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

运用高效液相色谱-电喷雾离子阱串联质谱(HPLC-ESI-MS/MS)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300 pg/mL,定量限(S/N≥20)为850 pg/mL。在300 pg/mL～1.28 μg/mL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0.9929)。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45 mg/kg,染毒4 d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) ng N7-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。

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

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.