4.0 pre-formulation studies - information and library...

Chapter 4 Pre-formulation Studies

School of Pharmaceutical Sciences 75

4.0 Pre-formulation Studies

4.1. Material & methods

4.1.1. Material

Drugs

Paracetamol and Diclofenac sodium were received as gift sample from Jackson

Laboratories Pvt. Ltd. Amritsar, India.

Chemicals

The chemicals used during the study are mentioned in Table 4.1.

Table: 4.1 List of chemicals.

Name Specification Manufacturer / Supplier

Acetone Laboratory Reagent Hi- Media

Hydrochloric acid Laboratory Reagent Hi- Media

Sodium hydroxide pellets Laboratory Reagent Hi- Media

Methanol Laboratory Reagent Hi- Media

Petroleum ether Laboratory Reagent Hi- Media

Methyl Paraben Laboratory Reagent CDH

Propyl paraben Laboratory Reagent CDH

Phosphate Buffer Saline

(pH- 7.4)

Laboratory Reagent Hi- Media

Chloroform Laboratory Reagent Hi- Media

Ether Laboratory Reagent Hi- Media

Lactose Laboratory Reagent CDH

Starch Laboratory Reagent CDH

Magnesium stearate Laboratory Reagent CDH

Talc Laboratory Reagent CDH

di-calcium Phosphate Laboratory Reagent CDH



Instruments and apparatus [1, 2]

The instruments and apparatus used during the study are mentioned in Table 4.2.

Table: 4.2 List of instruments and apparatus.

Name Model Manufacturer / Supplier

UV- Visible

Spectrophotometer UV-1700

Shimadzu Corporation Analytical

& Measuring Instrument Division.

FT-IR Spectrum BX Perkin Elmer.

Oven - Narang Scientific works Pvt. Ltd.

Analytical balance CX 220 Citizen.

Test tube shaker - Perfit India.

Melting point apparatus - Perfit India.

pH meter Perfit India.

4.2 Characterization of API

The drugs were characterized as per the specifications given in the monograph (IP) and

are tabulated in Table 4.3 and Table 4.4 for diclofenac sodium and paracetamol

respectively.

Determination of melting range

Open end capillary method was used to determine the melting range of the drug [3].

Table: 4.3: Characterization of diclofenac sodium [4]

S.No. Properties Observation As per monograph [5]

1

2

3

4

5

Appearance

Physical form

Odour

Moisture sensitivity

Melting point range

White

Crystalline powder

Odourless

slightly hygroscopic

281-285oC

White

Crystalline powder

Odourless

slightly hygroscopic

281-284oC



Table: 4.4: Characterization of Paracetamol

S.No. Properties Observation As per monograph[5]

1

2

3

4

5

Appearance

Physical form

Odour

Moisture sensitivity

Melting point range

White or almost white

Crystalline white powder

Odourless

slightely hygroscopic

168-1720C

White

White crystals

Odorless

hygroscopic

168-1700C

Identification test for diclofenac sodium

1. To 1 mL of a solution of diclofenac sodium in methanol (1 in 250), 1mL of nitric

acid was added. A dark red color was developed.

2. 5 mg of diclofenac sodium was subjected for flame coloration test. A light green

color on the flame was observed.

Identification test for Paracetamol

1. 0.1gm paracetamol was boiled with 1ml HCl for 3 minutes. 10 ml water was

added & cooled. No precipitates were formed.

2. 5 ml of 0.1N potassium dichromate was added to 1ml of 10% paracetamol

solution. Violet colour was developed slowly.

Infra Red Spectroscopy

The Infra red spectroscopy of the sample was carried out to ascertain identity of both the

drugs. A pellet of approximately 1 mm diameter of each drug was prepared by

compressing 3-5 mg of the drug with 100-150 mg of potassium bromide in KBr press

(Model M-15, Techno Search Instruments). The pellet was mounted in IR compartment

and scanned between wave number 4000-1

– 600 cm-1

. The FTIR spectra are represented in

Figure 4.1 and 4.2 for Diclofenac Sodium and in Figure 4.3and 4.4 for Paracetamol. The

interpretation of the spectra is presented in table 4.5.



Fig. 4.1: Reference FT-IR Spectrum of Diclofenac Sodium

Fig: 4.2 FT-IR Spectrum of Diclofenac Sodium



Fig. 4.3: Reference FT-IR Spectrum of Paracetamol.

Fig: 4. 4 : FT-IR Spectrum of Paracetamol



Table: 4.5 Interpretation of FT-IR spectrum of drug(s).

S.

No.

Drug Reported Peak

(cm-1)

Observed

Peak

(cm-1)

Inference

1 Diclofenac

Sodium [6]

3500-3100 (m) 3252.92 (m) N-H stretching

3150-3050 (s) 3252.92 (s) C-H (aromatic) stretching

1300-1000 (s) 1274.95 (s) C-O stretching

1350-1000 (m-s) 1195.19 (m) C-N (amines) stretching

785-540 (s) 765.97 (s) C-Cl stretching

2

Paracetamol

[6]

3500-3100 (m) 3162.09 (m) N-H stretching

3400-3200 (m) 3326.63 (m) O-H stretching

3150-3050 (s) 3162.09 (s) Aromatic ring

1450-1375 (m) 3170.53 (m) -CH3 bending

1300-1000 (s) 1259.63 (s) C-O stretching

4.3 METHODS

Solubility studies of drug [7]

Diclofenac Sodium

An excess of drug was dissolved in 10 ml buffer of different pH values (1.2, 4.5, 6.8 and 7.4)

in conical flask and was continuously shaken for 24 hours at room temperature with the help

of conical flask shaker. After 24 hours sample was filtered through Whatman filter paper

no.1, diluted appropriately and the drug was estimated using UV spectroscopy [8] (Shimadzu

1700). The value of solubility studies are shown in Table 4.6.

Paracetamol

An excess of drug was dissolved in 10 ml buffer of different pH values (1.2, 4.5, 6.8 and 7.4)

in conical flask and was shaken for 24 hours at room temperature with the help of conical



flask shaker. After 24 hours sample was filtered through Whatman filter paper no.1, diluted

appropriately and the drug was estimated using UV spectroscopy[8] (Shimadzu 1700). The

value of solubility studies are shown in Table 4.6.

Table 4.6: Solubility data of drugs

S.

No.

Drugs Solvents

0.1 N HCl Phthalate

buffer

pH 4.5

Phosphate

buffer

pH 6.8

Phosphate

buffer

pH 7.4

1 Diclofenac

Sodium

0.0163

gm/ml

0.0083

gm/ml

0.0069

gm/ml

0.0057

gm/ml

2 Paracetamol 0.0894

gm/ml

0.0061

gm/ml

0.0056

gm/ ml

0.0049

gm/ml

Preparation of standard solution

Paracetamol

Preparation of Stock Solution

Accurately weighed 100 mg of the drug was transferred to 100 ml volumetric flask. The

drug was dissolved in 5 ml methanol. The volume was made up to the mark with

methanol (stock solution I) to make a solution of 1000 µg/ml. One ml of stock solutions I

(1000 µg) diluted to 50 ml with methanol to give a stock solution of concentration 20

µg/ml (Stock solution II). Stock solution II was used to prepare a series of standard drug

solutions. From stock solution II different aliquots were transferred to a series of 10 ml

volumetric flasks and the volume was made up to the mark with 0.1 N hydrochloric acid

to get six standard dilutions of 2, 4, 8, 12, 16, 20 mcg/ml. The absorbance of standard

solutions [9-10] was measured at 249 nm and absorbance was recorded in table 4.7 and

the plot is shown in figure 4.5.



Standard curve of Paracetamol

y = 0.0433x + 0.0336

R2 = 0.9995

0

0.2

0.4

0.6

0.8

1

0 5 10 15 20 25

Concentration

Absorbance

Diclofenac Sodium

Accurately weighed 100 mg of the drug was transferred to 100 ml volumetric flask. The

drug was dissolved in 5 ml methanol. The volume was made up to the mark with

methanol (stock solution I) to make a solution of 1000 µg/ml. One ml of stock solutions I

(1000 µg) diluted to 50 ml with methanol to give a stock solution of concentration 20

µg/ml (Stock solution II). Stock solution II was used to prepare a series of standard drug

solutions. From stock solution II different aliquots were transferred to a series of 10 ml

volumetric flasks and the volume was made up to the mark with 0.1 N hydrochloric acid

[11-14] to get six standard dilutions of 5, 10, 15, 20, 25, 30 mcg/ml.. The absorbance of

standard solutions was measured at 276 nm and absorbance was recorded in table 4.8 and

the plot is shown in figure 4.6.

Table: 4.7: Standard curve data of Paracetamol at 249nm

S. No Concentration

(µg/ml)

Absorbance

1 2 0.11

2 4 0.215

3 8 0.38

4 12 0.56

5 16 0.723

6 20 0.896

Fig: 4.5: Standard curves for Paracetamol at 249 nm.



Standard curve of Diclofenac

y = 0.0196x - 0.0235

R2 = 0.9986

0

0.1

0.2

0.3

0.4

0.5

0.6

0 10 20 30 40

Concentration

Absorbance

Table: 4.8: Standard curve data of Diclofenac Sodium at 276nm

S. No Concentration

(µg/ml)

Absorbance

1 5 0.085

2 10 0.165

3 15 0.265

4 20 0.365

5 25 0.471

6 30 0.568

Fig: 4.6: Standard curves of Diclofenac sodium in Methanol at 276 nm

4.4 Drug-Excipients Compatibility Studies

FTIR spectra were recorded to assess the compatibility of the drugs and excipients.

drug(s) and excipients in the ration of 1:1 were mixed thoroughly and stored at 40 °C and

75% RH for and room temperature for 1 month [15]. The FTIR spectra are represented in

Figure 4.7 – Figure 4.10



Figure 4.7: IR Spectra: IR spectra of diclofenac sodium (a), Diclofenac sodium + A.

indica (b) and Diclofenac Sodium + P. persica

Fig.4.8: FTIR Spectra for Paracetamol formulation with G. optiva



Fig.4.9: FTIR Spectra for Paracetamol formulation with B. ceiba

Fig. 4.10: FTIR Spectra for Paracetamol formulation with Myrica



Diclofenac Sodium and Paracetamol were identified using different methods viz. melting

point determination, determination of absorption maxima (λmax) and FTIR spectroscopy.

The IR spectra of the diclofenac sodium, P. persica mucilage and A. indica mucilage

were portrayed in Figure 4.7. The IR spectra of diclofenac sodium exhibited distinctive

peaks at 3381.57 cm-1

due to NH stretching of the secondary amine, 1572.66 cm-1

owing

to -C = O stretching of the carboxyl ion and at 745.35 cm-1

because of C-Cl stretching.

FTIR spectra of paracetamol, showed characteristic O-H, N-H, C=O (amide) stretching

bands at 3326.98 cm-1

, 3413.77 cm-1

, 1654.81 cm-1

, respectively. Whereas, amide II

band, C-N-H group and para-disubstituted aromatic rings at 1560.30 cm-1

, 1259.43 cm-1

and 837.05 cm-1

, respectively were also observed. The observed FTIR spectra of both the

drugs were matched with reference spectra.

The solubility of both the drugs was determined in different media. Both drugs were

found to be sparingly soluble in acidic medium and slightly soluble in basic medium. The

solubility of diclofenac sodium in 0.1N HCl, phthalate buffer pH 4.5, phosphate buffer

pH 6.8, phosphate buffer pH 7.4 was found to be 0.0163 gm/ml, 0.0083 gm/ml, 0.0069

gm/ml, and 0.0057 gm/ml, respectively. The solubility of paracetamol in 0.1N HCl,

phthalate buffer pH 4.5, phosphate buffer pH 6.8, phosphate buffer pH 7.4 was found to

be 0.0894 gm/ml, 0.0061 gm/ml, 0.0056 gm/ml, and 0.0049 gm/ml, respectively.

FTIR spectra were recorded to assess the compatibility of the drugs and excipients. The

compatibility of drugs with mucilages was assessed by FTIR spectroscopy of the samples

kept at 40°C and 75% RH and at room temperature for 1 month. FTIR spectra of drug (s),

physical mixture of paracetamol & G. optiva mucilage, physical mixture of paracetamol

& B. ceiba, physical mixture of paracetamol & Myrica, physical mixture of diclofenac

sodium & A. indica, and physical mixture of diclofenac sodium & P. persica were

recorded and examined. In FTIR spectra of paracetamol, characteristic N-H stretching

band at 3413.77 cm−1

, O-H stretching band at 3326.98 cm−1

, and carbonyl stretching band

at 1654.81 cm−1

were noted and in case of diclofenac sodium, characteristic C = O

stretching of the carboxyl ion and at 745.35 cm-1

which are in agreement with the

reported values. All characteristic peaks of drug(s) were observed in the FTIR spectra of



physical mixture of paracetamol & G. optiva mucilage, physical mixture of paracetamol

& B. ceiba, physical mixture of paracetamol & Myrica, physical mixture of diclofenac

sodium & A. indica, and physical mixture of diclofenac sodium & P. persica. The results

showed no chemical interaction and changes took place in FTIR spectra of both the drugs

and various excipients alone or in combination exhibiting compatibility of the drugs with

all excipients.



4.5 References

[1] Instruction manual pharmaspec. UV-1700 series. User’s system guide. Shimadzu

Corporation. Kyoto Japan.

[2] Spectrum BX user’s guide Perkin Elmer.

[3] United State Pharmacopoeia 31. The National Formulary. Asian Edition.

Published by The Board of Trustees, 26. 2008 (I) pp 297-298.

[4] Indian Pharmacopoeia. The controlled publication, New Delhi. 2007, (3), pp

1514,1515,1814,1815.

[5] British Pharmacopoeia, The British Pharmacopoeia Commission offices, London,

2008, (1&2), pp 685, 686, 1653, 1654.

[6] Pavia, L. K, Introduction to Spectroscopy, 3rd

Ed, Thomoson Books Ltd. United

state, p 358.

[7] Synder, L.R.; Kirkland, J.J.; Glajch, J. L. Practical HPLC Method Development,

2nd

Ed, Johan Wiley & Sons, 1997, pp180-182.

[8] Seedher N, Bhatia S. Solubility Enhancement of Cox-2 Inhibitors Using Various

Solvent Systems. AAPS PharmSciTech. 2003; 4(3): article 33.

[9] Soma, S. M.; Vidyasagar, V.; Anandkumar, N.; Krishna, R. Indian J. Pharm. Sci.

2005, 67(3), 302-306.

[10] Panzade, P.D.; Mahadik, K.R. Indian drugs. 2000, 38(7), 368-370.

[11] ICH Harmonized Tripartite Guideline, “Text on Validation of Analytical

Procedure (Q2A)”, October 1994.

[12] Garatt, D. C. The quantitative Analysis of Drugs, 3rd

Ed, CBS publishers and

Distributors. 2005, p 876.

[13] Beckett, A. H.; Stenlake, J. B. Practical Pharmaceutical Chemistry, 4th

Ed (2),

2007, pp 282 – 288.

[14] Sharma, B. K. Instrumental Methods of Chemical Analysis, 23rd

Ed, Goel

Publishing House, 2004, pp 292-302.

[15] Biswajit Mukherjee, Kousik Santra, Gurudutta Pattnaik, and Soma Ghosh, Int J

Nanomedicine, 2008; 3(4): 487–496.

4.0 pre-formulation studies - information and library...

Documents