Monitoring mycotoxins and its metabolites in the blood of pigs or broiler chickens

Abstract

The present invention relates to a multi-screening method for the detection of a large number of mycotoxins and metabolites in broiler chickens and pigs, the method comprising collecting the blood of broiler chickens and pigs as a dried blood sample, preparing the dried blood sample for analysis and analyzing the prepared dried blood sample by a two-step liquid chromatography-tandem mass spectrometry LC-MS/MS process. In a first LS-MS/MS step, for a given mobile phase for the LC process, the mass spectrometer operates in negative electro-spray ionization mode, and for another mobile phase for the LC process, the mass spectrometer operates in positive electro-spray ionization mode. Such method can advantageously be used for screening and assessing the exposure of pigs or broiler chickens to feed contaminated with mycotoxins. Also, such method can be used for assessing the impact of the addition of mycotoxin detoxifying agents to animal feed.

Claims

1. A method for the detection of mycotoxins and metabolites in broiler chickens or pigs, comprising: collecting the blood of broiler chickens or pigs as a dried blood sample; preparing the dried blood sample for analysis; analyzing the prepared dried blood sample by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in two steps: a) detecting in an LC-MS/MS step, the reversed phase LC process using as mobile phase, a mixture of acetic acid, acetonitrile and water, the proportion of the acetonitrile solution over the water solution gradually increasing during the process, and at the end diminishing to recondition the LC column, the mass spectrometer operating in negative electro-spray ionization mode, and b) detecting in another LC-MS/MS step, the reversed phase LC process using as mobile phase, a mixture of ammonium formate, formic acid, water and methanol, the proportion of the methanol solution over the water solution gradually increasing during the process, and at the end diminishing to recondition the LC column, the mass spectrometer operating in positive electro-spray ionization mode.

2. A method according to claim 1 for the detection of the following mycotoxins and metabolites: Alternariol methyl ether (AME), Aflatoxin B1 (AFB1), Aflatoxin B2 (AFB2), Aflatoxin M1 (AFM1), Fumonisin (FB1), Fumonisin (FB2), Ochratoxin A (OTA), Deoxynivalenol (DON), T2 toxin (T2), HT-2-toxin (HT2), Zearalenone (ZEN) Alternariol (AOH), Tenuazonic Acid (TEA), Beauvericin (BEA), Enniatin A, A1, B, B1 (ENNA, ENNA1, ENNB, ENNB1).

3. A method according to claim 1 for the detection of the following mycotoxins and metabolites: de-epoxy-deoxynivalenol (DOM1), 15-acetyldeoxynivalenol (15ADON), 3-acetyldeoxynivalenol (3ADON), Zearalanone (ZAN), α-Zearalenol (AZEL), α-Zearalanol (AZAL), β-Zearalanol (BZAL), β-Zearalenol (BZEL), DON-glucuronide (DON-GlcA), DON-Sulphate (DON-S), ZEN-glucuronide (ZEN-GlcA), ZEN-sulfate (ZEN-S), α-zearalenol-glucuronide (A-ZEL-GlcA), β-zearalenol-glucuronide (B-ZEL-GlcA), Fumonisin B3 (FB3), Aflatoxin M2 (AFM2), Aflatoxin G 1 (AFG1), Aflatoxin G2 (AFG2).

4. A method according to claim 1 wherein any of the following mycotoxins and metabolites are detected by the mass spectrometer operating in negative electrospray ionization mode: zearalenone (ZEN), zearalanone (ZAN), α-zearalenol (AZEL) α-zearalanol (AZAL), β-zearalanol (BZAL), β-zearalenol (BZEL), tenuazonic acid (TEA), alternariol (AOH), alternariol methyl ether (AME), deoxynivalenol sulphate (DON-S), deoxynivalenol glucuronide (DON-GlcA), zearalenone sulphate (ZEN-S), zearalenone glucuronide (ZEN-GlcA), a-ZEL-glucuronide (AZEL-GlcA), b-ZEL-glucuronide (BZEL-GlcA).

5. A method according to claim 1 wherein any of the following mycotoxins and metabolites are detected by the mass spectrometer operating in positive electrospray ionization mode: deoxynivalenol (DON), de-epoxy-deoxynivalenol (DOM1), 3-acetyldeoxynivalenol (3ADON), 15-acetyldeoxynivalenol (15ADON), T2-toxin (T2), HT-2 toxin (HT2), aflatoxin B1 (AFB1), aflatoxin M1 (AFM1), ochratoxin A (OTA), enniatin A1 (ENNA1), enniatin A (ENNA), enniatin B (ENNB), enniatin B1 (ENNB1), beauvericin (BEA), fumonisin (FB1), fumonisin (FB2), aflatoxin B2 (AFB2), aflatoxin G1, (AFG1), aflatoxin G2 (AFG2), aflatoxin M2, (AFM2) fumonisin B3 (FB3).

6. A method according to claim 1, whereby in the LC-MS/MS step, using as mobile phase, a mixture of an acetic acid, acetonitrile and water solution, the overall process time amounts to 11 minutes or less, wherein the proportion of the acetonitrile solution over the water solution gradually increases from 5/95% to 60/40% in a time period of maximum 8 minutes, thereafter decreases to 5/95% for a 3 minutes time period to recondition the LC column, and/or, in the LC-MS/MS step, using as mobile phase, a mixture of ammonium formate, formic acid, water and methanol, the overall process time amounts 12 minutes or less, wherein the proportion of the methanol solution over the water solution gradually increases from 5/95% to 99/1% in a time period of maximum 9 min, thereafter decreases to 5/95% for a 3 minutes time period to recondition the LC column.

7. A method according to claim 1, wherein in the LC process a column is used comprising an hydrophobic stationary phase comprising silica with covalently bonded alkyl chains, preferably high strength silica with trifunctional C.sub.18 alkyl bonded chains.

8. A method according to claim 1, comprising, prior to analyzing, extracting the mycotoxins and the metabolites from the dried blood sample in an extraction solvent and subjecting the extraction solvent and the dried blood sample to an ultrasonic bath treatment.

9. A method according to claim 8, whereby the extraction solvent comprises a water/acetonitrile/acetone mixture.

10. A method according to claim 7, comprising drying the extraction solvent and reconstituting the dried mass in a reconstitution solvent.

11. A method according to claim 10, whereby the reconstituting solvent comprises a water/methanol/formic acid mixture.

12. A method according to claim 1, further comprising spiking the collected dried blood sample with one or more internal standards.

13. A method according to claim 12, comprising spiking the collected dried blood sample with one or more internal standards selected from the following: .sup.13C.sub.15-deoxynivalenol .sup.13C.sub.17-aflatoxin B1, .sup.13C.sub.17-aflatoxin M1, .sup.13C.sub.20-ochratoxin A, .sup.13C.sub.24-T2-toxin, .sup.13C.sub.34-fumonisin B1, .sup.15N.sub.3-enniatin B, .sup.13C.sub.6.sup.15N-tenuazonic acid, .sup.13C.sub.18-zearalenone.

14. Use of the method of claim 1 for screening and assessing the exposure of pigs or broiler chickens to feed contaminated with mycotoxins.

15. Use of the method of claim 1 for assessing the impact of the addition of mycotoxin detoxifying agents to animal feed.

Description

DETAILED DESCRIPTION OF THE INVENTION

Collection and Preparation of the Dried Blood Sample (DBS)

[0112] According to a preferred embodiment of the invention, collecting the dried blood sample comprises collecting a drop of blood on a filter paper, followed by drying at room temperature.

[0113] According to a further preferred embodiment of the invention, collecting the dried blood sample comprises isolating the dried blood sample from the filter paper by punching out a paper disk out of the filter paper, preferably round and about 8 mm in diameter, using a biopsy punch.

[0114] According to a further preferred embodiment of the invention, prior to analyzing, the blood from the dried blood sample is prepared for analysis, such preparation including extracting the dried blood sample in an extraction solvent.

[0115] The extraction solvent preferably comprises a water/acetonitrile/acetone mixture.

[0116] Thereupon, the extraction solvent is dried and reconstituted in a reconstitution solvent, such reconstituting solvent preferably comprising a water/methanol/formic acid mixture.

[0117] So, the collected dried blood sample is extracted in an extraction solvent, dried, reconstituted in a reconstitution solvent, whereupon the reconstituted dried blood sample can be analyzed by liquid chromatography tandem mass spectrometry.

LC-MS/MS

[0118] The basic analytical technique used in the method of the present invention is liquid chromatography with tandem mass spectrometry, hereinafter abbreviated as LC-MS/MS.

[0119] The term Liquid Chromatography is hereinafter referred to as LC.

[0120] The term Mass Spectrometry is hereinafter referred to as MS.

[0121] The term tandem Mass Spectrometry is hereinafter referred to as MS/MS.

[0122] The term High Resolution Mass Spectrometry is hereinafter referred to as HR-MS.

[0123] The term “Liquid Chromatography (LC)” as used in the context of the present invention should be understood as a laboratory technique for the separation of a mixture of compounds. The mixture is dissolved in a fluid called the mobile phase, which carries it through a structure holding another material called the stationary phase. The various constituents of the mixture travel at different speeds, causing them to separate. The separation is based on differential partitioning between the mobile and stationary phases. Subtle differences in a compound's partition coefficient result in differential retention on the stationary phase and thus affect the separation.

[0124] Chromatography may be preparative or analytical. The purpose of preparative chromatography is to separate the components of a mixture for later use, and is thus a form of purification. Analytical chromatography is done with smaller amounts of material and is for establishing the presence or measuring the relative proportions of analytes in a mixture. For the purpose of the present invention, analytical chromatography is applied.

[0125] Tables 4 and 5, included further in the present specification, illustrate the above: the retention times are set forth for the various components comprised in the DBS sample subjected to the LC process step as comprised in the method of the present invention.

[0126] The term “Mass Spectrometry (MS)” as used in the context of the present invention should be understood as an analytical technique that measures the mass-to-charge ratio of ions. The results are typically presented as a mass spectrum, a plot of intensity as a function of the mass-to-charge ratio. In the present case, mass spectrometry is applied to a quite complex organic mixture.

[0127] A mass spectrum is a plot of the ion signal as a function of the mass-to-charge ratio. The spectra are used to determine the elemental or isotopic signature of a sample, the masses of particles and of molecules, and to elucidate the chemical identity or structure of molecules and other chemical compounds.

[0128] In the MS procedure as applied in the present invention, a sample, being the collected dried blood sample, extracted, dried and reconstituted as described below, is ionized. This will cause some of the sample's molecules to break into charged fragments or simply become charged without fragmenting. These ions are then separated according to their mass-to-charge ratio, for example by accelerating them and subjecting them to an electric or magnetic field: ions of the same mass-to-charge ratio will undergo the same amount of deflection. The ions are detected by a mechanism capable of detecting charged particles, such as an electron multiplier. Results are displayed as spectra of the signal intensity of detected ions as a function of the mass-to-charge ratio. The molecules in the dried blood sample are then identified either through a characteristic pattern for the whole of the mycotoxin or phase I metabolite molecules or through a characteristic pattern for the fragments of the mycotoxin or metabolite molecules.

LC-MS/MS

[0129] The term “Liquid Chromatography tandem Mass Spectrometry (LC MS/MS)” as used in the context of the present invention should be understood as Liquid Chromatography, followed by Mass Spectrometry. In such a case the Dried Blood Sample, after being extracted in the extraction solvent, being dried and reconstituted in the reconstituting solvent, is injected in the column of the Liquid Chromatography apparatus; the various mycotoxins and phase I metabolites, exiting the Liquid Chromatography column, are then subjected to an identification and quantification analysis by a Mass Spectrometry apparatus, based on a targeted approach.

[0130] Liquid Chromatography with tandem mass spectrometry (LC-MS/MS) is a powerful analytical technique that combines the separating power of liquid chromatography with the highly sensitive and selective mass analysis capability of triple quadrupole mass spectrometry.

[0131] A sample solution containing analytes of interest are pumped through a stationary phase (LC column) by a mobile phase flowing through at high pressure. Chemical interaction between the components of the sample, the stationary phase and the mobile phase affects different migration rates through the LC column affecting a separation. The wide variety of stationary phase and mobile phase combinations allow for customizing a separation to suit many complex solutions.

[0132] After elution from the LC column, the effluent is directed to the mass spectrometer. The mass spectrometer for an LC-MS/MS system has an ionization source where the LC column effluent is nebulized, desolvated and ionized creating charged particles. These charged particles then migrate under high vacuum through a series of mass analyzers (quadrupole) by applying electromagnetic fields. A specific mass/charge precursor ion (or parent ion) is targeted to pass through the first quadrupole, excluding all other mass/charge ratio particles. In the collision cell, the selected mass/charge ions are then fragmented into product ions (or daughter ions) by collision with an inert gas. The third quadrupole is used to target specific product ion fragments. The resulting isolated product ions are then quantified with an electron multiplier. This transition of ions from the precursor to product ion (also referred to as MS.sup.2) is highly specific to the structure of the compound of interest and therefore provides a high degree of selectivity.

[0133] The strength of this technique lies in the separation power of LC for a wide range of compounds combined with the capability of the MS to quantify compounds with a high degree of sensitivity and selectivity based on the unique mass/charge (m/z) transitions of each compound of interest.

[0134] 36 Parent Mycotoxins and Phase I & II Metabolites

[0135] As set forth supra, a preferred embodiment of the present invention is directed to the detection of one or more of the 36 mycotoxins and their major phase I & I1 metabolites, set forth in the following list. In this list, the various compounds are grouped into either mycotoxins (parent), phase I metabolites, and phase I1 metabolites.

[0136] Also, the corresponding compounds detectable by the method of the prior art, described in the abovementioned reference (3) are also included in this table.

[0137] Finally the table indicates whether under the method of the present invention, the indicated compound is detected by the mass spectrometer either operating in positive (+) or negative (−) electrospray ionization (ESI−) mode.

[0138] Finally, the same table indicates whether according to the method of the present invention, as compared to the prior art method described in reference article (3), the indicated compound is detected quantitatively or qualitatively.

TABLE-US-00001 TABLE I List of Mycotoxins detected in DBS by LC-MS/MS Prior art list List acc. to invention 23 Analytes 36 Analytes Mycotoxins (parent) Mycotoxins (parent) ESI- fully quantified fully quantified Mode 1 Deoxynivalenol (DON) Deoxynivalenol (DON) + 2 3/15-acetyl deoxynivalenol 3-acetyl deoxynivalenol + (3/15ADON) (3/ADON) 3 Aflatoxin B1 (AFB1) Aflatoxin B1 (AFB1) + 4 Enniatin A (ENNA) Enniatin A (ENNA) + 5 Enniatin A1 (ENNA1) Enniatin A1 (ENNA1) + 6 Enniatin B (ENNB) Enniatin B (ENNB) + 7 Enniatin B1 (ENNB1) Enniatin B1 (ENNB1) + 8 Beauvericin (BEA), Beauvericin (BEA), + 9 Fumonisin B1 (FB1) Fumonisin B1 (FB1) + 10 Fumonisin B2 (FB2) Fumonisin B2 (FB2) + 11 Ochratoxin A (OTA) Ochratoxin A (OTA) + 12 T-2 toxin (T-2) T-2 toxin (T-2) + 13 Zearalenone (ZEN) Zearalenone (ZEN) − 14 Tenuazonic acid (TEA) Tenuazonic acid (TEA) − 15 Alternariol (AOH) Alternariol (AOH) − 16 Alternariol mono- Alternariol mono- − methylether (AME) methylether (AME) 17 Aflatoxin B2 (AFB2) − 18 Aflatoxin G1 (AFG1) − 19 Aflatoxin G2 (AFG2) − Phase I metabolites Phase I metabolites fully quantified fully quantified 1 de-epoxy-Deoxynivalenol de-epoxy-Deoxynivalenol + (DOM1) - phase I metabolite (DOM1) - phase I metabolite 2 Aflatoxin M1 (AFM1) - Aflatoxin M1 (AFM1) - + phase I metabolite phase I metabolite 3 α-Zearalenol (AZEL) - α-Zearalenol (AZEL) - − phase I metabolite phase I metabolite 4 β-Zearalenol (BZEL) - β-Zearalenol (BZEL) - − phase I metabolite phase I metabolite 5 α-Zearalanol (AZAL) - α-Zearalanol (AZAL) - − phase I metabolite phase I metabolite 6 β-Zearalanol (BZAL) - β-Zearalanol (BZAL) - − phase I metabolite phase I metabolite 7 Zearalanone (ZAN) - phase I Zearalanone (ZAN) - phase I − metabolite metabolite Phase II metabolites only Phase II metabolites only qualified: present/absent qualified: present/absent 1 15-acetyl deoxynivalenol + (15ADON) 2 DON-glucuronide + (DON-GlcA) 3 DON-sulphate (DON-S) + 4 ZEN-glucuronide + (ZEN-GlcA) 5 a-ZEL-glucuronide − (AZEL-GlcA) 6 b-ZEL-glucuronide − (BZEL-GlcA) 7 ZEN-sulfate (ZEN-S) + 8 Fumonisin B3 (FB3) − 9 Aflatoxin M2 (AFM2) − 10 HT-2 toxin (HT-2) +

[0139] Near to the name of each mycotoxin or phase I or II metabolite, the above table comprises also the name in abbreviated form (such as e.g. DON for Deoxynivalenol). In the description that follows, reference to the above components may be made by referring to such abbreviated name.

[0140] So, as compared to the LC-MS/MS part of the prior art technique disclosed in the article referenced above as (3), three additional parent mycotoxins can be detected by the method of the present invention.

[0141] Further, by applying the method of the present invention, 10 phase I1 metabolites can be detected in the LC-MS/MS method, without any need to apply the complicated LC-HRMS technique, as was the case for the prior art technique disclosed in reference article (3).

ESI Positive and Negative Mode

[0142] In the method of the present invention the overall procedure for analyzing a DBS sample is as follows: [0143] a) a first part of a given sample is submitted to a first LC step, the mobile phase in the LC apparatus being the mixture as set forth in Table 2 below (phase A—water+phase B methanol) followed by an MS procedure, operating in ESI positive mode; [0144] this yields results for the analytes listed in Table 4; (21 analytes); [0145] b) hereupon the remaining part of the same sample (or alternatively a separate identical sample) is submitted to a second LC step, the mobile phase in the LC apparatus being the mixture as set forth in Table 3 (phase A—water+phase B acetonitrile) again followed by an MS procedure, operating in ESI Negative mode; [0146] this yields results for the analytes listed in Table 5; (15 analytes).

[0147] The sequence as set forth above, whereby in the first step the MS operates in ESI positive mode, and whereby in the second step the MS operates in ESI negative mode, can be reversed. These is no technical requirement to have the MS apparatus work either in positive or negative mode in the first step; any of both these steps (ESI+, resp. ESI−) can be applied first, whereupon in the second step the other mode is used (ESI−, resp. ESI+).

[0148] So, what is applied in the method of the present invention is LC/MS step 1, followed by LC/MS step 2. Altogether, this 2-step-procedure takes 23 minutes for a complete analysis and detection.

TABLE-US-00002 TABLE 2 UPLC conditions for analytes determined in dried blood spots (DBS) using the ESI positive mode. Column + guard Acquity HSS-T3; column: 100 × 2.1 mm i.d., column dp: 1.8 μm; VanGuard pre-column: 5 × 2.1 mm i.d., dp: 1.8 μm Column temperature 40° C. Sample manager 8° C. temperature Gradient program Mobile phase A 0.3% formic acid + 10 mM ammonium formate in water Mobile phase B 0.3% formic acid + 10 mM ammonium formate in methanol Time Flaw rate % mobile % mobile (min) (ml/min) phase A phase B 0.0 0.3 95 5 0.5 0.3 95 5 1.5 0.3 40 60 2.5 0.3 40 60 5.0 0.3 20 80 6.0 0.3 1 99 8.9 0.3 1 99 9.0 0.3 95 5 12.0 0.3 95 5

[0149] As shown by the above table, the proportion of the methanol solution over the water solution gradually increases during the LC process. At the end, this trend is reversed, the proportion of the water solution over the methanol solution being increased again so as to recondition the LC column.

[0150] As compared to the prior art process disclosed in reference (3), the inventors have succeeded in reducing the overall process time to only 12 minutes (as compared to a required process time of 16 minutes in the prior art.)

TABLE-US-00003 TABLE 3 UPLC conditions for analytes determined in DBS using the ESI negative mode. Column + guard Acquity HSS-T3; column: 100 × 2.1 mm i.d., column dp: 1.8 μm; Vanguard pre-column: 5 × 2.1 mm i.d., dp: 1.8 μm Column temperature 40° C. Sample manager 8° C. temperature Gradient program Mobile phase A 1% acetic acid in water Mobile phase B 1% acetic acid in acetonitrile Time Flaw rate % mobile % mobile (min) (ml/min) phase A phase B 0.0 0.3 95 5 1.0 0.3 95 5 4.5 0.3 40 60 7.8 0.3 40 60 8.0 0.3 95 5 11.0 0.3 95 5

[0151] As shown by the above table, the proportion of the acetonitrile solution over the water solution gradually increases during the LC process. At the end, this trend is reversed, the proportion of the water solution over the acetonitrile solution being increased again so as to recondition the LC column.

[0152] As compared to the prior art process disclosed in reference (3), the inventors have succeeded in reducing the overall process time to only 11 minutes (as compared to a required process time of 12 minutes in the prior art.)

[0153] The term ‘parent’ mycotoxin as used in the above table and throughout the present specification means a mycotoxin that can be found in the (animal) feed, this to make the difference with a metabolite of such mycotoxin that can be formed in the animal (for example in the liver).

MS/MS ESI+ and ESI− Mode

[0154] Upon preparation of the DBS sample, the actual LS-MS/MS two-step process according to the present invention, can be applied.

[0155] The two subsequent steps are:

in one step, analyzing by LC-MS/MS, the mass spectrometer apparatus operating in negative electro-spray ionization (ESI−) mode, and using as mobile phase, a mixture of acetic acid, water and acetonitrile, the proportion of acetonitrile gradually increasing during the liquid chromatography process, a first series of mycotoxins and its phase I & II metabolites:
in another step, analyzing by LC-MS/MS, the mass spectrometer apparatus operating in positive electro-spray ionization mode, and using as mobile phase, a mixture of ammonium formate, formic acid, water and methanol, the proportion of methanol gradually increasing during the liquid chromatography process, a further series of mycotoxins and its phase I & II metabolites.

[0156] So, contrary to the prior art comprising detection of some phase I1 metabolites of mycotoxins by LC-HRMS, the method of the present invention comprises detection of such phase I1 metabolites of mycotoxins by the usual LC-MS/MS method.

TABLE-US-00004 TABLE 4 Analytes determined in DBS using the ESI positive mode: LC retention time, Internal standard, Quantitative (Quan) or Qualitative (Qual) detection Retention Name time (min) Internal standard Quan/Qual DON 3.27 [.sup.13C.sub.15]-DON Quan DOM-1 3.54 [.sup.13C.sub.15]-DON Quan 15-ADON 3.83 [.sup.13C.sub.15]-DON Qual 3-ADON 3.82 [.sup.13C.sub.15]-DON Quan [.sup.13C.sub.15]-DON 3.27 / / T-2 5.92 [.sup.13C.sub.24]-T2 Quan HT-2 5.29 [.sup.13C.sub.24]-T2 Qual [.sup.13C.sub.24]-T2 5.92 / / AFB1 4.47 [.sup.13C.sub.17]-AFB1 Quan AFB2 4.30 [.sup.13C.sub.17]-AFB1 Quan AFG1 4.05 [.sup.13C.sub.17]-AFB1 Quan AFG2 3.91 [.sup.13C.sub.17]-AFB1 Quan [.sup.13C.sub.17]-AFB1 4.46 / / AFM1 3.91 [.sup.13C.sub.17]-AFM1 Quan AFM2 3.78 [.sup.13C.sub.17]-AFM1 Qual [.sup.13C.sub.17]-AFM1 3.91 / / OTA 6.42 [.sup.13C.sub.20]-OTA Quan [.sup.13C.sub.20]-OTA 6.41 / / ENNA 8.50 [.sup.15N.sub.3]-ENNB or [.sup.13C.sub.20]-OTA Quan ENNA1 8.38 [.sup.15N.sub.3]-ENNB or [.sup.13C.sub.20]-OTA Quan ENNB 8.13 [.sup.15N.sub.3]-ENNB or [.sup.13C.sub.20]-OTA Quan ENNB1 8.25 [.sup.15N.sub.3]-ENNB or [.sup.13C.sub.20]-OTA Quan BEA 8.21 [.sup.15N.sub.3]-ENNB or [.sup.13C.sub.20]-OTA Quan [.sup.15N.sub.3]-ENNB 8.13 / / FB1 5.38 [.sup.13C.sub.34]-FB1 Quan FB2 6.52 [.sup.13C.sub.34]-FB1 Quan FB3 6.05 [.sup.13C.sub.34]-FB1 Qual [.sup.13C.sub.34]-FB1 5.39 / /

TABLE-US-00005 TABLE 5 Analytes determined in DBS using the ESI negative mode: LC retention time, Internal standard, Quantitative (Quan) or Qualitative (Qual) detection Retention Name time (min) Internal standard Quan/Qual TEA 5.82 [.sup.13C.sub.6.sup.15N]-TEA Quan AOH 5.93 [.sup.13C.sub.6.sup.15N]-TEA Quan [.sup.13C.sub.6.sup.15N]-TEA 5.81 / / AME 7.10 [.sup.13C.sub.18]-ZEN Quan ZEN 7.17 [.sup.13C.sub.18]-ZEN Quan A-ZEL 6.48 [.sup.13C.sub.18]-ZEN Quan B-ZEL 6.16 [.sup.13C.sub.18]-ZEN Quan ZAN 7.09 [.sup.13C.sub.18]-ZEN Quan A-ZAL 6.59 [.sup.13C.sub.18]-ZEN Quan B-ZAL 6.09 [.sup.13C.sub.18]-ZEN Quan [.sup.13C.sub.18]-ZEN 7.17 / / DON-S 3.80 [.sup.13C.sub.18]-ZEN Qual DON-GlcA 3.77 [.sup.13C.sub.18]-ZEN Qual ZEN-S ? [.sup.13C.sub.18]-ZEN Qual ZEN-GlcA 5.54 [.sup.13C.sub.18]-ZEN Qual A-ZEL-GlcA 5.24 [.sup.13C.sub.18]-ZEN Qual B-ZEL-GlcA 4.64 [.sup.13C.sub.18]-ZEN Qual

Spiking

[0157] According to a preferred embodiment of the present invention, the method comprises spiking the collected dried blood sample with one or more internal standards.

[0158] According to a further preferred embodiment of the present invention, the internal standard(s) are, resp. are selected from the following list of 8 compounds: [0159] .sup.13C15-deoxynivalenol, [0160] .sup.13C17-aflatoxin B1, [0161] .sup.13C20-ochratoxin A, [0162] .sup.13C24-T2-toxin, [0163] .sup.13C34-fumonisin B1, [0164] .sup.15N3-enniatin B, [0165] .sup.13C6.sup.15N-tenuazonic acid, [0166] .sup.13C18-zearalenone.
Practical use of the method according to the invention.

[0167] The method as set forth above, as well as any of the preferred embodiments of such method, can be used for screening and assessing the exposure of pigs and broiler chickens to feed contaminated with mycotoxins.

[0168] Further such method is also suitable for assessing the addition of mycotoxin detoxifying agents to animal feed.

[0169] Further aspects and advantages of the invention as well as of its preferred embodiments will appear from the following detailed description.

Further Detailed Description of the Invention

[0170] The term “monitoring” as used in the present specification is understood to mean determining the exposure of animals to mycotoxins with the use of so-called biomarkers of exposure.

[0171] The term “biomarkers” as used in the present specification is understood to mean molecules that are a measure for the exposure by the animal to the mycotoxins and their phase I and phase II metabolites, and that can be found in the biological matrices of the animals.

[0172] Two types of biomarkers should be distinguished: [0173] direct (exposure) and [0174] indirect (mechanism/effect) biomarkers.

[0175] The latter are mainly non-specific and associated with either the effect or the mechanism of mycotoxins. The effect-based biomarkers are even less specific than the mechanism-based. A typical example is the alteration in feed intake after administration of deoxynivalenol. The direct biomarkers are specific and directly linked to the exposure. This type of biomarker is often the mycotoxin itself or their phase I and II metabolites. In biomonitoring, the direct biomarkers are the preferred biomarkers.

[0176] The detection of biomarkers can occur in easily accessible matrices of the animal to be bio-monitored, such as blood, urine or feces.

In the context of the present invention, detection of the direct biomarkers or biomarkers for exposure, for example for rapidly absorbed mycotoxins, are to be detected in the blood.

[0177] Peak concentrations can be detected in the blood and the concentration in blood is directly related with the exposure. Plasma is thus a good matrix to measure mycotoxins a few hours after exposure.

[0178] The time at which mycotoxins are absorbed by the animal varies substantially between the various mycotoxins and between animal species. The inventors have determined after experiments that the optimal time slot for the detection of such mycotoxins, is to collect the dried blood sample after 30 minutes up to two hours after feeding, preferably approximately 1 hour after feeding the animal.

[0179] The experimental data for this preferential time frame are described in the following article by the inventors:

[0180] The Article entitled “Biomarkers for Exposure as a tool for efficacy testing of a mycotoxin detoxifier in broiler chickens and pigs”, by Marianne Lauwers, Siska Croubels, Ben Letor, Christos Gougoulias and Mathias Devreese, published on Mar. 28, 2019 by Toxins 2019, 11, 187; doi:10.3390/toxins 11040187, www.mdpi.com/journal/toxins.

Mycotoxins and Phase I & II Metabolites:

[0181] It is known to the person skilled in the art that animal feed may comprise a quite high number of different kinds of mycotoxins and the related metabolites.

[0182] As a first step, comprised in the method of the present invention, a selection should be made as to which mycotoxins and related metabolites should be the subject of the monitoring or assessment operation.

[0183] The mycotoxins targeted in the multi-method of the present invention comprise the regulated groups, i.e. aflatoxins, ochratoxin A and several Fusarium mycotoxins and two groups of unregulated or emerging mycotoxins, i.e. Alternaria mycotoxins and selected Fusarium mycotoxins (enniatins and beauvericin).

[0184] The selection of the abovementioned mycotoxins and their phase I & II metabolites has been based on a careful study of the regulations as presently in force in the European Union, and the occurrence data in animal feed. EFSA (European Food Safety Agency)-regulated mycotoxins in feed are AFB1, OTA, FB1+FB2, T2+HT2, ZEN and DON.

[0185] Non-regulated mycotoxins are e.g. ENNs, BEA, AOH, AME and TEA. As a result, in the method according to the present invention at least the following (10) mycotoxins and its metabolites are detected: [0186] Aflatoxin B1 (AFB1), [0187] Aflatoxin B2 (AFB2), [0188] Aflatoxin M1 (AFM1), [0189] Fumonisin (FB1), [0190] Fumonisin (FB2), [0191] Ochratoxin A (OTA), [0192] Deoxynivalenol (DON), [0193] T2 toxin (T2), [0194] HT-2-toxin (HT2), [0195] Zearalenone (ZEN).

[0196] According to a preferred embodiment, the method of the present invention comprises the detection of the following (8) additional mycotoxins and its metabolites:

Alternariol methyl ether (AME), [0197] Alternariol (AOH), [0198] Tenuazonic Acid (TEA), [0199] Beauvericin (BEA), [0200] Enniatin A, A1, B, B1 (ENNA, ENNA1, ENNB, ENNB1).

[0201] According to a further preferred embodiment, the method of the present invention comprises the detection of the following (18) additional mycotoxins and its metabolites: [0202] de-epoxy-deoxynivalenol (DOM1), [0203] 15-acetyldeoxynivalenol (15ADON), [0204] 3-acetyldeoxynivalenol (3ADON), [0205] Zearalanone (ZAN), [0206] α-Zearalenol (AZEL), [0207] α-Zearalanol (AZAL), [0208] β-Zearalanol (BZAL), [0209] β-Zearalenol (BZEL), [0210] DON-glucuronide (DON-GlcA), [0211] DON-Sulphate (DON-S), [0212] ZEN-glucuronide (ZEN-GlcA), [0213] ZEN-sulfate (ZEN-S), [0214] α-zearalenol-glucuronide (A-ZEL-GlcA), [0215] β-zearalenol-glucuronide (B-ZEL-GlcA), [0216] Fumonisin B3 (FB3), [0217] Aflatoxin M2 (AFM2), [0218] Aflatoxin G 1 (AFG1), [0219] Aflatoxin G2 (AFG2).

[0220] We will now describe in further detail the various steps of the method according to the invention:

Production of the Dried Blood Sample.

[0221] The present invention is based on the use of Dried Blood Samples, given its convenience in production and ease of conservation and transport.

The Dried Blood Sample is produced by taking a drop of blood sample, transferring same to a saver card (paper card), preferably followed by punching out the central 8 mm disk of the card.

[0222] The drop of blood can be taken from any part of the pig or broiler chicken, but preferably the blood is taken from the ear of the pig or from the leg of the broiler chicken.

[0223] As a next step, the drop of blood is transferred to a saver card.

As saver card, use can be made e.g. of a protein saver card marketed under the brand name Whatman 9034, available from Sigma-Aldrich.

[0224] A sample collection area of the 903 Protein Saver Card contains five half-inch circles. Each circle holds 50 to 80 μl of sample.

[0225] The optimal volume to cover a complete circle on the saver card was determined as 60 μl. On the saver card, the drop of blood spreads out over the card and fills a circle of at least 8 mm. As a next step, the 8 mm central disk on the saver card is punched out with a biopsy punch.

[0226] The so punched out saver paper comprising (part of) the drop of blood, constitutes the dried blood sample hereinafter referred to as the DBS.

[0227] Based on the following experiments, it appears that irrespective of the specific amount of the drop of blood taken yields the same results in terms of qualification and quantification of the mycotoxins are obtained.

Insertion of Internal Standards (IS) in the DBS

[0228] At this stage in the method according to the present invention, substances suitable to act as internal standard products are spiked into the DBS.

[0229] More in particular, according to a preferred mode of operation of the present invention, a combination of the following substances can be used as such internal standards and to that end are spiked into the DBS:

TABLE-US-00006 Theoretical accurate Name mass (g/mol) .sup.13C.sub.15-Deoxynivalenol 311.1763 .sup.13C.sub.17-Aflatoxin B1 329.1204 .sup.13C.sub.20-Ochratoxin A 423.1494 .sup.13C.sub.24-T2-toxin 490.3008 .sup.13C.sub.34-Fumonisin B1 755.5025 .sup.15N.sub.3-Enniatin B 642.4006 .sup.13C.sub.6.sup.15N-Tenuazonic acid 207.1387 .sup.13C.sub.18-Zearalenone 336.2071

[0230] The abovementioned substances are suited to act as internal standards in the method of the present invention as they comprise either carbon atoms comprising 13 neutrons or nitrogen atoms comprising 15 neutrons and are as such easily detectable by the mass spectrometer and have very similar characteristics as the .sup.12C and .sup.14N components.

[0231] These substances are used as reference standards in the LC-MS/MS apparatus and in the HRMS apparatus used in the present invention.

[0232] Since an isotopically labeled Internal Standard (hereinafter referred to as IS) for each single mycotoxin is too expensive and not commercially available, an IS labeled with [.sup.13C] or [.sup.15N] was used for each group of mycotoxins, as follows:

[.sup.13C.sub.15]-deoxynivalenol was used as IS for DON, DOM1 and 3/15ADON;
[.sup.13C.sub.24]-T2-toxin for T2 and HT2;
[.sup.13C.sub.17]-Aflatoxin B1 for AFB1, AFB2, AFG1, AFG2, AFM1 and AFM2;
[.sup.13C.sub.20]-Ochratoxin A for OTA;
[.sup.13C.sub.34]-Fumonisin B1 for FB1, FB2 and FB3;
[.sup.13C.sub.6.sup.15N]-Tenuazonic acid for TEA, AME and AOH;
[.sup.13C.sub.1]-Zearalenone for ZEN, AZAL, BZAL, AZEL, BZEL, ZAN, DON-GlcA, DON-S, ZEN-GlcA, ZEN-S, A-ZEL-GlcA and B-ZEL-GlcA;
[.sup.15N.sub.3]-Enniatin B for ENNA, ENNA1, ENNB, ENNB1 and BEA.

[0233] Hence, an optimal correction for matrix effects and losses during sample preparation was obtained, which was confirmed during method validation.

Extraction of the Mycotoxins in a Solvent

[0234] The next step in the method according to the invention is to extract the mycotoxins contained in the DBS, in a manner as pure and complete as technically feasible.

[0235] Differently phrased, in this step of the method according to the invention, the aim is to isolate the mycotoxins in the DBS from as much of the other blood constituents as possible.

[0236] To this end, the method comprises placing the DBS in a test tube, e.g. made of glass, and adding a suitable extraction solvent.

[0237] According to a preferred mode of operation, as extraction solvent, use can be made of a mixture of water/acetone/acetonitrile, preferably in the following respective volumes: 30/35/35 v/v/v.

[0238] The amount of solvent to be used can vary widely, but preferably an amount ranging from 0.1 up to 10 ml, preferably from 0.5 up to 5 ml, more preferably around 1 ml can be used.

[0239] The sample comprising the DBS and the extraction solvent is subjected to an ultrasonic treatment, preferably during a period of time ranging from 15 to 45 minutes, preferable around 30 minutes.

[0240] Upon completion of the ultrasonic bath step, the solvent is poured over in another test tube; the remaining saver card paper can be removed.

Drying and Reconstitution of the Solvent

[0241] The aim of the present step is to prepare a sample for injection into the liquid chromatography (LC) apparatus. This implies that the DBS sample should be further treated in view of the detectability as well in qualitative as in quantitative manner of the mycotoxins in the sample in the LC-MS/MS or LC-HRMS.

[0242] The latter can be realized on the one hand by an appropriate selection of the reconstitution solvent, and on the other hand by determining the appropriate amount of such reconstitution solvent to be used.

[0243] In this stage of the method of the present invention, the extraction solvent, containing the mycotoxins and related metabolites to be detected, is dried, e.g. under a gentle nitrogen stream at a suitable temperature, e.g. between 20 and 60° C., preferably between 30 and 50° C., more preferably at around 40° C. +/−5° C.

[0244] Upon drying, the dried material is reconstituted in a solvent, suitable for injection into the LC apparatus.

[0245] The inventors have found that to that end, according to the method of the invention, a solvent consisting of water/methanol/formic acid preferably can be used. Preferably the dried material is reconstituted in an appropriate amount of such solvent, being 60 μl (this amount corresponding to the same amount of the initial DBS, drop of blood).

[0246] By selecting the same amount of blood as of the original DBS, the concentration of mycotoxins in the sample to be analyzed is the same as in the original blood drop.

Experimental Support:

[0247] The following experiments, performed by the inventors, illustrate the above.

[0248] Three different reconstitution solvents (n=3 per condition) were evaluated: water/ACN/acetic acid (AA) (95/5/0.1, v/v/v); water/methanol/formic acid (60/40/0.1, v/v/v) and water/methanol (15/85, v/v). The first two mixtures have previously been applied for the detection of multiple mycotoxins or OTA alone in human DBS. The latter has been used by co-inventors Lauwers et al. in an LC-MS/MS method for multi-mycotoxin determination in pig and chicken plasma. The best results were obtained using methanol instead of acetonitrile in the reconstitution solvents (FIG. 1). Next, calibration curves were made with the two methanol containing reconstitution solvents and the lowest concentration achieved for each component as well as the peak shape were compared. The mixture of water/methanol/formic acid (60/40/0.1, v/v/v) showed the best results for both parameters and was therefore chosen as the preferred solvent. The same findings were also observed for DBS obtained from broiler chickens.

[0249] The FIGURE shows the evaluation of the analyte peak areas after LC-MS/MS analysis of DBS (60 μl), extracts which were re-dissolved in three different reconstitution solvents: water/acetonitrile (ACN)/acetic acid (AA) (95/5/0.1, v/v/v); water/methanol (MeOH)/formic acid (FA) (60/40/0.1, v/v/v) and water/MeOH (15/85, v/v). Individual mycotoxins were spiked in whole blood at a concentration of 10 ng.Math.ml-1. The mean (n=3) LC-MS/MS peak areas+standard deviation (SD) is shown in graph.

[0250] The following mycotoxins were the subject of the experiments:

Deoxynivalenol (DON), de-epoxy-deoxynivalenol (DOM1), 3/15-acetyl deoxynivalenol (3/15ADON), aflatoxin B1 (AFB1), aflatoxin M1 (AFM1), enniatin A (ENNA), enniatin A1 (ENNA1), enniatin B (ENNB), enniatin B1 (ENNB1), beauvericin (BEA), fumonisin B1 (FB1), fumonisin B2 (FB2), ochratoxin A (OTA), zearalenone (ZEN), α-zearalenol (AZEL), β-zearalenol (BZEL), α-zearalanol (AZAL), β-zearalanol (BZAL), zearalanone (ZAN), tenuazonic acid (TEA), alternariol (AOH), alternariol mono-methyl-ether (AME), T2 toxin (T2).

LC (Liquid Chromatography)

[0251] The next step of the method of the present invention is the qualitative and quantitative determination of the various mycotoxins and related phase I and II metabolites comprised in the blood sample.

[0252] This can be accomplished by subjecting the dried and reconstituted DBS sample to a separation by liquid chromatography. As set forth supra, such apparatus comprises a column comprising a stationary phase and a mobile (liquid) phase. In the column of such apparatus the mycotoxins and related metabolites are separated on the basis of their different affinity to on the one hand the stationary (or fixed) phase of the column, and on the other hand the mobile or liquid phase of such column. Given such differences in affinity, the residence time and hence the transfer time of the various mycotoxins and related metabolites through the column of the apparatus will be different, in term resulting in an appropriate separation of the individual mycotoxins.

[0253] A column suitable for use in the method of the present invention comprises HSS (High Strength Silica) Technology particles available from Waters® and its Benelux dealer, LabMakelaar Benelux BV Knibbelweg 18 C, Zevenhuizen, Nederland, e.g. the 1.8 μm High Strength Silica particle.

[0254] According to a preferred embodiment of the present invention, the composition of the liquid phase passing through the column of the liquid chromatography apparatus, is varied over time, according to the time of injection of the liquid phase into the column.

[0255] Tables 2 and 3 set forth supra illustrate the flow rate of the mobile phase as well as the gradient program applied.

[0256] The UPLC conditions for analytes determined using the ESI negative mode are as follows (see table 3):

at the start of the operation, the mobile phase comprises predominantly water (e.g. up to 95%) and a small proportion of acetonitrile (e.g. up to 5%), and as time goes by, the composition of the liquid phase is changed, whereby the amount of water gradually decreases (e.g. to approximately 40%) and the amount of acetonitrile gradually increases (e.g. up to approximately 60%).

[0257] Hereafter, the proportion of water again is increased substantially, up to 95%, so as to recondition the column for future use.

[0258] The UPLC conditions for analytes determined using the ESI positive mode are as follows (see table 2):

at the start of the operation, the mobile phase comprises predominantly water (e.g. up to 95%) and a small proportion of methanol (e.g. up to 5%), and as time goes by, the composition of the liquid phase is changed, whereby the amount of water gradually decreases (e.g. to approximately 1%) and the amount of methanol gradually increases (e.g. up to approximately 99%).

[0259] Hereafter, the proportion of water again is increased substantially, up to 95%, so as to recondition the column for future use.

[0260] As a result of the above, the various individual mycotoxins and related metabolites will exit the liquid chromatography column one after the other; at such stage, these compounds are suitable for being detected in the subsequent stage of the present invention, by the MS/MS apparatus.

[0261] Tables 4 and 5 set forth supra, illustrate the various retention times for the components comprised in the DBS sample to be analyzed.

Experimental Support:

[0262] Four different reversed phase columns (Hypersil Gold 50 mm×2.1 mm, dp: 1.9, Thermo Scientific, Breda, The Netherlands; Zorbax Eclipse C18 50 mm×2.1 mm, dp: 1.8, Agilent, Sint-Katelijne-Waver, Belgium; Acquity BEH-C18 50 mm×2.1 mm, dp: 1.7, Waters, Milford, Mass., USA; and Acquity HSS-T3 100 mm×2.1 mm, dp: 1.8, Waters, Milford, Mass., USA) were tested to achieve chromatographic separation of the selected mycotoxins. The best separation of all components was obtained on the HSS-T3 column.

[0263] The abbreviation HSS stands for High Strength Silica, the term T3 stands for a trifunctional C.sub.18 alkyl chain.

[0264] The latter column is available as Acquity UPLC HSS T3 column from Waters Corporation, 34 Maple Street, Milford, USA.

[0265] For further details regarding the chromatography apparatus suitable for use in the method of the present invention, reference is made to reference document (3) as identified supra, paragraph 4.5, page 23 of 30, the content whereof is incorporated herein by reference.

Detection of Mycotoxins by MS-MS

[0266] For the detection of the mycotoxins separated by the LC apparatus, the MS-MS method and apparatus are used.

[0267] When this technique is used, the detection is performed on the basic (‘precursor’) molecule in a first stage, as well as on some fragments of such molecule in a second stage.

[0268] For further details regarding the MS-MS apparatus suitable for use in the method of the present invention, reference is made to reference document (3) as identified supra, paragraph 4.6.1, page 23 of 30, the content whereof is incorporated herein by reference.

Method Validation:

[0269] Upon completion of the development of the method according to the present invention, the inventors have undertaken the task of validating the method according to the present invention with respect to the DBS from pigs and broiler chickens.

[0270] For the purpose of validation, calibration curves have been set up, on the one hand with respect to the DBS from pigs, on the other hand with respect to the DBS from broiler chickens, in both cases for each of the mycotoxins and their phase I metabolites as set forth above.

[0271] To that end, tests have been run on three different days as well within a day, and the mean values have been calculated based on these results.

[0272] The results of these tests are described hereinafter.

[0273] Reference is hereby made to the following document, paragraph 2.2, page 5 of 22, the content whereof is incorporated herein by reference:

the Article entitled “Assessment of dried blood spots for multi-mycotoxin biomarker analysis in pigs and broiler chickens” by Marianne Lauwers, Siska Croubels, Siegrid de Baere, Milena Sevastiyanova, Eva Maria Romero Sierra, Ben Letor, Christos Gougoulias and Mathias Devreese, published on Sep. 18, 2019 by Toxins 2019, 11, 541: doi:10.3390/toxins 11090541, www.mdpl.com/journal/toxins.

[0274] The linearity expressed as a correlation coefficient (r) and the goodness-of-fit (g) is shown in Table 1 for pig DBSs and Table 2 for chicken DBSs and is the mean±standard deviation of three curves across three different days of analysis. The r ranged between 0.991 and 0.999 for pigs and between 0.993 and 0.998 for broiler chickens. The g-value varied from 6% to 17% in pig and 8% to 19% in broiler chicken DBSs. Most of the calibration curves matched a linear model with a 1/x weighing factor, except for the ENNs and BEA which are best described by a quadratic model with a 1/x weighing factor. For most mycotoxins, the linearity ranged between 0.5 and 200 ng.Math.ml.sup.−1. However, the limit of quantification (LOQ) and thus the lowest concentration in the calibration curve was set at 1 ng.Math.ml.sup.−1 for ZEN, AZAL, BZEL, ZAN, DOM1 and AME in pigs and for ZEN, AZAL, BZAL, ZAN, AOH, T2, ENNA and FB1 in broiler chickens. In broiler chickens, AZEL had an LOQ of 4 ng.Math.ml.sup.−1 and FB2 2 ng.Math.ml.sup.−1 and in pigs AOH had an LOQ of 10 ng.Math.ml.sup.−1. The results for the LOQ and corresponding LOD values are found in Table 1 and Table 3, respectively.

[0275] The within-day and between-day precision and accuracy at a concentration of 10 and 100 ng.Math.ml.sup.−1 met the requirements for all mycotoxins for both species. The results of the within-day and between-day precision and accuracy experiments can be found in the Tables set forth hereinafter, respectively.

[0276] The results for signal suppression or enhancement (SSE) after the extraction of DBSs from broiler chickens and pigs showed acceptable results (range 60-112%) for most components. Moreover, in pig DBSs, good extraction recovery rates were also observed. For some components, SSE was more pronounced and extraction recovery was rather low (<60%). However, for all mycotoxins, an adequate internal standard (IS) and matrix-matched calibration curves were used, resulting in validation results for accuracy and precision matching the acceptance criteria. The results for matrix effects and extraction recovery are shown in the Tables set forth is said publication.

[0277] For the sake of completeness, we reproduce hereinafter the table regarding the test results for pig whole blood:

TABLE-US-00007 TABLE Validation results for linearity shown as the mean ± standard deviation of three curves across three different days of analysis (linear range, correlation coefficient (r) and goodness-of-fit coefficient (g)), limit of quantification (LOQ) and limit of detection (LOD) of 23 mycotoxins in dried blood spots of pig whole blood. Linearity (n = 3 Different Days) Linear Range LOQ LOD Analyte (ng .Math. ml.sup.−1) r ± SD ng .Math. mL.sup.−1 ng .Math. mL.sup.−1 ZEN .sup. 1-200 0.999 ± 0.001 1.0 0.09 AZEL 0.5-200 0.998 ± 0.000 0.5 0.20 AZAL .sup. 1-200 0.998 ± 0.001 1.0 0.38 BZAL 0.5-200 0.997 ± 0.001 0.5 0.11 BZEL .sup. 1-200 0.994 ± 0.004 1.0 0.21 ZAN .sup. 1-200 0.996 ± 0.004 1.0 0.10 TEA 0.5-200 0.996 ± 0.003 0.5 0.04 AOH  10-200 0.994 ± 0.005 1.0 0.74 AME .sup. 1-200 0.993 ± 0.002 1.0 0.01 DON 0.5-200 0.994 ± 0.003 0.5 0.11 DOM1 .sup. 1-200 0.997 ± 0.002 1.0 0.23 3/15ADON 0.5-200 0.994 ± 0.002 0.5 0.06 T2 0.5-200 0.996 ± 0.004 0.5 0.01 AFB1 0.5-200 0.994 ± 0.002 0.5 0.001 AFM1 0.5-200 0.991 ± 0.002 0.5 0.01 OTA 0.5-200 0.995 ± 0.003 0.5 0.01 ENN A1 0.5-200 0.997 ± 0.002 0.5 0.05 ENNA 0.5-100 0.998 ± 0.001 0.5 0.01 ENNB 0.5-100 0.999 ± 0.000 0.5 0.001 ENNB1 0.5-100 0.998 ± 0.001 0.5 0.001 BEA 0.5-200 0.997 ± 0.003 0.5 0.02 FB2 0.5-200 0.996 ± 0.002 0.5 0.35 FB1 .sup. 1-200 0.994 ± 0.004 0.5 0.23

[0278] For the sake of completeness, we reproduce hereinafter the table regarding the test results for broiler chicken whole blood:

TABLE-US-00008 TABLE Validation results for linearity shown as the mean ± standard deviation of three curves across three different days of analysis (linear range, correlation coefficient (r) and goodness-of-fit coefficient (g)), limit of quantification (LOQ) and limit of detection (LOD) of 23 mycotoxins in dried blood spots of broiler chicken whole blood. Linearity (n = 3 Different Days) LOQ LOD Linear Range ng .Math. ng .Math. Analyte (ng .Math. ml.sup.−1) r ± SD g ± SD mL.sup.−1 mL.sup.−1 ZEN .sup. 1-200 0.997 ± 0.001 17 ± 3 1.0 0.12 AZEL .sup. 4-200 0.997 ± 0.002  8 ± 3 4.0 1.10 AZAL .sup. 1-200 0.997 ± 0.002 16 ± 6 1.0 0.15 BZAL .sup. 1-200 0.996 ± 0.003 18 ± 2 1.0 0.27 BZEL 0.5-200 0.996 ± 0.003 16 ± 2 0.5 0.1 ZAN .sup. 1-200 0.997 ± 0.001 13 ± 5 1.0 0.21 TEA 0.5-200 0.998 ± 0.001 12 ± 6 0.5 0.001 AOH .sup. 1-200 0.996 ± 0.002 17 ± 3 1.0 0.02 AME 0.5-200 0.997 ± 0.001 17 ± 4 0.5 .001 DON 0.5-200 0.997 ± 0.000 15 ± 1 0.5 0.18 DOM1 0.5-200 0.998 ± 0.001 17 ± 1 0.5 0.16 3/15ADON 0.5-200 0.995 ± 0.003 16 ± 4 0.5 0.09 T2 .sup. 1-200 0.998 ± 0.000 13 ± 5 1.0 0.03 AFB1 0.5-200 0.997 ± 0.001 15 ± 4 0.5 0.01 AFM1 0.5-200 0.997 ± 0.001 15 ± 2 0.5 0.01 OTA 0.5-200 0.998 ± 0.001 19 ± 1 0.5 0.05 ENN A1 0.5-200 0.993 ± 0.002 14 ± 5 0.5 0.04 ENNA .sup. 1-200 0.995 ± 0.003 16 ± 2 1.0 0.11 ENNB 0.5-200 0.994 ± 0.001 12 ± 1 0.5 0.07 ENNB1 0.5-200 0.998 ± 0.002 12 ± 3 0.5 0.001 BEA 0.5-200 0.996 ± 0.000 15 ± 4 0.5 0.07 FB2 .sup. 1-200 0.997 ± 0.001 16 ± 3 1.0 0.96 FB1 .sup. 2-200 0.995 ± 0.001 15 ± 3 2.0 1.87

[0279] The present inventors have complemented the above results with data regarding linearity, limit of quantification (LOQ) and limit of detection (LOD) that were evaluated for the analytes that were added to the LC-MS/MS method as disclosed in this reference (2) and that were quantitatively determined, i.e. AFB2, AFG1 and AFG2.

[0280] These results are set forth in the table 7 below:

TABLE-US-00009 Linear range LOQ LOD Analyte (ng/ml) (ng/ml) (ng/ml) Pig AFB2 0.5-200 0.5 0.02 AFG1 0.5-200 0.5 0.03 AFG2 0.5-200 0.5 0.04 Chicken AFB2 0.5-200 0.5 0.03 AFG1 0.5-200 0.5 0.02 AFG2 0.5-200 0.5 0.05

Real Farming Conditions

[0281] Field DBS samples originating from chickens and pigs coming from the real farming production, where animals are encountering naturally contaminated feed, were analyzed using the updated LC-MS/MS method. This is a difference compared to the article described by Lauwers et al (reference article 3] were only DBS samples originating of a toxicokinetic study with DON, ZEN and AFB1 in pigs and OTA, AFB1 and DON in broiler chickens were analyzed.

[0282] The results of this multi-mycotoxin analysis in a real chicken and pig DBS sample demonstrate the applicability of the updated LC-MS/MS method analysis in ESI positive and negative mode, respectively.

Advantages of the Present Invention:

[0283] The advantages of the method according to the present invention:

As compared to monitoring the animal feed, the present invention offers the following advantages: [0284] 1) Biomonitoring measures the exposure at an individual level while feed analysis shows a general risk. Exposure at an individual level takes variation in food consumption and ADME parameters into account and integrates contamination from all different sources leading to a more accurate estimation and possible use in toxicokinetic studies. [0285] (ADME stands for Absorption, distribution, metabolism, excretion, all of these toxicokinetic parameters describing the effect of the animal body on the toxins concerned); [0286] 2) Although feed is an accessible matrix, it is difficult to sample correctly due to the presence of hotspots. Urine, feces and in particular plasma/blood in contrast are easy to sample correctly and also allow sequential sampling; [0287] 3) Measuring complete exposure: modified mycotoxins can convert back to their original forms during digestion and so contribute to the overall effect of the toxins. Biomonitoring includes these effects and gives thus a very accurate estimation of the risk; [0288] 4) Use of one LC-MS/MS apparatus is sufficient compared to the prior art method, requiring the additional use of an LC-HRMS equipment; [0289] 5) Broad screening or multi-mycotoxin biomonitoring directed to 36 components compared to the prior-art method, limited to 24 components.

Biomonitoring Analysis as a New Tool to Evaluate Mycotoxin Detoxifiers:

[0290] A common way to counter the harmful effect of mycotoxins comprises adding mycotoxins detoxifiers to the animal feed.

[0291] An example of such detoxifier is the product marketed by Innovad N.V., Cogels-Osylei 33,2600 Antwerp, Belgium, under the brand name Escent S®. A detoxifier product is a product that diminishes the effects associated with toxins. A possible way to prove the efficacy of these products is doing in vivo absorption tests. In these trials the concentration of mycotoxins in animal matrices is detected and the difference in concentration with and without detoxifier is determined. This difference is a measure for the efficacy of the product. This type of test gives accurate results and should in the future replace in vitro tests or in vivo test based on non-specific parameters such as growth performance and feed conversion.