色谱 ›› 2020, Vol. 38 ›› Issue (11): 1302-1307.DOI: 10.3724/SP.J.1123.2020.01003

• 研究论文 • 上一篇    下一篇

高效液相色谱法测定黑色素瘤小鼠尿液中达卡巴嗪

岳玉华, 周炳均, 艾佳媛, 封顺()   

  1. 西南交通大学生命科学与工程学院, 四川 成都 610031
  • 收稿日期:2020-01-04 出版日期:2020-11-08 发布日期:2020-12-11
  • 通讯作者: 封顺
  • 作者简介:封顺, E-mail:fengshunxd@hotmail.com
  • 基金资助:
    西南交通大学个性化实验项目(GX201913093);国家自然科学基金项目(21565023)

Determination of dacarbazine in the urine of mice with melanoma by high performance liquid chromatography

Yuhua YUE, Bingjun ZHOU, Jiayuan AI, Shun FENG()   

  1. College of Life Science and Engineering, Southwest Jiaotong University, Chengdu 610031, China
  • Received:2020-01-04 Online:2020-11-08 Published:2020-12-11
  • Contact: Shun FENG
  • Supported by:
    Personalized Experiment Project of Southwest Jiaotong University(GX201913093);National Natural Science Foundation Project(21565023)

摘要:

达卡巴嗪是治疗恶性黑色素瘤的一线化疗药物。它在体内主要经肝脏代谢,同时部分药物仍以原药形式经尿液排出。这就意味着可以通过监测尿液中达卡巴嗪的含量评估其在人体内的利用率和转化率,进而对其治疗效果进行评价。针对达卡巴嗪,人们发展了多种分析方法,但多基于高效液相色谱-质谱平台。然而达卡巴嗪为强极性弱碱性化合物,采用常规反相色谱法分析时会出现出峰时间过早、峰形拖尾的现象,导致定量不准确。基于此,该文建立了一种测定尿液中达卡巴嗪含量的高效液相色谱方法以克服上述问题。小鼠尿液经丙酮沉淀法去除蛋白后,采用Shimadzu-GL ODS柱(150 mm×4.6 mm,5 μm)分离,色谱条件如下:流动相为甲醇/乙腈(1:1,v/v)-0.01 mol/L磷酸二氢钠溶液(pH=6.5)(20:80,v/v),流速1 mL/min,检测波长280 nm,柱温35℃,洗脱时间15 min。在该色谱条件下,达卡巴嗪保留时间为5.3 min且峰形良好。其在0.25~1000 μg/mL范围内线性关系良好(r 2 =0.999)。基于信噪比(S /N )=3和S /N =10,计算出检出限和定量限分别为0.12 μg/mL和0.25 μg/mL。在低、中、高(50.0、375、500 μg/mL)3个添加水平下,加标回收率分别为98.9%、102%、99.1%,相对标准偏差(RSD)分别为3.2%、1.3%、1.2%(n =5)。日内与日间RSD分别小于3.8%和4.4%。将该法应用于不同发展阶段的黑色素瘤C57BL/6小鼠尿液中达卡巴嗪的监测,结果表明该方法操作简便,结果可靠,便于推广。

关键词: 高效液相色谱法, 达卡巴嗪, 尿液, 黑色素瘤, 蓝莓花青素, 营养辅助剂

Abstract:

Dacarbazine (DTIC) is a first-line chemotherapy drug that is widely used in clinical practice for malignant melanoma. DTIC is metabolized by the liver in vivo . Some drugs are excreted in urine in the form of a prototype. Hence, DTIC in urine can be monitored to evaluate its utilization and conversion rate in the human body, and then to determine its therapeutic effect. Urine is the only body fluid that can be obtained in large quantities without damage, and it plays an important role in the analysis of body functions. However, the composition of urine is complex and there is large matrix interference, because of which trace analysis or trace component analysis is difficult. At present, the main analytical methods for DTIC are high performance liquid chromatography (HPLC) with/without mass spectrometry (MS). HPLC and HPLC-MS have the advantages of good separation effect, good selectivity, high detection sensitivity, automatic operation, and wide application range. Unfortunately, DTIC is a strongly polar and weakly basic compound; thus, it is difficult to achieve good separation and obtain good peak shapes by conventional reversed-phase chromatography. To overcome these defects, it is necessary to develop a novel method for the analysis of DTIC. In this study, mice were subjected to 12 h of fasting; then, blueberry anthocyanin was administered by gavage, and DTIC was administered by intraperitoneal injection. Then, morning urine was collected in a metabolic cage. Urine collection was continued every 4 days for a total of 5 times. Within 2 h, the collected urine was centrifuged (3000 g, 4℃) for 10 min to remove solids. The supernatant was stored in a refrigerator at-80℃. Before analysis, the urine samples were removed from the refrigerator and thawed naturally at room temperature. Then, the samples were treated by the acetone-sediment method, freeze-dried, dissolved in the mobile phase, and subjected to HPLC analysis with isocratic elution. The separation was performed on a Shimadzu-GL ODS column (250 mm×4.6 mm, 5 μm). The mobile phase was methanol/acetonitrile (1:1, v/v)-0.01 mol/L NaH2 PO4 (pH 6.5; 20:80, v/v) at a flow rate of 1 mL/min. The detection wavelength, column temperature, and running time were 280 nm, 30℃, and 15 min, respectively. Under the optimized conditions, the retention time of DTIC was 5.3 min, and a good peak shape was obtained. The linearity ranged from 0.25 to 1000 μg/mL (r 2 =0.999). The limits of detection and quantification were calculated to be 0.12 μg/mL and 0.25 μg/mL based on signal-to-noise ratios of 3 and 10, respectively. At three spiked levels (50.0, 375, and 500 μg/mL), the average recoveries were 98.9%, 102%, and 99.1% with relative standard deviations (RSDs) of 3.2%, 1.3%, and 1.2% (n =5), respectively. The RSDs of the interday and intraday measurements were lower than 3.8% and 4.4%, respectively. The proposed method allowed for the accurate determination of DTIC in urine using a mixed organic solvent/phosphate buffer solution as the mobile phase, with equivalent elution for 15 min. This method was successfully applied to monitor the change in DTIC concentration in the urine of C57BL/6 mice in various stages of melanoma. The results demonstrate that the method is simple, reliable, and easy to apply.

Key words: high performance liquid chromatography (HPLC), dacarbazine, urine, melanoma, blueberry anthocyanin, nutritional adjuvant