We have 3 Non-polar compounds to be tested on rats. Their metabolites in the plasma and urine are polar derivatives. We need to quantify the 3 non-polar compounds and the 3 polar metabolites (6 compounds in one analyte). However, the detection limits of all the six compounds are significantly less. Can you suggest a method to quantify all the 6 compounds together?
You would have to use IPA as a mobile phase, since it has both polar and non-polar qualities. You will also have to use a 'gradient' method in order to separate the polar and non-polar constituants.
Without knowing the unique properties of the compounds such as LogP values, pKa, and ionic nature...it is hard to say a spot-on methodology. Since you are targeting the simultaneous extraction of the hydrophobic and polar compounds in a single pot, you may perform SPE (bi-modal or sequential) as sample pretreatment. Protein precipitation plates may also be beneficial at this stage. Extracted compounds should be analyzed by using a mix-mode column or polar embedded RP columns (e.g. aqua-c18) depending upon the molecule characteristic as I indicated above. It is typically hard work to combine two distinct-natured compounds into a single analysis platform. MS must be employed for sensitive analysis and besides chromatography, it is also arduous to adjust the best conditions for each compound.
Definitely, we should know more...We may narrow down the subject if you share some additional information regarding the molecules...
OK, I fail to see the issue. This is not an uncommon occurrence. You can separate both polar and non-polar compounds in the same run on both HPLC and GC. Are you worried about the sample prep? You may need to perform multiple extractions at differing pH or under differing conditions and then combine extracts.
Mark Krause Yes! I did not articulate the question and description properly. The problem we face is the detection limit is too low. We are working on cyclic hydrocarbon compounds. In every literature, they identified either the compound (non-polar) or its metabolite (polar). One or two articles have reported quantifying both but we don't get the result following their methods.
PS : The instruments, and sample prep cartridges are in good form as other results are fine
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