Chromatography Forum

Hi everybody,

I am facing again a problem,this time i spiked urine sample with orotic acid (10mM) and extracted the acidified and NaCl saturated urine sample with ethylacetate thrice but the recovery of orotic acid is very poor.In some urine samples it is negligible. What could be the reason of this poor recovery.If dilution effect plays any role here.

Thanks
Chandra

The polarity and water solubility of the analyte resulted in poor recovery, the better sample prep. method would be solid phase extraction using strong anion exchange sorbent which provides higher recovery and cleaner extract as well. If you are interested in SPE I can suggest you the SPE column and procedure.

Hi everybody,

I am facing again a problem,this time i spiked urine sample with orotic acid (10mM) and extracted the acidified and NaCl saturated urine sample with ethylacetate thrice but the recovery of orotic acid is very poor.In some urine samples it is negligible. What could be the reason of this poor recovery.If dilution effect plays any role here.

Thanks
Chandra

Yes please ....

Strong anion exchange sorbent, such as quaternary amine can be used to extract organic acids including orotic acid from urine or other matrices.

SPE cartridges: https://www.unitedchem.com/, UCT part# CHQAX123, or equivalent.

SPE procedure as a starting point:
1. Mix 1 mL urine sample with 1 mL pH 8 buffer, make sure sample pH > 7 to charge the acid.
2. Condition SPE cartridge (CHQAX123) with 3 mL methanol and 3 mL water.
3. Load sample at about 1 mL/min.
4. Wash the SPE cartridge with 2 mL 1:1 methanol:water, followed by 2 mL n-hexane.
5. Dry the cartridge by applying full vacuum for 1 min.
6. Elute the retained analyte with 2 aliquots of 1 mL of 2% formic acid in methanol or acetonitrile.
7. Evaporate the extract to dryness, reconstitute in mobile phase for LC analysis; or derivatized with 50 uL of BSTFA or MSTFA (UCT part#: SBSTFA-1-1, or SMSTFA-1-1) and 50 uL of acetonitrile for GC/MS analysis.

Let me know if you have additional questions.