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The Case of the Inseparable Opiates

I was developing a 78 drug Pain Panel and was having problems with the separation of my opiates; namely Morphine and Hydromorphone.

The chromatogram below illustrates what I was seeing:

I had diluted my standard in 50:50 Methanol/Water. My mobile phase was Water/Methanol starting at 90% Water. Opiates elute first in the aqueous phase. The Application Scientist told me to adjust the gradient to get the peaks to merge. However, after struggling for a few days I was unable to get any sort of satisfactory separation.

I watched Josh Hayden's presentation Getting going with mass spectrometry: Josh learns chromatography, and he had a similar issue, and he found that changing the solvent he was using to dilute the standard resolved his chromatography problem.

This made me think that I should look at my dilution solvent. I tested 100% methanol, 100% Water and 100% Urine Matrix. The 100% methanol matrix gave me unexpected separation and I saw two distinct peaks for morphine. The 100% Water and 100% Urine Matrix gave me perfect separation and chromatography.

In conclusion I had too much organic in my diluent and a simple change in matrix resolved my problem.