Precision Issues

[Martina]

Dear Experts,
I am noticing poor agreement between my replicate samples. I am analysing thyroxine and am finding typically that there is a 1% difference between the replicates (calibration samples) and up to 2% in the actual samples. The actual samples are the thyroxine extracted from tablets, using mobile phase and filtered (0.22u).

Peak purity analysis says that the peaks are pure but this loss of precision is worrying. What could I do to rectify this situation? I can't see any leaks.

Varian HPLC Prostar, PDA, C18, 0.2%Phosphoric acid/Acetonitrile 45:55, 255nm, 1mL/min, column heated:35deg.

kind regards
Martina

[tom jupille]

How many replicates?

Do 1% and 2% refer to range or relative standard deviation?

What kind of repeatability is expected based on the method validation?

[danko]

Hi Martina,

You can start the troubleshooting by discarding or confirming the possibility of faulty equipment.
The information provided, does not necessarily point to that direction. The problem might be due to the sample preparation/extraction of the API from the tablets. And I’m quite sure that this is the problem you’re dealing with. Otherwise there shouldn’t be any difference between injections of standards (calibration samples) and unknowns (actual samples). Another possibility is non homogenous distribution of the API in the different tablets.
My suggestion is: Inject the same number of injections from a single vial/sample as the number of replicates you do routinely (10? I assume you conduct a content uniformity test) and compare the calculated RSD with the one you feel is too large (2 %?).
Then get back to the board with the conclusion. And depending on, what the result is, I’m sure you’ll get many more qualified suggestions.

Best Regards

[Martina]

Thank you both for your comments. Much appreciated. Today I am going to try a few of your suggestions. I am going to work with the pure standard and assess these injection replicates. I definitely think API distribution is an issue for me as well. The tablet contains 100ug of API, total tablet weight being 130mg. I worry about my ability to prepare a representative sample from a crushing of ten tablets.

I will come back to the board probably with more questions.
kind regards
Martina Mangan

[Martina]

Good morning everyone,
While I was setting up to run my standard solution I decided to do some routine maintenance on the syringe. It was a possibility I thought that the needle could be cruddy. While carrying out multiple flushings I noticed that the integrity of the syringe seal was questionable. I could see leakage at the base of the rubber seal. I have ordered a new syringe and will go from there. This could certainly account for the woolly readings.

Thank you for your comments and I will surely return if this does not solve my problem.
kind regards
Martina

[danko]

Good morning Martina, (good night where I am)

If it is in fact the syringe that causes the problem, why should it only affect the unknowns and not the standards? To the same extend anyway. Or do you think the statistical material/number of determinations was too small to give reliable RSD estimations?

Best Regards

[Martina]

Dear Danko,
Yes a very good question. Really I have not done enough injecting for reliabile statistical information. I just wanted to fix what I saw as erratic/unstable results. When I install my new syringe I will be in a better position to comment.
Thank you for your comment
Martina

[Karan]

Dear Matrina,

Good morning,

Did you observe any trend in peak area of replicate injections for precision measurement? What is the concentration of analyte in solution and peak height?

These parameters can greatly influence the precision. I hope it will give some clue before going in depth of trouble shooting.

regards,