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- Joined: Mon Jan 07, 2013 8:54 pm
- Location: Western Kentucky
The transitions used are Cylindrospermopsin 416/194, Anatoxin-a 166/149, Uracil-d4 115/98, L-Phenylalanine-d5 171/125. Uracil-d4 is the internal standard for Chlindrospermopsin and L-Phenylalanine-d5 is the internal standard for Anatoxin-a. ESI positive mode used.
The column is the Waters X-Select HSS T3 3.5um 2.1x150mm at 40C and mobile phase is step gradient of A=5.8ml Acetic Acid/L H20 B=Methanol that steps from 100%A to 75%A at 0.8 minutes with flow of 0.375ml/min.(drops back to original percentages after elution of last peak at ~5 minutes and allow to equilibrate until 15 minutes)
The problem I am having is that l-Phenylalanine-d5 will begin with a huge peak, then after about 4 injections drop to about 1/2 the response, then continues to drop over a long run of about 35 injections. This last run l-Phenylalanine-d5 started with 160251 area counts for first blank(after 5 warmup injections) then fell to 121593 area counts at first calibration standard and on down to 55155 area counts at end of run. The run was queued to run again immediately after last injection where the area counts ranged from 54158 down to 39241 over the next 35 injections. The second run passes QC limits per the method since it falls less than 50% of the average of the calibration curve.
In the first set, the Uracil-d4 ranges from 139487 to 127594 on first run and 163564 to 147904 on second run.
The Cylindrospermopsin has recoveries of near 100% for all three calibration checks ran immediately after the calibration, mid run and end of run. The Anatoxin-a however has calibration check recoveries of about 120% on first check, 165% at mid check and 185% on last check. It seems that only the L-Phenylalanine-d5 suffers from the drop in sensitivity over time, which causes the increase in recovery of the Anatoxin-a. If I run the analysis the next day with the same mobile phase and samples the L-Phenylalanine-d5 will be even more steady, but slightly less response.
If this was charging of the quads, I would think that the Anatoxin-a would suffer nearly as much as the L-Phenylalanine-d5. So I tried adding a stirbar to the mobile phase and stirring during the run and it seems to drop a little less, plus letting the mobile phase sit over night also seems to stabilize the sensitivity, which this set of numbers comes from, when starting from fresh mobile phase made just before analysis the drop on the first run will be even greater.
Has anyone seen any similar problems with an analyte like this?
I did try increasing the acetic acid in the mobile phase but it lowers sensitivity for everything so maybe I need to try less. The peaks are needle sharp except for the Uracil which spreads a little at the bottom both front and back which I think causes the small amount of response shift it has. Overall the method works great, except for the rapid loss of response for the one internal standard. I always get a reportable run, but always as the second time through which wastes a lot of analysis time.
Thanks for any ideas anyone might have.