I dont quite understand the following and would like to clarify the meaning:
1. For Lower Limit of Quantification (LLOQ), the analyte response at the LLOQ should be at least 5 times the response compared to blank response.
2. The calibration curve should be 20% deviation of the LLOQ from nominal concentration.
3. If an analyte is unstable at the intended storage temperature, the stability sample should be frozen at -70 degrees during the three freeze and thaw cycles
Dear Sir. Concerning your issue about the HPLC validation protocol with respect to Lower Limit of Quantification (LLOQ) . Limit of detection (LOD) and limit of quantification (LOQ) are two important performance characteristics in method validation. LOD and LOQ are terms used to describe the smallest concentration of an analyte that can be reliably measured by an analytical procedure. There has often been a lack of agreement within the clinical laboratory field as to the terminology best suited to describe this parameter. Likewise, there have beenvarious methods for estimating it. The presented review provides information relating to the calculation of the limit of detection and limit of quantitation. HPLC assay which uses an ion-pairing reagent. One might investigate: sample sonication or mixing time; mobile phase organic solvent constituency; mobile phase pH; column temperature; injection volume; flow rate; modifier concentration; concentration of ion-pairing reagent; etc. It is through this sort of a development study that variables with the greatest effects on results may be determined in a minimal number of experiments. The actual method validation will ensure that the final, chosen ranges are robust. I think the following below links and the attached file may help you in your analysis:
http://www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2011/08/WC500109686.pdf
https://www.fda.gov/downloads/drugs/guidances/ucm368107.pdf
https://www.fws.gov/aah/PDF/QI-Terms%20and%20Defs.pdf
https://www.waters.com/webassets/cms/library/docs/720001826en.pdf
Thanks
You might probably need to read these two articles on LLOQ. Briefly, 1) and 2) are descriptions of a good quantification range. If your calibration curve produces 1) and 2) then you might be able to determine the LLOQ.
However, point 3) is a good laboratory practice tip. One of the reasons researchers obtain inaccurate LLOQs is poor handling of standards - they may degrade and result in gross inaccuracies during quantification.
For a quick summary on LLOQs I recommend this quick read:
http://www.quansysbio.com/lld-lloq-uloq/
I also recommend the attached document of my ex colleague Piotr Robouch (& other Authors): good examples and clear statistics.
I attach here the link because the button does not work
http://publications.jrc.ec.europa.eu/repository/bitstream/JRC102946/eur%2028099%20en_lod%20loq%20guidance%20document.pdf
I have an easy lecturer on ICH guidlines about method validation, hope this would help
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