RP-HPLC METHOD DEVELOPMENT AND VALIDATION

FOR ESTIMATION OF TRIAMCINOLONE ACETONIDE IN

INJECTABLE SUSPENSION USING USP-TYPE-IV
DISSOLUTION APPARATUS

Under the Supervision of:

Dr. M.Ajitha
M.Pharm, Ph.D
Associate Professor
BOS, Chairperson

Presented by:
D.Sumanth
14031S0405

Under the Guidance of:

K. Madhu
Manager

CONTENTS:
AIM & OBJECTIVE OF WORK
INTRODUCTION
LITERATURE REVIEW
DRUG PROFILE
PROGRESS OF WORK
VALIDATION
SUMMARY
REFERENCE

1.AIM & OBJECTIVE OF WORK

:

To develop a economical, simple, precise, accurate and

reproducible method for estimation of Triamcinolone
acetonide in injection suspension dosage form.

To validate the developed method as per ICH

guidelines.

2.INTRODUCTION:
DISSOLUTION:

Dissolution is the process by which a solid solute

enters in to a solution.i.e, mass transfer from solid
surface to liquid phase.
why dissolution studies?
To show that the rate of drug release is uniform batch
to batch.
To show that release is equivalent to those batches
proven to be bioavailable and clinically effective.

Various types of dissolution apparatus:

USP.APPARATUS

DESCRIPTION

DOSAGE FORM

TYPE 1

Basket apparatus

IR,DR,ER

TYPE 2

Paddle apparatus

IR,DR,ER

TYPE 3

Reciprocating cylinder

IR,ER

TYPE 4

Flow through cell

ER , Poorly soluble

TYPE 5

Paddle over disk

TRANSDERMAL

TYPE 6

Rotating cylinder

TRANSDERMAL

TYPE 7

Reciprocating holder

ER

APPARATUS-4 (FLOW THROUGH CELL)

Schematic representation of working of flow

through cell

(A)open system of a flow-through cell

(B) closed system of a flow-through cell

 DESIGN:

Reservoir : -For dissolution medium

Pump : -Forces dissolution medium through cell
Cells : -Holding a sample(12mm and 22.6mm i.d)
Beads: 5mm ruby bead and 1mm glass beads are used
Water bath:- Maintain at 370.5C
USE:

Low solubility drugs ,micro particulates ,implants,

suppositories controlled release formulations

Cell types:

Tablets 12 mm
Tablets 22.6 mm
Powders / Granules
Suppositories /
Soft gelatincapsules

Implants

HPLC : High performance liquid chromatography

Itis a technique in analytic chemistry
used to separate the components in a
mixture, to identify each component, and
to quantify each component
The basic principle involved is adsorption
Types :
Normal phase chromatography
Reverse phase chromatography

Schematic representation of HPLC:

Steps followed for method development

Literature survey
Study of physical properties of drug
Optimization of analytical method
Validation of developed method
Summary and conclusion
Results and discussion

3.LITERATURE REVIEW:
Literature review was done seeing for an method on
estimation of triamcinolone acetonide in injectable
suspensions
Pharmacopoeias like USP , B.P, I.P, E.P
Scholarly articles from pubmed , science direct
Internet

Assay procedures recommended in pharmacopoeias

USP:
Column:4mm30mm column containing packing L1
Wavelength:254
Retention time:14.5
Mobile phase : ACN:Water(30:70)
European pharmacopoeia:
50mg of drug in 50ml of alcohol and from this 2ml is diluted
to 100 with alcohol and absorbance is measured at 238.5
with specific absorbance to be 355
Indian pharmacopoeia:
Accurately weighed 25mg dissolved in ethanol to produce 100ml
and mix.dilute 5.0ml to 100.0ml with ethanol and measured at
239 with specific absorbance to be 354

4.DRUG PROFILE:
Triamcinolone acetonide:
Molecular weight:434.49

g/mol
Molecular formula:C24H31FO6
Chemical structure
Chemical name: 9 -fluoro-11 ,21dihydroxy16 ,17 -isopropylidenedioxy-l,4pregnadiene-3,20-dione.

TECHNICAL INFORMATION:
Appearance

:Crystalline

Physical State

:Solid

Solubility

:Insoluble in water

sparingly soluble in alcohol

Storage

:protected from light

Melting point

:292-294C

Specific rotation :+118 - +130

Refractive Index :99.38 m3mol-1(Predicted).

Mechanism of action:
Triamcinolone acetonide binds to specific cytosolic
glucocorticoid receptors and subsequently interacts with
glucocorticoid receptor response element on DNA and alters gene
expression. This results in an induction of the synthesis of certain
anti-inflammatory proteins while inhibiting the synthesis of
certain inflammatory mediators. Consequently, an overall
reduction in chronic inflammation and autoimmune reactions are
accomplished.

Category :Anti- inflamatory and Immunosuppressive

5.PROGRESS OF WORK:
Instruments and material used:
Dissolution tester :CE7 smart, Make : Sotax
HPLC systems
:Agilent with VWD/DAD Model:1200series

:waters with VWD/DAD Model:2690

Chemical

Water
Filters
sample
suspension

:Sodium dodecyl sulfate

:Acetonitrile(HPLC grade)
:Triamcinolone Acetonide (standard)
:Milli-Q grade
:Glass micro fiber GF/C(whatmann),25mm
:Triamcinolone Acetonide Injectable

METHOD(Flow through cell):

The flow through cell is transparent & inert mounted vertically
with filters.
Standard cell diameters are 12mm & 22.6 mm i.d
The bottom cone usually placed with Ruby beads of 5mm
diameter.
Place the glass beads of 7g into the cell
The weighed sample is placed over the beads and the sample is
sandwiched with another 6g of sample
Assemble the filter head and fix the parts together by means of a
suitable clamping device.
Introduce by the pump of the dissolution medium warmed to
370.5 through the bottom of the cell to obtain the flow rate
specified and measured with an accuracy of 5%.
Collected the eluate by fractions at each of the times stated.

Dissolution parameters
Media
:sodium dodecyl sulphate solution
Temperature
:370.5C
Flow rate
:4,8,16 ml per minute
Flow type
:laminar or turblent flow
Filters
:glass fiber and glass wool filters
Sample volume :20ml
Factors influencing drug release in dissolution apparatus
Media
Flow rate

: 0.35% & 0.5% of sodium dodecyl sulphate

:4,8ml per minute

Trial-1
Media
Flow rate

: 0.35% of sodium dodecyl sulphate

:4ml per minute
Observation:Drug release was less it is upto 80% only

Trial-2
Media
Flow rate

: 0.35% of sodium dodecyl sulphate

:8ml per minute
Observation:Drug release was attained upto 90% but
dissolution time is very long
Trial-3
Media
Flow rate

: 0.5% of sodium dodecyl sulphate

:8ml per minute
Observation:Drug release was attained but dissolution
time
is also optimised

Optimised dissolution parameters

Media
:0.50% sodium dodecyl sulphate
Temperature
:370.5C
Apparatus
:USP type IV open loop Offline
Type of cell
:22.6mm
Glass beads loading :13g
Type of filter
:glass micro fiber GF/C (whatmann)
Flow rate
:8 ml/minute
Sample volume :20ml
Time points
:5,10,15,20,30,40,50,60,70,80,90&120
minutes

PREPARATION OF SOLUTIONS:
Preparation of dissolution media:
Weigh and transfer 5.0g of sodium dodecyl sulphate into 1000ml of
water
Diluent preparation:
Water and Acetonitrile are mixed in ratio of 35:65% v/v
Standard stock preparation:

Weigh and quantitatively transfer about 45mg of triamcinolone

acetonide into 50ml volumetric flask. Add about 35ml of diluent
and sonicate to dissolve and the volume is made with dissolution
media
Standard preparation:
3ml of above solution was transferred to 200 ml volumetric flask
23

Steps followed in method development of RP-HPLC

Solubility analysis
Selection of wavelength
Selection of column
Selection of mobile phase
Solubility analysis:
Triamcinolone acetonide is insoluble in water
It is sparingly soluble in alcohol
Selection of wavelength:
The standard solution was scanned under uv spectrum to
know max the solution and it is found to be 254nm

Trial-1

Column
: ODS 2504.6mm,5m
Mobile phase
:30:70(Acetonitrile : Water)
Flow rate
:1.0ml/min
Injection volume
:10l
Wavelength
:254nm
Column temperature:30C
Runtime
:20min

Observation : The Rt of drug was found to be at

11.3min with plate count less than 2000

Trial-2

Column
:Zorbax Eclipse C181504.6mm,5m
Mobile phase
:30:20:50(ACN : methanol : water)
Flow rate
:1.2ml/min
Injection volume :10l
Wavelength
:254nm
Column temperature:30C
Runtime
:20min

 Observation : the Rt was at 5.03 min with tailing

factor greater than 2

Trial-3

Column
:Inertsil ODS 3V 1504.6mm,5m
Mobile phase
: 50:50 (ACN : water)
Flow rate
:1ml/min
Injection volume :10l
Wavelength
:254nm
Column temperature:30C
Runtime
:5 min

 Observation : the Rt was at 3.3 min with less number

of theoretical plates tailing factor greater than 2

Optimised method:

Column
:Inertsil ODS 3V 1504.6mm,5m
Mobile phase
:60:40(ACN : water)
Flow rate
:1ml/min
Injection volume :10l
Wavelength
:254nm
Column temperature:30C
Runtime
:5min

 Observation : the Rt was at 2.76min with 5592

theoretical plates and usp tailing of 1.2

6. Validation:
To perform Validation studies for the developed method and
the Validation parameters include:
Specificity
Linearity
Accuracy
Precision
LOD
LOQ
Robustness
System suitability

A. Specificity:
I. Blank interference:
Dissolution medium is used as blank sample solution and it should
not show any peak in the chromatogram.
II. Placebo interference:
Preparation of placebo:0.2 g of Polysorbate 80 was weighed and
transferred into 500ml volumetric flask and sonicated for 30min to
form clear solution and to it 7.5 g of Carboxymethyl cellulose, 1 g
of sodium chloride and 1 ml of benzyl alcohol was added and
sonicated to form clear solution.
Blank and Placebo samples are injected and analysed.

Chromatogram of Blank

Chromatogram of Placebo
Observation: No interference of Blank or Placebo is observed

B. Linearity:
Preparation of stock solution: Weighed and transferred 55.625 mg of
Triamcinolone Acetonide in to 50ml volumetric flask added 30ml of
diluent sonicate for 5min to dissolve and make up to the mark with diluent.
Dilutions are made from stock solution to get required ppm and made the
volume with dissolution media.
Dilution

ppm Obtained

Area Obtained

1 ml in 250 ml

4.45

65229

1 ml in 100 ml

11.12

156878

2 ml in 100 ml

22.24

314849

3 ml in 100 ml

33.36

462506

4 ml in 100 ml

44.48

625447

5 ml in 100 ml

55.60

753576

6 ml in 100 ml

66.72

911033

Typical chromatogram of Linearity 10%sample

Typical chromatogram of Linearity

25% sample

Typical chromatogram of Linearity 50%sample

Typical chromatogram of Linearity 75%

sample

Typical chromatogram of Linearity

100% sample

Typical chromatogram of Linearity 125%

sample

Typical chromatogram of Linearity

150%sample
Linearity of Triamcinolone Acetonide
1000000
f(x) = 13563.39x + 8678.99
R = 1

800000
600000
Response 400000
area
200000
0
0

10

20

30

40

50

60

70

Concentration in g/ml

Observation: Correlation Coefficient was found to be 0.999

80

C. Accuracy
A series of sample solutions were prepared in triplicate by
spiking the Triamcinolone Acetonide with placebo in the range
of 10% to 150% and injected into HPLC system and analyzed.
Amount
added(mg/ml)

Amount
recovered(mg/ml)

% Recovery

0.0045

0.00445

98.9

0.0045

0.00453

100.7

3.

0.0045

0.00458

101.8

1.

0.0453

0.0444

98.0

0.0453

0.0458

101.1

3.

0.0453

0.0448

98.9

1.

0.0542

0.0555

102.4

0.0542

0.0543

100.2

0.0542

0.0541

99.8

S. No.

% Spike
level

1.
2.

2.

2.
3.

10%

100%

120%

Mean
%recovery

%RSD

100.4

1.5

99.3

100.8

1.6

1.4

Observation: The amount of recovery is found to be 95 105 %.

Typical chromatogram of Accuracy 10%sample

Typical chromatogram of Accuracy

100%sample

Typical chromatogram of Accuracy

120%sample

D. Precision

S. No

% Sipe Level
Amount added
(mg/ml)

0.0453

0.0453

3
100

4
5
6

0.0453
0.0453
0.0453
0.0453

Amount
Recovered(mg/ml)

0.0445
0.0458
0.0452
0.0449
0.0448
0.045

% Recovery

Mean %Recovery

%RSD

98.23399558

0.974

98.23399558
101.1037528
99.77924945
99.11699779
98.89624724
99.33774834

Observation: The % RSD was found to be <2 at 6 replicates of

100% concentration.

Typical chromatogram of Precision

100%sample

E. Limit of Detection
A series of solution were prepared and injected till the Signal to
Noise ratio is greater than 3.

Observation: At a concentration of 0.18g/ml the Signal to Noise

ratio is observed to be 3.3

F. Limit of Quantification
Solutions approximately 3.3 times the concentration of LOD was
prepared and injected to observe Signal to Noise ratio of 10

Observation : At a sample concentration of 0.56g/ml the

Signal to Noise ratio is found to be 10.5

G. Robustness
Change in flow rate:

System suitability
parameters

As such

Results
+Flow

-Flow

(1.0
mL/min)

(1.2
mL/min)

(0.8
mL/min)

Acceptan
ce
criteria

% RSD for analyte peak

areas of five replicate
standard injections
Theoretical plates

0.09

0.17

0.14

NMT 2.0

5035

4113

5440

NLT 2000

Tailing factor

1.1

1.1

1.1

NMT 2.0

Change in temperature
Results
System suitability
parameters

As such
(25C)

-Column oven
temperature
(20C)

+Column
oven
temperature

Accepta
nce
criteria

(30C)

% RSD for analyte peak

areas of five replicate
standard injections

0.09

0.15

0.44

NMT
2.0

Theoretical plates

5035

4766

4776

NLT
2000

Tailing factor

1.1

1.1

1.1

NMT
2.0

H. System suitability
100% solution was prepared and injected as six replicates
System suitability parameters

Results

Acceptance criteria

% RSD for analyte peak areas of five replicate standard

injections

0.26

NMT 2.0

Theoretical plates

6123

NLT 2000

Tailing factor

1.24

NMT 2.0

Summary
Parameters

Linearity range (g / mL)

Triamcinolone Acetonide

4.5-66.72 ppm

Optimized wavelength

254 nm

Retention time

2.76

Regression equation (Y)

0.999

Correlation coefficient(r2)

y = 13563x+8679

Precision (% RSD)

3.5

Percentage Recovery

99.3

Limit of Detection (g/mL)

0.18

Limit of Quantification (g /mL)

0.56

7.REFERENCE
Pharmacopoeias USP , B.P , I.P
http://www.slideshare.net/shettyuc/dissolution-33496242/1
http://www.accessdata.fda.gov/scripts/cder/dissolution/dsp_g
etallData.cfm
http://en.chembase.cn/molecule-165706.html
www.dissolutiontech
.com/DTresour/201111Articles/DT200505_A05.pd
www.dissolutiontech
.com/DTresour/201111Articles/DT201111_A06.pd
www.dissolutiontech
.com/DTresour/201111Articles/DT201111_A02.pd

https://www.researchgate.net/publication/237445416
www.usp.org/sites/default/files/usp_pdf/EN/gc_1092.pdf
https://hmc.usp.org/sites/default/files/documents/HMC/GCs
-Pdfs/c
1225.pdf
www.us.edu.pl/uniwersytet/jednostki/wydzialy/chemia/acta
/ac18/.../11_AC18.pdf
www.chromacademy.com

Thank you