The study was focused on the analytic validation and further applied on pharmacokinetic parameter the prepared 5-FUNPs has been examined on the basis of analytic method validation, the HPLC analytic method was developed for 5-FUNPs in rat plasma. The result showed the HPLC-UV produced chromatogram wavelength at 267 nm (Image-4). The various chemical of the internal standard was included in thiadiazole, amiodarone, megestrol acetate (UV absorption at wavelength 262 nm). Megestrol acetate was at an IS compound was set to be for comparison and the analytical data was collected.

UV absorption retention time and extracted recovery, the mobile phase, the mixture of acetonitrile and distilled water (60:40; v/v) with p1 and 4.7 at a flow rate of 0.1 ml/min.

Method development and optimization process for extraction process ethyl acetate were used for recovery of samples. The plasma sample with ethyl acetate a simple liquid-liquid extraction process was used and highest recovery reported. The supported analytical date including resolution, symmetry remembrance extortion recovery through HPLC-UV method has been performed for 5-FUNPs. Method Validation:Specificity: Specificity was analyzed over blank rate plasma by comparing the resultant chromatogram, with rat blank plasma with 5-FUNPs and IS after oral administration the collected rat plasma sample were retention time for 5- FUNPs and IS were 8.5 and 8.8 mn respectively.Calibration curve and sensitivity: Calibration curve of drug sample validate the linearity of the concentration ranged from 0.01 to 8 mg/ml in the plasma sample.

The average regression equation for a y = 0.5530 × + 0.0133 ( r2= 0.9996, n=3) for the calibration curves.

Correlation coefficients for the linear regression ranged from 0.9379-0.9475. The 5-FUNPs for LLOQ 9.

8 mg/ml and LOD was detected 0.98 mg/ml in the plasma sample. The lowest concentration on the calibration curve (0.89 mg/ml) was found on LLOQ.Precision, accuracy and recovery: Precision and accuracy were evaluated for method validation on the basis to assay fine replicate in three concentration (0.025, 0.5, 4 µg/ ml) on the same day (intraday) and on 5 several days (inter-day). The accuracy was examined by – % deviation of the mean from the value and expressed or relative error (RE).

Precision was proliferated or variation value from the relative standard deviation (RSD). The basis of several variation values recorded ranges from 2.891 to 697.1mand 2.03 % to 6.46% for intraday to inter-day respectively.

The accuracy ranges from 1.98 % to 11.78% and 2.18% to 4.98% for intraday and inter-day respectively.

All the recorded values were within the acceptable ranges for the analysis. The extraction recovering within plasma sample for 5-FUNPs at different concentration medium 0.025, 0.5, 4 µg/mL, low, medium & high respectively were recorded from 96.1% to 98.90% in the acceptable ranges for the 5-FUNPs.

StabilityThe stability procurement of 5 FUNPs within rat plasma were determined by using analyzing quality control samples and stored at several storage conditions which is exposure of temperature, light radiation, past and pre proportion storage on 4oc for two days to 7 days, freezing at -20oc for 25 hrs thawing at room temperature and storage up to 3 months. The entire standard quality control samples on the standard stability assessment were analyzed and record the date in triplicate for assurance. The concentration (0.

025, 0.5,4 µg/mL) of quality control sample form rat plasma were also analyzing on the basis of three replicate. The different storage condition for stability has shown that the particle of 5- FUNPs was stable in plasma stored in different storage condition 24 hrs, 48 hrs, 1 week at 4oC followed by 2 weeks, 1 months, 2 months, 3 months at -200c deep freezing condition.Application of the method by the Pharmacokinetic study: Pharmacokinetic parameter of 5- FUNPs were analyzed through the validated analytical method and further this method examined the plasma drug concentration for 5- FUNPs in a single oral dose of 100 mg/kg in rats (n=6). The mean plasma drug concentration-time curve of 5- FUNPs at the different interval after the oral administration was detected in rat plasma.

At 72 and 96 hrs points, gradually lowering of concentration 5-FUNPs in plasma recorded (Image-5). The pharmacokinetic analyze of 5- FUNPs were analyzed by the non-compartment pharmacokinetic model on the parameter of Cmax, Tmax at Co- 96, whereas the recorded concentration of drug within plasma was maximum (Cmax) 3.235±0.78 mg/L at highest time course (Tmax) 22.58 hrs.

Furthermore the t1/2, area under secure for the concentration proved (Table -1).Acute toxicity study: There were no physical and behaviour changes seen in animals. The body weight of treated group animals was decreased as compared to control group animals, shown in table-2. Hematological parameter confirmed that the LD50 was evaluated to be ? 100 mg/kg (table-3). Conclusion: Our study concluded that 5-FU polymeric nanoparticles show an informative pharmacokinetic data to correlates with clinical findings, after the formulation of nanoformulation the HPLC-UV spectroscopic analytical method were developed and validated and further applied over pharmacokinetics study.

The developed polymeric nanoformulation would more helpful as potential drug delivery system to find out the controlled release of the encapsulated drug, by this proposed system can be capable to improve the bioavailability, reduce dose size and toxicity, avoid adverse effects by improving the ADME process within the drug delivery and monitored the toxicological window of 5-FU nanosystem.Acknowledgments: This study was support through Central Government research funding agency, Indian Council of Medical Research (ICMR), New Delhi to Mr Saurabh Srivastava, ICMR-SRF, (Project ID: 45/21/2013-NAN-BMS). We are grateful to ICMR, New Delhi, India for their financial support to carry out this research project.