Analysis of Quaternary Ammonium Compounds (QACs) as Possible Disinfectant Residues in Milk by LC-TOF

A P P L I C AT I O N N O T E
Liquid Chromatography/
Mass Spectrometry
Author:
Wilhad M. Reuter
PerkinElmer, Inc.
Shelton, CT
Analysis of Quaternary
Ammonium Compounds (QACs)
as Possible Disinfectant Residues
in Milk by LC-TOF
Introduction
Quaternary ammonium
compounds (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 particularly 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, these compounds may leach into the milk and, ultimately, may get into
the store-bought milk supplies at levels compromising personal health. Recent data
points to nearly 12% of all monitored milk to be tainted with QACs.1
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.
BAC 12 (expected mass in solution: 304.3004)
BAC 14 (expected mass in solution: 332.3317)
BAC 16 (expected mass in solution: 360.3630)
DDAC (expected mass in solution: 326.3781)
Figure 1. Chemical structures and expected masses of the four quaternary ammonium compounds (QACs) analyzed.
Regarding safety and regulations, the European Union
Reference Laboratory (EURL) has taken the following position:
All instrument control, analysis and data processing was performed
using the PerkinElmer Chromera® CDS software platform.
“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.
Method Parameters
The HPLC method parameters are shown in Table 1.
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 DDAC2 and BAC3 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.”4
Considering the above, this application note presents an
LC-TOF (time-of-flight) method for the analysis of the four
most common QACs that may be found in milk (a rather
complex matrix), with relatively little sample preparation. This
method takes advantage of the inherent mass accuracy and
high resolution afforded by TOF detection for specificity and
component identification. Method conditions and performance
data, including linearity and repeatability, are presented.
Experimental
Hardware/Software
For all chromatographic separations, a PerkinElmer Flexar
UHPLC System was used, including the Flexar FX-20 pump,
FX autosampler, column oven and AxION 2 TOF MS detector.
2
Table 1. HPLC Method Parameters.
HPLC Conditions
Column:
PerkinElmer Brownlee™ 2.7-µm 2.1 x 50-mm C18
(Part# N9308402)
Solvent A: 0.1% formic acid in water
Solvent B: 0.1% formic acid in acetonitrile
Solvent Program:
Mobile Phase:
Analysis Time:
Flow Rate:
Oven Temp.:
Detection:
Injection Volume:
Step
Time
Flow Rate
(mL/min)
%A
%B
Curve
0 (Equil.)
4.0
0.4
70
30
1
1
1.5
0.4
50
50
1
2
2.0
0.4
0
100
1
3
0.5
0.4
0
100
1
4 min.
0.4 mL/min. (maximum pressure: ~3900 psi)
25 ºC
Perkin Elmer AxION 2 TOF MS; positive ion mode;
mass range: 100-1250 MW
Ions 118.0862 and 922.0098 were used as lock mass
calibrants to compensate for any mass drift during
acquisition; spectral rate: 5 spectra/sec
MW channels (EICs): B AC 12: 304.300; BAC 14: 332.332;
BAC 16: 360.363; DDAC: 326.378
2 µL
Solvents, Standards and Samples
All solvents used were HPLC grade and filtered via 0.45-µm filters.
All QAC standards were purchased from Sigma-Aldrich
(St Louis, MO USA), including the following:
benzyldimethyldodecylammonium chloride (BAC 12),
benzyldimethyltetradecylammonium chloride (BAC 14),
benzyldimethylhexadecyl ammonium chloride (BAC 16), and
didecyldimethylammonium chloride (DDAC).
A 10-ppm stock standard solution was prepared by dissolving
10 mg of each of the four QAC components into 1 liter of
50:50 acetonitrile/water. This stock solution was then used to
prepare 1-ppm to 0.05-ppm standard solutions by serial dilution.
The analyzed product was a store-bought container of whole
milk. Using a 1-L solvent bottle, a spiked sample was prepared by
spiking 10 mg of each standard component into 1 liter of milk. Both
this spiked sample and an unspiked liter of milk were then placed on
a magnetic stirring plate and stirred for 30 minutes at 30 ºC. Both
samples were then capped and refrigerated until further use.
When ready for analysis, after allowing both samples to
equilibrate to room temperature, 20 mL of each sample was
transferred to a separate 50-mL polypropylene centrifuge tube.
20 mL of acetonitrile was then added to each tube, causing the
proteinaceous ingredients in the milk to precipitate (“protein
crash”). Both samples were then centrifuged at 8000 rpm for
five minutes. After centrifugation, 2 mL of each supernatant was
filtered through a 0.45 µm filter, transferred into a 2-mL sample
vial and subsequently injected.
Prior to injection, all calibrants were also filtered through 0.45-µm
filters, removing any small particles.
Results and Discussion
Figure 2 shows the chromatographic separation of the 0.5-ppm
QAC standard, separating the four QAC compounds in under
3.5 minutes. All displayed chromatograms consist of the overlays
of four EICs (extracted ion chromatograms), each corresponding
to the expected mass of the individual QAC components.
220
BAC 12
160
140
Intensity (Kcps)
DDAC
180
BAC 16
BAC 14
200
120
100
80
60
40
20
0
0.0
0.5
1.0
1.5
2.0
Time (min)
2.5
3.0
3.5
4.0
Figure 2. 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 3 shows the overlay of 10 replicate injections of a QAC standard mix, demonstrating exceptional reproducibility. The retention
time (RT) %RSDs ranged from 0.09 to 0.16.
350
300
250
150
Intensity (Kcps)
200
100
50
0
0.0
0.5
1.0
1.5
2.0
Time (min)
Figure 3. Overlaid chromatograms of 10 replicates of a preliminary standard mix.
2.5
3.0
3.5
4.0
3
Figure 4 shows the calibration plots over a concentration range of 0.05 to 1 ppm, with all four QAC components having quadratic
fit coefficients > 0.99 (n = 3 at each level).
BAC 12
BAC 14
R² = 0.99924
BAC 16
R² = 0.99975
DDAC
R² = 0.99882
R² = 0.99955
Figure 4. Plots of 5-level calibration set for all four QACs.
Figure 5 shows the averaged MS spectra for all four QAC components, highlighting the exceptional mass accuracy that was achieved
using the integrated lock mass option. These were based on the expected exact masses for each component in solution.
BAC 12
BAC 14
Expected Mass: 304.3004
Calculated Mass:304.3015
Mass Accuracy: 3.61 ppm
Expected Mass: 332.3317
Calculated Mass:332.3320
Mass Accuracy: 0.97 ppm
BAC 16
DDAC
Expected Mass: 360.3626
Calculated Mass:360.3617
Mass Accuracy: 2.51 ppm
Expected Mass: 326.3781
Calculated Mass:326.3777
Mass Accuracy: 1.23 ppm
Figure 5. Averaged MS spectra showing the mass accuracy achieved for each of the four QAC components.
4
The identity of the QACs was further confirmed with the help of
elemental composition matching via AxION EC ID software. Per
example shown in Figure 6, the accurate mass and isotope
information for DDAC was 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 the liquid-liquid extraction procedure described
earlier, an extracted sample of whole milk and the same whole
milk previously spiked with of 1-ppm QAC were analyzed.
The overlaid chromatograms (EICS) of both extracts are shown
in Figure 7.
The quantitative results for the spiked milk extract are shown in
Table 2. Considering that the liquid-liquid extraction procedure
involved a 1:1 sample dilution, the normalized amounts were
0.984, 0.730, 0.852 and 0.952 ppm, for BAC 12, BAC 14, BAC
16 and DDAC, respectively. Further refinements in the extraction
procedure may very well be able to improve on this recovery.
Also, in Figure 7, the expanded view of the four components
shows a small amount of QACs in the unspiked milk extract.
However, as shown in Figure 8, these amounts are very low and
are similar to levels observed for the 70% acetonitrile (ACN)
blank injections. Follow-up testing revealed this small amount of
QAC background to be due to slight column carryover. As this
level is far below the maximum allowable 0.5 mg/kg (ppm)
regulation QAC level set in the EU (the only global region that
has any regulations for this thus far), this background was
considered insignificant and quite acceptable.
Figure 6. Example of a database search for the formula confirmation of DDAC.
Table 2. Calculated/normalized amounts for QACs in 1 ppm-spiked whole milk extract.
Component
Calculated
Amount (ppm)
Normalized Amount
(ppm) – considering
1:1 dilution
% Recovery
0.492
0.365
0.426
0.476
0.984
0.730
0.852
0.952
98%
73%
85%
95%
BAC 12
BAC 14
BAC 16
DDAC
* All samples were run in duplicate, using averaged values
Whole Milk with 0.5ppm QAC : 5 PER(0:59800)PEIC(326.345:326.411),0.0326378,100, 3/24/2015
:1
11:43:06 AM
200
5.0
180
4.5
2.5
2.0
1.5
1.0
120
BAC 16
3.0
BAC 14
Intensity (Kcps)
Intensity (Kcps)
140
BAC 12
3.5
160
DDAC
4.0
0.5
0.0
100
-0.5
2.2
2.4
2.6
2.8
Time (min)
3.0
3.2
3.4
80
60
40
20
0
0.0
0.5
1.0
1.5
2.0
Time (min)
2.5
3.0
3.5
4.0
Figure 7. Chromatograms (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.
5
5.0
4.5
4.0
Intensity (Kcps)
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
-0.5
2.2
2.4
2.6
2.8
Time (min)
3.0
3.2
3.4
Figure 8. Chromatogram (EICs) of un-spiked milk extract overlaid with 70% ACN/water blank injection.
Conclusion
References
With rising health concerns and the large quantities of milk
that are consumed, it is important to have reliable procedures
for the monitoring of possible unhealthy contaminants in
dairy products. With this in mind, this work demonstrated the
fast/effective chromatographic separation for the quantitative
analysis of four quaternary ammonium compounds (QACs) in
milk by LC-TOF. The results exhibited exceptional reproducibility
with more than adequate sensitivity for monitoring down to
current regulated levels. In addition, by using a TOF detector,
the combination of averaged MS spectra, mass accuracy
checks and database search results allowed for the definitive
identification/confirmation of the four QAC components.
1. http://www.efsa.europa.eu/de/supporting/doc/483e.pdf
2.European Commission, 2012a. Guidelines as regards measures
to be taken as regards the presence of Didecyl Dimethyl
Ammonium Chloride (DDAC) in or on food and feed agreed by
the Standing Committee of the Food Chain and Animal Health
(SCoFCAH) on July 13, 2012 and modified on October 5, 2012.
3.European Commission, 2012b. Guidelines as regards measures
to be taken as regards the presence of Benzalkonium Chloride
(BAC) in or on food and feed agreed by the Standing
Committee of the Food Chain and Animal Health (SCoFCAH)
on July 25, 2012 and modified on October 5, 2012.
4.EU Reference Laboratory for Pesticides Requiring Single
Residue Methods, CVUA Stuttgart, Schaflandstr. 3/2, 70736
Fellbach, Germany.
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