Chapter 3 Preformulation Studies

T
he physicochemical properties of drug(s) play crucial role in the designing of

drug delivery system. These parameters allow a formulator to select various

additives and formulation conditions for the successful and effective drug

delivery. Preformulation study, an integral part of entire development process, may be

described as the process of optimization of a drug through the determination of those

physicochemical properties considered important in the formulation of a stable, effective

and safe dosage form. Thus, in order to establish optimum conditions for developing

suitable drug delivery system, preformulation studies were carried out. The

physicochemical parameters included test of identification, melting point, partition

coefficient, solubility, UV, IR spectroscopy, chromatography, etc.

3.1. DRUG IDENTIFICATION

Gift sample of RIF and INH were obtained from Lupin Laboratories, Pune, India and

evaluated for following parameters:

3.1.1. Physical Appearance and Melting Point

The physical examination of the drug(s) was performed in day-light by placing small

amount of the drug(s) sample on glass slide. Melting point was determined using a

melting point apparatus (Superfit, India). Data is presented in Table 3.1.

Table 3.1: Physical examination of gift sample of RIF and INH

Characteristics RIF INH

Physical Brick red to reddish crystalline

White crystalline powder
appearance powder

Color Brick red to reddish White

Odor Odorless Odorless

Melting Point 181°-184°C 170°-172°C

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3.1.2. UV Spectroscopy (Determination of  max)

The characterization and identification of an organic compound generally relies on

spectroscopy. Ultraviolet-Visible (UV-Vis) spectrophotometry has been used to obtain

specific information on the chromophoric part of the molecule. UV-Vis spectroscopy is

widely available and commonly used for both qualitative and quantitative experiments.

UV spectra were recorded by scanning the drug(s) solutions in the range of 200 – 400

nm. The absorption pattern of uv light of different wavelength are given in Fig. 3.1 and

Fig. 3.2.

3.1.3. Infrared (IR) Spectroscopy

IR spectrum of any compound or drug(s) provides information about the groups present
in that particular compound and thus provides a means of good qualitative
characterization of the drug(s) samples. IR spectra of drug(s) were taken by using KBr
pellet method, using Perkin Elmer IR spectroscope. The IR spectra of standard and
sample RIF and INH are shown in Fig. 3.3-Fig. 3.6.

3.2 PARTITION COEFFICIENT

The partition coefficient is defined as the ratio of unionized drug distributed between the
organic and aqueous phase. It is a measure of lipophilicity of drug(s) and an indirect
indication of its ability to cross cell membranes. Drugs having values of P much greater
than 1 are classified as lipophilic, whereas those with partition coefficients much less
than 1 are considered to be hydrophilic. The partition coefficient of the drug(s) was
measured in n-octanol: aqueous phase (distilled water and PBS 7.4). 10.0 mg of drug(s)
was accurately weighed and transferred into a screw capped glass vial containing 5 mL of
n-octanol and 5 mL of phosphate buffer saline (pH 7.4)/water. The mixture was shaken
on wrist action shaker (Yarco, New Delhi) for 24 hrs. Both the phases were separated
using separating funnel and aqueous phase was analyzed for the amount of drug after

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suitable dilution using Cintra GBC 10 UV/Vis spectrophotometer. The concentration in

the n-octanol phase was determined by subtracting the concentration of drug(s) in
aqueous phase from the initial concentration after 24 hours shaking. The partition
coefficient of RIF and INH are presented in Table 3.2.

Table 3.2: Partition coefficient of RIF and INH

Partition coefficient Partition coefficient

S.No. SOLVENT SYSTEM
of RIF of INH
1. n-octanol: Distilled Water 9.32 1.86
2. n-octanol: PBS (pH 7.4) 8.97 1.97

3.3. SOLUBILITY

Solubility of drug(s) was determined in water, phosphate buffer saline (PBS), methanol,

ethanol, ether, chloroform and benzene, etc. Accurately weighed amount of RIF and INH

(10 mg) was added separately to these solvents in screw cap test tubes. These capped

tubes after being tightly closed and packed, were shaken for about 72 h using a wrist

shaker (Yorko, India) and kept for 6 h. Then the supernatant was removed, filtered and

estimated spectrophotometrically. Solubility profile is presented in Table 3.3.

Table 3.3: Solubility profile of RIF and INH

S.No. Solvents RIF INH

1. Distilled Water + +++
2. Phosphate Buffer Saline (PBS) pH 7.4 + +++
3. Methanol ++ +
4. Chloroform +++ -
5. Ethylacetate +++ +
6. Ethanol ++ -
7. Acetone ++ -
+ + + Soluble; + + Sparingly Soluble; + Slightly Soluble; - Practically insoluble

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3.4. PREPARATION OF CALIBRATION CURVES

Phosphate buffer saline (pH 7.4) was prepared according to I.P. 1996. Disodium

hydrogen phosphate (1.38 g), potassium dihydrogen phosphate (0.19 g) and sodium

chloride (8.0 g) were dissolved in sufficient distilled water and the volume was made

upto 1 L with distilled water. The pH was adjusted to 7.4.

Accurately weighed quantity of INH and RIF (10 mg) was transferred into two

different clean and dried 100mL volumetric flasks and dissolved in minimum quantity of

methanol and water, respectively. The volumetric flasks were shaken gently to dissolve

whole amount of the drug(s). The volume was made to 100 mL with phosphate buffer

saline (pH 7.4) to obtain 100 µg/mL stock solutions. Various aliquots, i.e., 0.2, 0.4, 0.6

…… up to 2.0 mL of stock solution (100 g/mL) were transferred to a series of 10 mL

volumetric flasks and the volume of each volumetric flask was made upto 10 mL with

phosphate buffer saline (pH 7.4).

The absorbance of these solutions was determined at 479.0 nm for RIF and 262.0

nm in the case of INH. The standard curve of each drug(s) was prepared by plotting a

graph between absorbance and concentration. The standard curves were linearly

regressed and statistical parameters related to them were derived. The results are recorded

in Table 3.4 and Table 3.5 and linearly regressed curves are shown in Fig 3.7 and Fig.

3.8.

3.5 SIMULTANEOUS ESTIMATION OF RIF AND INH IN BLOOD PLASMA

AND ORGAN HOMOGENATES BY HPLC METHOD

HPLC method reported by Guillaumont et al., 1982 was selected for estimation of RIF

and INH in blood plasma and organ homogenates. Accurately weighed 5 mg of RIF and

INH were weighed and transferred separately in two clean and dry volumetric flasks and

dissolved in minimum volume of methanol and water respectively. The volume was

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made up to 50 mL with water. The two solutions were transferred into 100 mL

volumetric flasks and mixed thoroughly. 1 mL of this solution was taken in 10 mL

volumetric flask and 200 µL of plasma was added to it. The content was extracted with

2.5 mL of acetone by centrifugation at 2000 rpm for 10 min. 2 mL of supernatant was

taken in a boiling tube and vacuum evaporated at 45°C. The residue was dissolved in 0.5

mL of mobile phase [methanol: 0.5 mM ammonium formate (65:35) containing 5 mM

heptane sulfuric acid and 1.5 mL of butanol:chloroform (30:70)] and mixed thoroughly

for 10 min. An aliquot (0.5 mL) of organic layer was evaporated and the residue was

again dissolved in 2 mL of mobile phase. The sample solution was filtered and injected in

the loop of HPLC.

Table 3.4: Calibration curve of RIF in PBS (pH 7.4) at 479.0 nm

Concentration Regressed Statistical

S.No. Absorbance
(g/ml) Value Parameters

1 2 0.077 0.073

2 4 0.1476 0.1386

3 6 0.2147 0.2042

4 8 0.2473 0.2698
Equation of Line =
5 10 0.3359 0.3354
y = 0.0328x + 0.0074
6 12 0.39 0.401
R2 = 0.9969
7 14 0.4589 0.4666

8 16 0.5396 0.5322

9 18 0.611 0.5978

10 20 0.6635 0.6634

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Balb/c mice were sacrificed and required organs were isolated, washed with

saline, dried using tissue paper and weighed. One gram tissue from each organ or in case

organ weighing less than one gram whole organ was used, minced and homogenized with

1 mL of deionized water. To the resulting homogenate, 1 mL aliquot of RIF and INH

(100µg/mL) was added in a 10 mL volumetric flask and kept in dark for 30 min. Organ

homogenates were deproteinized with 100 µL of 10% v/v trichloroacetic acid. The

contents were vortexed for 5 min and centrifuged at 5000 rpm for 10 min. The

supernatant was suitably diluted with mobile phase and analyzed. Then, calibration

curves were constructed between the peak area and concentration. The calibration curves

in the concentration range of 200 ng/ML to 1000 ng/ML were prepared. The observations

are recorded in Table 3.6 and shown in Fig 3.9 and Fig. 3.10.

Table 3.5: Calibration curve of INH in PBS (pH 7.4) at 262.0 nm

Concentration Regressed Statistical Parameters

S.No. Absorbance
(g/ml) Value

1 2 0.0833 0.0881

2 4 0.1462 0.1573

3 6 0.2180 0.2265

4 8 0.2833 0.2957
Equation of Line =
5 10 0.3979 0.3649
y = 0.0346x + 0.0189
6 12 0.4386 0.4341
R2 = 0.9925
7 14 0.5216 0.5033

8 16 0.5835 0.5725

9 18 0.6426 0.6417

10 20 0.6785 0.7109

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Table 3.6: Calibration curve of RIF and INH in plasma by HPLC

Concentration Peak Area (x106) Statistical Parameters

S.No.
(ng/ml) RIF INH

1 100 0.658 0.478

2 200 1.188 1.088 RIF
3 300 1.864 1.68 y = 0.007x - 0.1908

4 400 2.576 2.28 R2 = 0.9978

5 500 3.142 3.042

6 600 3.932 3.732
7 700 4.638 4.238 INH
8 800 5.312 5.063 y = 0.0104x + 0.5136
9 900 6.068 5.568 R2 = 0.9962

10 1000 6.954 6.194

3.6. RESULTS AND DISCUSSION

The drug identification related studies showed that selected drug(s) match with the

standards described for their identity and purity in IP, 1996. The results are recorded in

Table 3.1. The UV-visible absorption maxima of RIF was recorded at 479.0 nm while

that of INH was found to be 262.0 nm which is in accordance with the values reported in

IP, 1996.The peaks in IR spectrum of RIF and INH were in accordance with those

reported in literature. The peaks match with the peaks as mentioned in IP, 1996 (Fig. 3.1

and Fig. 3.2).

The partition coefficient of RIF in n-octanol/PBS pH 7.4 and n-octanol/water was

found to be 8.97 and 9.32 and for INH, it was found to be 1.97 and 1.86, respectively

(Table 3.2). The values of partition coefficient of these drug(s) suggested that RIF is

lipophilic in nature while INH is hydrophilic in nature.

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Solubility of the drug(s) was determined in different solvents. RIF was found to

be freely soluble in ethanol, methanol and acetone, chloroform, ethylacetate and acetone

and slightly soluble in water and phosphate buffer saline (PBS) pH 7.4 (Table 3.3). INH

was found to be soluble in aqueous solvents (Table 3.3).

Spectrophotometric method was selected for the estimation of drug(s). For both

the drug(s), absorbance obeyed Beer-Lambarts’ law in the concentration range of 2-20

µg/mL (Fig. 3.7 and Fig. 3.8). The correlation coefficient of more than 0.99 for both the

drug(s) confirmed an excellent linear correlation between concentration and absorbance.

HPLC method reported by Guillaumont et al., 1982 was selected for estimation of

RIF and INH in blood plasma and organ homogenates. The calibration curve of RIF as

shown in Fig. 3.9 was linear between 100-1000 ng/mL (r2≥ 0.99). Similarly, the

calibration curve of INH as shown in Fig. 3.10 was linear between 100-1000 ng/mL (r2≥

0.998).

It can be concluded that the gift samples of RIF and INH were authentic and

results of the study were in accordance with the standards given in official monographs.

Various analytical techniques confirmed the chemical identity and authenticity of the

supplied drug(s). The data generated on the solubility, spectroscopy and standard curve

preparation were reproducible, as taken mean of three values and may be used reliably

for formulation studies in further work.

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Fig. 3.1: UV scan of RIF

Fig. 3.2: UV scan of INH

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 Chapter 3 Preformulation Studies

Fig. 3.3: IR spectrum of RIF (standard)

Fig. 3.4: IR spectrum of RIF (sample)

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 Chapter 3 Preformulation Studies

Fig. 3.5: IR spectrum of INH (standard)

Fig. 3.6: IR spectrum of INH (sample)

Department of Pharmaceutical Sciences, Dr. H. S. Gour Vishwavidyalaya, Sagar, M.P.

 Chapter 3 Preformulation Studies

Fig. 3.7: Standard curve of RIF in PBS (pH 7.4) at 479.0 nm

Fig. 3.8: Standard curve of INH in PBS (pH 7.4) at 262.0 nm

Department of Pharmaceutical Sciences, Dr. H. S. Gour Vishwavidyalaya, Sagar, M.P.

 Chapter 3 Preformulation Studies

Fig. 3.9: Standard curve of RIF using HPLC

Fig. 3.10: Standard curve of INH using HPLC

Department of Pharmaceutical Sciences, Dr. H. S. Gour Vishwavidyalaya, Sagar, M.P.