Sample clean up device and method

Abstract

The present invention relates to a flow-through device comprising at least one separation column wherein a first packing component, which comprises particles of alumina and/or silica, and a second packing component, which comprises a powder of one or more hygroscopic salts are provided. The two packing components may be blended or layered in the device, which may comprise a single tube or a plurality of tubes arranged in a plate format, such as the wells of a multiwall plate or tubes in a rack. In addition, the invention relates to a method for removing one or more matrix components, such as pigments, from a biological sample, by passing said sample across a first packing component, which comprises particles of alumina and/or silica, and a second packing component, which comprises a powder of one or more hygroscopic salts.

Claims

1. A flow-through device comprising at least one separation column, wherein a first packing component, which comprises particles of alumina and/or silica, and a second packing component, which comprises a powder of one or more hygroscopic salts, are provided such that at least a portion of the second packing component is downstream of a least a portion of the first packing component, wherein the second packing component is a mixture of magnesium sulphate and sodium acetate.

2. A device according to claim 1, wherein the first packing component comprises alumina and/or functionalised silica.

3. A device according to claim 2, wherein the silica comprises one or more functionalities selected from the group consisting of linear carbon chains having 4-18 carbon atoms; carboxylic groups; metal chelating groups; and ion-exchanging groups.

4. A device according claim 1, wherein the packing components are limited to a first packing component of silica; and the second packing component of magnesium sulphate and sodium acetate.

5. A device according claim 1, wherein the column comprises a bottom frit and a top frit arranged at opposite sides of the first packing component and the second packing component.

6. A device according to claim 1, wherein the first packing component and the second packing component are arranged as separate layers in the column.

7. A device according to claim 6, wherein the first packing component and the second packing component have been separated by a middle frit.

8. A device according claim 1, wherein the first packing component and the second packing component are provided as a blend in the column.

9. A device according claim 1, wherein said at least one column is a tube.

10. A device according to claim 1, which comprises a plurality of columns arranged on a plate.

11. A device according to claim 1, wherein the column comprises a hydrophobic top frit arranged upstream of the first packing component and the second packing component.

12. A device according claim 1, wherein the packing components are limited to a first packing component of alumina; and the second packing component of magnesium sulphate and sodium acetate.

13. A method of using of a device according to claim 1 for preparation of a urine sample prior to analysis.

14. A method for removing one or more matrix components from a biological sample, wherein said matrix components are one or more selected from the group consisting of urea; uric acid; phospholipids; amino acids; creatine, heme degradation products; and endogenous salts; which method comprises passing said sample across a first packing component, which comprises particles of alumina and/or silica, and a second packing component, which comprises a powder of a mixture of magnesium sulphate and sodium acetate wherein at least a portion of the second packing component is downstream of a least a portion of the first packing component.

15. A method according to claim 14, wherein the sample is urine.

16. A method according to claim 14, wherein the sample is an oral liquid.

17. A method according to claim 14, which method comprises passing the sample across a hydrophobic material before being passed across the first and second packing components.

18. A method according to claim 14, wherein the packing components are limited to a first packing component of alumina; and the second packing component of magnesium sulphate and sodium acetate.

19. A method of using of a mixture of a first packing component, which comprises removing one or more matrix components from a biological sample by passing the biological sample over particles of alumina and/or silica, and a second packing component, which comprises a powder of a mixture of magnesium sulphate and sodium acetate, wherein at least a portion of the second packing component is downstream of a least a portion of the first packing component.

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

21. The method according to claim 19, wherein the packing components are limited to a first packing component of alumina; and the second packing component of magnesium sulphate and sodium acetate.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIGS. 1a and b show a device according to the invention, which comprises a plurality of tubes arranged in a plate format in blended and layered format, respectively.

(2) FIGS. 2a and b show a device according to the invention, which comprises a single bed i.e. single tube format in blended and layered format, respectively.

(3) FIG. 3 is a chart demonstrating the composition i.e. the matrix components of normal urine.

(4) FIGS. 4a and b demonstrate raw hydrolysed urine without further processing on the left (a) and the resulting eluate after processing with the said flow through device on the right (b).

(5) FIGS. 5a and b demonstrate hydrolysed urine extract cleanliness in terms of pigment and salt removal, the left culture tube detailing precipitated urine followed by centrifugation and the eluate removed and evaporated; while the culture tube on the right details the same sample processed according to the invention.

(6) FIGS. 6a and b demonstrate bar chart comparisons of urea (a—top chart) and creatinine (b—bottom chart) content for a given non-hydrolysed urine sample varying component 1.

(7) FIG. 7 demonstrate a bar chart comparison of urea (horizontal pattern) and creatinine (diagonal pattern) content for a given non-hydrolysed urine sample varying component 2 with combinations of component 1.

(8) FIG. 8 demonstrates a bar chart comparison of urea (horizontal pattern) and creatinine (diagonal pattern) removal for a given hydrolysed urine sample when blending various ratios of alumina (acidic and neutral) with hygroscopic salts in 96 well plate and individual column format.

(9) FIGS. 9a and b demonstrate bar chart comparisons of urea (horizontal pattern) and creatinine (diagonal pattern) removal for a given non-hydrolysed (top chart) and hydrolysed urine sample (bottom chart) using layered vs blended alumina/hygroscopic salt combinations.

(10) FIGS. 10a and b demonstrate LC-MS/MS TICs of urea (top traces) and creatinine (bottom traces) removal for a given hydrolysed urine sample. The traces on the left demonstrate a simple precipitation with ACN and traces on the right are using the said flow through device.

(11) FIGS. 11a and b demonstrate bar chart recoveries from non-hydrolysed urine for a range of drugs of abuse obtained using the various protocols with the said flow through device.

DETAILED DESCRIPTION OF THE INVENTION

(12) A first aspect of the invention is a flow-through device comprising at least one separation column wherein a first packing component, which comprises particles of alumina and/or silica, and a second packing component, which comprises a powder of one or more hygroscopic salts are provided.

(13) Alumina is a well-known separation media occurring in acidic, neutral or basic form and widely used to remove water from gas streams, but also as a sorbent for chromatography columns and other lab equipment. The skilled person may either obtain alumina from commercial sources, or purchase alumina and control its pH by the addition of base in water slurry.

(14) Similarly, silica materials are widely used as sorbents for various separation and extraction purposes. For example, in solid phase extraction (SPE) silica materials functionalized e.g. with carbon are widely used, and such materials having carbon chains of any length between two and eighteen carbon atoms may be provided in the present device. Such materials are commonly referred to as “SPE phases”.

(15) In addition, other materials comprising lower proportions of silica may be provided as the first packing component in the present device, such as materials comprised of diatomaceous earth. These materials are commonly used in supported liquid extraction (SLE), and are therefore sometimes referred to as “SLE phases” and included herein within the term silica-comprising.

(16) In the present device, the particles of the first packing component may be porous particles. More specifically, if alumina is present in the device according to the invention, it may have a particle size of at least about 10 μm and up to about 200 μm, and it may have a pore diameter in the range of 40-200 Å, such as about 120 Å.

(17) Further, if silica materials are provided in the first packing component, they may have particle sizes in ranges equivalent to the above

(18) The particles of the first packing component may e.g. be spherical, or of any irregular shape.

(19) Further, the first packing component may comprise acidic or neutral alumina. The skilled person may consider for a specific application which form to use depending for example on the expected capacity. For different matrices and potentially different analyte panels, neutral alumina may be more selective.

(20) Further, if the first packing component comprises silica, it may advantageously be functionalized. Suitable functions may e.g. be any one or ones those commonly used in SPE phases. For example, functionalized silica may include one or more of the functionalities from the group consisting of carbon chains, such as linear C4-C18 chains; carboxylic groups; metal chelating groups, and ion-exchanging groups, such as cation exchangers or anion exchangers.

(21) In addition, the first packing component may comprise endcapped silica, which is often provided as an SPE phase. In brief, the capping therein refers to the capping of free silanol groups.

(22) Without wishing to impose any limitation to the invention as defined by the appended claims, the first packing component appears when combined with a solvent such as acetonitrile (ACN) provide a precipitation that removes certain matrix components, such as urinary salts, due to lower solubility in the solvent. The contacting of the sample with the first packing component also appears to remove some pigment, which has been difficult to remove using prior art methods.

(23) Thus, in the present device, the second packing component is used to in practise ‘dry out’ the sample, while in the prior art, hygroscopic salt(s) of the herein discussed sorts are commonly used to ‘salt out’ an analyte from one phase to another. Consequently, by the combining the powder of hygroscopic salt(s) with alumina and/or silica particles, the device according to the invention provides unexpectedly efficient and fast removal of matrix components from biological samples. Specifically, as shown in the Experimental part below, the present invention has been shown to efficiently remove even the more difficult matrix components from urine and other samples.

(24) More specifically, the powder of hygroscopic salt(s) may e.g. comprise one or more selected from the group consisting of magnesium sulphate; sodium sulphate; sodium acetate, sodium citrate; sodium citrate sesquihydrate; sodium chloride; and magnesium oxide. Other salts could be used as well, provided they achieve the above-discussed effect of the second packing component. Salts useful in the second packing components may be known as drying agents, or simply referred to as anhydrous salts.

(25) Inorganic salts such as magnesium sulphate (MgSO.sub.4) and sodium acetate (NaOAc, i.e. CH.sub.3COONa) are commercially available under the trade name QuEChERS (http://www.restek.com/). Thus, the skilled person may obtain the second packing component from commercial sources, or prepare it from the separate chemicals. In the device according to the invention, the second packing component is such a blend of magnesium sulphate and sodium acetate which may be in powder form, and the ratio may vary e.g. from 1 to 4 to about 4 to 1.

(26) In an advantageous format, the device of the invention includes no more than two packing components, wherein a first packing component comprises silica; and a second packing component comprises magnesium sulphate and sodium acetate. The two packing components may be provided as separated layers, or as a mixture, as will be discussed elsewhere in this specification.

(27) In the device according to the invention, the column may comprise a bottom frit and a top frit at opposite sides of the first packing component and the second packing component. In other words, viewing the device in flow-through mode, the upper frit is arranged upstream of the two packing components while the lower frit is arranged downstream of the two packing components. In this context, the term “frit” is used for any suitable divider, such as a filter, mesh or the like, which is capable of maintaining the two packing components in the column while liquids are allowed to pass. Such a frit may either be able to adsorb liquid; or be prepared as a mesh which is not capable of liquid adsorption at all, or only to a very little degree, in order to reduce the dead volume especially of small devices. As the skilled person will appreciate, all frits considered in the present device are able to allow liquid to pass during sample processing.

(28) In one embodiment of the present device, the first packing component and the second packing component are provided as separate layers in the column. The first packing component is then provided closer to the top frit, i.e. upstream of the second packing component and the bottom frit, as illustrated in FIGS. 1b and 2b.

(29) In order to provide for a clear separation of the layers, the first packing component and the second packing component may be separated by a middle frit, also as illustrated in FIGS. 1b and 2b. All features discussed above regarding the first and the second packing components are applicable to this embodiment.

(30) In an alternative embodiment, the first packing component and the second packing component are provided as a blend in the column, as illustrated in FIGS. 1a and 2a. Suitable blending ratios may e.g. be from 1:4 to 4:1 blend of alumina and/or silica particles:hygroscopic salts. All features discussed above regarding the first and the second packing components are applicable to this embodiment.

(31) The device according to the invention may be in a single bed format, in which case the column is a tube, a chromatography column or any other suitable barrel shaped container. All features discussed above regarding the two packing components and how they are blended or layered are applicable to this embodiment.

(32) Alternatively, the device according to the invention may be in a multi-bed format, in which case two or more, such as a plurality of columns, are arranged in a plate, e.g. as the fixed wells of a plate, such as a multiwall plate, or tubes or vials in a rack. All features discussed above regarding the two packing components and how they are blended or layered are applicable to this embodiment.

(33) As appears from other sections of this application, the device according to the invention is advantageously used for the removal of matrix components from biological samples.

(34) Thus, in one embodiment, the device according to the invention may comprise a hydrophobic top frit arranged upstream of the first packing component and the second packing component. All the device features discussed above such as the nature of the two packing components, the blend/layering thereof in a column and the formats may be combined with such a top frit within the scope of the invention.

(35) A second aspect of the invention is a method for removing one or more matrix components from a biological sample, wherein said matrix components are one or more selected from the group consisting of urea; uric acid; phospholipids; amino acids; creatine, heme degradation products such as urobillin; and endogenous salts. The method according to the invention comprises passing said sample across a first packing component, which comprises particles of alumina and/or silica, and a second packing component, which comprises a powder of one or more hygroscopic salts.

(36) The nature of the two packing components, the blend/layering thereof in a column and the illustrative formats discussed above may be applied within the scope of the method according to the invention.

(37) The biological sample may be any sample the complexity of which needs to be reduced for a subsequent successful analysis of an analyte. In this context, the term “biological” is understood as originating from a mammal, such as a human being or an animal, or any other biological source. The biological sample may be a biological liquid, such as urine or saliva.

(38) The matrix components removed from the sample may e.g. be any one or more selected from the group consisting of urea; uric acid; phospholipids; amino acids; creatine, heme degradation products such as urobillin; and endogenous salts; which are all urine components known to interfere with analytic methods such as MS.

(39) Alternatively, the biological sample may comprise a liquefied or mechanically finely divided tissue, such as a biopsy or other tissue originating from a human being or an animal.

(40) More specifically, and as will be illustrated in more detail in the experimental part below, a method according to the invention may encompass a simple workflow where e.g. raw urine, which may or may not have been hydrolysed, is diluted with a suitable solvent, such as acetonitrile. The resulting liquid is added to a column, such as a device according to the invention, positive pressure is applied in accordance to any well-known technology and an extract is collected. The resulting extract may then be evaporated and reconstituted in a solvent suitable for LC-MS, or even be directly injected onto the LC-MS.

(41) In an advantageous method according to the invention, the biological sample is hydrolysed before it is passed across the two packing components. This may be achieved e.g. by passage thereof across a hydrophobic frit.

(42) A third aspect of the invention is the use of a combination of a first packing component, which comprises particles of alumina and/or silica, and a second packing component, which comprises a powder of one or more hygroscopic salts for the removal of one or more matrix components from a biological sample.

(43) All details provided in this application regarding the nature of the two packing components, the blend/layering thereof in a column, the illustrative formats and the method may be applied in any combination or embodiment within the scope of the present use.

(44) The use according to the invention is advantageous for the purpose of removing matrix components from any biological sample, such as samples taken from individuals in order to identify or trace the use of drugs, biomarkers, metabolites etc. For example, the invention may be used e.g. in routine controls for the identification of individuals using illegal drugs, such as drugs that improve physical performance (dopants) during sport events. It may also be used to track drugs of abuse, and/or metabolites formed as a result of legal or illegal intake of chemical substances. Thus, the analyte targeted in e.g. MS subsequent to the present invention could e.g. be selected from the group consisting of amphetamines; opiates; cocaines; benzodiazepines: barbiturates; nonbenzodiazepine drugs known as z drugs; tetrahydrocannabinol (THC); steroids; and related metabolites.

(45) In an advantageous use according to the invention, at least one matrix component removed is a pigment and the sample is urine.

DETAILED DESCRIPTION OF THE DRAWINGS

(46) FIG. 1 illustrates the principles of a device according to the invention, which comprises a plurality of tubes arranged in a plate format: FIG. 1a shows how the two packing components are blended to provide a single bed format in each tube; while FIG. 1b shows the two packing components separated and into a layered format having the second packing component i.e. the hygroscopic salt(s) as the bottom layer and the first packing component i.e. alumina and/or silica as the top layer, and the layers separated by a frit.

(47) FIG. 2 illustrates the principles of a device according to the invention, which comprises a single bed clean up format: FIG. 2a shows how the two packing components are blended to provide a single bed format; while FIG. 2b shows a layered format having the second packing component i.e. the hygroscopic salt(s) as the bottom layer and the first packing component i.e. the alumina and/or silica as the top layer, where the layers are separated by a frit.

(48) FIG. 3 demonstrates normal urine composition. The majority (56.1%) of the matrix is made up of urea with various salts, small organic acids, creatinine making up the smaller proportions. From left to right the chart shows the proportion of: uric acid 1.3%, phospholipids 0.5%, amino acids 6%, creatinine 2%, others 0.2%, potassium 3%, sodium 8%, ammonia 1%, calcium 0.3%, sulphate 4%, chloride 14% and phosphate 3.6%.

(49) FIG. 4 demonstrates raw hydrolysed urine without further processing on the left and the resulting eluate after processing with the said flow through device on the right. It can be seen that when using the said flow through device full removal of the matrix pigment is achieved resulting in a clear liquid.

(50) FIG. 5 demonstrates hydrolysed urine extract cleanliness in terms of pigment and salt removal. Visual extracts shown are from evaporated samples. The left culture tube details precipitated urine followed by centrifugation and the eluate removed and evaporated; the culture tube on the right details the same sample processed using the said flow through device followed by eluate evaporation. Substantial residue and pigment is demonstrated on the left culture tube whereas a clear tube is shown on the right.

(51) FIGS. 6a and b demonstrate bar chart comparisons of urea (a—top chart) and creatinine (b—bottom chart) content for a given non-hydrolysed urine sample varying packing component 1. Extracts using modified hygroscopic salt combinations were compared to a dilute and shoot approach. The options from left to right: AOAC QuEChERs salts; EN QuEChERs salts; MgSO.sub.4 only; Na.sub.2SO.sub.4 only; 1:9 dilute and shoot. Urine crash ratios of 1:4 (diagonal pattern) and 1:9 (horizontal pattern) with ACN are detailed. Various QuEChERs salt combinations performed more reliably compared to single salt formats.

(52) FIG. 7 demonstrates a bar chart comparison of urea (horizontal pattern) and creatinine (diagonal pattern) content for a given non-hydrolysed urine sample varying packing component 2 with combinations of packing component 1. The options from left to right: AOAC salts only; layered C8-End capped above AOAC salts; layered C4 above AOAC salts; layered Si above AOAC salts; alumina-N only; 1:9 dilute and shoot. When using various modified silica sorbents only slight improvement was observed compared to using packing component 1 only. Using Alumina alone provided >40% removal while also eliminating pigment from the sample.

(53) FIG. 8 demonstrates a bar chart comparison of urea (horizontal pattern) and creatinine (diagonal pattern) removal for a given hydrolysed urine sample when blending various ratios of alumina (acidic and neutral) with hygroscopic salts in 96 well plate and individual column format. The options 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 was observed with various ratios.

(54) FIGS. 9a and b demonstrate bar chart comparisons of urea (horizontal pattern) and creatinine (diagonal pattern) removal for a given non-hydrolysed (top chart) and hydrolysed urine sample (bottom chart) using layered vs blended alumina/hygroscopic salt combinations. Both charts demonstrate various options from left to right: Layered plate 200/200 salt/Al—N; 200/200 salt/Al-A; 300/200 salt/Al—N; 300/200 salt/Al-A; Blended plate 400 mg salt/Al—N; 450 mg salt/Al—N; 400 mg salt/Al-A; 450 mg salt/Al-A. The comparison of layered vs blended formats demonstrate better creatinine and urea removal with the layered format while acidic alumina is also slightly better than the neutral equivalent.

(55) FIGS. 10a and b demonstrate LC-MS/MS TICs of urea (top traces) and creatinine (bottom traces) removal for a given hydrolysed urine sample. The traces on the left demonstrate a simple precipitation with ACN and traces on the right are using the said flow through device. High level of urea and creatinine removal was observed, typically greater than 90% when using the said flow through device compared to conventional processing.

(56) FIGS. 11a and b demonstrate bar chart recoveries from non-hydrolysed urine for a range of drugs of abuse obtained using the various protocols with the said flow through device, where the bars represent, starting from the left: morphine, amphetamine, codeine, MDMA, mephedrone, ketamine, cocaine, 7-amino flunitrazepam, PCP, oxazepam, zaleplon and ritalinic acid. The top chart (11a) details the standard method as detailed in the experimental section at the front with horizontal pattern; modification by addition of ACN as a further small aliquot in the middle with diagonal pattern; modification by addition of MeOH as a further small aliquot at the back with solid pattern. The bottom chart (11b) repeats the trend using urine modified with formic acid prior to extraction as detailed in the experimental section.

Experimental

(57) The present examples are provided for illustrative purposes only, and should not be interpreted as limiting the present invention as defined by the appended claims. All references provided below or elsewhere in the present application are hereby included herein by reference.

(58) Materials and Methods

(59) Drug standards and associated internal standards were purchased from LGC Standards (Teddington, UK) Ammonium acetate, ammonium hydroxide, HCl, formic and acetic acids and β-glucuronidase (helix pomatia) were purchased from Sigma-Aldrich Company ltd (Gillingham, UK). Urine was donated by healthy human volunteers. All solvents were LC/MS grade from Honeywell Research Chemicals (Bucharest, Romania). Water (18.2 MΩ.Math.cm) was drawn fresh daily from a Direct-Q 5 water purifier (Merck Millipore, Watford, UK).

(60) Hydrolysed Urine Procedure

(61) 1 mL of urine (blank or spiked) was diluted with 950 μL of 100 mM ammonium acetate pH 5 and 50 μL β-glucuronidase enzyme (equivalent to approximately 4500 U/mL of urine). Urine was hydrolysed at 60° C. for up to 2 hours then cooled prior to further processing. Alternative hydrolysis procedures will also work using various recombinant and non-recombinant enzymes. When using the proprietary treated hydrophobic fits the hydrolysis procedure can be performed on plate.

(62) Urine Extraction Procedure

(63) Non-hydrolysed or enzymatically hydrolysed urine (as above example), typically 100 μL is thoroughly mixed with 600 μL of ACN. Mixing can be performed offline in Eppendorf tubes or other suitable container or on-plate both using vortex action (20 seconds) or by repeat aspirate/dispense pipetting (3-4 cycles).

(64) Larger urine volumes are also possible.

(65) If processing offline in tubes these can be centrifuged for 10 minutes at 13,300 rpm to remove particulate matter. For on-plate processing the centrifugation step is not required due to adequate filtering capacity of the frit components.

(66) Apply the supernatant to the said flow through device if using offline processing.

(67) Apply positive pressure or vacuum to initiate flow. Flow recommendations will be different between the said flow-through device when using standard type top fits as opposed to using the proprietary treated hydrophobic top fits. Typical processing should use subtle conditions potentially ramping pressure or vacuum towards the end of the processing. Example processing could be: 1 PSI for 10 seconds, 3 PSI for 10 seconds, 5 PSI for 10 seconds and then 10 PSI for 10 seconds.

(68) The eluate can then be evaporated and reconstituted in appropriate mobile phase for LC/MS analysis. If chromatographic conditions and method sensitivity allow the eluate can be directly injected onto the LC/MS system with or without dilution with water.

(69) Method Optimization Strategies

(70) For difficult analytes, including some amphoteric analytes such as gabapentin and pregabalin and to increase recovery of some other drugs additional steps can be included:

(71) Addition of 10 μL of formic acid to urine sample directly prior to mixing with ACN.

(72) Post flow though of the sample/ACN mixture a further aliquot of organic solvent such as ACN or MeOH can increase recoveries. Here 100-200 μL can be effective. Processing should be performed using the previously stated vacuum or positive pressure procedure.