MS/MS of peruvoside

[jclark]

Fellow LC-MS users,
This is more of a mass spec question than a chromatography question. I'm trying to understand the MS/MS of my analyte, peruvoside. (It's a cardiac glycoside in yellow oleander; MW=548.7.) The analysis is done using ESI, positive mode, mobile phase is 0.1% FA / ACN (95%/5%). I get a nice peak on the chromatograph, which is made up mostly of the M+H ion, but also of an ion at 571, i.e. M+23. Hey, must be a sodium adduct, right? I'm not so sure.
The 571 peak always appears at a level of about 1/3 that of the M+H peak. I have done extractions with and without added NaCl (to enhance phase separation) and there is NO difference in the signal at 571. I would expect the 571 signal to increase, if it's due to a sodium adduct. Where the heck is the sodium coming from, anyway? This "adduct" shows up in my aqueous standards and well as in extracts from plant material.
The MS/MS of the M+H ion (549) consists mainly of ions at 371 (the aglycone fragment), 353, and 389. The MS/MS of the 571 ion consists only of an ion of 411. I would expect the MS/MS of a suspected sodium adduct to resemble the MS/MS of the molecular ion.
Summing up:
1: Wouldn't the amount of a sodium adduct in a MS vary in some way with the presence of added NaCl?
2: Wouldn't the MS/MS of a sodium adduct resemble the MS/MS of a M+H ion, or at least have some ions in common?

Thanks in advance for any insights or comments!
regards,
Jim

[lmh]

Thoughts:
(1) The fact you get a sodium adduct without putting any sodium in doesn't surprise me at all. The source of the world's sodium is somewhere in my LC-MS system. I'm sure. More seriously, sodium can leach out of glassware.
(2) The fact that adding sodium doesn't convert it all to a sodium adduct is slightly more concerning, but I wouldn't rely too heavily on that.
(3) Sodium adducts, in my hands, frequently fragment completely differently to hydrogen adducts, so I wouldn't worry unduly about the different fragment.

I'd check, in chromatography, that the +22 peak coelutes exactly with the hydrogen adduct peak, and if it does, I'd be tempted to assume it really is a sodium adduct.

Also the fragment you're losing is 18 different to the fragment from the hydrogen ion, which suggests a water to me, and one hydroxyl group that can't make up its mind what to do. You could look whether other (minor? Anything there?) ions in the sodium adduct MS2 spectrum look sensible for the structure you have.

[jclark]

lmh,
Thanks for the quick response.
1. Yes, I've been told that sodium can come from glassware in sufficient amounts to produce the adducts, it's just hard to believe!
2. The lack of increase in the supposed sodium adduct when NaCl is added must mean that the sample solution is, in effect, saturated with respect to sodium...which still seems weird. Then again, maybe not, since the solubility of NaCl in ACN can't be very high.
3. So it's not unusual for a sodium adduct to fragment differently than the molecular ion. This I did not know. (Another reason why I love this forum!)
The 571 ion correlates so well with the M+H ion in the MS that I am using the 571 ion as a confirmatory ion - even tho' I don't know exactly what it is.
thanks again,
Jim