Surface extraction system and method

Abstract

The lipid extraction system (100) includes a handle (102), a tube (104), and a solvent (106). The handle (10)2 includes a vacuum system. The tube (104) includes an aperture (108) configured to accept a lipid therethrough. The tube (104) is coupled to the handle (102). The tube (104) is configured to retain the solvent (106) while collecting the lipid from the sample. More specifically, the aperture (108) of the tube (104) retaining the solvent may contact a surface of a sample, thus extracting the lipid of the sample into the solvent (106) within the tube. The lipid collected in the retained solvent (106) may be dispensed in a testing container (110) for analysis, such as a multiple reaction monitoring (MRM) profiling analysis.

Description

SURFACE EXTRACTION SYSTEM AND METHODCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 638,251 filed April 24, 2024, the entirety of which is hereby incorporated by reference.FIELD

[0002] The disclosure generally relates to surface extraction systems and, more particularly, to systems for biomaterial and biofluid surface extraction.INTRODUCTION

[0003] This section provides background information related to the present disclosure which is not necessarily prior art.

[0004] Conventional liquid chromatography-mass spectrometry (LC-MS / MS) or shotgun lipidomics workflows involve biphasic liquid-liquid extraction methods such as Bligh & Dyer, Folch or based on methyl tert-butyl ether (MTBE). The Bligh & Dyer method is a widely used and an ingenious liquid-liquid technique based on creating a sample (water)-chloroform-methanol ratio to extract lipids as a one-phase solution and by changing the solvent ratios, the solution becomes biphasic, concentrating apolar, mostly lipid compounds into the bottom chloroform layer and polar molecules, represented by metabolites in the upper phase. The Folch method is similar to the Bligh & Dyer, and it uses a chloroform-methanol solvent mixture to extract lipids. One-phase methods are of interest for the lipidomics field since they are fast, cheaper, and easily automated. Nonetheless, they yield lipid extracts containing salts and polar metabolites that can compete with the lipids or create artifacts for the ionization and interferences in the full mass scan.

[0005] While traditional methods are effective, surface extraction methods that promote ionization and are directly coupled to mass spectrometry analysis allow for in situ sampling and near real-time analysis of small molecules. Such approaches are known as ambient mass spectrometry methods and desorption electrospray ionization (DESI), direct analysis in real-time (DART), liquid extraction surface analysis (LESA), rapid evaporative ionization mass spectrometry (REIMS), and the Mass Spec Pen are known examples of it. By DESI, a spray of primary charged droplets is directed to the sample’s surface, forming a microfilm where the surface desorptionoccurs. Sputtered secondary droplets containing molecules desorbed from the surface rapidly dry, generating ions in the gas phase. Additionally, DESI secondary microdroplets can work as microreactors, accelerating chemical reactions by up to 106times. DART is based on vibronically excited-state species from helium, argon, or nitrogen that ionize atmospheric molecules or dopant molecules, inducing ion-molecule reactions with the sample molecules that produce ions. LESA combines surface extraction followed by nano-electrospray ionization, while by REIMS samples are cauterized by diathermy and the resultant aerosols are collected, ionized, and analyzed by mass spectrometry. The Mass Spec Pen comprises a handheld sampling probe for gentle time- and volume-controlled extraction of molecules from a tissue sample using a discrete water droplet which is transferred by vacuum to a heated capillary for ionization and high mass resolution mass spectrometry.

[0006] Multiple reaction monitoring (MRM) profiling is a type of shotgun lipid profiling method since it does not include liquid chromatography. Instead of acquiring a full mass scan or product ion scan as for most lipid profiling methods, lipid class-diagnostic and fatty acid product ions or fatty acyl neutral losses typically obtained using neutral loss or product ion scan are combined as ion transition. Monitoring a list of ion transitions and looking at it as a lipid profile at lipid species level of structure definition is performed using more robust and affordable low mass resolution, while highly sensitive mass spectrometers. This approach is especially suitable to minute samples such as preimplantation mammalian embryos or exosomes.

[0007] So far, MRM profiling has been applied in samples processed by Bligh & Dyer lipid extraction. These known methods of lipid extraction require sample preparations and online mass spectrometry analysis. Another known method of lipid extraction is Liquid Extraction Surface Analysis (LESA). LESA dispenses a solvent onto a sample. Afterwards, the solvent may be recovered into a tube, thus collecting the lipid of the sample within the solvent. The sample containing solvent may then be dispensed into the testing tube for analysis. Lastly, one more known method of lipid extraction may include Rapid Evaporative Ionization Mass Spectrometry (REIMS). REIMS is an ionization method based on vaporizing the sample.

[0008] Accordingly, there is a continuing need for a lipid extraction system and method that may be more efficient than known lipid extraction systems. Desirably, the lipid extraction system may enable online mass spectrometry analysis.SUMMARY

[0009] In concordance with the instant disclosure, a lipid extraction system and method that is more efficient than known methodologies and also enables online mass spectrometry analysis, has surprisingly been discovered.

[0010] The present disclosure provides a lipid extraction system and method which may include a handle and a tube configured to collect a lipid from a sample. In certain circumstances, the lipid extraction system may be provided as a solvent touch extractor (STE). For instance, the tube may be configured to retain a liquid solvent. The tube may be coupled to a first opening in a first terminal end of the tube. A second terminal end of the tube may include a second opening. The handle may include a vacuum system. The vacuum system of the handle may be used to draw the liquid solvent into the second opening of the tube. The lipid may then be collected by touching the second terminal end of the tube to the surface of the sample, thus disposing the lipid of the sample into the liquid solvent disposed within the tube. The lipid of the sample may be dispensed from the tube by releasing the vacuum of the handle. The lipid of the sample may be released into a testing container for analysis. Provided as a non-limiting example, the tube may be provided as a pipette tip.

[0011] Further areas of applicability will become apparent from the description provided herein. The description and specific examples in this summary are intended for purposes of illustration only and are not intended to limit the scope of the present disclosure.DRAWINGS

[0012] The drawings described herein are for illustrative purposes only of selected embodiments and not all possible implementations and are not intended to limit the scope of the present disclosure.

[0013] FIG. 1A is a front elevational view of a lipid extraction system having a handle, a tube, and a solvent solution, according to one embodiment of the present disclosure;

[0014] FIG. IB is a front perspective view of the lipid extraction system utilizing the solvent touch extraction technique where the solvent is disposed within the tube and an opening of the tube containing the solvent contacts a surface of the sample, according to one embodiment of the present disclosure;

[0015] FIG. 1C is an enlarged front perspective view of the lipid extraction system utilizing the solvent touch extraction technique, as shown in callout A in FIG. IB, where the solvent is disposed within a tube and an opening of the tube containing the solvent contacts a surface of the sample, according to one embodiment of the present disclosure;

[0016] FIG. ID is a front perspective view of the lipid extraction system utilizing the solvent touch extraction technique, as shown in FIG. 1C, where the lipid is extracted from the sample through the opening of the tube while the solvent is retained in the tube, according to one embodiment of the present disclosure;

[0017] FIG. IE is a front elevational view of the lipid extraction system dispensing the lipid containing solvent solution, as shown in FIG. ID, into a testing container, according to one embodiment of the present disclosure;

[0018] FIG. 2 is a hierarchical clustering combined with heatmap for visualizing the three types of samples, where the samples represent the effect of the beef color or cooking, according to one embodiment of the present disclosure;

[0019] FIG. 3 is a bar graph illustrating the number of MRMs detected by MRM profiling in a diluted solvent used for surface touch extraction (STE) in beef and chicken samples pre- vs. postthawing samples, where two different types of solvent combinations were used for STE: 100% MeOH and 100% ACN, according to one embodiment of the present disclosure;

[0020] FIG. 4 is a hierarchical clustering combined with heatmap for the visualization of the discrimination of two types of samples, where pre- vs. post-thawed samples were compared using 100% MeOH and 100% ACN as the solvents for the STE, according to one embodiment of the present disclosure;

[0021] FIG. 5 is a hierarchical clustering combined with heatmap for visualizing the discrimination of two types of samples, where the sampled spots were related to the beef color [brown beef color (red label) and red beef color (green label)] and these were clustered according to the top 10 most significant features, according to one embodiment of the present disclosure;

[0022] FIG. 6 is a principal component analysis (PCA) scores plot showing 10 replicates collected by STE on the same beefsteak sample by three individuals and analyzed by MRM profiling, where variability within the same individual and within different individuals is observed, further depicting where it is clear that the blue-labeled subject was able to yield more similar lipidprofiles compared to the red-labeled individual, according to one embodiment of the present disclosure;

[0023] FIG. 7 is a bar graph illustrating the number of MRMs related to lipids related to different classes detected in beef samples using the samples and the lipid extraction system described in the present disclosure, according to one embodiment of the present disclosure; and

[0024] FIG. 8 is a flow chart of a method for using the lipid extraction system, according to one embodiment of the present disclosure.DETAILED DESCRIPTION

[0025] The following description of technology is merely exemplary in nature of the subject matter, manufacture and use of one or more inventions, and is not intended to limit the scope, application, or uses of any specific invention claimed in this application or in such other applications as may be filed claiming priority to this application, or patents issuing therefrom. Regarding methods disclosed, the order of the steps presented is exemplary in nature, and thus, the order of the steps can be different in various embodiments, including where certain steps can be simultaneously performed. “A” and “an” as used herein indicate “at least one” of the item is present; a plurality of such items may be present, when possible. Except where otherwise expressly indicated, all numerical quantities in this description are to be understood as modified by the word “about” and all geometric and spatial descriptors are to be understood as modified by the word “substantially” in describing the broadest scope of the technology. “About” when applied to numerical values indicates that the calculation or the measurement allows some slight imprecision in the value (with some approach to exactness in the value; approximately or reasonably close to the value; nearly). If, for some reason, the imprecision provided by “about” and / or “substantially” is not otherwise understood in the art with this ordinary meaning, then “about” and / or “substantially” as used herein indicates at least variations that may arise from ordinary methods of measuring or using such parameters.

[0026] Although the open-ended term “comprising,” as a synonym of non-restrictive terms such as including, containing, or having, is used herein to describe and claim embodiments of the present technology, embodiments may alternatively be described using more limiting terms such as “consisting of’ or “consisting essentially of.” Thus, for any given embodiment reciting materials, components, or process steps, the present technology also specifically includes embodimentsconsisting of, or consisting essentially of, such materials, components, or process steps excluding additional materials, components or processes (for consisting of) and excluding additional materials, components or processes affecting the significant properties of the embodiment (for consisting essentially of), even though such additional materials, components or processes are not explicitly recited in this application. For example, recitation of a composition or process reciting elements A, B and C specifically envisions embodiments consisting of, and consisting essentially of, A, B and C, excluding an element D that may be recited in the art, even though element D is not explicitly described as being excluded herein.

[0027] As referred to herein, disclosures of ranges are, unless specified otherwise, inclusive of endpoints and include all distinct values and further divided ranges within the entire range. Thus, for example, a range of “from A to B” or “from about A to about B” is inclusive of A and of B. Disclosure of values and ranges of values for specific parameters (such as amounts, weight percentages, etc.) are not exclusive of other values and ranges of values useful herein. It is envisioned that two or more specific exemplified values for a given parameter may define endpoints for a range of values that may be claimed for the parameter. For example, if Parameter X is exemplified herein to have value A and also exemplified to have value Z, it is envisioned that Parameter X may have a range of values from about A to about Z. Similarly, it is envisioned that disclosure of two or more ranges of values for a parameter (whether such ranges are nested, overlapping, or distinct) subsume all possible combination of ranges for the value that might be claimed using endpoints of the disclosed ranges. For example, if Parameter X is exemplified herein to have values in the range of 1-10, or 2-9, or 3-8, it is also envisioned that Parameter X may have other ranges of values including 1-9, 1-8, 1-3, 1-2, 2-10, 2-8, 2-3, 3-10, 3-9, and so on.

[0028] When an element or layer is referred to as being “on,” “engaged to,” “connected to,” or “coupled to” another element or layer, it may be directly on, engaged, connected, or coupled to the other element or layer, or intervening elements or layers may be present. In contrast, when an element is referred to as being “directly on,” “directly engaged to,” “directly connected to” or “directly coupled to” another element or layer, there may be no intervening elements or layers present. Other words used to describe the relationship between elements should be interpreted in a like fashion (e.g., “between” versus “directly between,” “adjacent” versus “directly adjacent,” etc.). As used herein, the term “and / or” includes any and all combinations of one or more of the associated listed items.

[0029] Although the terms first, second, third, etc. may be used herein to describe various elements, components, regions, layers and / or sections, these elements, components, regions, layers and / or sections should not be limited by these terms. These terms may be only used to distinguish one element, component, region, layer or section from another region, layer, or section. Terms such as “first,” “second,” and other numerical terms when used herein do not imply a sequence or order unless clearly indicated by the context. Thus, a first element, component, region, layer, or section discussed below could be termed a second element, component, region, layer, or section without departing from the teachings of the example embodiments.

[0030] Spatially relative terms, such as “inner,” “outer,” “beneath,” “below,” “lower,” “above,” “upper,” and the like, may be used herein for ease of description to describe one element or feature's relationship to another element(s) or feature(s) as illustrated in the figures. Spatially relative terms may be intended to encompass different orientations of the device in use or operation in addition to the orientation depicted in the figures. For example, if the device in the FIG. is turned over, elements described as “below”, or “beneath” other elements or features would then be oriented “above” the other elements or features. Thus, the example term “below” can encompass both an orientation of above and below. The device may be otherwise oriented (rotated 90 degrees or at other orientations) and the spatially relative descriptors used herein interpreted accordingly.

[0031] As shown in FIGS. 1A-1D, the lipid extraction system 100 may include a handle 102 and a tube 104 configured to collect a lipid from a sample. The tube 104 may include an aperture 106 configured to accept the lipid of the sample. The lipid extraction system 100 may be provided as a solvent touch extractor used in accordance to a solvent touch extractor (STE) method 200. For instance, the tube 104 may be configured to retain a liquid solvent 108. The tube 104 may be coupled to a first opening in a first terminal end of the tube 104. A second terminal end of the tube 104 may include the aperture 106, which may otherwise be named a second opening. The handle 102 may include a vacuum system. As shown in FIGS. 1A and FIG. 8, the vacuum system of the handle 102 may be used to draw the liquid solvent 108 into the aperture 106 of the tube 104. The aperture 106 may have a diameter between 0.5 pm to 200 pm. In a specific example, the aperture 106 may have a diameter between 0.5 pm to 150 pm. In a more specific example, the aperture 106 may have a diameter between 0.5 pm to 75 pm. As shown in FIGS. 1B-1D and 8, the lipid may then be collected by touching or otherwise contacting the second terminal end of the tube 104 to a surface of the sample, thus disposing the lipid of the sample through the aperture 106 into the liquidsolvent 108 disposed within the tube 104. As shown in FIGS. IE and 8, the lipid of the sample may then be dispensed from the tube 104 by releasing the vacuum of the handle 102. More specifically, the lipid of the sample may be released into a testing container for analysis. It is contemplated that the tube 104 may be provided as any containment structure having the aperture 106 and that may also retain the solvent 108 during the collection of the lipid. Provided as a non-limiting example, the tube 104 may be provided as a pipette tip. For instance, the solvent 108 may be kept inside a large-orifice tip while contacting the surface of the sample for a few seconds while molecules present in the surface of the sample are transferred to the solvent 108 inside the tip. A skilled artisan may select other suitable ways for providing the solvent touch extractor 100, within the scope of the present disclosure.

[0032] Provided as a non-limited example, the solvent touch extractor 100 was provided in the following manner for experimental testing. The solvent touch extractor 100 was utilized with manual collection using a 200 pL large orifice tip. For this, 20 pL of solvent 108 was loaded into the tip, and the tip was placed in contact with the sample, ensuring that the solvent 108 inside the tip came into contact with the sample for about 5 seconds, as shown in FIGS. 1 A-1D. This period of time was found to be sufficient for the molecules to be transferred to the solvent 108. It is contemplated that other periods of time may be selected. Then, as shown in FIG. IE, the sample was transferred to a testing tube 110 and diluted 10 times with ACN:MeOH:NH4Ac 300mM (3:6.5:0.35, v / v). Other dilutions methods may be selected, as desired. Finally, the sample was loaded into an autosampler (Agilent Technologies, G1377A) for flow injection as part of the MRM profiling data acquisition workflow. Using the input of the LESA experiment with different solvent combinations, STE 200 was performed using either ACN or MeOH to evaluate if lipid profiles would be affected by one freeze-thaw cycle of beef and chicken meat samples. For this, sample collection was first performed using STE 200 on fresh chicken and beef meat samples. Then, the samples were frozen at -20°C for forty-eight hours and thawed at 4°C for four hours before resampling. Ten different spots were sampled across each sample.

[0033] Also, the STE 200 was performed in a beef sample displaying regions of both brown and red color. The STE 200 experiments were performed using 20 pL of ACN, as described above. Five different spots across each sample were sampled.

[0034] In order to evaluate the reproducibility of the STE 200, three different individuals performed STE 200 in 10 different spots of the same sample beef using ACN as solvent 108.

[0035] Provided as a non-limiting example, the lipid extraction system 100 of the present disclosure was experimentally tested as outlined below and compared to known extraction methods. The MRM profiling lipid panel was performed with a discovery phase. In summary, 100 mg of beef was extracted using the Bligh & Dyer method. The discovery phase was used to screen lipid extracts from the meat sample for 3200 MRMs related to acyl-carnitines (AC), cholesterol esters (CE), ceramides (CER), diacylglycerols (DAG), free fatty acids (FFA), phosphatidylcholine (PC), lysophosphatidylcholine (LPC), phosphatidylethanolamines (PE), lysophosphatidylethanolamine (LPE), phosphatidylglycerols (PG), phosphatidylinositols (PI), lysophosphatidylinositols (LPI), phosphatidylserines (PS), and triacylglycerols (TG) at species level. Then, MRMs with ion signals less than 30% higher than those in the blank sample were considered noise and removed from the discovery phase. This resulted in the selection of 679 lipids, which were then used for the screening of each sample. Data acquisition was performed using flow-injection (no chromatographic separation) from 10 pL of the diluted lipid extract stock solution delivered using a microautosampler (G1377A) to the ESI source of an Agilent 6410 triple quadrupole mass spectrometer (Agilent Technologies, Santa Clara, CA, USA). A capillary pump was connected to the autosampler and operated at a flow rate of 8 pL / min and pressure of 150 bar. The capillary voltage on the instrument was 5 kV and the gas flow 5.1 L / min at 300°C.

[0036] The STE method 200 was performed with manual collection using a 200 pL large orifice tip. For this, 20 pL of solvent 108 was loaded into the tip, and the tip was placed in contact with the sample, ensuring that the solvent 108 inside the tip came into contact with the sample for about 5 seconds, as shown in FIGS. 1 A-1D. This period was found to be sufficient for the molecules to be transferred to the solvent 108. Then, the sample was transferred to a testing tube 110 and diluted 10 times with ACN:MeOH:NH4Ac 300mM (3:6.5:0.35, v / v). One skilled in the art may select other suitable dilution protocols, within the scope of the present disclosure. Finally, the sample was loaded into an autosampler for flow injection as part of the MRM profiling data acquisition workflow as described above. Using the input of the LESA experiment with different solvent combinations, STE 200 was performed using either ACN or MeOH to evaluate if lipid profiles would be affected by one freeze-thaw cycle of beef and chicken meat samples. For this, sample collection was first performed using STE 200 on fresh chicken and beef meat samples. Then, the samples were frozen at -20°C for 48 hours and thawed at 4°C for 4 hours before resampling. Ten different spots across each sample.

[0037] Also, the STE 200 was performed in a beef sample displaying regions of both brown and red color. The STE 200 experiments were performed using 20 pL of ACN, as described above. Five different spots across each sample were sampled.

[0038] In order to evaluate the reproducibility of the STE 200, three different individuals performed STE 200 in 10 different spots of the same sample beef using ACN as solvent 108.

[0039] Following MRMs profiling data processing as described above, the maximum ion signal obtained for a given MRM was divided by the ion signal of the blank sample. The MRMs that did not produce at least 30% higher ion signals than those of the blank sample in at least one of the samples were considered background and removed from the statistical analysis. Next, to obtain the relative amounts, the ion intensity value of each MRM was divided by the sum of ion intensity for the whole sample. MetaboAnalyst 5.0 was used for heatmap / cluster analysis. After relative ion amounts were uploaded, data were normalized by autoscaling (mean-centering and dividing by the standard deviation of each variable). Multivariate statistics included principal component analysis (PCA) and cluster analysis (heatmaps).

[0040] We observed a limitation of the LESA method for beef sampling which is the immediate dispersion of the solvent dispensed onto the sample surface due to the natural irregularities of the sample. Therefore, lower recovery of the solvent volume dispensed occurred. The variation in the volume of solvent recovered likely resulted in inconsistent results between not acidified and acidified solvents, as well as some disproportional results, such as the average low number of MRMs detected with ACN:MeOH:NH4Ac 300mM (3:6.5:0.35, v / v) vs. the high number of MRMs detected using ACN:MeOH:NH4Ac (3:6.5:0.35, v / v) + ACN 0.1%.

[0041] To avoid losing part of the solvent used in LESA due to the natural irregularities of the beef samples’ surface and to facilitate future automation, the solvent 108 was retained inside a tube with a large orifice tip and the tip was gently pressed against the sample for about 5 seconds. One skilled in the art may select other suitable timeframes to contact the retained solvent 108 with the surface of the sample, within the scope of the present disclosure.

[0042] As shown in FIG. 2, STE followed by MRM profiling was used to assess the number of detected lipids and the discrimination between beef and chicken breast meat which were fresh or after one freeze-thaw cycle as features. The volume of 20 pL of two types of solvents 108, namely 100% ACN or 100% MeOH were used. After STE 200, the solvent 108 volume was diluted 7 times (into 120 pL) of ACN: MeOH :NH4Ac 300mM (3:6.5:0.35, v / v) for sample injection.

[0043] For both beef and chicken samples, 100% ACN provided a higher number of detected MRMs (295 and 278 for beef and chicken meat samples, respectively) compared to 100% MeOH (213 and 153, respectively). Also, at proportion levels, 100% ACN was more effective than 100% MeOH in recovering TAGs, DAGs, and PCs for both beef and chicken breast samples, as shown in FIG. 3. These results may be related not only to the different physical properties of the solvents 108 since part of the MeOH used for STE 200 transferred to the sample due to its low viscosity and high volatility, likely causing loss of part of the lipids extracted in the solvent 108 inside the tip, which was used for the analysis. Using ratios of MeOH:water may avoid this limitation, but the extraction efficiency, especially for the neutral lipids such as TAG, DAG and CEs, may decrease.

[0044] To assess the efficiency of STE 200 followed by MRM profiling in discriminating the samples, a heatmap with cluster analysis was utilized as a visualization tool for pre vs. post freezing samples, as shown in FIG. 4. The use of pure ACN as a solvent 108 in beef and chicken breast samples led to better clustering and a higher number of lipids recovery compared to pure MeOH. This finding may be due to the fact that when the solvent-leaded large orifice tip touched the meat’s surface, part of the MeOH quickly spread through the sample along the tip’s end, due to MeOH lower viscosity and higher volatility compared to ACN.

[0045] Next, we used 100% ACN as the STE 200 solvent 108 to evaluate the types of lipids recovered and if these could provide discrimination between the brown and red regions in a beef sample. Panels of MRMs related to beef color were observed using cluster analysis and heatmap, as shown in FIG. 5.

[0046] Lastly, a reproducibility test was conducted to verify the impact of three individuals during sample collection using STE 200. In this experiment, the same beefsteak sample and solvent 108 used was pure ACN. The PC A (as shown in FIG 6; PCI 41.8% of the variability explained) indicated that manual collection by different individuals may be a source of variability in the small molecule profiles obtained. In that aspect, it is contemplated that a robotic arm could ensure that the process is carried out with a higher degree of consistency, precision, and reproducibility, which is essential for maintaining data uniformity and minimizing variations in the collected samples. One skilled in the art may select other suitable ways for minimizing variations in the small molecule profiles, within the scope of the present disclosure.

[0047] It is important to mention that a limited number of samples was used to evaluate analytical aspects of solvent type and technique used for the surface extraction. It is to be determined if the discrimination efficiency is maintained as different animals and types of meat are compared. Also, the number of ion transitions and to which lipid classes they are related are key aspects to the efficient discrimination and it can be optimized.

[0048] To summarize, the lipid information recovered using beef samples for the different methods evaluated, the average number of MRMs detected, and the lipids classes related were plotted, as shown in FIG. 7. As expected, Bligh & Dyer, used as a reference method, provided the highest number of MRMs detected. Next was LESA, which compared to STE 200, provides higher surface of contact with the sample. As practical observations, due to the natural surface irregularities of beef samples, part of the solvent used for LESA could not be recovered but the relative amount analysis was able to partially correct for that variable since feature discrimination was observed. Using STE 200, loss of solvent 108 is minimal since it is not dispensed, but the surface of contact between the solvent 108 and the sample is smaller.

[0049] Accordingly, surface extraction of lipids from meat samples followed by MRM profiling can provide informative profiles related to color, type of meat, and cooking state. The three types of surface extraction methods have advantages such as easiness and speed, as well as limitations such as sampling variability and the sampling only of superficial molecules. The power of discrimination is still to be accessed in a large number of samples as we just present non-limiting proof-of-concept experiments. Nonetheless, the results indicate that the use of composition information (i.e., relative amounts) compensates for some of the sampling variability. Also, STE 200 is especially suitable to automation, allowing for industrial on-site sampling workflows, which can be combined to shotgun lipidomics and real-time analysis software.

[0050] Example embodiments are provided so that this disclosure will be thorough and will fully convey the scope to those who are skilled in the art. Numerous specific details are set forth such as examples of specific components, devices, and methods, to provide a thorough understanding of embodiments of the present disclosure. It will be apparent to those skilled in the art that specific details need not be employed, that example embodiments may be embodied in many different forms, and that neither should be construed to limit the scope of the disclosure. In some example embodiments, well-known processes, well-known device structures, and well-known technologies are not described in detail. Equivalent changes, modifications and variations of someembodiments, materials, compositions, and methods can be made within the scope of the present technology, with substantially similar results.

Claims

CLAIMSWHAT IS CLAIMED IS:

1. A method of using a lipid extraction system configured to collect a lipid from a surface of a sample, the method comprising the steps of: providing a handle, a tube, and a solvent, the handle includes a vacuum system, the tube is coupled to the handle and having an aperture configured to accept the lipid from the sample; disposing the solvent within the tube via the vacuum system; and contacting the aperture of the tube with the surface of the sample while the solvent is retained within the tube, thus collecting the lipid into the retained solvent through the aperture of the tube.

2. The method of Claim 1, wherein the aperture of the tube contacts the surface of the sample for more than one second.

3. The method of Claim 2, wherein the aperture of the tube contacts the surface of the sample for at least five seconds.

4. The method of Claim 3, further comprising a step of dispensing the lipid containing solvent into a testing container.

5. The method of Claim 4, further comprising a step of performing a multiple reaction monitoring (MRM) profiling analysis.

6. The method of Claim 5, wherein the step of contacting the aperture of the tube with the surface of the sample while the solvent is retained within the tube is done with a robotic arm that is one of semi-autonomously controlled and autonomously controlled.

7. The method of Claim 5, wherein the step of contacting the aperture of the tube with the surface of the sample while the solvent is retained within the tube is done with a robotic arm that is one of semi-autonomously controlled and autonomously controlled.

8. The method of Claim 5, wherein the solvent is acetonitrile.

9. The method of Claim 5, wherein the solvent is methanol.

10. The method of Claim 5, wherein the aperture has a diameter between 0.5 pm to 200 pm.

11. The method Claim 10, wherein the aperture has a diameter between 0.5 pm to 75 pm.

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