aminoglycoside antibiotic.. rp-isocratic lc.. no retention?

[alicee!]

hellow all!
i'm working on a rp-hplc method for quantitation of an aminoglycoside (lots of OH/- groups and a few NH2/3+ groups) antibiotic in my lysate. for now i've only tried running a standard.. i got a paper which used spherisorb waters ODS column.. 80angstrom.. 5 micron.. % acn, 5%methonol, with some tfa + tca in it.. its an rp-isocratic lc..

what i did was used the same stuff.. just that the column they mention wasnt' available.. so i used a normal ODS, 80 angstorm.. 5 micron.. not waters. what i see is no retention:( my std elutes with the buffer. i had the method for ELSD detection.. i tried higher amounts on RI.. i see a dose response so i suppose the std is not binding (and not not-eluting). with literature i figured this is not reversed phase binding on the column but ion-pairing.

i tired to lessen and then remove the an and methanol % so has to make more polar mobile phase.. increase tfa.. etc.. i dint see binding. m i missing out something? how do i retain my antibiiotic here!! plss suggestt.. thank you!

[Uwe Neue]

This compound is not likely to be retained by the C18, but by the silanols of the packing. Since the procedure prescribed the Spherisorb ODS 1 column, I would use that one, and nothing else. Otherwise, you are wasting your time.

[SIELC_Tech]

Here is approach to aminoglycosides:
http://www.sielc.com/compound_313.html
http://www.sielc.com/compound_003.html
http://www.sielc.com/compound_222.html

For compounds with several amino groups you can use Obelisc R column and obtain retention from 2 min to 60 minutes just by controling pH of the mobile phase. You pH should be aroubd 3.

Tylosin structure is here:
http://de.wikipedia.org/wiki/Tylosin

Contact me by email and I will send you few more examples

P.S. You can also use ion-pairing chromatography or HILIC approach

[alicee!]

Here is approach to aminoglycosides:
http://www.sielc.com/compound_313.html
http://www.sielc.com/compound_003.html
http://www.sielc.com/compound_222.html

For compounds with several amino groups you can use Obelisc R column and obtain retention from 2 min to 60 minutes just by controling pH of the mobile phase. You pH should be aroubd 3.

Tylosin structure is here:
http://de.wikipedia.org/wiki/Tylosin

Contact me by email and I will send you few more examples

P.S. You can also use ion-pairing chromatography or HILIC approach

yess.. but i'd have to order these columns.. cannot wait till they come..
besides, ssince i'm using an RI detector.. i'd have to do something isocratic.. and would any way wanna know what could be done?

spherisorb.. i couldnt find out what this column has that any other similar c18 column wouldnt have.. similarly endcapped and large surface area. does any one know if anything else is implied?
thanks!

on second thoughts.. let me mention than i'm looking for kanamycin methods!! does that help more?

[SIELC_Tech]

here what you can try on columns you might have:
1. Regular bare silica column
ACN/water/any acid or buffer=90/10/0.1 HILIC mode. Play with pH and ACN to adjust selectivity, retention and peak shape.Watch for sulubilty of buffer, can try ion-exchange on silica column at low ACN concentrations. If retention is too long reduce amount of ACN and add more buffer
2. Regular C18 or C8 column
0% ACN with sodium phosphate pH 7. You will be using residual silanols on the column to retain your amino sugar by ion-exchange mechanism on weak and limited amount of acidic sites of the column. Use buffer concentration and pH to control peak shape and retention. This might be tricky on the long run as slight variation is silanol activity/amount will cause shift in retention time (which might be dramatic)
3. Any C18 column with HFBA or other ion-pairing reagent. this might be tricky because of RI detection
4. Derivatization of amino groups to create chromophors and add hydrophobicity (benzyl chloride). This might be tricky in terms of obtaining quantitative accurate results, because you hav to push this reaction to compliton

[AA]

This is a set of general conditions that work for most of the aminoglycosides. It can be tweeked depending on the exact one(s) you are looking at. This method was originally done using an ECD for detection, there is no reason thant a RI wouldnt work if your assay amounts are high enough.

Atlantis dC18 5 um 4.6X250

0.02 M phosphate + 35 g/L sodium sulfate + +0.5g/L OSA + 15 mL/L THF pH=3

1 mL/min

[alicee!]

here what you can try on columns you might have:
1. Regular bare silica column
ACN/water/any acid or buffer=90/10/0.1 HILIC mode. Play with pH and ACN to adjust selectivity, retention and peak shape.Watch for sulubilty of buffer, can try ion-exchange on silica column at low ACN concentrations. If retention is too long reduce amount of ACN and add more buffer
2. Regular C18 or C8 column
0% ACN with sodium phosphate pH 7. You will be using residual silanols on the column to retain your amino sugar by ion-exchange mechanism on weak and limited amount of acidic sites of the column. Use buffer concentration and pH to control peak shape and retention. This might be tricky on the long run as slight variation is silanol activity/amount will cause shift in retention time (which might be dramatic)
3. Any C18 column with HFBA or other ion-pairing reagent. this might be tricky because of RI detection
4. Derivatization of amino groups to create chromophors and add hydrophobicity (benzyl chloride). This might be tricky in terms of obtaining quantitative accurate results, because you hav to push this reaction to compliton

1. u mean ion-exchange using the NH3+ group? as oh- will be present on silica right? yes i shal try that..
2. i have tried only water.. that hasn't worked.. so i doubt if phosphate buffer with pH 7 would work.. i can try that too.. what say?
somehow i feel with 80% methanol it was bound.. but then i m not sure? could u explain that? is there a possibility?
3. i m not thinking of trying that for now. shal try it if nothing works.
4. i have tried OPA derivitization.. but i would face the problem in quantitation as you say.
besides, i m planning to try an anion exchange.. my aminoglycoside is kanamycin.. so i may try anion exchange with pH 4-10 gradient.. any experiences with that?

would RI be very less sensitive than ECL detector? any ranges? i need a more sensitive mehtod. in that case ECL / derivitization would be better?

thanks a ton!
importantly, i have understood the importance of pH or ion-pairing agents here.. but ACN.. i fail to understand.. could u help that? thanks!

[alicee!]

This is a set of general conditions that work for most of the aminoglycosides. It can be tweeked depending on the exact one(s) you are looking at. This method was originally done using an ECD for detection, there is no reason thant a RI wouldnt work if your assay amounts are high enough.

Atlantis dC18 5 um 4.6X250

0.02 M phosphate + 35 g/L sodium sulfate + +0.5g/L OSA + 15 mL/L THF pH=3

1 mL/min

thanks! i cud try this too.. with the c18 brand i have.
again.. would RI give me much lesser sensitivity as compared to ECD? any ranges? thank you!!

[SIELC_Tech]

running your application at pH 7 will help ionizing silanols and create ion-exchange mechanism of retention.
On HILIC I am talking about using acetonitrile not MeOH. Your compound is highly polar so retaining it on silica column by HILIC mode is a choice. You will need ions in the mobile phase too as your compound will interact by ion-exchange mechanism too due to the presense of amino groups
You can not try anion-exchange because your compound is cationic in nature. You can try cation-exclusion.

[alicee!]

running your application at pH 7 will help ionizing silanols and create ion-exchange mechanism of retention.
On HILIC I am talking about using acetonitrile not MeOH. Your compound is highly polar so retaining it on silica column by HILIC mode is a choice. You will need ions in the mobile phase too as your compound will interact by ion-exchange mechanism too due to the presense of amino groups
You can not try anion-exchange because your compound is cationic in nature. You can try cation-exclusion.

i probably can use anion exchange.. kanamycin has 4nh2 grps and 6 oh grps.. so oh groups can help. i think kanamycin's pka is 7.2 so this cud b a li'l difficult. thanks!!

[Uwe Neue]

I am confused why you want to depart from using the column that has been shown to work for this application. Spherisorb ODS is an unendcapped C18 with a low surface coverage. It contains a large number of silanols that interact with the aminoglycoside via ion-exchange. This is why it works, while the other ODS column that you tried did not work.

Of course, there are many ways in which you could redevelop the method. My personal first choice for an aminoglycoside would be HILIC on silica.

The proposal by AA using ion-pairing might work as well. Overall, ion-pairing is similar to what the original method on Spherisorb ODS was all about: reversed phase in combination with ion-exchange. Keep in mind that it may not be that straightforward to remove the ion-pair reagent from your column, in case that you want to use it for something else afterwards.