Stability indicating assay

STABILITY STUDIES OFSTABILITY STUDIES OF
PHARMACEUTICALSPHARMACEUTICALS
By.
Dr. Archana Naik

OUTLINE:Introduction.
Stability studies.
• International stability guidelines.
• Stages of stability studies
Stability indicating assay methods (SIAMs)
• Definition
• Need
• Types of SIAMs.
• Development of validated SIAMs.
Case studies.
Conclusion.
References.

INTRODUCTIONINTRODUCTION
“The capability of a particular formulation
( dosage form or drug product) in a
specific container/closure system to
remain within its physical, chemical,
microbiological, therapeutic and
toxicological specifications throughout its
shelf life.”1
To detect and measure the changes which occurs in the
product to check that the product is within limits of
specification- a tool called stability indicating method is
used.2
Current ICH stability guidelines- ‘Gold standard’ for
conducting stability studies.
1) G.T. Kulkarni,K. Gowthamarajan,B. Suresh, Indian Journal Of Pharmaceutical Education, Vol. 38,
issue 4, Oct-Dec. 2004 page no.194.
2) Manutosh M. Acharya, Eastern Pharmacist,vol.42, 1999 May, page no.31-36.

STABILITY STUDIESSTABILITY STUDIES
To ensure the efficacy, safety and quality of
active drug substance and dosage forms1
To establish re-test period for the drug
substance or shelf life for the drug product and
recommended storage conditions1
Physical, chemical or microbiological changes
impact the efficiency, integrity and security of the
final product1
Stability Studies are performed on
Drug Substances (DS)
And Drug Products (DP)1
issue 4, Oct-Dec. 2004 page no.194

INTERNATIONAL STABILITY GUIDELINESINTERNATIONAL STABILITY GUIDELINES11
ICH Guidelines
FDA (Food and Drug Administration)
WHO (World Health Organization)
ASEAN Guidelines
USP (US Pharmacopoeia)
issue 4, Oct-Dec. 2004 page no.194

.. AND HOW DOES ALL WORKS TOGETHER?.. AND HOW DOES ALL WORKS TOGETHER?1111
ICH Q1AICH Q1A
R2R2
WHOWHO
ASEANASEAN
FDAFDA
Stability Testing of New DS and DP
(Climatic Zone I and II)
ICHQ1B
Photo stability
ICHQ7
GMP
ICH
Q6A/B
Specifications
ICH
Q5
Biotechnogical
products
ICH
Q3A/B/C
Impurities ICH
Q2R1
Analytical
Validation
ICH
Q1E
Evaluation of
Stability Data
ICH
Q1D
Bracketing and
Matrixing
ICH
Q1C
New Dosage
Forms
11) www.ich.org.com

STAGES OF STABILITY STUDIESSTAGES OF STABILITY STUDIES1010
Early stage stress and accelerated testing with Drug
Substances
Stability on pre-formulation batches
Stress testing on scale-up batches
Long term testing for registration purposes
On-going Stability Testing
Follow-up Stabilities
10) Carstensen, J.T., Drug stability: Principles and Practices, New York,
Marcel Dekker,vol107, 1995.

STABILITY INDICATINGSTABILITY INDICATING
ASSAY METHODSASSAY METHODS
[SIAMs][SIAMs]

SIAMsSIAMs
‘Validated quantitative analytical methods that
can detect the changes with time in the chemical,
physical, or microbiological properties of the drug
substance and drug product, and that are speciﬁc
so that the contents of active ingredient,
degradation products and other components of
interest can be accurately measured without
interference.’5
5) S. Singh, Journal of pharmaceutical and biomedical analysis, vol.28(2002)
page no.1011-1040.

Need of SIAMsNeed of SIAMs
Non-specificity of assay procedure5
Essential for new drug molecule- unknown degradation products,
known/unknown impurities
structurally related byproducts
and intermediates5
Essential for new dosage forms - unstability of API
incompatibility with other actives
and inactives5
page no.1011-1040.

Types of SIAMs5
Methods Description Example
Titrimetry Non-aqueous titration
Oxidation-reduction
Iodometric assay
Amine,sulphonamides etc
Ferric ions
ß-lactam antibiotics
UV/VIS
Spectrophotometry
Spectral selectivity can be
increased by use of difference
spectrophotometry and dual
wavelength spectrophotometry
Dicloxacillin and Ampicilin
etc.
Chromatographic
TLC Used especially during initial
stress testing to study
the number of degradation
Products formed
Limitations: variability, non-
quantitative
Cephalosporins etc.
page no.1011-1040.

Types of SIAMsTypes of SIAMs55
Methods Description Example
HPTLC
GC
HPLC
Reliable, fast and accurate for quantitative drug
analysis and many samples can be run
simultaneously using a small quantity of mobile
phase
Is not very versatile, as the drug
substance may be nonvolatile or thermally
unstable
High-resolution capacity, sensitivity and
specificity
Non-volatile, thermally unstable or
polar/ionic compounds
Oxyphenbutazone
Impurities of
ampicilin sodium
etc
Atenolol, danozol,
Nifedipine,
Piroxicam,
Captopril,
Trimethoprim etc.
page no.1011-1040.

DEVELOPMENT OF VALIDATED SIAMsDEVELOPMENT OF VALIDATED SIAMs

Step IStep I: Critical study of the drug structure to assess: Critical study of the drug structure to assess
the likely decomposition route(s)the likely decomposition route(s)
Much information can simply be gained from the structure, by study of
the functional groups and other key components.5
Different
functional
groups
amides, esters,
Lactams
lactones, etc
thiols,
thioethers,
etc.
olefins, aryl
halo derivatives,
aryl acetic acids
hydrolysis oxidation photodecomposition
page no.1011-1040.

Step IStep I: Critical study of the drug structure to assess: Critical study of the drug structure to assess
the likely decomposition route(s)the likely decomposition route(s)
Most of the new drugs are congeners of existing drug molecules
Very few new drugs, which originate from absolutely new leads5
For example, there are more than 40 penicillins and almost all of them
follow the same degradation behavior at the beta- lactam moiety.5
Fig. 1. Known degradation behavior of penicillins under different hydrolytic conditions5
page no.1011-1040.

Step IIStep II: Collection of information on: Collection of information on
physicochemical propertiesphysicochemical properties55
PARAMETER IMPORTANCE
pka most of the pH-related changes in retention occur at pH
values within ±1.5 units of the pKa value.
Helps in selecting the pH of the buffer to be used in the
mobile phase.
LogP provides fair idea about the separation behavior likely to be
obtained on a particular stationary phase
Solubility The drug or degradation products should be soluble in HPLC
compatible solvents
useful in the selection of the sample solvent and the mobile
phase.
Wavelength
maxima
HPLC analysis employing a UV detector is usually carried out
at the wavelength maximum
page no.1011-1040.

Step IIIStep III: Stress (forced decomposition) studies: Stress (forced decomposition) studies
The ICH guideline Q1A(R2) suggests the following conditions to be
employed:
1) 10 °C increments above the accelerated temperatures (e.g. 50 °C,
60 °C, etc.),
2) humidity (e.g. 75% or greater),
3) hydrolysis,
4) oxidation and
5) Photolysis.3
3) International Conference On Harmonization (ICH) Q1A(R2) guidelines 2005

1) Acid and alkali hydrolysis
Figure No 2: Flow chart of acid/alkali induced hydrolysis8
8) S. Singh, M. Bakshi, Guidance on conduct of stress tests to determine inherent stability of drugs,
Pharm. Tech Asia online, April 2000.

2) Oxidation
Figure No 3: Flow chart of oxidation8

3) Photolytic degradation
Figure No 4: Flow chart of Photo stability8

4) Neutral hydrolysis
By refluxing the drug in water for 12 hours.5
5) Thermal Degradation
Heating the drug powder
at high temperature in oven5
Wet heat:Dry heat:
Refluxing the drug
solution for several hrs.5
page no.1011-1040.

Step IVStep IV: Preliminary separation studies: Preliminary separation studies
Done to study the number and types of degradation products formed
under various conditions.5
Observation of the fall in drug peak followed by a corresponding rise in
the degradation product peaks is important
In some situations, no additional peak appears in the chromatogram,
other than the drug.5
Fig. 5. Degradation of metronidazole in acidic conditions under light:
(a) initial sample (b) 3 day sample (c) 12 day sample5
page no.1011-1040.

Step VStep V: Analytical method development and optimization: Analytical method development and optimization
The RT and relative retention times (RRT) of all products formed
should be tabulated for each reaction condition specially for those
components whose RT or RRT is very close.5
In acidic condition drug peak definite RT
In alkali condition drug peak same RT
But appears different
By PDA
By LC-MS
To separate close or co-eluting peaks, the method is optimized,
by changing the mobile phase ratio, pH, flow rate, temperature, solvent
type and the column and its type.5
page no.1011-1040.

Step VStep V: Analytical method development and optimization: Analytical method development and optimization
Finally, mixture of the reaction solutions is again subjected to
resolution behavior study5
Fig.6. Example showing separation of different degradation
products of ornidazole in a mixture of reaction solutions5
page no.1011-1040.

Step VIStep VI: Identification and characterization of: Identification and characterization of
degradation productsdegradation products
Identification and
characterization
Conventional Modern
Isolation and determining
the structure by spectral
(MS,NMR,IR) and
Elemental analysis5
Use of HPLC and
hyphenated
techniques
(LC-MS and LC-MS-MS)5
page no.1011-1040.

Step VIIStep VII: Validation of SIAMs: Validation of SIAMs
Two stages
First stage- is early in the development cycle
when drug substance is subjected to forced
decomposition studies5
Second stage- when the SIAM developed and
the emphasis is to just prove the pertinence of
the established validation parameters in the
presence of excipients or other formulation
constituents5
9) ICH, Q2(R1), Validation of analytical procedure: Methodology, proceedings of International
Conference on Harmonization, London,2005.
5) S. Singh, Journal of pharmaceutical and biomedical analysis, vol.28(2002) page no.1011-1040.
Fig.7 Validation parameters9

Case study-1Case study-1
Validated stability-indicating HPLC method for simultaneous
determination of ampicillin and cloxacillin in combination drug
products6
Degradation studies were carried out in acidic, alkaline, neutral,
oxidative,
light and thermal conditions as per ICH.6
Developed SI HPLC method:
Acetonitrile: phosphate buffer (pH 5.0) in the ratio of 15:85 (v/v) for
1 min, then changed to 30:70 (v/v) for next 14 min finally equilibrated
back to the same ratio of 15:85 (v/v) from 15 to 20 min.6
6) V. kumar, H. Bhutani, S. Singh, Journal of pharmaceutical and biomedical analysis,
vol.43(2007) page no.769-773.

Fig. 8. Chromatograms showing resolution of components in (a) blank (b) mixture of stress samples
(c) degraded formulation6
Key: Acidic condition: II,III & IV alkaline condition: I,III& IV neutral condition: I,III,V& VII,
oxidative condition: I, IV&V light and thermal condition: II, V& VI
vol.43(2007) page no.769-773.

Validation of SI HPLC Method6
Drug Added
(ug/ml)
Intra-day precision
Found±S.D. (ug/ml),
R.S.D. (%)
Inter-day precision
Found±S.D. (ug/ml), R.S.D. (%)
Ampicillin 200
400
600
800
1000
204.64 ±0.24, 0.11
402.37 ±0.15, 0.03
602.11 ±3.52, 0.58
791.79 ±7.17, 0.90
1003.09 ±2.45, 0.24
207.79 ±2.01, 0.96
405.29 ±0.32, 0.08
600.54 ±2.37, 0.39
792.31 ±2.08, 0.26
1002.58 8.76, 0.87
Cloxacillin 200
400
600
800
1000
202.35 ±0.37, 0.18
400.15 ±1.25, 0.31
602.42 ±3.51, 0.58
792.91 ±7.38, 0.93
1003.56± 2.56, 0.25
202.57 ±2.76, 1.36
403.64 ±4.85, 1.20
600.09 ±5.03, 0.83
786.60 ±5.03, 0.63
994.57 ±1.86, 0.18
Intra-day and inter-day precision studies (n=3)
vol.43(2007) page no.769-773.

Case study-2Case study-2
Stability-indicating HPTLC determination of imatinib
mesylate in
bulk drug and pharmaceutical dosage form7
Advantages:
Lowers analysis time and cost per analysis
Mobile phase having pH 8 and above can be employed
Dirty or turbid samples can be directly applied
Simultaneous assay of several components in a
multicomponent
formulation is possible7
Degradation studies were carried out in acidic, alkali, oxidative
and thermal conditions as per ICH.7
7) G. Subramanian, P. Musmade, Journal of pharmaceutical and biomedical analysis,vol.43
(2007) page no.722-726.

Developed SI HPTLC method:
chloroform: methanol (6:4, v/v) gave a sharp and well defined
peak at Rf value of 0.53.7
Fig. 10. HPTLC chromatogram of acid
degraded imatinib mesylate7
Fig. 9. A typical HPTLC chromatogram
of imatinib mesylate (Rf = 0.53).7
(2007) page no.722-726.

Fig. 11. HPTLC chromatogram of
base degraded imatinib mesylate7
Fig. 12. HPTLC chromatogram of hydrogen
peroxide degraded imatinib mesylate7
(2007) page no.722-726.

CONCLUSIONCONCLUSION
A successful stability study establishes
shelf life of drug products
retest period of a drug substance and
appropriate storage conditions
But that’s not all- a successful stability study ensures that
Patients receive a safe and effective medicine
Assured byAssured by validated SIAMsvalidated SIAMs which meets ICH and regulatorywhich meets ICH and regulatory
requirementsrequirements
Stability is not all
But all comes to naught
Without stability

REFERENCESREFERENCES
1) G.T. Kulkarni,K. Gowthamarajan,B. Suresh, Indian Journal Of
Pharmaceutical Education, Vol. 38, issue 4, Oct-Dec. 2004 page no.194
2) Manutosh M. Acharya, Eastern Pharmacist,vol.42, 1999 May,
page no.31-36.
3) International Conference On Harmonization (ICH) Q1A(R2) guidelines.
4) Grimm W. ,Stability testing of pharmaceuticals, page no.141-153.
5) S. Singh, Journal of pharmaceutical and biomedical analysis,
vol.28(2002) page no.1011-1040.
6) V. kumar, H. Bhutani, S. Singh, Journal of pharmaceutical and
biomedical analysis, vol.43(2007) page no.769-773.

REFERENCESREFERENCES
7) G. Subramanian, P. Musmade, Journal of pharmaceutical and
biomedical analysis,vol.43 (2007) page no.722-726.
8) S. Singh, M. Bakshi, Guidance on conduct of stress tests to
determine inharent stability of drugs, Pharm. Tech Asia online,
April 2000.
9) ICH, Q2(R1), Validationof analytical procedure:
Methodology,proceedings of International Conference on
Harmonization, London,2005.
10) Carstensen , J.T., Drug stability: Principles and Practices,
New York, Marcel Dekker,Vol107, 1995.
11) www.ich.org.com

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