SMALL MW PEPTIDE BY ESI+LC-MS-MS
Posted: Wed Apr 17, 2013 9:05 pm
Hello all,
I am assaying a small molecular weight pentapeptide using ESI+LC-MS-MS, we do have an ion pairing agent (PFPA) to increase retention on the C18 column.
It seems that the stock solution (1mg/mL) is prepared in distilled water, and the working solutions are prepared in HEPES Buffer (pH 7.4) for a working stock of 10 ng/uL.
The mobile phase consists of a 5mM aqueous pentafluoropropionic acid (PFPA) and 5mM PFPA in Acetonitrile , at a (volume to volume of 87:13) under isocratic conditions.
More recently I took an ESI + MS2 fullscan spectra and observed the molecular ion but followed by a blob and unsymetric very large peak of the compound eluting over a period of 1.5min . I am assuming this was due to having injected the standard sample in the working solution (in hepes buffer pH 7.4). It seems the compound eluted as a huge blob. Probably this was due to poor sample conditions, such as having the nonvolatile hepes buffer as part of the 5uL injection volume?
My question is , should this compound be dried down from the aqueous working solution (hepes buffer pH 7.4), and reconstituted in (starting mobile phase 87:13 with the ion pairing reagent) in order to increase the peak symmetry and chromatographic condition?
Is it acceptable to dry down about 500uL of aqueous sample under low heat or do we worry about the peptide degradation? Also, does vortexing the sample cause degredation, and how stable are these working solutions typically?
I am thinking this is what caused the original poor chromatographic conditions?
Does anybody have any suggestions??
I am assaying a small molecular weight pentapeptide using ESI+LC-MS-MS, we do have an ion pairing agent (PFPA) to increase retention on the C18 column.
It seems that the stock solution (1mg/mL) is prepared in distilled water, and the working solutions are prepared in HEPES Buffer (pH 7.4) for a working stock of 10 ng/uL.
The mobile phase consists of a 5mM aqueous pentafluoropropionic acid (PFPA) and 5mM PFPA in Acetonitrile , at a (volume to volume of 87:13) under isocratic conditions.
More recently I took an ESI + MS2 fullscan spectra and observed the molecular ion but followed by a blob and unsymetric very large peak of the compound eluting over a period of 1.5min . I am assuming this was due to having injected the standard sample in the working solution (in hepes buffer pH 7.4). It seems the compound eluted as a huge blob. Probably this was due to poor sample conditions, such as having the nonvolatile hepes buffer as part of the 5uL injection volume?
My question is , should this compound be dried down from the aqueous working solution (hepes buffer pH 7.4), and reconstituted in (starting mobile phase 87:13 with the ion pairing reagent) in order to increase the peak symmetry and chromatographic condition?
Is it acceptable to dry down about 500uL of aqueous sample under low heat or do we worry about the peptide degradation? Also, does vortexing the sample cause degredation, and how stable are these working solutions typically?
I am thinking this is what caused the original poor chromatographic conditions?
Does anybody have any suggestions??