I am attempting to derivatize polymyxins using FMOC - Cl. I run the derivatives on UPLC, but unfortunately I am struggling to determine which peaks are polymyxins.
Briefly, using a vacuum manifold, I run 1 ml of acetone through a C18 prep column. I condition with 1 ml methanol, then equilibrate with 1 ml carbonate buffer, pH 10. I then add 100 ul of my sample my sample i.e., 1mg/ml colistin dissolved in dddH2O and rinse with 1 ml carbonate buffer. I always bring the volume in the column down to the meniscus. I then add 100 ul of FMOC - Cl (100mM) that is dissolved in acetonitrile. I react for 10 min at room temp. I then run the prep column dry and elute the derived Polymyxin with 900 ul Acetone. To the eluate, I add 600ul of 0.2M boric acid and 500 ul Acetonitrile.
The samples are run using a gradient of dddH2O and acetonitrile:THF:dddH2O (87:4:12). I run for 25 min, gradually increasing solvent. I detect using UV ~ 265nm
Various peaks appear, but there is not difference in my controls i.e., I do the same above, except instead of colistin I just use dddH2O.
The peaks that appear are the following: 10.8, 11.1, 12.0, 13.8, 15.3, 15.4, 18.4
The only difference that I observe are in the peaks at 18.4 and 15.4. Their areas differ by about 50 %. The colistin speak at 18.3 is 50 % less than that of the controls and the peak at 15.4 is 50 % more than that of the control.
Can anybody suggest something?
That's why I use MS. But, the first question I have: Is the C18 SPE cartridge, you are calling a prep column, specially suited to pH 10? Even though you don't have to worry too much about it with SPE, they usually have a stable pH range of 2 to 7.5. The second question: Is this on-column derivatization procedure known to work at room temperature? From what I can see, 50-60◦C is used for on-line pre-derivatization. Maybe you should derivatize it normally to verify. You may also want to rinse with water before running it dry. You also tell us the last peak is colistin. Is this the underivatized peak? I assume so since you see it in the control. If so, then the derivatized product would elute much later than this. This leads to the third question: what is the gradient on your UPLC column? I can't tell if you mean you are running a gradient of water to MeCN/THF/H2O or are you starting with the 87:4:12 and ending with something else?
Thanks John, I am not sure about the SPE? It is a waters product and I will have a look into that. Publications I have don't mention anything about temperature. I assume that if nothing is mentioned that it is at room temperature. Rinsing with water is a good idea. I am concerned this might produce FMOC - OH and affect my results. I don't know which peak is colistin. It cannot be seen if underivitized. I only assume which peak is colistin. As I mentioned in my question, i only see a difference at 18 and 15 min between controls. Those controls are derivitized colistin and FMOC - Cl only (no colistin). The article I have run isocratic using 87:4:13 ACN:THF:H2O. I observed peaks I wanted to separate so began running a gradient using water and the mentioned solvent. The peaks separated fine.
I prepared my samples as mentioned on the pre column. My controls are FMOC - Cl without colistin and colistin without FMOC - Cl. I sent it for MS and will hopefully get the results soon.
Please consider that acyl chloride derivatisation is rather unstable. Its ok if you will use online derivatisation (i autosampler) If sample has to wait hydrolysis of derivative will occure. Maybe another reagent like dansyl chloride - more stable derivatives.
Thanks, I have sorted the problem out. It looks like it was a stability issue. How could I improve the stability of the derivatized sample? We determined that it starts degrading as soon as 10 min after derivatization.
I thought the reason for adding 0.2M boric acid makes the solution acidic and thus more stable?
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