Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2024 Apr 15.
Published in final edited form as: J Chromatogr B Analyt Technol Biomed Life Sci. 2023 Mar 11;1221:123654. doi: 10.1016/j.jchromb.2023.123654

Development and Validation of a Liquid Chromatography-Mass Spectrometry Assay for Quantification of Z- and E-Isomers of Endoxifen and its Metabolites in Plasma from Women with Estrogen Receptor Positive Breast Cancer

Sarah A Buhrow a, Emily J Koubek a,1, Matthew P Goetz a,b, Matthew M Ames a,b, Joel M Reid a,b
PMCID: PMC10249430  NIHMSID: NIHMS1886844  PMID: 37004493

Abstract

The selective estrogen receptor modifier tamoxifen (TAM)2 is widely used for the treatment of women with estrogen receptor positive (ER+) breast cancer. Endoxifen (ENDX) is a potent, active metabolite of TAM and is important for TAM’s clinical activity. While multiple papers have been published regarding TAM metabolism, few studies have examined or quantified the metabolism of ENDX. To quantify ENDX and its metabolites in patient plasma samples, we have developed and validated a rapid, sensitive, and specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantitative determination of the E- and Z-isomers of ENDX (0.5–500 ng/ml) and the ENDX metabolites norendoxifen (1–500 and 0.5–500 ng/ml E and Z, respectfully), ENDX catechol (3.075–307.5 and 1.92–192 ng/ml E and Z, respectfully), 4’-hydroxy ENDX (0.33–166.5 and 0.33–333.5 ng/ml E and Z, respectfully), ENDX methoxycatechol (0.3–300 and 0.2–200 ng/ml E and Z, respectfully), and ENDX glucuronide (2–200 and 3–300 ng/ml E and Z, respectfully) in human plasma. Chromatographic separation was accomplished on a HSS T3 precolumn attached to an Poroshell 120 EC-C18 analytical column using 0.1% formic acid/water and 0.1% formic acid/methanol as eluents followed by MS/MS detection. The analytical run time was 6.5 min. Standard curves were linear (R2 ≥ 0.98) over the concentration ranges. The intra- and inter-day precision and accuracy, determined at high-, middle-, and low-quality control concentrations for all analytes, were within the acceptable range of 85% and 115%. The average percent recoveries were all above 90%. The method was successfully applied to clinical plasma samples from a Phase I study of daily oral Z-ENDX.

Keywords: Endoxifen, tamoxifen, LC-MS/MS, metabolite, validation

1. INTRODUCTION

Endoxifen (ENDX) is one of the active metabolites of the selective estrogen receptor modifier tamoxifen (TAM) which is widely used for the treatment of women diagnosed with estrogen receptor positive (ER+) breast cancer [1]. Metabolism plays a crucial role in TAM activity and cytochrome P450-catalyzed oxidation yields several metabolites that contribute to the antitumor activity of TAM [2]. ENDX and 4-hydroxy tamoxifen (4HT) are the most potent active metabolites of TAM with 100-fold greater binding of the estrogen receptor compared to TAM [3–5]. There are several reports of the extensive phase I and phase II metabolism of TAM [6–17]. However, few have been published on the metabolism of ENDX [6, 10–13, 18] and those have focused on ENDX glucuronidation [6–8, 11–13, 17].

ENDX metabolites that have been identified display a variety of in vitro activity. E-norendoxifen is a potent aromatase inhibitor while Z-norendoxifen displays high binding affinity for both Erα and Erβ [19, 20]. On the other hand, ENDX glucuronide does not promote E2-induced gene expression to the same extent as ENDX [17]. Based on the promising preclinical and clinical data showing that TAM anticancer activity is associated with ENDX, demonstrated biological activity of some ENDX and other TAM metabolites, and lack of data regarding the identity, activity and pharmacokinetics of ENDX metabolites, there is a critical need to develop a method to quantitate ENDX and its metabolites [1, 5, 21, 22].

Several HPLC and LC-MS methods have been published that separate and quantify tamoxifen, its principal metabolites N-desmethyltamoxifen (NDMT), 4HT, and ENDX from plasma [23–30]. However, these assays have several limitations with respect to analysis of ENDX and its metabolites. First, none of these assays identify or quantify metabolites of endoxifen (Figure 1). Second, 4-hydroxy metabolites of TAM, most notably 4HT and ENDX, undergo isomeric conversion via oxidation to yield E-isomers that may contribute to adverse effects. A few of these methods do separate the E- and Z- isomers of the 4HT and ENDX, but it is not a focus of the studies. Third, most TAM methods have long sample processing time that use either liquid/liquid extraction, which limits analysis to nonpolar metabolites [31–34], or protein precipitation with solvent evaporation to improve sensitivity [27, 35]. In addition to the long sample processing time, chromatographic separations frequently require 10 minutes or longer to achieve baseline resolution of metabolites that have small structural differences and vary over a broad range of polarity [9, 26, 27, 32–35]. Development of an assay for detection and quantification of TAM and ENDX metabolites is of particular importance as metabolites of both compounds have shown in vitro anti-tumor activity [11, 19]. A validated assay would allow in vivo identification of ENDX metabolites, exploration of metabolite activity, and determination of metabolite contribution to ENDX activity and toxicity.

Figure 1. Metabolic pathways and structures of ENDX and its metabolites.

Figure 1.

Open in a new tab

ENDX (N-desmethyl-4-hydroxy tamoxifen, endoxifen); Norendoxifen (N,N-didesmethyl-4-hydroxy tamoxifen); 4’-hydroxy ENDX (4,4’-dihydroxy-N-desmethyl tamoxifen); ENDX catechol (N-demethyl-3,4-dihydroxytamoxifen); ENDX methoxycatechol (N-demethyl-3-methoxy-4-hydroxytamoxifen hydrochloride) ENDX glucuronide (N-desmethyl-4-hydroxy tamoxifen ß-D-glucuronide).

Here, we developed and validated a rapid, sensitive, simple LC-MS/MS method that quantifies the E- and Z-isomers of ENDX and its metabolites norendoxifen, ENDX catechol, 4’-hydroxy ENDX, ENDX methoxycatechol, and ENDX glucuronide in plasma. To the best of our knowledge, this is the first single assay to quantify all these compounds. Our assay uses a simple protein crash with a 96-well filter plate method for easy, quick, multi-sample processing with no evaporation step. The small particle size of the stationary phase and the high-pressure capabilities of the UHPLC system allowed rapid separation and a short 6.5 minute run time. The method was successfully applied to plasma samples from a Phase I study and demonstrated acceptable performance for future continued support of clinical studies and therapeutic drug monitoring.

2. MATERIALS AND METHODS

2.1. Chemicals and Reagents

Z- and E-ENDX and norendoxifen (N-desmethylendoxifen) were synthesized and provided by the Developmental Therapeutics Program, National Cancer Institute (purity >99%). (E/Z)-4,4’-dihydroxy-N-desmethyl tamoxifen (4’-hydroxy ENDX, 97% purity), N-desmethyl-4-hydroxy tamoxifen ß-D-glucuronide (ENDX glucuronide, 95% purity), N-demethyl-3,4-dihydroxytamoxifen hydrochloride (ENDX catechol, 97% purity), N-demethyl-3-methoxy-4-hydroxytamoxifen hydrochloride (ENDX methoxycatechol, 99.97% purity), and the internal standard N-desmethyl-4-hydroxy tamoxifen-d5 (d5 ENDX, 96% purity) were purchased from Toronto Research Chemicals Inc. (Toronto, ON, Canada). HPLC grade formic acid, L-ascorbic acid, and 200 proof ethanol (EtOH) was purchased from Sigma Aldrich (St. Louis, MO). HPLC grade methanol (MeOH) was purchased from EMD (Billerica, MA). HPLC grade acetonitrile (ACN) was purchased from Fisher Scientific (Fairlawn, NJ). Drug-free human plasma was obtained from healthy volunteers.

2.2. LC-MS/MS Conditions

A Waters Acquity H class ultra-performance liquid chromatography (UHPLC) system containing a quaternary solvent manager and sample manager-FTN coupled with a Xevo TQ-S mass spectrometer equipped with an electrospray ionization (ESI) source (Waters, Milford, MA) was used. The LC-MS/MS data were acquired and processed with Waters MassLynx v4.1 software.

E- and Z-ENDX isomers and ENDX metabolites were separated using an Agilent Poroshell 120 EC-C18 analytical column (2.1 × 50mm, 2.7 μm particle size, Chrom Tech, Apple Valley, MN) which was guarded by a Waters VanGuard HSS T3 precolumn (2.1 × 5mm, 1.8 μm particle size, Milford, MA), maintained at 40°C, and eluted with a linear gradient at a flow rate of 0.5 mL/min. Solvent A consisted of HPLC grade water with 0.1% formic acid and Solvent B consisted of HPLC grade methanol with 0.1% formic acid. Following isocratic elution with 56% A and 44% B for 2.0 min, B was increased from 44 to 75% in a linear gradient over 3 min, held at 75% B for 0.2 min, and returned to initial conditions over 1.3 min. The injection volume of each sample was 2 μl and the autosampler temperature was 10°C. The total run time was 6.5 min with a void time of 0.264 min.

Detection of ENDX and its metabolites was accomplished using a Xevo TQ-S mass spectrometer with positive electrospray ionization (ESI). The MS conditions were as follows: capillary voltage, 1.0 kV; source temperature, 150°C; desolvation temperature, 500°C; cone gas flow, 150 L/hr; desolvation gas flow, 1000 L/hr. The cone voltages and collision energies were determined by MassLynx-Intellistart, v4.1, software and varied between 24–54 V and 18–26 eV, respectively. MS conditions for the individual metabolites are summarized in Table S1. The MRM quantifier precursor and product ions were monitored at m/z 374.3>58.1 for ENDX, 379.3>58.1 for d5 ENDX, 360.3>129.1 for norendoxifen, 390.3>58.1 for hydroxy ENDX, 404.3>58.1 for ENDX methoxycatechol, and 550.3>374.2 for ENDX glucuronide. The MRM qualifier transition precursor and product ions were monitored at m/z 374.3>129.05 for ENDX.

2.3. Stock Solution and Standard Curve Preparation

The primary stock solutions of Z-ENDX (1 mg/ml in EtOH), E-ENDX (1 mg/ml in EtOH), Z-norendoxifen (1 mg/ml in EtOH), E-norendoxifen (2 mg/ml in DMSO), (E/Z)-ENDX catechol (1mg/ml in MeOH), (E/Z)-ENDX methoxycatechol (1mg/ml in EtOH), (E/Z)-4’-hydroxy-ENDX (7.8 mg/ml in MeOH), (E/Z)-ENDX glucuronide (4 mg/ml in DMSO), and (E/Z)-d5-ENDX (IS) (2 mg/ml in EtOH) were prepared in amber silanized glass vials and stored at −20°C. Working standards were prepared by dilution of the stock solution with EtOH in amber silanized glass vials and stored at −20°C. All solutions were prepared in amber vials and procedures were performed under yellow light to minimize light exposure [36, 37].

Plasma standard curves were prepared daily containing Z- and E-ENDX (0.5–500 ng/ml), E-norendoxifen (1–500 ng/ml), Z-norendoxifen (0.5–500 ng/ml), E-ENDX catechol (3–307 ng/ml), Z-ENDX catechol (1.92–192 ng/ml), E-4’-hydroxy-ENDX (0.33–166 ng/ml), Z-4’-hydroxy-ENDX (0.33–333 ng/ml), E-ENDX methoxycatechol (0.3–300 ng/ml), Z-ENDX methoxycatechol (0.2–200 ng/ml), E-ENDX glucuronide (2–200 ng/ml), Z-ENDX glucuronide (3–300 ng/ml), and IS (45ng/ml).

2.4. Sample Preparation

A 125 uL plasma standard was prepared in a 1.7 mL microcentrifuge tube using 25X working stock solution (prepared as described in the previous section). 100 μl plasma sample was added to an Orochem 2 mL 0.2 μm protein crash plate (Chrom Tech, Apple Valley, MN) well containing a solution of 300 μl of MeOH containing IS (15 ng/ml) and ascorbic acid (66.67 μg/mL). The protein crash plate was shaken on a plate shaker for 15 minutes at 1100 × rpm. Samples in the crash plate were filtered using a vacuum apparatus and collected in a deep 96-well plate. Samples with concentrations above the highest concentration on the standard curves were diluted appropriately with blank human plasma to fit inside the standard curves and the concentrations were adjusted for the dilution accordingly.

2.5. Assay Validation Procedures

The method was validated for specificity, matrix effect, recovery, linearity, carryover, precision, accuracy, and stability, in accordance with the FDA guidance [38].

2.5.1. Specificity

Specificity was tested using six different sources of blank plasma which were individually analyzed for potential interference from endogenous compounds. Any interfering endogenous peak detected for specific MRM scans and retention times had to have less than 20% peak area of the lower limit of quantitation (LLOQ).

2.5.2. Matrix Effects and Percent Recovery

Initial qualitative matrix effect: ion signal suppression/enhancement experiments were carried out by visually assessing chromatograms of blank human plasma samples from 6 different sources for potential interferences from endogenous components. This method was carried out by simultaneously injecting blank plasma extract from the LC with post column infusion of 1 ug/ml of each analyte and IS at a rate of 10 μL/min from the mass spectrometer. Chromatograms were visually inspected for any troughs or peaks occurring in the chromatogram [39]. Matrix effects were investigated by calculating the ratio of the peak area of each analyte spiked into pre-extracted plasma extract to the peak area of each analyte spiked into 1:1 MeOH:H2O (v/v) and multiplied by 100. Samples were run in triplicate at low-, medium-, and high-quality assurance (QA) concentrations.

The percentage recovery was determined for each analyte (ENDX, norendoxifen, ENDX catechol, 4’-hydroxy ENDX, ENDX methoxycatechol, and ENDX glucuronide). Percent recovery was determined by comparing the peak areas of each analyte extracted from plasma to those from external standards prepared in 1:1 MeOH/H2O (v/v). The percentage recovery was determined by comparing the peak area of the analyte extracted from the plasma samples with the peak area of the analyte in neat solution and multiplied by 100%. Samples were run in triplicate at low-, medium-, and high-quality assurance (QA) concentrations, specifically: Z- and E-ENDX (3, 15, 75, and 300 ng/ml), Z- and E-norendoxifen (3, 15, 75, and 300 ng/ml), E-ENDX catechol (9.225, 46.125, and 184.5 ng/ml), Z-ENDX catechol (5.76, 28.8, and 115.2 ng/ml), E-4’-hydroxy-ENDX (2, 10, 50, and 200.1 ng/ml), Z-4’-hydroxy-ENDX (1, 5, 25, and 100 ng/ml), E-ENDX methoxycatechol (1.8, 9, 45, and 180 ng/ml), Z-ENDX methoxycatechol (1.2, 6, 30, and 120 ng/ml), E-ENDX glucuronide (6, 30, and 120 ng/ml), Z-ENDX glucuronide (9, 45, and 180 ng/ml)

2.5.3. Linearity and Sensitivity

Linearity was assessed for ENDX, norendoxifen, ENDX catechol, 4’-hydroxy ENDX, ENDX methoxycatechol, and ENDX glucuronide using plasma blank, internal standard only, and a minimum of six non-zero calibration standards. The LLOQ was at least 5 times the response compared to plasma blank. Triplicate standard curves were prepared daily and run over three days and included low, medium, and high QA samples. Percentage of deviation of the mean calculated concentrations of the calibration standards in comparison to nominal concentrations and coefficient of variation (CV) needed to be within 85% and 115%, except for the LLOQ in which a deviation of 20% was acceptable. The linearity of each standard curve was analyzed by plotting the peak area ratio of analyte/IS linear versus the nominal concentration of analyte. The standard curves were constructed by weighted (1/x) least-square regression and checked for goodness of fit (R2 > 0.98).

2.5.4. Sample Carryover

Carryover was assessed by injecting ENDX, norendoxifen, ENDX catechol, 4’-hydroxy ENDX, ENDX methoxycatechol, and ENDX glucuronide in 1:1 MeOH:H2O (v/v) at a two-fold higher concentration than the highest concentration on the standard curve. This was followed by injection of three consecutive blank 1:1 MeOH/H2O (v/v) samples. Blank injections need to be lower than 20% of the peak area of the LLOQ.

2.5.5. Precision and Accuracy

The precision and accuracy for this method was evaluated measuring the concentrations of ENDX, norendoxifen, ENDX catechol, 4’-hydroxy ENDX, ENDX methoxycatechol, and ENDX glucuronide in five replicate samples at low, medium, and high concentrations and triplicate samples for the LLOQ within a single-run analysis for intra-day assessment. This analysis was repeated on three separate days for inter-day assessment. Precision was reported as the coefficient of variation and was calculated as the relative standard deviation. Accuracy was measured as the mean calculated concentration of each analyte as a percentage of the nominal concentration. The precision and accuracy values had to be within 85% and 115%, except for the LLOQ which needed to be within 80% and 120%.

2.5.6. Sample Stability

Autosampler stability of ENDX, norendoxifen, ENDX catechol, 4’-hydroxy ENDX, ENDX methoxycatechol, and ENDX glucuronide was assessed in 1:1 MeOH/H2O (v/v) and extracted plasma samples over 24 hours at room temperature. Short term stability (bench top) of ENDX and metabolites was determined for up to 6 hours at 37°C in the dark. Freeze-thaw stability of ENDX and metabolites in plasma was obtained over three freeze-thaw cycles using a dry ice MeOH bath over a one-hour period. Long-term stability of analytes in plasma was assessed over 1 month at −70°C. All stability tests of ENDX, norendoxifen, ENDX catechol, 4’-hydroxy ENDX, ENDX methoxycatechol, and ENDX glucuronide were run in triplicates using concentrations of 10 and 400 ng/ml and repeated three times for a n = 3. ENDX and metabolites were considered stable if 85–115% of the nominal concentration was recuperated.

2.6. Clinical Application

The method was used to explore ENDX and its metabolite concentrations in endocrine-refractory, estrogen receptor-positive metastatic breast cancer patients during a phase I clinical trial (NCT02311933) of Z-endoxifen [1]. Participating institutions obtained study approval from their institutional review boards and filed assurances with the Department of Health and Human Services. Informed written consent was obtained from all participants. Protocol design and conduct followed applicable regulations, guidance, and local policies. Patients received Z-endoxifen by mouth daily in a 28-day treatment cycle. Blood samples for ENDX and metabolite analysis were collected 1 day prior to Z-endoxifen administration, 24 hours post administration, and on days 7, 14 and 28 before administration of daily Z-endoxifen. Samples were collected in K2-EDTA tubes (3 mL), immediately wrapped in aluminum foil, chilled to 4°C, and centrifuged within 20 minutes of sample collection at 2,000 rpm for 10 minutes at 4°C. Immediately after centrifugation the plasma was transferred to a tube wrapped in aluminum foil and stored at −70°C.

3. RESULTS AND DISCUSSION

3.1. Chromatographic Conditions

Using an ESI source in positive ion mode, ENDX, norendoxifen, ENDX catechol, 4’-hydroxy ENDX, ENDX methoxycatechol, ENDX glucuronide, and IS produced abundant protonated molecular precursor ions [M+H] at m/z 374.3, 360.3, 390.3, 390.3, 404.3, 550.3, and 379.3, respectively. Product ions for each compound were selected based on abundance (signal intensity) for MRM quantification. After fragmentation, the prominent product ion of ENDX, ENDX catechol, 4’-hydroxy ENDX, and ENDX methoxy catechol was found at m/z 58.1, norendoxifen at m/z 129.1, and ENDX glucuronide at m/z 374.2. Based on these data, quantification of ENDX and metabolites was performed in the multiple reaction monitoring (MRM) mode by monitoring the m/z 374.3>58.1 for ENDX, m/z 360.3>129.1 for norendoxifen, m/z 390.3>58.1 for ENDX catechol, m/z 390.3>58.1 for 4’-hydroxy ENDX, m/z 404.3>58.1 for ENDX methoxycatechol, m/z 550.3>374.2 for ENDX glucuronide, and m/z 379.3>58.1 for the IS. Satisfactory chromatography was achieved using a Waters VanGuard HSS T3 precolumn attached to an Agilent Poroshell 120 EC-C18 analytical column and solvent mixtures composed of water with 0.1% formic acid (A) and methanol with 0.1% formic acid (B, Figure 2). As shown in Figure 2B, the elution of the analytes was rapid with baseline peak separation between isomers within a 6.5 min run time and a void time of 0.264 min. The E- and Z-isomers of ENDX and all the metabolites except ENDX glucuronide were separated with baseline resolution as shown in the ion chromatograms. The resolution for E- and Z-ENDX glucuronide at the LLOQ was 0.825. The hydroxylated ENDX metabolites (ENDX catechol and 4’-hydroxy ENDX) which had the same MRM transition (m/z 390.3 > 58.1) were also separated with baseline resolution (Figure 2B).

Figure 2. LC/MS/MS analysis of ENDX and metabolites in human plasma extracts.

Figure 2.

Open in a new tab

A) Blank plasma with internal standard (transition not shown). B) Plasma containing ENDX and metabolite standards: 5 ng/mL E and Z Endx (374 m/z, 4.10 and 4.33 minutes, respectively), 5 ng/ml E and Z norendoxifen (360 m/z, 4.11 and 4.34 minutes, respectively), 1.67 ng/ml E-4’hydroxy ENDX (390 m/z, 0.92 minutes), 3.34 ng/ml Z-4’hydroxy ENDX (390 m/z, 1.91 minutes), 3.08 ng/ml E ENDX catechol (390 m/z, 3.28 minutes), 1.92 ng/ml Z ENDX catechol (390 m/z, 3.59 minutes), 3 and 2 ng/ml ENDX methoxycatechol (404 m/z, 4.16 and 4.37 minutes, respectively), and 2 and 3 ng/ml E and Z ENDX glucuronide (550 m/z, 2.62 and 2.81 minutes, respectively).

3.2. Sample Extraction

Protein precipitation utilizing MeOH containing ascorbic acid and subsequent crash plate filtration proved to be a fast and effective method to extract ENDX and its metabolites from plasma. Addition of ascorbic acid (66.67 ng/mL) in 300 μl MeOH stabilized the ENDX catechol in plasma extract [40]. Without ascorbic acid, over 50% of ENDX catechol degraded in plasma extract in the chilled autosampler in 2 hours.

3.3. Specificity

Six different lots of blank human plasma were extracted and injected on the LC-MS/MS to identify potential interfering endogenous peaks within the same MRM transition and retention times (Table S2). ENDX, norendoxifen, ENDX catechol, 4’-hydroxy ENDX, ENDX methoxycatechol, and ENDX glucuronide had very small interfering endogenous peaks. Except for ENDX glucuronide, all endogenous peaks were under 20% of the LLOQ peak area and 5% of the peak area of the internal standard. All lots of plasma contained an endogenous peak that spanned the retention time of the E- and Z-ENDX glucuronide peaks. These endogenous peaks integrated as a single peak and had a total area that was larger than 20% of the E-ENDX glucuronide LLOQ peak. However, this endogenous peak did not appear to affect quantification of ENDX glucuronides.

3.4. Matrix Effect and Recovery

Simultaneous injection of blank plasma extracts with post column infusion of ENDX, norendoxifen, ENDX catechol, 4’-hydroxy ENDX, ENDX methoxycatechol, ENDX glucuronide, or IS resulted in no visual dips or rises in individual drug chromatograms (Figure S1A). The blank plasma extract chromatograms were superimposable with the chromatogram from LC injection of 1:1 MeOH/H2O (v/v) with drug infusion (Figure S1B).

The matrix effect measurements using the peak area ratio of drug spiked into pre-extracted plasma extract divided by the ratio of drug spiked into 1:1 MeOH/H2O (v/v) are summarized in Table S3. There was little matrix effect observed on E- and Z-ENDX, E- and Z-norendoxifen, and E- and Z-4’-hydroxy ENDX with the mean and %CV within 15% for all concentrations. Little matrix effect was all observed for ENDX methoxycatechol with the exception of the 9 ng/mL standard for E- ENDX methoxycatechol. Large matrix effects of >20% were observed for low concentrations of E- and Z-ENDX catechol (enhancement with a mean of 209% and 154%, respectively) and E- and Z-ENDX glucuronide (suppression with a mean of 52% and 51%, respectively). Matrix effects were low (<15%) or absent at the higher concentrations.

High, reproducible recovery was observed for ENDX and all metabolites except the ENDX-catechol and ENDX methoxycatechol (Table S3). The average percent recovery (%CV in parentheses) was 96.9% (12.7%) for E-ENDX, 95.2% (8.4%) for Z-ENDX, 102.7% (9.3%) for E-norendoxifen, 99.8% (7.6%) for Z-norendoxifen, 93.3% (4.3%) for E-4’-hydroxy ENDX, and 101.2% (12.9%) for Z-4’-hydroxy ENDX. The percent recovery was higher than the theoretically best 100% recovery for low and medium concentrations of E-ENDX catechol (mean 212% and 131%, respectively), low Z ENDX catechol concentration (mean 162.0%), the two lowest values of E- and Z-ENDX methoxycatechol (mean 124% and 144%, respectively), and the 9 ng/ml values of Z-ENDX methoxycatechol (mean 129%) but moderate on all other concentrations. The high percent recovery was consistent with the high matrix signal enhancement in the plasma extracts. The low percent recovery for low concentrations of E- and Z-ENDX glucuronide (mean 62% and 63%, respectively) was associated with ionization suppression observed in the plasma extract matrix. The percent recoveries are summarized in Table S3.

3.5. Calibration Curve and Carryover

The LLOQ for Z- and E- ENDX, and each metabolite are shown in Table 1 and Supplemental Table S4. The LLOQ of 0.5 ng/mL for Z- and E- ENDX were within the range of the values of 0.4 – 3.0 ng/mL reported in previous studies [21–28, 41]. To our knowledge, the LLOQs for the other metabolites have not been reported previously, as this is the first report of the quantitative analysis of ENDX metabolites. The matrix effects did not appear to interfere with quantification of the ENDX and its metabolites. The calibration curves were linear over the range of 0.5–500 ng/ml for E-ENDX, Z-ENDX, Z-norendoxifen; 1–500 ng/ml for E-norendoxifen; 3.075–307.5 ng/ml for E-ENDX catechol; 1.92–192 ng/ml for Z-ENDX catechol; 0.3–300 ng/ml for E-ENDX methoxycatechol; 0.2–200 ng/ml for Z-ENDX methoxycatechol; 2–200 ng/ml for E-ENDX glucuronide; 3–300 ng/ml for Z-ENDX glucuronide; 0.333–166.5 ng/ml for E-4’-hydroxy ENDX; and 0.334–333.5 ng/ml for Z-4’-hydroxy ENDX. The intra-day and inter-day triplicate standard curve correlation coefficients (R2) were all greater than >0.997. The percent of coefficient of variation (% CV) and the percent accuracy for each of the standard curve concentrations were within 15% of the nominal concentrations for both the inter-day and intra-day statistics. This is within the assay validation acceptable range. The calibration curve statistics are summarized in Table S4. No carry-over effect was observed for ENDX, norendoxifen, ENDX catechol, 4’-hydroxy ENDX, ENDX methoxycatechol, and ENDX glucuronide after injection of a 2-fold higher concentration than the highest standard curve concentration in three subsequent 1:1 methanol:water (1:1, v/v) blank injections.

Table 1.

ENDX and metabolite precision and accuracy data using intra-day mean results.

Analyte Nominal Conc. (ng/ml) Mean Measured Conc. (ng/ml) Precision (% CV) Accuracy (% Difference)
E-ENDX 0.5 5 50 500 0.52 5.0 52.0 531.1 8.0 2.0 7.4 3.9 4.2 0.6 4.0 6.2
Z-ENDX 0.5 5 50 500 0.49 5.2 53.5 540.6 5.1 4.4 6.4 5.7 −1.4 3.2 6.9 8.1
E-Norendoxifen 1.0 5 50 500 1.03 4.8 48.8 527.9 3.6 4.7 9.5 5.2 2.7 −3.8 −2.5 5.6
Z-Norendoxifen 0.5 5 50 500 0.57 5.0 49.9 536.4 0.6 3.4 7.4 5.0 15.0 −0.3 −0.2 7.3
E-ENDX
Catechol		 3.1 30.8 308 2.8 29.6 320 19.7 7.9 1.4 −7.8 −3.7 4.1
Z-ENDX
Catechol		 1.9 19.2 192 1.8 18.6 202.1 16.7 5.8 2.5 −5.9 −2.9 5.3
E-ENDX
Methoxycatechol		 0.3 3 30 300 2.99 2.9 29.9 309.8 11.0 1.0 4.8 1.6 −2.8 −4.0 −0.4 3.3
Z-ENDX
Methoxycatechol		 0.2 2 20 200 1.96 2.0 20.4 206.6 7.2 5.0 2.4 3.0 2.0 −1.1 1.9 3.3
E-ENDX
Glucuronide		 2 20 200 2.3 19.6 211.0 8.4 4.3 0.7 13.4 −1.9 5.5
Z-ENDX
Glucuronide		 3 30 300 3.2 30.3 319.6 3.7 2.3 1.2 8.2 1.1 6.5
E-4’-Hydroxy
ENDX		 0.3 1.7 16.7 166.5 0.35 1.6 16.4 169.5 4.2 4.8 6.8 2.0 6.0 −5.1 −1.4 1.8
Z-4’-Hydroxy
ENDX		 0.3 3.3 33.4 333.5 0.36 3.4 33.9 348.8 4.1 1.6 2.9 0.9 7.9 0.5 1.6 4.6
Open in a new tab

3.6. Precision and Accuracy

The precision and accuracy for this method was evaluated by analyzing five replicates at low, medium, and high concentrations and triplicate replicates for the LLOQ within a single-run analysis for intra-day assessment and over three separate days for inter-day assessment and over three separate days for inter-day assessment. The method was precise and accurate, with intra- and inter-day statistics within the acceptable range of 85% and 115%, except for the LLOQ which was within 80% and 120%, for ENDX, norendoxifen, ENDX catechol, 4’-hydroxy ENDX, ENDX methoxycatechol, and ENDX glucuronide (Tables 1 and S4).

3.7. Stability

The stability of ENDX and its metabolites were examined under a range of conditions (Table S5). Autosampler stability was assessed over 24 hours at 10°C. Initial stability of analytes in 1:1 methanol:water (v/v) showed drug stability up to 24 hours. However, ENDX catechol concentrations fell to 50% of the initial concentration after 2 hours in plasma extracted using MeOH. To prevent degradation of ENDX catechol in the autosampler, ascorbic acid (66.67 μg/mL) in 300 μl methanol was added during extraction [40]. Addition of ascorbic acid to methanol ensured stability of all analytes in the autosampler for up to 24 hours. All analytes were stable in human plasma for at least 6 hours at 37°C and over three freeze/thaw cycles (Table S5). After 7 weeks of storage in human plasma, at approximately −80°C, ENDX and its metabolite concentrations were within 15% of their original concentrations. However, the concentration of the E- and Z-ENDX glucuronide low concentration sample increased over time (Table S5).

3.8. Application of the Assay

The presented method was successfully applied to measuring trough concentrations of Z-ENDX and its metabolites in 10 patients with endocrine-refractory, estrogen receptor-positive metastatic breast cancer who received 40 mg and 100 mg Z-ENDX in a phase I study of Z-endoxifen administered orally once daily for 28 days [1]. Z-ENDX and its metabolites were measured in plasma samples collected before Z-endoxifen administration and 24 hours post administration on day 1 and prior to dose administration on days 7, 14, and 28 (Figure 3 and Table S6). For patients receiving the 40 mg Z-ENDX dose, mean trough concentrations exceeded 290 ng/ml (776 nM) Z-ENDX, which was nearly 50-fold greater than the concentration of 16 nM Z-ENDX noted by some investigators as the threshold concentration associated with reduced risk of breast cancer recurrence following TAM treatment [42]. Consistent with previous studies, the glucuronide metabolites E- and Z- ENDX were found in greatest abundance, and approximately 20% of the Z-ENDX concentration. Four oxidative metabolites (E-ENDX, Z-norendoxifen, Z-ENDX catechol, Z-4’-hydroxy ENDX) were found in low abundance (≤2% for Z-ENDX). E-norendoxifen, E-ENDX catechol, and E-4’-hydroxy ENDX were not detected in patient samples. Of note, we found low concentrations (2% for Z-ENDX) of Z-ENDX methoxycatechol, but not E-methoxycatechol ENDX, in patient samples. To our knowledge, this is the first report to detect ENDX catechol and methoxycatechol in human samples. For patients receiving the 100 mg Z-ENDX dose, mean trough concentrations exceeded 726 ng/ml (1944 nM) Z-ENDX, which was over 120-fold greater than the threshold concentration of 16 nM Z-ENDX and exceeds concentrations of 100 – 1000 nM which are associated with maximum inhibition of estrogen-induced stimulation and ER transcription [21, 22]. An approximately dose proportional increase in trough plasma concentrations was observed for Z-ENDX, E-ENDX and each of the metabolites except for Z- and E- ENDX glucuronide which appeared to be lower at the higher dose and consistent with saturation of this metabolic pathway. Steady-state concentrations of Z-ENDX and its metabolites were achieved on day 7 of treatment for both doses and were maintained throughout the treatment course.

Figure 3. Plasma concentration versus time profiles of ENDX and its metabolites.

Figure 3.

Open in a new tab

Plasma concentrations of ENDX and its metabolites from six patients receiving 40 mg (A) and four patients receiving 100 mg (B) Z-ENDX orally once daily over 28 days. Data are represented as the mean ± standard error of the mean.

4. CONCLUSIONS

The tamoxifen metabolite ENDX is currently under development as a treatment option for estrogen receptor positive breast cancer. ENDX pharmacokinetics have been well characterized in two Phase I trials [1, 21]. However, the pharmacokinetics of ENDX metabolites have not been investigated and a rapid, sensitive method to identify and quantify ENDX metabolites in human plasma has yet to be developed. Here, we present a validated, sensitive, precise, accurate, and quick LC-MS/MS method that quantifies the E- and Z-isomers of ENDX, norendoxifen, ENDX catechol, 4’-hydroxy ENDX, ENDX methoxycatechol, and ENDX glucuronide in human plasma. The validated method has been successfully used to measure plasma concentrations of ENDX and its metabolites in samples from breast cancer patients treated with oral ENDX∙HCl in a phase I clinical trial. There are, however, a few limitations to the assay that illustrate the challenges associated with development of a sensitive, specific assay for multiple metabolites with a broad range of polarity. The limited availability of stable-isotope labelled standards did not allow us to incorporate an internal standard for each metabolite. The number of metabolites quantified in this assay limited the use of qualifier m/z transitions and led to reliance on the ENDX qualifier transition. It was not possible to remove ionization suppression and enhancement for all the metabolites and this appeared to affect the apparent recovery. With these limitations in mind, the method presented here will be crucial to further investigation of the in vitro metabolism of ENDX, ENDX metabolite pharmacokinetics including the role of metabolism on the interindividual variability of ENDX pharmacokinetics and the potential role of metabolism on the activity and toxicity of ENDX.

Supplementary Material

1

HIGHLIGHTS.

  • Few methods exist to identify and quantify endoxifen metabolites

  • Developed a LC-MS/MS method for identification and quantification of endoxifen metabolites

  • Method was validated and determined to be rapid, sensitive, and specific

  • Method was successfully applied to patient plasma samples

5. FUNDING

This work was supported in part by the Mayo Clinic Breast Cancer Specialized Program of Research Excellence [Grant Number P50CA 116201], The Mayo Clinic Cancer Center Support Grant [Grant Numbers P30 CA15083, UO1 CA69912] from the Mayo Clinic. The project was also funded in part with federal funds from the National Cancer Institute, National Institutes of Health [Contract Numbers HHSN261200800001E, N01-CM52206].

Footnotes

Declaration of interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

2

Abbreviations: 4’-hydroxy ENDX, 4,4’-dihydroxy-N-desmethyl tamoxifen; d5 ENDX, N-desmethyl-4-hydroxy tamoxifen-d5; ENDX, endoxifen; ENDX catechol, N-desmethyl-3,4-dihydroxytamoxifen hydrochloride; ENDX glucuronide, N-desmethyl-4-hydroxy tamoxifen β-D-glucuronide; ENDX methoxycatechol, N-desmethyl-3-methoxy-4-hydroxytamoxifen hydrochloride; ER+, estrogen receptor positive; norendoxifen, N-desmethylendoxifen; TAM, tamoxifen;

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

8. REFERENCES

  • [1].Goetz MP, Suman VJ, Reid JM, Northfelt DW, Mahr MA, Ralya AT, Kuffel M, Buhrow SA, Safgren SL, McGovern RM, Black J, Dockter T, Haddad T, Erlichman C, Adjei AA, Visscher D, Chalmers ZR, Frampton G, Kipp BR, Liu MC, Hawse JR, Doroshow JH, Collins JM, Streicher H, Ames MM, Ingle JN, First-in-Human Phase I Study of the Tamoxifen Metabolite Z-Endoxifen in Women With Endocrine-Refractory Metastatic Breast Cancer, Journal of clinical oncology : official journal of the American Society of Clinical Oncology, 35 (2017) 3391–3400. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [2].Desta Z, Ward BA, Soukhova NV, Flockhart DA, Comprehensive evaluation of tamoxifen sequential biotransformation by the human cytochrome P450 system in vitro: prominent roles for CYP3A and CYP2D6, Journal of Pharmacology & Experimental Therapeutics, 310 (2004) 1062–1075. [DOI] [PubMed] [Google Scholar]
  • [3].Jin Y, Desta Z, Stearns V, Ward B, Ho H, Lee KH, Skaar T, Storniolo AM, Li L, Araba A, Blanchard R, Nguyen A, Ullmer L, Hayden J, Lemler S, Weinshilboum RM, Rae JM, Hayes DF, Flockhart DA, CYP2D6 genotype, antidepressant use, and tamoxifen metabolism during adjuvant breast cancer treatment, Journal of the National Cancer Institute, 97 (2005) 30–39. [DOI] [PubMed] [Google Scholar]
  • [4].Jordan VC, Collins MM, Rowsby L, Prestwich G, A monohydroxylated metabolite of tamoxifen with potent antioestrogenic activity, The Journal of endocrinology, 75 (1977) 305–316. [DOI] [PubMed] [Google Scholar]
  • [5].Lim YC, Desta Z, Flockhart DA, Skaar TC, Endoxifen (4-hydroxy-N-desmethyl-tamoxifen) has anti-estrogenic effects in breast cancer cells with potency similar to 4-hydroxy-tamoxifen, Cancer chemotherapy and pharmacology, 55 (2005) 471–478. [DOI] [PubMed] [Google Scholar]
  • [6].Blevins-Primeau AS, Sun D, Chen G, Sharma AK, Gallagher CJ, Amin S, Lazarus P, Functional significance of UDP-glucuronosyltransferase variants in the metabolism of active tamoxifen metabolites, Cancer research, 69 (2009) 1892–1900. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [7].Hennig EE, Piatkowska M, Karczmarski J, Goryca K, Brewczynska E, Jazwiec R, Kluska A, Omiotek R, Paziewska A, Dadlez M, Ostrowski J, Limited predictive value of achieving beneficial plasma (Z)-endoxifen threshold level by CYP2D6 genotyping in tamoxifen-treated Polish women with breast cancer, BMC cancer, 15 (2015) 570. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [8].Johanning J, Kroner P, Thomas M, Zanger UM, Norenberg A, Eichelbaum M, Schwab M, Brauch H, Schroth W, Murdter TE, The formation of estrogen-like tamoxifen metabolites and their influence on enzyme activity and gene expression of ADME genes, Archives of toxicology, 92 (2018) 1099–1112. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [9].Jones RM, Yuan ZX, Lamb JH, Lim CK, On-line high-performance liquid chromatographic-electrospray ionization mass spectrometric method for the study of tamoxifen metabolism, Journal of chromatography. A, 722 (1996) 249–255. [DOI] [PubMed] [Google Scholar]
  • [10].Mazzarino M, de la Torre X, Di Santo R, Fiacco I, Rosi F, Botre F, Mass spectrometric characterization of tamoxifene metabolites in human urine utilizing different scan parameters on liquid chromatography/tandem mass spectrometry, Rapid Commun Mass Spectrom, 24 (2010) 749–760. [DOI] [PubMed] [Google Scholar]
  • [11].Murdter TE, Schroth W, Bacchus-Gerybadze L, Winter S, Heinkele G, Simon W, Fasching PA, Fehm T, Eichelbaum M, Schwab M, Brauch H, Activity levels of tamoxifen metabolites at the estrogen receptor and the impact of genetic polymorphisms of phase I and II enzymes on their concentration levels in plasma, Clinical pharmacology and therapeutics, 89 (2011) 708–717. [DOI] [PubMed] [Google Scholar]
  • [12].Poon GK, Chui YC, McCague R, Llnning PE, Feng R, Rowlands MG, Jarman M, Analysis of phase I and phase II metabolites of tamoxifen in breast cancer patients, Drug metabolism and disposition: the biological fate of chemicals, 21 (1993) 1119–1124. [PubMed] [Google Scholar]
  • [13].Romero-Lorca A, Novillo A, Gaibar M, Bandres F, Fernandez-Santander A, Impacts of the Glucuronidase Genotypes UGT1A4, UGT2B7, UGT2B15 and UGT2B17 on Tamoxifen Metabolism in Breast Cancer Patients, PLoS One, 10 (2015) e0132269. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [14].Sun D, Chen G, Dellinger RW, Duncan K, Fang JL, Lazarus P, Characterization of tamoxifen and 4-hydroxytamoxifen glucuronidation by human UGT1A4 variants, Breast cancer research : BCR, 8 (2006) R50. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [15].Teunissen SF, Rosing H, Schinkel AH, Schellens JH, Beijnen JH, Bioanalytical methods for determination of tamoxifen and its phase I metabolites: a review, Analytica chimica acta, 683 (2010) 21–37. [DOI] [PubMed] [Google Scholar]
  • [16].Teunissen SF, Rosing H, Seoane MD, Brunsveld L, Schellens JH, Schinkel AH, Beijnen JH, Investigational study of tamoxifen phase I metabolites using chromatographic and spectroscopic analytical techniques, Journal of pharmaceutical and biomedical analysis, 55 (2011) 518–526. [DOI] [PubMed] [Google Scholar]
  • [17].Zheng Y, Sun D, Sharma AK, Chen G, Amin S, Lazarus P, Elimination of antiestrogenic effects of active tamoxifen metabolites by glucuronidation, Drug Metab Dispos, 35 (2007) 1942–1948. [DOI] [PubMed] [Google Scholar]
  • [18].Liu J, Flockhart PJ, Lu D, Lv W, Lu WJ, Han X, Cushman M, Flockhart DA, Inhibition of cytochrome p450 enzymes by the e- and z-isomers of norendoxifen, Drug metabolism and disposition: the biological fate of chemicals, 41 (2013) 1715–1720. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [19].Lu WJ, Xu C, Pei Z, Mayhoub AS, Cushman M, Flockhart DA, The tamoxifen metabolite norendoxifen is a potent and selective inhibitor of aromatase (CYP19) and a potential lead compound for novel therapeutic agents, Breast cancer research and treatment, 133 (2012) 99–109. [DOI] [PubMed] [Google Scholar]
  • [20].Lv W, Liu J, Lu D, Flockhart DA, Cushman M, Synthesis of mixed (E,Z)-, (E)-, and (Z)-norendoxifen with dual aromatase inhibitory and estrogen receptor modulatory activities, J Med Chem, 56 (2013) 4611–4618. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [21].Takebe N, Coyne GO, Kummar S, Collins J, Reid JM, Piekarz R, Moore N, Juwara L, Johnson BC, Bishop R, Lin FI, Mena E, Choyke PL, Lindenberg ML, Rubinstein LV, Bonilla CM, Goetz MP, Ames MM, McGovern RM, Streicher H, Covey JM, Doroshow JH, Chen AP, Phase 1 study of Z-endoxifen in patients with advanced gynecologic, desmoid, and hormone receptor-positive solid tumors, Oncotarget, 12 (2021) 268–277. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [22].Wu X, Hawse JR, Subramaniam M, Goetz MP, Ingle JN, Spelsberg TC, The tamoxifen metabolite, endoxifen, is a potent antiestrogen that targets estrogen receptor alpha for degradation in breast cancer cells, Cancer research, 69 (2009) 1722–1727. [DOI] [PubMed] [Google Scholar]
  • [23].Antunes MV, Rosa DD, Viana Tdos S, Andreolla H, Fontanive TO, Linden R, Sensitive HPLC-PDA determination of tamoxifen and its metabolites N-desmethyltamoxifen, 4-hydroxytamoxifen and endoxifen in human plasma, Journal of pharmaceutical and biomedical analysis, 76 (2013) 13–20. [DOI] [PubMed] [Google Scholar]
  • [24].Heath DD, Flat SW, Wu AH, Pruitt MA, Rock CL, Evaluation of tamoxifen and metabolites by LC-MS/MS and HPLC methods, Br J Biomed Sci, 71 (2014) 33–39. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [25].Dahmane E, Boccard J, Csajka C, Rudaz S, Decosterd L, Genin E, Duretz B, Bromirski M, Zaman K, Testa B, Rochat B, Quantitative monitoring of tamoxifen in human plasma extended to 40 metabolites using liquid-chromatography high-resolution mass spectrometry: new investigation capabilities for clinical pharmacology, Anal Bioanal Chem, 406 (2014) 2627–2640. [DOI] [PubMed] [Google Scholar]
  • [26].Jaremko M, Kasai Y, Barginear MF, Raptis G, Desnick RJ, Yu C, Tamoxifen metabolite isomer separation and quantification by liquid chromatography-tandem mass spectrometry, Anal Chem, 82 (2010) 10186–10193. [DOI] [PubMed] [Google Scholar]
  • [27].Teunissen SF, Jager NG, Rosing H, Schinkel AH, Schellens JH, Beijnen JH, Development and validation of a quantitative assay for the determination of tamoxifen and its five main phase I metabolites in human serum using liquid chromatography coupled with tandem mass spectrometry, J Chromatogr B Analyt Technol Biomed Life Sci, 879 (2011) 1677–1685. [DOI] [PubMed] [Google Scholar]
  • [28].Maggadani BP, Harahap Y, Harmita SJ Haryono CWP. Untu, Analysis of tamoxifen and its metabolites in dried blood spot and volumetric absorptive microsampling: comparison and clinical application, Heliyon, 7 (2021) e07275. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [29].Rangel-Mendez JA, Graniel-Sabido MJ, Sanchez-Cruz JF, Moo-Puc R, Development of a high-performance liquid chromatography method with fluorescence detection for the routine quantification of tamoxifen, endoxifen and 4-hydroxytamoxifen in plasma from breast cancer patients, Biomed Chromatogr, 33 (2019) e4462. [DOI] [PubMed] [Google Scholar]
  • [30].de Krou S, Rosing H, Nuijen B, Schellens JH, Beijnen JH, Fast and Adequate Liquid Chromatography-Tandem Mass Spectrometric Determination of Z-endoxifen Serum Levels for Therapeutic Drug Monitoring, Ther Drug Monit, 39 (2017) 132–137. [DOI] [PubMed] [Google Scholar]
  • [31].Arellano C, Allal B, Goubaa A, Roche H, Chatelut E, An UPLC-MS/MS method for separation and accurate quantification of tamoxifen and its metabolites isomers, Journal of pharmaceutical and biomedical analysis, 100 (2014) 254–261. [DOI] [PubMed] [Google Scholar]
  • [32].Binkhorst L, Mathijssen RH, Ghobadi Moghaddam-Helmantel IM, de Bruijn P, van Gelder T, Wiemer EA, Loos WJ, Quantification of tamoxifen and three of its phase-I metabolites in human plasma by liquid chromatography/triple-quadrupole mass spectrometry, Journal of pharmaceutical and biomedical analysis, 56 (2011) 1016–1023. [DOI] [PubMed] [Google Scholar]
  • [33].Golander Y, Sternson LA, Paired-ion chromatographic analysis of tamoxifen and two major metabolites in plasma, Journal of chromatography, 181 (1980) 41–49. [DOI] [PubMed] [Google Scholar]
  • [34].Lee KH, Ward BA, Desta Z, Flockhart DA, Jones DR, Quantification of tamoxifen and three metabolites in plasma by high-performance liquid chromatography with fluorescence detection: application to a clinical trial, J Chromatogr B Analyt Technol Biomed Life Sci, 791 (2003) 245–253. [DOI] [PubMed] [Google Scholar]
  • [35].Dahmane E, Mercier T, Zanolari B, Cruchon S, Guignard N, Buclin T, Leyvraz S, Zaman K, Csajka C, Decosterd LA, An ultra performance liquid chromatography-tandem MS assay for tamoxifen metabolites profiling in plasma: first evidence of 4’-hydroxylated metabolites in breast cancer patients, J Chromatogr B Analyt Technol Biomed Life Sci, 878 (2010) 3402–3414. [DOI] [PubMed] [Google Scholar]
  • [36].Kikuta C, Schmid R, Specific high-performance liquid chromatographic analysis of tamoxifen and its major metabolites by “on-line” extraction and post-column photochemical reaction, Journal of pharmaceutical and biomedical analysis, 7 (1989) 329–337. [DOI] [PubMed] [Google Scholar]
  • [37].Fried KM, Wainer IW, Direct determination of tamoxifen and its four major metabolites in plasma using coupled column high-performance liquid chromatography, J Chromatogr B Biomed Appl, 655 (1994) 261–268. [DOI] [PubMed] [Google Scholar]
  • [38].Bioanalytical Method Validation -Guidance for Industry, in: U.S.D.o.H.a.H. Services, F.a.D. Administration, C.f.D.E.a.R. (CDER), C.f.V.M. (CVM), https://www.fda.gov/files/drugs/published/Bioanalytical-Method-Validation-Guidance-for-Industry.pdf (Eds.), 2018.
  • [39].Bonfiglio R, King RC, Olah TV, Merkle K, The effects of sample preparation methods on the variability of the electrospray ionization response for model drug compounds, Rapid communications in mass spectrometry : RCM, 13 (1999) 1175–1185. [DOI] [PubMed] [Google Scholar]
  • [40].Njus D, Kelley PM, Tu YJ, Schlegel HB, Ascorbic acid: The chemistry underlying its antioxidant properties, Free Radic Biol Med, 159 (2020) 37–43. [DOI] [PubMed] [Google Scholar]
  • [41].Yong YF, Tan SC, Liew MWO, Yaacob NS, Liquid chromatography-tandem mass spectrometry (LC-MS/MS) method development for screening of potential tamoxifen-drug/herb interaction via in vitro cytochrome P450 inhibition assay, J Chromatogr B Analyt Technol Biomed Life Sci, 1148 (2020) 122148. [DOI] [PubMed] [Google Scholar]
  • [42].Braal CL, Jager A, Hoop EO, Westenberg JD, Lommen K, de Bruijn P, Vastbinder MB, van Rossum-Schornagel QC, Thijs-Visser MF, van Alphen RJ, Struik LEM, Zuetenhorst HJM, Mathijssen RHJ, Koolen SLW, Therapeutic Drug Monitoring of Endoxifen for Tamoxifen Precision Dosing: Feasible in Patients with Hormone-Sensitive Breast Cancer, Clin Pharmacokinet, 61 (2022) 527–537. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

1
NIHMS1886844-supplement-1.docx (175KB, docx)

RESOURCES