Potentials of ion trap collisional spectrometry for liquid chromatography/electrospray ionization tandem mass spectrometry determination of buprenorphine and nor-buprenorphine in urine, blood and hair samples Donata Favretto, Giampietro Frison, Susanna Vogliardi and Santo Davide Ferrara * Forensic Toxicologyand Antidoping, University Hospital of Padova, Via Falloppio 50, I-35121 Padova, Italy Received 13 January 2006; Revised 15 February 2006; Accepted 15 February 2006 A liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) method has been developed for the analysis of buprenorphine (BUP) and nor-buprenorphine (NBUP) in biological fluids. Analytes are isolated from urine and blood, after addition of d 4 -buprenorphine (d 4 -BUP) as internal standard, by solid-phase extraction. Preparation of hair involves external decontamination, mechanical pulverization, overnight incubation in acidic medium, and neutraliza- tion prior to extraction. Enzymatic hydrolysis with b-glucuronidase may be performed to distinguish between free and total BUP. Chromatographic separation is accomplished by gradient elution on a cyanopropyl 2.1 T 150 mm column. Positive ion ESI and MS analyses are carried out in an ion trap mass spectrometer. The use of this mass analyzer allows effective collisional experiments to be performed on ESI-generated MH R species. Abundant product ions are produced, which can be monitored together with precursor ions without losing sensitivity. Thus, assay selectivity is defi- nitely increased with respect to LC/ESI-MS/MS methods in which only precursor ions are monitored. The method has good linearity (calibration curves were linear in the range 0.1–10 ng/mL in urine and blood, in the range 10–160 pg/mg in hair) and limits of detection of 0.05 ng/mL for both BUP and NBUP in blood and urine samples, of 4 pg/mg for both analytes in hair. Both intra- and inter-assay precision and accuracy were satisfactory at three concentrations studied: relative standard deviations were <13.7% in urine, <17.3% in blood, <17.8% in hair; percent deviation of the mean from the true value was always <10.5% in urine and blood, <16.1% in hair. The method can be used to determine both analytes in the urine and hair of drug addicts on replacement therapy, and in post-mortem blood specimens when there is suspicion of drug-related death. Copyright # 2006 John Wiley & Sons, Ltd. Buprenorphine (BUP) is a semi-synthetic, highly lipophilic opiate derivative, with a structure closely related to that of morphine. It is obtained from the alkaloid thebaine after a multistep chemical procedure. The drug has been classified as a powerful analgesic (25–40 times more potent than morphine) 1 with prolonged duration of action. 2 It shows both agonist and antagonist activity on central receptors: 3 at low doses (typically 0.3–0.6 mg, intravenous or intramus- cular), BUP acts as a partial agonist at m receptors, with action similar to that of morphine; at higher doses, it exhibits potent antagonistic effects at k opiate receptors, with activity like that of naloxone. For more than twenty years, BUP has successfully been used through the intramuscular, intrave- nous and sublingual routes to control both acute post- operative pain and the chronic pain associated with terminal cancer. 4–6 The drug has a low abuse potential, causes limited respiratory depressant activity, 7 and presents a less rapid and less intense withdrawal syndrome than pure m receptor agonists like morphine, 8 and it is now a valid alternative to methadone for treating opiate addiction. 9 After intramuscular injection or sublingual administra- tion, BUP rapidly reaches its peak plasma concentration while oral administration is subject to considerable first- pass metabolism in the liver. Metabolism occurs by N- dealkylation to N-dealkylbuprenorphine (nor-buprenorphine, NBUP) and conjugation; metabolites are eliminated mainly in the faeces, and 30% are excreted in the urine. Because of the clinical importance of BUP as an analgesic and for the treatment of opiate addiction, it has become advisable for a forensic laboratory to be able to assay it in biological samples. However, this determination is difficult due to: (1) very low therapeutic plasma levels (peak plasma concentrations of 0.45–0.84 ng/mL after a single sublingual administration of 0.4 mg; steady-state concentrations in the RAPID COMMUNICATIONS IN MASS SPECTROMETRY Rapid Commun. Mass Spectrom. 2006; 20: 1257–1265 Published online in Wiley InterScience (www.interscience.wiley.com) DOI: 10.1002/rcm.2444 *Correspondence to: S. D. Ferrara, Forensic Toxicology and Anti- doping, University Hospital of Padova, Via Falloppio 50, I-35121 Padova, Italy. E-mail: santodavide.ferrara@unipd.it Copyright # 2006 John Wiley & Sons, Ltd.