A Scoping Review on Fungus and Mycotoxin Studies in the Building’s Environment: Mycotoxin Analysis by Mass Spectrometry
Table 3
Various procedures for mycotoxin studies in indoor air.
No.
Country
Sample collection
Type of extraction
Protocol/modification
Advantages/disadvantages
Reference
1
France
Fungal aerosols were collected for mycotoxin quantification by using a sterile PTFE filter of 0.2 μm pore size mounted in 47 mm diameter filter holders and connected to personal air pumps calibrated to draw 2 L/min for 3 h. Sampling point was selected at room with visible damage and room without apparent damage
The mycotoxin from the fungal aerosol was extracted from the PTFE filters using 10 mL of methanol/water/formic acid (80/20/0.1), which was then maintained in an ultrasonic bath for 3 minutes before being agitated in a multitube vortex. Second extraction was performed with 10 mL of 50/50 dichloromethane/ethyl acetate
The extracts were combined and evaporated to dryness under nitrogen using a Buchi evaporator. The final residue was diluted in 0.5 mL of acetonitrile/water (10/90) and filtered through Millex-HV 0.45 m before being injected into the HPLC-MS/MS system
The study found that a multianalyte tandem mass spectrometry-based technology could be used to look for mycotoxins in fungal aerosols, allowing occupants’ exposure to indoor mycotoxins to be assessed. Future research is needed to better understand the amounts of mycotoxins in indoor aerosols over lengthy periods of time
Indoors, a low-volume universal XR pump, SKC deluxe 224-PCEX8, fitted with an aluminium cyclone, was utilized at a flow rate of 2.5 L·min1 (to collect PM4), while an outdoor dual-channel sampler (HYDRA dual sampler) was employed at a flow rate of 2.3 m3/h (38.3 L/min) to collect PM10. Private interior environment, public indoor environment, indoor workplace, and outdoor environment sampling points were chosen
ASE extraction and followed by SPE purification
Sampled filters, blank filters, and spiked filters were extracted by ASE using two cycles of a combination of H2O: ACN (10/90) at 100°C and 1500 psi. The extracted volume (about 20 mL) was evaporated to dryness and redissolved in 1 mL water before loading into an SPE C18-M Strata cartridge that had previously been conditioned with 4 mL MeOH and rinsed with 4 mL water. After washing with pure water, compounds loaded and retained in the cartridge were eluted with 9 mL of MeOH/H2O 90/10 and 4.5 mL of pure ACN (4 mL). The analytes were swiftly eluted from the SPE cartridge at a steady flow rate using a vacuum manifold. Before the HPLC-MS-MS analysis, the extracts were evaporated under nitrogen stream. Optimal ASE extraction conditions: ACN/H2O (90/10) solvent mixture with two static extraction cycles (heat-up time 5 min; static time 5; flush volume 60%; purge time 300 s; pressure 1500 psi; T 100°C). The target analytes recovered the best with the Strata C18-M cartridge
Sample cleanup and SPE purification step reduced ion suppression significantly and/or eliminate interferences before HPLC-MS-MS analysis
21 dust floor samples from two water-damaged buildings were collected using floor vacuum. Each floor was vacuum covered for a 2 m2 area for 5 minutes. Samples were homogenized after removing hair, fluff, and larger objects
Extraction with methanol followed by derivatization
HP-5 ms fused-silica capillary column (30 m i.d., 0.25 mm i.d.). The sample injection volume is 1 l. The oven was set to a 70°C beginning temperature, which was then ramped up to 280°C at a rate of 20°C/min. The verrucarol derivative precursor ion was m/z 638, which gave a target product ion of m/z 302 for quantification and two ions of m/z 262 and 213 for qualification (retention time: 9.8 min). Internal benchmark (ISTD, 1, 12-dodecanediol)
Matrix-matched standard curves could be effective for obtaining accurate MCT readings in dust. In the dust extracts, ISTD showed significantly larger matrix effects. None of the 21 dust samples obtained from water-damaged buildings could be detected using standard calibration curves with ISTD modification
Ambient air sampling was conducted 24 hours per day in the feeding corridor cattle shed of dairy farm, using a high-volume sampler with PM10 head and 150 mm microfiber quartz filters. Sampling point was selected at feeding corridor
Samples were extracted twice with 30 mL of methanol acidified with acetic acid (0.5%) from the quartz microfiber filter. The solutions were kept in an ultrasonic bath for 3 min and then shaken for 10 min in a multitube vortexer. After evaporation to dryness, the final residue was dissolved in 0.5 mL of a mixture of acetonitrile/water (10/90)
Two approaches were created for the analysis. At 60°C column temperature, the first approach used a Zorbax Eclipse Plus column with a rapid resolution HD-C18 column (1.8 m, 50 2.1 mm; Agilent Technologies). Deoxynivalenol-13C15 was used as an internal standard. The injection volume was 20 μL. Mixture of methanol (solvent A) and water (solvent B) as the mobile phase, linear gradient with 10%–100% solvent A for 10 min, and stay at 100% for 1 min, flow rate of 0.4 mL/min (positive and negative mode). In second approach, Zorbax SB, rapid resolution HT-C18 column (1.8 μm, 50 × 2.1 mm; Agilent Technologies) at 60°C column temperature. Fumonisin B1-13C34 was used as the internal standard. The injection volume was 10 μL. Mixture of acetonitrile (solvent A) and water added formic acid 1% (solvent B) as mobile phase, linear gradient with 10%–100% solvent A for 10 min, and stay at 100% for 1 min, flow rate of 0.4 mL/min (positive mode)
Settled airborne dust samples were collected from surfaces above floor levels using a conventional vacuum cleaner device and nylon dust collecting socks. To eliminate the coarse fraction, dust bag dust samples were size homogenized by filtering through a sterile strainer. Samples were dried in a desiccator prior to aliquoting and stored at 20°C. Sampling point selected in living room
The raw extracts were diluted and analyzed without further cleaning in a mixture of acetonitrile, water, and acetic acid (79/20/1), and the raw extracts were diluted and analyzed without further cleaning
A method for determining multimycotoxins in food and feed was created, and it was expanded to include a list of 159 fungal and 27 bacterial metabolites for the examination of naturally infected indoor samples. Its purpose is to demonstrate the usefulness of a multianalyte LC-MS/MS-based spiking at many levels for determining extraction performance parameters, matrix effects, and recoveries. Since dust samples absorb a substantial quantity of solvent relative to food and feed matrixes, the fraction of extraction solvent has been increased. In order to completely utilize instrument capacity for data gathering, each analyte’s availability time was defined in the sMRM mode, where retention time and dwell time were fiercely generated for each point in time from the target scan time, for each analyte
When compared to pure methanol and ethyl acetate, the acidified mixture of acetonitrile and water provided the optimum compromise for extracting chosen metabolites. Compared to mortar, carton-gypsum board and coarse-soil-containing splints, settled house dust caused severe matrix effects and incomplete extraction led to low recoveries of analytes due to complex composition from cell fragments, and many other organic compounds accumulated on particulate matter HPLC-MS-MS and GC-MS-MS have both been shown to be useful analytical methods for detecting some of the most toxic mycotoxins produced by molds that are commonly found in moist indoor environments. These technologies are sensitive that they can identify STRG, VER, and TRID in both mold-affected building materials and house dust
Airborne dust was collected by both the cotton swab and Petri dish. Settled dust samples were collected by swabbing 60 cm2 of surface (1 × 60 cm per swab) from the top frame of the blackboard in each classroom. The blackboard top frame was divided into a left and right part, with the left-side dust samples used for fungal DNA analysis and the right-side samples for mycotoxins analysis. Sampling point was selected at the blackboard frame in a classroom
Samples were extracted with methanol, dissolved with DCM, applied to PEI-bonded silica gel column, eluted, evaporated, and redissolved with methanol, and filtered into vials for HPLC injection. For GC-MS injection, methanolic sample extracts are mixed with IS, hydrolyzed and extracted with methanol, and evaporated to dryness. Dried extracts were later derivatized and heated prior to injection
Establishment on GC-MS and GC-MS-MS methods for determination of mycotoxins (verrucarol and trichodermol) and fungal biomass marker (ergosterol) in contaminated indoor environmental samples. Establishment of the LC-MS-MS method for determination of mycotoxins (sterigmatocystin, gliotoxin, aflatoxin B1, and satratoxins G and H) and water-damaged indoor environments samples. Application of different derivative reagents (trimethylsilyl, pentafluorobutyryl, and heptafluorobutyryl) for optimizing determination of mycotoxin with GC-MS and GC-MS-MS. Carryover and ghost peak formation were triggered by adsorption of nonderivatized or semiderivatized mycotoxin in the instrument injector and were overcome by regular injection of a mixture of derivatized reagent with solvent, avoid washing derivatized extracts with water and maximum injection of extracts, to minimize risk of injector contamination
In all samples, mycotoxins were discovered using MSMS and SIM (NICI). Peaks were found in SIM analysis at the correct retention duration but with a significant background noise level due to interference from a partially coeluting chemical. The MSMS analysis, on the other hand, resulted in significantly decreased background noise and higher detection specificity. Heptafluorobutyryl (HFB) derivatives of selected mycotoxins show negligible fragmentation and have good GC-MS and GC-MS-MS detection sensitivity. Combining CI and negative ion (NICI) detection with MS-MS yielded the best detection sensitivity and specificity. Because of its lower detection limit, higher noise reduction, and considerably increased detection specificity, the NICI mode analysis was frequently preferred. HPLC-MS-MS and GC-MS-MS have both been shown to be useful analytical methods for detecting some of the most toxic mycotoxins produced by molds that are commonly found in moist indoor environments. These technologies are so sensitive that they can identify STRG, VER, and TRID in both mold-affected building materials and house dust
Methanol-soaked foam swabs were pushed around the test area, and adherent dust was carried into methanol-filled vials. A few sampling regions are combined to provide a collective sample per site. The samples were collected at ambient temperature, sealed with parafilm, and stored at −20°C until they were analyzed. Classrooms, hallways, teacher’s lounges, libraries, dining halls, bath/shower rooms, storage rooms, and other areas of the school building were chosen as sampling points
The methanolic suspensions were shaken using a rotary shaker, allowed to settle and the clear upper methanolic layers were transferred into glass vials equipped with glass microinserts. The diluted raw extract was directly injected into the HPLC-MS/MS instrument. Then, the methanolic samples were evaporated and reconstituted in DCM. The liquids were applied to preconditioned PEI-bonded silica gel columns. The eluates were evaporated and redissolved in methanol. Methanolic materials were combined with IS, evaporated, hydrolyzed, and extracted with DCM and water. The extract was transferred to fresh vials, evaporated, and kept in a desiccator overnight before being derivatized with HFBI and heated before injection into the GC/MS-MS
For the measurement of 186 fungal and bacterial secondary metabolites, an HPLC-MS/MS technique was developed. A QTRAP 4000 LC-MS/MS system with a TurboIonSpray electrospray ionization (ESI) source and an 1100 series HPLC system were used for detection and quantification in the scheduled multiple reaction monitoring (sMRM) mode. Using a GC-triple-quadrupole MS/MS apparatus, establish GC-MS/MS analyses for the determination of mycotoxins (verrucarol and trichodermol). Carryover and ghost peak formation were triggered by adsorption of nonderivatized or semiderivatized mycotoxin in the instrument injector and were overcome by regular injection of a mixture of derivatized reagent with solvent, avoid washing derivatized extracts with water and maximum injection of extracts, to minimize risk of injector contamination
Since matrix-matched calibration was found to be insufficient for correction of these effects in the very heterogeneous dust matrix, HPLC-MS/MS findings were not corrected for incomplete extraction and/or signal suppression/enhancement due to coeluting matrix constituents. The acquisition of two sMRM transitions per analyte was required for positive identification, and the LC retention time and intensity ratio of the two sMRM transitions had to coincide with the associated values of a genuine standard within 0.1 min and 30% rel., respectively. High prevalence of mycotoxins due to high detection sensitivity offered by the triple-quadrupole mass spectrometers in MS-MS mode. HPLC-MS-MS and GC-MS-MS have proven to be complementary analytical tools for detecting some of the most potent mycotoxins produced by molds frequently encountered in damp indoor environments. These methods are so sensitive that STRG, VER, and TRID can be detected not only in mold-affected building materials but also in house dust
Using a MAS-100 Eco air sampler (Merck, Darmstadt, Germany) with 400 holes (hole to agar impactor) and dichloran 18 percent glycerol agar (DG18) plates, airborne fungi were collected in two-month intervals at apartments (APs), basements (BS), grain mill (GM), and open air (ODA) over a one-year period
Growth aspergilli were cut at 3 plugs of 6 mm diameter by using cylindrical drill and transferred into Eppendorf tubes containing 1000 μl of solvent mixture methanol-DCM-ethyl acetate with a ratio (1/2/3) supplemented with 1% (v/v) formic acid. The clean extracted sample was sonicated. Ultrasonically extraction was transferred into clean vials using syringe with 0.45 μm filters (Sartorius, Germany). Then, the extracted samples were dried under stream of nitrogen gas before kept at −20°C. The dried samples were redissolved in 500 μl of methanol/water (3/1) mixture and filtered through 0.45 μm filters into new vials. Collected dust samples were extracted with solvent mixture methanol-DCM-ethyl acetate with a ratio (1/2/3) supplemented with 1% formic acid. The sample mixture was shaken for 1 hour at room temperature and centrifuged. The supernatants were collected and evaporated to dryness. Dried residues were redissolved with ration of 5 mL of methanol/water (3/1). All samples were filtered through 0.45 μm filters into clean vials
Analysis by using ClassVP 6.2 software and control DGU-14A vacuum degasser, a quaternary LC-10ADVp pump, a CTO-10ASVp column thermostat, an SPD-10ADVp UV-VIS detector, and an SCL-10 system (HPLC Shimadzu). Separation was performed on a LiChroCART Purosphere STAR RP-18 250 mm × 4 mm column with 5 μm particle size (Merck, Hungary) coupled with a Lichospher 100 RP-18 guard column (Merck, Hungary) at 35°C. Mobile phase with setting of flow rate of 0.5 mL/min, and the injection volume was 20 μL. The gradient elution was achieved by changing the ratio of methanol and water. STC was quantified by measuring peak areas in an HPLC chromatogram and comparing them to STC calibration standards
Sharp peak of less polar metabolite eluted right before STC in A. jensenii and A. venenatus chromatograms was discovered. This study did not analyze it by LC/MS/MS analyses and considering STC-derived metabolites and/or precursors belonging to 5-methoxy-STC. Another research had compared with this study and found the metabolite detected by HPLC-DAD and TLC with AlCl3
Moldy surfaces in form of scrapings and airborne dust from 22 moldy dwellings in winter season
All filters were washed with 4 mL of a solvent ratio of acetonitrile/water/acetic acid (79/20/1). Then, the samples were extracted for 90 min with KL 2 Multipurpose Shaker (Edmund Bühler GmbH, Germany). The samples were centrifuged at 1000 for 15 min. The supernatant was dried under gentle stream of nitrogen at 40°C. 1 mL of acetonitrile was dissolved into dried residue
HPLC analysis was conducted using a symmetry C18 column (150 × 2.1 mm 5 μm) with column temperature set at 40°C and 10 μL of sample injection. Gradient elution using mixture of methanol: acetate buffer 0.1 mol/L pH 4.6 serves as mobile phase. Simultaneous determination was performed at an absorbance of 247 nm and 326 nm. UV spectra at an absorbance of 210–500 nm and retention time of the standards were used to analyze the samples
The method collection like scraping and airborne dust did not indicate quantities exceeding the limit of determination of the method. However, the study revealed that ST and RC were present in the fungi cultivated from scrapings. This demonstrates that toxinogenic strains of Aspergillus versicolor and Penicillium chrysogenum display this feature when cultivated on MEA medium in the laboratory. All the studies, including this study, used the HPLC approach; therefore, it is unclear why this study was not able to demonstrate the presence of mycotoxins in the air dust and scrape samples from flats
Settled dust samples were collected by swabbing 60 cm2 of surface (1 × 60 cm) from the top frame of the blackboard in each classroom. The blackboard top frame was divided into a left and right part, with the left-side dust samples used for fungal DNA analysis and the right-side samples for mycotoxin analysis. Sampling point was selected at the blackboard frame in a classroom
Three types of liquid extraction were performed. First, extraction with methanol with three different samples in which the pieces of agar (approximately 5 cm2), dust sample (∼0.4 g), and building material (0.3 to 3 g) were immersed with methanol at room temperature for 72 h. The sample was then centrifuged for 5 min at 3,200 rpm, and the supernatant was poured into new tubes. Second, extraction with heptane 100 μL of sterile water added and the mixture were extracted twice with 2 mL of heptane. Finally, methanolic phases were dissolved in DCM and applied to PEI (1 mL bonded silica gel columns) after being evaporated under a moderate stream of nitrogen. The PEI column already preconditioning the columns with 4 mL of methanol and DCM before eluted with sample. Then, 5 mL of DCM was eluted into the sample through the PEI column and had been evaporated under nitrogen. Prior to analysis, the sample was redissolved with 1 mL of methanol and filtered through 0.45 μm Millex syringe filters. The methodology was modified by the previously mentioned method [41]
HPLC-MS analysis was performed using a ProStar HPLC/1200L triple-quadrupole MS-MS system (Varian Inc., Walnut Creek, CA). 20 μL of each sample was injected, using an autosampler (model 410; Varian), into a Polaris 5 μM C18-A 150 by 2.0 mm RP-18 column equipped with a MetaGuard 2.0 mm Polaris 5-μM C18-A precolumn (Varian). Reserpine was used as the internal standard. GC-MS analysis sample was performed on a CP-3800 gas chromatograph equipped with a fused-silica capillary column (FactorFOUR™, VF-5 ms, 30 m × 0.25 mm i.d., 0.25 mm film thickness) and connected to a 1200 L triple-quadrupole MS-MS detector (Varian Inc., Walnut Creek, CA, USA). Derivatives were analyzed in both EI modes, at an energy of 70 eV and an ion source temperature of 250°C (TMS derivatives) or 200°C (HFB derivatives) and in NICI mode with methane as ionization gas at a pressure of 0.8 kPa and a source temperature of 200°C. Volumes of 1-2 mL were injected in the splitless mode with a helium carrier gas pressure of 69 kPa, using a CombiPAL autosampler (CTC Analytics AG, Zwingen, Switzerland). The MS-MS conditions were optimized by repeatedly injecting 0.1–1 ng amounts of standards at different collision energy, ion source temperature, and argon pressure in the collision cell. The parameters that gave the largest product ion peak area were selected
A supplement of 10 mM ammonium acetate and 20 μM sodium acetate was added to the methanol aqueous buffer to increase the cationization in the electrospray ionization mode. Ten microliters of methanol was injected in between samples to minimize cross-contamination. A mix of HFBI and acetone (1/3) was injected in between samples to eliminate any trace of underivatized or semiderivatized VER/TRID
Aerosolization of produced mycotoxins from contaminated wallpapers. Sampling point was selected at wallpapers
Four MCT (SG, SH, VerJ, and RL2), MPA, and STC were extracted from samples (wallpaper and fiberglass disks) by gentle mechanical agitation on an agitation table in chloroform-methanol (2/1). Mycophenolic acid-d3 and o-methyl sterigmatocystin were added at known concentrations before starting extraction in order to serve as internal standards for MPA and STC, respectively. For MCT, verrucarin A was chosen as an internal standard, as already described. After 4 h, extracts were centrifuged for 5 min at 3,500 rpm and passed through a phase separator (PS) filter (Whatman 1 PS). The filtered extracts were evaporated to dryness and suspended in 1 mL of methanol
Quantification was performed using an Acquity ultraperformance liquid chromatography (UPLC) system coupled to Xevo triple-quadrupole mass spectrometer ethanol. The desolvation temperature and nitrogen flow rate were set at 650°C and 800 liters/h, respectively. Argon was used as the collision gas at a flow rate of 0.12 mL/min. Mycotoxins (5 μL of samples) were eluted on an Acquity BEH C18 column (2.1 by 100 mm, 1.7 m; Waters), with ACN-H2O gradient (0 to 0.5 min), 10% ACN; (0.5 to 4 min), 90% ACN) at a flow rate of 0.35 mL/min. Quantification was carried out by multiple reaction monitoring (MRM) mode in positive electrospray ionization (ESI). Chromatographic data were monitored using the MassLynx 4.1 software
This study showed that three different toxinogenic species produce mycotoxins during their development on wallpaper. These toxins can subsequently be aerosolized, at least partly, from moldy material. This transfer to air requires air velocities that can be encountered under real-life conditions in buildings. Most of the aerosolized toxic load is found in particles whose size corresponds to spores or mycelium fragments. However, some toxins were also found on particles smaller than spores that are easily respirable and can deeply penetrate the human respiratory tract. All of these data are important for risk assessment related to fungal contamination of indoor environments
Dust was collected in the air using a CIP 10 sampler (Tecora, France) with an inhalable fraction selector
Mycotoxins were extracted from the porous polyurethane foam filter (PUF) using methanol, and the PUF capsules were rinsed to remove any dust particles
High-resolution mass spectrometry combined with ultrahigh-performance liquid chromatography (UPLC-Q-Orbitrap HRMS). A BEH C18 column (2.1 mm, 10 mm, 1.7 mm) and precolumn were used to separate the analytes (Waters). In positive electrospray ionization mode, mass spectrometry detection was performed using a Q Exactive mass spectrometer (Thermo Fisher Scientific™). Analytes were detected by comparing product ion retention durations and mass accuracy
Three waste management sectors were incorporated in the sample strategy, as well as both stationary and personal air samplings. The use of UPLC-HRMS for quantitative analysis of airborne mycotoxins is a very sensitive and specific technology that allowed for the detection of low mycotoxin concentrations in the air. The application of measuring results to the assessment of health risks
Samples were collected using a vacuum cleaner from different waste management units and houses inhabited by less than 5 people
Headspace volatile extraction procedure fully automated by an autosampler for microbial volatile organic compound using Agilent 6890 GC QTRAP 4000 LC-MS/MS with C18 column
The use of LC-MS-MS and GC-MS provides many microbial metabolite and volatile anthropogenic chemical presence in indoor environments. This is the first study to compare individual settled floor dust samples derived from relatively different indoor environments using both LC-MS/MS and GC-MS methods
