To improve bioavailability and provide resistance to deamination, an array of gemcitabine (dFdC) prodrugs carrying the acyl modifications has been successful in the optimization of pharmacokinetic properties of dFdC, but the reports about 4-N-carbobenzoxy-dFdC (Cbz-dFdC), a dFdC prodrug bearing alkyloxycarbonyl modification, are relatively rare. Notably, in vivo enzymatic hydrolysis was an absolutely essential factor for the activation of these prodrugs, which is correlated with the anti-tumor activity. Therefore, detailed metabolism studies of Cbz-dFdC should be carried out for a more authentic pharmacodynamic evaluation. In order to detect the pharmacokinetic characteristics of Cbz-dFdC, a selective, sensitive and accurate method for the simultaneous determination of Cbz-dFdC, along with dFdC and its major metabolite dFdU in rat plasma was developed and validated using UFLC–MS/MS techniques. Column was at 40 °C for separation using an eluent with acetonitrile and 0.1% formic acid, 1 mM ammonium formate at a flow rate of 0.2 mL/min. Detection was performed using ESI source in positive ion selected reaction monitoring mode by monitoring the following ion transitions m/z 398.1 → 202.2 (Cbz-dFdC), m/z 264.1 → 112.0 (dFdC), m/z 265.3 → 113.2 (dFdU) and m/z 246.1 → 112.0 (IS). Analytes were extracted by simple precipitation with acetonitrile containing internal standards followed by liquid-liquid extraction with ethyl acetate. The calibration curves of Cbz-dFdC, dFdC and dFdU were linear in the concentration range of 2 to 500 ng/mL, 2 to 500 ng/mL and 40 to 10,000 ng/mL, respectively. The assay ranges selected for the three analytes were appropriate and minimized the need for reanalysis. All the validation data, such as intra- and inter-day precision, accuracy, selectivity and stability, were within the required limits. In conclusion, the sensitive analytical assay was selective and accurate for the determination of rat plasma concentrations of Cbz-dFdC, dFdC and dFdU from a single LC–MS/MS analysis and well-suited to support pharmacokinetic studies.

为了提高生物利用度并提供抗脱氨性，一系列带有酰基修饰的吉西他滨（dFdC）前药已成功地优化了dFdC的药代动力学特性，但有关4- N-碳氧苯甲氧基-dFdC（Cbz-dFdC）的报道，带有烷氧基羰基修饰的dFdC前药相对较少。值得注意的是，体内酶水解是激活这些前药的绝对必要因素，这与抗肿瘤活性有关。因此，应进行Cbz-dFdC的详细代谢研究，以进行更真实的药效评估。为了检测Cbz-dFdC的药代动力学特征，开发了一种同时测定Cbz-dFdC以及dFdC及其主要代谢产物dFdU在大鼠血浆中的选择性，灵敏和准确的方法，并使用UFLC-MS / MS技术进行了验证。柱在40°C下分离，使用乙腈和0.1％甲酸，1 mM甲酸铵的洗脱液，流速为0.2 mL / min。检测是在正离子选择反应通过监测以下离子跃迁监测模式中执行使用ESI源中号/ z 398.1→202.2（Cbz-dFdC），m / z 264.1→112.0（dFdC），m / z 265.3→113.2（dFdU）和m / z246.1→112.0（IS）。通过用含有内标的乙腈简单沉淀来萃取分析物，然后用乙酸乙酯进行液-液萃取。Cbz-dFdC，dFdC和dFdU的校准曲线分别在2至500 ng / mL，2至500 ng / mL和40至10,000 ng / mL的浓度范围内呈线性。为这三种分析物选择的测定范围是适当的，并将重新分析的需要降到最低。所有验证数据，例如日内和日间精度，准确性，选择性和稳定性，均在要求的范围内。总而言之，灵敏的分析方法对于通过单次LC-MS / MS分析确定大鼠血浆Cbz-dFdC，dFdC和dFdU的血浆浓度具有选择性和准确性，非常适合于支持药代动力学研究。