WO2023153946A1

WO2023153946A1 - A system for dispersive solid-phase extraction and a method of dispersive extraction using the system

Info

Publication number: WO2023153946A1
Application number: PCT/PL2022/050092
Authority: WO
Inventors: Tomasz BĄCZEK; Mariusz BELKA; Pavel Georgiev; Dagmara SZYNKIEWICZ; Szymon ULENBERG
Original assignee: Gdański Uniwersytet Medyczny
Priority date: 2022-02-08
Filing date: 2022-12-08
Publication date: 2023-08-17
Also published as: PL440329A1

Abstract

The invention concerns a set for dispersive solid-phase extraction, characterised in that it is composed of a top cap (A), an extraction chamber (B) fitted with a replaceable filter (B1) in its lower part, a bottom closure (C), and a fraction collector (D), where all components of the set are produced by employing additive manufacturing technologies. The invention further concerns the method of performing dispersive extraction using the said set.

Description

Title of Invention: A system for dispersive solid-phase extraction and a method of dispersive extraction using the system

[0001] The invention concerns a set for extraction, particularly for dispersive solid-phase extraction, and a method of dispersive extraction using the set.

Technical Field

[0002] Extraction is a method of isolating fractions of substances which have similar properties from a sample being a mixture of many compounds. In spite of the fact that there are numerous methods of extracting analyte from a sample, researchers continue to seek for new opportunities and improvements in isolating individual compounds from challenging mixtures.

[0003] Dispersive extraction is one of many types of extraction, and dispersive solid-phase extraction is a specific type thereof. The dispersive solid-phase extraction method consists in placing powdered substance of sorptive properties in a liquid sample and its continuous stirring until the moment when the quantity of the analyte depositing on the surface of the sorbent particles dispersed in the solution reaches maximum value. Then, the fluid where the sorption occurred is separated from the sorbent, to be replaced with the appropriate volume of fluid where desorption occurs. The solid phase is the loose sorbent selected depending primarily on the properties of the substance intended for extraction, and the solvent used at the sorption stage differs from the one used for desorption primarily in its elution strength and frequently in its chemical nature too.

[0004] Dispersive extraction is one of the simpler preparative processes. However, extraction performed according to the method involves a certain difficulty related to the separation of the sorbent from the sample liquid fraction, which causes that the method is most frequently used to purify the sample of undesirable substances.

Background Art

[0005] A common analytical problem consists in effective and repeatable isolation of the target substance from its matrix. The biological matrix represents a specific case due to co-occurrence of numerous substances which interfere with the course of instrumental analysis.

[0006] In the event dispersive solid-phase extraction is used to purify a sample of undesirable substances, the easiest method of separating the phases is to centrifuge the sample vigorously and collect the liquid from above the deposit, where the liquid can be subject to subsequent tests. However, when dispersive extraction serves isolation of the target substance, the sorbent with the analyte should be additionally treated with desorption so as to free the substance of interest and make it ready for subsequent tests. To that aim, it is necessary to use another solvent, which necessitates separation of the sorbent from the liquid remains of the sample and any impurities. The standard steps consisting of evaporation of the remains prolong the analysis, increase its cost, and do not solve the problem altogether [Ghorbani, M. et al. (2019) ‘Dispersive solid phase microextraction’, TrAC - Trends in Analytical Chemistry. Elsevier B.V., pp. 793-809]. [0007] Known in the state of the art are methods and devices dedicated to dispersive extraction. Patent application US20200025755A1 discloses a device and method of automatised filtering of chemical preparations or biological samples, which make use of modified tips for automatic measuring pipettes to improve separation of analytes from matrices. The filtering method comprises solid phase extraction (SPE) steps and/or protein precipitation from the matrix. The device is characterised in that the lower part of the pipette tip is modified by placing at least one filter, and if two filters are used, a frit, sorbent, or semi-permeable screen can be placed in between them. The top part of the pipette tip serves retaining the sample or the solvent.

[0008] Patent US9040672B2 describes a device for reducing matrix effects in a bioanalytical sample, resulting from the presence of proteins, where the device comprises a bed and a bed-related sorbent capable of binding the matrix compounds present in the bioanalytical sample. The device further comprises filtering means which serve removal of the precipitated protein particles. The filtering means is selected from among exclusion filters: polymeric or inorganic bed having the maximum pore size less than or equal to the diameter of the particles to be removed from the sample.

[0009] Patent PL237582B 1 discloses an insert for a centrifugation container, test tube in particular, which serves separation of fractions of the desired density range by centrifugation at a density gradient. The device is suitable for swift and semi-automatic separation of fluids into fractions of different densities such as e.g. biological fluids, blood included. In addition, it may enable purification, isolation of the fractions, and preservation of biological samples.

[0010] Patent US7875462B2 comprises methods and devices for extracting the analyte from sample solution using packed extraction bed, e.g. the bed of gel beads derivatised with groups having an affinity towards the analyte of interest. One of the embodiments of the invention consists in an extraction column body constructed from a tapered pipette tip, while another embodiment consists in an extraction column built of two cylindrical members.

[0011] Patent application US2012107799A1 concerns an extraction apparatus comprising a filtration vessel which ends with a receiving tip on one end, and containing a membrane filter adjusted to bind a specific group of substances, e.g. nucleic acids, spread over the entire width of the filtration vessel and at least partially inside the vessel. The apparatus incorporates a mechanism for volumetric dosing of the fluid content, functionally connected to the filtration vessel containing the membrane filter, and to a collection container intended for receiving the filtered fluid.

[0012] Patent application US2019293526A1 discloses a set for swift extraction, composed of a column and a cap. The inner space of the column is filled with extraction bed. Located in the top part of the column is an opening surrounded with a flange, and there is a short pipe ending with an opening in the bottom part of the column. The lower end and the column flange are connected tight by screwing both parts together.

Technical Problem

[0013] The state of the art solutions provide ready extraction sets made of standard materials and of strictly defined volumes. No dispersive extraction sets have been developed to date which would enable their swift adjustment to the analysed sample and to the conditions of the analysis, including the equipment used to process the sample.

[0014] A solution to the problems related to dispersive extraction has been reached in the course of the research works conducted by the Inventors.

Solution to Problem

[0015] The subject matter of the invention is a system for dispersive solid-phase extraction, comprising an extraction chamber, a filter, and a fraction collector, characterised in that it is composed of a cap A, extraction chamber B fitted with a replaceable filter Bl in the lower part, and the bottom closure C plus fraction collector D, which constitute two variants of the closure of the extraction chamber B at the bottom, where all set components are produced by employing additive technologies.

[0016] In the set according to the invention, the extraction chamber B is threated on both ends so that the cap A and the bottom closure C or the fraction collector D can be connected detachably by thread to the extraction chamber B, and the fraction collector D is a container threaded in its upper part, where the thread sizes, the diameters of the caps and both ends of the extraction chamber B are fitted to one another so as to match their dimensions, thus forming detachable tight connections in between individual components.

[0017] According to the invention, alongside the tightness of the system, the screwing of the matching threads ensures free replacement of individual components and easy access to all elements of the set, which further enables their thorough cleaning.

[0018] The components of the extraction set according to the invention are produced by the additive manufacturing method in the technology of fused deposition modelling, layer after layer, or by other methods such as e.g. laser sintering of powders.

[0019] The production of the components of the extraction set according to the invention by additive manufacturing technologies, particularly deposition of melted material, ensures the possibility of defining the dimensions freely and scaling them to match the volume of the analysed sample.

[0020] The solution according to the invention further leaves the option of selecting the specific material to be used to form set components, matching the needs of the conducted analysis, where it is further possible to produce individual components of the set of different materials. Depending on the reagents employed, the material used to produce the set can be adapted to the needs of the conducted analysis. The solution according to the invention enables using the fraction collector D as the container for the sample subject to analysis, e.g. in the autosampler of the liquid chromatograph, without the need to transfer the fluid, which substantially facilitates the analysis and shortens its time.

[0021] Preferably, the set according to the invention is produced of materials of the thermoplastic group, polypropylene in particular, due to its chemical resistance and accessibility for the additive manufacturing process. Other possible materials include polyamide (PA, nylon) of high abrasion resistance, or polyether ether ketone (PEEK) of high resistance to temperature.

[0022] Also identified in the course of the research works were optimal parameters of additive manufacturing which ensure that the obtained model will be water-tight, the tightness also attained in between the layers of the deposited building material.

[0023] Preferably, according to the invention the model fill density is 100%, the number of the model contour paths is 3, and the thickness of the layer falls within the range of 0.1 mm to 0.3 mm, 0.1 mm being most preferable.

[0024] Preferably, according to the invention the temperature of extrusion of the melted material falls within the range of 205°C and 220°C, 205°C being most preferable

[0025] Preferably, according to the invention the temperature of the print bed falls within the range of 85°C to 100°C, 85°C being the most preferable.

[0026] Application of additive manufacturing technologies to produce elements of the extraction set according to the invention ensures precise fitting of all its members and opens the opportunity of producing the set ‘in situ’, to match the needs, i.e. rescaling the dimensions so as to match the conditions of the analysis.

[0027] Furthermore, the solution according to the invention eliminates the problem of separating the sorbent from the fluid.

[0028] An advantage of the solution according to the invention also consists in the possibility of matching the filter to the size of the sorbent particles and the chemical nature of the extracted analyte. Preferably, in the set according to the invention the diameter of the filter Bl is 9.5 mm.

[0029] Preferably, the capacity of the extraction chamber B falls within the range up to 2 ml, 1.5 ml being the most preferable. [0030] The dispersive extraction set according to the invention can be applied as disposable equipment; alternatively, it can be reused after cleaning.

[0031] Preferably, used to clean the set are detergents containing at least 5-15% of anionic surface-active substances and <5% of non-ionic surface-active substances; purified water.

[0032] Preferably, the method employed to clean the set according to the invention is that of mechanical cleaning, i.e. cleaning in ultrasonic bath for 60 minutes. According to the tests conducted by the Inventors, elements of the set remain fully functional after 20 washing cycles. Therefore, the set according to the invention is highly economical in operation and environmentally friendly.

[0033] Another subject matter of the invention is the dispersive extraction method which is characterised in that it is performed using the system according to the invention, and the procedure comprises the following successive stages, where: a. at stage one, the extraction set is prepared, composed of four members: the cap A, the extraction chamber B, the bottom closure C, the fraction collector D, and the filter Bl; b. at stage two, screwed into the lower part of the extraction chamber B, fitted with the filter Bl, is the bottom closure C, and the liquid sample and sorbent, plus an optional magnetic stirrer are placed in the extraction chamber B, whereupon the cap A is screwed on the upper part of the extraction chamber B c. at stage three, the content is stirred in the extraction chamber B closed on both sides, and during the stirring sorption of the analyte from the sample takes place; d. at stage four, the extraction chamber B is rotated 180° to the position where the bottom closure C is at the top, following which the bottom closure C is unscrewed and the fraction collector D is screwed in to replace it; e. at stage five, the extraction chamber B is rotated 180° to the position where the extraction chamber B finds itself above the fraction collector D, whereupon the fraction is collected; f. at stage six, the fraction collector D is unscrewed, and the obtained filtrate disposed of or left for further analysis.

[0034] In one of the variants of the method according to the invention filter B 1 with additional sorption properties is employed, where in the variant the sorbent and the magnetic stirrer are not placed in the extraction chamber B at stage b).

[0035] Preferably, in the method according to the invention the stirring of the sample at stage c) is carried out manually, or by mechanical shaking, or with a magnetic stirrer or ultrasound bath. In another variant of the method according to the invention the sample is left for some time undisturbed instead of being stirred.

[0036] Preferably, in the method according to the invention the collection of the fraction at stage e) is performed gravitationally or supported with centrifuging.

[0037] The dispersive extraction method performed using the set according to the invention allows its optimisation in terms of efficiency and repeatability. At the sorption stage, the analyte binds with the sorbent, then the sorbent is separated from the liquid sample remains, following which the analyte is recovered by desorption of the sorbent. The efficiency of the extraction is construed as the ratio of the analytical signal from the sample of the recovered analyte and the analytical signal from the original sample. In the case of multiple repetitions (a series of samples), the accuracy of the method is observed when the results obtained for the repetitions are similar. In the case of dispersive extraction, which is a multiple-step method, each stage (accuracy of the weighing, preparation of the solutions, the analyst’s work, sameness of the sets, etc.) may contribute to the repeatability error.

[0038] The advantage of the dispersive extraction performed using the set according to the invention consists in ensuring swift and successful separation of the sorbent from the fluid. Moreover, the method according to the invention does not require evaporation of the solvent remains, which consequently enables elimination of the problem of that lengthy and costly phase of the analysis, plus reduces the occurrence of matrix impurities in the extract.

[0039] Compared to the traditional extraction methods, the method according to the invention is easier to perform, more effective in terms of the quality of the analysis (efficiency and repeatability), takes less time, and is cheaper. A major advantage of the method according to the invention consists in that dispersive extraction can be performed in a simple and repeatable way, which substantially broadens application of the dispersive extraction method in analytics, where today its application is limited solely to sample clean-up.

Brief Description of Drawings

[0040] The subject matter of the invention described in its embodiment is shown on the figures where: Fig.l

[0041] [Fig-1] presents individual elements of the set in axial section in two variants of their arrangement;

Fig.2

[0042] [Fig.2] presents schematically the way dispersive extraction is performed using a magnetic stirrer as the stirring method, where Fig 2A presents the sorption stage, and Fig 2B presents the desorption stage; Fig.3

[0043] [Fig.3] presents the dispersive extraction set produced by the technique of fused deposition modelling with polypropylene material.

Description of Embodiments

[0044] The solution according to the invention is illustrated in an embodiment which does limit the scope of the invention.

Examples

[0045] [Embodiment 1]

[0046] The dispersive solid-phase extraction sets were prepared by the method of additive manufacturing in the technology of depositing melted polypropylene material. The dimensions of individual set members were matched to fit the standardised dimensions of the commercially available columns, where the capacity of the extraction chamber B was 2 ml.

[0047] The following printing parameters were employed: a. layer height: 0.1 mm; b. fill density: 100%; c. infill pattern: concentric; d. printing temperature: 205°C; e. print bed temperature: 85°C; f. printing speed: 25 mm/s; g. ventilator speed: 20%.

[0048] Once all parts of the set had been produced, the fitting of all its elements was checked.

[0049] Then, the set was used to perform solid phase extraction.

[0050] The extraction was performed using three solvents: water at the sorption stage, plus methanol and isopropanol at the desorption stage, and three powdery sorbents: silica (Sigma-Aldrich), C8-chain modified silica (Sigma-Aldrich), and Phree™ Phospholipid Removal (Phenomenex). Tested was the aqueous solution of imipramine at the concentration of 10 pg/ml.

[0051] The filter (Bl) was placed in the lower part of the extraction chamber (B) which was then screwed into the bottom closure (C). Used as the filter (Bl) was the SPE packed- bed filter of the frit type (filter diameter: 9.5 mm (Agilent Technologies)). Put in the extraction chamber closed from the bottom were: 995 pl of water as the sorption solvent, 5 pl of imipramine solution at the concentration of 10 pg/ml, 30 mg of sorbent, and a magnetic stirrer. The set was closed from the top with the cap (A) and placed on the magnetic stirrer which stirred the content of the extraction chamber for 15 min. (1000 rpm) with the sorption process occurring during the stirring. When the sorption had been completed, the set was rotated 180° so that the bottom closure (C) found itself at the top, which made it possible to unscrew and replace it with the fraction collector (D). Upon exchange of the set elements, the fluid was collected by rotating the set by 180° so that the fraction collector (D) found itself below the extraction chamber (B). The liquid was centrifuged (3000 RCF, 1 min.). Once the collection of the liquid was completed, the fraction collector (D) was unscrewed and replaced with the bottom closure (C). The fraction from the collector (D) was disposed of. Then, the cap (A) was unscrewed and 1 ml of desorption solvent was added to the extraction chamber (B). The set was closed again with the cap and placed on a magnetic stirrer for 15 min. (1000 rpm) where the desorption process occurred during the stirring. Following completion of the desorption stage, the set was rotated 180° so that the bottom closure (C) would come to the top to be replaced with the fraction collector (D). When the set elements had been exchanged, the liquid was collected by rotating the set 180° so that the fraction collector (D) would find itself below the extraction chamber (B). The fluid drained down by gravity. Then, the fraction collector (D) was unscrewed and the fraction collected for further chromatographic analysis.

[0052] In order to assess the dispersive liquid-solid extraction efficiency, the sample was subject to chromatographic analysis. The samples were analysed by the liquid chromatography technique combined with mass spectrometry (LC-MC), where concentration of imipramine in the samples before extraction and following desorption was measured, thus identifying the efficiency of the extraction performed using the set. The results are shown in table 1.

[0053] [Table 1]

[0054] In addition, a test was conducted to compare the efficiency of the dispersive extraction performed using the extraction set the invention concerns and that performed without the set. The sorbent used was the C18-chain modified silica (Sigma- Aldrich), and the solvents used were: water (at the sorption stage) and methanol (at the desorption stage), whilst the analyte was the remaining imipramine in the con- centration of 10 i tg/ml. The procedure of dispersive extraction performed using the set the invention concerns was as described above. The dispersive extraction without using the extraction set the invention concerns was performed in tubes of the Eppendorf type, 2 ml in volume.

[0055] Extraction without using the set according to the invention

[0056] Placed in an Eppendorf tube were: 995 pl of sorption solvent, 5 pl of imipramine solution, 30 mg of sorbent, and a magnetic stirrer. The tube was positioned on a magnetic stirrer and its content stirred for 15 min. (1000 rpm) with the sorption process occurring over the time. Following completion of the sorption stage the tube was centrifuged (15000 RCF, 7 min.) and the fluid collected from above the deposit, the latter fraction disposed of. Then, 1 ml of desorption solvent was added to the tube. The tube was again positioned on the magnetic stirrer and subject to stirring for 15 min. (1000 rpm) where desorption took place in the process. Upon completion of the desorption stage the tube was centrifuged (15000 RCF, 7 min.) and the fluid from above the deposit collected as the fraction intended for further chromatographic analysis preceded with repeated centrifuging (15000 RCF, 7 min.) to remove the remaining particles of the sorbent.

[0057] The samples, following extraction performed by the method according to the invention and using the extraction set, and without the set used, were analysed by the liquid chromatography technique combined with mass spectrometry (EC-MS), where the imipramine concentration in the samples was determined before extraction and after desorption, thus identifying the efficiency of the extraction performed with and without the extraction set. The %RSD denotes the percent standard deviation.

[0058] The repeatability of the method according to the invention was expressed as a standard deviation of the result series, divided by the average result for the series and expressed in percent (%RSD). The results are presented in table 2.

[0059] [Table 2]

[0060] The results obtained prove that efficiency of extraction performed by the method according to the invention, using the dedicated extraction set according to the invention, is higher compared to extraction performed without the set according to the invention. When comparing the conditions under which both methods were applied, one can note that imipramine extracted without using the set required higher rotation speeds and longer centrifugation, plus repeating the process to remove the sorbent particles which, if left in the sample, could damage the chromatograph during the analysis. Moreover, when extraction was performed without using the set according to the invention it was impossible to eliminate the remains of the solvent from the sorption stage altogether, and its maximum effective removal involved a loss of the sorbent. No such problem, on the other hand, was faced when using the set according to the invention.

[0061] Unlike in dispersive extraction performed in Eppendorf tubes, application of various sorbents and solvents of different densities does not require adjusting the conditions of dispersive extraction, if performed using the set according to the invention, which also testifies to its universality.

[0062] [Embodiment 2]

[0063] Solid phase extraction (SPE)

[0064] The extraction set according to the invention, employed in Embodiment 1 was cleaned with detergent, flushed with distilled water, and dried. The set was used again in solid phase extraction.

[0065] Once the elements of the set had been dried, the set was checked for the fitting of all its parts. Then, solid phase extraction and chromatographic analyses were performed to assess the efficiency of imipramine solid phase extraction in the variant using the extraction set according to the invention as the extraction column and in the variant using the commercially available SPE columns.

[0066] The extraction was performed using water as the solvent at the sorption stage and methanol as the solvent at the desorption stage. The bed was of C18-chain modified silica (Sigma- Aldrich), the same as the bed in the commercial SPE columns. Tested was aqueous solution of imipramine at the concentration of 100 ng/ml.

[0067] The procedure for each sample consisted in using the dispersive extraction set being the subject matter of this invention in the way the commercially available solid phase extraction (SPE) columns are used. The filter (Bl) was placed in the bottom part of the extraction chamber (B) and the latter was screwed onto the fraction collector (D). Used as the filter (Bl) was the SPE packed-bed filter of the frit type (9.5 mm (Agilent Technologies)). Placed in the extraction chamber (B) was 60 mg of the sorbent to form the bed. Then, 1.5 ml of methanol was added to the extraction chamber (B) to moisten the bed. The set was centrifuged briefly (3000 RCF, 1 min.) making sure that the bed remained moist. The fluid collected from the fraction collector (D) was disposed of. Then, the procedure was repeated using 1.5 ml of water, and the fluid collected from the fraction collector (D) was disposed of again. Placed on the bed in the extraction chamber (B) was 1.5 ml sample of aqueous solution of imipramine at the concentration of 100 ng/ml, centrifuged briefly once more, and the fluid collected from the fraction collector (D) was disposed of. The subsequent step consisted in placing 1.5 ml of water in the extraction chamber to flush the bed, centrifuging it briefly, and discarding the fluid from the fraction collector (D). Then, 1.5 ml of methanol was placed as eluent, then centrifuged, and the fraction from the fraction collector (D) was collected for further chromatographic analysis.

[0068] The SPE extraction using the commercially available SPE columns was performed following the manufacturer’s instructions, using the apparatuses dedicated to the method.

[0069] The obtained samples were analysed by the liquid chromatography technique combined with mass spectrometry (LC-MC), where concentration of imipramine in the samples before extraction and following desorption was measured, thus identifying the efficiency of the extraction performed using the set according to the invention and the commercial SPE columns. The results are shown in table 3.

[0070] [Table 3]

[0071] The efficiency of solid phase extraction of imipramine using the extraction set according to the invention and the commercially available SPE columns is comparable. This means that the set according to the invention may be an alternative to the commercially available set, where the set according to the invention ensures simplicity of the extraction method without the need to employ dedicated apparatuses, and lower costs, which in effect translates to broadened availability of dispersive extraction as an analytical method.

Claims

[Claim 1] A set for dispersive solid-phase extraction, comprising an extraction chamber, a filter, and a fraction collector, characterised in that it is composed of a top closure in the form of a cap (A), an extraction chamber (B) fitted with a replaceable filter (Bl) in the lower part, and a bottom closure (C) plus fraction collector (D), where all set components are produced by employing additive manufacturing technologies.

[Claim 2] The set according to claim 1 characterised in that the components of the set are produced by the additive manufacturing in the technology of gradual deposition of melted material, layer after layer, where the material is a thermoplastic material.

[Claim 3] The set according to claim 2 characterised in that the thermoplastic material includes polypropylene, polyamide, or polyether ether ketone.

[Claim 4] The dispersive extraction method using the set according to any of the claims 1 or 2, characterised in that it follows successive stages, where: a. at stage one, the extraction set is prepared, composed of four members: the cap (A), the extraction chamber (B), the bottom closure (C), the fraction collector (D), and the filter (Bl); b. at stage two, screwed into the lower part of the extraction chamber (B), fitted with the filter (Bl), is the bottom closure (C), and optionally, the liquid sample and sorbent, plus a magnetic stirrer are placed in the extraction chamber (B), whereupon the cap (A) is screwed on the upper part of the extraction chamber (B); c. at stage three, sorption of the analyte is performed; d. at stage four, the extraction chamber (B) is rotated 180° to the position where the bottom closure (C) is at the top, following which the bottom closure (C) is unscrewed and the fraction collector (D) is screwed in to replace it; e. at stage five, the extraction chamber (B) is rotated 180° to the position where the extraction chamber (B) finds itself above the fraction collector (D), whereupon the fraction is collected; f. at stage six, the fraction collector (D) is unscrewed, and the obtained filtrate disposed of or left for further analysis.

[Claim 5] The method according to claim 3 characterised in that at stage c), while the analyte is undergoing sorption, the sample is stirred by manual or mechanical shaking, or using a magnetic stirrer or ultrasound bath.

[Claim 6] The method according to claim 4 characterised in that filter B 1 with additional sorption properties is used, where no sorbent or the magnetic stirrer are added at stage b).

[Claim 7] The method according to claim 3 characterised in that the fraction at stage e) is collected gravitationally or supported with centrifuging.