Analytical independent prepared calibrated curves. The squares

Analytical Instrumentations:
HPLC-UV method has been performed to develop & analyze by waters HPLC
systems (Milford USA), which consist of the binary
pump (515 model), an automatic sampler
with temperature control facility (717 model), and an ultraviolet detector (UV). The wavelength of the UV is set around
267 nm. Chromatographic separation has
been achieved on the thermal accuacore hillic
column (150 × 4.6 mm, 5?m), and the mobile phase consisting the mixture of
acetonitrile and distilled water (60:40, v/v), with pH 4.7 at a flow rate of
0.1mL/min. The injection volume of injection was 30 ?L with a total run time
6.5 min. The HPLC data was collected and analyzed using breeze-version 3.1
software (Milano and Chamorey, 2002).

Preparation of standard stock solution,
calibration standard & quality control (QC) samples:

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The
standard stock solutions of 5-FUNPs & IS (internal standard solution) have
been prepared by accurately weighing (500 ?g/mL both 5-FUNP & IS
separately) and allowed to dissolve in methanol. The stock solution was taken
serial dilution in methanol to prepare the working solution, for 5-FUNPs, 0.12,
0.24, 0.4, 0.8, 1.6, 4, 8, 12, 16, 32, 64, 96, 128 ?g/mL & IS working
solution was prepared by diluting stock solution in concentration of 16 ?g/mL.
Calibration standard stock solutions for 5-FUNPs & IS were prepared by
adding the both prepared working solution (30 ?L) in to 400?L blank rat plasma
to achieve the final concentration for 5-FUNPs (0.01, 0.025, 0.05, .01, 0.25,
0.5, 1.0, 2.5, 4, 8 ?g/mL) & IS (1 ?g/mL). The samples for Quality control
were also prepared as calibration standard solution in low, medium, high
concentration as 0.025, 0.1 & 8 ?g/mL.

HPLC method validation: The
evaluation of HPLC-UV method was assessed by specificity, linearity,
sensitivity, precision as accuracy, recovery and stability (Rothenberg et al.,
2002). For the estimation of specificity, blank plasma samples were taken from
multiple sources for confirmation of nil interference in the retention time of
the 5-FUNPs & IS. The linearity of
the calibration curves has been assessed
(0.01 to 8?g/mL) by using five sets of independent prepared calibrated curves.
The squares linear regression parameters were calculated using weighted (1/x2)
least. Correlation coefficients (r2) have 0.99 or better within the calibration
curve. The lower limit of quantification (LLOQ) was
showing the ratio of sensitivity and able
to resolute where both precision and accuracy were not more than 20%. The
replicates of different samples of QC have been used to scrutinize the intra-inter precision and accuracy within QC
samples range from low, medium and high
concentration, 0.025, 0.5, and 4 ?g/mL respectively.

Plasma sample collection: The
animal ethical committee of the King George’s Medical University (KGMU),
Lucknow, India, allowed this animal study, the Sprague Dawley rats were housed in the institutional
animal house (room temperature, 20°C ±5°C &
relative humidity, 55 ± 15%) before experiments. The samples were collected by
sparse sampling technique with three blood samples from each rat. Animals were
fasted and procure 12 hrs before the experiment, aqueous suspension of 5-FUNPs
at suitable doses were administered orally 250 ug/mL, and the pre-dose plasma
sample was collected at regular time
interval 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, 5, 6,
8, 10, 12, 24, 48 hrs through retro-orbital
plexus afterword the samples were centrifuged at 2800 rpm for 15 min and plasma
was stored in freezer for further to be
analyzed.

Plasma sample preparation for HPLC-UV
loading: 5-FUNPs was extracted by dissolving plasma sample 400 uL,
25 uL IS solution and 2 ml extraction solvent and this mixture were vortexed for 2 min followed by centrifuging at
2800 rpm for around 15 min and organic layer taken in a glass tube. Liquid-liquid extraction processes have been performed
twice with ethyl acetate & upper organic layer was evaporated under the mild flow of N2. The loading sample was
prepared by addition of residue with 100 uL methanol and injected 20 uL into
HPLC for the collection of analytical
& bio-analytical process data.

Pharmacokinetics studies & statistic
analysis: The graph between Mean plasma drug concentrations in
specific time intervals has been evaluated through non-compartment models.
WinNonlin version 5.1 (Leichman et al.,
2002) were used for analysis of pharmacokinetics parameters included Cmax,
Tmax, area under the curve (AUC), half-life (t1/2), and clearance
(CL).

Acute toxicity study: The
acute toxicity study for formulated 5-FUNPs was evaluated in rats according to
OECD guidelines (Caldwell and Yan, 2006), through up & down method. The animals were
divided in two group (n=3) three in each groups and single dose of 100mg/kg
administered orally through gastric intubation. The animals were observed for
72 hrs for toxicological changes on the basis of like behavior, physical,
mortality, hematological & biochemical parameters. 

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