Chromatography Forum

I use an Agilent 1100 HPLC coupled to AB Sciex 4000 QTrap to analyze some samples for multi-analytes (approx. 16) in the sequence: Blank (50% mobile phase), AQC (standards spiked 50% mobile phase), NQC (blank urine), same blank urine sample spiked with one analyte, positive QC (urine spiked with all analytes).

Unexpectedly, an analyte not spiked was detected in the urine sample in addition to the spiked analyte. 2 MRM transitions were positive and in high count (AQC: 4e6, urine sample: 3e6). The compound identity was further confirmed by Enhanced Product Ion full mass spectrum.

I did not set needle wash. But there was no carryover detected at a concentration of 5000 ng/mL in the validation experiments before (the concentration was greater than the analyte's spiked concentration in the AQC (100 ng/mL).

No analyte was detected in the NQC sample immediately after the AQC.

I wonder if carryover can occur to the urine sample even after an NQC in between.

Please help.

I would run the single analyte spiking sol'n to verify that there really is only one analyte in it.

Thanks.

I analyzed the preparation for spiking without extraction. The unexpected analyte was not detected. I now suspect there was contamination due to washed glassware. I washed the used Toxi-tube A with a single rinse of methanol. Then soak in water. Then use an automatic glassware washer (detergent added).

I wonder if the glassware wasn't clean enough.