STABILITY INDICATING

TEST PROCEDURE

SUBMITTED BY
POOJA SINGH
M.PHARM.(1ST SEM)
QUALITY ASSURANCE
 INTRODUCTION
Stability indicating test procedure is a method which
differentiate and quantity the active ingredients being
assayed from other active ingredient, excipient,
synthetic impurities & degradation products present in
the dosage form.
Various factors contribute to the nonspecificity of an
assay procedure
1. If dosage form contain two or more active ingredients,
then this may interfere in assay, interference may
occur by excipient like preservative, diluents,
chelating agent, antioxidant etc.
2. Raw material may also introduce impurities in the
form of its starting material & its impurities,
intermediates or by products.
Product non specific also get introduced during its
processing & manufacturing finally degradation or
decomposition products of active ingredients may
arise due to drug-drug interaction, drug excipient
interaction, drug dosage form container-closure
interaction.
 Stability Indicating Assay
1.Conventional method
• Low cost
• Simplicity
2. Instrumental method
lesser analysis time & accurate for small amount sample.

A.Tritrimetry- Assay of amines, alkaloids, sulphonamides,

antihistamines and amino acids in pharmaceutical
preparation.
 Non aqueous titration
 Oxidation reduction titration- ferric iron, total iron in iron
tablet, capsule, in anemia related disorder.
 Iodometric- assay of β lactam antibiotics 9ampicillin,
benzylpenicilline potassium (penicillin G).
B.Ultra violet/ visible spectroscopy- spectral selectivity can
be enhanced by use of Differene spectrophotometry,
Derivative spectrophotometry and Dual wavelength
spectrophotometry.
When spectra of two or more compound overlape, than vierordt
multiwavelength technique, second or higher derivative
spectrophotometry etc. can be used to measure individual
cone.
Example. Second spectrophotometry- assay of mixing of
Dicloxacillin sodium and Ampicillin sodium, also mixing of
Cephalosporin lie Cephaloridine and cephalothin sodium.
Vierordt multiwavelength technique- limit test of Amphotericin a
in antifungal antibiotic.

C.Colorimetry- colorimetry is made specific by use of color

reagent which is selective for the drug molecules only and
not for degradation product, impurities and excipients.
The reaction time, pH, solvent temperature, reagent excess and
other factor require to obtain stable chromospheres must be
optimized for better selectivity and sensitivity.
Example. Tetrazolium assay for corticosteroids (diazotization and
coupling with CNCl- nephthyl) ethenediamine.
Isoniazide- bromimetrically but degradation product, succeptible to
oxidation, may interfere so 6,7-dichloroquinoline-5-8 dione as
chromogenic reagent to interact with hydrazide moiety – green
colour.

4. Thin layer chromatography- widely used for separation and

identification, low cost, simplicity, high sensitive can be for small
scale, as alternative mean to differentiate between active drug &
degradation product/ relative compound.
The mobile phase moves across a uniform thin layer of stationary
phase (silica gel, alumina) bonded to plate of aluminium foil, glass or
plastic.
The separation occur due to different rate of migration which depend
upon difference in solubility in mobile phase and the degree of
adsorption on stationary phase & denoted by Rf value.
Spots are located either by visual color, uv light, fluorescence
quenching, iodine vapour or spraying with chromogenic reagents
etc.
If drug and related substances with same Rf value & overlapping of
spots then two dimensional TLC used by chromatography the
plates by turning it at right angle & using different mobile phase.
Quantitative TLC is performed by spotting k/n amount of both
sample & standard solution along with appropriate blank on plate
after solvent reaches the desire light, the plate is removed,
mobile phase allowed to evaporate & then dried. Quantitation is
done by-
 Comparing intensity or size of spot on plate by photodensiometry
or fluorescence measurement.
 The spot is marked and scraped in to a vessel eluted with suitable
solvent, concentrated and absorbance.
High speed automated TLC scanner has improved quantitation
speed and sensitivity.
Example. Cephalosporin- by using acidic mobile phase, spraying
with floreseamin & quantitated into site by measuring
fluorescence emission or by using a silica.
5.High performance thin layer chromatography-

HPTLC is TLC, improved by use of new stationary phase,

chromatographic development modes, automated sample
application devices and use of micro process are controlled
densiometer & b/c plate have smaller particle size & narrow particle
size distribution, so it make faster, more reproducible, sensitive,
accurate, precise and having greater degree of resolution.
It is used to separate complex mixture for both qualitative or
quantitative analysis.
The developed plate are visualized, recorded by photography or
quantitatively analyzed using scanning densiometer or video
camera and an image processing system.

Advantage-
 Many samples can run simultaneously.
Disadvantage-
b/c volume of sample is very low so inability to use another
identification technique.

Example. Oxyphenbutazone- TLC not possible b/c poor

resolution, hence to separate and quantify its six potential
decomposition products HPTLC is carryout. Quantitation by-
reflectometer.
6. Gas chromatography- for volatile and thermal substances.
Separation occur by partitioning between the stationary phase 9solid,
liquid coated on solid support) or column maintained at definite
temperature and mobile phase flowing at a constant rate.
On injection sample are volatile by preheated, oven passed through
the column and after separation based on their retention time are
detected by FID or TCD detectors.

Advantages- high sensitivity, high selectivity can be achieved by

adjustment of carrier gas flow rate, column temperature, type or
column.

Disadvantage- possibility of changes in molecular structure as it

passes through chromatographic system due to thermal stress.
Drug high molecular weight poor volatility, relatively polar
substituent group & possessing thermal instability are derivatized
by accetylation, methylation to give thermal stable and volatile
derivatives.
Use-
 To detect and determine quantitatively the amount of residual
solvent or solvent of crystallization present in a drug.
Example- presence of methanol in streptomycin sulphate.
 To detect & quantify the amount of two k/n impurities of
Ampicillin sodium like Dimethyl aniline and Dichloromethane.
 Separating and disintegrating between closely related
compound.
Example- cis & trance isomer a tranylcypromine.
7. High pressure liquid chromatography-
Separation is done by partitioning between a mobile phase
(solvent system) and stationary column material and depends
mainly on the solubility of solute.
Mobile phase is pumped at high pressure through a column and
sample is injected and solute after separation enter the
detector (uv, fluorescence, IR.)
As compare to GC, non volatile, thermal unstable or polar/ ionic
compound can be analyzed easily b/c high resolution capacity,
sensitivity & specificity.

Special techniques used are-

1. Gradient elution- for compound with wide difference in
retention time.
2. Derivative formation- to enhance selectivity & sensitivity by
forming uv absorbing fluorescence derivatives.
HPLC can be used to resolve and determine the active drug
and small amount of its degradation product where in
existing official methods prove inadequate and non specific.

Example. Dextrose used in ph preparation, degrades on

autoclaving ar an storage which can determined by HPLC.
Differentiation between chlorpromazine & its degradation
products (chlorpromazine sulphoxide & chlorpromazine
sulphone).
Benzodiazepine( chlordizepoxide, diazepam) and their
pharmacological active metabolized can be analyzed
simultaneously.
 The stability assay was developed for used in determining

the integrity of the drug substances during accelerated shelf

life studies.
 Stability properties can often be predicted from inspection of

chemical structure and physical properties.

There can be several appropriate approaches to determine

drug stability or instability.
 Measure intact drug by a highly selective method.

 Measure total drug content nonselectively and estimate the

degradation products.
 Selectively measure both drug and decomposition product.
1.Selective determination of active drug
Example- atropine degradation would be ester hydrolysis

ATROPINE TROPINE + TROPIC ACID

Hydrolysis rate measured by modification of compendial state

of organic nitrogenous base method, where the amine is
separated by extraction alkali in to an organic solvent or
extracted into dilute aqueous acid and determined by uv
spectrophotometrically.

2. Phenazocine HBr
PHENAZOCINE DESPHENETHYL PHENAZOCINE
+
STYRENE
 Gas chromatography (as reference method) for methanol
sulphuric acid colorimetric assay for quinesterol, ethinyl
estradiol 3-cyclopentyl ether.
 Solubility analysis as a reference method for judging the

suitability of routine stability testing methods for a number

of drugs.
 Biological assay as reference method-

example. Column partition chromatography

CHLORAMPHENICOL
 Estimation of decomposition products

Example. For determination of formation of nephrotoxic 4-

epianhydro tetracycline from tetracycline – paper, thin layer
and gas chromatography used for monitoring decomposition
products.
Quantitation by mean of decomposition product
Example.

1. Salicylic acid has an uv absorbance bond at along

wavelength and more intense absorbance than aspirin and
salicylic acid form intensely colored chelate with ferric ion.

2. Stability of hydrochlorthiazide

HYDROCHLORTHIAZIDE 4-AMINO-6-CHLORO,

1-3DISULPHAMIE

FORMALDEHYDE