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Roshini ☆ 2008-01-21 13:36 (6732 d 22:07 ago) Posting: # 1534 Views: 4,706 |
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Hi All, I am a fresher in this field. During processing of plasma samples should we add the internal standard (IS) followed by the analyte in plasma or add analyte in plasma followed by IS. Since if IS is added first the chances of missing a vial (i.e not adding IS to a vial in between) can be avoided. Most of the methods state addition of analyte in plasma followed by addition of IS. Is there any guideline to indicate the same. R we supposed to prove the same during robustness experiment? Thanks and Regards, Roshini Singh |
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vpardhasaradhi ☆ 2008-01-25 05:39 (6729 d 06:03 ago) @ Roshini Posting: # 1554 Views: 3,760 |
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Dear Roshini, It is always a good practice to take plasma sample first into the vial and add internal standard to it. But your practical concern of missing of internal standard (IS) addition to some vials, if IS is added later, is valid. Hence, it is better to practically prove (as you said, as part of your Robustness experiments) that it makes no significant difference whether you add 'IS to plasma' or 'plasma to IS'. In addition, you need to state in your method protocol or method SOP, what you planned to do. For example, you may say, "take so and so volume of plasma sample into the vials containing so and so IS". With best regards, V.Pardhasaradhi |
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Helmut ★★★   Vienna, Austria, 2008-01-26 14:21 (6727 d 21:22 ago) @ vpardhasaradhi Posting: # 1562 Views: 3,893 |
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Dear V.Pardhasaradhi! ❝ It is always a good practice to take plasma sample first into the vial and ❝ add internal standard to it. True, because you easily notice whether you have missed a vial or not... ❝ ... Hence, it is better to practically prove (as you said, as part of ❝ your Robustness experiments) that it makes no significant difference ❝ whether you add 'IS to plasma' or 'plasma to IS'. I strongly disagree with you. IMHO (of analysing tenthousands of samples myself) missing IS addition is a rather rare event - I would guess <1/500... Any type of rubustness experiment does only make sense, if you get some meaningful insights of the influential parameters - in Roshini's case the order of IS addition. From a statistical point of view this would mean setting up an experiment being able to detect a difference in the occurance rate of a rare event. Not going into the details of sample size estimation this would mean analysing a couple of thousand samples under both conditions. Of course this is not feasible. On the other hand, if you set up a kind of rubustness study in just a few samples (let's say the usual batch size for three days), the chance of finding a difference in conditions is practically nil. But since the power of such a study is small you can't claim anything from it: Absence of evidence is not evidence of absence. ❝ ... better to practically prove ... BTW, in science it's not possible to prove something. We only reject or accept a hypothesis. Therefore I'm only with your second suggestion: ❝ ... you need to state in your method protocol or method SOP, what you ❝ planned to do.... — 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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