Chromatography Forum

Posted: **Fri Sep 24, 2004 3:27 am**

Dear Friends,
I am new to forum and new user for HPLC. Can some one help me in establishing protocol for validation of invitro samples and biological samples. I will give my protocol and please correct me where ever it is necessary at the same time update me with new regulatory requirements and limits.
1) Accuracy: Lower, intermediate and higher concentrations of analyte were prepared (n = 5). Measured as % Relative error.
2) Precision: Lower, intermediate and higher concentrations of analyte were prepared (n = 5). Measured as % Relative standand deviation. For intra-day and inter day varibility study n = 6 samples per day for 3 days.
3) Specificity and selectivity: Inject blank (n = 5) for checking of interference at analyte retention time.
4) Linearity range: Five different concentrations (n = 6). Apply one-way ANOVA for linearity checking.
5) Ruggedness: Change the instrument, analyst, column, brand of solvents and inject Lower, intermediate and higher concentrations of analyte (n = 5). Report as % Analytical recovery Â± SD.
6) Robustness: Change the pH of mobile phase (Â± 0.1) and inject Lower, intermediate and higher concentrations of analyte (n = 5). Report as % Analytical recovery Â± SD.
7) LLOQ LLOD: ?????????
8)System suitability: How to do it?????????????
Sorry for such long question. Expert comments will help me in designing protocol.
Thank you

Posted: **Sat Oct 02, 2004 2:13 am**

Please somebody respond to this question

Posted: **Mon Oct 04, 2004 1:30 am**

Accuracy:
Using simulation drug powder at Â± 20% of the lowest expected conc to Â± 20% of the highest expected conc. Min 3 conc levels, n = 3. Measured as % recovery.

Precision:
1. Repeatability: replicate measurements of std soln.
2. Intermediate precision: same sample is analyzed by at least two analysts, using diff instr, diff batch of mob phase or solvent, diff batch of column, etc.

Specificity:
inject blank, and impurities solution if possible, and compare it with chromatogram from std soln.

Linearity:
Prep at least 5 concs std solns of the drug, ranging in conc Â±20% below the lowest expected conc to Â± 20% above the highest. Use linear regression to evaluate the linearity.

Robustness:
Variation of the mobile phase composition, flow rate, pH changes, and column type, brand, lot or age.

Detection / Quantitation Limit:
Not always be necessary. See USP for details, there are 3 ways to determine it.

System suitability:
Repeated injection of std soln (n = 6) and check %RSD, peak shape, retention time repeatability.

Posted: **Mon Oct 04, 2004 3:17 am**

Dear syx,
thanks for your valuble suggestions.
But i have a small doubt.

Usually in experiments we don't know the concentration range first. In those cases what will you suggest.

Best regards

Sneha

Posted: **Mon Oct 04, 2004 7:24 am**

I usually use 80% - 100% - 120%

Posted: **Mon Oct 04, 2004 7:29 am**

Dear MSL,

for validation of bioanalytical methods, you should also consult relevant guidlines of the authorities, ie FDA Guidance for Industry, Bioanalytical Method Validation, U.S. Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research (CDER), Center for Veterinary Medicine (CVM), May 2001 or EMEA guidelines, available at http://www.emea.eu.int/

Regards Bert

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