HPLC/MS determination of buprenorphine and norbuprenorphine in biological fluids and hair samples

An original method, based upon HPLC (high performance liquid chromatography)/Ionspray-MS, has been developed for the identification of buprenorphine (BUP) and norbuprenorphine (norBUP) in biological fluids and hair samples. Biological fluids (2 mL) are extracted at pH 8.4 by CHCl3/2-propanol/n-heptane (25:10:65, v/v) after addition of deuterated BUP (BUP-d4, 10 ng). Hair samples (40 mg) are extracted in the same conditions after decontamination by CH2Cl2, mechanical pulverization, addition of BUP-d4 (1 ng), acidic incubation (1 mL 0.1 N HCl, 56 degrees C overnight), then neutralization by NaOH. Analytes are separated on a 4-microns NovaPak C18 (Waters) column (150 by 2.0 mm, ID) with a mobile phase of acetonitrile/2 mM NH4COOH buffer, pH 3.0 (80:20, v/v; flow rate 200 microL/min; post column split 1:3). Detection is done by a Perkin-Elmer Sciex API-100 mass analyzer equipped with an ISP interface (nebulizing and curtain gas:99.95-% N2; main settings: orifice + 50 V, electron multiplier + 2400 V). The mean retention times for BUP, BUP-d4, and norBUP are 5.84, 5.79, and 4.42 min, respectively. For all compounds, mass spectra exhibit a unique, protonated molecular ion [M + H]+ at m/z 414 (norBUP), 468 (BUP), and 472 (BUP-d4), without any significant fragmentation. The lower limits of detection are 0.10 and 0.05 ng/mL blood, and 4 and 2 pg/mg hair for BUP and norBUP, respectively. BUP and norBUP concentrations measured in hair from six addicts under substitutive therapy by BUP ranged from 4 to 140 pg/mg, and from nondetected to 67 pg/mg, respectively. The good performances of this method in terms of both sensitivity and specificity make it a convenient alternative to HPLC/coulometry and GC/MS for the separate analysis of BUP and norBUP in biological samples.