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Dr. Harish L. Rao ☆ India, 2007-09-20 11:42 (6056 d 07:22 ago) (edited by Dr. Harish L. Rao on 2007-09-20 11:56) Posting: # 1096 Views: 4,786 |
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Dear Group Members, During subject plasma sample analysis by LC/MS/MS, an extra peak (possibly due to metabolite) is observed which is not completely separated from the drug peak. But Method Validation was performed without the metabolite (as per OGD communication) & there was no interference peak during method validation. In such a situation, how should the analyst proceed with subject sample analysis? Thanks & regards, Dr. Harish L. Rao |
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Helmut ★★★   Vienna, Austria, 2007-09-20 14:56 (6056 d 04:09 ago) @ Dr. Harish L. Rao Posting: # 1098 Views: 4,439 |
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Dear Harish! ❝ .. Method Validation was performed without the metabolite (as per OGD ❝ communication) & there was no interference peak during method validation. The interference probably is a metabolite; you should check this by:
❝ In such a situation, how should the analyst proceed with subject sample ❝ analysis?  According to the Reviewer Guidance: Validation of Chromatographic Methods (Nov 1994) FDA is expecting a resolution Rs of adjacent peaks of >2. Rs=2×(t2-t1)/(w1+w2) where t1 and t2 are the retention times of peak 1 and peak 2, respectively, and w1 and w2 are the baseline peak widths measured between tangents drawn to the sides of the peak.Assuming a Gaussian peak, we may calculate the baseline width from the width at half-height: w~1.699×w0.5. By this we get an alternative calculation (more easily obtained from the chromatographic data system) based on peak width measured at half of the peak height, w0.5: Rs=2×(t2-t1)/[1.699×(w0.5,1+w0.5,2)] Consider modifying the method for better separation; only after (partial) re-validation continue with subjects' samples. — 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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Ohlbe ★★★ France, 2007-09-21 03:55 (6055 d 15:10 ago) @ Helmut Posting: # 1103 Views: 4,028 |
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Dear Harish and HS, ❝ The interference probably is a metabolite; you should check this by: ❝ (...) ❝ - the m/z ratio This may not work if you are dealing with an isomer (such as a cis/trans isomer). A typical example is isotretinoin, which is partially converted in vivo to tretinoin. You can separate them chromatographically, but not by m/z. ❝ Consider modifying the method for better separation; only after (partial) ❝ re-validation continue with subjects' samples. I would consider this the only way out... Regards Ohlbe |
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vamshi ● 2008-08-14 14:49 (5727 d 04:15 ago) @ Dr. Harish L. Rao Posting: # 2186 Views: 3,833 |
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Hari, you need to perform an additional experment during validation that, check for any impact of chromatography with co Concurrent medications, OTC drugs and drug metabolites and posible degradation products of analytes. Some times Anti coagulant effect and if for animals differnt Strain or beed plasma can be checked. |
Ing. Helmut Schütz