Pesticide is a general term that includes a variety of chemical and biological products used to kill or control living organisms such as rodents, insects, fungi and plants [1]. Chemical pesticides are conventionally synthetic materials that directly kill or inactivate unwanted organisms in crops, public areas, homes and gardens and parasites in medicine [2,3]. Biopesticides are pesticides derived from natural sources like animals, plants, bacteria, and certain minerals. They are environmentally safe and non-toxic to plants and animals. However, their use is limited due to poor social awareness, comparatively lower crops yields, need for frequent applications, and the fact they are generally poorly researched. On the contrary, application of chemical pesticides has proved to be economically beneficial and has increased globally especially after the advent of “Green Revolution” of the 1960s. The productivity of crops has been increased by use of suitable pesticides. Moreover, they ensure increased food production, a safe and secure food supply, and other secondary benefits. Another important advantage is the resulting reduction in costs and labor [2,3].

Even though pesticides play a significant role in agriculture, they are among the most important environmental pollutants. This is due to their widespread presence in water, soil, the atmosphere and agricultural products. Pesticides pose a major threat, not only to living organisms, but also to the environment, especially ground and surface waters. Worldwide consumption of pesticides for agriculture use is increasing constantly [2,3,4]. In the European Union (EU) alone approximately 320,000 tons of active substances are sold every year, which accounts for one quarter of the world market [5]. Humans and wildlife are continuously exposed to a number of pesticides due to human occupations, through dietary (food, drinking water) and environmental exposure (surface water, ground water, soil, air) [2,3]. Adverse effects on human health of pesticide residues are generally known to include acute neurologic toxicity, chronic neurodevelopment impairment, possibly dysfunction of the immune, reproductive systems or cancer and many other ill effects [2,4].

Residues in fruit and vegetables, cereals, processed baby food and foodstuffs of animal origin are controlled through a system of statutory maximum residue limits (MRLs). MRLs are defined as: “the maximum concentration of pesticide residue (expressed as milligrams of residue per kilogram of commodity (mg/kg)) likely to occur in or on food commodities and animal feeds after the use of pesticides according to good agricultural practice (GAP)” [6]. There are various organizations which set MRLs, such as the European Commission (EC), Codex Alimentarius or national governments in Australia, Canada, Japan, USA, etc. Individual limits for different active substance/food commodity combinations are being set. As an example around 30,000 different MRLs have been set by the EC [7]. MRLs vary ordinarily within the interval 0.0008–50 mg/kg [8], typically between 0.01 and 10 mg/kg for adult population. The lower values of MRLs are set for baby food—the EC has specified a MRL of 0.010 mg/kg [9], and the lowest levels are set for particular special residues [10]. In drinking water, the permissible maximum residue level in the EU is 100 ng/L, and in the case of some persistent chlorinated pesticides the limit is set to 30 ng/L [11]. Analyses close to these above-mentioned levels correspond to what is called ultra-trace analysis. Scientifically accepted validated analytical methods for determination of compounds at low concentration levels are essential for surveillance/compliance programs established with the terminal goal of minimizing the hazards and the risks to health and achieving more sustainable use of pesticides.

There is increasing concern about certain pesticides and other synthetic chemicals that may act as pseudo hormones which disrupt the normal function of the endocrine system in humans and wildlife [12,13]. This specific category of pollutants, known as endocrine disrupting chemicals (EDCs) or endocrine disrupters, is comprised of the compounds that may affect the normal hormonal function or possess endocrine-related functions. During the last decades the interest and concern related to endocrine disrupters among scientists, regulators and public has increased substantially. In the last years a great deal of concern has been expressed worldwide over the increasing levels of EDCs found in the environment. This anxiety is caused by the adverse effects of these pollutants on the hormone systems of humans and wildlife, even when present at levels below ppb [14]. The present safety assessments of pesticides do not take into account of possible endocrine disrupting properties of pesticides.

This globally increased concern about EDCs has induced a need to develop highly sensitive and specific analytical tools for their determination in food, environmental and biological samples at very low concentrations. The state-of-the-art of analytical methodologies that are currently used to screen for EDCs are limited and are often based on both biological assays and chromatographic or hyphenated techniques [15]. The enzymatic methods can serve as a tool for rapid, in situ screening of large numbers of samples in a short period of time [16]. Biological tests are too specific to cover a wide range of different EDCs and they achieve limits of detection in µg/L [16]. Therefore, for reliable identification and quantification of compounds at ultratrace level chromatographic methods are required. GC-MS (gas chromatography-mass spectrometry) and GC-tandem MS are used for volatile or volatilizable analytes, while LC-MS/MS (liquid chromatography with tandem MS) is aimed at determining more polar and less volatile compounds [17].

This review article focuses on pesticides that act, or may act as endocrine disruptors (endocrine disrupting pesticides (EDPs)) and their analysis by capillary GC combined with MS detection in various matrices. Some space is devoted to sample preparation methods. The main part of the paper deals with published results and methods developed for EDPs residues analysis from the point of view of separation method, mode of MS detection and ionization and obtained limits of detection and quantification. The contribution to the development of analytical methods for EDPs investigation with the utilization of fast GC with MS detection is shown. The benefits of developed fast GC methods for selected EDPs over conventional GC are discussed. Examples of real-life analyses are presented.

Various types of natural and synthetic chemical compounds have been identified as EDCs. They are broadly classified into several categories, such as hormones (natural and synthetic estrogens or steroids, thyroids), pharmaceuticals and personal care products, industrial chemicals, pesticides, surfactants, and others [18]. By definition adopted by European Commission, “an endocrine disrupter is an exogenous substance or mixture that alters function(s) of the endocrine system and consequently causes adverse health effects in an intact organism, or its progeny, or (sub) populations” [19]. It is important to distinguish this from a potential endocrine disrupter, which is an exogenous substance or mixture that possesses properties that might be expected to lead to endocrine disruption in an intact organism, or its progeny, or (sub) populations [19]. EDCs were defined by the United States Environmental Protection Agency (US EPA) as “an exogenous agent that interferes with synthesis, secretion, transport, metabolism, binding action, or elimination of natural blood-borne hormones that are present in the body and are responsible for homeostasis, reproduction, and developmental process“. According to Diamanti-Kandarakis et al. [20] it is necessary to broaden the term—an EDC is a compound, which through environmental or inappropriate developmental exposures, alters the hormonal and homeostatic systems that enable the organism to communicate and respond to environment. The European Union (EU) has done extensive work towards official designation of endocrine disrupting substances, collecting literature studies on many chemicals and establishing priority lists since the year 1999 [21]. The majority of substances registered in the list of chemicals adopted by the EC in 2007 are pesticides [22]. Pesticides are selected in Table 1, Table 2, Table 3 according to their Category based on the documented/potential endocrine effect and their chemical group.

In the United States, the US EPA has been authorized to screen all manufacturing or processing chemicals and formulations for potential endocrine activity. This was realized in a two-tiered screening and testing process, Tier 1, Tier 2. In 2009, EPA released the Final list of Chemicals for Tier 1 Screening [25], which is an update of Initial list from 2007 (some chemicals were removed). On November 2010 the US EPA published the second list of chemicals for further testing. This list of 134 chemicals includes a large number of pesticides [26].

Pesticides are often very persistent, with half-lifes lasting decades and are transported over long distances by global circulation. Some of the pesticides were withdrawn from general use many years ago, but are still found in the environment. For example, DDT has been found in remote areas such as the Arctic, and even in the Antarctic, even though it was banned for agricultural use in the USA in 1973 and worldwide under the Stockholm Convention [13,16]. According to Snyder and Benotti [27] who reviewed the occurrence of trace EDCs in U.S. source and finished drinking water, atrazine was present in at least 50 percent of distribution system samples at concentrations up to 50 ng/L.

EDCs act mainly by interfering with natural hormones because of their strong potential to bind to estrogen or androgen receptors, as reviewed by Mnif et al. [2]. In particular, EDCs can bind to and activate various hormone receptors and then mimic the natural hormone’s action (agonist action). EDCs may also bind to these receptors without activating them. This antagonist action blocks the receptors and inhibits their action. Finally, EDCs may also interfere with the synthesis, transport, metabolism and elimination of hormones, thereby decreasing the concentration of natural hormones. For example, thyroid hormone production is known to be inhibited by ten EDPs (amitrole, cyhalothrin, fipronil, ioxynil, maneb, mancozeb, pentachloronitrobenzene, prodiamine, pyrimethanil, thiazopyr, ziram, zineb).

At the environmental level, wildlife is particularly vulnerable to the endocrine disrupting effects of pesticides. Effects linked to endocrine disruption have been largely noted in invertebrates, reptiles, fish, birds, and mammals [28]. Most of them are linked to exposure to organochlorine pesticides (OCP) and affect the reproductive function [2].

At the human level, EDPs have also been shown to disrupt reproductive and sexual development, and these effects seem to depend on several factors, including gender, age, diet, and occupation [2]. Age is a particularly sensitive factor. Human fetuses, infants and children show greater susceptibility than adults [29,30]. However, the effects may not become apparent until adulthood. Infants are extremely vulnerable to pre- and post-natal exposure to EDPs, resulting in a wide range of adverse health effects including possible long-term impacts on intellectual function [31] and delayed effects on the central nervous system functioning, low birth weight, fetal death and childhood cancers [32].

Due to the large variety of EDCs it is probable that humans and animals are exposed not to a single agent, but to a mixture of multiple endocrine disrupting agents [33]. Furthermore, the combined actions of pesticides also need to be addressed in the risk assessment process, as mixtures of these substances may cause higher toxic effects than those expected from the single compounds [2]. A central feature of endocrine disruption is that may cause detrimental effects on organisms at very low chemical concentrations [34]. Effects of EDCs at very low concentrations can be different from effects of the same chemical at higher concentrations [35]. Traditional approaches to determining safe exposure levels (for example, chemical risk assessments) do not work with EDCs.

The most efficient approach to pesticide analysis involves the use of chromatographic methods. Gas chromatography-mass spectrometry (GC-MS) with electron ionization (EI) and the combination of liquid chromatography (LC) with tandem mass spectrometry (LC-MS/MS) were identified as techniques most often applied in multi-residue methods for pesticides by Alder et al. [36]. For GC-amenable volatile and semivolatile pesticides GC methods are still preferred over LC methods due to higher resolution and lower detection limits. Especially fast GC techniques satisfy the present-day demands on faster and cost-effective analysis [4].

All the separate steps which make up the whole analytical method: extraction, clean up, pre-concentration, injection, separation, detection, and even data evaluation are adjusted according to the specific demands in the pesticide residues analysis. The two special essential requirements of pesticide residue analysis are the following: high sensitivity and multiresidual character of the method [4]. The tendency to reduce the absolute amount of applied pesticides and an increase of the effectiveness of EDPs at very low concentrations leads to the continuous shift to the lower analyte concentration in a sample and thereby need for methods able to reach lower limits of detection (LODs) and quantification (LOQs). Multiresidual methods provide the capability of determining different pesticide residues in a single analysis. Multiresidue procedures deal with a wide variety of physico-chemical properties of pesticides of different chemical families. The occurrence of EDCs at ultratrace concentration and with extremely diverse groups makes the analysis procedures even more challenging [18]. To overcome difficulties in the analysis, various methods have been developed, such as methods based on the use of enzymes [16], sensors and biosensors [37,38] and immunoassays [39]. However, the need for positive identification can currently be satisfied only by MS detection. Generally speaking, the mass-based methods employing mass spectrometry show relatively low detection limits as compared to other methods. For example, comparing detection limits of enzymatic methods for the detection of organochlorine, organophosphate and carbamate pesticides with chromatographic methods and MS detection it was concluded that enzymatic methods achieve limits of detection in the µg/L range, whereas traditional chromatographic methods are often able to detect pesticides at ng/L levels [16].

The mass-based analytical methods generally comprise a pretreatment or extraction/sample cleaning step followed by an instrumental analysis with specific settings for each target compounds based on its chemical properties. In many cases the sample preparation step plays in practice an important role in determining the overall level of analytical performance [18].

Several reviews have been published, which overview analytical methods for multiple classes of EDCs utilizing MS detection (GC-MS, LC-MS, GC-MS/MS, LC-MS/MS) in aqueous and solid samples by Petrovič et al. [40], LaFleur and Schug [41] and EDCs including pesticides in food and the environment by Holland [39], water, wastewater by Comerton et al. [17], and aquatic environment by Wille et al. [42]. At present, a combination of LC/MS and GC/MS techniques appears to be the most powerful and comprehensive approach to multi-class compound analysis as it widens the range of EDCs that can be reliable measured [17,42]. The consensus of these reviews is that mass spectrometry is undeniably the best technique to examine trace levels and provide essential identification and quantification of EDCs in food and environmental samples. Methods for analysis of EDCs residues including pesticides are reviewed also from the point of view of sample preparation [17,18,39,42].

The tendency towards the use of more polar and less volatile degradable pesticides has stimulated the application of LC in pesticide-residues analysis in environmental matrices with MS detection and atmospheric pressure chemical ionization (APCI) and electrospray ionization (ESI) as ionization techniques [42,43]. Matrix effects are one of the major drawbacks of LC-MS/MS, particularly when working in the ESI mode [17]. In addition, GC-MS allows less costly and easier operation than LC-MS/MS, GC/MS and GC/tandem MS generally have lower LODs when compared to LC-MS/MS [18], but requires more complicated and time-consuming sample preparation owing to the need for derivatization of polar analytes. However, commonly determined pesticides are typically volatile/semivolatile and thermostable, so GC is still a more suitable and preferable technique as reported in the literature.

In order to assess the applicability of the developed method, several real-life samples analyses usually follow the method validation or the developed method is applied in a monitoring survey presented in a separate subsequent publication. Target compounds in the analysis were pesticide residues, or multicomponent multiclass residues of EDCs including pesticides. We present examples of occurrence of EDPs in various matrices of real samples analyzed by conventional and fast capillary GC with MS detection. In water samples, wastewaters, surface and ground waters, according to Baugros et al. [58] banned OCPs, DDT and its residues, and methoxychlor are still present in surface and ground waters at the detection limits, or between 0.084 and 0.302 µg/L. Domestic use pesticides, like dimethoate at concentrations 0.0021–0.0198 µg/L and propoxur 0.0045–0.0897 µg/L, respectively, were determined in wastewater treatment plant effluents. Among fungicides, tebuconazole was present in the majority of samples, even in groundwater at concentrations from 0.0258–0.8947 µg/L. Snyder and Benotti [27] published results on a U.S. drinking water quality. They surveyed the occurrence of 36 pharmaceuticals and EDCs in the source (n = 20) and finished drinking water (n = 20). Twelve compounds were detected in at least 50% of source water samples and among EDPs atrazine (28 ng/L), metalochlor (15 ng/L), TCEP (13 ng/L), triclosan (1.9 ng/L), trimethoprim (2.2 ng/L) were found (median concentrations are given in brackets). Eight compounds were detected in at least 50% of the finished drinking water samples, and atrazine, metalochlor and triclosan were confirmed among the EDPs there.

Lopez-Espinosa et al. [61] analysed OCPs in 150 placenta samples from Southern Spain. Residues of one or more pesticides were detected in all of samples, with a mean of eight pesticides per placenta (range, 3–15). The highest concentrations were those of p,p´-DDT (2.37 ng/g of placenta), isomers and metabolites, followed by endosulfans. Endosulfan isomers and metabolites were found in more than 98% of the searched series of placentas. Lindane was detected in 74.17% of the placenta samples. With respect to the aldrin group, the order of their frequency was endrin < aldrin < dieldrin.

To show the potential of fast GC-QMS in the EI mode for the utilization in the ultratrace analysis of pesticide residues with endocrine disruption behaviour, a survey of EDPs in non-fatty food was published by Hrouzková et al. [50]. An important objective was to assess the occurrence of pesticides from different chemical classes suspected or known to act as endocrine disrupters in fruit and vegetable samples available on the market in Slovakia. Thirty-four samples of 20 different commodities were analyzed. Twenty-one compounds at concentrations in the range of 0.003–2.14 mg/kg were detected in 28 samples. The MRL value was exceeded in the case of dimethoate (peachA). In the case of fenitrothion (peachB) the determined concentration was at the MRL level. Seven samples contained residues of three or more EDPs. The concentrations of EDPs residues determined also in the NCI mode were in a good agreement with EI mode (Table 6). The example of pearA and kohlrabi shows the ability of the NCI-MS method for the pesticide residue analysis at the low concentration level. The given pesticides were not detected in EI mode. For illustration, the chromatograms of the target ions of EDPs residues analyzed by fast GC-MS in the SIM mode in the real sample extracts, in NCI and EI ionization modes, are given in Figure 3 [50]. It is evident, that NCI mode produces very “clean” chromatograms with high responses of analytes without influence from the matrices (orange, strawberry) as compared to results using EI mode.

Many chemical pesticides are in use today and new ones are being developed. They contaminate the food chain and the environment, through groundwater, rivers and soil. Pesticides are intentionally toxic towards target pests, but unintentionally toxic towards humans and wildlife. Unintentional health effects of pesticides include their endocrine disrupting effects. EDPs are known as a class of EDCs of xenobiotic origin. The significance and importance of research on analysis methods for EDCs/EDPs and the current state of EDPs are discussed in the paper. The concentration of a single EDC, or mixture of EDCs may be surprisingly low, in some instances less than 1 ng/L in aqueous system [41]. These findings are driving the detection limits of the analytical methods used to monitor EDCs/EDPs lower than they have ever been before. A variety of sample preparations and analytical techniques that offer ultra-trace detection limits were investigated (Table 4). The use of chromatographic methods hyphenated with mass-spectrometric detection provide the excellent sensitivity and precision. GC-MS remains the most useful and sensitive method for the detection of trace levels of volatile EDCs/EDPs. The most often used MS detector has been the quadrupole MS with EI. By utilising tandem MS it is possible to reach even lower detection limits. The powerful technique GC-TOF-MS provides the the possibility of performing untargeted fast GC of pesticides in complex samples on commercial instruments. At present a combination of GC-MS and LC-MS/MS techniques appears to be the best approach to multi-compound class analysis [17].

The main part of the review paper is devoted to the contribution of capillary GC-MS for method development for EDP analysis with the utilization of conventional and fast GC. The search for different calibration approaches based on the matrix-matched standardization, the application of analyte protectants and the influence of different matrices with differing amounts of co-extractants was studied with the aim of eliminating the adverse effects caused by matrix interferences. According to several authors [47,58] the use of the standard addition method is the best approach to solving matrix effect phenomena. Isotopically labelled surrogate and internal standard compounds can also be used to compensate for matrix effects [17,55,56]. The combination of conventional and efficient fast GC separation and selective MS detection with NCI results to selectivity enhancement and decrease of the limits of detection and quantification.

For EDP residues analysis ultrasensitive analytical methods are required and there is still the need to improve the performance and ruggedness of analyses. Despite the significant progress in the analytical instrumentation development, for most of analytes/matrices combinations there is a continuous need to employ effective extraction and preconcentration methods to reach the ultra-trace concentration levels and to avoid adverse effects of the matrix co-extractants on the results of quantitative analysis and maintenance of GC instrumentation. Identification and determination of endocrine disrupting pesticides is a relevant research trend and a source of progress of analytical methods as a base for necessary changes in regulations of the quality of food and environment in the future is expected.