Chromatography Forum

Hello!

My last post led me to great success, so I hope this one can too.

I am attempting to optimize our mass spec (Thermo TSQ Quantum Ultra with positive mode HESI) for the detection of thymidine, but my initial attempts showed nada in the TIC. I had a solution made up of 1:1 H2O:MeOH + 0.1% formic (their LC mobile phase will be ammonium acetate, pH 5.6 with acetic acid and I plan on trying with it on Monday). I was curious if anyone can give me any pointers? Most of the papers I've seen mention MRM, but I don't think anything but a full scan is desired by the colleagues I'm working with.

Here are the spectra I was able to obtain with direct infusion of the samples:








Each analyte was ~100 ppm to give great initial signal. I also tried uracil and uridine, for example, and wasn't able to see them either. The sample labeled by my grad student as "madmix" contained all of the analyes in one where we only observe dC.

I did try to vary my source conditions. The source was held at positive polarity and using our software's optimization feature (TuneMaster) we examined:

spray voltage (optimium was around 4.9 kV).

Sheath gas (optimum was around 14).

Aux (drying gas on our system) was hit or miss but for most of the analytes 0 gave the highest intensity.

Tube lens (I can't recall its optimum at the moment)

Cappilary offset was also examined.

The parameters that weren't examined were the vaporizer temperature (ours is set to be ambient at the moment as so far elevated temps made the spectra look awful, but I do plan on examining it on Monday), and capillary temperature (ours is maintained at 350, I plan on examining it also on Monday).

I'll close by fully disclosing I am in no way a biochemist. I avoided it (sadly) like the plague in grad school.

Thanks to anyone that reads this!

MB,

It would be very helpful to us readers if you would provide the Mol. Wt for each of your analytes.

If I were given the spectrum of dG as an unknown, I would interpret m/z 252 as [M+Na] and m/z 268 as [M+K], but without MW those are just my best guesses.

MW info would reduce the amount of effort required on my/our part.

Regards,

JMB

MB,

It would be very helpful to us readers if you would provide the Mol. Wt for each of your analytes.

If I were given the spectrum of dG as an unknown, I would interpret m/z 252 as [M+Na] and m/z 268 as [M+K], but without MW those are just my best guesses.

MW info would reduce the amount of effort required on my/our part.

Regards,

JMB

I feel so silly that I forgot to include that, sorry!

Adenosine = 267
Cytidine = 243
Guanosine = 283
Thymidine = 242
Uracil = 112
Uridine = 244
Deoxyadenosine (dA) = 251
Deoxyguanosine (dG) = 267
Deoxycytidine (dC) = 227
Madmix = combination of all of the above.

Thanks!

MB,

Thanks for the info.

I’ll have a look at your spectra later .

Regards,
JMB

MB,

FULL DISCLOSURE: I am not a biochemist, and have never been within 100 miles of a nucleo(S/T)ide.

That said, I can see no reason for non-detection of thymidine/uracil/uridine, so could you post the spectra that you recorded.

Regards,

JMB

MB,

FULL DISCLOSURE: I am not a biochemist, and have never been within 100 miles of a nucleo(S/T)ide.

That said, I can see no reason for non-detection of thymidine/uracil/uridine, so could you post the spectra that you recorded.

Regards,

JMB

Touché! The spectra I was able to obtain/record are above in the original post. That’s my starting point, sadly.

MB,

I suspect that there is something amiss with the source parameters you are using for thymidine/uracil/uridine.

Perhaps the parameters of the LC-MS interface are too harsh for these analytes which leads to degradation. If possible, compare your settings to those given in the lit.

Your plan to look at capillary temp and vaporizer temp is a good idea.

Regards,

JMB