A Scoping Review on Fungus and Mycotoxin Studies in the Building’s Environment: Mycotoxin Analysis by Mass Spectrometry

Abdul Rahman, Salina; Ahmad, Nurul Izzah; Mohd Salim, Roshan Jahn; Muhamad, Nur Jannaim; Omar Hamdan, Anis Syuhada; Leong, Yin-Hui

doi:https://doi.org/10.1155/2024/8581029

International Journal of Analytical Chemistry

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Abstract Introduction Conclusions Conflicts of Interest Authors’ Contributions Acknowledgments References Copyright Related Articles

Review Article | Open Access

Volume 2024 | Article ID 8581029 | https://doi.org/10.1155/2024/8581029

A Scoping Review on Fungus and Mycotoxin Studies in the Building’s Environment: Mycotoxin Analysis by Mass Spectrometry

Salina Abdul Rahman,¹Nurul Izzah Ahmad,¹Roshan Jahn Mohd Salim,¹Nur Jannaim Muhamad,¹Anis Syuhada Omar Hamdan,²and Yin-Hui Leong²

Academic Editor: Waleed Alahmad

Received13 Jun 2022

Revised08 Dec 2023

Accepted23 Dec 2023

Published27 Jan 2024

Abstract

It has been well-established that mycotoxins are poisonous chemical metabolites secreted by certain molds. Some of them significantly affect the health of humans and livestock. Increasing attention is now being paid to uncovering and identifying mycotoxins’ presence in the building’s environment. However, the main challenge remains in suitable and reliable analytical methods for their identification and detection in infected structures. GC-MS and LC-MS/MS techniques have been used extensively for mycotoxin analysis, and advancement in these techniques enabled a more comprehensive range of mycotoxins to be detected. As such, this study aimed to address a brief overview of various phenomena of existing sample collection, preparation, and analysis to detect mycotoxins in the building’s environment. This scoping review includes articles from 2010 to 2020 available from PubMed, Scopus, Cochrane, Wiley, Google Scholar, and ScienceDirect. Duplicate articles were removed, and exclusion criteria were applied to eliminate unrelated studies, resulting in 14 eligible articles. The present study provides an overview of mycotoxin analysis by GC-MS and LC-MS/MS in buildings. Many techniques are available for analyzing and detecting multiple mycotoxins using these methods. Future efforts would focus on rapid assays and tools enabling measuring a broader range of mycotoxins in a single matrix and lower detection limits. In addition, it would assist future findings on new techniques and mycotoxins that existed in the building’s environment.

1. Introduction

Mycotoxins are secondary metabolites of fungi associated with various toxicities in humans and animals. They have long been studied because of their extensive exposure to food and feed commodities and their potential use as therapeutic drugs and biological warfare agents. Due to the prevalence of mycotoxin contamination in foods and feeds, many years of research have focused on ingesting these toxic compounds. Inhalation of mycotoxins has yet to attract much attention, and available reports point mainly to occupational and agricultural settings. In the 2000s, mycotoxins were hinted to be toxic agents adverse to human health through inhalation exposure in a nonagricultural indoor environment. Airborne mycotoxins have been reported in moldy buildings other than agricultural settings [1, 2]. They originate from the fungal pollution of the indoor environment, e.g., sterigmatocystin and aflatoxins produced mainly by Aspergillus spp. which include A. versicolor and A. flavus, and macrocyclic trichothecenes produced by Stachybotrys chartarum. A comprehensive review of fungal pollution in an indoor environment was documented by Khan and Karuppayil [3]. Wood or wood-based products are susceptible to infestation by Cladosporium and Penicillium (Penicillium brevicompactum and Penicillium expansum), Trichoderma, and Aspergillus [4, 5]. Paecilomyces variotii, Trichoderma harzianum, and Penicillium species attack polyurethanes used in composites for insulation [6]. Also, dust on the surfaces and inner wall materials used in buildings, such as prefabricated gypsum board, paper, and glue, represents an excellent substrate for fungal growth. According to D’Mello [7] and Ciegler and Bennett [8], one mold species can produce several mycotoxins, and vice versa, and different mold genera may produce the same mycotoxins.

Human health effects attributed to the inhalation of mycotoxins in workplaces include mucous membrane irritation, skin rash, nausea, immune system suppression, acute or chronic liver damage, acute or chronic central nervous system damage, endocrine effects, and cancer [9]. Furthermore, some nonspecific symptoms possibly related to mycotoxin production, such as cough, irritation of the eyes, skin, respiratory tract, joint aches, headache, and fatigue, have also been documented [10, 11]. Aflatoxin, trichothecenes, and ochratoxins are the most well-known mycotoxins found in the indoor environment [12, 13]. To date, several hundred mycotoxins have been discovered. Various methods, including high-performance liquid chromatography techniques such as gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS), are widely used for identifying and detecting mycotoxins. Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) is vital and central for mycotoxin analysis. In contrast, the molecular polymerase chain reaction (PCR) approach and the enzyme-linked immunosorbent assay (ELISA) were commonly used in fungi identification [14]. The ability and sensitivity of multiple mycotoxin quantifications in various matrices such as food, feed, and biological samples have expanded significantly since 2010, attributed to the progress in LC-MS and the combination of appropriate sample extraction and cleanup procedures [15–17]. This approach is highly relevant as several mycotoxins tend to co-occur with others, regardless of the similarity in their chemical structure [18–20]. The most common mycotoxin extraction method applied for an indoor environment is liquid-liquid extraction (LLE) with a wide variety of solvents such as methanol [21–24], acetonitrile [25–27], and dichloromethane [28, 29]. In addition, the combination of the solvent mixture, e.g., methanol, dichloromethane, and ethyl acetate and chloroform and methanol, in different ratios has also been adopted [23, 30–32].

This selected study is a scoping review that aims to provide insight into the recent mycotoxin study and analysis in the building’s environment using GC-MS and LC-MS/MS from the available literature. This review has also enabled us to identify the knowledge gaps and future potential research in this area. Validated and updated evidence from this review can assist professional bodies in the importance of this subject matter on human health and mitigation strategies.

2. Methodology

Scoping reviews provide an excellent approach to analyzing research findings on a particular topic. Scoping offers an overview of the literature, narrowing down the related study to match our targeted case, and finally summarising the main component, concepts, and the available data to give an insight into the gap that is available in the field [33, 34]. Hence, a scoping review was chosen to review research articles on the available analytical techniques that allow the optimum discovery and quantification of targeted mycotoxins relying on the mass and ion charge methods. The mass spectroscopy system offers sensitivity and specificity for challenging and matrix-complex samples. The selected topic accommodated the proposed research study in the Institute for Medical Research (IMR), Malaysia (NMRR-18-962-41809). For the scoping approach, we adopted Arksey and O’Malley’s [35] method consisting of six phases to guide the selection process of the suitable literature available for our review purposes. We also considered updated guidelines published by the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) 2020 statement [36].

2.1. Phase 1: Identifying the Research Question

Various methods have been adopted for determining mycotoxins, whether in the air, building materials, or dust, utilizing mass spectrometry or a more advanced tandem mass spectrometry method. As for our scope, we would like to answer “what are the recent methods of LC-MS/MS and GC-MS that are being used to determine the presence of mycotoxin in the targeted samples collected from indoor air in building environments?”

2.2. Phase 2: Identifying the Relevant Studies

During the search, the following criteria were set as guidance:(1)Articles were written in English only(2)Articles published from 2010 to 2020(3)Databases search from PubMed, Google Scholar, Cochrane, Scopus, Wiley, and ScienceDirect(4)Open-access research articles only, excluding review papers, book chapters, and conference papers(5)Keywords are indoor air, mycotoxin, LCMS/MS, and GCMS

2.3. Phase 3: Study Selection

PRISMA guidelines were used in our study selection to assist as a guided protocol for the search. At the initial stage, all team members were assigned to a specific database to conduct a web search based on the selected keywords. The search was then transferred to the Microsoft Excel spreadsheet and assigned to team members to scrutinize the obtained papers based on our agreed inclusion criteria. Team members will review the journal papers’ abstracts to determine their relevance to our review scope. Furthermore, the full articles will be retrieved, and the information will be transformed to highlight the findings to decide whether a particular paper will be included or excluded for scoping review purposes. When members were unsure about the acceptability of a particular paper during abstracts or full-text screening, a discussion session was conducted to finalize the decision. The flow of the process is shown in Figure 1.

2.4. Phase 4: Charting/Organizing the Data

The identified, sorted, and selected papers were charted into a table to organize the information systematically. Table 1 shows how the information was tabulated to meet the research questions and scoping review purposes.

2.5. Phase 5: Collating, Summarising, and Reporting the Results

The main aim of a scoping review is to collect the available data, results from findings, and research output into more tangible information to be well-versed on the research that had taken place, the area that can be improvised, and finally give an overview of the research gap that can be tackled for the future research direction (Figure 2).

3. Mycotoxins in Buildings

This review was started with a concurrent investigation on mycotoxins in Peninsular Malaysia’s healthcare institutions initiated by researchers from the Institute for Medical Research (IMR), Malaysia. A scoping review was published covering topics on fungal identification in hospital settings, with participants from many nations and locations [38]. The review was generally on fungus profiling in hospitals, with limited papers reviewed on mycotoxin distribution. The findings revealed that the most common fungal genera detected in such settings were Aspergillus sp., Cladosporium sp., Penicillium sp., and Fusarium sp. with the most identified Aspergillus sp. in hospital wards being A. flavus, A. fumigatus, and A. niger, when compared to several settings such as neonatal intensive care units (NICUs), labour rooms, laboratories, and others. The findings discovered that intensive care units (ICUs) and wards were home to various fungus species, primarily Aspergillus spp. (Sham et al., 2021). Additional information on analysis for secondary metabolites in indoor air in building environments reported in this current review believes that more studies in this area can be pursued to gain more knowledge and understanding to solve issues quickly and effectively.

In the past ten years (Jan 2010–Feb 2021), 14 published studies on analysis for secondary metabolites in indoor air in building environments were recorded. Five studies were published in 2016, followed by three earlier publications in 2011. Significant findings from analysis of secondary metabolites in indoor air and studies on building environments based on 14 selected articles are listed in Table 2. Most studies on these areas were conducted in European countries, with four studies in France [21–23, 32]. One of each study was conducted in Italy [39], Croatia [30], Poland [27], Denmark [31], and Germany [25], respectively. Two studies were conducted in Finland [25], but another was in collaboration with researchers from the Netherlands and Spain [29]. Three studies were reported from outside Europe, one from the USA [24], and the other two were conducted in Malaysia. Both studies collaborated with a researcher from Sweden [28, 40].

Indoor air samples and study locations were selected from water-damaged home buildings [21, 24, 25, 27], schools and kindergartens [28, 29, 31, 40], workplaces (farms and industries) [22, 23, 39], and few selected buildings and locations [30] (Vishwanat et al., 2011), but one study did not mention their location [32]. Regarding study scope and objective, most studies provided qualitative and quantitative descriptions of the microbial toxins in indoor air and their metabolites found in samples. Lanier et al. [22] conducted an in vitro study on a specific fungus to check on a specific mycotoxin produced. Jeżak et al. [27] determined the toxicogenic potential of fungus isolates from moldy surfaces. Other studies extended into mutagenic properties of bioaerosol samples [21, 22], health risk assessment [23, 28, 40], and cytotoxic potency [30]. The development of mycotoxin screening in airborne particulate matter and method performance using LC-MS was also described [39].

There are studies reported on mycobiota on building materials and bioaerosols collected from different selected locations [21, 22, 25, 30]. Pottier et al. [21] reported that nine out of twenty selected houses contained a fungus identified as Serpula lacrymans. Also found in the selected houses were ligninolytic strains like Donkioporia expansa, Serpula himantioides, and Coniophora puteana. Lanier et al. [22] identified 45 fungal species in the cattle shed where Stachybotrys chartarum was observed for the first time. Among the common fungal species identified were Aspergillus fumigatus, Cladosporium cladosporioides, Penicillium chrysogenum, Stachybotrys chartarum, Ulocladium chartarum, and Aspergillus glaucus. Stachybotrys chartarum was found to be the highest contribution of recurrent strain which was up to 41%. Fourteen fungus species (8 genera) and three yeast species (2 genera) were most frequently isolated on infected surfaces in residential rooms in Poland. They were identified as Aspergillus versicolor, Cladosporium cladosporioides, Penicillium chrysogenum, Ulocladium chartarum, and Acremonium charticola. Four identified genera were susceptible to humans (Aspergillus sp., Penicillium sp., Cladosporium sp., and Phoma sp.). These included two species capable of producing hazardous mycotoxins (Aspergillus versicolor and Penicillium chrysogenum) [27].

Aspergillus section Versicolores producing sterigmatocystin was found in an apartment’s basements and grain mill, the first study over a year in Croatia. The dominant and highest sterigmatocystin-producing species identified using the calmodulin sequence were A. jensenii (1.192–133.63 μg/mL), A. creber, and A. griseoaurantiacus (208.29 μg/mL). The study also showed that the Aspergillus extracts producing positive-sterigmatocystin exert cytotoxicity towards A549 and THP-1 macrophage-like cells in low concentrations [30]. A fungal toxicogenic evaluation was conducted from a mold-infected exterior found in a residential room in the urban agglomeration in Poland without the influence of an environmental factor such as a flood-affected building or area. Mycological analysis showed Aspergillus versicolor and Penicillium chrysogenum producing sterigmatocystin (Figure 3(a)) and roquefortine C at a range of 2.1–235.9 μg/g and 12.9–27.6 μg/g, respectively, and the detection from air dust and scrapped material was below the limit of detection [27]. Indoor air quality greatly affects respiratory illnesses in which Norbäck et al. [40] found the prevalence of rhinitis and sick-building syndrome among students (n = 462; 14–16 years) from the tropical country of Malaysia. Total fungal DNA and Asp/Pen DNA were detected in all classrooms from Petri dishes and swab samples. In the Petri dish samples, 70% detection was obtained for A. versicolor DNA, 13% for S. chartarum, and 87% for Streptomyces DNA. Meanwhile, for swab samples, the detection was recorded at 56% for A. versicolor DNA, 3% for S. chartarum DNA, and 28% for Streptomyces DNA.

(a)

(b)

An interesting risk association existed between mycotoxin growing on wallpaper and the transfer to indoor air. A study by Aleksic et al. [32] showed mycotoxins growing on wallpaper followed by aerosolization from the infected surfaces produced macrocyclic trichothecenes (112.1 mg/m²) of satratoxins G and H, roridin L2 (RL2), and verrucarin J (VerJ); mycophenolic acid (1.8 mg/m²); and sterigmatocystin (27.8 mg/m²) as shown in Figures 3(a) and 3(b). The mycelium branching from fungal species and conidial morphology contributed to the aerosolization of particles from a substrate with the macrocyclic trichothecenes requiring the highest airspeed, and the total aerosolized toxic load was 5-fold more than others. Stachybotrys chartarum required the highest velocity of 5.9 m/s compared to Aspergillus versicolor and Penicillium brevicompactum to transfer the contaminated substrates. Stachybotrys chartarum biomarkers were identified in pure fungal cultures and cotton-tipped swab extracts collected from kindergarten in Greater Copenhagen. The identification revealed 12 Stachybotrys metabolites with atranones and macrocyclic trichothecenes (Figure 3(b)) on the gypsum wallboard. Došen et al. [31] also reported that it was the first time the same mycotoxins were found on the contaminated gypsum wallboard and settled dust. Four mycotoxins were targeted at water recycling and recovery facilities in France. Ninety-four air samples revealed quantifiable aflatoxin B1 and sterigmatocystin, while gliotoxin and ochratoxin (Figure 3(b)) were not found in any samples. Mycotoxin exposure was reported to be insignificant and did not give any concerning threat to the workers in a study conducted at waste management facilities [23]. A comparative study using settled floor dust collected from waste management facilities in Germany and residential houses in Finland showed a wider range of metabolites in concentrations of 0.04–49, 1444.0 μg/kg (Vishwanath et al., 2011).

3.1. Sample Collection and Processing

The sample collection and extraction procedures for mycotoxin analysis are summarised in Table 3. Mold and mycotoxins were sampled using a variety of techniques. Building material and dust samples from damaged buildings were collected using a vacuum cleaner [25] to detect multiple microbial toxins from indoor samples and naturally infested materials. Two papers described airborne dust analysis by sampling samples using cotton swabs and Petri dishes. This is performed in a classroom where they are interested in the associations of respiratory symptoms with the levels of selected fungal DNA, furry pet allergens, and mycotoxins in schools [28, 40]. A vacuum cleaner was used to collect settled dust floored from houses inhabited by small groups of people, generally less than 5 (Vishwanath et al., 2011). A foam swab wetted with methanol and swiped across the sampling area was performed for settled dust and moldy spot swab surfaces at different sites in school buildings [29]. Scraping on moldy surfaces and airborne dust samples inside residential rooms were collected using the “aspirator and head with filter” sets. The set consisted of a GilAir-5 (Sensidyne, USA) aspirator, an elastic hose, and an open-measuring head (Two-Met, Poland), with 37 mm diameter and 0.7 μm pore diameter of the GF/F glass fiber filter (Whatman, UK) reported by [27]. Pottier et al. used two methods to collect fungal aerosols in a damaged house: a sterile polytetrafluoroethylene (PTFE) filter with a 0.2 μm pore size attached to a calibrated vacuum pump and a sterile liquid with a cyclonic air sampler Coriolis® (Bertin Technologies, France). PM4 and PM10 samplings were performed for indoor/outdoor environments [39]. Another study on ambient air PM10 sampling was reported where bioaerosol from a cattle shed monitored revealed the presence of mycotoxins without concentration data due to below quantification unit. Airborne fungi were collected using a MAS-100 Eco air sampler (Merck, Darmstadt, Germany) with 400 holes (hole to agar impactor) and dichloran 18% glycerol agar (DG18) plates [30]. Air sampling was carried out by collecting dust with a CIP 10 sampler. The sampler uses the rotative cup technique with rotation, maintaining a flow rate of 10 L·min⁻¹. The sampler cup had a porous polyurethane foam filter (PUF). After sampling, the rotating cup containing the PUF was removed from the sampler, closed by the cover, and stored at 4^○C before analysis [23].

Floor dust mycotoxins were reported by [24] where they used a vacuum attached to a polyethylene filter sock (Midwest Filtration Company, Fairfield, OH, USA) and a precleaned crevice tool on a L’il Hummer™ backpack vacuum sampler (100 ft3/min, 1.5 horsepower; ProTeam Inc., Boise, ID, USA). An Andersen multistage impactor (Tish 180 Environmental, OH, USA) was used for capturing particles according to 6 ranges of size and 181 aerodynamic characteristics. Each impactor stage had a fiberglass disk to collect particles [32]. Settled dust samples were collected from all available surfaces (shelves, tables, fridges, and tops of the hanging lamps) and other places (excluding the floor) that were regularly cleaned. Each sample was taken from an approximate surface area of 45 × 45 cm using a clean precision Kimwipes® Lite wipe (Kimberly-Clark, GA, USA). Pure agar cultures were extracted using a microscale method modified for Stachybotrys metabolites. Three agar plugs (6 mm ID) were cut from a 15-day-old colony from each agar medium (potato dextrose agar (PDA) or malt-extract agar (MEA)) and placed in a 2 mL screw-top vial. Extracts from pure fungal cultures and cotton tip swabs from infected gypsum wallboards were further processed in laboratory before analysis for detection of mycotoxin metabolites by injection directly to an ultrahigh performance liquid chromatography diode array detector quadrapole time-of-flight mass spectrometry method (UHPLC-DAD_QTOF/MS) [31].

Depending on the type of samples collected for sampling, it is important to note that sampling techniques may differ depending on the specific objectives, environment, and suspected mycotoxin contamination sources. The vacuum cleaner can cover a large sampling area and larger sample material. At the same time, cotton swabs and Petri dishes allow for targeted sampling of specific areas where mold growth is visible. The GilAir-5 aspirator is a portable air sampling pump commonly used to collect and analyze various contaminants, including gases, vapors, and aerosols.

The method of collection and the type of samples obtained are the main differences between a sterile PTFE filter with a 0.2 m pore size attached to a calibrated vacuum pump and a sterile liquid with a cyclonic air sampler Coriolis®. A sterile PTFE filter with a pore size of 0.2 m connected to a calibrated vacuum pump is commonly used to collect particulate matter such as dust, pollen, or other solid particles in the air. The filter serves as a barrier, trapping particles while allowing air to pass through. The filter can be removed and analyzed after sampling to determine the types and quantities of particles present. On the other hand, a sterile liquid with a cyclonic air sampler Coriolis® collects microorganisms, such as bacteria and fungi, from the air. The cyclonic action within the sampler separates and concentrates the airborne microorganisms onto a sterile liquid substrate. This liquid is then used for laboratory analysis to identify and quantify the microbial contamination present in the air.

The main difference between PM10 and PM4 air filters lies in the size range of particles they are designed to capture. A PM10 air filter is specifically engineered to capture particles 10 μm or smaller in diameter, including dust, pollen, mold spores, and larger airborne particles. By targeting this size range, the PM10 filter helps monitor and assess air quality, as these larger particles can potentially impact respiratory health and indoor or outdoor air pollution levels. On the other hand, a PM4 air filter is designed to capture particles that are 4 μm or smaller in diameter, including finer particles, such as combustion byproducts, soot, fine dust, and certain allergens. By focusing on this smaller particle size, the PM4 filter provides more detailed information about fine particulate matter, which is known to have potential health implications, especially when inhaled.

The MAS-100 Eco air sampler is an advanced air quality monitoring and analysis device. It is specifically designed to sample and measure microbial contamination in the air, including bacteria, fungi, and other microorganisms. The MAS-100 Eco air sampler utilizes a high-performance filtration system to capture and collect these microorganisms, permitting further analysis and identification in laboratories. A CIP 10 sampler is an individual sampler that traps respirable particles. The physical collection efficiency of CIP 10 equipped with the inhalable fraction selector is estimated to be 50% for particles with an aerodynamic diameter of 1.8 mm and more than 95% for particles with an aerodynamic diameter greater than 2.8 mm.

3.2. Instrumentation Analysis

After sampling, mycotoxins require sample preparation using appropriate analytical instruments. Sample preparation demands using a suitable solvent to extract toxins from the matrix, a cleanup procedure to remove interferences from the matrix, and, if necessary, sample preconcentration before analysis. Selecting an appropriate solvent for mycotoxin extraction depends on the toxin’s structure. The most common method is liquid-liquid extraction (LLE), as shown in Table 3. Different types of solvent were used for the extraction of mycotoxins, such as methanol [21–23] and acetonitrile [25–27], and dichloromethane [28, 29]. Alternatively, a combination of a solvent mixture such as methanol, dichloromethane, and ethyl acetate [30, 31] and chloroform and methanol (2 : 1) [23, 32] was performed to extract different metabolites from samples which are compatible with the solvent. One paper reported on the two-stage extraction (methanol followed by hexane) procedure [40]. One paper conducted sampling on headspace-solid-phase microextraction (SPME), which employs a fiber coated with an extracting phase, which can be a liquid (polymer) or a solid (sorbent), to extract various analytes (volatile and nonvolatile) from various media (Vishwanath et al., 2011). Another paper described an accelerated solvent extractor (ASE) step followed by solid-phase extraction (SPE), ultimately enhancing the purification of analytes, making it possible to eliminate, reduce, and suppress signals from interference [39].

Airborne and bioaerosol samples were analyzed for instrumentation analysis using two HPLC-MS/MS protocols to cover many mycotoxins. Positive and negative ion modes were chosen to obtain a good signal from mycotoxins [21, 22]. One study was reported on the ultraperformance liquid chromatography (UPLC) system connected to Xevo Triple Quadrupole to determine mycophenolic acid, sterigmatocystin, and macrocyclic trichothecenes [32], UPLC-Orbitrap [23], and UHPLC-QTOF [31]. One paper reported mycotoxins in dust analyzed by gas chromatography-MS/MS [24], and one paper described mycotoxins indoor/outdoor airborne particulate matter using LC-MS/MS both in positive and negative ion modes to achieve efficient ionization for the known analytes [39]. Combination analysis using GC-MS/MS and LC-MS/MS was competent to cover volatile and nonvolatile compounds. These instruments analyzed airborne dust, fungi, and moldy surface samples [25, 28, 29, 40]. Interestingly, one paper reported using headspace GC-MS, and detection and quantification were performed on QTRAP LC-MS/MS (Vishwanath et al., 2011). Two papers described the application of HPLC to detect mycotoxins. Concentrations were calculated based on peak areas of the analyte compared to calibrated standards [27, 30].

Table 4 shows that most of the mycotoxins are analyzed using LC-MS/MS compared to the GC-MS method. Twelve publications reported mycotoxin analysis by LC and differentiated by HPLC, HPLC-MS/MS, LC-MS/MS, HPLC/UV-VIS, UPLC, and UHPLC. In general, mycotoxins such as aflatoxins (AFB1, B2, G1, G2, and M1), gliotoxin, mycophenolic acid, ochratoxin, alternariol, zearalenone, sterigmatocystin, patulin, citrinin, fumagillin, and trichothecenes (neosolaniol, T-2 toxin, HT-2 toxin, nivalenol, satratoxin, and roridin) are frequently quantified using liquid chromatography with the detector of a mass spectrometer. All the data acquisition on LC-MS/MS in mycotoxin experiments was performed in the positive or negative ESI (electrospray ionization) mode, using multiple reaction monitoring (MRM) scans.

Although some of the mycotoxins like sterigmatocystin are analyzed using detectors other than MS, e.g., UV-VIS [30], one of the advantages of MS over the other detectors is that it is easier to distinguish coeluting compounds using extracted ion chromatograms. LC-MS/MS offers a sensitive, efficient, and multianalyte analysis which is of great importance, especially on mycotoxin determination in various matrices, including indoor environmental samples, for example, ambient air, settled dust, and moldy surface. Attempts to identify these toxins in dust particularly are challenging as it correlates to the amounts present in the sample. Many fungal metabolites possess the same elemental composition and coeluate at the same retention time. Thus, a specific and sensitive instrument is required to distinguish similar compounds which are normally difficult to separate chromatographically. Indeed, several LC-MS/MS multimethods (≥2 mycotoxins) have already been developed for indoor environmental samples [28] (Vishwanath et al., 2011). Developed methods were reported to produce good recovery ranging from 42 to 101.10% with CV around 10% and of 0.994–0.999 (Table 4).

3.3. Method Validation

GC-MS and LC-MS/MS are widely used techniques for mycotoxin analysis in various environmental samples, including building materials, due to their high sensitivity, selectivity, and ability to analyze complex matrices. These techniques have successfully identified and quantified a broad range of mycotoxins, even at low levels, and can differentiate between mycotoxin isomers and closely related compounds. However, the reliability and accuracy of these techniques depend on proper method development, validation, and quality control measures. The lack of such data in studies significantly impacts the interpretation and outcomes. Without proper validation and quality control, findings may be influenced by matrix interferences, extraction efficiency, instrument variability, and method biases. Thorough evaluation and reporting of method performance parameters, such as limit of detection (LOD), limit of quantitation (LOQ), linearity, accuracy, precision, and selectivity, are essential. Quality control measures, including calibration standards, matrix-matched standards, and internal standards, are crucial to ensure accuracy and reliability. In addition, using appropriate quality control samples, such as certified reference materials, helps assess measurement uncertainty and ensures comparability across studies.

In mycotoxin analysis, it is crucial to mitigate matrix effects to ensure accurate and reliable results. Matrix effects occur when the sample matrix interferes with the ionization and detection of analytes, resulting in signal suppression or enhancement. To ensure optimal performance, assessing the practices employed to mitigate matrix effects (e.g., matrix-matched calibration, internal standards, and different sample preparation techniques) is essential.

To obtain an accurate measurement, matrix-matched standards to reduce matrix effects [24], stable isotope-labelled internal standards such as the 13C standard [21], and efficient sample cleanup [39] are normally performed. The detection and quantification of an analyte are significantly influenced by matrix effects associated with heterogeneous components in environmental samples [44]. Coextracted matrix components may cause interference with active sites in the GC inlet liner and the column and produce differential analyte signals between the matrix-containing sample extract and the matrix-free standard extract [45–47]. Efficient sample preparation, for example, solid-phase extraction (SPE) or LLE, is essential and has been found to reduce matrix effects potentially [48]. The effectiveness of these techniques in reducing matrix effects can vary depending on the sample matrix and mycotoxin of interest. This step is crucial, and optimization is needed to minimize sample loss. An internal standard (IS) is frequently used to improve the precision of quantitative analysis in which it compensates for matrix effects or sample loss during preparative procedures. In other words, it monitors and corrects any variations during sample preparation, extraction efficiency, and instrument response. Therefore, the selected IS should be similar to the target analytes regarding ionization properties or chemical structures to ensure it always reacts the same way as the analytes of interest, especially with a matrix [49]. An IS labelled with (13C) or (15N) was commonly employed for each group of mycotoxins, for instance, Fumonisin B1-13C34 and Deoxynivalenol-13C15 [21, 22, 39]. However, a nonlabelled IS, such as reserpine, has also been used as an internal standard [40]. According to Saito et al. [24], the accuracy of the results is largely dependent on the matrix effects, the appropriateness of IS, or the combination of them. On the other hand, only five publications reported analyses have been performed using GC-MS with the negative chemical ionization (CI) mode. Trichodermol and verrucarol, which are in the group of trichothecenes, are the most common mycotoxins tested by GC-MS [24, 28, 29, 40] (Vishwanath et al., 2011).

4. Conclusions

In conclusion, various techniques are available for analyzing and detecting multiple mycotoxins using LC-MS/MS and GC-MS methods. Mold and mycotoxin analysis has evolved in sampling techniques, processing, preconcentration, and instrumentation over the past years. Technological advances are beginning to overcome many challenges posed by the complexity of detecting multiple mycotoxins. Mass spectrometry advancements such as ionization modes, sensitivity, and acquisition speed have increased throughput, the number of mycotoxins that can be simultaneously screened, and the discovery of novel compounds of mycotoxins. Modern technologies, such as hyphenated liquid or gas mass spectrometry, have enabled these analytical methods to be developed and validated for mycotoxin analysis. However, due to the variety of chemical structures, using a single method for mycotoxin analysis is impossible. Routine analysis faces significant challenges due to the demand for rapid, simultaneous, and accurate determination of multiple mycotoxins. Future efforts would concentrate on rapid assays and tools that measure a broader range of mycotoxins in a single matrix and lower detection limits. Highly sophisticated multianalyte methods based on liquid chromatography coupled with multiple-stage mass spectrometry have been developed to identify and determine multiple mycotoxins. This new era of various screening mycotoxin and detection technologies will benefit future research.

5. Future Perspectives

It is anticipated analytical techniques and technologies for mycotoxin detection are likely to advance. This could include creating more sensitive and specific methods, such as advanced chromatographic techniques or rapid screening methods based on biosensors or nanomaterials. These advances will allow for faster and more accurate detection of mycotoxins in a variety of samples.

Moreover, there will be a greater emphasis on developing portable and field-deployable mycotoxin analysis devices. This will enable on-site testing and real-time monitoring, which is especially important in monitoring building environments where rapid decisions are required to prevent mycotoxin contamination.

Conflicts of Interest

The authors declare that they have no conflicts of interest.

Authors’ Contributions

All the authors contributed equally to each subtopic, reviewing, and final editing of the manuscript.

Acknowledgments

The authors would like to thank the Director-General of Health Malaysia for approval to submit this paper for publication. The authors also thank the Environmental Health Research Center (EHRC) and the Institute for Medical Research (IMR) staff for their assistance. The research herein was funded by the Ministry of Health, Malaysia (NMRR-18-962-41809).

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Copyright © 2024 Salina Abdul Rahman et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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