Evaluation of quaternary ammonium compounds residues in food ...

Evaluation of Quaternary Ammonium Compounds Residues in Food PlantsSurfaces: Method Validation and Residue PersistenceZeinab E. Mousavi 1 *, Francis Butler 1 , Martin Danaher 21 School of Biosystems Engineering, Agriculture and Food Science Centre, UniversityCollege Dublin, Belfield Dublin 4, Ireland2Teagasc Food Research Centre, Ashtown, Dublin 15, Ireland*Corresponding author:E-mail: zeinab.moussavi@gmail.comAbstractA simple spectrophotometric method was validated and applied for the quantification ofQACs residues on stainless steel surfaces in food processing environments. QACresidues were recovered from surfaces using cotton swabs. Residues were subsequentlymeasured in isolated extracts using a spectrophotometer operating in the visible region at535 nm. The selectivity of the method was investigated through application of the methodto commercial cleaning products known to be free of QACs. The method wassubsequently validated where linearity, precision, accuracy, limit of detection (LOD), andlimit of quantification (LOQ) and stability were investigated. Calibration curves were foundto be linear over the range of 0.5 to 10 mg L -1 (r 2 > 0.99). The LOD and LOQ of themethod were determined to be 0.53 and 1.77 mg L -1 , respectively. The accuracy andprecision of the method was investigated through the application of the method tostainless steel surfaces sprayed with 0.48, 0.560, 0.686, 0.753 and 0.960 mg L -1 of QACon three different days. Recovery was typically in the range 81 to 97%. Intra and interdaymethod precision were 8.8% and 7.8 %, respectively. QAC residues were found to bestable on stainless steel surfaces for at least six days following disposition to stainlesssteel surfaces.Key words: Quaternary Ammonium Compounds, Residue, Food Plant Surfaces,Spectrophotometry1. IntroductionQuaternary ammonium compounds (often known as QACs) are economically importantindustrial biocides widely used in food processing plants to minimise bacterial growth andsubsequent biofilm formation (Oulahal et al., 2008). They are a class of salts derivedfrom ammonium in which a nitrogen atom is attached to four aryl or alkyl groups (Jia etal., 2001). QACs are positively charged cations, hence, their mode of action is related totheir attraction to negatively charged materials such as bacterial proteins (Caillier et al.,2009) and their antimicrobial activities depend on the alkyl chain length (Li & Brownawell,2009). In sanitizing procedures, these chemicals are generally sprayed on surfaces andtheir residues may persist over time resulting in a risk of chemical contamination of thefinal product. In addition, QACs residues are of concern because they can result in theformation of the resistant pathogens or biofilms, which are a potential risk to theconsumer(Bridier et al., 2011; Meyer & Cookson, 2010). Residue persistence hasparticular relevance for the powdered infant formula industry. Arising from concerns overthe persistence of Cronobacter spp. in wet processing environments in powdered infantformula manufacturing plants, many of these plants have switched to a ‘dry cleaning’protocol for the processing environment (Craven et al., 2010). This ‘dry cleaning’ protocolgenerally consists of regular vacuuming of the process environment followed by theapplication of a sanitizer often containing QAC. In the absence of a wet clean, QACresidues can potentially build up over a period of time.

Simple and reliable analytical methods are required for continuous monitoring of QACresidues in a factory environment. Several methods have been developed formeasurement of QAC residues.. A number of instrumental methods have beendeveloped to determine QACs using gas chromatography (GC), nuclear magneticresonance, thin-layer chromatography coupled with flame ionization detection, liquidchromatography (LC) and capillary electrophoresis (CE) and liquid chromatographycoupled with mass spectrometry (LC–MS). However, these methods require sophisticatedequipment, technical staff and are more suitable for application in a laboratoryenvironment (Bester & Lamani, 2010; Wulf et al., 2010). In addition, somespectrophotometric methods based on the application of ion-pairing compounds such asanionic dye (Eosin-Y) have been reported for the determination of QACs (Sakai & Hirose,2003; Schep et al., 1995; Yamamoto, 1995). The majority of the analytical methodsmentioned above have been developed for detecting and quantifying QACs in drinkingwater, waste water, effluents or sludges. The objective of this study was to validate andapply a simple but accurate analytical method that allows the recovery and determinationof low levels of quaternary ammonium compounds residues deposited on stainless steelsurfaces. The method would be useful to the quality control sector of food processingfactories. The instrument required for residues monitoring, a spectrophotometer, is widelyavailable in industrial plants which make this techniques readily applicable to the foodindustry.2. Materials and methods2.1. Reagents and apparatusReagents - Dimethyl tetradecyl benzalkonium chloride, dimethyl dodecyl benzalkoniumchloride, sodium tetraborate, Borax, Eosin Y, Triton 100-X were purchased from SigmaAldrich, Germany. Quadted Clear and Triquart (Johnson Diversy, Ireland) were used asthe commercial QAC containing biocides. RBS 25 (Chemical Products, R. Borghgraef,Brussels, Belgium) and Divaushaum (Jhonson Diversy, Ireland) were used as QAC freecommercial disinfectant and detergents. Cotton swabs ITW Text wipe, TX 775 (FisherScientific, Ireland) were used for swabbing surfaces. All spectrophotometricmeasurements were performed using a Unicam, UV-3 spectrophotometer (UK). AHannah HI 1280 (UK) was used for to measure pH.2.2. CalibrationA 100 mg L -1 stock solution of dimethyl tetradecyl benzalkonium chloride, 0.9 mg L -1 EosinY and 0.025% v/v Triton X-100 solutions were prepared in borate buffer (pH 8). Workingstandard solutions (0.5, 2, 4, 6, 8, 10 mg L -1 ) were prepared by stock solution dilution toget a calibration curve.2.3. Sample preparationA 1% solution of commercial biocides (% recommended in the Material Safety Data Sheetof Commercial Quadtet Clear and Triquart) was prepared using distilled water. By thestandard addition method, different volumes of working standard solutions (1, 2, 3, 4, 5mg L -1 ) were added to the commercial solutions was also added prior to be diluted up withborate buffer to volume of 10 mL. The blank solution contained borate buffer, 0.5 mLEosin Y and Triton X-100.The adsorbance was plotted against the correspondingamounts added to obtain a standard addition calibration graph. The x-intercept, was thenused for calculating the content of active substance in the commercial sample analysed.2.4. Spray and swabbing protocolA series of 100 mL standard solutions were prepared in distilled water (0.48, 0.56, 0.686,0.753 and 0.96 mg L -1 ). 10 × 10 cm stainless steel plates (2B finish, 0.9 mm thickness,AMARI Ireland) were thoroughly cleaned and dried before spraying. They were positioned

vertically by a clamp under a laminar hood. An adjustable spray gun operated withcompressed air (Radionics, Ireland) was connected to a clean air supply in the lab andthe air pressure was controlled by a pressure gauge. The pressure was set to 2 bars forall experiments. The distance between the plate and spray gun was fixed (45 cm) and thespray gun nuzzle was adjusted to get a uniform aerosol. The spray gun was filled with 100mL of prepared solutions. The stainless steel plates were weighed before and afterspraying using a high precision scale (±0.001 g Sartorius, BP161P, Germany) to evaluatethe weight of adheredsolution. The swabbing protocol was developed based on protocolsdeveloped by other groups (Akl et al., 2011; Klinkenberg et al., 2003; Madalina B. B.,2005; Menezes Filho et al., 2010; Qin et al., 2010). 10 mL of buffer solution wasdispensed into a plastic tube. A cotton swab was immersed into the borate buffer andimmediately used to swab the stainless steel surfaces. The entire stainless steel platewas swabbed in a horizontal and subsequently in a vertical direction, from the outsidetowards the centre. The protocol was repeated with a dry cotton swab. Both cotton swabswere returned to the buffer containing tube and sonicated for 15 min.2.5. Method validationThe analytical method reported here has been validated considering selectivity, linearity,limits of detection (LOD), quantitation (LOQ), suitability, accuracy and precision. For theswab sampling technique selected, the influence of swabbing on the recovery of QACsresidues and the precision of the analytical method was evaluated. The stability ofstandards and samples was also studied.2.6. StatisticsSamples were analysed in triplicate and analysis of variance (ANOVA) was performedusing SAS version 9.1. Mean analysis using Tukey’s multiple range tests at significancelevel of p

standard deviation (RSD%) ranged from 2.8 to 8.4 and 4.6 to 7.8%, respectively. Inaddition, repeatability (intra-day precision) of the method in the three different days wasgood and intermediate precision of the assay was 5.28-7.52%, confirming the suitability ofthe method for daily routine analysis.TABLE 1: Recovery results (Mean ±SD) of the swabbing technique after spraying QACsolutions with different concentrations on stainless steel sheets for intra-day and inter-dayprecision and accuracy evaluationSprayed solution concentration (mg L -1 )Day 480 560 686 753 960Day1 Sprayed QAC (µg) 21.5±1.6 24.6±1.4 29±0.8 25.6±1.2 36.5±2.7QAC recovered (µg) 17.5±2.1 21.7±1.0 26±3.3 26.1±2.7 33.9±3.4Recovery (%) 81±4.7 88±7.4 90±8.8 100±6.0 93±8.8RSD (%) 5.8 8.4 9.8 5.9 9.4Day 2 Sprayed QAC (µg) 17.9±2.0 24.3±2.0 27.1±2.2 28.4±2.7 34.2±4.1QAC found (µg) 14.8±1.0 22.6±2.1 25.5±4.9 25.5±2.5 34.7±3.2Recovery (%) 86±7.2 89±8.2 97±9.9 90±8.4 102±7.5RSD (%) 8.4 9.2 10.2 9.4 7.4Day 3 Sprayed QAC (µg) 19.2±4.1 19.2±4.8 24.5±3.3 23.2±2.3 34.2±0.9QAC found (µg) 15.8±0.3 17.3±1.9 22.2±3.2 20.7±1.0 32.8±1.1Recovery (%) 77±2.2 79±1.2 83±7.7 90±9.0 95±3.1RSD (%) 2.8 1.6 9.3 9.9 3.3Average recovery (%) 81±4.6 85±5.6 90±8.8 94±7.8 96±6.5Inter-day precision (RSD%) 5.6 6.4 9.8 8.4 6.73.1. Method suitabilityIn order to validate the method suitability, the volume of active substance QACs availablein the 1% commercial detergent Quadtet Clear and Triquart (which were further diluted100 time) was also quantified using the standard addition method by adding increasingknown amounts of pure standard solutions (Dimethyl tetradecyl benzalkonium chloride)(0, 1, 2, 3, 4 mg L -1 ) to the commercial ssolutions. Then, the absorbance of the preparedsolutions containing a constant amount of the commercial biocides spiked with differentlevels of standard solution was read in the spectrophotometer. According to theregression lines shown in Fig. 2, the slope of the two commercial solutions spiked withstandard solutions are approximately similar revealing that the method is reliable fordifferent commercial biocides. As observed in the Fig. 2, the absorbance of non-spikedcommercial samples is 0.12 and 0.28 respectively, revealing that in comparison withTriquart, the amount QACs available in Quadtet Clear is higher. By referring to regressionequation obtained Fig. 1, the concentration of active substance in Quadtet Clear andTriquart are 5.3 and 2.27 mg L -1 , respectively.3.2. Residue monitoring of commercial biocideThe swabbing method was applied for residue measurement of Quadtet Clear built up onstainless steel sheets within 6 days. The highest recommended concentration for usingQuadtet Clear was 1%, in which, 530 mg L -1 quaternary ammonium compounds, as activesubstance, was measured by the aid of the suggested protocol described in previoussections. As shown in Fig. 3, there was no significant change from the initial concentrationof active substance in Quadtet Clear. This indicated the QACs residues did not degradeafter spraying and remained stable on the stainless steel surface. Therefore, QACs maybe deposited on the surfaces after several sprays and this can be a potential risk ofchemical contamination of the final product and appearance of resistant pathogenicbacteria. It should be, however, noted that there are many different factors affecting thestability of QACs including relative humidity, surface properties and spraying conditionswhich were not considered in this study.

FIGURE 2: Absorbance of two selected 1 % commercial biocides (Quadtet Clear andTriquart) containing QACs, spiked with different levels of benyzl dimethyl tetradecylammonium chloride (1, 2, 3 and 4 mg L -1 ).The error bars represent standard deviation ofthree replications)FIGURE 3: Changes of the concentration of standard benyzl dimethyl tetradecylammonium chloride and Quadted Clear within 6 days sprayed on the stainless steel sheet(mean values with the same letter in individual group are not significantly different(P>0.05) by Tukey’s multiple Comparison Test (The error bars represent standarddeviation of three replications).4. ConclusionsResults showed that the spectrophotometric method proposed for the quantification ofQACs is a rapid and convenient method with a reasonable limit of detection (as low as0.53 mg L -1 ) and sensitivity for quantifying purposes that can be easily used as a routinemethod for residues analysis of quaternary ammonium compounds in food processingenvironments. Results also illustrated that QACs remained stable during time (up to 6days) and may accumulate on equipment surfaces after consecutive spraying forsanitizing purposes which can be harmful in terms of chemical toxicity and microbialresistance. More experiments are necessary to study the effect of different parameters onthe stability of QACs and to evaluate the residues in real plant conditions.5. ReferencesAkl, M.A., Ahmed, M.A., Ramadan, A. (2011). Validation of an HPLC-UV method for thedetermination of ceftriaxone sodium residues on stainless steel surface of pharmaceuticalmanufacturing equipments. Journal of Pharmaceutical and Biomedical Analysis, 55, 247-252.Bester, K., Lamani, X. (2010). Determination of biocides as well as some biocidemetabolites from facade run-off waters by solid phase extraction and high performance

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