USP Method for Hydrocortisone Assay

[michaelbarnes42]

The USP method requires a resolution of NLT 9.0 between the hydrocortisone and propylparaben peaks.

The column is a C18 L1 Column 4.6 mm x150 mm, 5 µm packing

the mobile phase is 50:25:25 Water:Acetonitrile:Methanol

Using the above mobile phase I have a resolution of 8.3

The best resolution I am getting is 8.8, I have changed the mobile phase to be 57.5:17.5:25

Anyone else have any tips for improving resolution?

[KM-USA]

Go to smaller 3µ particle size. Minimize your tubing diameters and lengths. Have you also tried other C18 L1 columns, there are a ton that fall into the L1 capacity. Have you tried raising column temperature 10C up, or lowering 10C down, see if that helps or hurts your resolution; USP procedure may be so old that column temperature is not detailed, like column heaters not so common back then.

May I say that NLt 9.0 is an incredibly large amount of resolution !!! Go USP !!!

[michaelbarnes42]

Thanks for the advice,

I have been trying different columns, just to see if I have a bad column, I have used 3 different 4.6 mm x150 mm x 5.0 µm L1 Columns from 2 different manufacturers, the 8.8 resolution has been the best I have gotten so far. I don't have a 3µm column, I think I might get one, the USP calls for a 5 µm, but <621> does allow for the use of different particle size.

You are correct, there is no specified temperature. I will try the column temperature suggestion, I am currently running at 25C. I just run all my methods at 25C that don't have a specified temperature. I will let you know what happens

Yeah, NLT 9.0 is a very large resolution spec. I actually have run this in the past, I found an old run that I had with 9.4 resolution. The peak widths are exactly the same as they were last time; however, the peaks are eluting slightly earlier and the peaks are closer together.

[nms]

I will go for one by one following step to increase resolution.

1)Reduce injection volume.
2)Prepare a new mobile phase and filter it and sonicate it.
3)Reinstall column to check to reduce the dead volume.
4)As KM suggested minimize tubing diameters and lengths.
5)Resolution mainly depends on peak sharpness and difference in retention time. Dirty frit gives broad peak. By reversing the column and cleaning it with either Methanol, Acetonitrile or any solvent which has been used in the previous analysis.
6)Still u are not getting resolution change to new column as some time column loose its efficiency and peak become broad.
6)Try to go for column with amide linkage or any other type of L1 column.
7)If ambient temperature written, you can go up to 30 degree centigrade. Temperature helps to sharpen the peak but at same time decrease retention time.
8)Play with Mobile phase composition as described adjusting USP method. As u have already done by increasing the water content as per USP, some time decreasing the water also make less retention difference but sharpen the peak and increase the resolution.
9)You can have a option for reducing the particle size, diameter of the column.
10) You can try and see to reduce Methanol as per USP criteria, as methanol has more viscosity it may broaden the peak.
As USP can achieve the resolution 9.0, you will definitely reach it. If someone has more ideas please give suggestions.

Thanks,
Nawaz

[michaelbarnes42]

I will go for one by one following step to increase resolution.

1)Reduce injection volume.
2)Prepare a new mobile phase and filter it and sonicate it.
3)Reinstall column to check to reduce the dead volume.
4)As KM suggested minimize tubing diameters and lengths.
5)Resolution mainly depends on peak sharpness and difference in retention time. Dirty frit gives broad peak. By reversing the column and cleaning it with either Methanol, Acetonitrile or any solvent which has been used in the previous analysis.
6)Still u are not getting resolution change to new column as some time column loose its efficiency and peak become broad.
6)Try to go for column with amide linkage or any other type of L1 column.
7)If ambient temperature written, you can go up to 30 degree centigrade. Temperature helps to sharpen the peak but at same time decrease retention time.
8)Play with Mobile phase composition as described adjusting USP method. As u have already done by increasing the water content as per USP, some time decreasing the water also make less retention difference but sharpen the peak and increase the resolution.
9)You can have a option for reducing the particle size, diameter of the column.
10) You can try and see to reduce Methanol as per USP criteria, as methanol has more viscosity it may broaden the peak.
As USP can achieve the resolution 9.0, you will definitely reach it. If someone has more ideas please give suggestions.

Thanks,
Nawaz

this is a very nice list of things to do.

1) I will try this, Injection volume is 10 µL, I will try 5 µL first to see if it has any effect.
2) I have done this several times, it was the first thing I did. But definitely good advice.
3) I didn't even consider dead volume being an issue, I did reinstall the column several times and switched it out, but just dumb luck that I tried this one.
4) I have already minimized all the tubing as much as I can.
5) I have actually reversed the column already and flushed with 100% Acetonitrile and 90:10 Acetonitrile:Water. This actually improved my resolution, but not enough, and further backflushing has not improved it at all.
6) I am currently using a Supelco Discovery C18 4.6 mm x 150 mm x 5 µm
7) Good advice, didn't realize I can go to 30C
8) I played with the mobile phase composition the more water and less acetonitrile gave me the best resolution, but I didn't try reducing the water from the 50:25:25 listed in the USP. I can try this.
9) I have a waters symmetry C18 3.9mm x 150 mm x 5 µm, I can try using the smaller diameter column to try to increase the resolution.
10) I may be misreading the USP, I thought it allowed for the change of only one of the minor components in a ternary mixture. If it allows the change of both minor components, I would change the methanol, but I can give the methanol change a try anyway, maybe do a 55:25:20 mobile phase.


Thanks, for this wonderful advice, I will give it a try.

[michaelbarnes42]

So, here is what I ended up with 3.9 mm x 150 mm x 3 µm column with 57.5:17.5:25 mobile phase, and a 0.7 mL/min flow rate, it is giving a resolution of 13.4.

Thanks for all the help.

[KM-USA]

Great.

I didn't realize that your QA would let you just make a change as listed in <621> without running some sort of studies, why I suggested temperature changes as many USP procedures don't detail that for HPLC. Our QA would makes us run studies of some kind, but we rarely run USP procedures, we have our own finished product validated methods, and QA says that <621> allowable changes only pertains to USP procedures, not house-validated procedures. Any one know that for sure?

[michaelbarnes42]

Great.

I didn't realize that your QA would let you just make a change as listed in <621> without running some sort of studies, why I suggested temperature changes as many USP procedures don't detail that for HPLC. Our QA would makes us run studies of some kind, but we rarely run USP procedures, we have our own finished product validated methods, and QA says that <621> allowable changes only pertains to USP procedures, not house-validated procedures. Any one know that for sure?

That is how I have always looked at it, but I am running the assay using the USP hydrocortisone Monograph (I am running it on our Raw Material Hydrocortisone not the finished product) it isn't an in-house validated method, if it were I wouldn't be able to make the changes without re-validating.

I have also looked at it as you need to do a verification of the USP method in order to use it. I am by no means an expert on the USP.

[qfiviera]

Did you looked up at USP page? There is a link which goes to Chromatography columns, and this tells you which column they used to develop the assay (brand and type). If you don´t have it, then check the characteristics, like type of silica, particle size, pore size, endcapping, etc, and try to use a similar. There´s a PDF from ACE that compares a lot of C18 columns and is really a useful quick guide.
But, if you don´t succeed, 8.8 is near USP asks, so make an FDE and stop lossing time. I agree, 9 is quite a large resolution.