高效液相色谱-电喷雾离子阱串联质谱法检测芥子气经皮肤染毒SD大鼠肺中的DNA加合物

Biomonitoring method of dermal exposure to sulfur mustard based on DNA adduct in lung of SD rats by high performance liquid chromatography-electrospray ionization tandem mass spectrometry

运用高效液相色谱-电喷雾离子阱串联质谱(HPLC-E S I-M S/M S)技术,建立了快速、简单、灵敏的SD大鼠肺中N7-(2-羟乙基硫代乙基)鸟嘌呤(N7-HETEG)的检测方法。以N7-苯甲基鸟嘌呤为内标,用甲醇和水为流动相进行梯度洗脱,正离子模式检测,方法的检出限(信噪比(S/N)≥10)为300pg/m L,定量限(S/N≥20)为850pg/m L。在300pg/m L^1.28μg/m L的质量浓度范围内,N7-HETEG浓度与N7-HETEG和内标的峰面积比呈良好的线性关系(线性相关系数为0.992 9)。高、中、低3个添加水平的日内测定精密度(以相对标准偏差(RSD)计)和日间测定精密度均小于10%(n=7),回收率为100%~132%。对SD大鼠背部皮肤染芥子气,剂量分别为5.5、11、22和45m g/kg,染毒4d后检测大鼠肺脏中N7-HETEG的含量。各个不同染毒剂量下,每克组织中分别检测到(0.56±0.16)、(0.67±0.12)、(1.36±0.68)和(5.14±0.92)ngN7-HETEG,N7-HETEG的含量随着染毒剂量的增大而增大,表明N7-HETEG可用作芥子气暴露的体内生物标志物。

The analysis of biological samples can provide qualitative and quantitative evidence of exposure to sulfur mustard（SM）.N7-（2-hydroxyethylthioethyl）guanine（N7-HETEG） is the most abundant DNA adduct and one of the biomarkers of SM exposure.For the detection and validation of SM exposure,a sensitive high performance liquid chromatography-tandem mass spectrometry（HPLC-ESI-MS/MS） method for the determination of N7-HETEG in a biomedical sample was developed,using N7-benzylguanine as the internal standard.The method was validated and showed the limit of detection of 300 pg/mL（S/N≥10） and limit of quantitation of 850 pg/mL（S/N≥20）.The linear range was 300 pg/mL-1.28 μg/mL,with both the values of intraday and interday relative standard deviations of less than 10%（n=7） over the calibration range.The recoveries varied from 100% to 132%.The method was applied for the determination of N7-HETEG in the lung of SD rats caused by dermal exposure to SM in vivo.Under the exposure to SM of 5.5,11,22 and 45 mg/kg,respectively,（0.56±0.16）,（0.67±0.12）,（1.36±0.68） and（5.14±0.92） ng of N7-HETEG per gram of SD rat lung tissue were found after four days of exposure.The amount of N7-HETEG increased with the exposed doses,and it can act as a biomarker of sulfur mustard exposure.