Fosfomycin assay by HPLC with DAD detector

[greyfield]

Hi everyone,

I'm on the way to develop the method of determination of fosfomycin calcium in dissolution test. Some methods such as UV spectroscopy (using reaction of phosphate group with molybdate) and volumetric titration (using reaction of diol group after transforming to the open ring form) show unexpected results. HPLC method in USP uses differential refractometer detector with amino column but I have only DAD detector and cyano column. I am trying with cyano column and some mobile phases containing phosphate buffer/ion-pair mobile phase with TEA.

I am happy to receive valuable experiences of yours!!!
Thanks so much!

[Vlad Orlovsky]

Fosfomcin is not UV active and you need to use ELSD, CAD, MS, RI or indirect detection. ID is a mess and would not recommend it. You also will have hard time retaining it on RP or Cyano column. You can try to use IP reagent in combination with indirect detection. Something like cetylpyridinium

Here are two methods we developed for our customer:
https://helixchrom.com/compounds/fosfom ... ode-column

Let me know if you have questions.

[greyfield]

Thank you very much!
I tried with the following program:
- DAD 200 nm (because fosfomycin hasnot UV absorption, so I choose short wavelength).
- Column: CN, 250 x 4,6 mm, 5 mcm
- MP: 10,89 g/L potassium dihydrogenphosphate (pH ~ 4,5)
- Sample solution: 1 mg/ml with injection 100 mcl
When using water as diluent, the accuracy is 100%, but peak didnot appear with diluent containing acetate, chloride or phosphate (ions in dissolution test).

And when using MP containg 10,89 g/L potassium dihydrogenphosphate and 3 ml/L triethylamine (pH ~ 6,2), peak didnot appear, too

I dont unterstand the reason TT ((

[Vlad Orlovsky]

I think that you are seeing a change in refractive index when you inject 100 uL. If you dilute sample with mobile phase it will be less pronounced. I would not rely on UV in this case. Any minor impurity with high UV activity will throw you off.