Determination of Nitrite and Nitrate in Brazilian MeatsUsing High Shear Homogenization

Elisabete Alves Pereira & João F. S. Petruci &Arnaldo Alves Cardoso

Received: 18 May 2011 /Accepted: 22 August 2011 /Published online: 6 September 2011# Springer Science+Business Media, LLC 2011

Abstract Nitrate and nitrite are usually added toprocessed meat products to protect against the growthof microorganisms. Two sample preparation methodolo-gies using either manual grinding (with a mortar andpestle) or mechanical high shear homogenization wereinvestigated and compared. The results showed that highshear homogenization was the most suitable for theextraction of nitrite and nitrate from ham, salami, andbacon samples, achieving high extraction recoveries(>98%) together with low relative standard deviations(RSDs) for the samples analyzed. Analyses wereperformed using capillary electrophoresis. A runningbuffer consisting of 60 mmol L−1 tetraborate and0.2 mmol L−1 cetyltrimethylammonium bromide enabledseparation of the analytes in <5 min. In validationexperiments, good repeatability was obtained for bothmigration times (<0.8% RSD) and peak areas (<1.1%RSD). Analytical curves for nitrite and nitrate were linear(r>0.998) in the 0.2- to 2.5-mg L−1 and 0.5- to 5-mg L−1

concentration ranges, respectively. The limits of detectionwere 0.15 mg L−1 for nitrite and 0.17 mg L−1 for nitrate.The method developed was applied to the analysis ofdifferent kinds of meats (sausage, ham, salami, bacon, andothers) produced in Brazil. The ranges of concentration

found were 17.3–46.4 mg kg−1 (nitrite) and 69.9–198.1 mg kg−1 (nitrate). The contents of nitrate and nitritein the samples were below the Brazilian legislation limitvalues (150 and 300 mg kg−1 for nitrite and nitrate,respectively).

Keywords Capillary electrophoresis . Nitrite–nitrate . Foodanalysis .Meat products . High shear homogenization

Introduction

For centuries, meat has been treated with crude salt as ameans of preservation. The presence of nitrite as animpurity in the salt resulted in an attractive red color ofthe product, and at the end of the nineteenth century,nitrite was recognized to be a principal active compo-nent in the curing process. Both nitrate and nitrite havesubsequently been used extensively for the curing ofmeats, such as ham, bacon, and some sausages (Watson2001). Nitrite acts as an inhibitor of microorganismgrowth and gives a pleasant flavor (Swuan 1977). In theearly 1970s, the use of nitrite was recognized to behazardous due to the formation of carcinogenic nitros-amines (Pegg and Shahidi 2004). The nitrite added to meatis partially oxidized to nitrate by sequestering oxygen,while in raw meat products, the nitrate may be reduced tonitrite by microorganisms. Nitrate can also be reduced tonitrite in the oral cavity and in the digestive system(Duncan et al. 1997). Once in the stomach, nitrite canreact with amines to form N-nitroso compounds, exposureto which has been associated with increased risks ofgastric and esophageal cancers (Walters 1980).

The Agência Nacional de Vigilância Sanitária(ANVISA), the federal regulatory agency responsible for

E. A. Pereira (*)Universidade Federal de São Carlos, Campus Sorocaba,Rodovia João Leme dos Santos Km 110, SP 264, Bairro Itinga,CEP 18052-780, Sorocaba, São Paulo, Brazile-mail: ealves@ufscar.br

J. F. S. Petruci :A. A. CardosoInstituto de Química, Departamento deQuímica Analítica, UNESP,CP 355,CEP 14801-970, Araraquara, São Paulo, Brazil

Food Anal. Methods (2012) 5:637–642DOI 10.1007/s12161-011-9294-1

the monitoring of food additives in Brazil, has establishedconcentration limits for nitrite and nitrate in meats of 150and 300 mg kg−1, respectively (ANVISA 2009).

The method commonly reported in the literature forthe analysis of nitrate and nitrite in meat consists of thesolubilization of the analytes and determination of theanions by considering the difference between theamount of nitrite and the amount of nitrate plus nitrite(AOAC 1995). In this procedure, nitrate is reduced tonitrite by passage through a cadmium column, and nitriteis measured using a colorimetric reaction. Another aliquotof the same sample is used to determine nitrite, withoutany treatment with reducing agent. The colorimetricreaction involves the diazotization of nitrite with sulfa-nilamide and coupling with N-(1-naphthyl)-ethylenedi-amine dihydrochloride (NED) to form a highly coloredazo dye. However, this method involves laboriousprocedures for sample preparation, uses a metal of highenvironmental toxicity, and allows the determination ofonly a single analyte at a time. Ion chromatography (IC)and high-performance liquid chromatography (HPLC)have been reported in the literature for the simultaneousdetermination of nitrate and nitrite in various matrices(Siu and Henshall 1998; Dennis et al. 1990; Muir andSoroka 1992; Kodamatani et al. 2009; Tahboub 2009;Ferreira and Silva 2008). Common to all of thesemeasurement methods is the sample preparation stepconsisting of the solubilization of the anions. Practicallimitations of these techniques include the need forexpensive columns, long separation times, high consump-tion of hazardous organic solvents, and pretreatmentsteps, such as protein precipitation procedures afterextraction or use of reversed-phase cartridges to removesample matrix interferences.

Capillary electrophoresis (CE) offers an attractivealternative for the determination of ions (Kaniansky etal. 1999; Paull and King 2003; Jackson and Haddad 1993;Merusi et al. 2011; Xu et al. 2008). Compared with IC andHPLC, the advantages include lower consumption ofelectrolytes and samples, use of a water-based electrolyte,high separation efficiency, short run time, and therelatively simple and direct sample injection with littlepretreatment. Only a few publications in the literaturecurrently describe the determination of nitrites and nitratesin meat products using CE. Marshall and Trenerry (1996)developed a method for the determination of nitrate andnitrite in a variety of meat products using an electrolytecontaining sodium chloride and osmotic flow modifier(OFM-Anion-BT) for reversal of the direction of theelectroosmotic flow (EOF).

Solid samples were blended for 2 min, filtered, andthe filtrate passed through a Sep-Pak C18 cartridge.Good recoveries of ions were observed using thiocyanate

as an internal standard. Öztekin et al. (2002) determinedthe nitrite and nitrate in meat products using a CE methodbased on the separation of the two anions in a capillarycoated with polyethyleneimine, which allowed the rever-sal of the electroosmotic flow. Solid samples were blendedfor 2 min with deionized water and then the suspensionwas incubated in a warm water bath and filtered. Goodreproducibility and recovery were obtained using thiocy-anate as the internal standard. Shiddiky et al. (2009)measured nitrite in ham and sausage using microchip CEwith extraction by either sonication or microwave irradi-ation. The technique provided an analysis time of 4 min,together with good sensitivity. More recently, theseauthors proposed the application of an isotachophoreticCE method to the simultaneous determination of nitrateand nitrite in meat products. Samples were minced andhomogenized using a plate with 3-mm diameter holes.The meat product samples were then extracted withredistilled water using an orbital shaker (Jastrzebska2010).

In this work, an alternative method for the analysis ofnitrite and nitrate has been developed and applied to meatsamples. The method is based on free solution CE withdirect UV detection (at 210 nm). Two different samplepreparation methodologies (manual and mechanical) wereinvestigated; the optimized technique was compared withthe standard methodology normally adopted in Brazil usinganalyses of real samples.

Experimental

Chemicals and Materials

NED, sulfanilamide, zinc sulfate, cadmium sulfate, sodiumnitrite, and sodium nitrate were obtained from Merck(Darmstadt, Germany). Sodium sulfite and sodium tetrabo-rate were obtained from Synth (São Paulo, Brazil).Cetyltrimethylammonium bromide (CTAB) was providedby Aldrich (St. Louis, MO, USA). All reagents used wereanalytical grade. Water was purified using a Milli-Q system(Millipore Corp., Bedford, MA, USA). The cadmiumcolumn was prepared as described in the literature (Senand Donaldson 1978).

Instrumentation

CE Analysis

All analyses were performed using a capillary electropho-resis system (model HP 3D CE, Agilent Technologies, PaloAlto, CA, USA) equipped with a diode array detector andtemperature control (to maintain a constant 29 °C). Data

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acquisition and treatment employed HP ChemStationsoftware (rev A.06.01). Fused silica capillaries (PolymicroTechnologies, Phoenix, AZ, USA) with dimensions48.5-cm total length, 40-cm effective length, 75-μm i.d.,and 375-μm o.d. were used. Samples were injectedhydrodynamically at 30 mbar for 12 s, and the detectionwavelength was set at 210 nm. The applied voltage was setto −10 kV. On a daily basis prior to the first analysis of theday, the capillary was conditioned by flushing with1 mol L−1 NaOH solution for 5 min, followed by a 5-minflush with deionized water and a 40-min flush withelectrolyte solution. The capillary was rinsed with freshelectrolyte solution for 3 min before each run.

Preparation of Stock Solutions and Reagents

Stock solutions of nitrite, nitrate, and sulfite were preparedby dissolving appropriate amounts of the chemicals indeionized water. These solutions were stable for at least3 months when stored under refrigeration in brown bottles.Fresh working standard solutions were prepared daily bydiluting the stock solutions appropriately with deionizedwater.

The sulfanilamide reagent was prepared by dissolving600 mg of sulfanilic acid in 50 mL of hot water. Aftercooling, 20 mL of glacial acetic acid was added and themixture diluted to 100 mL with deionized water. TheNED reagent was prepared by dissolving 20 mg of N-(1-naphthyl)-ethylene diammonium dichloride in 20 mLof glacial acetic acid and diluting to 100 mL withdeionized water.

The cadmium sulfate solution was prepared bydissolving 10 g of cadmium sulfate in 100 mL ofdeionized water.

Procedure

Sample Preparation

Meat products were purchased from local supermarkets.Sausage, pork sausage, bologna, and pepperoni sampleswere cut into pieces and homogenized manually using amortar and pestle. Ham, salami, and bacon sampleswere cut into pieces and homogenized mechanicallyusing a high shear mixer (ULTRA-TURRAX Q252-K28,Quimis, Brazil) at 6000 rpm for 3 min at roomtemperature (23 °C).

A representative amount (5 g) of each homogenizedsample was weighed out and placed in a Falcon tube.Fifty milliliters of deionized water at 80 °C was added andthe mixture stirred for 2 min and heated at 80 °C for 1 h.After cooling to room temperature, the sample was filteredand the volume adjusted to 50 mL with deionized water. A

1-mL aliquot was diluted to 10 mL, the solution filteredthrough a 0.45-μm cellulose acetate filter, and the filtratecollected for CE analysis. Quantification employed sulfiteas an internal standard.

Determination of Nitrate and Nitrite by the Official AOACMethod (993.03/973.31)

Sausage, bologna, and pepperoni samples were cut intopieces and homogenized manually. Ham, salami, andbacon samples were cut into pieces and homogenizedmechanically.

For the determination of nitrite, a 5-g portion ofeach sample was weighed out and transferred to aFalcon tube. Thirty milliliters of deionized water at80 °C was added and the mixture stirred for 2 min.The homogenized sample was heated and the temper-ature maintained at 80 °C for 2 h. After cooling toroom temperature, the sample was filtered (usingWhatman 41 filter paper) and the volume adjusted to500 mL with deionized water. A 5-mL aliquot of thefiltered solution was transferred to a 50-mL volumetricflask and 2.5 mL of the sulfanilamide reagent addedwith mixing. The mixture was left to stand for 5 min atroom temperature and then 2.5 mL of NED reagentwas added with mixing, followed by dilution tovolume. After 15 min, the absorbance was measuredat 540 nm.

For nitrate determination, 600 mg of zinc powder and4 mL cadmium sulfate solution were added to 25-mLvolumetric flasks to obtain a homogeneous mixture. Afterstanding for 10 min, 5 mL of the filtered solution sampleand 5 mL of ammonium buffer were added to the flash. Thesolution was stirred for 10 min and then filtered (usingWhatman 41 filter paper) and transferred to a 50-mLvolumetric flask, to which 2.5 mL of the sulfanilamidereagent was added with mixing. After standing for 5 min atroom temperature, 2.5 mL of the NED reagent was added,with mixing and dilution to volume. After 15 min, theabsorbance was measured at 540 nm. The nitrate concen-tration was calculated as the difference between the totalnitrite concentration after reduction and the initial nitriteconcentration.

Method Validation

Appropriate aliquots of the standard stock solutions ofnitrate and nitrite, together with a fixed amount of sulfite(internal standard, IS) were transferred to separate 10-mLvolumetric flasks. The volumes were completed withdeionized water. The concentration ranges were 0.2–2.5 mg L−1 (nitrite) and 0.5–5 mg L−1 (nitrate). Eachsolution was injected in triplicate. Peak area ratios (analyte/IS)

Food Anal. Methods (2012) 5:637–642 639

were plotted against the respective concentrations of eachanalyte.

The precision of the technique was estimated usingthe relative peak area and relative migration timerepeatability for ten consecutive injections of a standardsolution containing 20 mg L−1 of each analyte plus10 mg L−1 of the internal standard. The intra-day precisionfor each analyte was determined by assaying three controlsamples at low, medium, and high concentrations usingtriplicate injections on the same day.

The accuracy of the procedure was assessed byperforming recovery experiments. Two different concen-tration levels of the analytes, together with a fixedconcentration of the internal standard, were added to thesamples both before and after the extraction procedure.The results obtained for the samples spiked after theextraction procedure were considered to represent 100%recovery. The recoveries were calculated according toEq. 1.

Recovery %ð Þ ¼ Cbefore

Cafter� 100 ð1Þ

where Cbefore and Cafter are the analyte concentrations ofthe samples spiked before and after the extractionprocedure, respectively.

Results and Discussion

Optimization of the CE Conditions

Nitrate and nitrite absorb sufficiently over the wavelengthrange from 190 to 225 mm to allow direct UV detection.For sensitive detection of the analytes, the electrolyte bufferused should have low absorbance at the detection wave-length (Guan et al. 1996). In this work, tetraborate wasselected as the electrolyte anion because its mobility issimilar to that of the analytes of interest; it presents “hightransparency” and its solutions are stable during storage andcontinuous analysis.

The CE separation mechanism is based on the differencesbetween the electrophoretic mobilities of charged species inthe presence of an electric field. In a conventional CE system,the EOF is toward the cathode; anions with an electrophoreticmobility higher than the electroosmotic mobility of the bulkelectrolyte cannot reach the detector, and the polarity of thepotential applied must be reversed in order to detect theseanions. However, under these conditions, anions with mobi-lities lower than the electroosmotic mobility would neverreach the detector. Therefore, for most anion separations, it isnecessary to use a modifier to reverse the direction of the EOF(Paull and King 2003).

As nitrite and nitrate have reasonable UV absorptionsaround 210 nm, an electrolyte buffer of high transparency atthe detection wavelength is required. As the simplestchoice, tetraborate buffer containing CTAB (EOF modifier)was used as the running electrolyte. The influence oftetraborate concentrations in the range 20–80 mmol L−1

was studied using electrolytes containing a fixed concen-tration of CTAB (0.2 mmol L−1). A gain in resolution wasobserved with increasing tetraborate concentration. How-ever, the use of higher concentration buffers can lead tosignificant losses of resolution and peak efficiency ifexcessive heating occurs within the capillary. A bufferconcentration of 60 mmol L−1 was selected because itrepresented a compromise considering the analytical signal,current counts, and analysis time. The separation of nitrateand nitrite obtained under the optimized conditions isshown in Fig. 1.

Validation of Analytical Method

The precision of the method were better than 0.73% formigration time (n=10) and between 0.89% and 1.04% forrelative peak area repeatabilities. Intra-day precisionshowed RSDs ranging from 0.81% to 1.92% (Table 1).

For quantification purposes, analytical curves wereconstructed based on peak area versus concentration.Since much of the variance in precision is attributableto variable injection volume, voltage, or EOF, the use ofan appropriate internal standard is required to minimize

2 3 4 5

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Fig. 1 Electropherogram obtained under optimized conditions for theanalysis of nitrate and nitrite. Concentration of each anion, 10 mg L−1.Separation conditions: fused silica capillary column, 75-μm i.d.×360-μm o.d., 48.5-cm total length (40 cm to detector); running buffer,60 mmol L−1 borate and 0.2 mmol L−1 CTAB; hydrodynamicinjection, 12 s at 30 mbar; separation voltage, −10 kV; T=29 °C;direct UV detection at 210 nm. Peak labels: 1 Nitrite, 2 Nitrate, 3Sulfite (internal standard)

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these sources of error. The analytical curves of relativepeak area (anion/IS) versus concentration consisted ofsix points, with three replicate injections of standards ateach concentration level. The equations describing theanalytical are presented in Table 2, together with thelimits of detection. The results showed good linearity overthe concentration range used (r>0.998). The limits ofdetection were 0.15 mg L−1 (nitrite) and 0.17 mg L−1

(nitrate).The results obtained for the accuracy of the method,

using pork sausage, bologna, and pepperoni, are shown inTable 3. Samples were spiked with known quantities ofnitrate and nitrite and subsequently submitted to theextraction procedures. The recoveries were calculatedaccording to Eq. 1 by comparing peak area ratios of thesamples spiked before extraction with peak area ratios ofthe samples spiked after extraction and ranged from 96.8%to 101.9% for the concentration levels used. Meanrecoveries were >96% for both nitrate and nitrite in allthree of these food matrices. Recoveries were <80% in thecase of ham, salami, and bacon samples (results notshown).

Comparison of the two sample preparation methodolo-gies (manual and mechanical) showed that significantly

higher nitrate and nitrite contents were obtained when thesamples were homogenized mechanically (Table 4). The useof different homogenization times from 1 to 5 mindemonstrated that the extraction efficiency increased withtime. The homogenization time was optimized at 3 minsince increasing the time further did not significantlyimprove the recovery.

Comparison with Standard Methods

Seven cured meat products were analyzed by the proposedmethodology, as well as by the official standard method(AOAC 1995). The analytical results are summarized inTable 5. Figure 2 presents the electropherograms obtainedfor the sample extracts. Identification of the nitrate andnitrite was achieved using spiking techniques. The resultsobtained by CE were consistent with those obtained by theAOAC method in terms of both precision and accuracy, asestablished by F test and t test calculations (all values fellbelow the critical values for t of 2.98 and for F of 19.0, atp=0.05).

Conclusions

This work describes a rapid, simple, and efficientmethod based on capillary electrophoresis for thedetermination of nitrite and nitrate in meat products

Table 3 Results of the recovery study using two different concen-trations of nitrate and nitrite

Sample Analyte Amount added(mg kg−1)

Amount found(mg kg−1)

Recovery (%)

Sausage Nitrite 40.0 40.2 100.4±2.1

Nitrate 100.0 96.8 96.9±1.8

Sausage Nitrite 150.0 145.4 96.8±2.1

Nitrate 300.0 305.7 101.9±1.9

Table 4 Performance of sample preparation procedures using eithermechanical (A) or manual (B) sample homogenization

Sample Procedure Aa

concentration (mg kg−1)Procedure Bb

concentration (mg kg−1)

NO2− NO3

− NO2− NO3

−

Ham 126.3±2.5 46.5±0.9 70.4±1.5 35.7±0.5

Salami 83.9±1.7 <LOD 34.1±0.7 <LOD

Bacon 38.3±0.8 <LOD 27.8±0.8 <LOD

Table 1 Intra-day precision using different concentrations of nitriteand nitrate

Analyte Concentration(mg L−1)

Intra-daya

RSD (%)

Nitrite 0.2 1.9

1.5 1.9

2.5 1.1

Nitrate 0.5 1.6

3.0 1.1

5.0 0.8

a Based on n=3

Table 2 Method validation in terms of linearity, LOD, and LOQ

Anion Analytical curve LODa (mg L−1) LOQb (mg L−1)

Nitratec Y=0,16X–0,0032 0.17 0.56

Nitrited Y=0,15X+0,011 0.15 0.49

Six data points, three replicate injections at each concentration levela S/N=3b S/N=10c Concentration range 0.5–5 mg L−1 , based on peak area (nitrate/IS)d Concentration range 0.2–2.5 mg L−1 , based on peak area (nitrite/IS)

Food Anal. Methods (2012) 5:637–642 641

using a high shear mixer for homogenization. This isthe first time that the influence of the sample prepara-tion procedure has been evaluated for the determinationof nitrite and nitrate in meat samples. The techniqueshowed good migration time and area repeatability,excellent linearity, and adequate accuracy. In comparisonwith other standard methods, the proposed CE methodhas the advantage of not requiring a nitrate reductionstep procedure since the determination of nitrate andnitrite is carried out simultaneously, which reduces thegeneration of toxic residues and decreases the totalanalysis time. The method could be readily employedfor the analysis of sample matrices including vegetables,potable water and river water, as well as otherenvironmental materials.

Acknowledgments The authors wish to thank the Conselho Nacio-nal de Desenvolvimento Científico e Tecnológico (CNPq) and theFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) ofBrazil for financial support.

References

ANVISA http://anvisa.gov.br. Accessed September 2009AOAC (1995) AOAC Official methods of analysis of the Association

of Official Analytical Chemists, Method 973.31 and Method993.03. AOAC, Washington

Dennis MJ, Key PE, Papworth T, Pointer M, Massey RC (1990) FoodAddit Contam 7:455

Duncan C, Li H, Dykhuizen R, Frazer R, Johnston P, MacKnight G(1997) Comp Biochem Physiol Part A: Mol Integr Physiol118:939

Ferreira IMPLVO, Silva S (2008) Talanta 74(5):1598Guan F, Wu H, Luo Y (1996) J Chromatogr A 719:427Jackson PE, Haddad RP (1993) Trends Anal Chem 12:231Jastrzebska A (2010) J Anal Chem 65:1170Kaniansky D, Masar M, Marak J, Bodor R (1999) J Chromatogr A

834:133Kodamatani H, Yamazaki S, Saito K, Tomiyasu T, Komatsu Y (2009) J

Chromatrog A 1216:3163Marshall P, Trenerry VC (1996) Food Chem 57:339Merusi C, Corradini C, Cavazza A, Borromei C, Salvadeo P (2011)

Food Chem 120(2):615Muir AD, Soroka JJ (1992) J Agric Food Chem 40:1602Öztekin N, Nutku MS, Erim FB (2002) Food Chem 76:103Paull B, King M (2003) Electrophoresis 24:1892Pegg RB, Shahidi F (2004) Curing of meat: the N-nitrosamine

problem and nitrite alternatives. Wiley-Blackwell, LondonSen NP, Donaldson B (1978) J Assoc Off Anal Chem 61:1389Siu DC, Henshall A (1998) J Chromatogr A 804:157Shiddiky MJ, Lee KS, Son J, Park DS, Shim YB (2009) J Agric Food

Chem 57:4051Swuan PF (1977) Proc Roy Soc Med 70:113Tahboub YR (2009) Jordan J Chem 3:69Walters CL (1980) Oncology 37:289Watson DH (2001) Food chemistry safety, vol. 1: Contaminants.

Woodhead, LondonXu Z, Doi T, Timerbaev AR, Hirokawa T (2008) Talanta 77:278

(A) Bologna

(B) Ham

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-5

0

5

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20 3

2

1

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Fig. 2 Electropherograms of the meat extracts. Electrophoreticconditions as in Fig. 1. Peak labels: Unidentified impurity (numbersymbol). 1 Nitrite, 2 Nitrate, 3 Sulfite (internal standard)

Table 5 Comparison of the CE and AOAC methods for themeasurement of nitrite and nitrate in meat products

Sample CE method concentration(mg kg−1)

AOAC methodconcentration (mg kg−1)

NO2− NO3

− NO2− NO3

−

Sausage 36.2±0.8 86.9±1.9 35.6±2.4 86.7±2.2

Pork sausage <LOD 154.7±3.3 <LOD 155.4±6.1

Bacon 46.4±1.8 <LOD 47.7±2.4 <LOD

Salami 26.2±1.4 <LOD 23.3±1.7 <LOD

Ham 17.3±1.3 198.1±3.5 24.4±9.5 198.5±5.9

Bologna 25.1±0.7 69.9±2.0 25.5±0.8 69.6±1.7

Pepperoni <LOD 104.9±2.1 <LOD 105.4±6.6

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