**2.2.2.5 Fumagillin**

This is the active ingredient of the preparation Fumidil used by beekeepers to treat nosemosis. It could cause teratogenesis and have genotoxic effects (Stanimirovic et al., 2007). Nowadays, it is not permitted to use fumagillin in Europe and no MRLs have been established, neither for honey nor for any other products of animal origin.

#### **2.2.2.6 Monitoring of antibiotics in bee products**

Many other antibiotics have been used worldwide. One of these is tylosine, which got an approval for use in the U.S.A. in the form of preparation Tylan. Moreover, beta-lactams are suggested to be the ideal antibiotic group in terms of efficiency and lack of residues to the final product.

Fifty chestnut, pine, linden and multifloral honey samples from Southern Marmara region of Turkey were analysed for erythromycin residues by Liquid Chromatography-Mass Spectrometry. Four of the honey samples were contaminated with erythromycin residues at concentrations ranging from 50 to 1776 μg kg-1 (Gunes et al., 2008).

A percentage of 1.7% out of 3855 honey samples of European market, which was analyzed for antibiotic residues, were non compliant with the EU standards. Antibiotics were detected in the honey samples in a range of 3–10.820 μg kg-1, 5–4.592 μg kg-1, 5–2.076 μg kg-1, 0.1–169 μg kg-1, 0.3-24.7 μg kg-1, 2–18 μg kg-1, 1-504 μg kg-1 for streptomycin, sulfonamides, tetracyclines, chloramphenicol, nitrofurans, tylosine and quinolones respectively (Diserens, 2007).

In the period 2000-2001, samples of honey of Belgian market were monitored for the presence of residues of antibiotics. Streptomycin was detected in 4 out of 248 (1.6%) samples that, tetracycline in 2 (2.8%) and sulfonamides in 3 (4.2%) out of 72 samples analyzed. No residues of β-lactams and chloramphenicol were detected. In imported honey samples, streptomycin was detected in 51 out of 108 samples (47.2%), tetracyclines in 29 out of 98 samples (29.6%), sulfonamides in 31 out of 98 samples (31.6%) and chloramphenicol in 40 out of 85 samples (47.1%). Residues of β-lactams were not detected in any sample (Reybroeck, 2003).

A total of 57 samples of royal jelly were collected from beekeepers and the Chinese market. The royal jelly was analyzed for seven fluoroquinolones used in beekeeping (ciprofloxacin, norfloxacin, ofloxacin, pefloxacin, danofloxacin, enrofloxacin, and difloxacin). Ofloxacin, ciprofloxacin and norfloxacin residues were detected in concentrations ranging from 0.012 to 0.056 mg kg-1. Difloxacin was found at a concentration of 0.047 mg kg-1 in one sample (Zhou et al., 2009).

In 2002, sulfa drugs were detected in 3 out of 91 samples of honey collected from the Belgian market. Moreover, 12 out of 203 honey samples collected in 2003 were contaminated by

The problem with streptomycin is that it may cause ototoxicity and nephrotoxicity. It is considered more dangerous than oxytetracycline and less hazardous than sulfathiazole and chloramphenicol regarding side effects. According to the Food Standards Agency of UK, an Indian honey was found to be contaminated by streptomycin in 2003 (Mayande,

This is the active ingredient of the preparation Fumidil used by beekeepers to treat nosemosis. It could cause teratogenesis and have genotoxic effects (Stanimirovic et al., 2007). Nowadays, it is not permitted to use fumagillin in Europe and no MRLs have been

Many other antibiotics have been used worldwide. One of these is tylosine, which got an approval for use in the U.S.A. in the form of preparation Tylan. Moreover, beta-lactams are suggested to be the ideal antibiotic group in terms of efficiency and lack of residues to the

Fifty chestnut, pine, linden and multifloral honey samples from Southern Marmara region of Turkey were analysed for erythromycin residues by Liquid Chromatography-Mass Spectrometry. Four of the honey samples were contaminated with erythromycin residues at

A percentage of 1.7% out of 3855 honey samples of European market, which was analyzed for antibiotic residues, were non compliant with the EU standards. Antibiotics were detected in the honey samples in a range of 3–10.820 μg kg-1, 5–4.592 μg kg-1, 5–2.076 μg kg-1, 0.1–169 μg kg-1, 0.3-24.7 μg kg-1, 2–18 μg kg-1, 1-504 μg kg-1 for streptomycin, sulfonamides, tetracyclines, chloramphenicol, nitrofurans, tylosine and quinolones respectively (Diserens,

In the period 2000-2001, samples of honey of Belgian market were monitored for the presence of residues of antibiotics. Streptomycin was detected in 4 out of 248 (1.6%) samples that, tetracycline in 2 (2.8%) and sulfonamides in 3 (4.2%) out of 72 samples analyzed. No residues of β-lactams and chloramphenicol were detected. In imported honey samples, streptomycin was detected in 51 out of 108 samples (47.2%), tetracyclines in 29 out of 98 samples (29.6%), sulfonamides in 31 out of 98 samples (31.6%) and chloramphenicol in 40 out of 85 samples (47.1%). Residues of β-lactams were not detected in any sample

A total of 57 samples of royal jelly were collected from beekeepers and the Chinese market. The royal jelly was analyzed for seven fluoroquinolones used in beekeeping (ciprofloxacin, norfloxacin, ofloxacin, pefloxacin, danofloxacin, enrofloxacin, and difloxacin). Ofloxacin, ciprofloxacin and norfloxacin residues were detected in concentrations ranging from 0.012 to 0.056 mg kg-1. Difloxacin was found at a concentration of 0.047 mg kg-1 in one sample

established, neither for honey nor for any other products of animal origin.

concentrations ranging from 50 to 1776 μg kg-1 (Gunes et al., 2008).

residues of sulfonamides (Reybroeck et al., 2004).

**2.2.2.6 Monitoring of antibiotics in bee products** 

**2.2.2.4 Streptomycin** 

**2.2.2.5 Fumagillin** 

final product.

2007).

(Reybroeck, 2003).

(Zhou et al., 2009).

2007).

#### **2.2.3 Residues of volatile insecticides in bee products**

The greater wax moth *Galleria mellonella* is a serious pest of stored combs and weak colonies. Adult female wax moths enter hives and lay their eggs on wax combs or in small crevices between wooden parts of the hives not easily accessible to honey bees. After few days the larvae hatch and begin feeding on bees-wax, pollen, cast larval skins and other remains in cells. This devastating activity of wax moths leads to great financial losses every year in the field of beekeeping.

Strong colonies are the best control against the wax moth in the field. In comb storage chests, technical, physical, biological and chemical methods have been used to control the pest. The most effective method to avoid the destruction of combs from wax moth is their continuous maintenance in temperatures of the refrigerator, or their passing from the freezer for a short time. Cantwell and Smith (1970) confirmed that temperature lower than - 18 °C destroys all stages of the wax moth insect (egg, immature forms and adult). Although this treatment requires expensive facilities, it is successfully applied nowadays protecting the honeycombs from the wax moth without contaminating the beehive products.

In addition, biological and environment-friendly control method were developed such us the male sterile technique with gamma-rays (Jafari et al., 2010), the trapping of moths by using pheromone (Flint & Merkle, 1983) and the use of the bacterium *Bacillus thuringiensis* that kills the wax moth larvae when it ingests the spores (Burges & Bailey, 1968; Burges 1997; Charriere & Imdorf, 2004).

Chemical methods, includes substances that are considered friendly to environment like methyl salicylate, clove oil, formic acid, sulphur, acetic acid, basil oil and other have been used (Wilson, 1965; Williams, 1980; Owayss & Abd-Elgayed, 2007). Most of these compounds are dangerous for bee brood and human health, while they require repeated application and may react and destroy the metal parts of the combs. Besides these, 1,2-dibromo-ethane (DBE), 1,4 dichloro-benzene (p-DCB), naphthalene had been used for many years in different countries even though their use causes significant contamination of bee products.

DBE is a manufactured chemical. In nature, it is produced in small amounts in the sea water, where it is formed, probably by algae and kelp. It is dissolved in water and by this way it can stay in groundwater and in soil for a long time. In air it breaks down quickly. This substance has been used as a pesticide in soil, and on citrus, vegetables, and grain crops. EPA has banned most of these uses since 1984. The same organization has also set a limit of 0.05 μg.cm-3 of 1,2-dibromo-ethane in drinking water (ATSDR, 1992).

The compound p-DCB is one of the three di-chloro-benzene isomers (1,2-DCB, 1,3-DCB and 1,4-DCB), which is commonly used as a space deodorant i toilets and for moth control. It is a volatile colorless to white crystalline material with a mothball-like, penetrating odor and it is commercially, the most important isomer (ATSDR, 2006).

Naphthalene is a white solid substance that evaporates easily. Its major use is in the manufacture of polyvinyl chloride (PVC) plastics and it is also used in moth repellents and toilet deodorant blocks. That use of naphthalene accounted for 73% and 60% of commercial demand for naphthalene in Japan and the United States, respectively in 1999, (ATSDR, 2005).

No MRL's in honey for the above three compounds were defined until 2005 when the European regulation 396/2005 EC set the limit at 10 μg kg-1 for substances for which no MRL had been established. This limit for p-DCB was also the Swiss Tolerance Limit (STL)

Pesticide Residues in Bee Products 103

Contamination of bee products by chemicals that are used against wax moths was also noted in Greece by Tananaki et al. (2005). Initially a multi-method had been developed for the determination of DBE, p-DCB and naphthalene and then this method had been applied in twenty five honey samples produced in different areas of Greece. The 8% of the samples had detectable amounts of DBE, 92% had p-DCB and 88% had naphthalene residues. Concentrations of naphthalene, p-DCB and DBE that exceeded 10 μg kg-1 were measured in

After confirming the mass contamination, beekeepers had been informed to stop the treatment with those chemicals and to destroy all the combs that had been treated before. Meanwhile a monitoring program for the residues of volatile insecticides in Greek honey was initiated by laboratory of Apiculture – Sericulture of Aristotle University. A total of 1,519 samples were analyzed during the period 2004 – 2010 (Tananaki et al., 2006). From those, 209 samples were bought from Greek supermarkets (commercial) while 1,310 were collected from beekeepers or

Comparing the results of eight years' monitoring of p-DCB, a considerable reduction of residues is observed both in commercial and bulk honey samples. During the first year the 82.9% of commercial samples had residues more than 10 mg kg-1 which is the established action limit in Greece since 2005. In the following three years this percentage decreased gradually and finally p-DCB wasn't detected at concentrations more than 10 mg kg-1 in 2010. Similar behavior was observed for the samples collected from beekeepers. These results demonstrate that the Greek beekeepers' efforts to restrict the problem and to find alternative

The great percentage of commercial samples in all years of study have either no detectable amounts or below 10 μg kg-1 DBE. Only one sample was found exceeding 40 μg kg-1 in year 2003. This sample had 60.5 μg kg-1 DBE, which is the maximum concentration found in samples bought from stores. Samples that had been collected from beekeepers had higher concentration of DBE than the commercial ones. This is because commercial samples usually are mixtures from different producers. During year 2003, a percentage of 9,9% of the samples exceeded the level of 10 μg kg-1 and a maximum value of 132.5 μg kg-1 was found in one of them.During the following two years this percentage decreases to 1.9% and 2.8% respectively, but still some beekeepers continue to use the chemical as indicated

Figure 2 summarizes the results of naphthalene residues in honey from the Greek market and from beekeepers as well. Contrary to p-DCB and DBE, naphthalene was found in more commercial samples than in samples from beekeepers during the first year of monitoring program. This could be attributed to blending of Greek commercial honeys with imported honey originating from countries where naphthalene is still used to control wax-moth. During the following years the residues in commercial samples dropped below 10 μg.kg-1 and very few beekeepers' samples were contaminated at higher levels. The highest

Tananaki et al. (2006) also found differences in the level and the frequency of contamination among different types of honeys. Honey produced during the spring honey flow (blossom and fir honeys) was contaminated in a higher percentage than the honey produced later in the season (thymus and pine honey). Thymus and blossom honey have higher contamination in naphthalene than other types of honey. This might happen because both thymus and blossom are the types of Greek honey that are probably mixed with imported honey. Paleologos et al., (2006), Tsimeli et al., (2008) and Harizanis et al., (2008) have also

from their associations (bulk honey). Results of this research are indicated in Fig. 2.

solutions for the control of the wax-moth (*Galleria mellonela*) have been accomplished.

by the high concentration of 331.2 μg kg-1 detected in one sample in 2004.

concentration of naphthalene found in one sample was 523.6 μg.kg-1 in 2004.

analyzed samples of Greek honey with similar results.

6.7%, 32% and 8% of tested samples, respectively.

and was already used as action level in Greece. ADI values for DBE, p-DCB and naphthalene, range according to Table 3. Besides killing the moth those chemical are absorbed by the wax and when bees store honey into combs, they are transferred into the product. Laboratory comb-melting experiment showed that p-DCB is not removed from wax during the comb recycling (Bogdanov et al., 2004). Residues up to 0.002 mg kg−1 may be detected in honey due to the use of precontaminated wax. Residues of p-DCB exceeding 0.01 mg kg−1 indicate contamination of bee product by beekeeping practices. Countries that have reported problems with residues from the above volatile insecticides are Germany, Switzerland, Greece and Turkey (Wallner, 1992; Bogdanov et al., 2004; Tananaki et al., 2005; Beyoğlu & Omurtag 2007).

Wallner (1992), stated in his paper that the problem of p-DCB residues in Germany is serious, since 50% of the analyzed honey samples had been found contaminated from 3 to 50 μg kg-1. He noticed that p-DCB is very stable in honey and it cannot evaporate from the sealed glass containers. Finally, he stated that beeswax works like a sponge as it has large capacity for fat-soluble active compounds. The more the p-DCB crystals are added to combs the higher is the substance stored in the wax. The evaporation of p-DCB from wax is impossible even after prolonged ventilation.


Table 1. ADI of three compounds that have been used against wax moths

Bogdanov et al. (2004) analyzed Swiss commercial honey samples during five years period for p-DCB residues and they found that the contaminated samples ranged from 14% to 46% (fig. 1). The percentage of the imported samples was lower, on average 7%. Although there is no MRL for p-DCB, Switzerland has established a "Swiss tolerance value" (STV) for honey at 10 μg kg-1. From the total 173 Swiss and 287 imported honey samples, 13% and 0.8% exceeded the STV respectively.

Fig. 1. Residues of p-DCB in honey samples in Switzerland (Bogdanov et al., 2004)

and was already used as action level in Greece. ADI values for DBE, p-DCB and naphthalene, range according to Table 3. Besides killing the moth those chemical are absorbed by the wax and when bees store honey into combs, they are transferred into the product. Laboratory comb-melting experiment showed that p-DCB is not removed from wax during the comb recycling (Bogdanov et al., 2004). Residues up to 0.002 mg kg−1 may be detected in honey due to the use of precontaminated wax. Residues of p-DCB exceeding 0.01 mg kg−1 indicate contamination of bee product by beekeeping practices. Countries that have reported problems with residues from the above volatile insecticides are Germany, Switzerland, Greece and Turkey (Wallner, 1992; Bogdanov et al., 2004; Tananaki et al., 2005;

Wallner (1992), stated in his paper that the problem of p-DCB residues in Germany is serious, since 50% of the analyzed honey samples had been found contaminated from 3 to 50 μg kg-1. He noticed that p-DCB is very stable in honey and it cannot evaporate from the sealed glass containers. Finally, he stated that beeswax works like a sponge as it has large capacity for fat-soluble active compounds. The more the p-DCB crystals are added to combs the higher is the substance stored in the wax. The evaporation of p-DCB from wax is

**ADI (mg kg-1 bw day-1) Compound US EPA Canadian health** 

1,2-dibromoethane 0.009 0.009 1,4-dichlorobenzene 0.03 0.11 naphthalene 0.02 0.02

Bogdanov et al. (2004) analyzed Swiss commercial honey samples during five years period for p-DCB residues and they found that the contaminated samples ranged from 14% to 46% (fig. 1). The percentage of the imported samples was lower, on average 7%. Although there is no MRL for p-DCB, Switzerland has established a "Swiss tolerance value" (STV) for honey at 10 μg kg-1. From the total 173 Swiss and 287 imported honey samples, 13% and 0.8%

Fig. 1. Residues of p-DCB in honey samples in Switzerland (Bogdanov et al., 2004)

Table 1. ADI of three compounds that have been used against wax moths

Beyoğlu & Omurtag 2007).

exceeded the STV respectively.

impossible even after prolonged ventilation.

Contamination of bee products by chemicals that are used against wax moths was also noted in Greece by Tananaki et al. (2005). Initially a multi-method had been developed for the determination of DBE, p-DCB and naphthalene and then this method had been applied in twenty five honey samples produced in different areas of Greece. The 8% of the samples had detectable amounts of DBE, 92% had p-DCB and 88% had naphthalene residues. Concentrations of naphthalene, p-DCB and DBE that exceeded 10 μg kg-1 were measured in 6.7%, 32% and 8% of tested samples, respectively.

After confirming the mass contamination, beekeepers had been informed to stop the treatment with those chemicals and to destroy all the combs that had been treated before. Meanwhile a monitoring program for the residues of volatile insecticides in Greek honey was initiated by laboratory of Apiculture – Sericulture of Aristotle University. A total of 1,519 samples were analyzed during the period 2004 – 2010 (Tananaki et al., 2006). From those, 209 samples were bought from Greek supermarkets (commercial) while 1,310 were collected from beekeepers or from their associations (bulk honey). Results of this research are indicated in Fig. 2.

Comparing the results of eight years' monitoring of p-DCB, a considerable reduction of residues is observed both in commercial and bulk honey samples. During the first year the 82.9% of commercial samples had residues more than 10 mg kg-1 which is the established action limit in Greece since 2005. In the following three years this percentage decreased gradually and finally p-DCB wasn't detected at concentrations more than 10 mg kg-1 in 2010. Similar behavior was observed for the samples collected from beekeepers. These results demonstrate that the Greek beekeepers' efforts to restrict the problem and to find alternative solutions for the control of the wax-moth (*Galleria mellonela*) have been accomplished.

The great percentage of commercial samples in all years of study have either no detectable amounts or below 10 μg kg-1 DBE. Only one sample was found exceeding 40 μg kg-1 in year 2003. This sample had 60.5 μg kg-1 DBE, which is the maximum concentration found in samples bought from stores. Samples that had been collected from beekeepers had higher concentration of DBE than the commercial ones. This is because commercial samples usually are mixtures from different producers. During year 2003, a percentage of 9,9% of the samples exceeded the level of 10 μg kg-1 and a maximum value of 132.5 μg kg-1 was found in one of them.During the following two years this percentage decreases to 1.9% and 2.8% respectively, but still some beekeepers continue to use the chemical as indicated by the high concentration of 331.2 μg kg-1 detected in one sample in 2004.

Figure 2 summarizes the results of naphthalene residues in honey from the Greek market and from beekeepers as well. Contrary to p-DCB and DBE, naphthalene was found in more commercial samples than in samples from beekeepers during the first year of monitoring program. This could be attributed to blending of Greek commercial honeys with imported honey originating from countries where naphthalene is still used to control wax-moth. During the following years the residues in commercial samples dropped below 10 μg.kg-1 and very few beekeepers' samples were contaminated at higher levels. The highest concentration of naphthalene found in one sample was 523.6 μg.kg-1 in 2004.

Tananaki et al. (2006) also found differences in the level and the frequency of contamination among different types of honeys. Honey produced during the spring honey flow (blossom and fir honeys) was contaminated in a higher percentage than the honey produced later in the season (thymus and pine honey). Thymus and blossom honey have higher contamination in naphthalene than other types of honey. This might happen because both thymus and blossom are the types of Greek honey that are probably mixed with imported honey. Paleologos et al., (2006), Tsimeli et al., (2008) and Harizanis et al., (2008) have also analyzed samples of Greek honey with similar results.

Pesticide Residues in Bee Products 105

Residues of p-DCB were also detected in royal jelly. Tananaki et al. (2009) found that the concentrations of p-DCB in honey were significantly lower than in the royal jelly; in some cases, royal jelly had some hundred times more residues than honey from the same comb. The maximum concentration of p-DCB found in royal jelly was 1,520 μg kg-1. Bogdanov et al. (2004) checked the p-DCB residues in wax. They analyzed wax samples from manufactures during the years 1994 -1998 and 2002 and they found residues in 66% of the wax sample, in concentrations from 0.7 to 74.9 mg kg-1. Τhe concentrations of p-DCB in new wax after melting of old combs were the same with those of the old combs. This indicates

**2.3 Methods of analysis of pesticide and acaricide residues detected in bee products**  The need of monitoring residues of acaricides used by the beekeeper in conjunction with the need to monitor the contamination of bee products from other sources, such as pesticides used on crops and environmental pollutants, makes the development of appropriate methods of analysis obligatory. Furthermore, it is necessary to analyze products like honey and pollen randomly in order to find any violations of existing legislation on the part of producers or sellers. The suitability of each method lies in its ability to give a reliable result of the concentration of residues. A complete method should usually includes four sub-

The particular physicochemical properties of each product (moisture, fat, protein content etc.) in conjunction with specific physicochemical properties of each substance (polarity, volatility, etc.) do not permit the use of one methodology for the determination of all active substances in all products. Various techniques have been reported in order to clean –up the

The first step of an analysis is the sampling. Specifically, the meaning of a sample is to take a part of the product, which should be as representative as possible. The way of the sampling varies, depending on the type of sample. In homogeneous samples such as water, the sampling is simple and does not require complicated procedures. On the contrary, heterogeneous samples such as fruits, vegetables and animal products require additional measures during sampling in order to reduce the uncertainty. The contribution of sampling to the total uncertainty is so great that in some cases approaches 40%. The EU issued a special directive on the sampling of food (2002/63/EK) and requires the accurate

The second stage of the analysis is the preservation of the sample. The common practice of laboratories is the storage of collected samples for a period ranging from some hours to years. Storage conditions must ensure the preservation of the sample during the period required for the analysis. Food should normally be preserved under freezing conditions,

that p-DCB is not being removed from wax during the comb recycling process.

stages, which are described as:

sample and isolate the analyte.

**2.3.2 Sample preservation** 

implementation of the official control laboratories.

• Sample Preservation • Sample Preparation

• Sampling

• Analysis

**2.3.1 Sampling** 

#### 1,4-dichlorobenzene

Naphthalene

Fig. 2. Residues of volatile insecticides in Greek honeys

Fig. 2. Residues of volatile insecticides in Greek honeys

1,4-dichlorobenzene

1,2-dibromoethane

Naphthalene

Residues of p-DCB were also detected in royal jelly. Tananaki et al. (2009) found that the concentrations of p-DCB in honey were significantly lower than in the royal jelly; in some cases, royal jelly had some hundred times more residues than honey from the same comb. The maximum concentration of p-DCB found in royal jelly was 1,520 μg kg-1. Bogdanov et al. (2004) checked the p-DCB residues in wax. They analyzed wax samples from manufactures during the years 1994 -1998 and 2002 and they found residues in 66% of the wax sample, in concentrations from 0.7 to 74.9 mg kg-1. Τhe concentrations of p-DCB in new wax after melting of old combs were the same with those of the old combs. This indicates that p-DCB is not being removed from wax during the comb recycling process.

#### **2.3 Methods of analysis of pesticide and acaricide residues detected in bee products**

The need of monitoring residues of acaricides used by the beekeeper in conjunction with the need to monitor the contamination of bee products from other sources, such as pesticides used on crops and environmental pollutants, makes the development of appropriate methods of analysis obligatory. Furthermore, it is necessary to analyze products like honey and pollen randomly in order to find any violations of existing legislation on the part of producers or sellers. The suitability of each method lies in its ability to give a reliable result of the concentration of residues. A complete method should usually includes four substages, which are described as:


The particular physicochemical properties of each product (moisture, fat, protein content etc.) in conjunction with specific physicochemical properties of each substance (polarity, volatility, etc.) do not permit the use of one methodology for the determination of all active substances in all products. Various techniques have been reported in order to clean –up the sample and isolate the analyte.

#### **2.3.1 Sampling**

The first step of an analysis is the sampling. Specifically, the meaning of a sample is to take a part of the product, which should be as representative as possible. The way of the sampling varies, depending on the type of sample. In homogeneous samples such as water, the sampling is simple and does not require complicated procedures. On the contrary, heterogeneous samples such as fruits, vegetables and animal products require additional measures during sampling in order to reduce the uncertainty. The contribution of sampling to the total uncertainty is so great that in some cases approaches 40%. The EU issued a special directive on the sampling of food (2002/63/EK) and requires the accurate implementation of the official control laboratories.

#### **2.3.2 Sample preservation**

The second stage of the analysis is the preservation of the sample. The common practice of laboratories is the storage of collected samples for a period ranging from some hours to years. Storage conditions must ensure the preservation of the sample during the period required for the analysis. Food should normally be preserved under freezing conditions,

Pesticide Residues in Bee Products 107

• Supercritical Fluid Extraction (SFE). The SFE is a technique similar to the ASE with similar advantages and disadvantages, while the equipment used for this technique is rather expensive (Mitra, 2003). The difference between the two techniques lies in the type of solvent, which is carbon dioxide (CO2) for the SFE. Adding a small amount (1- 10%) of an organic solvent (such as methanol, ethanol, etc.) improves the efficiency of extraction of more polar compounds, which otherwise would be very small. Two types of supercritical fluid extraction techniques, called static and dynamic were developed. In the case of static SFE, the solvent enters the cell, which contains the lyophilized sample and remains an exact time at constant pressure and temperature conditions. However, in the dynamic SFE, the flow of solvent into the cell remains constant and stable for perfectly accurate time and at constant pressure and temperature conditions. The final extract is transferred to a vial containing an organic solvent. The SFE is a rapid technique that requires very small quantities of organic solvents and does not contaminate the environment significantly. Unlike ASE, there are several publications on the analysis of residues in honey using SFE. Rissatto et al. (2004) developed a method to analyze samples of honey combined SFE system and gas chromatography. The limit of quantification was 0.01 mg kg-1, while recovery rates ranged from 75% to 94 %. In a second study conducted by Atienza et al. (1993) the average recovery rates ranged from 53% -94% while the RSD of the method ranged from 1.3% to 1.6%. In one case, this technique was used for the analysis of organophosphorus and carbamate insecticide residues in bees. The recovery rate exceeded 75% for all substances except

• Gel Permeation Chromatography (GPC). This technique allows the separation of different components based on their size (larger particles move faster). Gels of various porosity and organic solvents are used in order to achieve the separation. Usually, this technique is used to remove lipids, proteins, polymers and other macromolecules contained in the sample. Especially for the pesticide analysis, the technique is suitable for removing high boiling point compounds, which are deposited to the inlet of gas chromatography. Rossi et al., (2001) have used the GPC on the analysis of residues in bees. The recovery was satisfactory for 25 of 29 substances analyzed (percentage recovery ranged from 70.9% to 106.8%). In contrast, the recovery rate for active substances pirimicarb, ethiofencarb, methiocarb and fenoxycarb was 38.7%, 48.6%,

and PFE (Pressurized Fluid Extraction).

omethoate (Jones & McCoy, 1997).

46.6% and 58.4% respectively.

this technique are very small compared to the SE and the automation of the extraction procedure much easier. Disadvantage of this technique is the high cost of required equipment. However, the small amount of solvent and the possibility of automation make it possible to recover the cost within a short period of time (especially for laboratories that analyze large numbers of samples). The ASE has been used successfully in many cases of food and water analysis by EPA (Chuang et al., 2001). There is only one study on the analysis of bee products with ASE. Results indicated good efficacy in the determination of acaricides in honey by the use of High Performance Liquid Chromatography (HPLC). The recovery rates of this method ranged from 58% to 103% and limits of quantification ranged from 0.01 mg kg-1 to 0.2 mg kg-1 (Korta et al., 2002). ASE is likely to be referred in the literature with the names of PLE (Pressurized Liquid Extraction), PSE (Pressurized Solvent Extraction)

until the day of analysis, in order to minimize the evaporation or chemical reactivity of these compounds.

#### **2.3.3 Sample preparation and analysis**

The next step includes the preparation and the analysis of the sample. The analysis is performed directly, i.e. without pretreatment of the sample, where a direct measurement is possible (e.g. measurement of moisture in honey). In most cases, however, a preparation of the sample should take place before the analysis. The preparation of the sample includes the removal of interferences and the isolation of compounds of interest. This step is necessary in methods of analysis for residues of pesticides and veterinary drugs. Especially in the case of residue analysis, this stage is divided into separate stages that vary in terms of the number and type. Typically, these steps are five and consist of:


The cleaning of the sample is the most complicated stage. That is the reason why various techniques have been used. The main techniques used for the preparation and analysis of honey samples are summarized in a review of Rial-Otero et al. (2007). Techniques that have been used in order to achieve the determination of acaricide and pesticide residues in bee products are:


until the day of analysis, in order to minimize the evaporation or chemical reactivity of these

The next step includes the preparation and the analysis of the sample. The analysis is performed directly, i.e. without pretreatment of the sample, where a direct measurement is possible (e.g. measurement of moisture in honey). In most cases, however, a preparation of the sample should take place before the analysis. The preparation of the sample includes the removal of interferences and the isolation of compounds of interest. This step is necessary in methods of analysis for residues of pesticides and veterinary drugs. Especially in the case of residue analysis, this stage is divided into separate stages that vary in terms of the number




The cleaning of the sample is the most complicated stage. That is the reason why various techniques have been used. The main techniques used for the preparation and analysis of honey samples are summarized in a review of Rial-Otero et al. (2007). Techniques that have been used in order to achieve the determination of acaricide and pesticide residues in bee

• Solvent Extraction (SE). This is the first technique developed in order to detect pesticide residues. In this technique, the sample is dissolved in water, or mixtures of water and alcohols. After the dilution of the sample, an extraction with suitable organic solvents takes place, in order to collect the analyte and remove a large portion of co-extractives components. Several methods of the SE used combined with acidification of the sample (Waliszewski et al., 1998; Waliszewski et al., 2003; Bernal et al., 1997) or the use of ultrasound (Jimenez et al., 2000; Rezic et al., 2005), in order to improve the efficiency. Due to the use of large quantities of organic solvents, the SE is particularly aggravating for the environment and the health of laboratory staff. Moreover, the cost is quite high due to the large quantity of supplies. Finally, many hours are required for analysis of a sample and the automation of the process is very difficult. Despite these drawbacks, the SE has been used with satisfactory results in various methods of analysis of honey (Jimenez et al., 2002; Menkissoglu-Spiroudi et al., 2000; Taccheo et al., 1988a), royal jelly (Balayannis, 2001), pollen and bees (Bernal et al., 1997) for the determination of

• Accelerated Solvent Extraction (ASE). This technique includes steps of extraction with organic solvents at predetermined conditions of pressure and temperature. In ASE, the extraction solvent is carried out in a special device and the extraction under steady environmental conditions (pressure and temperature) allows efficient and reproducible isolation and collection of analytes. The quantities of solvents used in

compounds.

products are:

**2.3.3 Sample preparation and analysis** 

and type. Typically, these steps are five and consist of:

of the sample easier and faster (optional step).

evaluation of chromatograms.

pesticide and acaricide residues.

minimization of the quantitative limits.



Pesticide Residues in Bee Products 109

• Matrix Solid Phase Dispersion (MSPD). The MSPD includes a stage of dilution of the sample in an organic solvent (e.g. methanol) and mixing a quantity of the solution with a sorbent, which is usually C18 or Florisil. Next phase involves addition of solvents (hexane, ethyl acetate, etc.), working as means of extraction and elution. After good homogenization in an ultrasonic bath and centrifugation, the extract is collected and analyzed in chromatography systems. The advantages of this technique include the limited use of solvents and the rapid process of the sample. Although MSPD was a promising technique, it is expected to be replaced by QuEChERS, which is a new method of analysis described in next paragraph. The MSPD is rarely used in the analysis of acaricide and pesticide residues in bee products. However, there are few studies used MSPD and gas chromatography for the detection of pesticides in honey. Limits of quantification in these studies were lower than 0.015 mg kg-1 for any pesticide, while the recovery ranged between 60% and 113% (Albero et al., 2001; Sanchez et al,

• QuEChERS. The name of the technique derives from the characteristics of this method, which is described as Quick, Easy, Cheap, Effective, Rugged and Safe (Schenck & Hobbs, 2004). The QuEChERS is a new technique used for the determination of pesticide residues in food analysis. This technique is based on solidphase dispersion extraction (Matrix Solid-Phase Dispersion). QuEChers developed and validated by Anastassiades (2005) and quickly began to be used by many laboratories. Nowadays, QuEChERS is the common sample preparation technique of official laboratories of European Union. This technique was developed primarily for the analysis of products with high water content. The addition of water to the sample makes possible the use of this technique for the analysis of products like honey. The disadvantage of this technique is the need of expensive equipment (GC/MS/MS, LC/MS/MS etc.), because of insufficient "cleanup step" of the sample. QuEChERS was used in order to detect residues of 36 pesticides in honey. Honey samples were extracted with acetonitrile. The extraction step was followed by the addition of acetic acid with the simultaneous addition of magnesium sulphate and sodium acetate. A mixture of primary/secondary amine (PSA) and magnesium sulphate was added as a second purification step. This step was followed by a change of solvent with a mixture of hexane and acetone. The quantification of organophosphorus compounds carried out using a nitrogen phosphorus detector (NPD), while an electron capture detector (ECD) was used for the determination of chlorinated hydrocarbons and pyrethroids. Recovery experiments were made at three levels (from 0.02 mg kg-1 to 5 mg kg-1) and the results ranged from 70% to 120%. Experimental repeatability was satisfactory, as the RSD ranged from 1% to 22%. Finally, the expanded uncertainty

and time of extraction obtained (Jimenez et al., 1998).

2002).

ingredients. The period of immersion of the fiber, as well as the temperature was strictly defined and determined by tests during the development of the method. The technique of solid phase microextraction has been applied for the determination of OCP, OPP, pyrethroid and acaricide residues in honey (Blasco et al., 2004; Yu et al., 2004; Jimenez et al., 1998). In a comparative study, two different types extraction fibers (PDMS 7 mm, PDMS 100 mm and PA 85mm) were tested. The fiber made of PDMS proved significantly superior in terms of reproducibility, sensitivity, linearity


• Stir Bar Sorptive Extraction (SBSE). This extraction technique is using an appropriate stirring bar, which adsorbs the analyte. The bar was either eluted with suitable organic solvents or placed directly to the inlet of gas chromatography systems (Baltussen et al., 1999). Particularly, the bar is made by stainless steel coated with a thin layer of glass and poly-dimethyl siloxane (PDMS), which adsorbs the analyte in the sample (Popp et al., 2001). It is very important for the efficiency of extraction to be accurate in temperature and extraction time. The greater the precision, the more improved the repeatability of the method. In the final phase, the bar is placed in a special unit, which in turn is attached to the inlet. The adsorbed substances led to the column and detector with a carrier gas flow rate increasing with temperature. There is also the option for the bar to be extracted with organic solvents (e.g. acetonitrile), which constitute the final sample for chromatographic analysis (Sanchez-Rojas et al., 2008). SBSE has been used on bee products with good results compared the SPME. Based on data given by Blasco et al. (2004), the SBSE is more efficient as a technique than SPME, while accuracy and repeatability are much better. More specifically, the limit of quantification was 0.04 mg kg-1 for SBSE technique, while those for SPME techinique ranged from 0.8 mg kg-1 to 3.0 mg kg-1. Moreover, the recovery of SBSE ranged from 40% to 64%. Finally, the relative standard deviation of repeatability did

• Solid Phase Micro Extraction (SPΜE). The SPME is a relatively modern technique, developed by Pawlyszin et al. (1997). The principle of this technique relies on the use of a fiber, which adsorbs the analyte, which then eluted to the inlet of gas chromatography systems. The SPME technique is suitable for the determination of volatile compounds in liquid or solid samples. SPME can be used in two ways. The first method involves an extraction by sinking the fiber into the sample solution directly. This is an advantage in terms of sensitivity and the number of identified substances. The second way relates to cases of extraction in the supernatant layer of sample. The advantage of this method is the higher level of purity of the final sample (Arthur & Pawliszyn, 1990; Louch et al., 1992; Zhang & Pawliszyn, 1993; Page & Lacroix, 1993). Both SPME and SBSE are based on the logic of the adsorption of chemicals in various absorbents, which in the first case is a fiber (SPME), and in the second a bar (SBSE). Another important parameter, which can greatly improve the results of SPME, is pH (Volante et al., 2001). The adjustment of pH by using buffers could improve efficiency or reduce the time of extraction. It should be noted that there is a variety of fibers, which differ in the type and thickness of the adsorbent material. The advantages of SPME include: (i) the lack of use of organic solvents, (ii) the purest final samples, (iii) the minimization of time, (iv) the good linearity of the method, (v) the non-requirement for full adsorption of the analyte & (vi) the relatively simple automation (Pawliszyn, 1997). The major disadvantage of SPME is the low efficiency for the semi-volatile or non-volatile compounds and the inability to repeat the analysis of a sample (same bottle). SPME was used for the detection of pesticide and acaricide residues in honey. More specifically, the fiber of SPME was immersed in an aqueous solution of honey and remained there until equilibrium of the analyte between the fiber and the environment was achieved. After this, the fiber was removed and placed in the inlet of gas chromatography in order to desorb the

not exceed 10% in both cases.

ingredients. The period of immersion of the fiber, as well as the temperature was strictly defined and determined by tests during the development of the method. The technique of solid phase microextraction has been applied for the determination of OCP, OPP, pyrethroid and acaricide residues in honey (Blasco et al., 2004; Yu et al., 2004; Jimenez et al., 1998). In a comparative study, two different types extraction fibers (PDMS 7 mm, PDMS 100 mm and PA 85mm) were tested. The fiber made of PDMS proved significantly superior in terms of reproducibility, sensitivity, linearity and time of extraction obtained (Jimenez et al., 1998).


Pesticide Residues in Bee Products 111

The analyst is able to detect many different compounds by preparing and analyzing a sample only once. However, there are certain compounds, which can be identified individually and only with the use of complex techniques (e.g. amitraz in honey or pear after derivatisation). In recent years, due to the significant improvement of the equipment (GC-MS-MS, LC-MS-MS, etc.) the number of identified substances has increased considerably and many laboratories can identify the majority of active ingredients. The improvement of the quality and quantity of results issued by laboratories has been particularly important. The development of methods

The gas chromatography was used more than any other method to determine pesticide and acaricide residues in bee hive products. As mentioned above, this technique is mainly used for determination of volatile and low molecular weight compounds, but there are cases where higher molecular weight compounds (e.g. amitraz), analyzed by gas chromatography after laborious and time consuming processes (e.g. derivatization). In these cases, the substances were converted into more volatile compounds and then analyzed using gas chromatographic system. Gas chromatographic systems consist of three main sections

a. The first main section is called inlet and ensure the entrance of the sample in a gas chromatograph. The types of inlets used in gas chromatography is the Cool On Column, purged packed and split/splitless. The type of inlet may be a problem for some classes of substances that are sensitive to high temperature (e.g. methamidophos and dichlorvos gave better results in Cool on Column inlets due to the lower temperature). The injection in a Cool on Column inlet takes place at low temperatures and benefits in repeatability and stability of analytes. The problem in this case is the more frequent maintenance of the column. Instead, the split/splitless inlet advantages in the purity of the sample, since the majority of high molecular weight substances are removed by a flow of gas and do not enter the column. The result is the extension of the lifetime of the column and the less frequent maintenance. In return, the above advantages of the split/splitless inlet may indicate the low reproducibility due to the

b. The second part consists of the oven and column at which the separation of analytes happens. The separation of substances achieved with the strictly programmed temperature and carrier gas flow within the oven and column respectively. The repeatability of retention time of an analyte depends on the repeatability of the above conditions. The column packing material is a very important factor for the separation and identification of various substances. Small to medium polarity columns are usually used for the detection of acaricides and pesticides. The use of more polar columns is necessary in some cases of single residue methods (e.g. determination of amitraz and its metabolites in honey and beeswax). Finally, a factor worth mentioning is the quality of the gases (carrier, auxiliary gas, etc.) that can substantially improve the sensitivity and

c. The inlet and the column associated with a suitable detector. Detector achieves the visualization of the result. In the case of beehive products the following detectors have

includes the use of chromatography described below.

removal of a quantity of analyte during cleaning.

lifetime of the column and the detector.

been used:

**2.3.4.1 Gas chromatography (GC)** 

outlined below:

was relatively high (30%), but within the limit of 50% provided in pesticide residues analysis (Barakat, 2007).

• Solid Phase Extraction (SPE). It is the most widely used technique of last decades, in the case of analysis for pesticide and veterinary drug residues. The solid phase extraction is the perfect choice for most researchers, since it requires a small amount of organic solvent (and thus is environmentally friendly), is easily automated and requires no expensive equipment. The disadvantages are the more expensive consumables (solid phase extraction microcolumns), the specialized staff, the differences between lots of microcolumns and the possible absorption of some substances on the polypropylene used in cartridges. Specifically, the sample is dissolved in water (Jimenez et al., 2000; Bernal et al., 1996), alcohol (Bernal et al., 2000) or mixtures of them (Karazafiris et al., 2008; Jimenez et al., 2008), followed by activation of the microcolumn with the same solvent. Subsequently, the sample is passed through a microcolumn containing a suitable solid material, which captures the analyte. The bound substances are eluted with the passage of an appropriate organic solvent. In the case of honey acetone (Bernal et al., 1996), dichloromethane (Jimenez et al., 1998), ethyl acetate (Tsigouri et al., 2001), hexane (Gomis et al., 1996), methanol (Bernal et al., 2000), a mixture of hexane- ethyl acetate (Tsigouri et al., 2001) have been occasionally used. With regard to the types of substrates used occasionally, the reverse phase C18 was the most appropriate and chosen by most researchers for the extraction of insecticides, acaricides, herbicides, fungicides and other pesticides (Jimenez et al., 2000; Bernal et al., 2000; Korta et al., 2001). Also microcolumn with Florisil gave good results in trials for determination of pyrethroid, OCP and OPP residues (Jimenez et al., 1998a) and C8 in the determination of tau fluvalinate (Tsigouri et al., 2001). The pH adjustment proved particularly important for the good recovery of some active substances. For example, coumaphos is unstable in an alkaline environment, as opposed to amitraz, the recovery increases with increasing pH values up to 11 (Korta et al., 2001). A comparison of the effectiveness of SPE and SE in pesticide residue analysis was conducted in two publications by Bernal et al. (1996 & 2000). According to the results of the comparison, it should be noted that the recovery rate with both techniques was similar, but SPE proved superior to the purity of the chromatograms. The analysis of royal jelly using the technique of solid phase extraction was first mentioned, by Karazafiris et al., (2008b). The method proved efficient for the determination of acaricide and insecticide residues.

#### **2.3.4 Determination of analytes**

The isolation of analytes from the matrix is followed by the necessary step of separation. The practices used on pesticide and veterinary drug residue analysis are based on chromatographic methods. The choice of gas or liquid chromatography is mainly based on the chemical properties of analytes. The technique of gas chromatography was proved suitable for the determination of volatile and low molecular weight compounds, in contrast to the technique of liquid chromatography, which was used in less volatile and high molecular weight substances. The methods of analysis are classified according to the number of compounds detected. The two main categories are multi residue methods (MRM) and single residue methods (SRM). The majority of compounds identified with multi residue methods.

• Solid Phase Extraction (SPE). It is the most widely used technique of last decades, in the case of analysis for pesticide and veterinary drug residues. The solid phase extraction is the perfect choice for most researchers, since it requires a small amount of organic solvent (and thus is environmentally friendly), is easily automated and requires no expensive equipment. The disadvantages are the more expensive consumables (solid phase extraction microcolumns), the specialized staff, the differences between lots of microcolumns and the possible absorption of some substances on the polypropylene used in cartridges. Specifically, the sample is dissolved in water (Jimenez et al., 2000; Bernal et al., 1996), alcohol (Bernal et al., 2000) or mixtures of them (Karazafiris et al., 2008; Jimenez et al., 2008), followed by activation of the microcolumn with the same solvent. Subsequently, the sample is passed through a microcolumn containing a suitable solid material, which captures the analyte. The bound substances are eluted with the passage of an appropriate organic solvent. In the case of honey acetone (Bernal et al., 1996), dichloromethane (Jimenez et al., 1998), ethyl acetate (Tsigouri et al., 2001), hexane (Gomis et al., 1996), methanol (Bernal et al., 2000), a mixture of hexane- ethyl acetate (Tsigouri et al., 2001) have been occasionally used. With regard to the types of substrates used occasionally, the reverse phase C18 was the most appropriate and chosen by most researchers for the extraction of insecticides, acaricides, herbicides, fungicides and other pesticides (Jimenez et al., 2000; Bernal et al., 2000; Korta et al., 2001). Also microcolumn with Florisil gave good results in trials for determination of pyrethroid, OCP and OPP residues (Jimenez et al., 1998a) and C8 in the determination of tau fluvalinate (Tsigouri et al., 2001). The pH adjustment proved particularly important for the good recovery of some active substances. For example, coumaphos is unstable in an alkaline environment, as opposed to amitraz, the recovery increases with increasing pH values up to 11 (Korta et al., 2001). A comparison of the effectiveness of SPE and SE in pesticide residue analysis was conducted in two publications by Bernal et al. (1996 & 2000). According to the results of the comparison, it should be noted that the recovery rate with both techniques was similar, but SPE proved superior to the purity of the chromatograms. The analysis of royal jelly using the technique of solid phase extraction was first mentioned, by Karazafiris et al., (2008b). The method proved

efficient for the determination of acaricide and insecticide residues.

The isolation of analytes from the matrix is followed by the necessary step of separation. The practices used on pesticide and veterinary drug residue analysis are based on chromatographic methods. The choice of gas or liquid chromatography is mainly based on the chemical properties of analytes. The technique of gas chromatography was proved suitable for the determination of volatile and low molecular weight compounds, in contrast to the technique of liquid chromatography, which was used in less volatile and high molecular weight substances. The methods of analysis are classified according to the number of compounds detected. The two main categories are multi residue methods (MRM) and single residue methods (SRM). The majority of compounds identified with multi residue methods.

analysis (Barakat, 2007).

**2.3.4 Determination of analytes** 

was relatively high (30%), but within the limit of 50% provided in pesticide residues

The analyst is able to detect many different compounds by preparing and analyzing a sample only once. However, there are certain compounds, which can be identified individually and only with the use of complex techniques (e.g. amitraz in honey or pear after derivatisation). In recent years, due to the significant improvement of the equipment (GC-MS-MS, LC-MS-MS, etc.) the number of identified substances has increased considerably and many laboratories can identify the majority of active ingredients. The improvement of the quality and quantity of results issued by laboratories has been particularly important. The development of methods includes the use of chromatography described below.

#### **2.3.4.1 Gas chromatography (GC)**

The gas chromatography was used more than any other method to determine pesticide and acaricide residues in bee hive products. As mentioned above, this technique is mainly used for determination of volatile and low molecular weight compounds, but there are cases where higher molecular weight compounds (e.g. amitraz), analyzed by gas chromatography after laborious and time consuming processes (e.g. derivatization). In these cases, the substances were converted into more volatile compounds and then analyzed using gas chromatographic system. Gas chromatographic systems consist of three main sections outlined below:


Pesticide Residues in Bee Products 113

This technique is used primarily for detecting drugs in biological samples. However, TLC was used to determine pesticide residues in food. More generally, the TLC requires sample extraction with a solvent mixture and separation of the components into blocks with a suitable coating material (e.g. Silica gel). The next step is an elution with suitable solvents. Special equipment is necessary in order to achieve the visualization and quantification of results. The TLC was used by Rezic et al. (2005) to detect residues of herbicides atrazine and simazine in honey. The recovery rate was estimated at 92.3% and 94.2% for atrazine and simazine respectively. The TLC was used in the above study in conjunction with the use of

A fact that has to be mentioned is that differences in the chemical synthesis of bee products may affect the efficiency of extraction (Blasco et al., 2004; Yu et al., 2004) and the response of the chromatographic systems to analytes (Volante et al., 2001; Jimenez et al., 1998; Karazafiris et al., 2008b). This is the reason why, solutions for calibration curve and recoveries should be prepared in an extract of the same sample analyzed. Specifically, the analyst applies the chosen technique in honey or other hive products containing no detectable residues of analytes. The final extract is derived from the overall process used in the construction of standard calibration curves. If it is not possible to find sample with no residues, an analyst can use an extract of the sample that gives a response 30% over the reference value. The response may be due to the presence of the analyte or an interference

**2.4 Methods for the determination of volatile insecticide residues in bee products**  The research on the detection of volatile insecticides residues from substances that are used against *Galleria mellonella* has been started twenty years ago. Various methods of isolation and analysis have been developed which are mainly based on chromatographic separation. Table 2 summarizes all those methods with some analytical information and the

During the first SMPE isolation method a small amount of honey was diluted with water and transferred to the vials. The p-DCB molecules were collected on PDMS-fiber (5 cm, 100 μm) and the adsorption process took place for 45 min at 20-25 oC. Desorption was performed by raising the fibre temperature to 250 °C for 15 min and the analytes transferred to the GC column (DB-5ms: 30m x 0,25mm, 0,25μm). The detection was achieved with a MS detector at the level of 1 μg kg-1 (Bogdanov et al., 2004). Tananaki et al. (2005) developed a sensitive method for the simultaneous determination of p-DCB, EDB and naphthalene residues in honey, using a purge and trap - gas chromatography – mass spectrometry system (P&T-GC–MS). In this research the analytes were extracted by He purging and then they absorbed onto the Tenax resin. With thermal desorption the isolated compounds were transferred to the GC – MS system. Separation was performed on a fused silica capillary column (30m×0.25mm I.D., 0.25 μm film thickness). The limits of detection were found to be 0.8, 0.15 and 0.05 μg kg−1 honey, while the limits of quantification were 2.4, 0.5 and 0.125 μg kg−1 for EDB, p-DCB and naphthalene

**2.3.4.3 Thin layer chromatography (TLC)** 

ultrasound during the extraction.

eluting at the same retention time.

corresponding references.

respectively.

**2.3.4.4 Matrix effect** 


Each chromatographic system may include components that automate the process and provide valuable assistance to the analyst. The most important component in optimizing the analytical procedure is the autosampler. The performance of a chromatographic system is maximizing by the use of autosampler, while it improves the reproducibility of injection volume and the number of samples, which can be analyzed daily.

#### **2.3.4.2 Liquid chromatography (LC)**

Unlike gas chromatography, which is limited to determining the most volatile compounds, liquid chromatography is used for the isolation of a widespread group of compounds. These compounds may not be sufficiently volatile or heat-resistant to analysis by gas chromatography. The most common types of detectors used in liquid chromatography were Diode Array Detectors (DAD) (Atienza et al., 1993; Blasco et al., 2004; Jones & McCoy, 1997; Martel & Zeggane, 2002), Ultraviolet/Visible Detectors (UVD) (Jimenez et al., 2000) and Fluorescence Detector (FLD) (Bernal et al., 1997). The detection technique that is gaining ground is mass spectrometry (MS) (Blasco et al., 2004; Chauzat et al., 2006; Fernandez et al., 2002). In particular, the mass spectrometer with a triple quadrupole is concerned the most suitable detector for pesticide and veterinary drug residue analysis. The above technique enables determination of the majority of active substances, combined with excellent sensitivity (LOQ of about 0.001 mg kg-1 for most of analyzed substances) and fewer requirements for the cleaning of the sample. The mass spectrometry is the ideal detector in conjunction with the QuEChERS method referred above. In each case, the high cost of the equipment and the need of qualified scientific staff should be noted. The packing material and the size of the column play an important role in the analysis with HPLC. The most widely used column is a C18 reverse phase with an internal diameter of 4.6 mm id. There are also columns with different packing material (C8, ODS, etc.) and columns with very small internal diameter (e.g. 2.1 mm id and 0.32 mm id), which help to increase the sensitivity and reduce the quantities of solvents used (Atienza et al., 1993). The mobile phase used in liquid chromatography is solvents such as water, methanol and acetonitrile or mixtures of them. Also, the adjustment of pH of the mobile phase plays an important role in the effectiveness of the method. In most cases a value of pH=9 is ideal for analysis of pesticide residues.

2008b; Menkissoglu-Spiroudi et al., 2000; Rissato et al., 2004).

• Flame Ionization (FID), to identify residual acaricides (Bernal et al., 2000). • Atomic Emission (AED), to detect acaricide residues (Jimenez et al., 1996).

1996; Bernal et al., 1996; Chauzat et al., 2006; Rissato et al., 2004).

et al., 1998b; Menkissoglu-Spiroudi et al., 2000).

volume and the number of samples, which can be analyzed daily.

2004).

pesticide residues.

**2.3.4.2 Liquid chromatography (LC)** 

• Electron Capture (ECD), to detect pyrethroid, organochlorine and organophosphorus insecticide and acaricide residues (Baltussen et al., 1999; Barakat et al., 2007; Jimenez et al., 1996; Jimenez et al., 1998a; Karazafiris et al.,

• Nitrogen-Phosphorus (NPD), to detect pyrethroid and organophosphorus insecticide and acaricide residues (Balayannis, 2001; Baltussen et al. 1999; Jimenez

• Mass Spectrometry (MSD), to detect pyrethroid, organophosphate, carbamate and organochlorine insecticide or acaricide residues (Albero et al., 2004; Baltussen et al.,

• Flame Photometric Detector (FPD) and Pulsed Flame Photometric Detector (PFPD), for detection of organophosphorus insecticide and acaricide residues (Yu et al.,

Each chromatographic system may include components that automate the process and provide valuable assistance to the analyst. The most important component in optimizing the analytical procedure is the autosampler. The performance of a chromatographic system is maximizing by the use of autosampler, while it improves the reproducibility of injection

Unlike gas chromatography, which is limited to determining the most volatile compounds, liquid chromatography is used for the isolation of a widespread group of compounds. These compounds may not be sufficiently volatile or heat-resistant to analysis by gas chromatography. The most common types of detectors used in liquid chromatography were Diode Array Detectors (DAD) (Atienza et al., 1993; Blasco et al., 2004; Jones & McCoy, 1997; Martel & Zeggane, 2002), Ultraviolet/Visible Detectors (UVD) (Jimenez et al., 2000) and Fluorescence Detector (FLD) (Bernal et al., 1997). The detection technique that is gaining ground is mass spectrometry (MS) (Blasco et al., 2004; Chauzat et al., 2006; Fernandez et al., 2002). In particular, the mass spectrometer with a triple quadrupole is concerned the most suitable detector for pesticide and veterinary drug residue analysis. The above technique enables determination of the majority of active substances, combined with excellent sensitivity (LOQ of about 0.001 mg kg-1 for most of analyzed substances) and fewer requirements for the cleaning of the sample. The mass spectrometry is the ideal detector in conjunction with the QuEChERS method referred above. In each case, the high cost of the equipment and the need of qualified scientific staff should be noted. The packing material and the size of the column play an important role in the analysis with HPLC. The most widely used column is a C18 reverse phase with an internal diameter of 4.6 mm id. There are also columns with different packing material (C8, ODS, etc.) and columns with very small internal diameter (e.g. 2.1 mm id and 0.32 mm id), which help to increase the sensitivity and reduce the quantities of solvents used (Atienza et al., 1993). The mobile phase used in liquid chromatography is solvents such as water, methanol and acetonitrile or mixtures of them. Also, the adjustment of pH of the mobile phase plays an important role in the effectiveness of the method. In most cases a value of pH=9 is ideal for analysis of

### **2.3.4.3 Thin layer chromatography (TLC)**

This technique is used primarily for detecting drugs in biological samples. However, TLC was used to determine pesticide residues in food. More generally, the TLC requires sample extraction with a solvent mixture and separation of the components into blocks with a suitable coating material (e.g. Silica gel). The next step is an elution with suitable solvents. Special equipment is necessary in order to achieve the visualization and quantification of results. The TLC was used by Rezic et al. (2005) to detect residues of herbicides atrazine and simazine in honey. The recovery rate was estimated at 92.3% and 94.2% for atrazine and simazine respectively. The TLC was used in the above study in conjunction with the use of ultrasound during the extraction.

#### **2.3.4.4 Matrix effect**

A fact that has to be mentioned is that differences in the chemical synthesis of bee products may affect the efficiency of extraction (Blasco et al., 2004; Yu et al., 2004) and the response of the chromatographic systems to analytes (Volante et al., 2001; Jimenez et al., 1998; Karazafiris et al., 2008b). This is the reason why, solutions for calibration curve and recoveries should be prepared in an extract of the same sample analyzed. Specifically, the analyst applies the chosen technique in honey or other hive products containing no detectable residues of analytes. The final extract is derived from the overall process used in the construction of standard calibration curves. If it is not possible to find sample with no residues, an analyst can use an extract of the sample that gives a response 30% over the reference value. The response may be due to the presence of the analyte or an interference eluting at the same retention time.
