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chiragkhatri ★ India, 2007-12-14 07:21 (6768 d 12:03 ago) Posting: # 1374 Views: 6,693 |
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Dear members, I have a combination of Drug 1+2+3. Since it is not possible to analyze all three together, I have validated method for drug 1 separately and drug 2and 3 will be validated in combination. The point here is since subject samples will be in combination of all three, I will be analyzing it twice with two different methods, but the validation is not done in presence of drug1 for drug2 and drug3 and vice versa. I nedd suggestions as to how to plan an experiment so that it can be proved that the method used for Drug1 does not have any interference of drug 2 and drug3 and vice-versa. Any reference to guidelines are welcome. Thanks in advance Chirag Khatri |
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Ohlbe ★★★ France, 2007-12-15 13:05 (6767 d 06:19 ago) @ chiragkhatri Posting: # 1378 Views: 5,548 |
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Dear Chirag, No guideline jumps to my mind on this topic. What I would suggest would be to prepare and analyse 3 different QC samples, each spiked at a low concentration (LLOQ or LQC) with one analyte (1, 2 or 3) and ULOQ with the other two. If there is any interference (either directly visible, or influencing the ionisation of your analyte or IS if you are using MS/MS) you would have maximum chances to see an impact on the accuracy. Regards Ohlbe |
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chiragkhatri ★ India, 2007-12-15 13:22 (6767 d 06:02 ago) @ Ohlbe Posting: # 1379 Views: 5,475 |
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Dear Ohlbe Thanks a lot, at leasts I got some clue on it. Regards Chirag |
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Helmut ★★★   Vienna, Austria, 2007-12-15 14:42 (6767 d 04:42 ago) @ Ohlbe Posting: # 1381 Views: 5,601 |
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Dear Ohlbe! ❝ No guideline jumps to my mind on this topic. Not quite; FDA (2001) states for chromatographic methods ( 'Potential interfering substances in a biological matrix include endogenous matrix components, metabolites, decomposition products, and in the actual study, concomitant medication and other exogenous xenobiotics. If the method is intended to quantify more than one analyte, each analyte should be tested to ensure that there is no interference.' ... and for ligand binding assays (Section V.A.1):'Cross-reactivity of metabolites, concomitant medications, or endogenous compounds should be evaluated individually and in combination with the analyte of interest.' ANVISA (2003) states (Sections 3.1.3/3.1.4): The interferents may be components of the biological matrix, metabolites, decomposition products and drug products used concomitantly with the study. ❝ What I would suggest... Your suggestions are quite reasonable, but metabolites may make the life of the analyst quite miserable. Since metabolites generally are more polar (less lipophilic) than the parent compound(s), they will show up in the commonly used RP-LC at earlier retention times. If analyzing combinations of drugs, interferences (let's say by the metabolite of a more lipophilic drug 1 with the less lipophilic parent drug 2) may occur. Looking at the parent compounds only may lead to surprises with 'real world' samples. The ideal solution would be checking the retention times of metabolites, but since they may not be available...
Edit: Link corrected for FDA’s new site. [Helmut] — 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, 2007-12-16 14:32 (6766 d 04:52 ago) @ Helmut Posting: # 1383 Views: 5,406 |
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Dear HS, ❝ Your suggestions are quite reasonable, but metabolites may make the life of the analyst quite miserable... 100 % agree. My answer was much too short there, and metabolites add more risks of interferences. But this is not specific to multiple co-administered drugs and it can happen with just one analyte. My worry is not really with UV or fluo detection, rather with MS. Many labs just forget that LC/MS/MS starts with LC and go for very short run times with a reduced sample preparation. If you don't separate your metabolite chromatographically you may end with real trouble, such as in the case of in-source dissociation of acyl-glucuronides or N-oxydes. And the interference in that case will be with the parent compound itself, not with a co-administered drug. Regards Ohlbe |
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Helmut ★★★ Vienna, Austria, 2007-12-16 14:50 (6766 d 04:34 ago) @ Ohlbe Posting: # 1384 Views: 5,515 |
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Dear Ohlbe! ❝ My worry is not really with UV or fluo detection, rather with MS. OK, but UV and fluorescence spectra change just a little by common phase-1 metabolic pathways (e.g. demethylation, hydroxylation, …). Unless you check peak purity (DAD) of ‘unknown samples’ you probably simply don’t get the interference. ❝ Many labs just forget that LC/MS/MS starts with LC and go for very short run times with a reduced sample preparation. Yes this is a major problem. The first-shot-approach of protein-precipitation-and-inject IMHO is just b……shit (sorry). Many analysts were caught in this pitfall set by vendors of LC/MS-MS systems. In my experience LC/MS-MS calls for more sophisticiated sample preparation (getting ‘cleaner’ extracts) – not less. ❝ If you don't separate your metabolite chromatographically you may end with real trouble,... Sure, but still the metabolite(s) may either not be available commercially, or even worse not possible to synthetize. At least if the drug is excreted renally, extraction form urine may be an option… — 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 |
Ing. Helmut Schütz