Frequently Asked Questions about Analytical Method Validation

Frequently asked Questions about Analytical Method Validation

1) What is Analytical Method validation?

Ans: Analytical method validation is the documented process of ensuring a pharmaceutical analytical method is suitable for its intended use. 

This is achieved by performing a series of experiments on the procedure, materials, and equipment that comprise the method being validated. The experimental results undergo statistical analysis, and a series of pre-defined acceptance criteria are applied to the results. 

Establishing that the analytical method consistently produces reliable analytical results is a critical element of assuring the quality and safety of pharmaceutical products.

A validated analytical method is one has been documented as selective, accurate, precise, and linear over a stated range. Additional parameters and performance characteristics are often evaluated for methods of greater complexity. These serve to establish the method ruggedness or robustness.

Analytical method validation is a requirement for entities engaging in the testing of biological samples and pharmaceutical products for the purpose of drug exploration, development, and manufacture for human use. It also of great value for any type of routine testing that requires consistency and accuracy.

2) What Analytical Methods need to be validated?

Ans: Generally, any method used to produce data in support of regulatory (e.g., FDA, EMA) filings or the manufacture of pharmaceuticals for human use must be validated. This includes bioanalytical methods of analysis for bioavailability (BA), bioequivalence (BE), pharmacokinetic (PK), toxicokinetic (TK), and clinical studies, as well as methods used for analytical testing of manufactured drug substances and products.

According to ICH Guidelines, the following four types of methods require validation:

• Identification tests
• Quantitative tests for impurities content
• Limit tests for the control of impurities
• Quantitative tests of the active moiety in samples of drug substance or drug product or other selected component(s) in the drug product

In addition, ICH Guidelines define these four types of methods:

“Identification tests are intended to ensure the identity of an analyte in a sample. This is normally achieved by comparison of a property of the sample (e.g., spectrum, chromatographic behavior, chemical reactivity, etc.) to that of a reference standard.

Testing for impurities can be either a quantitative test or a limit test for the impurity in a sample. Either test is intended to accurately reflect the purity characteristics of the sample. Different validation characteristics are required for a quantitative test than for a limit test.

Assay procedures are intended to measure the analyte present in a given sample. In the context of this document, the assay represents a quantitative measurement of the major component(s) in the drug substance. For the drug product, similar validation characteristics also apply when assaying for the active or other selected component(s). The same validation characteristics may also apply to assays associated with other analytical procedures (e.g., dissolution).”

3) Why Analytical Methods need to be validated?

Ans: The Food and Drug Administration (FDA), European and other regulatory authorities have requirements and expectations regarding the performance and documentation of an analytical method validation.

Method validations are required when a new method is developed, an existing method is significantly modified (optimized) or an existing, validated method is applied to a different sample matrix. These methods may include HPLC, GC, GC/MS, UV-Visible, FTIR, titration, etc. Additionally, many wet chemistry (non-instrumental) methods require some level of verification or validation.

Executing validation activities is not a one-step process.

There must be assurance that "the accuracy, sensitivity, specificity, and reproducibility of test methods employed by the firm are established and documented." (CFR Title 21-Part 211).

The FDA and other regulatory authorities have cited organizations for not providing assurance that their test methods have been appropriately validated or verified. Many firms use USP methodology for routine analyses but fail to verify that the methods are "suitable for their intended use." A few examples of key regulatory findings include:

• Company A - Failure to have laboratory controls which establish scientifically sound and appropriate specifications, standards, and test procedures to assure product identity, strength, purity, and quality.
• What does this mean?
• There was no "proof" that the test methods used for product disposition decisions had been properly validated.

• Company B - "The test methods performed for Product A have not been verified to ensure suitability under actual conditions of use."
• What does this mean?
• The firm was using USP methodology without any verification of suitability.

4) what is regulatory expectation regarding Analytical Method validation?

Ans: Understand the requirements (i.e., the validation characteristics required for the sample matrix).

• i. Understanding the expectation is key to a successful validation.

5) what points to be considered before Analytical Method validation?

Ans:   a) Test method is "ready" for validation.Method development should not occur at this time. 
                If there is a need for continued method optimization, your method is NOT ready for validation.

          b) Analyst must be qualified, has demonstrated proficiency and clearly understand how to perform a method validation.

          c) Instruments/Equipment is in a "qualified state."

          d) materials is in qualified state (i.e samples, standards, impurities etc.. should have proper COA.)

          e) protocol to be prepared and approved by concerned quality and management departments.

6) what is the difference Analytical Method validation and analytical method verification?

Ans: Method verification is the re-checking the existing validated method whether the method is suitable for particular site for inteded use.  

All validation characteristics are not required for verification of a validated test method or USP compendial procedure.

7) what are the parameters commonly to be performed in analytical method verification?

Ans: 

• a. Accuracy
• b. Precision
• i. Reproducibility
• ii. Repeatability
• iii. Intermediate Precision (Ruggedness)
• iv. Instrument Precision
• c. Specificity
• d. Selectivity
• e. Limit of Detection
• f. Limit of Quantitation
• g. Linearity
• h. Range
• i. Robustness
• j. Ruggedness

Other Validation Considerations:

• a. System Suitability - Used to verify that the resolution and reproducibility of the chromatographic system are adequate for the analyses performed.
• b. Solution Stability - Determines the stability of the solution after preparation in accordance with the test method.
• i. Consideration must also be given to reagents, chemicals, mobile phase, etc.
• c. Stress Testing - Demonstrates the specificity of the assay and analytical procedures for impurities. We need to ensure that "no interference with quantitation is evident from impurities and degradants from the active ingredient and excipients."
• d. Stability indicating nature of analytical method can be proved using specificity and stress testing by showing the peak purity.

8) what are Key elements of The Method Validation Summary Report:
Ans:

• i. Introduction
• ii. Scope
• iii. Summary of Results
• iv. Pre-validation activity (pre-requisites)
• v. Detail of all relevant materials, chemicals, reagents, etc.
• 1. Mfr. Lot#, Purity, Expiry, etc.
• vi. Listing of all instruments/equipment
• vii. Reference Standards
• 1. Sample and Blank Preparations
• viii. Test Method Detail (may also be an attachment)
• ix. Detail of each Validation Characteristic
• 1. Data Presentation (Table Format)
• x. Protocol Exceptions (if applicable)
• xi. Copies of key representation plots, chromatograms, etc.
• xii. Representative Calculations
• xiii. Approvals

All validation activities and results should be documented in a Method Validation Summary Report and approved by Quality and Management.

Minimizing and rectification of common analytical errors in laboratory.

Hai viewers........

Sorry for taking long gap to my blog.

But Today I come up with new and very important topic for all analysts and scientists....

 "Minimizing and rectification of common analytical errors"

Before going into the hot topic, I want give definitions of some common terms.

True value : A value compatible with the definition of a given particular quantity.

Error : An error is a " Deviation from a true value"

Mistake : Mistake is an error caused by a fault. The fault being a misjudgement, carelessness, forgetfullness etc...

     "errors occurs... Mistakes are made....."

"Machines have errors.... Humans makes mistakes.."

So from the above quotes, what can be concluded..?

Errors may occur mechanically... but mistakes were done by humans.

Machine or instruments may give errors, if a person makes a mistakes.

" Errors cannot be rectified but can be minimized.....

                          Mistakes can 100% rectified, because it is a human errors...."

                     "Errors can be minimized by rectifying human mistakes"

Why Acceptance limit or specification limit come into picture..?

As I already told,  errors cannot be rectified but it can be minimized. So definitely there should be certain limit for the analytical result.

So there a possibility to rectify mistakes which is in the hand of Analyst or Scientist.

" A best analyst or scientist cannot commit mistakes"

One analyst asked me some questions as follows. 

                    

                       "Yes.. I want to be one of the best analyst. But How..?"

My Answer:  

                       First of all, I will tell why mistakes will happen..

                             1) The person may have lack of experience.
                             2) The person may have lack of knowledge.
                             3) The person may lack of attitude, care ( means having laziness), interest, problem solving skills, self confidence, self thinking to settle an issue(plan), etc...(human errors).
                             4) The person may have lack of self checking.

So If you overcome all above points, you will become a best analyst or scientist.

 How to overcome..? How to become best best analyst.?

Answer:        A) know what you are going to do.( pre-plan).
                        B) Read the procedure carefully (Care).
                        C) Learn technique behind the test or process (knowledge).
                        D) put interest on analysis or work.
                        E) Self check each step you are doing.
                        F) Ensure each step whether you are doing correctly or not.
                        G) Build confidence on your work by ensuring above all.
                        H) Learn tips and tricks from experienced people.(Pipetting, Sample preparation & trouble shooting etc...).

Generally from where errors may occur..? (Sources of errors).

Answer: 
                Sources of errors:

                  a) Input Data: Standard & its potency used (wrong standard or wrong potency).
                                             Calibration values (example: KF factor)
                                             Physical constants etc....

                  b) Quantity measured: Titre value, volumes, Weights etc....
  
                  c) Instruments used: Example: If UV-VIS spectrometer is not suitable for assay analysis, we have to go for HPLC analysis for better accuracy and repeatability.( So, UV not suitable, HPLC suitable). Instruments used are not calibrated properly.

                  d) Observer fallability: Reading errors (example: analyst taken wrong STP or misunderstood wrongly.)

                  e) Environment: Method conditions, Temperature, Humidity etc....

                   f)  Theory assumed: Calculations, Approximations etc...


       What types of errors may occur..? (Types of errors).    

Answer: 
                      Random Error: errors that vary in an unpredictable manner in replicated measurements.

                      Systematic Error: the results of analytical measurements to one side, to higher or lower values which lead to false results.

                      Trend Error: A data set shows a trend when the chronologically ordered values move steadily downwards or upwards.

                      Gross Error: Gross errors result from human mistakes, or have their origins in

instrumental or computational errors. Frequently, they are easy to recognize and the

origins must be eliminated.

Statistical Evaluations to understand extent of errors:

      

Mean, Standard deviation, Variance, %RSD, Coefficient of variance, etc...... are some of the statistical evaluations for estimating the extent of error to the true value.

Parameters to Control Errors:

There are many parameters for control the Errors.

Examples : Precision, Linearity, Accuracy,Specificity etc…..

Instruments: Calibrations, Preventive maintanance, performance verification etc….

Methods: validations, Verifications, system suitability etc…,

Documents: Review, quality assurance, etc…,

Analysts: Trainings, Practise etc….

Why to control Errors : 

Answer is : If we control the errors it directly controls the quality of the product.

Thanks

V.Suresh

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.