DTCs represent one of the most frequently detected classes of plant protection products in the European Union. Various methods have been developed to analyse their residues and metabolites in food and environmental matrices [6].

DTCs constitute a complex pesticide group to be analytically determined due to their poor stability in vegetable matrices and low solubility in water and common organic extraction solvents. Several parameters, including temperature and pH, influence DTC analysis. Therefore, food and environmental monitoring requires the development of specific methods not compatible with multi-residue ones commonly used for the routine quantification of many plant protection product residues [41].

The analysis of food matrices requires the homogenisation of plant samples and the DTC extraction with organic solvents. Indeed, once DTCs come into contact with vegetable acid juices, they rapidly degrade and decompose into carbon disulfide (CS₂) and the respective amine [58]. Still, many methods are, in fact, based on the detection of CS₂ evolved after the acidic digestion of any dithiocarbamates present in the sample and its following detection by different techniques such as UV-Vis spectrophotometry, gas chromatography (GC) coupled to mass spectrometry or headspace GC [59,60,61,62].

Other authors have considered special techniques such as capillary electrophoresis and reverse-phase high-performance liquid chromatography (HPLC) coupled with optical, electrochemical or mass-spectrometry detectors [46] to focus on the detection of some single DTCs such as thiram [63], propineb and ziram [61,64].

Other methods include the use of biosensors based on enzymatic inhibition, stir bar sorptive extraction (SBSE), dispersant liquid–liquid microextraction (DLLME), solid-phase extraction (SPE) and solid-phase microextraction spectroscopy (SPME) [65], and Raman spectroscopy [46,66].

Many methods developed to analyse DTCs in different matrices are based on the official EPA method 630 [67], a colourimetric method applicable to determining DTC pesticides in municipal and industrial wastewater. The method is based on reducing DTC compounds to carbon disulfide released during hot acid digestion; the total dithiocarbamate concentration is measured from the amount of CS₂ produced and measured by spectrophotometric techniques.

The total DTCs concentration is expressed as the sum of CS₂, but fails to distinguish among the individual analytes. An aliquot of the sample of approximately 1 L is digested in a hydrolysis flask with a tin chloride solution dissolved in concentrated hydrochloric acid; bringing the liquid in the flask to a gentle boil, the reaction leads to the formation of carbon disulfide by hydrolysis of the dithiocarbamate moiety. The developed CS₂ is conveyed by a slight vacuum/stream of nitrogen into the hydrolysis apparatus. The CS₂ evolved is then subjected to purification; it is then absorbed in an ethanol solution and reaction with copper acetate in the presence of diethanolamine leads to the formation of a yellow complex [58]. The absorbance of the coloured complex can be measured at 435 nm using a UV–visible spectrophotometer [6] or, alternatively, CS₂ evolved from the acid treatment of the sample can be analysed by gas chromatography technique [68].

Method interferences may occur by contaminants in reagents, glassware and laboratory equipment; therefore, it is also essential to correctly evaluate the washing reagents and their power to eliminate any interference. The pairs of reagents proposed by the different authors, e.g., lead and sulphuric acid acetate, sodium hydroxide and sulphuric acid or sodium hydroxide and lead acetate, are environmentally and economically viable. Sulphuric acid has also been effective in reducing background interference [58].

Additional matrix interferences due to the formation of collateral compounds may also occur in the DTC analysis. For example, the acid hydrolysis of thiram (tetramethylthiram disulfide, TMTD) leads to the formation of reaction products carbonyl sulphide (COS) and hydrogen sulphide (H₂S) at the expense of CS₂ [69]. Amines may be used as absorbent agents to separate CS₂ and other reaction products [13]. It is important that the digestion/distillation flask′s temperature reaches boiling point faster. In addition, a significant problem concerns the quantification of phytogenic CS₂, which leads to false positives. In fact, plants of food interest, such as Brassica, produce glucosinolates [70]. Such natural substances, during the acidic digestion of DTCs, can produce CS₂ and thus can lead to an overestimation of the content of DTCs in these agricultural products [5]. This is a pervasive problem in raw materials used to prepare baby food [5].

Some authors have tried to evaluate the specific ranges of CS₂ naturally produced by Brassica under conditions of acid digestion. The evaluations were carried out during post-harvest treatments or in the processing of horticultural products belonging to this crop [71]. The researchers noted that natural plant components such as brassine or isothiocyanates favour the formation of CS₂ under certain conditions. Other authors evaluated the effect of the sulphurisation and sulphur residues on the traditional DTCs analysis method. Various agricultural foods are subjected to the treatment of sulphurisation that improves the use of the product, but, at the same time, it releases sulphur residues [35]. The evaluation was conducted on sulphur dioxide and dithiocarbamates present in apricots before and after the sulphurisation process. The study shows that measuring total CS₂ as the sum of DTCs after sulphurisation can lead to false positives because sulphur compounds (or also pesticides) are present in products and can be considered DTCs by mistake [72]. Consequently, the formation of CS₂ is not an unequivocal indication of the presence of the DTCs [71].

Therefore, CS₂ may not always be well correlated with DCTs and identifying the specific residues of this pesticide group would be necessary. Indeed, the European Food Safety Agency (EFSA) has expressed the need for analytical protocols specific to each active ingredient of the DTCs.

In a study of [73], the researchers proposed a new digestion/distillation protocol to overcome the interferences issues, with washing and absorption units positioned vertically on the reflux condenser. The apparatus was easier to assemble and less vulnerable to gas leaks from the joint points than the traditional one cited in the EPA method [73]. Moreover, the new vertical arrangement allowed a percentage of recovery of ziram, mancozeb and thiram extracted from plant matrices ranging from 82% to 120%. However, in other similar matrices, recoveries appeared to be the same as those obtained using the traditional digestion/distillation system [73].

Subsequently, attention was placed on the efficiency of washing in the vertical system, which improved the sensitivity of the method, particularly for the matrices subjected to background interference [74]. Some authors used a modified vertical system in which the two washing chambers (arranged vertically) with a sintered glass bottom contain approximately 10 g of boiling chips wet with 50% concentrated solutions of NaOH and H₂SO₄. A methanol KOH solution absorbs the gas. The system described allowed them to reach the limit of quantification (LOQ) in fruits and vegetables up to 0.01 mg/kg⁻¹ of CS₂ [74].

Although spectrophotometric determination is still widely used in DTC analysis, the chromatographic approach may become the accepted method because it allows better recoveries and high sensitivity [75]. Gas chromatography coupled to mass spectrometry (GC-MS) is considered one of the techniques indicated in the most widely used and standardised methods (EN 12396) for analysing dithiocarbamate fungicides.

The CS₂ derived from DTCs digestion was measured by gas chromatographic [68] coupled with more selective detectors such as electron capture, pulsed flame photometric and ion trap mass spectrometry detectors (GC-ECD, GC-PFPD and GC-ITD-MS). In a study of [75], the authors determined DTCs as the sum of CS₂ in soya by GC-PFPD and GC-ITD-MS. The method showed good recoveries (between 68 and 91%) and LOQ equal to 0.05 mg kg⁻¹ of CS₂ [75,76].

Another study conducted by [77] reported a study in which the authors determined separately millinebs, ethylene bisdithiocarbamate (EBDTC), propylene bisdithiocarbamate (PBDTC) and dimethyldithiocarbamate (DMDTC). The technique used was GC-MS after extracting fungicides from plant and zootechnical matrices in an alkaline ethylenediaminetetracetic acid (EDTA)/cysteine solution. DTCs were derived in their methyl esters with methyl iodide (LOQ 72 mg/kg) [77].

The analysis of individual DTCs in tap water is rather complex. Several methods have been developed (capillary-UV electrophoresis, HPLC-UV, HPLC ion pair, etc.) but need better selectivity and sensitivity. Some authors [76] analysed the polycarbamate, consisting of EBDC combined with zinc and DMDC, with a GC-MS method. The method involves the programmed injection at the temperature of the input column. The compound was analysed as methyl derivatives of dimethyldithio-carbamate (DMDC) and dimethyl ethylene bisdithiocarbamate (EBDC). The technique makes it possible to measure the polycarbamate in tap water better. In addition, the simultaneous analysis of DMDC-methyl and EBDC-dimethyl allows recognition from other DTCs, such as thiram and ziram, with DMDC side chains. The analysis showed a recovery of 79% with an RSD of approximately 6% [76].

Dithiocarbamates include neutral compounds such as thiram (dimethylf-thiocarbamate) and disulfiram, which are more easily analysed than the other ones due to their tendency to create complexes with different metals and polymers. Several methods detect DTCs ions after their complexation with specific agents such as ethylenediaminotetraacetic acid (EDTA).

Some authors analysed ziram, thiram and zineb by extraction and subsequent high-performance liquid chromatography coupled to a UV detector (HPLC-UV) on a reverse-phase column (Nucleosil RP-18). In the method, ziram and zineb were subjected to a double extraction from the vegetable matrix; the first extraction was performed through an EDTA/cysteine solution and the second one with an organic solvent (tetrabutylammonium hydrogen sulphate) for a final derivatisation with methyl iodide. Otherwise, thiram was extracted with chloroform and analysed by direct injection with HPLC-UV. Good recoveries were obtained for different crops and water samples (59–85%) with a limit of detection (LOD) equal to 0.01 mg/kg for ziram, 0.02 mg/kg for zineb and 0.01 mg/kg for thiram [58,78].

Other authors tried to extract thiram from soil and foods (apples and lettuce), with a dichloromethane solution. The analyte was loaded onto a reverse-phase column and then complexed with copper (II). Differently, zineb and maneb were extracted from the soil with an alkaline solution of EDTA, selectively pre-concentrated as ion pairs on a pre-column C18 and loaded online with cetyltrimethylammonium bromide. Thiram showed a LOD ranging from 0.005 to 0.01 mg/Kg for soil and apples, while lettuce showed a LOD equal to 0.05–0.1 mg/Kg [79].

Two different procedures have been described by [80], to quantify ziram in spinach grown in greenhouses. Both require determination with HPLC and ultraviolet detection. In both cases, the samples were freeze dried; in the first protocol, the sample was extracted with supercritical carbon dioxide, adding methanol as an organic modifier. In the second method, the extraction was performed using an EDTA–methanol solution (1:1). On the one hand, the extraction with supercritical carbon dioxide resulted acceptable for low ziram concentrations and a small amount of samples. On the other hand, the EDTA-methane-based extraction was more suitable for samples with a higher content of ziram [80].

Van Lishaut and Schwack [81] proposed the first work to detect four classes of DTCs (ethylene dithiocarbamates, N-methylthiocaramates, N,N-dimethyldithiocarbamates, propylene dithiocarbamates) in plant samples using a reverse-phase ion-pair chromatography method coupled to a UV and electrochemical detection. Several plant matrices showed recoveries ranging from 72 to 111% and LOD of 4–8 µg/L.

Perz and Schwack [82] added cysteine to the alkaline extracts of the samples to increase the stability of the DTCs. They also optimised the method of Van Lishaut and Schwack [81], using a new column, Xterra RP18. The method showed good recoveries for different plant matrices (93–120%) [82].

Neutral DTCs can be analysed separately by LC-MS with a source operating in atmospheric pressure chemical ionisation (APCI) in positive ion mode (APCI+). This ionisation resulted in more sensitivity than electrospray ionisation (ESI) in positive mode (ESI+) [83].

In a study of [84], it validated a quick but sensitive method to determine propineb (a representative from the PBDC group) and mancozeb (the representative of ethylenebis-dithiocarbamate EBDC group) simultaneously in real samples of fruits, vegetables and mushrooms. The analytes were decomposed in an alkaline medium and derivatised with dimethyl sulphate to PBDC-dimethyl and EBDC-dimethyl. After the extraction and cleaning with QuEChERS (quick, easy, cheap, effective, rugged and safe), the methyl derivatives of PBDC and EBDC were analysed with UHPLC-MS/MS and electrospray ionisation. The method allowed good recoveries in the samples examined (from PBDC to PBDC-dimethyl: 86.1–106.9%, while from EBDC to EBDC-dimethyl: 85.2–101.6%). The analysis of PBDC-dimethyl derivatives showed a LOQ of 0.5–1.5 µg/kg, while LOQ was 0.4–1.0 µg/kg for EBDC-dimethyl derivatives. In addition, the stability of the methyl derivatives of the two groups of fungicides was observed both in the extraction solvent (acetonitrile) and in the plant matrices [84].

Other researchers [85] characterised and analysed fungicides N,N–ethylene-dithiocarbamate (manzeb, maneb and zineb) in environmental water samples. The analytes were extracted in chloroform–hexane (3:1) and derivatised with methyl iodide. The derivatised products were determined in LC-MS with ESI ionisation, and the average recovery at the sub-ppb level was 79%.

The choice of the right DTC analysis technique, as for other pesticides, depends on the chemical–physical properties of the molecules and obviously on the economy, instrumental and labour resources available. The analysis of DTC fungicides has seen the transition from spectrophotometric to spectroscopic techniques to the success of chromatographic methods (HPLC-MS or GC-MS). In fact, the latter show optimal instrumental performance such as sensitivity and accuracy, achieving at the same time satisfactory LOQ.

However, the success of these techniques depends on the complexity of the matrix of the sample and the chemical–physical characteristics of the molecules belonging to the DTCs group. Therefore, in HPLC-MS or GC-MS analyses, cleaning and extracting analytes become essential to eliminate all interferences in food and environmental matrices. Since environmental and food samples can contain many components with different properties, the matrix′s complexity will affect the chromatographic techniques′ robustness [2].

The analysis of these fungicides requires a sample preparation step to remove the compounds that cannot be quantified chromatographically to avoid the matrix effect. For this reason, many authors, in their studies, refer to techniques of liquid or solid phase extraction (SPE). In this way, it is possible to increase the concentration of analytes of interest and significantly improve the sensitivity of the determination. Some authors have used liquid–liquid partitioning with dichloromethane or SPE on Extrelut columns, often obtaining poor recoveries [92].

In a study of [93], it was defined a simple method for analysing fungicidal polycarbamate in riverine and tap waters. The samples were placed in an alkaline solution of EDTA/cysteine and then subjected to SPE with the Oasis HLB cartridges (60 mg, 3 mL), eluting the final extract with distilled water. The LC-MS/MS analysis in positive atmospheric pressure ionisation followed the extraction. The LOD and LOQ for DMDC-methyl in water were, respectively, 0.061 and 0.21 µg/L. Otherwise, the analysis of EBDC-dimethyl showed LOD and LOQ of 0.032 and 0.11 µg/L, respectively.

Another study of [92] optimised two different extraction protocols to evaluate the best approach in terms of recovery in the analysis of DTCs and their metabolites (propilenthiourea or PTU, etilenthiourea or ETU) in commercial vegetable matrices. The first protocol assessed the SPE extraction, while the second one was based on solid-phase matrix dispersion methods (MSPD). The authors prepared preliminary tests using solutions of fungicides at different concentrations in water in order to understand the influence of different parameters, including salt addition, pH, homogenisation of samples before extraction and the solid phase (florisil, alumina, C18, C8, carbon and silica) on the SPE. Differently, they tested the better dispersion agent (C8, C18 and carbon) for optimal extraction based on the MSPD procedure. Both extraction procedures demonstrated several advantages, such as poorly used organic solvent, low cost, small amount of sample and good sensitivity. However, as far as the recoveries are concerned, the MSPD showed better recovery values than SPE. On the one hand, the MSPD showed recoveries in the range of 5–90% for DTCs and 64–89% for metabolites. On the other hand, SPE recoveries ranging from 3% to 90% were obtained for DTCs and, lower than the MSPD method, for the metabolites. The metabolites′ high polarity and water solubility probably do not allow good recoveries with SPE. However, the SPE could increase the sensitivity of the determination due to the possibility of extracting a large amount of sample (up to 10 g), leading to an improved LOQ with, at the same time, good recoveries. In addition, the pH and salt addition did not show an important effect on the SPE extraction. Vice versa, the homogenisation of the sample influenced the extraction and the best results, in terms of recoveries, were obtained by homogenising the samples with water, probably due to the loss of the compounds during evaporation using solvents [92].

An alternative analytical approach to DTC analysis concerns the electrochemical approaches. Several authors have deepened the application of electrochemical sensors as simpler and cheaper tools than spectroscopic and chromatographic techniques for analysing DTCs.

Using electrochemical instruments to determine DTCs has been a known technique for decades [94]. The DTCs lend themselves well to this analysis due to the presence of various electroactive sites on their surface. Even in water, their determination is favoured by the presence of thiol groups that dissociate from the metal, forming the carbamate anions. For example, in aqueous conditions, thiol groups of propineb lose interaction with zinc, forming their carbamate anion [95]. In the electrolytic analysis of DTCs, other factors such as the pH of the solvent electrolyte, the type of electrode and the waveform used for the analysis are involved in the control of the fraction of the molecule detected [35].

In this analytical context, the low solubility of these molecules in water improves the method′s sensitivity because a preconcentration of the analytes is to be determined before the quantitative phase takes place; this is because the molecules adsorb to the surface of the electrodes. Many methods studied used non-modified metal or carbon electrodes applying important voltages to determine DTCs effectively. The voltages could reduce the specificity of the signal due to the presence of other compounds that react electrolytically, influencing the signal and, thus, the selectivity of the method used. Despite the great attention shown towards these simple and economical methods, they cannot be used for routine analysis. In fact, electroanalytical methods do not have sufficient specificity and are conditioned by various interferences [35].

In order to optimise the separation process for complex mixtures to be analysed, two-dimensional chromatography is used. It leads to the enrichment and purification of analytes, increasing separation and method sensitivity. In a study of [96], the researchers conducted a monitoring study of prenatal exposure to mancozeb. The mothers′ urine was analysed to determine the metabolite ETU. Complex analysis was performed by two-dimensional liquid chromatography with a triple-quadrupole linear ion trap (QTRAP 5500; AB Sciex, Milan, Italy).

To improve analytical selectivity, emphasis has been placed on techniques involving the use of biosensors. These techniques associate a sensitive analysis method with a precise biorecognition element. They overcome the drawbacks of electrochemical sensors because they exploit electron transfer mediators or provide a direct passage of charges from enzymes to the electrode. Therefore, large voltages are not necessary.

Biosensors have been used more for other pesticides than DTCs due to the low commercial availability of molecular-compatible biological molecules (aptamers, antibodies and polymers). DTCs inhibit the enzymes laccase, tyrosinase and aldehyde dehydrogenase, and this ability has, therefore, been exploited to develop biosensors that use these proteins [97,98,99,100,101,102]. The application consists in exposing the sample (containing the active principle of the fungicide) to the sensor and measuring the signal before and after exposure. In the presence of DTCs that inhibit the activity of enzymes, the electrochemical signal is reduced proportionally to the amount of fungicides present [35].

The electrochemical detection method may be poorly selective because enzymes are inhibited by a group of DTCs but not by individual compounds belonging to this class of fungicides. Furthermore, other molecules can inhibit these proteins, e.g., heavy metals. Therefore, the detection by enzymatic inhibition based on biosensors could be used as a quick method to make a first screening. Several researchers are deepening their knowledge about them to solve the selectivity problem. Some authors investigated the potential use of multiplex sensors, others the use of enzymes extracted from more stable extremophiles and with greater specificity for the substrate. It is also interesting to use genetic engineering to create new enzymes that can be efficient for these applications. In addition to controlling enzymatic abilities, research today investigates new nanomaterials that can produce a better electrochemical signal. The goal is to create rapid, effective and sensitive devices to build biosensors suitable for both DTC analysis and sampling [35].