Abstract：
This study established a method to prepare and detect OPs adducts on butyrylcholinesterase (BChE) and human serum albumin (HSA). OPs (methyl paraoxon, ethyl paraoxon, methyl parathion, parathion) were incubated with BChE or HSA in vitro, and the adducts of OPs-BChE or OPs-HSA were prepared and qualitatively analyzed by ultra-performance liquid chromatography data-dependent high-resolution tandem mass spectrometry (UPLC-ddHRMS/MS). The amounts of BChE and HSA in the incubating systems were varied and the resulting amounts of the adducts were determined using linear regression. OPs-BChE in the blood were isolated by immunomagnetic separation (IMS), and then digested into the OPs-nonapeptide adduct by pepsin. The proteins in the remaining blood plasma were precipitated and digested by pronase to OPs-tyrosines(OPs-Tyr), which were quantified by UPLC-ddHRMS/MS. 4 OPs-nonapeptides and 4 OPs-Tyr adducts were obtained through the process above. The relative mass deviation of incubated adducts between the actual and theoretical exact masses was less than 10 ppm, and further confirmed by fragmentation mass spectra analysis. Calibration curves were linear for all adducts with a coefficient of determination value (R2) ≥0.995. The limits of detection (LOD) and limits of quantification (LOQ) for adducts detected by MS ranged from 0.05 to 1.0 ng/mL, and from 0.1 to 2.0 ng/mL, respectively. The recovery percentages for adducts ranged from 76.1 % to 107.1 %, matrix effects ranged from 83.4 % to 102.1 %. The inter-day and intra-day precision were 6.1-10.1 % and 6.9-12.9 % for adducts. This study provides a new reference method for the detection of organophosphorus pesticide poisoning. In addition, two blood samples with organophosphorus poisoning were tested by the designed method, and the corresponding adducts were detected in both samples.
摘要：
本研究建立了丁酰胆碱酯酶（BChE）和人血清白蛋白（HSA）OPs加合物的制备和检测方法。OPs（甲基对氧磷，乙基对氧磷,甲基对硫磷,对硫磷）与BChE或HSA体外孵育，制备OPs-BChE或OPs-HSA的加合物，并通过超高效液相色谱数据依赖性高分辨率串联质谱（UPLC-ddHRMS/MS）进行定性分析。改变孵育系统中BChE和HSA的量,并使用线性回归确定所得加合物的量。通过免疫磁性分离（IMS）分离血液中的OPs-BChE，然后被胃蛋白酶消化成OPs-九肽加合物。剩余血浆中的蛋白质被链霉蛋白酶沉淀并消化成OPs-酪氨酸（OPs-Tyr），通过UPLC-ddHRMS/MS定量通过上述方法获得4种OPs-九肽和4种OPs-Tyr加合物。实际和理论精确质量之间的孵育加合物的相对质量偏差小于10ppm，并通过碎片质谱分析进一步证实。所有加合物的校准曲线均为线性，测定系数值（R2）≥0.995。MS检测加合物的检测限（LOD）和定量限（LOQ）范围为0.05至1.0ng/mL，从0.1到2.0ng/mL，分别。加合物的回收率为76.1%至107.1%，基质效应从83.4%到102.1%不等。加合物的日间和日内精度分别为6.1-10.1％和6.9-12.9％。本研究为有机磷农药中毒的检测提供了一种新的参考方法。此外,用设计的方法检测了两个有机磷中毒的血液样本，并在两个样品中检测到相应的加合物。
