Shifting Analyte Retention Times or Matrix Interferences?

Occasionally, we will get a Technical Service call about peak retention times moving during a customer analysis.  Early eluting compounds are especially prone to this shifting, but the issue can often be attributed to matrix interferences that are also not well retained.  Pregabalin and Gabapentin are two antiepileptic drugs typically analyzed by LC-MS/MS.  Because of their small, polar nature, they may be difficult to retain using reversed phase chromatography.

In our example of an antiepileptic drug panel, Pregabalin and Gabapentin are well retained on the Raptor Biphenyl column.

 These two analytes can be accurately quantitated and results are reproducible when looking across different column lots:

However, when analyzing urine samples using a simple dilute & shoot method, quantitation becomes a bit more difficult.   Matrix interferences may give the illusion of peaks moving within the chromatogram.     In the example below, a sample chromatogram with 100 ng/mL of antiepileptic drugs in human urine was diluted 5x with 0.1% formic acid in water.  The retention time of the analytes is remaining the same, but there are clear interferences from the matrix components.

If we take closer look, and extract the MRM transitions for pregabalin, we can clearly see matrix interferences that could make quantitation difficult.

The next time you see your analyte “shifting” retention time, I would recommend running a solvent standard to rule out the possibility of matrix interferences.     For more information on this and other challenging analyses, be sure to come to our Monday morning workshop at MSACL 2015 US in San Diego, CA.