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drajasekhar ☆ India, 2009-12-27 11:43 (6024 d 07:30 ago) (edited on 2009-12-27 12:09) Posting: # 4524 Views: 7,813 |
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Dear Members, This is with regard to use of internal standards in LCMSMS bioanalytical techniques especially in polarity switching mode. For the determination of two drugs (one showing good response at positive ion mode and other at negative ion mode) in human plasma by LC-MS-MS; for this how many internal standards we should use? Using one internal standard for both the polarities (positive or negative) is it legitimate? Thanks in Advance Rajasekhar -- Edit: Subject line changed. [Jaime] |
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Ohlbe ★★★ France, 2009-12-28 02:00 (6023 d 17:12 ago) @ drajasekhar Posting: # 4527 Views: 6,966 |
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Dear Rajasekhar, I personally would go for two internal standards: one detected in the positive mode, and one in the negative mode. You will have better chances to compensate for variations in ionisation, due for instance to matrix effects. Nothing mentioned in the FDA or draft EMEA bioanalytical guidelines, though. Regards Ohlbe |
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drajasekhar ☆ India, 2009-12-28 07:32 (6023 d 11:40 ago) @ Ohlbe Posting: # 4530 Views: 6,978 |
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Dear Members, Please give me a conclusion for the following: Drug A (Positive ion mode) Drug B (Negative ion mode)
CASE I.Thanks in Advance, Rajasekhar. Edit: Your post was a mess! See here for Instructions. Please use the Preview before submitting your post. Don't open new threads which essentially deal with the same (your) problem. I linked your post to the old thread. [Helmut] |
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Ohlbe ★★★ France, 2009-12-30 14:51 (6021 d 04:21 ago) @ drajasekhar Posting: # 4536 Views: 6,757 |
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drajasekhar ☆ India, 2009-12-30 15:45 (6021 d 03:28 ago) @ Ohlbe Posting: # 4538 Views: 6,756 |
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Dear Ohlbe, The recovery of Drug A, Drug B and internal standards were determined by analyzing of six replicates of low, medium and high quality control concentrations for drugs along with internal standards. The percent recovery was evaluated by comparing the peak area of extracted samples and peak area of un-extracted (aqueous) samples. With regards, Rajasekhar |
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Ohlbe ★★★ France, 2009-12-30 18:36 (6021 d 00:36 ago) @ drajasekhar Posting: # 4540 Views: 6,713 |
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Dear Rajasekhar, ❝ The percent recovery was evaluated by comparing the peak area of extracted ❝ samples and peak area of un-extracted (Aqueous) samples. OK. Then I would not call this recovery but rather overall efficiency. What you have measured is a combination of the yield of your extraction or protein precipitation method (which is what I would call recovery) and matrix effects. If you want to evaluate the recovery of your extraction method when using LC/MS methods you should compare the response of extracted samples to the response of blank samples, extracted and then spiked post-extraction. That's an experiment you can combine with matrix effects, where you will compare the response in samples spiked post-extraction to the response in aqueous samples: - recovery: response in extracted samples / response in samples spiked post extraction - matrix factor: response in samples spiked post extraction / response in aqueous samples - overall efficiency: response in extracted samples / response in aqueous samples The low "recovery" (below 40 %) you had for analyte B with your protein precipitation method is probably due to matrix effects. As mentioned by Helmut protein precipitation is the worst sample preparation method you may think of when using LC/MS methods. Fast, cheap, easy, but terrible. Regards Ohlbe |
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drajasekhar ☆ India, 2009-12-30 19:17 (6020 d 23:55 ago) @ Ohlbe Posting: # 4541 Views: 6,810 |
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Dear Ohlbe, Thank you very much for your clarification. Please tell me the overall efficiency and absolute recovery are the same or not! And now, you please give me a conclusion for the following comparison i.e. Overall efficiency Vs Limit of Quantitation (here I included the LOQ and the instrumentation):
CASE I.With regards, Rajasekhar. Edit 1: Removed details of the method which were already given in a previous post. Learn how to format your posts and use the Preview - you would have noticed the mess immediately! Quote from the Instructions: Please do not include [...] series of blanks, or tabs in your text. [...] series of blanks and/or tabs will be stripped and cut down on a single blank. Both multiple blanks and tabs may look nice on your system, but awful on others. I'm not in the mood of removing your combinations of multiple blanks and tabs all the time! [Helmut]Edit 2: Since you reverted my first edit to your original version, I must assume that you want to start an edit-war. This is the first warning. Adhere to the Forum's Policy or your account will be blocked. [Helmut] |
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Ohlbe ★★★ France, 2009-12-30 20:16 (6020 d 22:56 ago) @ drajasekhar Posting: # 4542 Views: 6,784 |
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Dear Rajashekhar, Please read (and take into consideration !) Helmut's comments at the bottom of your post, if you want to continue this discussion ! ❝ Please tell me the overall efficiency and absolute recovery are the same or not! I can't say - I'm not sure what you call absolute recovery... ❝ And now, you please give me a conclusion for the following comparison i.e. ❝ Overall efficiency Vs Limit of Quantitation (here I included the LOQ and ❝ the instrumentation) Conclusion on what ? What are you trying to compare (overall efficiency vs. LLOQ  ) ? What will you be using your method for ? What LLOQ will you need ? No need to go to 0.1 ng/ml if the lowest concentration you expect in your study is 5 ng/ml...Regards Ohlbe |
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drajasekhar ☆ India, 2009-12-30 20:50 (6020 d 22:22 ago) @ drajasekhar Posting: # 4543 Views: 6,679 |
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Dear Ohlbe, Sorry, I'm not trying to compare Overall efficiencies and LOQ, but when we compare the two methods (CASE I and CASE II) in terms of sensitivity the first method is high sensitive method and when we comparing the overall efficiencies and extraction technique of both the methods, the second (CASE II) one will become a good method. Now, by considering all the above facts, you please tell me which of the above method is a better method! regards Rajasekhar |
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Helmut ★★★   Vienna, Austria, 2009-12-30 20:58 (6020 d 22:14 ago) @ drajasekhar Posting: # 4544 Views: 6,877 |
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Hi Rajasekhar! I would consider LLE with tBME better than protein precipitation when matrix-effects are considered. But still LLE is a last century's method. How do you remove the organic layer? Short freezing and pouring, removal with a pipette or ... I would guess that you loose more of the organic phase than you like. If you want to get a more 'clean' extract I would suggest to opt for solid-phase-extraction. SPE gives you much more options. One example: if your drug is basic (many are) it may bind to residual silanol-groups of RP (you also have a choice between C2/C8/C18). You may wash then with buffer followed by up to 100% methanol or acetonitrile and remove all neutral/acidic lipohilic compounds. Next elute with an acidic phase, where you add just as much organic modifier needed to remove your drug - stronger bases and more lipophilic compounds will be retained on the SP. Voilá! We must not consider validation as an end in itself, but as a demonstration of the method's suitability for use. As Ohlbe already pointed out - what LLOQ do you require? — 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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drajasekhar ☆ India, 2009-12-30 21:35 (6020 d 21:37 ago) @ Helmut Posting: # 4545 Views: 6,693 |
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Dear HS, Thank you very much for your clarification and conclusion. As pointed out by Ohlbe, my required LLOQ is 1 ng/mL and i was achieved this for both the analytes. Actually, the CASE I is a published method and CASE II is our developed and validated method. regards Rajasekhar |
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Helmut ★★★ Vienna, Austria, 2009-12-30 21:38 (6020 d 21:34 ago) @ drajasekhar Posting: # 4546 Views: 6,704 |
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Dear Rajasekhar! ❝ [...] my required LLOQ is 1 ng/mL and i was achieved this for both the ❝ analytes. Actually, the CASE I is a published method and CASE II is our ❝ developed and validated method. Fine, so start testing the matrix effect. — 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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Ohlbe ★★★ France, 2009-12-30 21:43 (6020 d 21:29 ago) @ drajasekhar Posting: # 4547 Views: 6,607 |
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Dear Rajasekhar, ❝ my required LLOQ is 1 ng/mL Then the method you have developed suits your needs. Good. ❝ Actually, the CASE I is a published method and CASE II is our ❝ developed and validated method. So you've been trying to have us say that your method is better than the one which has been published  Sorry, but I don't really have time to loose playing this kind of games.Regards Ohlbe — Regards Ohlbe |
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drajasekhar ☆ India, 2009-12-30 21:54 (6020 d 21:18 ago) @ Ohlbe Posting: # 4548 Views: 6,573 |
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Dear Ohlbe, I am really sorry, really i don't want to waste your valuable time. Really, i want to know all these things, which we discussed. Once again thank you very much for all your valuable clarifications. regards Rajasekhar |
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drajasekhar ☆ India, 2009-12-28 08:00 (6023 d 11:12 ago) @ Ohlbe Posting: # 4531 Views: 6,878 |
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Dear Members, This is with regard to LC-MS/MS bioanalytical method validation. What is the sufficient measure for matrix effect? Thanks in Advance, Rajasekhar |
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Helmut ★★★ Vienna, Austria, 2009-12-28 15:18 (6023 d 03:54 ago) @ drajasekhar Posting: # 4532 Views: 6,942 |
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Dear Rajasekhar! ❝ What is the sufficient measure for matrix effect? See one of my lectures (slides 9-10). Concerning your other post I would proceed with the second method, because:
— 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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Ohlbe ★★★ France, 2009-12-30 14:53 (6021 d 04:19 ago) @ drajasekhar Posting: # 4537 Views: 6,858 |
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Dear Rajasekhar, ❝ What is the sufficient measure for matrix effect? Have a look at the draft EMA guideline. Regards Ohlbe |
Ing. Helmut Schütz