Hello smart people. I can to you in my time of need. I am at wits end trying to validate my methodology to move forward with my thesis lab work dealing with environmental concentrations of antibiotics in marine sediment.

I was able to get very nice calibration curves for all 3 of my analytes (r^2 of .98 plus) using a series of 8 stardards in sequence from 0ng/uL to 2ng/uL. The issue I am finding is when I run my spiked samples to validate the method is reproducible. I have prepared both spiked sediment samples and samples solutions of analytes and internal standards to try and validate. I used Quan-Lynx to process my samples with my calibration curve and the measured concentrations of my analytes (and internal standards) are no where near my calculated spiking concentrations. But at least the sample triplicates are close together.

I am very new to analytical chem so I am really lost in the deep end here. I am unsure if my issue stems from a problem with my calibration, a problem with my processing method, problems with my stock solutions, or just because of my low experience.

For additional background: I had 2 spiked concentrations (high and low each in triplicate) for my spiked sediment samples along with a blank sediment sample. All samples were spiked with the same concentrations of Internal Standards prior to solvent extraction with SPE-cleanup. The final reconstitution is in 10:90 MeOH:MilliQ + 0.1% Formic Acid and this is what my original calibration samples were preppared in as well. I also made additional samples of just analytes and internal standards in the same MeOH:MilliQ solution to see if the issue was with my extraction process. However, I am still running into the same issue that my processed sample concentrations are far off from my expected calculated concentrations. This MeOH:MilliQ solution is different from my starting gradient eluent which is 5% ACN with 0.5% formic acid, but the rentention times I am getting all still look very nice.

I am ready to provide more background if needed. Any help is seriously appreciated 🙏🏼