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Dear All:
I have several problems on in vivo metabolite ID and appreciated someone can give me some advises.
1. I have encountered several compounds which their sulfation metabolites in urine, bile and plasma all have longer retention time than parent drug in the RP-HPLC system. Is it possible? If possible, can anyone tell me in what cases this phenomenon will happen? Our compounds are not with high polarity and is just a weak base. The mobile phase is ACN: H2O with 0.1% Formic acid and the column is just a RP C18 column.
2. We always found that in bile, the metabolites retention time always shifted backward to several minutes (about 10-20 min shifted in a 90 min run method) compared with urine and plsma. I know that it maybe due to the bile salt, but I have few experiences on how to resolve this problem. Can anyone help me?
Thanks!
Best regards,
Jian
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Hi, Jian,
1. It is possiple the more polar metabolites give longer retention time on RP-HPLC. The retention time depends on both polarity and molecular size.
2. You can try to use solid phase extraction (SPE) to decrease the matrix effects.
Best,
Shan
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Hi, Shan:
Thanks for your advise. Yes! SPE is a good choice to remove the matrix effects. But we don't know if there are some metabolites with high polarity can be eluted as well during the process. If it happened, we may miss important metabolites and get a incomplete metabolic profiles. How to resolve this problem?
Jian
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The following message was posted to: PharmPK
Hi jian
1. There are several cases where metabolites can elute after Parent drug, e.g. "Acidic metabolites can elute latter in acidic mobile phase"
"N-Oxides generally more nonpolar than parent and have higher retention time"
2. It seems that pH of the sample medium is creating the chromatographic disturbance! I believe you are using ACN crash method to precipitate the protein in the sample, after protein precipitation/dilution dry the sample to dryness and reconstitute the sample with your Mobile Phase (Initial composition of Mobile phase)/Use a buffer to provide stable pH condition to the sample
Hope this will solve your problem
Regards
Rajanikanta Sahu
Research Scientist
Saiadvantium Pharma Ltd
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I am recently doing my metabolism experiment with some nonpolar alkaloids (pyridine alkaloid). I do see metabolites in bile sample with retention time forward_shift (around 10min) compared to the parent compounds.
I also have a question that in my blank sample (without any treatment) I find several peaks distributing at the very nonpolar range, where the ACN is almost 90%. This also happened in my treated sample. The UV wavelenth 230nm resulted in very high peaks.
Did anyone have similar experience?
I treated the bile sample with Ethylacetate, and then centrifuge to get the upper layer (Ethylacetate phase).
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Hi, Jian,
I guess that you run RP-HPLC-MS/MS for the full metabolic profile, right?
You can try to use liquid-liquid extraction (LLE), then evaporate the organic solvent and reconstitute with methanol. LLE would give you lower recovery than SPE but more complete metabolites.
You can also try to add methanol or acetonitrile to the matrix and centrifuge, evaporate supernatant by Speed Vac, then reconstitute.
I hope this can help.
Shan
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