I am working on the determination of 5-fluorouracil and its degradation impurities (Barbituric acid, 5-hydroxyuracil, and Urea). I have to assay these compounds by the same method. The method that I found in the Pharmacopoeia for the determination of impurities (apart from urea) is reverse phase liquid chromatography. However, if I have understood correctly, reverse-phase HPLC is for non-polar molecules, and for polar molecules the normal-phase HPLC method or the HILIC method should be preferred.
I have several questions on this topic:
1) Why is the reversed phase used in most scientific articles and in the pharmacopoeia but not the HILIC method or normal phase HPLC. I have searched in vain but I can't find anything as an explanation. I even read in an article that 5-fluorouracil cannot be assayed by reverse phase HPLC because it is an extremely polar molecule. So i'm a bit lost.
2) Why is urea not measured by this method? And is this molecule measurable by the reverse phase HPLC with a UV detector? Because I see in certain articles written "Urea does not absorb in the UV" and then in the pharmacopoeia I see that it can be assayed by HPLC at a wavelength of 190 nm with a UV detector.
3) What method would you recommend for measuring all these molecules? I was thinking of using the HILIC method, but I'm a bit lost, I'm still a student and it's the first time I'm developing a method so I'm afraid of doing it wrong.
It would help me a lot if someone could answer my questions,
Thanks in advance.
Have a nice day.