**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 corresponding references.

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 respectively.


Table 2. Methods for the determination of volatile insecticides residues

Pesticide Residues in Bee Products 115

The acid-induced liquid-liquid phase separation of anionic surfactants in aqueous solutions and its applicability to cloud point extraction methodology were applied as a tool for the extraction of 1,4- dichlorobenzene (p-DCB) from aqueous honey samples. The analyte is extracted into the micelles of sodium dodecane sulfonate. For the separation of p-DCB a high-performance liquid chromatographic equipped with a UV detector system (225 nm) was used (Paleologos, et al. 2006). Dichlorobenzene and naphthalene residues in honey were investigated by solid-phase microextraction (SPME) coupled to gaschromatographic/mass spectrometry from Harizanis et. al (2008). The equilibration time and the sampling time for the extraction of the analytes by the fibre was 30 min and 60 min respectively, while the honey solution was kept at 60 °C. The LOD and LOQ for the pdichlorobenzene was 1 μg kg–1 and 5 μg kg–1, while for naphthalene 0.1 μg kg–1 and 1 μg kg–1

Tsimeli et al. (2008) developed a method for the determination of DBE, p-DCB and naphthalene based on SMPE extraction. Commercially available 100 μm film thickness polydimethylsiloxane (PDMS) fiber was employed for the extraction. The fibre was exposed to the headspace above the sample for 30 min, while the sample was kept at 40±2 °C and stirred at 900 rpm. The separation and detection are carried out using gas chromatography–

For the determination of naphthalene in honey, a high-performance liquid chromatography with a diode array detector method was also used. The compound was detected at 220 nm and the limit of detection and the limit of quantification were 0.023 μg g-1 and 0.078 μg g-1

The p-DCB molecules were extracted from the bee wax with ethanol and the sample clean up was accomplished by solid-phase extraction (C18 columns), while the determination was achieved by capillary GC and FID detector. The detection limits of the method were 0.7 mg kg-1 while average recovery was 74.8±5.5% (Bogdanov et al., 1998; 2004). For the isolation of p-DCB from the royal jelly a Purge and Trap system was used (Tananaki et al., 2009). The molecules of this compound extracted from the aqua royal jelly solution by He purging at 40 ml min-1 for 40 min keeping the sample temperature at 40 oC and were absorbed on Tenax resin. For the separation a fused silica capillary column (HP-5MS) has been used, while the detection was achieved using a mass spectrometer detector. The LOD and LOQ of

To maximize the production of agricultural products, extended amount of insecticides, herbicides, fungicides and bactericides are used which eventually lead to contamination of water, soil, crops, animals, even humans. Many environmental studies are concerned with the bioavailability of these pollutants and their subsequent introduction into food chain. Pesticides, Persistent Organic Pollutants (OCs, PCBs, PBBs), toxins and heavy metals have been investigated worldwide as substances that contaminate man's food. Chemicals contaminate hive products like honey, wax, pollen, propolis and royal jelly, while residues may exceed the established MRLs, either because of the improper use of the products or the

Honeybees forage over a circular area, with radius more than 6 Km, visiting numerous plant species and various sources of water and are notorious for collecting materials contaminated with chemicals and bringing them back to the hive. In anyway, pollutants may reach the

mass spectrometry (GC/MS) in selected ion monitoring mode (SIM).

respectively (Beyoğlu & Omurtag 2007).

the method was 0.3 μg kg-1 and 0.9 μg kg-1 respectively.

utilization of unauthorized products by the beekeepers.

respectively.

**3. Conclusion** 

Table 2. Methods for the determination of volatile insecticides residues

**Bee product Analytes Isolation Determination Analytical information References** Head space sampling GC – MS Column: Rtx-624 LOD: 1 μg kg-1

Honey p-DCB SPME (PDMS-fiber) GC - MS Column: J & W DB5ms LOD: 1 μg kg-1

Bogdanov et al., 2004 DBE, p-DCB, naphthalene Purge & Trap (Tenax TA) GC - MS Column HP-5MS LOD: 0.5, 0.15, 0.05 μg kg-1

LOQ: 2.4, 0.5, 0.125μg kg-1

18 LOD: 2.5 μg kg-1 LOQ: 7.5 μg kg-1

Paleologos et al., 2006 p-DCB, naphthalene SPME (VB/carboxen/PDMS) GC - MS Column: J & W DB5ms LOD: 1, 0.1 μg kg-1

LOQ: 5, 1 μg kg-

Harizanis et. al., 2008 DBE, p-DCB, naphthalene HS-SPME (PDMS) GC - MS Column: DB5 LOD: 2, 1, 0.1 μg kg-1

LOQ: 5, 4, 0,3 μg kg-1

Tsimeli et al., 2008 Naphthalene HPLC-DAD GC-MS LOD: 0.023 μg kg-1

LOQ: 0.078 μg kg-1

Beyoğlu and Omurtag 2007 Beeswax p-DCB ethanol, SPE (C18) GC - MS LOD: 0.7 mg kg-1

Royal jelly p-DCB Purge & Trap (Tenax TA) GC - MS Column HP-5MS

LOD: 0.3 μg kg-1

LOQ: 0.9 μg kg-1

Bogdanov et al., 2004

Tananaki

et al, 2009

Tananaki et al, 2005 p-DCB Acid-induced liquid-liquid phase separation of anionic surfactants HPLC - UV Column : LiChrospher-100 RP-

The acid-induced liquid-liquid phase separation of anionic surfactants in aqueous solutions and its applicability to cloud point extraction methodology were applied as a tool for the extraction of 1,4- dichlorobenzene (p-DCB) from aqueous honey samples. The analyte is extracted into the micelles of sodium dodecane sulfonate. For the separation of p-DCB a high-performance liquid chromatographic equipped with a UV detector system (225 nm) was used (Paleologos, et al. 2006). Dichlorobenzene and naphthalene residues in honey were investigated by solid-phase microextraction (SPME) coupled to gaschromatographic/mass spectrometry from Harizanis et. al (2008). The equilibration time and the sampling time for the extraction of the analytes by the fibre was 30 min and 60 min respectively, while the honey solution was kept at 60 °C. The LOD and LOQ for the pdichlorobenzene was 1 μg kg–1 and 5 μg kg–1, while for naphthalene 0.1 μg kg–1 and 1 μg kg–1 respectively.

Tsimeli et al. (2008) developed a method for the determination of DBE, p-DCB and naphthalene based on SMPE extraction. Commercially available 100 μm film thickness polydimethylsiloxane (PDMS) fiber was employed for the extraction. The fibre was exposed to the headspace above the sample for 30 min, while the sample was kept at 40±2 °C and stirred at 900 rpm. The separation and detection are carried out using gas chromatography– mass spectrometry (GC/MS) in selected ion monitoring mode (SIM).

For the determination of naphthalene in honey, a high-performance liquid chromatography with a diode array detector method was also used. The compound was detected at 220 nm and the limit of detection and the limit of quantification were 0.023 μg g-1 and 0.078 μg g-1 respectively (Beyoğlu & Omurtag 2007).

The p-DCB molecules were extracted from the bee wax with ethanol and the sample clean up was accomplished by solid-phase extraction (C18 columns), while the determination was achieved by capillary GC and FID detector. The detection limits of the method were 0.7 mg kg-1 while average recovery was 74.8±5.5% (Bogdanov et al., 1998; 2004). For the isolation of p-DCB from the royal jelly a Purge and Trap system was used (Tananaki et al., 2009). The molecules of this compound extracted from the aqua royal jelly solution by He purging at 40 ml min-1 for 40 min keeping the sample temperature at 40 oC and were absorbed on Tenax resin. For the separation a fused silica capillary column (HP-5MS) has been used, while the detection was achieved using a mass spectrometer detector. The LOD and LOQ of the method was 0.3 μg kg-1 and 0.9 μg kg-1 respectively.
