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Validation of a Simple Spectrophotometric Method for the Measurement of

Quaternary Ammonium Compound Residue Concentrations in Food
Production Facility

Article in Food Analytical Methods · October 2012

DOI: 10.1007/s12161-012-9537-9

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Validation of a Simple Spectrophotometric
Method for the Measurement of
Quaternary Ammonium Compound
Residue Concentrations in Food Production
Facility
Zeinab E. Mousavi, Francis Butler &
Martin Danaher

Food Analytical Methods

ISSN 1936-9751

Food Anal. Methods

DOI 10.1007/s12161-012-9537-9

1 23
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 Author's personal copy
Food Anal. Methods
DOI 10.1007/s12161-012-9537-9

Validation of a Simple Spectrophotometric Method

for the Measurement of Quaternary Ammonium Compound
Residue Concentrations in Food Production Facility
Zeinab E. Mousavi & Francis Butler & Martin Danaher

Received: 18 September 2012 / Accepted: 15 November 2012

# Springer Science+Business Media New York 2012

Abstract Quaternary ammonium compound (QAC) residues Keywords Quaternary ammonium compounds . Residue .
can increase with each application and build up over a period of Food plant surfaces . Spectrophotometry
time during sanitation of food plants. Therefore, it is crucial to
establish a simple but accurate method for routine measurement
of QAC residues in food plants. In this study, a spectrophoto- Introduction
metric method was validated and applied for the quantification
of QAC residues on stainless steel surfaces. This method was Arising from concerns over the persistence of Cronobacter
based on the formation of a colored ion pair between the spp. in wet processing environments in powdered infant
quaternary ions and Eosin-Y in the presence of Triton X-100. formula manufacturing plants, many of these plants have
QAC residues were recovered from surfaces using swabs and switched to a “dry cleaning” protocol for the processing
were subsequently sonicated, and the isolated extracts were environment (Craven et al. 2010). This “dry cleaning” pro-
analyzed with a spectrophotometer (535 nm). The method tocol generally consists of regular vacuuming of the produc-
was shown to be selective in the presence of interfering sub- tion environment followed by the application of a biocide
stances, and a linear relationship between the absorbance and often containing Quaternary Ammonium Compounds
concentration of QACs was detected in the concentration range (QACs). QACs are economically important industrial bio-
from 0.5 to 10 mgL−1 (r2 >0.99). The limit of detection and cides widely used in food processing plants to minimize
limit of quantitation were 0.53 and 1.77 mgL−1, respectively. bacterial growth and subsequent biofilm formation (Oulahal
Satisfactory accuracy (93–97 %) and precision (relative stan- et al. 2008).
dard deviation (RSD)<2.7 %) were obtained for the method. In a dry cleaning process, QACs residues can increase
Assessment of the effect of swabbing protocol on the recovery, with each application and build up over a period of time,
repeatability and intermediate precision of the method revealed increasing the risk of chemical contamination of the produc-
that the mean recovery of residues was 90 %. Moreover, the tion process environment and possibly the final food prod-
percent RSD was less than 8.8 and 9.4 % for repeatability and uct. In addition, QACs residues are of concern, because they
intermediate precision, respectively. QAC residues were found can select for resistant pathogens and biofilm containing
to be stable on stainless steel surfaces for at least 6 days populations of bacteria, which is a potential risk to the
following deposition, a treatment that could be a potential risk consumer (Meyer 2006; Meyer and Cookson 2010; Méchin
especially in dry cleaning processes. et al. 1999; Bridier et al. 2011). Therefore, it is very impor-
tant to establish an accurate method for the careful measure-
ment of QACs residues in food plants. However, the method
Z. E. Mousavi (*) : F. Butler
should potentially be used as a routine analytical procedure,
School of Biosystems Engineering, Agriculture and Food Science
Centre, University College Dublin, Belfield, and therefore, provide simplicity, ease of use and time effi-
Dublin 4, Ireland ciency for the user.
e-mail: zeinab.moussavi@gmail.com A number of instrumental methods have been developed to
determine QACs, using gas chromatography (GC), nuclear
M. Danaher
Teagasc Food Research Centre, Ashtown, magnetic resonance, thin-layer chromatography coupled with
Dublin 15, Ireland flame ionization detection, capillary electrophoresis (CE) and
 Author's personal copy
Food Anal. Methods

liquid chromatography coupled with mass spectrometry (LC– Borax, Eosin-Y and Triton X-100 were purchased from Sigma
MS). However, these methods require sophisticated and ex- Aldrich, Ireland. Quadted Clear and Triquart (Johnson
pensive equipment, skilled technical staff, and are more suit- Diversy, Ireland) were used as commercial biocides containing
able for application in a reference laboratory (Heinig et al. QACs. RBS 25 (Chemical Products, R. Borghgraef, Brussels,
1997; Ford et al. 2002; Liu and Ding 2004; Koike et al. Belgium) and Divaushaum (Johnson Diversy, Ireland) were
2007; Wulf et al. 2010; Bester and Lamani 2010; Li and used as QACs-free commercial biocides.
Brownawell 2009; Martínez-Carballo et al. 2007; Núñez et
al. 2004). Stock Solutions Preparation
Spectrophotometry is increasingly employed in process
control due to its simplicity, availability and adaptability. A A 100 mgL−1 stock solution of Tetradecyl Dimethyl Benzyl
few spectrophotometric methods based on ion-pair forma- Ammonium Chloride was prepared by dissolving 0.01 g of
tion with an anionic dye such as Bromthymol Blue, Bromo- the standard compound in 100 mL distilled water. Eosin-Y
phenol Blue and Eosin-Y have been reported for the stock solution was prepared by dissolving 0.018 mg Eosin-
determination of QACs in pharmaceuticals (Lowry 1979; Y powder in 100 mL Borate buffer. Triton X-100 stock
Petrocci et al. 1983; Yamamoto 1995; Schep et al. 1995; solution was made by pipetting 500 μl Triton X-100 into a
Sakai and Hirose 2003). According to a literature survey 100 mL volumetric flask and diluting with borate buffer.
carried out by the authors, none of the mentioned methods Volumes containing 1 % solutions of commercial biocides
have been validated for QACs monitoring in food processing (Quadtet Clear, Triquart, RBS 25 and Divaushaum) were
environments. prepared by adding 1 mL of each biocide solution to a
In this study, a simple but accurate spectrophotometric 100 mL volumetric flask followed by dilution with distilled
technique was validated for the determination of QACs water. A blank solution for spectrophotometric analysis was
residues on the surfaces of food production facilities. The prepared by pipetting 0.5 mL Eosin-Y and 0.5 mL Triton X-
method involved the formation of ion-pairs of QACs with 100 into a 10 mL volumetric flask and diluting with 9 mL
an anionic dye (Eosin- Y) in the presence of micelles of the Borate buffer.
non-ionic surfactant Triton X-100. The complex forms a
purple color with maximum absorbance at 535 nm (Jones Borate Buffer Solution
and Schep 1995). Since swab sampling was selected for the
recovery of QACs residues from surfaces, the influence of Borate buffer solution for adjusting the pH to 8, was
swabbing protocol on the precision and accuracy of the prepared by mixing a 70–30 volume ratio of 0.02 M
spectrophotometric method was also investigated. The boric acid (12.368 gL−1) and 0.05 M Sodium Tetraborate
method would be useful for quality control sectors, since it (19.071 gL−1), respectively.
provides rapid monitoring of low levels of QACs resi-
dues deposited on stainless surfaces after cleaning and Procedure for Calibration Curve
disinfection procedures (in particular dry cleaning pro-
cess). After protocol development, the method was employed For preparation of different concentrations of standard
for the evaluation of QACs persistence on stainless steel solutions in the range of 0.5–10 mgL−1, aliquots (0.05–
surfaces over time following spraying QACs solutions on 1 mL) of the prepared stock solution (100 mgL−1) were
the surfaces. transferred into series of 10 mL volumetric flasks, and
subsequently, 0.5 mL of 0.018 gL−1Eosin-Y and 0.5 mL
of 0.5 % Triton X-100 solutions were added from the
Materials and Methods stock solutions to the 10 mL volumetric flasks. After-
wards, the mixtures were diluted with Borate buffer and
Apparatus the absorbance was read against the blank sample containing
all compounds except QACs. The adsorbance at 535 nm was
All of the spectrophotometric measurements were per- plotted versus the amounts should read concentrations, to
formed with a Double beam Unicam, UV-3 spectrophotom- produce a calibration curve.
eter (UK). A pH meter (Hannah HI 1280, UK) was used to
adjust the pH. Determination of Quaternary Ammonium Compounds
in Commercial Biocides (Method Suitability)
Chemicals
Initially, 100 μL of commercial biocides (Quadtet Clear and
Tetradecyl Dimethyl Benzyl Ammonium Chloride, Dodecyl Triquart) were transferred from the stock solutions into 10 mL
Dimethyl Benzyl Ammonium Chloride, Sodium Tetraborate, volumetric flasks and, were subsequently spiked with 100 μL,
 Author's personal copy
Food Anal. Methods

200 μL, 300 μL, 400 μL and 500 μL of Tetradecyl Dimethyl Borate buffer solution was pipetted into a 10 mL plastic tube. A
Benzyl Ammonium Chloride stock solution. In the next step, cotton swab (Text wipe, TX 775, Fisher Scientific, Ireland) was
0.5 mL Eosin-Y and 0.5 mL Triton X-100 were pipetted into immersed into the Borate buffer solution, and applied for
the flasks and the mixture were diluted with Borate buffer to wiping the stainless steel plate surface. The entire stainless steel
10 mL. Finally, the solutions were transferred into polystyrene plate was wiped in a horizontal and subsequently in a vertical
cuvettes (VWR, Ireland) and their absorbance in the spectro- direction, from the outside towards the center. The cotton swab
photometer was recorded. The adsorbance was plotted against was then returned to the tube containing Borate buffer, and a
amounts should read concentrations to obtain a standard ad- similar swabbing operation was repeated using a dry cotton
dition calibration graph. The y-intercept, was then used for swab. Finally, the Borate solution enclosing cotton swabs was
calculating the content of active substance (QACs) in the placed in a sonication bath for 15 min allowing QACs recov-
commercial samples. ered by the cotton swabs to be delivered to the Borate buffer
solution.
Biocide Delivering and Swabbing Protocol
Method Validation
The biocides solutions were delivered on stainless steel
plates using a spraying method. The plates (10×10 cm, 2B The analytical method was validated considering different
finish, 0.9 mm thickness, AMARI Ireland) were firstly validation parameters including selectivity, linearity, limit of
cleaned with ethanol, rinsed with distilled water and dried detection (LOD), limit of quantitation (LOQ), suitability,
before spraying. accuracy, precision and stability. In addition, the influence
Standard solutions with different concentrations (480, of swabbing technique on the percentage recovery of QACs
560, 686, 753 and 960 mgL−1) were prepared using Tetra- residues, inter-day precision and intra-day precision of the
decyl Dimethyl Benzyl Ammonium Chloride stock solution. proposed analytical method was assessed.
All of the prepared solutions were made up with distilled
water. In the next step, the prepared solutions were trans- Statistics
ferred consecutively into the solvent jar of a spray gun
(Radionics, Ireland) to be delivered on the plates. In order Analysis of variance (ANOVA) was performed using SAS
to achieve an integrated and uniform delivery of the solution version 9.1. Mean analysis using Duncan's multiple range
on the plates, the air pressure (supplied by the connection of tests at significance level of p<0.05 was performed if needed.
pressure valve of spray gun to the lab air supply) was
adjusted with a pressure gauge at 14.5 psi for all experi-
ments. The spray gun was equipped with an adjustable Results and Discussion
nuzzle in order to control the liquid flow and provide a
uniform aerosol. The spray gun was always maintained at Method Validation
45 cm from the plates. The plates were positioned vertically
by a clamp under a laminar hood. The stainless steel plates A linear trend between the absorbance and QACs concen-
were weighed before and after biocide delivery using a high tration was observed in the range of 0.5–10 mgL−1. The
precision scale (±0.001 g Sartorius, BP161P, Germany) to coefficient of determination (r2) obtained from the regres-
evaluate the weight of sprayed solution on each plate. Since sion line was 0.999 (Fig. 1). The spectroscopic technique
the concentration of the sprayed solution was known, the studied in this work is based on a calibration curve for
following formula was employed to calculate the net weight
of the QACs delivered on the plates: 0.6
Absorbance (535 nm)

Ws 0.5 y = 0.044x + 0.023

Concentration of QACs ¼  Ci ð1Þ R² = 0.999
Vb 0.4

where: 0.3
Ws 0Weight of sprayed solution 0.2
Vb 0Volume of buffer in the tube (we used 10 mL) 0.1
Ci 0Initial solution concentration 0
After spraying the solution on the plates, the swabbing 0 2 4 6 8 10 12
procedure was performed for biocide recovery according to Concentration (mg L-1)
the protocol developed previously(Akl et al. 2011; Klinkenberg Fig. 1 Plot of absorbance (at 535 nm) as a function of Tetradecyl
et al. 2003; Madalina et al. 2005; Qin et al. 2010; Nozal et al. Dimethyl Benzyl Ammonium Chloride concentration (The error bars
2000, 2001; Menezes Filho et al. 2000). Initially, 10 mL of represent standard deviation of three replications)
 Author's personal copy
Food Anal. Methods

quantitative analysis, and the limit of detection (LOD) and Table 2 Determination of QACs in the presence of interfering substan-
ces. Three different levels of each interfering substance were studied and
limit of quantification (LOQ) were determined using the
the highest level exhibiting no interference is exhibited (Sucrose: 1.2, 2.4
approach previously reported (Sarkar et al. 2006). The LOD and 4.8 %; NaCl: 0.35, 0.7 and 2.1 %; Skimmed milk powder: 1, 3 and
and LOQ were determined to be 0.53 and 1.77 mgL−1, 5 %; Divaushaum: 0.5, 1 and 1.5 %; RBS 25: 0.25, 0.5 and 0.75 %)
respectively.
Interfering Amount added %QAC %RSD (n03)
The accuracy of the spectrophotometric method is pre- Substance (wt %) recovery
sented in Table 1. A 4.75 μgmL−1 standard solution was
spiked with known amounts of standard solutions (0.9, 2.06, Sucrose 9.6 98.2 0.58
2.73, and 4.75 μgmL−1) and the values measured were com- NaCl 3 93 2.74
pared with the known added values. It is evident from Table 1, Skimmed milk Powder 5 82 1.65
that the accuracy was in the range of 93–97 %. The results Divaushaum 1.5 96 2.16
showed that the spectrophotometric method developed for the RBS 25 0.75 98 0.14
determination of QACs can be considered as an accurate
method within the concentration range investigated.
The precision of the method, reported as relative standard protein and fat in the milk with QACs (Chinard 1948). How-
deviation (RSD), was estimated by measuring repeatability ever, the recovery of QACs in the presence of skimmed milk
of the measurement for three replicates at five different powder was also reasonable with a value of 82±1.65 %,
concentrations (0.9, 2.06, 2.73, and 4.75 μgmL−1). The % although an extraction procedure in a case were milk or dairy
RSD values were less than 3 % and illustrated a good products are present would enhance the measurement results.
precision of the analytical method (Table 2). In addition to the accuracy results which confirmed the
The specificity of the method to quantify QACs was selectivity of the method, relative standard deviation (%RSD)
assessed in the presence of probable interfering substances. values where lower than 2.74 %, confirming a good precision
Three different levels of potential commonly interfering sub- of the spectrophotometric method for the evaluation of QACs
stances in food processing facilities (NaCl, Sucrose, skimmed at the presence of interfering substances.
milk powder) and two QACs-free biocides (Divaushaum and The stability of analytical reagents and test samples were
RBS 25), were spiked into a solution with a known amount of determined following 1, 2, 3, 4, and 6 days storage at room
Tetradecyl Dimethyl Benzyl Ammonium Chloride (5 μg and refrigerated temperatures. Solutions were analyzed and
mL−1), and the relevant absorbance and concentrations were compared with a freshly prepared standard at each time inter-
recorded. Maximum concentration levels of each interfering val. No degradation was observed during this period, which
substances which did not interfere with the determination of indicates that samples can be stored for at least 1 week prior to
QACs in the standard solution are shown in Table 2. The analysis if necessary (data not shown).
recovery of the target QACs (Tetradecyl Dimethyl Benzyl
Ammonium Chloride) in the presence of all interfering com- Method Suitability
pounds was 93 % or higher, except in spiked skimmed milk
powder that had a lower percentage recovery compared to In order to evaluate the method suitability, the amount of
other substances. The lower recovery of the QACs detected in QACs available in 1 % solutions of commercial biocides
the presence of skimmed milk powder might be arising from (Quadtet Clear and Triquart) was quantified using the standard
different reasons including the interactions between the addition method by adding increasing known amounts of pure
standard solutions (Tetradecyl Dimethyl Benzyl Ammonium
Table 1 Results of recovery and repeatability of the spectrophotomet- Chloride) (0, 1, 2, 3, 4 mgL−1) to the commercial solutions.
ric method for the determination of quaternary ammonium compounds Then, the absorbance of the prepared solutions was recorded
in solutions
in the spectrophotometer and the absorbance was plotted
Amount added Amount found Accuracy(%)b Repeatability against concentration of spiked QACs (Fig. 2). The intercept
(ppm) (ppm)a (%RSD)c of regression lines of the plotted data would indicate the
concentration of QACs in the commercial solutions. Accord-
0.90 0. 83±0.03 92.96±0.03 0.57
ing to the regression lines shown in Fig. 2, the slopes of the
2.06 1.93±0.19 93.95±0.19 2.85
trend lines obtained for both commercial solutions spiked with
2.73 2.65±0.02 97.16±0.02 0.32
standard solutions are approximately similar, revealing the
4.75 4.56±0.26 96.07±0.26 2.81
suitability of the method for the determination of QACs in
a
Values are means of 3 replications± standard deviations individual biocides,. The absorbance of non-spiked commer-
b
Accuracy (%): (Obtained value/spiked value) × 100 cial samples was 0.12 and 0.28, respectively (Fig. 2), indicat-
c
Repeatability (%RSD) 0standard deviation of concentration mea- ing that in comparison with Triquart, the amount QACs
sured/mean of concentration measured available in Quadtet Clear is higher. From the regression
 Author's personal copy
Food Anal. Methods

0.5 20.0

Concentration change
y = 0.041x + 0.284 Quadtet Clear

Absorbance (535 nm)

0.4 R² = 0.997
10.0 BAC Quadted Clear
Triquart
0.3 aa a a

(%)
0.0
0.2 a a
a a
y = 0.046x + 0.1274 -10.0 a a a a
0.1
R² = 0.984

0 -20.0
0 2 4 6 0 1 2 3 4 5 6
Spiked level (mg L-1 )
Time (day)
Fig. 2 Plot of absorbance of two commercial biocides (Quadtet Clear
Fig. 3 Changes of the concentration of standard Tetradecyl Dimethyl
and Triquart) containing QACs (at 535 nm) as a function of different
Benzyl Ammonium Chloride and Quadted Clear within 6 days sprayed
spiked concentrations of Tetradecyl Dimethyl Benzyl Ammonium
on the stainless steel sheet. Mean values with the same letter in each
Chloride (1, 2, 3 and 4 mgL−1)
individual group are not significantly different (P>0.05) by Duncan's
multiple Comparison Test (The error bars represent standard deviation
equation obtained in Fig. 2, the concentration of active sub- of three replications)
stance in Quadtet Clear and Triquart could be calculated
which were 5.3 and 2.27 mgL−1, respectively. for different solution concentrations. The mean recovery of
QACs residues was approximately 90 %, revealing a suitable
Validation of QACs Recovery from Surfaces by Swabbing recovery from stainless steel with the applied swabbing proto-
Protocol col. Repeatability and intermediate precision of the swabbing
procedure were calculated from the same samples used for
After validation of the spectrophotometric method for the recovery studies. The results, expressed as %RSD, are shown
quantification of QACs, in the next step, the validity of the in Table 3. The % RSD values obtained were less than 8.8 and
swabbing technique to recover QACs residues from surfaces 9.4 % for repeatability and intermediate precision, respectively,
was evaluated. Therefore, the recovery, repeatability and intra- and revealed a good precision of the swabbing procedure.
day precision of the swabbing method was examined. Differ-
ent solutions containing 480, 560, 686, 753 and 960 μgmL−1 Application of the Validated Method to Routine Analysis
Tetradecyl Dimethyl Benzyl Ammonium Chloride (3 repli-
cates for each concentration level), were prepared and then The swabbing method was applied for residue measurement
sprayed onto the stainless steel plates with a predefined of Quadtet Clear, built up on stainless steel sheets, within
100 cm2 surface area (The weight of sprayed QACs on surfa- 6 days. The highest recommended concentration for using
ces (μg) was calculated using Eq.1). The plates were then Quadtet Clear was 1 %, in which, 530 mgL−1 QACs, as
stored at room temperature until the surface was dried. The active substance, was measured by the aid of the protocol
residues at the surfaces were collected by using cotton swabs described in previous sections. As shown in Fig. 3, there
according to the protocol described in the material and meth- was no significant change of active substance in Quadtet
ods section. Since higher recoveries are achieved by using two Clear after 6 days. This indicated that the QACs residues
cotton swab (Akl et al. 2011; Nozal et al. 2000), we collected were not degraded after spraying, and remained stable on
the residues using double swabbing technique. Analysis of the stainless steel surface for 6 days. Therefore, QACs may
variance (ANOVA, P00.05) was carried out for all concen- be deposited on the surfaces after several sprays and this can
tration levels to satisfy that the recovery was not dependent on be a potential risk of chemical contamination of the final
the concentration level and that the mean recovery could, product and appearance of resistant pathogenic bacteria. It
therefore, be calculated. Table 3 illustrates the recovery data should be, however, noted that there are many different

Table 3 The influence of swab-

bing protocol on the precision, Intraday studies (n03) Inter-day studies (3 days)
repeatability and intermediate
precision of the swabbing Sprayed QACs solution %Recovery %RSD %Recovery %RSD (Intermediate
procedure concentration (μgmL−1) (Repeatability) precision)

480 81±4.7 5.8 81±4.6 2.8

560 88±7.4 8.4 85±5.6 1.6
686 90±8.8 9.8 90±8.8 9.3
753 100±6.0 5.9 94±7.8 9.9
960 93±8.8 9.4 96±6.5 3.3
 Author's personal copy
Food Anal. Methods

factors affecting the stability of QACs including relative Bridier A, Briandet R, Thomas V, Dubois-Brissonnet F (2011) Bio-
fouling 27:1017
humidity, surface properties and spraying conditions which
Heinig K, Vogt C, Werner G (1997) J Chromatogr A 781:17
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Liu HY, Ding WH (2004) J Chromatogr A 1025:303
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