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Hi all
I have a question on Very short run time (~2 min) employed in LC-MS-MS for evaluation of bioequivalence.
I have observed Most Glucuronide metabolites are fragmented back to the parent in source.
In a 2 min or 1.5 min Isocratic method. Almost everything elutes at same Rt.
In study samples, different metabolites(including glucuronides) are also expected.
For example if a glucuronide metabolite is eluting at same Rt as that of parent drug, the probability of in-source fragmentation of Glu-metabolite to parent drug(Analyte) will lead to over estimation of the analyte(Drug)
Although we are comparing the data with reference Product, still we are not evaluating the real concentration of analyte
I request all to discuss whether, very short run time LC-MS-MS based method is good or bad for science.
Rajanikanta Sahu (M.Pharm)
Research Scientist
Saiadvantium Pharma Ltd
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Rajanikanta,
Good or bad is a relative term, does it meet your needs and is it prudent for your analytical situation may be a better question
If your aim is to get a simple and rapid quantification then your analytical method should separate out your analyte from other materials in your mixture, if this can be done in 2 minutes then you are good (in your sense) if not then you must adequately separate your analye from the mixture.
Alternately, if you are using a ballistic method you can employ differential MRM methods to separate out your analytes, depending on your instrumentation, this will either be possible or not. In certain cases, dependent upon your scan speed, you will either be able to build a method for up to 10 analytes in a single injection or not. There are some good literature references and instrument company publications that can assist you with this method.
I have always held that analytical separation is a better method than one that is ballistic in nature. Spend a little more time and be sure than to charge forward and have to back track to find the answer.
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Rajanikanta,
In your case you do need to resolve the glucuronide from the aglycone. Also, you need to confirm the stability of the glucuronide and ascertain that it does not degrade back into the aglycone during sample storage, sample thawing or while sample processing. If the glucuronide is very unstable you want to adjust the pH of the sample during sample collection to stabilize/minimize degradation.
We have had some experience in resolving glucuronide from the aglycone using a short LC/MS/MS method (column choice plays a big role) but it may not always be possible. In your case it seems like the glucuronide and the aglycone are not resolved and that is definitely a "bad case".
Best Regards,
Sandeepraj Pusalkar
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Dear Rajanikanta,
The focus should not be so much on the run time of the analysis than on its performance. There are now technologies (instruments, columns) that actually allow to do decent chromatography in a short time frame. On the other hand ballistic / crash and shoot approaches are, I believe, a generally bad idea for quantitative assays in biological matrices; 1.5-2 minutes LC run on classical instruments / columns generally fall under this category. One risk with specific MS methods (MRM) is that they give clean peaks above low background noise, and hence a false sense of confidence, even when all sort of crud co-elutes. Such method may suffer from matrix effect (e.g. ion suppression) and, as you rightly observe, from interference from unstable metabolites, if no separation exist these may be mistaken for the analyte of interest.
Rather than absolute length of time of the method you should look at capacity factor for your analytes (when using isocratic methods) or anyway by how much they are retained compared to dead volume. If you know you may have glucuronides (or other metabolites, co-drugs, metabolites thereof ...) in your samples and have standards available, check how these may interfere with your analysis, in terms of effect on response as well as specificity. You may also evaluate how matrix components (matrix, formulating agents, stabilizers ...) may co-elute with your analyte and cause matrix effects.
The more specific your sample extraction is (glucuronides, for example, are generally easily separated from parent drugs by SPE or LLE), the less strain you may put on your chromatography, but good LC remains essential for good LC-MS-MS.
Best regards
--
Patrice Larger
Pharmacokinetics, Metabolism & Dynamics
Translational Sciences & Pharmacokinetics Dpt
ROTTAPHARM | MADAUS patrice.larger.-at-.rottapharm.com
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The following message was posted to: PharmPK
Dear Rajanikanta,
First of all I would like you say thanks for dropping such a wonderful topic.
Typically, in BA/BE we generally perform generic drug analysis. You will get all information such as metabolites, active metabolites, solubility, pKa, Log P, Log D values from internet. Hence during chromatographic method development you can focus how and what to separate.
In case of glucuronides, which are very unstable and can easily be separated from analyte using SPE elution techniques (Focus should be on washing steps) and LLE techniques. We can also get an idea about this during matrix effect experiments (Both qualitative and quantitative).
But yes, still some question remains. Short run times some time may leads to over estimation of analyte. Hence my recommendation is to use gradient techniques so that both analyte and metabolite are well resolved. If you want short run time, advanced instrumentations should be used.
Regards,
S.Basu
Principal Scientist (DMPK & TOX)
Sai Advantium Pharma Ltd.,
E-Mail : sudipta.b.aaa.saiadvantium.com
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