Toxicokinetics Of Glufosinate-Ammonium In Rats

Glufosinate-ammonium [ammonium dl-homoalanin-4-(methyl) phosphinate] is abroad spectrum and nonselective herbicide developed by AgrEvo GmbH Corporation in1970s. The systemic action is not strong and glufosinate-ammonium conducts through planttranspiration in the xylem. The mainly reason for weeding is due to inhibition of glutaminesynthesis (GS) and gives excellent performance following glyphosate in weed. But canweed, can also serve as transfer screening agent for gene technology research. The resistantgene of bar gene screened out has been successfully transformed into many crops, whichenlarges the fields of using glufosinate. Research on glufosinate-ammonium basicallyfocused on the environmental behavior and application. However, toxicokinetics andpharmacology of glufosinate-ammonium in animals has been reported rarely at home andabroad in the current. So it is very important to clarify its toxicokinetics in the animal. Anew HPLC method for determination of glufosinate-ammonium in biological samples wasdeveloped and successfully used in the toxicokinetics kinetics study. The findings of thisinvestigation will provide the foundation for further study of toxicity and mechanism.Part Ⅰ The Development of a HPLC Method for Determination ofGlufosinate-ammonium in Biological SamplesObjective: To develop the method of high performance liquid chromatography (HPLC) todetermine glufosinate-ammonium in biological samples and assess this method.Methods: Combination of the glufosinate-ammonium chemical properties and derivativeagent conditions, the concentration of glufosinate-ammonium in biological samples wasdetermined by using changing the proportion of the derivative agent and buffering agent, different detection wavelength, detection wavelength, column temtemperature and flow rate.The recovery rate, the precision degrees within and among the day, the stability at normaltemperature after12hours and at-20℃after10days freezer were estimated.Results: Established the method of per-column derivatization high performance liquid-ultraviolet detection (HPLC-UVD) to determine glufosinate-ammonium in biologicalsamples. Boric acid buffer solution was used as the extract and9-chloroformate fluorenemethyl ester (9-FMOC-CL) as a derivative agent. The condition for the HPLC method todetermine glufosinate-ammonium after the optimized process were as follows: AgilentEclipse XDB-C18column250mm×4.6mm (i.d), grain diameter:5μm, the mobile phase:Acetonitrile-0.02mol/L ammonium acetate (0.1%formic acid) gradient elution by flowrate of1ml/min, detection was done at263nm, column temperature was40℃, and10μlsample was used. The retention time a（t4.8±0.1）min; the correlation coefficient is0.9996,the lowest detection limit is0.9μg/ml, the recovery rate was89.00%、87.66%and86.10%.The precision degrees within the day and among the day were fewer than4.9%and6.05%,respectively. The stability RSD was lower than3.35%at normal temperature after12hours,lower than04.28%at-20℃freezer after10days and lower than7.46%after3times offreezing and thawing.Conclusion: Using per-column derivatization HPLC-UVD to determineglufosinate-ammonium in biological samples. This method has the derivatives stability,higher sensitivity and better specificity characteristics; the approach is consistent with therequirements of the method argument, suitable for analyzing the biological sample ofglufosinate-ammonium. Part Ⅱ Investigation of Glufosinate-ammonium ToxicokineticsCharacteristics in RatsObjective: To observe the concentration change of glufosinate-ammonium with reactiontime of the serum and toxicokinetics characteristics in rats.Methods: The intravenous injection with glufosinate-ammonium was used to10adultnormal rats which divided into A and B groups. The dose to A group rat was80mg/kg andB group rat was16mg/kg. Blooding after intravenous injection in0，0.083,0.25,0.5,1,2,4,8,24and48hour. These serum deals with acetonitrile, boric acid buffer solution,derivatization reagent and ethyl acetate. And then was detected by HPLC. DAS2.1wasused to analysis the toxicokinetics parameters.Results:80mg/kg bw and16mg/kg bw glufosinate-ammonium by a single tail veininjection, the serum drug-time curve was consistent with two-compartment compartmentmodel. The rats kinetic equation was the t1/2βwas4.78±2.09and4.50±1.34h, the AUC0-twas313.0083.30and58.8443.59(μg·h/mL), the AUC0-was339.4697.45and66.60±43.65mg/L·h, CL was0.25±0.07and0.34±0.21L/h·kg, and Vd was0.53±0.16and0.71±0.38L/kg respectively.Conclusion: After intravenous injection, the diffuse of glufosinate-ammonium was rapid,both consistent with two-compartment compartment model. Both groups AUC with doseincreases and a positive correlation (R2=0.9993), while t1/2β is not obvious.Part Ⅲ Investigation of Glufosinate-ammonium Tissue Distributionand Excretion in RatsObjective: Investigation the concentration change of glufosinate-ammonium with reactiontime in different tissues, in feces and urine of rats. To determine the target organ and themainly discharge way of glufosinate-ammonium in rats.Methods:1.The intravenous injection to rat with glufosinate-ammonium (80mg/kg bw and16mg/kg bw respectively) was used to66adult rats, which were divided into2groups.Rats were killed by exsanguination at2,4,8,24and168h after iv. administration. Theconcentration of glufosinate-ammonium in different tissues, including heart, liver, spleen, lungs, kidneys, brain, testicular, fat and muscle tissue were detected after intravenousinjection in2,4,8,24and168h by HPLC.2. The intravenous injection to rat with glufosinate-ammonium (80mg/kg bw and16mg/kgbw respectively) was used to12adult rats. Then these rats put into the metabolic cages. Thefeces and urine of rats were collected at the same time during0-4,4-8,8-24,24-48,144-168h. The content of glufosinate-ammonium in these biological samples was detectedby HPLC after special treatment.3. The data were analyzed by Excel.Results:1.The glufosinate-ammonium was detected after intravenous injection in2h inmany tissues. Higher concentration is in the kidney and liver. The concentrations in alltissues were lower after8hours and only be detected in liver, kidney and spleen after24h.After7days, glufosinate-ammonium was not undetectable.2. The content of glufosinate-ammonium in rats’ feces and urine gradually decreased within48hours. Within4hours, glufosinate-ammonium only been detected in the urine. Within48hours, account for59.01%of the total inputs discharged from feces and urine and was notdetected after7days.Conclusion:1.The findings in this Investigation indicated that the distribution ofglufosinate-ammonium in rats was widely and kidney and liver may be the target organ.2. The glufosinate-ammonium in rat was mainly excreted by urine.