WO2025141233A1 - Device and method for microextracting compounds in samples - Google Patents

Abstract

The invention relates to a device and method for microextracting compounds in samples, wherein the device comprises a support (1) that carries a power source, a microcontroller, an interface (3), which comprises a fixed supporting element (5) comprising an electromagnet (6), designed to support a container in which the sample to be extracted is disposed, and an arm (7) that holds a motor which, by means of a shaft, moves a rotary magnet (8) arranged on the electromagnet (6) and spaced therefrom. According to the invention, the method is based on agitating a magnetic sorbent inside the container by means of the rotation of the magnet (8) and retaining the sorbent by means of the electromagnet (6) to prevent the accidental loss thereof when the one or more solutions are removed from the container.

Description

DESCRIPTION

Device and method for microextraction of compounds in samples

TECHNICAL SECTOR

The present invention relates to a device and method for microextracting compounds from liquid samples, especially when a small sample volume is available. These samples can be either directly liquid or solid samples previously dissolved in a small volume of solvent. It is a method for extracting compounds of interest present in liquid samples, or previously dissolved solid samples, without risk of contamination, with high reproducibility, and in an automated manner. The preferred device is small and battery-powered, which makes it highly portable.

The present invention falls within the field of methods for sample treatment, mainly within the context of clinical analysis and especially for small biological samples.

The invention also has application in the analytical laboratory sector when small samples are available and procedures are required where operator intervention is minimal, but without ruling out analytical laboratories in any other sector besides the clinical one mentioned above.

STATE OF THE ART

The determination of biomarkers in biological fluids using extraction techniques prior to their measurement by chromatographic techniques, coupled or not to more sensitive and selective detectors such as mass spectrometry, is a common practice that has been carried out for many years.

Conventional extraction techniques, i.e. liquid-liquid extraction (LLE) or solid-phase extraction (SPE), usually require large amounts of solvent or sorbent, respectively, in addition to a large amount of sample. Microextraction techniques, of which there are numerous modalities, are based on the same principles as these, but it should be noted that although the quantities of extracting phase (solvent or sorbent, respectively) have been drastically reduced to a few microlitres (solvent) or milligrams (sorbent), the same level of reduction has not been achieved with regard to the sample, which is still in the order of millilitres.

At this point, it is necessary to differentiate between dispersive microextraction techniques, those in which the extractant phase is dispersed within the donor phase (sample), and non-dispersive ones, the former being faster and requiring less time (minutes vs. hours) as a result of the increase in extraction kinetics by increasing the contact surface between the donor and acceptor phases.

There are devices on the market that allow the automation of some non-dispersive microextraction techniques, such as solid-phase microextraction (SPME). However, there are no known commercial automated devices for dispersive techniques such as the one presented in this invention to carry out dispersive solid-phase microextraction (DMSPE), whose features make it a widely accepted technique in the scientific community (Trends Anal. Chem. 112 (2019) 226).

However, it is worth mentioning the semi-automated approach published a few years ago by the research group that presents the present invention (J. Chromatogr. A 1362 (2014) 25), which was called stir bar dispersive microextraction (SBSDME). In this technique, the sorbent material, with magnetic properties, is manually added to the interior of a vial containing the solution to be extracted and a neodymium magnet, so that it adheres to the magnet due to the magnetic field. The magnet is then rotated by the action of a common laboratory stirrer plate, causing the material to disperse, which, after stirring stops, collects it again, this time together with the compounds of interest. Subsequently, it is manually removed to proceed with the desorption of the compounds of interest by liquid or thermal desorption. There are many researchers internationally who have used this technique in their publications (Anal. Chim. Acta 1153 (2021) 338271).

Also worth highlighting is the proposal by Turnes-Palomino et al. (Anal. Chem. 87 (2015) 7545), similar to the previous one but with a greater degree of automation by using articulated syringes with automatic pistons and computer-controlled valves to take the solutions and solvents, the extraction being carried out in the body of one of the syringes that contains the neodymium magnet inside.

Both proposals use extraction devices of varying capacities, allowing for the extraction of a relatively large sample volume (5-25 mL). These devices cannot be used for very small samples (<100 pL), as in the device presented in the present invention. As a result of using larger extraction devices in these approaches, higher solvent volumes (in the milliliter range) and sorbent quantities (in the tens/hundreds of milligrams) are used than in the invention presented (in the range of a few microliters and a few milligrams). Furthermore, in both proposals, the magnet is located inside the device, in direct contact with the solution (sample) to be extracted and the sorbent material. This can lead to contamination problems from one solution (sample) to another if it is not thoroughly cleaned. This cleaning can be tedious, especially when removing all the magnetic sorbent, even more so if the sorbent has a high magnetic character and is therefore strongly attracted to the magnet.

In the invention presented, this problem is eliminated because the magnet is located externally and does not come into contact with either the solution to be extracted (sample) or the magnetic sorbent. Furthermore, the invention presented is technically very simple, allowing it to be compact and easily portable, which also sets it apart from previous approaches.

The applicant is not aware of any method with the same characteristics as the invention, nor a device as effective as the one described and claimed below.

BRIEF EXPLANATION OF THE INVENTION

The invention relates to a device for microextraction of compounds in small-volume liquid samples, including solid samples previously dissolved in a small volume, according to the claims. In its various embodiments, it addresses the problems of the prior art. It also relates to the method of use for performing this microextraction. The present invention arises directly from the most current trends in sample preparation strategies in the field of analytical chemistry. On the one hand, there is a need to be able to extract traces of compounds of interest from small samples (microsamples), such as some biological fluids. Often, only a small volume of these is available, either due to the nature of the sample (saliva, semen, follicular fluid, cerebrospinal fluid, etc.), or due to the particular characteristics of the patients (newborns, immunosuppressed or anemic patients, etc.), or due to the context itself (remains in forensic analysis).

Considering the biological risk posed by these types of samples and, furthermore, to avoid cross-contamination when processing subsequent samples, it is extremely important that all materials in contact with them be thoroughly cleaned once each analysis is completed, or that disposable materials be used. Therefore, the amount of material in direct contact with the sample must be minimized to avoid analytical errors, as well as tedious and time-consuming cleaning procedures that pose a risk of introducing errors.

Furthermore, due to the complexity of these samples, as well as the large number of potential interferents and the low concentration levels of some of the compounds of interest, the use of microextraction techniques is required to remove the bulk of the matrix and perform a pre-concentration of the analytes that allows reaching the required detection/quantification limits.

Another important factor to consider is the degree of automation of the analysis methods, as a higher degree of automation will minimize operator involvement, providing several advantages, such as faster procedures, greater operator safety, a reduction in potential errors associated with direct operator intervention, and greater efficiency.

On the other hand, the portability of extraction devices is also one of the parameters to be taken into account when developing them. This is because portable devices allow sample processing to be carried out at the sample collection site, thus avoiding the need to transport the sample to the laboratory and therefore its transport, preservation, and storage, which, in addition to consuming energy, energy and time, poor performance can lead to sample deterioration and therefore erroneous results.

In order to cover all the aforementioned needs, a novel microextraction device has been developed that allows the treatment of small-sized samples and, unlike other devices, allows the agitation of the sorbent material added to the sample from an external magnet without coming into contact with the sample, thus avoiding the risks mentioned above.

This is an automated and portable microextraction device that allows the extraction of compounds of interest from small samples. The device is programmed so that all tasks are performed automatically. The main hardware components consist of a microcontroller, a power supply module, an electronic board, a motor (preferably DC), and a rotating magnet, usually cylindrical, suspended over an electromagnet and attached to a motor by means of a shaft that rotates it. All the components are integrated into a support.

This device extracts compounds of interest from very small samples (just a few microliters) by dispersing the magnetic extraction material in the sample. Dispersion is caused by the rotation of the motor-driven magnet, which is positioned a short distance above the sample, without coming into contact with either the sample or the sorbent material. Both are located in a small container placed over the electromagnet.

The advantages derived from the use of the invention can be summarized as:

- Automation of DMSPE (dispersive solid-phase microextraction), with less operator involvement and reduced times.

- Application of DMSPE to microsamples.

Low consumption of solvents and sorbents.

Generation of low amounts of waste and therefore less need for treatment, thus reducing the ecological footprint

Reducing the risk of contamination between samples.

Portability, by having miniaturized elements integrated into a small support. To this end, the device for microextraction of compounds from small samples comprises a support that carries a power supply, a microcontroller, and an interface. It also comprises a fixed support that holds an electromagnet, configured to hold a container in which to place the sample. It also carries an arm with a motor that moves a shaft with a magnet at one end located perpendicular to said axis, and the shaft and magnet assembly rotates on itself, arranged above the electromagnet and spaced from it. Thus, the rotation of the magnet produces the dispersion of a magnetic sorbent in the sample. When the motor stops, and therefore the rotating magnet stops, the electromagnet is activated, immobilizing the sorbent on the bottom of the container that contains it.

To facilitate container removal or any other operation on it, it is preferable for the arm to be movable between a position where the magnet is above the electromagnet (working position) and a position where it is removed (access position). This allows the magnet to be placed on the container before turning and removed after stopping.

Similarly, the magnet can be height-adjustable relative to the container holding the sample.

Thus, the method for microextraction of compounds from small samples comprises the following steps: a) Placing a liquid sample (either the starting liquid or a dissolved solid sample) and a magnetic sorbent material in a container. b) Placing the container over the electromagnet. c) Placing the magnet over the container. d) Rotating the magnet. e) Stopping the magnet and activating the electromagnet. f) Removing the sample from the container. g) Washing the sorbent by adding a neutral solvent, deactivating the electromagnet and repeating steps c)-f) with the neutral solvent. h) Performing liquid desorption by repeating step g) with a suitable solvent, or by removing the material and performing thermal desorption.

Some variants are set forth in the independent claims and are described below. DESCRIPTION OF THE FIGURES

In order to complete the description and to help better understand the characteristics of the invention, a set of figures and drawings is presented in which the following is represented for illustrative and non-limiting purposes:

Figure 1 shows a perspective view of an embodiment of the device.

Figure 2 shows a side view of the previous example with the arm in the working position.

DETAILED EXPLANATION OF AN EMBODIMENT OF THE INVENTION

Below, several embodiments of the invention are briefly described, as an illustrative and non-limiting example thereof.

The device of the invention consists of a support (1) and a container (2). The support (1) comprises the electronic equipment, a power supply (battery or mains cable), a microcontroller, an interface (3), generally by means of buttons and pilot lights or by means of a touch screen, one or more ports (4) for connecting other devices, whether physical (wired) or wireless, etc. These ports (4) will be used to transmit the status of the machine, receive software updates.

From the support (1) shown, a lower fixed support (5) emerges, which serves as a support for the small sample container (2). For example, the lid of a centrifuge microtube can be used as the container (2). This fixed support (5) materializes in an electromagnet (6) that forms the support surface of the container (2). An arm (7) allows a magnet (8), for example, a neodymium one, to be placed on the container and the electromagnet (6). The arm (7) is movable in order to be able to place the container with the sample on the electromagnet without risk of accident. That is, there is an access position, where the fixed support (5) is fully accessible because the arm (7) is withdrawn and a working position where the magnet (8) is close to the electromagnet (6) and on it. The arm (7) can be moved by a servomotor (9) for these movements, and a spring can be provided to facilitate one of the movements. The magnet (8) can be at a fixed height or move vertically to move away from or towards the electromagnet (6). In any case, will ensure that it does not come into contact with the contents of the sample container (2). A motor (10) moves a shaft that has a magnet at one end located perpendicular to said shaft and the assembly of the shaft and magnet rotates on itself, arranged on the electromagnet and distanced from it, to produce changing magnetic fields.

DMSPE is used as an extraction technique using this device. In this technique, a suitably functionalized sorbent material with magnetic properties is used as a sorbent. The functionalization of the material will be appropriately carried out according to the compounds to be extracted, in order to increase the extraction yield and selectivity. The magnetic susceptibility of this material must be sufficiently high (for example, 50-60 emu g ^-1 ) to ensure its attraction to the magnet (8) and to the electromagnet (6), although materials with lower magnetic susceptibility values may be used, suitably adjusting the height of the magnet (8). The particle size of this material must be sufficiently small (50 nm - 50 pm) to increase the contact surface with the donor phase (sample) and, consequently, the extraction kinetics.

Thus, the appropriate amount of sorbent is weighed and added to the container (2), together with the sample (a few microliters). The container (2) is placed on the fixed support (5) and the magnet (8) is placed on the container, at a measured distance, adjustable according to the type of material, sample, etc. The magnet (8) is rotated at a programmed speed for a predetermined time and, by means of magnetic interactions, the magnet (8) causes the magnetic sorbent to move, causing it to disperse within the sample. The motor (10) is then stopped, the electromagnet (6) is activated, thus attracting the magnetic sorbent, and the arm (7) is withdrawn to allow access to the container.

In the “access” position of the arm (7) the operator is allowed to carry out the necessary tasks, such as removing the sample, adding the desorption solvent, etc. It is possible to indicate to the operator when or what to do through the interface (3) (screen, coloured LEDs, LEDs next to written instructions...).

This sequence of operations is repeated as many times as necessary depending on the stages involved. It is important to note that the entire extraction/desorption process is carried out within the same container without the need for transfers, so that only the final extract is conveniently transferred to the extraction instrument. extent.

Example 1

In a test, an analytical method was developed for the determination of three acylcarnitines (C16, C18 and C18:1) in newborn serum samples. To carry out the extraction procedure, 0.74 mg of magnetic sorbent (oleic acid-coated cobalt ferrite magnetic nanoparticles, (CoFe2C>4@OA); 57 emu g ^-1 ; 50 nm) was weighed into the container. The container was placed on the electromagnet (6) and 50 pL of acetonitrile was added to condition the material, by stirring at 200 ua (approximately 7000 rpm) for 1 minute.

The acetonitrile was then removed, and 50 pL of the serum sample was added, followed by stirring at 140 a.u. (approximately 5400 rpm) for 5 minutes. The remaining sample was then removed, and 50 pL of ultrapure water was added for washing, which was then removed.

For analyte desorption, 20 pL of acetonitrile was added, followed by further stirring at 200 AU for 3.5 minutes. Finally, the acetonitrile extract was transferred to a 250 pL insert placed inside an injection vial for analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The concentration of acylcarnitines in ng mL ^-1 for five patients is shown in the following table.

Claims

1- Device for microextraction of compounds in samples, specifically liquid samples or solutions of solid samples, comprising a support (1) that carries a power supply, a microcontroller, an interface (3), characterized in that it has a fixed support (5) that comprises an electromagnet (6), configured to support a container where the sample is placed, and an arm (7) with a motor (10) that moves an axis that has at one end a magnet (8) located perpendicular to said axis and the assembly of the axis and magnet rotates on itself, arranged on the electromagnet (6) and spaced from it. 2- Device for microextraction of compounds in samples, according to claim 1, characterized in that the arm (7) is movable between a position where the magnet (8) is on the electromagnet (6) and a withdrawn position. 3- Device for microextraction of compounds in samples, according to claim 1, characterized in that it comprises one or more ports (4) for connecting other devices. 4- Device for microextraction of compounds in samples, according to claim 1, characterized in that the magnet (8) is adjustable in height on the electromagnet (6). 5- Device for microextraction of compounds in samples, according to claim 2, characterized in that the arm (7) is actuated by a servomotor (9). 6. Method for microextraction of compounds in samples, with the device of any of the preceding claims, characterized in that it comprises the steps of: placing a liquid sample or a solution of solid sample and a magnetic sorbent material in a container; placing the container on the electromagnet (6); placing and rotating the magnet (8) on the container; stopping the magnet (8) and activating the electromagnet (6); removing the sample from the container and washing the container with a neutral solvent; performing liquid or thermal desorption; removing and analyzing. 7- Method of microextraction of compounds in samples, according to claim 6, characterized in that it comprises placing the magnet (8) on the container prior to rotation and removing it after stopping it.