Study On Toxicokinetics Of Paraquat In Rat And Clinical Monitoring Of Plasma Concentration

Part 1 Establishment of method for determination of paraquat concentration and study on toxicokinetics of paraquat in ratObjective: To develop a high performance liquid chromatography method for determination of paraquat in rat plasma and study the toxicokinetics of paraquat in rat.Methods: The rat plasma sample were deproteimized with prechlorld acid(8%,V/V)and were determinated by high performance liquid chromatography.Chromatographic column: Diamonsil C18 column(4.6 mm×250 mm,5 ?m);Mobile phase: 0.1 mol·L-1 potassium dihydrogen phosphate buffer solution(involving 80 mmol·L-1 heptanesulfonate sodium,adjusted p H to 3.0 by phosphoric acid)-acetonitrile(80:20,V/V);Flow rate: 1.0 m L·min-1;Column temperature: 25?;Detection wavelength: 258 nm.The specificity,linearity,recovery,precision and stability were investigated.After being administrated intragastric at 35 mg·kg-1 of paraquat,the blood samples were taken at the preset time points,and the concentration of paraquat was determined.The toxicokinetics parameters were fitted by DAS2.1.1 software and calculated.Results: A good linearity was obtained in the concentration rang of 50~5000 ng·m L-1(r=0.9999),the absolut and relative recovery were 96.3%~99.2% and 96.1%~102.3%,respectively.The RSD values of intra-day and inter-day precisions were both less than 15%.The main toxicokinetics parameters: AUC0-24:(16.65±7.22)?g·m L-1·h;AUC0-?:(17.04±7.59)?g·m L-1·h;t1/2:(3.896±1.847)h;CL:(2.396±0.886)L·h-1·kg-1;V:(13.88±10.45)L·kg-1;Cmax:(3.387±1.097)?g·m L-1;Tmax:(0.987±0.574)h.Conclusions: This method was proved to be simple,highly sensitive and well reproducible.It is suitable to determine the concentration of paraquat in rat plasma.The toxicokinetics studies showed that paraquat was absorbed rapidly and cleared slowly.It could accumulate in the body easily.Part 2 Distribution and pathological damage of paraquat in different tissues of ratObjective: To establish a method to determine the concentration of paraquat in rat tissues and observe the pathological changes of tissues after exposure to paraquat.Methods: The rat tissue homogenate samples were pretreated with prechlorld acid(6%,V/V)and were determinated by high performance liquid chromatography.Chromatographic column: Diamonsil C18 column(4.6 mm×250 mm,5 ?m);Mobile phase: 0.1 mol·L-1 potassium dihydrogen phosphate buffer solution(involving 80 mmol·L-1 heptanesulfonate sodium,adjusted p H to 3.0 by phosphoric acid)-acetonitrile(82:18,V/V);Flow rate: 1.0 m L·min-1;Column temperature: 25 ?;Detection wavelength: 258 nm.The specificity,linearity,recovery,precision and stability were investigated.Rats were administrated intragastric at 35 mg·kg-1 of paraquat,and then the heart,liver,brain,lung and kidney tissue blocks were removed after 6 hours.Paraquat concentration was measured and the pathological changes of tissues were observed by H-E and Masson staining.Results: The paraquat concentration distributed in heart,liver,brain,lung and kidney were(313.92±88.68)ng·g-1,(281.46±126.76)ng·g-1,(67.47±30.45)ng·g-1,(1104.27±624.89)ng·g-1 and(1228.08±473.61)ng·g-1,respectively.Histopathological study revealed that the damage of lung and kidney was severe,including inflammatory cell infiltration,alveolar stenosis,epithelial proliferation,congestion in lung and glomerular congestion,mild dilatation of renal tubules,dilatation of renal interstitial vessels,and congestion of renal tubules.In addition,the heart,liver,brain tissue injury is mild with local inflammatory pathological changes.Conclusions: The results showed that the paraquat concentration of kidney and lung was the highest,the heart and liver was lower and the brain was the lowest at 6 hours after oraling paraquat of 35 mg·kg-1.Paraquat caused pathological damage to various tissues and organs,and that could provid reference for the treatment of paraquat poisoning.Part 3 Monitoring the concentration of paraquat in human plasma by high performance liquid chromatographyObjective: To establish a method for the determination of paraquat in human plasma which can be used in clinical practice.Methods: The human plasma samples were processed with solid phase extraction column and were determinated by high performance liquid chromatography.The chromatography determination was conducted on a Diamonsil C18 column(250mm×4.6mm,5 ?m)with the mobile phase consisted of 0.1 mol·L-1 phosphate buffer(containing 80mmol·L-1 sodium heptanesulfonate,p H 3.0)-acetonitrile(80:20,V/V),and the flow rate was 1.0m L·min-1.The detection wavelength was set at 258 nm,and the column temperature was 25?.Results: The plasma concentration of paraquat had good linearity over the range of 20~5000 ng·m L-1(r=0.9999).The extraction recovery ratio was 96.0%~98.0% and the RSD values of intra-day and inter-day precision were less than 5%.The method was applied to clinical determination of paraquat and the calculation of SIPP values,which was helpful in the clinical treatment of patients with paraquat poison and to predict the prognosis.Conclusions: This methed has a good performance in terms of precesion,sensitive and accuracy,it can be used to determinate paraquat in plasma and predict the prognosis of poisoning patients.