METHOD FOR DETECTING SKATOLE IN A SAMPLE OF PIG ADIPOSE TISSUE

Abstract

A method for detecting the presence of skatole in a sample of pig adipose tissue, wherein the method comprises at least the steps of: a) subjecting an organic extract of the adipose tissue sample to an electrochemiluminescence reaction; b) measuring the luminescence intensity during step a) and, if the measured luminescence intensity exceeds a threshold value, deducing the presence of skatole in the sample of adipose tissue. The method also makes it possible, if skatole is present in the organic extract, to determine the content thereof.

Claims

1. A method for detecting a presence of skatole in a sample of a pig adipose tissue, comprising at least the steps of: a) subjecting an organic extract of the sample of the adipose tissue to an electrochemiluminescence reaction; b) measuring an intensity of a luminescence produced during step a) and, if the measured luminescence intensity exceeds a predetermined threshold value, deducing the presence of skatole in the sample of the adipose tissue.

2. The method of claim 1, wherein the organic extract comprises an aprotic organic solvent, less than 1 wt. % water, and a ground salt.

3. The method of claim 2, which comprises, prior to step a), preparation of the organic extract, said preparation comprising the steps of: i) separating the fat of the sample of the adipose tissue from the non-fatty elements of the sample of the adipose tissue; ii) dehydrating the fat of the sample of the adipose tissue; iii) dissolving the fat obtained at the end of step ii) in the aprotic organic solvent to obtain an organic solution and heating the organic solution; iv) degreasing the organic solution obtained at the end of step iii); and v) adding the ground salt to the organic solution obtained at the end of step iv), the ground salt being an anhydrous ground salt; and wherein steps i) and ii) are carried out successively or simultaneously.

4. The method of claim 3, wherein steps i) and ii) are carried out simultaneously.

5. The method of claim 4, wherein steps i) and ii) comprise heating the sample of the adipose tissue to a temperature of 100° C. to 150° C. in an open container, or to a temperature lower than 100° C. with a vacuum drawing.

6. The method of claim 2, wherein the organic solvent is acetonitrile, dimethylsulfoxide, propylene carbonate or γ-butyrolactone.

7. The method of claim 2, wherein the heating of step iii) is carried out in a closed container, at a temperature of 50° C. to 80° C., for 10 minutes to 30 minutes and with stirring.

8. The method of claim 2, wherein step iv) comprises centrifugating the organic solution obtained at the end of step iii), then fixing the fatty part of the organic solution by maintaining the organic solution at a temperature lower than or equal to 4° C. but higher than a solidification temperature of the aprotic organic solvent, and removing the fatty part that has been fixed.

9. The method of claim 2, wherein the ground salt is a tetraalkylammonium tetrafluoroborate, hexafluorophosphate or perchlorate wherein the alkyl group comprises 1 to 4 carbon atoms.

10. The method of claim 2, wherein the organic extract comprises 0.01 mol/L to 1 mol/L of the ground salt.

11. The method of claim 2, wherein the organic extract further comprises a strong base.

12. The method of claim 11, wherein the preparation of the organic extract further comprises a step vi) consisting of adding the strong base to the organic solution obtained at the end of step v).

13. The method of claim 1, wherein step a) comprises introducing the organic extract in an electrochemical cell comprising at least one working electrode and one counter-electrode, and applying a cathodic potential followed by an anodic potential to the working electrode.

14. The method of claim 13, wherein the application of the cathodic potential followed by the anodic potential to the working electrode comprises a potential sweep, a potential jump or a series of alternate cathodic and anodic potential pulses.

15. The method of claim 13, wherein the working electrode and the counter-electrode are made of doped diamond.

16. The method of claim 13, wherein the electrochemical cell is for single use.

17. The method of claim 1, further comprising a quantification of the skatole present in the organic extract, the quantification comprising comparing a maximum of the luminescence intensity measured during step b) with a calibration curve.

18. A method for sorting carcasses of whole male pigs, comprising an implementation of a method of claim 1.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0109] FIG. 1 represents schematically a first electrochemical cell which has been used in the experiments described above, in cross-section.

[0110] FIG. 2 represents schematically a second electrochemical cell which has been used in the experiments described below, ¾ view.

[0111] FIG. 3 illustrates the voltammogram obtained by subjecting an organic extract of a sample of pig adipose tissue not contaminated with skatole, to which synthetic skatole has been added, to an ECL reaction by potential sweeping.

[0112] FIG. 4 illustrates the luminescence measured simultaneously with the recording of the voltammogram shown in FIG. 3.

[0113] FIG. 5 illustrates the chronoamperogram obtained by subjecting an organic extract of a sample of pig adipose tissue not contaminated with skatole, to which synthetic skatole has been added, to an ECL reaction by potential step.

[0114] FIG. 6 illustrates the luminescence measured simultaneously with the recording of the chronoamperogram shown in FIG. 5.

[0115] FIG. 7 illustrates the current response obtained by subjecting a solution comprising synthetic skatole and tetrabutylammonium hexafluorophosphate in acetonitrile to an ECL reaction by applying a repetition of alternating cathodic and anodic voltage pulses.

[0116] FIG. 8 illustrates the luminescence measured simultaneously with recording the current response shown in FIG. 7.

[0117] FIG. 9 illustrates the luminescence measured by subjecting a solution comprising synthetic skatole and tetrabutylammonium hexafluorophosphate in acetonitrile to an ECL reaction by potential sweep.

[0118] FIG. 10 illustrates the luminescence measured by subjecting a solution comprising indole and tetrabutylammonium hexafluorophosphate in the acetonitrile to an ECL reaction by potential sweep.

[0119] FIG. 11 illustrates the luminescence measured by subjecting an organic extract of a sample of pig adipose tissue contaminated by skatole to an ECL reaction by potential step.

[0120] FIG. 12 illustrates the luminescence measured by subjecting an organic extract of a sample of pig adipose tissue not contaminated with skatole to an ECL reaction by potential step.

[0121] FIG. 13 illustrates two standard curves produced by subjecting organic extracts of samples of pig adipose tissue not contaminated with skatole but to which synthetic skatole has been added in an amount of 0.1 μmol/L to 5 μmol/L (curve 1) and solutions comprising 0.1 μmol/L to 5 μmol/L synthetic skatole and tetrabutylammonium hexafluorophosphate in acetonitrile (curve 2) to an ECL reaction by potential step.

[0122] In FIG. 3, the y-axis corresponds to the intensity, denoted I and expressed in microamperes (μA), of the current measured at the working electrode, whereas the x-axis corresponds to the potential, denoted V and expressed in volts (V) in relation to the potential of the reference electrode, applied to the working electrode.

[0123] In FIGS. 5 and 7, the y-axis corresponds to the intensity, denoted I and expressed in milliamperes (mA) in FIG. 5 and in microamperes (μA) in FIG. 7, of the current measured at the working electrode, whereas the x-axis corresponds to time, denoted t and expressed in seconds (s).

[0124] In FIGS. 4, 6, 8 to 12, the y-axis corresponds to the number of counts emitted, denoted N.sub.C and expressed in arbitrary units (u.a.), whereas the x-axis corresponds to the time, denoted t and expressed in seconds (s).

[0125] In FIG. 13, the y-axis corresponds to the number of counts emitted, denoted N.sub.C and expressed in arbitrary unit (u.a.), whereas the x-axis corresponds to the concentration of skatole, denoted [C] and expressed in micromoles/L (μmol/L).

DETAILED DESCRIPTION OF PARTICULAR EMBODIMENTS

[0126] The experiments which are described below are performed using: [0127] either organic extracts of samples of pig adipose tissue, which is either contaminated with skatole or not; [0128] or solutions referred to as “standard solutions” in the following and comprising synthetic skatole in acetonitrile.

I—Preparation of the Organic Extracts of Samples of Pig Adipose Tissues and of the Standard Solutions

[0129] I.1—Organic Extracts:

[0130] The organic extracts of samples of pig adipose tissues, which are either contaminated with skatole or not, are prepared for each extract according to the following operating protocol.

[0131] A sample of adipose tissue from a pig, cut finely, is placed in a glass beaker and brought to a temperature of 120° C. for 1 hour. During this heating, the beaker is not covered to allow the water vapour to escape. The size of the sample of adipose tissue is selected such that at least 5 mL of melted fat is collected at the end of this step.

[0132] Then, 5 mL of this melted fat is transferred into a test tube to which 5 mL acetonitrile (purity: 99.8%) are added. The test tube is closed, then the mixture is brought to a temperature of 70° C. for 10 minutes. A solution is obtained which is agitated by means of a vortex stirrer for 5 minutes, then centrifuged at 4000 rpm for 15 minutes to obtain a fatty phase and a solvent phase.

[0133] The tube is then placed in a freezer at 18° C. for at least 2 hours to freeze the fatty phase. The solvent phase is recovered and transferred to a new test tube.

[0134] To this phase, 100 mg of magnesium sulphate are added to finish drying it, then 0.385 mg tetrabutylammonium hexafluorophosphate (TBAHFF) are added to give it a ground salt concentration of 0.1 mol/L.

[0135] The extracts prepared in this way are kept in the refrigerator until used.

[0136] I.2—Standard Solutions:

[0137] The standard solutions are prepared by dissolving, with stirring, synthetic skatole in acetonitrile in a concentration ranging from 0.1 μmol/L to 5 μmol/L, then adding TBAHFF to the resulting solutions to give them a ground salt concentration of 0.1 mol/L.

II—Detection of Skatole

[0138] II.1—Experimental Apparatus:

[0139] The ECL reactions are performed by using the two electrochemical cells, denoted 10 and 20 respectively, which are illustrated schematically in FIGS. 1 and 2, and a PalmSens4™ potentiostat (PALMSENS).

[0140] The cell 10 comprises a single use UV/visible spectroscopy cuvette, made of polystyrene, the lower part of which has a quadrangular cross-section and in which two electrodes 11 and 12 made of boron-doped diamond, used respectively as the working electrodes and counter electrode, are positioned on two opposite walls of this lower part such that these electrodes face one another. The electrodes 11 and 12 have a surface area of 1 cm.sup.2.

[0141] The cell 20 itself comprises a parallelepiped-shaped cuvette in which two boron-doped diamond electrodes 21 and 22, used respectively as the working electrode and the counter electrode, are positioned on the two opposite walls of this cuvette which have the largest surface area but offset from one another so that these electrodes are arranged parallel to one another but without facing one another. The electrodes 21 and 22 have a surface area of 4 cm.sup.2.

[0142] The cells 10 and 20 are further provided with a platinum wire, respectively 13 and 23, used as a pseudo-reference electrode.

[0143] The detection of photons emitted during the ECL reactions is carried out in the wavelength range of 450 nm to 550 nm, by means of a Fluoromax™ 4 or 4P spectrofluorometer (HORIBA JOBIN YVON) which makes it possible to monitor in real time the evolution of the luminescence by means of integrated software.

[0144] During the ECL reactions, the electrochemical cells are placed in absolute darkness to limit the background noise of the spectrofluorometer.

[0145] II.2—Deprotonation of Organic Acids:

[0146] The measurements of skatole in the organic extracts are performed after the deprotonation of the organic acids present in these extracts.

[0147] This deprotonation is obtained by adding to each extract sodium hydride (NaH) in the form of a 60% dispersion of NaH in mineral oil (CAS: 7646-69-7), in an amount of 60 mg of this dispersion for 5 mL of extract. During this addition, bubbling is observed, corresponding to a release of dihydrogen. The extract is then left to rest for 15 minutes during which a precipitate forms, corresponding to the saponification of organic acid present in the extract. This precipitate is removed.

[0148] II.3—Verification of the Possibility of Performing the ECL Reaction According to Different Electrochemical Protocols:

[0149] As previously indicated, the ECL reaction of skatole which is preferred in the context of the invention is a reaction caused by the application of a cathodic potential to the working electrode of an electrochemical cell to induce the formation of superoxide ions, in an organic extract obtained from a sample of pig adipose tissue, by reducing the dioxygen dissolved in this extract.

[0150] This reaction can be broken down into the following steps: [0151] application of a cathodic potential: [0152] (1) formation of superoxide ions by reduction of dissolved dioxygen:

O.sub.2+e.sup.−.Math.O.sub.2.sup.−. [0153] (2) formation of the conjugate base of skatole in the presence of superoxide ions:

3-MIH+O.sub.2.sup.−..fwdarw.3-MI.sup.−+HO.sub.2. [0154] (3) formation of hydroperoxyl radicals:

O.sub.2.sup.−.+HO.sub.2..fwdarw.O.sub.2+HO.sub.2.sup.− [0155] application of an anodic potential: [0156] (4) oxidation of skatole:

3-MI.sup.−-e.sup.−.fwdarw.3-MI.

[0157] with:

##STR00001## [0158] (5) radical coupling of oxidised skatole with HO involving the formation of an intermediate compound comprising a 1,2-dioxetane group followed by N-(2-acetylphenyl)formamide in an excited state:

##STR00002##

[0159] To obtain this reaction, three electrochemical protocols are tested: [0160] a potential sweep (cyclic voltammetry); [0161] a potential step (chronoamperometry); and [0162] a repeated application of alternating cathodic and anodic voltage pulses.

[0163] Potential Sweep:

[0164] For the potential sweep, the following are used: [0165] an organic extract of a sample of a pig adipose tissue not contaminated with skatole to which synthetic skatole has been added in a concentration of 5 μmol/L, and [0166] the electrochemical cell 10 shown in FIG. 1.

[0167] After introducing the organic extract into the electrochemical cell, a potential sweep from 0 to −2 V followed by a potential sweep from −2 V to +0.8 V (versus the reference electrode 13) are applied to the working electrode 11 of this cell with a sweep speed of 50 mV/s.

[0168] The luminescence measurement is started from the beginning of the potential sweep.

[0169] The thus obtained voltammogram and the thus measured luminescence are illustrated in FIGS. 3 and 4 respectively.

[0170] As shown in FIG. 4, a luminescence peak is observed in the anodic potential sweep zone, which can be related to the presence of skatole in the organic extract and the amplitude of which can be connected to the quantity of skatole present in this extract.

[0171] Potential Step:

[0172] For the potential step, the following are used: [0173] an organic extract of a sample of pig adipose tissue not contaminated with skatole to which synthetic skatole has been added in a concentration of 5 μmol/L, and [0174] the electrochemical cell 20 shown in FIG. 2.

[0175] Having introduced the organic extract into the electrochemical cell, a potential of −2 V (versus the reference electrode 23) for 30 seconds, followed by a potential of +1 V (versus the reference electrode 23) for a further 30 seconds are applied to the working electrode 21 of this cell.

[0176] Here also, the luminescence measurement is started from the beginning of the application of potential to the working electrode.

[0177] The thus obtained chronoamperogram and the thus measured luminescence are illustrated in FIGS. 5 and 6 respectively.

[0178] As shown in FIG. 6, an intense peak of luminescence is observed during the passage from the first potential to the second potential, this peak being able, there also, to be related to the presence of skatole in the organic extract and the amplitude of which may be related to the quantity of skatole present in this extract.

[0179] Pulse Regime:

[0180] The test which consists of applying to the working electrode a repetition of alternating cathodic and anodic voltage pulses is performed by using: [0181] a standard solution of 5 μmol/L skatole, and [0182] the electrochemical cell 20 shown in FIG. 2.

[0183] After introducing the standard solution into the electrochemical cell, a potential of −2 V (versus the reference electrode 23) followed by a potential of +1 V (versus the reference electrode 23) are applied to the working electrode 21 of this cell in a repeated manner at a frequency of 1 Hz.

[0184] Here too, the measurement of the luminescence is started from the beginning of the application of potential to the working electrode.

[0185] The thus recorded current response of the working electrode and the thus measured luminescence are illustrated in FIGS. 7 and 8 respectively.

[0186] As shown in FIG. 8, the emission of a variable luminescence signal but with an average intensity of around 14 000 counts is observed throughout the duration of the application of the pulse regime.

[0187] Another test is carried out in the same experimental conditions except that a frequency of 5 Hz is used instead of 1 Hz. A similar ECL response is obtained but with an average luminescence intensity of about 8 000 counts, that is to say weaker than the preceding one.

[0188] II.4—Verification of the Specific Nature of the Detection of Skatole:

[0189] Two experiments are carried out, namely: [0190] a first experiment which consists of subjecting a statistically significant series of organic extracts from samples of pig adipose tissues not contaminated with skatole (the absence of skatole in these extracts having been verified by gas chromatography coupled with mass spectrometry or GC/MS, which makes it possible to dose skatole to a concentration of 0.03 μg/g fat), in an ECL reaction and to measure the luminescence emitted during this reaction; [0191] a second experiment which consists of subjecting to an ECL reaction, on the one hand, a standard solution of 5 μmol/L skatole, and, on the other hand, solutions comprising 5 μmol/L indole and 0.1 mol/L TBAHFF in acetonitrile, in the same operating conditions and measure the luminescence emitted during this reaction.

[0192] In this regard, it should be noted that skatole and indole only differ structurally from one another in that the pyrrole cycle of skatole bears a methyl group contrary to the pyrrole cycle of indole.

[0193] The first experiment is performed in the electrochemical cell 10 shown in FIG. 1 and by applying a potential step, as described in point II.3 above.

[0194] The second experiment is performed in the electrochemical cell 20 shown in FIG. 2 and by applying a potential sweep, as also described in point II.3 above.

[0195] No significant luminescence signal is detected during the first experiment, the measured luminescence being of the type shown in FIG. 12.

[0196] As shown in FIGS. 9 and 10 which represent examples of the luminescence measured during the second experiment for a standard solution of skatole and a solution of indole respectively, an intense luminescence signal is observed for the standard solution of skatole, whereas no significant luminescence signal is observed for the indole solution, thus confirming the very specific nature of the detection of skatole by the method of the invention.

[0197] II.5—Blind Skatole Detection Tests:

[0198] Skatole detection tests are performed by subjecting organic extracts of samples of a pig adipose tissue contaminated with skatole (0.27 μg skatole/g adipose tissue) and organic extracts of a pig adipose tissue not contaminated with skatole to an ECL reaction in the electrochemical cell 20 shown in FIG. 2, by applying a potential step as described in point II.3 above and by measuring the luminescence emitted during this reaction.

[0199] The contamination and absence of contamination of organic extracts were checked beforehand by GC/MS.

[0200] The tests were carried out blind, i.e. the experimenter does not know when subjecting an extract to an ECL reaction whether this extract is contaminated or not with skatole.

[0201] FIG. 11 shows a luminescence signal as typically observed for an organic extract of a sample of pig adipose tissue contaminated with skatole, whereas FIG. 12 shows the absence of a significant luminescence signal as observed for an organic extract of a sample of pig adipose tissue not contaminated with skatole.

[0202] II.6—Calibration Curves:

[0203] In order to verify the possibility of quantifying the skatole present in an organic extract of pig adipose tissue, calibration curves are produced by subjecting: [0204] on the one hand, standard solutions of skatole comprising 0.1 μmol/L to 5 μmol/L skatole; and [0205] on the other hand, organic extracts of samples of a pig adipose tissue not contaminated with skatole but with the addition of synthetic skatole in an amount of 0.1 μmol/L to 5 μmol/L;
to an ECL reaction and measuring the maximum intensity of the luminescence emitted during this reaction.

[0206] The ECL reaction is performed in the electrochemical cell 10 shown in FIG. 1 and by applying to the working electrode 11 of this cell a potential of −2 V (versus the reference electrode 13) for 30 seconds, then of +1 V (versus the reference electrode 13) for another 30 seconds.

[0207] The calibration curves obtained in this way are illustrated in FIG. 13, curve 1 corresponding to the organic extracts with added synthetic skatole and the curve 2 corresponding to standard solutions of skatole.

[0208] This figure shows, for the two curves, an almost linear relationship between the amplitude of the peaks of luminescence and the concentration of skatole.

[0209] The slope of the curve 1 is approximately twice as low as that of the curve 2, which means that the detection sensitivity of the skatole in the case of organic extracts with added synthetic skatole is about twice as low as in the case of standard solutions of skatole.

[0210] This is explained experimentally, first of all, by the fact that the organic extracts with synthetic skatole added reabsorb 30% of the photons emitted (measurement carried out by UV/visible spectroscopy). The loss of the remaining 20% of signals is probably due to the fact that the organic extracts with synthetic skatole added have a higher viscosity than the standard solutions of synthetic skatole and therefore a lower ionic mobility which slows down the diffusion processes governing the electrochemical processes.

[0211] However, these two phenomena (reabsorption of photons and increase in viscosity) can be easily corrected from, on the one hand, the absorption coefficient of the organic extract analysed at the wavelength of interest (use of the average value which is not likely to vary significantly from one organic extract to another, or where necessary the measurement performed in parallel on each organic extract), and on the other hand diffusion coefficients of the ionic species involved in the analysed organic extract that have been evaluated previously.

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