Animals and in vivo experimental design
The study used eighteen eight-week-old male Wister rats weighing 200 g. Before beginning the study, rats were chosen at random, had their tails identified, and were placed in separate cages/groups. A week before the study began, all rats were acclimatized, fed regular food, and water was ad libitum.
Rats were divided into three groups. Group 1 was administered 0.1 mL of LEV intravenously; group 2 was administered an oral dose of 11.5 mg of LEV in 5 mL of water, and group 3 was kept only drinking for seven days' date syrup (Dates Molasses; 250 g mixed with 250 mL water) and then on day seven an oral dose of 11.5 mg of LEV in 5 mL of water was administered.
Chemicals: LEV was supplied from Hikma, Jordan, with a potency of 99.8%, whereas the following reagents and chemicals were obtained from Merck (Darmstadt, Germany): orthophosphoric acid (AR grade 85%), acetonitrile (HPLC grade), and Milli-Q purified water.
High-performance liquid chromatography (HPLC)
The HPLC system used was Thermo Finnigan Surveyor UV-VIS with a detector, LC Pump (SRVYR-LPUMP), and autosampler (SRVYR-AS). The column used was ACE C18-AR (250mm*4.6mm) 5µm, an average particle size.
The mobile phase, internal standard, and the standard solutions
The buffer solution was prepared by adding 3.4g KH2PO4 to 1000 mL of HPLC- grade water, and pH adjusted to 5.2 with orthophosphoric acid. The mobile phase was prepared by mixing 830 mL of the buffer solution with 170 mL of acetonitrile and orthophosphoric acid. The mixture was then filtered using a 0.45 µm membrane filter and degassed by sonication. The internal standard (IS) was prepared by weighing 3 mg of pure Metformin hydrochloride and dissolving it in 100 mL of freshly prepared acetonitrile. Then 1 mL of it was diluted with 200 mL of acetonitrile.
Preparation of stock solutions and working solutions
Stock solutions of LEV were prepared by weighing and transferring 20 mg of each of the active ingredients into a 100 mL volumetric flask and diluted up to the mark with ACN.
Preparation of working standard solution:
Step Dilution 1:2.5 mL of LEV solution was pipetted into a 20 mL volumetric flask.
Concentration, then diluted to Seven concentrations 10, 25, 50, 75, 100, 500, 1000 ng.
For the sample solution for LEV preparation, a volume of plasma was pipetted in Eppendorf and diluted with IS, then vortex for 1 minute, and then centrifuged for 15 minutes. The supernatant was injected into the HPLC.
UV-VIS scan applied for the solution of LEV was within a range of 200–400 nm. Maximum absorbance of 205 nm was obtained.
The method of development of the study was to best chromatographic conditions for assaying LEV using metformin as an IS, such as having a short retention time with good resolution and symmetric peaks. Different factors such as pH, ion pair, the composition of mobile phase, and column were evaluated.
After three trials for chromatographic conditions, in which two were rejected, we found the best resolution at pH5.2, wavelength: 210nm, for 10 µl injection volume, flow rate 1mL / min, and oven temperature 25°C.
Samples were drawn at different times: initial ,0.33,0.66,1,2,4,8,24,36,72and 96 Hours.
The maximum plasma LEV concentration (Cmax) and time to reach the maximum concentration (Tmax) were calculated by averaging the highest plasma concentration and its corresponding time of LEV in each rat. Besides, the area under the curve under the plasma concentration-time profile from time zero to time t (AUCt), zero to infinity (AUC0⟶∞), and elimination half-life (t1/2) were calculated based on non-compartmental pharmacokinetic calculations.
Furthermore, AUC0⟶∞ was calculated by adding AUCt to the value of dividing the last measurable concentration at time T over the elimination rate constant. The t1/2 was calculated from the slope of the semi-logarithmic of the last plasma concentration points vs. time.
The PK parameters; Cmax, Tmax, AUCt (AUC0⟶∞), and t1/2 were presented as mean (± SD). One-way Analysis of Variance was used to calculate the significant differences of each parameter between the groups, followed by Tukey as a post hoc test to find out the differences between each group.