MATERIAL

Technical field of the invention

The invention is related to the field of analytical chemistry and deals with equipment for the automated determination of the content of mycotoxins in analytes, prepared from solid samples, and specifically with a cartridge for isolation and obtaining samples from solid materials, and with a device for carrying out the fluorescence analysis of the obtained analyte to determine the content of mycotoxins.

Prior art

Mycotoxins are toxic substances synthesized by certain species of spore fungi that parasitize on crop plants in the field or on commodities obtained from plant raw materials after harvesting and may cause the human and animal intoxication. Therefore, the necessity of mycotoxins detection is relevant for many areas of application, including the food and agricultural industry in the manufacture and storage of food and feed, the protection of the environment by analyzing its state for the protection of plants, etc.

The most common types of mycotoxins are aflatoxins, ochratoxins, patulin, ergot fungi alkaloids, and toxins of fungi of the genus Fusarium which have toxic and carcinogenic effects or affects grains and fodder crops in fields and storage places, for example, in warehouses or mills.

Today, the most common method for determination of most mycotoxins in commodities is the reversed-phase liquid chromatography with optical detection, according to which mycotoxins are extracted from the milled solid samples, purified and concentrated by the immunoaffmity adsorbent, followed by elution of the concentrated analyte into the vessel for analysis - spectroscopic cell or waveguide. Further detection and evaluation of the mycotoxin concentration is carried out by analyzing changes in the optical properties of the eluate, such as absorption, fluorescence, phosphorescence, etc. and the compliance with standard requirements is then verified. Usually, the implementation of this technology requires numerous manual steps and the detection of mycotoxins with benchtop liquid chromatography devices (see, for example, http://www.analyt.com. ua/page/prod/name=lumch) and cannot be applied out of the analytical laboratory.

This drawback is partly solved in US4895808 A as of January 23, 1990, where an instrument for simplification of the preparation for the liquid chromatography via portable adsorption preparation of an analyte for mycotoxins detection in a pre-made and pre-extracted solution is disclosed. The device is composed of a single-use cartridge made of two adsorption columns connected by a fluidic channel for transferring their contents by pressurization.

The adsorption of the pre-made extract is carried by subsequent passage thereof through adsorption columns equipped with plungers. In the first column, pressurized by a plunger, isolation of the analyte from the impurities and particles is carried out with the subsequent flow of the purified analyte into a second column filled with a selective adsorbent specifically adsorbing the analyte. Then the content of the second column is transferred to the chromatographic device and the presence and/or concentration of mycotoxins in the purified extract of the test sample is evaluated by the changes of the adsorbent optical properties at the different wavelengths of light emitted from the light source.

The advantage of the described solution is a preparation of purified sample ready for analysis of mycotoxins, which is carried out in a standalone device and is an independent and potentially mobile stage. However, several preparation stages (for example, analyte extraction from milled sample and finalization of the analysis conduction) require laboratory equipment with a system for the detection of the fluorescence signal. This approach increases the duration of the analysis and probability of eluate contamination due to manual sample transfer to the detection stage.

This disadvantage is partly solved in a mobile analytical device Aokin Rapid Analysis made in accordance with patents US8173379 B2 as of May 8, 2012, and EP2048500 B2 as of April 6, 2016 (https://www.pathtech.com.au/site/wp- content/uploads/2017/07/Aokin-RapidAnalysisSystem.pdf). The device contains a fluorescence detection module, which consists of a light source, pair of polarization filters, detector and thermostated spectroscopic cuvette, alongside with a set of bottles filled with solvents and markers, a micropump and automatic sampler for fluid transfer, power supply, and control unit installed in the casing. The described analytical device can be powered from both mains power source and batteries, contains the tools for data transfer and analysis. So, the analysis of mycotoxin content can be carried out rapidly and precisely and does not require dedicated laboratory facilities.

However, the device is designed for low molecular weight compounds like mycotoxins by solid-phase immunoassay method, so-called“sandwich”-binding of analyte with immobilized antibodies and subsequent binding of fluorescent-labeled antibodies followed by analysis of fluorescence signal from label. Thus, the method is based not on direct fluorescence detection, but fluorescence polarization which eliminates background fluorescence. Such an approach complicates significantly the device as it requires additional fluorescent anti-analyte antibodies for signal amplification in samples with a low concentration of the analyte for its algorithm. Also, it requires specific technical skills and manual sample handling for several stages of the workflow and it increases the analysis duration as well as the probability of errors.

Thus, there is a strong demand to develop a portable device for continuous sample preparation process and analysis of mycotoxin content in single medium in single device with minimal probability of contamination and avoiding direct contact of operator with working fluids that allows simplifying and acceleration of the process, decreasing the errors from operator's manipulations and allows the outdoor usage where samples are collected.

Summary of the invention

The invention is based on a goal to develop the device comprising all the complete steps of sample preparation of purified extract of analyte ready for fluorescence and/or absorbance spectroscopy. The device is implemented as a single-use cartridge with interlinked components required for complete process of sample preparation for the fluorescence and/or absorbance spectroscopy analysis conduction, which is composed of analyte extraction from the milled sample, dilution (buffering) of the extract, affinity adsorption on the affinity adsorbent and elution of the concentrated analyte directly into a spectroscopic cell.

Also, the goal of the claimed device consists in the development of an autonomous device for absorbance and/or fluorescent spectroscopy detection of mycotoxin content at least in plant materials with option of integration of above mentioned single-use cartridge and with option of direct measurement of the fluorescence of the purified and concentrated analyte or the fluorescent analyte derivate.

The goal is achieved by the development of the single-use cartridge that contains the interlinked together vessel system, which consists of at least four vessels interlinked at their outlet parts with fluidic channel with opened terminals via 3 -position valves, switching flow direction, and at least two vessels located near the opened terminals are equipped with pumping means, typically implemented as plungers or pistons. One of the vessels with a plunger is dedicated for loading of plant material for the analysis and one contains affinity adsorbent for the target analyte. Other two of at least four other vessels are filled with solvent for extraction and with buffered solution and sealed.

Achieved thereby technical result consists of increasing of functionality and reliability of a cartridge with possibility of fast and complete sample preparation of analyte ready for the detection of mycotoxins. The single-use prefilled sealed cartridge, containing vessels with liquid reagents, affinity adsorbent, vessel for milled solid sample, which is equipped with means for fluid transfer, e.g. plungers, allows to perform complete continuous process of sample preparation with following spectroscopic analysis (absorbance- based and/or fluorescence-based) of analyte from solid milled plant materials. Aforesaid method is performed in the cartridge by the loading of the milled plant material sample into one of the vessels, equipped with a plunger, followed by the connection of the vessel with sample to a vessel, containing solvent for extraction, via 3 -position valves with corresponding positions and transfer of the required volume of the solvent into the vessel, containing a solid sample, by retraction of its plunger, starting extraction process for a required period with optional agitation.

After that, a portion of buffered solution is transferred to a sample vessel from a vessel, containing buffered solution, via fluidic channel with corresponding positions of 3 -position valves by retraction action of its plunger, resulting in dilution of the obtained extract with optional agitation.

The diluted extract is transferred from a sample vessel to a vessel, containing affinity adsorbent, via 3 -position valves with corresponding positions by the retraction of the plunger, and the analyte binds to the adsorbent with additional agitation by slow retraction of the plunger.

Extract unbound to the adsorbent is transferred in reverse direction from the adsorbent vessel to the sample vessel via 3 -position valves with corresponding positions by the corresponding plungers and the rest of buffer solution is transferred into the adsorbent vessel for the adsorbent washing from non-target substances. After that, the adsorbent vessel contains only analyte specifically bound to the adsorbent.

Then the extractant is transferred from the vessel filled with extractant into the vessel, containing adsorbent, under the control of the plunger and 3 -position valves with corresponding positions and the extractant disrupts electrostatic bonds between the affinity adsorbent and analyte molecules, resulting into analyte releasing into solution. Obtained analyte solution is eluted by the extrusion of the plunger from the adsorbent vessel into the analysis vessel (spectroscopic cell) via the opened terminal of the fluidic channel.

Herewith, one possible embodiment of the cartridge contains mechanical filters at the outlets of vessels, equipped with plungers, to avoid the passage of fine particles of the sample and adsorbent into the valves and fluidic channel.

According to another embodiment, the cartridge contains as 3 -position valves faucets, enabled for automatic control. Such valves performance allows operating the fluid flow inside the cartridge manually or via the control device, intended for a fully automated mode of sample preparation.

According to another embodiment, in the adsorbent vessel the cartridge contains a cell for the latter as physical stroke limiter for the plunger, intended to protect the adsorbent with bound analyte against possible compression or destruction with the plunger.

According to another embodiment, the cartridge contains as means for sealing vessels, containing extractant and buffer solution, membrane and/or sliding rubber plug to save the content while stored, and for allowing flowing of liquids after the elastic membrane (e.g. foil) puncture and/or move of the rubber plug in response for plunger movement in connected adjacent vessels during the solutions transfer.

According to another embodiment, the cartridge includes a mechanism for agitation (stirring) in a vessel dedicated for loading solid plant material sample in.

In special cases for enhancing or chemical modification of the analyte fluorescent features, it is derivatized. For this purpose, another embodiment of cartridge includes additional vessel, equipped with mechanisms for fluid transfer, typically performed as a plunger, and containing derivatization agent, into which the eluted analyte from affinity adsorbent is transferred.

The problem of development of the autonomous device for the performance of the analysis for a mycotoxin content determination is solved by the device comprising a housing, a docking stage inside the housing for placing the vessels filled with substances for the analyte preparation, the fluorescence detection module, that consists of a light source with a pair of optical filters, an optically transparent spectroscopic cell placed between the filters, and a light detector, a means for processing and measurement a signal of the fluorescence detection module, a power supply unit and a control unit. According to the invention, the docking stage holds the cartridge arranged therein and performed according to the presented invention, and means for pumping a fluid and 3 -position valves of the cartridge are connected to a corresponding actuators with electrical drives, herewith a terminal of the fluidic channel from the side of the affinity adsorbent vessel is linked to the optically transparent spectroscopic cell.

The technical result of the aforementioned is in the optimization of the device architecture, its portability and autonomy for fast analysis of mycotoxin content in plant materials via the continuous automatized process in single medium and reduced error susceptibility without operator contact with working solutions.

In the preferred embodiment, electrical actuators of 3 -position valves implemented as servomotors and electrical plunger actuators are driven by stepper motors equipped with means to engage plunger tail.

Another device embodiment assigns an ultraviolet light (UV) emitting diode with dominant wavelengths in 350 - 380 nm band as a light source for a fluorescent spectroscopic module.

Another device embodiment includes agitation (stirring) mechanism, consisting of ferromagnetic rotor placed in the sample vessel of cartridge and electromagnetic coils, mounted on device docking stage alongside the aforesaid vessel.

Brief description of the drawings

For a deeper understanding of the invention and its advantages, explanations of possible device embodiments, with references to figures in drawings attached, follow below. Each specific part is designated with its specific position number, herewith:

FIG. 1 is a schematic representation of the cartridge according to the independent claim and the embodiment thereof including limited volume for affinity adsorbent protection and additional agitation mechanism in corresponding vessels.

FIG. 2 is a schematic representation of the optional cartridge embodiment including limited volume for affinity adsorbent protection, an agitation mechanism in corresponding vessels and an additional vessel filled with derivatization agent.

FIG. 3 is a schematic representation of the device for mycotoxin content determination in plant materials with cartridge included.

FIG. 4 is a representation of the part of the device for mycotoxin content determination in plant materials with the cartridge installed and cartridge plungers and valves engaged with corresponding actuators of the device.

FIG. 5 is the electrical block diagram of the device.

Listed figures are included to the claim to improve understanding of the claimed device, represent its optional embodiments and serve to explain principles of device operation.

The detailed description of the drawings (information confirming the invention implementation possibility)

Cartridge 1 for analyte preparation consists of at least four vessels interlinked together at their outlet parts via tube 2 with 3 -position valves 3 which direct the fluid transfer. Two of aforementioned vessels are equipped with mechanisms, typically embodied as plungers or pistons 4, for extrusion fluids out or drawing them in, one of the aforementioned vessels 5 is dedicated for loading plant material for analysis and another one 6 contains affinity adsorbent. Other two vessels of at least four ones are filled with extractant 7 and buffer solution 8, sealed with sealing means 9. Mechanical filters 10 are installed at the outlets of vessels 5, 6 to avoid the particles of sample and beads of adsorbent passing into valves and fluidic channel, and a mechanism for rotational agitation 11 is placed in a vessel 5. Stroke limiter 12 for the plunger installed in vessel 6, is intended to protect the adsorbent against possible contact with plunger 4. Cartridge 1 may include additional vessel 13 equipped with means for fluids pumping 4 and containing derivatization agent. Device for automatic sample preparation and analysis of mycotoxin content consists of outer housing 14, dock 15 for a cartridge 1 inside the outer housing, fluorescent spectroscopic detection module 16, UV light source 17, bandpass optical filters 18, light detector 19, spectroscopic cell 20, signal acquisition/processing circuit of spectroscopic module 16, power source 21, power supply unit 22 and control unit 23. 3 -position valves 3 and plungers 4 are equipped with electrical actuators and 3 -position valves 3 are driven by servomotors 24, plungers 4 are driven by stepper motors 25 via leadscrew mechanisms 26. Plungers actuators are equipped with clutches 27 to engage and lock plungers 4 tail.

Cartridge operation algorithm

1. Sample loading, extraction.

Sample of milled plant material (typically 2 - 4 g, depending on expected contamination) is loaded into vessel 5. 4 ml of solvent (e.g. methyl alcohol) is transferred into the vessel 5 from the vessel 7 via 3-position valves 3 with positions allowing flow from vessel 7 to vessel 5, starting extraction process for 15 min with optional agitation by its plunger 4.

Then 12 ml of buffer solution (e.g. phosphate buffered saline pH 7.4) or distilled water is transferred into the vessel 5 from the vessel 8 via 3 -position valves 3 with positions allowing flow in this direction, diluting the extract.

2. Analyte binding and adsorbent washing.

The diluted extract is transferred from vessel 5 to vessel 6 for binding of analyte on affinity adsorbent and is mixed during 5 min. Unbound extract from vessel 6 is transferred into vessel 5. 8 ml of distilled water is transferred from vessel 8 to the vessel 6 for adsorbent washing from non-specifically bound substances, and then it is transferred in reverse direction to the vessel 8.

3. Elution.

Extractant (0.5 ml of methyl alcohol) is transferred from vessel 7 to vessel 6, for the analyte elution and is pressed out for analysis to vessel 20.

The device with installed cartridge operates in the following way. The enabled power supply unit 21 using battery 20 as power source provides power for the control unit 22, plungers 4 linear actuators, 3-position valves 3 rotary actuators, UV light source 17 with wavelengths in 350 - 380 nm band and total power 2 W, the light detector 19 photodiode with amplifier circuit.

In response on command signal sequence from the control unit 22, actuators configure 3 -position valves 3 into positions connecting fluidic channel 2 from the vessel 5 to the vessel 7 prefilled with the extractant. In response to the command from the control unit 22, linear actuator 25 displace plunger 4 of vessel 5, forcing a required volume of extractant to flow into vessel 5 from vessel 7 for the period required for extraction with optional agitation. Agitation may be performed using one of the varieties of known approaches. For example, via a vibration or via rotation of the ferromagnetic rotor 28, placed in the vessel, in the alternating magnetic field.

Then in response on command signal sequence on actuators 24 and 25 of plungers 4 and valves 3 specific volume of buffered solution is pumped from the vessel 8 into the vessel 5 via fluidic channel 2 by retraction of plunger 4 and position of 3 -position valves 3 of vessel with the sample 5 and vessel with buffer solution 8, providing their connection and possible mixing and resulting in dilution of obtained extract.

Command signal sequence on actuators 24 of 3 -position valves and actuator 25 of plunger induce by slow retraction action of plunger 4 flow of the diluted extract from vessel 5 through the fluidic channel 2 with the corresponding position of 3-position valves 3 to vessel 6 for analyte binding on affinity adsorbent.

Unbound to the adsorbent extract is transferred to the vessel 5 in the reversed direction in response on the command signal sequence from the control unit 22 to rotary actuators 24 and linear actuator 25, and remaining buffered solution is transferred to the vessel 6 for the adsorbent washing from non-specifically bound substances. As a result, the adsorbent vessel contains the only specifically bound analyte. In response to another command sequence from the control unit 22 on actuators 24 of the 3 -position valves and actuator 25 of the plunger, the extractant is transferred from vessel 7 to vessel 6 and disrupts electrostatic bonds between adsorbent and analyte, resulting in the analyte releasing into solution. Obtained analyte solution in eluent is transferred for analysis into the spectroscopic cell 20 via the opened side of fluidic channel 2 by extrusion action of plunger 4.

Once spectroscopic cell 20 is filled with the analyte, the command signal enables power supply for UV light source 17 and light detector 19. Fluorescence of analyte in a spectroscopic cell is induced by excitation irradiance. Excitation irradiance and fluorescence emission pass through the optical filters, blocking the excitation and passing through the emission light on the detector 19, where the electric signal, directly proportional to the flux of mycotoxins fluorescence emission, is induced.

The signal is acquired by the control unit 22, which processes it and displays on displaying means with possible transmission by other known means.

For using of the cartridge 1 with an additional derivatization vessel 13 in the device is operated by the following sequence.

The device with installed 5-vessel cartridge is operated by following sequence.

The power supply of the device is enabled. Sample of grinded plant material is loaded into vessel 5. Cartridge 1 is installed into the device. Sample preparation sequence starts. 3 -position valves 3 are positioned to allow flow from vessel 7 with extractant to vessel 5 for extraction. The extractant is transferred by retraction of plunger 4 of extraction vessel 5. Extraction is processed with optional agitation.

After extraction 3 -position valves 3 with positions blocking extractant flow into the fluidic channel 2, but allowing buffer solution flow from vessel 8 to vessel 5. Retraction of plunger 4 of extraction vessel 5 allows the transfer of buffer solution to vessel 5 for the extract dilution and buffering.

On the next stage, 3 -position valves 3 are positioned to block the buffer solution flow from vessel 8 into fluidic channel 2, but allowing the channel flow between vessels 5 and 6. Extrusion of the plunger 4 of the extraction vessel 5 and retraction of the plunger 4 of the adsorbent vessel 6 actuates the diluted extract, which is gradually passing through the adsorbent for the analyte binding with the adsorbent. Then the extract from vessel 6 discards by pumping out into vessel 5.

After that, the adsorbent with the bound analyte is washed with the buffer from the vessel 8. For this action, 3-position valves 3 are positioned to allow the buffer solution flow from vessel 8 to vessel 6 with the affinity adsorbent. A buffer solution is transferred into the vessel 6 with the adsorbent by retraction of the plunger 4 of the vessel. After passing of buffer solution through the adsorbent for elimination of contamination, the buffer solution is pumped out into the initial vessel 8 by extrusion of the plunger 4.

Elution of the analyte from affinity adsorbent is provided by transferring of the extractant via 3 -position valves 3 with positions, allowing flow from vessel 7 filled with the extractant to vessel 6 containing the adsorbent, by retraction of plunger 4 in vessel 6 containing the adsorbent. Added solvent disrupts electrostatic bonds between adsorbent and analyte inside the vessel 6, resulting in analyte releasing into solution. Then 3 -position valves 3 are positioned to allow flow through the fluidic channel 2 from vessel 6 with an adsorbent to vessel 13 with derivatization agent. Obtained eluate is pumped out from vessel 6 containing the adsorbent to vessel 13 filled with the derivatization agent by extrusion of the plunger 4 in the vessel 6 and retraction of the plunger in the vessel 13. The range of substances can be used as derivatization agents: for ochratoxins - aluminium (III) nitrate nonahydrate AI(Nq ₃ ) ₃ ·9H ₂ q water/methanol (1 :4) solution; for fumonisins - phthalic anhydride and L-acetylcysteine borate buffer solution. Derivatization reaction transforms mycotoxins into fluorescent derivatives which can be further detected by the spectroscopic module.

Derivatized analyte solution is eluted through the fluidic channel 2 via 3- position valves 3 with positions allowing flow from derivatization vessel 13 to spectroscopic cell 20. Inside the cell, 20 excitation of the fluorescent signal is processed similarly to 4-vessel cartridge algorithm. Thereby, the claimed group of the invention may be used for rapid detection of mycotoxins.

Following examples demonstrate a possibility of implementation of the claimed device purpose and achievement of the result, which is in the base of its development

Example 1

Calibration curve plotting for aflatoxin B1 concentration determination and aflatoxin B1 content determination in milled com kernel sample.

Aflatoxin B1 (Sigma Aldrich, USA) stock solution 1 mg/ml in absolute methyl alcohol is being diluted to following concentrations: 100 ng/ml (100 ppb), 25 ng/ml (25 ppb), 10 ng/ml (10 ppb), 2 ng/ml (2 ppb). Absolute methyl alcohol is used as a blank sample with no aflatoxin B 1 content. The volume of each standard solution is 3 ml.

Empty cartridge is used for injection of the standard solution into a spectroscopic module for fluorescence measurement. The cartridge contains 5 ml of absolute methyl alcohol in its solvent vessel alongside with injected aflatoxin B1 standard solution in sample vessel. Methyl alcohol in the solvent vessel is intended for spectroscopic cell purge after standard solution measurements have been completed. The standard solution flows from its vessel for analysis to spectroscopic cell via 3-position valves with positions allowing flow from a sample vessel to the cell by extrusion of plunger. Once a spectroscopic cell is filled with the standard solution, fluorescence signal acquisition sequence proceeds. Mean value of three subsequent measurements of each standard solution is calculated. A calibration curve is plotted using fluorescence intensity for each given aflatoxin B1 concentration in a set of standard solutions.

1 g of milled reference standard com kernel (EnviroLogix, USA) with the toxin content 4.5 ppb (4.5 ng per 1 gram of material) loads to the sample vessel of the cartridge. Sample vessel is then being closed with its plunger. Loaded cartridge installs into the device. The prefilled sealed cartridge contains 10 ml of methyl alcohol in the solvent vessel; 20 ml of distilled water in buffer vessel; 0.25 ml of immunoaffmity adsorbent (Aokin, Germany) secured with fine mesh filters in the adsorbent vessel; ferromagnetic agitation rotor in sample vessel. Automatic sample preparation procedure starts. 2 ml of methyl alcohol are transferred into the vessel, containing a solid sample. Extraction proceeds for 15 min with agitation. 8 ml of distilled water are transferred to the sample vessel to dilute the obtained extract. The diluted extract is transferred with flow rate 3 ml/min from a sample vessel to a vessel, containing affinity adsorbent, and then is transferred in reverse direction with the same flow rate from the adsorbent vessel into the extraction vessel. 10 ml of distilled water is transferred to the adsorbent vessel and then transferred in reverse direction, resulting in adsorbent washing from non-specifically bound substances. 0.5 ml of methyl alcohol is transferred into the vessel, containing washed adsorbent and bound to it analyte resulting in analyte releasing into solution. Obtained analyte solution is eluted from the adsorbent vessel into a spectroscopic cell for fluorescence measurement. With acquired fluorescence intensity value and previously plotted calibration curve, aflatoxin B1 content in com sample is determined.

Example 2

Calibration curve for ochratoxin A concentration determination and for ochratoxin A content determination in grinded com kernel sample.

Ochratoxin A (Sigma Aldrich, USA) stock solution 1 mg/ml in absolute methyl alcohol is being diluted to following concentrations: 25 ng/ml (25 ppb), 10 ng/ml (10 ppb), 2 ng/ml (2 ppb) and 0.5 ng/ml (0.5 ppb). Absolute methyl alcohol is used as a blank sample with no ochratoxin A content. The volume of each standard solution is 1 ml.

Empty cartridge is used for injection of the standard solution into a spectroscopic module for fluorescence measurement. The cartridge contains 5 ml of absolute methyl alcohol in the solvent vessel and derivatization solution (0.5 mM aluminium (III) nitrate nonahydrate methyl alcohol solution, Sigma Aldrich, USA) in additional derivatization vessel alongside with injected ochratoxin A standard solution in sample vessel. Methyl alcohol in the solvent vessel is intended for spectroscopic cell purge after standard solution measurements have been completed. The standard solution flows from its vessel to derivatization vessel via 3 -position valves with corresponding positions by synchronous retraction and extrusion of corresponding plungers. Derivatization proceeds for 5 min. The derivatized standard solution flows from its vessel for analysis to a spectroscopic cell via 3-position valves with positions allowing flow from derivatization vessel to the cell by extrusion action of plunger. Once a spectroscopic cell is filled with derivatized standard solution, fluorescence signal acquisition sequence proceeds. Mean value of three subsequent measurements of each derivatized standard solution is calculated. A calibration curve is based on fluorescence intensity for each given ochratoxin A concentration in a set of standard solutions.

1 g of milled reference standard wheat (BCR, Belgium) with ochratoxin A content 0 ppb (non-contaminated material) loads to the sample vessel of the cartridge. Sample vessel is then being closed with its plunger. Loaded cartridge installs into device. Prefilled sealed cartridge contains 10 ml of methyl alcohol in solvent vessel; 1 ml of 0.5 mM aluminium (III) nitrate nonahydrate A1(N03)3*9H20 (Sigma Aldrich, USA) methyl alcohol solution in derivatization vessel; 20 ml of distilled water in buffer vessel; 1 ml of molecularly imprinted polymer affinity adsorbent (Affmisep, France) secured with fine mesh filters in adsorbent vessel; ferromagnetic agitation rotor in sample vessel. Automatic sample preparation procedure starts. 2 ml of methyl alcohol are transferred into the vessel, containing a solid sample. Extraction proceeds for 15 min with agitation. 8 ml of distilled water is transferred to the sample vessel to terminate extraction and dilute obtained extract. The diluted extract is transferred with flow rate 3 ml/min from a sample vessel to a vessel, containing affinity adsorbent, and then is transferred in reverse direction with the same flow rate from the adsorbent vessel to a sample vessel. 10 ml of distilled water is transferred to the adsorbent vessel and then transferred in reverse direction, resulting in adsorbent washing from non- specifically bound substances. To elute the analyte, 1 ml of methyl alcohol is transferred into the vessel, containing washed adsorbent. Obtained analyte solution is eluted from its vessel to derivatization vessel. Derivatization proceeds for 5 min. The fluorescent derivative solution is transferred from the derivatization vessel into spectroscopic cell for fluorescence measurement. With acquired fluorescence intensity value and previously plotted calibration curve, ochratoxin A content in the sample is determined.