Help on unknown impurity (QTOF-ESI)

[Mattias]

Hi,

I am struggling to identify an unknown impurity in one of our products. The product is a peptide, but this impurity is definitely not peptide related. The product also contains chlorobutanol. The impurity increases during storage.

I do not manage to get any mass signal at all, have tested negative and positive mode and all different cone and capillary voltages (Waters QTOF Premier with ESI interface)

It elutes (RP column) at about 25% acetonitrile and have a very distinct UV-spectrum (one UV max at 243 nm, no absorption below 215 nm).

Any good ideas of how to get any mass data on this impurity (on our equipment), or any other ideas? Thanks!

[JMB]

Mattias,

One generally applicable strategy in cases like this is to isolate the unknown by multiple HPLC injections; collect and pool appropriate fractions, blow off the MeCN under N2, extract into CH2Cl2 and analyze by GC-MS.

It's interesting that the unknown increases with time (oxidation/hydrolysis/etc ??) but is NOT peptide-related. Does this imply that the peptide content remains constant ??

Unknowns have to be either process impurities or degradation products. The former are not expected to increase with time, while the latter are expected to increase. Can the level of the unknown be increased by accelerated stressing ??

Does the peptide contain any blocking groups/structural modifications etc that may be readily lost; if the degraded peptide then co-elutes with the original product, the peptide content will appear constant.

JMB

[Mattias]

Thanks for your reply!

The product is in a glass vial with a rubber stopper, and I start to to suspect that this peak is related to the stopper. We have never seen this peak increase before, and the content of the peptide is unchanged.

One problem is that the concentration of peptide is very low so the area% of this peak gets really high (5%). And it refuses to get ionised with my equipment.

Is there any option to change the ionisation mode on a QTOF Premier (I guess I should ask Waters about this).

The most obvious way forward is to do fraction collection and send to NMR lab, I guess.

[JMB]

My understanding is that leachables and extractables are much better analyzed by APCI.