Stability Testing Of Pharmaceutical Material Biology Essay

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Stability testing forms an integral part of the Quality Assurance program. The manufacturing process of a drug necessitates the stability testing of pharmaceutical materials in various stages. The information of the stable formulation obtained from the preformulation studies is used for the later stages of manufacturing procedure. But the assigned expiration date of the pharmaceutical product may vary from the shelf-life due to the improper storage conditions and even due to the influence of environmental factors.

The interaction between the ingredients or process of formulation or the containers and closure systems; and the environmental impact during the handling, storage and shipment of the drugs affect the stability of the pharmaceutical products. The inadvertent use of these products may be hazardous to the patient. Hence it is essential for a pharmacist to investigate the conditions for the long shelf-life of the pharmaceutical materials.

Aims and objectives of stability testing:

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To know the quality and efficacy of the pharmaceutical material under the influence of environmental conditions like temperature, humidity, incident light etc.

To predict the shelf life of the substance and storage conditions of the pharmaceutical materials.

Stress testing studies are performed to know the degradation pathways and the effect of elevated temperatures. Accelerated stability testing uses forced degradation at extreme conditions but these studies are costly and take more time to extrapolate the Arrhenius plots. Hence thermal analysis techniques are preferred to conventional methods. The main thermal analysis techniques include DSC, isothermal calorimetry. These values are precise and accurate. These methods explain the effect of temperatures and degradation pathways by using Arrhenius equations in very less time. The physical properties of the products like purity, colour etc. can also tested by assay methods like HPLC.

INTRODUCTION

Stability of a pharmaceutical material can be described as the capability of a formulation to remain within the standard specifications throughout the shelf life (Alfonso R.Gennaro et al, 2000).

The stability testing are performed to estimate proper storage conditions for long-life of the product and to calculate the shelf-life. The prediction of this shelf-life can be performed by Real time stability studies or Accelerated stability studies. The former studies include the testing in which the product is stored at standard and recommended storage conditions. The product is observed till it crosses the limits of standards. In the latter studies, the product is subjected to stress conditions (like temperature, humidity, pH, light) (Robert T.Mayaro, 2003).

The accelerated stability studies are suitable for the investigation of impact of environmental factors on pharmaceutical products. Thermal analysis methods are the recent advances in the prediction of temperature changes which influence the stability of the pharmaceutical products. They are the research techniques of my interest.

LITERATURE REVIEW

Requirements of stability studies:

The stability testing of the products are enclosed in the literature of current Good Manufacturing Practices (cGMPs), compendiums and FDA guidelines.

cGMPs: They cover the written testing program in order to estimate the stability of the products. It is used to assess the storage conditions and shelf-life. The shelf-life conform the purity, strength and quality of the product.

Compendiums: They are designed to provide broad information on stability and shelf-life. It gives the duties of manufacturer and pharmacist in dispensing. The storage conditions are defined as per the standards.

For example, as per USP 26, controlled room temperature was defined as the "temperature maintained thermostatically between 20 and 25ËšC (68 AND 77ËšF respectively).

FDA guidelines: They give the information on

The design of the stability testing in order to establish the relationship and storage conditions.

The stability requirements for biological, new drugs, new drug applications etc. (Alfonso R.Gennaro et al, 2000).

Evaluation of product stability:

The pharmaceutical product has to be tested for five categories of stability. They include:

Physical stability

Chemical stability

Microbiological stability

Therapeutic stability

Toxicological stability

Broadly, for the stability evaluation, the classification is done by considering the physical and chemical factors. The physical stability evaluation allows the consideration of physical factors like temperature, incident light, humidity etc. The chemical stability testing includes the degradation pathways like oxidation, reduction, hydrolysis, racemisation, precipitation, incompatibility etc. (Alfonso R.Gennaro et al, 2000).

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According to Lian Yu (200), the stability of amorphous solids depend on

Stability of thermolabile products during manufacturing and storage.

Selection of stable and appropriate storage conditions.

STRESS TESTING:

Stress testing is helpful in the identification of degradation pathways and products. It is used generally for accelerated stability studies (Klick, S., et al, 2005).

As per ICH guidelines, the stress testing involves the stress conditions like increase of temperature by increments (say 10Ëšc), humidity conditions (say 75Ëšc or more), pH (1-12) and extreme photolytic conditions based on individual monographs (Dr.S. Koop, 2006).

Effect of temperature on the shelf life:

In order to assess the effect of temperature on the product, the rate constants or the velocity of degradation should be considered.

Order of the reaction:

The number of collisions between the product molecules is directly proportional to the concentration of the products.

In the second order reaction,

The rate of the reaction (-d [D]/dt) is given as the rate, at which the drug is lost, is given as

-d [D]/dt α [D] [W]

Where, D,W are considered as molecules in the reaction

-d [D]/dt = k2 [D] [W]

Where, k2 is Rate constant

In the first order reaction,

The rate of reaction depends on the concentration of single reactant and the rate equation is given as

-d [D]/dt = k1 [D]

Where, [D] is the Concentration of Reactant.

Concentration-time profile can be calculated by the integration from t=0 to t=t where [D] at t=0 is [Do]

= - where k = rate constant

ln[D] = ln[D]0 - kt

log[D] = log[D]0 - kt/2.303

The half-life of the product is

ln[D]0 /2 = ln[D]0 - k t1/2 where [D] = [D]0/2

t1/2 = 0.693/ k

The shelf-life i.e. the time period for drug to undergo 10% decomposition, is given as

t90 = 0.105/k1

In the zero order reaction, r1

The rate equation is independent of concentration.

The rate equation is given as

-d [D]/dt = ko

On integration from t=0 to t=t where [D] at t=0 is [Do],

=

[D] = [D]0 - kot

The half-life of the product is

t1/2 = 0.5[D0]/k0

The shelf-life i.e. the time period for drug to undergo 10% decomposition, is given as

t90 = 0.1[D0]/k0

The effect of temperature on the rate of reaction is given by Arrhenius reaction which is explained later in the methods of investigation.

The effect of temperature depends on the mechanisms of degradation and even the other factors like diffusion and photolysis (Kenneth A. Connors et al, 1979).

Shaun etal. (1978) observed that the presence of moisture at elevated temperatures and humidity causes PVP to change its stable glassy form to rubbery state. It can be observed in accelerated stability studies in stress conditions rather than long term studies by modulated temperature Differential Scanning Calorimetry. The stability of nitrazepam in solid state by considering the factors like temperatue and relative humidity was studied. In this, the rate constants were identified by taking four temperatures and six relative humidity conditions. Thus the correlation of constants of nitrazepam is performed.

Effect of incident light on shelf life:

The incident light may cause the activation of the molecules in the pharmaceutical product, thus affecting the stability of it (Patrick J. Sinko & Alfred Martin, 2006). As the product absorbs the incident light at a particular radiation, the radiation is absorbed leading to the degradation pathways. It is referred to as photolytic reaction (Alfonso R.Gennaro et al, 2000).

Generally, oxidation occurs due to the exposure of light (Kenneth A. Connors et al, 1979). As the wavelength decreases, the light becomes more potential to damage. UV radiation is hastens photolysis to a larger extent (Hussain sheikh R. et al, 1996).

If the product absorbs light, the reaction is photochemical and if the absorbed molecules pass the energy to other molecules, it is photosensitisation (Lachmann L., 1986).

Chlorpromazine hydrochloride and prochlorperazine ethanedisulfoate shows colouration when placed in Warburg respirometer and exposed to light (Kenneth A. Connors et al, 1979). Photostability testing is performed by forced degradation and confirmation testing. Forced degradation testing is performed mainly to validate the process. The method development and degradation reactions are established by this technique. The product is exposed to different conditions and the exposure levels are studied. Then it is used for confirmation testing for the examination of degradation products, if they are formed (Klick, S., et al, 2005).

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Matsuda et al. investigated the effect of radiations of mercury vapour and fluorescent light on nifedipine. Among the four photoproducts, nitrosopyridine easily degrades in UV and visible light mainly at wavelength of 380 nm. On exposure to mercury vapour and fluorescent light, the degradation is hastened to a large extent. The photolysis in crystalline state of drug is used to measure the decomposition of nifedipine in solid state in order to establish photostability conditions (Patrick J. Sinko & Alfred Martin, 2006).

Accelerated stability testing:

Accelerated stability testing is the process in which the product is subjected under stress conditions changing the factors like temperature, pH, humidity etc. These experimental values are converted to find out the shelf-life of the product, stability and storage conditions.

The conditions maintained for the stability testing procedures are given below.

Type of study

Storage conditions

Minimum time period

Real time testing

25°C ± 2°C/60% RH ± 5% RH (I)

or

30°C ± 2°C/65% RH ± 5% RH (II)

12 months

Intermediate testing

30°C ± 2°C/65% RH ± 5% RH

6 months

Accelerated testing

40°C ± 2°C/75% RH ± 5% RH

6 months

Table 1: Time period for stability testing procedures (European Medicinal Agency, 2003).

In real time testing, conditions can be I or II. If condition II is followed, intermediate testing can be exempted. If condition I is performed and the significant changes are observed in accelerated studies, then all the tests are covered in intermediate stage. It should range from 6 months to a year.

The duration of study by accelerated stability testing differs based on zones.

In zone IV (hot climate), the conditions are 40±2˚C, 75±5% and 6 months. In zone II (temperate and subtropical climate), the conditions are 40±2˚C, 75±5% and 3 months. However, for drugs with low stability, the time period is enhanced to 6 months. At high temperatures i.e. 45-50˚C and 75% RH, the study can be minimised to 3 month (WHO, 1997).

Barbara A.Kozikowski (2003) tested the stability for DMSO in ambient conditions for a year. The amount of compound present was measured regularly and found that the compound gradually decreased with time. Based on the results obtained, the storage and shelf life of DMSO was identified.

Kenneth A.Jandik etal. (1995) have studied the accelerated stability studies of heparin from 0 to 4000 hours. Rapid degradation was observed after 500 hours due to acidic degradants and decrease of pH gradually. The degradation pathways of heparin were found from the data.

Thermal analysis:

It is a technique where in the effect of temperature changes on the physical property of pharmaceutical product are measured. It determines the stability calculations and shelf-life.(Remington). Different techniques of thermal analysis include Differential Scanning Calorimetry (DSC), Differential Thermal Analysis (DTA), Thermo gravimetric Analysis (TGA), Isothermal Calorimetry etc.

These techniques are helpful in estimation of melting point, weight loss of product, heat changes and kinetics of decomposition (Patrick J. Sinko & Alfred Martin, 2006).

Harold Jacobson etal. (1969) worked on penicillin-stearic acid complex by Differential Thermal Analysis (DTA). It was found to be compatible with Ampicillin trihydrate by the consideration of resulting thermograms.

Peter Simon etal. (2004) found the screening method for the stability detection of non-isothermal DSC. It was performed by taking the onset temperature of oxidation peaks into account which showed that the stability of compounds were similar to the classical methods.

RECENT LITERATURE:

Sumi Yoshioka (2007) conducted a study on the effect of molecular mobility on chemical stability of amorphous products during storage. Three factors were considered to establish the relationship. Firstly, the effect of temperature on global transition temperature (Tg). Secondly, the relation between chemical stability and global mobility was studied. Thirdly, importance of local mobility of drug in chemical reaction was explained.

Jun Han etal. (1999) developed a method to evaluate the physical stability of pharmaceutical product by Thermo gravimetric analysis and X-ray diffraction under controlled vapour pressure. Arrhenius plots show the stable phase of amoxicillin trihydrate. The humidity controlling device is used to estimate the stability at different temperatures.

Aman and Thoma (2005) proposed that the absorption of light into the drug is a limitation factor in solid dosage forms. The degradation is higher on the surface due to more exposure of light on that area. It leads to the degradation of drug on the surface more than the inner layers. For example, Nifedipine tablets cause degradation to a depth of 360 µm and 880 µm based on the dosing given.

Recent studies carried out by Anthony E.Beezer etal.(1999) uncover the application of calorimetric studies in real time stability testing to hasten any degradation pathway. This method utilizes the dependence of high temperatures on the products. The recent studies in the field permits the estimation of both kinetic and thermodynamic factors for long term reactions at suitable temperatures and environmental factors.

Though the methods of stability are established and well followed, the limitations also focus on the new techniques for the stability prediction. The prediction of room temperature stability from elevated temperatures may result in erroneous results. The products may evaporate due to elevated temperature, thus the nature of the product may be affected. Higher temperature may induce less relative humidity and oxygen solubility thus affecting the prediction of stability for products sensitive to these factors.

In my research, I would follow the simple techniques of thermal analysis like isothermal calorimetry. The techniques adapted are simple and easy to obtain the stability criteria. Minute samples may be required for analysis. It takes less time compared to conventional methods and results are accurate.

METHODS OF INVESTIGATION

For the prediction of shelf life and to know about the influence of environmental factors, the following methods of investigation can be adopted.

Effect of temperature:

Activation energy calculations:

Rate of reaction is proportional to number of collisions per unit time. As the number of collisions increase, the temperature factor also increases. As per rule of thumb, the rate of reaction doubles for every 10ËšC rise in temperature. The reaction rate constant is known from the Arrhenius equation.

K = Ae-Ea/RT

Where K = reaction rate constant of any order

log K = logA - Ea/2.303RT (1)

log (k2/k1) = -Ea/2.303R(1/T2 - 1/T1)

log (k2/k1) = Ea(T2 - T1)/2.303RT1T2

where k2, k1 are rate constants at temperatures T1 and T2

On plotting a graph between logK and 1/T, we get the slope of - Ea/2.303R.

This is referred to as Arrhenius plot and value of Ea can be determined from it.

In order to calculate half-life for the first order reaction, we can write

t1/2 = 0.693/K

By substituting this in equation (1), we get

log t1/2 = log0.693 - logA + Ea/2.303RT

log t1/2 = Ea/2.303RT + constant

Q10 calculations:

Q10 calculations show the effect of 10ËšC rise in temperature on stability of pharmaceutical products. The method was developed by Simonelli and Dresback.

Q10 = K(T+10)/KT

where Q10 is the factor by which rate constant increases for 10ËšC rise in temperature.

Q10 = exp[-Ea/K(1/T+10 -1/T)]

If Ea is known, this equation can be used for calculations. Otherwise, the approximation of Ea can be taken between 12-24 kcal/mole.

The effect of temperature in varying amounts can be calculated by

QΔT = K(T+ΔT)/KT = Q10(ΔT/10) (Kenneth A. Connors et al, 1979).

Photostability testing:

Photostability testing involves the estimation of degradation pathways and also make certain the physical changes in the product. In this process, the drug product is taken in sealed containers and physical changes like melting, evaporation etc. are taken care of. The precautions should be taken that no interaction takes place between sample and containers. The sample is exposed to irradiation in such a way that uniform exposure of light is provided. The physical changes (colour, appearance) should be examined for any degradation procedures.

Quinine chemical actinometry can be carried out for photostability testing. The sample is exposed to near UV fluorescence light in actinometrical system. The product solutions are placed in two separate ampoules, one of which is sealed hermetically and the other is sealed with aluminium foil to protect the sample from light. After the specified period of exposure, the change in the absorbance can be calculated as

ΔA = AT - A0

where AT = absorbance of the sample and A0 = absorbance of the control

The sample can be assayed by using techniques like HPLC, GC, titration etc.

This is a simple technique and easy to perform. Actinometry can be performed in less time and includes simple calculations (Federal Register, 1997).

Estimation of stability by DSC:

For the determination of stability, the method of Ozawa can be implemented. It is a non-isothermal method of DSC which is useful in the prediction of half life time and shelf-life. The conditions for storage can be calculated from the data obtained.

In the prediction of stability, variable heating method is implemented. The sample of drug is taken and heated at 10ËšC/20ËšC min-1 from normal room temperatures till the decomposition is observed.

If the degradation is endothermic, then it is referred to as thermostable. If it is exothermic, ASTM method is followed which uses Arrhenius rate constants. It provides the values for activation energy (E) and Arrhenius frequency factor (Z).

Using Arrhenius equation, the interpretation is done.

K = Z e-E/RT

where K = specific rate constants

Z = Arrhenius frequency factor

E = activation energy

R = gas constant

T = temperature (k)

From this equation, K value can be calculated. From this, t0.1 (shelf-life) and t1/2 (half-life) values are calculated for first order reactions.

To verify the results, isothermal ageing is done for predicted t1/2 at any temperature. The area under the exotherm for aged sample should be half of the unaged sample. If the data is already available, the DSC predictions can be done from them.

The method is advantageous because it takes very less time compared to isothermal methods. The quantity of material taken for analysis is very less, and the results are accurate and precise (Miller B., 1982).

Stability studies by Isothermal Calorimetry:

The equipment used for isothermal calorimetry is Thermal Activity Monitor (TAM). The rate of heat output can be recorded digitally by a recorder. Samples are sealed in ampoules (preferably stainless steel ampoules). The samples are sealed 18 - 24 hr before measurement to get steady baselines. Samples are then inserted into the calorimeter for 1 - 3 hours before measuring the rate of heat output. After this time, the sample is lowered to equilibrium position.

As 'q' decreases with time, extrapolation is done to get initial value. It is done at 30 min after inserting sample into calorimeter. The temperature range is selected such that 'q' is measurable in magnitude. 'q' can be positive (in exothermic conditions) or negative (in endothermic conditions). The measurable temperature can be started at 25ËšC. Then temperature is gradually increased 5 - 10ËšC till the temperature reaches 85ËšC. The extrapolation is then carried out back to the initial value of 'q'. Based on these values, Arrhenius plot can be constructed to estimate the stability conditions and temperature effects.

The products can be assayed by HPLC techniques. In this, a minute quantity of sample is taken in ampoule and stored at different temperatures for 4-8 weeks. After every 1-2 weeks, the weighed quantity of sample in ampoules is compared to the ampoule stored in 4ËšC and assayed using the technique. The amount of moisture can be detected by Karl-fischer titration.

The degradation rate is obtained from Arrhenius equation

K = Ae-Ea/RT at 25ËšC

In zero order reactions, the heat output 'q' is given as

q/D0 = -ΔHβK = C =constant

where D0 = initial amount of product

ΔH = change in enthalpy

β = reactive portion of product

If ΔH and β are not taken as the function of temperature, then rate constant k is proportional to initial rate of heat output 'q0'.

q0 = ck

ln q0 = ln C - Ea/RT

From Arrhenius equation, we can get

k1 = k2/ [exp{ Ea(T2 - T1)/R1R2T}]

The value of Ea can be calculated by isothermal calorimetry and the degradation rate (k2) is calculated from HPLC at temp T2.

Hydrated products may lose water at elevated temperatures and the degradation mechanism may differ from conventional techniques. The time required for study also affects the quality of the product. Previous studies required the rate of heat output to be calculated at portion of reaction and at several temperatures. This technique is advantageous and is useful to calculate Ea of various stable products. Ea and degradation rate (k2), calculated at single elevated temperatures by HPLC, can be used to estimate k1 (at 25ËšC) (Koenigbauer M.J. et al, 1992).