Liquid Chromatography-Mass Spectrometry

For determination of brain/blood concentration of tamoxifen and its metabolites, tamoxifen was administered to mice in one/two/five doses (100/200 mg/kg i.p.) 24 h apart. After 6 h, 2, 4, 6, and 8 days the mice were decapitated, and the brains were quickly dissected out/blood samples were collected and frozen at -80°C until sample processing. In the case of mice with FCI, tamoxifen was administrated in two doses (200 mg/kg i.p.) 24 h apart, and 3 days after the second tamoxifen injection, the FCI was induced, and 1 day after FCI induction, the brains were isolated. According to (Lien et al., 1991), the brains were homogenized (1:5, w:v) in ice cold 50 mM Tris-HCl (Sigma–Aldrich, St. Louis, MO, USA), pH 7.4 using a tissue ruptor homogeniser (Qiagen, Hilden, Germany). Brain homogenates were mixed with acetonitrile (1:1, v:v) and precipitated proteins removed by centrifugation (15 000 g for 6 min). The supernatants represented extracts for analysis by liquid chromatography coupled with mass spectrometry (LC-MS). The same procedure applied to tamoxifen non-treated mice was used to prepare analyte-free matrix. For each analyzed time-point we used 3–5 mice with the exception of aged mice treated with full dose of tamoxifen at time-point 2 days. At this time-point we analyzed two mice and due to high mortality of aged mouse we reduced the initial tamoxifen dose in the next experiments with aged mice.

Standards of tamoxifen, 4-hydroxytamoxifen, N-desmethyltamoxifen, (E/Z)-N-desmethyl-4-hydroxytamoxifen(endoxifen), (E/Z)-N,N-didesmethyl-4-hydroxytamoxifen (norendoxifen), and (Z)-4-hydroxytamoxifen-d5 were purchased from Toronto Research Chemicals INC (Toronto, ON, Canada). The ratio of Z and E isomers in N-desmethyl-4-hydroxytamoxifen (48% of Z isomer) and N,N-didesmethyl-4-hydroxytamoxifen (66% of Z isomer) was determined by liquid chromatography under isocratic conditions (35% B) with UV detection at 245 nm (further conditions are described below in the LC-MS analysis paragraph). The standards were dissolved in acetonitrile, at the concentration of 200 μg/ml and diluted with analyte-free matrix at the required concentration.

A validated method (Teunissen et al., 2011) for determination of tamoxifen and its main phase I metabolites was adopted for analysis of the samples. Four five-point calibration curves were constructed: (1) From limit of detection (LOD) (0.625 or 1.250 ng/ml) to 10 ng/ml; (2) from 5 to 160 ng/ml; (3) from 62.5 ng/ml to 1000 ng/ml; and (4) from 1 to 64 μg/ml. Calibration solutions, quality control (QC) samples and extracts for calibrations (3) and (4) were, prior to analysis diluted 100x with the analyte-free matrix. The calibration curves were fitted linearly or quadratically with the residual values lower than 15% (20% for a limit of quantification – LOQ – level). QC samples were measured in triplicates at the LOQ, low, middle and high concentration levels of the respective calibration curve. Recovery values of the QC samples between 85 and 115% (80–120% for LOQ) and relative standard deviation lower than 5% for all QC samples and all analytes demonstrated satisfactory accuracy and precision of the method. LOQ values were determined as follows: 1.25 ng/ml for 4-hydroxytamoxifen and N-desmethyl-4-hydroxytamoxifen; 0.625 ng/ml for tamoxifen, N-desmethyl-tamoxifen, and N,N-didesmethyl-4-hydroxytamoxifen. LOD values were determined as 0.313 ng/ml for all analytes.

(Z)-4-Hydroxytamoxifen-d5 was used as the internal standard for all determined analytes, at the final concentration of 2.5 (calibrations 1 and 3) or 40 ng/ml (calibrations 2 and 4). The extracts, calibration or QC samples were centrifuged (13 000 rpm, 5 min) and mixed with the solution of internal standard in acetonitrile [9:1 (v:v)] or diluted 100x with analyte-free extract and then mixed with the solution of internal standard in acetonitrile [9:1, (v:v)]. The samples were analyzed on an Acquity UPLC system with an LCT premier XE time-of-flight mass spectrometer (Waters, Milford, MA, USA) using the liquid chromatography column Acquity UPLC BEH C18 (50 mm × 2.1 mm I.D., particle size 1.7 μm, Waters, Milford, MA, USA) and a two-component mobile phase. The mobile phase A and B consisted of 0.1% formic acid in water and acetonitrile, respectively. The analyses were performed under a linear gradient program (min/%B) 0/30, 2/30, 6.5/52.5 followed by a 1 min column clean-up (95% B) and a 1.5 min equilibration (30% B). The total analysis time was 9 min. The column temperature was set at 30°C, and flow rate at 0.4 ml/min. The mass spectrometer operated in the V mode, the capillary voltage was set at +2600 V; cone voltage, +40 V; desolvation gas temperature, 350°C; ion source block temperature, 120°C; cone gas flow, 50 L/h; desolvation gas flow, 800 L/h; ion guide 1 and 2 RFs, 200 and 300 V, respectively; hexapole RF, 150 V. Ions in the m/z range 100–1000 were detected using a scan time of 0.15 s and an inter-scan delay of 0.01 s. The mass accuracy was kept below 5 ppm using lock spray technology, with leucine enkephalin as the reference compound (2 ng/μl, 5 μl/min). The data were processed with MassLynx software using the QuanLynx application manager (Waters, Milford, MA, USA). The chromatograms of ions corresponding to [M+H]⁺ adducts were extracted with the tolerance window of 0.05 Da, and smoothed (mean method, two smoothing iterations, width of 2 scans).