Development and validation of a liquid chromatography–tandem mass spectrometry method for the simultaneous quantification of tamoxifen, anastrozole, and letrozole in human plasma and its application to a clinical study

Abstract

There is substantial evidence that circulating estrogens promote the proliferation of breast cancer. Consequently, adjuvant hormonal treatment strategies targeting estrogen action have been established. Such hormonal therapies include selective estrogen receptor modulators, such as tamoxifen, which interfere at the estrogen receptors directly, or non-steroidal aromatase inhibitors, such as anastrozole and letrozole, which inhibit estrogen synthesis through blocking the aromatase, a key enzyme of estrogen production. Despite considerable therapeutic success, in several cases, the use of these drugs is limited by side effects that have been described to significantly impair the adherence of patients to endocrine treatment. However, objective data concerning patient adherence and its clinical relevance are limited. One promising approach to check patient-reported adherence is drug monitoring in human plasma. Therefore, a liquid chromatography-tandem mass spectrometry method to determine the plasma concentrations of tamoxifen, anastrozole, and letrozole has been developed and fully validated according to guidelines for clinical and forensic toxicology. The validation criteria evaluated were selectivity, linearity, accuracy and precision, limit of quantification, recovery and matrix effects, sample stability, and carryover. The six-point calibration curves showed linearity over the range of concentrations from 25 to 500 ng/ml for tamoxifen, 5 to 200 ng/ml for anastrozole, and 10 to 300 ng/ml for letrozole. The intra- and inter-day precision and accuracies were always better than 15%. The validated procedure was successfully applied to a clinical study (Patient-Reported Outcomes in Breast Cancer Patients undergoing Endocrine Therapy, PRO-BETh). A major aim of PRO-BETh study is the comprehensive evaluation of adherence to treatment in pre- and post-menopausal women with breast cancer. Plasma samples of 310 breast cancer patients undergoing anti-estrogen therapy were analyzed. Eight samples did not contain a quantifiable amount of drug, strongly indicating non-adherence of the corresponding patients to adjuvant breast cancer treatment. Furthermore, plasma concentrations at the lower end of the observed plasma level distribution might represent a hint but not a confirmation for non-adherence in terms of non-daily and irregular intake of the prescribed drug.

Simultaneous quantitative analysis of letrozole, its carbinol metabolite, and carbinol glucuronide in human plasma by LC-MS/MS

“…The extraction procedure was optimized for the sensitive quantification of carbinol, as carbinol concentrations in plasma were one order of magnitude lower than those measured for carbinol-gluc and close Δ is the difference between first and second analysis to two orders of magnitude lower when compared to letrozole. The observed influence of the matrix leading to a peak reduction of 70% is in line with previously reported strong ion suppression effects [12]. Repeated freeze-thaw cycles did not affect the concentrations measured for any compound (less than 10% deviation).…”

“…An improved method for sample preparation by solid phase extraction (SPE) and fluorescence detection led to a 20-fold enhanced sensitivity for letrozole in plasma, but yet, carbinol could not be detected [11]. Recently, a sensitive method for the quantification of letrozole, anastrozole, and tamoxifen has been validated to survey the compliance of breast cancer patients [12]. In addition to the oncology setting, several anti-doping laboratories developed analytical methods for the screening of the presence of letrozole, which is included in the Prohibited List of the World Anti-Doping Agency, or its carbinol metabolite in urine samples using rapid resolution LC-ESI-MS/MS [13], HPLC-ESI-TOF [14], or GC-MS [15].…”

“…In addition to the oncology setting, several anti-doping laboratories developed analytical methods for the screening of the presence of letrozole, which is included in the Prohibited List of the World Anti-Doping Agency, or its carbinol metabolite in urine samples using rapid resolution LC-ESI-MS/MS [13], HPLC-ESI-TOF [14], or GC-MS [15]. Although a few methods for the quantification of letrozole and/or carbinol have been established [9][10][11][12][13][14][15], as of yet, there is no method available for the determination of carbinol and/or carbinol-gluc in plasma.…”

A Review of Chromatographic Methods Used in the Determination of Anastrozole Levels

“…The method employed a mobile phase consisting of ACN:water containing N,Ndimethyloctylamine (pH adjusted to 3.0 with orthophosphoric acid) at a flow rate of 1.0 ml/min with an injection volume of 20 µl, monitored at 270 Omeprozole LC-MS/MS [30] Androsteroneglucuronide LC-MS/MS [41] Letrozole LC-MS/MS [43] Bunitrolol LC-MS/MS [44] ANA-d12 LC-MS/MS [45] Desmethyldiazepam LC-MS/MS [18,21] Verapamil LC-MS/MS [34] Diazepam GC-ECD [46,47] Clomipramine GC-FID [35] LC-MS/MS= LC coupled with tandem mass spectrometry; GC-ECD= GC with electron capture detection; GC-FID= gas chromatography with flame ionization detection; ANA-d12= deuterium-labeled anastrozole.…”

“…A group of researchers developed an LC-MS/ MS method, using a triple quadrupole ion trap spectrometry, for the simultaneous detection of TAM, letrozole and ANA using Si C18 column (200×0.5 mm, 5 µm) [44] . A linear gradient separation technique was employed for an analytical time of 16 min.…”