**2. Results and Discussion**

Sample preparation plays a key role for the quality of chromatographic results. The selection of extraction solvent and condition are very important for achieving the true value of the assigned analyte. Prior to validation of the method, we optimized the AF extraction efficiency conditions by testing the effect of different extraction solvents, the effect of extraction solvent amount, and the effect of shaking time (unpublished data). We tested five solvents: chloroform, ethyl acetate, acetone, petroleum ether, and methanol in six different sample to solvent ratios (1:1, 1:1.5, 1:2, 1:2.5, 1:3, and 1:5). We also assessed the effect of the shaking time by vortexing the samples for 30, 60, 90, 120, 150, and 300 s. Through it all, we found that chloroform and ethyl acetate were the best extraction solvents with the highest recovery values. The data revealed that the extraction yield with chloroform was a little higher, with no significant differences, when compared with ethyl acetate. We chose chloroform as the AF extraction solvent in this study because (1) higher recover values were achieved and (2) AFs are more stable and soluble in chloroform than in ethyl acetate [22]. Technically, complete obtaining of the lower organic layer (chloroform) was achievable and easier than those on the top (ethyl acetate). We also found that a minimum 1.5-fold volume of chloroform and 30 s shaking time produced the maximum AF recovery values. Results indicated that total transfer of AFs can be accomplished by two extractions with chloroform. Although there was no detectable AFs in the third chloroform extract, three extractions are recommended to preclude loss of toxin.

Method validation is a crucial prerequisite to performing an analysis [23]. Several methods are available for analysis of AFs in food and feed that have been validated and accepted by official authorities, such as the European Committee for Standardization, the Association of Official Analytical Chemists (AOAC), and the International Organization for Standardization (ISO). Here, we employed a reverse-phase chromatography for the analysis of AFs by using a nonpolar bonded silica surface column and a polar mobile phase. With this reversed phase mode, AFs were eluted in the order of AFG2, AFG1, AFB2, and AFB1 (Figure 2A,B). This order was confirmed by comparing the obtained retention times in an AF mixture with the retention times of the individual AFs. All separated AFs were then detected by DAD and FLD detectors, connected in series, at parts per billion (ppb; ng/mL) concentrations (Figure 2A,B). It needs to be noted that, in using the FLD detector, AFG2 and AFB2 could be detected even at lower levels, as they fluoresce 40-fold more than AFB1 and AFG1 (Figure 2B). The LOQ is defined as the minimum concentration or mass of analyte in a given matrix that can be reported as a quantitative result with a certain level of precision [24]. On the contrary, the LOD is defined as the lowest concentration of the analyte that can be detected, but not necessarily quantitated, under the stated experimental conditions [25]. The LOD and LOQ for all AFs as detected by the UV detector was 1.0 ng/mL and 2.5–5.0 ng/mL, respectively. Using an FLD detector, the LOD and LOQ for AFB1 and AFG1 were 1.0 ng/mL and 2.5 ng/mL, respectively. Importantly, the LOD and LOQ for AFB2 and AFG2 using our method were 0.01 and 0.025 ng/mL, respectively. This method was designed for detection and quantification of aflatoxins mixture in laboratory cultures medium of growing fungi and it is not intended to use for food or feed for regulatory purpose. We found that DAD could at most detect as low as 1.0 ng/mL and quantify as low as 2.5–5.0 ng/mL for all aflatoxins. However, we injected several concentrations below 2.5 ng/mL to check the sensitivity of the FLD for detection of aflatoxins, specifically B2 and G2. We found that AFB2 and AFG2 could be easily detected, as expected and previously proved, at parts per trillion (ppt) level by FLD because of the absence of a double bond in the furan ring. To the best of our knowledge with fungal and fungal genetic studies, 1.0–5.0 ng/mL as LOD is sufficient to help researchers to distinguish between aflatoxigenic and non-aflatoxigenic strains, as well as the relative amounts of aflatoxins B and G produced between aflatoxigenic strains.

**Figure 2.** High-performance liquid chromatography (HPLC) chromatograms of the standard solution **Figure 2.** High-performance liquid chromatography (HPLC) chromatograms of the standard solution containing four aflatoxins (100 ng/mL each of AFB1, AFB2, AFG1, and AFG2) detected by diode array (DAD) (**A**) and fluorescence (FLD) (**B**).

containing four aflatoxins (100 ng/mL each of AFB1, AFB2, AFG1, and AFG2) detected by diode array

(DAD) (**A**) and fluorescence (FLD) (**B**). Selectivity is defined as the ability to separate the analyte from other components (including impurities) that may be present in the sample [26]. Our method demonstrated a good separation ability and selectivity that allowed simultaneous quantification of four different AFs in the culture medium without interference between the AFs. Both detection methods (DAD and FLD) were able to differentiate the AF peaks in the same HPLC run with minimal background interference. In order to demonstrate a proportional relationship of response versus AF concentrations over the working range, the linearity of the method was tested from the calibration curves using seven points over the range of 5.0–1000 ng/mL for each AF and defined using the correlation coefficient (coefficients of determination, *R2*) and the slope. Calibration curves were constructed by plotting the peak area (*y*) versus the concentrations of the AFs (*x*) (Figure 3A,B). Calibration curves fitted by linear regression Selectivity is defined as the ability to separate the analyte from other components (including impurities) that may be present in the sample [26]. Our method demonstrated a good separation ability and selectivity that allowed simultaneous quantification of four different AFs in the culture medium without interference between the AFs. Both detection methods (DAD and FLD) were able to differentiate the AF peaks in the same HPLC run with minimal background interference. In order to demonstrate a proportional relationship of response versus AF concentrations over the working range, the linearity of the method was tested from the calibration curves using seven points over the range of 5.0–1000 ng/mL for each AF and defined using the correlation coefficient (coefficients of determination, *R 2* ) and the slope. Calibration curves were constructed by plotting the peak area (*y*) versus the concentrations of the AFs (*x*) (Figure 3A,B). Calibration curves fitted by linear regression showed *R 2* ranging from 0.9987 to 1.0 for both detectors, indicating an excellent linearity for all four AFs (Figure 3C).

showed *R2* ranging from 0.9987 to 1.0 for both detectors, indicating an excellent linearity for all four AFs (Figure 3C). The fraction or percentage of the analyte that is recovered when the test sample is analyzed using the entire method is referred to as the method recovery [27]. Table 1 shows the percentage of AF recovery at a low, three-point intermediate, and high concentration levels spiked in three culture conditions. Recovery of AFs in solid, submerged, and slant culture states showed similar retention times with an overall average recovery of 76%–88%, 77%–88.4%, and 77%–86%, respectively. All spiked samples were detected by both DAD and FLD in a series manner, and the mean of both was calculated. This recovery range was within the guideline of acceptable recovery limits of AOAC and the Codex Alimentarius. The AOAC guideline for the acceptable recovery at the 10 ng/mL level is 70%−125%. The Codex Alimentarius acceptable recovery range is 70%−110% for a level of 10−100 ng/mL and 60%−120% for a level of 1−10 ng/mL. The repeatability of the method for AF analysis, as evaluated by the percentage of the RSD, ranged from 0.8% to 8.9%. These values agree with the AOAC guideline for a validated analytical method. The AOAC guidelines for acceptable repeatability (RSD) at 10 ng/mL are less than 15% and less than 8% at 1000 ng/mL.

AFs (Figure 3C).

(DAD) (**A**) and fluorescence (FLD) (**B**).

**Figure 2.** High-performance liquid chromatography (HPLC) chromatograms of the standard solution containing four aflatoxins (100 ng/mL each of AFB1, AFB2, AFG1, and AFG2) detected by diode array

Selectivity is defined as the ability to separate the analyte from other components (including impurities) that may be present in the sample [26]. Our method demonstrated a good separation ability and selectivity that allowed simultaneous quantification of four different AFs in the culture medium without interference between the AFs. Both detection methods (DAD and FLD) were able to differentiate the AF peaks in the same HPLC run with minimal background interference. In order to demonstrate a proportional relationship of response versus AF concentrations over the working range, the linearity of the method was tested from the calibration curves using seven points over the range of 5.0–1000 ng/mL for each AF and defined using the correlation coefficient (coefficients of determination, *R2*) and the slope. Calibration curves were constructed by plotting the peak area (*y*) versus the concentrations of the AFs (*x*) (Figure 3A,B). Calibration curves fitted by linear regression

**Figure 3.** Calibration of aflatoxins. (**A**) Calibration curves of standard aflatoxin solutions (AFB1, AFB1, AFG1, and AFG2) over the concentrations of 5, 10, 50, 100, 500, and 1000 ng/mL as detected by DAD. (**B**) Those detected by FLD. (**C**) Linear relationship between aflatoxin concentrations and peak areas in the range of 5 to 1000 ng/mL. Correlation coefficient (*R 2* ) and regression equation values were determined by plotting area values (*y*-axis) against aflatoxin concentration (*x*-axis).


**Table 1.** Recovery (%) of spiked aflatoxins from three culture methods (solid, submerged, and slant cultures); mean with (RSD) in percentages.

To validate our method, AFs were extracted from known aflatoxigenic and non-aflatoxigenic *Aspergillus* strains grown in three different culture conditions. *A. flavus* NRRL 3357 was able to produce 879 and 7.8 ng/mL of AFB1 and AFB2 (Figure 4A,B), respectively, when the fungus was cultured in solid agar with a total amount of 13,302 ng per plate. This strain produced 2041.9 and 221.1 ng/mL of

AFB1 and AFB2, respectively, when grown in liquid culture medium. On a slant cultivation, NRRL 3357 yielded 1100 and 11.49 ng/mL of AFB1 and AFB2, respectively. *A. parasiticus* NRRL 2999 was able to produce 398.27, 2.98, 207.8, and 10.19 ng/mL of AFB1, AFB2, AFG1, and AFG2, respectively, when it was inoculated onto an agar plate with a total amount of 9288.6 ng per plate. In liquid cultivation, this fungus was able to produce 508.2, 24.21, 339.3, and 42.6 ng/mL for AFB1, AFB2, AFG1, and AFG2 (Figure 4C,D), respectively. It was able to produce 310, 10.1, 437.86, and 37.66 ng/mL of AFB1, AFB2, AFG1, and AFG2, respectively, when the fungus was cultured in a slant tube. No peaks were detected within the expected retention times for *Aspergillus oryzae* NRRL 3483 grown on any of three cultivation mediums. Representative chromatograms of *A. oryzae* NRRL 3483 grown in slant cultivation medium are shown in Figure 4E,F. In addition, *A. oryzae* M2040 and *A. oryzae* NRRL RIB40 were unable to produce any types of AFs when grown in different culture medium, as shown in previous studies [28,29]. *Toxins* **2020**, *12*, x FOR PEER REVIEW 6 of 11 liquid cultivation, this fungus was able to produce 508.2, 24.21, 339.3, and 42.6 ng/mL for AFB1, AFB2, AFG1, and AFG2 (Figure 4C,D), respectively. It was able to produce 310, 10.1, 437.86, and 37.66 ng/mL of AFB1, AFB2, AFG1, and AFG2, respectively, when the fungus was cultured in a slant tube. No peaks were detected within the expected retention times for *Aspergillus oryzae* NRRL 3483 grown on any of three cultivation mediums. Representative chromatograms of *A. oryzae* NRRL 3483 grown in slant cultivation medium are shown in Figure 4E,F. In addition, *A. oryzae* M2040 and *A. oryzae* NRRL RIB40 were unable to produce any types of AFs when grown in different culture medium, as shown in previous studies [28,29].

**Figure 4.** Analyses of aflatoxins from three different *Aspergillus* species. Representative HPLC chromatograms of aflatoxins in five-day potato dextrose agar (PDA) solid culture of *Aspergillus flavus* NRRL 3357 detected by DAD (**A**) and FLD (**B**), in five-day potato dextrose broth (PDB) submerged culture of *A. parasiticus* NRRL 2999 detected by DAD (**C**) and FLD (**D**), and in five-day PDB slant culture of *Aspergillus oryzae* M2040 detected by DAD (**E**) and FLD (**F**) are shown. Note that no aflatoxins are detectable from the culture of this food-grade strain. **Figure 4.** Analyses of aflatoxins from three different *Aspergillus* species. Representative HPLC chromatograms of aflatoxins in five-day potato dextrose agar (PDA) solid culture of *Aspergillus flavus* NRRL 3357 detected by DAD (**A**) and FLD (**B**), in five-day potato dextrose broth (PDB) submerged culture of *A. parasiticus* NRRL 2999 detected by DAD (**C**) and FLD (**D**), and in five-day PDB slant culture of *Aspergillus oryzae* M2040 detected by DAD (**E**) and FLD (**F**) are shown. Note that no aflatoxins are detectable from the culture of this food-grade strain.

In summary, in this work, we report a HPLC method coupled with DAD and FLD detectors that would be the first and only validated tool for simultaneous quantitation of AFB1, AFB2, AFG1, and AFG2 in three different laboratory culture conditions. This was an effective tool for quantitative screening of AFs in diverse *Aspergillus* strains. Chloroform was used as the extraction solvent to avoid emulsion formation—the mixture separates into two layers with AFs in the chloroform layer, thus reducing toxin loss and leaving other compounds in the aqueous layer. The extraction and cleanup procedures can be performed in less than 10 min and do not require the use of large amount of solvent or immune-affinity columns (IAC). The HPLC analysis is to be performed without any pre- or postcolumn derivatization reagents or any fluorescent enhancers. Peaks of the four AFs are separated in less than 10 min with high selectivity, linearity, and recovery. Finally, our method provides sufficient sensitivity to enable AF detection within mixtures at ppb levels for AFB1 and AFG1, and at parts per In summary, in this work, we report a HPLC method coupled with DAD and FLD detectors that would be the first and only validated tool for simultaneous quantitation of AFB1, AFB2, AFG1, and AFG2 in three different laboratory culture conditions. This was an effective tool for quantitative screening of AFs in diverse *Aspergillus* strains. Chloroform was used as the extraction solvent to avoid emulsion formation—the mixture separates into two layers with AFs in the chloroform layer, thus reducing toxin loss and leaving other compounds in the aqueous layer. The extraction and cleanup procedures can be performed in less than 10 min and do not require the use of large amount of solvent or immune-affinity columns (IAC). The HPLC analysis is to be performed without any pre- or post-column derivatization reagents or any fluorescent enhancers. Peaks of the four AFs are separated in less than 10 min with high selectivity, linearity, and recovery. Finally, our method provides sufficient sensitivity to enable AF detection within mixtures at ppb levels for AFB1 and AFG1, and at parts per

trillion (ppt) levels for AFB2 and AFG2 via FLD detection. In addition, our method can by readily

available and easily applied in most mycology laboratories.

**3. Materials and Methods** 

*3.1. Chemicals and Materials* 

trillion (ppt) levels for AFB2 and AFG2 via FLD detection. In addition, our method can by readily available and easily applied in most mycology laboratories.
