CHAPTER 5 MATERIALS AND METHODS

V. MATERIALS AND METHODS

MATERIALS:
Sr. No Chemicals/ Excipients Source
1 Sitagliptin Phosphate Gift sample from Wockhardt Ltd.,
Monohydrate (API) Aurangabad
2 Hibiscus rosa-sinesis Mucilage Prepared from Hibiscus rosa-sinesis
leaves, which are collected from Botanical
Garden of Y. B. Chavhan College of
Pharmacy.
3 Plantago ovata Forskal (husk) Purchased from a local shop.
Powder
4 Pearlitol 400DC ( Mannitol Roquette Pvt. Ltd.
Granules)
5 Magnesium Stearate Loba Chemie Pvt. Ltd.
6 Talc Loba Chemie Pvt. Ltd.
7 Lactose Fisher Scientific Pvt. Ltd.
Table 5.1: List of Drug and Chemicals/Excipients required for MDT

Sr.no Equipment and Instruments Source

1 UV Spectrophotometer Shimandzu Corp.,Japan
2 FTIR Spectrophotometer Jasco/FTIR-4100,Japan
3 Digital Balance Shimandzu Corp.,Japan
4 Dissolution Appartus Electrolab, Mumbai
5 Tablet Disintegration Tester Remi equipment, Mumbai
6 Digital oven Eli Electro lab, Mumbai
7 Tablet Compression Machine Karnawati, Mumbai
Table 5.2: List of Equipment and instruments

METHODS

5.1 Standardization and Characterization of Drug and Excipients.

5.1.1 Identification and characterization of Sitagliptin Phosphate Monohydrate39

5.1.1 (a) Appearance:

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Sitagliptin Phosphate Monohydrate was characterized for color, odor, and other
characteristics.

5.1.1 (b) Melting Point:

The melting point of Sitagliptin Phosphate Monohydrate was determined by the capillary
method.

5.1.1 (c) Solubility:

The saturated drug solution was prepared in different solvents and agitated on a rotary
shaker for 24 hours. The sample was analyses by UV spectrophotometer.

5.1.1 (d) FTIR Spectroscopy

The FTIR Spectrum of Sitagliptin Phosphate Monohydrate was obtained using the KBr
dispersion technique. The drug sample was mixed with IR grade KBr and Scanned in the
range of 4000-400cm-1.

5.1.2 Identification and Characterization of Excipients

Excipients were characterized for their physical characteristics, chemical characteristics,

and FTIR analysis.

5.1.2 UV Calibration Curve for Sitagliptin Phosphate Monohydrate.

5.1.2 (a) Determination of λmax:
Sitagliptin Phosphate Monohydrate was analyses by UV Spectrophotometer the λmax was
determined at 267nm the procedure adopted is given below.

5.1.2 (b) Preparation of Standard solution

Accurately weighed 100mg of Sitagliptin Phosphate Monohydrate was transferred to
100ml volumetric flask and the volume was made up to 100ml with 0.1N HCl to produce
a stock solution of 1000µg/ml. The 10ml of above stock solution of the drug was transferred
to 100ml volumetric flask and was made up to 100ml with 0.1N HCl to produce a stock 2
solution of 100µg/ml get standard solution of Sitagliptin Phosphate Monohydrate.

5.1.2 (c) Preparation of calibration curve

Dilutions of 2,4,6,8,10µg/ml were prepared using the above working stock solution and
were recorded λmax 267nm.

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5.1.3 Identification and characterization of Excipient

Excipients were characterized for their physical and chemical characterization, FTIR
analysis.

5.2 Drug-Excipient compatibility studies40

5.2.1 FTIR Studies
FTIR Spectrum of the physical mixture comprising drug along with Hibiscus rosa-sinesis
mucilage and Plantago ovata husk powder was recorded using FTIR-4100
Spectrophotometer from 400-4100cm-1.

5.2.2 Extraction of Hibiscus rosa-sinesis Mucilage41

1. Leaves of hibiscus rosa sinesis were collected from the botanical garden of Y. B.
Chavhan College of Pharmacy, Aurangabad and washed with distilled water.

2. Leaves were crushed in a mixer and soaked in Dist. Water for 4-5hrs.

3. Above mix. was boiled for 30 min and left for 24hrs.

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4. Mucilage was extracted via muslin cloth.

5. Acetone was added to it to precipitate mucilage and separated it.

6. Separated mucilage was air-dried and crushed into fine powder with the help of
Mortor and Pestle.

7. Crushed mucilage was collected and stored in air tight polybag in decicator.

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5.3 Formulation of MDT42

MDT Sitagliptin Phosphate Monohydrate is formulated considering the need for treatment
of naive diabetic patients.

5.3.1 Preliminary Formulation Studies

The powder blend comprising of drug and excipients considered for preliminary and
factorial formulations was evaluated for different flow properties as given below:

All ingredients were passed through mesh No. 60 required amount of each ingredient was
taken for each specified formulation and all the drug and excipients without lubricant were
mixed for 15 min. with mortar and pestle. After the addition of magnesium stearate (#40),
the procedure continued for 5 minutes.

5.3.1 (a) Angle of repose

The angle of repose was calculated using the equation

tanƟ = h/r

Ɵ=Angle of repose

H=Height r=Radius of the pile

5.3.1 (b) Bulk density43

It is the ratio of the total mass of powder to the bulk volume of powder. It was measured
by pouring the weight into a measuring cylinder and initial weight was noted. This initial
volume is called bulk volume. From this bulk density is calculated according to the formula
given below. It is expressed in g/ml.

Db= N/Vb

Where M is the mass of powder

Vb is the bulk volume of powder.

5.3.1 (c)Tapped density

It is the ratio of the total mass of powder to the bulk volume of powder It is expressed in
g/ml

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Dt=M/Vt

Where M is the mass of powder

V is the tapped volume of the powder.

Carr's index: Can be calculated as follows:

Carr's index=Dt-Db/Dt ˟ 100

Dt & Db are tapped and bulk density of powder respectively.

Cars index(%) Flow Character Housner’s ratio

≤10 Excellent 1.00-1.11
11-15 Good 1.12-1.18
16-20 Fair 1.19-1.25
21-25 Passable 1.26-1.34
26-31 Poor 1.35-1.45
32-37 Very poor 1.46-1.59
˃38 Very very poor ˃1.60
Table 5.3: Desirable standard values for flow properties

5.3.1 (d)Hausner's ratio44

Hausner's ratio is an indirect index of the case of powder flow. It is calculated by the
following formula:

Hausner's ratio= Dt/Db

Where D, & D, are tapped and bulk densities respectively.

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5.3.2 Preformulation studies:

The uniform blends of the composition prepared considering standard GRAS approved
excipients were as reported in the literature for MDT were considered (Table 5.4) and then
weighed and directly compressed using 8.5 mm punch, curve faced tooling to make the
tablets of said compression specifications using 12 stations Lab press compression
machine. The tablet press setting was kept constant across all formulations.

Sr. Category of Material Percent

No. Amount/tablet
1. Drug (Sitagliptin Phosphate Monohydrate) X
2. Natural Disintegrant 2-8%
3. Sweetener 28%
4. Diluent 4.5%
5. Glidant 4.5%
6. Lubricant 30-36%
Table 5.4: Generalized formulation of MDT

Ingredient in mg S1 S2 S3 S4 S5 S6
Sitagliptin 50 50 50 50 50 50
Phosphate
Monohydrate
Hibiscus 2% 4% 8% - - -
Mucilage
Plantago ovata - - - 2% 4% 8%
husk powder
Mannitol 56 56 56 56 56 56
(Pearlitol)
Talc 9 9 9 9 9 9
Magnesium 9 9 9 9 9 9
stearate
Lactose 72 68 60 72 68 60
Table 5.5: Formulation development of preliminary batches of MDT

Further preliminary formulations were developed by the EVOP technique considering

changes in independent variables (Natural disintegrants i.e., Hibiscus rosa-sinesis

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mucilage, and Plantago ovata husk Powder), and their effect on response disintegration
time was studied. (Table 5.5)

5.3.3 Optimization of MDT using 32 Full Factorial Design45

A 32 full factorial design was used, in this design 2 factors were evaluated each at 3 levels,
and experimental trials were performed at all 9 possible combinations as shown in Table
(5.7).

The amount of Natural disintegrants Hibiscus rosa-sinesis and Plantago ovata husk powder
were selected as independent variables.

Batch code X1 X2
F1 -1 -1
F2 -1 0
F3 -1 +1
F4 0 -1
F5 0 0
F6 0 +1
F7 +1 -1
F8 +1 0
F9 +1 +1
Table 5.6: Full Factorial Design matrixes for MDTs of Sitagliptin Phosphate
Monohydrate

Tablet F1 F2 F3 F4 F5 F6 F7 F8 F9
ingredient(mg)/formulation
Sitagliptin Phosphate 50 50 50 50 50 50 50 50 50
Monohydrate
Hibiscus rosa-sinesis 2% 2% 2% 4% 4% 4% 8% 8% 8%
Plantago ovata husk powder 2% 2% 2% 4% 4% 4% 8% 8% 8%
Mannitol 56 56 56 56 56 56 56 56 56
Talc 9 9 9 9 9 9 9 9 9
Magnesium stearate 9 9 9 9 9 9 9 9 9
Lactose 72 72 72 68 68 68 60 60 60
Table 5.7: Formulation Design of MDT as per Full Factorial Design (32)

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5.3.4: Evaluation of MDT:

5.3.4.1.a) Appearance: General appearance of a tablet involves the measurement of several

attributes such as the tablet's size, shape, color, presence or absence of odor, and surface
texture.

5.3.4.1.b) Weight Variation: 20 tablets were selected randomly and the average weight was.
determined. Then individual tablets were weighed and compared with the average weight.
As per IP, for tablets weighing 80 mg or less than 250mg, not more than 2 of individual
weights. deviate from the average weight by more than 7.5% and none deviate by more
than twice - that relevant percentage.

5.3.4.1.c) Hardness: The limit of crushing strength for an MDT is usually kept in a lower
range to facilitate early disintegration in the mouth. And it was measured using a Monsanto
tablet hardness tester.

5.3.4.1.d) Thickness: The crown thickness of 20 tablets was individually measured using

Vernier caliper, which permits accurate measurement and provides information on the

variation between the tablets. The tablet thickness should be controlled within 5%.

variation of a standard value.

5.3.4.1.e) Friability: It was determined by using Roche Friabilator. Usually, it should be

below 1%. An indication of good mechanical resistance of tablets. Pre weighed sample of
tablet was placed in the friabilator and subjected to 25 rpm. The tablets were dedusted using
a son muslin cloth and reweighed.

The friability (f) is given by the formula,

F=(1-Wo/W) x 100

Where Wo is the weight of the tablet after the test

W is the weight of the tablet after the test.

5.3.4.1.1) Wetting time: wetting time is relayed with the contact angle. A lower wetting
time implies a quicker disintegration of the tablets.

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Wetting time was determined by the method tissue paper folded twice was placed in a small
Petri dish (5.5cm diameter) containing 10 ml of water at room temperature A tablet was
put on the tissue paper and allowed to wet completely. The time required for complete
wetting of the tablet was then recorded.

5.3.4.1.g) Drug content: Twenty tablets were weighed and powdered. The blend equivalent
to 5mg of sitagliptin phosphate monohydrate was weighed and dissolved in a sufficient
quantity of 0.1N HCl. The solution was filtered through Whatman filter paper (no.41),
suitably diluted with 0.1N HCl, and assayed at 267nm, using a UV-visible double beam
spectrophotometer (UV-Shimadzu).

5.3.4.1.h) In vitro Disintegration Time46: For a drug to be adsorbed from a solid dosage
form after oral administration, it must first be in solution, and the first important step
towards this condition is usually the breakup of the tablet, a process known as
disintegration. The disintegration time of MDTs was determined in conventional
disintegration test apparatus following the official European Pharmacopoeia monograph,
orodispersible tablets stating a max disintegration time of 3 mins.( European
Pharmacopoeia 5.0,2005). Disintegration or more specifically dispersion times were
measured in 900 ml purified water according to the IP method without using a disc at room
temp. (25°C+2°C).

5.3.4.2: In vitro Dissolution Study: The development of dissolution, a method for MDT is
comparable to the approach taken for conventional tablets, and is practically identical.
Dissolution conditions for drugs are listed in many research papers. Other media such as
buffers (pH 6.8 and 0.1 N HCL) should be evaluated for MDT much in the same way as
their ordinary counterparts. The USP -2 paddle apparatus is the most suitable and common
choice for orally disintegrating tablets, with a paddle speed of 50 rpm commonly used. The
following method was used for dissolution studies. Dissolution studies of sitagliptin
phosphate monohydrate tablets were done using USP method 2 with paddle speed at 50
rpm. Dissolution was performed in 900 ml 0.1N HCl mentioned at 37+0.5°C. 2 ml of
sample was withdrawn at a specified time interval. The volume of dissolution fluid was
adjusted to 900ml, by replacing each 2ml aliquot withdrawn with 2 ml of 0.1N HCl
maintained at 370.5°C. Samples withdrawn were filtered with Whatman filter paper

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(no.41), suitably diluted with 0.1N HCl, and analyzed at 267nm, using a UV-visible
spectrophotometer(UV-1780 Shimadzu).47

5.3.5 Multiple regression analysis of 32 factorial batches: MDT

The responses obtained from 3² factorial batches were subjected to multiple regression
analysis. The polynomial equations were determined using the form

Yi=b0+b1X1+b2X2+b11X12+b12X22+b12X1X2+b12X1X2+b12X1X22+b12X12X2+b12X12X22

Where Yi is the dependent variable, b0 is the arithmetic mean responses of the 9 runs, and
b1, is the estimated coefficient for the factor X1 The main effects (X1 and X₂) represents.
the average results of changing one factor at a time from its low to high value. The term
X₁² and X₂² indicate curvilinear relationship. The interaction X₁X2 shows how the
dependent variable changes when two or more factors are simultaneously changed.

Further RSM (Response surface methodology) plots were used to get a picturesque

understanding of the influence of independent variables (Factors) on the various

responses studied.

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