Introduction to Analytical Method Validation

The analytical results you communicate can have far-reaching consequences and can form the basis for taking decision on safety of use of commercial products, foods and natural resources in addition to decisions involving legal matters. As a responsible analyst before you undertake analysis in the laboratory you should make sure that your results are authentic and are universally acceptable. This key objective can be realized only if the method selected for the purpose is duly validated.

Analytical method validation establishes documented evidence that the procedure adopted for a test is fit for the intended purpose in terms of quality, reliability and consistency of results.

Each and every method requires validation:

  • Before putting to routine use
  • When analytical conditions are changed such as change of technique, change in desired concentration range or change of sample matrix
  • Whenever changes are made to an existing procedure

It is important to collect relevant information on analysis requirements before you plan validation of the procedure

  • Components to be detected
  • Expected concentration levels
  • Required level of detection and quantification
  • Nature of sample matrix
  • Type of analytical technique to be used
  • Required degree of precision and accuracy

Parameters of Method Validation

  • Selectivity and specificity
  • Linearity
  • Range
  • Accuracy
  • Precision
  • Limit of quantification
  • Ruggedness
  • Robustness

We shall discuss each of the parameters in detail so that you can better understand analytical method validation

Selectivity and Specificity

Selectivity refers to ability of a method to determine a specific analyte or group of analytes without interference from other components of the sample matrix.

Specificity refers to ability of the method to respond to a single specific analyte only.

Selectivity is more frequently used as analytical techniques are seldom specific to only one analyte. Further selectivity should not be affected by interfering species such as degradation products, impurities and other matrix components.

Linearity

Linearity refers to the ability of analytical procedures to produce results in direct proportion to the concentration range of analyte in samples within the required concentration levels.

  • Linearity should be determined using a minimum of 6 standards whose concentration spans from 80% to 120% of expected concentration level.
  • Linearity report should include slope of line, linear range and correlation coefficient data. Correlation coefficient r should be greater than or equal to 0.99 in the working range

Range

Operating range is deduced from the calibration plot. It is the interval between the upper and lower concentration of analyte falling in the linear range. The results corresponding to this range demonstrate acceptable levels of precision, accuracy and linearity.

Accuracy

  • Degree to which the determine value of analyte corresponds to the true value.
  • Accuracy can vary over the expected concentration range.
  • It should be determined using a working or reference standards in the 80% – 120% level of expected range
  • Accuracy is determined by :
  1. Analyzing a sample of known concentration and comparing with the true value
  2. Spiking a blank (Sample having all components except the analyte) and comparing with the expected result.
  3. Standard addition method in which the sample concentration is determined. A known amount of analyte is added and the concentration is once again determined. The difference of the two concentration values is compared with the actual value of added analyte.
  • Accuracy is also defined by the comparison of test results with those obtained using another validated test procedure

Precision

  • Precision expresses closeness of a series of measurements of the same sample under identical conditions
  • High degree of precision does not necessarily means a high degree of accuracy
  • Precision is expressed as variance, standard deviation or as coefficient of variation of a series of measurements
  • Minimum of five replicate sample determinations should be carried out

Limit of Detection

  • Lowest amount of an analyte that can be detected but not necessarily quantified
  • Lowest concentration of calibration standard which produces a peak response corresponding to the analyte should be measured at least 6 to 10 times. Average response (X) and standard deviation (SD) are required to calculate limit of detection

Limit of detection = X + (3SD)

  • Signal to noise ratio at limit of detection should be at least 3:1

Signal to noise ratio should be greater than 3 at limit of detection and greater than 10 at limit of quantification

 Limit of Quantitation 

  • Lowest amount of the analyte that can be quantitatively determined with defined precision under the stated experimental conditions
  • 6 – 10 observations should be made for average response and standard deviation

Limit of quantitation = X + (10SD)

  • Signal to noise ratio should be at least 10:1 at the limit of quantitation

Ruggedness

Ruggedness measures reproducibility of test results under following conditions :

  • Results generated for same sample under identical conditions by different laboratories
  • Results generated for sample under identical conditions by different analysts
  • Same analysis using different instruments
  • Same analysis under different environmental conditions
  • Same analysis using test materials from different sources

Robustness

Robustness examines the effect of operational parameters changes on the analytical results

  • pH
  • Temperature
  • Operational conditions such as flow rate, injection volume, detection wavelength or mobile phase composition in chromatographic analysis

Variation should be deliberate but within realistic range to study the robustness of the method. The results of the analysis after making the deliberate changes should be within the method’s specified tolerance limits.

Now that you have been introduced to analytical method validation please feel free to ask any questions you have in the comments below.

About Dr. Deepak Bhanot

Dr Deepak Bhanot is a seasoned professional having nearly 30 years expertise beginning from sales and product support of analytical instruments. After completing his graduation and post graduation from Delhi University and IIT Delhi he went on to Loughborough University of Technology, UK for doctorate research in analytical chemistry. His mission is to develop training programs on analytical techniques and share his experiences with broad spectrum of users ranging from professionals engaged in analytical development and research as well as young enthusiasts fresh from academics who wish to embark upon a career in analytical industry.

Comments

  1. Do you have a similar summary for manufacturing a diagnostic based on biologic material ? (ie, process validation of immunoassays intended as IVDs?).
    Thanks!
    Susanne

    • Saurabh Arora says:

      Hi Susanne, we don’t have such a summary ready but we will consider it for the future and keep you posted if we do make it.

  2. K Vishwanath says:

    Please let me know acceptance criteria of Accuracy and Precision determinations.
    Thanks

    • Saurabh Arora says:

      Hi Vishwanath, the acceptance criteria have to be based on the application of your method. USP and other guidelines clearly state that you have to establish these based on what you will use the method for. You can refer to the IUPAC guidelines for a table which summarizes the criteria for different ranges of measurement and if you share your exact requirement I might be able to dig it out for you.

  3. Please let me know the acceptable range for change in pH, tempearture etc for robustness.

    Thanks

    Swati

  4. P.Dharmakkannu says:

    Dear,
    pH always ± 0.05 to the target and the robustness purpose we can study upto ± 0.1 in the validation. The temperature is based on the instrument temperature calibration limit. With in the range we need to the robustness test and shall be validated.

    Thanks and Regards
    Dharmakkannu

  5. Please, what can can increase in BOD and COD of an effluent treatment plant of a paint industry.
    Thanks

  6. Dharmendra Panchal says:

    I want to learn step by step procedure of analytical method validation and calculation

    • Dear Dharmendra,
      The topic is briefly covered in the certificate programmes but we do have plans to provide an elaborate treatment through a dedicated programme on the topic which we shall roll out sometime in future.

  7. Malgas says:

    Kindly help me on the application or use of HPLC in various fields/ industries (eg mining, agriculture, pharmacitical etc ) where analytical chemistry is applied. please

    • Hello Malgas,
      I would recommend our Certificate Course on HPLC to you as it would meet your requirements. It is an online programme and you would be able to complete it as per your convenience.On completion you would be eligible for our certificate as well.Please go to the site for joining details.

  8. Which concentration can be used for HPLC method validation of drug product either label claim or COA?

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