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Stability is defined as the time lapse during which the drug product retains the same properties and characterstics that it possessed at the time of manufacture. The stability of the product is expressed as the expiry period or technically as a shelf life. Stability is interpreted as the duration of time under specific conditions and storage that a product will remain within the specifications possessing all its important characteristics. The legal requirement of stability are aimed to ensure its identity, strength, quality and purity inorder to ensure that drug product remain within its predicted limits.
Instabilities occurs when they are stored under normal conditions for a long period of time. So.to determine the stability of a formulated product, it is recommended to expose it to high stress conditions and to reduce the time required for testing. Hence, it allows more data to be gathered in shorter time and reach the product to the market successfully.
The ICH guideline on stability testing of new drug substance and product provides guidance on stress testing or purposeful degradation. Stress testing helps to determine the intrinsic stability of the molecule by estabilishing degradation pathways in order to identify the likely degradation products and to validate the stability indicating power of the analytical procedure used.
The stability-indicating assay is a method that is employed for the analysis of stability samples in pharmaceutical industry. With the advent of International Conference on Harmonisation (ICH) guidelines, the requirement of establishment of stability-indicating assay method (SIAM) has become more clearly mandated.
An exact definition of a stability-indicating method according to 1987 guideline were defined as the 'quantitative analytical methods that are based on the characteristic structural, chemical or biological properties of each active ingredient of a drug product and that will distinguish each active ingredient from its degradation products so that the active ingredient content can be accurately measured.'
In short, the reason for requiring stability indicating assay method (SIAM) is as follows:
Pharmaceuticals of different dosage form
Change in pharmacological effect
Alteration in efficacy
Therapeutic and toxicological consequences
The ICH guidelines Q1A on stability testing of new drug substances and product emphasizes that the testing of these features which are susceptible to change during storage and are likely to influence quality, safety and/or efficacy must be done by validated stability indicating testing methods. That is, these stability indicating studies should estabilish the inherent stability characterstics of the molecule, such as the degradation pathways and leads to the identification of degradation products and hence support the suitability of proposed analytical procedure. The ICH guidelines have been incorporated as law in the EU, Japan and in the US.
An ideal stability indicating method is one that quantifies the drug product and also resolves its degradation products.
The practical steps involved in the development of SIAMs are discussed below.
Step I: critical study of the drug structure to assess the likely decomposition route(s):
Much information can simply be gained from the structure, by study of the functional groups and other key components. There are definite functional group categories, like amides, esters, lactams, lactones, etc. that undergo hydrolysis.
Step II: collection of information on physicochemical properties:
Before method development is taken up, it is generally important to know various physicochemical parameters like pKa, log P, solubility, absorptivity and wavelength maximum of the drug in question. The knowledge of pKa is important asmost of the pH-related changes in retention occur at pH values within 1.5 units of the pKa value. The ionization value also helps in selecting the pH of the buffer to be used in the mobile phase.
The knowledge of log P for the drug and the identified degradation products provides good insight into the separation behavior likely to be obtained on a particular stationary phase.
Step III: Stress ( Forced Decomposition) Studies:
The next step in the development of SIAM is the conduct of forced decomposition studies to generate degradation products of the drug. The ICH guideline Q1A suggests the following conditions to be employed: (i) 10 °C increments above the accelerated temperatures (e.g. 50 °C, 60 °C, etc.), (ii) humidity where appropriate (e.g. 75% or greater), (iii) hydrolysis across a wide range of pH values, (iv) oxidation and (v) photolysis.
DRUG SUBSTANCE DEGRADATION PRODUCTS
As per ICH guidelines, degradation of any product is the chemical change in the drug molecule brought about over time by action of various factors like light, temperature and pH, presence of water or by reaction with closure / container system.
FORCED DEGRADATION STUDIES
The ICH guideline specifically states,
Stress testing is likely to be carried out on a single batch of materials and to include the effect of temperature in 100c increments above the accelerated temperature test conditions (ex. 500c, 600c etc); humidity where appropriate (eg. 75%RH or greater) ; oxidation and photolysis across a wide range of pH values when in solution or in suspension. Specified stress condition should result in approximately10 - 20 % degradation of the drug substance or represent a reasonable maximum condition achievable for the drug substance.
Purposeful degradation studies of the drug substance include appropriate solution and solid state stress condition. They include
Acid / Base hydrolysis
Guidelines from the USP, ICH and FDA provide a brief outline of drug substance conditions.
ACID / BASE STRESS TESTING
Acid/ Base stress testing is performed to force the degradation by exposure to acidic / basic conditions over time. Functional groups which are likely to introduce acid/base hydrolysis are amides, esters, carbamates, imides, imines, alcohols and arylamines.
To initiate acid/base studies ,a preliminary solubility screen of the drug substance is performed. Solubility of 1 mg/ml in 1 N acidic and 1N basic condition is recommended for acid /base stress testing. Acid/Base reaction should be initiated at room temperature in the absence of light. If no degradation is observed at room temperature, then the temperature can be increased. If the 24 hr point shows 10 - 20 % degradation and the primary degradants are understood, there is no need to continue the reaction out to the 1 week point.
Another critical parameter in the acid/base hydrolysis experiment involves incorporation of the appropriate controls, the drug substance , drug product and placebo. The acid/base stress conditions should result in approximately, 10 - 20 % degradation of the drug substance or represent a reasonable maximum condition achievable. If this level of degradation is not achieved, additional hydrolysis experiments should be performed at not more than 700c for 1 week total reaction time. Going above this level is not recommended for typical drug substance material .
Oxidative studies are executed to force the degradation of drug substance to determine the primary oxidative degradation products. Oxidative degradation is a serious stability problem and can cause a major halt in pharmaceutical development. The 1987 stability guidelines states that a high oxygen atmosphere should be evaluated in stability studies on solution or suspension of bulk drug substance.
Drug substance functional groups which are susceptible to oxidation reaction include heteroatom,benzylic sites, aldehydes and ketones.
The main source of oxidative degradants for pharmaceutical drug candidate is the reaction of the drug substance and /or drug product with molecular oxygen, a complex reaction. Hydrogen peroxide is often used in the pharmaceutical industry for oxidative challenge. The major problem with H2O2 non predictive of molecular oxygen reaction. H2O2 stress testing can be useful in drug product studies where H2O2 is an impurity, in an excipient.
To prepare for oxidative degradation study, a preliminary solubility analysis of drug substance should be performed. Oxidative purposeful degradation studies typically require solubility of approximately 1- 10 mg/ml in unbuffered conditions to achieve reasonable levels of degradation.
Its goal is to force the degradation of drug substance over time to determine the thermal degradation products. Elevated temperature stress conditions are selected based on conservative estimate of Arrhenius expression- a quantitative relationship of reaction rate and temperature using average activation energy.
Where Kabs = Specific rate constant
A = pre-experimental factor
E = Activation energy
T = Temperature in degree Kelvin
R = Gas constant (1.987 cal K-1mol - 1)
A 100 C increase in temperature results in a doubling of reaction rate and a decrease in the reaction time by a factor of 2.
The goal of the photostability studies is to force the degradation of the drug substance via UV and fluorescent conditions over time to determine the primary degradation products. UV and visible lights are the most energetic electromagnetic radiation source to which pharmaceutical drug substance and drug product are typically exposed.
Functional groups likely to introduce drug photo reactivity are carbonyl, nitroaromatic, N-oxide, alkenes, aryl chloride, weak C-H and O-H bonds, sulphides and polyenes.
There are 2 types of studies used in pharmaceutical photo stability testing.
Stress testing (purposeful degradation)
Purposeful degradation is used to evaluate the overall photosensitivity of the material (unprotected drug substance and drug product) for method development purposes and for degradation pathway elucidation.
Confirmatory tests are used to determine if there is any need to protect the final product from light. The ICH guideline on stability testing of new drug substance and products note that photo stability should be an integral part of stress testing. The light source used should be continuous over the near UV and visible regions.
Usually one month is required to complete a purposeful degradation fluorescent study and approximately 5 days to perform a purposeful degradation UV study . ICH guideline specifies an exposure of 200 watth/m for UV light confirmatory testing and an exposure of 1.2 x 106 luxurious hours for fluorescence study.
DRUG PRODUCT DEGRADATION STUDIES
Drug product degradation cannot be predicted from the stability of the drug substance in the solid state or solution. The non active pharmaceutical ingredients (excipients) can also react with the drug substance or catalyse the degradation reactions. Impurities in the excipients can also lead to degradation in the drug product not originally observed in the drug substance. Purposeful degradation studies are performed to determine the physical and chemical compatibility of drug substance with excipients. The studies on the drug product depend on the chemical composition of the drug product formulation. For drug product formulation , heat, light and humidity are often used.
The drug product stress conditions should result in approximately, 10-20% degradation of the drug substance or represent a reasonable maximum condition achievable for a given formulation. The specific condition (intensity and length of time) used will depend on the chemical characterstics of the drug product. For all drug product studies, it is critical to run the proper controls , the drug substance, drug product and placebo.
For drug product, the following key experiments should be considered. These experiments will vary depending on whether the formulation is a solution or solid drug product. For a solid drug product, key experimentals are thermal, humidity, photostability and oxidation if applicable. The most common type of interaction in solid form is between water and drug substance. Hence thermal and humidity challenges are critical. For solution formulation , key experimentals are thermal, acid/base hydrolysis, oxidation and photostability . For a solution drug product, more emphasis should be placed on acid / base hydrolysis.
The meaningful evaluation of the stability of a drug is to use stability indicating method, an analytical method distinguishing the intact molecule from the degradation products. Therefore, it is essential to develop stability indicating assay methods for ensuring quality and therapeutic nature of active ingredient.
Basic kinetic principle:
According to the law of Mass action, the rate of a chemical reaction is proportional to the product of the molar concentration of the reactants each raised to a power usually equal to the number of molecules, a and b, of the substances A and B , undergoing reaction.
In the reaction,
aA + bB + ……= products
The rate of the reaction is:
Rate = (-1/a) - (d[A]/dt)
= (-1/b) - (d[B]/dt)
= …..k(A)a (B)b.....
Where, k is a rate constant.
Chemical kinetics involves the study of rate of chemical change from its initial state to its final state and the way in which this rate is influenced by the concentration of reactants, products and by factors such as solvents, pressure and temperature.
Zero - order reaction:
It is defined as the reaction in which the rate does not depend on the concentration of the reactant.
- dA = k0
Period of time required for one half of the material to initial concentration of the reactant to undergo reaction.
Zero-order kinetics are most applicable in suspensions.
t1/2 = a/2k
It is proportional to initial concentration (a).
First order reaction:
It is defined as the reaction in which the rate of reaction depend on the concentration of one reactant.
- dC = kc
Period of time required for a drug to decompose to one half the original concentration, t1/2. It is independent of concentration(a).
t1/2 = 0.693/k
Second - order reaction:
The rates of biomolecular reactions, which occur when 2 molecules come together.
A + B products
There are two forms of second-order reaction. For the first case, the rate of reaction is proportional to the concentration of reactant A raised to the power of two. That is, second order with respect to A.
It takes the form:
dA/dt = k[A]2
The second type occurs if the rate of reaction is proportional to the product of the concentrations of two reactants , each raised to the power of one. That is, first order with respect to both reactants.
-d[A]/dt = - d[B]/dt = k[A][B].
t1/2 = 1/ak
Pseudo - order reactions:
For some reactions, the rate of the reaction may be independent of the concentration of one or more of the reacting species over a wide range of concentrations. This may occur under these conditions:
One or more of the reactants enters into the rate equation in great excess compared to others.
One of the reactants is a catalyst.
One or more of the reactants is constantly replenished during the course of a reaction.
Rate = (k[A]n[B]m)…. = kapp [B]m…
EFFECTS ON CHEMICAL REACTION RATE:
The speed of many reactions increases about two-three times with each 100rise in temperature. Arrhenius equation explains the effect of temperature on the rate of reaction.
K = Ae-Ea/RT
Where, K - Specific Reaction Rate
A - Frequency Factor or Arrhenius factor
Ea - Energy of Activation
R - Gas constant (1.98cal/deg mole)
T - Absolute Temperature
Influence of light (Photodegradation):
Light energy like heat enhance the rate of reaction to occur. When a radiation of sufficient wavelength falls on a molecule, the molecule absorbs a quantum of radiant energy and collide with other molecules raising their kinetic energy and temperature of the system will increase. A best example is the irradaiation of ergosterol to produce Vitamin-D.
Photochemical reactions usually follow a Zero-order kinetics. Photo-oxidation of drugs is initiated by UV radiation according to one of two classes of reactions. The first is a free radical chain process and second is initiated by a dye-methylene blue.
Influence of oxidation:
Oxidation is the loss of electron from a molecule or removal of Hydrogen atom or addition of an oxygen. The presence of atmospheric oxygen, temperature, radiation and presence of a catalyst accelerate the rate of a reaction. Trace amounts of heavy metals promote the rate of reaction. Hydronium and Hydroxyl ions catalyse oxidative reactions. Reaction between drug and molecular oxygen is called Auto-oxidation.
Oxidation may be inhibited by the use of Antioxidants like Tocopherols,BHA, BHT. Synergists are generally organic compounds that chelate small amounts of Heavy metals ions and hence metal ions are not available to catalyse the oxidation. Eg: EDTA, Citric acid, Tartaric acid .
Oxygen free Environment: Air is replaced with inert gases like,Nitrogen, Carbondioxide.
Drugs with ester or amide group reacts with one molecule of water and undergoes hydrolysis . Hydrolysis are catalysed by H and OH ions. Amide groups are more stable than ester groups.
Rate of hydrolysis can be controlled by using Buffers, and by removal of water and inhibited by addition of complexing agent.
TOOL FOR STABILITY INDICATING ASSAY METHOD
HIGH PERFORMANCE THIN LAYER CHROMATOGRAPHY
HPTLC is one of the powerful, reliable and cost effective separation technique for both qualitative and quantitative analysis.
Features of HPTLC
Simultaneous processing of sample and standard - better analytical precision and accuracy less need for internal standard.
Lower analysis time and less cost per analysis.
Low maintenance cost.
Several analysts work simultaneously.
Simple sample preparation - handle samples of divergent nature.
Low mobile phase consumption per sample.
No prior treatment for solvents like filteration and degassing.
No interference from previous analysis - fresh stationary and mobile phase for each analysis - no contamination.
Visual detection possible - open analysis.
Non UV absorbing compounds detected by post- chromatographic derivatisation.
The principle of separation is adsorption. The mobile phase solvents flow through because of capillary action. The components are separated based on the affinity of the components towards the stationary phase.
Steps involved in HPTLC
Steps involved in HPTLC method development can be summarized as follows.
Selection of chromatographic layer
Pre coated plates with different support materials and different sorbents are available. The high performance silica gel is more efficient and reproducible than conventional grade of silica. Particle size is very small about 5 µm and uniform in size.
The sample preparation procedure is to dissolve the dosage form in a solvent with complete recovery of intact compound of interest and minimum of matrix with a suitable concentration of analytes for direct application on the HPTLC plate.
Layer pre treatments
Prior to chromatography it is a common practice to prepare the layer by any or all of the following steps; washing, activation, conditioning and equilibrium so as to avoid problems like irregular and drifting densitiometry baselines, ghost peaks and reduced sample detectability in post chromatographic derivatisation reactions. Activation can be done by placing in an oven at 110-120 0c for 30 minutes prior to spotting.
Application of the sample
Use of Linomat V (automatic application devices) is recommended for quantitative analysis. Usual concentration of sample is in the range of 0.1 - 1µg/ml. Preferably samples are applied as bands because it ensures better separation because of the rectangular area in which the compounds are present on the plate.
Mobile phase optimization
A solvent of correct strength for a single development separation will migrate the sample into the Rf range of 0.2 to 0.8. Mobile phase should chosen taking into consideration chemical properties of analytes and sorbent layer.
If the tank is saturated prior to development, solvent vapor soon get uniformly distributed throughout the chamber, hence less solvent shall be required to travel at a particular distance, resulting in the lower Rf values, as soon as the plate is placed in a saturated chamber.
Chromatographic development and drying
Ascending, Descending, Two dimensional, Horizontal, Multidimensional, gradient, radial etc are most common methods of development. Dry in vacuum dessicator.
Detection and visualisation
Detection under UV light is most preferred method as it is non-destructive, spots of non-fluorescent compounds can be seen at 254 nm(short wavelength) or at 366nm (long wavelength), spots of non fluorescent compounds can be seen using fluorescent stationary phase(silica gel GF).
The chromatographic development should clearly and completely separate all the compounds of interest. Sample and standard should be chromatographed on same plate-after development chromatogram is scanned.
ADVANTAGE OF HPTLC
Ability to analyse several samples
Visual chromatogram and simplicity
Multiple sample handling
Low running and maintenance cost
Disposable layer can be used
Quantification of crude drug
Simultaneous analysis of samples
Automatic sample application
Small quantity of mobile phase sufficient
Method validation is the process to confirm that the analytical procedure employed for a specific test is suitable for its intended use. Methods need to be validated or revalidated,
Before their introduction into routine use.
Whenever the conditions change for which the method has been validated, eg, instrument with different characteristics.
Whenever the method is changed, and the change is outside the original scope of the method.
The parameters as defined by the ICH and by other organisations.
Limit of detection
Limit of quantitation
It is the ability of the method to accurately measure the analyte response in the presence of all potential sample components. A specific method can accurately measure the analyte of interest even in the presence of potential sample components (placebo ingredients, impurities, degradation products etc).
The precision of an analytical procedure expresses the closeness of agreement between a series of measurements from multiple sampling of the same homogenous sample under prescribed conditions. Precision may be considered at three levels:
Repeatability expresses the precision under the same operating conditions over a short interval of time. It is obtained when analysis is carried out in one laboratory by one operator using one piece of equipment over relatively short time span atleast 5 or 6 determinations of three different matrices at 2 or 3 different concentrations.
Intermediate precision expresses within laboratories variations: Different days, different analysts, different equipments etc.
Reproducibility expresses the precision obtained between laboratories. It is determined by analysing aliquots from homogenous lots in different laboratories with different analysts with the specified parameters of method.
Accuracy of a method is the closeness of the measured value to the true value for the sample. Accuracy is often determined by recovery studies in which the analytes are spiked into a solution containing the matrix. The matrix should be found not to interfere with the assay of the compound of interest.
Linearity of an analytical procedure is its ability (within a given range) to obtain test results which are directly proportional to the concentration (amount) of the analyte in the sample. Acceptability of linearity data is often judged by examining the correlation coefficient and Y- intercept of the linear regression line for the response vs concentration plot.
Range of an analytical procedure is the interval between the upper and lower concentration (amount) of the analyte in the sample (including these concentrations) for which it has been shown that the analytical procedure has a suitable level of precision, accuracy and linearity.
Limit of detection(LOD)
The detection limit is the lowest analyte concentration that produces a response detectable above the noise level of the system, typically, three times the noise level(S/N=3).
Limit of quantitation(LOQ)
The Limit of quantitation is the lowest level of analyte that can be accurately and precisely measured. It is calculated as the analyte concentration that gives (S/N=10).
The robustness of a method is its ability to remain unaffected by small changes in parameters such as percentage organic content and pH of mobile phase, buffer concentration, temperature and injection volume.
Degree of reproducibility of test results obtained by analysing the same sample under variety of normal test conditions such as different analysts, different instruments, different lots of reagents etc.
Analyte should not decompose during the development of the chromatogram and should be stable in solution and on the sorbent for atleast 30 and 15 minutes respectively. The intensity of the spot on the chromatogram should be constant for atleast 60 min. Stability is checked for:
- Stability in sample solution
- Stability on the sorbent layer prior to development
- Stability during development
- Stability after chromatographic development
- Purity of reagents and solvents.