Mass balance during forced degradation study

Hai...... Good morning to all....

Yesterday I forgot to add Mass balance concept to forced degradation study.

So today my concept is.......

" Mass balance calculation during Forced degradation study"

 

 

There is no proper guidance from any regulatory authorities for mass balance calculation during forced degradation study.

The mass balance concept is based on the "law of conversion of mass" which states "Mass can neither created nor destroyed"

Mass balance means...?

I will give a simple example with a small story. Understand carefully.

   " A college is having exctaly 100 students. Oneday, campus interview was conducted in that college. Out of 100 students, 11 students were selected in campus interview, and 3 students were kept on hold for selection.

   Now,     Total no. of students                                    = 100
                 No. of students not selected in interview    = 86
                 No of students selected                               = 11
                 No. of students kept on hold for selection  = 3.

Total No. of students = (No. of students not selected + No. of students selected + No. of students on hold)

Before coming to the main point, Now lets compare the story with with chemistry.

     College                                                                    = Stability indicating Method
     Campus interview                                                   = Forced degradation study
      Total No. of Students                                             = amount of mass (analyte) taken for degradation
      No. of students not selected in interview               = amount of mass (analyte) remained after degradation
      No of students selected                                          =  amount of known degradants observed.
      No. of students kept on hold for selection             = amount of unknown degradants observed.

Now mass balance means,

  Total mass = (amount of mass remained  + amount of known degradants + amount of unknown degradants)


 Why Mass balance required...?

While developing stability indicating method for degradants ( generally called as RS method), we have to ensure whether the method is exactly quantifying all possible degradants or not. Hence, Mass balance is useful element for that.

How Mass balance calculated..?

As I already explained the mass balance equation above, it is
 Total mass = (amount of mass remained  + amount of known degradants + amount of unknown degradants)

So,  here I will convert the equation for better understanding. After each degradation study,

 Total % of drug = % of drug remained + % of known degradants + % of unknown degradants

Important : Don't ran away without knowing this points...

                  1) Relative response factors plays an important role to calculate exact mass balance.
                  2) Molecular weight of Drug and molecular weight of degradant formed plays an important role to calculate exact mass balance.

Importance of Relative Response Factor(RRF):

  Suppose for example, if the RRF of an impurity (RRF should be established while developing analytical method) is 0.6.
and the if amount of degradant formed as per area calculation is 3.5, . now the exact amount of impurity formed will be

                                            = 3.5 * 1/0 .6 =  5.83%

Importance of Molecular weight(MW):

1)  Suppose for example, MW of Main drug is 151, and MW of degradant formed is 195. If the % of degradant formed as per % area is 3.5, now the exact amount of impurity formed is

         3.5* 151/195 = 2.71%

2) Suppose for example, MW of Main drug is 151, and MW of degradant formed is 125. If the % of degradant formed as per % area is 3.5, now the exact amount of impurity formed is

         3.5* 151/125 = 4.23 %

 What is limit for mass balance.?

The ultimate goal is that the analytical method should achieve 100% mass balance. however, the acceptable limit for mass balance will be not less than 95%.

What to do if mass balance not acheived.?

Some times 100% mass balance may not be achieved because of
        1) The degradant formed may not be eluted in the developed method.(extend runtime or modify method conditions)
        2) The RRF may be zero at method wavelength.(select appropriate wavelength or develop separate method)
        3) Degradant may be UV inactive.( Identify the impurity by different techniques like RI, ELSD, LC-MS etc..)

Don't worry if the mass balance is not achieved, proper justification should be given for not achieving mass balance. as I shown in the brackets for each above points.



That's it for today........... have a nice day.........

V.Suresh


 

 



Forced Degradation Study for pharmaceutical products

Hai............. how is going..........

Sorry for not posing any article since last three days.......

But.....

Today I came with very interesting article and 

That is........

"Forced Degradation Study"

It is typical task to many scientists to perform Forced degradation study to the formulation products.

Here I am trying to clarify so many doubts about " Forced degradation study", and explain the practical approach in different cases.

Before going to practical approach I want to tell.......

What.............?

Why.............?

When...........?

How............?
How much........?

1) What is Forced degradation.?

Forced degradation is a ..............

" process of increasing the Degradation rate of a material or product by the application of additional forces like Oxidation, Reduction, excess heat, excess humidity, more intense light etc......."

2) Why Forced degradation studies are required.?

         a) To know the degradation pathways of drug substances and drug products.
    
             b) To differentiate degradation products that are related to drug products from those that are generated from placebo in a formulation. 

             c) To findout the structure of degradation products. 
     
             d) To determine the intrinsic stability of a drug substance in formulation.

             e) To reveal the degradation mechanisms such as hydrolysis, oxidation, thermolysis or photolysis of the drug substance and drug product.

             f) To establish stability indicating nature of a developed method. 

             g) To understand the chemical properties of drug molecules.

             h) To generate more stable formulations. 

              i) To produce a degradation profile similar to that of what would be observed in a formal stability study under ICH conditions. 

              j) To solve stability-related problems.

3) When Forced Degradation Studies Do.?

                                        a) During Formulation studies, forced degradation studies are useful to establish stability indicating nature of developed analytical method, to compare pre manufacturing and post manufacturing changes.
                                           
                                        b) During Pre-clinical studies, forced degradation studies are useful to identify degradants, toxic components like-body conjugates, etc...
     

                                        c)  During Clinical development,  Forced degradation studies are useful to compare Pre-clinical and clinical quality.

       Stability and forced degradation requirements in ICH quality guidelines

ICH guideline title                                                                                   Comments

Q1B: Photostability testing of new drug substances and products                  Provides guidance for the                                                                                                                               generation of photostability                                                                                                                             studies to support submission in                                                                                                                     registration applications for new                                                                                                                     molecular entities.

Q2(R1): Validation of analytical procedures: text and methodology                 Forced degradation studies are                                                                                                                       required for analytical method                                                                                                                         validation to demonstrate test                                                                                                                         specificity ( stability indicating                                                                                                                       nature)

Q3A(R2): Impurities in new drug substances
Q3B(R2): Impurities in new drug products                                                    Recommends the use of                                                                                                                                 appropriate stress conditions for                                                                                                                     validation of analytical                                                                                                                                   procedures applicable to new                                                                                                                         chemical entities.
                                                                                                                Biological/biotechnological                                                                                                                             products not covered in the                                                                                                                             guidelines.


Q5C: Stability testing of biotechnological/biological products                         Recommends the use of                                                                                                                                accelerated and stress                                                                                                                                  conditions to support the 
                                                                                                               establishment of expiration date                                                                                                                      and to support product                                                                                                                                  comparability. 
                                                                                                               Selection of stress conditions to                                                                                                                    be conducted on a case-by-case                                                                                                                    basis.

Q5E: Comparability of biotechnological/biological products subject 
to changes in their manufacturing process
                                                                                                                The manufacturing process and                                                                                                                       its changes may potentially                                                                                                                           produce different degradants and                                                                                                                     degradation pathways.                                                                                                                                   Recommends                                                                                                                                               forced degradation studies to                                                                                                                         establish potential product                                                                                                                             differences in the degradation                                                                                                                         pathways.

Q6B: Test procedures and acceptance criteria for biotechnological/
biological products                                                                                     Forced degradation studies can                                                                                                                     help to understand process related 
                                                                                                                degradants/impurities; justify and                                                                                                                   rationalize to meet the                                                                                                                                   requirements of setting up the                                                                                                                         acceptance criteria based on                                                                                                                         potency and the level of 
                                                                                                                product-related impurities.

Q8(R2): Pharmaceutical development                                                          Forced degradation studies usually                                                                                                                 include extended variations (ie, 
                                                                                                                temperature, pH, light, shear),                                                                                                                        container closure system                                                                                                                              compatibility, and suitability studies                                                                                                                under normal and stressed storage                                                                                                                  conditions.

Q11: Development and manufacture of drug substance                                Forced degradation studies will                                                                                                                       provide product knowledge and fulfill 
                                                                                                              quality requirements.

4) How Forced degradation do.?(Practical approach)

The practical approach to the forced degradation depends on requirement of forced degradation study.

The most common type of Dedradations are....

                          1) Hydrolytic Degradation : Degradation with acids, bases and water etc...

                          2) Oxidative degradation  : Degradation with oxidising agents like peroxide, potassium permanganate etc.....

                          3) Thermal Degradation : Degradation with heat

                          4) Photolytic Degradation : Degradation by exposing to UV, Visible light.

These are main degradation types and apart from that, metallic degradation also used in some cases.

Hydrolytic Degradation:

Hydrolysis is a solvolytic process in which drug reacts with water to yield breakdown products of different chemical compositions.

    Acid Degradation: Hydrolysis under acidic condition.

 

Above diagram shows how paracetamol undergoes hydrolysis in acidic condition. Most common acid used for acid hydrolysis is hydrochloric acid.

 

There are no specific guidance for how much concentration of acid to be taken for degradation. Hence most recommended range will be 0.1N to a maximum of 5N of acid in case of hydrochloric acid. If concentration of acid is more, it will impact badly on HPLC  column.

 

Drug substances(DS), Drug product(DP) and Placebo were degraded with Acid to attain required amount of degradation. Neutralise the sample with same concentration of base. then follow procedure as per analytical methodology.  Differentiate the peaks based on peaks obtained from DS, DP and placebo.

 

 



       Base Degradation: Hydrolysis under Basic condition.
                   

     

      
Above diagram shows how paracetamol undergoes hydrolysis in basic condition. Most common base used for base hydrolysis is Sodium hydroxide.            

Most recommended range will be 0.1N to a maximum of 5N of base in case of sodium hydroxide. If concentration of base is more, it will impact badly on HPLC  column.

Drug substances(DS), Drug product(DP) and Placebo were degraded with base to attain required amount of degradation. Neutralise the sample with same concentration of acid. Then follow procedure as per analytical methodology.  Differentiate the peaks based on peaks obtained from DS, DP and placebo.

Note: If sample mass is more, first dissolve the sample mass in diluent then proceed for degradation. Our final goal is to degrade the analyte. If no degradation achieved even with the addition highly concentrated acid/base, then reflux the sample after adding acid/base, cool to room temperature and proceed as per analytical methodology.

Humidity Degradation: Hydrolysis under high humidity condition.

Humidity under different temperatures is one of the possible cause for degradation during stability studies. Excess humidity (  over 90%) should be applied to the samples to degrade.

Saturated solution of Sodium nitrate or potassium nitrate under vacuum can be used to produce excess humidity.

Water Degradation: Hydrolysis with water under heat condition.
 
Samples can undergo hydrolysis with water in presence of high temperatures.

Drug substances(DS), Drug product(DP) and Placebo were degraded with water  at temperatures ( 70°C to 80°C) to attain required amount of degradation. Neutralise the sample with same concentration of acid. Then follow procedure as per analytical methodology. Differentiate the peaks based on peaks obtained from DS, DP and placebo.

Oxidative Degradation:

         Oxidative Degradation: Oxidation under strong oxidizing agents.

 



Above diagram shows how paracetamol undergoes oxidation under strong oxidizing agents. Most common oxidising agent is Hydrogen peroxide.

Maximum recommended concentration is 10% hydrogen peroxide.

Drug substances(DS), Drug product(DP) and Placebo were degraded with oxidizing agent to attain required amount of degradation. Then follow procedure as per analytical methodology. Differentiate the peaks based on peaks obtained from DS, DP and placebo.

Problem with hydrogen peroxide: Some times a peak due to hydrogen peroxide will interfere with degradant peaks. In such cases, an alternative oxidizing agent can be used. e.g potassium permanganate.

Note: If sample mass is more, first dissolve the sample mass in diluent then proceed for degradation. Our final goal is to degrade the analyte.

Thermal Degradation:
Temperature is one of the major cause for degradation of pharmaceutical products in stability studies.

DS, DP and Placebo can be exposed to excess heat ( probably more heat than Accelerated condition-40°C/75%RH) to get required degradation.

Note: Samples should not be exposed more than to its boiling points.

Photolytic Degradation:

As per ICH Q1B guideline, Samples should be exposed to overall illumination of not less than 1.2 million lux hours to visible light and integrated UV energy of not less than 200 watts/square meter.

Calculate the intensity of light with lux meter and load samples to expose to visible and UV light. After completion of required time, prepare samples as per analytical methodlogy and anlyse.


" If samples were not degraded in any one or more of the above degradation studies, declare the sample is stable with respect to particular type of degradation. "


5) How much to be  degraded .?
Now its a big question for all scientists that how much the sample to be degraded..?

This purely based on the purpose of degradation.

 If forced degradation study is to elucidate the structure of degradation product, % degradation to be achieved is based on how the structure is determined.(structure can be determined by LC-MS, LC-MS/MS and NMR spectrometry etc....).

If forced degradation study is to prove an analytical method is stability indicating, then the % degradation should be not less than specification of Total impurities. How ever it is not fixed.

Excessive degradation may lead to secondary degradation (i.e degraded products may further degrade).

Hence a moderate degradation from 5% to 20% may be good choice.

Some scientists may say slightly more than 10% degradation is acceptable  for stability indicating methods, because the specification to the % assay for major of the products is NLT 90% until unless justified.



"So that's it for today. I think after reading this, so many doubts may arise to the readers. so please post your questions as comments below.

My next topic will be " Stability indicating Methods "

Thanks and bye.........
V.Suresh.

About the Center for Drug Evaluation and Research The Biopharmaceutics Classification System (BCS) Guidance

The Biopharmaceutics Classification System (BCS) Guidance

Use of Internal Standards in HPLC

Dear All.........., How is going.........? all is well......

As a part of knowledge sharing, Today I want to explain you

" Use of Internal Standards in HPLC "

Yesterday, While travelling from Hyderabad to Hosur..., One of my beloved research associate asked about "Use of Internal standard in HPLC". 

So dear friend(s)....., Come we will jump into one region of knowledge ocean......

We all knows that HPLC can be used for " Identification, product characterization, Purity determination and quantification" purposes.

There are different types of Quantitation methods in HPLC analysis.

Internal Standard Method is one type of Quantitative method used in HPLC & GC analysis.

 Types of quantitative analysis using HPLC:

There are mainly    1) Area Normalization method.

                                 2) Linearity Curve method.

                                 3) External Standard method.

                                 4) Internal Standard method.

1) Area Normalization method:

The %Area Normalization procedure reports the area of each peak in the chromatogram as a 
percentage of the total area of all peaks. %Area does not require any standard and does not depend upon the amount of sample injected within the limits of the detector. 

If all components respond equally in the detector and are eluted, then %Area provides a 
suitable approximation of the relative amounts of components.

This is very simple and easy to do.

 But......,

There are certain Limitations to % Area normilization method.
             
               (-)  This method is applicable only when the responce of all peaks are equal.
               (-)  This method is applicable only when the all components of sample are eluted in single run.
               (-)  This method is applicable only when the all peaks are free from interferences and no carry-over.

2) Linearity curve method:

A Linearity curve is a graphical representation of the amount(concentration) versus response(area)  for a single analyte (compound) obtained from a series of standard injections.

The curve is usually constructed by injecting an aliquot of the standard solution of known concentration and measuring the peak area obtained. Peak height is sometimes used but only in exceptional circumstances.

Before injecting sample solution, a series of standard solutions covering target concentration of sample should be injected. Plot a linearity curve using Amount(concentration) versus response(area).

The general regression equation for linearity plot is Y = mX +C

         Here, C = Intercept represents systematic error
                   m = Slope represents analytical sensitivity

  Check correlation coefficient (R) and it should not be less than 0.999, then inject sample solution. 

From sample solution, you will get response(area) which is 'Y'. Calculate amount(concentration) which is 'X' by using regression equation.

   This quantitative method can be used where the response of the analyte is considerable low.

3) External Standard Method:

The external standard (ESTD) quantitation procedure is the most common quantification procedure in which both standard and samples are analysed under the same conditions.

Inject standard solution in replicates( No. of injections are based on requirement) and evaluate system suitability (%RSD, Tailing, Plate counts, etc......) and inject sample. Calculate results based on following formula

              Amount = (area of sample / avg. area of STD) x ( STD dilutions/ sample dilutions)

In case of Related substances, Relatitive Response Factor(RRF) should be established to the impurities with respect to main analyte.

AND NOW.............. 

we reached to our main part........

4) Internal Standard Method:

The Internal Standard (ISTD) method eliminates the disadvantages of the External Standard method by adding a known amount of a new component that serves as a normalizing factor.  

The way of analysis and caculation is similar to External Standard Method. 

 External Standard Method    :       Area of analyte

 Internal Standard Method      :       Ratio of anlyte area and Internal standard area

"The internal standard is added to both standard and unknown samples and ‘compensates’ for losses during sample preparation or variability during the analytical determination."

Internal Standard Method is used ....
                  
                        a) if there is a routine variation in injection volume( e.g Manual injectors)
                        b) if there is complex procedure for sample preparation ( large procedure in sample preparation, extraction techniques, etc...)
                        c) if there is small retention time shifts( RT variation from injection to injection).

Some Care and Considerations should be taken while using Internal Standards.

Care:      

          * Same quantity of Internal standard should be added to both standard and Sample solutions. Otherwise it may lead to errors.
         
          * Internal Standard concentration should be same in both sample and standard solutions.

         * If Large procedure for sample preparation or extraction procedure is used, Internal standard should be added at the first step itself.

Considerations:

        
                          * The internal Standard should be similar to analyte chemically and physically.

                          * The internal Standard should elute near to main analyte and well resolved.

                          * The internal Standard should not present in Orginal Sample matrix.
                       
                          * The internal Standard should be unreactive to sample matrix.

                          * The internal Standard should be availabel in high pure form
                           
                          * The concentration of Internal standard should be about to half (either in height or response) to the main analyte. (this is to distinguish two peaks instantly in case of RT shifting).

Thats it..................................

Is this clear to all.......?

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Thanks 

V. Suresh.

                   
                    

Mechanism of Dissolution

Hai guys....... Today I want to share you the details mechanism of Dissolution. But It is a huge ocean to expalin. So I will explain Dissolution in three different sessions.....

Lets start............................

What is Tablet Dissolution?

When a dosage form is swallowed, the rate at which it releases the active ingredient is critical to ensure that the drug is delivered properly.

                 

                        “The rate at which the drug is released is called the dissolution rate”

One of the problems facing pharmaceutical manufacturers is to how optimise the amount of drug available to the body, i.e. its bioavailability.

Inadequacies in bioavailability can mean that the treatment is ineffective and at worst potentially dangerous (toxic overdose). All kinds of factors affect this from the formulation of the dosage form, size, shape, excipients, bindings and other physical characteristics, to the pH, temperature and so on.

The actual drug release in the human body can be measured in-vivo by measuring the plasma or urine concentrations in the patient. However, there are certain obvious impracticalities involved in employing such techniques on a routine basis.

These difficulties have led to the introduction of official in-vitro tests which are now rigorously and comprehensively defined in the respective Pharmacopoeia and recent harmonisation between the various Pharmacopoeia (notably the USP, BP, EP and JP) has lead to global standardisation in the measurement of drug release rates.

Tablet Dissolution Testing

When it comes to measuring the release rates of drugs in a manufacturing environment then the technique of Tablet Dissolution testing is employed.

Tablet Dissolution is a standardised method for measuring the rate of drug release from a dosage form and the key word here is “standardisation" because for any results to be meaningful, it is essential that all the apparatus used for the testing, produces the same sets of results given all other parameters are equal.

The principle function of the dissolution test may be summarised as follows:

·         Optimisation of therapeutic effectiveness during product development and stability      assessment.

·         Routine assessment of production quality to ensure uniformity between production lots.

·         Assessment of ‘bioequivalence’, that is to say, production of the same biological availability from discrete batches of products from one or different manufacturers.

·         Prediction of in-vivo availability, i.e. bioavailability (where applicable).

Dissolution testing was initially developed for oral dosage forms, but the role of the test has now been extended to drug release studies on various other forms such as topical and transdermal systems and suppositories.

Why Test?

From a manufacturing objective, the aim is to:

"Manufacture a dosage form in such a way that the active ingredient is released from the dosage form in a predicatable way and within a reasonable time in order for it to be absorbed by the body".

Drugs also need to be released in the right area of the body - in the intestine instead of the stomach for example.

Most routine dissolution testing is used to confirm the statement above. 

When a dosage form is manufactured, there are a number of parameters which need to be checked:

·         That the active ingredient is released in the predicted way

·         That the manufactured batch is the same as previous batches and falls within the required levels.

·         That he product can be stored for the specified shelf life without deterioration

·         To ensure that the dosage form does not break up in transit

·         To confirm that the drug is stable over time.

The Dissolution Test is a very useful tool and the only standardised way to generate scientific data that enables comparison

In addition, standardised testing promotes globalisation and harmonisation and also acts as a referee to identify mis-branded or substandard products

Application of dissolution data

Testing the dosage form from production to the end of its shelf life produces data that

·         Confirms immediate quality control

·         Ensures that the drug is still pharmaceutically active throughout its shelf life

·         Includes stability testing within well defined and strict criteria for each drug

·         Validates the manufacturing process and confirms therapeutic equivalence

What is tested:

Dissolution testing is appropriate to a wide range of products:

·         ‘Traditional’ pharmaceuticals

·         Dietary supplements

·         Veterinary drugs

·         Other ‘remedies’

Theoretical Concepts of Dissolution

The basic definition of dissolution rate for a solid dosage form is as follows:

‘The amount of active ingredient in a solid dosage form dissolved in unit time under standardised conditions of liquid-solid interface, temperature and media composition.’

First it is helpful to look at the way that a tablet breaks down and begins to dissolve. This refers to disintegrating tablets which make up a good proportion of the type of dosage forms tested

It is not unusual during dissolution testing to see particles moving towards the base of the vessel  or moving around in the media. The behaviour of these particles effect the dissolution rate and so it is useful to look at this in more detail later.

Dissolution Rates of Dosage Forms

 

There are many kinds of dosage forms of course and all of them have a dissolution rate. The dissolution time can range from seconds to hours or even days for implants.

Of course there are other dosage forms such as patches, implants, creams etc. but the principles remain the same.

The interface between the dosage form, and in particular the particles after deaggregation, and the dissolution media is critical and is known as the Shear Rate.

Shear Rate & Sink Conditions

Shear Rate

This is a very complex relationship but includes the interface between the surface of the solid and the rate at which fresh solvent contacts it.

If a tablet particle were to be suspended in media with no agitation at all, the liquid immediately around the tablet would become saturated and dissolution would essentially stop.

As the media starts to move then the saturated film is ‘washed’ away and new media enables the dissolution to continue again. Logically therefore, anything that affects the fluid dynamics or the way in which a dosage form disintegrates and dissolves should be understood and controlled

The Shear Rate depends on many variables including flow pattern variables, turbulence, viscosity, surface tension and dissolved gasses, which are in turn effected by other system variables to do with physical parameters.

Before any of those can be examined however, it is essential to ensure that there is sufficient media present to allow free dissolution of the active ingredient into solution.

Sink Conditions

If you put a spoon of sugar into a beaker of water it will dissolve readily. A second spoon will also dissolve. But keep adding spoonfuls and it becomes slower for the sugar to dissolve until at some point it becomes impossible for any more to dissolve as the solution becomes saturated.

Relating this to the dissolution of drugs, it is essential that as a drug dissolves, the presence of the already dissolved drug in solution should not affect the ability of more drug to be dissolved in any way. i.e.  the concentration of drug in solution should not be anywhere other than the bottom of the saturation curve for that drug. Concentration should never be close to the saturation point.

If the concentration level were to rise too high, the dissolution rate of additional drug would be slowed and the data would cease to be reproducible.

In order to ensure that sufficient media is present in relation to the drug to be dissolved, typically 5 to 10 times greater volume of media is used in respect to that saturation point at which dissolution would slow. This is known as Sink Conditions – sufficient media to ensure un-impaired dissolution.

This is typically why dissolution is performed in larger volumes such as 900ml or 1litre. 500ml tests may be used where sink conditions permit and the measurable level of the drug is lower. In recent years, the introduction of microcapsules and very low dosage levels have led to mini vessel tests in volumes as low as 100mls or 200mls, but in all these cases, sink conditions are maintained. Conversely, if 1000mls is not enough volume, then larger 2000ml vessels can be used, and above that volume USP4 can be considered.