Page 13 - PROPOSAL LATIP Zanamivir_Neat 2

days (interday) will be done by repeating each of 6 times. The

relative standard deviation (RSD) values are between 1 and 2%
(Rohman, 2016).

3.8.4 Preparation of plasma samples

To 1ml of plasma in a glass-stoppered 15-ml centrifuge tube (tube

I), the following will be added: 40µl of 0.1M ascorbic acid; 0.1M
ethylenediaminetetraacetic acid disodium salt. To this mixture, 40µl of

internal standard solution (40µg/ml of sulphadimidine), 40µl of 0.1M
sodium hydroxide and 40µl 2,4`-dibromoacetophenone (pBPB) (1 mg/ml)

in methanol were added. The tube will be vortex mixed for 30s and will be

allowed to stand at room temperature for 30 min. A total of 0.4 ml of 1M
hydrochloric acid and 5ml ether were added. After vortexing for 2 min,

and after 15 min centrifugation at 2700×g, the organic phase will be
transfer into a new borosilicate glass tube (tube II) and will be evaporated

until completely dry under a nitrogen stream. In the aqueous phase (tube
I), 5ml dichloromethane will be added for the second extraction. After

vortexing for 2 min, and after 15 min centrifugation at 2700×g, the organic

phase will be transferred into tube II and evaporated until completely dry
under a nitrogen stream. The residue will be dissolved in 50µl acetonitrile

and 20µl of solution was injected into the liquid chromatograph.

3.9 In vivo oral absorption study

Male Sprague Dawley rats (weighing from 230 - 250 g) will be
obtain from Laboratory Animal Facility and Management (LAFAM),

UiTM Puncak Alam, and will be kept at 25℃ with unlimited supply of
food pellet and water when necessary. They will then be anaesthetized

with anesthetic solution. All animal experiments will be performed

according to guidelines of the Committee on Animal Research & Ethics
(CARE) of Faculty of Pharmacy, Universiti Teknologi MARA (UiTM)

(UiTM Care:43/2014). The animals will be fasted overnight, 12 h prior to
the start of experiment with water and will be prepared according to the

method previously reported (Mohd & K. A. Hamid., 2019). The rats will