Analysis of 8-hydroxy-2'-deoxyguanosine in human urine by ultra-high performance liquid chromatography mass spectrometer method
10.3760/cma.j.issn.0253-9624.2016.04.014
- VernacularTitle:人尿液中8-羟基脱氧鸟苷的超高效液相色谱-串联质谱联用检测方法
- Author:
Zhong LIU
1
;
Zhigang YU
;
Yanhua WANG
;
Ying CI
;
Zhan SUN
;
Haitao JIAO
;
Huawei DUAN
Author Information
1. 250021济南市疾病预防控制中心理化检验所
- Keywords:
Deoxyguanosine;
Urine;
Chromatography,supercritical fluid;
Oxidative damage;
Biomarker
- From:
Chinese Journal of Preventive Medicine
2016;50(4):357-361
- CountryChina
- Language:Chinese
-
Abstract:
Objective To develop an ultra?high performance liquid chromatography mass spectrometer method for the rapid determination of 8?hydroxy?2'?deoxyguanosine (8?OHdG) in human urines. Methods 8?OHdG standard solution with the concentration from 0.01 to 0.1μg/ml was formulated. The solution was implanted into ion source with a rate of 7μl/min, the mass?to?charge ratio of parent ion and product ions, and ion mass to charge ratio was identified. The mass spectrum parameters of each Ion pairs, such as DP, EP and EXP, were gradually optimized. The urine sample with a concentration of 10.0μg/L was detected, and the pH of the sample was adjusted using 1 mol/L ammonium formate and formic acid solution with a volume ratio of 5∶1, 4∶1, 3∶1, 2∶1, and 1∶1. It was tested using three different polarity of SPE, i.e.:HLB, MCX, and MAX. The elution effect of methanol and water mixture with the proportion of 90∶10, 80∶20, 50:50, 20:80, and 10:90 were tested, and then acetonitrile and water mixture with the proportion of 90∶10, 80∶20, 50∶50, 20∶80, 10∶90 were also tested. The standard curve was constructed using the ratio of a standard series application fluid concentration to corresponding compounds quantitative ion liquid concentration of the peak area. The detection limit was determined as 3 times of the signal to noise ratio corresponding to the concentration of 8?OHdG, and the quantitative lower limit was determined as 10 times of the signal to noise ratio corresponding to the concentration of 8?OHdG. The blank urine spiked recovery method was used to evaluate the precision and recovery rate. Results The mass to charge ratio of parent ion was 284.1 and the product ions was 168.1, 140.1, 123.0, and 112.0, respectively. The collision voltage of quantitative ion?pair 284.1/168.1 was 18 V, the 284.1/140.1 collision voltage was 42 V, the 284.1/123.0 collision voltage was 48 V, and the 284.1/112.0 collision voltage was 53 V. The recovery rate was the highest (87.9%-104.3%) when the pH of urine was adjusted by a 10 ml 1 mol/L ammonium formate solution, 2 ml of formic acid, 88 ml of water are mixed with the sample solution volume ratio of 1∶5, and then purified with 3 ml of methanol and 3 ml water activated HLB extraction column. Within 1.0-100.0 μg/L concentration range, 8?OHdG standard application solution test results showed a good linear relationship. The regression equation was y=1.25x+0.74, and the correlation coefficient was r=0.999 5. The detection limit was 0.2μg/L, and the limit of quantification was 0.7μg/L. The method of recovery rate was in the range of 87.9%to 104.3%, the precision was in the range from 1.5% to 3.7% and inter?assay precision was in the range from 1.6% to 5.4%. Conclusion The method developed in this study had high sensitivity, good precision and accuracy, and a wide range of testing concentrations.
