was blended with adsorbents such as C18 to be packed in a disposable plastic column. It was then extracted with a suitable organic solvent (Barker et al., 1989). The method has been applied to the extraction of pesticides, pharmaceuticals, and antibiotics since then (Barker, 2000a, 2000b). MSPD is also applied to the extraction of neonicotinoid insecticides from crop, honey, and fruit juice samples (Blasco et al., 2002a; Pous et al., 2001; Radišić et al., 2009; Totti et al., 2006).

In any event, the first obstacle in the development of analytical methods for pesticide residues is to secure extraction efficiency from measurement samples. It is probably important to select extraction conditions that are suitable for the physicochemical features of the target pesticide and for the characteristics of the measurement sample.

In the process of (2) clean-up, classical liquid–liquid partitioning and column chromatography have been used. However, because of concern over the health impact of the use of organic solvents in large quantities on analytical staff as well as the environmental load, the strong need exists for the reduction of organic solvents used in sample pretreatment procedures (Wan & Wong, 1996). SPE packed with widely various adsorbents such as silica gel, Florisil, C18, polymeric materials, graphitized carbon black (GCB), and ion exchange resin has come into common use these days, making a great contribution to reduction of the use of organic solvents (Fritz & Macka, 2000).

In the 2000s, marked technical innovation in analytical instruments has taken place: LC-MS and LC-MS/MS have been brought into use for the determination of neonicotinoid insecticides. These methods have higher measurement sensitivity than classical HPLC-UV and HPLC-DAD. Moreover, in many cases, sample pre-treatment procedures have reportedly included only the extraction and dilution of the extract, with subsequent measurement, without even the need for additional clean-up (Table 1). The most common extraction procedure is the following: a measurement sample is homogenized; then it is dehydrated and extracted with ethyl acetate and anhydrous sodium sulfate with subsequent solvent evaporation and then LC-MS determination (Blasco et al., 2002a, 2002b; Fernández-Alba et al., 2000) or LC-MS/MS determination (Agüera et al., 2004; Jansson et al., 2004; Venkateswarlu et al., 2007).

In 2003, Anastassiades et al. (2003) reported fast, simple and easy sample pre-treatment procedures by extraction with acetonitrile and dispersive SPE. The method that was presented is known as a quick, easy, cheap, effective, robust, and safe (QuEChERS) method, which is a breakthrough in which the sample pre-treatment is completed via acetonitrile extraction, dehydration, and salting-out with anhydrous magnesium sulfate and sodium chloride. Thereafter, dehydration and clean-up of the extract are done using dispersive SPE with anhydrous magnesium sulfate and primary secondary amine (PSA), which is a weak anion exchange adsorbent. Reportedly dispersive SPE using PSA is extremely effective for the removal of organic acids, polar dye components, and saccharides (Anastassiades et al., 2003). QuEChERS is very different from general sample pre-treatment procedures by organic solvent extraction, re-extraction with the organic solvent phase, and clean-up with SPE in that no process of concentration (evaporation of solvents) is needed. The concentration process, a procedure that must be conducted after processes such as extraction and clean-up, unexpectedly accounts for a large percentage of the sample pre-treatment time. Therefore, QuEChERS is a technology that contributes much to speeding up and simplification of sample pre-treatment procedures. In addition, the amounts of organic solvents used are extremely as small: about 10 mL of acetonitrile per sample. For those reasons, it can be concluded that the technology has met all of the requirements described above (Wan & Wong, 1996).

was blended with adsorbents such as C18 to be packed in a disposable plastic column. It was then extracted with a suitable organic solvent (Barker et al., 1989). The method has been applied to the extraction of pesticides, pharmaceuticals, and antibiotics since then (Barker, 2000a, 2000b). MSPD is also applied to the extraction of neonicotinoid insecticides from crop, honey, and fruit juice samples (Blasco et al., 2002a; Pous et al., 2001; Radišić et al., 2009;

In any event, the first obstacle in the development of analytical methods for pesticide residues is to secure extraction efficiency from measurement samples. It is probably important to select extraction conditions that are suitable for the physicochemical features of

In the process of (2) clean-up, classical liquid–liquid partitioning and column chromatography have been used. However, because of concern over the health impact of the use of organic solvents in large quantities on analytical staff as well as the environmental load, the strong need exists for the reduction of organic solvents used in sample pretreatment procedures (Wan & Wong, 1996). SPE packed with widely various adsorbents such as silica gel, Florisil, C18, polymeric materials, graphitized carbon black (GCB), and ion exchange resin has come into common use these days, making a great contribution to

In the 2000s, marked technical innovation in analytical instruments has taken place: LC-MS and LC-MS/MS have been brought into use for the determination of neonicotinoid insecticides. These methods have higher measurement sensitivity than classical HPLC-UV and HPLC-DAD. Moreover, in many cases, sample pre-treatment procedures have reportedly included only the extraction and dilution of the extract, with subsequent measurement, without even the need for additional clean-up (Table 1). The most common extraction procedure is the following: a measurement sample is homogenized; then it is dehydrated and extracted with ethyl acetate and anhydrous sodium sulfate with subsequent solvent evaporation and then LC-MS determination (Blasco et al., 2002a, 2002b; Fernández-Alba et al., 2000) or LC-MS/MS determination (Agüera et al., 2004; Jansson et al., 2004;

In 2003, Anastassiades et al. (2003) reported fast, simple and easy sample pre-treatment procedures by extraction with acetonitrile and dispersive SPE. The method that was presented is known as a quick, easy, cheap, effective, robust, and safe (QuEChERS) method, which is a breakthrough in which the sample pre-treatment is completed via acetonitrile extraction, dehydration, and salting-out with anhydrous magnesium sulfate and sodium chloride. Thereafter, dehydration and clean-up of the extract are done using dispersive SPE with anhydrous magnesium sulfate and primary secondary amine (PSA), which is a weak anion exchange adsorbent. Reportedly dispersive SPE using PSA is extremely effective for the removal of organic acids, polar dye components, and saccharides (Anastassiades et al., 2003). QuEChERS is very different from general sample pre-treatment procedures by organic solvent extraction, re-extraction with the organic solvent phase, and clean-up with SPE in that no process of concentration (evaporation of solvents) is needed. The concentration process, a procedure that must be conducted after processes such as extraction and clean-up, unexpectedly accounts for a large percentage of the sample pre-treatment time. Therefore, QuEChERS is a technology that contributes much to speeding up and simplification of sample pre-treatment procedures. In addition, the amounts of organic solvents used are extremely as small: about 10 mL of acetonitrile per sample. For those reasons, it can be concluded that the technology has met all of the requirements described

the target pesticide and for the characteristics of the measurement sample.

reduction of the use of organic solvents (Fritz & Macka, 2000).

Totti et al., 2006).

Venkateswarlu et al., 2007).

above (Wan & Wong, 1996).

Review on Current Analytical Methods with Chromatographic

Imidacloprid Tobacco leaf Ultrasonic extraction with ethyl

Thiamethoxam Honeybee Ultrasonic extraction with

Grape, cantaloupe, strawberry, cucumber, tomatillo, lettuce and bell pepper

Bee and filter paper

Water

Apple, carrot, cucumber, grape, peach, sweet pepper, spinach and tomato

Bovine milk

Coated seeds of wheat and corn

Orange juice, apple juice and mixture of pineapple and pear juice

> Pear and tomato

Strawberry, orange, potato, pear and melon

Imidacloprid

Imidacloprid and 2 metabolites

Acetamiprid, imidacloprid and thiamethoxam

Imidacloprid and 6 chloronicotinic acid

Acetamiprid, clothianidin, dinotefuran, imidacloprid, nitenpyram, thiacloprid and thiamethoxam

Acetamiprid, imidacloprid, thiacloprid and thiamethoxam

5 pesticides including imidacloprid

Imidacloprid

Acetamiprid, imidacloprid and thiacloprid

5 pesticides including imidacloprid

5 pesticides including imidacloprid

and Nonchromatographic Techniques for New Generation Insecticide Neonicotinoids 487

NH2 and Florisil SPE

Liquid–liquid

None

Hydromatrix SPE packed with diatomaceous earth material

Liquid–liquid

Chem Elut SPE packed with diatomaceous earth material and GCB/NH2 SPE

acetonitrile None HPLC-UV Bourgin et al., 2009 ASE with acetonitrile

> Dilution of 0.05 M sodium dodecyl sulfate

acetone/water (80:20, v/v) C18 SPE HPLC-UV Mohan et al.,

2006b Maize leaf

acetone None HPLC-ECD Rancan et al.,

HPLC-nitrogen chemiluminescent detector and DAD

partition HPLC-UV Liu et al.,

None HPLC-UV Zhou et al.,

partition HPLC-FLD García et al.,

None HPLC-DAD Seccia et al.,

None LC-MS Fernández-Alba

None LC-MS Pous et al., 2001

Ting et al., 2004

2005

2006a

2006

2007

al., 2007

2008

HPLC-DAD Chin-Chen et al., 2009

2010

et al., 2000

HPLC-DAD Watanabe et

HPLC-ECD Rancan et al.,

Homogenization with ethyl acetate and anhydrous Na2SO4

acetate

Ultrasonic extraction with acetone

Extraction with methanol from SPE packed with multiwalled carbon nanotubes

acetone

Mechanical shaking with acetone

Extraction with dichloromethane from Chem Elut SPE packed with diatomaceous earth material

Ultrasonic extraction with

None

Homogenization with ethyl acetate and anhydrous Na2SO4

Extraction with dichloromethane from MSPD with C8

HPLC equipped with MS or tandem MS

Analyte(s) Sample(s) Extraction Clean-up Determination Ref.

10 pesticides Orange Extraction with None LC-MS Blasco et al.,

Cotton seed Mechanical shaking with

Honeybee Ultrasonic extraction with

HPLC equipped with detectors except for MS or tandem MS Analyte(s) Sample(s) Extraction Clean-up Determination Ref.

C18 SPE None

acetonitrile/water (80:20, v/v) None

al., 1997 Soil Mechanical shaking with

Liquid–liquid partition and silica gel column chromatography

Liquid–liquid partition and C18 SPE

None

Liquid–liquid partition and Extrelut SPE packed with diatomaceous

C18 SPE and liquid–liquid partition

PSA SPE and

Liquid–liquid

dichloromethane None HPLC-DAD Galera et al.,

Homogenization with acetonitrile/water (80:20, v/v)

acetonitrile/water (80:20, v/v)

Homogenization with acetone

Mechanical shaking with acetonitrile/methanol/water (3:2:2, v/v)

Mechanical shaking with methanol/0.1 M NH4Cl (8:2, v/v)

Mechanical shaking with methanol/0.1 M NH4Cl (8:2, v/v) and methanol/0.5 M NaOH (8:2, v/v)

Trap with Amberlite XAD-2 and desorption with acetonitrile and phosphate buffer

> Homogenization with acetonitrile

Microwave-assisted extraction with acetone

Soil Ultrasonic extraction with

Water Extraction with methanol from

Groundwater Liquid–liquid extraction with

Imidacloprid

Imidacloprid

Imidacloprid

Imidacloprid and 6 chloronicotinic acid

Imidacloprid and 6 chloronicotinic acid

Acetamiprid and 2 metabolites

6 chloronicotinic acid

Imidacloprid and 6 chloronicotinic acid

Acetamiprid, imidacloprid and nitenpyram

3 pesticides including imidacloprid and thiamethoxam Japanese pear, apple, peach (pulp, peel), grape, radish (root, leaf), cucumber, eggplant, rice grain, rice green, rice straw and potato

Pepper, tomato and cucumber

Soil

Soil

Greenhouse air

Cucumber, potato, tomato, eggplant, Japanese radish and grape

Cabbage, tomato, chili, pepper and potato

al., 2003

HPLC-UV Ishii et al.,

HPLC-UV Baskaran et

HPLC-DAD

HPLC-pulsed amperometric detector

earth material HPLC-UV Tokieda et

None HPLC-DAD Frenich et al.,

silica gel SPE HPLC-DAD Obana et al.,

partition HPLC-UV Singh et al.,

1994

Fernandez-Alba et al., 1996

de Erenchun et al., 1997

1998

al., 1998

2000

2002

2004

Review on Current Analytical Methods with Chromatographic

Apricot, celery, courgette, peach and pear

Lemon, raisin, tomato and avocado

Honeybee

Potato, orange and cerealbased baby food

Honey

Green pepper, tomato, cucumber and orange

Raisin, red grape and red wine

Orange and wheat flour

Grape

Tomato, pear and orange

imidacloprid, thiacloprid and thiamethoxam Acetamiprid, imidacloprid, thiacloprid and thiamethoxam

52 pesticides including acetamiprid, imidacloprid and thiacloprid

6 pesticides and metabolites including imidacloprid

52 pesticides including acetamiprid, imidacloprid and thiacloprid

19 pesticides and metabolites including imidacloprid

101 pesticides and metabolites including acetamiprid, imidacloprid, nitenpyram and thiacloprid

42 pesticides including acetamiprid, clothianidin, dinotefuran, imidacloprid, nitenpyram, thiacloprid and thiamethoxam

10 pesticides including imidacloprid and thiamethoxam

160 pesticides including acetamiprid, dinotefuran, imidacloprid, nitenpyram, thiacloprid and thiamethoxam

and Nonchromatographic Techniques for New Generation Insecticide Neonicotinoids 489

Acetate buffered QuEChERS method

containing 1% acetic acid Dispersive PSA SPE

QuEChERS method

Shaking with acetonitrile Dispersive PSA SPE

acetate from C18 SPE None

QuEChERS method

acetonitrile None

QuEChERS method

11 pesticides Dried hop Homogenization with Polymeric styrene- LC-MS/MS Hengel &

acetonitrile Dispersive PSA SPE

2008 Shaking by hand with

acetonitrile Dispersive PSA SPE

HLB (hydrophilic– lipophilic balanced copolymer) SPE

2007 Shaking with acetonitrile

None LC-MS Di Muccio et

None LC-MS Totti et al., 2006

None LC-MS/MS Pirard et al.,

al., 2006

LC-MS/MS Hernández et al., 2006

UPLC-MS/MS Leandro et al.,

LC-TOF-MS Ferrer &

LC-MS/MS Payá et al., 2007

LC-MS/MS Kmellár et al.,

et al., 2007

None LC-MS/MS Venkateswarlu

2007

Thurman, 2007

Homogenization with acetone and extraction with dichloromethane from Extrelut SPE packed with diatomaceous earth material

Homogenization with methanol/water (80:20, v/v) containing 0.1% formic acid

Extraction with dichloromethane/methanol (85:15, v/v) from MSPD with C18

Extraction with ethyl acetate from Chem Elut SPE packed with diatomaceous earth material

Shaking by hand with

Shaking by hand with

Homogenization with ethyl acetate and anhydrous Na2SO4

Water Extraction with ethyl

2002a

2002b

2003

LC-MS/MS Bonmatin et al., 2003

2004

2004

2004

2005

2005

2005

LC-MS/MS Lehotay et al.,

None LC-MS/MS Jansson et al.,

None LC-MS/MS Sannino et al.,

None LC-MS/MS Agüera et al.,

None LC-MS Fidente et al.,

None LC-MS Seccia et al.,

acetate None LC-TOF-MS Ferrer et al.,

2005 Shaking by hand with

None LC-MS Blasco et al.,

methanol GCB SPE LC-MS Obana et al.,

None

C18 SPE

dichloromethane from MSPD with C8

Stir bar sorptive extraction after homogenization with methanol and water Homogenization with ethyl acetate and anhydrous Na2SO4

Homogenization with ethyl acetate and anhydrous Na2SO4

Homogenization with

Mixing with methanol/0.05% NH4OH (3:1, v/v)

Grinding with methanol/0.05% H2SO4 (4:1, v/v)

Homogenization with ethyl acetate and anhydrous Na2SO4

Homogenization with acetone and ethyl acetate/cyclohexane (50:50, v/v)

Homogenization with ethyl acetate and anhydrous Na2SO4 under alkaline

Extraction with dichloromethane from Extrelut SPE packed with diatomaceous earth material

Extraction with ethyl acetate/methanol (50:50, v/v) from poly(styrenedivinylbenzene) SPE

Homogenization with ethyl

QuEChERS method

acetonitrile Dispersive PSA SPE

(75:25, v/v) None

Pollen Mixing with ethanol/water

including imidacloprid

4 pesticides including imidacloprid

Acetamiprid, imidacloprid, nitenpyram, thiacloprid and thiamethoxam

Imidacloprid

57 pesticides including imidacloprid and acetamiprid

24 pesticides including imidacloprid

17 pesticides including acetamiprid, imidacloprid and thiacloprid

Acetamiprid, imidacloprid, thiacloprid and thiamethoxam

Acetamiprid, imidacloprid, thiacloprid and thiamethoxam

Acetamiprid, imidacloprid and thiacloprid

146 pesticides including acetamiprid,

Peach and nectarine

Bell pepper, cucumber, eggplant, grape, grapefruit, Japanese radish (root, leaf), peach, pear, potato, rice and tomato

Soil

Maize, rape, wheat and sunflower

Fruits and vegetables

Apple puree, concentrated lemon juice and tomato puree

Pepper, lettuce and eggplant

Honey

Drinking water

Cucumber, tomato, lettuce and pepper

> Lettuce and orange

Review on Current Analytical Methods with Chromatographic

spinach, apple, plum, watermelon, green bean, zucchini, broccoli, strawberry, grape and tomato

Wheat, cucumber and red wine

Tomato, pear and orange

Bovine muscle

Chestnut, shallot, ginger and tea

Greenhouse air

and metabolites including acetamiprid, clothianidin, dinotefuran, thiacloprid, thiamethoxam

93 pesticides and mycotoxins including acetamiprid, imidacloprid, thiacloprid and thiamethoxam

150 pesticides including acetamiprid, imidacloprid, nitenpyram, thiacloprid and thiamethoxam

70 pesticides including imidacloprid, thiacloprid and thiamethoxam

Acetamiprid, clothianidin, dinotefuran, imidacloprid, nitenpyram, thiacloprid and thiamethoxam

Acetamiprid, clothianidin, dinotefuran, imidacloprid, thiacloprid and thiamethoxam

Imidacloprid and 6-chloronicotinic acid

insecticides.

and Nonchromatographic Techniques for New Generation Insecticide Neonicotinoids 491

Acetate buffered QuEChERS method

Acetate buffered QuEChERS method

(optional), C18 SPE and Florisil SPE

None

Dispersive PSA SPE

Water None None LC-MS/MS Pareja et al.,

and liver ASE with water HLB SPE LC-MS/MS Xiao et al., 2011

Activated carbon

None

SPE and HLB SPE LC-MS/MS Xie et al., 2011

Capillary electrophoresis (micellar electrokinetic chromatography)- DAD

2010

Romero-González et al.,

2011

LC-MS/MS Kmellár et al., 2011

2011

Carretero et al., 2003

UPLC-MS/MS

acetonitrile containing 1% triethylamine

Vortex mixing with acetonitrile containing 1% acetic acid

Shaking by hand with acetonitrile containing 1% acetic acid

> Vortex mixing with acetonitrile

Trap with Amberlite XAD-2 and desorption with water

Table 1. Overview of chromatographic techniques for determination of neonicotinoid

With the presentation of QuEChERS as a turning point, many reports have described multiresidue analysis of various pesticides including neonicotinoid insecticides (Ferrer & Thurman, 2007; Frenich et al., 2008; Kamel, 2010; Kmellár et al., 2008, 2011; Leandro et al., 2007; Lehotay et al., 2005; Payá et al., 2007; Romero-González et al., 2011). Additionally,

Other chromatographic technique Analyte(s) Sample(s) Extraction Clean-up Determination Ref.

Review on Current Analytical Methods with Chromatographic and Nonchromatographic Techniques for New Generation Insecticide Neonicotinoids 491

490 Insecticides – Advances in Integrated Pest Management

Buffered QuEChERS method

Vortex mixing with acetonitrile containing 1% acetic acid

Vortex mixing with acetonitrile containing 1% acetic acid

Extraction with methanol

Ultrasonic extraction and shaking with methanol after soak in 1% acetic acid

Extraction with dichloromethane from MSPD with diatomaceous earth material

Extraction with methanol

Vortex mixing and homogenization with water, ethyl acetate/cyclohexane (9:1, v/v) and sodium chloride

acetonitrile

Vortex shaking with acetonitrile

Homogenization with water and acetonitrile containing 2% triethylamine

Ultrasonic extraction with acetonitrile containing 1% acetic acid

Modified QuEChERS method

22 pesticides Milk, orange, Homogenization with Salting out UPLC-MS/MS Kamel et al.,

Rice and tea Ultrasonic extraction with

acetonitrile divinylbenzene

methacrylate SPE and NH2 SPE

None

Florisil SPE

Chem Elut SPE packed with diatomaceous earth material and Florisil SPE

from HLB SPE None LC-MS/MS Economou et

from Florisil SPE None LC-MS/MS García-Chao et

Dispersive SPE with PSA, GCB and Florisil

C18 SPE

Dispersive SPE with

Miller, 2008

al., 2009

UPLC-MS/MS Frenich et al., 2008

LC-MS/MS Mayer-Helm, 2009

2009

al., 2010

LC-MS/MS Kanrar et al., 2010

LC-MS/MS Kamel, 2010

2010

None LC-MS/MS Radišić et al.,

HLB SPE UPLC-MS/MS Liu et al., 2010

PSA and GCB UPLC-MS/MS Zhang et al.,

including imidacloprid

53 pesticides including acetamiprid, imidacloprid, nitenpyram, thiacloprid and thiamethoxam

46 pesticides including acetamiprid and imidacloprid

52 pesticides including acetamiprid, imidacloprid and thiamethoxam

12 pesticides including acetamiprid

7 pesticides and metabolites including imidacloprid and thiamethoxam

42 pesticides including acetamiprid, clothianidin, imidacloprid, thiacloprid and thiamethoxam

Acetamiprid, clothianidin, dinotefuran, imidacloprid, nitenpyram, thiacloprid and thiamethoxam

Clothianidin, dinotefuran, imidacloprid, thiamethoxam and 8 metabolites

36 pesticides including acetamiprid and imidacloprid

Orange, strawberry and cucumber

Olive

White and red wines

Tobacco

Apple juice, peach juice, orange juice and raspberry juice

> Honey and pollen

Tea (made tea, tea infusion and spent leaf)

Apple, cabbage, potato, chicken, pork, milk and egg

Bee, bee pollen and bee honey

Tea

Table 1. Overview of chromatographic techniques for determination of neonicotinoid insecticides.

With the presentation of QuEChERS as a turning point, many reports have described multiresidue analysis of various pesticides including neonicotinoid insecticides (Ferrer & Thurman, 2007; Frenich et al., 2008; Kamel, 2010; Kmellár et al., 2008, 2011; Leandro et al., 2007; Lehotay et al., 2005; Payá et al., 2007; Romero-González et al., 2011). Additionally,

Review on Current Analytical Methods with Chromatographic

MS/MS, with which highly sensitive determination is possible.

treatment by QuEChERS and UPLC-MS/MS.

their respective chemical structures.

and Nonchromatographic Techniques for New Generation Insecticide Neonicotinoids 493

In the 2000s, a dramatic increase occurred in the number of cases reported on the residue pesticide analysis by HPLC equipped with quadrupole MS, ion-trap MS, tandem MS (MS/MS), and time-of-flight MS (TOF-MS). Utilization of MS enabled not only the detection of trace pesticide residues in various matrices with high accuracy but also the elucidation of

Obana et al. (2003) reported a method of extracting five neonicotinoid insecticides from 12 crop samples using methanol, clean-up with GCB SPE, and determination by LC-MS. They suggested that the analytical method that was developed was effective as a regular monitoring method. In determination methods including neonicotinoid insecticides by LC-MS/MS, good recovery is obtained in most reported cases. In one important case, quantification was achieved by direct injection of the filtered water sample in the LC-MS/MS determination of 70 pesticides including imidacloprid, thiacloprid, and thiamethoxam in paddy water samples (Pareja et al., 2011). It is exactly the benefit of LC-

Ultra-performance liquid chromatography (UPLC) was developed only a few years ago in which the mobile phase can be flown at a high pressure (about 15,000 psi) using a short column of about 50 mm packed with C18 of particle size of not more than 2 m. Its application to pesticide residue analysis has been examined because the utilization of UPLC enables not only the achievement of highly sensitive determination, but also improved high throughput attributable to reduced measurement time as well as substantially smaller amounts of organic solvents (mobile phase) used than in conventional HPLC (Frenich et al., 2008; Kamel et al., 2010; Leandro et al., 2007; Liu et al., 2010; Romero-González et al., 2011; Zhang et al., 2010). Liu et al. (2010) used UPLC-MS/MS to construct simultaneous analyses of seven neonicotinoid insecticides in crops and livestock products, obtaining good recovery except for the lower recovery of nitenpyram (not more than 70%) in some of samples including potatoes and cabbages. Romero-González et al. (2011) constructed highly sensitive ultra-rapid analysis of more than 90 pesticides including acetamiprid, imidacloprid, thiacloprid, and thiamethoxam as well as mycotoxins by the combination of sample pre-

As described above, the trend in the development of residue analysis of various pesticides including neonicotinoid insecticides by LC-MS and LC-MS/MS can be summarized via the overview of several cases. LC-MS and LC-MS/MS are suitable for highly sensitive determination of only slightly volatile and heat-unstable pesticides. They enable quantification only with extremely simple and rapid sample pre-treatment compared to those of HPLC-UV, HPLC-DAD, and GC with an element-selective detector, which requires complicated sample pre-treatment procedures. Particularly MS/MS can be characterized as an effective method for the structural analysis of the target pesticide and its confirmation because much chemical information can be acquired by obtaining product ions from the precursor ion. Moreover, little interference occurs by matrix components because ions can be selected at will, thereby enabling highly sensitive determination. Recently, newly marketed insecticides tend to be included in the subjects for HPLC determination.

Therefore, increasing need is expected for LC-MS and LC-MS/MS in the future.

pesticide residues that might be present in various samples.

Although many advantages of LC-MS and LC-MS/MS are described above, the matrix effect must also be noted, which is a problem in the chromatographic determination of

reagents and adsorbents for QuEChERS are marketed as kits, implying that the method is extremely practical as multiresidue analysis for pesticide residues.

#### **2.2 Determination of neonicotinoid insecticides based on chromatographic methods**

Chromatographic determination of neonicotinoid insecticides are classifiable into two groups that use HPLC or GC (Table 1). However, because neonicotinoid insecticides are generally degraded by heat, additional processes such as derivatization are necessary for GC determination, making sample pre-treatment procedures more complicated than HPLC determination. Vilchez et al. (1996) and Navalón et al. (1997) used a hydrolyzed compound of imidacloprid [1–(6-chloro-3-pyridylmethyl)imidazolidin-2-one] formed by heat treatment of a measurement sample under alkaline conditions for GC–MS determination of imidacloprid in soil, water, and crops. MacDonald and Meyer (1998) extracted imidacloprid from white pine with water-containing methanol under acidic conditions with diluted sulfuric acid, cleaned up with C18 SPE and a self-prepared Florisil minicolumn, heptafluorobutyryl-derivatized, and then subjected to GC–MS determination.

As presented above, it is presumed that utilization of HPLC rather than GC is more advantageous for the determination of neonicotinoid insecticides from the viewpoints of speedup and simplification of sample pre-treatment procedures. It also is readily apparent that most cases reported to date used HPLC for determination (Table 1). In determination by HPLC, both HPLC-UV and HPLC-DAD contributed greatly to analyses of neonicotinoid insecticides until the 2000s, when MS and MS/MS began to become popularly used. Since the report by Ishii et al. (1994), HPLC-UV and HPLC-DAD have been applied to the determination of neonicotinoid insecticides in various matrices (Table 1). In addition to UV and DAD, methods were developed in which imidacloprid and thiamethoxam were separated using a column and then converted into electrochemically active compounds by ultraviolet irradiation to be detected by an electrochemical detector (ECD) (Rancan et al., 2006a, 2006b), or converted into fluorescent substances to be detected using a fluorescence detector (FLD) (García et al., 2007). ECD and FLD are generally more sensitive than UV or DAD. They are applied to residue analysis in the bodies of bees, where determination at low concentrations must be done.

In fact, HPLC-UV and DAD have less measurement sensitivity and selectivity than either LC-MS or MS/MS, which are the most widely used methods today. Therefore, thorough clean-up is indispensable for the pre-treatment procedures of samples consisting of complicated matrices such as crops. Liquid–liquid partition has been used since the initial phase of the market release of imidacloprid (Fernandez-Alba et al., 1996; García et al., 2007; Ishii et al., 1994; Liu et al., 2005; Singh et al., 2004). However, that technique presents problems: e.g. a large amount of organic solvent is used; and an emulsion is formed at the liquid–liquid interface depending on the extraction sample that is used. For that reason, it has been increasingly replaced by clean-up mainly by SPE (Mohan et al., 2010; Obana et al., 2002; Ting et al., 2004; Watanabe et al., 2007). Watanabe et al. (2007) used re-extraction with diatomaceous earth SPE and clean-up with GCB/NH2 SPE in the development of simultaneous analysis by HPLC-DAD of seven neonicotinoid insecticides released on the market. However, the recovery of nitenpyram was not satisfactory (not more than 40%). According to their discussion, matrix components in the sample affected nitenpyram in some way, leading to the factor of reduced recovery.

reagents and adsorbents for QuEChERS are marketed as kits, implying that the method is

**2.2 Determination of neonicotinoid insecticides based on chromatographic methods**  Chromatographic determination of neonicotinoid insecticides are classifiable into two groups that use HPLC or GC (Table 1). However, because neonicotinoid insecticides are generally degraded by heat, additional processes such as derivatization are necessary for GC determination, making sample pre-treatment procedures more complicated than HPLC determination. Vilchez et al. (1996) and Navalón et al. (1997) used a hydrolyzed compound of imidacloprid [1–(6-chloro-3-pyridylmethyl)imidazolidin-2-one] formed by heat treatment of a measurement sample under alkaline conditions for GC–MS determination of imidacloprid in soil, water, and crops. MacDonald and Meyer (1998) extracted imidacloprid from white pine with water-containing methanol under acidic conditions with diluted sulfuric acid, cleaned up with C18 SPE and a self-prepared Florisil minicolumn,

As presented above, it is presumed that utilization of HPLC rather than GC is more advantageous for the determination of neonicotinoid insecticides from the viewpoints of speedup and simplification of sample pre-treatment procedures. It also is readily apparent that most cases reported to date used HPLC for determination (Table 1). In determination by HPLC, both HPLC-UV and HPLC-DAD contributed greatly to analyses of neonicotinoid insecticides until the 2000s, when MS and MS/MS began to become popularly used. Since the report by Ishii et al. (1994), HPLC-UV and HPLC-DAD have been applied to the determination of neonicotinoid insecticides in various matrices (Table 1). In addition to UV and DAD, methods were developed in which imidacloprid and thiamethoxam were separated using a column and then converted into electrochemically active compounds by ultraviolet irradiation to be detected by an electrochemical detector (ECD) (Rancan et al., 2006a, 2006b), or converted into fluorescent substances to be detected using a fluorescence detector (FLD) (García et al., 2007). ECD and FLD are generally more sensitive than UV or DAD. They are applied to residue analysis in the bodies of bees, where determination at low

In fact, HPLC-UV and DAD have less measurement sensitivity and selectivity than either LC-MS or MS/MS, which are the most widely used methods today. Therefore, thorough clean-up is indispensable for the pre-treatment procedures of samples consisting of complicated matrices such as crops. Liquid–liquid partition has been used since the initial phase of the market release of imidacloprid (Fernandez-Alba et al., 1996; García et al., 2007; Ishii et al., 1994; Liu et al., 2005; Singh et al., 2004). However, that technique presents problems: e.g. a large amount of organic solvent is used; and an emulsion is formed at the liquid–liquid interface depending on the extraction sample that is used. For that reason, it has been increasingly replaced by clean-up mainly by SPE (Mohan et al., 2010; Obana et al., 2002; Ting et al., 2004; Watanabe et al., 2007). Watanabe et al. (2007) used re-extraction with diatomaceous earth SPE and clean-up with GCB/NH2 SPE in the development of simultaneous analysis by HPLC-DAD of seven neonicotinoid insecticides released on the market. However, the recovery of nitenpyram was not satisfactory (not more than 40%). According to their discussion, matrix components in the sample affected nitenpyram in

heptafluorobutyryl-derivatized, and then subjected to GC–MS determination.

concentrations must be done.

some way, leading to the factor of reduced recovery.

extremely practical as multiresidue analysis for pesticide residues.

In the 2000s, a dramatic increase occurred in the number of cases reported on the residue pesticide analysis by HPLC equipped with quadrupole MS, ion-trap MS, tandem MS (MS/MS), and time-of-flight MS (TOF-MS). Utilization of MS enabled not only the detection of trace pesticide residues in various matrices with high accuracy but also the elucidation of their respective chemical structures.

Obana et al. (2003) reported a method of extracting five neonicotinoid insecticides from 12 crop samples using methanol, clean-up with GCB SPE, and determination by LC-MS. They suggested that the analytical method that was developed was effective as a regular monitoring method. In determination methods including neonicotinoid insecticides by LC-MS/MS, good recovery is obtained in most reported cases. In one important case, quantification was achieved by direct injection of the filtered water sample in the LC-MS/MS determination of 70 pesticides including imidacloprid, thiacloprid, and thiamethoxam in paddy water samples (Pareja et al., 2011). It is exactly the benefit of LC-MS/MS, with which highly sensitive determination is possible.

Ultra-performance liquid chromatography (UPLC) was developed only a few years ago in which the mobile phase can be flown at a high pressure (about 15,000 psi) using a short column of about 50 mm packed with C18 of particle size of not more than 2 m. Its application to pesticide residue analysis has been examined because the utilization of UPLC enables not only the achievement of highly sensitive determination, but also improved high throughput attributable to reduced measurement time as well as substantially smaller amounts of organic solvents (mobile phase) used than in conventional HPLC (Frenich et al., 2008; Kamel et al., 2010; Leandro et al., 2007; Liu et al., 2010; Romero-González et al., 2011; Zhang et al., 2010). Liu et al. (2010) used UPLC-MS/MS to construct simultaneous analyses of seven neonicotinoid insecticides in crops and livestock products, obtaining good recovery except for the lower recovery of nitenpyram (not more than 70%) in some of samples including potatoes and cabbages. Romero-González et al. (2011) constructed highly sensitive ultra-rapid analysis of more than 90 pesticides including acetamiprid, imidacloprid, thiacloprid, and thiamethoxam as well as mycotoxins by the combination of sample pretreatment by QuEChERS and UPLC-MS/MS.

As described above, the trend in the development of residue analysis of various pesticides including neonicotinoid insecticides by LC-MS and LC-MS/MS can be summarized via the overview of several cases. LC-MS and LC-MS/MS are suitable for highly sensitive determination of only slightly volatile and heat-unstable pesticides. They enable quantification only with extremely simple and rapid sample pre-treatment compared to those of HPLC-UV, HPLC-DAD, and GC with an element-selective detector, which requires complicated sample pre-treatment procedures. Particularly MS/MS can be characterized as an effective method for the structural analysis of the target pesticide and its confirmation because much chemical information can be acquired by obtaining product ions from the precursor ion. Moreover, little interference occurs by matrix components because ions can be selected at will, thereby enabling highly sensitive determination. Recently, newly marketed insecticides tend to be included in the subjects for HPLC determination. Therefore, increasing need is expected for LC-MS and LC-MS/MS in the future.

Although many advantages of LC-MS and LC-MS/MS are described above, the matrix effect must also be noted, which is a problem in the chromatographic determination of pesticide residues that might be present in various samples.

Review on Current Analytical Methods with Chromatographic

pesticide residues, especially in liquid samples.

et al., 2011).

method.

Imidacloprid

16 pesticides and metabolite including nitenpyram and thiacloprid

neonicotinoid insecticides.

Imidacloprid Water

Water, hemlock xylem fluid and grape

Orange, lemon, apple, green pepper, persimmon, grapefruit, tomato, pear and grape

Fruir and vegetable skin (peel)

and Nonchromatographic Techniques for New Generation Insecticide Neonicotinoids 495

FIA are generally inexpensive. Furthermore, the method is known to be capable of rapid, easy, and highly sensitive detection of trace substances (Lara et al., 2010; Llorent-Martínez

Reports on the determination of neonicotinoid insecticides by FIA have targeted imidacloprid to date. In all such studies, methods are constructed by which a measurement sample is irradiated with ultraviolet light for conversion into a fluorescent substance [1–(6 chloro-3-pyridylmethyl)-2-(hydroxyimino)-3,4-didehydroimidalozolidene] which is detected using a spectrofluorometer (Flores et al., 2007; Vílchez et al., 2001). Alternatively, nitrite is detached from imidacloprid to be reduced into nitric oxide by iodide, which is detected by chemiluminescence detection with ozone (Lagalante & Greenbacker, 2007). All FIA methods show measurement sensitivity that is equal to or better than the detection limit of HPLC or GC. For the determination of liquid samples such as water, direct injection is possible with filtration only (Flores et al., 2007; Lagalante & Greenbacker, 2007). Actually, FIA is regarded as making a great contribution to fast, simple, and easy determination of

García-Reyes et al. (2009) reported rapid in situ qualitative and quantitative analysis of 16 pesticides including nitenpyram and thiacloprid by desorption electrospray ionization MS (DESI-MS) and MS/MS (DESI-MS/MS). In their determination, crop samples were pretreated according to QuEChERS and the resulting sample solution was applied on the PTFE surface, while the skin of fruit and vegetable samples were fixed on glass slides, and electrospray was applied directly to the sample to ionize target pesticides in the sample. The measurement sensitivity was extremely high: on the order of g/kg, and the measurement accuracy was comparable to LC-MS. Although such an analytical method remains under development at present, it can be anticipated as a new, highly sensitive, and rapid screening

Analyte(s) Sample(s) Extraction Clean-up Determination Ref.

Only filtration None

QuEChERS method

None None

Table 2. Overview of nonchromatographic techniques with FIA and MS for determination of

None

Dispersive PSA SPE

FIA with photochemically induced fluorescence detection

FIA with chemiluminescence detection

FIA with photochemically induced fluorescence detection

Desorption ESI-MS and MS/MS

Vílchez et al., 2001

Lagalante & Greenbacker, 2007

Flores et al., 2007

García-Reyes et al., 2009

Liquid–liquid extraction with dichloromethane

> Shaking with acetonitrile

Honey Only dilution

Imidacloprid Water Only filtration None

It has been pointed out that, as in GC-ECD and GC-FPD determination, quantitative determination is not possible because of the matrix effect when the sample clean-up is insufficient. Lee and Wylie (1991) have reported interesting observations by which the susceptibility (to the matrix effect) of several GC detectors is examined for individual crop samples. The matrix effect is a phenomenon that is also observed in LC-MS and LC-MS/MS determination; it was shown earlier that when the target pesticide is eluted together with matrix components in the sample, ion suppression or ion enhancement occurs during the ionization process, engendering error in the determination result (Niessen et al., 2006). The matrix effect can be avoided using a matrix-matched standard method or using isotope dilution with isotopically labeled internal standards (Niessen et al., 2006), in addition to thorough sample clean-up.

Xie et al. (2011) reported observation of ion enhancement in dinotefuran, imidacloprid, and thiacloprid, as well as ion suppression in acetamiprid, clothianidin, and thiamethoxam by the matrix effect, and that acetamiprid and thiamethoxam among these were significantly affected. However, because the recovery was improved by correction using the matrixmatched standard method, they emphasized the effectiveness of the method in the avoidance of the matrix effect.

In any event, the matrix effect should be regarded as a common problem of pesticide residue analysis by HPLC and GC; it goes without saying that it is most important to evaluate the matrix effect that might be derived from the subject sample when the development of a new analytical method is attempted or when an established, existing analytical method is applied.

#### **3. Analytical methods based on nonchromatographic techniques**

Section 2 summarized the trends up to now in the development of the determination of neonicotinoid insecticides by chromatography. This section refers to analytical methods based on nonchromatographic techniques. The list in Table 2 shows that major nonchromatographic methods include flow injection analysis (FIA), direct MS analysis, and enzyme-linked immunosorbent assay (ELISA), which is an immunochemical determination using an antigen–antibody reaction with high specificity or selectivity. This section summarizes the trend and the current situation of the development of analytical methods for neonicotinoid insecticides using these.

#### **3.1 FIA or MS analysis for direct determination of neonicotinoid insecticides**

FIA is a method by which a predetermined amount of a sample solution is injected into carrier solution that flows continuously in a tube, and the target substance is detected or quantified using a detector, as might be done after a chemical reaction. Instruments used in

It has been pointed out that, as in GC-ECD and GC-FPD determination, quantitative determination is not possible because of the matrix effect when the sample clean-up is insufficient. Lee and Wylie (1991) have reported interesting observations by which the susceptibility (to the matrix effect) of several GC detectors is examined for individual crop samples. The matrix effect is a phenomenon that is also observed in LC-MS and LC-MS/MS determination; it was shown earlier that when the target pesticide is eluted together with matrix components in the sample, ion suppression or ion enhancement occurs during the ionization process, engendering error in the determination result (Niessen et al., 2006). The matrix effect can be avoided using a matrix-matched standard method or using isotope dilution with isotopically labeled internal standards (Niessen et al., 2006), in addition to thorough sample clean-up. In the matrix-matched standard method, the matrix effect is evaluated through the comparison of the responses of the standard solution and the target pesticide prepared in the measurement sample solution free of the target pesticide; this method is commonly used as a means to correct the matrix effect (Di Muccio et al., 2006; Economou et al., 2009; Ferrer et al., 2005; Ferrer & Thurman, 2007; Fidente et al., 2005; Frenich et al., 2008; Hernández et al., 2006; Kamel, 2010; Kamel et al., 2010; Kanrar et al., 2010; Kmellár et al., 2008; Leanro et al., 2007; Mayer-Helm, 2009; Payá et al., 2007; Pirard et al., 2007; Radišić et al., 2009; Romero-González et al., 2011; Totti et al., 2006; Venkateswarlu

Xie et al. (2011) reported observation of ion enhancement in dinotefuran, imidacloprid, and thiacloprid, as well as ion suppression in acetamiprid, clothianidin, and thiamethoxam by the matrix effect, and that acetamiprid and thiamethoxam among these were significantly affected. However, because the recovery was improved by correction using the matrixmatched standard method, they emphasized the effectiveness of the method in the

In any event, the matrix effect should be regarded as a common problem of pesticide residue analysis by HPLC and GC; it goes without saying that it is most important to evaluate the matrix effect that might be derived from the subject sample when the development of a new analytical method is attempted or when an established, existing

Section 2 summarized the trends up to now in the development of the determination of neonicotinoid insecticides by chromatography. This section refers to analytical methods based on nonchromatographic techniques. The list in Table 2 shows that major nonchromatographic methods include flow injection analysis (FIA), direct MS analysis, and enzyme-linked immunosorbent assay (ELISA), which is an immunochemical determination using an antigen–antibody reaction with high specificity or selectivity. This section summarizes the trend and the current situation of the development of analytical methods

FIA is a method by which a predetermined amount of a sample solution is injected into carrier solution that flows continuously in a tube, and the target substance is detected or quantified using a detector, as might be done after a chemical reaction. Instruments used in

**3. Analytical methods based on nonchromatographic techniques**

**3.1 FIA or MS analysis for direct determination of neonicotinoid insecticides**

et al., 2007; Xiao et al., 2011; Xie et al., 2011).

avoidance of the matrix effect.

analytical method is applied.

for neonicotinoid insecticides using these.

FIA are generally inexpensive. Furthermore, the method is known to be capable of rapid, easy, and highly sensitive detection of trace substances (Lara et al., 2010; Llorent-Martínez et al., 2011).

Reports on the determination of neonicotinoid insecticides by FIA have targeted imidacloprid to date. In all such studies, methods are constructed by which a measurement sample is irradiated with ultraviolet light for conversion into a fluorescent substance [1–(6 chloro-3-pyridylmethyl)-2-(hydroxyimino)-3,4-didehydroimidalozolidene] which is detected using a spectrofluorometer (Flores et al., 2007; Vílchez et al., 2001). Alternatively, nitrite is detached from imidacloprid to be reduced into nitric oxide by iodide, which is detected by chemiluminescence detection with ozone (Lagalante & Greenbacker, 2007). All FIA methods show measurement sensitivity that is equal to or better than the detection limit of HPLC or GC. For the determination of liquid samples such as water, direct injection is possible with filtration only (Flores et al., 2007; Lagalante & Greenbacker, 2007). Actually, FIA is regarded as making a great contribution to fast, simple, and easy determination of pesticide residues, especially in liquid samples.

García-Reyes et al. (2009) reported rapid in situ qualitative and quantitative analysis of 16 pesticides including nitenpyram and thiacloprid by desorption electrospray ionization MS (DESI-MS) and MS/MS (DESI-MS/MS). In their determination, crop samples were pretreated according to QuEChERS and the resulting sample solution was applied on the PTFE surface, while the skin of fruit and vegetable samples were fixed on glass slides, and electrospray was applied directly to the sample to ionize target pesticides in the sample. The measurement sensitivity was extremely high: on the order of g/kg, and the measurement accuracy was comparable to LC-MS. Although such an analytical method remains under development at present, it can be anticipated as a new, highly sensitive, and rapid screening method.

Table 2. Overview of nonchromatographic techniques with FIA and MS for determination of neonicotinoid insecticides.

Review on Current Analytical Methods with Chromatographic

Watanabe et al., 2004a, 2004b, 2006, 2007, 2011).

Analyte(s) Sample(s) Extraction

Coffee cherry and coffee bean

Water

green pepper, tomato and apple

Imidacloprid Apple Shaking by hand

Cucumber, eggplant, lettuce, green pepper and spinach

Acetamiprid Cucumber,

Imidacloprid

Imidacloprid

Imidacloprid

especially as a screening method, by solving this problem.

Water None None

Homogenization with methanol/1% sulfuric acid (3:1, v/v)

Extraction with ethyl acetate from C18 SPE

Shaking with methanol

with methanol

Shaking by hand

Ultrasonic extraction with methanol

with methanol Filtration and

Apple Homogenization with methanol

Thiamethoxam Water None Diluted with

effectively but also to reduce the influence of the organic solvent.

and Nonchromatographic Techniques for New Generation Insecticide Neonicotinoids 497

with water or phosphate buffer. The recovery from various measurement samples were generally good in all reports (Byrne et al., 2005; Eisenback et al., 2009; Kim et al., 2006; Ma et al., 2009; Watanabe et al., 2001; Watanabe et al., 2004a, 2004b, 2006, 2007, 2011; Xu et al., 2010). The measurement sensitivity of ELISA is apt to be affected by the concentration of extraction solvent (mainly methanol) coexisting in the sample solution during measurement (Nunes et al., 1998). The sample extraction is diluted not only to avoid the matrix effect

Recently, kit-based ELISA for neonicotinoid insecticides was developed and marketed by Horiba Ltd. (Kyoto, Japan) and Envirologix Inc. (Portland, ME). Kit-based ELISA package reagents needed for determination (96-well microplate pre-coated with antibody, washing solution, substrate solution, and stopping solution, etc.) can be used easily for monitoring tests of a specific neonicotinoid insecticide. However, it is important in the use of kit-based ELISA to remember that the matrix effect should be evaluated in advance (Byrne et al., 2005;

In any event, when ELISA is applied to a sample, it might be affected by the matrix effect. Therefore, it is important to examine in advance if the matrix effect is present, and to examine methods to avoid it. At least, ELISA can be applied sufficiently to routine analysis,

> Following sample preparation

Evaporation, extraction with ethyl acetate and reconstitution with buffer

Evaporation and reconstitution with buffer

buffer

Evaporation and reconstitution with buffer

Centrifugation and dilution with methanol/buffer (9:1, v/v)

Filtration and dilution with water

dilution with water

et al., 2001 Imidacloprid MoAb 3.3

None Dilution with

PoAb

PoAb

MoAb

buffer PoAb Direct competitive

Antibody Assay format IC50 Ref.

35 ppb Li & Li, 2000

> Lee et al., 2001

Kim et al., 2003

Watanabe et al., 2004a

Watanabe et al., 2004b

17.3 ng/mL

1.0

ng/mL

9.0 ng/mL

ng/mL Watanabe

Indirect competitive ELISA

Indirect competitive ELISA

Direct competitive ELISA

ELISA

ELISA kit 8 ng/g

ELISA kit -

MoAb Direct competitive

MoAb Direct competitive

#### **3.2 ELISA analysis for neonicotinoid insecticides as a rapid and simple preliminary screening method**

Since Yalow and Berson applied radioimmunoassay (RIA) to the determination of insulin in the 1950s (Yalow & Berson, 1959, 1960), RIA has been widely used mainly in the field of clinical laboratory tests. Although immunoassay for pesticide determination was not reported in two decades. Subsequently, antibodies selective to DDT and malathion were developed (Centeno et al., 1970). In addition, RIA for parathion determination was developed (Ercegovich et al., 1981). After Engvall and Perlmann (1972) proposed enzyme immunoassay (EIA, ELISA) using enzyme-labeled antigens instead of radioisotope-labeled antigens, a marked increase existed in the development of ELISA for various pesticides including organophosphorus and synthetic pyrethroid insecticides (Hennion et al., 1998; Meulenberg et al., 1995; Nunes et al., 1998; Shan et al., 2002).

The immunogenicity of small molecules such as pesticides themselves is extremely low. Therefore, it is necessary for the development of antigens to these compounds to design and synthesize hapten molecules that imitate the chemical structure of the target substances. It is known that the measurement sensitivity and selectivity of the resulting antibody is strongly dependent on the chemical structure of the designed hapten molecule (Shan et al., 2002; Szurdoki et al., 1995). However, the importance of hapten design is not explained in this chapter.

ELISA, based on an antigen–antibody reaction, is a method used to detect residual pesticides, etc., in various samples consisting of complex matrices such as food samples including crops. Therefore, fast analysis can be achieved because significant laborsaving is possible in complicated sample pre-treatment procedures before chromatographic determination. Moreover, ELISA is regarded as an economical, straightforward, and easy analytical method because only small amounts of organic solvents are used, instruments requiring expertise are not needed, and multisample treatment is possible using 96-well microplates (Ellis, 1996). In contrast, ELISA is disadvantageous compared to chromatographic determination in that it is limited to the determination of a single pesticide, it is incapable of identification, and it might produce a false positive result when it cross-reacts to a compound with similar chemical structure because it is a selective analytical method.

As shown in Table 3, ELISA for the determination of neonicotinoid insecticides was first developed in 2000 using polyclonal antibody (PoAb) specific to imidacloprid (Li & Li, 2000). It was followed by a report by Lee et al. (2001) in which the measurement sensitivity was improved to be approximately twice that obtained using another hapten. Watanabe et al. (2001) and Kim et al. (2004) developed monoclonal antibody (MoAb) specific to imidacloprid, and constructed ELISA that is 5-fold to 20-fold more sensitive than that obtained using PoAb. In addition to imidacloprid, ELISA using MoAb specific to acetamiprid (Watanabe et al., 2001) and thiamethoxam (Kim et al., 2003, 2006) were also developed.

As described above, ELISA uses highly specific antigen–antibody reaction. Theoretically, it responds sensitively only to the trace pesticide in matrices. In fact, however, ELISA is susceptible to the matrix effect described in Section 2.2, and it is important to evaluate the matrix effect for individual measurement samples (Jourdan et al., 1996; Nunes et al., 1998; Skerritt & Rani, 1996). While the matrix effect can be avoided or reduced by SPE, etc., such measures would eliminate advantages of ELISA shown above. Table 3 shows that the easiest method to avoid the matrix effect is dilution of the sample extract (mainly methanol extract)

Since Yalow and Berson applied radioimmunoassay (RIA) to the determination of insulin in the 1950s (Yalow & Berson, 1959, 1960), RIA has been widely used mainly in the field of clinical laboratory tests. Although immunoassay for pesticide determination was not reported in two decades. Subsequently, antibodies selective to DDT and malathion were developed (Centeno et al., 1970). In addition, RIA for parathion determination was developed (Ercegovich et al., 1981). After Engvall and Perlmann (1972) proposed enzyme immunoassay (EIA, ELISA) using enzyme-labeled antigens instead of radioisotope-labeled antigens, a marked increase existed in the development of ELISA for various pesticides including organophosphorus and synthetic pyrethroid insecticides (Hennion et al., 1998;

The immunogenicity of small molecules such as pesticides themselves is extremely low. Therefore, it is necessary for the development of antigens to these compounds to design and synthesize hapten molecules that imitate the chemical structure of the target substances. It is known that the measurement sensitivity and selectivity of the resulting antibody is strongly dependent on the chemical structure of the designed hapten molecule (Shan et al., 2002; Szurdoki et al., 1995). However, the importance of hapten design is not explained in this

ELISA, based on an antigen–antibody reaction, is a method used to detect residual pesticides, etc., in various samples consisting of complex matrices such as food samples including crops. Therefore, fast analysis can be achieved because significant laborsaving is possible in complicated sample pre-treatment procedures before chromatographic determination. Moreover, ELISA is regarded as an economical, straightforward, and easy analytical method because only small amounts of organic solvents are used, instruments requiring expertise are not needed, and multisample treatment is possible using 96-well microplates (Ellis, 1996). In contrast, ELISA is disadvantageous compared to chromatographic determination in that it is limited to the determination of a single pesticide, it is incapable of identification, and it might produce a false positive result when it cross-reacts to a compound with similar chemical structure because it is a selective

As shown in Table 3, ELISA for the determination of neonicotinoid insecticides was first developed in 2000 using polyclonal antibody (PoAb) specific to imidacloprid (Li & Li, 2000). It was followed by a report by Lee et al. (2001) in which the measurement sensitivity was improved to be approximately twice that obtained using another hapten. Watanabe et al. (2001) and Kim et al. (2004) developed monoclonal antibody (MoAb) specific to imidacloprid, and constructed ELISA that is 5-fold to 20-fold more sensitive than that obtained using PoAb. In addition to imidacloprid, ELISA using MoAb specific to acetamiprid (Watanabe et al., 2001) and thiamethoxam (Kim et al., 2003, 2006) were also

As described above, ELISA uses highly specific antigen–antibody reaction. Theoretically, it responds sensitively only to the trace pesticide in matrices. In fact, however, ELISA is susceptible to the matrix effect described in Section 2.2, and it is important to evaluate the matrix effect for individual measurement samples (Jourdan et al., 1996; Nunes et al., 1998; Skerritt & Rani, 1996). While the matrix effect can be avoided or reduced by SPE, etc., such measures would eliminate advantages of ELISA shown above. Table 3 shows that the easiest method to avoid the matrix effect is dilution of the sample extract (mainly methanol extract)

**3.2 ELISA analysis for neonicotinoid insecticides as a rapid and simple preliminary**

Meulenberg et al., 1995; Nunes et al., 1998; Shan et al., 2002).

**screening method**

chapter.

analytical method.

developed.

with water or phosphate buffer. The recovery from various measurement samples were generally good in all reports (Byrne et al., 2005; Eisenback et al., 2009; Kim et al., 2006; Ma et al., 2009; Watanabe et al., 2001; Watanabe et al., 2004a, 2004b, 2006, 2007, 2011; Xu et al., 2010). The measurement sensitivity of ELISA is apt to be affected by the concentration of extraction solvent (mainly methanol) coexisting in the sample solution during measurement (Nunes et al., 1998). The sample extraction is diluted not only to avoid the matrix effect effectively but also to reduce the influence of the organic solvent.

Recently, kit-based ELISA for neonicotinoid insecticides was developed and marketed by Horiba Ltd. (Kyoto, Japan) and Envirologix Inc. (Portland, ME). Kit-based ELISA package reagents needed for determination (96-well microplate pre-coated with antibody, washing solution, substrate solution, and stopping solution, etc.) can be used easily for monitoring tests of a specific neonicotinoid insecticide. However, it is important in the use of kit-based ELISA to remember that the matrix effect should be evaluated in advance (Byrne et al., 2005; Watanabe et al., 2004a, 2004b, 2006, 2007, 2011).

In any event, when ELISA is applied to a sample, it might be affected by the matrix effect. Therefore, it is important to examine in advance if the matrix effect is present, and to examine methods to avoid it. At least, ELISA can be applied sufficiently to routine analysis, especially as a screening method, by solving this problem.

Review on Current Analytical Methods with Chromatographic

Mechanical shaking with methanol

determinations for determination of neonicotinoid insecticides.

Dinotefuran Rice

**neonicotinoid insecticides**

necessary performance.

ISSN 0021-9673

ISSN 1060-3271

**5. References**

and Nonchromatographic Techniques for New Generation Insecticide Neonicotinoids 499

with methanol/buffer (1:9, v/v)

Centrifugation and dilution with water (if necessary, dilution with methanol/water (1:9, v/v))

MoAb Direct competitive ELISA kit

5.4 ng/mL Watanabe et al., 2011

dichloromethane reconstitution

Table 3. Overview of nonchromatographic techniques with immunochemical

**4. Overview of analytical methods for pesticide residues including**

Sections 2 and 3 summarize trends in the determination methods for neonicotinoid insecticides and the problems discovered during the development of analytical methods such as the matrix effect. At present, highly accurate and sensitive multiresidue analysis by GC or HPLC equipped with MS or MS/MS as a detector is the major trend in the determination not only of neonicotinoid insecticides but also of pesticides in general. It goes without saying that pesticide residue analysis is an indispensable basic technology that is useful to secure the safety of food, including crops, as well as in various research fields such as environment and ecological impact evaluation. However, it is questionable because it is entirely dependent only on GC–MS, LC-MS, and LC-MS/MS for pesticide residue analysis. Particularly when pesticide residues are analyzed to secure food safety, two situations are assumed: (1) testing of foods on the market, and (2) testing of crops before shipment. In the former situation, the history of pesticides that have been used is often unknown. Therefore multiresidue analysis using MS or MS/MS is suitable, in which as many pesticides as possible can be tested and unknown ingredients can be identified. On the other hand, in the latter situation, the subject pesticide can be selected based on the history of use. Therefore there might be cases in which it is more sensible to choose a rapid, simple, and easy method such as ELISA described in Section 3 over multiresidue analysis which has better than

In any event, among the numerous analytical methods, it is necessary to make a wise choice

Agüera, A.; López, S.; Fernández-Alba, A.R.; Contreras, M.; Crespo, J. & Piedra, L. (2004).

Anastassiades, M.; Lehotay, S.J.; Štajnbaher, D. & Schenck, F.J. (2003). Fast and easy

One-year routine application of a new method based on liquid chromatography – tandem mass spectrometry to the analysis of 16 multiclass pesticides in vegetable samples. *Journal of Chromatography A*, Vol. 1045, No. 1-2, (August 2004), pp. 125-135,

multiresidue method employing acetonitrile extraction/partitioning and "Dispersive solid-phase extraction" for the determination of pesticide residues in produce. *Journal of AOAC International*, Vol. 86, No. 2, (March 2003), pp. 412-431,

for a suitable method via accurate comprehension of the analytical objectives.

Table 3. Overview of nonchromatographic techniques with immunochemical determinations for determination of neonicotinoid insecticides.

#### **4. Overview of analytical methods for pesticide residues including neonicotinoid insecticides**

Sections 2 and 3 summarize trends in the determination methods for neonicotinoid insecticides and the problems discovered during the development of analytical methods such as the matrix effect. At present, highly accurate and sensitive multiresidue analysis by GC or HPLC equipped with MS or MS/MS as a detector is the major trend in the determination not only of neonicotinoid insecticides but also of pesticides in general. It goes without saying that pesticide residue analysis is an indispensable basic technology that is useful to secure the safety of food, including crops, as well as in various research fields such as environment and ecological impact evaluation. However, it is questionable because it is entirely dependent only on GC–MS, LC-MS, and LC-MS/MS for pesticide residue analysis. Particularly when pesticide residues are analyzed to secure food safety, two situations are assumed: (1) testing of foods on the market, and (2) testing of crops before shipment. In the former situation, the history of pesticides that have been used is often unknown. Therefore multiresidue analysis using MS or MS/MS is suitable, in which as many pesticides as possible can be tested and unknown ingredients can be identified. On the other hand, in the latter situation, the subject pesticide can be selected based on the history of use. Therefore there might be cases in which it is more sensible to choose a rapid, simple, and easy method such as ELISA described in Section 3 over multiresidue analysis which has better than necessary performance.

In any event, among the numerous analytical methods, it is necessary to make a wise choice for a suitable method via accurate comprehension of the analytical objectives.

#### **5. References**

498 Insecticides – Advances in Integrated Pest Management

MoAb

MoAb

water - Direct competitive

2004 Cucumber Extraction with

Potato, 2006

Indirect competitive ELISA

Indirect competitive ELISA

MoAb Flow fluorescent immunoassay

MoAb Direct competitive ELISA kit

ELISA kit

Indirect competitive ELISA

Indirect competitive ELISA

Indirect competitive chemiluminescent ELISA

> Indirect competitive ELISA

buffer MoAb Direct competitive

0.8 g/L

> 6.82 ppb

30 pg/mL

> 0.6 ng/g

3.9 g/L

ELISA kit - Eisenback

6.5 ng/mL Ma et al.,

ng/mL

6.2

ng/mL

14.8 ng/mL

0.0875 mg/L

ng/mL Xu et al.,

Girotti et al., 2010

Fang et al.,

ELISA kit - Byrne et

Kim et al.,

al., 2005

Xu et al., 2006

Kim et al.,

Watanabe et al., 2006

Watanabe et al., 2007

et al., 2009

buffer

Centrifugation, filtration and dilution with buffer

Dilution with

Filtration, evaporation, centrifugation, liquid–liquid partition, reconstitution and dilution with water

Filtration and dilution with buffer

Centrifugation, filtration and dilution with buffer

Filtration and dilution with water

Centrifugation and dilution with water

2009 Thiamethoxam 0.57

2010 Thiamethoxam 0.5

Dilution with

Dilution with methanol/buffer

Centrifugation, evaporation and

buffer MoAb

buffer MoAb

water PoAb

(2:8, v/v) MoAb

2011 Soil and

None Dilution with

None Dilution with

Water None Dilution with

methanol

Homogenization with water

Ultrasonic extraction with methanol/0.04% sulfuric acid (4:1, v/v)

Shaking with methanol/water (7:3, v/v)

Vortex mixing with methanol

Shaking by hand with water

Liquid–liquid extraction with dichloromethane after homogenization with acetone and coagulation

Ultrasonic extraction with

Water None

Honey None Dilution with

Water None

Imidacloprid

Thiamethoxam

Acetamiprid

Imidacloprid

Imidacloprid

Imidacloprid

Imidacloprid Apple juice,

Imidacloprid Honeybee

Imidaclothiz

Imidacloprid Avocado

Imidacloprid Wiliwili leaf

leaf

cucumber and apple

Peach, apple, strawberry, cucumber, eggplant and tomato

Apple juice, grape juice and orange juice

Hemlock wood and needle tissues

grape juice, orange juice and peach juice

cabbage

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**23**

*Colombia*

**IPM Program to Control Coffee Berry Borer**

*Beauveria bassiana***, to Overcome Insecticide**

Coffee is cultivated by some 20 millions farmers in more than 50 countries in Africa, Asia and America, and generates an industry that surpasses USD 70 billion annually. Coffee cultivars are planted in a very wide range of ecological and social conditions and may be grown using few culture practices where almost no changes occur to the natural environment or by means of mechanization, irrigation, fertilization and pest control with chemical insecticides which often leads to a complete destruction of the surrounding vegetation and changes in the ecosystem. Small farmers are the predominant coffee growers in all countries and technology in most cases is not readily available to them. This coffee world situation results in a great variety of sanitary problems, which varies according to the country even in similar ecological regions. All agricultural production systems have to deal with plant protection problems and coffee is no exception. Since the early 20th century, coffee production suffers from numerous insect pests, being the coffee berry borer, the white stem borer, leaf miners and mealybugs, the most serious examples since they can cause coffee farmers to lose up to 20% of a crop and reduce the coffee value by 30 to 40%. These coffee pests could be kept below economic threshold levels by adopting integrated management strategies such as anticipation and continuous monitoring of pest outbreaks, maintenance of optimum shade, pruning of coffee bushes, good harvesting and processing of the berries, conservation and augmentation of indigenous natural enemies, introduction of exotic natural enemies and timely use of need based chemical or bio insecticides. This chapter deals with the most important insect pest affecting coffee plantations in all coffee producing countries, the Coffee Berry Borer, *Hypothenemus hampei* (Ferrari) (Coleoptera: Curculionidae: Scolytinae), a brief description of an integrated pest management program that can help reducing the insect populations, the appearance of resistant insects in the field after spraying inadequately endosulfan for several years and a description of a novel strategy to select a highly pathogenic mixed strains of fungi in order to overcome this

resistance and maintain infestation in the field under threshold levels.

**1. Introduction**

*Hypothenemus hampei***, with Emphasis**

**on Highly Pathogenic Mixed Strains of**

Pablo Benavides, Carmenza Góngora and Alex Bustillo

**Resistance in Colombia**

*Centro Nacional de Investigaciones de Café - Cenicafé*

### **IPM Program to Control Coffee Berry Borer**  *Hypothenemus hampei***, with Emphasis on Highly Pathogenic Mixed Strains of**  *Beauveria bassiana***, to Overcome Insecticide Resistance in Colombia**

Pablo Benavides, Carmenza Góngora and Alex Bustillo *Centro Nacional de Investigaciones de Café - Cenicafé Colombia*

#### **1. Introduction**

510 Insecticides – Advances in Integrated Pest Management

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and acetamiprid in environmental water samples with solid-phase extraction packed with multiwalled carbon nanotubes prior to high-performance liquid chromatography. *Analytical and Bioanalytical Chemistry*, Vol. 385, No. 8, (August Coffee is cultivated by some 20 millions farmers in more than 50 countries in Africa, Asia and America, and generates an industry that surpasses USD 70 billion annually. Coffee cultivars are planted in a very wide range of ecological and social conditions and may be grown using few culture practices where almost no changes occur to the natural environment or by means of mechanization, irrigation, fertilization and pest control with chemical insecticides which often leads to a complete destruction of the surrounding vegetation and changes in the ecosystem. Small farmers are the predominant coffee growers in all countries and technology in most cases is not readily available to them. This coffee world situation results in a great variety of sanitary problems, which varies according to the country even in similar ecological regions. All agricultural production systems have to deal with plant protection problems and coffee is no exception. Since the early 20th century, coffee production suffers from numerous insect pests, being the coffee berry borer, the white stem borer, leaf miners and mealybugs, the most serious examples since they can cause coffee farmers to lose up to 20% of a crop and reduce the coffee value by 30 to 40%. These coffee pests could be kept below economic threshold levels by adopting integrated management strategies such as anticipation and continuous monitoring of pest outbreaks, maintenance of optimum shade, pruning of coffee bushes, good harvesting and processing of the berries, conservation and augmentation of indigenous natural enemies, introduction of exotic natural enemies and timely use of need based chemical or bio insecticides. This chapter deals with the most important insect pest affecting coffee plantations in all coffee producing countries, the Coffee Berry Borer, *Hypothenemus hampei* (Ferrari) (Coleoptera: Curculionidae: Scolytinae), a brief description of an integrated pest management program that can help reducing the insect populations, the appearance of resistant insects in the field after spraying inadequately endosulfan for several years and a description of a novel strategy to select a highly pathogenic mixed strains of fungi in order to overcome this resistance and maintain infestation in the field under threshold levels.

IPM Program with Emphasis in Biological Control

been affected with the presence of this pest.

**Coffee Berry Borer in Colombia**

**3. Distribution and dispersion of** *Hypothenemus hampei*

and a third introduction was evident in Peru and Colombia (Benavides 2005).

with a Highly Pathogenic *Beauveria bassiana* Mixed Strains as a... 513

The coffee berry borer dispersal through the world has been documented (Benavides 2005; Benavides *et al.* 2005), using molecular tools based on AFLP. Results suggested that invasion in Asia and America came from West Africa. The distribution of the fingerprints and its genetic relationship determined by a neighbor - joining analysis, showed that there were two introductions of Coffee Berry Borer in Brazil, then dispersed into American countries

*Hypothenemus hampei* has now invaded all coffee producing countries worldwide (Table 1). It was first detected in Colombia in 1988 and is found now in all Colombian coffee plantations infesting near 800.000 ha and affecting the assets of more than a half million of Colombian coffee producing families. The coffee cultivars in Colombia have been kept free of important insect pests since the beginning of its development as a commercial exploitation. Only a few records exists on the attack of minor pests such as: *Leucoptera coffeellum* (Guerin - Méneville), *Coccus viridis* (Green), *Planococcus citri* (Risso), *Dysmicoccus brevipes* (Cockerell), *Puto barberi* (Murillo), and the red spider mite *Oligonychus yothersi* McGregor (ICA 1989; Cárdenas 1983, 1985). These arthropods have not became serious pests due to the fact that these agroecosystems are quite stable with a great biodiversity, which favors the development of beneficial agents and maintain in equilibrium the potential pests present in the farms. On the other hand, in the coffee growing areas, insecticides were not used indiscriminately and it is recognized that Colombia is the only country in the world where the coffee plantations were handled without the use or with very low use of insecticides up to the arrival of *H. hampei* (Bustillo 1991). This situation of equilibrium has

**4. Implementation of an Integrated Pest Management for the control of the**

Different strategies are needed to control the Coffee Berry Borer, such as: cultural practices, crop agronomical management, which can reduce insect populations, the protection of beneficial fauna, and the introduction of exotic natural enemies and entomopathogens. Among these are the parasitoids: *Cephalonomia stephanoderis* Betrem, *Prorops nasuta* Waterston, *Phymastichus coffea* La Salle and the fungus *Beauveria bassiana* (Báls.) Vuillemin (Bustillo 1991, 1995; Benavides *et al.* 1994; Orozco 1995; Orozco y Aristizábal 1996).  The IPM focuses in a series of principles and concepts on pest control which are integrated and in a theoretical way are proposed to establish an ecological guideline in the solution of a pest problem. So the IPM is flexible, dynamic and always susceptible of improvement, even though its comprehension and adoption by the farmers may be difficult. In the case of *H. hampei* the IPM program has been defined as: the use of a series of control measures (cultural, biological and chemical) to reduce coffee berry borer populations to levels which can not cause economical damage and which allows the farmers the production of coffee for exportation in a competitive way. The control measures used must be compatible and should not cause deleterious effects to the farmers living in the coffee zone, nor to the fauna, and do not contaminate the coffee ecosystem. (Bustillo *et al.* 1998).This concept is now extended to the Integrated Crop Management

#### **2. Life cycle and damage caused by** *Hypothenemus hampei*

The life cycle of Coffee Berry Borer has been studied by several authors (Corbett 1933; Bergamin 1943; Ticheler 1963; Baker 1984; Muñoz 1989; Decazy 1990a; Baker *et al.* 1992; Jaramillo *et al.* 2009), under different temperature and ambient conditions. In Colombia several studies have been carried out under laboratory and field conditions (Montoya y Cárdenas 1994; Gaviria *et al.* 1995; Ruíz *et al.* 1996; Ruíz 1996). The adult female coffee berry borer is a small black beetle, 1.5 mm in length, longer and slightly wider than the male.  Males mate inside the berry with females and never emerge. Males live between 50 and 75 days, while females from 100 to 150 days. The female beetle enters the coffee berry and borers a tunnel inside the coffee bean where lays eggs at a rate of 2-3 per day up to 20 days. The average number of offspring produced per female is 74 and the total life cycle was calculated to be 27.5 days (24.5°C), however, a complete development of coffee berry borer from egg to adult in Colombia is estimated between 45 to 60 days under conditions of 21ºC and 19ºC, respectively (Ruiz 1996, Bustillo 2002). The life cycle in degree–days is 237.2 with a threshold temperature development of 16.5 ºC. Even though there are reports of non-mated females giving origin to fertile eggs (Montoya y Cárdenas 1994, Muñoz 1989, Barrera *et al.* 1995), this has not being verified experimentally (Alvarez y Cortina 2004). Mated females emerge to fly and look for a new berry. Host finding is achieved initially by responding to volatiles emitted by the coffee berry during its development and close orientation to the berries may be assisted by vision preferring the red berries (Bustillo *et al.* 1998).

*Hypothenemus hampei* has a reproductive behavior that ensures a high degree of inbreeding.  Females mate with their flightless male siblings when still inside the berry, so they leave the infested berry already fertilized. Although cytological examination of somatic cells in males proved that they were diploid, males failed to express paternally derived alleles and then transmitted only their maternally derived chromosomes; thus, *H. hampei* is functionally haplo-diploid (Brun *et al.* 1995). It has been suggested that the unusual sex determination and skewed sex ratios favoring females is caused by the infection of *Wolbachia* in *H. hampei* (Vega *et al.* 2002, Benavides 2005); However, the presence of males in the coffee berry borer populations can be explained by the presence of an extra chromosome in male cells (Bergamin and Kerr 1951).

The damage caused by *H. hampei* is mainly a decrease of coffee yield due to abscission of berries, loss of weight, and a decrease on coffee quality and, therefore, coffee price. It has been estimated that there is a weight loss of 55% on coffee grains attacked by *H. hampei* (Montoya 1999); however, the decrease of weight of the total coffee production is about 18% Borbón 1990). *Hypothenemus hampei* also attacks young berries in formation (less than 20 weeks after flowering) which results in the abscission of 32% of coffee berries (Mendoza 1996). Furthermore, *H. hampei* causes yield losses as high as 40-80% at a field infestation of 90% (Le Pelley 1968). Coffee prices are greatly reduced when the beans exhibit *H. hampei* damage. International marketing policies do not allow coffee for exportation that have more than 1.5% damage caused by insects. Thus, the price of coffee in producing countries is severely reduced if the levels of infestation with *H. hampei* are greater (Duque and Baker 2003).

The life cycle of Coffee Berry Borer has been studied by several authors (Corbett 1933; Bergamin 1943; Ticheler 1963; Baker 1984; Muñoz 1989; Decazy 1990a; Baker *et al.* 1992; Jaramillo *et al.* 2009), under different temperature and ambient conditions. In Colombia several studies have been carried out under laboratory and field conditions (Montoya y Cárdenas 1994; Gaviria *et al.* 1995; Ruíz *et al.* 1996; Ruíz 1996). The adult female coffee berry borer is a small black beetle, 1.5 mm in length, longer and slightly wider than the male.  Males mate inside the berry with females and never emerge. Males live between 50 and 75 days, while females from 100 to 150 days. The female beetle enters the coffee berry and borers a tunnel inside the coffee bean where lays eggs at a rate of 2-3 per day up to 20 days. The average number of offspring produced per female is 74 and the total life cycle was calculated to be 27.5 days (24.5°C), however, a complete development of coffee berry borer from egg to adult in Colombia is estimated between 45 to 60 days under conditions of 21ºC and 19ºC, respectively (Ruiz 1996, Bustillo 2002). The life cycle in degree–days is 237.2 with a threshold temperature development of 16.5 ºC. Even though there are reports of non-mated females giving origin to fertile eggs (Montoya y Cárdenas 1994, Muñoz 1989, Barrera *et al.* 1995), this has not being verified experimentally (Alvarez y Cortina 2004). Mated females emerge to fly and look for a new berry. Host finding is achieved initially by responding to volatiles emitted by the coffee berry during its development and close orientation to the berries may be assisted by vision

*Hypothenemus hampei* has a reproductive behavior that ensures a high degree of inbreeding.  Females mate with their flightless male siblings when still inside the berry, so they leave the infested berry already fertilized. Although cytological examination of somatic cells in males proved that they were diploid, males failed to express paternally derived alleles and then transmitted only their maternally derived chromosomes; thus, *H. hampei* is functionally haplo-diploid (Brun *et al.* 1995). It has been suggested that the unusual sex determination and skewed sex ratios favoring females is caused by the infection of *Wolbachia* in *H. hampei* (Vega *et al.* 2002, Benavides 2005); However, the presence of males in the coffee berry borer populations can be explained by the presence of an extra chromosome in male cells

The damage caused by *H. hampei* is mainly a decrease of coffee yield due to abscission of berries, loss of weight, and a decrease on coffee quality and, therefore, coffee price. It has been estimated that there is a weight loss of 55% on coffee grains attacked by *H. hampei* (Montoya 1999); however, the decrease of weight of the total coffee production is about 18% Borbón 1990). *Hypothenemus hampei* also attacks young berries in formation (less than 20 weeks after flowering) which results in the abscission of 32% of coffee berries (Mendoza 1996). Furthermore, *H. hampei* causes yield losses as high as 40-80% at a field infestation of 90% (Le Pelley 1968). Coffee prices are greatly reduced when the beans exhibit *H. hampei* damage. International marketing policies do not allow coffee for exportation that have more than 1.5% damage caused by insects. Thus, the price of coffee in producing countries is severely reduced if the levels of infestation with *H. hampei* are greater (Duque and Baker

**2. Life cycle and damage caused by** *Hypothenemus hampei*

preferring the red berries (Bustillo *et al.* 1998).

(Bergamin and Kerr 1951).

2003).

#### **3. Distribution and dispersion of** *Hypothenemus hampei*

The coffee berry borer dispersal through the world has been documented (Benavides 2005; Benavides *et al.* 2005), using molecular tools based on AFLP. Results suggested that invasion in Asia and America came from West Africa. The distribution of the fingerprints and its genetic relationship determined by a neighbor - joining analysis, showed that there were two introductions of Coffee Berry Borer in Brazil, then dispersed into American countries and a third introduction was evident in Peru and Colombia (Benavides 2005).

*Hypothenemus hampei* has now invaded all coffee producing countries worldwide (Table 1). It was first detected in Colombia in 1988 and is found now in all Colombian coffee plantations infesting near 800.000 ha and affecting the assets of more than a half million of Colombian coffee producing families. The coffee cultivars in Colombia have been kept free of important insect pests since the beginning of its development as a commercial exploitation. Only a few records exists on the attack of minor pests such as: *Leucoptera coffeellum* (Guerin - Méneville), *Coccus viridis* (Green), *Planococcus citri* (Risso), *Dysmicoccus brevipes* (Cockerell), *Puto barberi* (Murillo), and the red spider mite *Oligonychus yothersi* McGregor (ICA 1989; Cárdenas 1983, 1985). These arthropods have not became serious pests due to the fact that these agroecosystems are quite stable with a great biodiversity, which favors the development of beneficial agents and maintain in equilibrium the potential pests present in the farms. On the other hand, in the coffee growing areas, insecticides were not used indiscriminately and it is recognized that Colombia is the only country in the world where the coffee plantations were handled without the use or with very low use of insecticides up to the arrival of *H. hampei* (Bustillo 1991). This situation of equilibrium has been affected with the presence of this pest.

#### **4. Implementation of an Integrated Pest Management for the control of the Coffee Berry Borer in Colombia**

Different strategies are needed to control the Coffee Berry Borer, such as: cultural practices, crop agronomical management, which can reduce insect populations, the protection of beneficial fauna, and the introduction of exotic natural enemies and entomopathogens. Among these are the parasitoids: *Cephalonomia stephanoderis* Betrem, *Prorops nasuta* Waterston, *Phymastichus coffea* La Salle and the fungus *Beauveria bassiana* (Báls.) Vuillemin (Bustillo 1991, 1995; Benavides *et al.* 1994; Orozco 1995; Orozco y Aristizábal 1996).  The IPM focuses in a series of principles and concepts on pest control which are integrated and in a theoretical way are proposed to establish an ecological guideline in the solution of a pest problem. So the IPM is flexible, dynamic and always susceptible of improvement, even though its comprehension and adoption by the farmers may be difficult. In the case of *H. hampei* the IPM program has been defined as: the use of a series of control measures (cultural, biological and chemical) to reduce coffee berry borer populations to levels which can not cause economical damage and which allows the farmers the production of coffee for exportation in a competitive way. The control measures used must be compatible and should not cause deleterious effects to the farmers living in the coffee zone, nor to the fauna, and do not contaminate the coffee ecosystem. (Bustillo *et al.* 1998).This concept is now extended to the Integrated Crop Management

IPM Program with Emphasis in Biological Control

Table 1. Worldwide distribution of *Hypothenemus hampei.*

with a Highly Pathogenic *Beauveria bassiana* Mixed Strains as a... 515

(ICM), which includes besides all the above mentioned tools, all the agronomical practices which are not directly pointed to the borer control, but if they are implemented can contribute indirectly to reduce borer populations (Bustillo 2002).

The implementation of an IPM program for the control of the Coffee Berry Borer in Colombia begins with sampling and determining an economic threshold level. The damage caused by Coffee Berry Borer creates the necessity to take efficient control measures, in the right moment when the insect menaces the coffee crop. Therefore, an important requirement in an IPM program is to measure in the field the insect population and correlate this population with the final damage to the crop. In the case of *H. hampei,* the sampling consists on taking, from a hectare (sampling universe), 30 trees randomly (sample size), selecting a productive branch containing 30 to 100 coffee berries (sampling unit) and then counting total number of berries in the branch and total number of berries infested by the Coffee Berry Borer. The infestation level is the result of dividing the total number of infested berries over the total counted coffee berries (Bustillo *et al.* 1998, Decazy 1990b, 1990c, Baker 1989, Baker *et al.* 1989, Muñoz 1988). The sampling should be done in a monthly basis in each coffee plot to follow up the borer populations and deciding control measures timely (Bustillo *et al.* 1998). By going through the coffee plots, allows to the evaluator localize sites where there is a high concentration of insect borer population, and in these marked places the farmer should intensify the control measures. On the other hand, when evaluation takes place, a sample of 2 – 3 infested berries per tree should be taken, to determine the borer internal population and mortality, recording also the position, inside or outside the coffee berry (Bustillo *et al.* 1998). The level of infestation, the position of the borer inside the berry, and the location inside the plot, allows the farmer to take good insect control decisions (Bustillo *et al.* 1998, Bustillo 2002). The infestation levels cannot surpass 2% in field conditions during the critical period which is described as the moment when the coffee berries are most susceptible to the insects attack such as 120 days after flowering.