Key Laboratory of New Drug and Clinical Application, Xuzhou Medical College, No. 209 Tongshan Road, Xuzhou 221004, Jiangsu, China.
J Chromatogr B Analyt Technol Biomed Life Sci. 2013 Mar 1;919-920:10-9. doi: 10.1016/j.jchromb.2012.12.027. Epub 2013 Jan 9.
The cytotoxic agent Gemcitabine (2',2'-difluoro-2'-deoxycytidine) has been proved to be effective in the treatment of malignant gliomas. A rapid, sensitive and specific ultra performance liquid chromatography with tandem mass spectrometry (UPLC-MS/MS) assay using microdialysis sampling was developed and validated to quantify gemcitabine and its major metabolite 2',2'-difluoro-2'-deoxyuridine (dFdU) in Sprague-Dawley rat bearing 9L glioma. Microdialysis probes were surgically implanted into the area of rat brain tumor in the striatal hemisphere, and artificial cerebrospinal fluid was used as a perfusion medium. The samples were analyzed directly by UPLC-MS/MS after the addition of 5-bromouracil as an internal standard (IS). Separation was achieved on Agilent SB-C(18) (50 mm × 2.1mm I.D., 1.8 μm) column at 40 °C using an isocratic elution method with acetonitrile and 0.1% formic acid (4:96, v/v) at a flow rate of 0.2 mL/min. Detection was performed using electrospray ionization in positive ion selected reaction monitoring mode by monitoring the following ion transitions m/z 264.0→112.0 (gemcitabine), m/z 265.1→113.0 (dFdU) and m/z 190.9→173.8 (IS). The calibration curves of gemcitabine and dFdU were linear in the concentration range of 0.66-677.08 ng/mL and 0.31-312.00 ng/mL, respectively. The lower limit of quantification of gemcitabine and dFdU were 0.66 ng/mL and 0.31 ng/mL, respectively. The lower limit of detection of gemcitabine and dFdU were calculated to be 0.2 ng/mL and 0.1 ng/mL, respectively. All the validation data, such as intra- and inter-day precision, accuracy, selectivity and stability, were within the required limits. The validated method was simple, precise and accurate, which was successfully employed to determinate the concentrations of gemcitabine and dFdU in the extracellular fluid of rat brain tumor.
细胞毒性药物吉西他滨（2',2'-二氟-2'-脱氧胞苷）已被证明可有效治疗恶性脑胶质瘤。本研究建立并验证了一种快速、灵敏和特异的超高效液相色谱-串联质谱（UPLC-MS/MS）分析方法，采用微透析采样技术，可定量检测荷 9L 脑胶质瘤 Sprague-Dawley 大鼠脑中吉西他滨及其主要代谢物 2',2'-二氟-2'-脱氧尿苷（dFdU）。微透析探针通过手术植入大鼠大脑肿瘤纹状体半球区，以人工脑脊液作为灌流介质。样品直接通过 UPLC-MS/MS 分析，加入 5-溴尿嘧啶作为内标（IS）。采用 Agilent SB-C(18)（50mm×2.1mmID，1.8μm）色谱柱，在 40°C 下以等度洗脱方式，以乙腈和 0.1%甲酸（4:96，v/v）为流动相，流速为 0.2mL/min。采用电喷雾电离，正离子选择反应监测模式，监测以下离子对的转换：m/z264.0→112.0（吉西他滨）、m/z265.1→113.0（dFdU）和 m/z190.9→173.8（IS）。吉西他滨和 dFdU 的校准曲线在 0.66-677.08ng/mL 和 0.31-312.00ng/mL 的浓度范围内呈线性。吉西他滨和 dFdU 的定量下限分别为 0.66ng/mL 和 0.31ng/mL。吉西他滨和 dFdU 的检测下限分别计算为 0.2ng/mL 和 0.1ng/mL。所有验证数据，如日内和日间精密度、准确度、选择性和稳定性，均在要求范围内。该验证方法简单、准确、精确，成功应用于测定荷瘤大鼠脑肿瘤细胞外液中吉西他滨和 dFdU 的浓度。
