Device for solid phase extraction and use method thereof

Abstract

Devices for solid phase extraction are disclosed that include two or more sorbents that remove phospholipids and salts from a sample, thereby eliminating matrix effects during mass spectrometry. In particular, the adsorbents include at least one adsorbent that is water-wettable and contains at least one hydrophobic component and at least one hydrophilic component, and at least one adsorbent that has a specific affinity for matrix interferents such as phospholipids. Methods of using the apparatus of the invention are also disclosed.

Description

[0001] This application is a divisional application of the Chinese invention patent application with the application date of December 16, 2015, application number 201580075258.0, and invention name “Device for solid phase extraction and method of use thereof”.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of and priority to U.S. Provisional Patent Application Nos. 62 / 093,286, filed December 17, 2014, and 62 / 168,559, filed May 29, 2015. The entire application is incorporated herein by reference in its entirety. Technical Field

[0004] The present invention relates to a device for extraction. In particular, the device comprises a variety of adsorbents, thereby providing a simple solid phase extraction and preparation method before analysis (especially by mass spectrometry). Background Art

[0005] Accurate analysis of molecules (such as agents or metabolites associated with diseases or symptoms) is very critical in medical diagnosis or clinical chemistry. Since most biological samples or clinical samples (such as blood samples, urine samples, serum, plasma, etc.) are mixtures of molecules, compounds and cell debris, the target molecule may be interfered by other matrix materials in the sample. Therefore, cleaning the sample and removing interfering matrix materials before biological analysis is essential for reproducible and reliable results.

[0006] Solid phase extraction (SPE) has been widely used in clean-up procedures for biological, food, and environmental samples that include various interfering components. For example, for biological samples, SPE applications are primarily used to separate salts, phospholipids, and / or proteins from the sample to eliminate interference during mass spectrometry (MS) analysis. The disadvantage of SPE is that the typical protocol requires at least 5 steps and, depending on the target analyte, method development may be required to eliminate interference. Although technicians in the field of SPE method development are able to perform this task, the technology is becoming increasingly difficult to find. This leaves room for other sample preparation methods that do not require method development.

[0007] The pass through method is one of the simplest methods to perform. However, the pass through method still cannot provide an extracted sample that is substantially free of mass spectrometer (MS) matrix interferences to meet the sensitivity and reproducibility according to FDA guidelines. In particular, polar matrix components and salts remaining in the sample can disrupt MS ionization by forming polyion complexes or inhibiting or enhancing the ionization of the target analyte depending on the interfering components. The factors lead to reduced accuracy of the analysis, which is caused by variations in the concentration and type of interfering components between samples.

[0008] The present invention therefore addresses the need for producing clean samples and sample extracts containing high recovery of target molecules using a simple, general method that has been developed for biological or environmental samples to be analyzed by mass spectrometry or any other technique that uses nebulization and ionization of sample components prior to detection. Summary of the invention

[0009] The present invention provides a separation device and chromatographic column. In particular, the device or chromatographic column can be used to remove interfering components such as phospholipids, proteins and salts in the sample before mass spectrometry analysis, thereby reducing or eliminating matrix effects. Unless a "universal" selective adsorbent provides retention of analytes but not matrix interferences, a single solid phase extraction method for all possible analytes cannot be developed. However, the problem with universal selective / retaining adsorbents is that they can also retain matrix interferences (matrix interferences) that elute with the analyte and hinder high sensitivity, or produce non-reproducible and / or inaccurate quantification of the analyte.

[0010] The present invention combines a highly retentive adsorbent with a specific selective adsorbent designed to remove at least one major matrix interferent from a sample prior to mass spectrometry (MS) analysis. By providing retention for an extremely diverse set of analytes while simultaneously targeting the removal of well-known specific matrix interferents, a single "universal" method can provide clean extracts for analysis (by MS) of samples containing common interferents. By targeting the removal of specific endogenous matrix interferents, the same method can provide clean extracts for a previously or a priori undefined set of analytes with properties similar to those of pharmaceutically or environmentally relevant analytes.

[0011] The strategy can also be applied to clean-up of pass samples. In such cases, at least two adsorbents are combined to remove more than one major matrix interferent from a sample prior to mass spectrometry (MS) analysis. In the art, single adsorbent devices have been provided for clean-up and removal of pass samples of at least one major matrix interferent that adversely affects quantification by MS. A combination of judiciously selected adsorbents can remove more than one major matrix interferent for a more comprehensive set of analytes that are not defined a priori.

[0012] Multiple sorbent devices are known in the art, but these multiple sorbent devices are intended to retain specific analytes or specific groups of analytes, such as for the analysis of volatile organic compounds (VOCs) in Methods 5972 or 5973N (similar to EPA SW846 Method 8260B, NIOSH Method 2549, and equivalent to EPA Compendium Method TO-17). Existing multiple sorbent devices have been developed to retain application specific groups of analytes, such as disulfide from serum as described previously. In the present invention, the invention relates to the detection of dioxins and polychlorinated biphenyls (J Chromatogr A. 2006 Oct 13; 1130 (1): 97-107, 2006 Jun 30, "High-throughput biomonitoring of dioxins and polychlorinated biphenyls at the sub-picogram level in human serum", Focant JF1, Eppe G, Massart AC, Scholl G, Pirard C, De Pauw E.). Other applications of multi-sorbent devices include the fractionation of foods to accommodate various analytical methods for food analysis as described above (Food Contaminants and Residue Analysis, By Yolanda Picó, Elsevier, 2008). Coupling of different sorbent devices has also been used for this purpose.

[0013] With the advent of newer and more sensitive MS instrumentation and the shortage of skilled personnel in the field of SPE method development, there is room for other sample preparation devices and methods that do not require method development and are more versatile in their application.

[0014] In one aspect, a separation device is provided which may include: at least two or more adsorbents. In particular, for aqueous samples, the separation device may include at least one adsorbent containing at least one hydrophobic component and at least one hydrophilic component and at least one adsorbent having a specific affinity for phospholipids. For samples containing >50% organic solvent, the adsorbent may include at least one hydrophobic adsorbent (such that the hydrophobic adsorbent does not substantially interact with the aqueous sample), and at least one adsorbent having a specific affinity for phospholipids. The adsorbent may also be selected from silica, modified silica, alumina, modified alumina, titanium oxide and zirconium oxide, graphitized carbon or activated carbon, magnesium-silica (e.g. ), polymers, modified polymers and combinations thereof. The adsorbent may also be porous, non-porous or film-like.

[0015] In certain embodiments, for aqueous samples, the adsorbent can be layered. In particular, the top layer can be an adsorbent containing at least one hydrophobic component and at least one hydrophilic component. Alternatively, the adsorbent can be mixed in a single layer in the separation device. In particular, an adsorbent containing at least one hydrophobic component and at least one hydrophilic component and an adsorbent having a specific affinity for phospholipids can be mixed in a weight ratio of about 1:2, respectively.

[0016] In certain embodiments, the separation device may further include at least one frit or filter. The frit or filter may be disposed at the bottom of the device to retain the adsorbent. In addition, the separation device may further include an inlet and an outlet; or a housing. Exemplary housings may be syringes, cartridges, columns, multiple chambers, 4 to 8 well racks, 4 to 8 well strips, 48 ​​to 96 well plates, 96 to 384 well micro elution plates, micro-elution tips, 96 to 384 micro-elution tip arrays, 4 to 8-tip micro-elution strips, single micro-elution pipette tips or rotating tubes or rotating containers. In addition, the separation device may further include a vacuum chamber, a reservoir, or a structure (which allows use in a centrifuge or with a vacuum or pressure source).

[0017] In an exemplary embodiment, the adsorbent containing at least one hydrophobic component and at least one hydrophilic component may be water wettable. It is understood that HLB adsorbents may provide a suitable choice for water wettable adsorbents. Adsorbents with specific affinity for phospholipids may contain modified or unmodified C 8 To C 30 Alkyl or octadecyl (C 18 ) component.

[0018] In one aspect, a chromatographic column is also provided. The chromatographic column may include: a column having a cylindrical interior for receiving an adsorbent bed and at least two or more adsorbents. In particular, the chromatographic column may include at least one adsorbent containing at least one hydrophobic component and at least one hydrophilic component and at least one adsorbent having a specific affinity for phospholipids. In addition, the adsorbent containing at least one hydrophobic component and at least one hydrophilic component may be water wettable. The adsorbent may also be selected from silica, modified silica, alumina, modified alumina, titanium oxide and zirconium oxide, graphitized carbon or activated carbon, magnesium silicate Modified polymers and combinations thereof. The adsorbents can also be porous, nonporous or film-like.

[0019] In certain embodiments, the adsorbent can be layered. In particular, the top layer can be an adsorbent containing at least one hydrophobic component and at least one hydrophilic component. Alternatively, the adsorbent can be mixed in a monolayer. In particular, an adsorbent containing at least one hydrophobic component and at least one hydrophilic component and an adsorbent having a specific affinity for phospholipids can be mixed in a weight ratio of about 1:2, respectively. In certain embodiments, the chromatographic column may also include at least one glass frit or filter. The glass frit or filter may be arranged at the bottom of the adsorbent. In addition, the chromatographic column may also include an inlet and an outlet. In addition, the chromatographic column may also include a vacuum chamber, a reservoir or a structure (which allows use in a centrifuge or use with a vacuum or pressure source).

[0020] In certain exemplary embodiments, the chromatography column can be contained in a syringe, a cartridge, a column, a multi-chamber, a 4 to 8 well rack, a 4 to 8 well strip, a 48 to 96 well plate, a 96 to 384 well micro-elution plate, a micro-elution tip, an array of 96 to 384 micro-elution tips, a 4 to 8-tip micro-elution strip, a single micro-elution pipette tip, a spinning tube, or a spinning container.

[0021] In an exemplary embodiment, the adsorbent containing at least one hydrophobic component and at least one hydrophilic component may be HLB adsorbents and adsorbents with specific affinity for phospholipids can be modified or unmodified C 8 To C 30 Alkyl or octadecyl (C 18 ) component.

[0022] In addition, in one aspect, a chromatographic device is provided. The chromatographic device may include: a column having a cylindrical interior for receiving an adsorbent bed; and at least two or more adsorbents. In particular, at least one adsorbent may include at least one hydrophobic component and at least one hydrophilic component, and at least one adsorbent may have a specific affinity for phospholipids. In addition, the adsorbent containing at least one hydrophobic component and at least one hydrophilic component may be water wettable.

[0023] In certain embodiments, the adsorbent may be layered. In particular, the top layer may be an adsorbent containing at least one hydrophobic component and at least one hydrophilic component. Alternatively, the adsorbents may be mixed in a single layer. In particular, an adsorbent containing at least one hydrophobic component and at least one hydrophilic component and an adsorbent having a specific affinity for phospholipids may be mixed in a weight ratio of about 1:2.

[0024] In certain exemplary embodiments, the chromatographic device may further include at least one glass frit or filter. The glass frit or filter may be disposed at the bottom of the adsorbent. In addition, the chromatographic device may further include an inlet and an outlet; or a housing. An exemplary housing may be a syringe, a cartridge, a column, a multi-chamber, a 4 to 8-hole rack, a 4 to 8-hole strip, a 48 to 96-hole plate, a 96 to 384-hole micro-elution plate, a micro-elution tip, a 96 to 384 micro-elution tip array, a 4 to 8-tip micro-elution strip, a single micro-elution pipette tip, a rotating tube, or a rotating container. In addition, the chromatographic device may further include a vacuum chamber, a reservoir, or a structure (which allows use in a centrifuge or use with a vacuum or pressure source).

[0025] In an exemplary embodiment, the adsorbent containing at least one hydrophobic component and at least one hydrophilic component may be HLB adsorbents and adsorbents with specific affinity for phospholipids can be modified or unmodified C 8 To C 30 Alkyl or octadecyl (C 18 ) component.

[0026] In addition, a solid phase extraction method is provided. The method may include: using the device or chromatographic column as described above. In particular, the device or chromatographic column may include an adsorbent bed, which includes an adsorbent containing at least one hydrophobic component and at least one hydrophilic component and an adsorbent having a specific affinity for phospholipids. In addition, the adsorbent containing at least one hydrophobic component and at least one hydrophilic component may be water wettable.

[0027] In certain embodiments, adsorbent can be layered. In particular, the top layer can be an adsorbent containing at least one hydrophobic component and at least one hydrophilic component. Alternatively, adsorbent can be mixed in a monolayer or single adsorbent bed. In particular, an adsorbent containing at least one hydrophobic component and at least one hydrophilic component and an adsorbent having a specific affinity for phospholipids can be mixed in a weight ratio of about 1:2. It can be determined that the weight ratio provides the retention of the analyte to be quantified and provides sufficient retention of phospholipids in the elution solvent, so that the analyte is eluted in an elution solvent that is substantially free of phospholipids, salts, proteins and other high-polarity matrix components, which causes mass spectrometry ion suppression or enhances or damages the life of the analytical column used for analyte resolution before quantification in front of a mass spectrometer.

[0028] For pass-through applications, the adsorbent weight ratio is determined by the mass capacity required to remove at least two major matrix interferents.

[0029] In an exemplary embodiment, the adsorbent containing at least one hydrophobic component and at least one hydrophilic component may be HLB adsorbents and adsorbents with specific affinity for phospholipids can be modified or unmodified C 8 To C 30 Alkyl or octadecyl (C 18 ) component.

[0030] In an exemplary embodiment, the device can be a syringe, a cartridge, a column, a multi-chamber, an 8-well rack, an 8-well strip, a 96-well plate, a 96-well micro-elution plate, an elution tip, a 96-micro-elution tip array, an 8-tip micro-elution strip, a single micro-elution pipette tip, a spinning tube, or a spinning container.

[0031] In certain exemplary embodiments, the method may further include conditioning the adsorbent bed, and methanol may be used for the conditioning.

[0032] In certain exemplary embodiments, the method may further include balancing the sorbent bed, and water may be used for balancing.

[0033] In certain exemplary embodiments, the method may further include loading the sample onto the sorbent bed without conditioning or equilibrating the sorbent bed.The sample may be a biological fluid, tissue, food, or environmental sample.

[0034] Alternatively, when the adsorbent comprising at least one hydrophobic component and at least one hydrophilic component is water wettable, the sample can be loaded onto the adsorbent bed without conditioning and equilibrating the adsorbent bed.

[0035] In certain exemplary embodiments, the method may further include eluting the analyte from the adsorbent bed. For example, a mixture of acetonitrile and methanol is used for elution at a mixing ratio of about 90:10. In particular, after eluting the analyte, the eluent obtained from the method may contain less than about 2wt% of phospholipids in the loaded sample. In addition, liquid chromatography-ultraviolet light, liquid chromatography-mass spectrometry / mass spectrometry (LC-MS / MS), mass spectrometry (MS), MALDI-MS, electrospray ionization (ESI), sector, time of flight (ToF) or ion trap may be used.

[0036] In certain exemplary embodiments, a vacuum chamber, reservoir, or structure (which allows for use in a centrifuge or with a vacuum or pressure source) may be applied to the device to increase flow rates.

[0037] In other aspects, the present invention provides a separation device, a chromatography column or a chromatography device comprising two or more adsorbents, wherein

[0038] Compared to a separation device comprising only one adsorbent, matrix interferents retained on one of the adsorbents are substantially removed by the other adsorbent;

[0039] The flow rate of the combined adsorbents is substantially increased compared to a separation device comprising only one adsorbent; or

[0040] The backpressure of the combined adsorbents is substantially reduced compared to a separation device comprising only one adsorbent.

[0041] In another aspect, the present invention provides a method for removing matrix interferents retained by an adsorbent, comprising mixing the adsorbent with at least one or more additional adsorbents of different types. In certain embodiments, the adsorbent and at least one or more additional adsorbents are mixed in a monolayer. In other embodiments, the adsorbent is a water wettable adsorbent and at least one additional adsorbent is not a water wettable adsorbent. In other embodiments, the adsorbent is not a water wettable adsorbent and at least one additional adsorbent is a water wettable adsorbent.

[0042] In another aspect, the present invention provides a method of increasing the flow rate of an adsorbent, comprising mixing the adsorbent with at least one or more additional adsorbents of different types. In certain embodiments, the adsorbent and at least one or more additional adsorbents are mixed in a monolayer. In other embodiments, the adsorbent is a water wettable adsorbent and at least one additional adsorbent is not a water wettable adsorbent. In other embodiments, the adsorbent is not a water wettable adsorbent and at least one additional adsorbent is a water wettable adsorbent.

[0043] In another aspect, the present invention provides a method of reducing the back pressure of an adsorbent, comprising mixing the adsorbent with at least one or more additional adsorbents of different types. In certain embodiments, the adsorbent is mixed with at least one or more additional adsorbents in a monolayer. In other embodiments, the adsorbent is a water wettable adsorbent and at least one additional adsorbent is not a water wettable adsorbent. In other embodiments, the adsorbent is not a water wettable adsorbent and at least one additional adsorbent is a water wettable adsorbent.

[0044] Other aspects of the apparatus and methods of the present invention may be disclosed hereinafter. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 An exemplary adsorbent is shown. For example, a sorbent containing a hydrophobic component and a hydrophilic component may be used according to an exemplary embodiment of the present invention. HLB adsorbent. In addition, other adsorbents containing ion exchange groups can be used without restriction according to user requirements. Adsorbent.

[0046] Figure 2A-2B Exemplary housings of devices according to exemplary embodiments of the present invention are shown.

[0047] Figure 3 is an exemplary method of analyzing a biological sample according to an exemplary embodiment of the present invention.

[0048] Figure 4 Example mass spectrometry analysis of a milk sample showing the phospholipid region. HLB adsorbents and Exemplary mixed adsorbents or HLB adsorbents and An exemplary layered adsorbent of an adsorbent) was used to obtain mass spectrometry analysis after extraction of a milk sample.

[0049] Figure 5 An exemplary mass spectrometric analysis of a milk sample is shown. Using an apparatus according to an exemplary embodiment of the present invention (comprising C 18 , HLB adsorbent, Z-Sep / C18 and containing HLB adsorbents and adsorbent) and mass spectrometry was obtained after extraction of milk samples.

[0050] Figure 6 Example mass spectrometry analysis of a milk sample showing the region of ciprofloxacin and enrofloxacin.18 , HLB adsorbent, Z-Sep / C 18 and containing HLB adsorbents and adsorbent) and mass spectrometry was obtained after extraction of milk samples.

[0051] Figure 7 Example mass spectrometry analysis of a milk sample showing the region of oxytetracycline. 18 , HLB adsorbent, Z-Sep / C 18 and containing HLB adsorbents and adsorbent) and mass spectrometry was obtained after extraction of milk samples.

[0052] Figure 8 Exemplary MRM transitions monitored by ESI are shown. DETAILED DESCRIPTION OF THE INVENTION

[0054] The present invention provides a device and method for developing a sample for cleaning before subsequent analysis. Such cleaning may also require the use of a separation method such as liquid chromatography (LC) or supercritical fluid chromatography (SFC) with mass spectrometry / mass spectrometry (MS / MS), mass spectrometry (MS), MALDI-MS or ESI, etc. Therefore, the device and method of the present invention can provide high recovery for analytes and minimize matrix effects by removing interfering matrix materials. In particular, such objects of the present invention can be achieved by combining adsorbents, which provide analyte retention with specificity for components such as phospholipids, salts, etc. for removing MS interfering substances from extracted analytes. Adsorbents are selectively combined to provide suitable retention of analytes from aqueous samples without the need to adjust or balance the adsorbents before sample loading, thereby reducing the number of steps in the SPE method. In addition, the adsorbent combination is selected to elute polar matrix interfering substances in the washing step while maintaining analyte retention, and to adequately retain at least one matrix interfering substance in the elution step, thereby removing multiple interfering substances from the eluent containing the extracted analyte.

[0055] definition

[0056] The term "analyte" is a target component, substance or chemical constituent in an analytical procedure. Specific examples may include, but are not limited to, drugs, pesticides, herbicides, toxins and environmental pollutants, metal organic compounds, biologically active compounds such as metabolites, proteins, peptides, hormones, polynucleotides, vitamins, cofactors, metabolites, lipids and carbohydrates.

[0057] As used herein, the term "sample" refers to a complex fluid mixture containing soluble and insoluble components. Specific examples include, but are not limited to, food samples (e.g., milk, fortified food matrices), biological samples, including samples from humans or animals (e.g., blood, plasma, urine, mucosal tissue secretions, tears, semen, and breast milk), and environmental samples (e.g., groundwater, wastewater, soil, and ocean, river, pond, or bay water). The sample may also include macromolecules, such as substances, such as biopolymers, such as proteins, such as proteolytic proteins or lipophilic proteins, such as receptors and other membrane-bound proteins and peptides. The sample may also contain one or more lipid or phospholipid molecules.

[0058] As used herein, the term "matrix" refers to components of a sample other than the target analyte. When separating, extracting, and analyzing analytes, the matrix can have substantial interference with the analytes during analysis by ESI or any other technique that uses nebulization and ionization of sample components prior to detection, such as APCI and APPI.

[0059] As used herein, the term "matrix interferents" refers to those components of a sample that produce substantial signal enhancement or suppression to an analyte during analysis by mass spectrometry. Substantial signal enhancement or suppression that interferes with analyte quantification is also referred to as substantial interference. In certain embodiments, "substantial interference" refers to matrix effects greater than 20% (for targeted analysis) and 50% (for screening analysis). If most of the analytes in the analysis have matrix effects below the threshold, matrix interferences below these values ​​are considered acceptable.

[0060] As used herein, the term "targeted analysis" refers to the analysis of a predetermined set of analytes expected to be present in a sample to be analyzed. Targeted analysis generally does not rely on full scan mode data from MS, but relies on full scan mode data from selected ion monitoring of target ions of interest. An example of targeted analysis is the quantification of tacrolimus (an immunosuppressive drug used after organ transplantation and given to reduce the risk of rejection). Discrete concentration values ​​for these analyses are reported.

[0061] As used herein, the term "screening analysis" refers to the analysis of a large set of analytes that may or may not be present in a sample to be analyzed. An example of a screening analysis is determining whether pesticides are present in food. These reports contain information about the presence of analytes at or above a certain threshold.

[0062] As used herein, the term "log P" refers to the octanol: water partition coefficient. The formula for the partition coefficient is Log P = [analyte concentration in octanol] / [analyte concentration in water]. The log P value is used as a measure of the hydrophobicity or hydrophobicity of the analyte (wherein a value less than 1 indicates that the analyte concentration is higher in water than in octanol). Therefore, the analyte will be considered a more hydrophilic or polar analyte. A log P value greater than 1 indicates that the analyte is more hydrophobic or non-polar. The higher the log P value relative to a lower value on the same scale, the higher the hydrophobicity. The lower the log P value relative to 1, the more hydrophilic or polar the analyte is than other values ​​with higher values.

[0063] As used herein, the term "matrix effect" or "ME" refers to the quantitative enhancement or suppression of an analyte signal. The matrix effect of a target analyte is calculated according to the following formula:

[0064]

[0065] In certain embodiments, the matrix effect is calculated using the following formula:

[0066]

[0067] Matrix effects can be caused by, but are not limited to, small molecules, proteins, peptides, polymers, surfactants, particles, cells, lipids or phospholipids, pharmaceutical excipients, and salts.

[0068] As used herein, the term "solid phase extraction (SPE) method or protocol" refers to a method comprising the following steps:

[0069] 1. Conditioning: Addition of an organic-containing wetting solvent (usually methanol), which is required for subsequent use of non-aqueous wettable adsorbents such as silica gel C18.

[0070] 2. Equilibrate the adsorbent: Add water or buffer to replace the organic solvent used to condition the adsorbent. Removal of the conditioning solution is required for retention to occur in the next step.

[0071] 3. Loading sample: Add sample as received or prepared by dilution and / or centrifugation or filtration.

[0072] 4. Washing the adsorbent: Add a solution that removes matrix salts, proteins, especially albumin, and other polar matrix interferents. The polarity of the washing solution enables the target analyte to be retained on the adsorbent. The washing step can be repeated multiple times to remove specific interferents.

[0073] 5. Elution of analyte: Add solution that elutes the target analyte from the sorbent while minimizing co-elution of matrix interferences.

[0074] As used herein, the term "recovery" refers to the amount of analyte recovered from an extracted sample. The calculation for recovery is as follows:

[0075]

[0076] The term "biological sample" refers to any solution or extract containing molecules or mixtures of molecules, which includes at least one biological molecule from a biological source (such as, humans and animals) that has been subjected to extraction or analysis. Biological samples are intended to include crude or purified, for example, isolated or commercially obtained samples. Biological samples can be, but are not limited to, inclusion bodies, biological fluids, biological tissues, biological matrices, embedded tissue samples, cells (e.g., one or more types of cells), and cell culture supernatants. Specific examples may include plasma, urine, cerebrospinal fluid, synovial fluid, and other biological fluids, including tissue extracts, such as liver tissue, muscle tissue, brain tissue, and heart tissue.

[0077] The term "biological matrices" is intended to include any material contained or produced by cells, such as bone, inclusion bodies, blood components, cell fragments such as cell lysates, and the like.

[0078] As used herein, the term "biological fluid" is intended to include fluids obtained from biological sources. Exemplary biological fluids include, but are not limited to, blood, plasma, urine, cerebrospinal fluid, mucosal tissue secretions, tears, interstitial fluid, synovial fluid, semen, and breast milk.

[0079] As used herein, the term "chromatographic" method refers to a method involving a physical separation method that partitions components into two phases, one fixed (stationary phase), the other moving in a certain direction (mobile phase) to separate between the two phases.

[0080] As used herein, the term "eluent" refers to the mobile phase that leaves the sample preparation device or column. In certain embodiments, the eluent may include the target analyte, or the eluent may not include the analyte, which may be retained by the resin or resin matrix. In such cases, the resin or the matrix of the resin may also be further eluted in a subsequent step to elute the retained target analyte.

[0081] The term "solid phase extraction (SPE)" refers to a common chromatographic technique used to separate analytes from a sample for quantitative analysis, particularly a chromatographic technique used in conjunction with high performance liquid chromatography (HPLC) or gas chromatography (GC) (McDonald and Bouvier, eds. Solid Phase Extraction Applications Guide and Bibliography, 6th ed., Milford, MA: Waters (1995)). Solid phase extraction can be useful for separating target components from potential interfering elements in complex solutions and concentrating target analytes to sufficient levels for detection and measurement. For example, solid phase extraction has been widely used to prepare food or beverage samples, environmental samples, and pharmaceutical agents or metabolites for analysis.

[0082] As used herein, the term "water wettable" describes a material that is partially or completely solvated by water. Water wettable materials form energetically favorable or attractive interactions with water molecules, so that even if the adsorbent dries out, it still maintains its ability to retain high retention and excellent recovery, which means that no special steps need to be taken to prevent the adsorbent bed from drying out in the critical step before sample loading. Examples of water wettable materials are, but are not limited to, those described in U.S. Patent No. 5,882,521. As observed in the examples of U.S. Patent No. 5,882,521, adsorbents that do not participate in favorable energetics or interactions with water require organic adjustment and then moisture balance to maintain retention of target analytes during sample loading. In certain embodiments, when a water-wettable material is partially solvated, less than about 1%, less than about 3%, less than about 5%, less than about 7%, less than about 10%, less than about 15%, even less than about 20%, less than about 25%, less than about 30%, less than about 35%, less than about 40%, less than about 45%, less than about 50%, less than about 55%, less than about 60%, less than about 65%, less than about 70%, less than about 75%, less than about 80%, less than about 85%, less than about 90%, less than about 95% or less than about 99% of the material can be solvated or wetted by water.

[0083] As used herein, the term "monomer" refers to a molecule and a repeating unit of a polymer that contains one or more polymerizable functional groups prior to polymerization. A polymer may contain two or more different monomers, in which case it may also be referred to as a copolymer. The "mole percent" of a given monomer contained in a copolymer is the mole fraction (expressed as a percentage) of the target monomer relative to the total number of moles of the various (two or more) monomers that make up the copolymer.

[0084] The term "sorption" or "sorbing" describes the ability of a material to take up and hold another material by absorption or adsorption. Without the adsorption of matrix components and target analytes on the surface of the sorbent material, retention will not be maintained when the system is subjected to the flow of certain fluids.

[0085] The term "adsorbent" refers to a material or molecule that is capable of absorbing or adsorbing. In certain embodiments, the adsorbent can adsorb analytes or other matrix molecules.

[0086] As used herein, the term "hydrophobic" refers to the physical or chemical property of repelling water or polar molecules. In certain embodiments, the hydrophobic groups in the resin have a substantial interaction or affinity with the hydrophobic analytes or matrices in the sample and adsorb the hydrophobic species.

[0087] As used herein, the term "hydrophilic" refers to a physical or chemical property that favors water or polar molecules. In certain embodiments, the hydrophilic groups in the resin substantially interact with hydrophilic or polar analytes or matrices in the sample and adsorb the hydrophilic species.

[0088] The term "phospholipid" refers to a lipid containing a phosphate group and one or more glycerides. Phospholipids are the main components of cell membranes in the form of lipid bilayers. Therefore, phospholipids are mainly included in biological samples and foods and other by-products produced from animal products. In certain embodiments, phospholipids can cause matrix effects that substantially disrupt the qualitative and quantitative analysis because they can interact with or adhere to the analyte. Therefore, it is preferred to remove phospholipids before analysis.

[0089] The terms "analysis" or "analyzing" are used interchangeably and refer to any of a variety of methods for separating, detecting, isolating, purifying, solubilizing, detecting, quantifying and / or characterizing a chemical or biological composition. In certain embodiments, analysis may also refer to various methods for determining the degree of purification of a sample. Examples of various methods include, but are not limited to, solid phase extraction, solid phase microextraction, electrophoresis, mass spectrometry such as MALDI-MS or ESI, liquid chromatography such as high performance chromatography, such as reverse phase, normal phase or size exclusion, ion pair liquid chromatography, liquid-liquid extraction such as accelerated fluid extraction, supercritical fluid extraction, microwave assisted extraction, membrane extraction, Soxhlet extraction, precipitation, clarification, electrochemical detection, staining, elemental analysis, Edmund degradation, nuclear magnetic resonance, infrared analysis, flow injection analysis, capillary electrochromatography, ultraviolet detection and combinations thereof.

[0090] The term "mass spectrometry detection" refers to any of a variety of mass spectrometry methods. Examples include, but are not limited to, electrospray ionization (ESI), surface desorption ionization, and atmospheric pressure chemical ionization (APCI). In certain embodiments, mass spectrometry detection also includes the use of a tandem mass spectrometer, a quadrupole time-of-flight mass spectrometer, or a sector magnetic field mass spectrometer.

[0091] The term "high purity analyte / sample" refers to a prepared or extracted analyte that may have reduced contamination and / or non-degraded chromatographic properties prior to quantitative or qualitative analysis, such as chromatography and mass spectrometry.

[0092] As used herein, the term "substantially" (as in "substantially higher," "substantially greater," or "substantially increased") refers to an increase in an effect of about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% of the original extent of a particular effect. Alternatively, a "substantially" amount may refer to an amount greater than about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% of the original or total amount. Similarly, as used herein, "substantially reduced" refers to a reduction in an effect of about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90% or about 95% of the original extent of a particular effect.

[0093] As used herein, the term "substantially free" (as in "substantially free of phospholipids, salts, proteins and other highly polar matrix components") refers to the elution of the analyte so that the analyte is eluted in the elution solvent so that the recovered analyte contains an amount of the component that is less than 20% by weight of a specific component compared to the total elution mixture. In certain embodiments, "substantially free" refers to less than 10%, less than 5%, less than 2%, less than 1%, less than 0.5% or less than 0.1% by weight of a specific component compared to the total elution. In certain other embodiments, "substantially free" refers to less than 1.0%, less than 0.7%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2% or less than 0.1% by weight of a specific component compared to the total elution. In other embodiments, "substantially free" refers to less than 1.0%, less than 0.7%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2% or less than 0.1% by weight of a specific component compared to the total preparation.

[0094] Adsorbent

[0095] The present invention provides a device for preparing a high purity sample, and the device is used for solid phase extraction or by a method. In particular, the device includes an adsorbent that can selectively adsorb or retain matrix components. In addition, the device of the present invention includes at least two or more adsorbents for solid phase extraction. The adsorbent can be provided in a filled adsorbent bed.

[0096] In one aspect, when the sample is aqueous or contains less than about 50% organic solvent by volume, at least one adsorbent may contain at least one hydrophobic component and at least one hydrophilic component. In certain exemplary embodiments, the adsorbent may include a water-wettable polymer having an optimal balance between hydrophobic and hydrophilic properties, such as described in U.S. Pat. No. 5,882,521, U.S. Pat. No. 5,976,367, U.S. Pat. No. 6,106,721, U.S. Pat. No. 6,254,780, U.S. Pat. No. 6,332,695, 6,468,422, U.S. Pat. No. 6,726,842, 6,723,236, and U.S. Pat. No. 6,773,583 (each of which is incorporated herein by reference in its entirety). Thus, an adsorbent containing at least one hydrophobic component and at least one hydrophilic component can maintain a high retention of hydrophobic species such as lipids or non-polar organic molecules and hydrophilic species such as polar organic molecules or salts in water, aqueous solutions or solutions miscible with water. In addition, the retained species in the adsorbent can be acidic, basic and neutral compounds. It should also be understood that a range of choices for adsorbents containing at least one hydrophobic component and at least one hydrophilic component are provided by Provided by HLB (Waters Corporation, Milford MA).

[0097] In certain embodiments, the adsorbent containing at least one hydrophobic component and at least one hydrophilic component may be further modified to include at least one ion exchange functional group. The functional group may be capable of selectively retaining cations, anions, or other polar species in solution, as described, for example, in U.S. Pat. No. 7,442,299, U.S. Pat. No. 7,232,520, U.S. Pat. No. 7,731,844, U.S. Pat. No. 7,790,038, and U.S. Pat. No. 8,574,433 (each of which is incorporated herein by reference in its entirety). It should also be understood that a range of options for adsorbents containing at least one hydrophobic component, at least one hydrophilic component, and at least one ion exchange functional group are also provided. MAX adsorbent, MCX adsorbent, WCX adsorbent or WAX adsorbent (Waters Corporation, Milford MA) was provided.

[0098] In one embodiment, at least one adsorbent may contain at least one component having a specific affinity for a lipid or lipid-like species. The adsorbent may have a high hydrophobicity to remove such lipophilic substances from the sample solution. In certain exemplary embodiments, the adsorbent may have an affinity for phospholipids, as described in U.S. Patent Application No. 2013 / 0053588 (which is incorporated herein by reference in its entirety). In an exemplary embodiment, the adsorbent may contain a highly hydrophobic group, such as an octadecyl (C 2+) group bonded to silica. 18 ) or C 8 To C 30 Alternatively, the adsorbent may be further modified with other chemical groups to provide greater specificity for phospholipids. It is also understood that a range of choices of adsorbents containing at least one hydrophobic component and at least one hydrophilic component are provided by (Waters Corporation, Milford MA).

[0099] In certain exemplary embodiments, adsorbents containing at least one hydrophobic component and at least one hydrophilic component, adsorbents having affinity for phospholipids, and many combinations of other adsorbents based on user requirements may also be provided (but not limited to). In certain exemplary embodiments, the adsorbent may also be selected from silica, modified silica, alumina, modified alumina, titanium oxide and zirconium oxide, graphitized carbon or activated carbon, magnesium silicate, Modified polymers and combinations thereof. The adsorbent can also be porous, non-porous or film-like.

[0100] In an exemplary embodiment of the present invention, at least one of the adsorbents may be a porous resin as described in U.S. Patent No. 5,882,521 (which is incorporated herein by reference in its entirety). The porous resin may be formed by copolymerizing the monomers contained in the resin composition, and a porous structure may be formed to penetrate the copolymer. The porous structure may provide a path or channel through which a sample solution may diffuse. In certain exemplary embodiments, an adsorbent containing at least one hydrophobic component and at least one hydrophilic component may be formed by copolymerizing at least one hydrophobic monomer and at least one hydrophilic monomer. In certain exemplary embodiments, the pores in the resin may have a diameter ranging from about 0.5 nm to about 100 nm. In certain exemplary embodiments, the porous resin may be in the form of porous particles or monoliths.

[0101] In certain exemplary embodiments, the adsorbent may be layered and may thereby form at least two or more layers in the device. In an exemplary embodiment, an adsorbent containing at least one hydrophobic component and at least one hydrophilic component may be layered on top of the adsorbent layer. Other layers containing adsorbents having an affinity for phospholipids, etc. may be layered under the bottom or top layer, but are not limited to. However, the order or number of layers may not be limited within the scope of the present invention.

[0102] In an exemplary embodiment, each adsorbent can be used independently in an amount of (but not limited to) about 0.1 mg, about 0.2 mg, about 0.3 mg, about 0.4 mg, about 0.5 mg, about 0.6 mg, about 0.7 mg, about 0.8 mg, about 0.9 mg, about 1 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 7 mg, about 8 mg, about 9 mg, about 10 mg, about 20 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg or about 100 mg based on user requirements, device capacity, solution sample volume, phospholipid and salt amounts, etc.

[0103] In an exemplary embodiment, the adsorbents in each layer can be used in a specific weight ratio therebetween. For example, the weight ratio between the adsorbent containing at least one hydrophobic component and at least one hydrophilic component and the adsorbent having an affinity for phospholipids can be about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, or about 1:10. In particular, the adsorbent containing at least one hydrophobic component and at least one hydrophilic component and the adsorbent having a specific affinity for phospholipids can be mixed at a weight ratio of about 1:2, respectively.

[0104] For adsorbents of different densities, the appropriate weight ratio between the adsorbents of the layered bed may vary. For example, as an adsorbent containing at least one hydrophobic component and at least one hydrophilic component HLB adsorbent has a density of about 1.2 g / cm3; and as an adsorbent with specific affinity for phospholipids The adsorbent has a density of about 2.2 g / cm3. Thus, the appropriate weight ratio between the two different adsorbents can be determined by the volume requirements in a chromatography column or apparatus or the like.

[0105] In addition, a suitable weight ratio can be determined to provide retention of the analyte to be quantified and to provide sufficient retention of phospholipids in the elution solvent, so that the analyte is eluted in an elution solvent that is substantially free of phospholipids, salts, proteins, and other high-polarity matrix components (which cause ion suppression or enhancement of mass spectrometry or impair the life of the analytical column used for analyte resolution in front of the mass spectrometer before quantification). For example, after the first wash, the second wash, or elution with a mobile phase, most of the phospholipids may remain in an adsorbent having a specific affinity for phospholipids to provide sufficient retention of phospholipids in the elution solvent, so that the analyte can be eluted in an elution solvent that is substantially free of phospholipids. In addition, after elution, other high-polarity matrix components such as salts, proteins, etc. that cause ion suppression or enhancement of mass spectrometry may remain in an adsorbent containing at least one hydrophobic component and at least one hydrophilic component or a water-wettable adsorbent to provide sufficient retention thereof, so that the analyte eluted in the elution solvent may be substantially free of polar matrix components.

[0106] Alternatively, in certain exemplary embodiments, the adsorbents may be mixed and formed in a monolayer or single packed bed. In an exemplary embodiment, the adsorbent may be prepared by mixing an adsorbent containing at least one hydrophobic component and at least one hydrophilic component and an adsorbent having an affinity for phospholipids. The adsorbent mixture may be formed homogeneously, partially homogeneously, or heterogeneously (randomly mixed). In addition, the adsorbent mixture may be formed with a gradient of one adsorbent or with independent gradients of each adsorbent. A mixed adsorbent bed may be used to reduce the back pressure of an adsorbent bed composed of adsorbent particles. In other words, the mixed bed may be a charge-stabilized or in-situ formed monolith so that the flow rate of water may be increased while reducing the back pressure. For example, the back pressure of a mixed adsorbent in a monolayer bed may be a vacuum of about 5"Hg, compared to 8-15"Hg for a layered bed using the same adsorbent.

[0107] In an exemplary embodiment, each adsorbent may be mixed in an amount of, but not limited to, about 0.1 mg, about 0.2 mg, about 0.3 mg, about 0.4 mg, about 0.5 mg, about 0.6 mg, about 0.7 mg, about 0.8 mg, about 0.9 mg, about 1 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 7 mg, about 8 mg, about 9 mg, about 10 mg, about 20 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, or about 100 mg based on user requirements, the capacity of the device, the volume of the solution sample, the amount of phospholipids and salts, etc.

[0108] In an exemplary embodiment, the adsorbents may be mixed to form a monolayer single absorbent at a specific weight ratio therebetween. For example, the weight ratio between the adsorbent containing at least one hydrophobic component and at least one hydrophilic component and the adsorbent having an affinity for phospholipids may be about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, or about 1:10. In particular, the adsorbent containing at least one hydrophobic component and at least one hydrophilic component and the adsorbent having a specific affinity for phospholipids may be mixed at a weight ratio of about 1:2, respectively. However, the order or method of mixing may not be limited within the scope of the present invention.

[0109] For adsorbents of different densities, the appropriate weight ratio between the adsorbents of the mixed bed may vary. For example, as an adsorbent containing at least one hydrophobic component and at least one hydrophilic component The density of HLB adsorbent is about 1.2 g / cm3; and as an adsorbent with specific affinity for phospholipids The adsorbent has a density of about 2.2 g / cm3. Therefore, the appropriate weight ratio between the two different adsorbents can be determined by the packaging volume, back pressure, other volume requirements in the chromatography column or device, etc.

[0110] In addition, a suitable weight ratio can be determined to provide retention of the analyte to be quantified and to provide sufficient retention of the phospholipids in the elution solvent, so that the analyte is eluted in an elution solvent that is substantially free of phospholipids, salts, proteins, and other high-polarity matrix components (which cause ion suppression or enhancement of mass spectrometry or impair the life of the analytical column used for analyte resolution in front of the mass spectrometer before quantification). For example, after the first wash, the second wash, or elution with a mobile phase, most of the phospholipids may remain in an adsorbent having a specific affinity for phospholipids to provide sufficient retention of the phospholipids in the elution solvent, so that the analyte can be eluted in an elution solvent that is substantially free of phospholipids. In addition, after elution, other high-polarity matrix components such as salts, proteins, etc. that cause ion suppression or enhancement of mass spectrometry may remain in an adsorbent containing at least one hydrophobic component and at least one hydrophilic component or a water-wettable adsorbent to provide sufficient retention thereof, so that the analyte eluted in the elution solvent may be substantially free of polar matrix components.

[0111] On the other hand, when the sample is non-aqueous or contains greater than about 50% organic solvent by volume, an adsorbent containing at least one hydrophobic component and at least one hydrophilic component or a water-wettable adsorbent may not be needed, thereby eliminating the need for a hydrophilic component, but at least one adsorbent may still contain at least one component having a specific affinity for a lipid or lipid-like species. In an exemplary embodiment, when the sample is non-aqueous or contains greater than about 50% organic solvent by volume, an adsorbent having a specific affinity for a phospholipid or lipid-like species may be combined with a non-polar adsorbent such as a high coverage C 18 -Silica and divinylbenzene adsorbents are used in combination, and those adsorbents may be mixed in a single layer or singly packed adsorbent bed, or may be provided in each layer to provide a layered adsorbent bed. The amount and weight ratio of the adsorbent for non-aqueous samples may be used as described above (but not limited thereto).

[0112] Device

[0113] The present invention provides a device for preparing a high-purity sample, and the device is used for solid phase extraction. The device disclosed in the present invention may include: an inlet, an outlet, an adsorbent, and a shell capable of confining the adsorbent and withstanding sufficient mechanical strength applied during extraction.

[0114] In one aspect, the separation device comprises at least two or more adsorbents provided in packed adsorbent beds.

[0115] In one embodiment, the device may include: at least one water-wettable adsorbent containing at least one hydrophobic component and at least one hydrophilic component; and at least one adsorbent having an affinity for lipids or lipid-like species such as phospholipids (as described above). Alternatively, when the sample is non-aqueous or contains greater than about 50% organic solvent by volume, the device may include: a non-polar adsorbent and at least one adsorbent having an affinity for lipids or lipid-like species such as phospholipids. Most non-polar adsorbents used in SPE are wettable by solutions containing greater than about 50% organic solvent.

[0116] In certain embodiments, the adsorbent containing at least one hydrophobic component and at least one hydrophilic component may be further modified to include at least one ion exchange functional group. In certain exemplary embodiments, many combinations of adsorbents containing at least one hydrophobic component and at least one hydrophilic component, adsorbents having affinity for phospholipids, and other adsorbents may also be provided based on the user's requirements (but not limited thereto). Exemplary adsorbents may also be selected from silica, modified silica, alumina, modified alumina, titanium oxide and zirconium oxide, graphitized carbon or activated carbon, magnesium silicate, Modified polymers and combinations thereof. The adsorbent can also be porous, non-porous or film-like.

[0117] In certain exemplary embodiments, the adsorbent provided as a packed adsorbent bed may be layered, thereby forming at least two or more layers. For example, an adsorbent containing at least one hydrophobic component and at least one hydrophilic component may be layered on top of the adsorbent layer. Other layers containing adsorbents with affinity for phospholipids, etc. may be layered below (but not limited to) the bottom layer or top layer. However, the order or number of layers may not be limited within the scope of the present invention.

[0118] In certain exemplary embodiments, the adsorbents may be mixed and provided in a single layer or single packed bed. For example, the adsorbent may be prepared by mixing an adsorbent containing at least one hydrophobic component and at least one hydrophilic component and an adsorbent having an affinity for phospholipids. The mixture of adsorbents may be formed homogeneously, partially homogeneously, or heterogeneously (randomly mixed). In addition, the mixture of adsorbents may be formed with a gradient of one adsorbent or a separate gradient of each adsorbent.

[0119] In certain exemplary embodiments, each adsorbent may be independently included in the device in an amount of, but not limited to, about 0.1 mg, about 0.2 mg, about 0.3 mg, about 0.4 mg, about 0.5 mg, about 0.6 mg, about 0.7 mg, about 0.8 mg, about 0.9 mg, about 1 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 7 mg, about 8 mg, about 9 mg, about 10 mg, about 20 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, about 100 mg, or about 1 g based on user requirements, the capacity of the device, the capacity of the adsorbent used, the volume of the solution sample, the amount of phospholipids and salts, etc.

[0120] Each adsorbent in the device can be included in the layered and mixed adsorbent beds at a specific weight ratio therebetween. For example, the weight ratio between the adsorbent containing at least one hydrophobic component and at least one hydrophilic component and the adsorbent having an affinity for phospholipids can be about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, or about 1:10. In particular, the adsorbent containing at least one hydrophobic component and at least one hydrophilic component and the adsorbent having a specific affinity for phospholipids can be mixed at a weight ratio of about 1:2, respectively. The weight ratio is based on matching the capacity of the adsorbent containing at least one hydrophobic component and at least one hydrophilic component for the phospholipids that are removed by the adsorbent having a specific affinity for phospholipids. However, the order or method of mixing may not be limited within the scope of the present invention.

[0121] For adsorbents of different densities, the appropriate weight ratio between the adsorbents may vary. For example, as an adsorbent containing at least one hydrophobic component and at least one hydrophilic component The density of HLB adsorbent is about 1.2 g / cm3; and as an adsorbent with specific affinity for phospholipids The adsorbent has a density of about 2.2 g / cm3. Therefore, the appropriate weight ratio between the two different adsorbents can be determined by the packaging volume, back pressure, other volume requirements in the chromatography column or device, etc.

[0122] In addition, the suitable weight ratio between the adsorbent can be determined by the balance of the retention of analyte during sample loading and washing and the continuation of the retention of phospholipids in the eluent in the elution process of analyte. For example, after first washing, second washing or third washing, most of phospholipids can remain in the mixed adsorbent bed. Suitable weight ratio provides the sufficient retention of phospholipids in the elution solvent between the adsorbent, so that the elution analyte can be eluted in the elution solvent without phospholipids in fact. In addition, other high polar matrix components such as salt, protein etc. that cause the ion suppression of mass spectrometry or enhancement can remain in the mixed adsorbent, or are removed by washing, and its enough mass capacity and retention are provided simultaneously, so that the analyte of wash-out in the elution solvent can be substantially free of polar matrix components.

[0123] For pass-through applications, the adsorbent weight ratio is determined by the mass capacity required to remove at least two major matrix interferents.

[0124] In one embodiment, the device may further include a housing. As used herein, the housing may have a chamber for receiving an adsorbent or contain an adsorbent bed of the present invention. The housing in the present invention may also be used for a chromatography column or device (but is not limited thereto).

[0125] In some exemplary embodiments, the housing of the device can be a single chamber, or the housing has a plurality of chambers. In addition, the housing can also be constructed and arranged to present chambers in the plate format described in the U.S. Publication No. US 2013 / 0053588A1 and U.S. Patent No. 6,723,236 (each of which is incorporated herein by reference in its entirety). In some exemplary embodiments, the housing can be but is not limited to a syringe, a cartridge, a column, a multi-chamber, an 8-hole rack, an 8-hole bar, a 96-well plate, a 96-well micro-elution plate, a micro-elution tip, a 96-micro-elution tip array, an 8-tip micro-elution bar, a single micro-elution pipette tip, a rotating tube or a rotating container. However, the shape of many chambers or chambers can be obtained based on the requirements of the user.

[0126] In certain embodiments, the solution received in the housing can flow spontaneously into the adsorbent bed, for example, by capillary action. Alternatively, flow can be generated through the device by an external force such as gravity, a vacuum chamber, a reservoir, or a structure that allows for use in a centrifuge or with a vacuum or pressure source, an external force, etc., but is not limited thereto.

[0127] In certain exemplary embodiments, housing can be configured to have at least one cap or lid for packaging housing after receiving sample. In addition, cap can provide pressure source for efficient extraction method. In certain exemplary embodiments, cap can be connected with vacuum chamber, reservoir or structure (it allows to use in centrifuge, or use together with vacuum or pressure source), so as to apply vacuum or suction, to promote solution or liquid to flow out to outlet through described device. In particular, as known in the art, vacuum chamber, reservoir or structure (it allows to use in centrifuge or use together with vacuum or pressure source) can also be connected with other parts (but not limited to) of housing.

[0128] In certain exemplary embodiments, the housing may be formed of a material that is rigid in nature and withstands sufficient mechanical strength applied during extraction. Exemplary materials may be, but are not limited to, glass, metal, plastic, etc. In an exemplary embodiment, molded plastics such as polyetheretherketone (PEEK), polycarbonate (PC), etc. (as known in the art) may be used (but are not limited to).

[0129] In certain exemplary embodiments, based on the user's requirements, the capacity of the housing may vary according to the volume of the adsorbent, sample, etc. Exemplary housings of the present invention may have a capacity of about 1 mL, about 2 mL, about 3 mL, about 4 mL, about 5 mL, about 6 mL, about 7 mL, about 8 mL, about 9 mL, about 10 mL, about 15 mL, about 20 mL, about 30 mL, about 40 mL, about 50 mL, or about 100 mL (but not limited thereto).

[0130] In one embodiment, the device may further comprise an inlet or an outlet.

[0131] In one embodiment, the device of the present invention may include one or more glass frits or filters. For example, the glass frit or filter may be arranged at the bottom of the adsorbent, or the glass frit or filter may be arranged at the top of the adsorbent. As used herein, the glass frit or filter may maintain the stability or balance of the adsorbent before sufficient flow is generated through the device. In other words, the glass frit or filter may prevent the adsorbent from being penetrated by the sample solution before sufficient external force is applied. In certain exemplary embodiments, porous glass frit may be used. Exemplary glass frit may be, but is not limited to, a hydrophobic glass frit material such as Teflon, which may be in the form of a membrane, a filter, a screen, or a monolithic structure or a combination thereof. In certain exemplary embodiments, the glass frit may not be arranged in the adsorbent. In other certain exemplary embodiments, the glass frit may be arranged between the adsorbent layers without limiting the quantity or order.

[0132] In an exemplary embodiment, the device of the present invention may include: an adsorbent bed containing at least two adsorbents, a housing, a glass frit at the bottom of the adsorbent bed; an inlet and an outlet. In particular, at least one adsorbent may contain at least one hydrophobic component and at least one hydrophilic component, and at least one adsorbent may have an affinity for phospholipids, thereby efficiently removing salts and phospholipids from a sample solution and eliminating matrix effects.

[0133] In various exemplary embodiments of the present invention, the device can be used to separate and eliminate interfering species such as phospholipids and salts from analytes in a sample solution by using a combination of adsorbents. In other words, the matrix effect can be minimized. In addition, the analyte in the sample can be recovered with a high recovery rate and provided in a concentrated solution. Therefore, the analyte in the concentrated solution may not require an additional cleaning process before further analytical methods such as liquid chromatography, liquid chromatography-mass spectrometry / mass spectrometry (LCMS / MS), mass spectrometry (MS), MALDI-MS or ESI, etc. In certain exemplary embodiments, the analyte can be, but is not limited to, a small molecule, a protein, a peptide, a nucleic acid, an oligonucleotide and / or other compounds.

[0134] method

[0135] In another aspect, the present invention can provide an improved solid phase extraction method by using the device of the present invention having various advantages over conventional protein precipitation (PPT) and supported liquid extraction (SLE). In particular, the method of the present invention can substantially reduce the matrix effects that can be generally caused by salts, phospholipids, small molecules, proteins, peptides, particles, cells and salts, etc.

[0136] In one embodiment, the method may include the use of a separation device of the present invention. In particular, the device includes at least one adsorbent containing at least one hydrophobic component and at least one hydrophilic component and at least one adsorbent having an affinity for lipids or lipid-like species such as phospholipids (as described above). In addition, the adsorbent containing at least one hydrophobic component and at least one hydrophilic component can be water wettable. In certain exemplary embodiments, many combinations of (but not limited to) adsorbents containing at least one hydrophobic component and at least one hydrophilic component, adsorbents having an affinity for phospholipids, and other adsorbents based on the user's requirements may also be provided. Various other exemplary adsorbents used in the methods of the present invention are also described above.

[0137] In an exemplary embodiment, the device further comprises: an inlet, an outlet, and / or a housing capable of confining the adsorbent and capable of withstanding sufficient mechanical strength during extraction. For example, the device may comprise: a housing; an inlet; an outlet; at least one or more glass frits; and at least two or more adsorbents, and the housing may be, but is not limited to, a syringe, a cartridge, a column, a multi-chamber, a 4 to 8-well rack, a 4 to 8-well strip, a 48 to 96-well plate, a 96 to 384-well micro-elution plate, a micro-elution tip, a 96 to 384-micro-elution tip array, a 4 to 8-tip micro-elution strip, a single micro-elution pipette tip, a rotating tube, or a rotating container. Various non-limiting exemplary devices for the methods of the present invention are described above.

[0138] In an exemplary embodiment, the SPE method may include: providing a device; conditioning an adsorbent in the device; equilibrating the adsorbent; loading a sample onto the adsorbent; washing the adsorbent; eluting the analyte from the adsorbent. Alternatively, when the adsorbent containing at least one hydrophobic component and at least one hydrophilic component is water wettable, conditioning and equilibration may not be performed before loading the sample.

[0139] In certain exemplary embodiments, conditioning may be performed with methanol, ethanol, propanol, isopropanol, water, and mixtures thereof.

[0140] In certain exemplary embodiments, the equilibration solution may have a similar or identical ionic strength to the sample solution to provide a balanced partitioning environment in the adsorbent when the sample solution is loaded. Exemplary equilibration solutions / solvents may be, but are not limited to, water, a buffer solution such as phosphate buffer (PBS), or a solvent. 3 PO 4 solution), aqueous solution, organic solution miscible with water, etc.

[0141] In an exemplary embodiment, conditioning of the adsorbent bed may be performed with methanol, and the adsorbent bed may also be subsequently equilibrated with water.

[0142] Sample can be loaded on adsorbent in solution form.In certain exemplary embodiments, sample solution can be aqueous solution or organic solution miscible with water.In certain exemplary embodiments, sample can be in solid form, such as particle, freeze-dried solid, powder, film etc.The sample of solid type can be prepared in suitable solution using water, buffer solution or other organic solvent miscible with water before loading.The sample of solid type can also be prepared in homogeneous solution by using technology (such as vortex, ultrasound, shaking etc.) generally known in the art.Prepared sample can contain the analyte of further analytical method (such as mass spectrum (MS), chromatogram, MALDI-MS, ESI etc.) of experience.

[0143] In certain exemplary embodiments, the sample can be a biological sample. Exemplary biological samples can be, but are not limited to, inclusion bodies, biological fluids, biological tissues, biological matrices, embedded tissue samples, cells (e.g., one or more types of cells) and cell culture supernatants. Specific examples can include plasma, urine, cerebrospinal fluid, synovial fluid and other biological fluids, including extracts of tissues (such as liver tissue, muscle tissue, brain tissue and heart tissue, etc.). Biological samples can be suitably prepared before being loaded onto an adsorbent using techniques generally known in the art (but not limited thereto).

[0144] In one embodiment, the adsorbent can be washed with a suitable washing solvent. In certain exemplary embodiments, the solvent can be the same solvent as the solvent used in the conditioning step. Exemplary solvents for washing can be, but are not limited to, methanol, ethanol, propanol, isopropanol, water, and mixtures thereof.

[0145] In one embodiment, the analyte can be eluted with a suitable elution solution. In certain exemplary embodiments, the elution solution can be a neutral, acidic or alkaline mixture of acetonitrile and methanol; a neutral, acidic or alkaline methanol solution; a neutral, acidic or alkaline acetonitrile solution; a neutral, acidic or alkaline mixture of methanol and water, a neutral, acidic or alkaline mixture of acetonitrile and water; any of the above as a mixture of acetone or a neutral, acidic or alkaline solution of acetone.

[0146] In one embodiment, the method may further include analyzing the analytes from the SPE using analytical instruments and / or techniques (e.g., liquid chromatography, LC-MS / MS, mass spectrometry, MALDI-MS, ESI-MS, UHPLC, HPLC, nuclear magnetic resonance, infrared analysis, flow injection analysis, capillary electrochromatography, ultraviolet detection, or a combination thereof). In certain exemplary embodiments, the analysis and identification of the analytes may be performed by mass spectrometry. In certain other exemplary embodiments, mass spectrometry analysis may be obtained by comparing peaks with known compounds in a computer database.

[0147] According to various exemplary embodiments, the method using the device of the present invention can provide an efficiently clean biological sample analyte for analysis using liquid chromatography (e.g., UHPLC, HPLC), liquid chromatography-mass spectrometry / mass spectrometry (LC / MS-MS), mass spectrometry (MS), MALDI-MS or ESI, etc. In certain exemplary embodiments, the eluate obtained after eluting the analyte contains less than about 10 wt%, about 9 wt%, about 8 wt%, about 7 wt%, about 6 wt%, about 5 wt%, about 4 wt%, about 3 wt%, about 2 wt% or about 1 wt% of the phospholipids contained in the sample. In addition, a wide range of analytes can be recovered at a recovery rate of greater than about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100%. Alternatively, the matrix effect can be reduced to less than about 20%, about 19%, about 18%, about 17%, about 16%, about 15%, about 14%, about 13%, about 12%, about 11%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, about 1% compared to a method not using the apparatus of the present invention. Example

[0148] The following examples illustrate the present invention and are not intended to limit the scope of the present invention.

[0149] Experimental conditions used for sample analysis:

[0150] Unless otherwise stated, the LC-MS / MS or LC-MS data in the examples were generated using the following conditions. Unless otherwise specified, analyte concentrations, identities and abbreviations are as follows.

[0151] (2)LC conditions:

[0152] LC system: ACQUITY UPLC Class I, fixed-loop syringe, 2 μL loop

[0153] Detection: Xevo TQ-S

[0154] Column: ACQUITY UPLC CSH C18, 2.1×100 mm, 1.7 μm (PN Mobile phase A: 0.1% formic acid in water (v / v)

[0155] Mobile phase B: 0.1% formic acid in acetonitrile (v / v)

[0156] Flow rate: 500 μL / min

[0157] The solvent gradient table is as follows:

[0158]

[0159] Column temperature: 40°C

[0160] Sample temperature: 10°C

[0161] Injection volume: 1 μL

[0162] Strong Needle Wash: 70 / 30 ACN / water with 2% formic acid

[0163] Weak needle wash: 70 / 30 ACN / water with 2% formic acid

[0164] Injection mode: Partial loop with needle overfill

[0165] Vials / Plate: 1cc 96-well collection plate (PN 186002481)

[0166] (2)MS conditions:

[0167] MS system Waters Xevo TQ-S conditions, ESI+

[0168] Capillary voltage: 2.5kV

[0169] Desolvation temperature: 500°C

[0170] Conical airflow: 150L / hour

[0171] Desolvation gas flow: 1000L / hour

[0172] Monitored MRM transitions: See table below

[0173] Exemplary MRM transitions monitored by ESI are shown below:

[0174] Analytes Precursor ion m / z Product ion m / z Cone voltage (V) Collision energy (eV) Azidothymidine 268.08 127.10 22 8 7-Hydroxycoumarin 163.06 107.06 58 20 Phenacetin 180.13 110.06 26 20 Betamethasone 393.17 373.15 32 8 Protriptyline 264.20 155.08 46 20 Alprazolam 309.11 281.17 38 26 Amitriptyline 278.22 91.04 44 22 Naproxen 231.12 185.06 20 16 Propranolol 260.19 116.15 48 16

[0175] Additionally, an exemplary chromatogram for the method is shown in Figure 8 middle.

[0176] (3) The MS parameters of phospholipids are shown below.

[0177]

[0178]

[0179] Analyte concentrations, characteristics, and therapeutic relevance

[0180]

[0181] Reagents

[0182] Formic acid 88% was from JT Baker. All other reagents and solvents were purchased from Sigma-Aldrich unless otherwise specified.

[0183] Example 1: μElution plate with layered adsorbent combination of the present invention

[0184] A μElution plate was prepared in which each well contained 2 mg of adsorbent and layered on top with 1 mg of 30 μm HLB. The adsorbent plates of the invention were compared with commercially available plates of the same format from Waters Corporation (pn 186001828BA containing 2 mg HLB).

[0185] Diluted human plasma (1:1, containing 4% H 3 PO 4 ) were loaded onto a μElution plate containing the adsorbent combination of the present invention or HLB. The loaded sample was washed with 400 μL of 5% methanol in water and eluted by using a mixture of acetonitrile:methanol (90:10) as the elution solution under the optimal conditions for analyte recovery and phospholipid removal for the above adsorbent combination.

[0186] As shown in Table 1 below, the average recovery and standard deviation are similar to those of The amount of phospholipids in the extracted analyte samples was compared to the HLB plates and 1:3 plasma:acetonitrile PPT supernatant in Table 2. The plates of the present invention had 74% to 93% less phospholipids than the commercially available plates and 95.7% to 99.8% less phospholipids than the 1:3 PPT supernatant. This example demonstrates that for a wide range of analytes, recoveries and standard deviations remain comparable to commercially available plates and a substantial reduction in phospholipids is achieved.

[0187] Table 1: Comparison of analyte recoveries and standard deviations (STD)

[0188]

[0189] Table 2: Comparison of phospholipid removal

[0190]

[0191] Example 2: Comparison of adsorbents of the present invention in a 1 cc device

[0192] This example shows that the layered format of the present invention provides recoveries comparable to a 1 cc HLB cartridge alone, whether or not the layered selected adsorbents are separated by a frit. However, the 1 cc cartridge of the present invention separated by a frit (as is typically done for multi-adsorbent devices) shows substantially higher relative standard deviations (RSDs) than the other configuration.

[0193] Device

[0194] • Three device formats were prepared using 1 cc cartridges. Each format was configured as follows.

[0195] ● Device 1: (Top) Glass frit - about 10 mg in amount HLB-Glass Frit (Bottom)

[0196] • Device 2: (Top) Glass frit - about 10 mg of HLB - about 10 mg - Glass frit (bottom)

[0197] • Device 3: (Top) Glass frit - about 10 mg in amount HLB-(glass frit)-amount of about 10 mg - Glass frit (bottom)

[0198] method

[0199] Solid phase extraction was performed using each device with a 1 cc cartridge. The adsorbent in each cartridge as described above was conditioned with methanol in an amount of about 400 μL and equilibrated with water in an amount of about 400 μL. After equilibration, 400 μL of a 1:1 ratio of plasma and 4% by volume of concentrated H 3 PO 4 The sample of the solution was then washed at least once with 400 μL of 5% methanol. The analytes in the sample were eluted with 400 μL of an acetonitrile / methanol mixture (80 / 20).

[0200] Experimental conditions

[0201]

[0202]

[0203]

[0204] result

[0205] (1) Eluates from each device format (n=6) were collected and analyzed as shown in Table 3. Each analyte was detected using a 254 nm UV spectrophotometer and quantified for percent recovery calculations.

[0206] [Table 3: Analyte recovery and relative standard deviation]

[0207]

[0208] According to the results in Table 3, devices 1, 2 and 3 showed similar results in the recovery of each analyte. HLB and When extraction was performed with Apparatus 2 using the adsorbent without a separation frit, most analytes had recoveries ranging from about 93% to 108%. The %RSDs for analyte recoveries in Apparatus 3 were substantially greater than those in Apparatuses 1 and 2, which may be due to the presence of a sorbent in the extract and a separation frit in the extract. HLB and The additional glass frits added between the adsorbents cause non-uniform flow rates. Most multi-adsorbent SPE devices currently on the market use glass frits to separate the different adsorbents.

[0209] (2) The eluate from each device was collected and analyzed as shown in Table 4. In particular, MS / MS was used to determine the percentile of phospholipid removal by comparison with the untreated sample. The amount of phospholipids in the sample and the eluate can be obtained by the sum of m / z 496, 520, 522, 524, 704, 758, and 804.

[0210] [Table 4: Data on removal of phospholipids from the eluate]

[0211]

[0212] Device 1 removed approximately 80% of the phospholipids, while Devices 2 and 3 removed approximately 95% or more of the phospholipids. Additionally, the %RSD for Device 1 was substantially higher compared to Devices 2 and 3.

[0213] Overall, Device 2 provided the best results for the extraction and cleanup process prior to mass spectrometry analysis. Device 3, containing the adsorbent combination of the present invention in 1 cc cartridges separated by a frit (as is typically done for multi-adsorbent devices), showed substantially higher relative standard deviations (RSDs) than the other configurations.

[0214] Example 3: Method for determining MS / MS matrix effects

[0215] One method for determining potential matrix effects utilizes post-column injection of the eluate generated from the proposed SPE method using blank (unspiked) plasma (see figure below). A clean standard (1) containing the target analyte is injected into the column. A tee that injects the eluate is combined with the flow from the column outlet. The combined stream is then directed into the MS for analysis.

[0216] The MS detector analyzes each analyte as it elutes from the column in the presence of blank plasma from the various sample preparation methods to be compared. The method shows differences in matrix effects due to matrix materials remaining in the eluent. By the method cited, the MS response of each analyte when a matrix-free eluent solution is injected is compared with the MS response of each analyte when an eluent from processed blank plasma is injected. The method of obtaining matrix effects is known to those skilled in the art. An example of a reference for the method is C. Polson et al. / J. Chromatogr. B 785 (2003) 263-275.

[0217] Each eluate obtained from SPE or using each device sample preparation method was injected into line (3) for MS analysis. In the case described, rat plasma was used as the sample and was not spiked with analytes. Table 5 contains a description of the devices used to obtain blank plasma eluates and the MS reactions relative to the infusion of the eluate solutions.

[0218] As shown in Table 5, with 10 mg HLB and 10mg The combined device had the lowest average matrix effect. Since the most likely contributors to matrix effects are salts, other polar components, and PL, the larger MEs seen in Table 5 for each sample preparation technique except the present invention are likely caused by one or more of these matrix components.

[0219] Other matrix components not listed in Table 6 were utilized The combination of SPE of HLB with the sorbent of the present invention reduces the expected sources of matrix effects for each sample preparation method in Table 6. The average of the absolute values ​​of matrix effects is reported, as both suppression and enhancement are considered equally detrimental to accurate quantitation.

[0220] [Table 5: Matrix effect data of blank plasma eluates prepared by various sample preparation schemes]

[0221]

[0222] *Mean of the absolute values ​​of ME

[0223] Table 6: Common causes of matrix effects in eluents

[0224]

[0225] Procedure for 1cc cartridge SPE of eluents in the above table: ● Condition with 400 μL MeOH

[0226] ●Balance with 400 μL of water

[0227] ● Load 400 μL of 1:1 plasma:4% H 3 PO 4 sample

[0228] ● Wash with 400 μL 5% MeOH

[0229] ● LC conditions for elution with 400 μL 80 / 20 MeCN / MeOH

[0230]

[0231] The solvent gradient table is as follows:

[0232]

[0233] MS conditions

[0234]

[0235] Analytes Capillary(kV) Cone voltage (V) Collision (eV) Precursor product Clozapine 3 30 25 327.02 270.1 Desloratadine 1.5 28 20 311.2 259.25 Procainamide 2 20 35 236.4 120.4 Risperidone 3 30 30 411.3 191.6 9-OH Risperidone 2.5 35 30 427.2 207.25

[0236] Example 4: Removal of phospholipids using layered and mixed adsorbents

[0237] Each device is prepared with a 1 cc cartridge comprising 1) a HLB and about 40 mg The mixed adsorbent contains about 10 mg of HLB and about 20 mg The mixed adsorbent 3) contains about 10 mg of HLB as top layer and about 20mg The adsorbent was layered as the bottom layer. The apparatus was conditioned with 80:20 acetonitrile / water.

[0238] The milk was extracted and precipitated with pH 5 succinic acid / EDTA buffer. After centrifugation, the supernatant was collected and loaded onto the conditioned device. The device was washed with 5% methanol / water. The pellet after centrifugation was resuspended, extracted with 80:20 acetonitrile / water (0.2% formic acid) and centrifuged. After centrifugation, the supernatant was collected and loaded onto the device to elute the device.

[0239] Figure 4 The results of mass spectrometry analysis of a milk sample in the phospholipid region are shown. When the mixed adsorbent and the layered adsorbent are used in the same amount, the phospholipid removal rate may be similar. When a larger amount of adsorbent is used, the phospholipid removal rate may be increased.

[0240] Example 5: Detection of veterinary drugs in milk

[0241] A device with a 1 cc cartridge containing approximately 10 mg of HLB with about 20 mg The device was conditioned with 80:20 acetonitrile / water.

[0242] Milk was extracted and precipitated with pH 5 succinic acid / EDTA buffer. After centrifugation, the supernatant was collected and loaded onto the conditioned device. The device was washed with 5% methanol / water. The pellet after centrifugation was resuspended, extracted with 80:20 acetonitrile / water (0.2% formic acid) and centrifuged. After centrifugation, the supernatant was collected and loaded onto the device for elution.

[0243] For comparative examples, each containing about 100 mg C 18 Adsorbent, about 30 mg HLB and about 20 mg Z-Sep (Sigma-Aldrich, USA) and about 50 mg C 18 The extraction and SPE were performed according to the manufacturer's manual of each sorbent product.

[0244] exist Figure 5 In the embodiment, it is shown that the HLB and The results of removing phospholipids from milk samples by the device using the mixed adsorbent of and other devices in the comparative examples are shown. In addition, in Table 7, the removal rate of each device is shown. In particular, compared with non-polar adsorbents (such as C 18 ), the device of the present invention removes phospholipids with a substantially improved efficiency of greater than about 90%.

[0245]

[0246]

[0247] *Affected by nonspecific binding losses in solvent-only standards.

[0248] exist Figure 6-7 In the present invention, veterinary drugs contained in milk were analyzed by mass spectrometry after extraction using the apparatus according to the exemplary embodiment of the present invention and other apparatuses in the comparative examples. Figure 6 As shown in FIG. , ciprofloxacin and enrofloxacin in milk samples were recovered by using a method containing HLB and After extraction with the mixed adsorbent device, the sample was clearly analyzed by mass spectrometry, while Z-sep did not recover enrofloxacin from the sample. Figure 7As shown in FIG. , in the use of the HLB and After extraction with the mixed adsorbent device of , oxytetracycline was recovered and clearly analyzed by mass spectrometry, while Z-sep did not recover oxytetracycline from the milk sample. The average recovery rate of each adsorbent-containing device was compared. For the device containing the adsorbent according to the exemplary embodiment of the present invention, HLB and The highest average recovery was achieved with the mixed adsorbent. The average of the absolute values ​​of ME showed that the Zsep device gave the lowest value of 28%. However, three of the veterinary drugs were not recovered at all. The ME of the other three devices was essentially the same.

[0249] Example 6 - Analysis of steroids in human plasma

[0250] Corticosteroid analysis has always been a challenge requiring time-consuming derivatization prior to GC-MS methods. Efforts have been made to move away from GC-MS or to favor immunoassays for LC-MS (M. Ohno et al. / J. Chromatogr. B 746 (2000) 95–101 and U. Ceglarek et al. / Clinica Chimica Acta 401 (2009) 114–118). In the following, the use of the device of the present invention and the provided SPE method greatly reduces the time required for the analysis of cortisol, androstenedione and 17α-hydroxyprogesterone. Corticosteroids were extracted from plasma using a μElution plate format containing 2 mg of and 1mg Mixed adsorbent composed of HLB.

[0251] method

[0252] Solid phase extraction was performed as follows. 1 mg / mL stock solutions of cortisol, androstenedione (A-dione), and 17α-hydroxyprogesterone (17-OHP) were purchased from Cerilliant Corporation (Round Rock, TX, USA). Solutions containing 10 ng / mL of each analyte were prepared in double charcoal stripped human plasma (MSG 4000 from Golden West Biological Suppliers). 300 μL of a 4:1 ratio of methanol (MeOH) and 89 g / L ZnSO 4 150 μL of blank plasma or pre-spiked plasma was precipitated with a solution consisting of a solution. The sample was pipetted several times and centrifuged at 3220 rcf for 10 minutes. The supernatant of each replicate (n=4) was then mixed with 900 μL of 4% H3 PO 4 The entire sample was then loaded onto the μElution plate in 2 equal portions without any pretreatment or equilibration. The plate was then washed with 2×200 μL aliquots of 25:75 MeOH:H 2 Wash each well of the plate with 2 x 25 μL aliquots of a solution consisting of 90:10 ACN:MeOH. Wash all wells with 2 x 25 μL aliquots of 90:10 ACN:MeOH by passing 2 x 200 μL aliquots of 25:75 MeOH: 2 O-washed wells were used to prepare solvent samples for matrix effect calculations.

[0253] Use 50 μL of 25:75 MeOH:H 2 Samples pre-spiked with 10 ng / mL of the steroid mix were post-spiked with 50 μL of 25:75 MeOH:H 2 Blank plasma samples that were not pre-spiked were post-spiked with 50 μL aliquots of 25:75 MeOH:H 2 O solution (equal to the mass loaded in each sample). 2 O solution spiked solvent samples for matrix effects.

[0254] The recovery rate was calculated according to the following formula:

[0255]

[0256] The matrix effect is calculated according to the following formula:

[0257]

[0258] Chromatographic conditions

[0259] The separation was achieved on a Waters' ACQUITY I-Class system with a fixed loop autosampler. Details are listed in the table below.

[0260] LC system: Waters ACQUITY I-Class UPLC system

[0261] Column: ACQUITY UPLC HSS T3 column, 1.7 μm, 2.1 × 50 mm (p / n1860035358)

[0263] Column temperature: 40℃

[0264] Sample temperature: 10℃

[0265] Mobile phase A (MPA): water containing 0.1% formic acid

[0266] Mobile phase B (MPB): ACN containing 0.1% formic acid

[0267] Purge solution: 25:25:50ACN:MeOH:water Washing solution: 10% ACN

[0268] Injection volume: 7.5 μL

[0269] Mobile phase gradients for steroid applications.

[0270]

[0271]

[0272] Mass spectrometry conditions

[0273] Samples were analyzed in a WATERS TQ-S mass spectrometer in MRM mode. The conditions are listed below.

[0274] MS system: Waters Xevo TQ-S mass spectrometer

[0275] Ionization mode: ESI positive

[0276] Capillary voltage: 1.0kV

[0277] Cone Voltage: Optimized for each analyte Desolvation gas: 1000L / hour

[0278] Cone gas: 150L / hour

[0279] Desolvation temperature: 500℃

[0280] Source temperature: 150℃

[0281] use Data were acquired and analyzed using the software (V4.1; SCN 876). Carry out quantitative analysis.

[0282] The table below lists the retention times and individualized MS parameters for the steroids, including MRM transitions, cone voltages, and collision energies. Two MRM transitions were used for each compound, a primary transition (listed first) and a confirming transition (listed second).

[0283]

[0284] Several phospholipid transitions were also monitored to determine the extent of phospholipid removal. Their MRM transitions and MS parameters are listed in the table below. The same LC parameters used for corticosteroid analysis were used for phospholipid analysis, with the addition of an additional 3 minutes hold at 95% B to completely elute the phospholipids.

[0285]

[0286] result

[0287] The extraction recoveries and matrix effects are listed in the table below. The average recovery of all three compounds was 72.2% and the average matrix effect was -10.1%.

[0288] Table 8: Summary of corticosteroid recoveries, standard deviations, and matrix effects.

[0289]

[0290] Phospholipid (PL) removal was compared to samples that had been subjected to the same protein precipitation method as the extracted samples but without solid phase extraction. The total peak area of ​​all monitored phospholipids is listed below and shows 97% removal of the phospholipids present in the plasma samples. In view of the fact that the concentration of the extracted samples was 6 times that of the precipitated samples, the actual phospholipid removal was close to 99.5%.

[0291] Table 9: Comparison of total peak areas of phospholipids

[0292] Total PL peak area Relative abundance Protein precipitation only 22152370 100％ μElution Extraction 690579 3.12％

[0293] Example 7 - Extraction of different sets of analytes from the 1 cc device of the present invention

[0294] Prepare a device with 1 cc cartridges, each containing 30 mg of a mixture of the following adsorbents: Approximately 10 mg HLB and about 20mg Follow this 3-step plan:

[0295] 1. Load 1 mL of plasma (with 4% H 3 PO 4 1:1 dilution of

[0296] 2. Wash with 1 mL 5% MeOH

[0297] 3. Elute with 1 mL 90 / 10 MeCN / MeOH (v / v)

[0298] LC-MS / MS analysis was performed using the chromatographic and MS conditions described below. Analyte recoveries were ≥91% with relative standard deviations between 4% and 10%. These recoveries were obtained for a wide range of ionic analytes and the neutral analyte betamethasone with log P values ​​of 0.05-4.9 and pKa values ​​of 2.2-9.7. For targeted quantitative analysis of drugs, matrix effects (ME) of less than ±20% are considered acceptable. The ME values ​​in the following table are well within the acceptable standards, and on the device of the present invention, the average analyte recovery was 98.8% with %RSD≤10%.

[0299] Table 10: SPE results for plasma extracted using the three-step SPE protocol.

[0300]

[0301]

[0302] Example 8: Analysis of synthetic cannabinoids in whole blood

[0303] This example uses whole blood, which is often used in forensic toxicology cases. Whole blood samples contain higher concentrations of phospholipids due to increased release from lysed blood cells. For the same reason, matrix effects are increased in whole blood samples.

[0304] experiment

[0305] LC conditions:

[0306] LC system: ACQUITY UPLC Class I

[0307] Column: CORTECS UPLC C 18 ,1.6μm;2.1×100mmP / N 186007095

[0308] Column temperature: 30℃

[0309] Injection volume: 5 μL

[0310] Flow rate: 0.6mL / min

[0311] Mobile phase A: 0.1% formic acid in MilliQ water

[0312] Mobile phase B: 0.1% formic acid in ACN

[0313] Gradient: Initial conditions started at 30% B. % B increased to 50% in 2 minutes and held at 50% B for 1 minute, increased to 90% B in 4 minutes, then returned to 30% in 0.2 minutes. The system was allowed to re-equilibrate for 1.3 minutes. The total cycle time was 8.5 minutes.

[0314] MS conditions:

[0315] MS system: TQD Mass Spectrometer

[0316] Ionization mode: ESI positive

[0317] Acquisition mode: MRM (transitions see Table 2)

[0318] Capillary voltage: 1kV

[0319] Synthetic Cannabinoid Standards:

[0320] AM2233, JWH-015, RCS-4, JWH-203, RCS-8, JWH-210, JWH-073, and JWH-018 were purchased from Cerilliant (Round Rock, TX). All other compounds and metabolites were purchased from Cayman Chemical (Ann Arbor, MI).

[0321] Individual stock solutions (1 mg / mL) were prepared in methanol, DMSO, or 50:50 DMSO:methanol. Combined stock solutions of all compounds (10 μg / mL) were prepared in methanol. Working solutions were prepared daily in 40% methanol. Calibrators and quality control (QC) samples were prepared by spiking various concentrations of working standards and into matrix (whole blood). Calibrator concentrations for all analytes ranged from 0.2-100 ng / mL. Quality control samples were prepared in rat whole blood at 2.5, 7.5, and 75 ng / mL.

[0322] The 22 compounds analyzed are listed in Table 10 and constitute a panel that includes various types of forensically relevant synthetic cannabinoids. These include adamantoylindoles (AM 1248 and AKB48), naphthoyl indole (JWH022), phenylacetylindole (RCS-4 and RCS-8), and tetramethylcyclopropyl indole (UR-144 and XLR11). The major metabolites of JWH-073 and JWH-018 are also included, as some of these compounds are structural isomers with identical mass spectral fragments that require adequate chromatographic separation for accurate quantification.

[0323] The table below lists the retention times and individualized MS parameters for the steroids, including MRM transitions, cone voltages, and collision energies. Two MRM transitions were used for each compound, a primary transition (listed first) and a confirmatory transition (listed second).

[0324] Table 11: Retention times and MS parameters for synthetic cannabinoids

[0325]

[0326]

[0327]

[0328] Sample preparation:

[0329] Use approximately 10 mg in a 96-well plate HLB and about 20mg The sample was extracted with a combination of sorbents (30 mg / well in total). 0.1 mL of 0.1 M zinc sulfate / ammonium acetate solution was added to 0.1 mL of rat whole blood and vortexed for 5 seconds to lyse cells. All samples were then precipitated by adding 400 μL ACN. The entire sample was vortexed for 10 seconds and centrifuged at 7000 rcf for 5 minutes. The supernatant was subsequently diluted with 1.2 mL of water and then loaded. The sample was directly loaded on the 30 mg sorbent mixture without adjustment or balance. All holes were then washed with 2 × 500 μL 25:75 MeOH: water and eluted with 2 × 500 μL 90 / 10 ACN / MeOH (v / v). The eluent was then evaporated under nitrogen and reconstructed with 100 μL 30% ACN. 5 μL was injected onto the UPLC system.

[0330] The analyte recovery was calculated according to the following formula:

[0331]

[0332] Where A equals the peak area of ​​the extracted sample and B equals the peak area of ​​the extracted matrix sample (where the compound was added after extraction).

[0333] result

[0334] The extraction protocol resulted in nearly complete recovery of all compounds and minimized matrix effects for most analytes. All compounds except one had 80% or greater recoveries with an average recovery of 91%. The recoveries were consistent with an average %RSD value of 5% for all compounds. Matrix effects were very good across the panel. Only two compounds had matrix effects of more than 40% and all remaining compounds had matrix effects of less than 25%. The average magnitude of the matrix effect was only 17%.

[0335] Table 12: Results of the extraction protocol

[0336]

[0337] The PL of the protein precipitation (PPT) samples was analyzed before the 1.2 mL dilution. However, even without dilution, the supernatant had a 6-fold reduction in phospholipid concentration relative to the eluate (PPT volume = 600 μL, eluate volume = 100 μL). The plates of the present invention removed more than 95% of the phospholipids in whole blood samples compared to the protein precipitation (PPT) supernatant, resulting in a cleaner extract.

[0338] Table 13: Comparison with PPT

[0339] The sum of PL areas Elution using 90 / 10 ACN / MEOH 1443 PPT - 600 μL supernatant 4617806

[0340] Example 9: Comparison of phospholipids in the final extraction samples of 10-15 mg 96-well plates

[0341] 96-well plates are used to increase the throughput of the SPE method. Since such LC analysis of samples prepared using 96-well plates is affected by the PL that continues to accumulate on the analytical column, a constantly changing background of various phospholipids carried from previously analyzed samples is exhibited. In order to prevent this accumulation on phospholipids, various solvent combinations can be used at the end of the analytical gradient as described in Little et al. (JL Little et al. / J. Chromatogr. B833 (2006) 219-230). Unfortunately, it takes time to "wash" phospholipids from the column, which is contrary to the goal of rapid high-throughput analysis. In practice, stable isotope-labeled analogs or target analytes are able to correct for most matrix effects, but problems with such approaches have been reported (E. Stokvis, H. Rosing, JH Beijnen, Rapid Commun. Mass Spectrom. 19 (2005) 401-407, and S. Wang et al. / Journal of Pharmaceutical and Biomedical Analysis 43 (2007) 701-707).

[0342] The 15 mg 96-well plate of the present invention was compared with various state-of-the-art polymeric adsorbents that appear to target, directly or indirectly, SPE Product Sales. The following table contains a description of the commercially available adsorbents in the comparison. Included are the part numbers (PN) of the products used in the evaluation, the PN of the brochure provided by the product manufacturer, and a brief description of the adsorbent. These are provided so that the relevance of the comparison can be understood.

[0343] Table 14: Adsorbents used

[0344]

[0345] The concentration of total phospholipids was compared between the above-mentioned prior art polymer adsorbents. Each well of the 96-well plate of the present invention contained 5 mg HLB with 10mg Combination of absorbents.

[0346] SPE Solution

[0347] Each well of a 96-well plate was conditioned with 500 μL of methanol and then equilibrated with 500 μL of water. 3 PO 4 500 μL of blank rat plasma was diluted 1:1, loaded and washed with 500 μL of 5% methanol in water. Each well was then eluted with 500 μL of 100% acetonitrile and the following MRM phospholipid transitions were quantified: 496.4→184.3, 520.4→184.3, 522.4→184.3, 524.4→184.3, 704.4→184.3, 758.4→184.3 and 806.4→184.3.

[0348] result

[0349] As shown in Table 15, the eluates from the four commercially available polymer adsorbents tested contained 11 to 26 times more phospholipids than the present invention. Although the total mass of the plates of the present invention was 15 mg compared to 10 mg for all other plates, the difference is expected to produce high phospholipid amounts in their eluates.

[0350] Table 15:

[0351]

[0352] In this way, the device according to various exemplary embodiments of the present invention can substantially improve the phospholipid removal rate and further improve the recovery rate of the target analyte for mass spectrometry analysis. Therefore, when the analyte in the sample (such as food sample and biological sample) is extracted by the device or method of the present invention as described herein, the matrix effect is reduced or eliminated, and no additional cleaning process is required before mass spectrometry analysis.

[0353] Example 10: μElution Plates Containing Adsorbent Mixtures of the Invention with HLB and Supported Liquid Extraction (SLE) recovery, %RSD, matrix effects and phospholipid comparison

[0354] The following example demonstrates that the μElution plate containing the adsorbent combination of the present invention is superior to the 2 mg Performance benefits of HLB μElution plates and ISOLUTE SLE+ 96-well plates (pn 820-0200-P01). The adsorbent combination of the present invention is 1 mg HLB and 2 mg mixture.

[0355] step

[0356] The ISOLUTE SLE+ plate scheme used is as recommended by the manufacturer. Each well (n=8) is loaded with 200 μL of 1:1 rat plasma: water. The flow of loading solution is started by applying a vacuum of 0.2 bar for 2-5 seconds until the sample is loaded and gravity fed into diatomaceous earth. The sample is then left to stand for 5 minutes to completely absorb and form a layer for extraction. The sample is eluted using 1 mL of 95:5 dichloromethane (DCM): isopropanol (IPA) v / v. The solution is gravity fed through the plate for 5 minutes, and then a vacuum of 0.2 bar is applied for 10-30 seconds to complete elution. The sample eluent is evaporated to dryness and reconstructed in 200 μL of 40% acetonitrile aqueous solution. Due to the use of a solvent that is immiscible with water, evaporation and reconstruction steps are required before analysis by reversed phase LC-MS / MS. Four of the wells are loaded with spiked plasma, and four wells have blank plasma. These are used to calculate recovery and RSD. Four solvent blanks are prepared to calculate matrix effects. Phospholipids were calculated from spiked and unspiked plasma wells (n=8 total).

[0357] For the μElution plate, each well (n=8) was loaded with 200 μL of 1:1 rat plasma:4% H 3 PO 4 Samples. Each well was washed with 200 μL of 5% aqueous methanol and then eluted with 200 μL of 90 / 10 acetonitrile / methanol (v / v). Four wells / plate were loaded with spiked plasma and four wells were loaded with blank plasma. These were used to calculate recoveries and RSDs. Four solvent blanks were prepared to calculate matrix effects. Phospholipids were calculated from spiked and unspiked plasma wells (n=8 in total).

[0358] The values ​​for phospholipids are the signal areas of the following MRM phospholipid transitions: 496.4→184.3, 520.4→184.3, 522.4→184.3, 524.4→184.3, 704.4→184.3, 758.4→184.3 and 806.4→184.3.

[0359] result

[0360] Comparison of recoveries, %RSD, matrix effects and phospholipids found for the final extracts are shown in the table below. Similar recoveries were obtained for the 3 different plates, except for naproxen (an acid) which was not effectively recovered along with the basic and neutral analytes by SLE. The lowest relative standard deviation was obtained on the plates of the present invention. The mean of the absolute values ​​of the ME for each analyte was about the same for the HLB and the plates of the present invention, 2.3 and 1.9 respectively. For the SLE+ plates, the mean ME for all analytes was 18%, about 8-9 times higher. The μElution plates of HLB and ISOLUTE SLE+ plates showed 14 and 9 times higher total PL than the plates of the present invention.

[0361] Table 16: Utilization Comparison of SPE recovery, %RSD, matrix effect and phospholipids between HLB, the adsorbent plate of the present invention and ISOLUTE SLE+ plate

[0362]

[0363]

[0364] Example 11: Performance comparison of different adsorbent combinations of the present invention

[0365] In Example 4, it is shown that HLB and The mixed adsorbent combination and the layered form give substantially the same performance. However, the results of the examples may not be obvious.

[0366] method

[0367] In these experiments, μElution plates were used as SPE devices. Three plates were prepared using three combinations of sorbents. HLB and Plate A was prepared with 3 mg of the mixture. (at the bottom), 1mg Plate B was prepared with HLB (on top). HLB (at the bottom), 2mg (On top) Prepare plate C by loading 500 μL of 1:1 diluted plasma:4% H 3 PO 4 The plates were tested by washing with 500 μL of 5% aqueous methanol and then eluting with 2×50 μL of 90 / 10 acetonitrile / methanol (v / v) solution. Unless otherwise stated, the eluates were analyzed using the methods contained in the experimental section.

[0368] result

[0369] The following table shows an overview of the total phospholipids for plates A, B and C. The total value for phospholipids is the total signal area of ​​the following MRM phospholipid transitions: 496.4→184.3, 520.4→184.3, 522.4→184.3, 524.4→184.3, 704.4→184.3, 758.4→184.3 and 806.4→184.3. As can be seen from the table, the mixed adsorbent eluates and those of plate B (with HLB on top) gave equivalently lower values ​​for total PL. The eluates from plate C (with ) gave substantially higher values ​​for the total PL value.

[0370] Table 17: Overview of total phospholipids for 3 different adsorbent combinations in 96-well μElution plates

[0371]

[0372] Unlike phospholipids, recoveries and standard deviations were not affected by differences in sorbent combinations. However, matrix effects did reflect The added eluent cleaning provided by the adsorbent.

[0373] Table 18: Overview of recoveries, standard deviations, and matrix effects for 3 different sorbent combinations in a 96-well μElution plate

[0374]

[0375] Example 12: Other adsorbent combinations

[0376] The popularity of HLB has driven the development of similar products that have been patented around it. The adsorbent has also been imitated in the market. HLB and The most similar products are Strata X polymeric hydrophobic adsorbent and Phree. Both of these clones (used here to describe products introduced after the original product) are sold by Phenomenex. Strata X has been mentioned in earlier examples. The description of Phree provided by Phenomenex is "Phree Phospholipid Removal Solution provides a fast and effective method to remove proteins and phospholipids from plasma samples without negatively affecting the recovery of your target analytes. To understand whether our inventive adsorbent combination is effective for HLB and Is unique for use HLB and A similar combination was prepared using the same 1:2 weight ratio of Strata X and Phree adsorbents.

[0377] method

[0378] A μElution plate was used as the device for these experiments. 2 mg Plates were prepared with HLB, 2 mg StrataX, Strata X and Phree (at a weight ratio of 1:2). The source of Phenomenex adsorbent was a commercially available product.

[0379] By loading 500 μL of 1:1 diluted plasma:4% H3 PO 4 The plates were tested by washing with 500 μL of 5% aqueous methanol and then eluting with 2×50 μL of 90 / 10 acetonitrile / methanol (v / v) solution. Unless otherwise stated, the eluates were analyzed using the methods included in the experimental section.

[0380] result

[0381] The table below shows a summary of the total phospholipids for plates A, B, and C. The total value for phospholipids is the total signal area for the following MRM phospholipid transitions: 496.4→184.3, 520.4→184.3, 522.4→184.3, 524.4→184.3, 704.4→184.3, 758.4→184.3, and 806.4→184.3. As can be seen from the table below, the single adsorbent showed approximately 15-20 times the total PL of the mixed adsorbent plate containing STRATA X and Phree.

[0382] Table 18: Summary of the total signal area of ​​phospholipids from 4 different adsorbents in a 96-well μElution plate

[0383]

[0384] The table below shows a comparison of recoveries and standard deviations for the same plates. The best recoveries were obtained for HLB in this experiment. This example highlights the problem that not all batches of rat plasma (or human plasma) are identical and that differences in recovery can also occur for PL, which appears to be higher than usual for all the different adsorbents. With the exception of the recovery of AZT, which was lower, the recoveries for the mixed adsorbent plates were similar to those of their single adsorbent counterparts.

[0385] Table 19: Overview of recoveries and standard deviations for 3 different adsorbents in a 96-well μElution plate

[0386]

[0387] Example 13: Flow rate increase

[0388] Chromatographic adsorbents, especially reversed phase (RP) adsorbents, are commonly used in solid phase extraction (SPE). Reversed phase adsorbents are usually polymer-based, e.g. HLB, or by combining silica particles with hydrophobic ligands (e.g. ) bonding. RP particles can vary considerably in their degree of hydrophobicity, ranging from relatively hydrophilic to well-balanced hydrophilic / lipophilic adsorbents such as HLB), changes to hydrophobic Waters manufactures many other adsorbents covering a wide range of hydrophobicity.

[0389] Those skilled in the art who control the flow rate of liquids through SPE devices have observed that certain percentages of their aqueous samples or subsequent aqueous solutions may be more difficult to pass through the device than other aqueous solutions. The mechanism that causes the flow variability is not fully understood, but lies in the adverse interaction between the device, adsorbent and sample / solution, especially under low vacuum / pressure settings. This problem is most significant when a variety of highly aqueous samples / solutions are passed through reverse phase devices, whether these devices contain purely hydrophobic or water-wettable hydrophilic / lipophilic balanced particles. This unpredictable flow behavior usually requires some form of user intervention to force the fluid through and avoid sample loss. These activities increase cost, time and complexity. Therefore, any improvement that leads to faster and more repeatable flow characteristics will lead to improved devices.

[0390] Since analysts often work with biological samples (which tend to contain various forms of endogenous particles) - the lack of predictable flow was attributed to the sample. Although endogenous particles do not help the flow rate performance of the SPE device, the analyst's experience has been reproduced by sequentially flowing two separate aliquots of filtered pure Milli-Q water through an unused device using a vacuum setting of 6" Hg. The problem solved by the present invention is that although the first aqueous solution / sample passing through the device may flow well, problems are encountered in maintaining similar flow rates for subsequent aqueous solutions (whether it is sample or pure water).

[0391] Chromatographic adsorbents, especially reversed phase (RP) adsorbents, are frequently used in SPE. Reversed phase adsorbents are usually polymer-based, such as HLB (HLB), or by combining silica particles with hydrophobic ligands such as ) bonding. RP particles can vary considerably in their degree of hydrophobicity, from hydrophilic ( HLB) changes to hydrophobic Waters manufactures a number of adsorbents covering a range of hydrophobicity.

[0392] One aspect of the invention uses a mixture of adsorbent types in a packed bed apparatus, wherein one adsorbent type is substantially more hydrophilic ( HLB), another substantially more hydrophobic It has been found that by mixing the two powders together in a chromatography device such as an SPE device, the flow characteristics are enhanced when compared to the flow properties using either adsorbent alone, or when compared to a device in which the two adsorbents have been layered separately in the same device. This improvement results in a more permeable bed structure that flows faster and more consistently, and also minimizes undesirable flow blockages when low vacuum / pressure driving forces are used.

[0393] The flow rates of water or aqueous samples through the mixed adsorbent type bed of the present invention are much faster than those through a mixture of the same particle size combination of the same amount of HLB adsorbent. The following is a table of water flow rates obtained on a 30 mg packed bed apparatus after conditioning with water to displace the air in the bed. Various weight combinations of HLB and HLB 30μm and 20μm compared with those of the combination.

[0394] method

[0395] The 1cc devices were prepared using the same bottom and top frits used in all other 1cc devices of the present invention. The filling process involved inserting the bottom frit into a 1cc cartridge, adding 30 mg of various combinations of adsorbents, and then stacking the device with the top frit, which was used to confine the combined adsorbents in a packed bed with an external porosity of about 0.4. This porosity is defined as the ratio of the volume of fluid in the fluid-filled bed (which excludes the volume of the particle skeleton structure and its pore volume) to the volume occupied by the entire bed. Sufficient amounts of bulk adsorbent combination were used to prepare four 1cc packed bed devices for each mass ratio tested. For example, a batch combination of four 1cc devices each containing 10 mg HLB 30μm:20 mg HLB 60μm was prepared by weighing 100 mg HLB 30μm on an analytical balance and adding 200 mg HLB 60μm. The powders were mixed thoroughly and the excess mixture allowed the preparation of four 1cc devices.

[0396] Each 1cc contained a total of 30 mg of combined adsorbents in various ratios. Each device was tested one at a time on a standard 20-position vacuum manifold. The vacuum was monitored using a calibrated vacuum gauge. The vacuum in the manifold was adjusted to 6" Hg. 1 mL of Milli-Q water was added to condition each device and displace the air. A second aliquot of water was added to each device, and the time taken for 1 mL of water to flow through at 6" Hg was monitored and recorded using a calibrated stopwatch. The time was converted to flow rate using the following formula: 1 mL / recorded time in minutes.

[0397] result

[0398] The table below lists the water flow rates for various adsorbent combinations. The first flow rate entry in the table is for 100% of the additional adsorbent used in the combination. The last flow rate entry in the table is for 100% HLB 30μm. As shown in the table, all 3 flow rates for 100% HLB 30μm are fairly close. When the time to empty the device exceeds 10 or 20 minutes, the flow is entered as less than the value. For the combination of 30 and 60μm HLB, when the amount of 30μm HLB exceeds 5mg, the flow rate on the device becomes higher than that at 30μm HLB and 20μm Slower flow rates are achieved over the adsorbent.

[0399] Prepared from 30 and 20 μm HLB with 30 μm HLB:20 μm These HLB:HLB devices gave very low flow rates in all combinations. Once 20 μm When the amount of 30 μm HLB in the device is 5 mg or more, the flow rate through the device increases dramatically until the bed consists of only 30 μm HLB. The combination of 20 μm and 60 μm HLB flows faster than the combination of 30 and 60 μm HLB in a similar weight ratio range.

[0400] Table 20: Water flow rates for various adsorbent compositions compared to the apparatus of the present invention

[0401]

[0402] The results in the above table are completely unexpected based on the average particle size in the combined adsorbents. The well-known and understood relationship between particle size and expected back pressure in a packed bed device or LC column is theoretically expressed in the Kozeny-Carmen equation.

[0403] Based on the Kozeny-Carmen equation (1), for a given delta pressure (ΔP) (which in this case is a vacuum of 6" Hg), the flow rate through the packed bed is proportional to the square of the particle size. In the experimental data shown above, the radius of the apparatus (r), the length of the bed (L), the viscosity of the solution (water) (η), and the external porosity of the packed particle bed (εe) were held essentially constant.

[0404]

[0405] Example 14: Layered Comparative Combination Adsorbent Packed Bed Back Pressure

[0406] The present invention will not only allow state of the art SPE devices containing ~30 μm particles to operate more stably at commonly used vacuum settings (5.5-6.5" Hg), but will also allow the use of smaller particle sizes (which may be more compatible with the increasingly popular microsampling (<10 μL) devices). It is expected that smaller SPE particles will enable smaller elution volumes, which can prevent excessive dilution of very low volume samples (<10 μL).

[0407] Using a 1 cc 30 mg packed bed device, water flow rate was measured as described in Example 13. In this example, each device contained 20 mg and 10 mg HLB. However, in this case, the adsorbent is separated into layers by a device, The flow rate of the HLB at the bottom and the HLB at the top) is compared to the flow rate through the device in which the adsorbents are combined.

[0408] The water flow rates obtained in the same manner as above but at various vacuum settings are listed in the table below. The flow rate of water through the mixed adsorbent type bed of the present invention is substantially faster than the flow rate of water through the same amount and type of adsorbent simply layered in the device. As can be seen from the table below, at 5" Hg vacuum, no flow was obtained over the layered bed device, while >1 mL / min was achieved for the same delta pressure through the mixed adsorbent bed. The adsorbents in the layered and mixed adsorbent devices were the same two adsorbents, and each device contained the same weight of each.

[0409] At higher delta pressures, flow was obtained on both the layered and mixed bed units. However, substantially (more than 2 times) faster flow rates were achieved for the mixed adsorbent bed units which exhibited higher permeabilities.

[0410] Table 21: Comparison of water flow rates between layers in 1cc and mixed adsorbent beds

[0411]

[0412] Example 15: Study of External Porosity of Mixed Adsorbent Beds

[0413] There are many ways to define the tendency of a solid to interact favorably with water. If the solid surface is completely flat and free of contamination, and the contact angle between a water drop on the surface is less than 90°, the surface is considered hydrophilic. If the contact angle is greater than 90°, the surface is considered hydrophobic. Unfortunately, the measurement of the contact angle between porous particles and water is not as straightforward. The contact angle is increased by the roughness / irregularities of the hydrophobic surface and / or the presence of trapped air in surface defects. In addition to providing an understanding of wettability, the contact angle is also used to understand adhesion and solid surface free energy.

[0414] The physics or phenomena behind the increased flow rates of the hybrid adsorbent beds of the present invention are not fully understood at this time. One factor is the higher porosity of the hybrid adsorbent bed. For this experiment, both adsorbents were chosen to be 20 μm to exclude particle size differences (30 μm HLB and 20 μm ) contributes to the lower back pressure of the device observed. It has been shown that the increase in bed porosity is independent of mixing different particle sizes.

[0415] The bed heights for the hybrid adsorbent devices were measured and reported in the table below. Based on 30 mg of 20 μm HLB and 30 mg of 20 μm The bed height of the layered device was calculated using the device diameter of 0.221". The volume of the cylinder (πr 2 L), calculate the volume of the layered versus mixed bed device. A single adsorbent device assembled as described above typically has an external porosity (the space between the particles where flow occurs) of 0.40, and for single adsorbent HLB and This is assumed for the device.

[0416] The calculated and measured data are shown in the table below. Based on our calculations, the external porosity of the mixed adsorbent bed was increased to 0.47. The experiments were performed using particles of similar size (20 μm) to avoid any effect of combining particles of multiple sizes. The data show that increased flow rates were obtained for the higher porosity mixed adsorbent bed. Under 6" Hg vacuum conditions, reasonable water flow rates (<0.1 mL / min) were not achieved for the single adsorbent device.

[0417] Table 22: Height, volume, external porosity and water flow rate (mL / min) at 6" Hg for mixed adsorbent bed

[0418]

[0419] *Based on assumptions: 30 mg packaged in 1cc cartridges at 0.151" or 0.091" bed heights respectively HLB or It has a bed external porosity of 0.40.

[0420] Example 16: Comparison of the polymer adsorbents of the present invention with those of the prior art

[0421] To perform the comparison, four state-of-the-art commercially available 10 mg 96-well plates were purchased. In addition to the Biotage 10 mg Information on all plates except the ABN 96-well plate (PN 600-0010-RP) is given in the previous examples. Biotage describes the sorbents as "advanced polymeric SPE for acidic, basic, and neutral analytes." Biotage states that their sorbents are "specifically developed for analyte quantification by LC-MS / MS, and the EVOLUTE products provide an efficient solution to ion suppression and matrix effect issues."

[0422] In this example, 10 mg plates were used to prepare whole blood samples for LC-MS / MS analysis of synthetic cannabinoids. The same volumes were used in the 10 mg plates as were used in the 30 mg plates in Example 8. The importance of providing a device that substantially reduces phospholipids becomes apparent in this application.

[0423] method

[0424] The samples were prepared using the same protocol as described in Example 8, except that methanol was used to adjust the Commercially available plates other than HLB plates were equilibrated with water prior to loading the PPT and diluted samples.

[0425] The MS / MS conversion, MS conditions and LC conditions are given in Example 8 and were subsequently used to acquire the data.

[0426] result

[0427] As shown in the table below, the best average percent recoveries (%REC) were obtained in the plates containing the adsorbent mixture of the present invention (approximately 1:2 by weight). The recovery for the HLB plate was 93%. For the Strata X and Plexa plates, retention of the most polar cannabinoid (AM2233) was problematic.

[0428] Table 23: Recovery rates of synthetic cannabinoids

[0429]

[0430]

[0431] The standard deviations (STDs) of the recoveries are listed in the table below. For all plates except the plates of the present invention, unexpectedly high STDs were obtained for the cannabinoid JWH-203. Close inspection of the data showed that this cannabinoid completely co-eluted with one of the common PLs.

[0432] Table 24: Standard deviations of synthetic cannabinoids

[0433]

[0434]

[0435] The matrix effects of JWH-203 and total phospholipids were calculated and quantified separately. These data, along with the percent recovery of JWH-203, are included in the table below. Keep in mind that the same sample was loaded on all devices. The expected result was 100%, and the deviation from this appears to be related to the phospholipids in the sample, without a method to eliminate them from the final analyte extract (as performed in the present invention).

[0436] Table 25: Summary of data on JWH-203

[0437]

[0438]

[0439] All patents, published patent applications, and other references cited herein are expressly incorporated by reference in their entirety.

[0440] Those skilled in the art will recognize or be able to ascertain numerous equivalents to the specific procedures described herein using no more than routine experimentation. Such equivalents are considered to be within the scope of the present invention and are covered by the appended claims.

[0441] This application also includes the following specific implementation schemes:

[0442] 1. A separation device comprising two or more adsorbents, wherein:

[0443] Compared to a separation device comprising only one adsorbent, matrix interferents retained on one of the adsorbents are substantially removed by the other adsorbent;

[0444] The flow rate of the adsorbent is substantially increased compared to a separation device comprising only one adsorbent; or

[0445] Compared to a separation device comprising only one adsorbent, the back pressure of the adsorbent is substantially reduced.

[0446] 2. The separation device of embodiment 1, wherein at least one adsorbent contains at least one hydrophobic component and at least one hydrophilic component.

[0447] 3. The separation device of embodiment 2, wherein the adsorbent comprising at least one hydrophobic component and at least one hydrophilic component is water wettable.

[0448] 4. The separation device of embodiment 1, wherein at least one of the adsorbents has a retention of the analyte in aqueous solution of a log P of 0.05 or greater.

[0449] 5. The separation device of embodiment 4, wherein the remainder of the adsorbent collectively has a specific affinity for the matrix interferent in greater than 40% organic solvent solution.

[0450] 6. The separation device of embodiment 1, wherein at least one adsorbent has a specific affinity for phospholipids.

[0451] 7. The separation device of specific embodiment 1, wherein the adsorbent is selected from silica, modified silica, alumina, modified alumina, titania and zirconia, graphitized carbon or activated carbon, magnesium silicate, modified polymers and combinations thereof.

[0452] 8. The separation device of embodiments 1-7, wherein the adsorbent is layered.

[0453] 9. The separation device of embodiment 8, wherein the top layer is water wettable.

[0454] 10. The separation device of embodiment 9, wherein the top layer is an adsorbent comprising at least one hydrophobic component and at least one hydrophilic component.

[0455] 11. The separation device of embodiments 1-7, wherein the adsorbents are mixed in a single layer.

[0456] 12. The separation device of embodiment 11, wherein the monolayer is water wettable.

[0457] 13. The separation device of specific embodiments 8-12, wherein the adsorbent containing at least one hydrophobic component and at least one hydrophilic component and the adsorbent having a specific affinity for phospholipids are mixed in a weight ratio of about 1:2.

[0458] 14. The separation device of embodiment 1, further comprising at least one frit or filter.

[0459] 15. The separation device of embodiment 1, further comprising an inlet and an outlet.

[0460] 16. The separation device of embodiment 1, further comprising a shell.

[0461] 17. The separation device of embodiment 14, wherein the glass frit or filter is disposed at the bottom of the adsorbent.

[0462] 18. The separation device of specific embodiment 16, wherein the shell is a syringe, a cartridge, a column, a multi-chamber, a 4 to 8-well rack, a 4 to 8-well strip, a 48 to 96-well plate, a 96 to 384-well micro-elution plate, a micro-elution tip, a 96 to 384-well micro-elution tip array, a 4 to 8-tip micro-elution strip, a single micro-elution pipette tip, a rotating tube or a rotating container.

[0463] 19. The separation device of embodiment 1 further comprising a vacuum chamber, reservoir, or structure that allows for use in a centrifuge or with a vacuum or pressure source.

[0464] 20. The separation device of embodiment 3, wherein the adsorbent is a reverse phase water wettable adsorbent.

[0465] 21. The separation device of embodiment 20, wherein the adsorbent is HLB.

[0466] 22. The separation device of embodiment 5, wherein the adsorbent is a modified or unmodified octadecyl (C 18 ) or C 8 To C 30 Alkyl component silica.

[0467] 23. The separation device of embodiment 5, wherein the adsorbent is capable of retaining one or more matrix interferents during the elution step.

[0468] 24. A chromatographic column comprising:

[0469] a column having a cylindrical interior for receiving an adsorbent bed; and

[0470] At least two or more adsorbents.

[0471] 25. The chromatography column of embodiment 24, wherein at least one adsorbent comprises at least one hydrophobic component and at least one hydrophilic component.

[0472] 26. The chromatography column of embodiment 25, wherein the adsorbent comprising at least one hydrophobic component and at least one hydrophilic component is water wettable.

[0473] 27. The chromatographic column of embodiment 24, wherein at least one of the adsorbents has a retention of the analyte in aqueous solution of a log P of 0.05 or greater.

[0474] 28. The chromatographic column of embodiment 27, wherein the remainder of the adsorbent collectively has a specific affinity for matrix interferents in greater than 40% organic solvent solution.

[0475] 29. The chromatographic column of embodiment 24, wherein at least one adsorbent has a specific affinity for phospholipids.

[0476] 30. The chromatographic column of embodiment 24, wherein the adsorbent is selected from silica, modified silica, alumina, modified alumina, titania and zirconia, graphitized carbon or activated carbon, magnesium silicate, modified polymers and combinations thereof.

[0477] 31. The chromatography column of embodiments 24-30, wherein the adsorbent is layered.

[0478] 32. The chromatography column of embodiment 31, wherein the top layer is water wettable.

[0479] 33. The chromatography column of embodiment 32, wherein the top layer is an adsorbent comprising at least one hydrophobic component and at least one hydrophilic component.

[0480] 34. The chromatography column of embodiments 24-30, wherein the adsorbents are mixed in a monolayer.

[0481] 35. The chromatography column of embodiment 34, wherein said monolayer is water wettable.

[0482] 36. The chromatography column of embodiments 31-35, wherein the adsorbent containing at least one hydrophobic component and at least one hydrophilic component and the adsorbent having a specific affinity for phospholipids are mixed in a weight ratio of about 1:2.

[0483] 37. The chromatography column of embodiment 24, wherein the chromatography column is a syringe, a cartridge or a column.

[0484] 38. The chromatography column of embodiment 24, further comprising at least one frit or filter.

[0485] 39. The chromatography column of embodiment 24, further comprising an inlet and an outlet.

[0486] 40. The chromatography column of specific embodiment 24, which is contained in a multi-chamber, a 4 to 8-well rack, a 4 to 8-well strip, a 48 to 96-well plate, a 96 to 384-well micro-elution plate, a micro-elution tip, a 96 to 384-micro-elution tip array, a 4 to 8-tip micro-elution strip, a single micro-elution pipette tip, a rotating tube or a rotating container.

[0487] 41. The chromatography column of embodiment 38, wherein the frit or filter is disposed at the bottom of the adsorbent.

[0488] 42. The chromatography column of embodiment 24, further comprising a vacuum chamber, reservoir, or structure that allows use in a centrifuge or with a vacuum or pressure source.

[0489] 43. The chromatographic column of embodiment 25, wherein the adsorbent is a reverse phase water wettable adsorbent.

[0490] 44. The chromatographic column of embodiment 43, wherein the adsorbent is HLB.

[0491] 45. The chromatographic column of embodiment 29, wherein the adsorbent is a modified or unmodified octadecyl (C 18 ) or C 8 To C 30 Alkyl component silica.

[0492] 46. ​​A chromatography device comprising:

[0493] a column having a cylindrical interior for receiving an adsorbent bed; and

[0494] At least two or more adsorbents.

[0495] 47. The chromatographic device of embodiment 46, wherein at least one adsorbent comprises at least one hydrophobic component and at least one hydrophilic component, and at least one adsorbent has a specific affinity for the phospholipids contained in the adsorbent bed,

[0496] The adsorbent comprising at least one hydrophobic component and at least one hydrophilic component is water wettable.

[0497] 48. The chromatography device of embodiment 46, wherein the adsorbent is layered.

[0498] 49. The chromatography device of embodiment 46, wherein the top layer is water wettable.

[0499] 50. The chromatographic device of embodiment 49, wherein the top layer comprises an adsorbent comprising at least one hydrophobic component and at least one hydrophilic component.

[0500] 51. The chromatography device of embodiment 46, wherein the adsorbents are mixed in a monolayer.

[0501] 52. The chromatography device of embodiment 46, wherein the adsorbent comprising at least one hydrophobic component and at least one hydrophilic component and the adsorbent having a specific affinity for phospholipids are mixed in a weight ratio of about 1:2.

[0502] 53. The chromatography device of embodiment 46, further comprising a housing.

[0503] 54. The chromatography device of embodiment 46, further comprising at least one frit or filter.

[0504] 55. The chromatography device of embodiment 46, further comprising an inlet and an outlet.

[0505] 56. The chromatographic device of embodiment 53, wherein the housing is a syringe, a cartridge, a column, a multi-chamber, a 4 to 8 well rack, a 4 to 8 well strip, a 48 to 96 well plate, a 96 to 384 well micro-elution plate, a micro-elution tip, an array of 96 to 384 micro-elution tips, a 4 to 8-tip micro-elution strip, a single micro-elution pipette tip, a rotating tube or a rotating container.

[0506] 57. The chromatography device of embodiment 54, wherein the frit or filter is disposed at the bottom of the adsorbent.

[0507] 58. The chromatographic device of embodiment 46, wherein the chromatographic device further comprises a vacuum chamber, a reservoir, or a structure that allows use in a centrifuge or with a vacuum or pressure source.

[0508] 59. The chromatographic device of embodiment 46, wherein said adsorbent comprising at least one hydrophobic component and at least one hydrophilic component is a reverse phase water wettable adsorbent.

[0509] 60. The chromatographic device of embodiment 59, wherein the adsorbent is HLB.

[0510] 61. The chromatographic device of embodiment 46, wherein the adsorbent having a specific affinity for phospholipids is a modified or unmodified octadecyl (C 18 ) or C 8 To C 30 Alkyl component silica.

[0511] 62. A method for removing matrix interferences from a sample, comprising

[0512] The adsorbent is mixed with at least one or more additional adsorbents of a different type, and the sample is contacted with the mixed adsorbent.

[0513] 63. The method for removing matrix interferents from a sample according to embodiment 62, wherein the adsorbent and the at least one or more additional adsorbents are mixed in a monolayer.

[0514] 64. The method for removing matrix interferents from a sample according to embodiment 62, wherein the adsorbent is a water wettable adsorbent and at least one additional adsorbent is not a water wettable adsorbent.

[0515] 65. The method for removing matrix interferents from a sample according to embodiment 62, wherein the adsorbent is not a water wettable adsorbent and at least one additional adsorbent is a water wettable adsorbent.

[0516] 66. A method for increasing the flow rate of an adsorbent, comprising

[0517] The first adsorbent is mixed with at least one or more additional adsorbents of a different type.

[0518] 67. The method for increasing the flow rate of an adsorbent according to embodiment 66, wherein

[0519] The first adsorbent and the at least one or more additional adsorbents are mixed in a monolayer.

[0520] 68. The method for increasing an adsorbent flow rate of embodiment 66, wherein the first adsorbent is a water wettable adsorbent and the at least one additional adsorbent is not a water wettable adsorbent.

[0521] 69. The method for increasing the adsorbent flow rate of embodiment 66, wherein the first adsorbent is not a water wettable adsorbent and the at least one additional adsorbent is a water wettable adsorbent.

[0522] 70. A method of reducing back pressure of an adsorbent, comprising:

[0523] The first adsorbent is mixed with at least one or more additional adsorbents of a different type.

[0524] 71. The method for reducing the back pressure of an adsorbent according to embodiment 70, wherein

[0525] The first adsorbent and at least one or more additional adsorbents are mixed in a monolayer.

[0526] 72. The method for reducing back pressure of an adsorbent according to embodiment 70, wherein the first adsorbent is a water wettable adsorbent and the at least one additional adsorbent is not a water wettable adsorbent.

[0527] 73. The method for reducing the back pressure of an adsorbent according to embodiment 70, wherein the first adsorbent is not a water wettable adsorbent and the at least one additional adsorbent is a water wettable adsorbent.

[0528] 74. A solid phase extraction method comprising:

[0529] A device is used, wherein the device comprises an adsorbent bed comprising an adsorbent comprising at least one hydrophobic component and at least one hydrophilic component and an adsorbent having a specific affinity for phospholipids.

[0530] 75. The method of embodiment 75, wherein the adsorbent comprising at least one hydrophobic component and at least one hydrophilic component is water wettable.

[0531] 76. The method of embodiment 75, wherein the adsorbent bed is layered.

[0532] 77. The method of embodiment 76, wherein the top layer is an adsorbent comprising at least one hydrophobic component and at least one hydrophilic component.

[0533] 78. The method of embodiment 75, wherein said adsorbent comprising at least one hydrophobic component and at least one hydrophilic component and said adsorbent having a specific affinity for phospholipids are mixed in a monolayer of said adsorbent bed.

[0534] 79. The method of embodiment 76 or 78, wherein the adsorbent comprising at least one hydrophobic component and at least one hydrophilic component and the adsorbent having a specific affinity for phospholipids are mixed in a weight ratio of about 1:2.

[0535] 80. The method of embodiment 77, wherein the adsorbent of the top layer is HLB.

[0536] 81. The method of embodiment 76, wherein the adsorbent having a specific affinity for phospholipids comprises a modified or unmodified octadecyl (C 18 ) or Octyl C 8 To C 30 Alkyl component silica.

[0537] 82. The method of specific embodiment 76, wherein the device is a syringe, a cartridge, a column, a multi-chamber, a 4 to 8 well rack, a 4 to 8 well strip, a 48 to 96 well plate, a 96 to 384 well micro-elution plate, a micro-elution tip, an array of 96 to 384 micro-elution tips, a 4 to 8-tip micro-elution strip, a single micro-elution pipette tip, a rotating tube or a rotating container.

[0538] 83. The method of embodiment 76, further comprising conditioning the adsorbent bed.

[0539] 84. The process according to embodiment 83, wherein methanol is used for the conditioning.

[0540] 85. The method of embodiment 76, further comprising balancing the adsorbent bed.

[0541] 86. The method of embodiment 85, wherein water is used for the balancing.

[0542] 87. The method of embodiment 76, further comprising loading a sample onto the adsorbent bed.

[0543] 88. The method of embodiment 87, wherein the sample is a biological sample.

[0544] 89. The method of embodiment 87 or 88, wherein the sample is loaded onto the adsorbent bed without conditioning or equilibration.

[0545] 90. The method of embodiment 89, further comprising the step of washing the adsorbent bed.

[0546] 91. The method of embodiment 76, further comprising eluting the analyte from the adsorbent bed.

[0547] 92. The method of embodiment 91, wherein a mixture of acetonitrile and methanol in a mixing ratio of about 90:10 is used for elution.

[0548] 93. The method of embodiment 91, wherein the eluate obtained from said method contains less than about 2 wt % of the phospholipids in said sample.

[0549] 94. The method of embodiment 91, wherein the analyte from the adsorbent is further analyzed using liquid chromatography, liquid chromatography-mass spectrometry / mass spectrometry (LC / MS-MS), mass spectrometry (MS), MALDI-MS or ESI.

[0550] 95. The method of embodiment 76, wherein a vacuum chamber, reservoir, or structure that allows use in a centrifuge or with a vacuum or pressure source is applied to the device.

Claims

1. A separation device comprising two or more adsorbents, the adsorbents comprising (a) a polymer adsorbent comprising at least one hydrophobic component and at least one hydrophilic component, wherein the polymer adsorbent is water wettable, and (b) an additional adsorbent having a specific affinity for phospholipids, the additional adsorbent being a modified or unmodified octadecyl (C 18 ) or C8 to C 30 Silicon-based particles of an alkyl component, wherein the polymeric adsorbent is homogeneously mixed with the additional adsorbent to form an adsorbent bed.

2. The separation device of claim 1, wherein the weight ratio of the polymeric adsorbent to the additional adsorbent in the adsorbent bed is about 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9 or 1:

10.

3. The separation device of claim 2, wherein the weight ratio of the polymeric adsorbent to the additional adsorbent in the adsorbent bed is about 1:1, 1:2 or 1:

3.

4. The separation device of claim 3, wherein the weight ratio of the polymeric adsorbent to the additional adsorbent in the adsorbent bed is about 1:

2.

5. The separation device of claim 1, wherein at least one of the adsorbents has a retention of the analyte in aqueous solution of a log P of 0.05 or greater.

6. The separation device of claim 5, wherein the remainder of the adsorbent collectively has a specific affinity for the matrix interferent in greater than 40% organic solvent solution.

7. The separation device of claim 1, wherein the at least one hydrophobic component comprises a phenyl moiety and the at least one hydrophilic component comprises a pyrrolidone moiety.

8. The separation device of claim 1, wherein the modified or unmodified octadecyl (C 18 ) or C8 to C 30 The alkyl component includes C 18 .

9. The separation device of claim 1, further comprising at least one frit or filter.

10. The separation device of claim 1, further comprising a housing having an inlet and an outlet.

11. The separation device of claim 9, wherein the at least one frit or filter is disposed below the adsorbent bed.

12. The separation device of claim 10, wherein the housing is a syringe, a cartridge, a column, a multi-chamber, a 4 to 8 well rack, a 4 to 8 well strip, a 48 to 96 well plate, a 96 to 384 well micro-elution plate, a micro-elution tip, a 96 to 384 micro-elution tip array, a 4 to 8-tip micro-elution strip, a single micro-elution pipette tip, a rotating tube, or a rotating container.

13. The separation device of claim 1 further comprising a vacuum chamber, reservoir, or structure that permits use in a centrifuge or with a vacuum or pressure source.

14. The separation device of claim 1, wherein the adsorbent bed is capable of retaining one or more matrix interferents during the elution step.

15. A method for removing matrix interferents from a sample, comprising: The sample is contacted with two or more adsorbents of the separation device of claim 1.

16. A solid phase extraction method, comprising: loading the sample onto the adsorbent bed of the separation device of claim 1; washing the adsorbent bed; and The analyte is eluted from the sorbent bed.

17. The method of claim 16, wherein the at least one hydrophobic component comprises a phenyl moiety and the at least one hydrophilic component comprises a pyrrolidone moiety.

18. The method of claim 16, wherein the modified or unmodified octadecyl (C 18 ) or C8 to C 30 The alkyl component includes C 18 .

19. The method of claim 16, wherein the weight ratio of the polymeric adsorbent to the additional adsorbent in the adsorbent bed is about 1:1, 1:2, or 1:

3.

20. The method of claim 19, wherein the weight ratio of the polymeric adsorbent to the additional adsorbent in the adsorbent bed is about 1:

2.

21. The method of claim 16, further comprising conditioning the adsorbent bed.

22. The process of claim 21, wherein methanol is used for conditioning.

23. The method of claim 16, further comprising equilibrating the adsorbent bed.

24. The method of claim 23, wherein water is used for the balance.

25. The method of claim 16, wherein the sample is a biological sample.

26. The method of claim 16, wherein a mixture of acetonitrile and methanol in a mixing ratio of about 90:10 is used for elution.

27. The method of claim 26, wherein the eluate obtained from said method contains less than about 2 wt% of the phospholipids in said sample.

28. The method of claim 27, wherein the analyte from the adsorbent is further analyzed using liquid chromatography, liquid chromatography-mass spectrometry / mass spectrometry (LC / MS-MS), mass spectrometry (MS), MALDI-MS, or ESI.

29. A solid phase extraction method, the method comprising: loading the biological sample onto an adsorbent bed of a solid phase extraction device without conditioning and / or equilibrating the adsorbent bed prior to loading; washing the adsorbent bed; The analyte is eluted from the adsorbent bed, wherein the eluate obtained after elution contains less than about 10% by weight of the phospholipids included in the biological sample.

30. The solid phase extraction method of claim 29, wherein the eluate obtained after the elution contains less than about 5% by weight of the phospholipids included in the biological sample.

31. The solid phase extraction method of claim 29, wherein the eluate obtained after the elution contains less than about 2% by weight of the phospholipids included in the biological sample.

32. The solid phase extraction method of claim 29, wherein the eluate obtained after the elution contains less than about 1% by weight of the phospholipids included in the biological sample.

33. The solid phase extraction method of claim 29, wherein the adsorbent bed comprises (a) a polymer adsorbent comprising at least one hydrophobic component and at least one hydrophilic component, wherein the polymer adsorbent is water wettable, and (b) an additional adsorbent having a specific affinity for phospholipids, wherein the additional adsorbent is a modified or unmodified octadecyl (C 18 ) or C8 to C 30 Silicon-based particles of alkyl components.

34. The process of claim 33, wherein the polymeric adsorbent is homogeneously mixed with the additional adsorbent across the adsorbent bed.

35. The method of claim 33, wherein the polymeric adsorbent is layered on top of the additional adsorbent to form the adsorbent bed.

36. The process of claim 33, wherein the additional adsorbent is layered on top of the polymeric adsorbent to form the adsorbent bed.

37. The method of claim 33, wherein the polymeric adsorbent and the additional adsorbent are included in the separation device in a weight ratio of about 1:

2.

38. A separation device comprising two or more adsorbents, the adsorbents comprising (a) a polymer adsorbent comprising at least one hydrophobic component and at least one hydrophilic component, wherein the polymer adsorbent is water wettable, and (b) an additional adsorbent having a specific affinity for phospholipids, the additional adsorbent being a modified or unmodified octadecyl (C 18 ) or C8 to C 30 Silicon-based particles of an alkyl component, wherein the polymeric adsorbent forms a top layer and the additional adsorbent forms a bottom layer.

39. The separation device of claim 38, wherein the weight ratio of the polymeric adsorbent to the additional adsorbent included in the separation device is about 1:1, 1:2 or 1:

3.

40. The separation device of claim 39, wherein the weight ratio of the polymeric adsorbent to the additional adsorbent in the adsorbent bed is about 1:

2.

41. The separation device of claim 38, wherein at least one of the adsorbents has a retention of the analyte in aqueous solution of a log P of 0.05 or greater.

42. The separation device of claim 41, wherein the remainder of the adsorbent collectively has a specific affinity for the matrix interferent in greater than 40% organic solvent solution.

43. The separation device of claim 38, wherein the at least one hydrophobic component comprises a phenyl moiety and the at least one hydrophilic component comprises a pyrrolidone moiety.

44. The separation device of claim 38, wherein the modified or unmodified octadecyl (C 18 ) or C8 to C 30 The alkyl component includes C 18 .

45. The separation device of claim 38, further comprising at least one frit or filter.

46. ​​The separation device of claim 38, further comprising a housing having an inlet and an outlet.

47. The separation device of claim 38, further comprising a vacuum chamber, reservoir, or structure that permits use in a centrifuge or with a vacuum or pressure source.

48. A separation device comprising two or more adsorbents, the adsorbents comprising (a) a polymer adsorbent comprising at least one hydrophobic component and at least one hydrophilic component, wherein the polymer adsorbent is water wettable, and (b) an additional adsorbent having a specific affinity for phospholipids, the additional adsorbent being a modified or unmodified octadecyl (C 18 ) or C8 to C 30 Silicon-based particles of an alkyl component, wherein the polymeric adsorbent forms a bottom layer and the additional adsorbent forms a top layer.

49. The separation device of claim 48, wherein the weight ratio of the polymeric adsorbent to the additional adsorbent included in the separation device is about 1:1, 1:2 or 1:

3.

50. The separation device of claim 49, wherein the weight ratio of the polymeric adsorbent to the additional adsorbent in the adsorbent bed is about 1:

2.

51. The separation device of claim 48, wherein at least one of the adsorbents has a retention of the analyte in aqueous solution of a log P of 0.05 or greater.

52. The separation device of claim 48, wherein the remainder of the adsorbent collectively has a specific affinity for the matrix interferent in greater than 40% organic solvent solution.

53. The separation device of claim 48, wherein the at least one hydrophobic component comprises a phenyl moiety and the at least one hydrophilic component comprises a pyrrolidone moiety.

54. The separation device of claim 48, wherein the modified or unmodified octadecyl (C 18 ) or C8 to C 30 The alkyl component includes C 18 .

55. The separation device of claim 48, further comprising at least one frit or filter.

56. The separation device of claim 48, further comprising a housing having an inlet and an outlet.

57. The separation device of claim 48, further comprising a vacuum chamber, reservoir, or structure that permits use in a centrifuge or with a vacuum or pressure source.

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