Annual reports on mycotoxins from EU countries by Rapid Alert System for Food and Feed (RASFF) [11].
\r\n\tThe book will be focused on special topics suggested as below but not limited:
\r\n\t• Original ideas for GIS applications in coastal environments
\r\n\t• GIS applications for coastal disasters monitoring, modeling, planning, and policy-making
\r\n\t• GIS applications in coastal disasters, coastal resources, coastal social systems, and coastal urban environment
\r\n\t• GIS applications of new algorithms, big data processing, and deep learning to global or regional coastal zones in both developed and developing countries
\r\n\t• Spatial and temporal prediction and validation
\r\n\t• Global/regional coastal database
Mycotoxins are secondary metabolites of molds (low-molecular-weight organic compounds) that have adverse effects on human, animals, and crops resulting in various illnesses, termed as mycotoxicosis [1]. Mycotoxins are colorless, odorless, and tasteless compounds with diverse characteristic structures and molecular weight, examples are deoxynivalenol (DON), fumonisins, ochratoxins, patulins, zearalenols, trichothecenes, and so on, but the most common of them are the aflatoxins. The name “aflatoxin” is known as toxins that are produced by
Street-vended foods are very important to human health as many people derive pleasure in quick ready-to-be-eaten food without stress especially on the road, streets, and other public places due to their unique flavors and conveniences. Most people that are involved in street food vending are the poor and the lower class people, most of whom are ultimately aimed in getting profit other than meeting people’s needs, and some hygienic handlings might be too expensive; they are therefore capable of spreading food-borne diseases if not hygienically handled, and aflatoxins are one of the most frequently reported toxins in street-vended foods.
Aflatoxin contamination of foods can, however, occur at different points in the food chain depending on the time of mold’s evasion. It may be produced during preharvest, harvest, drying, or storage period but ultimately depends on the method and handlings, packaging, or transport conditions of the food materials. Once any of the conditions favor the fungal growth, aflatoxins may be secreted. Rice and Ross [4] reported that FAO estimated that approximately 25% of the world’s cereal products are contaminated with mycotoxins. Rapid urbanization and population growths increase the labor force, and demand for survival with these street food trades gained its momentum. Majority of the population and labor force belong to the lower class group, and the street food trade is becoming viable informal-sector industry especially for the developing entrepreneurs. Consequently, people residing in aflatoxin-endemic countries are more reported with increased incidence of acute hepatic necrosis, resulting later in cirrhosis, or hepatocellular carcinoma, and this may be increasing due to different cultural systems of food preparation and storage [2, 5–8].
However, when considering different production methods adopted for different street-vended foods, it becomes very important to understand the chemical and health implications of aflatoxin contamination of these foods above stipulated limits and with this, we would be able to design adequate control measures against aflatoxin occurrence in the street-vended foods. Many countries have enacted regulations to prevent mycotoxins in foods due to their effect on human’s heath and the world trade, according to the annual report of the Rapid Alert System for Food and Feed (RASFF) [9], mycotoxins were the main hazard in border rejection notifications in the European Union (Table 1).
Mycotoxin | 2008 | 2009 | 2010 | 2011 | 2012 | Total |
---|---|---|---|---|---|---|
Aflatoxins | 902 | 638 | 649 | 585 | 484 | 3258 |
Deoxynivalenol (DON) | 4 | 3 | 2 | 11 | 4 | 24 |
Fumonisins | 2 | 1 | 3 | 4 | 4 | 14 |
Ochratoxin A | 20 | 27 | 34 | 35 | 32 | 148 |
Patulin | 3 | – | – | – | – | 3 |
Zearalenone | 2 | – | – | – | 4 | 6 |
Total | 933 | 669 | 688 | 635 | 525 | 3450 |
Annual reports on mycotoxins from EU countries by Rapid Alert System for Food and Feed (RASFF) [11].
Street food vending can make good contribution to the economy of developing countries, for example, the Indian National Policy for Urban Street Vendors/Hawkers reported street vendors of about 2% of the metropolis population [10, 11]. However, street-vended foods are perceived to pose some major public health risks mostly in developing countries, and this is due to unavailability of basic infrastructures and difficulty in controlling large number of food-vending operators. There are several reported incidences of food poisoning due to consumption of street-vended foods. For example, the Shandong Province in China has recorded about 691 outbreaks of food poisoning from vended foods which are responsible for over 49 deaths during the period of 1983–1992 [12–19]. Aflatoxins are mostly reported in stored cereals, legumes, and nuts, and their derivatives (European Food Safety Authority (EFSA) report [20]). Marin et al. [21] stated that different aflatoxin contents were detected in 34,326 food samples from different European Union countries (Table 2) and also in some food materials from African and Asian countries; selected examples are presented in Table 3.
Food category | No. samples | No. samples > LOD | Median AFB1/AFT | Mean AFB1/AFT | Maximum AFB1/AFT |
---|---|---|---|---|---|
Almonds | 1766 | 471 (27%) | 0.20/0.28 | 1.46/1.82 | 575/579 |
Brazil nuts | 622 | 271 (43%) | 0.20/0.40 | 22.2/39.6 | 1897/3337 |
Hazelnuts | 3163 | 940 (30%) | 0.16/0.30 | 0.95/1.70 | 200/200 |
Cashews | 336 | 33 (10%) | 0.10/0.20 | 0.42/0.60 | 36/39 |
Peanuts | 8929 | 1830 (20%) | 0.10–0.20 | 1.93/2.69 | 935/985 |
Pistachios | 4069 | 1783 (44%) | 0.20/0.40 | 16.8/19.4 | 2625/2680 |
Other nuts | 1131 | 158 (14%) | 0.10/0.20 | 1.16/1.41 | 385/402 |
Figs | 2067 | 618 (30%) | 0.15/0.24 | 1.36/2.22 | 130/151 |
Other dried fruits | 1396 | 114 (8%) | 0.10/0.24 | 0.26/0.51 | 20/90 |
Maize | 943 | 136 (14%) | 0.12/0.24 | 0.26/0.41 | 8/9 |
Other cereals | 3010 | 207 (7%) | 0.20/0.40 | 0.35/0.51 | 109/117 |
Spices | 4698 | 1988 (42%) | 0.20/0.40 | 1.46/1.88 | 96/96 |
Baby foods | 592 | 23 (4%) | 0.02/0.04 | 0.07/0.14 | 1/2 |
Other foodstuffs | 1604 | 303 (19%) | 0.10/0.20 | 0.53/0.75 | 99/99 |
Detected aflatoxin contents in food sample (μg/kg) from different EU countries as reported by the European Food Safety Authority [23].
Country | Commodity | Mycotoxin | Level | Source |
---|---|---|---|---|
Saudi Arabia | Peanuts | Aflatoxins | 28 μg/kg | [26] |
Nigeria | Groundnuts | Aflatoxins | 10–176 ppb | [27] |
South Africa | Cowpeas | Fumonisins | 0.6–25.30 μg/kg | [27] |
Iran | Walnuts | Aflatoxins | 14.4 ± 8.4 μg/kg | [29] |
Iran | Peanut (roasted) | Aflatoxins | 17.99 ± 18.70 μg/kg | [29] |
Benin Republic | Cowpea | Aflatoxins | 3.52 μg/kg | [28] |
Nigeria | Yam chips | Aflatoxins | 4–18 μg/kg | [25] |
Nigeria | Maize | Aflatoxins | 3–138 μg/kg | [31] |
Nigeria | Shelled melon | Aflatoxins | 5–20 μg/kg | [28] |
Pakistan | Chili | Aflatoxins | 0.1–96.2 μg/kg | [29] |
Pakistan | Milk and sweets | Aflatoxin M1 | 0.05–0.48 μg/kg | [30] |
Mycotoxins contamination in some foodstuffs in Afro-Asia.
Insufficient drying and humid storage environmental conditions may result in high mood invasion and concurrent aflatoxin contamination of foodstuffs. Aflatoxin contamination of street-vended foods is of a great concern especially when it occurred above the tolerance limit. Aflatoxin outbreak, such as the Kenya 2004 and 2005 aflatoxin outbreak from locally stored maize [22], may also have economic implications. Therefore, there is a need to recognize the aflatoxin biosynthesis associated with different handlings of street-vended foods as to human tolerance levels of these aflatoxins. Some countries already developed some specific regulations against aflatoxin contaminations with tolerance limit for aflatoxin B1 in foodstuffs ranging between 0 and 30 μg/kg and total aflatoxin contents of 0–50 μg/kg (worldwide regulations for mycotoxins in food and feed in 2003 [23]). However, prevention of disease in street-vended food in many developing countries was difficult due to uncontrollable environmental factors. The street vendors did not have significant knowledge of epidemiology and their safety measures.
Aflatoxins were first isolated from the Turkish X-disease patients that consumed the mold-contaminated food as a result of production of aflatoxins. Later these toxins were also found in
Aflatoxin B1 is one of the potent, mutagenic, and carcinogenic toxins [32–35]. It was present mostly in conjugation with other aflatoxins like B2, G2, and G1. Structurally, it consists of either five rings along with furofuran moiety or five aromatic carbon rings or six lactone rings. Another parasitical aflatoxin was produced from
Structure of aflatoxins.
The chemical characteristics of these aflatoxins are presented in Table 4.
Aflatoxin | Molecular formula | Molecular weight | Melting point | UV absorption max (e ), nm, methanol | |
---|---|---|---|---|---|
265 | 360–362 | ||||
AFB1 | C17H12O6 | 312 | 268–269 | 12,400 | 21,800 |
AFB2 | C17H14O6 | 314 | 286–289 | 12,100 | 24,000 |
AFG1 | C17H12O7 | 328 | 244–246 | 9600 | 17,700 |
AFG2 | C17H14O7 | 330 | 237–240 | 8200 | 17,100 |
AFM1 | C17H12O7 | 328 | 299 | 14,150 | 21,250 (357) |
AFM2 | C17H14O7 | 330 | 293 | 12,100 (264) | 22,900 (357) |
AFd1 | C16H14O5 | 286 | 250–300 | 11,200 | 20,120 |
AFRO | C17H14O6 | 314 | 280–290 | 12,100 (310) | 22,900 (320) |
Chemical characteristic of different aflatoxins.
Metabolism of aflatoxins usually form many other metabolites, examples are aflatoxins R0, RB1, RB2, and H. These aflatoxins have hydroxyl group in their ring’s carbonyl group, D ring may also be formed in some other cases such as in aflatoxins RB1 and RB2 while an opened E ring may be formed in B3.
Two pathways known for the formation of all these aflatoxins are:
Microbial transformation
Chemical reduction of sodium borhydride
Aflatoxins are highly toxic and carcinogenic. The disease caused by aflatoxins is known as aflatoxicosis. It mainly targets the liver of humans and animals. Toxicity of aflatoxins depends upon the number of factors such as age, sex, species, and national factors [24]. The aflatoxin metabolic pathways are well studied in animals, but in human, data was very limited. In rats, the DNA/RNA synthesis was inhibited by ingestion of aflatoxins (5 mg/kg). The B1 aflatoxin binds with the N7 guanine by covalent bonding and formed AFB1-N7-guanine adducts. It results in the transversion of G to T which caused the DNA mutations and carcinogenic. In animals or in humans, the pathway of aflatoxins is described as in Figure 2. While in humans (AFB1-N7-guanine) transverse the nucleotides G to T at the position of codan 249. These suppress the p53 tumor gene. The reactive epoxide formed which hydrolyzed to form AFB1-8,9-dihydrodiol. The dihydrodiol ionizes and formed a Schiff’s base with primary amine groups in the proteins. The AFB1 also inhibits the phosphodiesterase activity in the kidney, liver, and brain [36].
Aflatoxin metabolism in humans.
In the 1980s, aflatoxin B1 and sum total aflatoxin tolerance level in foodstuffs range from 0 to 50 μg/kg. Some countries have a zero tolerance which practice the limit of detection depending on the analytical procedures and standards (Table 3), for example, Austria and Switzerland have the lowest tolerance limit for aflatoxin B1. And aflatoxin B1 is the most important aflatoxin, based on the occurrence frequency and its toxicity. For detection of many aflatoxins, thin layer chromatography (TLC) or high-performance liquid chromatography (HPLC) is commonly used, but in some cases, mini-column, gas chromatography/mass spectrophotometry and qualitative radioimmunoassay (RIA) methods may be adopted. These methods are frequently coupled with the analytical methods of AOAC or AOAC-derived methods. It may be expected here that the immunoassay detection such as the enzyme-linked immunosorbent assay (ELISA) method will also gain attention, but this methods is presently still undergoing intensive validation processes to be used as a generalized tool for regulatory analysis.
Aflatoxin acts as toxic secondary metabolites, carcinogenic, hepatotoxic, immunotoxic, and teratogenic in humans and many animal species. High ingestion and accumulation of ‘aflatoxins’ also pose toxic effects on human cell and tissues and even on genes. Aflatoxin poisoning is common in some areas of the world, and different types of this case have been documented. Many cases of aflatoxicosis have also been reported in Africa and Asia, most of which involve the ingestion of contaminated cereals such as maize, rice, or other foods like cassava or dried powdered food materials; some other cases were reported in food products such as pasta or peanut meals. An example of such case is the 1990 Malaysia aflatoxin infection of over 40 adult humans and 13 children deaths associated with the consumption of aflatoxin and borate contamination of noodles. The autopsies of the heart, brain, spleen, liver, kidney, and lung showed damages from aflatoxin interference. Autopsy of brain (cerebrum) specimens from 18 kwashiorkor children and 19 other children who had died from a variety of other diseases in Nigeria showed aflatoxin present in 81% of the cases [37].
The human body could be exposed to mycotoxins through skin contact or by direct inhalation of spore-borne toxins, and these tend to accumulate within the body organs or tissues during their metabolism [24]. Infections caused by fungi such as
Diagnostic features of mycotoxicosis:
Non-transmissible.
Seasonal outbreak.
Disruption associated with specific foodstuff.
Drug treatments have little or no effect.
Many aflatoxins can cause acute mycotoxicosis (i.e., when the symptoms show within a short period after infection say like 7 days, it may lead to death if not treated on time) and/or chronic mycotoxicosis (i.e., when they appear and persist for a long period of time). In 1993 International Agency for Research on Cancer (IARC) has classified four types of naturally occurring aflatoxins AFs (AFB1, AFB2, AFG1, AFG2) as the most active substances that cause mutagenicity and carcinogenicity [45]. Aflatoxin AFB1 is the most prevalent among all types of aflatoxins due to long-term chronic exposure to even very small amount of aflatoxin in food items, and it is an important concern for human health [10]. Its chronic exposure can lead to malnutrition, suppressed immune response, centrilobular necrosis, proliferation of bile duct, hepatic lesions and fatty infiltration of liver, and even hepatomas [41, 42] as shown in Figure 2.
Symptoms of mycotoxicosis depend on the quantity or concentration of the toxin, time of exposure, type of the mycotoxin involved, degree of toxin combination, host resistant capacity, physiological status of the host, and so on. Mycotoxin can cause heath effect in contact with the skin and alimentary canal, by inhalation or by other means. The toxins can enter into blood streams and the lymphatic system to inhibit the process of protein synthesis, damage macrophage system, or affect the lung’s ability to clear particles or cause immune suppression.
Carcinogenic chemicals like aflatoxin can bring different modifications in DNA sequence or protein structure which causes DNA adduct formation and finally cancer in effected people. AFB1 is a micro-component of nutrition that causes genetic alteration by inducing adduct formation, leading to DNA strand break. This DNA or oxidative damage can turn out to be hepatocellular carcinoma (HCC) or cancer [43–48].
Studies have demonstrated that HCC caused by AFB1 is due to
Biotransformation from AFB1 comprises CYP450-mediated reaction resulting in nucleophilic genotoxic reactive intermediate (AFBO), hydroxylation (to AFM1 and AFQ1), or demethylation (to AFP1). AFBO binds to the liver cell and causes mutation in
Aflatoxins are major factor causing suppression in the immune system which further affects humoral and cellular response. Animal exposure to aflatoxin showed dose-dependent response in percentage of splenic CD8+ T cells, CD3− CD8a+ cells, natural killer (NK) cells [51, 55, 56]. Reduced expression level of cytokine mRNA in the intestine was observed in a study that might be due to reduced percentage of T cells in broiler. This phenomenon directly affects the immune function of intestinal mucosa [49].
Many fungal strains are genetically capable to produce mycotoxins, but they may not do so until certain conditions are met, and it is known that aflatoxin contents in food vary directly with the processing and storage methods and the associated producer microbe. Physical, chemical, and biological factors can affect the formation of aflatoxins in street-vended food including conditions such as temperature, moisture content, the location of vendors, the storage of utensils, personal hygiene, and method of reheating and storage of food. Availability of nutrients to enhance the fungal growth or energy sources such as sugar and vegetable oil will enhance the toxin production [26], for example, there was a higher aflatoxin produced by
The location and conditions set for preparing and storing street food may contribute to the production of fungal toxins. Jonathan et al. [31, 55, 56] reported variations in biodeteriorating fungi and aflatoxin contents of particular foodstuff collected from different locations in Nigeria. Most of the vendors are illiterate and do not have knowledge about the sanitary conditions [57]. A case reported in Africa showed that 85% of food stalls were located near garbage dumping sites [58]. Some storage molds such as
Many traditional ways used for food preparation such as heating and roasting can reduce the aflatoxin contents in foods; ranging values in sausage rolls prepared from different locations in Nigeria were observed by Jonathan et al. [55] due to different methods adopted for its preparation; Fandohan et al. [62] determined the fates of aflatoxin and fumonisin in different traditional ways of preparing maize and maize foods based on washing, sorting, winnowing, and hulling combined with crushing of the grains; this is also supported by Park [63] and Lopez-Garcia and Park [64]. The presence of other microorganisms either bacteria or fungi may alter elaboration of mycotoxins on food materials, for example, there was reduced aflatoxin production when
Aflatoxin-producing fungi can contaminate food crops during cultivation, harvest, transport, storage, or food preparation [70–72]. Iaha et al. [70] reported that insect damage of maize enhances
Effect of aflatoxin on the economy require good imparts assessment model experts and data sets [80]; this may include income losses due to deaths of livestock, weight loss, reductions in productivity, and the yield of eggs, meat, and milk. It affects aves (poultry) such as ducks, chicken, and turkeys; mostly prime example is that of the 1960 outbreak of “Turkey-X disease” in the United Kingdom caused about 100,000 turkey deaths; it may also lead to loss in fishes, birds, rabbits, dogs, and other mammals. The effect of aflatoxin on economy, storage loss, and generally output loss in animal husbandry due to aflatoxin incidence would be threatened here. Aflatoxin lethal dose (LD-50) is generally between 0.5 and 10 mg/kg of livestock body weight; however, high dose of mycotoxin was responsible for, another case with more economic loss may still occur if mycotoxins are not controlled. There is a need for capacity building to enhance the analysis economic impact of mycotoxin and trade analysis.
Mycotoxin contaminations may also affect other sectors of food production and agriculture. Infected commodities can be rejected from shipments, and some prices may become reduced due to loss in quality; this can devastate export markets especially in developing countries. The consumers may be indirectly affected by increased price/costs of food materials as the sellers/producers levied extra cost due to maintenance against contamination or health risks if they are only able to afford contaminated products which are always cheaper. Farmers may suffer reduced income, feed loss, or low outputs due to aflatoxin/mycotoxin interference most especially on the stored products, and economy impact of mycotoxins stems from high mortality, immunity, weight loss, fertility, and quality of dairy products in livestock.
However, we need to checkmate the mycotoxin preventive cost over economic cost; preventive cost in most cases saves many products as the saying “prevention is better than cure” and yields better production and storage practices. Most African countries such as Gambia, Ghana, Mali, Niger, Nigeria, Senegal, as well as Sudan have reported about 7.5 million US$ cost for mycotoxin control programs due to high-reported mycotoxin incidences on foodstuffs like peanuts and many cereals. In addition, fungi biodeterioration of foodstuffs has caused many countries economic loss due to rejection of exported foodstuffs and affected relationships with trading partners. Implementation of credible food safety controls by exporting countries is needed to foster their smooth exportation of food and agricultural produce. Quality assurance by importing countries has to be routine, and by so doing, the manufactured food will have insignificant mycotoxin levels. The exporting country must be able to comply with this requirement and demonstrate that compliance has been realized. Effectiveness of control measures requires important elements such as administrative structures, resource management, scientific and technical infrastructure, and financing and human capital. Lack of efficient management of resources in many countries has been verified to compromise the credibility of food safety controls. In 2010, foods worth more than US$200,000 were destroyed by regulatory agencies in Nigeria as a result of contamination by mycotoxins.
Aflatoxin (B1, B2, G1, and G2) level in product (ng/g) can give a mark of quality and can be used as a threshold for distinguishing low-, medium-, and high-quality product. Africa could have accrued an estimate of 67 million dollars annually, but this is lost due to export rejection of its food and agricultural produce contaminated with high levels of mycotoxins. Over the years, the European Union Rapid Alert System has notified Nigeria on its alarming export rejects. Contamination by mycotoxins is owed to hot and humid conditions, soil condition, storage method, crop variety, cultural practices, harvesting procedures, etc.
Adequate practices such as pest and disease control management are needed for safe and healthy food production; this must be judiciously implemented to tackle the menace of aflatoxin spoilage of storage and dairy agricultural produce. Early harvest of agricultural produce may avert aflatoxin/mycotoxin contaminations as the fungi that produce these toxins are usually associated with deterioration of the organic compounds. More also, mycotoxin prevention starts right from the cultivation and rearing of livestock. The use of contaminant-free tools, sterile water for washing, and some other hygienic measures needs to be taken seriously either the products are to be consumed or for sale. Some scientists suggest harvesting at dry period, and storage of agricultural produce in dry environment as wet condition improves the growth of molds [77]. Proper cleaning of harvested products, removal of spoilt products (most especially grains), and proper storage methods are good for mycotoxin control. Good transport and storage mechanisms and frequent check for daily temperature and humidity are good for mycotoxin control.
Many scientists have suggested the use of living organisms to nullify or reduce mycotoxin contamination [78]; this is called
Physical methods such as sorting out bad or infected products from healthy ones, hygienic handling, and maintenance of clean environment can reduce aflatoxin/mycotoxin incidence in street-vended foods or industrial food products [67]. A center control point for testing all street-vended food may be set up by the government to reduce mycotoxin incidence. Regulatory measure on mycotoxin should be strengthened in every country. Food management system point will save a lot from food poisoning [80]. Conduction of test at different steps in food production is also important in big food industries; this may be coupled with the use of supplier’s schemes. Hazard and analysis critical control point (HACCP) for pre- and postharvest stage plan has save many commodities from contaminants; this has been used on coconuts and corn in South East Asia and on nuts in South and West Africa; it has also been used on apple juice and pistachio nut in South America [79]. Detailed information on HACCP was reported by FAO [79] and suggested the implementation of HACCP for adequate fungal toxin management.
Legislation and food inspections are very important for food security. For thousands of years back, food has always been subjected to legislation and inspection as the need was felt for some control on the quality of food materials. This control was intended to safeguard the health of consumers and as well prevent cheating in terms of the composition of food; the principle that food contaminated with a hazardous substance is unfit for human consumption and shall not be sold or offered for sale was not always applied as intended. Before, local and municipal affair/ordinances were used for regulated food quality as there were no advanced scientific methods and tools, but today, advances in science of bacteriology, microscopy, and chemistry have aided official legislations on food. Today, we have legislations that prohibit adulterated or misbranded foodstuffs and incidence of contaminants in food particularly incidence of these contaminants above the tolerance limit in either humans or animals.
Mycotoxins as a food toxic substance have recently been considered in food regulation (Table 5). It started shortly after the aflatoxin discovery in the 1960s, and some other specific mycotoxins such as ochratoxin, zearalenone, deoxynivalenol, patulin, and phomopsin were later considered. An attempt to study the whole world’s legislation on mycotoxins was made by the International Union of Pure and Applied Chemistry (IUPAC) and the Food and Agriculture Organization which led to an updated paper that was published by Schuller et al. [80] in the 1987 (second) mycotoxin joint International annual Conference of FAO/WHO/UNEP. The mycotoxin legislation has since then grow exponentially in various countries. Many countries are known to enforce or propose certain aflatoxin regulations for foodstuffs to continue to increase, and many countries expanded their regulations to specify more types of foodstuffs. As in 1981, the maximum limits for aflatoxins in food (aflatoxin B1 or the sum of aflatoxins B1, B2, G1, and G2) vary from zero detectable to 50 jtg/kg. The strategies which may be employed to limit the establishment of mycotoxins in food should include both on-field and post-field measures. The regulation of a country is to ensure that any food contaminated in an amount that is intolerable from a public health point of view, particularly, at a toxicological level, is not tradable in that country. Contaminant levels are required to be kept as low as possible by proper measures. Regulations are established in many countries to control food contamination so as to protect human health; these regulations may include specific maximum limits for several contaminants for different foods and a reference to the sampling methods and methods of analysis used [81, 82]. Report by FAO in 2003 shows that about 100 countries have existing regulations on mycotoxins in specific foods and feeds; this was about 30% increase over 1995 report.
Country | Food material | Tolerance level (μg/kg) | Authority | |
---|---|---|---|---|
B1 | B2 + G1 + G2 | |||
Argentine | Sugar-coated nuts | – | 5 | Ministry of Public Health, Ministry of Agriculture |
Peanuts, peanut products, maize products | 5 | 20 | Ministry of Public Health, Ministry of Agriculture | |
Infant foods based on cereals and AH foods | 2 | Ministry of Public Health, Ministry of Agriculture | ||
Australia | All foods except peanut products | 3 | – | National Health and Medical Research Council, 1982 (recommendation) |
Australia | Peanut (products), all foods, except milling and shelled products | 15 1 | 5 | Not official Ministry of Public Health |
Milling and shelled products and derived products children foods | 2 0.02 | 5 0.02 | Ministry of Public Health Ministry of Public Health | |
Belgium | All foods | 5 | Ministry of Public Health and Ministry of Agriculture (not official) | |
Brazil | Industrially prepared foodstuffs for children from 0 to 2 years and for school meals Imported foodstuffs | 5 | 3 10 | Proposed |
Other foodstuffs | 15 | 30 | Proposed | |
Canada | Nuts and nut products | 15 | Health and Welfare Canada (official) | |
China | Rice, peanuts, maize, sorghum, beans, wheat barley, oats | 50 | Ministry of Public Health, Council of Agriculture and Local Authorities (official) | |
Colombia | Sesame seed | 20 | Publication official del Instituto Colombiana de normas technicas “Icontec.” Norma Colombiana nr. 536, edicion 1981 | |
Oil seeds (peanuts) | 10 | Ministry of Public Health (official) | ||
Cereals, grains (sorghum, millet) | 30 | Ministry of Public Health (official) | ||
Cuba | Cereals, grains, peanuts | 0 | Ministry of Agriculture | |
Czechoslovakia | Cereals, grains, peanuts All foods except infant and children foods | 5 | 10 | Ministry of Health (official) |
Infant foods on milk basis (calculated on basis of reconstituted product) Other infant foods and children foods | 0.1 1 | 0.2 2 | Ministry of Health (official) | |
Denmark | Peanuts | 10 | Ministry of the Environment (official) | |
Dominican Republic | Maize and maize products, peanuts, soya, tomatoes, and products thereof | 0 | Ministry of Agriculture and Ministry of Public Health | |
Finland | All foods | 5 | Decision of the Ministry of Trade and Industry on some food contaminants (762), 1984, Ministry of Trade and Industry; National Board of Trade and Consumer Interests | |
France | All foods Infant foods Dietary milk Foods | 10 5 O-1l^g/100 kilocalories (=0–024 ^g/100 kj) | Conseil Supérieur d’Hygiène Publique de France, séance 25.10.1975 Interests, Ministry of Consumption Arrêté 5.01.1981 Journal Officiel de la Republique Française; 11.01.1981, Ministry of Consumption Arrêté 30.03.1978 Journal Officiel de la Republique Française; 11.01.1981, Ministry of Consumption | |
Federal Republic Of Germany | Peanuts, peanut products, hazelnuts, walnuts, brazil nuts, pistachio nuts, apricot and peach pits, poppy and sesame seeds, cereals, cereal products, grated coconut, almonds | 5 | 10 | Aflatoxinverordnung Bundesgesundheitsblatt IS 3313 of 30.11.1976, Various Not ministries, official depending on the State |
German Democratic Republic | All foods | 5 | 10 | Circular of Ministry of Health 1970, Ministry of Health |
Hong Kong | Infant foods and children foods. Peanuts and peanut products | 20 | Municipal Services Branch, Goment Secretariat, Hong Kong Government Ministry of Ministry Health of Ordinance Health No. 4 of 25.06.1978 | |
Hungary | All foods | 5 | Ministry of Health of Ordinance Health No. 4 of 25.06.1978 | |
India | All foods Israel Ireland Italy Grains and nuts, peanuts | 30 | Ministry of Health and Family Welfare, Department of Health Ministry of Health (official) | |
Israel | Grains and nuts | 20 | Ministry of Public Health | |
Ireland | All foods | 5 | 30 | |
Italy | Peanuts | 50 | Ministry of Public Health | |
Japan | All foods | 10 | Food Sanitation Investigation Council, April 1974, Ministry of Health and Welfare (official) | |
Jordan | Almonds, cereals, maize, peanuts, pistachio nuts, pine nuts, rice | 15 | 30 | Minister of Finance and Customs Instructions (5/35/8251) 11.03.1981; letter of Minister of Health (48/37/2049) 03.03.1981, Ministry of Health |
Kenya | Peanuts and other vegetable oils, peanut products | 20 | Food, Drugs, and Chemical Substances Regulations Kenya Gazette, 01.07.1978, Ministry of Health | |
Luxembourg | Peanuts and peanut products | 5 | Règlement Grand-Ducal of 22.09.1978 Loi 25.09.1953, art.7f.ll and 12 Benelux Arrêté M(77) 5-03.05.1977 Ministry of Public Health | |
Malawi | Peanuts (export) | 5 | Letter of Malawi Bureau of Standards BS/1/1 of 24.06.1976 | |
Malaysia | All foods | 35 | Food Regulations 1985 | |
Mauritius | Groundnuts Others | 5 5 | 15 10 | Food and Drug (Control of Aflatoxins) Regulations, 1979. Government 1: Notice No. 222 of 19.9.1979 |
Mexico | All foods | 20 | Ministry of Official Public Health, Ministry of Agriculture | |
The Netherlands | Peanuts and peanut products All foods and food ingredient | 5 5 | Algemeen Besluit (Warenwet) art.3 quinquies. Staatsblad van het Koninkrijk der Nederlanden 46, artikel 1, 19.01.1974, Ministry of Welfare, Public Health and Cultural Affairs Ministry of Welfare, Public Health and Cultural Affairs Official | |
New Zealand | Peanut butter, shelled nuts and nut portion of products containing nuts, other foods | 15 | Food Regulations 1984, section 257 | |
Nigeria | All foods Infant foods | 20 0 | Food and Drug Administration (official) Food and Drug Administration (official) | |
Norway | Nuts, buck wheat, other foodstuffs | 5 | Rundskriv IK-1/85 of 08.02.1985 | |
Peru | Maize and peanuts | 5 | Code of practice | |
Philippines | Coconut, peanut products (export) | 20 | Ministry of Health | |
Poland | All foods | 0 | Ministry of Public Health | |
Portugal | Peanuts Infant foods Other foods | 25 5 20 | Decreto-Lei no.6/83 nr. Diärio da Républica of 14.01.1983, Ministry of Public Health, Ministry of Agriculture, Ministry of Commerce (official) | |
Romania | All foods | 0 | Joint papers of veterinary specialists and doctors of medicine 1978, Ministry of Health Ministry of Agriculture | |
Singapore | All foods | 0 | 0 | Food Regulations 1974, art. 2, para 3c Government Gazette no. 4959-16.01.1976 Ministry of the Environment |
South Africa | All foods | 5 | 10 | Government Gazette no. 4959-16.01.1976 |
Surinam | Peanuts, peanut products, pulses | 5 | Gouvernementsblad van Suriname nr. 199, 1971, Food Inspection Service (not official) | |
Sweden | All foods | 5 | National Food Administration’s Ordinance (SLV FS 1983: 1) on Foreign Substances in Food, SLV FS 1985: 16, Swedish National Food Administration | |
Switzerland (existing regulations) Proposed regulations | Almonds, peanuts, hazelnuts, walnuts, brazil nuts, pistachio, apricot and peach kernels, grated coconut, poppy, sesame, peanut butter, peanut flips, arachis oil (not refined, bottled), pumpkin, kernels, maize, cereals All foods, except maize and cereals | 1 2 1 | 5 5 5 | Verordnung über die hygienisch-mikrobiologischen Cantons Anforderungen an Lebensmittel, Gebrauchs-und Verbrauchsgegenstände 817.024 of 14.09.1981 |
Thailand | All foods | 20 | Ministry of Public Health Notification no. 98.B.E. 2529: Standard for Food Containing Contaminants, Ministry of Public Health (official) | |
Union of Socialist Soviet Republics | All foods | 5 | Methodic Documents Minszdrav USSR 2273-80 of 10.12. 1980 and 4082-86 of 20.03.1986, Ministry of Health and State Agric Industrial Committee of the USSR | |
The United Kingdom | Nuts and nut products | 10 | Proposal, MAFF press release 181 of 08.07.1986, Ministry of Agriculture, Fisheries and Food | |
Yugoslavia | Wheat, maize, rice and other cereals, beans | 5 | 1 | Article 57, Federal Register no. 2, 1980, Slüzbeni list Socijalisticka Federation Republika Jugoslavija 2/1980, Federal Committee for Labour, Health and Social Welfare (official) Article 57, Federal Register no. 2, 1980, Slüzbeni list Socijalisticka Federation Republika Jugoslavija 2/1980, Federal Committee for Labour, Health and Social Welfare (official) |
Zimbabwe | Groundnuts, maize, sorghum | 5 | 4 | Ministry of Agriculture |
Legal aflatoxin tolerance levels in foodstuffs for humans [80].
However, studies have shown that most African countries have adequate mycotoxin regulations due to the fact that many countries in Africa account for highest countries with massive aflatoxin incidence. Morocco had the most detailed regulations on mycotoxins with about 15 nations out of about 99 countries with known mycotoxin regulations in 1995. Nigeria, for example, adopted its regulatory system from the European commission used primarily on export commodities. However, mycotoxin regulatory status quo in African countries still needs to be improved upon especially for effective implementation.
In this chapter, it would be understood how historical events should gear enforcement of regulatory limits for foods produced locally in order to maintain a high standard especially in foreign trading. This chapter elucidated the need to devise principal methods of mycotoxin control in food and human. It would be understood that mycotoxins in food commodities are a result of fungi infection. It would reiterate the need for proper cultural practice, sanitation, and good storage procedures, among others, as possible measures.
This chapter deals with regulatory considerations related to radiopharmaceutical precursors within Europe. Outside, different aspects may apply, with the exception of certain harmonized documents. Radiopharmaceuticals are considered a safe class of medicinal products. Due to the small chemical quantities administered they are not expected to exhibit any measurable pharmacological effect [1]. However, since they are radioactive, the rules for minimizing the risk associated with the use of ionizing radiation to the patients and to the personnel must be observed. Depending on the chemical and physical properties, radiopharmaceuticals are used in major clinical areas for diagnostics and/or therapy [2]. As defined by the European Pharmacopeia (Ph. Eur.) general monograph (0125)
Radiopharmaceutical precursors according to Ph. Eur.
Given the complex nomenclature used in various regulations and guidance documents, the understanding of radiopharmaceutical precursor’s definition might be challenging. Depending on the context it could be interpreted as the substance which becomes a radiopharmaceutical after radiolabeling with a radionuclide of choice or a radionuclide which is used for radiolabeling of that substance. Therefore, the quality requirements and test methods specifications of precursors for use in preparation of theranostic radiopharmaceuticals can be discussed only in the light of current regulatory framework.
The preparation and use of radiopharmaceuticals are regulated by number of directives, regulations and rules. These documents may be classified with respect to the status of radiopharmaceutical preparation:
radiopharmaceuticals with marketing authorization (MA), regulated by:
radiopharmaceuticals to be used in clinical trials (CT), regulated by:
unlicensed radiopharmaceuticals extemporaneously (just before use) prepared, not for CT [12, 13].
Radiopharmaceuticals with marketing authorization (MA) meet the requirements of GMP Annex 3 (Manufacture of Radiopharmaceuticals) [8] and EMA Guideline on Radiopharmaceuticals [12]. For the small scale preparation of radiopharmaceuticals outside the marketing authorization the guide of the Pharmaceutical Inspection Convention and Pharmaceutical Inspection Co-operation Scheme (PIC/S) [14], the Guidelines on Good Radiopharmacy Practice (CRPP) issued by the Radiopharmacy Committee of European Association of Nuclear Medicine (EANM) [13] and the Chapter 5.19. Extemporaneous preparation of radiopharmaceutical preparations of the Ph. Eur. [15] are setting standards for good practices.
The translation of new radiopharmaceuticals from the preclinical stage into clinical trials requires appropriate quality assessment essential to ensure efficacy and safety of both drug substance and drug product [16, 17]. The specific regulatory framework for the use of radiopharmaceuticals in clinical trials has been established in Europe [9, 11, 18]. From the radiopharmaceutical development perspective, the essential step is the preparation of an Investigational Medicinal Product Dossier (IMPD). This document includes information related to the chemical and pharmaceutical quality of the drug substance and drug product, as well as non-clinical data related to pharmacology, pharmacokinetics, radiation dosimetry and toxicology [19]. IMPD contains two main sections related to the production and quality control of the radiopharmaceutical: the drug substance (the active pharmaceutical ingredient, or API) and the drug product.
An
The manufacture of APIs should be carried out following general GMP requirements. In a GMP-based system, all processes are defined, systematically reviewed, and shown to be capable of consistently providing medicinal products of the required quality and complying with their specifications [20]. Written and approved protocols specifying critical steps, acceptance criteria, must be in place. Process validation is a crucial part of GMP, meaning that all critical steps of manufacturing processes as well as significant changes to these processes are validated. It should be noted that the requirements for validations differ depending whether marketing authorization, clinical trials or in-house preparation of radiopharmaceuticals are planned (see also Figure 2.) [21]. The qualification and validation aspects related to the small-scale “in house” preparation of radiopharmaceuticals are covered in the EANM guidance [22].
Requirements for chemical precursors used in preparation of radiopharmaceuticals depending on their regulatory status.
In the process of IMPD preparation the prime challenge is to establish quality specifications for radiopharmaceutical precursors. They are supposed to comprise a set of tests that are necessary to confirm identity, purity and strength of the drug substance. Issues under consideration are the definition of release criteria, analytical procedures and especially their validation. Main references to address these issues are the European Pharmacopeia and guidance provided by the International Conference on Harmonization (ICH). Ph. Eur. provides general requirements for quality control of radiopharmaceutical precursors, in addition, a number of monographs for individual radiopharmaceuticals and chemical precursors are available in the Ph. Eur.
The use of analytical methods described in the pharmacopeia allows to reduce the work load related to analytical method validation. This does not mean that a pharmacopeia method may be implemented without any preliminary testing and verification. As a minimum, the most critical parameters should be verified, depending on the intended method. If no pharmacopeia monograph exists, analytical methods need to be fully validated. As stated by the general reference document issued by ICH the objective of validation of an analytical procedure is to demonstrate that it is suitable for its intended purpose [23]. To validate an analytical method, the following characteristics may be considered: specificity, accuracy, linearity range, precision (repeatability and intermediate precision), limit of detection (LOD), limit of quantitation (LOQ) and robustness. Recently, recommendations for the validation of analytical methods which are specific for radiopharmaceuticals has been published by EANM [24].
Chemical precursors for radiopharmaceutical preparations, are non-radioactive substances obtained by chemical synthesis for combination with a radionuclide in contrast to precursors manufactured using substances of human or animal origin [4].
The quality specification for chemical precursors is built upon three elements: exact methods, test limits and selection of reference standard. Pharmacopeia monographs comprise a set of critical attributes categorized into three subdivisions: identity, tests (related substances, residual solvents, metal catalyst or metal reagent residues, microbial contamination, bacterial endotoxin) and assay of the active substance. To ensure the appropriate quality, reference substances (like primary standards e.g. Ph. Eur. Chemical Reference Substance, CRS, or Pharmaceutical Secondary Standard, PSS) are used as a standard in an assay, identifications, or purity test. CRS or PSS are often characterized and evaluated for its intended purpose by additional procedures other than those used in routine testing [25].
For in-house prepared radiopharmaceuticals the confirmation of the chemical identity and purity of the precursor are the minimum quality control required, in order to qualify the material for subsequent clinical radiolabeling. Additional testing may apply if necessary for the specific process. For example, testing of trace metals content may not be necessary when the material will be subsequently radiolabeled with halogens, but is absolutely critical when the material is intended for labelling with radiometals [26].
To bring a novel radiopharmaceutical into the clinic it is needed that specific quality requirements for the radiopharmaceutical precursor are established, the range of testing would depend on their status and/or intended use. It is worth noting that for Phase I clinical trials full analytical validation is not necessary (only method suitability should be confirmed) [21]. While analytical methods used to evaluate a batch of API for clinical trials may not yet be validated, they should be scientifically sound [27].
There are some specific requirements for the large-sized molecules (e.g. proteins or monoclonal antibodies) as radiopharmaceutical precursors [28]. Monoclonal antibodies are immunoglobulins (Ig) with a defined specificity derived from a monoclonal cell line. Their biological activities are characterized by a specific binding characteristic to a target ligand (e.g. antigen) and they may be generated by recombinant DNA (rDNA) technology, hybridoma technology, B lymphocyte immortalization or other technologies. Generally, when chemical precursors are manufactured using substances of human or animal origin, the requirements of Ph. Eur. chapter 5.1.7. Viral safety [29] and the general monograph Products with risk of transmitting agents of animal spongiform encephalopathies (1483) [30] apply.
Stability testing is part of the chemical precursor’s characterization. Detailed requirements for carrying out stability studies are included in the ICH guideline Q1A (R2) [31]. The purpose of stability testing is to provide evidence on how the quality of a substance varies with time under the influence of a variety of environmental factors such as temperature, humidity, and light, and to establish a re-test period and recommended storage conditions. Stability studies should be carried out on at least three batches and include testing parameters of the chemical precursor that are susceptible to changes during storage and may affect quality, safety and efficacy (e.g. chemical purity and/or assay). The validated analytical methods should be used in these tests. For method validation, it is essential to investigate degradation products and establish degradation pathways under stress conditions (e.g. heat, humidity, light, acid/base hydrolysis and oxidation).
Peptides are an emerging class of compounds that have application in theranostics of several diseases, mainly in cancer [32, 33, 34, 35, 36]. These chemical precursors are positioned between the classic small organic molecules and the high molecular weight biomolecules. The interest of the scientific community for peptide drugs has been continuously growing. Currently, more than 60 peptide-based pharmaceuticals are marketed, over 150 peptides are in active clinical trials and estimated 500 more are in preclinical stages of development [37, 38]. Chemically, peptides have poly-amino acids structure ranging from 3 to 100 amino acids (less than 10 kDa) linked by a peptide (amide, –CONH–) bond, and are lacking a tertiary structure. From the biological point of view, peptides are important regulators of growth and cellular functions in normal tissue and tumors. They can act as cytokines, chemokines, neurotransmitters, hormones and growth factors. Generally, they offer many advantages over other groups for radiopharmaceutical applications. Peptides demonstrate high receptor specificity and selectivity, as well as binding affinity, good tissue penetration and favorable pharmacokinetic profiles. Most of them is characterized by low toxicity and immunogenicity [39, 40]. Their compact size results in rapid targeting and blood clearance. As a consequence low nonspecific uptake in non-targeted tissues and high target-to-background ratios are achieved. Moreover, peptides can be easily chemically synthesized in high purity, modified and stabilized to obtain optimized pharmacokinetic parameters. These all attributes together with ability to attach different chelating agents, prosthetic group and availability of various bioconjugation techniques make peptides an important target platform for theranostic radiopharmaceuticals [41, 42].
Peptide-based radiopharmaceuticals were introduced into the clinic more than three decades ago [43]. Since that time, several theranostic radioligand platforms are used for diagnosis and peptide receptor radionuclide therapy (PRRT) of different cancer types. In this concept, peptide analogs directed against somatostatin receptors (SSTR) play a crucial role [44]. The most prominent example of the theranostic pair of radiolabeled peptides are DOTA-conjugated SSTR agonist DOTA-(D-Phe1, Tyr3, Thr8)-octreotate (DOTA-TATE) labeled with 68Ga and 177Lu (Figure 3). The marketing authorization of NETSPOT® ([68Ga]Ga-DOTATATE) in 2016 and LUTATHERA® ([177Lu]Lu-DOTATATE) in early 2018 [45] encouraged the research in this field to develop improved radiolabeled peptides targeting other receptor/antigen families, exemplified by the prostate specific membrane antigen (PSMA) [46], gastrin-releasing peptide receptor (GRPr) [47] and cholecystokinin-2 receptor (CCK2R) [48, 49]. Some of these peptides are currently under clinical investigation.
Structure of DOTA-TATE for labelling with theranostics pair of radionuclides: Gallium-68 (68Ga) and lutetium-177 (177Lu).
Peptides as precursors for radiopharmaceutical preparations, similarly to other chemical precursors, require adequate specification as a part of their quality assurance in order to demonstrate the safety and efficacy of the final radiopharmaceutical preparation. Currently, no individual pharmacopeia monograph of peptide used as radiopharmaceutical precursors is available. Thus, the quality specification should be established according to the general requirements [4, 50]. Herein, we provide an overview of recommended methods and test limits for the characterization of peptides. The set of analytical procedures that need to be considered is presented in Table 1. However, it should be noted that new analytical methods and modifications to existing ones are continuously being developed and should be utilized where appropriate.
Parameters | Typical methods | Typical acceptance criteria |
---|---|---|
- Appearance/color | Visual inspection | White or almost white powder |
- Solubility | Visual inspection | Solubility in water, ethanol and dilute acid or alkali |
- Active moiety | RP-HPLC-UV | Retention time |
MS or | Mass spectrum | |
NMR | NMR spectrum | |
IR | IR spectrum | |
AAA (GC) | AA: theoretical content ±20% | |
- Related substances | HPLC-UV | Individual, unidentified: < 2.0% Total: ≤ 3.0% |
- Residual solvents | (Headspace) GC | Acetonitrile: ≤ 0.5% |
- Residual metals | AAS/ICP-AES/ICP-MS | Pt, Pd, Ir, Rh, Ru, Os, Mo, Ni, Cr, V, Pb, Hg, Cd, Tl: ≤ 0.01% |
- Residual reagents | HPLC-UV/IC/GC | Trifluoracetic acid: ≤ 1.0%* |
HPLC-UV/IC/GC | Acetic acid: target ±5% Trifluoracetic acid: target ±5% | |
Karl-Fisher | ≤ 10.0% | |
RP-HPLC-UV or CHN | ≥ 75.0% | |
TAMC plate count | ≤ 103 CFU/g for bulk ≤ 102 CFU per container | |
TYMC plate count | ≤ 102 CFU/g for bulk ≤ 101 CFU per container | |
Gel-clot | ≤ 100 IU/g for bulk ≤ 10 IU per container |
Summary of the recommended quality parameters for peptides used as radiopharmaceutical precursors.
The residual TFA content is determined when AcOH or HCl are used as counter-ions.
The preliminary quality evaluation of peptides is based on the visual inspection of the appearance/color and solubility. This parameter is given only for information, it is not a requirement in a strict sense. If any of the characteristics change during storage, this change should be investigated and appropriate action taken. A typical description of peptide appearance is: white to almost white, freeze-dried powder and solubility is stated in water, ethanol and dilute solutions of acids and alkali [38, 51].
According to the ICH Q6A guideline [25] identification testing should allow to discriminate between compounds of closely related structure which are likely to be present (e.g. peptides with altered sequences or functional groups that may be formed during the synthesis). The identification test should include combination of different procedures (mostly two) and should be specific and unequivocal. Several techniques are currently in use for confirmation of peptide identity: HPLC-UV, nuclear magnetic resonance spectrometry (NMR), mass spectrometry (MS), infrared absorption spectrophotometry (IR), amino acid analysis (AAA) or peptide sequencing [51]. The method of choice is typically HPLC-UV based on retention time by comparison with reference standard, since the separation by RP-HPLC is often utilized and the method is widely available. UV detection of peptides is realized at 210–220 nm and 250–290 nm for aromatic side chains of phenylalanine, tyrosine and tryptophan. Identification solely by a chromatographic retention time is not regarded as specific and should be complemented by spectrometric techniques. The NMR spectroscopy is the method that allows to unequivocally define the structure of a peptide in the terms of amino acid composition, sequence and chirality. Identification by NMR spectrometry is usually limited to peptides comprising up to 15 amino acids and requires complex data interpretation. For this reason NMR technique is primarily replaced by mass spectroscopy (MS). This technique provides highly accurate molecular weight information on intact molecules, which is an advantage of MS for peptide identification. The peptide molecular mass is most commonly determined by using the electrospray ionization method (ESI), which occurs through the addition or removal of protons and appears as singly or doubly charged ions. As alternative for the more sophisticated spectroscopic methods, amino acid analysis (AAA) could be considered. This technique involves the hydrolysis of the peptide (usually in acidic conditions) to its individual amino acid residues, followed by chromatographic separation and detection/quantification. The method also enables the determination of the enantiomeric purity with the use of appropriate reference standards. However, this method may not be applicable to peptides containing unnatural amino acids and/or specific chelators. The NMR and AAA as well as peptide sequencing techniques are generally used for characterization of PSS.
In the two recently published papers the identity of DOTA-TATE has been confirmed using suitable instrumental techniques; Sikora et al. [52] confirmed the identity of DOTA-TATE using three different methods: MS, IR and HPLC. Similarly, in the work by Raheem at al [53] the final product was analyzed using high resolution mass spectrometry for identification and analytical HPLC for purification; it was detected via analytical HPLC at a retention time of 9.52 min and detected by HRMS-ESI (calc m/z for [(DOTA-TATE +2H)/2]+: 718.3028, found: 718.3046 with −0.1144 ppm error).
In our experience ESI-MS in positive ionization mode was used to confirmed whether the masses of ions at m/z 1435.6 ± 1.0 [M + H]+ and 718.3 ± 1.0 [M + 2H]2+correspond to the monoisotopic mass of peptide [M] as presented in Figure 4. DOTA-TATE PSS was used as reference in IR analysis. Also a gradient HPLC-UV (220 nm) served as identity test of DOTA-TATE by comparison with the reference standard (Rt ± 5.0%). The same HPLC method was used for determination of peptide purity and assay. The representative HPLC chromatograms of DOTA-TATE and DOTA-TATE PSS are given in Figure 5.
ESI-MS spectrum for DOTA-TATE.
HPLC-UV (220 nm) chromatograms of (I) DOTA-TATE Rt = 19.831 min and (II) DOTA-TATE PSS Rt = 19,936 min. HPLC method: Luna C18(2) column; Mobile phase - A: water with 0.1% TFA, B: Acetonitrile with 0.1% TFA; gradient profile – From 0 to 25 min: 0–50% B; flow - 0.8 mL/min, oven temperature - 30°C.
Peptides are usually chemically synthesized using solid-phase peptide synthesis (SPPS) [54]. In this multi-stage process, amino acids are linked to each other during individual coupling steps, thus constructing the desired peptide sequence. This occurs when the carboxylic end of the sequence is covalently attached to a solid support matrix. The complexity of the peptide production process results in a greater diversity of potential impurities. Heterogenicity of the impurity profile is observed even among peptides manufactured by the same synthetic route. The impurities can originate from raw materials, the manufacturing process, degradation or may be formed during storage. Although protecting groups, scavengers or activated functional groups are used to prevent undesired side-chain reactions the peptide manufacturing process leads to formation of closely related impurities. The most common impurities are products of racemization, deamidation, amino acid deletion or insertion, acetylation, oxidation, β-elimination, cyclization, reduction and incomplete deprotection [51]. The presence of related peptide impurities is typically determined using gradient reversed-phase HPLC method with UV detection, because of its selectivity, high sensitivity, low limit of detection, quantification and robustness. The developed HPLC method should allow sufficient separation of potential impurities from manufacturing process as well as degradation products. The acceptance criteria for related substances according to the Ph. Eur. General Monograph 2902 [4] are presented in Table 2.
Specific thresholds should be applied for impurities known to be unusually potent or to produce toxic or unacceptable pharmacological effects.
The presence of inorganic impurity should also be considered, in particular when radiolabeling of the peptide with radiometals is concerned. According to the Ph. Eur. general monograph (2902), the metal residues in peptides should be determined if the manufacturing process is known or suspected to lead to its presence, e.g. due to the use of specific metal catalyst (e.g. Pd) or metal containing reagents. The content for each of the following metals: Pt, Pd, Ir, Rh, Ru, Os, Mo, Ni, Cr, V, Pb, Hg, Cd, Tl in the peptide precursors are limited to 0.01%. The metal impurities are typically examined using atomic absorption spectrometry (AAS), inductively coupled plasma with atomic emission spectrometry detection (ICP-AES) or mass spectrometry detection (ICP-MS) techniques. Determination of residual metals in peptides can be crucial for precursors intended for radiometal labeling [55]. It has been proven that the presence of certain metals can significantly affect the labeling efficiency through competitive chelation.
In addition to related substances the residual solvents are required to be examined as impurities in peptide precursors. Residual solvents in pharmaceuticals are defined as organic volatile chemicals that are used in the manufacturing process. The solvents are not completely removed by practical manufacturing techniques (e.g. lyophilization process). General guidelines established by the ICH divide solvents into three classes [56]. The Class 1 solvents should not be used in the final step of the manufacturing process of chemical precursors, because of toxicity and environmental impact. The use of the Class 2 solvents should be limited due to potential toxicity and Class 3 solvents are regarded as posing a lower risk to human health. Based on the permitted daily exposure (PDE), Class 2 and 3 solvents are limited to 0.5%. Residual solvents are typically determined using chromatographic techniques such as gas chromatography (GC) coupled with static headspace sampling. Many solvents are usually used in the peptides synthetic process. However, as the advantage of the SPPS and lyophilization process, the most frequently detected solvent is only acetonitrile (Class 2 solvent), used as the component of the mobile phase in the final purification process by preparative HPLC.
Synthetic peptides usually contain counter-ions on protonated amino functional groups (N-terminus, Arg, His, Lys, etc.). The presence of counter-ions such as acetate, chloride or trifluoroacetate results from the peptide post synthetic cleavage and/or purification process. Depending on the peptide sequence they reduce the net peptide content by 5 to 25%, but are not considered as impurity. Radiopharmaceutical preparations for diagnostic or therapeutic purposes are based on the net peptide content and thus the amount of residual counter-ions needs to be assessed. To determine counter-ion amounts different method are being used such as: GC, HPLC-UV or ion chromatography (IC). Trifluoroacetic acid (TFA) determined by IC at the level of ca. 20% in DOTA-TATE [52], corresponded to three TFA molecules associated to single peptide molecule. TFA is commonly used as a chemical reagent to remove residual protecting groups during purification of peptides and also as a mobile-phase modifier in a reversed-phase chromatography. Therefore, when the counter-ion finally is AcOH or HCl, determination of the TFA residual content is mandatory.
In order demonstrate a lot-to-lot consistency the test for water content (residual moisture remaining from the lyophilization process) should be also performed. This parameter may affect the stability of the peptide. For residual water Karl-Fischer titration method as well as GC method with thermal conductivity detector (TCD) [57] are commonly used and water content is limited to max. 10%.
Generally, assay is defined as a net peptide content. The lyophilized peptide contains also water, counter ions and residual solvents. The net peptide content is referred to percentage of peptide material in the lyophilized peptide. According to ICH guideline Q6A, a specific stability-indicating procedure should be included in the specifications to determine the content of the drug substance. There are two main approaches to determine net peptide content. The first method is a relative assay against a well-defined chemical reference substance, performed using comparative chromatographic procedures. Usually the same RP-HPLC method is used for both assay, identification and related substances. The second approach is an absolute assays involving a functional group (e.g. AAA or titration methods) or a nitrogen content analysis. The nitrogen content is determined from the results of elemental analysis CHN. The calculation of the net peptide content is based on the relation between determined %N to the theoretical content in the peptide structure. For example, this method was used to DOTA-TATE assay determination. Peptide content calculated from elemental analysis was ca. 78.0%, which was in agreement with the generally accepted limit ≥75% [52].
The presence of microorganisms may affect the stability of drug substances due to their propensity to degrade/metabolize peptides. Microbiological examinations involve the bioburden control (Ph. Eur 2.6.12) and content of bacterial endotoxins (Ph Eur. 2.6.14). The microbial enumeration tests for total aerobic microbial counts (TAMC) and total yeast and mold counts (TYMC) must adhere to the acceptance criteria of 103 CFU/g and 102 CFU/g for bulk material and 102 CFU/g and 101 CFU per container for chemical precursors packed in single and multi-dose containers, respectively. Bacterial endotoxin can be determined by the gel-clot or photometric methods (turbidimetric and chromogenic techniques) and acceptance criteria are limited to a maximum 100 IU/g for bulk material or maximum 10 IU per container for chemical precursors packed in single-dose and multidose containers.
Radionuclide precursors are offered as solutions for radiolabeling with MA, they are also locally produced for the in-house preparation of radiopharmaceuticals. There is an ongoing debate whether radionuclide precursors always have to be considered as medicinal product, or also can be provided as a starting material [58]. Unlike for chemical precursors for radiopharmaceutical preparation, up to date there is no monograph in the Ph. Eur. that sets out general requirements for radionuclide precursors. This is due to the fact that the quality requirements for radionuclides used to obtain diagnostic and therapeutic preparations are highly varying and depend on the irradiation route and chemical processing involved, which mainly affect the parameters of radionuclide purity or specific activity.
However, there are several individual Ph. Eur. monographs for radionuclide precursors. Two of these concern radionuclide precursors used to prepare radiopharmaceuticals for therapeutic use. These are:
Focusing attention on theranostic radiopharmaceuticals, herein the quality requirements only for metallic radionuclide precursors used in diagnostics and therapy are compared. Table 3 shows the exemplary quality requirements for radionuclide precursor for therapeutic use (177Lu) and a matching radionuclide precursor for diagnostic use (68Ga).
Reporting threshold | 0.2 per cent |
Identification threshold | 2.0 per cent |
Total unspecified impurities | Maximum 3.0 per cent |
Acceptance criteria for related substances [4].
Lutetium (177Lu) solution for radiolabelling (Ph. Eur. 2798 [59]) | Gallium (68Ga) chloride solution for radiolabelling (Ph. Eur. 2464 [60]) |
---|---|
RADIONUCLIDIC PURITY Gamma-ray spectrometry. - the total radioactivity due to ytterbium-175 (impurity B) is not more than 0.1 per cent; – the total radioactivity due to lutetium-177 m (impurity A) is not more than 0.07 per cent; – the total radioactivity due to radionuclidic impurities other than A and B is not more than 0.01 per cent. | RADIONUCLIDIC PURITY A. Gamma-ray spectrometry. B. Germanium-68 and gamma-ray-emitting impurities. Gamma-ray spectrometry. |
RADIOCHEMICAL PURITY | RADIOCHEMICAL PURITY |
Comparison of Ph. Eur. requirements for selected radionuclide precursors.
Comparing the requirements of these two monographs there are apparently large differences in numerical values seen, especially for metal ion content and radiochemical purity. However, when the radioactivity of these radionuclides (different for therapeutic or diagnostic use) is considered, there are basically no differences in quality requirements for both radionuclides. This can be demonstrated on the example of the DOTA-TATE preparations with 177Lu and 68Ga. For therapy 7.4 GBq of [177Lu]Lu-DOTA-TATE is used and this preparation contains ca. 0.2 mg of DOTA-TATE. Typical dose of [68Ga]Ga-DOTA-TATE is 200 MBq and the ligand content in the preparation should not exceed 0.05 mg. Therefore, when analyzing the limit of metallic impurities, e.g. Zn in the radionuclide precursor, similar values are obtained in both cases, i.e. maximum 37 ng and 40 ng per microgram of DOTA-TATE for lutetium-177 and gallium-68, respectively.
When the radiochemical purity is compared, the higher limit of permissible other forms of diagnostic radionuclide ([68Ga]gallium(III) ion: minimum 95%) than for the therapeutic radionuclide ([177Lu]Lutetium(III) ion: minimum 99%) does not result in a higher risk to the patient. Thus, 5% of other forms of a trivalent gallium-68 ion may result in the deposit of 10 MBq of this radionuclide in undesirable chemical form in non-target organs, while for 1% lutetium-177 it is as much as 74 MBq of uncontrolled chemical form. However, it must be noted that a stricter limit for the latter radionuclide is difficult to achieve due to the limitations of the analytical methods, which are characterized by an approximate 1% uncertainty of determination.
Bearing in mind that the differences in the profile of radionuclide contamination depend on the radionuclide production process [67], it is unlikely that uniform quality requirements for radionuclide precursors will be set in numerical terms. Each radionuclide precursor should be evaluated on a case-by-case basis, taking into account the physical characteristics of the radionuclide, its mode of irradiation and chemical processing as well as the envisaged clinical use and the dose planned for administration to the patient. This is clearly reflected in monographs referred in this Chapter. The monograph for 177Lu [59] applies to both the direct and indirect production routes of 177Lu in nuclear reactors and covers all quality aspects regardless the different specific radioactivity and impurity profiles. The decision is left to the producer of the final radiopharmaceutical preparation to use the appropriate solution for radiolabeling. However, the relevant information needs to be stated on the label. This is different in case of 68Ga, there are two different monographs specifying its quality requirements depending whether it’s generator [65] or accelerator produced [66]. One can expect that a similar individual approach applies to the future monographs for new theranostic radionuclides, for example 47Sc, which can be either accelerator or reactor produced [68].
Are the requirements for radiopharmaceutical precursors overregulated? With the development of new theranostic procedures involving radiopharmaceuticals, there is a need for proper qualitative evaluation of the final radiopharmaceutical preparation and both of the radiopharmaceutical precursors to ensure efficacy and safety of the treatment. An excellent example of the long pathway of a radiopharmaceutical, 111In-CP04, a peptide targeting the cholecystokinin-2 receptor, from the preclinical development over establishing the required pharmaceutical documentation to designing and submitting a clinical trial in patients with Medullary Thyroid Carcinoma, was recently presented [16]. All the quality aspects of CP04 as chemical precursor have been addressed in the IMPD in view of the quality and suitability of the radiolabeled preparation, 111In-CP04, in order to bring it to the clinic.
In this Chapter, the quality requirements applicable to radiopharmaceutical precursors in the context of their regulatory status in Europe were reviewed. EMA and Ph. Eur. provide public standards for manufacture and quality control of these precursors by establishing specifications and acceptance criteria. While in the case of radiopharmaceuticals with MA and CT regulations quite strictly define the quality and documentation requirements, such standards for in-house produced radiopharmaceuticals are still awaited.
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He is an academic staff member of the Department of Reproduction and Artificial Insemination, Selçuk University, Turkey. He manages several studies on sperms and embryos and is an editorial board member for several international journals. His studies include sperm cryobiology, in vitro fertilization, and embryo production in animals.",institutionString:"Selçuk University, Faculty of Veterinary Medicine",institution:null},{id:"90846",title:"Prof.",name:"Yusuf",middleName:null,surname:"Bozkurt",slug:"yusuf-bozkurt",fullName:"Yusuf Bozkurt",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/90846/images/system/90846.jpg",biography:"Yusuf Bozkurt has a BSc, MSc, and Ph.D. from Ankara University, Turkey. He is currently a Professor of Biotechnology of Reproduction in the field of Aquaculture, İskenderun Technical University, Turkey. His research interests include reproductive biology and biotechnology with an emphasis on cryo-conservation. He is on the editorial board of several international peer-reviewed journals and has published many papers. Additionally, he has participated in many international and national congresses, seminars, and workshops with oral and poster presentations. He is an active member of many local and international organizations.",institutionString:"İskenderun Technical University",institution:{name:"İskenderun Technical University",country:{name:"Turkey"}}},{id:"61139",title:"Dr.",name:"Sergey",middleName:null,surname:"Tkachev",slug:"sergey-tkachev",fullName:"Sergey Tkachev",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/61139/images/system/61139.png",biography:"Dr. Sergey Tkachev is a senior research scientist at the Institute of Fundamental Medicine and Biology, Kazan Federal University, Russia, and at the Institute of Chemical Biology and Fundamental Medicine SB RAS, Novosibirsk, Russia. He received his Ph.D. in Molecular Biology with his thesis “Genetic variability of the tick-borne encephalitis virus in natural foci of Novosibirsk city and its suburbs.” His primary field is molecular virology with research emphasis on vector-borne viruses, especially tick-borne encephalitis virus, Kemerovo virus and Omsk hemorrhagic fever virus, rabies virus, molecular genetics, biology, and epidemiology of virus pathogens.",institutionString:"Russian Academy of Sciences",institution:{name:"Russian Academy of Sciences",country:{name:"Russia"}}},{id:"310962",title:"Dr.",name:"Amlan",middleName:"Kumar",surname:"Patra",slug:"amlan-patra",fullName:"Amlan Patra",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/310962/images/system/310962.jpg",biography:"Amlan K. Patra, FRSB, obtained a Ph.D. in Animal Nutrition from Indian Veterinary Research Institute, India, in 2002. He is currently an associate professor at West Bengal University of Animal and Fishery Sciences. He has more than twenty years of research and teaching experience. He held previous positions at the American Institute for Goat Research, The Ohio State University, Columbus, USA, and Free University of Berlin, Germany. His research focuses on animal nutrition, particularly ruminants and poultry nutrition, gastrointestinal electrophysiology, meta-analysis and modeling in nutrition, and livestock–environment interaction. He has authored around 175 articles in journals, book chapters, and proceedings. Dr. Patra serves on the editorial boards of several reputed journals.",institutionString:null,institution:{name:"West Bengal University of Animal and Fishery Sciences",country:{name:"India"}}},{id:"53998",title:"Prof.",name:"László",middleName:null,surname:"Babinszky",slug:"laszlo-babinszky",fullName:"László Babinszky",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/53998/images/system/53998.png",biography:"László Babinszky is Professor Emeritus, Department of Animal Nutrition Physiology, University of Debrecen, Hungary. He has also worked in the Department of Animal Nutrition, University of Wageningen, Netherlands; the Institute for Livestock Feeding and Nutrition (IVVO), Lelystad, Netherlands; the Agricultural University of Vienna (BOKU); the Institute for Animal Breeding and Nutrition, Austria; and the Oscar Kellner Research Institute for Animal Nutrition, Rostock, Germany. In 1992, Dr. Babinszky obtained a Ph.D. in Animal Nutrition from the University of Wageningen. His main research areas are swine and poultry nutrition. He has authored more than 300 publications (papers, book chapters) and edited four books and fourteen international conference proceedings.",institutionString:"University of Debrecen",institution:{name:"University of Debrecen",country:{name:"Hungary"}}},{id:"201830",title:"Dr.",name:"Fernando",middleName:"Sanchez",surname:"Davila",slug:"fernando-davila",fullName:"Fernando Davila",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/201830/images/5017_n.jpg",biography:"I am a professor at UANL since 1988. My research lines are the development of reproductive techniques in small ruminants. We also conducted research on sexual and social behavior in males.\nI am Mexican and study my professional career as an engineer in agriculture and animal science at UANL. Then take a masters degree in science in Germany (Animal breeding). Take a doctorate in animal science at the UANL.",institutionString:null,institution:{name:"Universidad Autónoma de Nuevo León",country:{name:"Mexico"}}},{id:"309250",title:"Dr.",name:"Miguel",middleName:null,surname:"Quaresma",slug:"miguel-quaresma",fullName:"Miguel Quaresma",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/309250/images/9059_n.jpg",biography:"Miguel Nuno Pinheiro Quaresma was born on May 26, 1974 in Dili, Timor Island. He is married with two children: a boy and a girl, and he is a resident in Vila Real, Portugal. He graduated in Veterinary Medicine in August 1998 and obtained his Ph.D. degree in Veterinary Sciences -Clinical Area in February 2015, both from the University of Trás-os-Montes e Alto Douro. He is currently enrolled in the Alternative Residency of the European College of Animal Reproduction. He works as a Senior Clinician at the Veterinary Teaching Hospital of UTAD (HVUTAD) with a role in clinical activity in the area of livestock and equine species as well as to support teaching and research in related areas. He teaches as an Invited Professor in Reproduction Medicine I and II of the Master\\'s in Veterinary Medicine degree at UTAD. Currently, he holds the position of Chairman of the Portuguese Buiatrics Association. He is a member of the Consultive Group on Production Animals of the OMV. He has 19 publications in indexed international journals (ISIS), as well as over 60 publications and oral presentations in both Portuguese and international journals and congresses.",institutionString:"University of Trás-os-Montes and Alto Douro",institution:{name:"University of Trás-os-Montes and Alto Douro",country:{name:"Portugal"}}},{id:"38652",title:"Prof.",name:"Rita",middleName:null,surname:"Payan-Carreira",slug:"rita-payan-carreira",fullName:"Rita Payan-Carreira",position:null,profilePictureURL:"https://s3.us-east-1.amazonaws.com/intech-files/0030O00002bRiFPQA0/Profile_Picture_1614601496313",biography:"Rita Payan Carreira earned her Veterinary Degree from the Faculty of Veterinary Medicine in Lisbon, Portugal, in 1985. She obtained her Ph.D. in Veterinary Sciences from the University of Trás-os-Montes e Alto Douro, Portugal. After almost 32 years of teaching at the University of Trás-os-Montes and Alto Douro, she recently moved to the University of Évora, Department of Veterinary Medicine, where she teaches in the field of Animal Reproduction and Clinics. Her primary research areas include the molecular markers of the endometrial cycle and the embryo–maternal interaction, including oxidative stress and the reproductive physiology and disorders of sexual development, besides the molecular determinants of male and female fertility. She often supervises students preparing their master's or doctoral theses. She is also a frequent referee for various journals.",institutionString:null,institution:{name:"University of Évora",country:{name:"Portugal"}}},{id:"283019",title:"Dr.",name:"Oudessa",middleName:null,surname:"Kerro Dego",slug:"oudessa-kerro-dego",fullName:"Oudessa Kerro Dego",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/283019/images/system/283019.png",biography:"Dr. Kerro Dego is a veterinary microbiologist with training in veterinary medicine, microbiology, and anatomic pathology. Dr. Kerro Dego is an assistant professor of dairy health in the department of animal science, the University of Tennessee, Institute of Agriculture, Knoxville, Tennessee. He received his D.V.M. (1997), M.S. (2002), and Ph.D. (2008) degrees in Veterinary Medicine, Animal Pathology and Veterinary Microbiology from College of Veterinary Medicine, Addis Ababa University, Ethiopia; College of Veterinary Medicine, Utrecht University, the Netherlands and Western College of Veterinary Medicine, University of Saskatchewan, Canada respectively. He did his Postdoctoral training in microbial pathogenesis (2009 - 2015) in the Department of Animal Science, the University of Tennessee, Institute of Agriculture, Knoxville, Tennessee. Dr. Kerro Dego’s research focuses on the prevention and control of infectious diseases of farm animals, particularly mastitis, improving dairy food safety, and mitigation of antimicrobial resistance. Dr. Kerro Dego has extensive experience in studying the pathogenesis of bacterial infections, identification of virulence factors, and vaccine development and efficacy testing against major bacterial mastitis pathogens. Dr. Kerro Dego conducted numerous controlled experimental and field vaccine efficacy studies, vaccination, and evaluation of immunological responses in several species of animals, including rodents (mice) and large animals (bovine and ovine).",institutionString:"University of Tennessee at Knoxville",institution:{name:"University of Tennessee at Knoxville",country:{name:"United States of America"}}},{id:"251314",title:"Dr.",name:"Juan Carlos",middleName:null,surname:"Gardón Poggi",slug:"juan-carlos-gardon-poggi",fullName:"Juan Carlos Gardón Poggi",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/251314/images/system/251314.jpeg",biography:"Juan Carlos Gardón Poggi received University degree from the Faculty of Agrarian Science in Argentina, in 1983. Also he received Masters Degree and PhD from Córdoba University, Spain. He is currently a Professor at the Catholic University of Valencia San Vicente Mártir, at the Department of Medicine and Animal Surgery. He teaches diverse courses in the field of Animal Reproduction and he is the Director of the Veterinary Farm. He also participates in academic postgraduate activities at the Veterinary Faculty of Murcia University, Spain. His research areas include animal physiology, physiology and biotechnology of reproduction either in males or females, the study of gametes under in vitro conditions and the use of ultrasound as a complement to physiological studies and development of applied biotechnologies. Routinely, he supervises students preparing their doctoral, master thesis or final degree projects.",institutionString:null,institution:{name:"Valencia Catholic University Saint Vincent Martyr",country:{name:"Spain"}}},{id:"309529",title:"Dr.",name:"Albert",middleName:null,surname:"Rizvanov",slug:"albert-rizvanov",fullName:"Albert Rizvanov",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/309529/images/9189_n.jpg",biography:'Albert A. Rizvanov is a Professor and Director of the Center for Precision and Regenerative Medicine at the Institute of Fundamental Medicine and Biology, Kazan Federal University (KFU), Russia. He is the Head of the Center of Excellence “Regenerative Medicine” and Vice-Director of Strategic Academic Unit \\"Translational 7P Medicine\\". Albert completed his Ph.D. at the University of Nevada, Reno, USA and Dr.Sci. at KFU. He is a corresponding member of the Tatarstan Academy of Sciences, Russian Federation. Albert is an author of more than 300 peer-reviewed journal articles and 22 patents. He has supervised 11 Ph.D. and 2 Dr.Sci. dissertations. Albert is the Head of the Dissertation Committee on Biochemistry, Microbiology, and Genetics at KFU.\nORCID https://orcid.org/0000-0002-9427-5739\nWebsite https://kpfu.ru/Albert.Rizvanov?p_lang=2',institutionString:"Kazan Federal University",institution:{name:"Kazan Federal University",country:{name:"Russia"}}},{id:"210551",title:"Dr.",name:"Arbab",middleName:null,surname:"Sikandar",slug:"arbab-sikandar",fullName:"Arbab Sikandar",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/210551/images/system/210551.jpg",biography:"Dr. Arbab Sikandar, PhD, M. Phil, DVM was born on April 05, 1981. He is currently working at the College of Veterinary & Animal Sciences as an Assistant Professor. He previously worked as a lecturer at the same University. \nHe is a Member/Secretory of Ethics committee (No. CVAS-9377 dated 18-04-18), Member of the QEC committee CVAS, Jhang (Regr/Gen/69/873, dated 26-10-2017), Member, Board of studies of Department of Basic Sciences (No. CVAS. 2851 Dated. 12-04-13, and No. CVAS, 9024 dated 20/11/17), Member of Academic Committee, CVAS, Jhang (No. CVAS/2004, Dated, 25-08-12), Member of the technical committee (No. CVAS/ 4085, dated 20,03, 2010 till 2016).\n\nDr. Arbab Sikandar contributed in five days hands-on-training on Histopathology at the Department of Pathology, UVAS from 12-16 June 2017. He received a Certificate of appreciation for contributions for Popularization of Science and Technology in the Society on 17-11-15. He was the resource person in the lecture series- ‘scientific writing’ at the Department of Anatomy and Histology, UVAS, Lahore on 29th October 2015. He won a full fellowship as a principal candidate for the year 2015 in the field of Agriculture, EICA, Egypt with ref. to the Notification No. 12(11) ACS/Egypt/2014 from 10 July 2015 to 25th September 2015.; he received a grant of Rs. 55000/- as research incentives from Director, Advanced Studies and Research, UVAS, Lahore upon publications of research papers in IF Journals (DR/215, dated 19-5-2014.. He obtained his PhD by winning a HEC Pakistan indigenous Scholarship, ‘Ph.D. fellowship for 5000 scholars – Phase II’ (2av1-147), 17-6/HEC/HRD/IS-II/12, November 15, 2012. \n\nDr. Sikandar is a member of numerous societies: Registered Veterinary Medical Practitioner (life member) and Registered Veterinary Medical Faculty of Pakistan Veterinary Medical Council. The Registration code of PVMC is RVMP/4298 and RVMF/ 0102.; Life member of the University of Veterinary and Animal Sciences, Lahore, Alumni Association with S# 664, dated: 6-4-12. ; Member 'Vets Care Organization Pakistan” with Reference No. VCO-605-149, dated 05-04-06. :Member 'Vet Crescent” (Society of Animal Health and Production), UVAS, Lahore.",institutionString:"University of Veterinary & Animal Science",institution:{name:"University of Veterinary and Animal Sciences",country:{name:"Pakistan"}}},{id:"311663",title:"Dr.",name:"Prasanna",middleName:null,surname:"Pal",slug:"prasanna-pal",fullName:"Prasanna Pal",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/311663/images/13261_n.jpg",biography:null,institutionString:null,institution:{name:"National Dairy Research Institute",country:{name:"India"}}},{id:"202192",title:"Dr.",name:"Catrin",middleName:null,surname:"Rutland",slug:"catrin-rutland",fullName:"Catrin Rutland",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/202192/images/system/202192.png",biography:"Catrin Rutland is an Associate Professor of Anatomy and Developmental Genetics at the University of Nottingham, UK. She obtained a BSc from the University of Derby, England, a master’s degree from Technische Universität München, Germany, and a Ph.D. from the University of Nottingham. She undertook a post-doctoral research fellowship in the School of Medicine before accepting tenure in Veterinary Medicine and Science. Dr. Rutland also obtained an MMedSci (Medical Education) and a Postgraduate Certificate in Higher Education (PGCHE). She is the author of more than sixty peer-reviewed journal articles, twelve books/book chapters, and more than 100 research abstracts in cardiovascular biology and oncology. She is a board member of the European Association of Veterinary Anatomists, Fellow of the Anatomical Society, and Senior Fellow of the Higher Education Academy. Dr. Rutland has also written popular science books for the public. https://orcid.org/0000-0002-2009-4898. www.nottingham.ac.uk/vet/people/catrin.rutland",institutionString:null,institution:{name:"University of Nottingham",country:{name:"United Kingdom"}}},{id:"283315",title:"Prof.",name:"Samir",middleName:null,surname:"El-Gendy",slug:"samir-el-gendy",fullName:"Samir El-Gendy",position:null,profilePictureURL:"https://s3.us-east-1.amazonaws.com/intech-files/0030O00002bRduYQAS/Profile_Picture_1606215849748",biography:"Samir El-Gendy is a Professor of anatomy and embryology at the faculty of veterinary medicine, Alexandria University, Egypt. Samir obtained his PhD in veterinary science in 2007 from the faculty of veterinary medicine, Alexandria University and has been a professor since 2017. Samir is an author on 24 articles at Scopus and 12 articles within local journals and 2 books/book chapters. His research focuses on applied anatomy, imaging techniques and computed tomography. Samir worked as a member of different local projects on E-learning and he is a board member of the African Association of Veterinary Anatomists and of anatomy societies and as an associated author at local and international journals. Orcid: https://orcid.org/0000-0002-6180-389X",institutionString:null,institution:{name:"Alexandria University",country:{name:"Egypt"}}},{id:"246149",title:"Dr.",name:"Valentina",middleName:null,surname:"Kubale",slug:"valentina-kubale",fullName:"Valentina Kubale",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/246149/images/system/246149.jpg",biography:"Valentina Kubale is Associate Professor of Veterinary Medicine at the Veterinary Faculty, University of Ljubljana, Slovenia. Since graduating from the Veterinary faculty she obtained her PhD in 2007, performed collaboration with the Department of Pharmacology, University of Copenhagen, Denmark. She continued as a post-doctoral fellow at the University of Copenhagen with a Lundbeck foundation fellowship. She is the editor of three books and author/coauthor of 23 articles in peer-reviewed scientific journals, 16 book chapters, and 68 communications at scientific congresses. Since 2008 she has been the Editor Assistant for the Slovenian Veterinary Research journal. She is a member of Slovenian Biochemical Society, The Endocrine Society, European Association of Veterinary Anatomists and Society for Laboratory Animals, where she is board member.",institutionString:"University of Ljubljana",institution:{name:"University of Ljubljana",country:{name:"Slovenia"}}},{id:"258334",title:"Dr.",name:"Carlos Eduardo",middleName:null,surname:"Fonseca-Alves",slug:"carlos-eduardo-fonseca-alves",fullName:"Carlos Eduardo Fonseca-Alves",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/258334/images/system/258334.jpg",biography:"Dr. Fonseca-Alves earned his DVM from Federal University of Goias – UFG in 2008. He completed an internship in small animal internal medicine at UPIS university in 2011, earned his MSc in 2013 and PhD in 2015 both in Veterinary Medicine at Sao Paulo State University – UNESP. Dr. Fonseca-Alves currently serves as an Assistant Professor at Paulista University – UNIP teaching small animal internal medicine.",institutionString:null,institution:{name:"Universidade Paulista",country:{name:"Brazil"}}},{id:"245306",title:"Dr.",name:"María Luz",middleName:null,surname:"Garcia Pardo",slug:"maria-luz-garcia-pardo",fullName:"María Luz Garcia Pardo",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/245306/images/system/245306.png",biography:"María de la Luz García Pardo is an agricultural engineer from Universitat Politècnica de València, Spain. She has a Ph.D. in Animal Genetics. Currently, she is a lecturer at the Agrofood Technology Department of Miguel Hernández University, Spain. Her research is focused on genetics and reproduction in rabbits. The major goal of her research is the genetics of litter size through novel methods such as selection by the environmental sensibility of litter size, with forays into the field of animal welfare by analysing the impact on the susceptibility to diseases and stress of the does. Details of her publications can be found at https://orcid.org/0000-0001-9504-8290.",institutionString:null,institution:{name:"Miguel Hernandez University",country:{name:"Spain"}}},{id:"350704",title:"M.Sc.",name:"Camila",middleName:"Silva Costa",surname:"Ferreira",slug:"camila-ferreira",fullName:"Camila Ferreira",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/350704/images/17280_n.jpg",biography:"Graduated in Veterinary Medicine at the Fluminense Federal University, specialist in Equine Reproduction at the Brazilian Veterinary Institute (IBVET) and Master in Clinical Veterinary Medicine and Animal Reproduction at the Fluminense Federal University. She has experience in analyzing zootechnical indices in dairy cattle and organizing events related to Veterinary Medicine through extension grants. I have experience in the field of diagnostic imaging and animal reproduction in veterinary medicine through monitoring and scientific initiation scholarships. I worked at the Equus Central Reproduction Equine located in Santo Antônio de Jesus – BA in the 2016/2017 breeding season. I am currently a doctoral student with a scholarship from CAPES of the Postgraduate Program in Veterinary Medicine (Pathology and Clinical Sciences) at the Federal Rural University of Rio de Janeiro (UFRRJ) with a research project with an emphasis on equine endometritis.",institutionString:null,institution:null},{id:"41319",title:"Prof.",name:"Lung-Kwang",middleName:null,surname:"Pan",slug:"lung-kwang-pan",fullName:"Lung-Kwang Pan",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/41319/images/84_n.jpg",biography:null,institutionString:null,institution:null},{id:"125292",title:"Dr.",name:"Katy",middleName:null,surname:"Satué Ambrojo",slug:"katy-satue-ambrojo",fullName:"Katy Satué Ambrojo",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/125292/images/system/125292.jpeg",biography:"Katy Satué Ambrojo received her Veterinary Medicine degree, Master degree in Equine Technology and doctorate in Veterinary Medicine from the Faculty of Veterinary, CEU-Cardenal Herrera University in Valencia, Spain.Dr. Satué is accredited as a Private University Doctor Professor, Doctor Assistant, and Contracted Doctor by AVAP (Agència Valenciana d'Avaluació i Prospectiva) and currently, as a full professor by ANECA (since January 2022). To date, Katy has taught 22 years in the Department of Animal Medicine and Surgery at the CEU-Cardenal Herrera University in undergraduate courses in Veterinary Medicine (General Pathology, integrated into the Applied Basis of Veterinary Medicine module of the 2nd year, Clinical Equine I of 3rd year, and Equine Clinic II of 4th year). Dr. Satué research activity is in the field of Endocrinology, Hematology, Biochemistry, and Immunology in the Spanish Purebred mare. She has directed 5 Doctoral Theses and 5 Diplomas of Advanced Studies, and participated in 11 research projects as a collaborating researcher. She has written 2 books and 14 book chapters in international publishers related to the area, and 68 scientific publications in international journals. Dr. Satué has attended 63 congresses, participating with 132 communications in international congresses and 19 in national congresses related to the area. Dr. Satué is a scientific reviewer for various prestigious international journals such as Animals, American Journal of Obstetrics and Gynecology, Veterinary Clinical Pathology, Journal of Equine Veterinary Science, Reproduction in Domestic Animals, Research Veterinary Science, Brazilian Journal of Medical and Biological Research, Livestock Production Science and Theriogenology, among others. Since 2014 she has been responsible for the Clinical Analysis Laboratory of the CEU-Cardenal Herrera University Veterinary Clinical Hospital.",institutionString:null,institution:null},{id:"201721",title:"Dr.",name:"Beatrice",middleName:null,surname:"Funiciello",slug:"beatrice-funiciello",fullName:"Beatrice Funiciello",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/201721/images/11089_n.jpg",biography:"Graduated from the University of Milan in 2011, my post-graduate education included CertAVP modules mainly on equines (dermatology and internal medicine) and a few on small animal (dermatology and anaesthesia) at the University of Liverpool. After a general CertAVP (2015) I gained the designated Certificate in Veterinary Dermatology (2017) after taking the synoptic examination and then applied for the RCVS ADvanced Practitioner status. After that, I completed the Postgraduate Diploma in Veterinary Professional Studies at the University of Liverpool (2018). My main area of work is cross-species veterinary dermatology.",institutionString:null,institution:null},{id:"291226",title:"Dr.",name:"Monica",middleName:null,surname:"Cassel",slug:"monica-cassel",fullName:"Monica Cassel",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/291226/images/8232_n.jpg",biography:'Degree in Biological Sciences at the Federal University of Mato Grosso with scholarship for Scientific Initiation by FAPEMAT (2008/1) and CNPq (2008/2-2009/2): Project \\"Histological evidence of reproductive activity in lizards of the Manso region, Chapada dos Guimarães, Mato Grosso, Brazil\\". Master\\\'s degree in Ecology and Biodiversity Conservation at Federal University of Mato Grosso with a scholarship by CAPES/REUNI program: Project \\"Reproductive biology of Melanorivulus punctatus\\". PhD\\\'s degree in Science (Cell and Tissue Biology Area) \n at University of Sao Paulo with scholarship granted by FAPESP; Project \\"Development of morphofunctional changes in ovary of Astyanax altiparanae Garutti & Britski, 2000 (Teleostei, Characidae)\\". She has experience in Reproduction of vertebrates and Morphology, with emphasis in Cellular Biology and Histology. She is currently a teacher in the medium / technical level courses at IFMT-Alta Floresta, as well as in the Bachelor\\\'s degree in Animal Science and in the Bachelor\\\'s degree in Business.',institutionString:null,institution:null},{id:"442807",title:"Dr.",name:"Busani",middleName:null,surname:"Moyo",slug:"busani-moyo",fullName:"Busani Moyo",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",biography:null,institutionString:null,institution:{name:"Gwanda State University",country:{name:"Zimbabwe"}}},{id:"439435",title:"Dr.",name:"Feda S.",middleName:null,surname:"Aljaser",slug:"feda-s.-aljaser",fullName:"Feda S. Aljaser",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",biography:null,institutionString:null,institution:{name:"King Saud University",country:{name:"Saudi Arabia"}}},{id:"423023",title:"Dr.",name:"Yosra",middleName:null,surname:"Soltan",slug:"yosra-soltan",fullName:"Yosra Soltan",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",biography:null,institutionString:null,institution:{name:"Alexandria University",country:{name:"Egypt"}}},{id:"349788",title:"Dr.",name:"Florencia Nery",middleName:null,surname:"Sompie",slug:"florencia-nery-sompie",fullName:"Florencia Nery Sompie",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",biography:null,institutionString:null,institution:{name:"Sam Ratulangi University",country:{name:"Indonesia"}}},{id:"428600",title:"MSc.",name:"Adriana",middleName:null,surname:"García-Alarcón",slug:"adriana-garcia-alarcon",fullName:"Adriana García-Alarcón",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",biography:null,institutionString:null,institution:{name:"National Autonomous University of Mexico",country:{name:"Mexico"}}},{id:"428599",title:"MSc.",name:"Gabino",middleName:null,surname:"De La Rosa-Cruz",slug:"gabino-de-la-rosa-cruz",fullName:"Gabino De La Rosa-Cruz",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",biography:null,institutionString:null,institution:{name:"National Autonomous University of Mexico",country:{name:"Mexico"}}},{id:"428601",title:"MSc.",name:"Juan Carlos",middleName:null,surname:"Campuzano-Caballero",slug:"juan-carlos-campuzano-caballero",fullName:"Juan Carlos Campuzano-Caballero",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",biography:null,institutionString:null,institution:{name:"National Autonomous University of Mexico",country:{name:"Mexico"}}}]}},subseries:{item:{id:"3",type:"subseries",title:"Bacterial Infectious Diseases",keywords:"Antibiotics, Biofilm, Antibiotic Resistance, Host-microbiota Relationship, Treatment, Diagnostic Tools",scope:"