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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
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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
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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.
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Fig. 4.1: Reference FT-IR Spectrum of Diclofenac Sodium
Fig: 4.2 FT-IR Spectrum of Diclofenac Sodium
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Fig. 4.3: Reference FT-IR Spectrum of Paracetamol.
Fig: 4. 4 : FT-IR Spectrum of Paracetamol
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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
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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.
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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.
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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
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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
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Fig.4.9: FTIR Spectra for Paracetamol formulation with B. ceiba
Fig. 4.10: FTIR Spectra for Paracetamol formulation with Myrica
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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
Chapter 4 Pre-formulation Studies
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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.
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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.