of Applied Chemistry, University of Debrecen, H-4032 Debrecen, Egyetem te ´r 1, Hungary. E-mail: keki@tigris.unideb.hu Rapid Commun. Mass Spectrom. 2011, 25, 41–49 Copyright ß 2010 John Wiley & Sons, Ltd. 41