[DLK]

Hello every body,
i m struggling with a problem i have already for a week, i hope somebody could help me figuring it out. the question is , what are the possible protonation sites on tetracycline in positive mode electrospray ionization-mass spectrometry(ESI/MS), when using 0.1% formic acid and water/methanol. specifically do the oxygen atoms of the carbonyl group can also get protonated? and why? I want to be able to identify all the possible charge localization on this compound.

the first rule In electrospray positive ionization is that, protonation occurs specifically on the most basic site on the molecule to produce the protonated species which are thermodynamically favored.
beside the amino group and N(cH3)2 which are protonable while using electrospray ionization, the other potential protonable sites which comes in question would be the Oxygen atom of the carbonyl groups present in tetracycline structure, due to 2 free ion pairs available on the oxygen. the problem to confirm my assumption is first that i know Protonation of oxygen end of carbonyl can ONLY occur in presence of STRONG acid. in the mobile phase is but only a very low concentration of formic acid available(0.1%), which wouldn't result to a very strong acidic condition. on the other hand the protonation of this oxygen atom makes the compound very unstable, and rather activates this group towards nucleophilic attack and in this case a Nucleophilic acyl substitution should happen due to presence of formic acid, resulting to a peak of (M+HCOO)^+ in the mass spectra. so that if this substitution takes place, I would say the rapid protonation step of oxygen end of carbonyl group wouldn't be recognizable in the spectra and therefore tetracycline just have two potential protonation site(the amino group and N(cH3)2 ) using electrospray ionization and it won't get further triple and quadruple protonated because of two carbonyl groups. 
I would be very thankful for any correction or further discussion.

[Babcock_Hall]

It is a Forum rule that you must give your answer or your thoughts before we can help you.  You have started to do so, by discussing the carbonyl oxygen atoms.  So try to flesh this out a bit.

[DLK]

many thanks for your tip,

the first rule In electrospray positive ionization is that, protonation occurs specifically on the most basic site on the molecule to produce the protonated species which are thermodynamically favored.
beside the amino group and N(cH3)2 which are protonable while using electrospray ionization, the other potential protonable sites which come in question would be the Oxygen atom of the carbonyl groups present in tetracycline structure, due to 2 free ion pairs available on the oxygen. the problem to confirm my assumption is first that i know Protonation of oxygen end of carbonyl can ONLY occur in presence of STRONG acid. in the mobile phase is but only a very low concentration of formic acid available(0.1%), which wouldn't result to a very strong acidic condition. on the other hand the protonation of this oxygen atom makes the compound very unstable, and rather activates this group towards nucleophilic attack and in this case a Nucleophilic acyl substitution should happen due to presence of formic acid, resulting to a peak of (M+HCOO)^+ in the mass spectra. so that if this substitution takes place, I would say the rapid protonation step of oxygen end of carbonyl group wouldn't be recognizable in the spectra and therefore tetracycline just have two potential protonation site(the amino group and N(cH3)2 ) using electrospray ionization and it won't get further triple and quadruple protonated because of two carbonyl groups. 
I would be very thankful for any correction or further discussion.

[AWK]

Ions in MS decompose in the vicinity of positive charge. For tetracyclines the first elimination is MH⁺-H₂O for molecules that possess OH group at C(6) or MH⁺-NH₃ for others.

[DLK]

many thanks for your tip,

 
beside the amino group and N(cH3)2 which are protonable while using electrospray ionization, the other potential protonable sites which come in question would be the Oxygen atom of the carbonyl groups present in tetracycline structure, due to 2 free ion pairs available on the oxygen. the problem to confirm my assumption is first that i know Protonation of oxygen end of carbonyl can ONLY occur in presence of STRONG acid. in the mobile phase is but only a very low concentration of formic acid available(0.1%), which wouldn't result to a very strong acidic condition. on the other hand the protonation of this oxygen atom makes the compound very unstable, and rather activates this group towards nucleophilic attack and in this case a Nucleophilic acyl substitution should happen due to presence of formic acid, resulting to a peak of (M+HCOO)^+ in the mass spectra. so that if this substitution takes place, I would say the rapid protonation step of oxygen end of carbonyl group wouldn't be recognizable in the spectra and therefore tetracycline just have two potential protonation site(the amino group and N(cH3)2 ) using electrospray ionization and it won't get further triple and quadruple protonated because of two carbonyl groups. 
I would be very thankful for any correction or further discussion.