Chromatography Forum

Hello,
I am looking into methods for detecting Maraviroc by HPLC with a UV detector. There is a paper that lists an interesting looking method (A validated high-performance liquid chromatography-ultraviolet method for quantification of the CCR5 inhibitor maraviroc in plasma of HIV-infected patients) but I have some concerns with it that I wanted addressed. It appears that the authors are monitoring maraviroc at 193nm but they are running the sample in acetonitrile that has a UV cutoff of 190nm. This seems to me to be way to close to the cutoff and results could be impacted.

I wanted to get those more experienced with this type of analysis to tell me if my concerns are reasonable or not. The challenge is that maraviroc doesn't have much of a uv signal above 220nm (see fig 5 of the paper). Would it be a better choice to shift the detection wavelength up into the 200 range? Thank you very much for the help.

UV cutoff is sort of arbitrary; usually defined as the wavelength where the absorbance in a 1-cm cell is 1.0. What you'll see 3 nm up from there depends on the shape of the spectrum (steep or shallow drop-off) and the bandpass of your monochromator.

If you run at a longer wavelength, you'll have a lower background (so, less noise) but a lower signal as well. You would have to try to see who wins. That said, I suspect the authors of that paper already did the comparison. If it really was validated, information on the wavelenght dependence should be somewhere in the robustness data.

If your detector is amenable to it (and few are these days), you may want to consider nitrogen sparging it.

Probably more helpful - run low UV scans on your ACN bottles from different lots and cherry pick those with the lowest absorbances in the low UV range for your mobile phase. You might be surprised at how readily visible lot to lot variation in low UV absorbance is, especially if you use a 5 or 10 CM cell.

The detector wavelength accuracy is probably +/- 2 to 5 nm.
So…. 193 might mean 198 nm. Besides it is a band covering some nanometers and not a precise 193.00 nm ray. However, I don’t think the wavelength is lower than 190 due to specific light source capability.
I do a couple of tests as Tom suggested and see what suits me best – if I were you.

Best Regards