Extraction and Identification Techniques for Quantification of Carbamate Pesticides in Fruits and Vegetables

Abstract

The usage of carbamate pesticides in agriculture is increasing year by year. Carbamate pesticides are thioesters and esters, which are derived from aminocarboxylic acid. Carbamates are commonly utilized to improve agricultural production and protect humans and animals from disease. They were also used to control and prevent agricultural pests. However, carbamate can be highly toxic if not applied properly. Therefore, carbamate pesticides need to be monitored in fruits and vegetables. Sensitive and selective detection of carbamate pesticides using nanotechnology helps overcome the drawback of conventional methods of detecting carbamates. Nowadays, the demand for rapid, highly sensitive, and selective pesticide detection techniques is expanding to facilitate detection without complicated equipment. Due to this, this chapter focuses on nanotechnology and current detection methods for detecting residual carbamate pesticides in fruits and vegetables more precisely and faster.

1. Introduction

A pesticide is a hazardous chemical compound or a mixture of biological agents or chemicals that are deliberately presented into the environment to prevent, dissuade, eliminate, or control populations of insects, rodents, weeds, fungus, or other unwanted pests. Pesticides play an important role in attracting, enticing, and killing or repelling organisms. Generally, pesticides are widely applied and reported at approximately 5.2 billion pounds per year to reduce various harmful species such as microscopic fungi, weeds, rodents, and insects. Pesticide is highly applicable for pest control in agricultural areas and households to control mosquitoes, ticks, cockroaches, rats, fleas, and other dangerous creatures [1]. Using pesticides improves crop yields by controlling pathogenic microorganisms, resulting in better consumption of fresh fruits and vegetables [2]. There are four types of pesticides, namely organochlorines, carbamates, organophosphates, and pyrethroids, illustrated in Figure 1 with their chemical structures.

Figure 1.

Classification of pesticides with chemical structure.

Carbamate pesticides are known as esters of carbamic acid (R¹-S-CO-NR²R³), which are not structurally complex. They are commonly employed in farming to protect many crops, including fruits, cotton, rice, and vegetables, due to their broad biological activity, less mammalian toxicity, and minimal bioaccumulation potential [3]. Besides, it was applied as a therapeutic drug in human medicine and veterinary medicine. Carbamate has a high polarity, is water-soluble and thermodynamically unstable, which contains insecticides like carbaryl, acaricides, and fungicides [4]. Previous research found that carbamate pesticides are capable absorb in the food source’s tissues such as fish, poultry, and meat, in processed foods such as vegetables, nuts, dehydrated fruits, and vegetable oils [5]. Based on FAO and WHO, in 2016, Codex Alimentarius Commission for carbamate maximum residue levels was set up to 4844 but required the presence of different combinations of pesticides. However, in European Union, carbaryl was banned in most countries [6]. This is because the carbamate residual in foods functions as acetylcholinesterase inhibitors, which can damage the brain, nervous systems, liver, muscles, and pancreas over the long term [7, 8].

It is essential to track and measure the carbamate amounts in fresh products and improve the sensitivity of the detection methods that have been developed. Previously, conventional methods such as chromatography, immunoassay, and surface-enhanced Raman spectroscopy (SERS), have been applied and show reliability and sensitivity to determine the presence of carbamate. However, these techniques are typically insufficient for real-time and on-site detection, which necessitates advancements in terms of preparation time and cost of machinery and highly skilled workers [9]. Hence, the development of advanced nanotechnology is one of the alternative methods that show rapid, low-cost, easy to use, and capable of detecting low concentrations of carbamate in food samples. This chapter focuses on the latest information on sample pretreatment and analytical detection strategies available from 2000 to 2021. Also, we highlight the reader with an understanding of some innovative ways to increase carbamate pesticides detection in food products.

2. Types of carbamate pesticides

Carbamate is an N-methyl produced from carbonic acid, responsible for the carbamylation of acetylcholinesterase at neuromuscular junctions in the brain and spinal cord and at neuronal synapses. Carbamate is classified as an insecticide that is physically and mechanistically comparable to organophosphate (OP) insecticides in both structure and mechanism of action. Carbamates have a reversible binding to acetylcholinesterase and do not cause the irreversible phosphorylation of the enzyme that occurs when organophosphates interact with it [10]. Consequently, carbamates are toxicologically similar to OP poisoning, with a toxic period of fewer than 24 hours [11]. Aldicarb, carbaryl, carbofuran, bendiocarb, fenobucarb, methomyl, oxamyl, propoxur, and methiocarb are the most common agents that lead to dangerous exposure. Figure 2 below illustrates the chemical structure of carbamate pesticides available in agriculture applications.

Figure 2.

Chemical structure of types of carbamate pesticides.

Carbaryl is a member of the chemical family N-methyl carbamate and was discovered in 1959 for use as a carbamate pesticide in cotton in the United States. Carbaryl is a popular insecticide in agriculture, specialist turf control, ornamental production, and residential settings. Carbaryl is mildly toxic when taken orally and has low toxicity when applied topically or inhaled [12]. In outdoor conditions, carbaryl has a low persistence rate. Human exposure occurs by ingestion of residues in food, skin contact, and inhalation of airborne particles. Carbaryl blocks acetylcholinesterase in the neurological system, causing acetylcholine buildup and cholinergic hyperstimulation. In contrast to adults, immature organisms are more sensitive to the inhibition of cholinesterase (ChE). In addition to reproductive and developmental toxicity, carbaryl can also alter the immune system. It may also cause cancer in humans and be highly harmful to non-target organisms [13, 14].

Aldicarb is a carbamate insecticide active against insects, mites, and nematodes belonging to the chemical family of N-methyl carbamates. Aldicarb is water-soluble at pH 7 and a colorless crystalline substance that acts as a cholinesterase inhibitor, soil contaminant, carcinogen, and a possible endocrine disruptor. Aldicarb is acutely toxic and causes cholinergic symptoms by inhibiting acetylcholinesterase (AChE), neither genotoxic nor cancer-causing. Much information about toxicity includes developmental, long-term, short-term, reproductive, and neurotoxic studies. They are dose-dependent, rapidly reversible, and do not manifest at levels of human exposure predicted [15]. The toxicity of aldicarb is evident in even small doses with stomach cramping, dizziness, nausea, diarrhea, and convulsions [16, 17].

Carbofuran is a wide-spectrum of N-methyl carbamate insecticide commonly used in farming to combat insects, nematodes, and mites in soil or protect forest crops, fruit, and vegetables. It is incredibly toxic to birds, mammals, fish, and wildlife due to its anticholinesterase action that inhibits acetylcholinesterase and butyrylcholinesterase. Carbofuran can disrupt the neuroendocrine system, cause reproductive disorders, and be genotoxic and cytotoxic to humans [18]. However, it did not affect a humoral immune response [15]. Besides, it is a relatively unstable chemical that degrades in weeks or months. Recently, Amatatongchai et al. [19] found carbofuran in potatoes, corn, soybean, fruits, and vegetables. Similarly, Lan et al. [20] detected carbofuran in watermelon, long bean, mango, and chives samples.

Methomyl is known as metomil or mesomile, commonly used to treat crops. It is a colorless crystalline structure soluble in organic solvents and water, which may pollute the environment. It has a wide application in biological activities and is efficient against insects [21]. Methomyl is categorized as a harmful and dangerous pesticide by the World Health Organization and the European Union [22]. Acetylcholinesterase (AChE) is inhibited by methomyl lead in a reduction of the ability of the enzyme to hydrolyze acetylcholine that buildup in the body. The most common signs of methanol include tearing of the eyes, vomiting, nausea, stomach pain, diarrhea, loss of consciousness (coma), and death due to respiratory failure [23, 24, 25]. The endocrine system is also affected by methomyl because of its capability to influence estrogen production and reproductive capabilities [26]. Presently, Guo et al. [27] identified methomyl residue in barley, millet, wheat, and rice grains. Besides, Rasolonjatovo et al. [28] found methomyl residues in tomatoes.

Methiocarb is a carbamate pesticide that colorless, crystalline substance sparingly soluble in water and xylenes. However, it is unstable in alkaline media (pH 9). Methiocarb is a contact wide-spectrum, a residual insecticide which acts as a molluscicide, acaricide, and bird repellent since the 1960s [13]. Methiocarb is used on fruit crops and orchids to control snails and rice insects [29]. Sivaperumal et al. [30] found the methiocarb residues in mango fruits. The molecule is oxidized sequentially to sulfoxide and sulfone in the vertebrate liver. Methiocarb sulfoxide is also available in methiocarb sulfone in the form of iocarb sulfone and the combination known as methiocarb [31].

The chemical name for propoxur is 2-isopropoxyphenyl-N-methylcarbamate with a molecular weight of 209.24, which is hydrolyzed by strong alkali. Propoxur is unstable in alkaline media and has a half-life at a pH of 10 for 40 minutes. It is a non-systemic insecticide primarily used against household insect pests and domestic animal pests [32]. However, propoxur causes neurotoxicity by inhibiting acetylcholinesterase in a significant reversible manner [33]. Based on Borahan et al. [34], propoxur has been detected in raisins by gas chromatography-mass spectrometry (GC-MS). Besides, Xiao-Xue et al. [35] found propoxur in fruit samples such as plum, pear, and loquat by employing the molecularly imprinted photoelectrochemical sensor.

Through the oral pathway, oxamyl is highly toxic. Like other carbamates, exposure to oxamyl can result in cholinesterase inhibition over a short period [36]. The pure compound has a slightly sulfurous odor and is a white crystalline solid, which melts at 100–102°C and shifts to a different crystalline structure between 108 and 110°C [32]. Yaseen et al. [37] found oxamyl in peach fruit using a surface-enhanced Raman scattering. Bendiocarb is a carbamate insecticide efficient against a broad spectrum of agricultural pests. Bendiocarb is poisonous to fish, birds, and bees, and research has demonstrated that bendiocarb is unable to bioaccumulate in animals [38]. Kowalska et al. [39] stated that terbucarb residues were found in plants, and HP-LC detected it with tandem mass spectrometry (HPLC-MS/MS). Liquid fenobucarb pesticides are pale yellow or pale red. Pelle et al. [40] found fenobucarb residues in grain samples.

3. Physical and chemical properties of carbamate pesticide

The straightforward technique to identify carbamate pesticides is to look at their carbamic acid N- or S-substitutions. The carbamates are classified into nine major groups: dithiocarbamates, thiocarbamates, benzimidazole carbamates, N-phenyl carbamates, ethylenebisdithiocarbamate, N,N-methyl carbamates, N-methyl carbamates, aminophenyl N-methylcarbamates, and oxime N-methylcarbamates [41]. Carbamates are typically insoluble in water molecules because it has low solubility in polar organic solvents, ethanol, or acetone. Carbamate is a polar molecule soluble in solvents with a medium polarity, including benzene, chloroform, toluene, xylene, dichloromethane, or 1,2-dichloromethanebut are insoluble in nonpolar organic solvents [42, 43]. Pure carbamate pesticides are crystalline, white, practically odorless solids with low vapor pressure and high melting point. Carbamate pesticide features include physical form, melting point, vapor pressure, and solubility [41].

4. Toxicology of carbamate pesticide

Carbamates are carbamic acid esters substituted for N-methyl carbamic acid that act as AChE inhibitors to catalyze acetylcholine (ACh). The reaction enhanced the ACh level at a nerve synapse or neuromuscular junction, raising nerve-ending stimulation by reversible cholinesterase inhibition [44]. In contrast to organophosphates, the cholinesterase-inhibiting action of carbamates is reversible. Carbamates are toxic to rodents in doses ranging between LD50 > 200 mg/kg and LD50 > 50 mg/kg [45]. According to the classification system, the US Environmental Protection Agency and the World Health Organization (WHO) have classified carbamate as class II (moderate). Several additional factors, such as route and frequency of exposure, interactions with other impurities, and compromised physiological conditions, such as liver impairment, may all impact the level of toxicity [25, 46]. Besides, WHO includes carbamates on its endocrine-disrupting chemicals (EDCs), potentially harmful to animals and human health [47]. They discovered that EDCs might disrupt hormone production, transport, metabolism, and elimination, with developmental, behavioral, and reproductive effects resulting from these hormone-active compounds. De Coster and Van Larebeke [48] examined the endocrine-disrupting properties of chlorpropham, carbaryl, benomyl, methiocarb, pirimicarb, and propamocarb by highlighting various pathways, including nuclear receptor activation, estrogen-associated receptor activation, and membrane-bound estrogen-receptor activation, among others.

High-potential AChE-inhibitors have been utilized as toxicants, but low-potential AChE-inhibitors have been used as prevention agents against nerve agents or as therapeutic agents in treating illnesses such as glaucoma, Alzheimer’s disease, and myasthenia gravis, among other things [49]. The primary benefits of carbamate are its intense insecticidal action and poor durability since it degrades swiftly within weeks or months after being applied to crops. Carbamates are effective against a wide range of pests by blocking the enzyme cholinesterase, causing neurotoxicity, and interfering with the nervous system of the pests [50]. These chemicals also exhibit a range of neurotoxic effects not mediated by a cholinergic mechanism. Carboxylated acetylcholinesterase enzyme is a volatile version of the enzyme, and regeneration of this enzyme is comparatively quick when contrasted with the regeneration of a phosphorylated form of the enzyme [51]. Carbamates produce mild eye irritation and moderate skin irritation, depending on the specific vehicle employed, the duration of contact, and the substance applied directly to the skin that has been harmed or is in good condition, according to the manufacturer [51, 52].

5. Extraction techniques of carbamates pesticides

The separation of pesticides is necessary from the sample before introducing into the instrument. This approach is expected to limit measuring interferences while enhancing the analyte concentration for research. Besides, the extraction method is a standard procedure that begins with releasing a preferred analyte from matrices and ends with a purification procedure, which directs to a series of stages via the analytical approach wherein a high proportion of potential interference co-extracts is eliminated using chemical or physical means [53]. Liquid-liquid extraction (LLE), solid-phase extraction (SPE), solid-phase microextraction (SPME), quick, easy, cheap, effective, rugged, and safe, microwave-assisted extraction (QuEChERS), and microwave accelerated selective Soxhlet extraction are among the extraction technologies available.

5.1 Liquid-liquid extraction (LLE)

Liquid-liquid extraction (LLE) has become a standard procedure in sample preparation due to its convenience and efficacy for insecticide contamination of food [54]. However, LLE requires a lot of solvents, which is terrible for the environment compared to solventless extraction technologies like solid-phase microextraction. On the other hand, the LLE approach is poor in yield analyte concentration, laborious, and requires a significant volume of toxic organic solvents [55]. Previously, liquid-liquid extraction/low-temperature purification incorporated with HPLC-UV was applied for determining aldicarb, carbofuran, and carbaryl in water samples. The separation for the carbamates aldicarb, carbofuran, and carbaryl show a high recovery rate. Although in small amounts of material and solvent, the extraction method was selective, with a limit of detection was found 5.0 and 10.0 g L⁻¹ [56].

5.2 Solid-phase extraction (SPE)

Solid-phase extraction (SPE) was initially presented during the 1970s, then widely accessible in 1978. At the moment, the most often used widely is SPE procedures for the pretreatment of environmental materials [55]. SPE is simpler, acceptable, and convenient than traditional LLE. Wang et al. [57] recently published an SPE technique utilizing porous organic polymers as an absorbent to extract isoprocarb, metolcarb, bassa, carbaryl, and lastly, diethofencarb, from white wine, milk, and juice before HPLC-diode array detection. The findings showed that milk and white wine samples have excellent linearity, with low detection limits for milk, white wine, and juice samples.

Earlier, Li et al. [58] used a simple one-step synthesis technique to make graphene-based magnetic nanoparticles by using MSPE to detect trace carbamate insecticides in tomatoes. Under ideal conditions, this technique has high enrichment factors, good linearities, low detection, and satisfactory spiking recoveries. The findings show that this approach was an adequate preparation and enhancement approach that may be used to extract and determine trace carbamate pesticides in complicated matrices. Besides that, Shi et al. [59] used graphene-based solid-phase extraction with ultra-HPLC-tandem mass spectrometry to analyze carbamate in ambient water samples. The LOD ranged from 0.5 to 6.9 ng L⁻¹, with relative standard deviations of 5.54%. The graphene-packed SPE cartridge may be reused over 100 times for a typical solution after proper regeneration with no appreciable performance degradation. The target analytes’ has good enrichment values, which indicate that the developed approach successfully determined carbamate pesticide residues in ambient water samples.

5.3 Solid-phase microextraction (SPME)

Solid-phase microextraction (SPME) is a technology that is a highly selective, sensitive, and solvent-free sample and is frequently used to extract volatile and semi-volatile chemicals by its absorption fibers. The range of SPME coatings available, dependent on the analytes’ polarity, results in high sensitivity and selectivity because of the strong coating affinity for particular analytes that build up in the environment until they reach equilibrium [60]. Zhou and Fang [61] developed a graphene-modified TiO₂ nanotube array by electrodeposition utilizing a cyclic voltammetric reduction approach to detect carbamate. When utilized in TiO₂ nanotube arrays for MSPE, the combination of graphene’s adsorptive solid properties and its higher extraction capabilities results in remarkable sample preconcentration performance. These results indicate that graphene-modified TiO₂ nanotube arrays have a high capacity for adsorption of contaminants. The technique demonstrates a quick and efficient alternative analytical solution for detecting and quantifying carbamate in fruits and vegetables.

5.4 QuEChERS (quick, easy, cheap, effective, rugged, safe) extraction

Quick, easy, cheap, effective, rugged, safe (QuEChERS) is a sensitive food analysis technology that has undergone numerous revisions and advancements. QuEChERS is a two-stage technology employed to detect carbamate residues in foods that includes salting-out partitioning, which involves the transition between an aqueous and an organic layer. This technique necessitates further cleaning to remove interfering chemicals by combining magnesium sulfate with various sorbents like C18, graphitized carbon black (GCB), or primary-secondary amines (PSA). It may be used to clean a variety of complex substances like food products while also allowing for a less organic solvent [62]. Due to its numerous advantages, the QuEChERS technique has gained massive attention and is widely utilized and regarded as a preferable approach for measuring toxic contaminants in foods.

Previously, Anastassiades et al. [63] introduced the QuEChERS technique to extract carbamate from food matrices by using a small quantity of acetonitrile, followed by a clean-up step employing DSPE. This method was first used to examine fruits and vegetables. Nonetheless, recent research adapted QuEChERS and used dried samples, animal-based food, cereal, milk-based products, and soil-sediment analysis [64]. The approach is based on analyte extraction in buffered acetonitrile (MeCN) and subsequent separation by salting out and d-SPE. The primary disadvantage of this technique is that the natural elements of the sample must be removed. Based on a study by Zhang et al. [65], they adopted LC-MS/MS to assess 60 different insecticide contaminants in cinnamon bark using a repeated dispersive SPE with QuEChERS.

Some studies reported that almost 54 pesticides residues were extracted and analyzed by acetonitrile. Furthermore, Reddy and Reddy [66] employed QuEChERS to extract pesticides from sunflower oil using modified charcoal to reduce fat and pigment thermal deterioration during analysis. Furthermore, according to Neufeld et al. [67], QuEChERS extraction has a high sensitivity to organophosphates and carbamates. Besides, the QuEChERS technique combined with magnetic SPE and DLLME was developed to remove pesticides from high-solid vegetable, fruit, and nectar samples [68].

5.5 Microwave-assisted extraction (MAE)

Environmental Canada pioneered microwave-assisted extraction (MAE), which is currently used in research applications and industrial settings. This approach employs microwave radiation to induce polar molecules and ions to migrate and dipoles to spin to heat solvents and assist the transfer of the target from the food matrix to the extractant [69]. According to Wang et al. [70], the significant edges of adopting MAE are reducing the time extraction, which could be assigned to the differences in the microwave and traditional heating performance. MAE also allows for on-the-fly connection to different analytical processes and the simultaneous execution of several samples. A quick and straightforward analytical method based on LC-MS/MS has been established to measure carbamate residues and mycotoxins in apples using MAE simultaneously. In the recovery rate range of 70–116%, the technique displayed strong linearity with high acceptable accuracy and a lower limit of detection [71].

5.6 Microwave accelerated selective Soxhlet extraction (MA-SSE)

Microwave accelerated selective Soxhlet extraction (MA-SSE) is a technique similar to traditional Soxhlet extraction but employs microwaves to improve the procedure [72]. Although MA-SSE is fast and effective, its poor selectivity requires additional cleaning operations. Besides, a selective MA-SE approach is required due to its time-consuming and labor-intensive nature. Zhou et al. [72] employed MA-SSE as a selective extraction strategy in their investigation to detect the carbamate contaminants in ginseng. The MA-SSE extracts the sample’s target analytes and interfering components using microwave-irradiated extraction solvent. After the solvent passed through the extraction container, the sorbent adsorbed the interfering elements in the solvent and collected the target analytes. Because of the effect of microwave irradiation, MA-SSE outperformed conventional extraction processes significantly. According to the findings, MA-SSE has much potential as a fast and reliable method for preparing samples to detect pesticide residue in complex matrices.

6. Conventional techniques for detection of carbamate pesticides

Various techniques for identifying carbamate residues are summarized in Table 1.

Detection method	Carbamate pesticides	Food products	Reference(s)
Surface-enhanced Raman spectroscopy (SERS)	Carbaryl	Orange juice, grapefruit, milk	[73]
ELISA immunoassay (IA)	Carbofuran	Cucumbers, apples, leek, sweet potato, potato	[74, 75]
Terahertz time-domain spectroscopy (THz-TDS)	Methomyl	Wheat, rice flour	[76]
Gas chromatography-mass spectroscopy (GC-MS/MS)	Methiocarb	Cabbage	[77]
High performance liquid chromatography (HPLC)	Propoxur	Lemonade, grape juice	[78]
Liquid chromatography-tandem mass spectrum (LC-MS/MS)	Aldicarb	Vegetable	[79]
Surface-enhanced Raman spectroscopy (SERS)	Oxamyl	Peach, milk	[37, 80]
High performance liquid chromatography with tandem mass spectrometry (HPLC-MS/MS)	Terbucarb	Plants	[39]
High performance liquid chromatography (HPLC)	Fenobucarb	Lemonade, grape juice	[57]

Table 1.

Detection techniques of carbamate pesticides.

6.1 Capillary electrophoresis (CE)

Capillary electrophoresis (CE) is a proper analytical method that could also be applied in various situations and is expected to offer several advantages, including fewer chemicals and samples, higher removal efficiency, and time efficiency. The capillary’s inner diameter (50–75 m) is tiny, allowing only a limited sample volume to be injected into the system, thus limiting sensitivity detection. Due to the small volume of sample that can be injected into such a capillary system, CE has been combined with sensitive detection [81] and combined with internet-based-concentration methods. Attig et al. [82] described a microextraction technique for selective preconcentration of N-methyl carbamate in water prior to CE analysis using temperature-controlled IL-DLPME in an alkaline buffer. Microextraction with ionic liquid and elution with a trace amount of dichloromethane was used to obtain the samples. MMWCNTs enhanced ionic liquid-analyte binding and recovery compared to using simple nanomaterials as a sorbent. Cheng et al. [83] developed a CE with amperometric detection based on a polyamide-modified carbon paste electrode to determine carbamate in alkaline water solutions. According to Zhang et al. [84], an efficient method for simultaneous determination of carbamate pesticides in vegetables included solid-phase microextraction for purification and enrichment, followed by CE separation. Standard addition recoveries of 86.1–115.8% for vegetable samples are quick and accurate. The presence of carbamates has been determined using nanomaterials such as graphene and gold nanoparticles in pesticide biosensors [85]. Direct electrodeposition of electrochemically reduced graphene oxide-gold nanoparticles-cyclodextrin and Prussian blue-Chitosan modified glass carbon electrodes was used to identify pesticides. Carbamate pesticides inhibit AChE activity, with malathion having a LOD of 4.14 pg mL⁻¹ and carbaryl having a LOD of 1.15 pg mL⁻¹.

6.2 Micellar electrokinetic capillary chromatography (MEKC)

Micellar electrokinetic capillary chromatography (MEKC), a hybrid methodology incorporating chromatographic and electrophoretic extraction principles, extends the usability of capillary electrophoretic procedures to neutral analytes. Surfactants are added to the buffer solution at quantities remarkably different from their essential micellar concentrations, producing micelles that move electrophoretically like any other charged particle. The separation is based on the differential partitioning of an analyte between two-phase systems: the moving aqueous phase and the micellar pseudo stationary phase [86]. Using MEKC with a UV-Vis detector, the best separation conditions were 20 mM phosphate buffer (pH 8.0) and 15 mM sodium dodecyl sulfate. The detecting wavelength was set at 200 nm, with a voltage of 12.5 kV supplied. Baseline separation of five pesticides took 15 minutes under these circumstances with low detection limits. This method produced high repeatability, reproducibility, separation efficiency, and a reasonable recovery rate in rice samples [87]. MEKC has evolved into an effective separation technology for neutral and ionic chemicals in complex mixtures, including a broad spectrum of analytes. MEKC is based on the separation of the micellar and aqueous phases. See et al. [88] originally described a technique for determining glyphosate and aminomethylphosphonic acid in tap and river water using a dynamic supported liquid membrane tip extraction approach followed by MEKC with capacitively linked contactless conductivity detection. Besides, Sung et al. [89] used in-line LLE surface analysis with CE to detect pesticides on solid surfaces of apples. Other research used the SPE-MEKC approach to identify trifloxystrobin, tebufenozide, and halofenozide in foods with detection limits ranging from 0.088 to 0.094 mg/kg [90]. Moreover, Santalad et al. [91] described an SPE-MEKC approach for determining the presence of six carbamate pesticides with low detection limits. Water-soluble CdTe/CdSe core-shell quantum dots were employed to enhance pesticides selective fluorescence enhancement [92]. The baseline separation took 12 minutes, and the detection limits obtained varied from 50 to 180 μg/kg [93]. DLLME coupled with sweeping in MEKC, a quick, easy, and sensitive approach for detecting certain neonicotinoid pesticides in cucumber samples has been devised. Under optimal circumstances, enrichment factors ranging from 4000 to 10,000 were obtained. The method’s linearity ranged from 2.7 to 200 ng g⁻¹ for thiacloprid, acetamiprid, and imidacloprid in cucumber samples and from 4.0 to 200 ng g⁻¹ for imidaclothiz, with the limit of detection varied from 0.8 to 1.2 ng g⁻¹. The new approach successfully analyzed neonicotinoid pesticides in cucumbers, promising outcomes [94].

6.3 Enzyme-linked immunosorbent assay (ELISA)

Immunochemical techniques, such as enzyme-linked immunosorbent assay (ELISA), have recently gained interest and recognition as rapid and low-cost extraction and detection procedures for pesticide compounds. Based on the antigen-antibody interaction, this analytical technique can give high sensitivity and specificity (selectivity) for particular kinds of pesticides. Additionally, since it can load many samples concurrently, it enables rapid and precise assessment of pesticide residues in agricultural items prior to shipping. Indeed, the primary advantage of ELISA for identifying pesticide residues is the convenience of sample preparation methods [95]. Bellemjid et al. [96] created a rapid ELISA to detect carbamates such as carbendazim and carbofuran using synthetic compounds with acid functions linked with BSA protein and injected into rabbits with antibodies collected for the immunoanalytical test. Zhang et al. [97] used nanobody Nb316 to develop an indirect competitive enzyme-linked immunosorbent test (ELISA) to detect carbofuran in vegetable and fruit samples. A phage display platform was used to extract and characterize unique nanobodies against the pesticide carbofuran from an immunized library. The average recovery rate of spiked samples was 82.3–103.9%, comparable to the conventional UPLC-MS/MS approach.

6.4 Gas chromatography-mass spectroscopy (GC-MS)

James and Martin [98] devised the gas chromatography (GC) technology in 1952. The fundamental working concept of gas chromatography is the volatilization of the sample in the input or injector of the gas chromatograph, followed by the separation of the mixture’s components in a specially designed column. Pesticide residues were recently found in Chinese liquor using gas chromatography-mass spectrometry [99]. In general, Chinese liquor is an extraction of fermented food. They are a trendy alcoholic beverage in China. In Chinese liquor, ethyl carbamate was found at a detection limit of 0.56 μg/L and a limit of quantification of 1.87 μg/L. Ethyl carbamate was also discovered in Chinese rice wine using gas chromatography-mass spectrometry [100]. According to Yao et al. [101], GC-MS detected ethyl carbamate in grain co-products. A gas chromatography-mass spectrometry assay with the limit of detection of 0.7 ng/g was developed to measure ethyl carbamate extracted from different distillers grains co-products. It was identified in all of the co-products of distillers grains examined in this investigation. The greatest concentration of ethyl carbamate was found in corn condensed distillers solubles, ranging from 1618 to 2956 ng/g. Other kinds of distillers grains co-products exhibited ethyl carbamate concentrations ranging from 17 to 917 ng/g.

7. Advanced techniques for detection of carbamate pesticides

In pesticide analysis, advanced technologies are presented as an alternative to the conventional chromatographic methods combined with selective sensors. The chromatographic procedures yielded sensitive, specific, and dependable analytical findings. However, they are time-consuming, complicated, and costly, with a high organic solvent usage, which is unsuitable for analyzing large samples [102]. New approaches are challenging to implement in most developing countries. The advancement of improved methodologies has resulted in promising instruments for easy and fast operation, affordable cost, and suitable for in-situ evaluation. Furthermore, they perform well in terms of pesticide detection accuracy and precision.

7.1 Molecular imprinted polymer (MIP) biosensor

Biosensors based on molecularly imprinted polymers (MIP) are widely used as sensitive sensing materials because they detect molecules with many biological weights. MIP has effectively created artificial materials that behave similarly to biological receptors; however, it has limited stability. MIP has also been indicated as a biosensing breakthrough due to its ability to overcome the drawbacks of current specific molecular elements such as antibodies, peptides, and enzymes [103]. MIP is used to detect pesticides by imitating biological receptors, polymerizing a functional monomer in the analyte, and finally removing the template using a polymer matrix [104]. Hence, this approach can detect pesticide residues in food since they are inexpensive, simple to use, and have excellent chemical and physical stability. Recently, Li et al. [105] published a work that demonstrated the construction of a MIPs biosensor to detect pesticides utilizing a carbon paste electrode modified with surface MIP microspheres and evaluated using cyclic voltammetry. The approach used on vegetable samples showed high sensitivity, with significant recoveries ranging from 97.2 to 101%. Additionally, Wang et al. [106] used a MIP sensor modified with polyquercetin(Qu)-polyresorcinol(Re)-AuNPs to assess methyl parathion in waters, juice drinks, and vegetable juice. Nevertheless, the analytical performance of sensors created to detect methyl parathion was lower. Xie et al. [107] detected pesticides in brown rice using MIP sensors and linear sweep voltammetry. Additionally, the MIPs sensor was produced via free-radical polymerization of p-vinylbenzoic acid on the surface of a modified glassy carbon electrode. The study demonstrated that the approach could detect thiamethoxam residues with an 88.7–94.0% recovery range. Li et al. [108] used differential pulse voltammetry to build a MIP-based sensor to analyze paraoxon and exhibited excellent stability after 3 months.

7.2 Optical biosensors

Optical biosensors have attracted considerable interest and are being applied in various fields, including food safety and security, biological sciences, environmental sensing, and medical science. The optical characteristics of the optical transducers, including absorption, reflectance, and fluorescence emission, will change in response to the analyte. In many instances, optical biosensors have been used to detect pesticides, especially enzyme-based biomolecules Yotova and Medhat [109] developed an optical biosensor to identify pesticides contaminants based on the parallel immobilization of AChE and choline oxidase enzymes in silicon dioxide hybrid membranes. The bioactive component of the sensor is a multi-enzyme system that includes AChE and choline oxidase covalently immobilized on new hybrid membranes. It demonstrates a constant value of acetylcholine at concentrations ranging from 2.5 to 30 mM. Previously, Xavier et al. [110] studied an optical fiber biosensor for assessing propoxur and carbaryl in vegetable crops, employing chlorophenol red as an optical transducer of the analyte’s inhibitory impact on the AChE enzyme. The linear dynamic ranges of carbaryl and propoxur are 0.8–3.0 mg L⁻¹ and 0.03–0.50 mg L⁻¹, respectively. However, propoxur has a lower detection limit (0.4 ng) than carbaryl in the biosensor (25 ng). Ultrasonic extraction was utilized to detect propoxur in spiked onion and lettuce, with recovery rates ranging from 93 to 95% for onion samples at the different concentration levels studied.

7.3 Electrochemical biosensor

Electrochemical biosensors are gaining traction as a novel detection principle, increasing sensitivity, specificity, and repeatability [111]. Biosensors, in theory, are made up of two or three-electrode systems, comprising auxiliary, reference, and working electrodes, that create electrical signals when a target biomolecule interacts with a recognition element [112, 113]. For example, Chauhan and Pundir [114] used iron oxide nanoparticles and carboxylated multi-walled carbon nanotubes nanocomposite-based AChE enzymes. The enzyme AChE was isolated from maize seedlings and covalently attached to a modified gold electrode as a working electrode. The modified gold electrode was developed to measure the presence of different pesticides, including malathion, chlorpyrifos, monocrotophos, and endosulfan in water and milk samples with LODs as low as 0.1 nmol L⁻¹.

Similarly, Zhao et al. [84] established direct electrodeposition of electrochemically based reduced graphene oxide-gold nanoparticles-cyclodextrin and Prussian blue-Chitosan modified glass carbon electrodes for pesticide determination. The AChE enzyme was immobilized via adsorption with a low detection limit for carbaryl. An AChE enzyme-based biosensor based on rGO-coated GCE was also created to detect carbamate herbicides in tomatoes with a detection limit of 1.9 nmol L⁻¹ [115]. Additionally, Sun et al. [116] have created an amperometric AChE biosensor-based poly (diallyldimethyl-ammonium chloride)-multi-walled carbon nanotubes-graphene hybrid film to evaluate carbaryl in vegetables. Besides, Cesarino et al. [117] used polyaniline and multi-walled carbon nanotubes core-shell modified glassy carbon electrode to construct electrochemical AChE biosensors to measure carbamate pesticides in apple, broccoli, and cabbage. The detection limits for carbaryl and methomyl were 1.4 and 0.95 mol L⁻¹, which shows lower than the allowed concentrations indicated by Brazilian regulatory regulations for the pesticides tested in the samples. Besides that, Song et al. [118] detected the carbamate pesticides using citrate-capped gold nanoparticles. The biosensor was made by first creating 3D MPS networks on an Au electrode and then adding citrate-capped AuNPs via an Au–S bond. Based on the inhibitory effect of carbamate insecticides on AChE activity, the pesticide’s action may be evaluated at a shallow potential. It was also demonstrated that the method could detect carbamate pesticides in real-world samples.

8. Conclusion

Pesticides and other environmental pollutants are being extensively monitored due to their potential threat to humans and agriculture. As a result, multiple methods for assessing pesticide residues in various matrices have arisen. Scientifically, capillary electrophoresis, immunoassay, GC, HPLC, and fluorescence detectors have high sensitivity. On the other hand, the earlier approaches are time-consuming, costly, and need highly skilled personnel. As a result, newer technologies have developed as a feasible choice for determining insecticide contaminant levels. Pesticides are increasingly analyzed using enzyme-based biosensors instead of analytical methods. Experts seek to build low-cost, ecologically friendly technologies as pesticide residues become increasingly urgent. The established enzymatic biosensor methods must be used to detect pesticide residuals below the approved safety level. Nanobiosensors allows for simultaneous monitoring of food products such as packaged food components, fruits, vegetables, juices, and the environment. A single and miniature biosensor that employs nanomaterials has a bright future in pesticides detection.

Acknowledgments

The authors would like to thank all the researchers involved in the project. This work was supported by the Grant from Universiti Malaysia Sabah (Grant No: PHD0024-2019).

Conflict of interest

The authors declare no conflict of interest.