# Extraction and quantification of aflatoxins and ochratoxin A in dried tamarind from Côte d'Ivoire by LC-MS/MS

Christine Devouge-Boyer, Balla Ballo, Leonie Moyon, Juliette Vievard, Mathilde Lauzent, Tiemélé Laurent-Simon Amoikon, Solange Aka-Gbezo, Mélanie Mignot

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## To cite this version:

Christine Devouge-Boyer, Balla Ballo, Leonie Moyon, Juliette Vievard, Mathilde Lauzent, et al. Extraction and quantification of aflatoxins and ochratoxin A in dried tamarind from Côte d'Ivoire by LC-MS/MS. Journal of Food Composition and Analysis, 2025, 148, pp.108348. 10.1016/j.jfca.2025.108348. hal-05471740

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Submitted on 22 Jan 2026

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# Extraction and quantification of aflatoxins and ochratoxin A in dried tamarind from Côte d'Ivoire by LC-MS/MS

Christine Devouge-Boyer<sup>a</sup>, Balla Ballo<sup>b</sup>, Leonie Moyon<sup>a</sup>, Juliette Vievard<sup>a</sup>, Mathilde Lauzent<sup>a</sup>, Tiemélé Laurent-Simon Amoikon<sup>c,d</sup>, Solange Aka-Gbezo<sup>b,d</sup>, Mélanie Mignot<sup>a,*</sup> <sup>D</sup>

## ARTICLE INFO

### Keywords:

- Mycotoxin contamination
- Ochratoxin A
- Food safety
- LC-MS/MS
- West Africa

## ABSTRACT

Tamarind, a widely consumed fruit in Côte d'Ivoire, is often sold in open markets where storage conditions may promote fungal growth. Despite its popularity, little attention has been given to the health risks of mycotoxin contamination in this product. To our knowledge, this is the first study investigating simultaneous aflatoxin and ochratoxin A contamination in tamarind sold in Côte d'Ivoire. This study aimed to develop a simple and reliable method to extract and measure mycotoxins in tamarind. Forty samples were collected from the wholesale markets in three municipalities of Abidjan in Côte d'Ivoire. Using LC-MS/MS analytical method, levels of aflatoxins B1, B2, G1, G2, and ochratoxin A were quantified, following European Commission limits to mitigate health risks associated with this food contamination. The results indicate that aflatoxin levels in all analyzed tamarind samples remained below the acceptable threshold (10 µg/kg for aflatoxins), posing no significant health risk. However, 16 samples exceeded the permitted level for ochratoxin A (above 2 µg/kg). Although improvements in the extraction method and post-clean-up procedures are required to reduce the matrix effect for ochratoxin A, these findings highlight the need for continued monitoring of ochratoxin A contamination in tamarind to ensure consumer safety.

## 1. Introduction

The tamarind tree (Tamarindus indica L.) is one of the chief leguminous plant species of Fabaceae family native from Madagascar and East Africa, but is now widespread throughout tropical, semi-arid Africa and India (Devi and Boruah, 2020; Garba et al., 2019; Kuru, 2014). Tamarind fruit pulp is rich in soluble sugar, pectin, B and C vitamins and minerals (Hamacek et al., 2013; Jacqueline et al., 2022). It also contains organic acids, especially tartaric acid (98 %) and carbohydrates, proteins (Hamacek et al., 2013; Van Der Stege et al., 2011) and is source of polyphenols, flavonoids, procyanidin, triterpenes (Devi and Boruah, 2020; Gupta et al., 2014; Mathurin et al., 2021). This tree is considered as a multi-purpose tree given the different virtues attributed to the fruits, leaves, flowers, trunk and bark of the tree. Therefore, it has a real economic activity throughout the country (Bakayoko et al., 2024; Kouassi et al., 2018) and finds many applications in food, medicine, cosmetics and the textile industry (Bhadoriya et al., 2011; Kuru, 2014).

However, despite its nutritional and economic importance, tamarind pulp is particularly vulnerable to microbial contamination due to traditional post-harvest handling practices comparing to other dried fruits. After being extracted from the pods, the pulp is usually sun-dried outdoors, directly on the ground or on basic materials like mats, regardless of the season whether rainy or dry. This drying method does not change throughout the year. Once dried, tamarind is stored in unsealed containers such as sacks or baskets and sold in open markets without any control over temperature or humidity. These artisanal practices, combined with the pulp's high sugar and organic acid content, create ideal conditions for mold growth, especially from Aspergillus species known to produce mycotoxins like aflatoxins and ochratoxin A. Other genera such as Penicillium also contribute, as they are major producers of mycotoxins in food (Ghomfo et al., 2024; Kedjebo et al., 2014). These toxins can cause a range of serious health issues, including kidney damage, immune suppression, and cancer (Diop et al., 2022; Ghomfo et al., 2024; Kedjebo et al., 2014; Kouadio et al., 2014; Liew and

<footnote><sup>a</sup> INSA Rouen, INSA Rouen Normandie, Univ Caen Normandie, ENSICAEN, CNRS, Institut CARMeN UMR 6064, Saint Etienne du Rouvray, F-76800, France</footnote>

<footnote><sup>b</sup> Unité de Formation et de Recherche en Sciences et Technologie des Aliments (UFR-STA), Université Nangui Abrogoua, O2 BP 801 Abidjan 02, Côte d'Ivoire</footnote>

<footnote><sup>c</sup> National Institute of Public Health (INSP), Pierre Richet Institute of Bouaké (IPR), Bouaké BP. 1500, Côte d'Ivoire</footnote>

<footnote><sup>d</sup> Centre Suisse de Recherches Scientifiques, Côte d'Ivoire</footnote>

<footnote>* Corresponding author.</footnote>

<footnote>E-mail address: melanie.mignot@insa-rouen.fr (M. Mignot).</footnote>
Mohd-Redzwan, 2018). Among them, ochratoxin A, like aflatoxins, is recognized as a significant health hazard. Although regulations exist for certain products like cereals, coffee and cocoa (Banahene et al., 2024; Nazareth et al., 2024) little attention has been given to tamarind, despite its long storage periods under conditions favorable to fungal growth. In the European Union, maximum levels for aflatoxins and ochratoxin A in dried fruits like tamarind have been established to limit consumer exposure (Regulation - 2023/915 - EN - EUR-Lex, s. d.). In Côte d'Ivoire, no regulations currently exist regarding these toxins in food, particularly in tamarind. This absence of regulatory oversight raises important public health concerns. This issue extends beyond Côte d'Ivoire as in many other tropical regions where tamarind is widely consumed, there are also no clear regulations or monitoring systems in place to control mycotoxin contamination, including ochratoxin A. This raises broader public health concerns, particularly in low- and middle-income countries where food safety oversight is often limited. Tamarind is widely consumed across the country, including by vulnerable groups like children and pregnant women, who are more susceptible to the effects of these mycotoxins. Ochratoxin A and aflatoxins, recognized for their nephrotoxic and carcinogenic effects, represents a particularly significant risk. Continuous exposure, even with low levels, may contribute to long-term health problems such as kidney disorders, weakened immunity, and increased cancer (Agriopoulou et al., 2020b; Khan et al., 2024). In the absence of regulations and systematic monitoring, populations consuming potentially contaminated tamarind may unknowingly be at risk of long-term health effects. Therefore, assessing the presence of these toxins in tamarind is essential to know potential health risks and strengthen food safety measures if some toxicity was proven. Research work in Côte d'Ivoire has focused on the food and therapeutic uses of tamarind fruit (Bakayoko et al., 2024; Jacqueline et al., 2022), on the nutritional value of tamarind pulp (Kouassi et al., 2018) and on the biochemical and microbiological characterization of juice from tamarind fruit pulp (Jacqueline et al., 2022; Kouassi et al., 2018). To date, no study on the determination of mycotoxins especially for ochratoxin A and aflatoxins in tamarind pulp has been carried out knowing that tamarind pulp is preserved for a long time and during this conservation, molds are likely to develop there.

Mycotoxins can be detected by a variety of analytical and immunological methods. Traditional chromatographic techniques, including thin-layer chromatography (TLC), gas chromatography (GC) and high-performance liquid chromatography (HPLC), are widely used for qualitative and quantitative mycotoxin analysis. TLC is a cost-effective option for rapid screening but lacks the sensitivity required for accurate measurements. GC, although accurate, requires a derivatization step to analyze non-volatile mycotoxins, increasing its complexity. HPLC, especially when coupled with fluorescence or UV detectors, is highly effective for the detection of specific mycotoxins such as aflatoxins (AFB1, AFB2, AFG1, and AFG2), ochratoxin A (OTA) and zearalenone (ZEA), offering excellent reliability and sensitivity in rather simple matrices. Immunological methods such as enzyme-linked immunosorbent assays (ELISA), lateral flow assays (LFA) and biosensors have recently gained popularity. These approaches provide rapid and cost-effective alternatives to chromatographic methods, making them suitable for on-site or high-throughput mycotoxin detection (Khan et al., 2024). Of all the techniques, liquid chromatography-tandem mass spectrometry (LC-MS/MS) is the most advanced and reliable for mycotoxin analysis. It combines sensitivity, specificity and versatility, allowing the simultaneous detection and quantification of multiple mycotoxins in complex food matrices. LC-MS/MS has become the gold standard for mycotoxin analysis, widely applied in diverse food matrices (Agriopoulou et al., 2020a).

This study reports the comparison of extraction parameters as well as LC-MS/MS developments to quantify multiple mycotoxins—aflatoxins (AFB1, AFB2, AFG1, AFG2), and ochratoxin A (OTA)—in 40 tamarind fruit pulp samples from different regions of Côte d'Ivoire. The methodology could be transposed to other food matrices and the data provide

critical input for future exposure assessments and risk characterization studies, especially considering the high consumption of tamarind in the population. By comparing the measured levels to international standards, this study offers a first step towards understanding the possible health risks associated with tamarind consumption in Côte d'Ivoire., It emphasizes the need for targeted monitoring and regulatory measures to protect consumers.

## 2. Materials and methods

### 2.1. Chemicals and reagents

For the extraction method, methanol, acetonitrile (LC-MS gradient grade), and magnesium sulphate (anhydrous) were obtained from Fisher Scientific (UK). Sodium chloride and octanol (LC grade), and formic acid (p.a.) were purchased from Sigma-Aldrich (Switzerland). The QuEChERS extraction and cleanup kit were sourced from Thermo Scientific (France). Ultrapure water (resistivity 18.2 MΩ.cm) was prepared using a PURELAB® Flex 1 system (Veolia, France). The mycotoxin standard was obtained from LGC (France) as a mixture of AFB1, AFB2, AFG1, AFG2 and ochratoxin A at 10 µg/mL in acetonitrile.

A certified reference material (feedingstuff standard spiked with aflatoxins AFB1, AFB2 and AFG1 at concentrations of 12.9, 0.68, and 5.2 µg/kg, respectively) was obtained from LGC (France) to optimize the extraction step.

### 2.2. Samples

A total of 180 tamarind samples were collected from wholesale markets in three municipalities of Abidjan, Côte d'Ivoire: Koumassi, Abobo, and Adjamé (Fig. 1). Approximately 60 samples of 500 g were obtained from different wholesalers at each site. Abidjan is one of the main supply hubs for tamarind in the country (Bakayoko et al., 2024).

Following preliminary microbiological and molecular analyses of the full sample set, fungal species known to be potentially mycotoxigenic, such as Aspergillus aflatoxiformans (aflatoxin producer) and Aspergillus niger (ochratoxin A producer), were detected in a subset of samples (data not shown). Based on these findings, 40 samples were selected for targeted LC-MS/MS analysis. These 40 samples included 20 from Koumassi, 14 from Abobo, and 6 from Adjamé. The selection was specifically focused on samples in which potentially toxigenic fungi had been identified. After collection, all samples were stored at around 4 °C during transport to the laboratory. Before analysis, tamarind samples were stored in liquid nitrogen at -80°C and lyophilized using a Lab-conco FreeZone 4.5 L freeze dryer (cryostat temperature: -80°C). The dried samples were then crushed and sieved to a particle size of 500 µm.

### 2.3. Extraction optimization

Due to the unavailability of a certified reference material (CRM) for tamarind, method optimization was performed using a certified reference material for feedingstuffs. Various extraction techniques were evaluated to determine the most efficient method. A representative homogenized sample (2.5 g) was extracted either by microwave extraction as described by Du et al. (2018) (80°C for 10 min) or ultrasonic bath extraction (room temperature for 30 min). The extraction solvent, a 50/50 (v/v) mixture of methanol and acetonitrile, was tested with and without the addition of salt (1 g sodium chloride and 4 g magnesium sulphate). Additionally, the QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) extraction method explained by Sadighara et al. (2024) was tested on 2 g of feedingstuff. This method involved extraction with 10 mL of water and 10 mL of acetonitrile, followed by the addition of a salt mixture containing 4 g magnesium sulfate, 1 g sodium chloride, 1 g disodium citrate trihydrate, and 0.5 g disodium hydrogen citrate sesquihydrate. After stirring and centrifugation, a portion of the supernatant was purified using a solid-phase dispersive
<figure><img src="image_4.png" /><figcaption>Fig. 1. Map of Côte d'Ivoire, with a red border indicating the 3 markets from which the tamarind samples came. Map modified from (https://commons.wikimedia.org/wiki/File:CommunesAbidjan.svg).</figcaption></figure>

clean-up mixture composed of 900 mg magnesium sulfate, 150 mg primary and secondary amines (PSA), and 150 mg C18 phase. The recovery rates were calculated by Eq. 1:

$$ \text{Recovery rate}(\%) = \left( \frac{\text{Experimental content}}{\text{Theoretical content in CRM}} \right) \times 100 \quad (1) $$

## 2.4. Final extraction protocol

The optimal extraction method was determined as follows: 2.5 g of the sample was weighed into a 100 mL microwave vessel, followed by the addition of 10 mL of a 50/50 (v/v) methanol/acetonitrile extraction solvent. Microwave-assisted extraction was performed at 80°C for 10 min (150 W power) using a THERMO Milestone One microwave oven. Next, the extract was transferred into a 50 mL polypropylene tube, rinsed with 30 mL of methanol/acetonitrile (50/50, v/v), and centrifuged at 4000 rpm for 5 min. The extract was then concentrated by heating at 42°C under 200 mbar pressure, reconstituted in 2 mL of acetonitrile, filtered through a 0.22 µm PTFE filter, and stored at -20°C until analysis by LC-MS/MS. All extractions were performed in triplicate (i.e. 3 independent samples of each type) to ensure reproducibility.

## 2.5. Chromatographic conditions

The LC-MS/MS system consisted of a UPLC (Waters, ACQUITY) coupled with a mass spectrometer (Xevo, TQS). Chromatographic separation was performed at 30°C using an Acquity BEH C18 column (100 mm × 2.1 mm, 1.7 µm particle size; Waters, UK). Elution was performed in binary gradient mode, with various compositions of eluents A and B tested to optimize mycotoxin separation. The optimized eluent A consisted of water with 0.1 % formic acid, while eluent B was a mixture of methanol/acetonitrile (60/40, v/v) with 0.1 % formic acid. The gradient started with 30 % eluent B for 6 min, which then linearly increased up to 100 % over 6 min. This was followed by a 2-minute hold at 100 % B and a 3-minute column re-equilibration of the column at 30 % B. The flow rate was 400 µL/min. Acquisition was performed using MassLynx V4.2.

## 2.6. Mass spectrometer parameters

The mass spectrometer was operated in positive ESI mode, with a capillary voltage of 1.5 kV and a cone voltage of 10 V. The source temperature was set to 150°C, and the desolvation temperature was

500°C. Gas desolvation and nebulization were carried out using nitrogen at flow rates of 800 and 50 L/h, respectively. Signal acquisition was performed in multiple reaction monitoring (MRM) mode. Data were processed using DataAnalysis (version 5.1). A mycotoxin standard containing all target compounds was used to determine two MRM transitions (one quantifier and one qualifier) per compound, along with LC retention times and MRM intensity ratios. Identification criteria included a retention time within ± 0.1 min of the reference standard and an MRM intensity ratio within ± 30 %. The optimized MRM transitions and voltage parameters are detailed in Table 1.

# 3. Results and discussion

## 3.1. LC-MS/MS method validation

For each mycotoxin, the acquisition parameters, including eluent composition, MRM transitions, cone and collision voltages, were optimized based on the study by Han and al (Han et al., 2012).

In their study, the organic mobile phase was composed of methanol/acetonitrile/ (20/80, v/v). After conducting several experiments, it was found that a mobile phase of methanol/acetonitrile/ (60/40, v/v) provided better separation between aflatoxins B1 and B2, as well as between aflatoxins G1 and G2.

According to the literature, the MRM transitions *m/z* 403.8 → *m/z* 238.9 and *m/z* 403.8 → *m/z* 341.2 with cone and collision voltages set to 25 V, were selected for ochratoxin A analysis. With the aim of increasing the sensitivity in OTA detection, the parent ion search was conducted for product ions *m/z* 238.9, *m/z* 341.2, and *m/z* 358.2. The parent ion *m/z* 404.1 was identified for the transition of the product ions *m/z* 238.9 and *m/z* 341.2. A SIM (Single Ion Monitoring) mode spectrum was then obtained for *m/z* 404.1 to identify the corresponding product ions. Cone and collision voltages were optimized to maximize the area under the OTA peak, with the optimal cone voltage at 25 V and collision voltage at 25 V for the transitions *m/z* 404.1 → 238.9 and *m/z* 404.1 → 341.2.

For each mycotoxin, the MRM chromatogram for both the qualifier and quantifier ions was obtained for all standard solutions, CRM extracted solutions and tamarind extracted solutions. The area under the peak was noted (Fig. 2).

For aflatoxins, a five-point calibration curve has been established by diluting the standard solution in acetonitrile between 0.5 and 20 µg/L for a linearity better than 0.995. For ochratoxin A two curves were established between 0.5 and 20 µg/L and between 20 µg/L and 200 µg/L.

The limit of detection (LOD) and the limit of quantification (LOQ) on
<table><caption>**Table 1**
LC-MS/MS identification parameters.</caption><thead><tr><th>Compound</th><th>Retention time (min)</th><th>Ion quantifier transition</th><th>Ion qualifier transition</th><th>Cone tension (V)</th><th>Collision tension (V)</th><th>Ratio ion qualifier/ion quantifier</th></tr></thead><tbody><tr><td>Aflatoxin B1</td><td>7.5</td><td>313.2 &gt; 241.2</td><td></td><td>50</td><td>37</td><td>1.00</td></tr><tr><td>Aflatoxin B2</td><td>6.1</td><td>315.2 &gt; 287.2</td><td>313.2 &gt; 285.2</td><td>50</td><td>23</td><td></td></tr><tr><td>Aflatoxin G1</td><td>5.3</td><td>329.2 &gt; 243.2</td><td>315.2 &gt; 259.2</td><td>50</td><td>30</td><td>0.94</td></tr><tr><td>Aflatoxin G2</td><td>4.03</td><td>331.2 &gt; 245.2</td><td>329.2 &gt; 283.2</td><td>25</td><td>26</td><td>0.32</td></tr><tr><td>Aflatoxin G2</td><td>4.03</td><td>331.2 &gt; 245.2</td><td>329.2 &gt; 283.2</td><td>25</td><td>25</td><td>0.32</td></tr><tr><td>Aflatoxin G2</td><td>4.03</td><td>331.2 &gt; 245.2</td><td>331.2 &gt; 257.2</td><td>50</td><td>30</td><td>0.66</td></tr><tr><td>Aflatoxin G2</td><td>4.03</td><td>331.2 &gt; 245.2</td><td>331.2 &gt; 257.2</td><td>50</td><td>30</td><td>0.66</td></tr><tr><td>Ochratoxin A</td><td>10.2</td><td>404.1 &gt; 238.9</td><td>404.1 &gt; 341.2</td><td>25</td><td>20</td><td>0.14</td></tr><tr><td>Ochratoxin A</td><td>10.2</td><td>404.1 &gt; 238.9</td><td>404.1 &gt; 341.2</td><td>25</td><td>20</td><td>0.14</td></tr></tbody></table>

<figure><img src="image_5.png" /><figcaption>**Fig. 2.** MRM chromatograms of AFG2, AFG1, AFB2, AFB1, OTA for the standard solution at 2.5 µg/L.</figcaption></figure>

the standard solution were based on the calibration curves. These values in µg/L were calculated for each day of injection by Eqs. 2 and 3.

$$ LOD(\mu g/L) = 3 \times \frac{sa_0}{a} \qquad (2) $$

where a is the slope of calibration curve and $sa_0$ is the standard deviation of y-intercept

$$ LOQ(\mu g/L) = 10 \times \frac{sa_0}{a} \qquad (3) $$

The method performance characteristics are presented in Table 2.

For aflatoxins, calibration linearity was verified between 0.5 and 20 µg/L, with a correlation coefficient (R²) of at least 0.995. This

calibration allowed for a detection limit of less than 1 µg/L (equivalent to 1 µg/kg, based on the extraction of 2.5 g of tamarind in 2 mL of acetonitrile) and a limit of quantification of 2 µg/kg, meeting European regulatory requirements (Regulation - 2023/915 - EN - EUR-Lex, s. d.). For OTA, two linearity zones were identified: 0.5–20 µg/L for low concentrations, and 20 and 200 µg/L for high concentrations. For sample analysis, both calibration ranges were used, and quantification was performed using the linear equation derived from the appropriate range, depending on the OTA concentration in each sample.

To assess repeatability, a 10 µg/L aflatoxin standard was injected three times. The coefficient of variation (CV) ranged from 1.6 % to 4 % for aflatoxins and 8.6 % for OTA. The first step to the variability of final

<table><caption>**Table 2**
Method performance characteristics.</caption><thead><tr><th>Compound</th><th>Concentration range (µg/L)</th><th>Equation</th><th>Linearity R<sup>2</sup></th><th>Limit of detection (µg/kg)</th><th>limit of quantification (µg/kg)</th></tr></thead><tbody><tr><td>Aflatoxin B1</td><td>0.5–20</td><td>Y = 174.06 x + 29.38</td><td>0.9983</td><td>0.47</td><td>1.56</td></tr><tr><td>Aflatoxin B2</td><td>0.5–20</td><td>Y = 150.47 x + 0.04</td><td>0.9995</td><td>0.25</td><td>0.82</td></tr><tr><td>Aflatoxin G1</td><td>0.5–20</td><td>y = 171.10 x – 33.70</td><td>0.9991</td><td>0.33</td><td>1.12</td></tr><tr><td>Aflatoxin G2</td><td>0.5–20</td><td>y = 66.03x + 22.28</td><td>0.9975</td><td>0.56</td><td>1.87</td></tr><tr><td>Ochratoxin A</td><td>0.5–20</td><td>Y = 53.24 x –7.71</td><td>0.9991</td><td>0.34</td><td>1.14</td></tr><tr><td>Ochratoxin A</td><td>20–200</td><td>Y = 115.90 x –997.35</td><td>0.9993</td><td></td><td></td></tr></tbody></table>
results is the variability of the chromatographic system. Detection and quantification limits were determined daily based on the injection calibration curve. Due to variations in background noise, these limits fluctuated throughout the study ranging from 0.5–2 µg/kg for detection and 1–8.5 µg/kg for quantification. These limits are close to those set by the European Commission, and exceed them in the case of OTA quantification.

Significant variation in background noise on the chromatogram was observed for OTA during the experiment, even by cleaning the column after each injection and performing daily column washing. The influence of the complex matrix is evident and has been described by Prasain (2012). All of these parameters impact the determination of mycotoxins contents.

## 3.2. Extraction method optimization

According to Du et al. (2018), microwave extraction involves rapidly heating a solvent in contact with the sample to extract the compounds of interest. In contrast, the QuEChERS method is widely used for contaminant extraction at room temperature. These techniques were compared with ultrasonic bath extraction at room temperature, using certified feedingstuff reference material as a benchmark.

The addition of salts increased extraction yields (Table 3) in both QuEChERS and ultrasonic bath extraction, even when performed at room temperature. Salts aid in the separation of organic and inorganic compounds of varying polarities in QuEChERS-based methods, as described in other studies (Han et al., 2012).

Only aflatoxins B1 and G1 were identified in the CRM, as the content of aflatoxin B2 was very low (~0.7 µg/kg). Microwave extraction, both with and without salts, produced similar results (Table 3).

Extraction efficiency was assessed by calibrating with reference standards, and the peak areas of each mycotoxin were used to calculate extraction yields. The tested extraction methods resulted in yields ranging from 80 % to 120 %, except for salt-free ultrasonic bath extraction and QuEChERS without purification for G1. Overall, salts did not significantly improve extraction efficiency. For example, microwave extraction with salts yielded 85 %, while extraction without salts resulted in 97 %, showing minimal differences in B1 extraction performance. For G1, the differences were even smaller (83 % with salts vs. 85 % without salts).

These yields were similar to those obtained with QuEChERS extraction without any specific sample treatment. Additionally, it allows for high-throughput extraction (10 positions compared to just 1 for the ultrasonic bath). Therefore, microwave extraction without salts was selected for all tamarind samples.

The repeatability of microwave extraction of the CRM without salts was evaluated, yielding to a variation coefficient of 3 % for B1 and 5 % for G1.

## 3.3. Matrix effect

The matrix effect on tamarind was evaluated by spiking a tamarind sample with 10 and 25 µg/L of AFB1, AFB2, AFG1, AFG2, and OTA.

<caption>**Table 3**</caption>

Comparison of AFB1 and AFG1 recoveries in a CRM, depending on the extraction process (microwave, ultrasonic, Quechers) with or without salts.

<table><thead><tr><th rowspan="2">Extraction mode yields (%)</th><th colspan="2">Aflatoxin yields (%)</th></tr><tr><th>Aflatoxin B1</th><th>Aflatoxin G1</th></tr></thead><tbody><tr><td>Microwave with salts</td><td>85</td><td>83</td></tr><tr><td>Microwave without salts</td><td>97</td><td>85</td></tr><tr><td>Ultrasonic bath with salts</td><td>94</td><td>93</td></tr><tr><td>Ultrasonic bath without salts</td><td>44</td><td>31</td></tr><tr><td>QuEChERS with purification</td><td>102</td><td>86</td></tr><tr><td>QuEChERS without purification</td><td>88</td><td>62</td></tr></tbody></table>

Recovery was estimated by comparing the results of the spiked sample to those of a standard prepared at the same concentration by dilution in solvent (Eq. 4). Storage of extracted samples in the freezer caused flocculation of lipids, even after filtering the samples through a 0.2 µm filter. Returning the samples at 20°C helped restore homogenization of the extract. The highly colored brown extract indicated the presence of a significant number of compounds derived from the tamarind plant, confirming the complex matrix of these extracts. The recovery rates for aflatoxins were between 83 % and 97 %, confirming the absence of a matrix effect on tamarind (Table 4). However, for OTA, the recovery was not acceptable (higher than 200 %), indicating a real matrix effect for the determination of this mycotoxin in tamarind. The results of OTA contents are only an assessment.

$$R (\%) = 100 \times \frac{\text{(spiked sample area} - \text{sample area)}}{\text{(standard area)}} \quad (4)$$

Tamarind extracts represent a complex matrix making the simultaneous analysis of five mycotoxins particularly challenging. As written by Monaci and Palmisano (2004), purification using solid phase extraction (SPE) columns or immunoaffinity column preconcentration can improve the clean-up of complex matrices. However, these methods are costly and add extra steps when analyzing a large number of samples. Some molecularly imprinted polymers (MIPs) have been developed for OTA detection in samples, offering a highly specific and more cost-effective alternative to SPE method. The importance of sample homogeneity was also highlighted, as it affects the content and recovery. Another approach described by Monaci and Palmisano (2004) involves using liquid chromatography coupled with an immunoaffinity column to enhance the specificity of the analytical method. By considering these purification and selectivity techniques, interferences could have been eliminated, the background could have been reduced and the limits of quantification improved, but at a much higher cost of analysis. Moreover, the addition of an internal standard during extraction could improve the extraction efficiency. An isotopically labeled (e.g deuterated) standard (ochratoxin A-d5 solution) would have the same behavior as OTA during extraction, the similar transition in the mass spectrometer: it would have been the most appropriate, but is expensive.

## 3.4. Characteristic performances of published analytical methods

The performance of the proposed method was compared with that of published methods, as shown in Table 5. It demonstrated a short extraction time and good repeatability. The recoveries were better than in other methods. Although the analysis time was not the shortest, it remained acceptable. Linearity was confirmed, and the LOD and LOQ complied with the requirements of European Commission regulations.

## 3.5. Mycotoxin contents in tamarind

The concentration of each mycotoxin is expressed in µg/kg of lyophilized tamarind in Table 6. The standard deviation was provided

<table><caption>**Table 4**
Recoveries for spiked tamarind extract (%).</caption><thead><tr><th>Spiked samples</th><th>Aflatoxin B1</th><th>Aflatoxin B2</th><th>Aflatoxin G1</th><th>Aflatoxin G2</th><th>Ochratoxin A</th></tr></thead><tbody><tr><td>Tamarind extract doped with 10 µg/L</td><td>97.0</td><td>94.2</td><td>91.2</td><td>95.0</td><td>260.9</td></tr><tr><td>Tamarind extract doped with 25 µg/L</td><td>89.6</td><td>92.3</td><td>83.2</td><td>97.4</td><td>262.0</td></tr></tbody></table>
<table><caption>**Table 5**

Characteristic performances of extraction and analytical methods.</caption><thead><tr><th rowspan="2">Authors</th><th rowspan="2">Matrix</th><th colspan="2">Extraction</th><th colspan="6">Analysis by LC-MS/MS</th></tr><tr><th>Method and time</th><th>Repeatability %</th><th>Analysis time (min)</th><th>Linear ranges(µg/L)</th><th>R<sup>2</sup></th><th>LOD (µg/Kg)</th><th>LOQ(µg/Kg)</th><th>Recovery (%)</th></tr></thead><tbody><tr><td>Proposed method</td><td>Tamarind</td><td>Microwave extraction (10 min)+ Concentration by evaporation</td><td>Between 3 % and 5 %</td><td>16</td><td>0.5-20</td><td>&gt; 0.9970</td><td>AF B1: 0.47AFB2: 0.25AFG1: 0.33AFG2: 0.56OTA: 0.34</td><td>AF B1: 1.56AFB2: 0.82AFG1: 1.12AFG2: 1.87OTA: 1.14</td><td>83-97Except OTA</td></tr><tr><td>(Han et al., 2012)</td><td>Rhizomes, roots, seeds, grasses and leaves, flowers</td><td>ASE method(15 min)+ Concentration by rotatory evaporator+ Purification on SPE Cartridge</td><td>&lt; 16.7</td><td>17</td><td>0.2-600</td><td>&gt; 0.9990</td><td>AF B: 0.01AFB2: 0.01AFG1: 0.04AFG2: 0.04OTA: 0.03</td><td>AF B1: 0.02 AFB2: 0.02AFG1: 0.05AFG2: 0.06OTA: 0.04</td><td>71.4-119.3</td></tr><tr><td>(Du et al., 2018)</td><td>Peach seed, milk powder, corn flour</td><td>Microwave extraction (10 min)+ SPE process with nanoparticles</td><td>around 2.17</td><td>9</td><td>0.034-2.54</td><td>&gt; 0.9982</td><td>AF B1: 0.024OTA: 0.022</td><td>AFB1: 0.073OTA: 0.066</td><td>84-105</td></tr><tr><td>(Hidalgo-Ruiz et al., 2019)</td><td>Nuts</td><td>QueEChERS method42 min</td><td>&lt; 16.3</td><td>7.5 min</td><td>5-50</td><td>undefined</td><td>undefined</td><td>AFB1: 0.5AFB2: 0.5AFG1: 0.5AFG2: 0.5</td><td>70-117.5 for AF</td></tr><tr><td>(Cunha et al., 2018)</td><td>Nuts</td><td>QueEChERS method (42 min)+ evaporation under nitrogen</td><td>&lt; 14.0</td><td>25 min</td><td>1-10</td><td>Between 0.9787 and 0.9942</td><td>AF B: 0.4AFB2: 0.4AFG1: 0.5AFG2: 0.5OTA: 2</td><td>AF B: 1.25AFB2: 1.25AFG1: 1.25AFG2: 1.25OTA: 5</td><td>56-102</td></tr></tbody></table>

when all three extractions from a sample were valid. Note that the average moisture content of tamarind was around 10 %.

Finally, no aflatoxins were quantified as all measured values were below the established limits of quantification for this method determined in Table 2.

However, for OTA, 15 samples (37.5 % of samples) contained quantifiable levels ranging from 3 to 13.3 µg/kg in lyophilized tamarind, while one sample from the Abobo market (2.5 % of samples) had a significantly higher concentration of 46.9 µg/kg. OTA was also found in other dried fruits in Europe, Africa and South of America. For example in Cameroun, OTA was found in cocoa beans after drying in 84 % the cases studied (Ghomfo et al., 2024) and in italian cocoa derived products (Monaci and Palmisano, 2004) the levels (around 0.5 µg/kg) remained below the European Union's maximum allowable limit of 2 µg/kg. According to Cunha et al. (2018), OTA levels in nuts from Portugal range from 5.3 to 10.9 µg/kg for 25-50 % of the samples analysed. Another study (Monaci and Palmisano, 2004) added that uruguayan cereals such as rice, barley, maize and coffee contain OTA levels of up to 50 µg/kg. Vartolomei et al. (2014) had also developed the liquid chromatographic-tandem mass spectrometry (LC-MS/MS) method for quantification of ochratoxin A (OTA) in foods such as bread, dry fruit, breakfast cereals, spices, beer, cereals, and seeds. They concluded that this method is simple and efficient, and can be applied in the monitoring of food safety and assessment of exposure to OTA through food.

Another study on ochratoxin A contamination in cocoa beans found that rapid treatment and proper fermentation led to low OTA levels in safe beans (Kedjebo et al., 2014). Since no specific regulations exist in Côte d'Ivoire, the results are discussed in reference to the Official Journal of the European Union (Regulation - 2023/915 - EN - EUR-Lex, s. d.). According to the Commission Regulation, the maximum allowable level of 5 µg/kg of aflatoxin AFB1 in dried fruits placed on the market, with a sum of AFB1, AFB2, AFG1 and AFG2 of 10 µg/kg. Even if the limit of quantification vary slightly for aflatoxins, the level of 10 µg/kg was never exceeded. For OTA, the Commission Regulation sets a maximum limit of 2 µg/kg in dried fruits. The method used for the identification and quantification of aflatoxin and ochratoxin A in tamarind complies with this regulation, as the limits of quantification were equal to or

lower than the maximum permissible levels. In a study by Kouadio et al. (2014) on the assessment of mycotoxin exposure in Côte d'Ivoire OTA was found in 13 % of maize flour samples analyzed. Their study provided data on mycotoxin risk in Côte d'Ivoire, revealing a frequent co-exposure to the major mycotoxins such as AFS, OTA and fumonisins, which appeared to be related to the frequency of peanuts, maize, millet and attiéké consumption. Other authors had also found that Aflatoxin B1 (AFB1) and ochratoxin (OTA) were present in maize, millet, sorghum, soya and multigrain (mix of different cereals) flour samples collected from different localities of Côte d'Ivoire and analyzed (N'zi et al., 2023). AFB1 was detected in 100 % of maize (range 1.2-120.5 µg/kg; mean: 44.2 µg/kg) and 50-83.3 % of millet flours (range 0.2-31.5 µg/kg; mean: 31.5 µg/kg). Its level in all maize and rice flour samples exceeded EU standard (0.1 µg/kg). For OTA and fumonisins, millet and maize flours showed the highest levels of sample exceeding the EU standard. Thus, these authors concluded that artisanal infant flours marketed in Côte d'Ivoire, mainly maize and rice flours, represented potential health risks for children. In this study, 16 of the 40 samples (40 %) of freeze-dried tamarind analyzed contained more than 3 µg/kg of ochratoxin A, a level that makes tamarind unsafe to eat. However, it was proven in this study that OTA values may be overestimated due to matrix effects, justifying the need for internal standards or matrix-matched calibration in future studies. The potential OTA contamination warrants risk assessment and improved storage methods. Interestingly, the absence of aflatoxins in our tamarind samples may be influenced by the natural interactions among fungi present in the environment or on the fruit itself. Research has shown that some fungi, like *Trichoderma harzianum*, *Trichoderma viride*, and even non-toxigenic strains of *Aspergillus flavus*, can strongly inhibit the growth and toxin production of toxigenic A. flavus strains (Mostafa et al., 2013). These fungi can act through direct parasitism or by releasing enzymes and volatile compounds that disrupt aflatoxin biosynthesis. Such biological competition may help explain why aflatoxins were not detected, despite conditions that favor mold growth and the presence of other mycotoxins like ochratoxin A (Mostafa et al., 2013). This natural fungal antagonism highlights the complexity of microbial communities on tamarind and suggests potential avenues for biological control to reduce mycotoxin contamination.
<table><caption>**Table 6**
Contents of mycotoxins in lyophilized tamarind.</caption><thead><tr><th rowspan="2">Sample Code</th><th rowspan="2">Origin</th><th colspan="5">Mycotoxin contents (in µg/kg)</th></tr><tr><th>Aflatoxin B1</th><th>Aflatoxin B2</th><th>Aflatoxin G1</th><th>Aflatoxin G2</th><th>Ochratoxin A</th></tr></thead><tbody><tr><td>TAM 1</td><td>Abobo</td><td>&lt; 1</td><td>&lt; 1</td><td>&lt; 1.5</td><td>&lt; 1</td><td><b>3.2</b></td></tr><tr><td>TAM 2</td><td>Abobo</td><td>&lt; 1</td><td>&lt; 1</td><td>&lt; 1.5</td><td>&lt; 1</td><td>&lt; 1</td></tr><tr><td>TAM 3</td><td>Abobo</td><td>&lt; 1</td><td>&lt; 1</td><td>&lt; 1.5</td><td>&lt; 1</td><td>&lt; 1</td></tr><tr><td>TAM 4</td><td>Abobo</td><td>&lt; 1</td><td>&lt; 1</td><td>&lt; 1.5</td><td>&lt; 1</td><td>2.0</td></tr><tr><td>TAM 5</td><td>Abobo</td><td>&lt; 1</td><td>&lt; 1</td><td>&lt; 1.5</td><td>&lt; 1</td><td>2.1 ± 1.4</td></tr><tr><td>TAM 6</td><td>Abobo</td><td>&lt; 1</td><td>&lt; 1</td><td>&lt; 1.5</td><td>&lt; 1</td><td>2.4 ± 1.4</td></tr><tr><td>TAM 7</td><td>Abobo</td><td>&lt; 1</td><td>&lt; 1</td><td>&lt; 1.5</td><td>&lt; 1</td><td>&lt; 1</td></tr><tr><td>TAM 8</td><td>Abobo</td><td>&lt; 1</td><td>&lt; 1</td><td>&lt; 1.5</td><td>&lt; 1</td><td><b>3.9</b></td></tr><tr><td>TAM 9</td><td>Abobo</td><td>&lt; 1</td><td>&lt; 1</td><td>&lt; 1.5</td><td>&lt; 1</td><td>&lt; 1</td></tr><tr><td>TAM 10</td><td>Abobo</td><td>&lt; 0.5</td><td>&lt; 1</td><td>&lt; 1</td><td>&lt; 2</td><td><b>13.6 ± 8.5</b></td></tr><tr><td>TAM 11</td><td>Abobo</td><td>&lt; 0.5</td><td>&lt; 1</td><td>&lt; 1</td><td>&lt; 2</td><td>&lt; 3</td></tr><tr><td>TAM 12</td><td>Koumassi</td><td>&lt; 0.5</td><td>&lt; 1</td><td>&lt; 1</td><td>&lt; 2</td><td>&lt; 3</td></tr><tr><td>TAM 13</td><td>Koumassi</td><td>&lt; 0.5</td><td>&lt; 1</td><td>&lt; 1</td><td>&lt; 2</td><td><b>5.5</b></td></tr><tr><td>TAM 14</td><td>Koumassi</td><td>&lt; 0.5</td><td>&lt; 1</td><td>&lt; 1</td><td>&lt; 2</td><td>&lt; 3</td></tr><tr><td>TAM 15</td><td>Koumassi</td><td>&lt; 0.5</td><td>&lt; 1</td><td>&lt; 1</td><td>&lt; 2</td><td><b>4.1</b></td></tr><tr><td>TAM 16</td><td>Adjamé</td><td>&lt; 0.5</td><td>&lt; 1</td><td>&lt; 1</td><td>&lt; 2</td><td>&lt; 3</td></tr><tr><td>TAM 17</td><td>Adjamé</td><td>&lt; 0.5</td><td>&lt; 1</td><td>&lt; 1</td><td>&lt; 2</td><td>&lt; 3</td></tr><tr><td>TAM 18</td><td>Adjamé</td><td>&lt; 0.5</td><td>&lt; 1</td><td>&lt; 1</td><td>&lt; 2</td><td>&lt; 3</td></tr><tr><td>TAM 19</td><td>Koumassi</td><td>&lt; 0.5</td><td>&lt; 1</td><td>&lt; 1</td><td>&lt; 2</td><td>&lt; 3</td></tr><tr><td>TAM 20</td><td>Abobo</td><td>&lt; 0.5</td><td>&lt; 1</td><td>&lt; 1</td><td>&lt; 2</td><td><b>47.4 ± 8.0</b></td></tr><tr><td>TAM 21</td><td>Koumassi</td><td>&lt; 2</td><td>&lt; 3</td><td>&lt; 2</td><td>&lt; 2</td><td>&lt; 1</td></tr><tr><td>TAM 22</td><td>Koumassi</td><td>&lt; 2</td><td>&lt; 3</td><td>&lt; 2</td><td>&lt; 2</td><td>&lt; 1</td></tr><tr><td>TAM 23</td><td>Koumassi</td><td>&lt; 2</td><td>&lt; 3</td><td>&lt; 2</td><td>&lt; 2</td><td>&lt; 1</td></tr><tr><td>TAM 24</td><td>Abobo</td><td>&lt; 2</td><td>&lt; 3</td><td>&lt; 2</td><td>&lt; 2</td><td>&lt; 1</td></tr><tr><td>TAM 25</td><td>Abobo</td><td>&lt; 2</td><td>&lt; 3</td><td>&lt; 2</td><td>&lt; 2</td><td>&lt; 1</td></tr><tr><td>TAM 26</td><td>Adjamé</td><td>&lt; 2</td><td>&lt; 3</td><td>&lt; 2</td><td>&lt; 2</td><td>&lt; 1</td></tr><tr><td>TAM 27</td><td>Adjamé</td><td>&lt; 2</td><td>&lt; 3</td><td>&lt; 2</td><td>&lt; 2</td><td>&lt; 1</td></tr><tr><td>TAM 28</td><td>Adjamé</td><td>&lt; 2</td><td>&lt; 3</td><td>&lt; 2</td><td>&lt; 2</td><td>2.0</td></tr><tr><td>TAM 29</td><td>Koumassi</td><td>&lt; 2</td><td>&lt; 3</td><td>&lt; 2</td><td>&lt; 2</td><td>&lt; 1</td></tr><tr><td>TAM 30</td><td>Koumassi</td><td>&lt; 2</td><td>&lt; 3</td><td>&lt; 2</td><td>&lt; 2</td><td>&lt; 1</td></tr><tr><td>TAM 31</td><td>Koumassi</td><td>&lt; 2</td><td>&lt; 1</td><td>&lt; 1</td><td>&lt; 2</td><td><b>&lt; 8.5</b></td></tr><tr><td>TAM 32</td><td>Koumassi</td><td>&lt; 2</td><td>&lt; 1</td><td>&lt; 1</td><td>&lt; 2</td><td><b>&lt; 8.5</b></td></tr><tr><td>TAM 33</td><td>Koumassi</td><td>&lt; 2</td><td>&lt; 1</td><td>&lt; 1</td><td>&lt; 2</td><td><b>&lt; 8.5</b></td></tr><tr><td>TAM 34</td><td>Koumassi</td><td>&lt; 2</td><td>&lt; 1</td><td>&lt; 1</td><td>&lt; 2</td><td><b>&lt; 8.5</b></td></tr><tr><td>TAM 35</td><td>Koumassi</td><td>&lt; 2</td><td>&lt; 1</td><td>&lt; 1</td><td>&lt; 2</td><td><b>&lt; 8.5</b></td></tr><tr><td>TAM 36</td><td>Koumassi</td><td>&lt; 2</td><td>&lt; 1</td><td>&lt; 1</td><td>&lt; 2</td><td><b>&lt; 8.5</b></td></tr><tr><td>TAM 37</td><td>Koumassi</td><td>&lt; 1</td><td>&lt; 1</td><td>&lt; 0.5</td><td>&lt; 2</td><td><b>8.3 ± 0.7</b></td></tr><tr><td>TAM 38</td><td>Koumassi</td><td>&lt; 1</td><td>&lt; 1</td><td>&lt; 0.5</td><td>&lt; 2</td><td><b>10.8 ± 6.3</b></td></tr><tr><td>TAM 39</td><td>Koumassi</td><td>&lt; 1</td><td>&lt; 1</td><td>&lt; 0.5</td><td>&lt; 2</td><td><b>8.6</b></td></tr><tr><td>TAM 40</td><td>Koumassi</td><td>&lt; 1</td><td>&lt; 1</td><td>&lt; 0.5</td><td>&lt; 2</td><td><b>12.6 ± 7.7</b></td></tr></tbody></table>

Note: Values in bold are above the European norm. OTA values may be overestimated due to matrix effects, for more information see matrix effect section.

## 4. Conclusion

An extraction method based on microwave extraction and a LC-MS/ MS method were developed for the simultaneous screening of four aflatoxins and ochratoxin A in 40 tamarind samples collected from 3 different markets in Côte d'Ivoire. This study confirmed the absence of aflatoxins in tamarind; however, some samples contained ochratoxin A at high levels, significantly exceeding the 2 µg/kg limit set by European Commission regulations. These results highlight the potential health risks associated with tamarind consumption, particularly in the absence of local regulations and systematic monitoring. The complex composition of tamarind (rich in proteins, lipids, and sugars) presents challenges for accurate analysis. Indeed, matrix effects for OTA were observed and can lead to overestimation in tamarind samples quantification. This can be mitigated through the use of purification techniques before LC-MS/ MS analysis, by adding internal standards or by using matrix-matched calibration to enhance accuracy and reliability. Moreover, the development of a tamarind-specific certified reference material containing OTA would be suitable to improve the accuracy of the extraction and LC-MS/MS method. Other studies should be done in a larger dataset with samples coming from different markets to better evaluate the potential OTA contamination. Beyond this challenge, our findings underline the need for action at the production and post-harvest levels to reduce contamination. Producers should dry tamarind pulp on clean, non-absorbent surfaces such as plastic sheets or metal trays, away from

direct contact with soil, and under shade or controlled sunlight to avoid excessive humidity. Drying should ideally last 2–3 days, depending on weather conditions. After drying, tamarind should be stored in sealed containers like plastic barrels or hermetic bags to protect it from moisture and insects. Regular checks during storage could help detect early mold development. In addition, awareness-raising among producers and sellers about good handling practices is essential. Local authorities should consider establishing national limits for OTA in tamarind and implementing regular monitoring programs to protect consumers.

### CRediT authorship contribution statement

**Mélanie Mignot:** Writing – review & editing, Validation, Supervision, Conceptualization. **Solange Aka-Gbezo:** Writing – review & editing, Investigation, Conceptualization. **Balla Ballo:** Resources. **Christine Devouge-Boyer:** Writing – original draft, Validation, Methodology, Investigation. **Tiémélé Laurent-Simon Amoikon:** Investigation, Conceptualization. **Mathilde Lauzent:** Writing – review & editing, Resources. **Juliette Vievard:** Writing – review & editing, Validation, Methodology. **Leonie Moyon:** Writing – review & editing, Validation, Methodology.

### Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence
the work reported in this paper.

## Acknowledgements

This work was partially supported by Université de Rouen Normandie (URN), Normandie Université (NU), INSA Rouen Normandie, The Région Normandie, The Centre National de la Recherche Scientifique (CNRS), Labex SynOrg (ANR-11-LABX-0029), the graduate school for research XL-Chem (ANR-18-EURE-0020 XL CHEM), European Regional Development Fund (ERDF), and Innovation Chimie Carnot (I2C). The LC-MS/MS instrument was funded by project 2017NU01FOBC02 CBSB-14. Access to the CNRS research infrastructure Infranalytics (FR2054) is gratefully acknowledged. The collection of tamarind samples was carried out by members of the Biotechnology and Food Microbiology Laboratory, Department of Food Science and Technology, Nangui Abrogoua University, 02 BP 801 Abidjan 02, Côte d'Ivoire.

## Data availability

Data will be made available on request.

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