Quantification Of Letrozole In Human Plasma And Its Application In A Bioequivalence Study

Background Letrozole is a modern third generation aromatase inhibitorwhich effectively blocks the production of estrogen in postmenopausalwomen with estrogen receptor positive breast cancer. To date, the mainmethods for determining letrozole in human plasma are high–performanceliquid chromatography–fluorescence and liquid chromatography–tandemmass spectrometry （LC-MS/MS）.Objective To improve previously reporeted liquid chromatographic tandemmass spectrometric method for the determination of letrozole in humanplasma and apply it to a bioequivalence and pharmacokinetics study.Method A rapid, selective, and sensitive liquid chromatography–tandemmass spectrometric method in negative ionization mode was developed andvalidated to determine letrozole in human plasma using d4-letrozole as theinternal standard （IS）. Following protein precipitation, effectivechromatographic separation was accomplished on a Capcell PAK C₁₈MGcolumn （100mm×4.6mm,5μm） with a mobile phase consisting ofmethanol–10mM ammonium acetate （65:35, v/v） at a flow rate of0.6mL/min. The running time for each sample was4.0min. The detectionof the analyte and IS was carried out in selective reaction monitoring modevia negative electrospray ionization interface. Quantitation was performedusing seclective reaction monitoring （SRM） of the transitions of m/z284→m/z （215+242） and m/z288→m/z （219+246） for letrozole and theinternal standard d4-letrozole, respectively. Results The linear concentration range of the calibration curve forletrozole was0.400–50.0ng· mL^-1, the lower limit of quantification was0.400ng· mL^-1. The intraday and interday precisions were less than8.9%in terms of the relative standard deviation （RSD）, and the accuracy waswithin±4.4%in terms of the relative error （RE）. For the parameters ofC_max, AUC_0-tand AUC_0-∞in the bioequivalence study, the90%confidenceinterval for the ratio of the test and reference formulations were100%–116%,97.8%–105.1%, and97.3%–105.2%, respectively. Thepharmacokinetic profile of letrozole in the healthy, postmenopausalChinese women was similar with those of the Japanese populaiton ratherthan the Europeans.Conclusion It is the first time that quantification of letrozole wasperformed in a negative ionization mode, and the sensitivity under negativeionization mode was about150times higher than that under positiveionization mode. The method was successfully applied to a bioequivalenceand pharmacokinetics study in the healthy, postmenopausal Chinesewomen.