determination of hydroperoxides in nonaqueous solvents or mixed

Sensors and Actuators B 95 (2003) 321327

Determination of hydroperoxides in nonaqueous solvents or mixedsolvents, using a biosensor with two antagonist enzymes

operating in parallelL. Campanella, D. Giancola, E. Gregori, M. Tomassetti

Department of Chemistry, University La Sapienza, Piazza Aldo Moro 5, 00185 Rome, Italy

Abstract

The development and characterisation of a new organic phase enzyme electrode (OPEE) for hydroperoxides is described. The biosensorwas obtained by combining an oxygen gas diffusion amperometric electrode and two immobilised enzymes (peroxidase and tyrosinase)working in parallel and competing for the same substrate (catechol). LOD was about 0.1103 mol l1. The biosensor was applied for thedetermination, using decane as solvent, of the pool of hydroperoxides released during the heating (to about 130140 C) of extravirginolive oil, or used to determine the hydrogen peroxide content of lipophilic cosmetic products, working in a dioxanewater mixture. 2003 Elsevier B.V. All rights reserved.

Keywords: Organic phase enzyme electrode; Hydroperoxide; Analysis

1. Introduction

Over the past few years a number of reports have beenpublished, especially by Turner and coworkers [1,2], or byWang et al. [3], as well as by Popescu et al. [4], concern-ing the construction of organic phase enzyme electrodes(OPEEs) capable of determining hydrogen peroxide in or-ganic solvents. These biosensors have been applied mainlyto hydrogen peroxide determination in some comparativelyinsoluble drug matrixes [3]. The OPEEs developed for thispurpose were all based on the peroxidase enzyme coupledto amperometric transducers.

This type of OPEE performs well although, as weobserved on previous occasions [5,6], they have onelimitationthey can only operate in organic solvents that arenot particularly hydrophobic, such as acetonitrile, or at bestchloroform; the conductivity of this type of solvent can ac-tually be enhanced by dissolving special electrolytes in them[13] in order to allow the amperometric transducer to func-tion correctly. Unfortunately, however, partially water misci-ble solvents like acetonitrile, etc. especially in the anhydrousform, tend rapidly to deactivate the enzymes they come intocontact with [7] and so their use is generally not suitable forenzymatic processes. In order to overcome this kind of diffi-culty, over the past few years our research group has devel-

Corresponding author. Tel.: +39-06-49913744;fax: +39-06-49913725.

oped a large number of OPEEs [59], the majority of which,however, use a gaseous diffusion amperometric transducer,in practice, a Clark electrode (made of Teflon in order to re-sist attack by organic solvents). It is nevertheless clear that,in order to use this type of transducer, it is necessary, in theenzymatic reaction used, for oxygen to be consumed or pro-duced. However, in the case of the enzymatic reaction catal-ysed by the peroxidase this condition was not satisfied; thismeant it was impossible to develop a peroxidase OPEE basedon a gaseous diffusion transducer which would have the ad-ditional advantage of greater selectivity than OPEEs basedon the simple amperometric transducers mentioned above.

To overcome this difficulty, over the past few years,Wang et al. [10] and, at practically the same time, also ourgroup [11] have developed a catalase biosensor operatingin nonaqueous solvents that could be used to determine thehydrogen peroxide contained in real matrixes. However,not all the features of this type of biosensor are completelysatisfactory [12].

Nevertheless, Uchiyama and Sano recently proposed aninteresting type of biosensor consisting of two antagonisticenzymes operating in parallel which can use a gaseous dif-fusion amperometric oxygen electrode as transducer [13].Using precisely this type of geometry and two enzymes(peroxidase and tyrosinase), which compete for the samesubstrate, catechol, it was possible to construct a biosen-sor for hydrogen peroxide that could be used to determinehydrogen peroxide in aqueous solutions [14]. The aim ofthe present research was to develop a bienzymatic OPEE of

0925-4005/$ see front matter 2003 Elsevier B.V. All rights reserved.doi:10.1016/S0925-4005(03)00433-7

322 L. Campanella et al. / Sensors and Actuators B 95 (2003) 321327

this type for the determination also of hydroperoxides otherthan hydrogen peroxide contained in different real lipophilicmatrixes. The biosensor would thus be able to operate inorganic solvents or else in mixtures, that is, in solventsable in any case to dissolve the real matrix being tested,which is hydrophobic or in any case only slightly soluble inwater.

2. Experimental

2.1. Reagent

Hydrogen peroxide 30% (w/v) from Merck (Darm-stadt, Germany); monobasic potassium phosphate, diba-sic sodium phosphate, sulphuric acid 96% (analyticalgrade), potassium permanganate RPE-ACS, were suppliedby Carlo Erba; potassium chloride, kappa-carrageenan,tert-butylhydroperoxidedecane solution 5.5 mol l1,cumene hydroperoxidecumene (80 + 20)% (v + v) so-lution, were all supplied by Fluka, lauroylperoxide, ben-zoylperoxide, tert-butylperoxide and dicumylperoxide,were supplied by Sigma (St. Louis, USA). All reagentswere analytical grade. The enzymes used, horseradishperoxidase (1510 U/mg, EC 1.11.1.7) and mushroom ty-rosinase (6680 U/mg, EC 1.14.18.1), were supplied bySigma (St. Louis, USA). The solvents used were: extrapure 1,4-dioxane from Merck (Darmstadt, Germany), pureanalytical grade decane from Fluka (Switzerland).

2.2. Cosmetic and drug samples used

The following is a list of cosmetics or drug samples usedin the analysis, together with their nominal hydrogen perox-ide as declared by the manufacturers:

Sample no. 1: emulsion, H2O2 (40 volumes) (samplestored for a long time in the laboratory without any par-ticular precautions).

Sample no. 2: emulsion, H2O2 (40 volumes). Sample no. 3: emulsion, H2O2 (30 volumes). Sample no. 4: emulsion, H2O2 (20 volumes). Sample no. 5: bicomponent cream (nominal value of H2O2

not declared by the manufacturer). Sample no. 6: cream (nominal value of H2O2 not declared

by the manufacturer).

2.3. Olive oil samples

The olive oil tested was purchased at a local shop and clas-sified as extravirgin olive oil, which was heated to about130140 C. An amount of 3.0 ml aliquots of the olive oilwere taken before and during heating at fixed time inter-vals of 1 h (or several hours). Immediately after taking andcooling to room temperature, the samples were analysed to

determine the content of the pool of hydroperoxides andof the pool of polyphenols.

2.4. Apparatus

The following apparatus was used in the present research:a biosensor amperometric detector and a model 4000-1electrode for oxygen measurement, both supplied by Univer-sal Sensor INC (New Orleans, USA) and a model 868 Amel(Milan) recorder. The electrode used was of the gaseousdiffusion type, which allows oxygen to be determined am-perometrically; it is composed of a platinum cathode andan Ag/AgCl/Cl type anode, both immersed in a solutionof phosphate buffer (1/15 mol l1) and KCl (0.1 mol l1) atpH 6.6. The internal solution was contained in a cylindri-cal Teflon cap, the lower extremity of which was sealed bya Teflon gas-permeable membrane, secured to the cap by aTeflon O-ring, which prevented the passage of electrolytesand the solution but allowed that of oxygen. The cap, filledwith internal solution, was screwed on to the body of theelectrode. The Teflon gas-permeable membrane was sup-plied by Universal Sensor INC (New Orleans, USA). TheD-9777 type dialysis membrane used was supplied by Sigma(St. Louis, USA). The biosensor experiments were carriedout at 20 C in a 15.0 ml thermostatable glass cell suppliedby Marbaglass (Rome), connected to a Julabo C thermo-stat. The solvent used for the tests was kept under constantstirring using a magnetic microstirrer supplied by Velp Sci-entifica (Italy). Titrations were performed using a 25.0 mlburette (with 1/10 ml 0.03 ml graduations).

3. Methods

The biosensor tested in the present research was basedon two oxidation reactions involving the oxidation of thediphenol compound to quinone. The reactions were respec-tively catalysed by the enzyme tyrosinase, with consump-tion of oxygen, and by peroxidase, with consumption ofhydroperoxides. Catechol was selected (also in view of itsgood solubility in organic solvents) as common substrate,that is, for both the peroxidase-catalysed reaction and forthe tyrosinase-catalysed reaction. Both enzymes were immo-bilised in the gel membrane. The transducer was an amper-ometric gaseous diffusion oxygen electrode made of Teflon[6].

3.1. Enzyme immobilisation and biosensor assembly

The immobilisation of the two enzymes in the kappa-carrageenan gel-like membrane was carried out as describedin detail in a previous paper [15]. In order to optimise theeffect on biosensor response of the ratio between the en-zymatic units of the two enzymes used, quantities of 1.8,3.6 and 7.1 mg of peroxidase, respectively, were weighedout, while the quantity of weighed tyrosinase remained

L. Campanella et al. / Sensors and Actuators B 95 (2003) 321327 323

constant at 0.7 mg. In this way three different enzymaticsolutions were prepared, all obtained by dissolving the fol-lowing quantities of enzymes in 25.0l of phosphate buffer(1/15 mol l1 pH 6.5): (a) 0.7 mg of tyrosinase plus 1.8 mg ofperoxidase, corresponding to a ratio of 0.5 enzymatic units(peroxidase units/tyrosinase units); (b) 0.7 mg of tyrosinaseplus 3.6 mg of peroxidase, corresponding to a ratio of 1.0 en-zymatic units (peroxidase units/tyrosinase units); (c) 0.7 mgof tyrosinase plus 7.1 mg of peroxidase, corresponding toa ratio of 2.0 enzymatic units (peroxidase units/tyrosinaseunits). Each kappa-carrageenan disk, prepared as describedin a previous paper [15] was placed before use in a smallcontainer having a diameter of the same order of magnitudeas the disk, in which one of the two-enzyme solutions, pre-pared as described above, was placed; the small containerwas sealed and stored in a refrigerator at 4 C overnight.

In assembling the biosensor the disk containing the im-mobilised enzymes was positioned at the extremity of theTeflon cap of the amperometric gaseous diffusion oxygenelectrode, between the gas-permeable membrane and a dial-ysis membrane; the whole assembly was secured to the elec-trode cap by means of a Teflon O-ring [6].

3.2. Measures

Measures were performed in two stages:

1. The electrode response was allowed to stabilise (first sta-tionary state), after which a first addition of catechol wasmade. The addition of this substrate caused a reductionin dissolved O2 concentration in the solution due to ox-idation of the catechol, catalysed by the tyrosinase (re-action (1)). This produced a decrease in the current andthen, when the O2 consumption rate at the electrode sur-face became equal to the O2 diffusion rate from the at-mosphere to the solution, a new current stationary stateoccurred (second stationary state).

2. At this stage a fixed quantity of a hydroperoxide(tert-butylhydroperoxide, or H2O2, or some other hy-droperoxide) was added to the solution. After thisaddition, the catechol was oxidised not only by theO2 present in solution, but also by the hydroperoxideadded, according to the peroxidase-catalysed reaction

Fig. 1. Biosensor response to tert-butylhydroperoxide, working in decane.

(reaction (2)). This reaction led to an increase (i.e. apartial restoration) of the dissolved O2 concentration inthe solution as the hydroperoxide added competed withthe O2 in oxidising the catechol and so also a partialrestoration of the current occurred and a third stationarystate was reached. The difference in the current value,of the order of several tens of nA, between the lasttwo stationary states, is proportional to the quantity oftert-butylhydroperoxide added:

catechol+ 12 O2(tyrosinase) quinone+ H2O (1)

catechol+ 12 H2O2(peroxidase) quinone+ 2H2O (2)

A typical biosensor response to tert-butylhydroperoxide,working in decane solvent, is shown in Fig. 1.

3.3. Construction of a calibration curve usingthe biosensor

According to the scheme described in the precedingsection, the calibration curves were constructed using 8.0 mlof solvent contained in a 25.0 ml cell and maintained underconstant stirring using a magnetic microstirrer. A specialstock solution of hydroperoxide of known titre and one ofcatechol at 6.0 103 mol l1 in the same solvent werethen prepared. Once the signal had stabilised, 1.0 ml ofthe catechol solution was added. The signal variation wasrecorded, and the signal then allowed to stabilise again,after which a series of additions of 25.0l of the standardhydroperoxide test solution were made. After each additionthe signal was again allowed to stabilise and the correspond-ing current variation recorded. At the same time the signalvariation was constantly recorded on an analogue recorder.The standard solutions of the various hydroperoxides testedand used to construct the calibration curves case by case,were as follows: hydrogen peroxide 2.7 102 mol l1 indistilled water; or in water + dioxane (30 + 70)% (v + v);tert-butylhydroperoxide 9.2 101 mol l1 in decane;tert-butylhydroperoxide 2.8 101 mol l1 in water +dioxane (30 + 70)% (v + v); cumene hydroperoxide 8.7 101 mol l1 in decane; lauroylperoxide 5.6 102 mol l1in decane; di-tert-butylperoxide 2.1 101 mol l1 in


decane; dicumylperoxide 2.5 102 mol l1 in decane;benzoylperoxide 3.6 102 mol l1 in decane.

3.4. Hydrogen peroxide determination in drug samples

The method used to determine the hydrogen peroxide con-tent of the samples consisted of a direct comparison witha standard solution of known concentration, similar to thatof the test sample, both of which were suitably diluted sothat their final concentrations lay within the linear range ofthe method. Also in this case 8.0 ml of solvent was used,first adding 1.0 ml of standard catechol solution followed byfurther 25.0l additions, alternating additions of standardsolution with samples of the test solution, each time record-ing the current variations after each addition. By comparingthe latter after both the addition of sample and of standardsolution, at least three results were obtained referring to thesample concentration, which were then averaged.

3.5. Recovery tests on drug samples

Recovery trials using the standard addition method wereperformed on several of the drug samples tested. In eachcase the addition of a known hydrogen peroxide standardwas made in order to increase the initial concentration ap-proximately two-fold. The samples, before and after the ad-ditions of standard, were analysed using the procedure usedfor individual samples and described above.

3.6. Determination of hydrogen peroxide content of drugsolutions by means of titration

The reaction between permanganate and hydrogen per-oxide is used, adopting a decinormal solution of perman-ganate, the titre of which has been determined by titrationwith sodium oxalate. By suitably diluting the drug sample25.0 ml of an approximately decinormal solution of hydro-gen peroxide is prepared. From this solution 5.0 ml are takenand placed in a 150.0 ml flask to which are added 30.0 ml ofdistilled water and 10.0 ml of concentrated sulphuric acid,diluted 1+4 (v+v); lastly 15.0 ml of chloroform are added.Titration is performed at room temperature by dropping thepermanganate solution of known titre into the solution tobe determined. The organic phase remains at the bottom ofthe flask, separated from the aqueous phase in which thetitration takes place, so that the aqueous phase gradually be-comes depleted in peroxide due to its reaction with the per-

Table 1Comparison of analytical data for biosensor response to tert-butylhydroperoxide in decane using different P/T ratios (peroxidase units/tyrosinase units)P/T ratios Equation of calibration graph in the first day Correlation coefficient (r2) Linear range (mol l1) LOD (mol l1)0.5 y = 0.355(0.027)x+ 0.090(0.010) 0.9617 (0.21.5) 103 0.15 1031.0 y = 0.579(0.054)x 0.032(0.015) 0.9938 (0.21.5) 103 0.10 1032.0 y = 0.789(0.049)x 0.021(0.041) 0.9897 (0.21.4) 103 0.10 103

manganate; the hydrogen peroxide passes from the organicphase into the aqueous phase where it is titrated without anyloss [16]. At the equivalence point the solution takes on apale pink colour for about 30 s.

4. Result and discussion

In the chosen solvent. the biosensor was characterisedelectrochemically, enzymatically and analytically.

Great care was taken in characterising the biosensorin organic solvent (decane) according to three enzymaticratios (P/T = 0.5, 1.0, 2.0, respectively; P/T denotes per-oxidase units/tyrosinase units). Characterisation was per-formed using a standard solution of tert-butylhydroperoxide9.2 101 mol l1 in decane and immobilising the en-zymes in kappa-carrageenan. Decane was chosen as solventbecause it is highly hydrophobic, while immobilisation inkappa-carrageenan was suggested by the excellent repeatedresults obtained using this method, recently developed inour laboratory, in the construction of numerous OPEEs ca-pable of working efficiently in hydrophobic solvents liken-hexane, benzene, decane, etc. [15,17,18].

In Table 1 the analytical data referring to biosensor re-sponse to tert-butylhydroperoxide during the first day ofworking life are compared for the three different enzymaticratios (P/T) considered. The equations of the linear rangeof the calibration curves, the values of the slope, the linearrange and the lower detection limit (LOD) in the table arethe result of the mean of at least three experimental tests.

Using the same table a comparison can be made of sensi-tivity values, that is, of calibration curve slopes, during thefirst day of the working life of the biosensor prepared usingthe three different enzymatic ratios described above. It wasfound that the highest calibration sensitivity occurred witha P/T enzymatic ratio = 2 and that this sensitivity improve-ment was obtained by increasing the P/T ratio, although itwas not possible to test enzymatic ratios higher than 2 as,since the quantity of tyrosinase had to be kept constant (toavoid a strong reduction in signal strength), it would havebeen necessary to use quantities of peroxidase that were toohigh for the immobilisation method chosen in order to ob-tain P/T ratios higher than 2.

Furthermore, the period of use of the biosensor, whichwas assembled with three enzymatic P/T ratios, is about 34days in all three cases when operating in decane, althoughbiosensor sensitivity practically decreased by half as earlyas day two. Fig. 2 shows the biosensor slope variation trend


Fig. 2. Variation of sensitivity, as slope of calibration graph, as a function of time (days) for tert-butylhydroperoxide, working in decane or in dioxanewater(70+ 30)% (v+ v). Enzymatic P/T ratio = 2.

for P/T = 2, the ratio at which the greatest sensitivity wasobtained, throughout its entire lifetime (45 days).

Working in decane, also the biosensor response toother commercially available hydroperoxides soluble inorganic solvents was investigated. In Table 2 a compari-son is made of the analytical data referring to biosensorresponse to cumene hydroperoxide with those referring totert-butylhydroperoxide, working in decane. Lastly, it wasverified that the biosensor does not respond to peroxides sol-uble in decane, such as di-tert-butylperoxide, dicumylperox-ides and lauroylperoxides, or to benzoylperoxide, over theconcentration range tested (5 103 to 5 101 mol l1).

The biosensor was then used to determine the pool ofhydroperoxides released during the heating of extravirginolive oil to temperatures approaching those used to fry food-stuffs (=130140 C). Results are shown in Fig. 3. Alsothese measurements were performed working directly in de-cane, in which the olive oil is found to be highly soluble.

It is interesting to compare (Fig. 3) the peroxide concen-tration trend with that of the polyphenol obtained usinga tyrosinase biosensor (OPEE) as described in previous pa-pers [15,18]. In practice, it can be seen how, after the firsthour of heating, the polyphenol concentration is reduced toless than 1/5 of its initial value and it is at this stage thatthe hydroperoxides begin to appear, although in barely de-tectable concentrations, in the heated sample. However, after2 h of heating, they increase 16-fold, while the concentra-tion of the polyphenol pool is reduced to less than 1/10 of itsinitial value. Lastly, after 3 h of heating, as the polyphenolshave practically disappeared, a rapid increase in hydroper-oxides is observed, the concentration of which continues to

Table 2Comparison of analytical data for biosensor response to different hydroperoxides in decane or in dioxanewater (20+ 30)% (v+ v) mixtureTested substances Solvent Equation of calibration graph in the first day Correlation coefficient (r2) Linear range (mol l1)Tert-butylhydroperoxide Decane y = 0.355(0.027)x+ 0.090(0.010) 0.9617 (0.21.5) 103Cumene hydroperoxide Decane y = 0.027(0.003)x 0.022(0.003) 0.9702 (3.919.5) 103Tert-butylhydroperoxide Dioxanewatera y = 0.383(0.034)x+ 0.007(0.005) 0.9720 (0.31.3) 103Hydrogen peroxide Dioxanewatera y = 0.231(0.013)x+ 0.082(0.014) 0.9874 (1.35.2) 104

a (20+ 30)% (v+ v) mixture.

increase rapidly for about 5 h, after which it remains practi-cally constant before beginning to increase again, althoughmuch more slowly, after about 8 h of heating.

In conclusion, there is a close inverse relationship betweenpolyphenol concentration and hydroperoxide concentration:after the polyphenols have disappeared, the hydroperoxideconcentration increases rapidly.

Lastly the biosensor was used to determine the hydrogenperoxide content of cosmetic emulsions or creams, operatingin dioxanewater medium (70+ 30)% (v+ v). In this casethe choice of solvent was made not on theoretical groundsbut essentially for practical reasons. Indeed, after a series oftrials aimed at evaluating both the solubility of the productsto be analysed and the biosensor response in distilled waterand in pure dioxane, or else in different dioxanewater mix-tures; the dioxanewater mixture was chosen as it seemed tobe a good compromise between biosensor response and thesolubility of the tested products; the solubility actually variesfrom product to product, but is usually sufficiently good inmixtures containing around 70% dioxane. All the analyti-cal data concerning the standardisation of the biosensor totert-butylhydroperoxide, or to hydrogen peroxide workingin dioxanewater (70+30)% (v+v) mixture, have also beensummarised in Table 2, while the lifetime of the biosensorworking in this mixture is shown in Fig. 2.

The cosmetic samples were analysed, as well as with thebiosensor, also using classical titration with a standard per-manganate solution [16,19]. The good correlation of the re-sults obtained using both analytical methods is shown inTable 3 and the recovery values using the standard additionmethod are reported in Table 4.


Fig. 3. Reverse correlation between the trend of polyphenols and hydroperoxides concentration during the heating process of an extravirgin olive oilsample as a function of time.

Table 3Analysis of cosmetic and pharmaceutical samples containing hydrogen peroxide using biosensor in dioxanewater mixture; comparison with data obtainedby titration method

Sample no. Nominal value(%, w/v) (a)

Value obtained by titration(%, w/v) (RSD, %) (b)

Value obtained by biosensor(%, w/v) (RSD, %) (c)

[(b a)/a] (%) [(c a)/a] (%) [(c b)/b] (%)

1 Unknowna 9.80 (0.8) 9.32 (5.3) +4.92 12.31 12.23 (1.1) 11.84 (4.2) +0.65 3.8 +3.23 9.24 9.74 (0.8) 9.45 (3.6) +5.4 +2.3 3.04 6.15 5.96 (0.9) 5.93 (5.6) 3.1 3.6 +0.55 Unknown 3.52 (1.1) 3.63 (5.7) +3.16 Unknown 15.56 (0.6) 14.14 (5.3) 9.1

a Original nominal value 12.31% (w/v).

Table 4Recovery data for different cosmetic and pharmaceutical samples containing hydrogen peroxide using biosensor in dioxanewater mixture

Sampleno.

Found H2O2(%, w/v) (RSD, %)

Added H2O2(%, w/v)

Total H2O2(%, w/v)

Total found H2O2(%, w/v) (RSD, %)

Recovery(%)

1 9.32 (5.3) 9.00 18.32 18.37 (2.6) 100.33 9.45 (3.6) 9.50 18.95 19.06 (1.6) 100.64 5.93 (5.6) 5.55 11.48 11.46 (2.7) 99.85 3.63 (5.7) 4.00 7.63 7.41 (5.9) 97.1

5. Conclusions

Using the biosensor certainly makes it easier from boththe practical point of view and as regards rapidity andcost-effectiveness to assay the hydroperoxide content ofcosmetic preparations or of other samples containing hy-droperoxides that are poorly soluble in aqueous solution.

The precision of these determinations is generallygood (RSD 6%), the recoveries obtained using thestandard addition method are between about 97.1 and100.6%. Agreement is good also with the classical (per-manganate) titration method as in only one of the sixsamples considered does the difference between valuesexceed 5%.


Despite the apparent complexity of the previously de-scribed competitive mechanism, the method is actually veryrobust, versatile and reproducible. Furthermore if the OPEEdescribed is compared with the peroxidase amperometricOPEEs described in the literature [14], it may also be saidto have the advantage, as we have seen, of being able to op-erate not only in hydrophilic solvents, but also in hydropho-bic solvents, which favours the proper conservation of theenzymatic activity and also makes it possible to determinealso the hydroperoxides contained in highly hydrophobicreal matrixes, which are thus easily soluble only in highlyhydrophobic real matrixes (such as decane). This is true, forexample, in the case of the analysis of the hydroperoxidecontent of olive oil, as is shown by the encouraging resultsobtained in the determination of the pool of hydroperox-ides released during the heating of extravirgin olive oil. Themethod proved to be simple fast and very cheap.

Acknowledgements

This work was financially supported by ConsiglioNazionale delle Ricerche (CNR) of Italy, targeted project(Solid State Electronic Materials) MADESS and the Na-tional Group for Protection against Chemical IndustrialEcological Risk.

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Determination of hydroperoxides in nonaqueous solvents or mixed solvents, using a biosensor with two antagonist enzymes operating in parallelIntroductionExperimentalReagentCosmetic and drug samples usedOlive oil samplesApparatus

MethodsEnzyme immobilisation and biosensor assemblyMeasuresConstruction of a calibration curve using the biosensorHydrogen peroxide determination in drug samplesRecovery tests on drug samplesDetermination of hydrogen peroxide content of drug solutions by means of titration

Result and discussionConclusionsAcknowledgementsReferences

determination of hydroperoxides in nonaqueous solvents or mixed

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