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Geokad ☆ Canada, 2009-04-14 20:16 (6283 d 00:28 ago) Posting: # 3522 Views: 3,784 |
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Hi, Does anyone test or evaluate the impact of different injection volumes? If yes, is it done during method validation? — Regards, Geokad |
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Helmut ★★★   Vienna, Austria, 2009-04-14 20:56 (6282 d 23:49 ago) @ Geokad Posting: # 3523 Views: 3,100 |
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Dear Geokad! ❝ Does anyone test or evaluate the impact of different injection volumes? Yes.  The smaller the volume, the less dispersion of the solution in the column head - and less peak broadening. If you have to inject larger volumes for any reason, try this trick: reconstitute the residue not in mobile phase, but in a mixture of lower elution power (more buffer, less organic modifier). The analyte will concentrate on the column head during injection and will be flushed with mobile phase resulting in sharper/higher peaks. You have to find a compromise between this effect and limited solubility of the analyte in solutes with very reduced percentages of organic modifier. In most cases 10%-20% less organic will be sufficient. ❝ If yes, is it done during method validation? No - during method development. Once you finished your experiments, your method should be laid down in a working instruction or SOP. Then prepare a validation protocol - and start working. — Dif-tor heh smusma 🖖🏼 Довге життя Україна! ![[image]](https://static.bebac.at/pics/Blue_and_yellow_ribbon_UA.png) Helmut Schütz ![[image]](https://static.bebac.at/img/CC by.png) The quality of responses received is directly proportional to the quality of the question asked. 🚮 Science Quotes |
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Geokad ☆ Canada, 2009-04-14 21:43 (6282 d 23:02 ago) @ Helmut Posting: # 3524 Views: 3,003 |
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Thank you HS, What happens if within the same study, we have different injection volumes between the batches. The method SOP allows the scientist to change the injection volume for optimization, sensitivity purposes. Would not having the same injection volume throughout the same study, be an issue? — Regards, Geokad |
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Helmut ★★★ Vienna, Austria, 2009-04-14 22:21 (6282 d 22:23 ago) @ Geokad Posting: # 3526 Views: 3,068 |
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Dear Geokad! ❝ What happens if within the same study, we have different injection volumes ❝ between the batches. The method SOP allows the scientist to change the ❝ injection volume for optimization, sensitivity purposes. I think you have to distinguish between anything which was planned (i.e., before the analysis was done), and anything which was done after an insufficient result was obtained. Do you mean you have a loop of let's say 250 µl and are normally working with a partial loop technique injecting just 50 µl - and since the peak is to small for reliable quantification, inject 200 µl? I would by very wary doing that. Even if you use the same solution of the analyte (mobile phase or from protein precipitation) the peak shape will never be the same. If you are dealing with LC/MS-MS remember that the matrix effect may change as well! Your calibration / QC data come from only one (the lower) injection volume, wright? ❝ Would not having the same injection volume throughout the same study, be ❝ an issue? I am not a regulator, but I would guess so. IMHO the only 'way out' would be collecting all the samples which gave an insufficient response for reanalysis. In the next step analyse these samples together with calibrators and QCS using the same (higher) volume. Of course you have to have validation data for this modified procedure beforehand. There's a major drawback: in some regulations the percentage of repeated samples is limited (e.g.., Brazil's ANVISA 20%). If you repeat too many samples - for any reason - the study may be rejected. — 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 |
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NewInPK ☆ 2009-04-29 07:41 (6268 d 13:04 ago) @ Helmut Posting: # 3632 Views: 2,937 |
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Good Evening, Our practice is that as long as the peak shape is acceptably symmetric and the injection volume is consistent within a batch (run), then there is no problem from a regulatory point of view with changing the injection volume. A statement in the method description along the lines "injection volume may vary in order to maintain the assay performance, but it must be consistent throughout a run" may help. It is a good idea to test in method development the extent to which increasing the injection volume may help (nice to have a backup plan). Cheers! |
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Ohlbe ★★★ France, 2009-04-29 13:11 (6268 d 07:34 ago) @ NewInPK Posting: # 3633 Views: 2,964 |
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Dear all, I would not worry too much about a small difference in injection volume (e.g. 40 vs. 50 µl), but I'm not sure what the interest of such small variations could be. Better stick to what has been validated. For higher variations I would agree with Helmut that this should be tested during the method validation. Remember also that with MS methods, matrix effects can vary depending on the amount of matrix injected: you can have a sort of threshold effect, where you will have no effect for low amounts of matrix (low volume injected) and suddenly see matrix effects appear for higher amounts of matrix (higher volumes injected). There is a very nice figure in this paper, where they obtained a higher signal after a 1/10 dilution of their extracts (well, that's plant material and not plasma, but the principle will be the same): M. Villagrasa, M. Guillamon, E. Eljarrat, D. Barcelo Matrix effect in liquid chromatography–electrospray ionization mass spectrometry analysis of benzoxazinoid derivatives in plant material Journal of Chromatography A, 1157 (2007) 108–114 Regards Ohlbe |
Ing. Helmut Schütz