Exp. No - Date:

STABILITY STUDIES OF DRUGS IN DOSAGE

FORMS AT 60 OC AND 80 OC.

Aim :– To determine the shelf life of drugs in dosage forms at 60ºC and 80ºC by accelerated test conditions.

Objective :– The purpose of stability testing is to provide evidence on how the quality of a drug substance

Under the influence of variation of external factor Temperature with time.

To determine the shelf life of the product. To recommend storage conditions of the product.

Apparatus :– Hot air oven, Uv-Visible Spectrophotometer.

Chemicals :– Acetyl salicylic acid, Salicylic acid, Ferric nitrate, 1N HCl.

Principle :–

UV Visible spectroscopy :– UV Visible spectrophotometer works on the principle that exposure of compounds to light of ultraviolet and visible region causes electronic transitions by absorption of energy, this energy absorbed from the incident radiation is calculated and read out as absorbance by the molecule. According to Beer and Lambert’s law absorbance of a substance is proportional to the concentration of the absorbing species, with increase in the concentration of the absorbing species absorbance increases.

A=A1 %1cmbc Eq – 1

Where A is absorbance

A1%1cm ismolar absorptivity

b is path lengthc is concentration

Ester hydrolysis of aspirin :– Aspirin undergoes ester hydrolysis in the presence of water to form salicylic acid and acetic acid which are the raw materials used in the preparation of aspirin.

Salicylic acid reacts with ferric nitrate solution and produces violet colored complex. This solution absorbs light of the wavelength nearer to 530 nm. Aspirin in presence of water forms salicylic acid, at higher temperatures the amount of salicylic acid is more than that produced by the solution at lower temperatures. The absorption produced is proportional to the amount of salicylic acid present in the given solution.

Theory :–

Order of reaction :– According to law of mass action the rate is proportional to the

product of molar concentrations raised to the power equal to number of molecules. The order of the equation is the sum of the raised powers. In the reaction

It follows first order kinetics. And if one of the concentration say NaOH is increased then order depends only on one concentration and the order is apparent or pseudo first order equation

Order Explanation Rate equation Half lifeZero Rate is independent of

the concentration of the reactants

c=co−k ot t 12

=ao

2k o†

First Rate is dependant on one of the concentration of the reactants logc=log co−

kt2.303

t 12

=0.693k1

‡,

t 90=0.105k e

Second

Rate of equation is dependant on the two reactants. Hydrolysis reactions of drugs

xa(a−x )

=kt t 12

= 1ak2

‡

Note: † ao, ko are initial amount, zero order rate constant

‡ k1and K2 are rate constants at first and second order respectively.

Determination of order of reaction –

CH3COOC2H5 NaOH C2H5COONa C2H5OH

1. Substitution method – The data from the kinetic study is fit in to the integrated equation of different order and the equation in which the calculated k value remains constant the reaction is considered to be of that order.

2. Graphical method – The data is plotted in different plots and a) Straight line with concentration and time it follows zero

order.b) Straight line with log concentration and time it follows first

order.c) Straight line with inverse of concentration and time it

follows second order.3. Half life method - In general half life is given as in Eq – 2.

From this the order of the reaction is found out.

t 12

∝ 1

an−1 Eq – 2

where n is order of reaction.

Half life :–

It is the time required for one half of material to disappear i.e., A

to 12A. For a first order equation whose equation is given as Eq – 3.

Half life is given as

log c= log co−kt

2.303Eq -3

t 12

=0.693k Eq - 4

Shelf life and expiration date :–

It is the time period during which a drug product is expected to remain within the approved specifications if stored under defined conditions and after which it must not be used. The shelf life for a reaction following first order kinetics is given by the following equation.

t 90=0.105k25

Eq – 5

Stability of pharmaceuticals :–

Different processes like oxidation, hydrolysis, isomerization, epimerization, solvolysis, photolysis causes the degradation of the active pharmaceutical ingredient and inturn effects the quality of the finished product. Different factors like concentration of reactants, pH

radiation, catalysts, raw material used, time lapse play role in drug degradation.

Examples – Drugs like Aspirin, Chloramphenicol, Erythromycin, Procaine undergoes hydrolysis, Benzaldehyde, Ascorbic acid, Furosemide undergoes photodegradation, etc.,

Importance of stability testing :–

Poor patient acceptance Dose of drug less than required for therapeutic effect. To determine container closure systems. To determine shelf life. Storage conditions.

Methods of stability testing :–

Stability testing is performed for the new products or new formulations of existing products to predict and establish different conditions. The major aim of stability testing is to ensure that the product retains its specifications in acceptable range when stored at specified conditions.

1. Real time stability testing – This test is performed at recommended storage conditions for longer durations till no measurable degradation is observed. The testing data is collected at regular intervals such that slight changes can also be observed. Its reliability is increased by using a standard whose stability is already established.

2. Accelerated stability testing – This is done by subjecting the product to conditions that would accelerate the degradation. In this testing higher temperatures are employed and it gives early detection of the shelf life. Other factors that can be included in to accelerated stability testing is moisture, light, agitation, gravity, pH and package. This method is based on Arrhenius equation(equation 2). This can be modified as

logk2

k1

=log A−Ea

2.303 R( 1T2

− 1T1

) Eq – 6

where k1and k2 are rate constants T1 and T2 are two temperatures.

3. Retained sample stability testing – It is of usual practice that stability data are required for manufactured products. Atleast one batch a year or two if batches exceed 50 are

selected for retained samples. Stability studies are studied at 3, 6, 9, 12, 18, 24, 36, 48 and 60 for products with shelf life of 5 years.

4. Cyclic temperature stress testing – in this method stress conditions are designed on the knowledge of the product to mimic market storage conditions.

Guidelines for stability testing :–

Data of stability studies need to be submitted to the regulatory authorities in accordance to the guidelines. International conference of harmonization (ICH) has harmonized the guidelines, World health organization (WHO) has modified the guidelines in 1996 because ICH do not address extreme climatic conditions. Some of the codes under ICH guidelines are as follows

Code

Title of guideline

Q1A Stability testing of new drug substances and productsQ1B Stability testing: photostability testing of new drug substances

and productsQ1C Stability testing of new dosage formsQ1D Bracketing and matrixing design for stability testing of drug

substances and productsQ1E Evaluation of stability dataQ1F Stability data package for registration applications in climatic

zone III and IVQ5C Stability testing of biotechnological/biological products

In case of products of different strengths, multiple sizes reduced stability testing plan like bracketing method, matrixing statistical design can be employed. Bracketing is the design such that only samples on the extremes of certain design factors are tested at all time points as in full design. Matrixing involves testing of subset of total number of samples for all combinations at specific time points.

Parameters that cause the loss in quality, purity, potency, identity like appearance assay, degradation products, microbiological testing, dissolution, moisture are studied.

Accelerated stability testing :–

According to FDA(food and drug administration) drug substances or the products that are susceptible to change during storage and are likely to influence quality, safety, and efficacy need to be controlled. Accelerated stability testing should cover areas like physical, chemical, biological, microbiological and functionality test. It is tedious to perform real time or long-term stability studies and

ascertain the expiration date or the shelf life of the product, accelerated stability tests helps to support the tentative expiration of the product. Accelerated stability studies are designed to increase the rate of chemical degradation or physical change of the drug substance or product by exaggerated storage conditions. Stability testing should be done with all the committed batches and the stability data need to be submitted to the authorities. Stress testing uses conditions which are more severe than accelerated stability testing. The different conditions for accelerated stability testing are determined based different climatic zone conditions.

The method of stability testing is based on the principle of chemical kinetics, increased rate at exaggerated conditions and plotting concentrations against time or temperature or reciprocal of temperature. Different methods uses different plots, Arrhenious uses plot of log concentration vs time. From these plots k values at elevated temperatures are obtained and k value at room temperature (25oc) is obtained by extrapolation of the plot between log of concentration and inverse of absolute temperature. From the rate constant at room temperature the shelf life of the product is determined by using the equation – 5. According to Free and Blyth fractional life period is plotted against reciprocal of temperature and the time in days required to decompose to some fraction at room temperature is obtained.

Arrhenius Method :–

Aspirin ester hydrolysis follows second order equation where it depends on the concentration of both aspirin and water. Water is taken in excess amounts to make the reaction follow first order kinetics. According to Arrhenius the rate constant of a equation is given by the following equation

k=Ae−E aRT Eq – 7

Applying logarithm log k=log A−Ea

2.303 RT Eq – 8

Where log A is Arrhenius factork is rate constant (time -1) k=slope×−2.303

0 5 10 25 45 650

0.20.40.60.81

1.21.41.61.82

log c vs time plot

time (min)

log

conc

entr

ation

slope = -k/2.303

2.8 3.01 3.550.5970.5980.599

0.60.6010.6020.6030.6040.6050.606

log K vs 1/T plot

1/T x 106

(log

k)+c

From the second plot rate constant for the reaction at room temperature is obtained by extrapolation. Shelf life of the product is calculated using the Eq – 5.

Free and Blythe method :–

According to free and Blythe shelf life of a product can be predicted directly from the remaining concentrations of the drug. Shelf life is obtained1 from the following plots.

0 30 60 90 120 1500

0.5

1

1.5

2

2.5

log %drug remaining vs time

60°c

80°c

time (min)

% dr

ug re

main

ing i

n lo

g sca

le

26 28 30 32 340

0.51

1.52

2.53

3.54

4.55

t90 vs 1/T1/T x 106

t90

Climatic zones :–

Based on the prevalent annual climatic conditions of the different parts of the world the climatic conditions are divided in to four zones. Based on the conditions stability conditions are derived

Zones

Climate Region temp/relative humidity

Long term testing

conditionsI Temperate UK, North ≤15o c /≤11hPa 21o c /45 %RH

Europe, Russia, US

II Subtropical, Mediterania

n

Japan, Southern Europe

¿15−22o c /¿11−18hPa25o c /60 %RH

III Hot and dry India, Iraq ¿22o c /≤15hPa 30o c /35 %RHIVa Hot and

HumidIran, Egypt ¿22o c /¿15−27 hPa 30o c /65 %RH

IVb Hot and very Humid

Brazil, Singapore

¿22o c /¿27hPa 30o c /75 %RH

Procedure :–

Preparation of 5% FeNO3 solution :– 50 g of ferric nitrate was dissolved in 20 ml of 1N HCl and the volume is made up to 1000 ml with distilled water.

Preparation of standard curve of salicylic acid in Distilled water :– Accurately weigh 100 mg (0.1 g) of pure salicylic acid and prepare different concentrated solutions (5, 10, 15, 20. 25, 30, 35 µg/ml). The λmax of salicylic acid in distilled water was obtained by scanning for wavelength at which maximum absorption of light takes place. The wavelength is set to 525 nm. To 1 ml of different concentrated solutions of salicylic acid 5 ml of 5% FeNO3 was added and the volume was made up to 10 ml. The absorbance of the solution was measured and noted. From the values of absorbance (ordinate) and concentration (abscissa) the calibration curve for salicylic acid in distilled water is obtained.

Accelerated stability testing :– Accurately weigh 100 mg of acetyl salicylic acid (Aspirin) and place in two volumetric flasks make up the volume with distilled water. Place the volumetric flasks in hot air ovens maintained at 60ºC and 80ºC each. At regular time intervals of half an hour collect the samples. Place 1 ml of sample in a test tube and add 5 ml of 5% FeNO3 and then make up the volume to 10 ml with distilled water. Determine the absorbance at 525 nm using UV-Visible spectrophotometer. Calculate the amount of drug remaining in the solution, rate constant (K), t90 at different temperatures and report.

Reference :–

1. Martin’s physical pharmacy and pharmaceutical sciences, 6 edition, Patrick. J. Sinko

2. Sanjay Babaj et al. Stability testing of pharmaceutical products, Journal of applied pharmaceutical sciences 02 (03);2012 pg 129 – 138.

Results and discussions :–

Exp. No - Date:

Effect of Hydrochloric acid, sodium hydroxide solution on the stability of drugs in solution at

elevated temperatures and room tempertatures.

Aim – To determine the effect of Hydrochloric acid, sodium hydroxide on the stability of Acetyl salicylic acid at elevated temperature.

Objectives –

To study the effect of solvent on stability.To study the effect of PH on the stability.To study the combined effect of PH and temperature on stability

Apparatus – Hot Air Oven, Uv-Visible Spectrophotometer.

Chemicals – Salicylic acid, Acetyl Salicylic acid, Ferric nitrate, Sodium hydroxide, Hydrochloric acid.

References –

1.2.

Principle –

Procedure –

Preparation of 0.1 N, 0.01 N NaOH – Dissolve 4, 0.4 g of NaOH in small amounts of distilled water in a volumetric flask and make up the volume to 1000 ml.

Preparation of 0.1 N HCl, 0.01 N HCl - Add 8.5, 0.85 ml of HCl solution to small amount of distilled water in a volumetric flask and make up the volume to 1000 ml.

Preparation of standard curve of salicylic acid in 0.1 N, 0.01 N NaOH, 0.1 N, 0.01 N HCl – Accurately weigh 100 mg of salicylic acid and dissolve in 100 ml of 0.1 N NaOH, 0.01 N NaOH, 0.1 N HCl, 0.01 N HCl. To 10 ml of above solution and dilute it to 100 ml with solutions of same PH. pipette out required amounts of solution and prepare different concentrated solutions of 5,10,15,20,25,30 µg/ml. To one ml of above concentrated solutions add 5 ml of 5 %

ferric nitrate solution and make up the volume to 10 ml with the same PH solution and measure absorbance at their respective λmax.

Stability testing in different PH solutions - Accurately weigh 100 mg of acetyl salicylic acid (Aspirin) and place in volumetric flasks, make up the volume with different PH solutions in duplicates. To one set subject the solutions to 60°C and the other to 80°C. Collect sample at 15, 30, 45, 60, 75, 90 min and to one ml of sample add 5 ml of ferric nitrate solution and make up the volume with same PH solution and measure the absorbance.

Results and discussions –