retention time shift with Ion pairing LC

[Kostas Petritis]

DL,

Not continuasly... just several volumes is enough so 20-30 minutes is just fine.

What's your detection?

Both the amino acids that you indicate have stability problems with Cys oxidizing to Cystine and Gln to Glu.

I would inject large amounts of my Gln to see if it is partially degraded and for Cys always use fresh solutions (it would also be good to have fresh solutions for Gln although it is much more stable than Cys). If you are using ELSD you can extent Cys life with the addition of some mercaptoethanol (you won't detect it with the ELSD).

Uwe,

Yes DL said that, but at the same time he said that just last week he recovered the retention times just by filtering the mobile phase (which I think it has nothing to the present problem). If it was loss of the bonding then he wouldn't be able to recover the initial retention times...

[dlliu]

I see.... Thank you for pointing it out! I'm using MS as my detector. I also saw Asn becomes two peaks in my chromatogram. Any explanations?

Thanks, DL

[Kostas Petritis]

DL,

As with Gln, Asn has the same problem of tranforming overtime in to Asp but as you are doing MS detection there are maybe other reasons that you see two peaks.

So, if you extract your XIC at 133 (or 133 87 if you are doing MS/MS) and you see two peaks then (assuming that your instrument is tuned correctly -you have at least unit resolution)- you can only get additional peaks from the 13C isotopic ions [monoisotopic] of Ile and Leu which M+H is one unit down (i.e. 132 or 132 86 if you are doing MS/MS).

Anyway, I do not know the setting of your MS, and when you see the additional peaks (i.e. TIC or XIC) but it can only be from Asp, Ile and Leu if you look at the TIC and from Ile and Leu only if you are looking at the XIC (assuming correct tuning). It is easy to pinpoint out which one is which, from the retention time of these ions...

Hope the above helps...

[dlliu]

I'm using 133 -> 87 for Asn, but you bring my attention to the two peaks that come from 13C of Ile and Leu... I need to take a very closer look at this.

Thank you so much for your comments.

DL

[Kostas Petritis]

Have a look in Figure 6 at J. Chromatogr A. 855, 1999, 191 where you can see all the additional peaks you get for Asn for MS only analysis. Met, Ile and Leu can interfere with your Asn (CID fragmentation of Met leads to loss of NH3 which ends up to have the m/z as Asn (i.e. 150-17=133). Likely when you are doing MS/MS you can not further fragment that ion to immonium (i.e. H2N=CH-R) as you have already lost the NH3 function.

However, even in MS/MS you still get interferences from Ile and Leu that is why you need to separate them chromatographically.

Actually I have a complete table of several amino acids that needs to be separated before analysis by MS due to several intrinsic problems when analyzing by MS or MS/MS (i.e. CID fragmentation, monoisotopic interference or combination of the two). I can try to list it here if you are interested...

[dlliu]

Yes having the table listed here would be very helpful. It's always good to learn new things every day!

Thank you very much!

DL

[Kostas Petritis]

DL,

Here you are... Unfortunately the forum does not let me to format very well so normally there are two columns; one is the amino acid mixture and the other is the justification of the problem that can be a or b or c or combination of them... Probably you are not going to understand all the abbreviations but I can explain if ever any of these amino acids are of your interest...

Amino acid mixtures that have to be separated by LC before analysis by tandem mass spectrometry. Amino acids in bold cause the undetermination but they can specifically determined.


Amino acid mixutres Justification
Leu, Ile, Nle, Hyp a
Val, Iva, Nva a
o, m, p Tyr a
Gln, Lys a
a-aba, a-aiba a
Ser, Ise a
Thr, Hse a
ACPCA, Pia a
2-PhGly, 3- AphAc a
1-mHis, 3-mHis a
SeMet, Dopa a
b-Ala – Ala, Sar a
Asn – Orn a
a,b,g-ABA, a-AiBA – b-AiBA a
Ans – 1, 3-mHis b
AAA – ACHCA b
CysT – Asp b
Thr, Glu – ACC b
Cre, Hcys – Ala, Sar b
Glu, Gln, Lys – Pia, ACPCA b
Arg, Orn, Asp, AAA – Pro b
Met – g-aba b
Met – Asn, Orn b
Car – His b
SeMet, Dopa – 2PhGly b
Leu, Ile, Nleu, Hyp – Asn c
Asn – Asp c
Arg – Cit c
p-Aphe – o,m,p Tyr c
MetS – 3-AphA c
a-mTyr – 3-Atyr c
3-Atyr – SeMet c
SeMet, 3-Atyr, a-mTyr, 5-Ftyr c, d


Justification explanation:

a) Isomers or isobars with identical (major) collisionally activated dissociation (CAD) fragmentation.
b) The in-source CID and its corresponding CAD fragment of one correspond to the selected parent and product-ion of the other(s).
c) [M+1+H]+ of one [isotopic amino acid (13C)] and its corresponding CAD fragment correspond to the parent and product-ion of the other(s).
d) Same problem as in c, but enhanced by the isotopic distribution of Se.