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Technical Note Letrozole (Femara®) Lois C. Geer* and Patrick A. Hays ABSTRACT: Analytical data (GC/MS, FTIR, H1-NMR, C13-NMR) are reported for Letrozole (Femara), an anti-cancer drug which was submitted for a case involving androgenic steroids. KEYWORDS: Letrozole, Femara, Anti-Cancer Drug, Analysis, Forensic Chemistry. Introduction This laboratory occasionally receives unusual unknowns that were seized as suspected controlled substances. Recently, we received 4.46 grams of a flocculent white powder (see Photo 1), that had been included in a group of steroids submitted from a U.S. Customs seizure in Anchorage, Alaska. The steroids included various exhibits of boldenone, methandrostenolone, oxymetholone, stanazolol, testosterone, and trenbolone; tamoxifen (an anti-cancer drug) was also seized. The shipment is believed to have originated in Nanjing, China. The original analysis was performed by the U.S. Customs Laboratory, San Francisco, California, and determined that no controlled substance was present; however, an unknown substance was determined to be present. Preliminary analyses by Gas Chromatography/Mass Spectrometry (GC/MS, Figure 1) and Fourier Transform Infrared Spectroscopy (FTIR, Figure 2) gave no matches when searched in reference databases. The infrared spectrum showed an unusually strong peak in the region 2230 cm-1, suggesting strong carbon nitrogen triple bond (nitrile) stretching. Based on the GC/MS, the molecular weight was 285 atomic mass units (amu); this was confirmed by Liquid Chromatography/Mass Spectrometry (LC/MS) with chemical ionization. Further analysis by proton and carbon-13 Nuclear Magnetic Resonance (NMR) Spectroscopy (Figures 3 and 4) with quantitative analysis suggested a molecule with the chemical formula C17H11N5. An internet search for compounds with that formula returned letrozole as a possibility. Letrozole is an anti-cancer drug (Femara®) produced by Novartis Pharmaceuticals Corporation (East Hanover, NJ). Further analysis and comparison to spectral data and reference standard material (provided by Novartis) confirmed that the sample was letrozole (see structural formula below). Letrozole
Letrozole is commercially available as Femara® tablets containing 2.5 mg of letrozole. These are dark yellow, coated, slightly biconvex with beveled edges, and imprinted with “FV” on one side and “CG” on the other (see Photo 2). Experimental GC/MS GC/MS spectral data were collected on an Agilent 6890 GC/5973 MSD with a J&W 30 m length x 250 µm diameter column with a 0.25 µm film thickness of DB-1. The carrier gas was Helium with a constant flow of 1.0 mL/minute. The inlet was at 280 °C, with a split ratio of 25:1. The temperature program was 250 °C for 2 minutes, 15 °C per minute ramp to 300 °C with a 10 minute hold. The transfer line was at 280 °C, the quadrupole at 150 °C, and the source at 230 °C. The mass range was 29-550 amu. FTIR Infrared spectra were collected on a Nicolet Nexus 570 Infrared Spectrophotometer with KBr beam splitter and DTGS KBr detector equipped with a SensIR Technologies Durascope Attenuated Total Reflectance (ATR) accessory with a single bounce ATR element with KRS-5 focusing element. The 32 scans were collected between 4000 cm-1 and 400 cm-1 with a resolution of 4 cm-1. NMR One dimensional NMR analyses were performed on a Varian Mercury 400 MHz NMR using a 5 mm Nalorac Indirect Detection probe. The sample was prepared at 17 mg/mL in deuterated chloroform (CDCl3) containing TMS (tetramethylsilane) as the reference standard (Aldrich Chemical Co., Milwaukee, WI). The proton spectrum of the standard was obtained with 8 scans using a 45 second delay, 90° pulse, 2 second acquisition time, and oversampling by a factor of 6. The carbon spectrum of the standard was obtained with 256 scans using a 1 second delay, 45° pulse, 1.2 second acquisition time, and oversampling by a factor of 3. The sample was maintained at 25 °C. Gradient versions of the 2-Dimensional NMR experiments HSQC (correlation of hydrogens directly bonded to carbons) and HMBC (correlation of hydrogens 2, 3, or 4 bonds from carbons) were performed to make assignments (listed in Table 1). Prior to the arrival of the reference standard, structural elucidation was performed utilizing Applied Chemistry Developments (ACD, Toronto, Canada) software (HNMR Predictor, CNMR predictor, and Structure Elucidator). Results and Discussion This seizure represents the first time that letrozole has been submitted to a DEA laboratory as a drug exhibit. Letrozole is a non-steroidal inhibitor of the aromatase enzyme system, and acts to inhibit the conversion of androgens to estrogens (1). It is used in the first line treatment of breast cancer in post-menopausal women. It has potential for use in association with the abuse of androgenic steroids, both to prevent their conversion to estrogens, and to prevent or diminish the side effects of androgenic steroid abuse such as gyneocomastia (breast enlargement). Tamoxifen, another estrogen blocker, has long been associated with androgenic steroid abuse as it is also believed to prevent gynecomastia associated with that abuse; this explains why it (Tamoxifen) was also found in this seizure. References 1. http://www.us.femara.com
Figure 1. Mass Spectrum of Letrozole. Figure 2. FTIR of Letrozole. Figure 3. H1-NMR Spectrum of Letrozole (400.2 MHz). Figure 4. C13-NMR Spectrum of Letrozole (100.6 MHz).
Table 1. NMR Assignments of Protons and Carbons for Letrozole.
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