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.

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.......?

Please respond to my article by posting comments to this.......

" Comments are steps to know mistakes.........

                                             mistakes are steps to learn...... "

Thanks 

V. Suresh.