Chromatography Forum

Hello every one,
I'm working now in method development using API5000 mass spectrometry ... but because the analyte has very poor sensetivity as it is , I do dervtization using dansyl chloride , my question first is what he best way to prepare dervitized standard for tuning purposes..
and the other one that I have interfernce peak at the retention time of the analyte , I study all contamination sources which give me clean results .
I inject dansyl it self which is clean then NaHCO3 or Na2CO3 also give clean but when I inject mix sample of dansyl and NaHCO3 or Na2CO3 it gives me very large peak .. why this happened??? any one can explain it to me please

Can you provide more details please, I assume you are running in pos ion, are you scanning or using MRM, and what the mass of your target? Also, HPLC conditions would be helpful and a chromatogram.

about the first question: we did the same and solved the problem for optimization as follows: We derivatized the (a lot of) the compound and injected it on a 'normal'HPLC with UV detection. We collected the peak from the derivatized compound and used that for optimization of the MS. Without this step you are optimizing in 0.1M salt, dansylchloride and the MS doesn't like this.
The second question: no idea

peter

About your unknown peak: I would bet on dansyl.Cl hydrolysis to dansylic acid in your buffer. Try to reduce DNS.Cl or time before injection and see if your unknown signal diminishes.

I reduce the concentration of dansyl chloride from 1 mg/mL to100ug/mL
the peak is reduced but still appear which give me linearity from 6.0 pg/mL in the time I need to start from 1.0 pg/mL
I'm working in positive MRM mode.
I f Isuccess in developing method , what about studying absolute recovery and stock solution stability in the validation stage at the time there is no authentic dansylated standard?

As I said, it is difficult to give a good advice without knowing more about your method. The compound that you are derivitizing must have an amino group, so it should ionize fairly well. I would try and optimize sample prep first before going with derivatization (which opens its own can of worms).

You will have to have the std custom synthesized and characterized for you if you want to have a validated method.

Another option is to have an internal std (you have to have one anyway for a LC-MS/MS mathod), which would be similar to the target compound and would undergo derivatization as well. That way, you can use underivatized compound as your standard (or rather as a pair with your internal standard).

Hello peterS
I read your reply and I think that you can help me in solving problem but I didn't understand the meaning of this

"We collected the peak from the derivatized compound and used that for optimization of the MS"

Please can you explain to me in detail
thanks alot.

hello smkh

what I mean is this:
When you derivatize your compound it is probably in a pH 9 NaHCO3 salt, and 1 mg/ml dansylchloride in acetone. When you are doing direct infusion from this solution it is differenet than when the compound comes from the column. E.g. in our case the eluent was acetonitril/water/formic acid. So we injected the derivatized compound in NaHCO3/dansylchloride soluntion on an HPLC system with UV detection and looked for the peak of the derivative, it was let's say 10 min. Then we did a second injection and collected the peak at 10 min in a glass tube and this solution was used for direct infusion into the MS with a syringe. The derivate was then in eluent and separated from the salt and dansyl chloride. Then we could optimize the MS.

Hope this is more clear,
PeterS

Thank you PeterS

Yes this is more clear , the point that I want to share it with you to get your opinion , that when I prepared dervetized standard I do back extraction by adding hexane to the mixture of dansyl and Na2Co3 , mix it then get organic layer , evaporate and reconstitute .
dansyl prepared in acetonitrile rather than Acetone and the PH of Na2Co3 is 10.6.. is that OK
what you do it using UV to get standard AS IS without any other interfernces which I think that will make sense.
Can I use any C18 column(15 cm) and mobile phase of (1:1) (H2O:ACN) with 0.1% FA and what about wave length used on UV.

Thank you again for your reply.

Stepping back briefly to the original experimental problem... you said your original analyte has sensitivity problems on this MS. What sort of mobile phase and additives were you using when you experienced this poor sensitivity? I know ESI-MS can be very finicky about what else is in the mobile phase. Is it possible that optimizing these conditions could give you good enough response without derivatization?

And if you must derivatize, I agree that you must be mindful of what else is in the matrix you're injecting on the column. Ideally you want your analyte to be retained on the column long enough for all the early-eluting matrix components to pass (and while the matrix elutes, it could be helpful to divert to waste instead of MS).

Hello smkh

About your questions:
Are you sure that by the exctraction proecedure with hexane the compound is extracted and that the dansyl chloride is not? Otherwise you still optimize your MS in the presence of dansyl chloride which may disturb.

For me it is possible to use any C18 column as log as it gives a separation between the NaHCO3, the dansyl chloride and the derivative. For wavelength we used 254 nm.
The mobile phase is probable comparable to that used in your LC_MS so you will be able to tune the API5000.

Hello PeterS

About this:
"Are you sure that by the exctraction proecedure with hexane the compound is extracted and that the dansyl chloride is not? Otherwise you still optimize your MS in the presence of dansyl chloride which may disturb."
I agree with you 100%
And the picture now is more than clear for me , this is the first time I do something like this and I feel it is too intresting.
So I can do it in Revesre phase and when you say 'Normal' HPLC that is not mean Normal phase
I will try it and share the result with you.

Thanks alot.

Hello PeterS

I do the experiment using HPLC- UV and with c18 column (150*4.6) and mobile phase of (1:1)(H2O:ACN) with 0.1% FA and flow rate of 1ml/min using standrd prepared by evaporate 10ug/ml cocentration in methanol then add dansyl chloride, Na2CO3 mix , incubate and back extract , reconstitute it with mobile phase (0.500ml) and inject 100ul on UV
two main peaks appear at retintion time of 2, 4 and small peaks at 6, 10
when inject dansylated methanol proceesed as previous , it dosen't give me that of 4,6& 10
I collect that of 4 because it is main
and tune it using API5000, the tuning didn't give me high intensity but I get new parameters.
Inject stsndard and calibration and still I have apeak at the blank same of that at low concentratins
any thing to be considered?
by the way the analyte is Ethinyl Estradiol.

Hello smkh

If you still have a peak in the blank is there anything contaminated?
I think you are measuring now in MRM mode on the API5000 with m/z 530-171 as mass transition and an collision energy of about 50V.

Peter

Thanks for clarifying your analytes. I was once trying to optimize signal for dienestrol and diethylstilbestrol on ESI-MS. They were nearly impossible to see in positive mode, but I got a molecular ion in negative-ion mode and reasonably good sensitivity by MS/MS, no derivatization. I saw ion suppression with formic acid or ammonium hydroxide as an additive. Sensitivity was better with dilute ammonium acetate in the mobile phase (0.25 mM) - anything stronger led to suppression again.

I don't know if ethynylestradiol would follow the same trend but I figured it's worth bringing up, since you didn't mention under which conditions you had concluded the sensitivity would not suffice without derivatization.