forced degradation

RELATED SUBSTANCES METHOD DEVELOPMENT.

Forced degradation studies or Stress testing studies:

What are the Objectives ?:

To investigate the likely degradation products, which in turn

helps to establish the degradation pathways and

the intrinsic stability of the drug molecule.

To provide foundation for developing a suitable stability

indicating method.

RS MD - Forced degradation studies or Stress testing studies.

What we Stress?.

DRUG SUBSTANCE DRUG PRODUCT. PLACEBO.

All three to be stressed separately.

RS MD - Forced degradation studies or Stress testing studies.

What we get to Know from this?.

Differenciation between

Drug related and non-drug related degradation products

Discrimination between

Synthetic process impurities, excipients,

degradation products derived from excipients alone and

drug related degradation products including the drug excipient

combinations.

Forced degradation studies or Stress testing studies.

Key Point to be kept in mind :

The degradation products generated in the stressed samples are

termed as “potential” degradation products

that may or may not be formed under relevant storage conditions.

Forced degradation studies

Four major forced degradation studies are

Thermolytic Degradation,

Hydrolytic degradation,

Oxidative degradation, Photolytic degradation.


THERMOLYTIC DEGRADATION :

Determine the Melting Point of the Analyte.

Drugs for which melting point is <150°C,

Stress at 70°C or about 40°C below the melting point, which ever is higher.

Drugs for which melting point is >150°C,

Stress the samples at 105°C.



How long to stress?.

What if the degradation is not achieved?.



Stress for a week time, Observe the sample. Test the sample in between. Achieve a degradation between 5 to 20%.

If no degradation is achieved even after harsher stress, justification can be provided that the molecule is stable.


HYDROLYTIC DEGRADATION :

Drug degradation that involves reaction

with water is called Hydrolysis.

Degradation which is due to hydrolysis is called Hydrolytic Degradation.



Hydrolysis reactions are typically

Acid or Base catalyzed.

Apart from water, Acidic and basic conditions should also be employed in order to induce potential hydrolytic reactions.



As these hydrolytic stress studies are to be conducted in aqueous solutions,

estimate the solubility of the drug molecule of interest in water.

If drug is hydrohobic & found to be not soluble in water,

use a co-solvent to dissolve the required quantity.



Two most commonly used co-solvents are Acetonitrile and Methanol.

Methanol has the potential of participating in the degradation chemistry, it should be used with caution especially under acidic conditions when the compound being tested contains a carboxylic acid, ester or amide.


Acetonitrile is typically preferable to methanol.

However,

Acetonitrile is not completely inert and can participate in the degradation reactions.

For example,

acetonitrile is known to contribute to base catalyzed epoxidation reactions in the presence of peroxides.

Acetonitrile will also degrade in presence of base(at pH 13) and /or acid (at pH1) under elevated temperatures to detectable levels of Acetamide or Acetic acid which can show up as early eluting peaks in RP-HPLC when monitored at lower wavelengths.

The size of the HPLC peaks from these two products is relatively small and use of stressed blank solutions permits ready identification of these peaks.


In acidic acetonitrile/water solutions,

Tertiary alcohols can undergo Ritter reaction to form amides.

In the presence of radicals acetonitrile cab be oxidized to small amounts of formyl cyanides that will readily react with nucleophiles (such as amines) resulting in a formylation reaction.

Nonetheless, most of these side reactions of acetonitrile are relatively minor and acetonitrile remains the most frequently use co-solvent for hydrolysis studies.


HYDROLYTIC DEGRADATION :Co-solvents that are recommended for the hydrolytic stress testing studies are shown below.

Acidic pH Neutral pH Basic pH

Acetonitrile Acetonitrile Acetonitrile

DMSO N-methyl pyrrolidine DMSO

Acetic acid Diglyme

Propionic acid p-Dioxane.



The hydrolytic degradations are recommended to be performed

at a temperature of about 70°C

with a reflux condenser installed to avoid

the loss of evaporation and

use few glass beads or porcelain pieces to

avoid bumping.

Conduct the stress reflux always in fume hood.



Typical conditions :

Reflux using water/ 0.1M HCl/ 0.1M NaOH for stress testing with or without co-solvent at a temperature of about 70°C.

Reflux for 12 hours or until about 5 to 20% degradation is achieved or which ever is earlier.

Neutralize the stressed solutions before injection.

Prepare a stressed solution at a higher concentration than that of test concentration.

After the stress, dilute in diluent to get the test concentration, so that peak shapes are good.



HUMIDITY STRESS :

Stress the samples to 90% Humidity for 1 week.

Why humidity stress, when we are performing water stress?.

This is typically to know the influence of humidity on the drug.


OXIDATIVE DEGRADATION :one of the most common mechanisms of drug degradation.

Typically oxidative degradations are to be performed at room temp (about 25°C to 30°C) in dark.

Use of higher temperatures >30°C is not recommended, because the reaction rate in solution may actually be reduced at higher temp due to decrease in oxygen content of the solvent.

But for some compounds, degradation may occur at higher temperature due to free radical reaction initiation.Typical stress conditions :

Stress with 3% hydrogen peroxide in dark at room temperature with constant stirring.

Stress for 24 hours or until about 5 to 20% degradation is achieved or which ever is earlier.


PHOTOLYTIC DEGRADATION :

Degradation that results from exposure to UV or visible light.

Typical Exposure conditions :

Expose the samples to 3 times to

1.2 million lux-hr visible and

200 W-hr/m2 UV.


MULTI COMPONENT PRODUCT DEGRADATION :

Stress testing of placebo with each actives separately shall be performed in order to know the information about which degradant is from which active.

Alternatively, these can be identified by the UV spectra.

Stress testing of placebo with other actives excluding the one at a time, shall be performed, in order to know the non-interference from each other.

Alternatively, these can be identified by the Peak purity.

If methods are different, placebo shall include the other actives.

EVALUATION OF FORCED DEGRADATION :

Evaluate the peak purity of the Analyte peak and

the major degradants.

Evaluate the peak purity for the analyte peaks for which

peak heights are less than 1000 m AU.

If more than 1000 mAU, solutions may be diluted to

check Peak Purity.

Peak purity Criteria : Software specific.

If any of the major degradants are not passing peak purity, modify the conditions and see that the peak purity is passing.


For GC methods and methods where RI, ELSD and FLD detectors are used, peak homogeneity needs to be established by doing Mass spectral study.

Evaluate the peak purity of the Analyte peak and

the major degradants.

Method needs be Mass compatible.


In case any known impurity is observed to be increasing in stress,

Examine whether it is process impurity or a possible degradant.

In case a known compound is a process impurity and the peak is found to be increasing in forced degradation, it indicates that

A degradant is eluting at the same RT as that of the process impurity » or

There could be a secondary pathway of formation of process impurity via some other degradation route.

Eg : 9-Hydroxy Respiridone and N-oxide of Respiridone Dimethyl derivative of Gatifloxacin

Observe any peaks merging wrt main peak (shoulders) and or any critical pair of peaks.


Based on the structure of the Drug, Look for possible formation of degradants which are not UV active.

Eg : Phosphenytion degrades to give Phenytion which is UV active and the Phoshate which is not UV active at all and elutes in viod.

Employ RI detector or ELSD in such cases to see whether those degradants does form or not.

Examine the chromatograms at different wavelengths, especially at Lower wavelengths such as 200 or 205 or at 210 for poorly absorbing degradants.

Try to establish the structure of the Major degradants by doing a mass study.


If any Major degradant is having a different absorption maxima,

Preferrably it should be identified & estimated at that wavelength.

If not identified, it is difficult to assess the exact quantification, when the active ingradient has different absorption characteristics.

Eg : Respiridone, Only one unknown degradant is having absorption maxima at 260 nm, where as Respirodone & other impurities & degradants are having maximum at 280 nm.


If any of the Peaks are found to be not separating ,

Optimise the seperation using the forced degradation samples.

Mass balance study :

The process of adding together the assay value and levels of degradation products to see how closely these add up to 100% of the initial value.

Estimate the assay of the final force degradation samples and assess the mass balance. Mass balance is to be achieved at least up to 95% level.

If the mass balance is less than the required criteria investigation to be done and justified.


Reasons for not achieving the mass balance :

(1). Degradation products are not eluted from the HPLC column

(2). Degradation products are not detected by the detector used.

(3). Degradation products lost from the Sample matrix, due to

insolubility, volatility or adsorption losses.

(4). Parent compound lost from the sample matrix, due to insolubility,

volatility or adsorption losses.

(5). Degradation products are co-eluted with the parent compound.

(6). Degradation products are not integrated due to poor

chromatography.

(7). Inaccurate quantification due to differences in response factors.


Finalization of Detector wavelengths:

After finalization of the separation of all impurities and degradants,

take the overlaid spectra of all impurities for each of the stress conditions, along with the spectra of the analyte.

Select a wavelength where all impurities are detected and have maximum absorbance.

In case if this is not feasible, select different wavelengths to estimate all impurities.

Exclusion criteria is to be specified at the other wavelengths.


Finalization of Chromatograhic conditions & System suitability :

Can be done only after the study of the Robustness of the method.

Perform the robustness of the method with regard to

mobile phase composition (10%),

pH ( 0.2),

gradient ( 0.2 % per min) : Verify in different brand of HPLC’s,

flow rate 0.2 ml/min),

temperature ( : 5°C ).

Fine-tune the method in the range where it is most robust.

In case any parameter is sensitive, specify the same in the test method so that it will be monitored.


Finalization of System suitability :

based on the criticality of separation.

In general, resolution factor for the closely eluting compounds is selected as a system suitability requirement.

Try to avoid injecting the impurities solutions by improving the separations

If the separation is found to be satisfactory, there is no need to keep a resolution factor as a system suitability parameter.

In such a case, only a diluted standard reproducibility can be adopted as a system suitability requirement.

Reproducibility can be measured with two injections also, by monitoring the ratio of peak areas of two injections.

Ratio should be between 0.9 to 1.1

RS METHOD DEVELOPMENT

RRF (Relative Response Factor) :

Establish the stability of solutions & Filter suitability studies before conducting RRF study.

Response Factor = Area / amount (Concentration).

RRF = Ratio of Response factors of impurity to API.

Correction is given in denominator or Numerator depending on the way the RRF is calculated.

IF RRF =(RF of Impurity/ RF of API), Then RRF is used in Denominator.

IF RRF =(RF of API/ RF of Impurity), Then RRF is used in Numerator.Typically called as ‘correction factor ‘also.



Response Factor = Area / amount (Concentration).

While Calculating the Concentration, use potencies of Complete molecule for both API and for Impurities.

For Example,

If API / impurity is a salt or a complex, calculate the concentration as salt or as drug complex.

Concen. In mg/ml of fluoxetine HCL ( Not as fluoxetine base)Concen. In mg/ml of Omeprazole Magnesium ( Not as Omerazole base)

Consideration is same for Impurities also.



Best way to establish the RRF is by “Slope method”(By linearity curve).Where Slope = RF.

Typically using 5 Concentrations ranging between 0.1% to 1.0%."Do not include Zero" in linearity plot.

NOTE: If impurity quantity is a constraint, draw the curve with 2 or 3 points instead of five points.

RRF =(SLOPE of Impurity/ SLOPE of API), Then RRF is used in Denominator.

RRF =(SLOPE of API/ SLOPE of Impurity), Then RRF is used in Numerator.

Conduct recovery studies to prove that the established RRF's are correct. Acceptance criteria : 90% to 110%.

QUANTITATION METHODS :Area normalization:

If the Impurities RRF close to the Analyte (0.9 to 1.1)

Diluted Standard Method:

If the impurities RRF are different from the analyte.

External standard Method :

If the impurities are estimating with out Standard peak in same chromatogram.

Internal standard method:

If the sample preparation procedure involves different extraction steps (Normally when Derivitisation techniques or Liquid/Liquid Extraction techniques are used)

QUANTITATION METHODS :

For Multi Component products,

If method is common ,

Use the diluted standard area for which the response is low

If the methods are different,

Ensure that impurities are not estimated twice.

QUANTITATION METHODS :

DISREGARD CRITERIA :

Disregard any peak which is from Blank, Placebo.

Disregard any impurity which is less than 0.01%.

Placebo degradants can be disregarded if found not toxic.

QUANTITATION EQUIVALENCY :

Between API method and Formulation methods :

Analyse API in both methods.

Compare the % of known & number & % of unknown impurities.

Assess the difference & its impact.

Typically, it should be for all impurities individually.

RS METHOD DEVELOMENT

THANKSTHANKS

forced degradation

Documents

drug degradation

degradation reactions

oxidative degradation

photolytic degradation

degradation pathways

degradation chemistry

potential degradation

likely degradation products