Sample purification apparatus and method

Abstract

This invention relates to a flow-through device comprising at least one separation column, wherein a first packing component comprising alumina and / or silica particles and a second packing component comprising a powder containing one or more hygroscopic salts are provided. The two packing components may be co-mixed or layered within the device, which may comprise a single tube or multiple tubes arranged in a plate-like manner, such as tubes in the pores of a multi-walled plate or in a support. Additionally, this invention relates to a method for removing one or more matrix components, such as pigments, from a biological sample by passing the biological sample through the first packing component comprising alumina and / or silica particles and the second packing component comprising a powder containing one or more hygroscopic salts.

Description

[0001] This application is a divisional application. The original application was filed on October 17, 2018, with application number 2018800609459, and was entitled "Sample Purification Apparatus and Method". Technical Field

[0002] This invention relates to the field of sample preparation, and more particularly to providing an apparatus for rapidly and easily removing matrix components from biological samples. The invention also includes a method for removing matrix components from biological samples such as urine. Background Technology

[0003] The process of handling complex biological samples to remove or at least reduce substances present in the sample that could affect subsequent measurements of the analyte is commonly referred to as sample preparation, or simply "sampleprep." This term is used for a variety of methods and apparatuses, including solid-phase extraction (SPE), supported liquid-liquid extraction (SLE), and protein precipitation. When analyzing small organic molecules (e.g., drugs, prodrugs, their metabolites, etc.), the primary objective of sample preparation is to provide mass spectrometry (MS) with sufficient purity for analysis.

[0004] Solid phase extraction (SPE) is a chromatographic technique widely used for the preconcentration of analytical samples, the purification of various chemicals, and the removal of toxic or valuable substances from aqueous solutions. However, despite its widespread use in certain applications, it has proven to be both expensive and time-consuming when less precise purification of samples is required, such as in the routine testing of large volumes of urine samples to detect illicit substances, for example, during various sporting events.

[0005] Supported liquid-liquid extraction (SLE) uses a water-immiscible solvent for analyte extraction. More specifically, the aqueous phase is typically immobilized on an inert phase (e.g., diatomaceous earth) onto which the water-immiscible phase is loaded. After a short wait, the eluent is pushed into or blown through a column, producing an extract that is inherently cleaner than other simpler sample preparation techniques such as protein precipitation or "dilute and shoot" (D&S).

[0006] "Dilution and injection" (D&S) is a relatively rapid method widely used in clinical and forensic applications. The principle of D&S is to simply dilute the sample and then inject it directly into a liquid chromatography-mass spectrometry (LC-MS) system, for example.

[0007] By definition, the D&S method inherently still contains interfering components, although their concentration is lower than in the original sample due to dilution. Therefore, impure D&S extracts can increase column maintenance requirements and / or shorten the lifespan of subsequent LC systems, and low sample purity also increases LC-MS processing time. Furthermore, for certain known "difficult" analytes (such as certain opiates like buprenorphine and norbuprenorphine), D&S is even impossible.

[0008] US 6,541,273 (Aventis) relates to the field of cartridges for solid-phase extraction. More specifically, it discloses methods and apparatus for detecting pesticides (e.g., pyrazoles, including fipronil) present in soil, water, blood, tissue, urine, and other types of agricultural, medical, or pharmaceutical samples. The apparatus includes a column having a first opening for receiving a sample and a second opening for discharging eluent, the column comprising a first separation zone containing amino-functionalized silica, a second separation zone containing activated carbon, a third separation zone containing magnesium-silica gel or silica gel, and a fourth separation zone containing magnesium-silica gel or silica gel not used in the third separation zone.

[0009] WO 2007 / 030847 relates to a packing material for a microadsorption column used for drying and / or purifying dissolved organic or biological analytes, such as toxins, antibiotics, vitamins, hormones, pesticides, etc., wherein the packing material comprises at least one desiccant; and relates to a microadsorption column filled with the packing material and its uses. More specifically, the packing material comprises magnesium sulfate and at least one other desiccant selected from: alumina, calcium chloride, calcium hydride, calcium oxide, calcium sulfate, potassium hydride, silica gel, copper sulfate, magnesium oxide, magnesium perchlorate, molecular sieve, sodium hydroxide, phosphorus pentoxide, sulfuric acid supported on silicate, phosphorus pentoxide supported on silicate, and 0.5 to 90% by weight of a naturally occurring or synthetic carrier with a large internal surface area, such as zeolite, diatomaceous earth, bentonite, silica, etc.

[0010] WO 2008 / 103828 (William Brewer) describes pipette tips for extraction, sample collection, and sample purification (e.g., purification of gastric contents and extraction of basic drugs from urine). More specifically, the pipette tip incorporates a sieve or filter at its narrow lower end to contain a solid-phase adsorbent and barrier at its wide upper end. According to WO 2008 / 103828, the mixing of the solution with the adsorbent produces a gel, i.e., a homogeneous solution, thus providing allegedly unparalleled extraction efficiency and rapid equilibration. The adsorbent particle size can be 50-200 μm, small enough to provide efficient extraction, but requires high pressure. In addition to high-pressure adsorbents, other adsorbents are mentioned for removing unwanted sample matrices, such as immunoaffinity adsorbents, ion exchangers, and porous materials for molecular weight separation. Specific examples of adsorbents include functionalized or non-functionalized styrene-divinylbenzene; alumina (basic, acidic, or neutral); magnesium silicate; small-particle-size silica gel; C8, Q18, and functionalized C8 or C18 materials; Na2SO4, MgSO4, or CaSO4 (for drying); diatomaceous earth, cross-linked dextran (Sephadex), and polyethylene.

[0011] Due to the ever-growing need for rapid control of chemicals (e.g., the use of legal or illegal drugs), there is a constant demand in the field of sample purification methods to improve these methods in terms of speed, ease of use, and / or cost. Summary of the Invention

[0012] One object of the present invention is to reduce the complexity of biological samples by removing at least some of their matrix components more efficiently over a given time period compared to at least some of the prior art.

[0013] More specifically, the present invention relates to a flow-through device comprising at least one separation column, wherein a first packing component comprising alumina and / or silica particles and a second packing component comprising a powder comprising one or more hygroscopic salts are provided.

[0014] Furthermore, another objective of the present invention is to reduce the complexity of biological samples through a method that is suitable for routine testing of large numbers of samples due to its simplicity of processing.

[0015] More specifically, the present invention relates to a method for removing one or more matrix components from a biological sample, wherein the matrix components are selected from one or more of urea, uric acid, phospholipids, amino acids, creatine, heme degradation products (e.g., urobillin), and endogenous salts, the method comprising passing the sample through a first filler component comprising alumina and / or silica particles and a second filler component comprising a powder comprising one or more hygroscopic salts.

[0016] Finally, another object of the present invention is to provide rapid and simple analysis of pharmaceuticals and other chemicals, regardless of whether they are considered "difficult" in the prior art.

[0017] More specifically, the present invention relates to the use of a combination of a first filler component comprising particles of alumina and / or silica with a second filler component comprising powder of one or more hygroscopic salts for removing one or more matrix components from a biological sample.

[0018] Further details, embodiments, and advantages of the invention will become apparent from the detailed specification, claims, and other parts thereof.

[0019] definition

[0020] The term "flow-through device" is used herein to refer to a device that is capable of receiving liquids, such as samples, at its upper end but is not suitable for extraction at its lower end.

[0021] The term "alumina" is used in this document for Al2O3, also known as aluminum oxide or aluminum oxide.

[0022] The term "hygroscopic salt" is used herein for salts defined by their ability to absorb / adsorb water from the sample.

[0023] The term "packing material" is used herein for any material used for separation, i.e., the material contained in a column, to separate a sample into fractions by adsorption and / or retention. Packing materials are known in the art under various terms, such as medium, particularly chromatographic medium; adsorbent; absorbent, or simply packing material. The term "packing material" is used herein for the contents of a column, regardless of how they are provided within the column, i.e., whether they are pressurized or simply placed within the column.

[0024] The term "packing component" is used herein to more easily distinguish the different packing materials included in the apparatus of the present invention. Thus, each packing component can be any conventional chromatographic adsorbent, such as those exemplified in this specification, such as silica; a mixture of magnesium sulfate and sodium sulfate, etc.

[0025] The term "matrix component" is used herein in the sense commonly used in the field of sample preparation. Therefore, the matrix of a sample includes components or elements other than the target analyte. For example, a urine sample would include matrix components such as urea, phospholipids, amino acids, and many other components, such as... Figure 3 As shown, all of these components can make it difficult to identify the analyte, and therefore should be removed during sample preparation. Attached Figure Description

[0026] Figure 1a Figures b and c illustrate the apparatus of the present invention, which comprises a plurality of tubes arranged in a plate-like manner, respectively in the form of blended and layered tubes.

[0027] Figure 2a Figures b and c illustrate the apparatus of the present invention, which comprises a single bed, namely a single tube in the form of blending and layering, respectively.

[0028] Figure 3 This is a diagram illustrating the composition, specifically the matrix components of normal urine.

[0029] Figure 4 a and b show unprocessed hydrolyzed urine on the left (a) and the wash liquid obtained after treatment with the flow-through device on the right (b).

[0030] Figure 5 a and b show the purification of hydrolyzed urine extract in terms of pigment and salt removal. The left culture tube details the precipitated urine, which was then centrifuged, and the eluent was removed and evaporated. The right culture tube details the same sample processed according to the present invention.

[0031] Figure 6 a and b show bar graphs comparing the contents of urea (a - top) and creatinine (b - bottom) for a given non-hydrolyzed urine sample under varying component 1 conditions.

[0032] Figure 7 The bar graphs show a comparison of urea (horizontal line graph) and creatinine (slant line graph) contents for a given non-hydrolyzed urine sample, with changes to component 2 and component 1.

[0033] Figure 8 The bar graphs show a comparison of the removal of urea (horizontal graph) and creatinine (slant graph) after blending different proportions of alumina (acidic and neutral) with hygroscopic salts for a given hydrolyzed urine sample in 96-well plates and single-column configurations.

[0034] Figure 9 a and b show bar graph comparisons of removed urea (horizontal line graph) and creatinine (diagonal line graph) for a given non-hydrolyzed urine sample (top) and hydrolyzed urine sample (bottom) using layered and blended alumina / hygroscopic salt combinations.

[0035] Figure 10 a and b show the LC-MS / MS TIC of removed urea (upper trace) and creatinine (lower trace) for a given hydrolyzed urine sample. The trace on the left shows a simple precipitation using ACN, and the trace on the right shows the trace using the aforementioned flow-through apparatus.

[0036] Figure 11a and b show bar graphs of recoveries from unhydrolyzed urine obtained using the flow-through device with different protocols for a range of abused drugs. Detailed Implementation

[0037] A first aspect of the invention is a flow-through device comprising at least one separation column, wherein a first packing component comprising particles of alumina and / or silica and a second packing component comprising powders of one or more hygroscopic salts are provided.

[0038] Alumina is a well-known separation medium, existing in acidic, neutral, or basic forms, and is widely used to remove water from gas streams. It is also used as an adsorbent in chromatographic columns and other laboratory equipment. Technicians can obtain alumina from commercial sources or purchase it and control its pH by adding alkali to the water slurry.

[0039] Similarly, silica materials are widely used as adsorbents for various separation and extraction purposes. For example, in solid-phase extraction (SPE), silica materials, such as those functionalized with carbon, are widely used, and such materials with carbon chains of any length between two and eighteen carbon atoms can be provided in the apparatus. Such materials are commonly referred to as "SPE phases".

[0040] Alternatively, other materials containing a lower proportion of silica may be provided as the first packing component in this apparatus, such as materials containing diatomaceous earth. These materials are commonly used in supporting liquid-liquid extraction (SLE) and are therefore sometimes referred to as the “SLE phase,” which is included herein within the scope of the term “silica-containing.”

[0041] In the apparatus of the present invention, the particles of the first filling component may be porous particles. More specifically, if alumina is present in the apparatus of the present invention, it may have a particle size of at least about 10 μm and up to about 200 μm, and it may have... For example, about The aperture.

[0042] Furthermore, if silica material is provided in the first filler component, it may have the same particle size range as described above.

[0043] The particles of the first filler component may be, for example, spherical or any irregular shape.

[0044] Furthermore, the first packing component may contain acidic or neutral alumina. For a specific application, a technician can consider, for example, the desired capacity, which form to use. Neutral alumina may be more selective for different matrices and potentially different analyte groups.

[0045] Furthermore, if the first filler component comprises silica, it can be advantageously functionalized. Suitable functionalizations may be any one or more of those commonly used in SPE phases, for example. For instance, functionalized silica may contain one or more functional groups selected from: carbon chains, such as linear C4-C18 chains; carboxyl groups; metal chelating groups; and ion exchange groups, such as cation exchange groups or anion exchange groups.

[0046] Additionally, the first filler component may comprise capped silica, which is typically provided as an SPE phase. In short, capping refers to capping with free silanol groups.

[0047] Without intending to impose any limitation on the invention as defined in the appended claims, when the first filler component is used in combination with a solvent such as acetonitrile (ACN), the first filler component provides for the removal of precipitates of certain matrix components, such as urate, due to their lower solubility in the solvent. Contact between the sample and the first filler component also appears to remove some pigments that are difficult to remove using existing methods.

[0048] Therefore, in the apparatus of the present invention, the second filling component is used in practice to "dry" the sample, whereas in the prior art, hygroscopic salts of the type described herein are typically used to "salt out" analytes from one phase to another. Thus, by combining powders of one or more hygroscopic salts with alumina and / or silica particles, the apparatus of the present invention unexpectedly achieves efficient and rapid removal of matrix components from biological samples. Specifically, as shown in the experimental section below, the present invention has demonstrated its effectiveness in removing even more difficult-to-remove matrix components from urine and other samples.

[0049] More specifically, the powder of one or more hygroscopic salts may include, for example, one or more selected from: magnesium sulfate, sodium sulfate, sodium acetate, sodium citrate, sodium citrate sesquihydrate, sodium chloride, and magnesium oxide. Other salts may also be used, provided they achieve the aforementioned effects of the second filler component. The salts useful in the second filler component may be referred to as desiccants, or simply anhydrous salts.

[0050] Inorganic salts such as magnesium sulfate (MgSO4) and sodium acetate (NaOAc, i.e., CH3COONa) are commercially available under the trade name QuEChERS (http: / / www.restek.com / ). Therefore, those skilled in the art can obtain the second filler component from commercial sources or prepare it from separate chemicals. In the apparatus of the present invention, the second filler component is a mixture of magnesium sulfate and sodium acetate, which may be in powder form, and the ratio may vary, for example, from 1:4 to about 4:1.

[0051] In an advantageous form, the device of the present invention comprises no more than two filler components, wherein the first filler component comprises silicon dioxide; and the second filler component comprises magnesium sulfate and sodium acetate. As will be discussed elsewhere in this specification, the two filler components may be provided as separate layers or as a mixture.

[0052] In the apparatus of the present invention, the column may comprise a bottom glass frit and a top glass frit on opposite sides of the first and second packing components. In other words, considering the flow pattern of the apparatus, the upper glass frit is arranged upstream of the two packing components, while the lower glass frit is arranged downstream of the two packing components. In this context, the term "glass frit" is used for any suitable separator, such as a filter, screen, etc., capable of holding both packing components in the column while allowing liquid to pass through. Such glass frits are capable of adsorbing liquid, or are prepared as screens that do not adsorb liquid at all or only to a very small extent, to reduce dead volume, particularly in small devices. As those skilled in the art will understand, all glass frits considered in the apparatus of the present invention are capable of allowing liquid to pass through during sample processing.

[0053] In one embodiment of this apparatus, the first filler component and the second filler component are each provided as separate layers in the column. Then, the first filler component is provided closer to the top glass frit, i.e., upstream of the second filler component and the bottom glass frit, as... Figure 1b and 2b As shown.

[0054] To more clearly separate the layers, the first filler component and the second filler component can be separated by an intermediate glass frit, as shown below. Figure 1b and 2b As shown. All the characteristics of the first and second filler components described above apply to this embodiment.

[0055] In an alternative embodiment, the first and second filler components are provided as a blend in the column, such as Figure 1a and 2a As shown. A suitable blending ratio is, for example, alumina and / or silica particles: hygroscopic salt blended at a ratio of 1:4 to 4:1. All the characteristics of the first and second filler components described above apply to this embodiment.

[0056] The apparatus of the present invention can be in the form of a single bed, in which case the column is a tube, a chromatographic column, or any other suitable barrel-shaped container. All the characteristics described above regarding the two packing components and how they are blended or separated apply to this embodiment.

[0057] Alternatively, the device of the present invention can be in the form of a multi-bed assembly, in which case two or more (e.g., multiple) columns are arranged on a plate, for example as fixing holes in the plate (e.g., a multi-walled plate), or as tubes or vials in a support. All the features described above regarding the two filling components and how they are blended or layered apply to this embodiment.

[0058] As can be seen from other parts of this application, the apparatus of the present invention is advantageously used for removing matrix components from biological samples.

[0059] Therefore, in one embodiment, the device of the present invention may include a hydrophobic top glass frit disposed upstream of the first and second filler components. Within the scope of the invention, all the device features discussed above, such as the properties of the two filler components, their blending / stratification and form in the column, can be combined with such a top glass frit.

[0060] A second aspect of the invention is a method for removing one or more matrix components from a biological sample, said matrix component being selected from one or more of the following: urea, uric acid, phospholipids, amino acids, creatine, heme degradation products (e.g., urobilin), and endogenous salts. The method of the invention comprises passing the sample through a first filler component comprising alumina and / or silica particles and a second filler component comprising a powder containing one or more hygroscopic salts.

[0061] The properties of the two filling components described above, their blending / stratification in the column, and exemplary forms can be applied within the scope of the method of the present invention.

[0062] Biological samples can be any sample whose complexity needs to be reduced in order to subsequently and successfully analyze the analyte. In this document, the term "biological" should be understood as originating from mammals, such as humans or animals, or any other biological source. Biological samples can be biological fluids, such as urine or saliva.

[0063] Matrix components removed from the sample may be, for example, one or more of the following: urea, uric acid, phospholipids, amino acids, creatine, heme degradation products (e.g., urobilin), and endogenous salts; these are urine components known to interfere with analytical methods (e.g., MS).

[0064] Alternatively, biological samples may contain liquefied or mechanically subdivided tissues, such as biopsies or other tissues derived from humans or animals.

[0065] More specifically, and as will be described in more detail in the Experimental Section below, the method of the present invention can comprise a simple workflow in which unprocessed urine, which may be hydrolyzed or unhydrolyzed, is diluted, for example, with a suitable solvent (e.g., acetonitrile). The resulting liquid is added to a column, such as the apparatus of the present invention, positive pressure is applied according to any known technique, and the extract is collected. The resulting extract can then be evaporated and reconstituted in a solvent suitable for LC-MS, or even injected directly into the LC-MS.

[0066] In an advantageous method of the invention, the biological sample is hydrolyzed before passing through two filler components. This can be achieved, for example, by passing it through a hydrophobic glass frit.

[0067] A third aspect of the invention is a combination of a first filler component comprising particles of alumina and / or silica and a second filler component comprising powder of one or more hygroscopic salts, for use in removing one or more additional matrix components from a biological sample.

[0068] All details provided in this application regarding the properties of the two filler components, their blending / stratification in the column, exemplary forms, and methods are within the scope of this invention and may be used in any combination or applied to any implementation.

[0069] Using this invention facilitates the removal of matrix components from any biological sample (e.g., a sample collected from an individual) to identify or track drug use, biomarkers, metabolites, etc. For example, this invention can be used, for instance, in routine controls to identify individuals using illicit drugs, such as performance-enhancing drugs (adulterated substances) in sporting events. It can also be used to track drug abuse and / or metabolites resulting from legal or illicit chemical intake. Therefore, analytes targeted in subsequent MS tests of this invention can be selected, for example, from amphetamines; opiates; cocaine; benzodiazepines; barbiturates; non-benzodiazepine drugs, referred to as Z-drugs; tetrahydrocannabinol (THC); steroids; and related metabolites.

[0070] In an advantageous application of the invention, at least one matrix component removed is a pigment, and the sample is urine.

[0071] Detailed description of the attached figures

[0072] Figure 1 illustrates the principle of the device of the present invention, which comprises a plurality of tubes arranged in a plate-like manner: Figure 1a This demonstrates how two filler components can be blended to provide a single-bed form in each tube; while Figure 1b This shows two separate and layered filler components, with the second filler component, i.e., hygroscopic salt, as the bottom layer and the first filler component, i.e., alumina and / or silica, as the top layer, and the layers are separated by glass frit.

[0073] Figure 2 illustrates the principle of the device of the present invention, which includes a single bed for clearing the arrangement of elements: Figure 2a This demonstrates how two filler components can be blended to provide a single-bed form; while Figure 2b The diagram shows a layered form with a second filler component, namely a hygroscopic salt, as the bottom layer and a first filler component, namely alumina and / or silica, as the top layer, wherein the layers are separated by glass frit.

[0074] Figure 3 This diagram shows the composition of normal urine. The majority (56.1%) of the matrix is ​​composed of urea, along with various salts, small amounts of organic acids, and a relatively small proportion of creatinine. The figure shows the following proportions from left to right: uric acid 1.3%, phospholipids 0.5%, amino acids 6%, creatinine 2%, others 0.2%, potassium 3%, sodium 8%, ammonia 1%, calcium 0.3%, sulfate 4%, chloride 14%, and phosphate 3.6%.

[0075] Figure 4 The left side shows unprocessed hydrolyzed urine without further treatment, and the right side shows the eluent obtained after treatment using the aforementioned flow-through device. It can be seen that when using the flow-through device, complete removal of matrix pigments is achieved, resulting in a clear liquid.

[0076] Figure 5 The purification of the hydrolyzed urine extract is shown in terms of removing pigments and salts. The visually approximate extract shown is from the evaporated sample. The left culture tube details the precipitated urine, which was then centrifuged, and the eluent was removed and evaporated. The right culture tube details the same sample processed using the flow-through apparatus described above, followed by eluent evaporation. The left culture tube shows a large amount of residue and pigment, while the right tube shows a clear tube.

[0077] Figure 6 a and b show bar graphs comparing urea (a - top) and creatinine (b - bottom) content for a given non-hydrolyzed urine sample, with variations in filler component 1. Extracts using modified hygroscopic salt compositions are compared to dilution and injection methods. Items from left to right: AOAC QuEChERs salt; EN QuEChERs salt; MgSO4 only; Na2SO4 only; 1:9 dilution and injection. Details of urine crash ratios with ACN are shown at 1:4 (slant graph) and 1:9 (horizontal line graph). Using different QuEChERs salt compositions is more reliable compared to single-salt forms.

[0078] Figure 7The bar graphs show a comparison of urea (horizontal line graph) and creatinine (slant graph) contents for a given non-hydrolyzed urine sample under altered filler component 2 and filler component 1 conditions. Items from left to right: AOAC salt only; layered C8-terminated on AOAC salt; layered C4 on AOAC salt; layered Si on AOAC salt; alumina-N only; 1:9 dilution and injection. Only slight improvements were observed when using various modified silica adsorbents compared to using filler component 1 alone. Alumina alone provides a removal rate of >40%, while also eliminating pigments from the sample.

[0079] Figure 8 This paper presents a bar graph comparison of urea (horizontal line graph) and creatinine (slant graph) removal when different ratios of alumina (acidic and neutral) are mixed with hygroscopic salts in 96-well plates and single-column configurations for a given hydrolyzed urine sample. Items from left to right: Al-N / salt 1:1 plate; Al-N / salt 1:1 column; Al-N / salt 3:4 plate; Al-N / salt 2:3 plate; Al-N / salt 2:3 column; Al-A / salt 1:1 plate; Al-A / salt 1:1 column; Al-A / salt 3:4 plate; Al-A / salt 3:4 column; Al-A / salt 2:3 plate; Al-A / salt 2:3 column. Good creatinine and urea removal rates were observed at different ratios.

[0080] Figure 9 Figures a and b show bar graphs comparing the removal of urea (horizontal line) and creatinine (slant line) for a given non-hydrolyzed urine sample (top) and a hydrolyzed urine sample (bottom), using stratified and blended alumina / hygroscopic salt combinations. The two figures show different items from left to right: stratified plates 200 / 200 salt / Al-N; 200 / 200 salt / Al-A; 300 / 200 salt / Al-N; 300 / 200 salt / Al-A; blended plates 400 mg salt / Al-N; 450 mg salt / Al-N; 400 mg salt / Al-A; 450 mg salt / Al-A. The comparison between the stratified and blended forms shows that the stratified form achieves better removal of creatinine and urea, while acidic alumina is also slightly superior to the neutral equivalent.

[0081] Figure 10 a and b show the LC-MS / MS TIC of removed urea (upper trace) and creatinine (lower trace) for a given hydrolyzed urine sample. The trace on the left shows simple precipitation using ACN, and the trace on the right shows the trace using the described flow-through device. High levels of urea and creatinine removal, typically greater than 90%, were observed when using the described flow-through device compared to conventional processes.

[0082] Figure 11Figures a and b show bar graphs of recoveries from unhydrolyzed urine obtained using the flow-through device with various protocols for a range of abused drugs. The bars, from left to right, represent: morphine, amphetamine, codeine, MDMA, methoxyephedrine, ketamine, cocaine, 7-aminoflunitrazepam, PCP, oxazepam, zaleplon, and ritalinic acid. The upper figure (11a) details the standard method as described in the experimental section, represented by a horizontal line graph in the foreground; modifications by adding additional small amounts of ACN are represented by a diagonal line graph in the middle; and modifications by adding additional small amounts of MeOH are represented by a solid graph in the background. The lower figure (11b) uses urine modified with formic acid prior to extraction, as described in the experimental section, and replicates the aforementioned trends.

[0083] experiment

[0084] The embodiments provided in this application are for illustrative purposes only and should not be construed as limiting the invention as defined by the appended claims. All references provided below or elsewhere in this application are incorporated herein by reference.

[0085] Materials and methods

[0086] Pharmaceutical reference materials and related internal standards were purchased from LGC Standards (Teddington, UK). Ammonium acetate, ammonium hydroxide, HCl, formic acid, acetic acid, and β-glucuronidase (Burgundy snail) were purchased from Sigma-Aldrich Company Ltd (Gillingham, UK). Urine was donated by healthy human volunteers. All solvents were LC / MS grade, purchased from Honeywell Research Chemicals (Bucharest, Romania). Fresh water (18.2 MΩ·cm) was drawn daily from a Direct-Q 5 water purifier (Merck Millipore, Watford, UK).

[0087] Methods of hydrolyzing urine

[0088] Dilute 1 mL of urine (blank or spiked) with 950 μL of 100 mM ammonium acetate (pH 5) and 50 μL of β-glucuronidase (equivalent to approximately 4500 U / mL urine). Hydrolyze the urine at 60°C for up to 2 hours, then cool and proceed with further processing. Alternative hydrolysis methods using different recombinant and non-recombinant enzymes are also effective. When using specially treated hydrophobic glass frit, the hydrolysis method can be performed on plates.

[0089] Urine extraction methods

[0090] • Thoroughly mix 100 μL of unhydrolyzed or enzymatically hydrolyzed urine (as in the example above) with 600 μL of ACN. Mixing can be performed offline in an Eppendorf tube or other suitable container or plate by vortexing (20 seconds) or by repeated aspiration / dispensing pipetting (3-4 cycles).

[0091] Larger urine output is also possible.

[0092] • If processed offline in tubes, they can be centrifuged at 13,300 rpm for 10 minutes to remove particulate matter. For on-plate processing, a centrifugation step is not necessary because the glass frit components have sufficient filtration capacity.

[0093] • If offline processing is used, the supernatant is applied to the above-mentioned flow-through device.

[0094] • Apply positive pressure or vacuum to initiate flow. The flow recommendations for the flow-through device will differ when using a standard type of top glass frit compared to using a specially treated hydrophobic top glass frit. Typical processing should use fine conditions, possibly with gradual changes in pressure or vacuum near the end of the processing. An exemplary processing could be: 1 PSI for 10 seconds, 3 PSI for 10 seconds, 5 PSI for 10 seconds, then 10 PSI for 10 seconds.

[0095] The eluent can then be evaporated and redissolved in a suitable mobile phase for LC / MS analysis. If the chromatographic conditions and method sensitivity allow, the eluent can be diluted with water or injected directly into the LC / MS system without dilution.

[0096] Method optimization strategy

[0097] For difficult analytes, including certain amphoteric analytes (such as gabapentin and pregabalin), and to improve the recovery of certain other drugs, the following additional steps may be included:

[0098] • Add 10 μL of formic acid to the urine sample just before mixing with ACN.

[0099] • Adding an additional equal volume of organic solvent (e.g., ACN or MeOH) after the sample / ACN mixture has passed through can improve the recovery rate. 100-200 μL is effective in this study. The vacuum or positive pressure method described earlier should be used for processing.

Claims

1. A flow-through device comprising at least one separation column, wherein a first packing component comprising alumina and / or silica particles and a second packing component comprising a powder of a plurality of hygroscopic salts, wherein the powder of the plurality of hygroscopic salts comprises magnesium sulfate, sodium citrate, sodium citrate sesquihydrate and sodium chloride.

2. The apparatus according to claim 1, wherein, The first filler component comprises alumina and / or functionalized silicon dioxide.

3. The apparatus according to claim 2, wherein, The silica contains one or more functional groups selected from the following: linear carbon chains having 4-18 carbon atoms, carboxyl groups, metal chelating groups, and ion exchange groups.

4. The apparatus according to any one of the preceding claims, wherein, The filler component is limited to silicon dioxide as the first filler component.

5. The apparatus according to any one of claims 1 to 3, wherein, The filler component is limited to aluminum oxide as the first filler component.

6. The apparatus according to any one of claims 1 to 3, wherein, The column comprises a bottom glass frit and a top glass frit arranged on opposite sides of the first filler component and the second filler component.

7. The apparatus according to any one of claims 1 to 3, wherein, The first filler component and the second filler component are arranged as separate layers in the column.

8. The apparatus according to claim 7, wherein, The first filler component and the second filler component are separated by an intermediate glass frit.

9. The apparatus according to any one of claims 1 to 3, wherein, The first filler component and the second filler component are provided as a blend in the column.

10. The apparatus according to any one of claims 1 to 3, wherein, The at least one separation column is a tube.

11. The apparatus according to any one of claims 1 to 3, comprising a plurality of columns arranged on a plate.

12. The apparatus according to any one of claims 1 to 3, wherein, The column comprises a hydrophobic top glass frit disposed upstream of the first filler component and the second filler component.

13. A method for removing one or more matrix components from a biological sample, wherein the matrix components are selected from one or more of urea, uric acid, phospholipids, amino acids, creatine, heme degradation products, and endogenous salts, the method comprising passing the sample through a first filler component comprising alumina and / or silica particles and a second filler component comprising a powder comprising a plurality of hygroscopic salts, wherein the powder comprising the plurality of hygroscopic salts comprises magnesium sulfate, sodium citrate, sodium citrate sesquihydrate, and sodium chloride.

14. The method according to claim 13, wherein, The degradation product of heme is urobilinogen.

15. The method according to claim 13, wherein, The sample was urine.

16. The method according to claim 13, wherein, The sample was oral fluid.

17. The method according to claim 16, wherein, The oral fluid is saliva.

18. The method according to any one of claims 13-17, the method comprising passing the sample through a hydrophobic material and then through the first and second filler components.

19. The use of a mixture of a first filler component comprising particles of alumina and / or silica and a second filler component comprising powders of a plurality of hygroscopic salts for removing one or more matrix components from a biological sample, wherein the powders of the plurality of hygroscopic salts comprise magnesium sulfate, sodium citrate, sodium citrate sesquihydrate and sodium chloride.

20. The use according to claim 19, wherein at least one matrix component removed is a pigment, and the sample is urine.

21. Use of the apparatus according to any one of claims 1 to 12 for preparing a urine sample prior to analysis.

22. The use according to claim 21, wherein the analysis is mass spectrometry.

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