CHAPTER VIII

MATERIALS AND METHODS FOR

CITALOPRAM HYDROBROMIDE FAST
DISSOLVING ORAL FILMS
8.1. LIST OF MATERIALS AND EQUIPMENT USED
Table 8.1: List of the materials used

S.No Name Manufacturer

Citalopram
1 Hetero drugs Ltd, Hyderabad, India
Hydrobromide
HPMC E 50 Signet Chemicals Corporation Pvt.
2
Ltd. Mumbai
Signet Chemicals Corporation Pvt.
3 Maltodextrin
Ltd. Mumbai
Signet Chemicals Corporation Pvt.
4 Glycerol
Ltd. Mumbai
Lactose Signet Chemicals Corporation Pvt.
5
Ltd. Mumbai
Aspartame Signet Chemicals Corporation Pvt.
6
Ltd. Mumbai
Flavor Signet Chemicals Corporation Pvt.
7
Ltd. Mumbai
Signet Chemicals Corporation Pvt.
8 Water
Ltd. Mumbai

Table 8.2: Equipment used for Formulation and Analysis

S.NO EQUIPMENT MANUFACTURE
Electrolab(TDT-08L),
1 Franz Diffusion Cells
Mumbai.
2 HPLC apparatus Shimadzu, Japan.

3 UV-Visible spectrophotometer Shimadzu, Japan.

4 Differentialscanning calorimeter Shimadzu (DSC -822) (Japan)

5 FT-IR Shimadzu 8400S, Japan,

6 SEM HITACHI, S-3700N, Japan

Mitutoyo (absolute digimatic),
7 Screw Gauge
Mumbai

8 Digital pH meter Electro lab, Mumbai

9 Digital weighing balance Shimadzu, Japan

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8.2. STANDARD GRAPH OF CITALOPRAM HYDROBROMIDE:
8.2.1. The PB prepared as per the procedure mentioned in 5.2.1.
8.2.2. Standard stock solution of drug: Exactly 100 mg of Citalopram
Hydrobromide and dissolve in 100 mL distilled water. This gives 1000 µg/mL
concentration of standard stock solution.
8.2.3. Working stock solution: From the standard stock, 10 mL of was diluted up to
100 mL with DLW to obtain 100 µg/mL.
8.2.4. Working dilutions: From the working standard stock solution, 1- 6 mL were
diluted to 10 mL with distilled water to produce 2 - 12 µg/mL concentrations,
respectively.
8.2.5. Standard calibration curve: All working standards were scanned against
distilled water at 227 nm and graph plotted as per procedure mentioned in 5.2.5.
8.3. DETERMINATION OF DOSE OF CITALOPRAM
HYDROBROMIDE (Dinge et al., 2008; Arun et al., 2010;Gavaskar et al.,
2010)
Amount of drug required per film =20 mg of Citalopram Hydrobromide
Therefore, 4 films require 80 mg of drug
Area of the petridish (πr2) ==3.14*4.5*4.5=63.5cm2
6 films of 4 cm2 each i.e (2cm*2cm) can be obtained freely per petridish
Area not required is the one remaining after cutting the films from the centre of
petridish.
This is obtained as
Area considered= Sum of the areas of number of films taken= 4 cm2*6=24 cm2
Amount of drug in area considered=80 mg
Area not considered= Total area of petridish-Area considered
= 63.5-24= 39.5 cm2
4 cm2 film contains 80 mg of drug therefore 39.5 cm2 contains 40 mg of drug
Amount of drug in area not considered=40 mg
Therefore,
Total drug dose = (Amount of drug in area considered) + (Amount of drug in Area
not considered) = 80+40 =140
Therefore, an approximate amount of 140 mg drug was considered per petridish.

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8.4. FORMULATION AND DEVELOPMENT OF CITALOPRAM
HYDROBROMIDE FDOF
Procedure
Citalopram Hydrobromide FDOFs were prepared by the solvent-casting method.
(Khatoon et al., 2014; Saadya et al., 2016; Sayed et al., 2014)

Figure 8.1: Procedure for preparation of Citalopram Hydrobromide FDOFs

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8.5. RESPONSE SURFACE METHODOLOGY (Sed et al., 2011;
Montgomery and Douglas, 2005)
Study type: Response surface
Design type: central composite
Design mode: quadratic

Figure 8.2: CCD design

Twenty seven formulations (CF1-CF27) were prepared by solvent-casting method
using 33 response surface method where 33 indicates 3 variables and 3 levels of
polymers (HPMC E50, Maltodextrin) of different grades and plasticizer (Glycerol)
(low, middle & high concentrations) by using Design of experiment software.

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 Table 8.3: Citalopram Hydrobromide FDOF Composition of one Film (2X2 cm2)
Citalopram HPMC
Maltodextrin Glycerol Lactose Aspartame Flavor Water
F.NO Hydrobromide E50
(mg) (mg) (mg) (mg) (ml) (ml)
(mg) (mg)
CF1 20 45 25 10 10 04 0.1 10
CF2 20 45 20 10 10 04 0.1 10
CF3 20 45 30 15 10 04 0.1 10
CF4 20 45 30 10 10 04 0.1 10
CF5 20 45 20 20 10 04 0.1 10
CF6 20 45 25 10 10 04 0.1 10
CF7 20 35 30 20 10 04 0.1 10
CF8 20 45 30 20 10 04 0.1 10
CF9 20 35 25 15 10 04 0.1 10
CF10 20 45 25 15 10 04 0.1 10
CF11 20 40 20 15 10 04 0.1 10
CF12 20 40 30 15 10 04 0.1 10
CF13 20 40 25 10 10 04 0.1 10
CF14 20 40 25 20 10 04 0.1 10
CF15 20 40 25 15 10 04 0.1 10
CF16 20 40 30 20 10 04 0.1 10
CF17 20 35 30 10 10 04 0.1 10
CF18 20 40 25 20 10 04 0.1 10
CF19 20 35 20 15 10 04 0.1 10
CF20 20 35 25 10 10 04 0.1 10
CF21 20 40 30 10 10 04 0.1 10
CF22 20 35 30 15 10 04 0.1 10
CF23 20 35 25 20 10 04 0.1 10
CF24 20 35 20 10 10 04 0.1 10
CF25 20 35 20 20 10 04 0.1 10
CF26 20 35 30 20 10 04 0.1 10
CF27 20 40 20 20 10 04 0.1 10

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8.6. EVALUATION OF FILMS
All the formulated films are evaluated as per the procedure mentioned in 5.5
(lai et al., 2015;Patil et al., 2011; Vishwakarma et al., 2011)
In vitro drug release studies
Dissolution studies of Citalopram Hydrobromide FDOF were carried out as per the
procedure 5.5.8.Samples were analyzed for cumulative percentage drug release
spectrophotometrically at 227 nm. Three trials were carried out for all the samples and
average was taken.
8.7: CHARACTERIZATION OF CITALOPRAM HYDROBROMIDE
FDOF
The optimized formulation characterized for FTIR as per the procedure adopted in 5.7.
8.8: PHARMACOKINETIC STUDIES OF CITALOPRAM
HYDROBROMIDE IN RABBIT PLASMA
8.8.1. Animal dose calculation for Citalopram in rabbit plasma
The rabbit dose was calculated based on the conversion factor of human dose to rabbit
dose as shown below for Citalopram.
The rabbit surface area considered for this research was 0.2sq.m and the human surface
area is 1.6sq.m. The human dose of Citalopram 20mg. Dose calculation is done as
follows:

Surface area of animal

Dose of the animal= ……………………………………X Human dose
Surface area of Human
Rabbit surface area =0.2sq.m
Human surface area=1.6sq.m
Human dose of Citalopram =20mg
0.2
Animal dose =………………………………X20
1.6
Citalopram animal dose=2.5mg

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8.8.2. Animal preparation
As per the procedure adopted in 5.8.2.
8.8.3. Blood sampling done as per the procedure adopted in 5.8.3.
8.8.4. Estimation of Citalopram in Rabbit plasma by HPLC method
Table 8.4: Instrumentation

Stock and Standard solutions

Figure 8.3: Procedure for preparation of stock solutions

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 To the above plasma aliquots in centrifuge tubes, 10 ml of standard solution of salbutamol sulphate as
internal standard (IS) was added to give a final concentration of 100 µg ml-1

. Contents of the centrifuge tubes were made alkaline with 0.2 ml of 1M sodium hydroxide.

An aliquot of 10 ml hexane containing 1% v/v isoamyl alcohol was added and mixture was sonicated
for 5 min using a sonicator

After centrifugation at 1500 g for 10 min, the aqueous layers were frozen by immersing the tubes into a
cooling bath consisting of dry ice and ethanol

The hexane layers were decanted into another centrifuge tubes and 0.2 ml of 0.05 M hydrochloric acid
was added

The mixture was shaken centrifuged and frozen as mentioned above, and the organic layer was
discarded and 50 ml of acid extract was injected into the chromatograph

Figure 8.4: Procedure for HPLC Sample Preparation

Preparation of calibration curve in rabbit plasma
The calibration curve was plotted between the ratio of peak areas of citalopram to IS and
concentration of citalopram by replicate analysis (n=6) at all concentration levels and the
linear relationship was evaluated using the least square method using Microsoft Excels
program.
8.8.5. Pharmacokinetic analysis
Pharmacokinetic analysis carried out as per the procedure mentioned in 5.8.5

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