Hello everyone,

i have encountered a problem lately, that has first occured when trying to validate my method.

I am working on a HILIC-MS/MS method for some different amines. When trying to validate the linearity, i have observed a strange development of responses.

I am validating the linearity by doing 11 calibration levels and measuring them in triplicate, non-randomised. I have observed for some analytes (ethylamine, dimethylamine), that responses will decrease for every replicate while each 11-fold replicate itself will still give a linear calibration.
In the same samples I am also observing the opposite (increasing responses) for some other analytes (agmatine, cadaverine, histamine).
Also in the same samples, i have several similar analytes (trimethylamine, dimethylamine, putrescine, ethanolamine), which are linear over all replicates combined.

Any idea on why this occurs?

My method details are as follows:

Column: Agilent Poroshell 120 HILIC-Z (150x2.1 mm; 2.7 µm) / Accucore HILIC (100x2,1 mm; 2,6 µm) --> tried it on both, no difference.
A: 90:10 ACN:H2O + 50 mM ammonia formate + 2.5% formic acid
B: H2O + 50 mM ammonia formate + 2.5% formic acid
Flow rate: .5 ml/min
Injection volume: 1 µl
Column temperature: 40°C

My analytes are soluted in eluent A.

The instrument I'm working on is an Agilent Technologies 1260 Infinity II LC system equipped with an Agilent Technologies 6495C triple quadrupole mass spectrometer.

I'd be happy for any input on why this is happening and how to prevent it. If any info is missing or unclear I'll be happy to provide it.

Thanks in Advance!