Ohlbe
★★★

France,
2019-09-04 19:03

Posting: # 20541
Views: 2,193
 

 IS response variations [Regulatives / Guidelines]

Dear all,

There have been some questions and discussions several times on the forum on the monitoring of internal standard variations. The FDA has just published a Q&A on this topic.

Regards
Ohlbe
nobody
nothing

2019-09-06 16:21

@ Ohlbe
Posting: # 20547
Views: 1,523
 

 IS response variations

Hi France, many thanks!

"The approach(es) for investigation might include, but are not limited to, ... or any other scientifically justified approach"

Absolutely my kind of humour!

Kindest regards, nobody
Ohlbe
★★★

France,
2019-11-12 11:32

@ Ohlbe
Posting: # 20786
Views: 1,087
 

 More on IS response variations

Dear all,

If you feel there is still more to discuss, Bioanalysis has just published a whole issue on this topic.

Regards
Ohlbe
ElMaestro
★★★

Belgium?,
2019-11-12 12:27

@ Ohlbe
Posting: # 20787
Views: 1,074
 

 More on IS response variations

Hi Ohlbe and all,

» If you feel there is still more to discuss, Bioanalysis has just published a whole issue on this topic.

For sure, I am going to look into this with great interest - thanks.
Am OoO at the moment so can't look everything up, do you know if any of these or other publications deal with between-batch variation in IS response?

I am asking because I am seeing a tendency for CROs to define within-batch criteria for IS responses and trends, but I am also seeing at certain CROs how the IS response can vary enormously between runs. At other CROs the IS may be rock solid between runs, and it is of course somewhat equipment and analyte-dependent and factors like column age play a role. The phenomenon is really striking in some cases, but you will only see it if you look for it. I do not have a reference to discuss it numerically and I did not see this matter discussed on the forum here. In some cases I can almost predict the method is about to go out of whack, but it is always the same "But this run passes so there is no issue..."

I am looking for something that is a little better than the bare "But if the run passes...", "perhaps it is the ion source blah" and which has the quantitative aspect in it.

Am in dialogue with a few sponsors to see if we can publish on this matter if noone else has done it. But few sponsors seem willing to make public stories about how enormous the between batch IS response can be, - perhaps because it may reflect badly on their products?

I could be wrong, but...
Best regards,
ElMaestro
Helmut
★★★
avatar
Homepage
Vienna, Austria,
2019-11-12 13:24

@ ElMaestro
Posting: # 20789
Views: 1,070
 

 More on IS response variations

Hi ElMaestro,

» […] At other CROs the IS may be rock solid between runs, and it is of course somewhat equipment and analyte-dependent and factors like column age play a role.

I don’t see how that can affect the IS (and analyte) response unless someone works with peak heights (who does it nowadays?)…

» […] "perhaps it is the ion source blah"

If a stable isotope labeled internal standard is used, that’s the only reasonable explanation for me.

Cheers,
Helmut Schütz
[image]

The quality of responses received is directly proportional to the quality of the question asked. 🚮
Science Quotes
ElMaestro
★★★

Belgium?,
2019-11-12 13:38

@ Helmut
Posting: # 20790
Views: 1,071
 

 More on IS response variations

Hi Hötzi,

» I don’t see how that can affect the IS (and analyte) response unless someone works with peak heights (who does it nowadays?)…

As a general rule the S:N become more unfavourable as the column ages and peak separation suffers. This affects the identifcation of peaks and their integration. It will be more pronounced for the analyte near the LLOQ than for the IS.

I could be wrong, but...
Best regards,
ElMaestro
Helmut
★★★
avatar
Homepage
Vienna, Austria,
2019-11-12 13:41

@ ElMaestro
Posting: # 20791
Views: 1,060
 

 More on IS response variations

Hi ElMaestro,

» As a general rule the S:N become more unfavourable as the column ages and peak separation suffers. This affects the identifcation of peaks and their integration. It will be more pronounced for the analyte near the LLOQ than for the IS.

I stand corrected. :-D

Cheers,
Helmut Schütz
[image]

The quality of responses received is directly proportional to the quality of the question asked. 🚮
Science Quotes
Ohlbe
★★★

France,
2019-11-12 13:56

@ Helmut
Posting: # 20792
Views: 1,059
 

 More on IS response variations

Dear Helmut,

» » […] "perhaps it is the ion source blah"
»
» If a stable isotope labeled internal standard is used, that’s the only reasonable explanation for me.

Well, it could also be a wrong dilution during the preparation of a new working solution, a variable recovery...

Regards
Ohlbe
ElMaestro
★★★

Belgium?,
2019-11-12 16:25

@ Ohlbe
Posting: # 20793
Views: 1,038
 

 More on IS response variations

Ahem,

» » » […] "perhaps it is the ion source blah"
» »
» » If a stable isotope labeled internal standard is used, that’s the only reasonable explanation for me.
»
» Well, it could also be a wrong dilution during the preparation of a new working solution, a variable recovery...

it could be a ton of things. Matrix effect, variation stemming from different sources of plasma ("But is passed the selectivity experiments blah blah"). Even solar flares can under the right circumstances be detected by LC-MS/MS.

Different stocks, different storages, different equipment, pipetting errors, robot programming, different this, different that. And you get some info by also looking at the slope of the cal. curve in those cases.
But still, let us discuss how to establish the presence of the phenomenon before addressing causes? Let us say the average IS of cals+QCs in one run is 8x the average IS of cals+QC's in another run and all passing the usual criteria.
Would you be concerned?
Yes? Why?
No? Why not?
Not enough info? Which questions would you ask? (I would e.g. consider matrix sources used for QC's+cals, pipetting, volumes, station, method settings, columns, possibly grid anomalies if I can get such info, but all that will usually leave a blank)

I could be wrong, but...
Best regards,
ElMaestro
Ohlbe
★★★

France,
2019-11-12 18:14

@ ElMaestro
Posting: # 20794
Views: 1,032
 

 More on IS response variations

Dear ElMaestro,

» Different stocks, different storages, different equipment, pipetting errors, robot programming, different this, different that. And you get some info by also looking at the slope of the cal. curve in those cases.

Sure. This paper seems to have some interesting discussions on slope variations, unfortunately I don't have access to the full paper.

» But still, let us discuss how to establish the presence of the phenomenon before addressing causes? Let us say the average IS of cals+QCs in one run is 8x the average IS of cals+QC's in another run and all passing the usual criteria.
» Would you be concerned?
» Yes? Why?
» No? Why not?
» Not enough info? Which questions would you ask? (I would e.g. consider matrix sources used for QC's+cals, pipetting, volumes, station, method settings, columns, possibly grid anomalies if I can get such info, but all that will usually leave a blank)

First I would compare the slopes of the two runs. If the slope is comparable: response is seriously decreased in one run, which may make it difficult to still pass the LLOQ.

If you find the same variation in the slope as in the IS response, and there is no IS response difference between CC / QC samples on the one hand and subject samples on the other hand, and assuming they used the same frozen CC samples in both runs: I would check the preparation of the IS working solution and pipetting, mobile phase preparation, HPLC and MS settings, retention times differences between the two runs, peak shape, anything that may result in (or be an indicator of) differences in matrix effects. If the IS is not a stable isotope: I would also check anything which may result in differences in recovery (solvents, SPE column batch...).

I would also look at the ISR data, if available. If there is a high systematic bias in the samples analysed in one of these 2 runs, there is something seriously wrong in your Kingdom, even if the QCs passed.

Regards
Ohlbe
ElMaestro
★★★

Belgium?,
2019-11-12 18:39

@ Ohlbe
Posting: # 20795
Views: 1,029
 

 More on IS response variations

Hi Ohlbe,

please please please go back to the my initial question: When in the first place is the IS variation between runs so high that you start asking those questions?
Can you say anything generally or specifically about the level of variation at which all those wonderfully relevant question come into play?

I could be wrong, but...
Best regards,
ElMaestro
Ohlbe
★★★

France,
2019-11-13 00:06

@ ElMaestro
Posting: # 20796
Views: 1,017
 

 Not more on IS response variations

Hi ElMaestro,

» Can you say anything generally or specifically about the level of variation at which all those wonderfully relevant question come into play?

Honestly ? No. Sorry. Not a question I gave enough thought to have a valid opinion. Not a question I have heard discussed at the bioanalytical conferences I have attended.

Regards
Ohlbe
nobody
nothing

2019-11-13 10:06

@ Ohlbe
Posting: # 20797
Views: 990
 

 Not more on IS response variations

Not a key opinion leader in this field, but:

I like discussions along the line "problem -> solution". So: if the LLOQ is rock solid, the calibration function stable and the QCs don't indicate a problem, why should I take care of day-to-day variations in IS response too much?

If have had cases where different lab people (in the good old days of liquid-liquid extraction, though...) could produce different IS response with absolute same equipment, solutions, protocols to follow. Why bother to much, as long the quality of the method/validation covers that?

Kindest regards, nobody
ElMaestro
★★★

Belgium?,
2019-11-13 17:35

@ nobody
Posting: # 20799
Views: 962
 

 the run passes = there is no problem?

Hi nobody,

» I like discussions along the line "problem -> solution". So: if the LLOQ is rock solid, the calibration function stable and the QCs don't indicate a problem, why should I take care of day-to-day variations in IS response too much?

Using the same kind of argumentation why would you worry about IS at all then? If the run passes why would IS responses in any way be a worry under any circumstance?
If the purpose of a car is to take you from A to B why would anyone care if it starts making sudden clonking noises?

I could be wrong, but...
Best regards,
ElMaestro
nobody
nothing

2019-11-13 19:17

@ ElMaestro
Posting: # 20800
Views: 958
 

 the run passes = there is no problem?

» if the LLOQ is rock solid, the calibration function stable and the QCs don't indicate a problem

:-)

Kindest regards, nobody
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