Within the food industry, chemicals are routinely used to disinfect and sanitize food contact surfaces. These chemicals are necessary to ensure the continued safety of the food and drinks we consume—preventing microorganisms that could cause illness. There is a balance to strike; too little chemicals applied can result in inadequate efficacy, whilst too much can yield residues that do not meet regulatory standards.
Quaternary ammonium compounds (QACs) are surface-active substances often used as disinfectants. They are also used as biocides, pesticides, and as additives for technical applications.
QACs have the basic structure NR4+. Those possessing R groups with long alkyl chains are known to be especially effective as antimicrobial agents and particularly useful for the disinfection of containers and surfaces. This is especially relevant in the milk industry, as QACs are typically used to disinfect the insides of tanks used for transporting milk from farms to processing plants. If significant QAC residues are left behind after tank disinfection, allowing these compounds to leach into the milk and, ultimately, getting into the store-bought milk supplies at levels compromising personal health. Recent data points to nearly 12 percent of all monitored milk to be tainted with QACs. The primary QACs that may be found in milk are benzyldimethyldodecylammonium chloride (BAC 12), benzyldimethyltetradecylammonium chloride (BAC 14), benzyldimethylhexadecyl ammonium chloride (BAC 16), and didecyldimethylammonium chloride (DDAC). Their chemical structures and expected parent masses in solution are shown in Figure 1.
Regarding safety and regulations, the European Union Reference Laboratory has taken the following position: “Because no specific maximum limit for residues of DDAC and BAC was established under EU Regulation No. 396/2005, the general residue limit of 0.01 mg/kg applies. In October 2012, the Standing Committee on the Food Chain and Animal Health (SCoFCAH) endorsed guidelines on measures to be taken regarding the presence of DDAC and BAC in or on food and feed. It was recommended that EU Member States carry out investigations on the possible causes of BAC/DDAC contamination and to put in place a monitoring program to get an overview of the BAC and DDAC levels in all food and feed of plant and animal origin. Considering that the current default MRLs for DDAC and BAC (of 0.01 mg/kg) are not a health standard, a temporary enforcement level of 0.5 mg/kg was agreed upon. As no specific residue definition was defined, there is still uncertainty as to how residues are to be expressed. Based on the first results of the monitoring program, a lower enforcement level for QACs is under discussion.”
The use of QACs is also regulated in the U.S. by the EPA under “Code of Federal Regulations (CFR)—40 CFR part 180. Once applied, the allowable residues and their subsequent monitoring are the responsibility of the U.S. FDA. Of course, the task of ensuring that the chemicals are prepared and applied properly avoiding inappropriate residues rests with the food processors.
With regulatory guidance in place, we pursued a LC-TOF (liquid chromatography time-of-flight) method for the analysis of the four most common QACs that may be found in milk. This technology takes advantage of the inherent mass accuracy and high resolution afforded by TOF detection for specificity and component identification.
As milk itself is a rather complex matrix (containing many components including proteins, fats, vitamins, and minerals), a further objective was to develop a rapid analysis method requiring relatively little sample preparation.
Path to Accurate Results
Standards and samples were analyzed using a PerkinElmer Flexar UHPLC System and AxION 2 TOF Mass Spectrometry detector. The analyzed product was a store-bought container of whole milk, spiked with standard solutions from 1 parts per million (ppm) to 0.05 ppm. Samples were injected after protein precipitation, centrifugation and filtration. Figures 2a and 2b show the chromatographic separation of the 0.5-ppm QAC standard, single injection and the replicates, separating the four QAC compounds in under 3.5 minutes.
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.
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.
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.
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@example.com. Reuter is senior LC and LC-MS strategic applications leader at PerkinElmer. He can be reached at firstname.lastname@example.org.