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Dr. Harish L. Rao ☆ India, 2008-07-03 09:29 (6564 d 18:59 ago) (edited on 2008-07-04 03:00) Posting: # 1990 Views: 5,983 |
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Dear Group Members, Let's assume that we are performing the Bioequivalence study of the formulation of a highly protein bound drug which is 98% bound to plasma proteins. After direct Liquid-Liquid extraction or Solid Phase Extraction of the plasma samples followed by LC-MS/MS analysis, do we need to apply a correction factor for the protein binding? Eg: If a plasma concentration of a time point comes to 50ng/mL, do we need to calculate the bound form as 50*100/(100-98) = 2500ng/mL & use 50 (Free Form) + 2500 (Bound Form) = 2550 ng/mL, for Winnonlin data analysis? In case we initially precipitate the plasma proteins by addition of Acetonitrile / Methanol before Liquid-Liquid or Solid Phase Extraction, do we need to consider the results as sum of the free & bound concentrations? Can anyone provide any reference in this regard? Thanks & regards, Dr. Harish L. Rao -- Edit: capitals changed to mixed case in title [Ohlbe] |
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martin ★★ Austria, 2008-07-08 16:51 (6559 d 11:37 ago) @ Dr. Harish L. Rao Posting: # 2005 Views: 4,554 |
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dear Dr. Harish L. Rao ! chapter 4 of the following book: Källén A. (2008). Computational Pharmacokinetics. Chapman and Hall / CRC Biostatistics Series. provides concepts and examples regarding protein binding affecting volume of distribution. hope this helps martin |
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Helmut ★★★   Vienna, Austria, 2008-07-08 21:21 (6559 d 07:07 ago) @ martin Posting: # 2006 Views: 4,958 |
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Dear Martin & Harish! ❝ Källén A. (2008). Computational Pharmacokinetics. Chapman and Hall / CRC ❝ Biostatistics Series. Yes, this is a nice one!  Quoting pp 66 from Chapter 4: This reaction [the formation of the drug-protein complex] is usually very fast (milliseconds) and we can therefore assume that it is always in equilibrium [...] The reasons for such fast binding/release are twofold:
There are a few exceptions where molecules are trapped in precipitated protein - but this would mainly affect recovery and both accuracy/precision of the method. There was a lengthy debate about measuring total vs. free drug in BE studies more than 15 years ago which ended in the consensus of measuring total even for highly bound drugs whithout any correction factors whatsoever. BTW your example should read like this: measured: 50ng/mL (this is total!) free: 50 x 2% = 1ng/mL bound: 50 x 98% = 49ng/mLYour BE assessment wil not be affected whether you apply such a factor or not. Introduction of a factor from the literature does not make any sense. If you really want to start fiddeling around with ultrafiltration consider sneeking around at Millipore, but keep in mind:
— 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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vamshi ● 2008-08-14 13:29 (6522 d 14:59 ago) @ Dr. Harish L. Rao Posting: # 2183 Views: 4,460 |
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Hari, For Bioequivalence study of the formulation, you need to Compare the Test and reference (innovator) product. You can refer orange book and OGD (office of generic drugs of FDA). Even if your formulation Bioequivalence/ NCE you need prove Recovery in method validation is satisfactory. As your Calibration standards, Quality Controls and study sample, reference study samples will be processed under same conditions. You may consider same values for Statistics. Not required to calculate free and bound form. |
Ing. Helmut Schütz