Calibration curve by changing injection volume, validation?

Discussions about HPLC, CE, TLC, SFC, and other "liquid phase" separation techniques.

17 posts Page 1 of 2
We are working on a method on HPLC-UV where we are currently moving toward making the calibration curve by varying the injection volume due to different reasons. By proceeding with this calibration approach we would have different injection volumes for standards and samples (sample inj.vol. significantly higher than standards).
We are working according to GMP and we are a bit stumped by how we should move on with the upcoming validation.
Has anyone used this approach before in a validation? Does anyone have any ideas about this approach? Pros/Cons?

Thank you
I haven't had to take that approach during method development or validation. That said, there obviously isn't a regulation which states that your method must have the same injection volume for the standards and samples. It doesn't affect the experimental design of your validation, in my opinion.

A very meticulous auditor or reviewer will naturally ask why there is a difference, since it is unusual. If you have a sound reason for doing so and can show that your method met all your validation requirements, then it's a moot point as far as I would be concerned. That said, if you end up with acceptance criteria that are atypical for an LC validation in order to accommodate the difference in injection volume, you may get some pushback from a regulatory authority.
Blazer wrote:
If you have a sound reason for doing so and can show that your method met all your validation requirements, then it's a moot point as far as I would be concerned.


I tend to agree with above, as long as you can document that it meets all requirements. Document, document, document...
I have used increasing/decreasing volumes for generating standard curves millions of times.
Have done comparisons between different volumes versus different concentrations, injecting the same volume. And have shown – within reasonable ranges – that the results are the same.
Here is important to keep in mind the fact that the main task in such cases is determination of the amount or concentration of a certain substance. This opposite to the purity determinations where peak shape, width, height and in fact resolution are indeed much more important than in the case of assay.
The other important thing is that in assays, one typically determines concentrations from 80 to 120% of the target. So, in case of – let’s say injection of 20 μL – one can generate a relevant, 3-level, calibration curve by injecting 16, 20 and 24 μL standard solution.
Now who could object that?
Keeping in mind the method should be validated in any case, being a GMP method, one can include a validation test of the above showing no difference between volume and amount decrease/increase.
Best Regards
Learn Innovate and Share

Dancho Dikov
danko wrote:
I have used increasing/decreasing volumes for generating standard curves millions of times.


Wow, Danko - millions of times - you must be REAL old.
:D Old enough to know a thing or two about the matter - he, he....
Learn Innovate and Share

Dancho Dikov
Thank you for your input! We've proceeding with this procedure for the moment
Danko: I'll be sure to use your helpful input on this. Feels good to know someone with a lot of experience with this have had a say about it :)

Best regards
/Perre
Hi,

I have done this few times, not a million though (Respect) and what I have observed is; let me tell you this pointwise, please bear my rambling:

1. when I run a calibration run with a FAME (Fatty acid methyl esters) mix, in known amounts, and the area under the curve or response(the area/height ratio fixed within a fixed limit. The Retention factors of all the fatty acids from c:9 to c:24 fatty acids get established, along with the internal standard for quantification,with these calibrations and with the above mentioned responses we can quantify them.

2. I have seen that the ratio of the the response at 1 microliter/5 microliter are lower with the ratio being 1.9. With, ratio of RT/pA (signal) under the limit.

3. About internal standard peaks; At 5 microliter I get them named (as their RT matches with the fatty acid present in the calibration mix,and with aforementioned ratios under limits) but not at 1 or 2 microliters of injection. I could see the bump at the RT of internal standard on the signal panel during the run but,because of the limitations in the aforementioned ratios i run my at 5 microliter; this made sense to me.

So, theoretically speaking we are heating 5 microliter of the sample so we have to multiply the response at 5 microliters with 0.2 (1/5). But, practically I have see that the ratio is 1.9 (arrived after taking average over 15 samples)
In the case of Internal standard it can not be applied. Because, it would be 0. Anyways, I have see that the ratio of the internal standards response, when see in 1 microliter injection too, is 1.4.

Does it make sense to divide both the ratios and carry on with the regular quantification process using internal standards amounts and multiply with the ratio? Or What should I do?
Also, how reliable the R factors that are obtained from the calibration mix would be, if it is a commercially available mix and the software used in naming the fatty acids and in quantifying them is built around its dependence? Should I be changing the volume of sample injection after running the calibration mix at all?

I am sorry for those who have not read this post ;))
I am so sorry, the ratios must be 1/1.9
If the standard and sample is dissolved in the starting mobile phase it should work. If the standard and sample are in say 100% methanol while the starting mobile phase is 20% methanol/80%water, then the increasing injection volume will increase the "peak surfing" effect and at some point will result in a drastic loss of response so the curve will not be linear.

When working in the EPA methods as I do, all parameters must be the same for all injections including injection volume so we are restricted to using varying standard concentrations instead of varying injection volume.

You just have to show that the varying injection volume does not effect the result, especially if you are injecting a much larger volume of sample than standard.
The past is there to guide us into the future, not to dwell in.
anvesh4411 wrote:
Hi,

I am sorry for those who have not read this post ;))


The lack of response is probably because you are posting abut a GC method in an HPLC thread. Changing injection volume in GC is very poor practice.

Peter
Peter Apps
Peter Apps wrote:
The lack of response is probably because you are posting abut a GC method in an HPLC thread.


No wonder I can't get teriyaki chicken at the hardware store!
If the method performs well with changing injection volumes for the calibration standards and the samples and the method can be validated, What's the point of doing it then? Why not use large injection volume for your calibration standards just like your samples? The reasons behind using much larger injection volume for the samples than the calibration standards are the key here?

In method development, the simpler the method, the better.
hi Perreman :
2. LINEARITY
A linear relationship should be evaluated across the range (see section 3) of the
analytical procedure. It may be demonstra
ted directly on the drug substance (by
dilution of a standard stock solution) and/or separate weighings of synthetic mixtures
of the drug product components, using the
proposed procedure. The latter aspect can
be studied during investigation of the range.
Linearity should be evaluated by visual inspe
ction of a plot of signals as a function of
analyte concentration or content. If there is a linear relationship, test results should
be evaluated by appropriate statistical methods, for example, by calculation of a
regression line by the method of least squares. In some cases, to obtain linearity
between assays and sample concentrations, the test data may need to be subjected to
a mathematical transformation prior to
the regression analysis. Data from the
regression line itself may be helpful to pr
ovide mathematical estimates of the degree
of linearity.
The correlation coefficient, y-intercept, slope of the regression line and residual sum of
squares should be submitted. A plot of the data should be included. In addition, an
analysis of the deviation of the actual data points from the regression line may also be
helpful for evaluating linearity.
Some analytical procedures, such as imm
unoassays, do not demonstrate linearity
after any transformation. In this case, the
analytical response should be described by
an appropriate function of the concentration (amount) of an analyte in a sample.
For the establishment of linearity, a minimum of 5 concentrations is recommended.
Other approaches should be justified.

this is the linearity section in ICH guidlines for analytical method validation ,
it states that it should done by either dilution of standard stock or by weighing differents weights from the sysnthetic mixture of sample components , and for me it make sense as the test you aim to do is method validation , not injector OQ and PQ , performing linearity with different concentrations shows if there is any interaction between the component molecules and the matrix solvent and different conc. if present ,this issue cant be noticed by injecting different volumes from the same solution ,
i hope that i understood your question correctly and helped you
While I have never done linearity by changing injection volumes, I would imagine that would also require every injection volume to undergo a precision check to ensure that as you change injection volumes, your system is capable of reproducibility at each injection volume.

It just seems more convenient to spend the time doing the dilutions and not have to worry about one of the multiple injection volumes not meeting reproducibility %RSD and thus throwing a curve into the validation process.

At least this is how I would look at it from an auditor's perspective - how can you prove that your system is precise at all of the various injection volumes? That would be the biggest question to answer if you were to do it your way - in my opinion.
17 posts Page 1 of 2

Who is online

In total there is 1 user online :: 0 registered, 0 hidden and 1 guest (based on users active over the past 5 minutes)
Most users ever online was 1117 on Mon Jan 31, 2022 2:50 pm

Users browsing this forum: No registered users and 1 guest

Latest Blog Posts from Separation Science

Separation Science offers free learning from the experts covering methods, applications, webinars, eSeminars, videos, tutorials for users of liquid chromatography, gas chromatography, mass spectrometry, sample preparation and related analytical techniques.

Subscribe to our eNewsletter with daily, weekly or monthly updates: Food & Beverage, Environmental, (Bio)Pharmaceutical, Bioclinical, Liquid Chromatography, Gas Chromatography and Mass Spectrometry.

Liquid Chromatography

Gas Chromatography

Mass Spectrometry