JOURNAL OF CHROMATOGRAPHY B: BIOMEDICAL APPLICATIONS ELSEVIER Journal of Chromatography B, 674 (1995) 197-204 Synthesis of 5,6,6-[2H3]finasteride and quantitative determination of finasteride in human plasma at picogram level by an isotope-dilution mass spectrometric method A. Guarna a'*, G. Danza b, G. Bartolucci a, A. Marrucci b, S. Dini b, M. Serio b "Dipartimemo di Chimica Organica 'Ugo Schiff' e Centro di Studio SuUa Chimica e la Strunura dei Composti Eterociclici e loro Applicazioni, Universitgl di Firenze, Via G. Capponi, 9, 1-50124, Firenze, Italy t'Unitgl di Endocrino/ogia, Dipartimento di Fisiopatologia Clinica, Universit,i di Firenze, Viale G. Pieraccini, 6, 1-50134, Firenze, Italy First received 12 May 1995; revised manuscript received 14 July 1995; accepted 24 July 1995 Abstract Finasteride is a potent inhibitor of the enzyme steroid 5a-reductase now approved as a drug for the treatment of benign prostatic hyperplasia. We describe an original method for the quantitative determination of finasteride at picogram level in human plasma by isotope-dilution gas chromatography mass spectrometry. 5,6,6-[2H3]Finasteride was synthesized with an high ratio of trideuteration (finasteride/[2H3]finasteride = 0.007) allowing its optimal use as internal standard. Plasma samples were purified in a single-step procedure on solid-phase extraction C~8 columns with a recovery />90%. Samples were injected in the GC-MS instrument without any derivatization and the minimum detection level of finasteride was 50 pg with a signal-to-noise ratio of 6:1. The coefficients of variation for the 5 and 10 ng/ml (plasma) concentrations were 5.8% and 4%, respectively. The method has been applied to the determination of the plasma pharmacokinetic of finasteride in five male volunteers treated with a single 5-mg dose of the drug, affording kinetic parameters which are in good agreement with the values previously reported with a different methodology. The present method results accurate, specific, sensible and reliable for a routinely determination of finasteride at picogram levels. Keywords: Isotope-dilution mass spectrometry; Finasteride L Introduction high inhibitory activity of finasteride towards the human prostatic isozyme and its low toxicity Finasteride (N-tert.-butyl-3-oxo-4-aza-androst- allowed its use as a drug for the treatment of 1-ene-17/3-carboxamide) is a 4-azasteroid syn- DHT dependent pathologies. Several clinical thesized by Merck Sharp & Dohme [1] that studies have demonstrated the efficacy of finas- selectively inhibits steroid 5a-reductase, the en- teride in reducing the pathologic effects of zyme responsible of the conversion of testoster- benign prostatic hyperplasia (BPH) [2-4]. Finas- one (T) into dihydrotestosterone (DHT). The teride has been recently approved, and is now widely used, for the treatment of BPH. Other * Corresponding author. applications of this drug are now being studied, 0378-4347/95/$09.50 (~) 1995 Elsevier Science B.V. All rights reserved SSDI 0378-4347(95)00323-1 198 A. Guarna et al. / J. Chromatogr. B 674 (1995) 197-204 for the treatment of prostatic cancer [5,6] and employed for the synthesis were analytical grade male pattern baldness [7,8]. To support clinical and were purchased from Carlo Erba (Milan, studies it is therefore important to have a pre- Italy); all the reagents used for the synthesis cise, accurate and sensitive method to determi- were purchased from Sigma (St. Louis, MO, nate finasteride in biological fluids. The methods USA). The solvents employed for the purifica- developed since now are based on the use of tion procedure were analytical grade and were HPLC coupled with an UV detector [9,10] or, purchased from J.T. Baker (Phillipsburg, NJ, recently, with an atmospheric pressure chemical USA); columns used for the purification were ionization (APCI) tandem mass spectrometric SPE C18 (500 mg) IST Isolute (Mid Glamorgan, (MS-MS) detector [11]. The sensitivity limit UK); GC capillary column used was an HPl-12 reached with the UV detector is 1 ng/ml and, m x 0.2 mm × 0.33 /zm obtained from Hewlett- due to the low wavelength of detection (210 nm), Packard (Palo Alto, CA, USA). All analyses it results scarcely selective and requires an effi- were performed using a Hewlett-Packard GC- cient and highly controlled sample preparation MS system composed by a 5890 series II gas with a complex chromatographic system: finas- chromatograph equipped with a 5971A Mass teride is purified from biological samples using a Spectrometry Detector and a 7673A automatic solid-phase extraction (SPE) CN column and injector. analyzed with an HPLC system equipped with two coupled reversed-phase columns maintained 2.2. Methods at room temperature. The recent use of the mass spectrometric detector [11] has increased both Synthesis of deuterated internal standard the sensitivity (200 pg/ml) and the selectivity of 5, 6,6-[e H3]finasteride (4) the assay but, in the method described, the chromatographic system is still complex requir- 5,6,6-[2 H3]-3-Oxo-4-aza-androstane-17~- ing a series of two reversed-phase columns main- carboxylic acid (1). tained at 70°C; moreover the MS-MS detector 3-Oxo-4-aza-5-androstene-17fl-carboxylic acid used is quite expensive and the complexity of the (0.81 g, 2.55 mmol), prepared according to the apparatus requires an accurate management to procedure described by Rasmusson et al. [1,12] perform a quantitative routinely assay. was stirred in 2H20 for 16 h at room tempera- In this paper we describe a new method based ture and, after filtration, in CH3COO2H (12 ml) on isotopic dilution gas chromatography-mass for 3 h. Then, the CH3COO2H solution was spectrometry for the determination of finasteride maintained for 48 h under 2H z (3 atm) at 60°C in in human plasma using a GC-MS bench-top the presence of Pt20 (0.12 g, 0.53 mmol). The instrument. The present method results accurate, solution was then filtered, evaporated and specific, sensible and reliable for a routinely washed with water affording 5,6,6-[ H3]-3-oxo-4- determination of finasteride at picogram levels, aza-androstane-17fl-carboxylic acid (1) (90% and was applied to the determination of the yield, 2.29 mmol). pharmacokinetic profile of finasteride in plasma of five male volunteers treated with a 5-mg oral dose of the drug. 5,6,6-[2 H~]-3 - Oxo-4-aza-androst- 1-ene- 17fl- carboxylic acid (2). Bis(trimethylsilyl)trifluoroacetamide (BSTFA) 2. Experimental (3.8 ml, 14.3 mmol) was added to a stirred suspension of 5,6,6-[2H3]-3-oxo-4-aza-andros - 2.1. Materials tane-17fl-carboxylic acid (1) (1.1 g, 3.41 mmol) and 2,3-dichloro-5,6-dicyano-l,4-benzoquinone Finasteride was a kind gift of Merck Sharp & (DDQ) (0.79 g, 3.48 mmol) in 11 ml of an- Dohme (Dr. ElisaLzth Stoner). The solvents hydrous dioxane. The reaction mixture was A. Guarna et al. / J. Chrornatogr. B 674 (1995) 197-204 199 stirred at room temperature under N z for 4 h and 373.2 (2H 1 = 4.57%), m/z = 372.2 (2H 0 = then was refluxed for 18 h. The resulting dark 0.45%). red solution was poured into a mixture con- taining 20 ml of CH2C1 ~ and 5.3 ml of a 1% aqueous solution of sodium bisulphite. After Instrumental conditions vigorous stirring the mixture was filtered to Analyses were performed using a 12 m x 0.2 eliminate the precipitated hydroquinone, the mm x 0.33/zm GC capillary column with a 100% organic layer was separated, washed with 7 ml of methylsilicone phase. The temperature program 2 M HC1 and dried over Na2SO 4. After removal was: 70°Cx 1 min; then 30°C/min to 200°C; of the solvent, the residue was washed with 200°C x 1 min; then 10°C/min to 280°C; 280°C x CH3CN and dried under vacuum affording 5,6,6- 10 min; then 20°C/min to 300°C for 5 min. The [ZH3]-3-oxo-4-aza-androst-l-ene-17fl-carboxylic transfer line temperature was 280°C. The carrier acid (2) (70% yield, 2.39 mmol). gas was helium with an inlet pressure of 35 KPa. In these conditions the retention time of finas- teride and its trideuterated analogue was 19.7 S-2-Pyridyl-3-oxo- 4-aza-androst- 1-ene- 17~-thio- min. Injections were performed in the splittless carboxylate (3). mode with a purge-off time of 1 min and an A solution of the 5,6,6-[ZH3]-3-oxo-4-aza-an - injector temperature of 280°C. SIM analyses drost-l-ene-17/3-carboxylic acid (2) (0.75 g, 2.34 were made acquiring the molecular ions at m/z mmol), with triphenylphosphine (1.03 g, 4.67 372.2 and 375.2 for finasteride and mmol) and 2,2'-dithiopyridine in 5 ml of toluene [2H3]finasteride respectively. was stirred at room temperature for 6 h, then the solvent was evaporated under vacuum and the residue purified on a short silica gel column Analytical method (70-230 mesh, 7 cm x 4 cm I.D.), using acetone- Extraction and purification of finasteride and dichloromethane 1:1 (v:v) as eluent, to give S-2- I.S. from plasma samples were performed using pyridyl-3-oxo-4-aza-androst-l-ene-17/3-carboxy- SPE C j8 columns (500 mg). Columns were pre- late (3) (56% yield, 1.31 mmol). washed with 2 x 3 ml of ethyl acetate and 3 ml of methanol, then conditioned with 3 ml of metha- nol and 3 ml of water.