Carryover in LC-MS/MS (MRM) Ceramides

[Eddy_unisa]

Dear All
I am writing you on a carryover problem with the analysis of c16, c18, c24:1 Ceramides. I am working on a nexera coupled to lcms 8050 shimadzu, my column Is a kinetex evo c18, 150 x 2.1 mm, 2.6 um
Mobile phase A: 10mM HCOONH4 0.1%HCOOH in ACN/H2O 60:40, B: 0.1%HCOOH IN IPA/ACN 90:10, as told before i am working in mrm mode, samples are plasma and Cell lysates
I observe the transitions of all Ceramides at the same RT with blank (meoh ipa) or no injection (-1 on shimadzu), vial with cap or not
I tried:
Autosampler exclusion by Direct connection to mixer
Manuale injector
Different column (same new)
No precolumn
Column wash with thf
Autosampler multiple rinse (ipa, thf, dcm, magic mix etc)
New mobile phases (bottle) new reservoir
New ESI needle and peek connections/viper connectors
Autosampler performed manutention with change of rotor, stator, loop, inj Port,
I am getting Crazy
Please help me

[trozen]

as ceramides are very hydrophobic, the choice of the column does not seem optimal (even though the mobile phase is really strong)

why not C8, and a shorter one, 100 mm max length?
what is your gradient? column temperature?

if you inject a blank or air without any column attached, will you still see the signal for your transitions?

[Eddy_unisa]

Hi
Several lipidomics papers use C18 so I not considered C8 in first instance
Gradient is
0 min 5% B
2.50 min 50%B
7 min 99%B
9.50 min 99%B
returning to 5% in 0.1 min B mobile phase is IPA/ACN 90/10 0.1%HCOOH
Column temperature 55°C, the method is taken from literature
Flow 0.5 mL/min, injection volume 5 uL
The peaks are observed only with column, using a union (no column) i have no signals. Moreover their peak shape is good so it seems they are "chromatographic peaks"
Thanks
Eddy

[trozen]

okay, so it is the column that creates carryover
the method from literature, was it also a Kinetex C18 Evo, 150 mm length?
columns from different vendors may perform very differently, even with formally identical sorbent chemistry

if you increase the isocratic part of 99% B from 2.5 min to say 5 min, will you see any difference in the abundance of those ghost peaks?

also, when you say - return to 5% B in 0.1 min, I presume there is a longer equilibration period at 5% B before the next injection, correct?