I am standardizing a UHPLC method for separation of thioguanine phosphate metabolites. For this, I want to use 5-Bromouracil (5-BU) as an internal standard, however I am unable to prepare solution of higher molarity (1M or 0.5M) as the solubility specification for 5-BU is 'less than 1 mg/mL at 75° F' in water.
Please suggest solvents that would allow higher solubility of 5-BU?
You don't need high concentration of the internal standard, as the internal standard should be comparable to the concentration of your analytes. However, your stock solution concentration should be much higher, but I doubt you need more than 1 mg/mL. If you need more, use methanol or acetonitrile. Also the detection should be at the maximum wavelength in UV ~300 nm.
Hi Andrei Blasko ,
Thank you for your answer.
Like you rightly said I need a higher stock concentration, which I have been trying to prepare in water. Presently, I am adding 150ul of the prepared 5-BU stock (concentration - 314uM) to the samples. Final volume of the prepared sample is 1000ul which ends up diluting the IS 10 times. I have set detection wavelength at 280nm and injection volume is 5ul.
I will prepare stock concentration as per your suggestions and try out the experiment.
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