Department of Internal Medicine II, University Hospital of Würzburg, Oberdürrbacher Str. 6, 97080 Würzburg, Germany.
Institute for Pharmacy and Food Chemistry, University of Würzburg, Am Hubland, 97074 Würzburg, Germany.
Clin Biochem. 2022 Jul-Aug;105-106:35-43. doi: 10.1016/j.clinbiochem.2022.04.011. Epub 2022 Apr 26.
Liquid chromatography tandem mass spectrometry (LC-MS/MS) is a highly selective and sensitive method for the quantification of kinase inhibitors, yet not widely available in clinical routine for therapeutic drug monitoring (TDM). To provide a more accessible alternative, a high-performance liquid chromatography method with ultraviolet/diode array detection (HPLC-UV/DAD) to quantify cabozantinib, dabrafenib, nilotinib and osimertinib, was developed and validated. Results were compared to LC-MS/MS.
After liquid-liquid-extraction and reconstitution of the residue in 20 mM potassium dihydrogen phosphate (KH2PO4) (pH4.6), acetonitrile and methanol (50:25:25,v/v/v), chromatographic separation was achieved in 20.0 min using a Luna® C18(2)-HST column (100 × 2 mm, 2.5 μm), protected by a C18 guard column (4 × 2 mm) (column temperature: 30 °C, autosampler: 10 °C). Mobile phase A and B consisted of 20 mM KH2PO4 (pH4.9) and acetonitrile (9:1,v/v) and acetonitrile:20 mM KH2PO4 (pH4.9) (7:3,v/v), respectively. Gradient elution was performed at 200 µL/min. Analytes were quantified at 250, 280 and 330 nm, using sorafenib as internal standard.
Calibration curves were linear (35-2,000 ng/mL). Method validation assays met requirements by U.S. Food and Drug Administration and European Medicines Agency. Compared to the more sensitive and specific LC-MS/MS, HPLC-UV/DAD showed a good correlation and a strong positive association (Kendall's tau 0.811¬-0.963, p < 0.05). Bland-Altman-plots revealed 100% (cabozantinib), 98.6% (dabrafenib), 98.6% (nilotinib) and 96.2% (osimertinib) of relative differences inside the limits of agreement. Regulatory agency criteria for sample reanalysis and cross validation were met (±20%-criterion:100% (cabozantinib), 94.3% (dabrafenib), 92% (nilotinib) and 84.6% (osimertinib).
The developed HPLC-UV/DAD method is "fit-for-TDM" in clinical routine and serves as a genuine alternative to LC-MS/MS.
液相色谱串联质谱法（LC-MS/MS）是一种高度灵敏和选择性的激酶抑制剂定量方法，但在治疗药物监测（TDM）的临床常规中尚未广泛应用。为提供更易获得的替代方法，开发并验证了一种采用高效液相色谱法与紫外/二极管阵列检测（HPLC-UV/DAD）定量卡博替尼、达布拉非尼、尼洛替尼和奥希替尼的方法。并将结果与 LC-MS/MS 进行了比较。
在液-液萃取后，将残留物在 20mM 磷酸二氢钾（KH2PO4）（pH4.6）中复溶，乙腈和甲醇（50:25:25，v/v/v），使用 Luna® C18(2)-HST 柱（100×2mm，2.5μm）在 20.0min 内实现色谱分离，柱前采用 C18 保护柱（4×2mm）（柱温：30°C，自动进样器：10°C）。流动相 A 和 B 分别由 20mM KH2PO4（pH4.9）和乙腈（9:1，v/v）以及乙腈：20mM KH2PO4（pH4.9）（7:3，v/v）组成。以 200µL/min 的流速进行梯度洗脱。采用索拉非尼作为内标，在 250、280 和 330nm 处进行定量分析。
校准曲线呈线性（35-2000ng/mL）。美国食品药品监督管理局和欧洲药品管理局的验证试验符合要求。与更灵敏、更特异的 LC-MS/MS 相比，HPLC-UV/DAD 显示出良好的相关性和强正相关性（Kendall's tau 0.811¬-0.963，p<0.05）。Bland-Altman 图显示，在一致性限内，相对差异的 100%（卡博替尼）、98.6%（达布拉非尼）、98.6%（尼洛替尼）和 96.2%（奥希替尼）均在 100%以内。（±20%标准：100%（卡博替尼）、94.3%（达布拉非尼）、92%（尼洛替尼）和 84.6%（奥希替尼）符合监管机构对样品再分析和交叉验证的要求。
开发的 HPLC-UV/DAD 方法适用于临床常规 TDM，并可作为 LC-MS/MS 的真正替代方法。
