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

Abstract

Letrozole is an efficient endocrine treatment of postmenopausal breast cancer, however, not all patients benefit from this treatment, and moreover, severe side-effects like arthralgia frequently lead to discontinuation. To better understand inter-individual variability in drug response and side-effects, plasma analysis of steady-state concentrations of letrozole and its major metabolites is crucial. We developed a rapid, sensitive, and specific method for the simultaneous quantification of letrozole and its metabolites 4,4'-(hydroxymethylene)dibenzonitrile (carbinol) and bis(4-cyanophenyl)methyl hexopyranosiduronic acid (carbinol-gluc) by UHPLC-ESI-MS/MS using in-house synthesized, stable isotope-labeled internal standards. Following solid-phase extraction in BondElut C18 96-well plates, the analytes were separated on a ZORBAX Eclipse XDB-C18 column (1.8 μm, 4.6 × 50 mm) with a gradient of acetonitrile in 0.1% acetic acid in water and detected on a triple quadrupole mass spectrometer with electrospray ionization in the multiple reaction monitoring mode. Lower limits of quantification were 20, 0.2, and 2 nM for letrozole, carbinol, and carbinol-gluc, respectively. The assay has been validated according to FDA guidance and applied to the analysis of 20 plasma samples of postmenopausal breast cancer patients treated with 2.5 mg of letrozole per day. Mean plasma levels (±SD) were 366 ± 173, 0.38 ± 0.09, and 34 ± 12 nM for letrozole, carbinol, and carbinol-gluc, respectively. Our rapid and sensitive mass spectrometry based method enables future pharmacokinetic investigations of letrozole outcome.

Prediction of Response to Aromatase Inhibitors in Breast Cancer

“…At the same time, challenges in measurements of ultra-low oestrogen concentrations in AI-treated post-menopausal patients (reviewed by Per Lonning in another chapter of this book) might complicate the inclusion of such measurements in the large trails. However, recent development of mass-spectrometry tools means that AIs and oestrogen metabolites can now be measured accurately and simultaneously in large cohorts of AI treated patients [6,166]. The trial conducted by Ingle et al [6] reported concentrations of oestrone, oestradiol, androstenedione, testosterone, anastrozole and its metabolites in 649 patients receiving adjuvant anastrozole [6].…”

Resistance to Aromatase Inhibitors in Breast Cancer

“…At the same time, challenges in measurements of ultra-low oestrogen concentrations in AI-treated post-menopausal patients (reviewed by Per Lonning in another chapter of this book) might complicate the inclusion of such measurements in the large trails. However, recent development of mass-spectrometry tools means that AIs and oestrogen metabolites can now be measured accurately and simultaneously in large cohorts of AI treated patients [6,166]. The trial conducted by Ingle et al [6] reported concentrations of oestrone, oestradiol, androstenedione, testosterone, anastrozole and its metabolites in 649 patients receiving adjuvant anastrozole [6].…”

The Letrozole Phase 1 Metabolite Carbinol as a Novel Probe Drug for UGT2B7

“…Carbinol, carbinol-gluc, and d 4 -carbinol-gluc were synthesized as described previously (Precht et al, 2012). Purity, as determined by high-performance liquid chromatography-UV analysis, exceeded 99, 98, and 92% for carbinol, carbinol-gluc, and d 4 -carbinol-gluc, respectively.…”

“…For the quantification of carbinol-gluc, a recently developed LC-MS/MS method for the determination of letrozole, carbinol, and carbinolgluc in human plasma was adapted (Precht et al, 2012). Sample preparation was done by protein precipitation with acetonitrile.…”

“…All participants gave informed consent. In the plasma samples, letrozole and its metabolites carbinol and carbinol-gluc were quantified by LC-MS/MS, as previously described (Precht et al, 2012).…”