Elevated baseline for certain peaks

<p>Hello! I am developing a method for benzodiazepines on the Acquity TQD and am having problems with the chromatography for two peaks. I currently have 15 benzodiazpeines in the run with 3 internal standards. My mobile phase A is 0.1% formic acid in water and mobile phase B is 0.1% formic acid in methanol. My run time is 8.50 minutes with all peaks separated nicely. All my peaks look great except for two: alprazolam and alpha-hydroxyalprazolam (I attached the chromatogram containing the alprazolam function). </p><p></p><p>My problem is that the baseline jumps up about two minutes prior to the peak elution and stays that way until the peak comes out. Once the peak elutes, it goes back down to the regular baseline. At first I thought it was peak fronting, but the baseline still stays relatively flat instead of sloping upward. I'm thinking it may be related to the actual analytes since the two are related compounds and I don't observe it with any of the other 16 peaks. </p><p style="height: 8pt"/><p>Has anyone seen this before with these compounds or any other compounds? Any ideas on how to fix it?</p><p style="height: 8pt"/><p>Thanks!</p><p>~ Kate </p>


  • dougm

    Hello. What is the column (chemistry) and flow rate you are using? If you inject a blank, what does the baseline look like?

  • I have seen an alprazolam chromatogram that is the mirror image of what you're seeing; the peak tail was long and nearly flat until it dropped back to baseline. The method was adopted from AOAC and was an isocratic LC-UV elution on a CN column using 0.01% H3PO4:ACN:MeOH. Our solution was to develop a new method. There seems to be something about alprazolam that lends itself to this strange peak shape.

  • I am using an HSS C18 1.8 um, 2.1x150 mm with a flow rate of 0.4 mL/min. I run a gradient from 5% organic to 60% organic. My standards/samples are in 5/95 B/A (the mobile phase starting conditions).

    My blanks are clean with no baseline problems.

  • Thanks for the info! I'll try to do some research to find out what other people suggest for methods. Hopefully a new method won't affect the 16 good peaks in the run too much Gotta love the fun of developing methods! :)

  • dougm

    Thanks for the response. The reason I asked about your column chemistry was do see what your pH operating range could be. Since you have the silica-based HSS column, high pH operation (e.g., pH 10) is not possible. I performed a quick search of the Waters website and I found that high pH provides pretty good peak shape for alprazolam using an older XTerra IS Column Separation and XTerra Analytical Column Separation. Both of these separations are at pH 10. If you have an ACQUITY UPLC BEH C18 or Shield RP18 column, perhaps one of these chemistries might provide better peak shape.

    There is a Waters Bioanalysis App Note that also shows how a method was developed using UPLC/MS. Interestingly, if you look at that App note (Fig. 4), the alprozolam peak tails on the HSS T3 chemistry, but not on any of the BEH chemistries.

    Even elevating the pH on your existing HSS C18 column to pH 5 - 6 (using acetate buffer/additive) may also provide improved peak shape as well based upon several applications on the Waters website and used conventional silica-based LC columns with an acetate buffer.

  • Thanks, Doug! That's really helpful. I was going along your thinking that it was column/pH related, too. I do have a BEH column, so I'll give the a whirl and will let you know how it goes.

    Thanks again! :)