Ohlbe ★★★ France, 2019-09-04 21:03 (1918 d 04:56 ago) Posting: # 20541 Views: 7,491 |
|
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 18:21 (1916 d 07:38 ago) @ Ohlbe Posting: # 20547 Views: 6,315 |
|
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 12:32 (1849 d 12:27 ago) @ Ohlbe Posting: # 20786 Views: 5,861 |
|
Dear all, If you feel there is still more to discuss, Bioanalysis has just published a whole issue on this topic. — Regards Ohlbe |
ElMaestro ★★★ Denmark, 2019-11-12 13:27 (1849 d 11:32 ago) @ Ohlbe Posting: # 20787 Views: 5,851 |
|
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? — Pass or fail! ElMaestro |
Helmut ★★★ Vienna, Austria, 2019-11-12 14:24 (1849 d 10:34 ago) @ ElMaestro Posting: # 20789 Views: 5,814 |
|
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. — Dif-tor heh smusma 🖖🏼 Довге життя Україна! Helmut Schütz The quality of responses received is directly proportional to the quality of the question asked. 🚮 Science Quotes |
ElMaestro ★★★ Denmark, 2019-11-12 14:38 (1849 d 10:21 ago) @ Helmut Posting: # 20790 Views: 5,870 |
|
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. — Pass or fail! ElMaestro |
Helmut ★★★ Vienna, Austria, 2019-11-12 14:41 (1849 d 10:17 ago) @ ElMaestro Posting: # 20791 Views: 5,853 |
|
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. — Dif-tor heh smusma 🖖🏼 Довге життя Україна! Helmut Schütz The quality of responses received is directly proportional to the quality of the question asked. 🚮 Science Quotes |
Ohlbe ★★★ France, 2019-11-12 14:56 (1849 d 10:03 ago) @ Helmut Posting: # 20792 Views: 5,800 |
|
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 ★★★ Denmark, 2019-11-12 17:25 (1849 d 07:34 ago) @ Ohlbe Posting: # 20793 Views: 5,859 |
|
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) — Pass or fail! ElMaestro |
Ohlbe ★★★ France, 2019-11-12 19:14 (1849 d 05:45 ago) @ ElMaestro Posting: # 20794 Views: 5,790 |
|
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 ★★★ Denmark, 2019-11-12 19:39 (1849 d 05:20 ago) @ Ohlbe Posting: # 20795 Views: 5,767 |
|
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? — Pass or fail! ElMaestro |
Ohlbe ★★★ France, 2019-11-13 01:06 (1848 d 23:52 ago) @ ElMaestro Posting: # 20796 Views: 5,761 |
|
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 11:06 (1848 d 13:53 ago) @ Ohlbe Posting: # 20797 Views: 5,745 |
|
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 ★★★ Denmark, 2019-11-13 18:35 (1848 d 06:24 ago) @ nobody Posting: # 20799 Views: 5,682 |
|
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? — Pass or fail! ElMaestro |
nobody nothing 2019-11-13 20:17 (1848 d 04:41 ago) @ ElMaestro Posting: # 20800 Views: 5,703 |
|
❝ if the LLOQ is rock solid, the calibration function stable and the QCs don't indicate a problem — Kindest regards, nobody |