sera
☆    

Turkey,
2019-01-02 13:30

Posting: # 19721
Views: 713
 

 Resolution value in BE studies [Bioanalytics]

Dear Members,

In BE studies, what are the acceptance criteria for the resolution value for analytical procedure of analyte of interest and internal standard? A resolution value >2 between the peak of interest and internal standard is desirable in analytical method validation but there are no acceptance criteria about resolution value in EMA and FDA bioanalytical method validation guidelines.

In a completed HPLC study, the resolution value was about 1.2 during the study. Analyte and internal standard peaks were free of distortions and splitting. Internal standard behaved as the analyte at the every stage of the analysis. IS response variation was monitored in each batch regularly. No problem was observed between spiked samples and study samples. Full validation was performed prior to analysis of study samples and the parameters were successfully validated.
During analysis of study samples; QC samples were within acceptance limits. ISR was performed and the results were acceptable.

How can the resolution value between analyte of interest and internal standard in the chromatograms for the above study be justified to be appropriate? What is your opinion for the baseline resolution lower limit?

Thanks in advance.
Regards
Ohlbe
★★★

France,
2019-01-02 18:21

@ sera
Posting: # 19722
Views: 609
 

 Resolution value in BE studies

Dear sera,

» In BE studies, what are the acceptance criteria for the resolution value for analytical procedure of analyte of interest and internal standard? A resolution value >2 between the peak of interest and internal standard is desirable in analytical method validation but there are no acceptance criteria about resolution value in EMA and FDA bioanalytical method validation guidelines.
»
» In a completed HPLC study, the resolution value was about 1.2 during the study. Analyte and internal standard peaks were free of distortions and splitting.

» How can the resolution value between analyte of interest and internal standard in the chromatograms for the above study be justified to be appropriate? What is your opinion for the baseline resolution lower limit?

It really depends on the method used.

If you are using UV or fluorimetric detection: resolution is crucial and the two peaks should be clearly separated.

If you are using LC-MS/MS, a perfect resolution is not required thanks to the selectivity of the detection. Co-elution of the analyte and of the IS can actually help to compensate for matrix effects, if both are affected similarly (that's one of the reasons why stable-isotope labelled internal standards usually perform better than structural analogues or unrelated molecules, though the couple of seconds of difference in retention times often seen with deuterium labelling has been described to create problems). However, during method development you should ensure that there is no ion suppression of the analyte by the IS and vice-versa.

Regards
Ohlbe
ElMaestro
★★★

Denmark,
2019-01-02 23:32

@ sera
Posting: # 19723
Views: 598
 

 Resolution value in BE studies

Hello sera,

» How can the resolution value between analyte of interest and internal standard in the chromatograms for the above study be justified to be appropriate? What is your opinion for the baseline resolution lower limit?

Sounds like you have classical HPLC quantification without MS/MS? If this is not the case, stop reading now :-D

The resolution makes extremely good sense...if the peak is symmetric and even more so if it obeys certain mathematical rules. If the peaks are Gaussian (and there is not the slightest theoretical reason to think any peak is) then the theory behind resolution has a visually appealing significance and results in some fine rules of thumb.

But departure from Gaussianism is difficult to quantify - in theoretical maths you have skewness and curtosis and a palette of asymmetric distributions which all seem to be lacking practical application in the grand scheme of chromatolophystic things. Therefore, the resolution metric quickly loses its significance if the peaks are non-Gaussuan.
So it isn't quite easy to define any practical rules for resolution that fit all cases. When you can clearly see tailing or fronting offset "a lot" from the baseline then calculation of resolution may be ... well.... something you do if or when a regulator asks for it but not because it is something that makes particularly sense.
At the end of the day, if your peaks with or without t or f and so on are separated so much that you can resolve them quantitatively (the area of one peak does not overlap [=confound the area calculation of] the other) then you have a good case regardless of the resolution metric. You just need to convince the powers that be about this mere fact.

I would be inclined to take a practical approach here: If you pass validation and if your study sample chromatograms truly look like (and note, I do not quantify the meaning of this) the validation chromatograms then how could a resolution metric issue really be a problem in your study?

if (3) 4

x=c("Foo", "Bar")
b=data.frame(x)
typeof(b[,1]) ##aha, integer?
b[,1]+1 ##then let me add 1


Best regards,
ElMaestro

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