 The body is thought to be possessed by an "evil spirit," an illness that manifests itself in the patient by chronic anemia and the seizures of pains in the back and feet. These symptoms inform the traditional healer about the extent of possession and persecution of the sick child's body by wizards. In response to this possession/persecution, the traditional healer provides bodily care to the patient.

Jules says that he was wounded on the body.  He does this by administering a powder with magical powers, effective against sorcerers. Therefore, the care of the patient has a curative aim of "*freeing*" the patient from this possession and persecution by wizards. Thus, not only is the blood treated here, but also the body too.

#### *4.4.2 Care of the brothers, sisters and the mother*

Jules' mother and his paternal uncle accompanied Paulette to the traditional healer. The brothers and sisters, who were absent from the consultation, received through the uncle the treatment given by the traditional therapist. Jules refers to the fact that his paternal uncle administered on him and his other siblings scarifications and he applied the powder from the traditional healer. Because the father is deceased, the paternal uncle went accompanied his mother and the sick sister to the traditional practitioner. This shows the involvement of the extended family in traditional care and support for the mother in this process apart from acting as an intermediary between the traditional healer and the family. He is also responsible for monitoring the application of the traditional medication.

At the requestion of the traditional healer, his paternal uncle scarified Jules just as he did to his sister. The treatment was administered orally and via scarification of the back. "*Even my mother and my older sister ate the same medicine. After eating it, the remaining was put in the blood*." Thus, Paulette's siblings and her mother who were considered at risk of becoming, and of being persecuted by witches were also subjected to the traditional treatment against sickle cell anemia. The goal of this treatment is to protect them against the ailment.

The traditional practitioner, through these treatments, sought to domesticate the harm represented by the disease. He did not seek to exclude this evil from the family, but to transform it into an entity likely to cohabit in harmony with family members.  This therapy does not aim at excluding the "evil" from the family, but to make the ailment an entity of the family and to bring the family members to accept it as such [43].

#### **4.5 The experience of traditional treatment**

With Jules, there is a before and an after of traditional treatment. An "unsecured" pre-treatment period where the threat of contamination by the disease

**47**

*Sickle Cell Anemia, Representations and Care: Experience of a Brother of a Sick Child…*

looms, and a more "secure" post-treatment period when the threat is contained by

Jules thinks of the traditional treatment as a protection against its contamination by the disease. Taking this treatment is associated with a reduction of fear regarding the disease and possible contamination. He says, "*I'm not too scared. Before I was very scared. Now, with the remedy in my body, I'm not as afraid as I was before*." It is interesting to note that although the fear of his sister's death has diminished, it still lingers in his mind. He experiences his body as less vulnerable and more protected against the disease. This indicates that at this point, Jules feels safer with the disease

The receiving of traditional treatment against sickle cell anemia thus helps Jules contain the fear and anguish that arises in him because of his sister's illness. It helps him to contain his feelings of persecution by the wizards and the fear of infection

For Jules, the blood of the patient is a dead blood, a blood possessed by the wizards. In this sense, "a bad blood" attacks the "good blood" transfused into the patient during hospitalizations. According to him, this gives meaning to the chronic

The traditional treatment is thought by Jules to be effective against wizards.  His treatment reduces the feeling of persecution by wizards, even in the event that his sister dies. "*They will not look for me anymore*," he says. The traditional treatment is thus invested as a protection against the attacks of wizards, against the disease and, of

Jules does not associate the death of a patient with the end of the disease as in the case of his mother's youngest daughter, which preceded that of their father. According to him, the disease survives and invests another child or family member. This leads Jules to think of himself as the next potential victim of the disease in the event of Paulette's death. "*If Paulette dies after her little sister, that means we're going to get another person to kill. If she dies, another person will die. That's what scares me*." Jules has a fear of death concerning his sister. He suffers more from this fear of death that he feels threatened by the disease, this "death". He thinks of his sister's imminent death and, therefore, his infection with the disease

The administration of the traditional treatment leads Jules to say this about the wizards, "*it's over for them, everyone ate the cure, everyone is armored*," and specifically, "*when wizards enter the family they do not leave*." In connection with receiving traditional treatment, he says one thing and then contradicts it. He expresses ambivalent feelings of protection against and vulnerability to wizards. This suggests that the sense of security against witchcraft remains feeble, as the feelings of

The drawing was made, after the interview, on the dining table in the living

anemia and the iterative crises of pain that his sick sister experiences.

with the disease even if it does not totally eliminate this fear.

*DOI: http://dx.doi.org/10.5772/intechopen.90995*

*4.5.1 Treatment reduces feelings of insecurity*

*4.5.2 Treatment makes blood bad for wizards*

course, against Paulette's and his own death.

insecurity were never really eliminated.

**4.6 The contribution of the drawing**

receiving traditional treatment.

and with the wizards.

and his own death.

room, lit by sunlight.

*Sickle Cell Anemia, Representations and Care: Experience of a Brother of a Sick Child… DOI: http://dx.doi.org/10.5772/intechopen.90995*

looms, and a more "secure" post-treatment period when the threat is contained by receiving traditional treatment.

#### *4.5.1 Treatment reduces feelings of insecurity*

*Human Blood Group Systems and Haemoglobinopathies*

family.

*4.4.1 The care of the sick child*

provides bodily care to the patient.

*4.4.2 Care of the brothers, sisters and the mother*

**4.4 The traditional healer treats the patient and his family**

The traditional healer cares for the sick child, her siblings, her parents and her extended family because the illness is considered an ailment of the patient and his

The traditional healer cares for the sick child's body. The body is thought to be possessed by an "evil spirit," an illness that manifests itself in the patient by chronic anemia and the seizures of pains in the back and feet. These symptoms inform the traditional healer about the extent of possession and persecution of the sick child's body by wizards. In response to this possession/persecution, the traditional healer

Jules says that he was wounded on the body.  He does this by administering a powder with magical powers, effective against sorcerers. Therefore, the care of the patient has a curative aim of "*freeing*" the patient from this possession and persecution by

Jules' mother and his paternal uncle accompanied Paulette to the traditional healer. The brothers and sisters, who were absent from the consultation, received through the uncle the treatment given by the traditional therapist. Jules refers to the fact that his paternal uncle administered on him and his other siblings scarifications and he applied the powder from the traditional healer. Because the father is deceased, the paternal uncle went accompanied his mother and the sick sister to the traditional practitioner. This shows the involvement of the extended family in traditional care and support for the mother in this process apart from acting as an intermediary between the traditional healer and the family. He is also responsible

At the requestion of the traditional healer, his paternal uncle scarified Jules just as he did to his sister. The treatment was administered orally and via scarification of the back. "*Even my mother and my older sister ate the same medicine. After eating it, the remaining was put in the blood*." Thus, Paulette's siblings and her mother who were considered at risk of becoming, and of being persecuted by witches were also subjected to the traditional treatment against sickle cell anemia. The goal of this

The traditional practitioner, through these treatments, sought to domesticate the harm represented by the disease. He did not seek to exclude this evil from the family, but to transform it into an entity likely to cohabit in harmony with family members.  This therapy does not aim at excluding the "evil" from the family, but to make the ailment an entity of the family and to bring the family members to accept it as such [43].

With Jules, there is a before and an after of traditional treatment. An "unsecured" pre-treatment period where the threat of contamination by the disease

wizards. Thus, not only is the blood treated here, but also the body too.

for monitoring the application of the traditional medication.

treatment is to protect them against the ailment.

**4.5 The experience of traditional treatment**

**46**

Jules thinks of the traditional treatment as a protection against its contamination by the disease. Taking this treatment is associated with a reduction of fear regarding the disease and possible contamination. He says, "*I'm not too scared. Before I was very scared. Now, with the remedy in my body, I'm not as afraid as I was before*." It is interesting to note that although the fear of his sister's death has diminished, it still lingers in his mind. He experiences his body as less vulnerable and more protected against the disease. This indicates that at this point, Jules feels safer with the disease and with the wizards.

The receiving of traditional treatment against sickle cell anemia thus helps Jules contain the fear and anguish that arises in him because of his sister's illness. It helps him to contain his feelings of persecution by the wizards and the fear of infection with the disease even if it does not totally eliminate this fear.

#### *4.5.2 Treatment makes blood bad for wizards*

For Jules, the blood of the patient is a dead blood, a blood possessed by the wizards. In this sense, "a bad blood" attacks the "good blood" transfused into the patient during hospitalizations. According to him, this gives meaning to the chronic anemia and the iterative crises of pain that his sick sister experiences.

The traditional treatment is thought by Jules to be effective against wizards.  His treatment reduces the feeling of persecution by wizards, even in the event that his sister dies. "*They will not look for me anymore*," he says. The traditional treatment is thus invested as a protection against the attacks of wizards, against the disease and, of course, against Paulette's and his own death.

Jules does not associate the death of a patient with the end of the disease as in the case of his mother's youngest daughter, which preceded that of their father. According to him, the disease survives and invests another child or family member. This leads Jules to think of himself as the next potential victim of the disease in the event of Paulette's death. "*If Paulette dies after her little sister, that means we're going to get another person to kill. If she dies, another person will die. That's what scares me*." Jules has a fear of death concerning his sister. He suffers more from this fear of death that he feels threatened by the disease, this "death". He thinks of his sister's imminent death and, therefore, his infection with the disease and his own death.

The administration of the traditional treatment leads Jules to say this about the wizards, "*it's over for them, everyone ate the cure, everyone is armored*," and specifically, "*when wizards enter the family they do not leave*." In connection with receiving traditional treatment, he says one thing and then contradicts it. He expresses ambivalent feelings of protection against and vulnerability to wizards. This suggests that the sense of security against witchcraft remains feeble, as the feelings of insecurity were never really eliminated.

#### **4.6 The contribution of the drawing**

The drawing was made, after the interview, on the dining table in the living room, lit by sunlight.

#### *4.6.1 The complexity of the instructions*

Following the instruction "*I would like you to draw your family*" Jules says he does not know how to draw human beings. Regarding his family, he thinks he is too tall to be represented on a sheet of paper and he asked for a second drawing sheet. He was told that for this drawing, it is recommended that he use a single sheet.

Jules persisted in his request by asking whether he could draw the other people on the back of the paper. To this question, the answer was negative. Following these requests for clarification of the instructions, he resigned himself to drawing according to the rules. This behavior is informative about the limits of family design in this context where the family is not limited to the father, the mother and the children. It is an extended family leading to the need to (re)think the handover, analysis and interpretation of this drawing in a sub-Saharan African context following [42].

#### *4.6.2 The drawing of the extended family*

The eldest daughter in the family ("grande soeur," on the drawing) is the first character drawn by Jules, followed by his father ("papa" on the drawing) the second character. This can signify in Jules that this sister is the most important person in the family because she provides a home near the hospital and money for medical bills. She took the role of the father. That can be why the father is then drawn second.  All the characters in the drawing are real. There are no fictional characters. The drawing is invested as a projective support on which Jules illustrates what he lives and thinks about his family in connection with the illness of his sister.

That Jules's drawing includes his extended family reflects his sense of belonging to it and the support it brings to his immediate family in the face of his sister's illness and the successive deaths of his father and youngest sister. It is thus a drawing on which is projected the family solidarity surrounding his sister's illness (**Figure 1**).

The characters are not very invested by Jules, who drew them in a very minimalist way. It is possible that he wanted to draw all the members of the extended family, which would have led him to drawing his characters in a minimalist way on the sheet. It may also reflect an inhibition of affects in Jules concerning his family ties.

**49**

of his later father.

*Sickle Cell Anemia, Representations and Care: Experience of a Brother of a Sick Child…*

Moreover, this is the first time that he drew human characters without drawing on a predefined model as he often does at school. However, a large part of the sheet is empty. The drawing is in the "upper central part" high center of the sheet, in a portrait orientation. The drawing location in the upper part of the document can signify the escape of the present, the escape towards the unreal and the distance from oneself according to [41]. It is therefore possible that this drawing signifies Jules' avoidance concerning his family, which is "inhabited" by death and disease.

No character is in contact with others as they do not touch each other. This can be interpreted as lack of family support and the isolation of family members according to [41]. This interpretation is questionable, however, in a cultural context where affection and support do not necessarily manifest themselves through physical contact [26] through touch, caress or hug, for example, thus reflecting the

Jules says he drew "*these people*" in his father's house, at his father's funeral in his village. Of all these characters, the elder sister ("la grande soeur" on the drawing) is designated as the happiest person. This is because, according to Jules, she bought the coffin for the burial of his father. The mother is referred to as the least happy person in the drawing because "*she was crying because she had lost her husband*." The elder sister is invested psychologically and symbolically as the father of the family. She is the one who provided financial support for the funeral. She is designated as the nicest person in the drawing because she looks after her siblings. The fact that Jules first drew his older sister might reflect her emotional over-investment. She is invested as a mother emotionally and symbolically. The least kind person is the paternal aunt (paternal aunt, on the drawing). Jules justifies himself by declaring, "*It is they who caused the village to be fired. They are mean.  When asked what role he would like to take within the family dynamic in the drawing, if he had the opportunity, Jules points to his mother saying, "*She is kind. As I go to school, she always buys me lunch*." This reveals the mother's nurturing role in

*DOI: http://dx.doi.org/10.5772/intechopen.90995*

This may justify his lack of drawing.

complexity of this child's life.

*4.6.3 The elder sister, a father figure*

the family and invests her as an identification figure.

therefore thought to be inhabited by death.

*4.6.4 The absence of Jules on the drawing of a mourning scene*

Jules said he had drawn his family members in his father's house during his father's funeral in his village. This tells us that he is still suffering grieving the death of his father. It is possible that Jules represents himself and associates his family with death, due to the past deaths of his father and his youngest sister, along with the imminent and distressing risk of the death of his sick sister. The family is

Jules's absence from the drawing may reflect his avoidance of the family, which he associates with disease and death. With this, he avoids living psychologically in this family dynamic, where he feels insecure about the circumstances.  This can translate, for Jules, the anguish regarding the potential death of his sister. The sick sister is thus excluded from the family dynamic, thought of as not making/leaving her because of an illness that makes her a "dead" person. It could also be a way of distancing himself from this sister who reminds him of the suffering of the family. This absence contrasts with the presence

The presence of the father in the drawing can explain in Jules the incomplete mourning process of this father who is still invested as alive. He remains present

**Figure 1.** *Jules Family drawing.*

#### *Sickle Cell Anemia, Representations and Care: Experience of a Brother of a Sick Child… DOI: http://dx.doi.org/10.5772/intechopen.90995*

Moreover, this is the first time that he drew human characters without drawing on a predefined model as he often does at school. However, a large part of the sheet is empty. The drawing is in the "upper central part" high center of the sheet, in a portrait orientation. The drawing location in the upper part of the document can signify the escape of the present, the escape towards the unreal and the distance from oneself according to [41]. It is therefore possible that this drawing signifies Jules' avoidance concerning his family, which is "inhabited" by death and disease. This may justify his lack of drawing.

No character is in contact with others as they do not touch each other. This can be interpreted as lack of family support and the isolation of family members according to [41]. This interpretation is questionable, however, in a cultural context where affection and support do not necessarily manifest themselves through physical contact [26] through touch, caress or hug, for example, thus reflecting the complexity of this child's life.

### *4.6.3 The elder sister, a father figure*

*Human Blood Group Systems and Haemoglobinopathies*

Following the instruction "*I would like you to draw your family*" Jules says he does not know how to draw human beings. Regarding his family, he thinks he is too tall to be represented on a sheet of paper and he asked for a second drawing sheet. He was told that for this drawing, it is recommended that he use a single sheet.

Jules persisted in his request by asking whether he could draw the other people on the back of the paper. To this question, the answer was negative. Following these requests for clarification of the instructions, he resigned himself to drawing according to the rules. This behavior is informative about the limits of family design in this context where the family is not limited to the father, the mother and the children. It is an extended family leading to the need to (re)think the handover, analysis and interpretation of this drawing in a sub-Saharan African context following [42].

The eldest daughter in the family ("grande soeur," on the drawing) is the first character drawn by Jules, followed by his father ("papa" on the drawing) the second character. This can signify in Jules that this sister is the most important person in the family because she provides a home near the hospital and money for medical bills. She took the role of the father. That can be why the father is then drawn second.  All the characters in the drawing are real. There are no fictional characters. The drawing is invested as a projective support on which Jules illustrates what he lives and thinks about his family in con-

That Jules's drawing includes his extended family reflects his sense of belonging to it and the support it brings to his immediate family in the face of his sister's illness and the successive deaths of his father and youngest sister. It is thus a drawing on which is projected the family solidarity surrounding his sister's illness (**Figure 1**). The characters are not very invested by Jules, who drew them in a very minimalist way. It is possible that he wanted to draw all the members of the extended family, which would have led him to drawing his characters in a minimalist way on the sheet. It may also reflect an inhibition of affects in Jules concerning his family ties.

*4.6.1 The complexity of the instructions*

*4.6.2 The drawing of the extended family*

nection with the illness of his sister.

**48**

**Figure 1.**

*Jules Family drawing.*

Jules says he drew "*these people*" in his father's house, at his father's funeral in his village. Of all these characters, the elder sister ("la grande soeur" on the drawing) is designated as the happiest person. This is because, according to Jules, she bought the coffin for the burial of his father. The mother is referred to as the least happy person in the drawing because "*she was crying because she had lost her husband*." The elder sister is invested psychologically and symbolically as the father of the family. She is the one who provided financial support for the funeral. She is designated as the nicest person in the drawing because she looks after her siblings. The fact that Jules first drew his older sister might reflect her emotional over-investment. She is invested as a mother emotionally and symbolically. The least kind person is the paternal aunt (paternal aunt, on the drawing). Jules justifies himself by declaring, "*It is they who caused the village to be fired. They are mean.

When asked what role he would like to take within the family dynamic in the drawing, if he had the opportunity, Jules points to his mother saying, "*She is kind. As I go to school, she always buys me lunch*." This reveals the mother's nurturing role in the family and invests her as an identification figure.

#### *4.6.4 The absence of Jules on the drawing of a mourning scene*

Jules said he had drawn his family members in his father's house during his father's funeral in his village. This tells us that he is still suffering grieving the death of his father. It is possible that Jules represents himself and associates his family with death, due to the past deaths of his father and his youngest sister, along with the imminent and distressing risk of the death of his sick sister. The family is therefore thought to be inhabited by death.

Jules's absence from the drawing may reflect his avoidance of the family, which he associates with disease and death. With this, he avoids living psychologically in this family dynamic, where he feels insecure about the circumstances.  This can translate, for Jules, the anguish regarding the potential death of his sister. The sick sister is thus excluded from the family dynamic, thought of as not making/leaving her because of an illness that makes her a "dead" person. It could also be a way of distancing himself from this sister who reminds him of the suffering of the family. This absence contrasts with the presence of his later father.

The presence of the father in the drawing can explain in Jules the incomplete mourning process of this father who is still invested as alive. He remains present beyond his death; unlike the other daughter who died before him. While the dead father remains present (illustrated on the drawing), the sick sister' s absence from the drawing may signify Jules' avoidance of this sister and therefore, avoidance of the threat of death that accompanies her illness. On a psychological level, Jule's drawing may signify that, the living sister is dead, while the dead father remains alive. This complexity of Jules's experiences is illustrated within his drawing.


**Chapter 2**

*Light-Emitting Diodes and Photodetectors - Advances and Future Directions*

Organic Light Emitting Diodes

com, "No Title."

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[44] H.-W. Chen, J.-H. Lee, B.-Y. Lin, S. Chen, and S.-T. Wu, "Liquid crystal display and organic light-emitting diode display: present status and future perspectives," Light Sci. Appl., vol. 7, no. 3, pp. 17168-17168, Mar. 2018, doi:

[45] N. Sun, C. Jiang, Q. Li, D. Tan, S. Bi, and J. Song, "Performance of OLED under mechanical strain: a review," J. Mater. Sci. Mater. Electron., vol. 31, no. 23, pp. 20688-20729, Dec. 2020, doi: 10.1007/s10854-020-04652-5.

[46] R. J. Holmes *et al.*, "Blue organic electrophosphorescence using exothermic host–guest energy transfer," Appl. Phys. Lett., vol. 82, no. 15, pp. 2422-2424, Apr.

[47] C. W. Tang, S. A. VanSlyke, and C. H. Chen, "Electroluminescence of doped organic thin films," J. Appl. Phys., vol. 65, no. 9, pp. 3610-3616, May 1989,

[48] B. W. D'Andrade, J. Brooks, V. Adamovich, M. E. Thompson, and S. R. Forrest, "White Light Emission Using Triplet Excimers in Electrophosphorescent Organic Light-Emitting Devices," *Adv. Mater.*, vol. 14, no. 15, p. 1032, Aug. 2002, doi: 10.1002/1521-4095 (20020805)14:15<1032::AID-ADMA1032>3.0.CO;2-6.

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[50] N. 2018. W. 17 A. 2021. <https:// www. ukessays. com/essays/engineering/ advantages-and-disadvantages-oforganic-light-emitting-diodesengineering-essay. php?vref=1. "Advantages And Disadvantages Of

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**20**


**Multiple Mycotoxins Determination in Food by LC-MS**/**MS: An International Collaborative Study**

### **Thomas Bessaire \*, Claudia Mujahid, Pascal Mottier and Aurélien Desmarchelier**

Nestlé Research, Route du Jorat 57, Vers-chez-les-Blanc, 1000 Lausanne 26, Switzerland<EMAIL_ADDRESS>(C.M<EMAIL_ADDRESS>(P.M<EMAIL_ADDRESS>(A.D.)

**\*** Correspondence<EMAIL_ADDRESS>Tel.: +41-21-785-8323

Received: 4 October 2019; Accepted: 5 November 2019; Published: 12 November 2019

**Abstract:** An intercollaborative study was organized to evaluate the performance characteristics of a liquid chromatography tandem mass spectrometry procedure for the simultaneous determination of 12 mycotoxins in food, which were ochratoxin A, aflatoxins B1, B2, G1, G2, and M1, deoxynivalenol, zearalenone, fumonisins B1 and B2, and T-2 and HT-2 toxins. The method combined the simplicity of the QuEChERS (Quick, Easy, Cheap, Efficient, Rugged and Safe) approach with the efficiency of immunoaffinity column cleanup (the step used to enhance sensitivity and sample cleanup for some matrices only). Twenty-three entities were enrolled and were European reference laboratories for mycotoxin analysis, U.S. and European service laboratories, and Nestlé laboratories. Each participant analyzed 28 incurred and/or spiked blind samples composed of spices, nuts, milk powder, dried fruits, cereals, and baby food using the protocol given. Method performances were assessed according to ISO 5725-2. Relative standard deviations of repeatability and reproducibility and trueness values for each of the 115 mycotoxin/sample combinations ranged from 5% to 23%, 7% to 26%, and 85% to 129%, respectively, in line with requirements defined in EC 401/2006. The overall set of data gathered demonstrated that the method offered a unique platform to ensure compliance with EC 1881/2006 and EC 165/2013 regulations setting maximum limits for mycotoxins in food samples, even at low regulated levels for foods intended for infants and young children. The method was applicable regardless of the food, the regulated mycotoxin, and the concentration level, and thus is an excellent candidate for future standardization.

**Keywords:** LC-MS/MS; mycotoxins; collaborative study; isotopic dilution; compliance; infant food

**Key Contribution:** Validation data gathered through this international collaborative study demonstrated that the analytical method proposed was suitable to ensure full compliance with regards to EC 1881/2006 and EC 165/2013 regulations, even at low regulated levels for foods intended for infants and young children. The method was applicable regardless of the food commodity, the regulated mycotoxin, and the concentration level, and thus is an excellent candidate for future standardization.

### **1. Introduction**

Mycotoxins are a group of toxic chemical substances produced by filamentous fungi (molds) that commonly grow on a number of food commodities such as cereals, nuts, spices, fruits, oil seeds, or coffee. These toxins can be produced before harvest in the crop and even after harvest if climate conditions are favorable for further fungal growth. Mycotoxins are undoubtedly one of the most severe food safety hazards amplified by global climate change. Indeed, the ability of fungi to produce mycotoxins is largely influenced by temperature, precipitation, relative humidity, and stress conditions in the plants. For instance, aflatoxin-producing fungi (mainly *Aspergillus flavus* and *Aspergillus parasiticus*),

usually more prevalent in tropical and sub-tropical regions, are expected to be found in areas such as southern and eastern Europe or the USA, where temperatures >30 ◦C (close to the optimum for aflatoxin production) may become usual [1,2].

Among several hundreds of mycotoxins identified so far, a few are of concern from a food safety perspective. To protect consumer health, maximum levels (MLs) for mycotoxins in foodstuffs have been established worldwide. In particular, the European Union legislation (often considered as the most stringent one) has established MLs for aflatoxins (AFLAs), ochratoxin A (OTA), zearalenone (ZEN), fumonisins (FBs), and deoxynivalenol (DON) (EC 1881:2006 [3]) and recently indicative levels for T-2 and HT-2 toxins (EC 165/2013 [4]) in a broad range of food commodities. Lower MLs have been also established for food intended for infants and young children.

International organizations such as ISO (International Organization for Standardization), CEN (European Committee for Standardization), or AOAC International have continued gathering experts over the years to develop internationally recognized analytical standards. The goal is to avoid discrepancies in results that may arise from the use of different analytical approaches, with the risk to distorted global food trade. Today, there are 72 official methods available from these organizations for the monitoring of mycotoxins in food (Table 1). These methodologies are often limited to a single compound or to certain family of mycotoxins only and generally validated for a single food category. Indeed, current regulations were established on toxicological data from studies taking into account only one mycotoxin exposure at a time and did not consider the combined effects of mycotoxins [5]. Noteworthy, the co-occurrence of mycotoxins has been extensively described over the last decade. In 2016, data from 107 publications were compiled to summarize the findings on mycotoxins and their co-occurrence in various foods and feeds from all over the world [5]. AFLAs + FBs, DON + ZEN, AFLAs + OTA, and FBs + ZEN were the most observed combinations. Another study on barley and wheat (*n* = 72 and *n* = 83, respectively) evidenced that among the mycotoxin-positive samples, 70% of barley samples and 54% of wheat samples were co-contaminated with at least two mycotoxins [6]. The need to develop methods able to screen several mycotoxins at once was justified in a large-scale global survey in feed where mycotoxin co-contamination was common [7]. Concentrations of aflatoxin B1 (AFB1), ZEN, FBs, OTA, DON, and T-2 toxin were analyzed in 74,821 samples of feed and feed raw materials (e.g., maize, wheat, soybean) collected from 100 countries from 2008 to 2017. In total, a large fraction of samples (64%) was co-contaminated with more than two mycotoxins, whilst 88% of the samples were contaminated with at least one mycotoxin. In that regard, moving from single residue analysis toward "multi-analyte and multi-matrix" ones is of interest to speed up efficiently and rationalize mycotoxin analysis in high-throughput routine environments.

**Table 1.** Official methods (*n* = 72) for the determination of mycotoxins in food (AOAC, CEN, ISO). ELISA, enzyme-linked immunosorbent assay; HPLC-FLD, high performance liquid chromatography with fluorescence detection; IAC, immunoaffinity column; TLC, thin layer chromatography; HPLC-UV, high performance liquid chromatography with ultra violet detection; GC, gas chromatography.

**Table 1.** *Cont.*

1 Including foods intended for infants and young children; 2 aflatoxin M2; 3 citrinin; 4 fumonisin B3; 5 ochratoxin B; 6 α-zearalenol.

Most of the current reference methods still make use of non-confirmatory approaches for quantitative analysis of mycotoxins, e.g., TLC, HPLC-UV, and HPLC-FLD. Use of IAC cleanup often compensates for the lack of specificity of these detection techniques, as authorized in EC 2002/657 [8]. However, LC-MS/MS is currently considered as the state-of-the-art technique to analyze hundreds of contaminants (pesticides, veterinary drugs, etc.) in various food commodities, including mycotoxins, as emphasized in a recent review [9]. The first official LC-MS based methodologies for the determination of mycotoxins in foods were published in 2017 by the CEN and are limited to ZEN [10], and T-2/HT-2 toxins' [11] determination. Indeed, the need for standardized LC-MS methods for mycotoxins' determination was highlighted only recently by regulatory authorities and scientific advisory bodies, triggering the CEN organization to establish European standards within the M/520 standardization mandate [12]. Among the several LC-MS methods still under development or at the final approval

stage at CEN, only two are multi-mycotoxin methods for food analysis [13]. The first one is devoted to the screening of OTA, AFB1, DON, ZEN, FB1, FB2, T-2, and HT-2 toxins and excludes foods for infants and young children from the scope. The second one does not include regulated aflatoxins, zearalenone, and fumonisins and is limited to cereals and cereal based products. Such limited analyte and/or matrix scopes are constraints for food business operators willing to use efficient standard protocols to ensure the safety and compliance of a broad range of food commodities.

To fill this gap on our side, an analytical method was developed and internally validated in 2013 [14] before its deployment in 10 Nestlé Quality Assurance Centers (NQACs, control laboratories) in Brazil, Chile, the USA, France, Italy, Poland, Russia, China, Singapore, and India. The procedure enables the quantitative LC-MS/MS determination of regulated mycotoxins (OTA, AFB1, AFB2, AFG1, AFG2, AFM1, DON, ZEN, FB1, FB2, and T-2 and HT-2 toxins) in a broad range of food items including cereals and cereal based baby foods (infant cereals, biscuits), spices, nuts, coffee, tea, cocoa, vegetable oils, dried fruits, infant formula, dairy products, feed/pet food, etc. This routine method has the advantage of offering a unique platform to ensure full compliance with the EC 1881/2006 regulation [3], even at low regulated levels for foods intended for infants and young children. Its sample preparation combines the simplicity of the QuEChERS approach (widely used for pesticide residues analysis; European Norm EN 15662:2018 [15]) with the efficiency of IAC cleanup (this step being used for some matrices only). Quantification is performed by the isotopic dilution approach using 13C-labeled mycotoxins as internal standards (ISTD). The collection of thousands of validation data over time [16], the high number of analyses conducted each year (15,000 samples analyzed in 2017 [17]), and the regular enrollment of NQACs to internal (under ISO 17043:2010 accreditation [18]) and external proficiency tests have demonstrated the high robustness of this method.

In this context, we set up an international collaborative study to support its standardization. Twenty-three (23) laboratories from authorities and private sectors were involved in this study, representing entities from 14 countries. This collaborative study was organized according to the AOAC International guideline [19], and statistical evaluation was performed following ISO 5725-2 document [20]. The present paper summarizes the overall set of data gathered and demonstrates the method's applicability over a broad range of concentrations across different food categories, including foods for infants and young children.

### **2. Results and Discussion**

### *2.1. Samples and Homogeneity Testing*

Providing blank matrices to be further spiked by each participant as done in previous studies [21] was not considered to avoid potential analytical bias introduced by operators. Alternatively, the preference was to use blind duplicates of former proficiency test samples still available as quality control (QC) materials (paprika, black pepper, almonds, hazelnuts, dried raisins, dried figs, wheat, maize (2), milk powder (2), maize (2), rice, and wheat based infant cereals samples). The homogeneity of such materials was extensively tested by their providers. However, since the study samples were repacked from 200-g aluminum bags to 25-g units before shipping, a small-scale testing was performed (six replicates from six different units for each sample) to ensure sample homogeneity. The resulting coefficients of relative standard deviation of repeatability RSDr ranged from 0.5% to 12%, in line with internal validation data, evidencing the satisfactory homogeneity of the set of samples. The exception was a maize sample with unacceptable RSDr for ZEN and HT-2, at 40% and 21%, respectively. The particle size distribution of this material was heterogeneous (evidence by visual inspection), which led us to re-open and merge all individual units. The resulting bulk material (ca. 2 kg) was ground in our laboratory by means of a cryogenic grinder (SPEX 6875D Freezer/Mill, Stanmore, U.K.) and further dispatched again into 25-g units. Subsequent homogeneity testing (duplicate analysis of six different units) successfully validated this new preparation with a global decrease of RSDr for the twelve mycotoxins (ZEN and HT-2 at 6.7 and 10.6%, respectively).

### *2.2. Participants Instrumental Method Setup*

Knowing the diversity of laboratory equipment available worldwide, participants were free to select their instrumental setup, meaning choice of LC columns, mobile phases, MS parameters, etc. Typical LC-MS/MS conditions were provided by the study director for information purposes only. The use of HR-MS instruments was accepted, but none of the twenty-three participants reported its usage. As shown in Figure 1, 13 different LC columns and 11 models of MS instruments were engaged. Mobile phases and LC gradient provided in the guidance document were chosen by 16 and 18 out the 23 participants, respectively (sometimes with a reduction of the final equilibration time). Various column temperatures (from 30 ◦C to 50 ◦C) and injection volumes (from 4 μL to 20 μL) were considered. All instruments operated in MS/MS mode using polarity switching, except three labs for which ESI was only used in positive mode. In this last case, ZEN was analyzed as its [M + H]+ adduct as already reported [22,23]. The diversity of the instrumentation (i.e., different generations, varying degrees of performance) used by participants was an additional proof of the applicability of the method.

**Figure 1.** Instrumental setup used by the participating laboratories (*n* = 23).

To facilitate lab work, calibrant solutions (nine levels) were provided as ready-to-use to each participant. The extent of mycotoxin contaminations is known to be unpredictable and variable; thus, the calibration range of each analyte was set broad enough to avoid reinjection or re-analysis of highly contaminated samples. Participants were asked to consider the two highest levels (calibration point 7 (CAL 7) and CAL 8) only when facing such highly contaminated samples with concentrations out of the classical calibration range (CAL 0 to CAL 6). Use of a weighing factor (1/x or 1/x2) for drawing calibration curves was strongly recommended, or alternatively to force regression lines through the origin (i.e., intercept = 0), as done elsewhere [14], this to maintain good precision of data at low contamination levels. Typically, such an approach enabled the direct quantification of either AFB1 from 0.025 μg/kg to 32 μg/kg or OTA from 0.125 μg/kg to 32 μg/kg in cereals within one single analysis, avoiding a tedious re-extraction of the sample using a reduced test portion.

### *2.3. Laboratory Qualification*

Participants were first asked to analyze one single sample (practice sample) to get familiar with the protocol and to communicate generated results to the study director. This was to ensure that the method was correctly set up before engaging laboratories in the second part of the study, consisting of

the analysis of 28 samples. This practice sample being a maize based infant cereal, an IAC cleanup was required to get extra sensitivity for AFLAs and OTA. Other mycotoxins were extracted using the "QuEChERS" procedure (Figure 2). The 11 assigned values derived from the proficiency test were 0.26 μg/kg AFB1, 0.28 μg/kg AFB2, 0.15 μg/kg AFG1, 0.15 μg/kg AFG2, 0.81 μg/kg AFTOT, 0.71 μg/kg OTA, 138 μg/kg DON, 31 μg/kg ZEN, 31 μg/kg T-2, 27 μg/kg HT-2, 56 μg/kg T-2 + HT-2, 61 μg/kg FB1, 79 μg/kg FB2, and 140 μg/kg FBTOT, thus very close to the low regulated levels for foods intended for infants and young children [3]. Twenty (20) out of 23 participants successfully reported data with z-scores (Z) and recoveries (Rec) within −2 < Z < +2 and 70% < Rec < 130% for these 11 mycotoxins and were thus qualified for the second part of the study. At this stage, assistance provided by the study director was limited to a few participants facing issues in using the correct ISTD concentrations or eliminating FBs' carry-over on their MS instrument. One laboratory did not see any peak for ZEN, OTA, FBs, HT-2, T-2, and DON, which are compounds to be detected by the QuEChERS stream. Investigations to identify the root cause failed. As the method could not be properly implemented in this lab, all data further generated were not considered for method performance evaluation. Another laboratory missed reporting practice sample data before starting the full study. Unfortunately, the z-scores for ZEN and AFG2 and recoveries for AFB1, AFB2 and AFG1 were unsatisfactory, which highlighted a lack of control of the procedure together with a possible instrumental issue. A third laboratory reported both technical and organizational problems and did not provide data for the practice sample. For consistency with other participants, the decision was made to remove these three laboratories for the final statistical evaluation. Consequently, 20 laboratories were eventually approved in the full collaborative study.

**Figure 2.** Overview of the sample preparation.

### *2.4. Full Collaborative Study*

Among the 3700 individual data tentatively collected, only a few of them were missing or excluded (*n* = 84, i.e., 2.3% of the data) before statistical evaluation for the following reasons: (a) one lab did not report DON and HT-2 toxin concentration data in the wheat based baby food due to ion ratios out of tolerance; (b) OTA level in the black pepper sample was discarded for one laboratory facing a low signal-to-noise-ratio (<3) along with a very bad peak shape; (c) one lab did not report data for ZEN in maize and wheat samples due to important baseline interference for ZEN ISTD; (d) all data for black pepper were removed for one lab because the sample preparation was not followed; (e) data for T-2

and HT-2 toxins were not considered for another laboratory reporting major instrumental issues for these two toxins; (f) an error during the ISTD spiking step (DON, ZEN, T-2, HT-2) of the wheat based infant cereal was mentioned by one laboratory.

The precision of the method was characterized by the repeatability RSDr and the reproducibility RSDR, after removal of outliers as recommended in ISO 5725-2 [20]. As shown in Tables 2–4 and Figure 3, RSDr ranged from 3% to 22% (average 7%) and RSDR from 5% to 28% (average 12%), most values being largely below 20%. All these data fulfilled Commission Regulation (EC) No. 401/2006 [24], which has established method performance criteria for the official control of the levels of mycotoxins in foodstuffs. As expected, reproducibility values were higher than the repeatability ones for all combinations except three.

**Table 2.** Method performance evaluation for AFB1, AFB2, AFG1, AFG2, Total AFLAs and OTA in twelve samples.

**Table 2.** *Cont.*

S1: paprika; S2: hazelnuts, S3: dried raisins; S4: black pepper; S5: almond powder; S6: Figs; S7: maize; S8: maize; S9: wheat; S10: wheat based infant cereal; S11: maize based infant cereal; S12: rice based infant cereal; SDr: standard deviations of repeatability; RSDr: relative standard deviations of repeatability; SDR: standard deviations of reproducibility; RSDR: relative standard deviations of reproducibility; Rec: recovery; n.d.: not detected.

**Table 3.** *Cont.*

**Table 4.** Method performance evaluation for AFM1 in two milk powder samples (S13 and S14).

**Figure 3.** Method performances on 115 mycotoxin/sample combinations: (**a**) repeatability; (**b**) reproducibility; (**c**) Horwitz ratio (HorRat) values; (**d**) trueness. Rec, recovery.

The Horwitz ratio (HorRat) is a useful index of method performance with respect to precision and is calculated as the ratio between the RSDR as obtained during the collaborative study and the RSDR as predicted by the modified Horwitz equation [25,26]. Method reproducibility is considered as normal when the HorRat value is between 0.5 and 1.5 [19]. In this study, HorRat values ranged from 0.2 to 1.3 for the 115 mycotoxin/sample combinations (Tables 2–4, Figure 3). Approximately 50% of HorRat values were even lower than 0.5. This might be explained by: (a) the high robustness of the method (already known since it has been heavily used for >5 years in routine environments at Nestlé), (b) by the use of isotopically labelled internal standards for quantification purpose, and (c) by the analytical skills of participating laboratories.

The trueness of the method was then assessed by calculating the recovery (Rec, %) for each of the 115 mycotoxin/matrix combinations. Rec figures ranged from 84% to 129% with eleven data slightly above the requirements defined in EC 401/2006 [24]. We mention that three of them concerned FB1, FB2, and FBs in the maize sample for which levels were out of the calibration range (data reported for information purposes only). The overall set of data unambiguously demonstrated the high confidence level of the method regardless of the sample/concentration/mycotoxin combinations.

Focusing on individual participant data, all mycotoxins were correctly detected of the sample except in two cases. While all laboratories detected AFG2 at 0.31 μg/kg in the wheat based infant cereal, five of them could not detect AFG2 at a lower level in the rice based infant cereal (0.052 μg/kg), which

could jeopardize the conclusion with respect to method sensitivity. However, neither AFG2 nor AFTOT (sum of the four AFLAs) are regulated for infant cereals. AFM1 in each of the two milk powders was detected by 16 and 20 out of the 20 laboratories, respectively. According to the related supplier recommendation, respective concentration levels in reconstituted milk correspond to 0.0034 μg/kg and 0.0117 μg/kg. Consequently, the highest AFM1 validated level that was detected by all participants was still half of the ML set for AFM1 in milk-based products (0.025 μg/kg [3]).

As shown in Table 5, the lowest level of the sum of T-2 and HT-2 validated in this study was 18.6 μg/kg (i.e., 10.6 μg/kg and 8.0 μg/kg for T-2 and HT-2 as individual toxin, respectively). This is slightly higher than the 15 μg/kg level set for cereal based foods for infants and young children as an indicative level for which subsequent investigations should be performed according to EC 165/2013 [4]. However, internal validation data at 10 μg/kg for the sum of T-2 and HT-2 (i.e., 5 μg/kg for each T-2 and HT-2 toxins) [14,16] demonstrated the good performance of this method and its compliance with EC 165/2013 [4]. For all other mycotoxins considered, the lowest validated level in this study was lower than the lowest EU MLs [3], evidencing the method as fit for purpose to ensure compliance of food materials with regards to this regulation.

**Table 5.** Fitness-for-purpose of the method with regards to EU regulations for foods intended for infants and young children.

a Commission Regulation (EC) No. 1881/2006; b Commission Regulation (EC) No. 165/2013; c refers to the products ready to use (marketed as such or after reconstitution as instructed by the manufacturer).

### *2.5. Output: Future Perspective*

Whilst LC-MS/MS based methods are recognized as state-of-the-art approaches for chemical contaminants' analysis in food, current standards for mycotoxin monitoring are still mainly based on TLC or HPLC-UV/FLD and/or often limited to a single compound, or family at best [13]. This study proved that the proposed methodology is an excellent candidate for future standardization since it offers a highly trustful and efficient approach for multi-residue analysis of regulated mycotoxins. Its applicability whatever food type, mycotoxin, and concentration level is well adapted for food control environments, today facing shorter and shorter turn-around times.

The cost effectiveness of any routine method is not to be neglected, and the price of 13C-labeled compounds used as ISTD might impede their usage. However, our own estimation showed that the sample throughput by using this isotopic dilution approach was increased by a factor of three, which was balanced with the additional cost of the ISs. The matrix matched calibration curve (which is restricted to one or two sample types analyzed per batch with the additional requirement to have blank matrices available for spiking purposes) or standard addition (requiring several extractions for one single analysis) quantifications are indeed time consuming, not user friendly, and not adapted for laboratories dealing with a broad range of food matrices on a daily basis. The use of labelled ISTD also improves method precision and accuracy, as recently highlighted in a study evaluating different approaches (extraction, clean-up, quantification) for mycotoxins determination in cereals [27]. The best performances were achieved by a method making use of 13C-labeled internal standards for quantification.

Finally, the proposed extraction procedure (QuEChERS) is generic and already known to be efficient for a wide range of compounds with different polarity. Our trials already demonstrated that such an extraction approach is valid as well for deoxynivalenol acetylated or modified forms (e.g., 3-acetyl-DON, 15-acetyl-DON, DON-3-glucoside). These compounds are not regulated yet, but the European Commission initiated discussions to review the existing maximum levels for DON, taking into account DON acetylated and modified forms [28]. Related isotopically labelled compounds are now commercially available, making these compounds good candidates for future scope extension.

### **3. Conclusions**

Standardized and internationally accepted methods for food compliance testing are of utmost importance to avoid discrepancies in results, as this may generate unsubstantiated disputes and ultimately distort global food trade. The current standardized methodologies devoted to the analysis of mycotoxins in food are not adapted anymore to high-throughput routine environments, facing today a broader range of items to monitor, an increasing pressure to shorten turn-around time, and cost constraints. This paper proposes an efficient analytical approach for multi-residue analysis of regulated mycotoxins, even at low regulated levels for foods intended for infants and young children. The overall set of data derived from this study proved that the proposed methodology is an excellent candidate for future standardization.

### **4. Materials and Methods**

### *4.1. Study Organization*

### 4.1.1. Study Materials

Each participant was supplied with AFLAOCHRA PREP® IACs (R-Biopharm, Darmstadt, Germany, *n* = 26), ready-to-use QuEChERS salt mixtures (Agilent, Geneva, Switzerland, *n* = 35), and ready-to-use PBS tablets (Oxoid, Basingstoke, U.K., *n* = 10).

### 4.1.2. Study Samples

Each participant was given 30 bottles containing ca. 25.0 g of foods. Samples were composed of 28 blind duplicate samples randomly coded from A to β and of one "practice sample" in duplicate. Samples A to L (*n* = 12) belonged to the group "nuts, spices, and dried fruits" and were paprika, black pepper, almonds, hazelnuts, dried raisins, and dried figs. Samples M to R were "raw cereals" composed of wheat or maize (*n* = 6). Samples S to X were either rice, wheat, or maize based "infant cereals" (*n* = 6). Samples Y to β were "milk powder" (*n* = 4). The "practice sample" was a maize based infant cereal. All samples were in powdered form except hazelnuts and dried raisins, which were provided as slurry paste.

These samples were prepared by Fapas (York, U.K.) or BIPEA (Paris, France). All items but one were former proficiency test materials from the last 2 years, still commercially available as QC materials. They were initially packaged in 200-g aluminum bags, but further re-dispatched in smaller 25-g units after extensive mixing. Mycotoxins were either naturally present in the material and/or spiked by the supplier to reach the level of interest. Only the rice based infant cereal sample was specifically prepared by BIPEA using a mycotoxin-free product available from a supermarket in France. The item was further spiked at levels close to the first calibration point (i.e., at the limit of quantification).

### 4.1.3. Study Analytical Standards Solutions for Spiking Purposes

Five ready-to-use 13C-labelled mycotoxin mixtures to be used as internal standards were prepared by Romer Labs (Tulln, Austria): (a) the 13C-AFLA mixture was composed of (13C17)-AFB1, (13C17)-AFB2, ( 13C17)-AFG1, and (13C17)-AFG2, each at 0.5 μg/mL in acetonitrile; (b) the 13C-AFM1 solution was composed of (13C17)-AFM1 at 0.1 μg/mL in acetonitrile; (c) the 13C-[DON, T-2, HT-2, ZEN] mixture was composed of (13C15)-DON, (13C24)-T-2 toxin, (13C22)-HT-2 toxin, and (13C18)-ZEN, at 5, 2.5, 2.5, and 2 μg/mL in acetonitrile, respectively; (d) the 13C-FBs solution was composed of (13C34)-FB1 and

( 13C34)-FB2, each at 10 μg/mL in acetonitrile/water (50 + 50); (e) 13C-OTA was composed of (13C20)-OTA at 10 μg/mL in acetonitrile.

These prepared solutions were dispatched as such to all participants with the exception of 13C-OTA, which was subsequently diluted at 0.1 μg/mL in methanol-water (85 + 15) by the study organizer.

### 4.1.4. Study Analytical Standard Solutions for External Calibration Curves

Unlabeled mycotoxin working standard solutions were provided by Romer Labs and were: (a) the AFLA mixture composed of AFB1, AFB2, AFG1, and AFG2, each at 1 μg/mL in acetonitrile; (b) AFM1 at 0.1 μg/mL in acetonitrile; (c) the DON, T-2, HT-2, and ZEN mixture at 5, 2.5, 2.5, and 2 μg/mL in acetonitrile, respectively; (d) the FB mixture composed of FB1 and FB2, each at 5 μg/mL in acetonitrile-water (50 + 50); (e) OTA at 10 μg/mL in acetonitrile. The AFLA mixture was then diluted at 0.1 μg/mL and 0.01 μg/mL in acetonitrile and OTA at 0.1 μg/mL in methanol-water (15 + 85).

To ease lab work, a set of nine individual "ready-to-be injected" calibration points (named CAL 0 to CAL 8) was also provided. Related concentrations are given in the Supplementary Data. The same batch of each labelled mycotoxin mixture was intended to be used for both making calibration solutions and spiking test portions. These ready-to-use calibration solutions were all prepared at Nestlé Research Lausanne (Lausanne, Switzerland). A total of 30 mL of each of individual calibration solution was prepared and aliquoted in crimp vials before being stored at −20 ◦C. A previous internal stability study evidenced that such calibration solutions are stable for at least 4 months when stored at −20 ◦C.

Before use, all calibration solutions were brought to room temperature, extensively vortexed (minimum 1 min), and sonicated (approximately 10 min) to ensure efficient (re)solubilization of all analytes.

### 4.1.5. Shipping Study Materials

Initially, 26 laboratories enrolled in this study, but two labs could not receive materials due to customs issues (Romania, Russia). One lab withdrew its participation due to a lack of available time. Thus, 23 packages were finally dispatched to laboratories in Austria, France, Germany (2), Hungary, India, Ireland (2), Italia (5), Poland, Serbia, Singapore, Switzerland, The Netherlands (2), The United Kingdom (2), and the USA (2) in June 2019.

Samples and mycotoxin standards' solutions were all sent in a frozen state (dry ice), whereas other materials were sent at room temperature.

### *4.2. Sample Preparation*

Participants were asked to follow strictly the provided protocol. Samples were extracted as previously reported [14] with minor modifications.

Test portions (5 g for cereals and cereal based products and 2 g for milk powders, nuts, spices, and dried fruits) were weighed in 50 mL polypropylene tubes and each subsequently spiked with 50 μL of 13C-labelled working standard solutions of interest. Water (10 mL) was added, and the tube was vigorously shaken by hand until complete dissolution. Acetonitrile containing 1% acetic acid (10 mL) was added, and the tube was then mechanically shaken for 10 min at approximately 300 rpm. Ready-to-use QuEChERS salt mixture containing 4.0 ± 0.1 g of MgSO4 and 1.0 ± 0.1 g of NaCl was supplemented to initiate phase separation. The tube was immediately hand shaken to prevent any lump formation and then placed onto a mechanical shaker for 10 min. After centrifugation (4000× *g*, 10 min, room temperature (RT)), 5 mL of the supernatant acetonitrile phase were mixed with 5 mL of n-hexane and shaken for approximately 10 min on a mechanical shaker. After centrifugation (4000× *g*, 1 min, RT), the upper n-hexane phase was discarded.

The sample extract was then divided into two portions and submitted to two different clean-up protocols, named "QuEChERS" and "IAC", depending on the mycotoxin/matrix combination and the sensitivity required for AFLAs and OTA. A general scheme is presented in Figure 1:

### *4.3. LC-MS*/*MS Analysis*

Instrumental conditions described hereafter were given to the participants, who were free to adapt them for their own instrument.

LC analysis was performed with an Agilent 1290 binary pump system (Agilent, Geneva, Switzerland). Optimal LC conditions were obtained using a Waters Acquity BEH C18 column (2.1 × 100 mm, 1.7 μm) equipped with a BEH C18 VanGuard precolumn (2.1 × 5 mm, 1.7 μm), both thermostated at 50 ◦C. The mobile phases were constituted of formic acid (0.15%) in water containing 10 mM of ammonium formate (Solvent A) and formic acid (0.05%) in methanol (Solvent B). A gradient program was set up as follows: 0–0.3 min with 85% A; 0.3–4.0 min linear gradient down to 0% A; hold at 0% A for 3 min; return to 85% A in 0.05 min and hold at 85% A for 3.95 min (total run time 10 min). The flow rate was 0.4 mL/min, and the injection volume was 10 μL. The LC flow was directed into the MS detector between 1.0 and 6.0 min.

MS detection was performed using a Sciex TRIPLE QUAD 6500+ instrument (Foster City, USA) equipped with a Turbo V™ ion source. MS parameters were first obtained by syringe-infusing (each individual standard solution in electrospray ionization (ESI) mode at concentrations of ca. 0.1 μg/mL to 1.0 μg/mL along with the LC flow (0.4 mL/min, constituted of 50% Aqueous Mobile Phase A and 50% Organic Mobile Phase B) using a T connector. Analyses were conducted using tandem MS in scheduled selected reaction monitoring (scheduled MRMTM) mode alternating two transition reactions for each compound with an acquisition window of 40 s and a target scan time of 250 ms. The block source temperature was maintained at 550 ◦C, and gas values were set as follows: curtain gas 35 psi, GS1 40 psi, GS2 40 psi, and collision activated dissociation (CAD) gas at 10 psi. Other parameters were ion spray voltage (5.0 kV or −4.0 kV), entrance potential (±10 eV), and collision exit potential (±15 eV). MS/MS operated in positive/negative ionization switching mode. Individual MS parameters for the 24 compounds are provided in the Supplementary Data. Data acquisition was carried out using Analyst software 1.7 and subsequent data processing done using Multiquant software 3.0 (both from Sciex).

### *4.4. Identification of Mycotoxins*

Mycotoxins were considered as positively identified in the sample when all confirmation criteria defined in the SANTE/12089/2016 document [29] were fulfilled: (a) a signal visible at least at two diagnostic transition reactions selected for each mycotoxin and each corresponding IS; (b) the retention time of the analyte in the sample extract corresponds to that of the average of the calibration standards measured in the same sequence with a tolerance of ±0.2 min; (c) the retention time of the analyte corresponds to that of its labelled internal standard with a tolerance of ±0.05 min; (d) the peak area ratio from the different transition reactions recorded for each analyte is ±30%.

### *4.5. Quantification*

Quantification was performed by the isotopic dilution approach using 13C-labeled mycotoxins as internal standard. For each mycotoxin, the calibration curve was built by plotting the peak area ratio of each mycotoxin and its ISTD using the transition reaction for quantitation (= *y* axis) against the concentration ratio of each mycotoxin and its ISTD (= *x* axis) using calibration solutions from CAL 0 to CAL 6. CAL 7 and CAL 8 were only considered in the case of a highly contaminated sample when mycotoxin levels were out of the classical calibration range.

To improve the precision of the results at the low calibration points, participants were encouraged to use a 1/x or 1/x2 weighing factor for drawing calibration curves. Alternatively, the regression line was forced through the origin (i.e., intercept = 0) as initially validated [14]. Deviations of back calculated concentrations of calibrant standards from the true concentration were checked to be below ±20% [30].

The mass fraction of each analyte in the sample (*wa*) in μg/kg was calculated using the following equation:

$$\mathcal{W}\_a = \frac{\left(\frac{A\_d}{A\_{\bar{n}}}\right) - I}{S} \times \frac{m\_{\bar{n}}}{m\_a} \tag{1}$$

where *Aa* is the peak area of a given analyte; *Ais* is the peak area of the corresponding ISTD; *I* is the intercept of the regression line; *S* is the slope of the regression line; ma is the mass of the test portion, in g (either 2.0 g or 5.0 g); *mis* is the mass of *ISTD* added to the test portion, in ng.

### *4.6. Statistical Evaluation*

Each participating laboratory was first asked to analyze a "practice sample" by strictly following the protocol given and to report results to the study director. Such a preliminary trial was intended to familiarize the participant with the procedure and to ensure that the method was correctly set up before starting the full study. The practice sample was an old proficiency test sample. Therefore, for each laboratory, z-scores and recoveries were calculated based on the *p*-test assigned values (BIPEA, Babyfood, round 12-3931, December 2018). When z-scores (Z) and recoveries (Rec) were within −2 < Z < +2 and 70% < Rec < 130% ranges, the laboratory was qualified for the second part of the study. When a laboratory did not return satisfactory results, technical support was provided to fix issues.

Method performances were then assessed based on ISO 5725-2 [20] and AOAC International [19] procedures using the data from the 28 blind duplicate samples that were analyzed over a maximum of four different days by the qualified labs. After data collection, outliers and stragglers were detected using Cochran and Grubbs tests (with critical values set at 1% and 5%, respectively). Outliers were removed prior to calculations, but stragglers were retained. Statistics for AFTOT, FBTOT, and T2 + HT-2 toxins were derived from the sum of individual mycotoxin concentrations. Averaged concentrations, standard deviations of repeatability (SDr), and relative standard deviations of repeatability (RSDr) were estimated from blind duplicates for each sample and each mycotoxin. Standard deviations of reproducibility (SDR), relative standard deviations of reproducibility (RSDR), and HorRat values were also calculated. Trueness was evaluated based on the assigned values given by proficiency test manufacturers for each individual material, except for Sample S12 (Tables 2 and 3) for which reference values were obtained using our internal method (six replicates).

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2072-6651/11/11/658/s1: Document 1: Determination of mycotoxins by liquid chromatography-tandem mass spectrometry (LC-MS/MS), standard operation procedure for the method validation study, 3 July 2019.

**Author Contributions:** Conceptualization, T.B.; Methodology, A.D., T.B. and C.M.; Validation, C.M. and T.B.; Formal analysis, T.B.; Investigation, T.B. and C.M.; Supervision, P.M.; Writing—original draft, T.B.; Writing—review & editing, T.B., C.M., P.M. and A.D.; Project administration, T.B.

**Funding:** This research received no external funding.

**Acknowledgments:** The authors would like to thank R-Biopharm AG (Germany), who kindly provided the immunoaffinity columns for this study, Romer Labs Division Holding GmbH (Austria) for their help in preparing ready-to-use analytical standard mixtures, and FAPAS (United Kingdom) and BIPEA (France) for their support to prepare the set of samples. The authors wish to warmly thank the following people for their strong involvement in this collaborative study: Sonia Colicchia, Helmut Rost, Ervin Tanyi, and Daniel Piller (Romer Labs Diagnostic GmbH, Austria); Nina Tomcic, Dragana Marinkovic, and Biljana Marosanovic (SP LABORATORIJA, Serbia); Daniel Tan and Quentin Baslé (Nestlé Quality Assurance Center, Singapore); Marta Ferro and Silvia Anselmo (Arpal Dipartimento Laboratorio Regionale, Italy); Jozsef Kereszturi and Gabor Kiraly (WESSLING, Hungary); Aengus O'Briain, Audrey Nugent, and Julie Tierney (The State Laboratory, Ireland); Ernest Capraro and Kimberly Bowman (Nestlé Quality Assurance Center Dublin, USA); Phillip D Anderson, Dan Hengst, and John Zulkoski (Eurofins Food Integrity and Innovation, USA); Wim Broer and Manuska Ganpat (NOFALAB, The Netherlands); Joanna Oleniacz, Justyna Kłeczek-Panek, and Magdalena Migut (Nestlé Quality Assurance Center Rzeszow, Poland); Elisabetta Caprai, Giuseppe Luca Polonini, and Simonetta Menotta (Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia Romagna, Italy); Manfred Schneider and Marco Feyer (CVUA Rheinland, Germany); Amrit Kaur, Gurpreet Raj, and Mohit Mogla (Nestlé Quality Assurance Center Moga, India); Ed Boers, and Ruud van Dam (Wageningen Food Safety Research, The Netherlands); Marc Zollinger, Daniela Matera, and Thomas Gude (SQTS, Swiss Quality Testing Services, Switzerland); Alessandro Scantamburlo (Nestlé Quality Assurance Center Padova, Italy); Cristina Poligani, Gaia Alice Romeo and Marco Meschiari (Neotron S.p.A, Italy); Elaine Marley, Carol Donnelly and Dave Leeman (R-Biopharm Rhone LTD, United Kingdom); Nicolas O'Sullivan, Alison Brazil, and Patrick English (Public Analyst's Laboratory, Ireland); Florence Bouveret, Martine Briand, Fiona Gasparini, Marie-Thérèse Renon, and Vincent Dufailly (Nestlé Quality Assurance Center Cergy, France); Barbara De Santis, Carlo Brera, and Gabriele Moracci (Italian National Institute of Health, Italy); Simone Staiger, Christina Pille, and Niels Eick (Eurofins WEJ Contaminants GmbH, Germany); Susan MacDonald and Joanna Stratton (Fera Science Ltd., U.K.).

**Conflicts of Interest:** The authors declare no conflict of interest.

### **References**

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*Editorial* **Contextualized Behavior for Improving Managerial and Entrepreneurial Decision-Making**

**Matteo Cristofaro 1,\*, Maria José Sousa 2, José Carlos Sanchéz-Garcia 3 and Aron Larsson 4,5**

	- Department of Social Psychology, University of Salamanca, 37008 Salamanca, Spain<EMAIL_ADDRESS>
**Keywords:** editorial; decision making; management; entrepreneurship


**6. Survivin splice variants**

192 Advances in Cancer Therapy

Functional annotation analysis indicates that genes related with dorso-ventral axis formation or epidermal growth factor receptor signaling pathway (EGFR) are significantly enriched in the shared genes associated with LSC and Survivin signaling in the KEGG database (http://www.genome.jp/kegg/) (P<0.03). Similarly, genes associated with regulation of EGFR signaling pathway are enriched in the Gene Ontology database (P<0.02). Our analysis shows that molecules shared by LSC and Survivin signaling are mapped on a functional signaling network that connects through the EGFR pathway (Figure 4). EGFR signaling

**5. Survivin modulates gene expression in LSC that connects through an**

Fig. 4. Genes regulated by Survivin in ITD-Flt3+ KSL cells and deregulated in LSC are mapped on a functional signaling network associated with the epidermal growth factor receptor signaling pathway. Epidermal growth factor receptor signaling pathway (EGFR) is significantly enriched in the shared genes associated with LSC and Survivin signaling by pathway enrichment analysis in KEGG and Gene Ontology databases. Our analysis shows that 13 molecules shared by LSC and Survivin signaling are mapped on a functional signaling network associated with EGFR. The network was created based on the KEGG database using Cytoscape software. Gray and black circle represents down regulation and up regulation by Survivin gene deletion, respectively. The hatched circle

**EGFR signaling pathway**

shows EGFR.

Survivin, like many genes, is alternatively spliced and variants having both similar or opposite activities have been identified, some of which are of prognostic significance (Fukuda & Pelus, 2006; Sampath & Pelus, 2007) . Spliced variants identified to date include Survivin 2 (Caldas et al., 2005; Mahotka et al., 2002) , 2B, Ex3 (Ling et al., 2005; Mahotka et al., 1999; Mahotka et al., 2002; Song & Wu, 2005) and 3B (Badran et al., 2004), and some of these variants have been evaluated for their anti- or pro-apoptotic function and ability to interact with wild-type Survivin and other chromosomal passenger proteins (CPC) in some model systems. More recently, several new Survivin splice variants were found in the expressed sequence tag (est) database or described based on RT-PCR using RNA from Molt-4 cells (Li & Ling, 2006; Mola et al., 2007), however the biological functions of these new variants are not known. Overall, with most all the variants reported to date, there is still a considerable lack of understanding of their biology and their function and prognostic value is only now being realized (Sampath & Pelus, 2007). In order to better understand the biology of human Survivin splice variants, we cloned each variant and evaluated their antiapoptotic functions. In the process, we identified a new splice variant we termed Survivin 3 that is similar to Survivin 3B (Figure 5). To determine if this was truly a novel variant, a forward primer specific for all splice variants and a splice variant specific reverse primer were designed and used to amplify cDNA using a polydT reverse transcribed cDNA library from HL60, K562 and Mo7e-Bcr-abl cells, the products resolved on an agarose gel and ~400bp bands excised, cloned and sequenced. Sequence analysis confirmed the presence of the novel splice variant. Survivin 3 contains 12 new amino acids at the Cterminus compared to wild-type Survivin and differs from all of the previously reported Survivin splice variants. Secondary structure prediction using Jpred suggests that Survivin 3 is structurally similar to wild-type Survivin, with the new amino acids predicted to be capable of forming a C-terminal helix that could potentially form the coiled coil domain, even though the length of this helix would be smaller than wild-type Survivin.


Pathogenesis of Colorectal Cancer

*Multidisciplinary Approach for Colorectal Cancer*

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et al. K-ras mutations in

10.1093/jnci/72.6.1323

**24**

**27**

**Chapter 3**

**Abstract**

A Genetic Perspective on

*and Wilson Vilela Medeiros-Filho*

the development an efficient therapy.

**1. Introduction**

**Keywords:** colorectal cancer, molecular biology, carcinogenesis

economic changes that are associated with cancer development [1].

diversity in types of colorectal cancer and genetic involvement.

Colorectal Cancer Progression

*Lenuce Ribeiro Aziz Ydy, Willian Ricardo Camarço-Silva*

The colorectal cancer is one of most frequent neoplasia in adult population. Since was described the adenoma-carcinoma sequence for the first time in 1978, the elucidation of the molecular mechanisms involved in the pathogenesis of colorectal cancer became studied. The study of colorectal cancer has been one of the most important elements for understanding the mechanisms involved in the genesis of malignant neoplasms. Several genes, like DCC, APC, p53 were identified as participants of the adenoma-carcinoma. The annexin A1 protein (ANXA1) is related with some types of tumor and its study in colorectal cancer in scientific literature is been done, but discreetly yet. The increasing advancement in molecular biology research have contributes for understanding the carcinogenesis, with the possibility of detecting earlier pre-malignant lesions and a proper diagnosis, more over it allow

Colorectal cancer (CCR) is the third most common cancer in men, the second in women and the third most prevalent cause of mortality in the world. It was estimated more than 1.8 million new cases in 2018 according to American Institute for Cancer Research and Continuous Update project panel. It is linked to the transition nutrition to the western lifestyle, consuming processed meat, red meat and alcoholic drinks, greater body fatness and adult height increase the risk of the disease. The incidence of the disease continues to rise especially in low and middle income countries and it is considered one of the clearest markers for rapid societal and

Genetic knowledge is essential for understanding of carcinogenic colorectal cancer, and can develop strategies for prevention, diagnosis and treatment. A list of genes in which mutations are capable of interfering with both cancer creation and treatment, has grown a lot in recent years, helping to understand risks for cancer formation and identifying several promising therapeutic targets. Today focused on a best treatment, but research on colorectal cancer genetics began with a focus on diagnosis, much of our understanding of pathogenesis came from study of hereditary syndromes of colorectal diseases that advanced into cancer, showing a huge