In HPLC, what is the validation of data? How to do it?
Quoting USP 1225, you need to address the following to validate a method (including HPLC): "accuracy, precision, specificity, detection limit, quantitation limit, linearity, range, and ruggedness"
You need to show that the method can accurately, precisely and at specified limits detect, identify and quantify the analyte(s) of interest. The steps you take to do this depend heavily upon your method and what you are analyzing (an API in clean saline, for example, versus a vitamin in an oil matrix) but you must address all of the items in order to truly validate your method.
Generally, if the retention time of the standard sample and the retention time of the peak measured by HPLC are the same, it is identified as the same compound. However, it can not be validated that this alone guarantees 100% as exactly the same compound. It is the best measurement data guarantee to investigate using the equipment analyzer which can clarify the molecular structure such as NMR after fractionating and isolating the peak measured by HPLC. If it seems difficult to separate, simultaneously measure the data by comparing each wavelength intensity ratio using a detector that can measure multiple wavelengths by UV measurement and comparing with each standard sample. Likewise, if analysis is performed by HPLC-MAS, strong data validation will be obtained if the peak measured by HPLC is a single compound or molecular weight. In any case, I think that verification of analytical data only with HPLC should be done carefully enough.
Quoting USP 1225, you need to address the following to validate a method (including HPLC): "accuracy, precision, specificity, detection limit, quantitation limit, linearity, range, and ruggedness"
You need to show that the method can accurately, precisely and at specified limits detect, identify and quantify the analyte(s) of interest. The steps you take to do this depend heavily upon your method and what you are analyzing (an API in clean saline, for example, versus a vitamin in an oil matrix) but you must address all of the items in order to truly validate your method.
Dear Lalrinzuali
HPLC is generally considered a quantitative technique and is a very good technique due to being able to separate analytes into individual compounds eluting at specific retention times. However retention time is not a unique characteristic for compounds and cannot be used to provide proof of the identity of the eluting analytes. This is one of the problems that is attempted to be addressed by the process of method validation.
The most comprehensive guidelines for analytical method validation that is also recommended by the FDA, EMEA and also by the Japanese is given by the ICH Harmonised Tripartite Guidelines titled:
VALIDATION OF ANALYTICAL PROCEDURES: TEXT AND METHODOLOGY Q2(R1)
and can be found at:
https://www.ich.org/fileadmin/Public_Web_Site/ICH_Products/Guidelines/Quality/Q2_R1/Step4/Q2_R1__Guideline.pdf
A good review specific to HPLC is give at:
http://chromsoc.jp/Journal/pdf/33-2_65.pdf
The outcome of method validation is to ensure that the method is fit for purpose so that the analytes being quantified can be done so confidently.
A method validated for standards in defined matrix such as from a pharmaceutical formulation may not be applicable to the same analysis performed on complex matrices like plasma or urine. The sample preparation steps are often ignored when validating analytical methods, but these sample preparation steps are absolutely critical to ensure that the method is in fact applicable to the actual samples being analysed.
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