Chromatography Forum

Hi,

What does it mean „repetition” when we validate method (especially precision)? I’m not sure if it should be two injections from one sample (solution) or two independent injections from different analytical samples (but the same laboratory sample). Currently I analyse my laboratory sample by dividing it by sampler divider for 6 independent analytical samples and then I analyse them by one injection per sample. Can I say that I make 6 repetitions or not?

In general "repeatability" refers to multiple injections from the same sample solution. The idea is to quantify variability in the chromatography system. Multiple injections from different sample solutions (prepared separately) would introduce additional variability from the sample preparation procedure. "Reproducibility" would incorporate results from different days/analysts/systems.

You have to look at the context to decide what is meant by "repetitions".

In general "repeatability" refers to multiple injections from the same sample solution. The idea is to quantify variability in the chromatography system. Multiple injections from different sample solutions (prepared separately) would introduce additional variability from the sample preparation procedure. "Reproducibility" would incorporate results from different days/analysts/systems.

You have to look at the context to decide what is meant by "repetitions"

Don't you think that if you have multiple injections from the same sample solution you only measure injection precision? I'm not sure but I think that in addition to the system precision (or injection precision obtained from multiple injections), during repeatability measurement we should also remember about the contributions from the sample preparation, such as weighing, dilution, extraction, homogenisation, and etc. It is essential to apply the whole analytical procedure , rather only to injecting the same sample solution few
times. That's why I' m not sure if I can name result of multiple injections as a "repeatibility" in validation report

It *is* a bit ambiguous. I have always treated "different sample preps" under "intermediate precision" per ICH, but I could see it included in "repeatability" if all run on the same day. As long as you document what you *actually* did, you should be Ok.

Here are the definitions:

a. Repeatability
Repeatability expresses the precision under the same operating
conditions over a short interval of time. Repeatability is also termed
intra-assay precision

b. Intermediate precision
Intermediate precision expresses within laboratories variations: different
days different analysts, different equipment, etc.

c. Reproducibility
Reproducibility expresses the precision between laboratories
(collaborative studies usually applied to standardization of methodology).

per: https://www.fda.gov/media/71724/download

My rationale is that "single prep / repeat injections" establishes the performance of the *chromatography*, including injection volume, but also detector response, integration settings, etc. "multiple prep / repeat injections incorporates the performance of the entire method, including the chromatography and sample prep.

A lot of stuff even in USP is ambiguous and open to interpretation. Volumetric solutions chapter has some that state "standardize frequently" and give no guidance as to what they mean by that.

Some make a reference solution once and make dilutions from that to document linearity, some weigh out different amounts for each level.

Etc.

Thanks for all answers, as I can see you are experienced in validation so maybe you can help me with my method.
Aim of method: determination of additive. Compound is sputtered on the surface of product (granules). Amount of product which is send to sputtering is about 1 kg. I get about 120 g of it (6 samples after sample divider). On the basis of analysis we get information about efficiency of sputtering and homogeneity of sample (confidence interval for mean from 6 samples). I determined accuracy by standard addition (addition of standard solution to solution of matrix). I’m not quite sure how run experiments for estimation of repeatability and reproducibility in such a way that I will get information only about uncertainty of analytical method ( without uncertainty of sputtering because it will be changeable during tests).

Aim of method: determination of additive. Compound is sputtered on the surface of product (granules).

Do you mean that active is sprinkled on top of your granules, then mixed and then split?

Anyway, solid and semi-solid samples are notoriously difficult to either spike/fortify for lab testing or to sample real-life products. Splitters didn't work all that great for non-homogenous products.

We had bar soaps, solid antiperspirants, dry laundry products, etc. For validation tests, not easy to make a uniform sample of known active content. Typically, we prepped placebo sample into solvent, adding the active/analyte of interest to that, and proceeded from there. We used blenders, homogeniers, etc.

Samples like solid antiperspirants could vary in active from bottom to top, was quite dependent on the cooling process.

We use different methods to modify surface. Typically we use three methods:
- Solution of active ingredient is added to granules, than we use rotary evaporator to evaporate solvent
- Impregnation of granules in solution of active compound (than drying)
- compound is added by nebulizer