Saturday, 21st May, 2022.

ADVISOR STUDENT
Prajeet Khedekar
NMIMS, SDSOS Dr. Gurudas Mane and
M.Sc. Analytical
Mr. Vishwanath Nilkanth
Chemistry Part II

DISSOLUTION TEST METHOD DEVELOPMENT OF

DRUG PRODUCT ‘F’ BY
HPLC AND UV-VIS SPECTROSCOPY”
Table of
Contents 1. Overview
2. Drug Profile
3. Uses
4. Mechanism of Action
5. Basics of Dissolution
6. Instruments used for Preparation and Analysis
7. BCS Solubility
8. HPLC Development Trials
9. Selection of Discriminatory Media
10. Co-relation of HPLC and UV
11. Dissolution Development Trials
12. Conclusion
 Overview

Dissolution Method Development of Drug Product 'F' which is an antihyperlipidemic

BCS class II drug.
Understanding the need for Development.
Instruments used in the process.
Comparison of the Release Profile in media containing different concentration of SLS.

Selecting a Discriminatory Media to detect changes made in Formulation.

Performing HPLC method development and selecting appropriate method for routine
support and Dissolution Development
To Perform Dissolution Study of Various Batches of Drug Product 'F' and compare it
with RLD.
 Drug Profile
Name: Drug Molecule “F” 120mg
Pharmacologic classification: Hypolipidemic agent.
Therapeutic classification: Antilipemic Agent.
Solubility: It is soluble in Methanol, and practically insoluble in water.
PKa: -4.9
Dosage Form: Tablets or Capsules
Approved therapeutic use: Drug molecule “F” tablet is used to help lower bad cholesterol
and fats such as LDL and triglycerides.
Route of administration: Oral
Route of elimination: Drug molecule “F” is 5-25% is excreted in faeces and 60-88% is
excreted in urine.
Side effects: Respiratory Disorder, Abdominal pain, Headache, Constipation, Nausea.
 Uses

Increases
Decreases
Mechanism of Action
 Increases
Decreases
 What is Dissolution?
Dissolution testing measures the extent and rate of
solution formation from a dosage form, such as tablet,
capsule, ointment, etc.
The dissolution of a drug is important for its
bioavailability and therapeutic effectiveness. Dissolution
and drug release are terms used interchangeably.
Dissolution testing is an important tool for characterizing
the performance of oral solid dosage forms.
Its significance is based on the fact that for a drug to be
effective, it must first be released from the product and
dissolve in the gastrointestinal fluids before absorption
into the bloodstream can happen.
 Instruments used for Preparation
and Analysis
Hot Melt Extrusion (HME)
HME is a continuous pharmaceutical method that
involves pumping compound materials with a rotating
screw at higher temperatures to attain molecular level
combining of the active compounds and thermoplastic
binders, polymers, or both.
This molecular combining converts the parts into an
amorphous product with a regular form and density,
thereby increasing the dissolution profile of the poorly
soluble drug.
 Instruments used for Preparation
and Analysis
Dissolution Test Apparatus
In the pharmaceutical industry, drug dissolution testing
is routinely used to provide critical drug release
information.
Several dissolution apparatuses exist. In United States
Pharmacopeia (USP) General Chapter <711>
Dissolution, there are four dissolution apparatuses
standardized and specified
The vessels of the dissolution method are usually either
partially immersed in a water bath solution or heated
by a jacket. An apparatus is used on solution within the
vessels for a predetermined amount of time which
depends on the method for the particular drug.
Instruments used for Preparation
and Analysis
 Instruments used for Preparation
and Analysis
UV-Vis Spectrophotometry

It is the most frequently applied technique in

pharmaceutical analysis.
It involves measuring the amount of ultraviolet or
visible radiation absorbed by the substance in the
solution
Qualitative analysis through spectrophotometric
method achieves fast and accurate results using only
small sample quantities.
 Instruments used for Preparation
and Analysis
High Pressure Liquid Chromatography (HPLC)
High-performance liquid chromatography (HPLC) is
renowned for its high precision and accuracy. Despite the
high costs that are associated with the method, it’s a perfect
match for the kind of precise identification and
quantification required for pharmaceuticals.
HPLC is used to analyse pharmaceutical products (drug and
medicine products) for the ingredients they contain. The
method is utilised to separate, quantify and identify the
various components along with their quantities within
products.
Doing so gives developers a better idea of a drug’s
properties, with each component’s quantities affecting the
overall performance and strength of a product, for example.
However, it also allows them to identify and quantify any
impurities within pharmaceutical products.
 BCS Solubility
The BCS (Biopharmaceutics Classification System) is a scientific
approach based on the aqueous Solubility and intestinal
permeability characteristics of the drug substances. The BCS
categorizes Drug substances into one of four BCS classes as
follows:

Class I: high solubility, high permeability

Class II: low solubility, high permeability
Class III: high solubility, low permeability
Class IV: low solubility, low permeability

Solubility: A drug substance is classified as highly soluble if the

highest single therapeutic dose is completely soluble in 250 ml
or less of aqueous media over the pH range of 1.2–6.8 at
37±1°C.

Permeability: The ability of a drug to pass across biological

membrane is defined as drug permeability
 BCS Solubility in 0.30% SLS in Water
Mobile Phase Preparation:
Preparation of pH 2.9 Buffer – Weigh 2.767gm of Potassium Dihydrogen
Phosphate in 1L of water, adjust pH to 2.90 using dilute OPA.
Mobile Phase ratio: 85:15 (Methanol: Buffer)

Media Preparation:
0.30% SLS in water – Weigh 15gm of SLS and dissolve it in 5L of water.

Sample Preparation: Weigh 200mg of API and dissolve it in 250ml of

0.30%SLS in water media

Standard Preparation: Weigh 25mg of API and transfer it into 100ml of

volumetric flask. Add 50ml of ACN to it and sonicate to dissolve. Add 50ml of
0.30% SLS media. Further pipette out 5ml and transfer it to 100ml
volumetric flask, dilute it with 0.30% SLS dissolution media.
 Chromatogram:

Formula for Calculation: Conclusion: 0.30% SLS media gives solubility of 0.0796
mg/ml of Drug “F” API, therefore it will dissolve a maximum
amount of 71.64mg in 900ml which will be Non sink
condition since the strength of the Drug Product under
development is 120mg.
 BCS Solubility in 0.45% SLS in Water
Mobile Phase Preparation:
Chromatogram:
Preparation of pH 2.9 Buffer – Weigh 2.767gm of
Potassium Dihydrogen
Phosphate in 1L of water, adjust pH to 2.90 using dilute
OPA.
Mobile Phase ratio: 85:15 (Methanol: Buffer)
Media Preparation:
0.45% SLS in water – Weigh 22.5gm of SLS and dissolve
it in 5L of water.

Formula for Calculation:

Conclusion: 0.45% SLS media gives solubility of 0.1660
mg/ml of Drug “F” API, therefore it will dissolve a maximum
amount of 149.4mg in 900ml which will satisfy the sink
condition since the strength of the Drug Product under
development is 120mg.
 BCS Solubility in 0.60% SLS in Water
Mobile Phase Preparation:
Chromatogram:
Preparation of pH 2.9 Buffer – Weigh 2.767gm of
Potassium Dihydrogen
Phosphate in 1L of water, adjust pH to 2.90 using dilute
OPA.
Mobile Phase ratio: 85:15 (Methanol: Buffer)
Media Preparation:
0.60% SLS in water – Weigh 30gm of SLS and dissolve it
in 5L of water.
Conclusion: 0.60% SLS media gives solubility of 0.2387
Formula for Calculation:
mg/ml of Drug “F” API, therefore it will dissolve a maximum
amount of 214.83mg in 900ml which will satisfy the sink
condition since the strength of the Drug Product under
development is 120mg.
 BCS Solubility in 0.75% SLS in Water
Mobile Phase Preparation:
Chromatogram:
Preparation of pH 2.9 Buffer – Weigh 2.767gm of
Potassium Dihydrogen
Phosphate in 1L of water, adjust pH to 2.90 using dilute
OPA.
Mobile Phase ratio: 85:15 (Methanol: Buffer)
Media Preparation:
0.75% SLS in water – Weigh 37.5 gm of SLS and dissolve
it in 5L of water.

Formula for Calculation:

Conclusion: 0.75% SLS media gives solubility of 0.3296
mg/ml of Drug “F” API, therefore it will dissolve a maximum
amount of 296.64mg in 900ml which will satisfy the sink
condition since the strength of the Drug Product under
development is 120mg
 HPLC Development Trial-1
Chromatographic Conditions: Standard Chromatogram:
Column: Xterra, C18, 150mm ×4.6,5µ
Wavelength: 285 nm
Flow rate: 1.0 mL/min
Injection volume: 20 µL
Column temperature: 35°C
Run time: 8 minutes
Autosampler temperature: 25°C

Observation:
The retention time of analyte was observed at 3.973 minutes.

Conclusion:
In order to reduce the retention time of analyte Drug Product “F”, method
needs to be modified.
 HPLC Development Trial-2
Chromatographic Conditions: Standard Chromatogram:
Column: Xterra, C18, 150mm ×4.6,5µ
Wavelength: 285 nm
Flow rate: 2.0 mL/min
Injection volume: 20 µL
Column temperature: 30°C
Run time: 20 minutes
Autosampler temperature: 25°C

Observation:
The retention time of analyte was observed at 3.803 minutes.

Conclusion:
In order to reduce the retention time of analyte Drug Product “F”, method
needs to be modified.
 HPLC Development Trial-3
Chromatographic Conditions: Standard Chromatogram:
Column: Waters symmetry C18, 150mm ×4.6,5µ
Wavelength: 285 nm
Flow rate: 2.0 mL/min
Injection volume: 20 µL
Column temperature: 30°C
Run time: 6 minutes
Autosampler temperature: 25°C

Observation:
The retention time of analyte drug molecule “F” was observed at 2.520
minutes. A new column with same parameters but less particle pore size was
used which led to decrease in retention time.

Conclusion:
This is a tentatively developed method which can be used for routine
analysis. The method is yet to be validated.
 Selection of Discriminatory Media
Aim: To find percentage release of RLD of drug product “F” in 0.45% and 0.75% SLS dissolution media by HPLC.

Preparation of Dissolution Media:

1. 0.45% SLS in water – Weigh 22.5gm of SLS and dissolve it
in 5L of water, sonicate for complete dissolution.
2. 0.75% SLS in water – Weigh 37.5gm of SLS and dissolve it
in 5L of water, sonicate for complete dissolution.

Dissolution Release Profile Comparison:

The dissolution profile results showed a significant difference
in the dissolution profile of the 120 mg RLD tablet in the two
dissolution media namely:0.45% SLS in water and 0.75% SLS in
water. The results showed that in both the media 100%
dissolution was achieved, but in the 0.45% SLS in water media
the rate of dissolution in the initial time points was slower
compared to that of 0.75% SLS in water.

Conclusion: Therefore it is concluded that media of 0.45%

SLS in water can be used as a discriminatory media to detect
changes in the formulation during development of the drug.
 Co-Relation of HPLC and
UV-Vis Spectroscopy
Release profile of Drug Product ‘F’ RLD in 0.75% SLS in
water media was compared using two methods viz. HPLC
and UV-VIS Spectroscopy.
Release % in both the methods was found around 100%
at the end of 1 hour. Even when compared in different
media i.e. 0.45% SLS in water the results highly correlate
to each other for both the methods used.
Therefore, it can be concluded that both the methods
(HPLC and UV-VIS Spectroscopy) were highly co-related
for the Drug Product ‘F’ formulation.
Though UV-VIS Spectroscopy is a simple method for
analysis it is time consuming and tedious. While HPLC is
widely used because of its accuracy.
The plot above for both the methods show high
correlation (R2= 0.999) in the results given by both the
methods.
 Dissolution Development Trial-1
Aim: To determine and compare solubility of Drug “F” API and Blend of Drug “F” API passed through HME using UV-VIS
Spectrophotometer. (Batch – 1A and 1X). (A- HME, X-Non HME).
Media Preparation:
0.75% SLS in water – Weigh 37.5gm of SLS and dissolve it in 5L of water, sonicate for complete dissolution.
Standard Preparation: Weigh 25mg of API and transfer it into 100ml of volumetric flask. Add 50ml of ACN to it and
sonicate to dissolve. Add 50ml of 0.75% SLS media. Further pipette out 5ml and transfer it to 100ml volumetric flask, dilute it
with 0.75% SLS dissolution media.
Sample Preparation:
1. Weigh 240mg of Blend and place it in 900ml of 0.75%SLS in water media.
2. Weigh 120mg of API and place it in 900ml of 0.75%SLS in water media.
3. Filter the aliquot with 0.45 µ Nylon filter in dry test tube by discarding 3-4 mL of initial filtrate.
4. Further 2ml of this solution in 20 mL volumetric flask dilute it with dissolution media.
5. Record absorbance on UV-VIS spectrophotometer.
Dissolution Parameters: Release Profile:
Dissolution Media: 900 ml of 0.75% SLS in water
Apparatus: USP Type II (Paddle)
RPM: 75
o
Temperature: 37 ± 0.5 C
Time interval: 1 Hour
Media Volume: 900ml
Withdrawn Volume: 10ml aliquots and 3.5ml discard

Result and Conclusion:

Release of blend passed through HME is more than
that of pure API, hence it proves that using HME
method results in increasing the solubility of Drug
product “F”.
 Dissolution Development Trial-2
Aim: To determine release of Inhouse Formulated Drug Product “F” and compare it with RLD. (Batch - 3A and 3X). (A- HME,
X- Non-HME).
Media Preparation:
0.45% SLS in water – Weigh 45 gm of SLS and dissolve it in 10L of water, sonicate for complete dissolution.
Standard Preparation: Weigh 25mg of API and transfer it into 100ml of volumetric flask. Add 50ml of ACN to it and
sonicate to dissolve. Add 50ml of 0.45% SLS media. Further pipette out 5ml and transfer it to 100ml volumetric flask, dilute it
with 0.45% SLS dissolution media.
Sample Preparation:
1. Weigh each tablet and place it in 900ml of 0.45%SLS in water media.
2. Filter the aliquot with 0.45 µ Nylon filter in dry test tube by discarding 3-4 mL of initial filtrate
3. Further 2ml of this solution in 20 mL volumetric flask dilute it with dissolution media.
4. Inject it in HPLC
Dissolution Parameters: Release Profile:
Dissolution Media: 900 ml of 0.75% SLS in water
Apparatus: USP Type II (Paddle)
RPM: 75
Temperature: 37 ± 0.5 C
Time interval: 1 Hour
Media Volume: 900ml
Withdrawn Volume: 10ml aliquots and 3.5ml discard

Result and Conclusion:

The batch passed through HME shows comparatively
more release when compared to the Non-HME
batch. Still the release profile is not up to the mark of
RLD, hence more modification in the batch is
required
 Dissolution Development Trial-3
Aim: To determine release of In-house Formulated batch in 0.45% SLS and 0.75% SLS, and compare it with RLD. (Batch 4A).
Media Preparation:
0.45% SLS in water – Weigh 45 gm of SLS and dissolve it in 10L of water, sonicate for complete dissolution.
0.75% SLS in water - Weigh 37.5 gm of SLS and dissolve it in 5L of water, sonicate for complete dissolution.
Standard Preparation:
Weigh 25mg of API and transfer it into 100ml of volumetric flask. Add 50ml of ACN to it and sonicate to dissolve. Add 50ml of
0.45% SLS media. Further pipette out 5ml and transfer it to 100ml volumetric flask, dilute it with 0.45% SLS dissolution
media.
Weigh 25mg of API and transfer it into 100ml of volumetric flask. Add 50ml of ACN to it and sonicate to dissolve. Add 50ml of
0.75% SLS media. Further pipette out 5ml and transfer it to 100ml volumetric flask, dilute it with 0.75% SLS dissolution
media.
Sample Preparation:
1. Weigh each tablet and place it in 900ml of 0.45%SLS in water media.
2. Filter the aliquot with 0.45 µ Nylon filter in dry test tube by discarding 3-4 mL of initial filtrate
3. Further 2ml of this solution in 20 mL volumetric flask dilute it with dissolution media.
4. Inject it in HPLC
Dissolution Parameters: Release Profile:
Dissolution Media: 900 ml of 0.45% and 0.75% SLS in
water
Apparatus: USP Type II (Paddle)
RPM: 75
Temperature: 37 ± 0.5 C
Time interval: 1 Hour
Media Volume: 900ml
Withdrawn Volume: 10ml aliquots and 3.5ml discard

Result and Conclusion:

Batch 4A shows release which is closer to the RLD
compared to previous batches. Still more
optimization is needed.
 Dissolution Development Trial-4
Aim: To determine release of In-house Formulated batch in 0.45% SLS and 0.75% SLS, and compare it with RLD. (Batch 6A).
Media Preparation:
0.45% SLS in water – Weigh 45 gm of SLS and dissolve it in 10L of water, sonicate for complete dissolution.
0.75% SLS in water - Weigh 37.5 gm of SLS and dissolve it in 5L of water, sonicate for complete dissolution.
Standard Preparation:
Weigh 25mg of API and transfer it into 100ml of volumetric flask. Add 50ml of ACN to it and sonicate to dissolve. Add 50ml of
0.45% SLS media. Further pipette out 5ml and transfer it to 100ml volumetric flask, dilute it with 0.45% SLS dissolution
media.
Weigh 25mg of API and transfer it into 100ml of volumetric flask. Add 50ml of ACN to it and sonicate to dissolve. Add 50ml of
0.75% SLS media. Further pipette out 5ml and transfer it to 100ml volumetric flask, dilute it with 0.75% SLS dissolution
media.
Sample Preparation:
1. Weigh each tablet and place it in 900ml of 0.45%SLS in water media.
2. Filter the aliquot with 0.45 µ Nylon filter in dry test tube by discarding 3-4 mL of initial filtrate
3. Further 2ml of this solution in 20 mL volumetric flask dilute it with dissolution media.
4. Inject it in HPLC
Dissolution Parameters: Release Profile:
Dissolution Media: 900 ml of 0.45% and 0.75% SLS in
water
Apparatus: USP Type II (Paddle)
RPM: 75
Temperature: 37 ± 0.5 C
Time interval: 1 Hour
Media Volume: 900ml
Withdrawn Volume: 10ml aliquots and 3.5ml discard

Result and Conclusion:

Release profile of Batch 6A matches with RLD and
hence it can be concluded that the Drug Product ‘F’
formulation can be finalized and further studied for
stability.
 Conclusion
Drug ‘F’ is an anti-hyperlipidemic drug which is used to lower the cholesterol in body. API of Drug ‘F’ was
dissolved in different concentration of SLS i.e. 0.30%, 0.45%, 0.60%, 0.75% because it is a class II drug
and hence highly insoluble in water.
It was found that solubility is directly proportional to the amount of SLS added and hence the media of
0.75% SLS in water was chosen as OGD media and 0.45% SLS in water was chosen as discriminatory
media since the changes done in formulated could be detected. API of Drug ‘F’ is pH independent hence
there no effect observed when pH of the media is altered.
A tentative HPLC method was developed by referring to USP guidelines and the developed method was
reproducible as it gave release of Drug Product ‘F’ within the acceptance criteria. The developed method
is yet to be validated.
Different trials were taken which included various batches and it was concluded that batches passed
through HME showed more dissolution. After a number of changes were done in formulation the
developed Drug Product ‘F’ matched with RLD and the batch was finalized. Further study is still being
done on the product.
Thank you
for listening!