-
- Posts: 1
- Joined: Wed May 01, 2013 7:54 pm
Hi Everyone
I am developing a method for diclofenac on UHPLC-MS/MS (Flexar -15 connected with API Qtrap 5500) using Eclipse plus C18 RRHD column (Agilent Technologies). I was running an isocratic method (methanol:0.1% formic acid::90:10) but now I am trying to develop a gradient method. The gradient is set up with varying % of methanol and 0.1% formic acid in water: 0-0.5 min:0.1%FA (100%), 0.5-1.5 min: the methanol percent changes from 0 to 90%, 1.5-3.5: methanol 90%, 3.5-4.0 min: methanol back to 0 % and 4-5 min: 0.1% FA (100%).
In the isocratic method, the mobile phase injections are clean; however, I see a peak of very high intensity (4.00 e+04 peak height) when I run gradient method. I made several injections (100 injections) of the blank but the peak is consistent in every injection. I tried following needle wash solutions: (a) 50% methanol in water (b) 50% methanol in water with 0.1% formic acid (c) methanol:acetone:water::50:25:25 (d) methanol:acetonitrile:water::50:25:25 (e) DMSO:methanol:water:: 5:50:45 . None of these solution helped to remove the peak. I also removed the needle from autosampler and cleaned with acetonitrile by sonicating for 15 mintues. I washed column with methanol:water (90:10) for 2 hours but this did not alter the intensity of the peak. Even I injected air out of empty vial and this also gave the peak with the same intensity. Any idea what could be the problem and how can I solve it? Any input is greatly appreciated.
I am developing a method for diclofenac on UHPLC-MS/MS (Flexar -15 connected with API Qtrap 5500) using Eclipse plus C18 RRHD column (Agilent Technologies). I was running an isocratic method (methanol:0.1% formic acid::90:10) but now I am trying to develop a gradient method. The gradient is set up with varying % of methanol and 0.1% formic acid in water: 0-0.5 min:0.1%FA (100%), 0.5-1.5 min: the methanol percent changes from 0 to 90%, 1.5-3.5: methanol 90%, 3.5-4.0 min: methanol back to 0 % and 4-5 min: 0.1% FA (100%).
In the isocratic method, the mobile phase injections are clean; however, I see a peak of very high intensity (4.00 e+04 peak height) when I run gradient method. I made several injections (100 injections) of the blank but the peak is consistent in every injection. I tried following needle wash solutions: (a) 50% methanol in water (b) 50% methanol in water with 0.1% formic acid (c) methanol:acetone:water::50:25:25 (d) methanol:acetonitrile:water::50:25:25 (e) DMSO:methanol:water:: 5:50:45 . None of these solution helped to remove the peak. I also removed the needle from autosampler and cleaned with acetonitrile by sonicating for 15 mintues. I washed column with methanol:water (90:10) for 2 hours but this did not alter the intensity of the peak. Even I injected air out of empty vial and this also gave the peak with the same intensity. Any idea what could be the problem and how can I solve it? Any input is greatly appreciated.
