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目的建立适用于代谢组学研究的人体尿液保存方法及其代谢物气相色谱-质谱(GC-MS)联用分析方法。方法向尿样中分别加入1%HNO3、1%HCl、2%抗坏血酸作为保存剂,并设空白对照,置于-80℃保存0、5、39 d后连续3次反复冻融,每次取样100μl,加入20 g/L尿素酶50μl,于37℃酶解2 h,加入去氧保护剂甲氧基胺盐处理后,再加入衍生剂(MSTFA)于37℃反应过夜,取上清液进行GC-MS测定,并以马尿酸为指标测定方法学参数。结果以原尿在-80℃保存,在39 d内测得所选8个不同保留时间色谱峰面积的RSD均小于10%。以马尿酸为指标,在0.005~1.75 g/L的线性范围内,所得线性方程为y=0.127 4x+0.473 8,r=0.999 2;该方法的检出限为1.26 mg/L,加标回收率为75%~125%,RSD为1.1%~8.5%。对22名膳食镉暴露人群与12名对照人群尿样进行检测发现,两组人群尿液中代谢产物的种类和含量均有较大变化。结论该方法具有尿样稳定性良好,尿液分析方法学参数优良的优点,适用于膳食重金属暴露人群尿样中内源性代谢物的检测。
OBJECTIVE To establish a human urine preservation method suitable for metabolomics research and its metabolite GC-MS method. Methods 1% HNO3, 1% HCl and 2% ascorbic acid were added to the urine samples as preservatives, respectively, and blank control was set. The samples were stored at -80 ℃ for 0, 5 and 39 days, 50μl of 20g / L urease, enzymolysised at 37 ℃ for 2h, treated with deoxygenating agent methoxyamine salt, then added derivative reagent (MSTFA) and reacted at 37 ℃ overnight, the supernatant was taken GC-MS determination, and hippuric acid as an index to determine the methodological parameters. The results of the original urine stored at -80 ℃, measured within 39 d selected eight different retention time peak area RSD were less than 10%. The linear equation was y = 0.127 4x + 0.473 8 and r = 0.999 2 in the linear range of 0.005 ~ 1.75 g / L, with the detection limit of 1.26 mg / L, Rates ranged from 75% to 125% with RSDs ranging from 1.1% to 8.5%. Twenty-two dietary cadmium exposure groups and 12 control population urine samples were tested and found that the two groups of urine metabolites in the species and content have greater changes. Conclusion The method has the advantages of good stability of urine samples and good analytical method parameters in urine analysis. It is suitable for the detection of endogenous metabolites in urine samples of dietary heavy metal exposed people.