Chromatography Forum

Dear,

I found following guidelines to evaluate the freeze/thaw stability of an LC-MS/MS method:

"Analyse 6 replicates (2 concentration levels) before and after 3 freeze/thaw cycles. Calculate the percentage of mean concentration in treated samples as compared to mean concentration in control samples, calculation of respective 90% confidence interval. Stability is assumed when mean of treated samples within 90-110% and 95% confidence interval within 80-120% of control mean"

I am not sure if I understood the calculations well.
So I calculate the mean of treated and control samples.
I calculate 90% and 110% of the mean control sample, the mean treated sample should be within this.
I calculate 80% and 120% of the mean control sample, the 95% confidence interval of the mean treated sample should be within this.
Is this it?

Thanks!

I red it as follows:

Calculate the means control and treated samples

Mean [treated] x100/mean [control] should fall within the range 90 - 110%

Calculate the 95% confidence limit of the mean for the treated samples.

For the mean [treated] ± 95% confident interval both the upper and lower value should fall within the range of the mean [control] ± 20%

However, I would suggest that you design your experiment such that you can analyse a set samples after each freeze thaw cycle. If you only do 3 cylces and it fails you have no data and have to repeat the exercise. Whereas if you analyse after each cycle you have the data available to calculate for 2 or 1 cycle if 3 cycles fails.

Thanks! I will do it as you suggested

Are your controls fresh spiked?
Or have they been frozen and thawed themselves?

We used to have to do 3 freeze thaw cycles compared against both fresh spiked controls AND frozen controls.

It happened often enough to keep an eye out for it: that just ONE freeze thaw cycle (ie, the contols) was enough to lower recover of analyte as compared to fresh spiked and successive freeze thaws gave either little change or continued loss in recovery.


Alp