|Malgorzata Jaremko1, Kevin McCann1, M-P George2, Myra Barginear3, George Raptis3, Yumi Kasai1, Robert Desnick1, Chunli Yu1|
1Department of Genetics and Genomic Sciences, Mount Sinai School of Medicine, New York, NY
2Diagnostics, Life Sciences and Chemical Analysis, Agilent Technologies, Inc, Santa Clara, CA
3Department of Hemotology and Medical Oncology, Mount Sinai School of Medicine, New York, NY
|Tamoxifen (TAM) has been widely used to treat breast cancer patients who are estrogen receptor (ER)-positive to prevent relapse and improve survival. However, the clinical outcome varies widely among patients receiving the standard 20 mg/day dose of this drug. CYP2D6 is a key enzyme of the CYP450 family involved in the hydroxylation of TAM and N-desmethyl-TAM to pharmacologically active forms. It has been shown to play an important role in TAM responsiveness and outcome. However, variants of other TAM metabolizing and excretion enzymes and CYP2D6 inhibiting drugs will also affect TAM metabolism and active metabolites levels. Complexity of TAM metabolism makes the direct measurement of TAM metabolites equally important in guiding TAM treatment and studying the correlation of genetic polymorphisms to TAM metabolism.|
LC/MS/MS method was developed to quantitate TAM metabolites in patients who reached their steady states on standard TAM dose. After deproteinization, plasma samples were extracted with methanol containing a d3-TAM internal standard, they were then purified using cation exchange/nonionic SPE column before mass spec analysis. Tamoxifen (TAM) and its metabolites which include 4-OH-tamoxifen (4OHTam), endoxifen (Endox) and N-desmethyltamoxifen (NDT) were measured by multiple reaction monitoring (MRM) with positive jet stream electrospray ionization (ESI) using Agilent 1200 series fast resolution LC and QQQ 6460 tandem mass spectrometer system. The metabolites were well separated with a gradient program on Zorbax SB-C18 column (2.1 mm x 50 mm, 1.8 um particle size). E and Z isoforms products of both 4OHT and Endox were separated as well in our assay. Analytical performance characteristics were evaluated for specimen stability, AMR, reproducibility/precision, accuracy, matrix effects, and reference interval of steady states tam metabolites with standard TAM dose. The limits of quantization for 4 metabolites ranged from 0.1-1.0 ng/ml and linearity up to at least 2000 ng/ml. Recovery rate at 10, 500 and 1500 ng/ml spiked plasma ranged from 87.3-103.8%. Intra-day (n=10) and inter-day (n=10) CV% of all metabolites at three levels ranged from 1.8-8.7% and 2.4-10% respectively. Short term stability study showed that TAM and NDT remained stable up to 7 days. There were 10-15% drop in 4OHTam and Endox after 1-2 day and 25% drop after 7 day storage. In summary, we have developed and validated a sensitive and reliable analytical method for determination of TAM metabolites for clinical use. This clinical test is complementary to our CYP2D6 genotyping assay in improving individualized TAM treatment.