Simultaneous quantification of palbociclib, ribociclib and letrozole in human plasma by a new LC-MS/MS method for clinical application

A novel LC-MS/MS method was developed for the quantification of the new cyclin dependent kinase inhibitors (CDKIs) palbociclib and ribociclib and the aromatase inhibitor letrozole used in combinatory regimen. The proposed method is appropriate to be applied in clinical practice due to the simple and fast sample preparation based on protein precipitation, the low amount of patient sample necessary for the analysis (10 μL) and the total run time of 6.5 min. It was fully validated according to FDA and EMA guidelines on bioanalytical method validation. The linearity was assessed (R2 within 0.9992–0.9983) over the concentration ranges of 0.3–250 ng/mL for palbociclib, 10–10000 ng/mL for ribociclib and 0.5–500 ng/mL for letrozole that properly cover the therapeutic plasma concentrations. A specific strategy was implemented to reduce the carryover phenomenon, formerly known for these CDKIs. This method was applied to quantify the Cmin of palbociclib, ribociclib and letrozole in plasma samples from patients enrolled in a clinical study. The same set of study samples was analysed twice in separate runs to assess the reproducibility of the method by means of the incurred samples reanalysis. The results corroborated the reliability of the analyte concentrations obtained with the bioanalytical method, already proved by the validation process. The percentage differences were always within ±10% for all the analytes and the R2 of the correlation graph between the two quantifications was equal to 0.9994.

本研究建立了一种新型液相色谱-串联质谱法（LC-MS/MS），用于定量分析联合给药方案中使用的新型细胞周期蛋白依赖性激酶抑制剂（cyclin dependent kinase inhibitors, CDKIs）帕博西尼（palbociclib）、瑞博西尼（ribociclib）以及芳香化酶抑制剂来曲唑（letrozole）。该方法基于蛋白沉淀法实现简便快速的样品前处理，仅需10 μL的微量患者样本，总分析时长仅6.5分钟，因此适用于临床实践场景。本方法已按照美国食品药品监督管理局（FDA）与欧洲药品管理局（EMA）的生物分析方法验证指南完成全面验证。针对上述三种待测物，本研究考察了其线性范围：帕博西尼为0.3~250 ng/mL，瑞博西尼为10~10000 ng/mL，来曲唑为0.5~500 ng/mL，上述范围均覆盖了对应的治疗血浆浓度，其决定系数（R²）介于0.9992~0.9983之间。为减少此类CDKIs此前报道的残留现象，本研究采用了专属优化策略。本方法已被用于定量分析一项临床研究纳入患者的血浆样本中帕博西尼、瑞博西尼与来曲唑的谷浓度（Cmin）。采用受试样品重分析（incurred samples reanalysis）法，将同一批研究样本分两次独立上机检测，以评估本方法的重现性。检测结果证实了本生物分析方法所得待测物浓度的可靠性，这一结论已通过前期验证流程得到印证。所有待测物的百分比偏差均处于±10%以内，且两次定量结果的相关性曲线决定系数（R²）为0.9994。