Overpressured layer chromatographic determination of aflatoxin B1, B2, G1 and G2 in red paprika

Determination of aflatoxin B1 and total aflatoxin (B1 + B2 + G1 + G2) in red paprika powder is described using column chromatographic sample clean-up, overpressured layer chromatography (OPLC) separation and fluorescence densitometric evaluation. Two OPLC methods were developed for separation of the four aflatoxins. The detection limit and quantification limit of aflatoxins in red paprika were 0.5 and 1 μg/kg in both methods, respectively. Recovery experiment was carried out with sample containing 1.74 μg/kg aflatoxin B1 and 3.56 μg/kg total aflatoxins measured by European standard HPLC method. Mean recovery amounted to 78.5% (SD 16.1%, n = 5) for aflatoxin B1 and 81.8% (SD 17.1%, n = 5) for total aflatoxins in the case of method 1. It was 105.3% (SD 10.7%, n = 5) for aflatoxin B1 and 97.4% (SD 18.6%, n = 5) for total aflatoxins using the method 2. Despite of that the Hungarian climate is not proper for the toxin production of moulds high aflatoxin B1 contaminated red paprika purchased from the market was found, which may originate from mixing of imported paprika containing very high level toxin with Hungarian one.     

Simultaneous determination of aflatoxins B1, B2, G1, G2, M1 and M2 in peanuts and their derivative products by ultra-high-performance liquid chromatography-tandem mass spectrometry

A reliable ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method for simultaneous determination of aflatoxins B1, B2, G1, G2, M1 and M2 in peanuts and their derivative products was developed. The sample was extracted by 84% of acetonitrile aqueous solution and the extract was purified by a reliable solid phase extraction-based clean-up method. Then, the analytes were separated on Acquity UPLC HSS T3 column (100 mm × 2.1 mm, 1.8 μm particle size), and eluted with a mobile phase consisting of (A) water containing 0.1% formic acid and (B) acetonitrile/methanol (50/50, v/v). The separated compounds were detected with a Waters Micromass Quattro Ultima Pt tandem quadrupole mass spectrometer operating in positive electro-spray ionization using multiple reaction monitoring mode. The established method was extensively validated by determining the linearity (R² ≥ 0.9990), average recovery (74.7-86.8%) and precision (relative standard deviation ≤ 10.9%). It was shown to be a suitable method for simultaneous determination of the six aflatoxins in peanuts and their derivative products. Finally, a total of 73 samples randomly collected from different areas in Zhejiang province were screened for aflatoxins with the proposed method. The results showed that 31 samples of peanut butter, 14 samples of fresh peanut and 5 samples of musty peanut were contaminated with aflatoxins. Meanwhile, this was the first report on aflatoxins M1 and M2, which were found in unprocessed peanuts and their derivative products.     

Determination of aflatoxins B1, G1, B2 and G2 in medicinal herbs by liquid chromatography-tandem mass spectrometry

An easy method for the determination of aflatoxins B1, G1, B2 and G2 in Rhammus purshiana by LC coupled to mass spectrometry has been developed. Aflatoxins were extracted with a mixture of methanol and water and then it was purified by solid-phase clean-up using a polymeric sorbent, not described previously, for the determination of these toxins. The eluted extract was injected into the chromatographic system using a reversed-phase C18 short column with an isocratic mobile phase composed of methanol-water (30:70). A single-quadruple mass spectrometry using an electrospray ionization source operating in the positive ion mode was used to detect aflatoxins due to derivatization presenting several disadvantages. Recoveries of the full analytical procedure were 110% for aflatoxin B1, 89% for aflatoxin B2, 81% for aflatoxin G1 and 77% for aflatoxin G2. Detection limit (S/N = 3) was 10 ng and quantification limit (S/N = 10) was 25 ng, calculated as amount in medicinal herb.     

Simultaneous determination of aflatoxins B2 and G2 in peanuts using spectrofluorescence coupled with parallel factor analysis

In the present study a method for the simultaneous determination of aflatoxins B₂ and G₂ in peanuts has been developed. The method uses second order standard addition method and excitation–emission fluorescence data together with parallel factor analysis (PARAFAC). The aflatoxin analysis was based on extraction with methanol–water and carried out using immunoaffinity clean-up. The results of PARAFAC on a set of spiked and naturally contaminated peanuts indicated that the two aflatoxins could be successfully determined. The method was validated and analytical figures of merit were obtained for both analytes. The limits of detection (LOD) were 0.05 and 0.04 μg kg⁻¹ for aflatoxins B₂ and G₂, respectively. The limits of quantification (LOQ) were 0.16 and 0.12 μg kg⁻¹ for aflatoxins B₂ and G₂, respectively. Coupling of spectrofluorimetry with PARAFAC can be considered as an alternative method for quantification of aflatoxins in the presence of unknown interferences obtained through analysis of highly complex matrix of peanuts samples at a reduced cost per analysis.     

Simultaneous determination of aflatoxins B1, B2, G1, G2 in Fructus Bruceae by high‐performance liquid chromatography with online postcolumn photochemical derivatization

A simple, reliable, and highly sensitive method for the simultaneous determination of aflatoxin B₁, B₂, G₁, G₂ in Fructus Bruceae was developed using high‐performance liquid chromatography coupled to online postcolumn photochemical derivatization and fluorescence detection. Aflatoxins were first extracted by a methanol/water mixture and then cleaned up with an AflaTest? immunoaffinity column. Different clean‐up and derivatization methods were compared and optimized. The established method was extensively validated to show satisfactory performance of linearity (R² ≥ 0.9997), recovery (74.3–100.8%), and precision (RSDs ≤ 2.8%) for the investigated aflatoxins. This proposed method was also applied to 11 F. Bruceae samples and the results showed that 10 out of 11 were contaminated with aflatoxins ranging from 0.26 to 27.52 μg/kg and the occurrence of aflatoxin B₁, the most toxic one, was as high as 91% in all the samples, highlighting the severe contamination and the necessity to set legal limits for aflatoxins in F. Bruceae.     

Development of a microwave-assisted-extraction-based method for the determination of aflatoxins B1, G1, B2, and G2 in grains and grain products

This article describes the use of microwave-assisted extraction (MAE) as a pretreatment technique for the determination of aflatoxins B₁, G₁, B₂, and G₂ in grains and grain products. The optimal operation parameters, including extraction solvent, temperature, and time, were identified to be acetonitrile as the extraction solvent at 80 °C with 15 min of MAE. The extracts were cleaned up using solid-phase extraction followed by derivatization with trifluoroacetic acid and were determined by liquid chromatography–fluorescence detection. A Sep-Pak cartridge was chosen over Oasis HLB and Bond Elut cartridges. By the use of aflatoxin M₁ as an internal standard, relative recoveries of the aflatoxins ranged from 90.7 to 105.7 % for corn and from 88.1 to 103.4 % for wheat, with relative standard deviations between 2.5 and 8.7 %. A total of 36 samples from local markets were analyzed, and aflatoxin B₁ was found to be the predominant toxin, with concentrations ranging from 0.42 to 3.41 μg/kg.

Fluorescent properties of aflatoxins in organized media based on surfactants,cyclodextrins, and calixresorcinarenes

Fluorescent properties aflatoxin B1 (AfB1) and its metabolites, aflatoxins B2, G1, and G2 in the presence of surfactants, cyclodextrins, and calix[4]resorcinarenes are studied. It is found that surfactants and cyclodextrins enhance the fluorescence of aflatoxins B1 and G1. Using the example of AfB1, it is shown that the fluorescence intensity in solution attains a maximum in the presence of 2-hydroxypropyl-β-cyclodextrin. The effects observed increase the sensitivity of the fluorimetric determination of AfB1: the detection limit in water is 3.1 × 10^?8 M and decreases to 2.1 × 10^?9 M in a solution of cyclodextrin.     

Determination of aflatoxins B1, B2, G1 and G2 by high-performance liquid chromatography with electrochemical detection

A new approach to the determination of afiatoxins B₁, B₂, G₁ and G₂ is given; the method involves high-performance liquid chromatography with amperometric detection in the differential-pulse mode at the dropping mercury electrode with 1-s drop time. These aflatoxins can be determined simultaneously with good resolution but with some compromise in sensitivity. The detection limit of underivatized aflatoxin standards is around 5 ng. Average recoveries of aflatoxins from peanut butter by the Beebe method were G₂ 81%, G₁ 87%, B₂ 77% and B₁ 76%.     

High-performance liquid chromatography tandem mass spectrometry for simultaneous detection of aflatoxins B1, B2, G1 and G2 in Indian medicinal herbs using QuEChERS-based extraction procedure

Since the discovery of aflatoxins (AFs) in the 1960s, much research has focused on detecting the toxins in contaminated food and feedstuffs. But the quality determination in medicinal plant matrices with respect to AFs is scare. Hence, a simple, accurate and sensitive high-performance liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) method was developed for simultaneous determination of AFs AFB₁, AFB₂, AFG₁ and AFG₂ in two Indian popular medicinal herbs i.e. senna (Cassia angustifolia) and kalmegh (Andrographis paniculata). AFs have been extracted from herb matrix using a QuEChERS (quick, easy, cheap, effective, rugged and safe)-based extraction procedure followed by applying primary secondary amine and C18 for further clean-up step and then were quantified under the multiple reaction monitoring together with positive ionisation modes. Matrix-matched calibration was used for quantification in order to reduce the matrix effect. Validation of the method was carried out in herbs by recovery experiments. Recoveries of the spiked samples were in the range of 61.9–111.5% with an inter-day and intraday relative standard deviation lower than 20.0%. Limits of detection and quantification ranged from 0.41 to 0.95 ng mL^?1 and 1.2 to 3.8 μg kg^?1, respectively. The expanded uncertainty of the method was <21% for all the toxins in both the herbs. Finally, the proposed method was successfully applied to determine AF residues in real field samples of senna and kalmegh obtained from different locations in India.     

Application of dispersive liquid-liquid microextraction for the determination of aflatoxins B1, B2, G1 and G2 in cereal products

The application of dispersive liquid-liquid microextraction (DLLME) technique for the rapid analysis of aflatoxins B(1), B(2), G(1) and G(2) in maize, rice and wheat products has been evaluated. After extraction of aflatoxins from cereal matrices with a mixture of methanol/water 8:2 (v/v), the analytes were rapidly transferred from the extract to another small volume of organic solvent, chloroform, by DLLME. Aflatoxins were determined using high performance liquid chromatography with florescence detection and photochemical post-column derivatization. Parameters affecting both extraction and DLLME procedures, such as extraction solvent, type and volume of DLLME extractant, volume of water and salt effect, were systematically investigated and optimized to achieve the best extraction efficiency. Under the optimal experimental conditions, the whole analytical method provides enrichment factors around 2.5 times and detection limits (0.01-0.17 μg kg(-1)) below the maximum levels imposed by current regulation for aflatoxins in cereals and cereal products intended for direct human consumption. Recoveries (67-92%) and repeatability (RSD<10, n=3), tested in three different cereal matrices, meet the performance criteria required by EC Regulation No. 401/2006 for the determination of the levels of mycotoxins in foodstuffs. The proposed method was successfully applied to the analysis of retail cereal products with quantitative results comparable to the immunoaffinity chromatography (IAC). The main advantages of developed method are the simplicity of operation, the rapidity to achieve a very high sample throughput and low cost.     

免疫亲和柱高效液相色谱法快速测定牛奶和奶粉中黄曲霉毒素M1、B1、B2、G1、G2

采用单克隆抗体免疫亲和技术作为直接从样品中分离提取黄曲霉毒素的特效手段,提取液挥干后,经衍生用HPLC荧光检测器测定.对鲜奶和高脂奶粉(脂肪含量25%)在0.01,0.05,0.1和0.1,0.5,1.0 μg·L-1水平对黄曲霉毒素M1、B1、B2、G1、G2测定平均回收率(n=10)为66.0%～97.0%;相对标准偏差(n=10)为1.04%～14.0%.方法的检出限低于0.01 μg·L-1(鲜奶), 0.1 g·kg-1(奶粉).     

Simultaneous determination of aflatoxin B1, B2, G1, and G2 in corn powder,edible oil,peanut butter,and soy sauce by liquid chromatography with tandem mass spectrometry utilizing turbulent flow chromatography

A novel fully automated method based on dual column switching using turbulent flow chromatography followed by liquid chromatography with tandem mass spectrometry was developed for the determination of aflatoxin B₁, B₂, G₁, and G₂ in corn powder, edible oil, peanut butter, and soy sauce samples. After ultrasound‐assisted extraction, samples were directly injected to the chromatographic system and the analytes were concentrated into the clean‐up loading column. Through purge switching, the analytes were transferred to the analytical column for subsequent detection by mass spectrometry. Different types of TurboFlow^TM columns, transfer flow rate, transfer time were optimized. The limits of detection and quantification of this method ranged between 0.2–2.0 and 0.5–4.0 μg/kg for aflatoxins in different matrixes, respectively. Recoveries of aflatoxins were in range of 83–108.1% for all samples, matrix effects were in range of 34.1–104.7%. The developed method has been successfully applied in the analysis of aflatoxin B₁, B₂, G₁, and G₂ in real samples.     

Determination of aflatoxins in food samples by automated on-line in-tube solid-phase microextraction coupled with liquid chromatography–mass spectrometry

A simple and sensitive automated method for determination of aflatoxins (B1, B2, G1, and G2) in nuts, cereals, dried fruits, and spices was developed consisting of in-tube solid-phase microextraction (SPME) coupled with liquid chromatography–mass spectrometry (LC–MS). Aflatoxins were separated within 8 min by high-performance liquid chromatography using a Zorbax Eclipse XDB-C8 column with methanol/acetonitrile (60/40, v/v): 5 mM ammonium formate (45:55) as the mobile phase. Electrospray ionization conditions in the positive ion mode were optimized for MS detection of aflatoxins. The pseudo-molecular ions [M+H]⁺ were used to detect aflatoxins in selected ion monitoring (SIM) mode. The optimum in-tube SPME conditions were 25 draw/eject cycles of 40 μL of sample using a Supel-Q PLOT capillary column as an extraction device. The extracted aflatoxins were readily desorbed from the capillary by passage of the mobile phase, and no carryover was observed. Using the in-tube SPME LC–MS with SIM method, good linearity of the calibration curve (r > 0.9994) was obtained in the concentration range of 0.05–2.0 ng/mL using aflatoxin M1 as an internal standard, and the detection limits (S/N = 3) of aflatoxins were 2.1–2.8 pg/mL. The in-tube SPME method showed >23-fold higher sensitivity than the direct injection method (10 μL injection volume). The within-day and between-day precision (relative standard deviations) at the concentration of 1 ng/mL aflatoxin mixture were below 3.3% and 7.7% (n = 5), respectively. This method was applied successfully to analysis of food samples without interference peaks. The recoveries of aflatoxins spiked into nuts and cereals were >80%, and the relative standard deviations were <11.2%. Aflatoxins were detected at <10 ng/g in several commercial food samples.     

Surveillance and control of aflatoxins B1, B2, G1, G2, and M1 in foodstuffs in the Republic of Cyprus: 1992-1996.

Aflatoxins (AFs) B1, B2, G1, and G2 in locally produced and imported foodstuffs (nuts, cereals, oily seeds, pulses, etc.) were monitored and controlled systematically and effectively from 1992-1996. Samples (peanuts, pistachios, etc.) with total AFs above the Cyprus maximum level (ML) of 10 micrograms/kg fluctuated between 0.7 and 6.9%. The results indicate the effectiveness of monitoring, as well as the need for constant surveillance and control, especially at critical control points (sites of import, primary storage, etc.), to prevent unfit products from entering the Cyprus market. The control included sampling, retainment, analysis, and destruction of foodstuff lots with AF levels above MLs. The highest incidence of aflatoxin contamination was observed in peanut butter (56.7%) and the highest level of AF B1 was found in peanuts (700 micrograms/kg). Levels of AF M1 in raw and pasteurized milk analyzed in 1993, 1995, and 1996 were within both the Cyprus ML (0.5 microgram/L) and the lower ML (0.05 microgram/L) of some European countries. Only 12% of samples had detectable levels of AF M1. Analyses were performed by immunochemical methods. When recoveries were lower than 80%, the AF levels were corrected for recovery.     

Immunoaffinity column cleanup with liquid chromatography using post-column bromination for determination of aflatoxins in peanut butter, pistachio paste, fig paste, and paprika powder: collaborative study

A collaborative study was conducted to evaluate the effectiveness of an immunoaffinity column cleanup liquid chromatography (LC) method for the determination of aflatoxin B1 and total aflatoxins at European regulatory limits. The test portion is extracted with methanol-water (8 + 2) for dried figs and paprika, and with methanol-water (8 + 2) plus hexane (or cyclohexane) for peanut butter and pistachios. The sample extract is filtered, diluted with phosphate buffer saline, and applied to an immunoaffinity column. The column is washed with water and the aflatoxins are eluted with methanol. Aflatoxins are quantitated by reversed-phase LC with post-column derivatization (PCD) involving bromination. PCD is achieved with either an electrochemical cell (Kobra cell) and addition of bromide to the mobile phase or pyridinium hydrobromide perbromide. Determination is by fluorescence. Peanut butter, pistachio paste, dried fig paste, and paprika powder samples, both naturally contaminated with aflatoxins and containing added aflatoxins, were sent to 16 collaborators in 16 European countries. Test portions of samples were spiked at levels of 2.4 and 9.6 ng/g for total aflatoxins which included 1.0 and 4.0 ng/g aflatoxin B1, respectively. Recoveries for total aflatoxins ranged from 71 to 92% with corresponding recoveries for aflatoxin B1 of 82 to 109%. Based on results for spiked samples (blind duplicates at 2 levels) as well as naturally contaminated samples (blind duplicates at 4 levels, including blank), the relative standard deviation for repeatability ranged from 4.6 to 23.3% for total aflatoxins and from 3.1 to 20.0% for aflatoxin B1. The relative standard deviation for reproducibility ranged from 14.1 to 34.2% for total aflatoxins, and from 9.1 to 32.2% for aflatoxin B1. The method showed acceptable within-laboratory and between-laboratory precision for all 4 matrixes, as evidenced by HORRAT values <1, at the low levels of determination for both total aflatoxins and aflatoxin B1.     

Development of a multiplex UPLC-MRM MS method for quantification of human membrane transport proteins OATP1B1, OATP1B3 and OATP2B1 in in vitro systems and tissues

OATP1B1, OATP1B3 and OATP2B1 are important members of the organic anion transporting polypeptides (OATP) family and are implicated in the hepatic disposition of endobiotics and xenobiotics. Quantitating the expression levels of human OATP1B1, OATP1B3 and OATP2B1 in in vitro systems and tissue samples could significantly improve attempts to scale up in vitro data and result in more effective in vitro–in vivo correlation of transporter-mediated effects on drug disposition, such as hepatic clearance. In the present study, a quantification method was developed, characterized, and implemented for simultaneous determination of human OATP1B1, OATP1B3 and OATP2B1 in HEK cells transfected with OATP-expressing plasmid vectors (SLCO1B1, SLCO1B3, and SLCO2B1, respectively), human hepatocytes, human brain capillary endothelial cells, and humanized mouse liver tissue using UPLC-MRM MS. Purified membrane protein standards prepared and characterized as previously reported (Protein Expr. Purif. 2008, 57, 163-71) were first used as standards for absolute quantification of the expression levels of the three human OATP membrane proteins. The specificity of the optimized MRM transitions were characterized by analyzing the tryptic digests of the membrane protein fraction of wild type HEK cells and control mouse liver tissue using the herein reported UPLC-MRM MS method. The linearity of the calibration curve spanned from 0.2 μg mL⁻¹ (0.040 μg mg⁻¹) to 20 μg mL⁻¹ (4.0 μg mg⁻¹), with accuracy (% RE) within 15% at all concentrations examined for all three OATPs of interest in this study. The intra- and inter-day assay accuracy (% RE) and coefficient of variations (% CV) of triplicates are all within 15% for all levels of quality control samples prepared by mixing the membrane fraction of control mouse liver tissue with the required amount of purified human OATP1B1, OATP1B3 and OATP2B1.     

薏苡仁中黄曲霉毒素G1的动态表面增强拉曼光谱检测

中药具有不良反应少,疗效独特等优点,越来越受到国内外的关注。但中药在生产、采集、加工、运输、贮藏等方面极易发生霉变而污染黄曲霉毒素。黄曲霉毒素具有很强的毒性,不仅影响中药质量和疗效还会对人体肝脏等器官造成极大损害。该文建立了中药材薏苡仁中黄曲霉毒素G1(Aflatoxin G1,AFG1)的动态表面增强拉曼光谱(Dynamic Surface-enhanced Raman Spectroscopy,D-SERS)的快速分析方法。采用简单擦拭法提取薏苡仁表面的黄曲霉毒素G1,因此SERS图谱中干扰峰较少。并且以金纳米颗粒为增强基底,优化了检测条件,进行了黄曲霉毒素G1的D-SERS的检测与分析。结果表明,方法的线性范围为8~320μg/kg,线性方程为y=0.0293x+1.7307,相关系数(r2)为0.9884。检出限(LOD)为5.5μg/kg,相对标准偏差(RSD)为11%。该方法的线性良好,灵敏度和重现性均能满足薏苡仁中黄曲霉毒素G1的快速检测要求,且操作简单、快速、节约成本,对中药材中黄曲霉毒素G1的现场实时快速分析具有良好的应用潜力。     

Safety evaluation of Semen Sojae Preparatum based on simultaneous LC–ESI–MS/MS quantification of aflatoxin B1, B2, G1, G2 and M1

Semen Sojae Preparatum (SSP) is one of the most widely used traditional Chinese medicines, and is also a functional food. However, contamination with aflatoxins may occur in the fermentation process. To evaluate its safety, an accurate and rapid LC–ESI–MS/MS analytical method was developed and validated for the simultaneous determination of AFB₁, AFB₂, AFG₁, AFG₂ and AFM₁ in SSP. After a simple ultrasonic extraction of SSP samples, chromatographic separation was achieved on an Agilent Zorbax SB‐C₁₈ column (2.1 × 50 mm, 3.5 μm) with a flow rate of 0.50 mL/min. The gradient elution program was performed using a mobile phase consisting of water and acetonitrile, both containing 0.1% formic acid. Detection of five aflatoxins was based on triple quadrupole mass spectrometry using a multiple reaction monitoring mode with an electrospray ionization source. SSP is likely to be contaminated by aflatoxins in the processes of fermentation, storage, transportation and usage, and it is necessary to strictly monitor it. Artemisia annua L. and Morus alba L. may inhibit the production and growth of AFB₁‐ and AFB₂‐producing fungi, which has a certain detoxification effect on contamination with aflatoxins in SSP.     

Determination of aflatoxins B1, B2, G1, and G2 and ochratoxin A in ginseng and ginger by multitoxin immunoaffinity column cleanup and liquid chromatographic quantitation: collaborative study

The accuracy, repeatability, and reproducibility characteristics of a method using multitoxin immunoaffinity column cleanup with liquid chromatography (LC) for determination of aflatoxins (AF; sum of aflatoxins B1, B2, G1, and G2) and ochratoxin A (OTA) in powdered ginseng and ginger have been established in a collaborative study involving 13 laboratories from 7 countries. Blind duplicate samples of blank, spiked (AF and OTA added) at levels ranging from 0.25 to 16.0 microg/kg for AF and 0.25 to 8.0 microg/kg for OTA were analyzed. A naturally contaminated powdered ginger sample was also included. Test samples were extracted with methanol and 0.5% aqueous sodium hydrogen carbonate solution (700 + 300, v/v). The extract was centrifuged, diluted with phosphate buffer (PB), filtered, and applied to an immunoaffinity column containing antibodies specific for AF and OTA. After washing the column with water, the toxins were eluted from the column with methanol, and quantified by high-performance LC with fluorescence detection. Average recoveries of AF from ginseng and ginger ranged from 70 to 87% (at spiking levels ranging from 2 to 16 microg/kg), and of OTA, from 86 to 113% (at spiking levels ranging from 1 to 8 microg/kg). Relative standard deviations for within-laboratory repeatability (RSDr) ranged from 2.6 to 8.3% for AF, and from 2.5 to 10.7% for OTA. Relative standard deviations for between-laboratory reproducibility (RSDR) ranged from 5.7 to 28.6% for AF, and from 5.5 to 10.7% for OTA. HorRat values were < or = 2 for the multi-analytes in the 2 matrixes.     

Aflatoxin B1 and M1 in milk

The aflatoxin M₁ (AFLAM₁) is a mycotoxin that results from the hydroxylation of the aflatoxin B₁ (AFLAB₁). It contaminates the milk of animals fed with a diet containing its precursor. In this work, we determined the occurrence of AFLAB₁ and AFLAM₁ in milk, as well as the chromatographic conditions to quantify these mycotoxins. The extraction and quantification of AFLAB₁ and AFLAM₁ in naturally contaminated and artificially spiked milk samples which are produced and marketed in the state of RS were performed using the AOAC official method and UHPLC with fluorescence detection. We obtained a separation factor of 2.3 for AFLAB₁ and AFLAM₁ using a mobile phase consisting of 1% acetic acid:acetonitrile:methanol (55:10:35). The analytical curves had a wide linearity range and the limit of quantification (LOQ_m) concentrations of AFLAB₁ and AFLAM₁ were equal to 0.5 and 0.25 μg L⁻¹, respectively. Samples of pasteurized and ultra-high-temperature processed (UHT) milk showed natural contamination, and the levels for both aflatoxins ranged from 0.7 to 1.5 μg L⁻¹. Raw and concentrated milk samples only contained AFLAM₁, with a maximum average concentration of 1.7 μg L⁻¹. These concentrations, higher than permitted by legislation, confirm the existence of a health risk, as well as highlight the relevance of searching for alternatives to reduce this contamination.