Establishment And Clinical Application Of Paraquat Detection Methods In Biological Samples

Part one Ultra performance liquid chromatography coupled high resolution mass spectrophy method for the quantification of paraquat in plasma and urineObjective:In order to precisely measure paraquat concentration within internal systems after paraquat intoxication,and to provide guidelines for related clinical treatment,a method using ultra performance liquid chromatography coupled to electrospray tan-dem high resolution mass spectrophy(UPLC-ESI-HRMS/MS)has been optimized to measure paraquat concentration within plasma and urine.Method:This method can be simply operated with high sensitivity and specificity.Sample was prepared by one-step protein precipitation with acetonitrile in which the sample was loaded onto(HILC)hydrophilic interaction liquid chromatography and paraquat was then separated.After paraquat separation,Targeted-MS/MS scan mode was used to complete sample measurement,the whole method took about 10 min.Result:As to improve measuring sensitivity within this method,methodological parameters have been modified and optimized within this method.Those parameters included ammonium formate concentration,formic acid concentration,spray voltage,capillary temperature,and heater temperature and normalized collision energy.After methodological optimization,LOD was 0.1 and 0.3 ng/ml and LOQ was 0.3 and 0.8 ng/ml for plasma and urine.Better linearity was demonstrated within measurable range inside this method.The intra-and inter-day precisions for urine is lower than 4.78%,intra-and inter-day precisions for plasma is lower than 7.97%.The accuracy range for this method is 93.51-100.90%.Conclusion:This studies using ultra performance liquid chromatography coupled to electrospray tan-dem high resolution mass spectrophy(UPLC-ESI-HRMS/MS)has been validated to be a simple,sensitive and specific method for measuring paraquat concentration on human and porcine models,which could provide testing method guidelines for following clinical studies.Part two Predict the prognosis of paraquat intoxicated patients by severity index of paraquat poisoning and paraquat concentrationParaquat is classified as virulent chemicals which is fetal to human body.Paraquat intoxication leads to multiple organ dysfunction syndrome.Even though Medical technologies has demonstrated dramatic progression,and increased survival rate within paraquat toxication,the death rate still remains up to 50%which is an unsatisfying results for clinical survivability.The economic burdens that many families carry for treating intoxicated patient were heavy,but the recovery rate for those patients is disappointed.Purpose:using an ultra performance liquid chromatography coupled to electrospray tan-dem high resolution mass spectrophy(UPLC-ESI-HRMS/MS)method designed by previous research group to measure paraquat concentration within plasma samples to predict the prognosis of paraquat intoxicated patients,to evaluate the severity of paraquat intoxication,in which to provide guidelines for clinical treatment in order to reduce economic burdens for paraquat intoxicated patients'families.Methods:35 paraquat intoxicated patient hospitalized in the first affiliated hospital of Nanjing Medical University from August 2015 to December 2016 has been selected for clinical samples in this study,these samples have been tested for routine blood parameters,hepatic and renal function,blood coagulation,blood gas analysis,etc.They were then been treated with gastric lavage,hemoperfusion,water electrolyte balance maintenance,fluid replacement therapy,etc.Ultra performance liquid chromatography coupled to electrospray tan-dem high resolution mass spectrophy(UPLC-ESI-HRMS/MS)method was then used to measure paraquat concentration within plasma,and to evaluate the severity of patients' paraquat intoxication in which to predict the prognosis of intoxicated patients.Using serum paraquat concentration as a standard to divide these samples into 3 concentration groups,and then to analyze data differences between each concentration group in order to provide guidelines for paraquat intoxication grouping.Results:Paraquat intoxication death rate predicated by area under ROC curve is 0.957,P<0.0001.SIPP predicated by area under ROC curve is 0.928,P<0.0001.Within 3 concentration groups,values range from high,medium to low,the differences between numeration of leukocyte,serum potassium,serum sodium and myoglobin are statistically significant.Conclusion:Serum paraquat concentration and CTSI of paraquat intoxication can be used to predict the prognosis of paraquat intoxicated patients,and serum paraquant concentration can provide guidelines for intoxication clinical classification and clinical treatment.Part three sodium dithionite ultraviolet spectrophotometric method for the quantification of paraquat in gastric juiceParaquat has a widespread usage in developing countries which lead to many paraquat intoxication incidents within economically weak countries and regions.The most common intoxication pathway is through mouth ingestion,and gastric lavage becomes the most important methods for treating paraqaut intoxication patients,but the evaluation over the effect of gastric lavage remains insufficient for clinical studies.Even though paraquat concentration of gastric juice is a crucial parameter for evaluating the effect of gastric lavage,but many regions with epidemic paraquat intoxication failed to provide complicated and expensive methods for evaluating gastric paraquat concentration,and the cost for evaluation methods is as well unaffordable for patients within those regions.Objective:to establish a simple,easy material available,economic,quick and worthy for future promotion method using sodium dithionite ultraviolet spectrophotometry to monitor paraquat concentration inside gastric juice within porcine models.Method:The gastric juice samples stored at-80?C were thawed at room temperature,after fully vortex,samples were centrifuged at 3000 rpm for 10 min.900ul supernatant was pipetted to in cuvette for scanning;100ul measuring solutions were then added and were slightly mixed;optimize measuring wavelength has been chosen for sample scanning;The differences of optical density before and after adding the measuring solution have been calculated for each sample.Result:The calibration curve was linear over the concentration range of 1-500 ug/mL(1,2,5,10,20,50,100,200,500ug/ml).The intra-day precisions is 4.70-6.23%,and inter-day precisions is 4.82-6.67%.The accuracy range for this method is 93.51-100.90%.Conclusion:Method established by sodium dithionite ultraviolet spectrophotometric in this study for measuring paraquat concentration of gastric juice within porcine model and method for gastric lavage are sensitive and accurate which can be applied to evaluate paraquat concentration of gastric juice and gastric lavage solution.