Changing RT of Cefuroxime during HPLC Run

[ghie malig]

Thanks to those who replied on my query regarding cefuroxime stability. I have a new problem though:

1. We run system suitability first before proceeding with any validation run and in the result, the RT of cefuroxime is repeatable with RSD of less than 2.
2. During the validation run, we noticed a change in RT for both cefuroxime and IS sulfamethoxazole.
3. RT changed from 6.2 to 6.6 minutes for Cefuroxime and 7.5 to 8.0 minutes for sulfamethoxazole.

M.P.: MeOH/ 0.005 M KH2PO4 buffer (pH 3.6), 30:70
Sample treament: Analytes are in plasma, precipitated with Acetonitrile and back-extracted with DCM/Hexane (1:1)
Stock and Working Solutions are dissolved in 0.01M KH2PO4 buffer, pH 6.5

Is this change in RT significant? Is there any part in the sample preparation or Mobile Phase that can influence the change in RT?

I will appreciate any feedback!

Thank you.
Ghie Malig

[Srinivas SK]

Hello

Several factors can influence RT : flow rate, mp composition, column chemistry, temperature, injection technique, to name some. Sample prep won't usually affect RT significantly, unless your sample prep method is either extracting the wrong analyte or is changing the analyte structurally, (usually because of changes in pH )

However, assuming this is not happening and since you're using an internal standard, keep an eye on the relative retention time of your analyte w.r.t. the IS. If the RRT is repeatable within limits of RSD and if your system suitability is otherwise OK, don't worry too much about the occasional shift in the analyte's uncorrected RT - provided of course, the relative retention time remains constant.

Hope this is useful.

[amaryl]

Thanks to those who replied on my query regarding cefuroxime stability. I have a new problem though:

1. We run system suitability first before proceeding with any validation run and in the result, the RT of cefuroxime is repeatable with RSD of less than 2.
2. During the validation run, we noticed a change in RT for both cefuroxime and IS sulfamethoxazole.
3. RT changed from 6.2 to 6.6 minutes for Cefuroxime and 7.5 to 8.0 minutes for sulfamethoxazole.

M.P.: MeOH/ 0.005 M KH2PO4 buffer (pH 3.6), 30:70
Sample treament: Analytes are in plasma, precipitated with Acetonitrile and back-extracted with DCM/Hexane (1:1)
Stock and Working Solutions are dissolved in 0.01M KH2PO4 buffer, pH 6.5

Is this change in RT significant? Is there any part in the sample preparation or Mobile Phase that can influence the change in RT?

I will appreciate any feedback!

Thank you.
Ghie Malig

Hello ghie

Good to see you are proceeding. Isn't the buffer molarity of your mobile phase very low 0.005 M or else 5 mM ...check this. I find it strange.

As stated by Srinivasan sir,

A variation in RT reflects the chromatographic parameters such as mobile phase composition (% B, pH ), temperature, flow rate, use of different column, solvents chemicals, different HPLC equipment (for ruggedness) affecting your separation. RT is a parameter to qualitative identification of analyte. Analyzing a single analyte may provide you wider range of varriation in RT but your method is less rugged and robust. A variation of +/- 0.1 min makes your method more robust and easily transferable to other labs (rugged) with reproducible results.

In case of impurity profile and stability studies a more strict control in variation of RT is needed.


Refer LCGC article over Retention time changes.

If you don't find this article let me know I will send you the link.

Good Luck!

Regards,

Amaryl.

[ghie malig]

Thanks Amaryl! The low molarity of the buffer solution in my mobile phase also caught my attention. I'll raise it up and see if there would be improvement with the shifting of RT. I really appreciate your feedback.

Best regards,
Ghie Malig

[amaryl]

Hello ghie,

Good Morning! Here is your link over retention time changes.

http://www.lcgcmag.com/lcgc/data/articl ... rticle.pdf


Good Luck.

Regards,

Amaryl.

[Okkie]

do you stir your mobile phase?
Once I had the problem, the mobil phase separated (invisible)
and so my retentiontimes shifted, run by run.

[ghie malig]

To Amaryl and to all those who replied to my query!

Thank you so much for answering all my queries. It will really be of help. Our next project will be beta blockers atenolol, propranolol and metoprolol. will also be developing method for diltiazem. All in plasma solutions. So I hope you will be able to help me again. I hope i'll also be of help to you in the future.Thank you!

Ghie Malig