**1. Introduction**

Aflatoxins (AFs) are a group of mycotoxins that are toxic, carcinogenic, and mutagenic. Amongst them, aflatoxin B1 (AFB1) is the most potent carcinogen found in nature and thus is classified as a group 1 carcinogen to humans by the International Agency for Research on Cancer (IARC) [1,2]. AFs are produced mainly by a common fungus *Aspergillus flavus*in fields, transportation, and storage conditions. AFs consistently and increasingly contaminate both human food and animal feed, and thus have been strictly regulated by the government authorities in over 100 countries in the world [3–5]. Trace levels of AFs, 4–20 parts per billion (ppb), can be considered hazardous, and foods with higher amounts are not fit for human consumption [6]. As global warming progresses, AF-producing molds will expand their growing regions, leading to an increased burden of AF contamination in the world [7–9].

AFs are fluorescent heterocyclic secondary metabolites with molecular weights of 286 to 346 Da. Although more than 13 types of AFs have been discovered, AFB1, AFB2, AFG1, AFG2, and AFM1 (in milk) are particularly hazardous to humans and animals, as they have been commonly present in food and feed. The "B" and "G" refer to the blue and green-blue fluorescent colors emitted under ultraviolet (UV) light (Figure 1A), and the numbers represent the travelled position from the front line

on the thin layer chromatography (TLC); moreover, AFB2 and AFG2 are the dihydroxy derivatives of AFB1 and AFG1, respectively [10,11]. Due to their oxygenated pentaheterocyclic structure, which is known as coumarinic nucleus, AFs have natural fluorescence properties (Figure 1A,B). This ability to fluoresce has paved the way for most analytical methods for the detection and quantification of these toxins [12]. Because of the absence of a double bond in the furan ring, AFB2 and AFG2 have a higher fluorescence quantum yield of fluorophore than the unsaturated compounds AFB1 and AFG1 [13]. derivatives of AFB1 and AFG1, respectively [10,11]. Due to their oxygenated pentaheterocyclic structure, which is known as coumarinic nucleus, AFs have natural fluorescence properties (Figure 1A,B). This ability to fluoresce has paved the way for most analytical methods for the detection and quantification of these toxins [12]. Because of the absence of a double bond in the furan ring, AFB2 and AFG2 have a higher fluorescence quantum yield of fluorophore than the unsaturated compounds AFB1 and AFG1 [13].

line on the thin layer chromatography (TLC); moreover, AFB2 and AFG2 are the dihydroxy

**Figure 1.** Aflatoxin (AF) thin layer chromatography (TLC) and structures. (**A**) A thin layer chromatograph of standard aflatoxin mixture containing AFB1, AFB2, AFG1, and AFG2. Note the color and the separation order. The photo was taken in a UV chamber at 365 nm. (**B**) Chemical structure of AFB1, AFB2, AFG1, and AFG2. **Figure 1.** Aflatoxin (AF) thin layer chromatography (TLC) and structures. (**A**) A thin layer chromatograph of standard aflatoxin mixture containing AFB1, AFB2, AFG1, and AFG2. Note the color and the separation order. The photo was taken in a UV chamber at 365 nm. (**B**) Chemical structure of AFB1, AFB2, AFG1, and AFG2.

*Aspergilli* residing in field soil of *A. flavus* specifically, is considered as the main source of AF contamination of agricultural products; however, not all strains of *A. flavus* produce AFs [14]. Communities of AF-producing fungal residents in varying agricultural environments are complex groups of diverse individuals. Thus, knowing the AF-producing potential of *A. flavus* populations is an important factor for the predicting the incidence and severity of AF contamination. On the other hand, although it was thought that *A. flavus* only produced B type AFs, recent reports have *Aspergilli* residing in field soil of *A. flavus* specifically, is considered as the main source of AF contamination of agricultural products; however, not all strains of *A. flavus* produce AFs [14]. Communities of AF-producing fungal residents in varying agricultural environments are complex groups of diverse individuals. Thus, knowing the AF-producing potential of *A. flavus* populations is an important factor for the predicting the incidence and severity of AF contamination. On the other hand, although it was thought that *A. flavus* only produced B type AFs, recent reports have demonstrated that several *A. flavus* strains can also produce the G type AFs [15–18].

demonstrated that several *A. flavus* strains can also produce the G type AFs [15–18]. To detect and differentiate aflatoxigenic and non-toxigenic *Aspergilli*, several methods have been developed including molecular marker-based methods and fungal culture methods [19,20]. Currently, in most cases, aflatoxigenic fungi are being identified by culture methods coupled with thin layer chromatography (TLC) or high-performance liquid chromatography (HPLC). However, to the best of our knowledge, no methods have been optimized and validated for simultaneous quantifications of aflatoxin cocktail (AFB1, AFB2, AFG1, AFG2) in the fungal cultures. Gell and Carbone have used HPLC-FLD (fluorescence) for quantification of AFB1 from fungal mycelium culture after sample purification by solid phase extraction tubes (SPE), and they were able to achieve a limit of detection (LOD) and limit of quantification (LOQ) of 2 and 3.9 ng/mL, respectively [21]. Culture method has a number of advantages over others, including it being inexpensive, rapid, available in most labs, and requiring minimal technical skills. However, due to the lack of verifications of these methods, they are generally regarded as being less precise than the other methods. Here, we report a new method for a rapid, sensitive, and simultaneous detection of AFB1, AFB2, AFG1, and AFG2 produced in laboratory culture conditions using HPLC equipped with a To detect and differentiate aflatoxigenic and non-toxigenic *Aspergilli*, several methods have been developed including molecular marker-based methods and fungal culture methods [19,20]. Currently, in most cases, aflatoxigenic fungi are being identified by culture methods coupled with thin layer chromatography (TLC) or high-performance liquid chromatography (HPLC). However, to the best of our knowledge, no methods have been optimized and validated for simultaneous quantifications of aflatoxin cocktail (AFB1, AFB2, AFG1, AFG2) in the fungal cultures. Gell and Carbone have used HPLC-FLD (fluorescence) for quantification of AFB1 from fungal mycelium culture after sample purification by solid phase extraction tubes (SPE), and they were able to achieve a limit of detection (LOD) and limit of quantification (LOQ) of 2 and 3.9 ng/mL, respectively [21]. Culture method has a number of advantages over others, including it being inexpensive, rapid, available in most labs, and requiring minimal technical skills. However, due to the lack of verifications of these methods, they are generally regarded as being less precise than the other methods. Here, we report a new method for a rapid, sensitive, and simultaneous detection of AFB1, AFB2, AFG1, and AFG2 produced in laboratory culture conditions using HPLC equipped with a conventional diode array (DAD) and a fluorescence detector (FLD) without using any pre- or post-column derivatization reagents, SPE and

conventional diode array (DAD) and a fluorescence detector (FLD) without using any pre- or post-

immune affinity column (IAC), or fluorescent enhancers. Moreover, we have optimized and validated the method through a series of experiments to meet the research laboratory needs for a robust, fast, easy to use, cheap, and environmentally friendly protocol with minimum organic solvents waste.
