Preliminary results using a kit to measure tamoxifen and metabolites concentrations in capillary blood samples from women with breast cancer

Participants and sampling

This study was performed in accordance with the Declaration of Helsinki and was approved by the Regional Ethics Review Board of Karolinska Institute (Dnr 2019-02745).

Participant selection and study design

Patients who were participants in the single-center Tailor Dose II study (EudraCT No. 2017-000641-44) at the Karolinska University Hospital Breast Centre were eligible for this small pilot study. From January 17, 2020 to January 30, 2020, eligible patients were informed of the pilot study and written informed consent was obtained from those electing to participate. Patients were included who had BC and had been receiving an ongoing course of adjuvant TAM 20 mg daily for at least a month. Sampling occurred once for each participant, between Monday and Friday, from 0900 to 1500 h, at the Breast Centre. For each participant, time and date of sampling, age, and duration of TAM treatment were noted. In addition, participant functioning, and TAM side effects were identified using a variety of questionnaires.

Participant sampling techniques

Whole venous blood, plasma, and capillary blood samples were taken at the same session for each patient to allow direct comparisons of the results from each sampling technique. First, two venous samples were collected in EDTA tubes. One tube was centrifuged, and the isolated plasma was set aside and frozen (− 20 °C), while the other tube of whole blood was frozen (− 20 °C) without processing. Next, two capillary samples (50 µL each) were obtained. For this process, the finger was disinfected with a 70% isopropanol cloth, air-dried, and warmed. The fingertip was then pricked using a single-use automatic 1.8 mm safety lancet (Sarstedt AG & Co KG, Germany) and capillary blood was collected using a capillary Minivette POCT 50 µL (Sarstedt AG & Co KG, Germany). Capillary blood samples were immediately transferred into sampling vessels, in which they were mixed with 150 µL of the extraction solution. The vessels were vigorously shaken by hand, sealed with an O-ring, and then stored in the dark at room temperature (22 °C). All items used to obtain the capillary blood samples were from a rhelise kit. This kit is a patent-pending sampling kit intended to be used by laymen in a home environment. The kit consists of a lancet, a capillary, and a vial with an extraction solution. The extraction solution is optimized to keep analytes stable for more than 14 days at room temperature without the need for cold storage or additional extraction procedures at the laboratory.

Materials and methods

Chemicals

Reference standards

For the preparation of stock solutions to be used in the study, TAM (tamoxifen > 99%, product #T5648, batch #BCBW6527) and 4HT (4-hydroxytamoxifen ≥ 98% Z-isomer, product #H7904, batch #067M4003V) were both obtained from Sigma-Aldrich (Sweden). END (Z-endoxifen 99.3% E/Z Mixture 50/50, product #D21865, batch #HY-18719A/CS-5098) was obtained from Med Chem Express Europe (Sweden).

Internal reference standards

¹³C₆ TAM (¹³C₆ tamoxifen 99%, product #T006002, batch #7-JMR-144-1) was obtained from Toronto Research Chemicals (Canada). ¹³C₆ END (¹³C₆ Z-endoxifen 99% E/Z Mixture 50/50, batch #NC029-39-2) and ¹³C₆ 4HT (¹³C₆ 4-hydroxytamoxifen ≥ 98%, batch #NC029-39-2) were custom-synthesized by Novandi Chemicals (Sweden) and obtained from Redhot Diagnostics (Sweden).

Preparation of solutions for LC–MS/MS method

Stock standard solutions (Solution I)

Standard solutions of 1.00 mg/mL TAM, END, and 4HT were prepared by dissolving approximately 5 mg of each solid chemicals in an exact volume of acetonitrile (ACN) fortified with 0.2% formic acid to yield the desired concentration. Aliquots from each stock solution were further diluted in aqueous ACN (1/4 v/v) to yield 1.00 µg/mL solutions of TAM, END, and 4HT.

Stock internal standard solutions (Solution II)

Internal standard solutions of 1.00 mg/mL ¹³C₆ TAM, ¹³C₆ END and ¹³C₆ 4HT were prepared from the solid components dissolved in ACN with 0.2% formic acid to yield the desired concentrations, as described in the previous step.

Protein extraction solutions (Solution III)

Extraction solutions were prepared by diluting 20 µL aliquots of the stock internal standard solutions (consisting of ¹³C₆ TAM, ¹³C₆ END, and ¹³C₆ 4HT) into ACN fortified with 200 mL of 0.2% formic acid, to yield 10.0 ng/mL extraction solutions of each isotope-substituted standard.

Quality control (QC) solutions (Solution IV)

QC solutions were prepared by diluting 10 µL of each stock standard of TAM, END, and 4HT, respectively, in 970 µL frozen-thawed human whole blank blood (commercially available, from healthy donors), to yield 10.0 µg/mL solutions. Each 10.0 µg/mL solution was further sequentially diluted with blank blood to yield control blood samples of 100 ng/mL, 10.0 ng/mL, and 1.00 ng/mL for each of the 3 analytes.

Calibration solutions (Solution V)

Calibration solutions were prepared by first diluting 10 µL of each stock standard of TAM, END, and 4HT, respectively, in 970 µL frozen-thawed human whole blank blood to yield calibration stock solutions containing 10.0 µg/mL of each analyte. These calibration stock solutions were further sequentially diluted with lysed whole blank blood to yield calibration solutions of 200, 100, 20.0, 10.0, 2.00, 1.00, 0.200 and 0.100 ng/mL for each analyte.

High-performance LC–MS/MS method

A Waters Premier Triple Quadrupole Mass Spectrometer (Waters Ltd, United Kingdom) was used for all analyses. Separation was performed using a Rheos Alliance UPLC Binary Pump (Flux Instruments AG, Switzerland) with an XSelect HSS T3 column (50 mm × 2.1 mm, 2.5 µm) controlled by MassLynx software (both Waters Ltd, United Kingdom). The mobile phases of processing involved 0.1% formic acid in water (eluent A) and 0.1% formic acid in ACN (eluent B), running at a flow rate of 0.40 mL/min. The initial composition of 95% eluent A was maintained for 0.5 min and then the composition was decreased to 40% eluent A for the next 6.5 min and to 5% eluent A for another 2 min, resulting in a time of analysis of 9 min. Mass spectrometer conditions were optimized with a capillary voltage of + 3000 V, desolvation temperature 300 °C, desolvation flow 600 L/h and ESI+, 30 eV (for END, ¹³C₆ END) and 27 eV (for all others), with m/z values of 372 → 72.3 for TAM, 374 → 58.2 for END, 388 → 72.3 for 4HT, 378 → 72.3 for ¹³C₆ TAM, 380 → 58.2 for ¹³C₆ END, and 394 → 72.3 for ¹³C₆ 4HT.

Analysis of stock standard solutions (Solutions I and II above)

Stock solutions for the calibration curves were prepared in duplicates by separate weigh-ins of TAM, END, 4HT, ¹³C₆ TAM, ¹³C₆ END, and ¹³C₆ 4HT, mixed with human blank blood to final concentrations of 200, 100, 20.0, 10.0, 2.00, 1.00, 0.200 and 0.100 ng/mL, which were then analyzed with the LC–MS/MS method described above to obtain the calibration curves.

Sample preparation—protein extraction

Protein extraction was performed in tubes by adding 50 µL of blood samples from participants to 150 µL of the protein extraction solutions (Solutions III above) containing 1.00 ng/mL of the standards ¹³C₆ TAM, ¹³C₆ END, and ¹³C₆ 4HT, respectively. The tubes were then vortexed for 10 s and centrifuged in an Eppendorf centrifuge for 10 min at 10 °C. After centrifugation, 150 µL of each supernatant was transferred to a conical autosampler glass vial, evaporated to dryness under a gentle stream of nitrogen, and reconstituted in 60 µL of 20% ACN in water.

LC–MS/MS method validation

Method validation was done in accordance with the document Bioanalytical Method Validation Guidance for Industry³³. Selectivity, carry-over, calibration curves, lower limit of quantification (LLOQ), accuracy, precision, matrix effects, and stability were evaluated.

Preparation of batches

Triplicate validation batches of TAM, END, and 4HT were prepared, and these batches were then analyzed with the described LC–MS/MS method on three separate occasions. Each validation batch consisted of QC solutions (Solutions IV above), calibration solutions (Solutions V above), and blank solvent solutions to measure carry-over effects between different runs.

Selectivity and carry-over

As part of the validation process, experiments were performed using patient blood samples to determine whether TAM, END, and/or 4HT co-eluted or interfered with any other metabolites of TAM. The mass spectrometric peaks for TAM, END, and 4HT were well separated without overlap (Fig. 4). The selectivity and carry-over were determined to be adequate for the intended analyses.

Calibration curves

Concentration–response calibration curves for TAM, END, and 4HT were constructed based on two QC samples (Solutions IV above) and two analyses of each calibration solution (Solutions V above) at each concentration (Fig. 5). The prepared calibration solutions were analyzed and a regression line was fitted to the data for each analyte. Linearity of the calibration curves was identified from 1 to 200 ng/mL for TAM and from 0.2 to 200 ng/mL for both END and 4HT.

Lower limit of quantification (LLOQ)

Based on the linearity results of the calibration curves, the LLOQ for the method was determined to be 1.0 ng/mL for TAM and 0.20 ng/mL for both END and 4HT.

Precision and accuracy

Using the QC solutions (Solutions IV) and the calibration standard solutions (Solutions V above) for TAM, END, and 4HT, the precision, accuracy, and deviations from the nominal concentrations of the calibration standard solutions were determined. The concentration range for TAM was 1 ng/mL to 100 ng/mL, and the concentration ranges for both END and 4HT were 0.20 ng/mL to 100 ng/mL. The coefficients of variation were all less than 10%. The levels of precision and accuracy fulfilled FDA requirements for quantitative bioanalysis³³.

Stability testing

To determine the stability of each particular analyte (TAM, END, or 4HT) in a whole blood sample, a 50 µL sample aliquot was transferred to an Eppendorf 0.5 mL tube containing 150 µL of extraction solution (without internal standards for TAM, END, and 4HT). Each tube was then vortexed for 10 s and placed in an Eppendorf Thermomixer, where it was agitated for 10 min at 10 °C. Each tube was stored in the dark at either room temperature (20 °C) or in a refrigerator (8 °C), for either 7 or 14 days. After retrieval from storage, 150 µL of protein precipitation solution (Solutions III above) containing the internal standards of either TAM, END, or 4HT was added to the respective tube and the solution was briefly mixed. The solutions were then centrifuged at 20,100×g for 10 min at 10 °C. After centrifugation, 300 µL of the supernatant was collected and transferred to a conical autosampler glass vial. The protein precipitation solution was evaporated to dryness and reconstituted in 60 µL of 20% ACN in water, after which each was analyzed. The stability period that was selected for analysis was an estimate, based on what was deemed to be adequate time to accomplish the logistics of getting samples from patients, shipping them, and then analyzing them at the laboratory. It was estimated that a period of 7 days between sampling and analysis, including shipping, should be sufficient, but in case of unforeseen events, samples should ideally remain stable for up to 14 days.

Statistical methods

Demographic and clinical data, as well as ratios, are reported using either medians or means and either ranges or interquartile ranges (IQR), which were obtained using Excel. Plasma, venous whole blood, and capillary blood concentrations of TAM, END, and 4HT were presented as ratios for individuals, group means, group mean ratios, standard deviations (SD), and standards of the mean (SEM). Comparisons of the means were performed using the two-sided t-test and P values reported. Coefficient of variation (CV) was calculated by dividing SD by the mean and multiplying by 100. To evaluate correlations between analyte concentrations in plasma, whole blood, and capillary blood, a non-parametric Passing-Bablok regression analysis was performed for each analyte separately, using MedCalc (MedCalc Software bvba, Belgium). Statistical significance was defined at the 5% (P ≤ 0.05) level.