Li Yuan-dong, Li Yan, Liang Ning-sheng, Yang Fan, Kuang Zhi-peng
Research Department, Affiliated Tumor Hospital of Guangxi Medical University, Nanning, 530021 Guangxi, People's Republic of China.
Research Department, Affiliated Tumor Hospital of Guangxi Medical University, Nanning, 530021 Guangxi, People's Republic of China.
J Chromatogr B Analyt Technol Biomed Life Sci. 2015 Oct 1;1002:107-12. doi: 10.1016/j.jchromb.2015.08.017. Epub 2015 Aug 20.
A simple, rapid and sensitive reversed-phase high performance liquid chromatography (HPLC) method has been developed for the determination of methotrexate in human serum. After deproteinization of the serum with 40% silver nitrate solution, methotrexate and internal standard (IS) were separated on a reversed-phase column with a mobile phase consisting of 10mM sodium phosphate buffer (pH6.40)-methanol (78:22%, v/v) and ultraviolet detection at 310nm. The linearity is evaluated by a calibration curve in the concentration range of 0.05-10.0μg/mL and presented a correlation coefficient of 0.9995. The absolute recoveries were 97.52±3.9% and 96.87±3.7% for methotrexate and ferulic acid (internal standard), respectively. The intra- and inter-day precision were less 6.19 and 5.89%, respectively (n=6). The limit of quantitation was 0.02μg/mL and the limit of detection was 0.006μg/mL. The complete analysis was achieved less than 10min with no interference from endogenous components or 22 examined drugs. This method was validated by using serum samples from high-dose methotrexate treated patients with osteosarcoma, breast cancer, acute leukemia and lymphoma. The method was demonstrated to be a simple, rapid and reliable approach in quantification of methotrexate in serum samples from patients with high-dose methotrexate therapy.
已开发出一种简单、快速且灵敏的反相高效液相色谱（HPLC）法，用于测定人血清中的甲氨蝶呤。用40%硝酸银溶液使血清脱蛋白后，甲氨蝶呤和内标（IS）在反相柱上分离，流动相为10mM磷酸钠缓冲液（pH6.40）-甲醇（78:22%，v/v），并在310nm处进行紫外检测。通过在0.05-10.0μg/mL浓度范围内的校准曲线评估线性，相关系数为0.9995。甲氨蝶呤和阿魏酸（内标）的绝对回收率分别为97.52±3.9%和96.87±3.7%。日内和日间精密度分别小于6.19%和5.89%（n=6）。定量限为0.02μg/mL，检测限为0.006μg/mL。不到10分钟即可完成完整分析，且不受内源性成分或22种受试药物的干扰。该方法通过使用来自接受高剂量甲氨蝶呤治疗的骨肉瘤、乳腺癌、急性白血病和淋巴瘤患者的血清样本进行了验证。该方法被证明是一种简单、快速且可靠的方法，可用于定量高剂量甲氨蝶呤治疗患者血清样本中的甲氨蝶呤。
