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Bioactive paper platform for detection of hydrogen peroxide in milk
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy xxx (xxxx) xxx
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Bioactive paper platform for detection of hydrogen peroxide in milk Liliane Spazzapam Lima, Eduardo Luiz Rossini, Leonardo Pezza, Helena Redigolo Pezza ⁎ Instituto de Química, UNESP – São Paulo State University, R. Prof. Francisco Degni 55, P.O.Box 355, 14800-900 Araraquara, SP, Brazil
a r t i c l e
i n f o
Article history: Received 2 August 2019 Received in revised form 4 November 2019 Accepted 5 November 2019 Available online xxxx Keywords: Hydrogen peroxide Milk adulteration Bioactive paper platform Digital image
a b s t r a c t This paper describes the development and application of a paper-based analytical device (μPAD) for the determination of hydrogen peroxide, an important adulterant in milk. The method employs the reaction between hydrogen peroxide and guaiacol, catalyzed by peroxidase, producing a red product, which is then quantified by digital imaging. Experimental design methodology was used to optimize the experimental conditions. The linear concentration range was from 12.5 × 10−4 to 150 × 10−4 mol L−1, resulting in the regression equation AB = 0.02466 (±0.00192) + 17.053 (±0.750) C, with an excellent correlation coefficient (r = 0.986). The relative standard deviations obtained were 1.1 and 1.3% (intra-day), and 4.8 and 2.9% (inter-day), for 25.0 × 10−4 and 100 × 10−4 mol L−1 of hydrogen peroxide, respectively. The limits of detection and quantification were 3.54 × 10−4 and 11.8 × 10−4 mol L−1, respectively, with standard deviation of the blank of 0.002012. The proposed method was successfully applied for the determination of peroxide in milk samples, with recoveries between 92.2 and 109%. The proposed device constitutes a valuable analytical tool for the identification of hydrogen peroxide adulteration and offers advantages including low cost, simplicity, portability, and no (or minimal) requirement for sample pretreatment. © 2019 Elsevier B.V. All rights reserved.
1. Introduction Milk is one of the most complete foods for humans, containing nutrients including proteins, minerals, fats, carbohydrates, and vitamins, and is widely marketed and consumed by the population [1,2]. The composition of bovine milk is a combination of several solid components (12–13%) in water (approximately 87%). These components, their distributions, and interactions determine the structure and functional properties of the milk, together with its suitability for processing and consumption [3]. This rich composition makes milk an excellent substrate for the growth of various groups of microorganisms, both desirable and undesirable [2]. Due to their metabolism, microorganisms can easily degrade milk. To prevent this from happening, preservative substances are fraudulently added to decrease microbial growth and thus increase the product shelf life. These substances include hydrogen peroxide, sodium hypochlorite, formaldehyde, salicylic acid, and even potassium dichromate [4,5]. Hydrogen peroxide is one of the most versatile industrial chemicals. It is an environmentally-friendly oxidant that is widely employed as a bleaching agent in foods, textiles, and personal care products. Other applications include its use as an oxidant in wastewater treatment, and as a catalyst, antiseptic, and disinfectant [4,6]. The adulteration of milk by the addition of hydrogen peroxide is used to increase the shelf life and facilitate marketing of the product, due to the preservative ⁎ Corresponding author. E-mail address: [email protected] (H.R. Pezza).
characteristic of the contaminant [4]. Hydrogen peroxide is used fraudulently to halt microbial activity [6] in milk close to the expiry date or already unsuitable for consumption. The use of hydrogen peroxide as a preservative in fluid milk is forbidden in several countries, due to the toxic effects, while in places where treatment with this additive is permitted, such as the United States (only for the production of cheeses), its percentage may not exceed 0.05% of the weight of the milk [7,8]. Its addition leads to a decrease in the nutritional value of the food, due to the destruction of vitamins A and E, and it can have harmful effects on the health of the general population [8], especially individuals who are immunologically more fragile, such as children, the elderly, and immunocompromised persons [4,9]. Current milk quality control procedures aim to prevent fraud and the adulteration of the product in natura, but the decomposition of hydrogen peroxide makes it difficult for the contaminant to be detected and quantified in these matrices. Analytical procedures [4] including HPLC-UV [10,11], spectrophotometry [12], amperometry [13], fluorimetry [8], and portable spectroscopy using chemometric tools [8,14] have been described in the literature. The fluorimetric method [7] is highly sensitive, but the procedure requires a prior sample preparation step, which is a limitation of this method for routine application. Methods employing chemometric tools usually present high limits of detection and quantification and low sensitivity [15], while chromatographic methods require a trained operator, a laboratory to perform the analysis, expensive and sophisticated instruments, and clean-up steps. Therefore, it is necessary to develop new and sensitive methods for the rapid
https://doi.org/10.1016/j.saa.2019.117774 1386-1425/© 2019 Elsevier B.V. All rights reserved.
Please cite this article as: L.S. Lima, E.L. Rossini, L. Pezza, et al., Bioactive paper platform for detection of hydrogen peroxide in milk, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, https://doi.org/10.1016/j.saa.2019.117774
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L.S. Lima et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy xxx (xxxx) xxx
and reliable detection of this kind of adulteration. In addition, portability and simplicity of the method are very important features for in situ analysis. Paper is emerging as a simple, low-cost, disposable, widely available, and versatile platform for the development of new analytical methodologies [16]. The paper-based analytical device (μPAD) is a promising technology, due to its inherent advantages: no external pump or force is required to transfer the sample to the device; it provides a highcontrast background for colorimetric detection; paper allows simple or no preparation steps; and it is easy to use, portable, and requires low volumes of reagent and sample [16–18]. Consequently, μPAD devices are used in different areas, including biological [19–21], environmental [22,23], and food safety applications [23,24]. To the best of our knowledge, there is a lack of reports concerning the use of a bioactive paper platform, combined with digital imaging, for the detection of adulteration by hydrogen peroxide in milk samples. The present work describes the development of a microfluidic paper-based analytical device (μPAD) for the determination of hydrogen peroxide in milk, with colorimetric detection by digital imaging. The method employs the reaction between hydrogen peroxide and guaiacol, catalyzed by peroxidase, producing a red product. Experimental design methodology was used to optimize the experimental conditions. The proposed device constitutes a valuable analytical tool for the identification of hydrogen peroxide adulteration, offering advantages including low cost, simplicity, minimal environmental impact, portability, and no requirement for sample pretreatment, which are highly desirable for in-situ analysis. 2. Materials and methods 2.1. Reagents and standard solutions Preparation of all the solutions and dilutions employed deionized water (18.2 MΩ cm), obtained from a Milli-Q system (Millipore), and Class A glassware. All reagents used were analytical grade. A standard solution of sodium oxalate (9.40 × 10−3 mol L−1) was prepared by dissolving 0.3149 g of the dry salt (Sigma-Aldrich) in 250.00 mL of 1.00 mol L−1 sulfuric acid. This solution was used for standardization of a solution of 0.02505 mol L−1 potassium permanganate, as described elsewhere [25]. A standard solution of 8.10 × 10−2 mol L−1 hydrogen peroxide was prepared by dilution of 4.27 mL of stock solution (Êxodo Científica) to 500.00 mL with water. This solution was standardized with potassium permanganate solution [26] and was subsequently used to prepare the other working standard solutions (12.5, 15.0, 25.0, 50.0, 75.0, 85.0, 100, and 150 × 10−4 mol L−1). A solution of 5.50 × 10−2 mol L−1 vanadium pentoxide was prepared by dissolution of 1.00 g of the compound in sulfuric acid (6% v/ v). The solution was kept in an ultrasonic bath until total solubilization of the salt and was then transferred to a 100 mL volumetric flask, completing the volume with the same solvent. Guaiacol (99%, Dinâmica) solutions were prepared in 1:1 ethanol: water. The solutions were always freshly prepared and were kept in an ice bath. 2.2. Obtaining the enzymatic substrate The peroxidase enzyme was obtained from a plant source (zucchini), as described previously by Fatibello [27], with minor modifications. After washing and drying, the part closest to the bark was collected and 25.00 g of this raw plant tissue was weighed and chopped. These pieces were homogenized in a household blender, together with 100 mL of 0.1 mol L−1 phosphate buffer (pH 6.5) and 2.50 g of polyvinylpyrrolidone (PVP). The homogenate was then filtered through four layers of gauze and centrifuged twice for 10 min at 9000 rpm, increasing the speed gradually, in order to avoid heating the enzyme. The solution
obtained was divided into several aliquots, in 1.5 mL polystyrene tubes, and was stored in a refrigerator at 4 °C. 2.3. Determination of peroxidase activity in the crude plant extract The peroxidase activity in the crude extract was determined following the spectrophotometric procedure described in the literature [28], which uses guaiacol as substrate. 2.4. Fabrication and preparation of the μPADs The preparation of the μPADs was based on previous work [18], with some modifications. CorelDRAW X5 was used to design the μPAD. The analytical device consisted of a circular channel with internal diameter of 7 mm. The design was printed on a Whatman No. 1 qualitative filter paper with wax toner (Genuine Xerox Solid Ink Black), using a wax printer (ColorCube 8580). After printing, the paper was heated at 120 °C, for 120 s, in order to form the hydrophobic barrier. 2.5. Stability of the colored product The stability of the red product on the surface of the paper platform was evaluated by monitoring the spot every 5 min, during 1 h. 2.6. Optimization of variables The effects of the main significant parameters, namely the guaiacol concentration and the volume of the plant extract, were evaluated using response surface methodology. The pH of the reaction medium was maintained at 6.5, due to the buffer used in enzyme extraction, and was the optimum pH for peroxidase [29]. All the statistical calculations were performed using Statistica 7.0 software. 2.7. Analytical method The colorimetric determination of hydrogen peroxide was based on the reaction of the analyte with guaiacol, using peroxidase as a catalyst. For this, a 1.6 μL volume of guaiacol solution was added to the circular channel of the device, followed by addition of 2.8 μL of the plant extract. After total absorption of the added aliquots, 0.8 μL of sample was added, resulting in a red tetraguaiacol product. For quantitative determination, the device was digitized and the intensity of the blue color (RGB format) obtained in the reaction was determined using ImageJ software [18]. The effective intensity of the blue color (AB) was calculated using the equation AB = − log (BS/BB), as defined by Abbaspour [30], where BS and BB are the blue color intensities of the sample and the blank, respectively. The effective intensities could be used to quantify hydrogen peroxide using the analytical curves. 2.8. Qualitative analysis Three commercial samples and one raw milk sample were tested for the presence of hydrogen peroxide, following the procedure recommended by AOAC [31]. For qualitative analysis, 10 mL of the sample and six drops of 1% vanadium pentoxide were added to a test tube. The non-formation of a red coloration indicated the absence of hydrogen peroxide in the sample. 2.9. UHT sample preparation The UHT (ultra-high temperature) process is a continuous flow heat treatment, where the milk is preheated, homogenized (before or after sterilization), sterilized, cooled, and aseptically packed. The process uses temperatures of between 140 and 150 °C, for up to 8 s, in order to sterilize the product [32].
Please cite this article as: L.S. Lima, E.L. Rossini, L. Pezza, et al., Bioactive paper platform for detection of hydrogen peroxide in milk, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, https://doi.org/10.1016/j.saa.2019.117774
L.S. Lima et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy xxx (xxxx) xxx
The samples were purchased locally in the city of Araraquara (São Paulo State, Brazil). Three samples of UHT milk with different percentages of fat (whole, reduced fat, and low fat milk) and free from hydrogen peroxide were spiked by addition of a suitable aliquot of 8.10 × 10−2 mol L−1 hydrogen peroxide standard solution, in order to obtain a concentration of 85.0 × 10−4 mol L−1. The fortified concentration was selected based on the legislation and was below the maximum limit established by the FDA for the presence of peroxide in milk [7]. All the samples were analyzed according to the recommended procedure. No pretreatment steps were required. 2.10. Raw sample preparation The sample of raw milk was obtained from a local producer. Since the milk naturally contained the peroxidase enzyme in its composition, the sample was heated at boiling for 20 min, in order to deactivate and degrade this enzyme [33]. After cooling to room temperature, the sample was then prepared for analysis, as described in Section 2.9.
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Fig. 2. Example of spot test reaction between guaiacol and H2O2, in the absence (A) and presence (B) of peroxidase. The red compound (tetraguaiacol) formed only in the presence of the enzyme was the reaction product used for colorimetric determination of hydrogen peroxidase in milk samples. Order of reaction: guaiacol + H2O2 for A; guaiacol + peroxidase + H2O2 for B. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
3.1. Determination of the peroxidase activity of the crude plant extract
Validation of the proposed method was performed using a standard additions procedure that enabled evaluation of the presence of possible matrix effects associated with the commercial and raw milk samples. The samples with 85.0 × 10−4 mol L−1 of H2O2 were spiked with 50, 100, 150, and 200% of the initial concentration. The samples were then analyzed as described in Section 2.4.
The activity of the peroxidase present in the crude extract was determined according to the spectrophotometric procedure described in the literature [28], which uses guaiacol as substrate. In this procedure, the peroxidase activity is determined by measuring the change in absorbance at 470 nm, due to the formation of tetraguaiacol in the enzymatic reaction. One unit of peroxidase activity represents the amount of enzyme that catalyzes the oxidation of 1 μmol of guaiacol in 1 min. In the present case, 1 mL of the crude extract could catalyze 64 × 10−6 mol of guaiacol in 1 min. The enzymatic activity, in U mL−1, was determined as follows:
3. Results and discussion
U mL−1 ¼
2.11. Standard additions
This work reports the development of a methodology for the determination of hydrogen peroxide in milk samples. The colorimetric method was based on the oxidation of guaiacol to tetraguaiacol (a red compound) by hydrogen peroxide, in the presence of peroxidase (Fig. 1) [28]. In preliminary tests, the reaction between guaiacol and H2O2 did not occur in the absence of peroxidase (Fig. 2), so it was necessary to add the enzyme. The zucchini plant is an inexpensive, readily available, renewable, and eco-friendly source of peroxidase enzyme that can be extracted using a simple and fast method. The paper platform impregnated with the peroxidase enzyme showed no changes for at least 2 months, when stored in a refrigerator at 4 °C. The formation of the colored product occurred immediately after addition of the analyte, although the spot readings were performed after complete drying (10 min). The stability of the colored product was evaluated as described in Section 2.5, which showed that the effective intensity measurement remained constant for at least 1 h at room temperature (25 °C). The blue channel of the RGB color format was selected for the detection method, due to its higher sensitivity.
ð1Þ
where U mL−1 is the units of peroxidase activity per mL, A is the absorbance of the colored product at 470 nm, ε is the molar absorptivity of tetraguaiacol (26,600 L mol−1 cm−1), Ve is the plant extract volume (mL), t is the reaction time, and DF is the dilution factor of the crude enzyme extract. 3.2. Optimization of variables A central composite design was used to obtain the optimal conditions for the reaction. In these experiments, the effects of the guaiacol concentration and the volume of the enzymatic extract were studied at five levels (−√2, −1, 0, 1, √2). The points (levels) of a central composite design are coded units √2 distant from the central point (coded as zero point), so all the peripheral points lie on the same circumference. The extreme points (±√2) were established for a two-factor design [34]. Table 1 shows the matrix of the central composite design. The Table 1 Matrix of the central composite design. N°
1 2 3 4 5a 6a 7a 8 9 10 11 Fig. 1. Reaction between guaiacol and hydrogen peroxide, catalyzed by peroxidase.
A 1 1 : :DF: :1000 ∈ Ve t
a
Uncoded variable levels
Coded variable levels
[Guaiacol] (%)
Enzymatic extract (μL)
[Guaiacol]
Enzymatic extract
0.33 1.73 0.33 1.73 1.03 1.03 1.03 0.05 2.00 1.03 1.03
0.9 0.9 3.1 3.1 2.0 2.0 2.0 2.0 2.0 0.5 3.5
−1 +1 −1 +1 0 0 0 −1.41 +1.41 0 0
−1 −1 +1 +1 0 0 0 0 0 −1.41 +1.41
Central point in triplicate (n = 3).
Please cite this article as: L.S. Lima, E.L. Rossini, L. Pezza, et al., Bioactive paper platform for detection of hydrogen peroxide in milk, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, https://doi.org/10.1016/j.saa.2019.117774
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experiment corresponding to the central point was carried out using three replicates [35]. The resulting response surface is shown in Fig. 3, as a function of the guaiacol concentration and the volume of the enzymatic extract. It can be seen from the surface shape that the optimal region was found and that the maximum response was achieved when the guaiacol concentration was 1.39% and the volume of the enzymatic extract was 2.80 μL. The quadratic regression model describing the effective intensity in the blue channel (AB) was as follows: AB = −0.0716 + 0.168[Guaiacol] - 0.0609[Guaiacol]2 + 0.0961Vperoxidase - 0.0174V2peroxidase + 0.000539 [Guaiacol] Vperoxidase. It should be noted that although the pH was not studied as an experimental parameter in this experimental design, the medium was maintained at pH 6.5 during the entire extraction process. This pH was used for H2O2 determination, because it is the optimum pH for peroxidase [29]. 3.3. Analytical data The proposed methodology was validated by determination of the linear dynamic range, precision, limit of detection (LOD), limit of quantification (LOQ), and the influence of the matrix, using the standard additions technique [36,37]. After optimization of the variables, a linear calibration curve was constructed for hydrogen peroxide concentrations from 12.5 × 10−4 to 150 × 10−4 mol L−1. The calibration data (n = 8) resulted in the regression equation: AB = 0.02466 (±0.00192) + 17.053 (±0.750) C, where AB is the blue channel effective intensity and C is the hydrogen peroxide concentration (in mol L−1). The correlation coefficient was 0.986, indicating excellent agreement between the data and the regression model for the calibration curve (Fig. 4). LOD and LOQ were calculated using the expressions: LOD = 3 × SDBlank/B and LOQ = 10 × SDBlank/B, where SDBlank is the standard deviation of ten measurements of the blank (n = 10) and B is the slope of the analytical curve [36]. The LOD and LOQ were 3.54 × 10−4 and 11.8 × 10−4 mol L−1 (0.001 and 0.004% m/m), respectively, with standard deviation of the blank of 0.002012. The repeatability of the method was assessed by repetition of two points of the analytical curve, at different times on the same day (intra-day) and on different days (inter-day). The coefficients of variation obtained were 1.1 and 1.3% (intra-day), and 4.8 and 2.9% (interday), for 25.0 × 10−4 and 100 × 10−4 mol L−1 of hydrogen peroxide, respectively. These results demonstrated that the proposed method was
Fig. 4. Spots for the H2O2 concentrations of 12.5, 15.0, 25.0, 50.0, 75.0, 85.0, 100, and 150 × 10−4 mol L−1 used to construct the analytical curve.
repeatable and that the analytical device could be used for the quantification of hydrogen peroxide in milk, since all the values were lower than 10%. 3.4. Analytical application The effectiveness of the proposed procedure using the microfluidic device for determination of hydrogen peroxide in real samples was evaluated using UHT and raw milk samples. The values obtained (Table 2) showed excellent agreement with the spiked amounts of hydrogen peroxide. In the case of the raw milk, the sample was heated at boiling for 20 min, in order to degrade and deactivate the peroxidase naturally present in this type of sample. This treatment was necessary because the presence of peroxidase in the sample could lead to reaction conditions different from those established following the response surface experiments. The UHT milk samples did not require such pretreatment, since the industrial processing of this type of milk eliminates this enzyme. The raw milk samples previously spiked with the hydrogen peroxide standard were analyzed as described in Section 2.9. The results presented in Table 2 demonstrated that the proposed procedure using the microfluidic device could be successfully applied for the determination of hydrogen peroxide in raw milk. 3.5. Standard addition and recovery The proposed procedure was validated in recovery experiments performed with the UHT and raw milk samples. The sample treatment is described in Section 2.9 and the analysis procedure is presented in Section 2.7. The results (Table 3) showed recoveries in the range from 92.2 to 109%, indicating the absence of any significant matrix effects in the application of the technique for the determination of hydrogen peroxide in milk samples. There have been few reports in the literature concerning methods for the detection and quantification of hydrogen peroxide in milk [8,10,12–15]. Methodologies involving chemometric techniques have Table 2 Determination of hydrogen peroxide added to milk samples.
Fig. 3. Central composite design response surface obtained for the intensity of the blue color as a function of the guaiacol concentration and the enzymatic extract volume. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Samples
Hydrogen peroxide added (mol L−1)
Hydrogen peroxide founda (mol L−1)
Whole Reduced fat Low fat Raw
85.0 × 10−4 85.0 × 10−4 85.0 × 10−4 85.0 × 10−4
86.9 × 10−4 83.6 × 10−4 84.4 × 10−4 85.2 × 10−4
a
Average of four determinations.
Please cite this article as: L.S. Lima, E.L. Rossini, L. Pezza, et al., Bioactive paper platform for detection of hydrogen peroxide in milk, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, https://doi.org/10.1016/j.saa.2019.117774
L.S. Lima et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy xxx (xxxx) xxx Table 3 Recovery data for hydrogen peroxide spiked in milk samples. Samples
Whole
Reduced fat
Low fat
Raw
a
Standard added (mol L−1)
Found valuea (mol L−1)
Recovery (%, mean ± RSD)
0 42.5 × 10−4 85.0 × 10−4 128 × 10−4 170 × 10−4 0 42.5 × 10−4 85.0 × 10−4 128 × 10−4 170 × 10−4 0 42.5 × 10−4 85.0 × 10−4 128 × 10−4 170 × 10−4 0 42.5 × 10−4 85.0 × 10−4 128 × 10−4 170 × 10−4
87.8 × 10−4 46.2 × 10−4 84.4 × 10−4 133 × 10−4 174 × 10−4 85.0 × 10−4 44.0 × 10−4 86.4 × 10−4 118 × 10−4 175 × 10−4 88.9 × 10−4 42.9 × 10−4 88.5 × 10−4 116 × 10−4 171 × 10−4 86.4 × 10−4 45.3 × 10−4 89.2 × 10−4 128 × 10−4 166 × 10−4
– 108.8 ± 5.0 99.3 ± 0.7 104.2 ± 6.4 102.2 ± 5.0 – 103.6 ± 5.6 101.6 ± 1.9 92.2 ± 0.9 103.0 ± 3.2 – 100.9 ± 5.8 104.1 ± 0.7 98.7 ± 2.4 100.4 ± 6.0 – 106.6 ± 4.2 105.0 ± 1.9 99.6 ± 5.2 97.7 ± 6.1
Average of four determinations.
been reported, which are rapid and simple, without requirement for sample pretreatment, but the levels of adulteration detected were much higher than those used here, and were also much higher than the maximum limit established in USA legislation [15,38]. Other methodologies with lower sensitivity require sample preparation employing hazardous chemicals [8,14]. Several methodologies require specialized operators and the use of much higher quantities of reagent solutions and samples, compared to the paper platform-based method. The sample preparation and analysis procedures involve several steps that are time consuming, and the large volumes of liquid waste generated may be hazardous for the environment and for the operator. Furthermore, such techniques do not enable in situ analysis, since the equipment is not portable [8,10,12–15]. The advantage of the device described here is that the analysis can be performed in situ by a person with minimal training, using a low-cost platform, without the need for expensive equipment. The digital image could be acquired and analyzed using a smartphone, or could be transmitted to a specialist via the internet. Due to the simplicity of the paper platform, a semi-skilled person at a farm with minimal infrastructure, or at an industrial site, could use the test to detect hydrogen peroxide adulteration.
4. Conclusions This work presents a validated bioactive paper platform for determination of an important adulterant of milk. Chemometric tools were used to obtain the optimized conditions for the quantification of hydrogen peroxide, and the methodology was successfully validated. The use of a μPAD, together with colorimetric detection by digital imaging, provides a simple, portable, inexpensive, and eco-friendly procedure for the determination of hydrogen peroxide in UHT and raw milk samples. The proposed methodology is fast, accurate, and precise. UHT milk samples can be analyzed without the need for any sample preparation steps, while a simple boiling step is required for raw milk. The method does not produce liquid waste and the paper platform can be easily incinerated after analysis. The simplicity of the methodology enables in situ analysis using a camera or a smartphone to obtain an image of the paper platform. The analytical device developed in this work is suitable for use as a screening tool, since the LOD obtained for determination of hydrogen peroxide (11.8 × 10−4 mol L−1, or 0.004% m/m) is lower than the value permitted according to current legislation.
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Bioactive paper platform for detection of hydrogen peroxide in milk Liliane Spazzapam Lima, Eduardo Luiz Rossini, Leonardo Pezza, Helena Redigolo Pezza ⁎ Instituto de Química, UNESP – São Paulo State University, R. Prof. Francisco Degni 55, P.O.Box 355, 14800-900 Araraquara, SP, Brazil
a r t i c l e
i n f o
Article history: Received 2 August 2019 Received in revised form 4 November 2019 Accepted 5 November 2019 Available online xxxx Keywords: Hydrogen peroxide Milk adulteration Bioactive paper platform Digital image
a b s t r a c t This paper describes the development and application of a paper-based analytical device (μPAD) for the determination of hydrogen peroxide, an important adulterant in milk. The method employs the reaction between hydrogen peroxide and guaiacol, catalyzed by peroxidase, producing a red product, which is then quantified by digital imaging. Experimental design methodology was used to optimize the experimental conditions. The linear concentration range was from 12.5 × 10−4 to 150 × 10−4 mol L−1, resulting in the regression equation AB = 0.02466 (±0.00192) + 17.053 (±0.750) C, with an excellent correlation coefficient (r = 0.986). The relative standard deviations obtained were 1.1 and 1.3% (intra-day), and 4.8 and 2.9% (inter-day), for 25.0 × 10−4 and 100 × 10−4 mol L−1 of hydrogen peroxide, respectively. The limits of detection and quantification were 3.54 × 10−4 and 11.8 × 10−4 mol L−1, respectively, with standard deviation of the blank of 0.002012. The proposed method was successfully applied for the determination of peroxide in milk samples, with recoveries between 92.2 and 109%. The proposed device constitutes a valuable analytical tool for the identification of hydrogen peroxide adulteration and offers advantages including low cost, simplicity, portability, and no (or minimal) requirement for sample pretreatment. © 2019 Elsevier B.V. All rights reserved.
1. Introduction Milk is one of the most complete foods for humans, containing nutrients including proteins, minerals, fats, carbohydrates, and vitamins, and is widely marketed and consumed by the population [1,2]. The composition of bovine milk is a combination of several solid components (12–13%) in water (approximately 87%). These components, their distributions, and interactions determine the structure and functional properties of the milk, together with its suitability for processing and consumption [3]. This rich composition makes milk an excellent substrate for the growth of various groups of microorganisms, both desirable and undesirable [2]. Due to their metabolism, microorganisms can easily degrade milk. To prevent this from happening, preservative substances are fraudulently added to decrease microbial growth and thus increase the product shelf life. These substances include hydrogen peroxide, sodium hypochlorite, formaldehyde, salicylic acid, and even potassium dichromate [4,5]. Hydrogen peroxide is one of the most versatile industrial chemicals. It is an environmentally-friendly oxidant that is widely employed as a bleaching agent in foods, textiles, and personal care products. Other applications include its use as an oxidant in wastewater treatment, and as a catalyst, antiseptic, and disinfectant [4,6]. The adulteration of milk by the addition of hydrogen peroxide is used to increase the shelf life and facilitate marketing of the product, due to the preservative ⁎ Corresponding author. E-mail address: [email protected] (H.R. Pezza).
characteristic of the contaminant [4]. Hydrogen peroxide is used fraudulently to halt microbial activity [6] in milk close to the expiry date or already unsuitable for consumption. The use of hydrogen peroxide as a preservative in fluid milk is forbidden in several countries, due to the toxic effects, while in places where treatment with this additive is permitted, such as the United States (only for the production of cheeses), its percentage may not exceed 0.05% of the weight of the milk [7,8]. Its addition leads to a decrease in the nutritional value of the food, due to the destruction of vitamins A and E, and it can have harmful effects on the health of the general population [8], especially individuals who are immunologically more fragile, such as children, the elderly, and immunocompromised persons [4,9]. Current milk quality control procedures aim to prevent fraud and the adulteration of the product in natura, but the decomposition of hydrogen peroxide makes it difficult for the contaminant to be detected and quantified in these matrices. Analytical procedures [4] including HPLC-UV [10,11], spectrophotometry [12], amperometry [13], fluorimetry [8], and portable spectroscopy using chemometric tools [8,14] have been described in the literature. The fluorimetric method [7] is highly sensitive, but the procedure requires a prior sample preparation step, which is a limitation of this method for routine application. Methods employing chemometric tools usually present high limits of detection and quantification and low sensitivity [15], while chromatographic methods require a trained operator, a laboratory to perform the analysis, expensive and sophisticated instruments, and clean-up steps. Therefore, it is necessary to develop new and sensitive methods for the rapid
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Please cite this article as: L.S. Lima, E.L. Rossini, L. Pezza, et al., Bioactive paper platform for detection of hydrogen peroxide in milk, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, https://doi.org/10.1016/j.saa.2019.117774
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and reliable detection of this kind of adulteration. In addition, portability and simplicity of the method are very important features for in situ analysis. Paper is emerging as a simple, low-cost, disposable, widely available, and versatile platform for the development of new analytical methodologies [16]. The paper-based analytical device (μPAD) is a promising technology, due to its inherent advantages: no external pump or force is required to transfer the sample to the device; it provides a highcontrast background for colorimetric detection; paper allows simple or no preparation steps; and it is easy to use, portable, and requires low volumes of reagent and sample [16–18]. Consequently, μPAD devices are used in different areas, including biological [19–21], environmental [22,23], and food safety applications [23,24]. To the best of our knowledge, there is a lack of reports concerning the use of a bioactive paper platform, combined with digital imaging, for the detection of adulteration by hydrogen peroxide in milk samples. The present work describes the development of a microfluidic paper-based analytical device (μPAD) for the determination of hydrogen peroxide in milk, with colorimetric detection by digital imaging. The method employs the reaction between hydrogen peroxide and guaiacol, catalyzed by peroxidase, producing a red product. Experimental design methodology was used to optimize the experimental conditions. The proposed device constitutes a valuable analytical tool for the identification of hydrogen peroxide adulteration, offering advantages including low cost, simplicity, minimal environmental impact, portability, and no requirement for sample pretreatment, which are highly desirable for in-situ analysis. 2. Materials and methods 2.1. Reagents and standard solutions Preparation of all the solutions and dilutions employed deionized water (18.2 MΩ cm), obtained from a Milli-Q system (Millipore), and Class A glassware. All reagents used were analytical grade. A standard solution of sodium oxalate (9.40 × 10−3 mol L−1) was prepared by dissolving 0.3149 g of the dry salt (Sigma-Aldrich) in 250.00 mL of 1.00 mol L−1 sulfuric acid. This solution was used for standardization of a solution of 0.02505 mol L−1 potassium permanganate, as described elsewhere [25]. A standard solution of 8.10 × 10−2 mol L−1 hydrogen peroxide was prepared by dilution of 4.27 mL of stock solution (Êxodo Científica) to 500.00 mL with water. This solution was standardized with potassium permanganate solution [26] and was subsequently used to prepare the other working standard solutions (12.5, 15.0, 25.0, 50.0, 75.0, 85.0, 100, and 150 × 10−4 mol L−1). A solution of 5.50 × 10−2 mol L−1 vanadium pentoxide was prepared by dissolution of 1.00 g of the compound in sulfuric acid (6% v/ v). The solution was kept in an ultrasonic bath until total solubilization of the salt and was then transferred to a 100 mL volumetric flask, completing the volume with the same solvent. Guaiacol (99%, Dinâmica) solutions were prepared in 1:1 ethanol: water. The solutions were always freshly prepared and were kept in an ice bath. 2.2. Obtaining the enzymatic substrate The peroxidase enzyme was obtained from a plant source (zucchini), as described previously by Fatibello [27], with minor modifications. After washing and drying, the part closest to the bark was collected and 25.00 g of this raw plant tissue was weighed and chopped. These pieces were homogenized in a household blender, together with 100 mL of 0.1 mol L−1 phosphate buffer (pH 6.5) and 2.50 g of polyvinylpyrrolidone (PVP). The homogenate was then filtered through four layers of gauze and centrifuged twice for 10 min at 9000 rpm, increasing the speed gradually, in order to avoid heating the enzyme. The solution
obtained was divided into several aliquots, in 1.5 mL polystyrene tubes, and was stored in a refrigerator at 4 °C. 2.3. Determination of peroxidase activity in the crude plant extract The peroxidase activity in the crude extract was determined following the spectrophotometric procedure described in the literature [28], which uses guaiacol as substrate. 2.4. Fabrication and preparation of the μPADs The preparation of the μPADs was based on previous work [18], with some modifications. CorelDRAW X5 was used to design the μPAD. The analytical device consisted of a circular channel with internal diameter of 7 mm. The design was printed on a Whatman No. 1 qualitative filter paper with wax toner (Genuine Xerox Solid Ink Black), using a wax printer (ColorCube 8580). After printing, the paper was heated at 120 °C, for 120 s, in order to form the hydrophobic barrier. 2.5. Stability of the colored product The stability of the red product on the surface of the paper platform was evaluated by monitoring the spot every 5 min, during 1 h. 2.6. Optimization of variables The effects of the main significant parameters, namely the guaiacol concentration and the volume of the plant extract, were evaluated using response surface methodology. The pH of the reaction medium was maintained at 6.5, due to the buffer used in enzyme extraction, and was the optimum pH for peroxidase [29]. All the statistical calculations were performed using Statistica 7.0 software. 2.7. Analytical method The colorimetric determination of hydrogen peroxide was based on the reaction of the analyte with guaiacol, using peroxidase as a catalyst. For this, a 1.6 μL volume of guaiacol solution was added to the circular channel of the device, followed by addition of 2.8 μL of the plant extract. After total absorption of the added aliquots, 0.8 μL of sample was added, resulting in a red tetraguaiacol product. For quantitative determination, the device was digitized and the intensity of the blue color (RGB format) obtained in the reaction was determined using ImageJ software [18]. The effective intensity of the blue color (AB) was calculated using the equation AB = − log (BS/BB), as defined by Abbaspour [30], where BS and BB are the blue color intensities of the sample and the blank, respectively. The effective intensities could be used to quantify hydrogen peroxide using the analytical curves. 2.8. Qualitative analysis Three commercial samples and one raw milk sample were tested for the presence of hydrogen peroxide, following the procedure recommended by AOAC [31]. For qualitative analysis, 10 mL of the sample and six drops of 1% vanadium pentoxide were added to a test tube. The non-formation of a red coloration indicated the absence of hydrogen peroxide in the sample. 2.9. UHT sample preparation The UHT (ultra-high temperature) process is a continuous flow heat treatment, where the milk is preheated, homogenized (before or after sterilization), sterilized, cooled, and aseptically packed. The process uses temperatures of between 140 and 150 °C, for up to 8 s, in order to sterilize the product [32].
Please cite this article as: L.S. Lima, E.L. Rossini, L. Pezza, et al., Bioactive paper platform for detection of hydrogen peroxide in milk, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, https://doi.org/10.1016/j.saa.2019.117774
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The samples were purchased locally in the city of Araraquara (São Paulo State, Brazil). Three samples of UHT milk with different percentages of fat (whole, reduced fat, and low fat milk) and free from hydrogen peroxide were spiked by addition of a suitable aliquot of 8.10 × 10−2 mol L−1 hydrogen peroxide standard solution, in order to obtain a concentration of 85.0 × 10−4 mol L−1. The fortified concentration was selected based on the legislation and was below the maximum limit established by the FDA for the presence of peroxide in milk [7]. All the samples were analyzed according to the recommended procedure. No pretreatment steps were required. 2.10. Raw sample preparation The sample of raw milk was obtained from a local producer. Since the milk naturally contained the peroxidase enzyme in its composition, the sample was heated at boiling for 20 min, in order to deactivate and degrade this enzyme [33]. After cooling to room temperature, the sample was then prepared for analysis, as described in Section 2.9.
3
Fig. 2. Example of spot test reaction between guaiacol and H2O2, in the absence (A) and presence (B) of peroxidase. The red compound (tetraguaiacol) formed only in the presence of the enzyme was the reaction product used for colorimetric determination of hydrogen peroxidase in milk samples. Order of reaction: guaiacol + H2O2 for A; guaiacol + peroxidase + H2O2 for B. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
3.1. Determination of the peroxidase activity of the crude plant extract
Validation of the proposed method was performed using a standard additions procedure that enabled evaluation of the presence of possible matrix effects associated with the commercial and raw milk samples. The samples with 85.0 × 10−4 mol L−1 of H2O2 were spiked with 50, 100, 150, and 200% of the initial concentration. The samples were then analyzed as described in Section 2.4.
The activity of the peroxidase present in the crude extract was determined according to the spectrophotometric procedure described in the literature [28], which uses guaiacol as substrate. In this procedure, the peroxidase activity is determined by measuring the change in absorbance at 470 nm, due to the formation of tetraguaiacol in the enzymatic reaction. One unit of peroxidase activity represents the amount of enzyme that catalyzes the oxidation of 1 μmol of guaiacol in 1 min. In the present case, 1 mL of the crude extract could catalyze 64 × 10−6 mol of guaiacol in 1 min. The enzymatic activity, in U mL−1, was determined as follows:
3. Results and discussion
U mL−1 ¼
2.11. Standard additions
This work reports the development of a methodology for the determination of hydrogen peroxide in milk samples. The colorimetric method was based on the oxidation of guaiacol to tetraguaiacol (a red compound) by hydrogen peroxide, in the presence of peroxidase (Fig. 1) [28]. In preliminary tests, the reaction between guaiacol and H2O2 did not occur in the absence of peroxidase (Fig. 2), so it was necessary to add the enzyme. The zucchini plant is an inexpensive, readily available, renewable, and eco-friendly source of peroxidase enzyme that can be extracted using a simple and fast method. The paper platform impregnated with the peroxidase enzyme showed no changes for at least 2 months, when stored in a refrigerator at 4 °C. The formation of the colored product occurred immediately after addition of the analyte, although the spot readings were performed after complete drying (10 min). The stability of the colored product was evaluated as described in Section 2.5, which showed that the effective intensity measurement remained constant for at least 1 h at room temperature (25 °C). The blue channel of the RGB color format was selected for the detection method, due to its higher sensitivity.
ð1Þ
where U mL−1 is the units of peroxidase activity per mL, A is the absorbance of the colored product at 470 nm, ε is the molar absorptivity of tetraguaiacol (26,600 L mol−1 cm−1), Ve is the plant extract volume (mL), t is the reaction time, and DF is the dilution factor of the crude enzyme extract. 3.2. Optimization of variables A central composite design was used to obtain the optimal conditions for the reaction. In these experiments, the effects of the guaiacol concentration and the volume of the enzymatic extract were studied at five levels (−√2, −1, 0, 1, √2). The points (levels) of a central composite design are coded units √2 distant from the central point (coded as zero point), so all the peripheral points lie on the same circumference. The extreme points (±√2) were established for a two-factor design [34]. Table 1 shows the matrix of the central composite design. The Table 1 Matrix of the central composite design. N°
1 2 3 4 5a 6a 7a 8 9 10 11 Fig. 1. Reaction between guaiacol and hydrogen peroxide, catalyzed by peroxidase.
A 1 1 : :DF: :1000 ∈ Ve t
a
Uncoded variable levels
Coded variable levels
[Guaiacol] (%)
Enzymatic extract (μL)
[Guaiacol]
Enzymatic extract
0.33 1.73 0.33 1.73 1.03 1.03 1.03 0.05 2.00 1.03 1.03
0.9 0.9 3.1 3.1 2.0 2.0 2.0 2.0 2.0 0.5 3.5
−1 +1 −1 +1 0 0 0 −1.41 +1.41 0 0
−1 −1 +1 +1 0 0 0 0 0 −1.41 +1.41
Central point in triplicate (n = 3).
Please cite this article as: L.S. Lima, E.L. Rossini, L. Pezza, et al., Bioactive paper platform for detection of hydrogen peroxide in milk, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, https://doi.org/10.1016/j.saa.2019.117774
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experiment corresponding to the central point was carried out using three replicates [35]. The resulting response surface is shown in Fig. 3, as a function of the guaiacol concentration and the volume of the enzymatic extract. It can be seen from the surface shape that the optimal region was found and that the maximum response was achieved when the guaiacol concentration was 1.39% and the volume of the enzymatic extract was 2.80 μL. The quadratic regression model describing the effective intensity in the blue channel (AB) was as follows: AB = −0.0716 + 0.168[Guaiacol] - 0.0609[Guaiacol]2 + 0.0961Vperoxidase - 0.0174V2peroxidase + 0.000539 [Guaiacol] Vperoxidase. It should be noted that although the pH was not studied as an experimental parameter in this experimental design, the medium was maintained at pH 6.5 during the entire extraction process. This pH was used for H2O2 determination, because it is the optimum pH for peroxidase [29]. 3.3. Analytical data The proposed methodology was validated by determination of the linear dynamic range, precision, limit of detection (LOD), limit of quantification (LOQ), and the influence of the matrix, using the standard additions technique [36,37]. After optimization of the variables, a linear calibration curve was constructed for hydrogen peroxide concentrations from 12.5 × 10−4 to 150 × 10−4 mol L−1. The calibration data (n = 8) resulted in the regression equation: AB = 0.02466 (±0.00192) + 17.053 (±0.750) C, where AB is the blue channel effective intensity and C is the hydrogen peroxide concentration (in mol L−1). The correlation coefficient was 0.986, indicating excellent agreement between the data and the regression model for the calibration curve (Fig. 4). LOD and LOQ were calculated using the expressions: LOD = 3 × SDBlank/B and LOQ = 10 × SDBlank/B, where SDBlank is the standard deviation of ten measurements of the blank (n = 10) and B is the slope of the analytical curve [36]. The LOD and LOQ were 3.54 × 10−4 and 11.8 × 10−4 mol L−1 (0.001 and 0.004% m/m), respectively, with standard deviation of the blank of 0.002012. The repeatability of the method was assessed by repetition of two points of the analytical curve, at different times on the same day (intra-day) and on different days (inter-day). The coefficients of variation obtained were 1.1 and 1.3% (intra-day), and 4.8 and 2.9% (interday), for 25.0 × 10−4 and 100 × 10−4 mol L−1 of hydrogen peroxide, respectively. These results demonstrated that the proposed method was
Fig. 4. Spots for the H2O2 concentrations of 12.5, 15.0, 25.0, 50.0, 75.0, 85.0, 100, and 150 × 10−4 mol L−1 used to construct the analytical curve.
repeatable and that the analytical device could be used for the quantification of hydrogen peroxide in milk, since all the values were lower than 10%. 3.4. Analytical application The effectiveness of the proposed procedure using the microfluidic device for determination of hydrogen peroxide in real samples was evaluated using UHT and raw milk samples. The values obtained (Table 2) showed excellent agreement with the spiked amounts of hydrogen peroxide. In the case of the raw milk, the sample was heated at boiling for 20 min, in order to degrade and deactivate the peroxidase naturally present in this type of sample. This treatment was necessary because the presence of peroxidase in the sample could lead to reaction conditions different from those established following the response surface experiments. The UHT milk samples did not require such pretreatment, since the industrial processing of this type of milk eliminates this enzyme. The raw milk samples previously spiked with the hydrogen peroxide standard were analyzed as described in Section 2.9. The results presented in Table 2 demonstrated that the proposed procedure using the microfluidic device could be successfully applied for the determination of hydrogen peroxide in raw milk. 3.5. Standard addition and recovery The proposed procedure was validated in recovery experiments performed with the UHT and raw milk samples. The sample treatment is described in Section 2.9 and the analysis procedure is presented in Section 2.7. The results (Table 3) showed recoveries in the range from 92.2 to 109%, indicating the absence of any significant matrix effects in the application of the technique for the determination of hydrogen peroxide in milk samples. There have been few reports in the literature concerning methods for the detection and quantification of hydrogen peroxide in milk [8,10,12–15]. Methodologies involving chemometric techniques have Table 2 Determination of hydrogen peroxide added to milk samples.
Fig. 3. Central composite design response surface obtained for the intensity of the blue color as a function of the guaiacol concentration and the enzymatic extract volume. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Samples
Hydrogen peroxide added (mol L−1)
Hydrogen peroxide founda (mol L−1)
Whole Reduced fat Low fat Raw
85.0 × 10−4 85.0 × 10−4 85.0 × 10−4 85.0 × 10−4
86.9 × 10−4 83.6 × 10−4 84.4 × 10−4 85.2 × 10−4
a
Average of four determinations.
Please cite this article as: L.S. Lima, E.L. Rossini, L. Pezza, et al., Bioactive paper platform for detection of hydrogen peroxide in milk, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, https://doi.org/10.1016/j.saa.2019.117774
L.S. Lima et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy xxx (xxxx) xxx Table 3 Recovery data for hydrogen peroxide spiked in milk samples. Samples
Whole
Reduced fat
Low fat
Raw
a
Standard added (mol L−1)
Found valuea (mol L−1)
Recovery (%, mean ± RSD)
0 42.5 × 10−4 85.0 × 10−4 128 × 10−4 170 × 10−4 0 42.5 × 10−4 85.0 × 10−4 128 × 10−4 170 × 10−4 0 42.5 × 10−4 85.0 × 10−4 128 × 10−4 170 × 10−4 0 42.5 × 10−4 85.0 × 10−4 128 × 10−4 170 × 10−4
87.8 × 10−4 46.2 × 10−4 84.4 × 10−4 133 × 10−4 174 × 10−4 85.0 × 10−4 44.0 × 10−4 86.4 × 10−4 118 × 10−4 175 × 10−4 88.9 × 10−4 42.9 × 10−4 88.5 × 10−4 116 × 10−4 171 × 10−4 86.4 × 10−4 45.3 × 10−4 89.2 × 10−4 128 × 10−4 166 × 10−4
– 108.8 ± 5.0 99.3 ± 0.7 104.2 ± 6.4 102.2 ± 5.0 – 103.6 ± 5.6 101.6 ± 1.9 92.2 ± 0.9 103.0 ± 3.2 – 100.9 ± 5.8 104.1 ± 0.7 98.7 ± 2.4 100.4 ± 6.0 – 106.6 ± 4.2 105.0 ± 1.9 99.6 ± 5.2 97.7 ± 6.1
Average of four determinations.
been reported, which are rapid and simple, without requirement for sample pretreatment, but the levels of adulteration detected were much higher than those used here, and were also much higher than the maximum limit established in USA legislation [15,38]. Other methodologies with lower sensitivity require sample preparation employing hazardous chemicals [8,14]. Several methodologies require specialized operators and the use of much higher quantities of reagent solutions and samples, compared to the paper platform-based method. The sample preparation and analysis procedures involve several steps that are time consuming, and the large volumes of liquid waste generated may be hazardous for the environment and for the operator. Furthermore, such techniques do not enable in situ analysis, since the equipment is not portable [8,10,12–15]. The advantage of the device described here is that the analysis can be performed in situ by a person with minimal training, using a low-cost platform, without the need for expensive equipment. The digital image could be acquired and analyzed using a smartphone, or could be transmitted to a specialist via the internet. Due to the simplicity of the paper platform, a semi-skilled person at a farm with minimal infrastructure, or at an industrial site, could use the test to detect hydrogen peroxide adulteration.
4. Conclusions This work presents a validated bioactive paper platform for determination of an important adulterant of milk. Chemometric tools were used to obtain the optimized conditions for the quantification of hydrogen peroxide, and the methodology was successfully validated. The use of a μPAD, together with colorimetric detection by digital imaging, provides a simple, portable, inexpensive, and eco-friendly procedure for the determination of hydrogen peroxide in UHT and raw milk samples. The proposed methodology is fast, accurate, and precise. UHT milk samples can be analyzed without the need for any sample preparation steps, while a simple boiling step is required for raw milk. The method does not produce liquid waste and the paper platform can be easily incinerated after analysis. The simplicity of the methodology enables in situ analysis using a camera or a smartphone to obtain an image of the paper platform. The analytical device developed in this work is suitable for use as a screening tool, since the LOD obtained for determination of hydrogen peroxide (11.8 × 10−4 mol L−1, or 0.004% m/m) is lower than the value permitted according to current legislation.
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Please cite this article as: L.S. Lima, E.L. Rossini, L. Pezza, et al., Bioactive paper platform for detection of hydrogen peroxide in milk, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, https://doi.org/10.1016/j.saa.2019.117774