HPLC assay for Neomycin sulfate

[Nay Win Kyaw]

Hello everyone,
My lab don't have HPLC with ELSD detector and I found that some researcher use HPLC - ELSD for Neomycin sulfate assay. And also, don't have ion exchange chromatrography with PAD detector.
Now , I use the following condition for Neomycin sulfate assay
1. Mobile phase - 84% 0.2 mol trifluoro acetic acid in water and 16% Acetonitrile
2. Flow rate - 0.7 mL/min
3. Detector - HPLC with Refractive index detector
I already tried the different ratio of mobile phase (50:50, 60:40, 70:30, 80:20 and 90:10 of 0.2 mol Trifluoro acetic acid and Acetronitrile). Eventhough, I got the sharp peaks, I can't separate Neomycin C and B and they are still co-elute. Please someone, could you please suggest me about that? Should I change the condition or use derivatization method because of the problem of lacking chromophore in Neomycin sulfate.? May I kindly request to help and advice me.Thank you in advance.

[mattmullaney]

Good Morning,

If you do not have ELSD, CAD, MS or Integrated Pulsed Amperometric Detection, you could try pre-column derivatization and NP separation:

Journal of Chromatography A, Volume 369, 1986, Pages 105-115, "Derivatization of primary amines by 2-naphthalenesulfonyl chloride for high-performance liquid chromatographic assay of neomycin sulfate", by Kiyoshi Tsuji and Kevin M. Jenkins.

http://www.sciencedirect.com/science/ar ... 730090102X

[Nay Win Kyaw]

Thanks so much Matt for your kind suggestion.

[mattmullaney]

Hi again,

Let us know if you get to try the derivatization out--best wishes!

[Nay Win Kyaw]

Hi Matt,
I would like to try derivatization method also. Could you please suggest me for that?Thanks so much.

[tkubowicz]

Hello

If you think of methods with derivatization for primary amines I'd try to reagents:

-OPA o-phthalaldehyde
-FOMC Fluorenylmethoxy chloroformate
*reactions are fast and in room temperature

You will find a lot of aplications in internet (Agilent/Thermo/Waters websites plus journals)
The advantage is that you can set up all steps for reaction in sequence so everything is taking place in LC sampler which is good if you have highthroughput methods.

Regards

Tomasz Kubowicz

[mattmullaney]

Hi Nay,

Actually, the link I included is to a derivatization method for neomycin sulfate, followed by a Normal Phase separation. Thomas' suggested derivatization reactions have an advantage in that they may be used with Reverse Phase separation afterwards.

If you're interested in that paper, I may be able to get a copy form the library and send it to you if you provide an e-mail address. As noted, Thomas' proposal(s) may be easier to act on than what the paper suggests.

Please see what you think and thank you.

[Nay Win Kyaw]

Dear Matt,
I interest the way which you suggest me.May I kindly request you to give the paper which you told me.My email address is naywinkyaw.pharmacy@gmail.com. Thanks so much for your kind support and suggestion.

[Sallybeetle]

Years ago, I developed a method on Hypersil Gold column with TFA+solvent gradient for Neomycin and Gentamicin. I seem to remember that it took a 'much' stronger TFA concentration to separate Neomycin B and C, and Sisomicin from the Gentamicins.

[mattmullaney]

Hi again,

I should have time to head to the library tomorrow 18 Mar 2017...I'll see to sending the paper along.

[Nay Win Kyaw]

Thanks so much Matt for your kind support.

[Nay Win Kyaw]

Thanks so much Sally also for your kind suggestion.

[mattmullaney]

Hi Nay,

Was not feeling well this past weekend--please find the paper at your gmail account.

[Nay Win Kyaw]

No problem Matt. Thanks so much for your kind help.

[colettenc1@gmail.com]

Good afternoon. I know it's a few years after this posted but do you still have access to the referenced method? If so, will you forward it to colette.griffin@basf.com? Thank you very much.

Hi Nay,

Actually, the link I included is to a derivatization method for neomycin sulfate, followed by a Normal Phase separation. Thomas' suggested derivatization reactions have an advantage in that they may be used with Reverse Phase separation afterwards.

If you're interested in that paper, I may be able to get a copy form the library and send it to you if you provide an e-mail address. As noted, Thomas' proposal(s) may be easier to act on than what the paper suggests.

Please see what you think and thank you.