Chromatography Forum

Hello, my name is bas and i am new to this forum.

I hope somebody can help me with a problem.

I am working on an method for the determination of L-Carnintine by UPLC/MS/MS
I discoverd some problems with the blank. The blank gives a peak with the same retention time as L-Carnitine and d3-Carnitine (internal std)
on both transitions.
I think the ammonium acetate is the problem, because without the ammonium acetate there is nothing to see.
The peak area of ammonium acetate is 1800 and lowest standard has a peak area of 150000.

Conditions:

Gradient:
MPa: water(milliQ) + 10 mM ammonium acetate (10 min ultrasonic bath)
MPb: 97/3 ACN/water + 10 mM ammonium acetate (10 min ultrasonic bath)

column: BEH HILIC 150 mm from Waters
Transitions: 162,1>84,45+162,1>102,93 m/z
Instrumentation: UPLC+TQD from waters
Ammonium acetaat: LC-MS Ultra; eluent additive for UHPLC-MS (Fluka) from Sigma

I hope this is enough info

Greetz

Are you using gradient elution?
Maybe you're struggling with carry-over? Do you see the mentioned peaks while tuning your analytes?
A new paper on acylcarnitine analysis:
Anal Chim Acta. 2011 Mar 9;689(1):77-84. Epub 2011 Jan 18.
“Ultra-high performance liquid chromatography tandem mass spectrometry for comprehensive analysis of urinary acylcarnitines”
Feel free to send me an e-mail or ask more questions, as I’m working on acylcarnitines as well
/Rune

Do I understand you?
The peak area in your (ammonium acetate) blank is 1800.
The peak area in your lowest standard is 150000.
100*1800/150000 = 1.2%

This should not pose a major problem in your method unless you wish to lower the LOQ by about a factor of ten.

How's your precision and accuracy at the low end compared to this?

What is the source of the blank sample? Is it biological or is it made up of "pure solution"? If your blanks are made up from anything that MIGHT normally contain L-Carnatine then you just might find they contain some.
I would agree that the peak may be caused by gradient. You might try a similar column that has never seen L-Carnatine and see how the blanks look on this column. Sometimes a compound can stick to the column head/frit and come off each time the gradient runs. You can modify your gradient (or try some experiments with isocratic) and see if that helps, at least it might help verify if the problem really is in the blank. Or perhaps the drug is getting caught up in the injection port or sticking to to a (dirty) filter in the mobilephase path.
Also look at the solution makeup of your sample and compare to the mobile phase. Are they similar in composition or do they differ? Difference in sample makeup/mobile phase can cause such problems.

Alp