Log in
Register|
Ladi ☆ Thailand, 2016-02-23 12:18 (2979 d 00:01 ago) Posting: # 16024 Views: 3,812 |
|
|
Dear forum members, I have questions regarding interference peak observed during sample analysis. We are doing study of itopride in plasma usisng LC-MS/MS. We use gradient and our extraction method is protein precipitation. During method validation only single peak was observed for itopride at RT 1.3 min. However, during study samples analysis two extra peaks (same MRM transition and same qualifier ratio as itopride) were observed. First interference peak RT is 1.6 min and second one is 2.0 min. Peak resolution between itopride (RT 1.3 min) and interference at RT 1.6 min is >2.5. The area of interference at RT 1.6 min is around LQC which is quite distinct at elimination zone. The ISR of samples we did passed beautifully. However, after talking with sponsor, we decided to put everything on hold and investigate. After further review of literature, we learned that itopride main metabolite is itopride N-oxide which can back convert to itopride in MS source (we use ESI), hence different RT but same MRM transition. To get more information, we added another MRM transition of itopride n-oxide (get this from precursor ion calculation from molecular formula and product ion scan to get major product ion). When we re-injected some study samples with this new method, a huge peak was observe for itopride n-oxide MRM transition at RT 1.6 min where it exactly overlapped with interference peak at RT 1.6 min. I understand that the n-oxide is more polar and should come out first in reverse phase column but not sure what's going on here. We are waiting for itopride n-oxide reference standard to do test further. And what about third peak at 2.0 min? Not sure but we also learned during literature review that these n-oxide compounds can easily form dimers. Anyway, my questions here to the members are: 1.Since we using MS/MS, will it be acceptable to see more than one peak in study sample? 2.If the inference peak is well separated, is it necessary to identify what those peaks are? 3. I have adjusted my gradient table and flow rate so that the two interference peaks are farther away (real itopride 1.4 min, first interference at 2.0 min and second interference at 2.2 min). Will it be OK if I set up my method to stop acquiring itopride at RT 1.9 so that the two interference are no longer visible. What else we have to do to be able to use this new gradient method for study. 4. Once we get our hands on the n-oxide, we will add it to the QC to test for selectivity, back conversion during extraction and long term storage. Is that enough or should we do more. I really appreciate your time and the valuable knowledge you share. Regards, Ladi (Thailand) |
|
Ohlbe ★★★ France, 2016-03-03 17:22 (2969 d 18:58 ago) @ Ladi Posting: # 16044 Views: 2,689 |
|
|
Dear Ladi, ❝ However, after talking with sponsor, we decided to put everything on hold and investigate. Great ! ❝ I understand that the n-oxide is more polar and should come out first in reverse phase column... Not necessarily. I worked on some compounds in the past where the N-oxide was eluted late. ❝ And what about third peak at 2.0 min? Not sure but we also learned during literature review that these n-oxide compounds can easily form dimers. Could be. Glucuronides are also known to give extra peaks due to collision-induced in-source dissociation - but this time I would expect them to be eluted earlier. ❝ 1.Since we using MS/MS, will it be acceptable to see more than one peak in study sample? Yes, sure, as long as the peaks are well separated and do not interfere with the analyte/IS peak. ❝ 2.If the inference peak is well separated, is it necessary to identify what those peaks are? There we come to the difference between "nice to know" and "need to know". As an analyst I'd say it is very, very helpful to know what these additional peaks are: it helps to ensure whether there is a risk of back-conversion, potential stability issues etc. ❝ 3. I have adjusted my gradient table and flow rate so that the two interference peaks are farther away (real itopride 1.4 min, first interference at 2.0 min and second interference at 2.2 min). Will it be OK if I set up my method to stop acquiring itopride at RT 1.9 so that the two interference are no longer visible. What else we have to do to be able to use this new gradient method for study. It is possible. Some people even use multiplexing i.e. two HPLC systems connected to a single MS/MS system. Two runs injected in parallel, one on each HPLC system. A sample is injected from HPLC 2 during the "non-interesting" part of the chromatogram from HPLC 1. This being said, you have to be sure that the retention times are stable enough. Personally, unless using multiplexing I would acquire the full chromatogram. ❝ 4. Once we get our hands on the n-oxide, we will add it to the QC to test for selectivity, back conversion during extraction and long term storage. Is that enough or should we do more. It sounds good ! — Regards Ohlbe |
Ing. Helmut Schütz