I've been trying to learn (newbie) about LC-MS and our machine is having major contamination issues.
It seems that some steroids have somehow fouled the machine:
The UPLC is an Acquity H-Class w/ Xevo-TQS
The problem persists with a new column (Beh C18, 1.7um x 50mm; no guard column). The problem seems to be confined to the Acquity (direct injection in the MS did not show the contamination).
MW for some of the compounds- Ethinyl estradiol 297.30; Estriol 288.35; Estrone 270.36; Estradiol 272.38

We usually run a gradient from 5% acetontrile:h20 to 95% acetonitrile:h20 with .1% formic acid. The first figure below is from a troubleshooting run with straight methanol (no gradient, methanol as both A+B solvents)

Things that have been tried following the guide “Controlling Contamination 715001307, REV. D” Italics are things tried or comments.

Check mobile phase. Done multiple times.
a. Check the solvent, water, and acid used for dilution. Done
c. Change the injection size. Done.
d. Check the needle wash solutions. I think the non-H class has a weak and strong wash, but the H-class only has one. I’m not sure how to do this.

For the figure below:
A. 0 ul injection of ethanol
B. 1 ul of Estrogen mix
C. 0 ul of Estrogen mix
D. 0 ul of ethanol – w/ 60 s needle wash
E. 0 ul of Estrogen mix – w/ 60 s needle wash

The next day the 297.30 peak was back at .5 min when injecting 1ul of ethanol.
What happened in C,D and E where the .5 min peak disappeared? And why would it reappear the next day?
What are the typical counts for a 0 ul injection?


For the second figure:
I had read on the forum about how to estimate column volume, and decided to run a longer gradient (around 2 min @ .5ml/min flow @95% acetonitrile. Runs C and D have longer gradient. A new sample vial with freshly acquired ddH20 was injected.

A= 0 ul dH20, B=1ul dH20, C&D = 2ul dH20


For the third figure:
Increasing injection volume A=0, B=1, C=2, D=4, E=10 ul injection of straight methanol. The 269 peak is an anabolic steroid


The Troubleshooting guide also mentions:
Check the tubing and fittings on the injector, especially the injector outlet to column inlet. If there is dead volume, contamination can accumulate in those spaces. Replace the tubing and fittings. Make another injection and check for contamination. Which sounds good, but there is no mention on how to do this.

We've been thinking about ordering a new sample needle. Can the current sample needle be removed and sonicated? If the needle is the source of contamination and is replaced, what are some steps that can be taken so that it won't reoccur?

Any suggestions or explanations will be appreciated, the more basic the explanation or suggestion the better. Thank you.