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Analytical method limitations

An analytical method limitation is a boundary on what an analytical procedure can reliably report, set by what the procedure can distinguish, the smallest amounts it can detect or measure, the range over which it was validated and the conditions under which it is run. ICH Q2(R2) describes validation as the demonstration that an analytical procedure is fit for its intended purpose, and names specificity or selectivity, range, accuracy and precision among the characteristics that may be considered.[1]

What can a method fail to distinguish?

ICH Q2(R2) describes specificity and selectivity as terms for the extent to which other substances interfere with the determination of an analyte. Specificity describes the ultimate state of measuring a desired analyte unequivocally; selectivity is a relative term for the extent to which particular analytes can be measured without interference from other components with similar behavior.[1]

Where one procedure does not provide sufficient discrimination, ICH Q2(R2) recommends a combination of two or more procedures, and states that specificity can be verified by comparing the measured result with that of a second, well-characterized procedure.[1]

For synthetic peptides, D'Hondt and colleagues describe closely related impurities such as diastereomers arising from racemization of amino acid residues, and Lian and colleagues discuss liquid chromatography-mass spectrometry techniques for structural isomers and stereoisomers such as peptide epimers, together with potential pitfalls in that characterization.[2],[3]

FDA guidance on endotoxin testing states that bacterial endotoxins assays are subject to a variety of interferences related to the physical and chemical properties of the test article.[4]

What are detection and quantitation limits?

ICH Q2(R2) defines the detection limit as the lowest amount of an analyte in a sample that can be detected but not necessarily quantitated as an exact value, and the quantitation limit as the lowest amount that can be quantitatively determined with suitable precision and accuracy.[1]

The guidance states that the quantitation limit is used particularly for the determination of impurities and degradation products. By these definitions a component can be detectable at a level below that at which it can be quantitated.[1]

What does the range of a method cover?

ICH Q2(R2) defines the range of an analytical procedure as the interval between the lowest and the highest results in which the procedure has a suitable level of precision, accuracy and response. It states that the reportable range is typically derived from the specification and should include the upper and lower specification or reporting limits, as applicable.[1]

Where an analyte has a different response from the reference material, for example a different specific UV absorbance, ICH Q2(R2) states that relative response factors should be calculated, and that a correction factor should be applied if the relative response factor is outside the range 0.8 to 1.2.[1]

Why can results differ between runs and between laboratories?

ICH Q2(R2) describes precision at three levels: repeatability, under the same operating conditions over a short interval; intermediate precision, which expresses variation within a laboratory across different days, environmental conditions, analysts and equipment; and reproducibility, which expresses precision between laboratories.[1]

Robustness, as defined in ICH Q2(R2) with reference to ICH Q14, is a measure of a procedure's capacity to meet its expected criteria during normal use, tested by deliberate variations of procedure parameters. The parameters it lists for separation techniques include column lot, mobile phase composition and pH, column temperature, flow rate and detection wavelength.[1]

FDA guidance on analytical procedures states that analytical method transfer studies usually involve two or more laboratories and evaluate accuracy and precision, especially with regard to interlaboratory variability.[5]

Bolden and colleagues report an endotoxin spike and hold study in which endotoxin activity appeared to be lost until the study design was changed so that mixing time and sampling matched the routine method, after which the added endotoxin was consistently recovered over time.[6]

Why is a purity figure described as method dependent?

ICH Q6B states that the determination of absolute, as well as relative, purity presents considerable analytical challenges and that the results are highly method dependent, so purity is assessed by a combination of analytical procedures.[7]

ICH Q6B also states that critical quality attributes can be assessed by multiple analytical procedures, each yielding different results, and that it is not unusual for analytical technology to evolve in parallel with a product during development.[7]

ICH Q2(R2) describes a validated quantitative procedure as stability indicating when it can detect changes in relevant quality attributes of a product during storage, which is demonstrated with samples containing relevant degradation products.[1]

When does a validated method need to be checked again?

ICH Q7 states that analytical methods should be validated unless the method employed is included in the relevant pharmacopoeia or other recognized standard reference, and that the suitability of all testing methods used should nonetheless be verified under actual conditions of use and documented.[8]

ICH Q7 also states that complete records should be maintained of any modification of a validated analytical method, including the reason for the modification and data to verify that it produces results as accurate and reliable as the established method.[8]

The FDA guidance states that when an analytical procedure is changed, replaced with a new method or transferred to a new testing site, revalidation, a new validation exercise, an analytical method comparability study or a combination of these should be considered, and that changes to the manufacturing process may also warrant revalidation.[5]

Limitations

A reported result carries the limits of the procedure that produced it. Under the ICH Q2(R2) definition of a detection limit, a result of not detected means the analyte was not detected by that procedure at that limit, not that none was present.[1]

ICH Q2(R2) and ICH Q6B describe what validation and specifications should address. ICH Q6B states that it does not recommend specific test procedures or specific acceptance criteria, so these documents do not set the limits for any particular material.[1],[7]

This page describes what the cited documents say. It is not a statement that any PepGenex material has been manufactured, tested or released under any of them.

Compound profiles

Research peptide profiles in this library. What this page describes applies to peptide lots in general; none of these profiles reports a result of it for any lot.

References

  1. ICH Q2(R2) Validation of Analytical Procedures. U.S. FDA guidance for industry, March 2024. FDA docket FDA-2022-D-1503
  2. D'Hondt M, Bracke N, Taevernier L, et al. Related impurities in peptide medicines. J Pharm Biomed Anal. 2014;101:2-30. PMID 25044089 · DOI 10.1016/j.jpba.2014.06.012
  3. Lian Z, Wang N, Tian Y, Huang L. Characterization of Synthetic Peptide Therapeutics Using Liquid Chromatography-Mass Spectrometry: Challenges, Solutions, Pitfalls, and Future Perspectives. J Am Soc Mass Spectrom. 2021;32(8):1852-1860. PMID 34110145 · DOI 10.1021/jasms.0c00479
  4. U.S. FDA. Pyrogen and Endotoxins Testing: Questions and Answers. Guidance for industry. FDA docket FDA-2013-S-0610
  5. U.S. FDA. Analytical Procedures and Methods Validation for Drugs and Biologics. Guidance for industry, July 2015. FDA docket FDA-2015-N-0007
  6. Bolden JS, Warburton RE, Phelan R, et al. Endotoxin recovery using limulus amebocyte lysate (LAL) assay. Biologicals. 2016;44(5):434-440. PMID 27470947 · DOI 10.1016/j.biologicals.2016.04.009
  7. ICH Q6B Specifications: Test Procedures and Acceptance Criteria for Biotechnological/Biological Products. U.S. FDA guidance for industry, August 1999. FDA docket FDA-1998-D-0003
  8. ICH Q7 Good Manufacturing Practice Guidance for Active Pharmaceutical Ingredients. U.S. FDA guidance for industry, September 2016. FDA docket FDA-1995-D-0288