Figure 2A: Chromatogram of 0.5-ppm QAC standard; EIC channels: BAC 12: 304.300; BAC 14: 332.332; BAC 16: 360.363; DDAC: 326.378.
Figure 2B: Overlaid chromatograms of 10 replicates of a preliminary QAC standard mix.
So how accurate are the measurements at the lower levels of detection? Figure 3 shows the calibration plot of BAC 14 over a concentration range of 0.05 to 1 ppm. A quadratic fit R2 value of >0.999 demonstrates good linearity, assuring accuracy of results within the calibration range. Similar results were observed for the remaining three QACs.
Figure 3: Plots of 5-level calibration set for the BAC 14 (n=3 at each level).
The averaged MS spectra for all four QAC components are shown in Figure 4, highlighting the mass accuracy that was achieved using the integrated lock mass option. These were based on the expected exact masses for each component in solution.
Figure 4: Averaged MS spectra showing the mass accuracy achieved for each of the four QAC components.
The identity of the QACs was further confirmed with the help of elemental composition matching via AxION EC ID software. The accurate mass and isotope information for DDAC was simply entered into the software and searched against a selected database, in this case, PubChem. The search resulted in an elemental composition that perfectly matched DDAC.
Following a liquid-liquid extraction procedure, an extracted sample of whole milk and the same whole milk previously spiked with of 1-ppm QACs were analyzed. The overlaid chromatograms (EICS) of both extracts are shown in Figure 5. As shown in the expanded view, though trace levels of QACs were detected in the unspiked milk, none of them were above quantifiable limits.
Figure 5: EICs of 1 ppm-spiked whole milk extract (four large peaks) overlaid with that of the unspiked milk extract. Due to the very low levels, the EICs of the unspiked milk extract can only be seen in the expanded view.
Secured Milk Safety
With rising health concerns and the large quantities of milk that are consumed, it is imperative to have reliable procedures for the monitoring of possible unhealthy contaminants in dairy products. With this in mind, we have demonstrated the fast and effective chromatographic separation for the quantitative analysis of four QACs in milk by LC-TOF, with minimal sample preparation. The results exhibited exceptional reproducibility with more than adequate sensitivity for monitoring down to the current regulated levels in both the U.S. and in Europe. By using a TOF detector, the combination of averaged MS spectra, mass accuracy checks, and database search results allowed for the definitive identification and confirmation of the four QAC components.
In addition, such methodology has the benefits of offering the potential for horizon scanning. Not only are the compounds of interest identified and quantified, but as the whole mass spectrum of data is collected, any unexpected compounds can also be identified helping to prevent contamination scares before the milk is widely distributed.
Dr. Vosloo is the senior leader of strategy and global applications at PerkinElmer. Reach her at [email protected]. Reuter is senior LC and LC-MS strategic applications leader at PerkinElmer. He can be reached at [email protected].
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