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By Anonymous on Monday, June 28, 2004 - 10:26 pm:
Dear All,
1.We would like to know about the MS/MS of isomeric compounds. Is the MS/MS pattern is similar for isomeric compounds or different?.
2.In the case of Quantification of Drug by LC-MS/MS, by MRM mode as per theory onely one peak detection is possible (based on parent mass and fragment mass), suppose I get more than one peak is it means that it is due to Isomers or something else?
(we confirmed that the extra peak is not eluting from column at that particular RT).
Expecting your suggestion.
Advance thanks...
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By MG on Tuesday, June 29, 2004 - 09:06 am:
1. That depends on what type of isomer, and how the molecule fragments. If they have the same product ions, then you must chromatographically separate them if you want to quantitate them separately.
2. It is quite common to use only one MRM transition to quantitate a compound. Depending on the level of confirmation needed, I have seen some methods use more than one per compound.
Regarding the extra peak, I assume you mean you are seeing two chromatographic peaks for your MRM transition, one for your drug, and one for something else? The extra peak could be an isomer. If you are quantitating in a biological matrix, it is possible to have something completely unrelated that just happens to give the same precursor and product, because there are so many compounds in a biological sample. As long as they are chromatographically separated and your chromatography is stable, you should be fine, as long as your QC's and blanks pass your method criteria.
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By Anonymous on Tuesday, June 29, 2004 - 08:03 pm:
Dear MG,
Thanks for your suggestion.
Further we would like to hear your suggestion regarding the following points,
1. In the case of Quantification by LCMS, in the absence of drug (drug may degrade to metabolite)how can we detect the unknown metabolite without knowing the Mass number?
2.As the metabolite Mass number is unknown, in LCMS we have to scan for the full scan, where the sensitivity is very less compared to MRM mode. In that case how can we detect the mass number of the metabolite present in biological sample at ng level.
Advance thanks,
-------------------------------------------------------------------------------------------------------
By MG on Wednesday, June 30, 2004 - 06:43 am:
Sorry, I don't do that type of work. From things I've read, there are certain metabolites that are expected, e.g. hydroxylation, glucuronidation, etc. In addition to full scan, people use precursor ion and neutral loss scans to look for them, based on previous knowledge (or assumptions?) of MS/MS fragments or neutral losses observed from these type of metabolites. Unfortunately, none of these will be as sensitive as MRM. At least for AB/Sciex instruments, there is a software plugin you can buy, that is supposed to assist in looking for metabolites.
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By Anonymous on Wednesday, June 30, 2004 - 08:07 pm:
Dear MG,
Thanks for your suggestion, we have to think about the possibility of that software from Applied Biosystem.
Thanks
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By Kevin on Friday, July 2, 2004 - 04:36 pm:
Dear Anonymous,
Are you expecting one particular metabolite to be formed? If not I would suggest first trying the full scan to see what metabolites, if any, you can detect. Include a blank matrix analysis and then background substract to get the best picture of any metabolites that might be present. Next you can gain a little more sensitivity by running a precursor scan, looking for the major product ion produced from your parent compound. This might reveal additional metabolites that you didnt see in the full scan. Ultimately you should be able to use this information to use MRM detection on a specific metabolite(s).
A better bet if you have access to an ion-trap or time-of-flight instrument would be to first run a full scan on one of these instruments. You will have better sensitivity in full-scan mode and be better able to identify any putative metabolites that form.
Dear All,
1.We would like to know about the MS/MS of isomeric compounds. Is the MS/MS pattern is similar for isomeric compounds or different?.
2.In the case of Quantification of Drug by LC-MS/MS, by MRM mode as per theory onely one peak detection is possible (based on parent mass and fragment mass), suppose I get more than one peak is it means that it is due to Isomers or something else?
(we confirmed that the extra peak is not eluting from column at that particular RT).
Expecting your suggestion.
Advance thanks...
-------------------------------------------------------------------------------------------------------
By MG on Tuesday, June 29, 2004 - 09:06 am:
1. That depends on what type of isomer, and how the molecule fragments. If they have the same product ions, then you must chromatographically separate them if you want to quantitate them separately.
2. It is quite common to use only one MRM transition to quantitate a compound. Depending on the level of confirmation needed, I have seen some methods use more than one per compound.
Regarding the extra peak, I assume you mean you are seeing two chromatographic peaks for your MRM transition, one for your drug, and one for something else? The extra peak could be an isomer. If you are quantitating in a biological matrix, it is possible to have something completely unrelated that just happens to give the same precursor and product, because there are so many compounds in a biological sample. As long as they are chromatographically separated and your chromatography is stable, you should be fine, as long as your QC's and blanks pass your method criteria.
-------------------------------------------------------------------------------------------------------
By Anonymous on Tuesday, June 29, 2004 - 08:03 pm:
Dear MG,
Thanks for your suggestion.
Further we would like to hear your suggestion regarding the following points,
1. In the case of Quantification by LCMS, in the absence of drug (drug may degrade to metabolite)how can we detect the unknown metabolite without knowing the Mass number?
2.As the metabolite Mass number is unknown, in LCMS we have to scan for the full scan, where the sensitivity is very less compared to MRM mode. In that case how can we detect the mass number of the metabolite present in biological sample at ng level.
Advance thanks,
-------------------------------------------------------------------------------------------------------
By MG on Wednesday, June 30, 2004 - 06:43 am:
Sorry, I don't do that type of work. From things I've read, there are certain metabolites that are expected, e.g. hydroxylation, glucuronidation, etc. In addition to full scan, people use precursor ion and neutral loss scans to look for them, based on previous knowledge (or assumptions?) of MS/MS fragments or neutral losses observed from these type of metabolites. Unfortunately, none of these will be as sensitive as MRM. At least for AB/Sciex instruments, there is a software plugin you can buy, that is supposed to assist in looking for metabolites.
-------------------------------------------------------------------------------------------------------
By Anonymous on Wednesday, June 30, 2004 - 08:07 pm:
Dear MG,
Thanks for your suggestion, we have to think about the possibility of that software from Applied Biosystem.
Thanks
-------------------------------------------------------------------------------------------------------
By Kevin on Friday, July 2, 2004 - 04:36 pm:
Dear Anonymous,
Are you expecting one particular metabolite to be formed? If not I would suggest first trying the full scan to see what metabolites, if any, you can detect. Include a blank matrix analysis and then background substract to get the best picture of any metabolites that might be present. Next you can gain a little more sensitivity by running a precursor scan, looking for the major product ion produced from your parent compound. This might reveal additional metabolites that you didnt see in the full scan. Ultimately you should be able to use this information to use MRM detection on a specific metabolite(s).
A better bet if you have access to an ion-trap or time-of-flight instrument would be to first run a full scan on one of these instruments. You will have better sensitivity in full-scan mode and be better able to identify any putative metabolites that form.
