Ochratoxin Determination in Food Samples by Fabric Phase Sorptive Extraction Coupled with High- Performance Liquid Chromatography Technique

doi:10.21203/rs.3.rs-1478781/v1

The sample preparation step is very useful in results precision and accuracy. Achievement to a quickly performed, inexpensive, sensitive, and accurate analytical approach for measuring extremely small amounts of a dangerous compound such as mycotoxin, in addition to environmental issues, is always required and important. In this paper, fabric phase sorptive extraction (FPSE), a novel sorbent-based microextraction method, was used for the first time to obtain a basic and quick ochratoxin A extraction from cereals and legumes. To achieve maximum efficiency, key experimental factors in FPSE process were optimized. At the extraction stage, volume and type of solvent, time of extraction and salt effect, and in the desorption step, volume and pH of back-extraction solvent and time of desorption were evaluated. Extracted ochratoxin A was analyzed by HPLC-FLD method. The chromatographic separation was performed using mobile phase H₂O: ACN: acetic acid (49:49:2, v/v/v) at a flow rate of 1.0 mL/min on C18 column with fluorescence detection (λ_ex = 333 nm and λ_em = 447 nm). Limit of detection was found to be 0.49 ng/mL, whereas absolute acceptable recoveries (76.34-112.96%) with low relative standard deviations within day and between-day precision were (≤ 9.2% and ≤ 11.0, respectively) achieved.

Ochratoxin A

Fabric phase sorptive extraction

Sample preparation

High-performance liquid chromatography

Mycotoxins are secondary fungal metabolites, and nowadays, among fungal toxins, ochratoxins are very at the center of attention (Atanda et al. 2011). Various agricultural products can contain this mycotoxin, such as coffee and cocoa beans, soybeans, and especially cereals and legumes (Swanson 2011). Based on the chemical structure, ochratoxins are classified into different types of A, B, C, α, and 4-hydroxyochratoxin (Abrunhosa et al. 2010). Among them, ochratoxin A is the most toxic (Nogaim et al. 2020). Ochratoxin A is a relatively heat-resistant compound transmitted to the human body through the food chain (Ráduly et al. 2020). The most toxic effects of ochratoxin A are kidney toxicity (Bui-Klimke and Wu 2015), carcinogenicity (Organization and Cancer 1993), nephrotoxic, hepatotoxic, neurotoxic, immune toxic, and also teratogenic agents (Bostan et al. 2017). Therefore, according to the World Health Organization's motto, “healthy food is for everyone,” the responsibility of health authorities is very high.

There are many sample preparation methods used normally include solid-phase extraction (SPE) (Jahed et al. 2020; Sitterley et al. 2020; Yang et al. 2020), solid-phase microextraction (SPME) (Trujillo-Rodríguez and Anderson 2019; Zhang et al. 2018), liquid-liquid microextraction (LLME) (Arpa et al. 2018; Shishov et al. 2020) and stir bar sorptive extraction (SBSE) (Atoi et al. 2019; Khoshkbar et al. 2020; Locatelli et al. 2019b). These preparation approaches display various disadvantages, such as high consumption content of organic solvents in the conditioning as well as elution step (Kozlík et al. 2011; Zhou et al. 2013), small sorbent loading, poor sample capacity (Kabir et al. 2013), considerable cost, instability, and the fiber swelling in organic solvents (Nerín et al. 2009). Thus, in the current work, ochratoxin A pretreatment was prepared in a new method, fabric phase sorptive extraction (FPSE), which has recently been addressed by Kabir and Furton (Kabir and Furton 2013). In this technique, a fabric (polyester, fiberglass, or cellulose) is selected as the substrate, and a sol-gel sorbent is coated on its surface. An efficient extraction medium is provided by the strong covalent bond between the sol-gel and the fabric substrate. It helps the FPSE media expose to any extreme chemical condition (pH, 1–13) or organic solvent without chemical/structural integrity of the microextraction device being compromised. This method has successfully addressed two main drawbacks of all sorptive extraction techniques, including longer sample preparation time and low sample capacity (Kabir and Furton 2020).

FPSE has been used to analyze a broad spectrum of analytes, for instance, non-steroidal anti-inflammatory medicines (Tartaglia et al. 2020), triazine herbicides (Gülle et al. 2019; Ulusoy et al. 2020) or emerging contaminants (Agadellis et al. 2020; Roldán-Pijuán et al. 2015), amphenicols (Samanidou et al. 2015), androgens and progestogens (Guedes-Alonso et al. 2016), residual sulphonamides (Karageorgou et al. 2016), benzodiazepines (Samanidou et al. 2016), alkyl phenols (Kumar et al. 2015), PAHs (Gazioglu et al. 2021a; Gazioglu et al. 2021b), and UV stabilizers or plastic additives (Locatelli et al. 2019a; Montesdeoca-Esponda et al. 2015) found in various matrices, for instance, water, milk, and biological liquids and samples (Kabir et al. 2018; Locatelli et al. 2020; Locatelli et al. 2021; Locatelli et al. 2018) have recently been issued.

The current study aimed to explicate an FPSE- HPLC- FLD technique for the ochratoxin A quick isolation in different foodstuff, including cereals and legumes, through an optimized method for sample preparation procedure. Finally, along with considering the principles of green chemistry, we reached a fast, simplified, and practical approach for ochratoxin A analysis.

Materials and reagents

FPSE media substrates were obtained from Ardakan textile company (Yazd, Iran). Poly-tetrahydrofuran, methyltrimethoxysilane (MTMS), acetone, dichloromethane, sodium hydroxide, hydrochloric acid, HPLC grade acetonitrile, and methanol were purchsed from Merck (Darmstadt, Germany). Trifluoroacetic acid (TFA) and glacial acetic acid were obtained from Carlo Erba (Milan, Italy) and were used without further purification. Ochratoxin A powder was prepared from Sigma (St. Louis, USA). In all the experiments, double distilled water was used.

Instrumentation and chromatographic conditions

Different solutions centrifugation was performed to achieve solutions free of particles in an ortoalresa Centrifuge Model digicen 21(ortoalresa, Madrid, spain). A Fisher Scientific Digital Vortex Mixer (Fisher Scientific, USA) was used to mix different solutions thoroughly. A 2510 BRANSON Ultrasonic Cleaner (Branson Inc., USA) was used to provide sol solution free of bubbles. Deionized water (18.2 M) was obtained by a homemade deionized water system (Iran) for sol-gel synthesis. An AVATAR FT-IR spectrometer equipped with a universal ATR sampling accessory (Thermo, USA) was applied to carry out FT-IR characterization of fabric substrates, and sol-gel coated FPSE media. An LMU TESCAN BRNO- Mira3 Field Emission Scanning Electron Microscope (Oxford Instrument, England) equipped with an EDAX detector was employed to obtain SEM images of coated and uncoated FPSE media. HPLC analyses were performed on a Waters Breeze (Waters, Milford, MA, USA), HPLC system equipped with a binary HPLC pump (Waters 1525), with attached20 µL Rheodyne sample injector and a fluorescence detector with 333 nm excitation and 443 nm emission wavelength. The reverse-phase column was a Waters Nova-pak® C₁₈analytical column (250×4.6 mm), the temperature was adjusted to 30˚C. The mobile and flow rate was acetonitrile: water: acetic acid (49:49:2, v/v/v) and 1 mL/min, respectively. Analyzing and collecting the data as well as controlling the system operation was performed by Breeze software.

Standards preparations

Its solid powder was dissolved in methanol to prepare ochratoxin A stock standard solution (1000 ng/mL). The stock standard solutions were kept at -20 °C and working standard solutions at 4°C. Spectrophotometrically check was performed to assess the stock solution stability. Shortly before use, all working standard solutions were diluted and prepared.

Sample preparation

Samples were purchased from Mashhad (Khorasan, Iran) and tested for no contamination. A required specimen size of 1,000 g was applied, then they underwent 10 min mixing; 500 g of each sample was crushed into a fine powder with the resulting particle size of below 0.3 mm and stored at -20 ºC for later analysis. For ochratoxin A determination, 5 g ground samples, spiked with a determined volume of ochratoxin A stock standard solution and stored for 30 min in the dark. After this time, the sample was mixed with 20 mL methanol: water (50: 50, v/v) containing 5% (w/v) NaCl salt and stirred for 30 min. Once mixing was performed, we used a filter paper to filter (Whatman No. 4) the slurry, and 10 mL of this mixture were used for the next step in the fabric phase sorptive extraction method.

Preparation of sol-gel polytetrahydrofuran (Poly-THF) coated FPSE media

Fabrics pretreatment for sol-gel coating

One critical step to prepare the sol-gel sorbent coating is the selection and pretreatment of fabric (Kabir and Furton 2020). Due to the nature and polarity of the extracted target analyte, cotton fabric (100% cellulose) was chosen for sol-gel sorbent coating as the substrate. A significant disadvantage of commercial fabric is that they contain some finishing materials to impart glossiness and keep the fiber from collecting dust particles. To optimize the sorbent loading capacity on the fabric substrate, we need to remove these residuals and make the surface hydroxyl groups active before the sol-gel sorbent coating on the fabric surface. A fabric pretreatment was necessary for cleaning it and activating hydroxyl groups. This process has been described elsewhere in detail (Kabir et al. 2017; Kumar et al. 2014).

Sol solutions preparation for coating on the substrate and the matrix surface

The next step is to maximize the selectivity of the sol solution toward the target analyte found in the sample matrices. The nature of the sample matrix and target analyte determine how the sol solution needs to be designed. The selection of the organic polymer and sol-gel precursor has gained special attention. Selecting a proper polymer is vital for a better sorbent design ,thus, the type of organic polymer is highly important in the final selectionof the FPSE membrane (Kabir and Furton 2020). To coated 100% cotton cellulose fabric, a sol solution was prepared, using poly-THF as the polymer, methyl tri methoxy silane (MTMS) as the sol-gel precursor, trifluoro acetic acid (containing 5% water) as the sol–gel catalyst, and methylene chloride: acetone (50:50, v/v) as the organic solvent.

The sol-gel coating and sol solution preparation process was described in detail elsewhere (Kumar et al. 2014). After the synthesis of the sol solution, the pretreated clean fabric was carefully put into it and kept in the reaction bottle for four h. Since the solvent has to be evaporated and the sol-gel coating needs to aging, after removing the substrate from the solution, it was kept inside the desiccator overnight. Finally, methylene chloride: acetone (50:50, v/v) mixture was used to rinse the FPSE membrane and sonicated foe 30 min. Then, it was air-dried for 1 h and kept inside an airtight packaging for later use.

The substrate and sol-gel poly-THF coating chemistry

There are three building blocks for fabric phase sorptive extraction membranes : (1) a sol-gel active inorganic or organic polymer; (2) a fabric substrate; (3) a sol-gel inorganic precursor/organically modified inorganic precursor (Kabir and Furton 2020). The FPSE membrane is mainly built by Fabric substrate. The general selectivity of the FPSE membrane is affected by the substrate through hydrophilic/hydrophobic interactions (Lioupi et al. 2019). Since cellulose fabric has abundant sol-gel active functional groups compared to other fabric, there is a great deal of sorbent loading per unit on cellulose fabric (Kabir and Furton 2020). Due to the medium polar nature of the selected target analyte, hydrophilic cotton cellulose seems to be a suitable substrate. Inorganic sol-gel precursor establishes sol-gel sorbent in 3D networks and exhibits a linking role to bind the networks with the fabric substrate. So it can be said that it is central to the sol-gel sorbent coating process (Celeiro et al. 2020). The selectivity and extraction affinity regarding the target analyte directly depend on sol-gel active organic polymers. Due to the structure of polymer used in the sol-gel coating reaction, various intermolecular interactions were created (Lioupi et al. 2019). As the selected analyte possesses medium polarity, we chose polytetrahydrofuran (poly-THF), a medium polar polymer containing repeating units of tetramethylene oxide and terminal hydroxyl groups the organic polymer. Moreover, trifluoroacetic acid (with 5% v/v water), methylenechloride:acetone (50/50, v/v), and methyltrimethoxysilane (MTMS) were utilized as sol–gel catalyst, solvent system and the inorganic precursor, respectively.

Hydroxyl groups are formed as a result of the three-methoxy groups in MTMS being hydrolyzed during catalytic hydrolysis and start creating a 3D network through polycondensation. Sol-gel active organic polymer incorporates in into the network randomly when the 3D network is forming. Eventually, as a result of polycondensation, there is a chemical bond between the sol-gel sorbent network and the fabric substrate via hydroxyl groups of the fabric (Kabir and Furton 2020). As the sol-gel sorbent is very porous and the fabric substrate is so permeable, the aqueous sample can travel through the FPSE membrane countlessly during the process of extraction and guarantees rather full extraction of the target compound within a reasonably short time (Alampanos et al. 2019).

Characterization of sol-gel PTHF coated FPSE media

Scanning electron microscopy

The characterization of sol-gel sorbent-coated fabric phase sorptive extraction membranes is often done in different ways one of which is scanning electron microscopy (SEM). SEM image reflects important morphological properties of the fabric substrate surface. To compare the surface morphology before and after the coating process, SEM was used. Fig. 1 shows SEM micrographs of (a) the surface of uncoated cellulose fabric substrate; and (b) sol-gel poly-THF coated fabric phase sorptive extraction media. An SEM image reveals numerous micro-fibrils as well as bundles of micro-fibrils integrated to possess well-structured macropores even after sol-gel PTHF coating. These macropores cause faster and near exhaustive extraction kinetic which allow the sample matrix easily flow as the analyte extraction is performing. The SEM images show homogeneous and sponge-like porous of thin PTHF sorbent coating around the cellulose micro-fibrils that provides quicker permeation of the sample matrix which contains the target analyte and eventuallyleads to shortening the extraction time of equilibrium (Celeiro et al. 2020).

Insert Fig. 1

FT-IR spectroscopy

FT-IR spectra provide information on how various sol solution components chemically integrate into the sol-gel sorbent network and the fabric substrate. Fig. 2 shows two FT-IR spectra; uncoated cellulose substrate (a) and sol-gel poly-THF coated FPSE media (b). Characteristic absorption in two FT-IR spectra appears in three peak areas between 3000 and 3400 cm^-1; 1307 and 2900 cm^-1; and at 1090 cm^-1correspond to O–H, C–H, and C–O bending vibration. The indication of a transparent band in the FT-IR spectrum at 1096cm⁻¹ is that Si–O–C bonds could have established from the successfully incorporated sol-gel poly-THF network into the cellulose substrate. Also, regarding the systematic reduction in the absorption bands between 3000 and 3400 cm^-1in FT-IR spectrum of sol-gel coated FPSE media correspond to O–H bending vibrations, it can be said that this reduction is due to the involvement of hydroxyl groups of cellulose substrate to the sol-gel network (Montarsolo et al. 2013). There is no other clear signal of sol-gel poly THF coating as absorption bands overlaps with those of uncoated cellulose fabric. The sharpest peak in FT-IR spectra of sol-gel coated FPSE membrane compared with cellulose substrate shows the uniform coverage of sol-gel around the cellulose substrate that can be seen in the SEM image.

Insert Fig. 2

FPSE procedure

The fabric phase sorptive extraction protocol involves three steps: a) cleaning of FPSE membrane, b) extraction, c) back extraction (Kabir and Furton 2020). Before the extraction, the FPSE membrane was conditioned for its use. This work is done by immersing the FPSE membrane into 2 mL of methanol: acetonitrile (50:50, v/v) for 5 min, and subsequently in 2 mL of ultrapure water for 3 min, to remove any undesirable impurities and the remains of organic solvents. For extraction or the sorption of the target analyte by the sorbent, a clean FPSE membrane was drowned in 5 mL of the representative aliquot of the sample containing five ng/mL ochratoxin A, the vial was sealed, and then the mixture solution was magnetic stirring for1 h so that the analyte can be thoroughly absorbed into it. Once absorption was completed, the FPSE membrane was taken from the vial and air-dried on a watch glass at room temperature. Subsequently, the back extraction solvent containing 5 mL of acetonitrile/water/acetic acid (49: 49: 2, v/v/v) solution was used for one h in contact the substrate. The collected analyte was eventually centrifuged in a microtube at 13000 rpm for 5 min to gain solution free from particulate matter. After centrifuge and before injection into the HPLC system, a syringe filter (0.45 µm) was used to filter the solution.

After optimizing the experimental parameters, the final FPSE conditions were set using a 5 cm² (2.0 × 2.5 cm) FPSE device with PTHF based sol-gel coating for the extraction of 10 mL of methanol: water (50:50, v/v) containing 5% NaCl salt, for 40 min. Analyte desorption was reached with 1 mL of back extraction solution at pH= 6 for 15 min under sonication.

Optimization of efficiency FPSE conditions

Some key variables were studied to maximize extraction performance of analyte, including (a) extraction solvent solution, (b) effect of addition of salt into the extraction solvent solution, (c) volume of desorption/back extraction solvent, (d) pH of desorption solvent, (e) time of desorption, (f) time of extraction and, (g) volume of extraction solvent.

Extraction solvent solution effect

Different solvent mixtures were tried to optimize the analyte's sorption from the sample matrix on the FPSE media. According to the obtained results (Fig 3), the methanol: water (50:50, v/v) mixture exhibits better performance than other mixtures. This is because the selected target analyte is medium polar. Therefore, due to the polarity, it demonstrated higher solubility in the same ratio of methanol: water in comparison with other solutions. In addition, probably this solution concerning the FPSE membrane's polarity can provide better conditions to help the target analyte stay in the proper state of polarity and ionic state (pk_a) for effective adsorption on the FPSE media. The other reason for better efficiency of this solution as the sorption solution can be explained in this way that, methanol: water (50:50, v/v) mixture has better performance to raise the activity and ability of the sol-gel network of FPSE membrane for sorption the target analyte from the sample matrix in comparison with other solution. In this step, the volume of solvent in all samples was 5 mL, and the volume of the sorption solvent was optimized in further experimentation.

Insert Fig. 3

Ionic strength Effect

For the evaluation of adding sodium chloride salt effect on the adsorption attributes of ochratoxin A, several sodium chloride concentrations, including 0%, 5%, 10%, 15%, 20%, and 30% (w/v), were added to the extraction solvent solution to study their impact on the analyte adsorption on the FPSE membrance. An increase in sodium chloride salt concentration from 0% to 5% caused to higher extraction efficiency. Nevertheless, as NaCl concentration reached 30%, the extraction efficiency experienced a decline. Therefore, the addition of 5% of NaCl was considered as a suitable alternative to be examined in further works. The observed difference in extraction efficiencies is probably due to the contradictory effects displayed as salt was added to the solution. On the one hand, the analyte gradually loses its solubility in the aqueous solution as the ionic strength improves, and a salting-out effect is produced, which makes the target analyte more available in the sorption media. it is noteworthy that viscosity increases when NaCl concentrations are high and leads to the incomplete mass transfer of the analyte, which negatively affects the compound's sorption by the FPSE media (Gülle et al. 2019).

Desorption solvent volume effect

For determining the optimized volume of desorption solvent, five different volume of desorption solvent solution (acetonitrile: water: acetic acid (49: 49: 2, v/v/v)) including 1, 2, 3, 4, and 5 mL were used and, the best result was obtained with 1 mL. Thus, this volume was regarded as the back extraction solvent solution's optimized volume in future experimentation.

Effect of pH on desorption solvent

The impact of solvent pH on the efficiency of desorption was investigated at pH 2, 4, 6, and 8. However, we had achieved the best results under the approximately neutral condition at pH=6. Although FPSE media apply between pH 1 to 12, in the current study because of the efficient elution that was obtained in pH=6 in comparison with the acidic and basic condition, this pH value was regarded as the optimum condition. To explain this matter, it can be said that since the elution of analyte probably is due to its polarity (log K_ow) and ionic state (pK_a) at the elution conditions, probably in this pH value, suitable conditions was provided for the elution concerning the nature of target analyte (Zilfidou et al. 2019). In addition, the sol-gel network of the FPSE membrane remains so active for the highest interaction with the back extraction solution. Furthermore, we did not utilize harsh pH to maintain this technique as simple and easily applicable as possible laboratory routines.

Desorption time effect

After the sorption step, the FPSE media was placed in contact with the desorption solvent for 5, 10, 15, 20, 25, and 30 min under sonication to obtain the optimized desorption time. A rapid increase was observed in the peak areas from 5 min to 15 min. There was no significant increase in desorption value when the extraction time extended to 30 min. The equilibrium state reached for the FPSE coating media and the working solution after 15 min. Thus, the optimization time was reported as 15 min for back extraction in subsequent works.

Extraction time effect

The analyte's distribution coefficients determine extraction and elution times, which creates the adsorption equilibrium between the sample solution and the FPSE medium and affects the FPSE procedure performance (Zilfidou et al. 2019). To reach the maximum extraction of the analyte with FPSE membrane and evaluated the optimized extraction time, the analyte was placed in contact with the FPSE media from 10, 20, 30, 40, 50, and 60 min. The findings indicated that the extraction efficiency enhanced with time, and in 40 minutes, it reached the maximum value; however, no significant improvement was observed as contact time extended. Moreover, the shorter the extraction time, the more efficient the routine analysis approach. Thus, the shortest extraction time is intended. Overall, to achieve better results, 40 min extraction time was considered as the optimum time for extraction.

Effect of extraction solvent volume

For the optimization of the extraction solvent volume, the FPSE media was in contact with 2, 4, 6, 8, and 10 mL of a standard solution containing 5 ng/mL of ochratoxin A. As we increased the sample volume from 2 mL to 10 mL, extraction was improved; therefore, 10 mL was selected as the optimized volume of extraction solution. A high volume of extraction solution can help the analyte diffusion and mass transfer through the sample matrix to the extraction media. It shortens the equilibrium creation time for making the extraction more efficient. It can be concluded that the solution in high volume can lead to greater extraction values (Alampanos et al. 2019).

Method Validation

A calibration curve was prepared using the optimized FPSE-HPLC conditions. The linear range of the calibration curve was 1.5-15 ng/mL, and correlation coefficients were greater than that of 0.987. The characteristics of calibration curves are presented in Table 1. The limit of detection (LOD) and the limit of quantification (LOQ) were defined as signal to noise 3:1 and 10:1, respectively. FDA requires that the intensities at LOQ must have precision of 20% at most and trueness of ±20%.

The recovery of the ochratoxin A was used to determine the trueness. In order to perform the recovery stage a comparison was made between the peak area in each sample and the respective non-extract standards (ochratoxin in solution). The peak area in every sample was divided by the relative area below the curve corresponding to the quality control treatment multiplied by 100. Recovery of ochratoxin from samples was in an acceptable range (Table 2). The relative standard deviation for within the laboratory repeatability (RSD_r) was 3.29% (Table 1). Ochratoxin A was quantified in real samples, indicating the effectiveness of the presented method.

Insert Table 1

Application for real samples

Real samples, including rice and lentil were evaluated following the spiking with ochratoxin A for validation of the developed FPSE-HPLC technique. At the retention time corresponding to ochratoxin A, no interfering peak was observed during its determination in cereals or legumes samples. Acceptable recovery was obtained for rice (80.3-102.5%) and lentil (80.3–109.2%), which were in the range recommended by AOAC (Locatelli et al. 2019b). The percentage recovery from two type samples was different at various concentrations, probably due to unpredictable matrix behavior. FPSE-HPLC-FLD typical chromatogram of spiked rice samples at the level of 5 and 10 ng/g are depicted in Fig. 4 (R1 and R2), respectively and spiked lentil samples at the level of 5 and 10 ng/g are shown in Fig. 4 (L1 and L2), respectively. The blanks chromatograms for rice and lentil are shown in Fig. 4 (RB and LB), respectively. The determination results are given in Table 2.

Insert Fig. 4

Insert Table 2

Comparison with other reported methods

Characterization of ochratoxin A in food samples by FPSE-HPLC-FLD was compared to some microextraction techniques food and biological samples, such as DLLME-LC-MS/MS, SPME-HPLC-FLD, HF-LPME-HPLC-FLD, SPME-LC-MS/MS, and MI-μSPE-HPLC-FLD (Table 3) (Campone et al. 2011; González-Peñas et al. 2004; Lee et al. 2012; Vatinno et al. 2008a; Vatinno et al. 2008b). Since the cotton fabric coated PTHF displays higher mass transfer of analyte to the coating surface from the bulk phase, it can be concluded that our method needs considerably shorter extraction time in comparison with various techniques in this area. Data regarding the RSD and recovery obtained here can simply be compared with those from previous methods. Moreover, the calculated LOD was acceptable compared to other approaches. These findings reveal that our novel FPSE-HPLC-FLD technique is a quick, convinient and highly efficient way without harsh extraction conditions and time-consuming protein precipitation steps that can be readily used to determine ochratoxin A in food samples.

Insert Table 3

Measurement of compounds, elements, and toxins in trace amounts in aquatic, biological, food samples, etc., in addition to environmental issues is always required and important. Sample preparation is usually regarded as a sophisticated and demanding stage, especially when the analyte concentrations level is trace and ultra-trace in environmental matrices. There has been a growing trend towards sensitivity and miniaturization of analytical methods. One way to achieve this goal is to use rapid, inexpensive, sensitive and accurate analytical methods to measure trace amounts of the compound. The sample preparation step in the analytical methods often involves an extraction step to separate and concentrate the target analyte from the sample matrix. Choice of the method and extraction conditions has a special role in the analysis time, easiness of application and selectivity.

In this research, we developed and validated a novel FPSE-HPLC-FLD approach to extract/pre-concentrate ochratroxin A in food samples. The results show that fabric phase sorptive extraction technique can be effective in reducing solvent volume and analysis time, these advantages make this technique more economic, green and environmentally friendly. This technique had high validate and experienced the range, linearity, and at the same time was precise and accurate. Therefore, this study demonstrates that the FPSE method displays acceptable level of speed, precision, reproducibility, and sensitivity for determining ochratoxin A in food samples.

Compliance with Ethical Standards

Funding: Not Applicable

Conflict of Interest: The authors declare that they have no conflict of interest.

Ethics approval: Human samples have not been used in this study.

Consent to participate: Not Applicable

Consent for publication: Not Applicable

Code availability: Not Applicable

Author Contributions: 1. Aida Esmati Arze Olia: did the laboratory work, wrote the draft of this report, reviewed the final version of this report, and supplied all figures and tables. 2. Dr. Alireza Mohadesi was the supervisor of Aida Esmati Arze Olia, designed and directed the project. 3. Dr. Javad Feizy was the supervisor of Aida Esmati Arze Olia, conceived and planned the experiments. Wrote the final version of this report, and evaluated all data. All authors read and approved the final version of the manuscript.

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Table 1. FPSE-HPLC-FLD characteristics of ochratoxin A.

Ochratoxin A	Parameter
0.9876	Correlation coefficient (R²)
2.5-15	Linear range (ng/mL)
3.29	RSD (%)
0.49	LOD (ng/mL)
1.49	LOQ (ng/mL)

Table 2. Mean recoveries of ochratoxin A in cereal and legume samples.

RSD (%) n=3))	Recovery (%)	Spiked level (ng/g)	Sample	Analyte
6.62	102.5	5	Rice	Ochratoxin A
7.09	80.3	10	Rice
4.75	109.2	20	Lentil
1.90	80.3	40	Lentil

Table 3. Comparison of reported analytical methods with the current study.

Reference	RSD%	Mean Extraction Recovery%	LOQ (ng/ mL)	LOD (ng/ mL)	Extraction Time (min)	Sample	Sample Preparation Technique	Analytical Technique	analyte
(Lee, Saad, Ng, & Salleh, 2012)	<2.3 (n=5) < 1.0 (n=5) <0.9 (n=5)	91-99 99- 101 96-99	0.19 0.06 0.08	0.06 0.02 0.02	30 30 30	Coffee Grape juice Urine	MI- µSPE	HPLC-FLD	Ochratoxin A
(R Vatinno, Vuckovic, Zambonin, & Pawliszyn, 2008)	<14.3 (n=5)	91-109	0.70	0.30	60	Urine	SPME	LC-MS/MS
(González-Peñas et al., 2004)	<7 (n=3)	74-79	0.25	0.20	120	Wine	HF-LPME	HPLC-FLD
(Rosa Vatinno, Aresta, Zambonin, & Palmisano, 2008)	<3.3 (n=5)	93-103	2.00	0.30	60	Coffee	SPME	HPLC-FLD
(Campone, Piccinelli, & Rastrelli, 2011)	<5.8 (n=6)	97-102	0.015	0.005	-	Wine	DLLME	LC-MS/MS
This work	6.8 (n=3) 3.3 (n=3)	80.3-102.5 80.3-109.2	1.49 1.49	0.49 0.49	40 40	Rice Lentil	FPSE	HPLC-FLD

No competing interests reported.

Ochratoxin Determination in Food Samples by Fabric Phase Sorptive Extraction Coupled with High- Performance Liquid Chromatography Technique

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Abstract

Figures

Introduction

Experimental

Results And Discussion

Conclusion

Declarations

References

Tables

Additional Declarations

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