Accepted Manuscript Analytical Methods Bottled water: analysis of mycotoxins by LC-MS/MS A.T. Mata, J.P. Ferreira, B.R. Oliveira, M.C. Batoréu, M.T. Barreto Crespo, V.J. Pereira, M.R. Bronze PII: DOI: Reference:

S0308-8146(14)02001-9 http://dx.doi.org/10.1016/j.foodchem.2014.12.088 FOCH 16937

To appear in:

Food Chemistry

Received Date: Revised Date: Accepted Date:

12 February 2014 1 December 2014 19 December 2014

Please cite this article as: Mata, A.T., Ferreira, J.P., Oliveira, B.R., Batoréu, M.C., Barreto Crespo, M.T., Pereira, V.J., Bronze, M.R., Bottled water: analysis of mycotoxins by LC-MS/MS, Food Chemistry (2014), doi: http:// dx.doi.org/10.1016/j.foodchem.2014.12.088

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Bottled water: analysis of mycotoxins by LC-MS/MS A.T. Mata a, J.P. Ferreiraa, B.R. Oliveirab, M.C. Batoréua, M.T. Barreto Crespob,c, V.J. Pereirab,c, M.R. Bronze*a,b,c a

iMED, Faculdade de Farmácia Universidade de Lisboa, Av. Prof. Gama Pinto, 1649-019 Lisboa Portugal; email [email protected]

b

c

IBET - Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2780-901 Oeiras, Portugal

Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Av. da República, EAN, 2780-157 Oeiras, Portugal *Corresponding author. Phone: +351 217946400 Ext. 14329

Abstract The presence of mycotoxins in food samples has been widely studied as well as its impact in human health, however, information about its distribution in the environment is scarce. An analytical method comprising a solid phase extraction procedure followed by liquid chromatography tandem mass spectrometry analysis was implemented and validated for the trace analysis of mycotoxins in drinking bottled waters. Limits of quantification achieved for the method were between 0.2 ng L-1 for aflatoxins and ochratoxin, and 2.0 ng L-1 for fumonisins and neosolaniol. The method was applied to real samples. Aflatoxin B2 was the most frequently detected mycotoxin in water samples, with a maximum concentration of 0.48±0.05 ng L-1 followed by aflatoxin B1, aflatoxin G1 and ochratoxin A. The genera Cladosporium, Fusarium and Penicillium were the fungi more frequently detected. These results show that the consumption of these waters does not represent a toxicological risk for an adult.

Keywords: Drinking water, Cladosporium, Fusarium, Penicillium, Mycotoxins, LC-ESIMS/MS

1

Chemical compounds studied in this article Aflatoxin B1 (PubChem CID: 14403); Aflatoxin B2 (PubChem CID: 2724360); Aflatoxin G1 (PubChem CID: 14421); Aflatoxin G2 (PubChem CID: 2724362); Fumonisin B1 (PubChem CID: 3431); Fumonisin B2 (PubChem CID: 2733489); Fumonisin B3 (PubChem CID: 3034751); Neosolaniol (PubChem CID: 13818797); Ohratoxin A (PubChem CID: 442530);

1. Introduction Mycotoxins are toxic chemicals formed as secondary metabolites of fungi. They are a potential threat to human and animal health, and are frequently consumed in food products from crops origin, such as cereals, nuts, dried fruit, spices, oil seeds, beans, fruit, beer and wine (Turner, Subrahmanyam, & Piletsky, 2009). More than one hundred filamentous fungi are known, producing a plethora of mycotoxins with a structural diversity and thus, presenting different chemical and physical properties, and a diversity of toxic effects to invertebrates, plants, and microorganisms (Bennett, 1987). In general, exposure to mycotoxins is more likely to occur in countries that face malnutrition problems, due to poor methods of food handling and storage, as well as lack of food safety control. Nevertheless, in developed countries, certain groups may also be vulnerable to mycotoxin exposure due to a higher consumption of certain type of food products (Bennett & Klich, 2003). Aflatoxins are produced by many strains of Aspergillus (e.g. Aspergillus flavus, Aspergillus parasiticus, Aspergillus bombycis, Aspergillus ochraceoroseus, Aspergillus nomius, and Aspergillus pseudotamari) (Bennett & Klich, 2003) and have been described as hepatotoxic, mutagenic, and carcinogenic. However, the toxigenic abilities displayed by different strains are widely variable. Other species of Aspergillus (Aspergillus alliaceus, Aspergillus auricomus, Aspergillus carbonarius, Aspergillus glaucus, Aspergillus melleus, and Aspergillus

2

niger) and Penicillium verrucosum (Bennett & Klich, 2003) produce also other toxins but only ochratoxin is classified as nephrotoxic, teratogenic, immunotoxic, genotoxic, mutagenic and carcinogenic (Creppy, 1999). Fusarium species and Alternaria alternata have been reported to produce fumonisins (Rheeder, Marasas, & Vismer, 2002) which have been associated with pulmonary edema and hydrothorax in swine (Harrison, Colvin, Greene, Newman, & Cole, 1990), hepatotoxic and carcinogenic effects in rats (Gelderblom, Kriek, Marasas, & Thiel, 1991) and may cause neural tube defects in animals (Bennett & Klich, 2003). Trichothecene type mycotoxins (such as neosolaniol) may be produced by strains of different fungal genera (e.g. Fusarium, Myrothecium, Phomopsis, Stachybotrys, Trichoderma, Trichothecium) and are extremely potent inhibitors of eukaryotic protein synthesis (Bennett & Klich, 2003). B-type trichothecenes and zearalenone are two groups of mycotoxins found in food and feed (Cavaliere, Foglia, Guarino, Motto, Nazzari, Samperi, et al., 2007; Turner, Subrahmanyam, & Piletsky, 2009) and although not frequently detected, contents from 0.01 to 2.0 mg kg-1 were reported in cereals, rice and dried fruits (Cavaliere, et al., 2007). EU Commission set the maximum concentration levels for mycotoxins in foodstuffs (EU, 2006) as 4 and 10 µg kg-1 for the sum of B1, B2, G1 and G2 aflatoxins, and 2000 µg kg-1 and 200 µg kg-1 were set for the sum of fumonisins B1 and B2 in unprocessed food and processed food, respectively. For ochratoxin limits were set at 5 and 10 µg kg-1 in processed and unprocessed food, respectively, and 2 µg kg-1 in wine and juices. The occurrence of mycotoxins in food and feed has been evaluated extensively, but studies concerning their presence in the environment are scarce. The possibility of water contamination with xenobiotics and pathologic microorganisms has lead to an increasing concern over drinking water quality. The occurrence of bacteria, filamentous fungi and yeasts in surface, spring and groundwaters, was recently reported (Pereira, Basilio, Fernandes, Domingues, Paiva, Benoliel, et al., 2009) and forty nine fungal species were identified, most

3

of which had never been described to occur in water sources. Fungi were also reported to occur in water distribution systems (Doggett, 2000). The few works published about the occurrence of mycotoxins in surface (Gromadzka, Waśkiewicz, Goliński, & Świetlik, 2009; Laganà, Bacaloni, De Leva, Faberi, Fago, & Marino, 2004), ground (Gromadzka, Waśkiewicz, Goliński, & Świetlik, 2009)

and wastewaters (Gromadzka, Waśkiewicz,

Goliński, & Świetlik, 2009; Laganà, Bacaloni, De Leva, Faberi, Fago, & Marino, 2004) focused their attention on deoxynivalenol and zearalenone type mycotoxins. Bottled mineral water has long been consumed as a safe alternative and sales have been increasing all over the world. The occurrence and identification of fungi in this type of waters has already been discussed (Cabral & Fernández Pinto, 2002) and has been related to organoleptic defects concerning taste and odour and some allergenic reactions (De Hoog, 2000; Doggett, 2000). In this study, we describe the implementation and validation of an analytical method used for the quantification of nine mycotoxins (aflatoxin B1, aflatoxin B2, aflatoxin G1, aflatoxin G2, fumonisin B1, fumonisin B2, fumonisin B3, neosolaniol and ochratoxin A) in commercially available bottled water samples. The method used includes an initial sample preparation procedure using solid phase extraction (SPE) followed by LC-MS/MS using electrospray ionization (ESI) in positive mode. The filamentous fungi present in the water samples were quantified using the membrane filtration technique and identified based on their macroscopic and microscopic characteristics. Results are evaluated in terms of risk assessment.

2. Materials and methods 2.1 Chemicals and material The target mycotoxins aflatoxin B1, aflatoxin B2, aflatoxin G1, aflatoxin G2, fumonisin B1, fumonisin B2, fumonisin B3, neosolaniol, ochratoxin A, and the four isotope labeled internal standards (ILISs; [13Cx]):

13

C17-aflatoxins B2,

13

C17-aflatoxins G2,

13

C34-fumonisin B1 and

4

13

C20-ochratoxin A were purchased from Sigma-Aldrich® as standards of the highest grade

available (≥98%). Acetonitrile and methanol LC-MS grade were obtained from Fisher Scientific®. Milli-Q water (18.2 MΩ.cm resistivity) was obtained from a Millipore-Direct Q3 UV system (Millipore®, USA). Ammonium formate, used in the mobile phase, was purchased from Fluka (Sigma-Aldrich®) and formic acid 99%, was purchased from Carlo Erba (Carlo Erba reagents spa, Arese-Milano, Italy). OASIS HLB (6mL, 200 mg) were obtained from Waters (Waters, Milford, MA).

2.2 Preparation of stock and working solutions Individual stock standard solutions (PS) at 100 µg mL-1 were prepared by dissolving each standard in acetonitrile, except fumonisin B1, fumonisin B2, and fumonisin B3, which were dissolved in a acetonitrile/water (1:1, v/v) solution (Schenzel, Schwarzenbach, & Bucheli, 2010). Individual working standard solutions (Pt1) were prepared from PS, diluting 1:10 with acetonitrile (10 µg mL-1). A mixture of all the mycotoxins was also prepared at a concentration of 1 µg mL-1 using acetonitrile (PMix). For ILISs, individual standard solutions were prepared at a concentration of 250 µg L-1 for the two

13

C17-aflatoxins and 500 µg L-1 for

13

C34-fumonisin B1 and

13

C20-ochratoxin A. All

13

solutions were prepared with acetonitrile, except for

C34-fumonisin B1 where an

acetonitrile/water (1:1, v/v) solution was used. In order to evaluate the linearity of the method mixed standard solutions of all mycotoxins at a concentration range from 0.5 µg L-1 to 1000 µg L-1 and ILISs (5 µg L-1 for B2 and G2 and 100 µg L-1 for

13

C34-fumonisin B1 and

13

C17-aflatoxins

13

C20-ochratoxin A) were prepared

using acetonitrile/water (1:3, v/v). A Quality Control standard solution (QC solution) was prepared from Pt1 at concentration levels near the instrument limits of quantification (ILOQ), 5 µg L-1 for aflatoxins, ochratoxin and neosolaniol, and 25 µg L-1 for fumonisins using acetonitrile/water (1:3, v/v).

5

All standard solutions were stored at -20oC in amber glass vials after preparation.

2.3 Samples Natural bottled mineral waters and spring waters (n=26) from 9 different commercially available brands (A-I), comprising volumes from 1.5 L to 6 L, were purchased at local stores, between June 2012 and June 2013 (see supplementary data). All bottled waters are, according to the hydrochemical types of bottled waters distributed in Portugal (Lourenço, Ribeiro, & Cruz, 2010), classified as low mineralization (product ion] transitions signal. The optimized conditions were confirmed when performing the LC-MS/MS analysis of the standard solutions. Analytes were quantified using the internal standard method, except for neosolaniol that was quantified using external calibration as there was no labelled internal standard. All analysis were performed in multiple reaction monitoring (MRM) mode in order to achieve a higher selectivity and sensitivity.

2.6 Method Validation Method validation was performed in accordance with the International Conference on

Harmonization (ICH) guidelines (ICH, 2005) as described. Criteria

requirements were based on Commission Decision 2002/657/EC (EC, 2002). Water samples from brands A and B were used in the validation process as they presented the lowest and the highest levels of mineralization, 39 mg L-1 and 198 mg L-1 respectively.

2.6.1 Specificity Individual standard solutions of each compound at 10 µg mL-1 were infused into the mass spectrometer in order to obtain two product ions with the highest signal. These transitions were used as the quantification transition (MRM1) and the confirmation transition (MRM2). In order to evaluate specificity of the method, the MRM1/MRM2 signal transition ratios were determined for each mycotoxin standard solution and results were compared with values obtained when mycotoxins were detected in samples. The use of two transitions gives 4 identification points, corresponding to one precursor and two product ions. This parameter fulfils the European Commission requirements for specificity (at least 3 identification points) for the confirmation of compounds listed in group B in the Annex I (EC, 2002). According to the Commission Decision 2002/657/EC for identification methods it is allowed a 20%

9

maximum deviation for the MRM1/MRM2 ratio (EC, 2002). The retention time of the standard compounds and peaks detected in samples are also compared (see supplementary data).

2.6.2 Linearity and linear range To evaluate the linearity of the method, mixed standard solutions prepared in the concentration range from 0.5–1000 µg L-1 were analysed and the determination coefficient (r2) was calculated. Acceptable linearity was achieved when r2 was higher than 0.995 (ISO, 1990). Two statistical test methods were used to investigate the linearity within the working range, the Mandel test and the Rikilt test. The Mandel test was used according to ISO 8466-1 (ISO, 1990) to investigate if the linear equation provides a better fit to the calibration curve and can be used instead of the quadratic equation. PG values calculated and F values were compared. The Rikilt test is used to evaluate if instrument calibration can be done with a response factor instead of a calibration curve. It was considered that if the calibration points fall within a specified percentage range (90%-110%) (Van Trijp & Roos, 1991), it can be assumed that instrument calibration can be done using a response factor (ICH, 2005). 2.6.3 Limits of detection (LOD) and quantification (LOQ) The instrumental limit of detection (ILOD) values were estimated as the concentration of each mycotoxin that gives a signal that corresponds to three times the noise (S/N= 3). The instrumental limit of quantification (ILOQ) was defined as the concentration of each mycotoxin that gives a signal that corresponds to ten times the noise (S/N= 10). ILOQ values were confirmed by preparing solutions in acetonitrile/water (1:3, v/v) corresponding to these concentrations and were tested for precision and accuracy. Method limit of quantification (MLOQ ) was estimated considering a 5000 times concentration factor for the samples and values were confirmed by preparing water samples spiked with the target analytes at different concentration levels of mixed standard solutions, 0.2 ng L-1

10

(aflatoxins and ochratoxin) and 2.0 ng L-1 (fumonisins and neosolaniol) corresponding to the ILOQ (Schenzel, Schwarzenbach, & Bucheli, 2010).

2.6.4 Accuracy In order to evaluate the accuracy of the method, absolute and relative recoveries experiments were carried out in Milli-Q water, tap water and bottled water A and B. One litre of unfortified water of each matrix was previously analysed in order to confirm that no mycotoxins were detected. For the absolute method recovery, samples (500 mL) were spiked with mycotoxins prior to SPE procedure in order to obtain concentration levels of 10, 50 and 100 µg L-1. Samples were prepared as duplicates, and the absolute method recovery was determined for all the compounds and defined as the ratio between the quantified and the spiked amount. Relative method recovery was determined for those mycotoxins with ILIS available using the same procedure described for the absolute method recovery, but adding the internal standard solutions prior to SPE experiments. The relative method recovery was defined as the ratio between the quantified (taking into account the signal from the standard and the corresponding ILIS) and the spiked amount (Hartmann, Erbs, Wettstein, Schwarzenbach, & Bucheli, 2007; Schenzel, Schwarzenbach, & Bucheli, 2010). Criteria requirements were based on Commission Decision 2002/657/EC (EC, 2002).

2.6.5 Precision Instrument precision and intermediate precision (inter-day) of the method of analysis were determined. Instrument precision was determined by injecting the QC standard solution for 10 times. Intermediate precision was evaluated in Milli-Q water, tap water, bottled water A and B, spiking samples at a concentration level of 50 µg L-1 of each mycotoxin, for 3 days and performing six assays for each matrix (n=6).

11

For

both cases precision was calculated and expressed as relative standard deviation

(RSD%) (ISO, 1990; RELACRE, 2000). Criteria requirements were based on Commission Decision 2002/657/EC (EC, 2002).

2.6.6 Matrix effect In order to evaluate the effect of each water matrix on the signal obtained in the mass spectrometer, standard solutions prepared in acetonitrile/water (1:3, v/v) and using

an

extracted matrix blank were analysed and results were compared. Briefly, 2500 mL of each matrix, namely Milli-Q water, tap water, bottled water A and B, were prepared following the procedure described in sample preparation. A standard addition was carried out to the SPE eluates in order to obtain different concentrations corresponding to 5, 10, 25, 50 and 100 µg L-1 of each analyte, and assays were prepared as replicates. The matrix effect (expressed in percentages) was quantified as 1 minus the ratio between the slope of the curve for the different matrices analysed and the slope for the acetonitrile/water standards (Hartmann, Erbs, Wettstein, Schwarzenbach, & Bucheli, 2007; Schenzel, Schwarzenbach, & Bucheli, 2010).

2.7 Application of the method Water samples were analysed for mycotoxins content, immediately after filamentous fungi identification and as previously described in the mycotoxin content analysis section. Briefly 500 mL of water were spiked with 100 µL of the ILISs mixture and pH was adjusted between 2 and 3 by adding 0.1% formic acid. The water samples passed through the SPE column at a flow rate of 10 mL min-1. The retained compounds were eluted with 5 mL of methanol, the solution was dried up under a nitrogen gas stream, re-dissolved in 100 µL of acetonitrile and analyzed by LC-MS/MS.

12

The uncertainty in the measurements were calculated using the accuracy and precision values (IPAC, 2007).

3. Results and discussion Different fungi have been found in water matrices and are able to produce mycotoxins. Methodologies used for the screening of mycotoxins in food products and water samples must be selective and sensitive as these compounds are found at trace levels and have different physicochemical properties namely a wide range of polarities. In this work, the conditions of analysis by SPE-LC-MS/MS were optimized and the method was validated for the determination of nine mycotoxins in different types of bottled water matrices and tap water. The main reasons for selection of the mycotoxins studied was based on the fact that they are produced by fungi that were described to occur frequently in water samples, they are monitored to ensure compliance with food regulations and are considered among the most important associated with veterinary and human diseases.

3.1 Optimization of the solid phase extraction procedure Oasis HLB cartridges were used in the mycotoxin concentration process and acetonitrile and methanol were tested as eluting solvents. The results obtained showed that methanol was the best solvent to use since fumonisins were not eluted, even when using up to 10 mL of acetonitrile. These results were in agreement with the ones reported by other authors (Hartmann, Erbs, Wettstein, Schwarzenbach, & Bucheli, 2007; Schenzel, Schwarzenbach, & Bucheli, 2010). Samples of Milli-Q water (pH 6.3) were spiked with the target analytes at three different concentration levels, 0.2, 2.0, 20 ng L-1, concentrated 5000 times (1, 10, 100 µg L-1) and the average recovery values were determined. In order to improve the recovery of the acidic analytes, namely fumonisins, the sample pH was set below 3, using formic acid. There were

13

differences in the absolute SPE recoveries obtained for fumonisins and ochratoxin at pH below 3 and pH above 6 (see supplementary data). For pH below 3 the average of the recovery values were higher and range from 69-105%. The high RSD value for aflatoxin B1 is due to the poor recovery value obtained at the lowest concentration level (53%). Also for aflatoxin G2 recovery values below 79% were obtained for 1 and 10 µg L-1. Data on pKa are important for the evaluation of the effect of pH on the mycotoxins ionization. For fumonisins no values were found in the available literature, but pKa values for tricarballylic acid described are 3.49, 4.56 and 5.83(National toxicology program, 2001). For aflatoxins there was no pKa value reported either, however it is reasonable to assume that due to the chemical structure of those compounds, pKa values are higher than those of fumonisins. Oasis HLB cartridges were considered adequate for the intended purposes of the analysis and were according to previously reported data (Schenzel, Schwarzenbach, & Bucheli, 2010). Results showed that before sample treatment by SPE, the pH should be adjusted between 2 and 3, using formic acid, in order the optimize retention conditions in the cartridge.

3.2 Liquid chromatography tandem mass spectrometry method The positive ionization mode (ESI+) was selected because higher signals were obtained for the precursor ions, for all the mycotoxins studied. Due to the addition of 5 mM ammonium formate (Romero-Gonzalez, Martinez Vidal, Aguilera-Luiz, & Garrido Frenich, 2009) to the mobile phase used in the chromatographic separation, neosolaniol formed [M + NH4]+ adducts presenting higher precursor signal intensities than the related [M + H]+ species. The monitored product ions from these adduct precursors were analyte-specific and selected to fulfil the requirement for the confirmation of substances according to the Annex I of Directive 96/23/EC (EC, 2002). The optimized analytical conditions are presented in table 1.

14

3.3 Method validation

3.3.1 Specificity Selectivity expresses the extent to which a particular method can determine an analyte under given conditions in the presence of interferences from other components present in the matrix. In the evaluation of the specificity, blank assays were performed by analysing the solvents in the same conditions and no interfering peaks were detected. The retention time for each mycotoxin standard solution were determined, as well as the ratio between signals obtained in the two transitions MRM1/MRM2. The corresponding RSD% are presented in table 2. According to the results the method was considered specific for the mycotoxins studied.

3.3.2 Linearity The linearity study was performed using eight standard solutions prepared and analysed according to the procedure previously described. It measures how well a calibration plot of response against concentration approximates a straight line. Calibration curves were obtained and the least-square regression method was used to calculate the determination coefficient (r2). Mandel test was used to evaluate if the linear equation provides a better fit to the calibration curve and can be used instead of the quadratic equation within a working range. The test values (PG) obtained were compared with the F tabulated values (α=0.01) (ISO, 1990). Linearity was observed over a concentration range of 0.5–100 µg L-1 for aflatoxins, 10-250 µg L-1 for fumonisins and 5.0-250 µg L-1 for neosolaniol and ochratoxin A, with a determination coefficient between 0.99874287 332>303

313>241 315>259

[M+H] + [M+H] + [M+H]

+

329 331 348

283, 243 313, 245 259

40 40 40

25/35 25/35 25

329>243 331>313 348>259

329>283 331>245

+

722 756 706 706

352, 334 374 336, 318 336, 318

50 50 50 50

35/40 35 35/40 35/40

722>352 756>374 706>336 706>336

722>334

400

215, 305

20

15/15

400>215

400>305

404 424

239, 358 250

20 20

20/15 15

404>239 424>250

404>358

ion

AFT B 1 AFT B 2 13 C17-AFT B2 AFT G1 AFT G2 13 C17-AFT G2 FB1 13 C 34-FB1 FB2 FB3

7.75 7.55 7.52

[M+H] + [M+H] + [M+H]

+

7.54 7.33 7.34 7.25 7.24 7.59 7.7

[M+H] + [M+H] + [M+H] + [M+H]

NEO

6.64

[M+NH4]

9.20 9.20

[M+H]+ + [M+H]

A or B-Type Trichothecene Ochratoxin A

source potential (V) 40 40 40

MRM1 transition

313 315 332

product ions (m/z) 285, 241 287, 259 303

RT (min)

Compound

OTA C20-OTA

13

precursor ion (m/z)

+

706>318 706>318

RT – retention time; MRM - multiple reaction monitoring

26

Table 2. Results of the linearity, specificity, instrumental limits of detection (ILOD) and quantification (ILOQ) and repeatability, instrument precision, method limit of quantification (MLOQ) and uncertainty (U) of the method for each matrix.

Calibration Y=ax+b

Mandel Test PG value

F-Test (α α =0.01; n=5,6,8)

RIKILT test [min%max%]

MRM1/MRM2 (RSD)

ILOD -1 (µg L )

ILOQ -1 (µg L )

Repeatability of the ILOQ (RSD)

Instrument precision (RSD)

0.9998

Y=1.366x-0.087

0.34

16.3

[95; 106]

0.63(0.03)

0.10

0.30

6.96

3.02

0.9999

Y=1.455x+0.244

0.12

16.3

[97; 109]

0.88(0.02)

0.20

0.50

5.06

2.92

0.50-100

0.9998

Y=0.608x-0.038

0.00

16.3

[95; 109]

1.4(0.18)

0.30

1.0

5.49

10.00

Compound

Concentration -1 Range (µg L )

r

AFT B1

0.50-100

AFT B2

0.50-100

AFT G 1

2

AFT G 2

0.50-100

0.9997

Y=1.341x-0.219

4.53

16.3

[97; 108]

2.3(0.17)

0.30

1.0

8.92

6.02

FUM B1

10.0-250

0.9987

Y=0.314x+1.201

17.43

98.5

[93; 110]

0.6(0.09)

3.0

10.0

8.53

16.68

FUM B2

10.0-250

1.0000

Y=1.174x-0.324

8.09

98.5

[97; 102]

2.1(0.12)

3.0

10.0

7.57

13.23

FUM B3

10.0-250

0.9996

Y=1.207x+6.964

5.06

98.5

[91; 107]

1.7(0.14)

3.0

10.0

5.17

12.90

NEO

5.0-250

1.0000

Y=23.504x-15.229

0.01

34.1

[93; 105]

1.6(0.17)

2.0

5.0

8.96

6.33

OTA

5.0-250

1.0000

Y=2.188x-0.363

0.66

34.1

[95; 107]

2.6(0.13)

0.30

1.0

6.39

18.86

MLOQ -1 (ng L )*

Uncertainty of the Method (U, %) Milli-Q water

Bottled water A

Bottled water B

Tap water

AFT B1

0.20

10

8

18

-

AFT B2

0.20

4

8

10

14

AFT G 1

0.20

11

3

10

-

AFT G 2

0.20

8

13

5

-

FUM B1

2.0

9

9

12

9

FUM B2

2.0

8

5

17

26

FUM B3

2.0

6

21

16

24

NEO

2.0

24

23

28

28

OTA

0.20

10

13

6

1

*5000x concentration factor; U: uncertainty was determined based on precision and accuracy of the analytical method

27

Table 3. Intermediate precision (IP), absolute method recoveries (AR), relative method recoveries (RR) for water matrices spiked with solutions at 10, 50, 100 µg L-1 of each mycotoxin. Concentration -1 (μg L )

AFT B1

AFT B2

AFT G1

AFT G2

FUM B1

FUM B2

FUM B3

NEO

OTA

10 50 100 10 50 100 10 50 100 10 50 100 10 50 100 10 50 100 10 50 100 10 50 100 10 50 100

Milli-Q water IP RSD 2

4

10

8

7

8

4

24

10

(a)

AR % 59 (1) 43 (9) 36 (1) 36 (1) 42 (4) 38 (1) 79 (14) 42 (13) 40 (3) 76 (14) 46 (7) 40 (4) 20) 77 (19) 20) 20) 35 (17) 100 (2) 103 (4) 103 (2) 32 (16) 33 (4) 40 (16)

Bottled water B (a)

RR % 114 (20) 100 (1) 102 (1) 100 (1) 103 (1) 101 (1)