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WO2025036981A1 - Dispositif - Google Patents

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Publication number
WO2025036981A1
WO2025036981A1 PCT/EP2024/073021 EP2024073021W WO2025036981A1 WO 2025036981 A1 WO2025036981 A1 WO 2025036981A1 EP 2024073021 W EP2024073021 W EP 2024073021W WO 2025036981 A1 WO2025036981 A1 WO 2025036981A1
Authority
WO
WIPO (PCT)
Prior art keywords
composition
sample
sample vessel
internal standard
end cap
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/EP2024/073021
Other languages
English (en)
Inventor
Elham HEDAYATI
Per Rydberg
Djavad HEDAYATI
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
True Dose AB
Original Assignee
True Dose AB
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from GB2312444.9A external-priority patent/GB2633004B/en
Application filed by True Dose AB filed Critical True Dose AB
Publication of WO2025036981A1 publication Critical patent/WO2025036981A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/15—Devices for taking samples of blood
    • A61B5/150007—Details
    • A61B5/150015—Source of blood
    • A61B5/150022—Source of blood for capillary blood or interstitial fluid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/15—Devices for taking samples of blood
    • A61B5/150007—Details
    • A61B5/150343—Collection vessels for collecting blood samples from the skin surface, e.g. test tubes, cuvettes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/15—Devices for taking samples of blood
    • A61B5/150007—Details
    • A61B5/150351—Caps, stoppers or lids for sealing or closing a blood collection vessel or container, e.g. a test-tube or syringe barrel
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/15—Devices for taking samples of blood
    • A61B5/150007—Details
    • A61B5/150755—Blood sample preparation for further analysis, e.g. by separating blood components or by mixing
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/508—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above
    • B01L3/5082—Test tubes per se
    • B01L3/50825—Closing or opening means, corks, bungs
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/04—Closures and closing means
    • B01L2300/041—Connecting closures to device or container
    • B01L2300/042—Caps; Plugs

Definitions

  • the present invention is directed towards a fluid sample collection apparatus comprising an end cap comprising a substance suitable for use as an Internal Standard, which composition is sealed within the end cap in an atmosphere that protects the substance for use as an Internal Standard against degradation and wherein the end cap is configured to be couplable to a sample vessel and to controllably release the composition to the interior of the sample vessel.
  • the present invention also relates to a sample vessel for fluid sample collection, which comprises a substance suitable for use as an Internal Standard that is sealed in an atmosphere that protects the substance for use as an Internal Standard against degradation and wherein the sample vessel comprises means for removing the composition from the protective atmosphere and introducing it into the sample vessel concurrently with a provision of a sample, or before the provision of a sample.
  • the present invention also relates to a kit-of-parts comprising the fluid sample collection apparatus and/or the sample vessel, along with methods and uses of the same.
  • dose-ranging is an integral part of phase I and/or phase II clinical trials.
  • the main aim of dose-ranging studies is to analyse the half-lives of drugs in healthy volunteers in both plasma and urine, and this requires multiple bodily fluid samples being taken over wide spanning time ranges and the concentrations of the drugs in these samples being measured.
  • some patients require ongoing bodily fluid sampling to analyse target compound concentrations, for example recovering drug addicts.
  • Personalized medicine being the tailoring of medical treatment to the individual characteristics of each patient, is gaining prominence as it allows for drug dosing adjustments based on an individual's specific blood or plasma levels, leading to more effective treatment with fewer side effects
  • subjects need to go to a clinical setting where a medical professional obtains a sample and sends this to a laboratory for analysis.
  • blood samples these are often obtained by venous puncture and drawing large volumes of blood which are then sent for analysis.
  • the half-life of drugs in blood varies and even on being drawn out of the body drugs often continue to degrade. Therefore, during the time between the sample being taken and analysis in the laboratory, the drug degrades often at unknown rates meaning that any reliable quantification of drug levels at the time of sampling cannot be achieved.
  • the drug within can degrade due to factors such as transport time, temperature variations, exposure to oxygen, hydrolysis, and photodegradation. Additionally, the yields of analytes may vary between samples from different individuals.
  • the standard procedure for analytical labs is to add the internal standard just before analysis. However, any analyte loss during sample transport and yield loss during analytical sample preparation cannot be compensated for, potentially leading to an underestimation of the true blood concentration.
  • WO 2020/156849 describes a method involving blood sampling, the internal standard is added directly to the extraction solution during the production of kits.
  • chemicals such as internal standards are generally unstable in solutions, especially at low concentrations.
  • Our invention involves protecting the internal standard by keeping it separate from the solvent, light, and other potential degradative factors until the moment of use.
  • kits have utility in that only a small amount of blood sample is required.
  • the sample must be processed as quickly as possible after being taken, meaning that for accurate analysis the sample must be taken in a clinical setting with quick access to analysis instruments. Any delay in analysis will ultimately result in the analysis losing accuracy.
  • a fluid sample collection apparatus comprising an end cap, wherein the end cap comprises a composition comprising a substance suitable for use as an Internal Standard, wherein the composition is sealed within the end cap in an atmosphere that protects the substance suitable for use as an Internal Standard from degradation and wherein the end cap is configured to be couplable to a sample vessel and to controllably release the composition to the interior of the sample vessel, which apparatus is referred to hereinafter as "the apparatus of the invention".
  • the substance suitable for use as an Internal Standard may, interchangeably, be referred to herein as "the Internal Standard” or "IS”.
  • the Internal Standard in the context of chemical analyses, we refer to compounds that are used for the detection of sample losses of the analyte, e.g. the analyte in the sample, to be determined.
  • Internal Standards are known substances with similar analytical behaviour to the substance to be evaluated (i.e. the target compounds/moieties present in a sample), e.g. a stable or analogue isotope of the analyte.
  • Internal Standards may also be, for example, stable isotope substituted chemicals, or near identical, to the chemicals being measured in the sample.
  • the end cap may be couplable to the sample vessel in any way to achieve a seal, such as being couplable to the top or the bottom of the sample vessel.
  • the end cap is couplable to the top of the sample vessel.
  • sample vessel we refer to a container having a reservoir that is suitable for holding and retaining a fluid sample. Such a vessel may have an opening to which the end cap as defined herein may be coupled.
  • the sample vessel may comprise an analysis/extraction solution, (e.g., protein precipitation solutions, anticoagulants and/or other solvent mixtures employed to preserve the analytes in biological fluids and/or facilitate the analysis).
  • the analysis/extraction solution may comprise 2-propanol, methanol, acetonitrile, ZnSCh, acetone, water, formic acid and mixtures thereof.
  • the analysis/extraction solution comprises at least two of these components, for example the analysis/extraction solution may consist essentially of two or three of these components.
  • the analysis/extraction solution may further comprise one or more pH modifier.
  • pH modifier should be understood to include any chemical compound that adjusts the acidity or alkalinity of the solution to minimize acid/base catalysed degradation of the analytes and/or to obtain increased yields in the solvent protein precipitation step.
  • Suitable pH modifiers that may be included in the analysis/extraction solution may be selected from the list consisting of formic acid and acetic acid.
  • the analysis/extraction solution may comprise, or consist of, a mixture of 2-propanol and methanol in a ratio of from about 1: 10 to about 10: 1, such as from about 1:5 to 5: 1, for example about 1: 1, optionally with formic acid also being present in an amount of from about 0.01 vol.% to about 0.5 vol.%, such as about 0.05 to about 0.2 vol.%.
  • the analysis/extraction solution may alternatively comprise, or consist of, a mixture of acetonitrile and methanol in a ratio of from about 1: 20 to about 20: 1, such as from about 1: 10 to 10: 1, for example about 90: 10, optionally with formic acid also being present in an amount of from about 0.01 vol.% to about 0.5 vol.%, such as about 0.05 to about 0.2 vol.%.
  • the analysis/extraction solution may alternatively comprise, or consist of, a mixture of ZnSO4 and water in a ratio of from about 1 : 20 to about 20: 1, such as from about 1 : 10 to 10: 1, for example about 10:90, optionally with formic acid also being present in an amount of from about 0.01 vol.% to about 0.5 vol.%, such as about 0.05 to about 0.2 vol.%.
  • the analysis/extraction solution may alternatively comprise, or consist of, a mixture of acetonitrile and acetone in a ratio of from about 1: 20 to about 20: 1, such as from about 1: 10 to 10: 1, for example about 8:2, optionally with formic acid also being present in an amount of from about 0.01 vol.% to about 0.5 vol.%, such as about 0.05 to about 0.2 vol.%.
  • the volume of analysis/extraction solution in the sample vessel may be from about 100 pL to about 2000 pL, such as about 200 pL to about 1000 pL, for example about 250 pL to about 750 pL.
  • the sample vessel may be configured to be compatible with standard laboratory analysis equipment.
  • the sample vessel may have a size and shape that is configured to be compatible with a centrifuge apparatus and/or a high-performance liquid chromatography (HPLC) apparatus and/or liquid chromatography single or tandem mass spectrometry apparatuses such as (LC-MS/MS), mass spectrometry (MS/MS), or gas chromatography (GC-MS/MS).
  • HPLC high-performance liquid chromatography
  • GC-MS/MS gas chromatography
  • the sample vessel may be a micro tube, such as a micro tube that is configured to hold an internal liquid volume of from about 0.5 mL to about 5 mL, such as about 0.5 mL, to about 3 mL, for example about 1 mL to about 2 mL.
  • the sample vessel may be substantially cylindrical in shape.
  • the end cap may be reversibly couplable or permanently couplable to the sample vessel.
  • the end cap when reversibly couplable when in use the user may be able to couple the end cap to the sample vessel and, once the composition has been released into the interior of the sample vessel, the end cap may be removed.
  • the end cap may be reversibly couplable to the sample vessel by any means known in the art.
  • the end cap may be a screw cap comprising a threaded portion and the sample vessel may comprise an opposing threaded portion such that the end cap can be screwed onto the sample vessel in use and then removed by unscrewing.
  • the end cap may be configured such that it is push-fit onto the sample vessel and can be removed.
  • the end cap When providing to the user the end cap may already be coupled to a sample vessel with the sample vessel comprising an analysis/extraction solution as defined above. That is, the apparatus of the invention may comprise an end cap coupled (either reversibly or permanently) to a sample vessel, with the sample vessel comprising an analysis/extraction fluid.
  • the end cap and/or the sample vessel may comprise locking means (e.g., a locking mechanism) such that when the end cap is coupled to the sample vessel it locks into place and the user is not able to remove the end cap without, for example, specialist equipment or breaking apart the apparatus. That is to say, when stating that the end cap is permanently couplable to the sample vessel, the end cap may still be removed, or the interior of the sample vessel may still be accessed so that the sample can be analysed, but in doing so the apparatus will be required to be damaged such that it is not reusable.
  • the apparatus may, therefore, be for single use.
  • the apparatus may be sterilized and be provided in a sealed package for single use.
  • the composition being sealed within the end cap in an atmosphere that protects the Internal Standard from degradation
  • the composition comprising the Internal Standard is stored within the end-cap that is protected both from the atmosphere external to the end cap and from sunlight.
  • the composition may be stored in an area within the end cap which is surrounded by materials which light is not able to penetrate.
  • the atmosphere in which the Internal Standard is stored may be an inert atmosphere that prevents degradation of the Internal Standard.
  • the atmosphere may be an inert gas atmosphere (e.g., a nitrogen atmosphere or an argon atmosphere).
  • the components within the composition and the environment within which the composition is stored in the end cap may also protect the Internal Standard from degradation via photochemical, oxidation or solvolysis means.
  • the end cap may comprise an aperture for receiving a sampler, or a rod member attached to a lid, with the composition comprising the Internal Standard being positioned within the aperture and wherein the end cap is configured to release the composition into the interior of the sample vessel by introducing the sampler or the rod member of the lid, through the aperture.
  • the sampler may be configured to sample a defined volume of sample fluid.
  • the sampler may be a capillary tube which is configured to sample a defined volume of sample fluid.
  • the aperture may be configured to be capable of receiving a sampler and the rod member of the lid, with the rod member first being used to release the composition into the sample vessel, followed by the rod member being removed and the sampler being passed through the aperture to introduce the sample into the sample vessel.
  • the sampler may comprise a handle portion and the sampler may be removable from the handle portion such that once the sampler has been used to introduce the sample into the sample vessel it can be removed from the handle.
  • the handle portion may also function as a lid for attaching/coupling to the top of the end cap once the sampler has been introduced through the aperture.
  • the handle portion may comprise a threaded portion with the end cap comprising an opposing threaded portion such that once the sampler has been introduced through the aperture the handle portion can be screwed onto the end cap to function as a lid.
  • Other means of coupling the handle portion to the end cap are envisaged, such as using push-fit means.
  • the sampler may be a pipette or a syringe which is configured to sample a defined volume of fluid and is configured to pass through the aperture thus releasing the composition to the interior of the sample vessel, or for introducing the sample into the sample vessel following releasing the composition through use of the rod-member of the lid.
  • the fluid sample may be released from the pipette or syringe into the interior of the sample vessel by squeezing the pipette bulb or advancing the plunger through the syringe.
  • the sampler may be removed from the sample vessel and the end cap closed with a lid, such as a lid as defined above with respect to the capillary handle, or a lid comprising a rod member. Therefore, the apparatus may also comprise a lid that is couplable, either reversibly or permanently, to the end-cap.
  • the lid does not necessarily need to be coupled to the sampler and form a handle for the sampler. The lid, therefore, may be separate to the sampler.
  • samplers may be configured to sample a defined volume of fluid that the user cannot adjust, or, alternatively, the samplers may comprise volume markings such that the user can sample a certain volume of fluid and judge this by eye, meaning that a range of volumes can be sampled if required.
  • the sampler may be configured to sample any predetermined volume of fluid.
  • the sampler may be configured to sample from 10 to 500 pL, such as 10 to 200 pL, for example from 10 to 100 pL, and preferably 20 to 50 pL of fluid.
  • sampler being configured to sample a predetermined volume of fluid, this reduces the risk of sampling error and increases the accuracy of the sampling and subsequent analysis.
  • the fluid to be sampled may be, for example, a bodily fluid, such as blood, urine or saliva.
  • the fluid to be sampled may come from any source, such as an animal subject (e.g. a mammal, such as a human or animal).
  • the source may also be, for example, a water sample (e.g. drinking water sample or waste water sample) or any other sample that may comprise, or be suspected of comprising, target compounds.
  • the lid may comprise a rod member, rod-protruding element and/or pin, for example a member that protrudes and/or extends from the lid.
  • rod member this may be understood as a member that extends from the lid, for example wherein the rod member extends in a direction essentially perpendicular to the plane of the lid, aperture and/or penetrable film.
  • essentially perpendicular this may be understood as an angle of from about 60 to 120 degrees, such as from about 70 to about 110 degrees, for example from about 80 to about 100 degrees, such as from about 85 to about 95 degrees, for example from about 88 to about 92 degrees.
  • the rod member may extend above and/or below, i.e. on the underside of, the lid, for example wherein the rod member extends from the top (e.g. outer side) or underside (e.g. inner side) of the lid. That is, when the rod member is on the underside the rod member may reside within the aperture of the end cap once the lid is coupled to the end cap.
  • the rod member may have a length of from about 1 mm to about 10 cm, for example from 2 mm to about 5 cm, such as from about 0.5 cm to about 4 cm, for example from about 1 cm to about 3 cm.
  • the lid comprising a rod member may be configured to penetrate through the aperture, for example to pierce the penetrable films, thereby releasing the composition into the interior of the sample vessel.
  • the lid comprising a rod member may be used in a preactivation step to release the composition into the interior of the sample vessel prior to introducing the fluid sample into the interior of the sample vessel.
  • the activation step of breaking the penetrable films may be carried out using minimal force and reducing risk of sample loss.
  • the rod may be conical shaped, for example it is wider at the point where it adjoins the lid than the tip furthest away from the lid. When being conical shaped and on the underside of the lid this aids in sealing the aperture and closing the sample vessel with the lid following the addition of the sample to the interior of the sample vessel.
  • the lid comprising a rod member may comprise other features as defined herein.
  • the lid may comprise a handle portion and/or may be couplable, either reversibly or permanently, to the end cap.
  • the lid comprising a rod member is reversibly or permanently couplable to the end cap.
  • the composition may be sealed on both sides of the aperture by an upper and a lower penetrable film, such that on the sampler or rod member of the lid, being introduced through the aperture the penetrable films are broken resulting in the composition being released into the interior of the sample vessel.
  • Any film that provides a seal, but is able to be penetrated by the sampler or the rod member may be used.
  • the film may be an aluminium foil film, a plastic film or a plastic-coated aluminium film.
  • the composition may have a mass of from about 1 mg to about 20 mg, such as about 1 mg to about 10 mg, for example about 1 mg to about 5 mg.
  • the composition may have a diameter in its longest dimension of from about 5 mm to about 15 mm, such as about 5 mm to about 10 mm.
  • the composition comprising the substance suitable for use as an Internal Standard may be a matrix composition.
  • the purpose of the matrix is to absorb the Internal Standard and to release the standard to the solution simultaneously with the biological sample by the catch and release principle.
  • the purpose with the matrix is also to protect the analyte from un-desired by reactions such as solvolysis, reaction between solvents in the tube or atmosphere (water, alcohol or other reactive solvents).
  • the matrix composition can comprise, for example, a reverse phase silica stationary phase or a polymeric stationary phase that retains the Internal Standard(s) by hydrophobic interactions delivered from the incorporated aliphatic groups (C1-C18 chains, phenyl, diphenyl or other functional groups used within the art of liquid chromatography).
  • These matrix compositions may also or alternatively comprise a mixed-mode stationary phase, reversed silica or a polymeric phase, modified with polar groups such as aromatic or aliphatic hydroxyl groups, aromatic or aliphatic amines or other groups that retain the Internal Standard by both hydrophobic interactions and polar interactions, with anionic interactions and cationic interactions.
  • the matrix composition may also or alternatively comprise a reversed phase silica gel or a polymer (e.g., polypropylene, polyethylene, polyamide etc., including non-woven polypropylene, preferably polypropylene, polyethylene, polyamide etc.) which absorbs the Internal Standard.
  • the matrix composition may also comprise a cosolvent (e.g., Pure C8 MTC oil, mineral oil, dimethyl sulfoxide (DMSO), dimethyl formamide (DMF), natural oils and methyl esters of their constitutive fatty acid such as rapeseed methyl ester and others), preferably added in small amounts to increase the release reaction rate and increase the solubility of a poorly soluble compound.
  • a cosolvent e.g., Pure C8 MTC oil, mineral oil, dimethyl sulfoxide (DMSO), dimethyl formamide (DMF), natural oils and methyl esters of their constitutive fatty acid such as rapeseed methyl ester and others
  • the matrix composition may also or alternatively comprise a solid phase that is soluble in any solution, such as an analysis/extraction solution, used in the tube (e.g., protein precipitation solutions, anticoagulants or other solvent mixtures employed to preserve the analytes in biological fluids and/or facilitate the analysis).
  • an analysis/extraction solution used in the tube
  • the composition may comprise excipients into the matrix of which the substance suitable for use as an Internal Standard may be incorporated.
  • the matrix composition may further comprise a solid (e.g. a solid made of tert-butanol, carboxymethylcellulose, Arabic gum, coconut wax etc.) containing the Internal Standard but being soluble in the solvents used for protein precipitation (i.e. the solvents in the analysis/extraction solution as defined below).
  • antioxidants such as antioxidants (vitamin-E, beta carotene, 2 6-diisopropylphenol) can be comprised in the composition to further protect the Internal Standard.
  • antioxidants such as antioxidants (vitamin-E, beta carotene, 2 6-diisopropylphenol)
  • antioxidants such as antioxidants (vitamin-E, beta carotene, 2 6-diisopropylphenol)
  • anti-oxidants may be selected from the list consisting of vitamin-E (Tocopherol), beta carotene, 2 6-diisopropylphenol, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT) and ascorbic acid (vitamin C).
  • antioxidants When incorporating antioxidants into the composition, such anti-oxidants may be sterically hindered.
  • they may be configured to be sterically hindered so as to not interact with components in the matrix that comprise oxidising groups, but are still able to act as anti-oxidants against free oxygen and reactive oxygen species (ROS) that are generated/introduced during storage.
  • Suitable sterically hindered anti-oxidants include butylated hydroxytoluene (BHT), and butylated hydroxyanisole (BHA).
  • the matrix composition comprises a non-woven polypropylene fabric, which may have a thickness of from about 0.05 mm to about 0.5 mm, such as from about 0.1 mm to about 0.3 mm.
  • the matrix composition may comprise silica particles onto which the substance suitable for use as an Internal Standard may be adsorbed such that on entry into the sample vessel interior the substance may be released from the surface of the silica particles.
  • the composition may further comprise one or more beads, balls, gems, bullets, mixers and/or wires, for example metal balls or otherwise, such as stainless steel balls, or any related material.
  • the composition comprises a plurality of stainless steel balls.
  • the composition refers to any composition suitable for being stored within the aperture of the end cap and is not necessarily limited to a matrix composition, although any aspects of the composition as defined herein may be incorporated into the matrix composition defined above.
  • the one or more beads, balls, gems, bullets, mixers and/or wires may be provided within the interior of the sample vessel, such as residing within the analysis/extraction solution.
  • composition comprising beads, or the sample vessel interior comprising beads
  • mixing of the sample fluid, composition and/or analysis/extraction solution is improved due to enhanced mixing when agitating (e.g. shaking) the sample apparatus following introduction of the sample.
  • This may reduce sample preparation time, enhance homogeneous precipitation of proteins and provide quantitative release of the internal standard from the composition during mixing, while having a negligible negative impact on the analysis results.
  • Stainless steel balls may be particularly advantageous to provide higher yields and reduced variation in measured results.
  • the matrix composition comprises polymers, such as non-woven polypropylene
  • the inventors have found that the additional weight provided by the beads, in particular stainless steel balls, prove highly effective in achieving quick preparation time and enhanced precipitation.
  • the mass of the beads comprised within the composition, or incorporated into the interior of the sample vessel may be from about 50 mg to about 500 mg, such as about 100 mg to about 200 mg, for example about 150 mg to about 250 mg.
  • composition or the interior of the sample vessel may comprise from about 1 to about 20 beads, such as from about 2 to about 10 beads, such as from about 3 to about 7 beads.
  • the beads may have a diameter of from about 1 mm to about 1 cm, such as from about 2 mm to about 5 mm, such as from about 2.5 to about 3.5 mm.
  • the composition, or the interior of the sample vessel comprises one or more beads and a cosolvent as defined above.
  • the combination of beads and solvent may prove particularly advantageous in protein precipitation from the analysis/extraction solution, leading to significantly improved accuracy of the analysis results.
  • composition or the interior of the sample vessel comprises beads
  • significantly better results i.e. improved precipitation, higher yields and reduced variation in measured results
  • the composition may further comprise indicator compound.
  • indicator compound should be understood to include any chemical compound that causes a change of colour (i.e. visual change) when added to the analysis/extraction solution, for example a chemical compound that causes the colour of the analysis/extraction solution to change upon release of the composition into the interior of the sample vessel, and mixing of the indicator (i.e. the composition) with the analysis/extraction solution.
  • the indicator compound may act as a visualisation aid, and allow the user to visually monitor the activation process as the colour of the analysis/extraction solution changes following addition of the composition.
  • the indicator compound may provide for visual identification of early, unintentional or accidental release of the composition into the analysis/extraction solution, for example in the case where the bottom seal is damaged prior to sampling (e.g. during storage or transport).
  • the indicator compound may further act as an antioxidant.
  • antioxidant this may be understood as comprising means to minimise oxidation of the Internal Standard, and thereby protect the Internal Standard from excessive oxidation so as to extend the shelflife of the apparatus, sample vessel and/or kit-of-parts.
  • the addition of antioxidants to the standard will, in addition to protect the standard, also protect the collected blood sample, including its components, from oxidation.
  • the indicator compound may be any suitable dye compound or colorant. Suitable indicator compounds that may been included may be selected from one or more of triarylmethane basic dyes, flavin basic dyes, auramine basic dyes, safranine basic dyes, Phloxine basic dyes, xanthene basic dyes and methylene blue bases.
  • Further suitable indicator compounds may be selected from one or more of Methyl Violet, Crystal Violet, Magenta, Basic Cyanine 6G, Basic Cyanine EX, Victoria Pure Blue BO, Victoria Blue B cone., Brilliant Green GX, Malachite Green, Basic Yellow 1, Basic Red 2, Basic Red 12, Basic Blue GO, New Methylene Blue NX, Methyl Viologen, Methyl Red, Methylene Blue, Methyl Orange, tris(bipyridine)ruthenium(II) chloride, Rhodamine B, Rose Bengal, Brilliant Blue, Indigo Carmine, Patent Blue, Direct Yellow, Serva Blue, Prussian Blue, triarylmethane, flavine, xanthene, bikaverin, fluorescein and eosin.
  • the indicator compound is selected from one or more of Methylene Blue and Rhodamine B. More preferably, the indicator compound is Methylene Blue.
  • the indicator compound may be included in the composition in an amount of from about 0.001 wt.% to about 0.1 wt.%, such as from about 0.001 wt.% to about 0.05 wt. %, for example from about 0.001 wt.% to about 0.01 wt.%.
  • the Internal Standard may be included in the composition in an amount of from about 0.001 wt.% to about 0.05 wt. %, for example from about 0.001 wt.% to about 0.01 wt.%.
  • the Internal Standard may be included in the composition in an amount of from about 0.001 wt.% to about 0.1 wt.%, such as from about 0.001 wt.% to about 0.05 wt. %, for example from about 0.001 wt.% to about 0.01 wt.%.
  • composition may further comprise one or more radical inhibitor or one or more oxygen radical inhibitor.
  • radical inhibitor and “radical inhibitor” should be understood to include any chemical compound that prevents the formation of free radicals from oxygen or others, and/or terminates radical chain reactions, thereby stabilizing materials within the composition.
  • Suitable radical inhibitors may be selected from the list consisting of hydroquinone, tertiary butylhydroquinone (TBHQ), phenols, polyphenols, gallic acid, naringin and quercetin.
  • TBHQ tertiary butylhydroquinone
  • phenols phenols
  • polyphenols polyphenols
  • gallic acid naringin and quercetin.
  • the composition may further comprise one or more pH modifier.
  • pH modifier should be understood to include any chemical compound that adjusts the acidity or alkalinity of a solution to stabilize sensitive chemicals that are prone to degradation in certain pH environments.
  • the pH modifier acts to modify the solution in the sample vessel when the composition is introduced into the sample vessel.
  • Suitable pH modifiers may be selected from the list consisting of citric acid, formic acid, acetic acid, sodium hydroxide, hydrochloric acid, TRIS (tris(hydroxymethyl)aminomethane) and similar buffers.
  • the composition may further comprise one or more scavengers.
  • scavenger should be understood to include any chemical compound that reacts with and/or neutralizes reaction species within the composition, such as oxygen, peroxides or metal ions that could catalyse degradation reactions.
  • Suitable scavengers may be selected from the list consisting of ethylenediaminetetraacetic acid (EDTA), activated charcoal and sodium bisulfite.
  • the composition may further comprise one or more stabilising agents.
  • stabilising agent should be understood to include any chemical compound that aids in maintaining the integrity and functionality of the chemicals and materials within the composition over time by preventing degradation through various mechanisms.
  • Suitable stabilising agents may be selected from the list consisting of polyvinylpyrrolidone (PVP) and glycerol.
  • the composition may further comprise one or more preservatives.
  • preservatives should be understood to include any chemical compound that prevents microbial growth (e.g., bacteria growth, fungi growth and other microorganism growth) that could potentially lead to degradation of materials within the composition.
  • microbial growth e.g., bacteria growth, fungi growth and other microorganism growth
  • preservative also encompasses antimicrobial agents.
  • Suitable preservatives/antimicrobial agents may be selected from the list consisting of sodium benzoate, parabens (e.g., methylparaben and propylparaben), sorbic acid, silver ions, triclosan and chlorhexidine.
  • parabens e.g., methylparaben and propylparaben
  • sorbic acid e.g., silver ions, triclosan and chlorhexidine.
  • composition may further comprise one or more humectants.
  • humectant should be understood to include any chemical compound that retains moisture to prevent drying out and degradation of products.
  • Suitable humectants that may be included may be selected from the list consisting of glycerin, 2,5-hexanediol.
  • the composition may be a dry pellet and may also be shaped so as to enable the dry pellet to penetrate the films on the application of pressure.
  • the end of the dry pellet that is towards the lower penetrable film may be shaped to a tapered point, such that when the sampler, or the rod member of the lid, is passed through the aperture of the end cap this places pressure on the top of the dry pellet and, with the bottom end of the pellet being tapered, this pierces the lower film causing the pellet to enter the interior of the sample vessel.
  • kit-of-parts comprising a sample vessel, a sampler configured to sample a defined volume of sample fluid and an end cap as defined above, wherein the sample vessel and end cap are configured to be couplable together.
  • the kit-of-parts may comprise a plurality of sample vessels, samplers and end caps. By the term plurality we envisage at least 10, such as at least 20, 30, 40, 50 or 100 of each component of the kit-of parts.
  • the kit-of-parts may further comprise a lid comprising a rod member, for example a lid comprising a rod member as defined above.
  • the sample vessel may comprise an analysis/extraction solution, which may be an analysis/extraction solution as defined above.
  • the kit-of-parts may further comprise a lancet.
  • a lancet we refer to a skin-puncturing device which comprises needles or narrow, sharp blades that poke a small hole in the skin obtain capillary blood.
  • the lancet is used to extract blood of the subject through the skin for the sampler to draw up.
  • the sampler included in the kit-of-parts may be a sampler as defined above.
  • sample vessel for fluid sample collection coupled to an end-cap as defined above, which sample vessel is referred to hereinafter as "the sample vessel of the invention".
  • the sample vessel for fluid sample collection may be for future analysis of the sample, with the sample vessel comprising: a reservoir; a composition comprising a substance suitable for use as an Internal Standard in said analysis; wherein the composition is sealed within an atmosphere that protects the substance suitable for use as an Internal Standard from degradation; and wherein the sample vessel comprises means for removing the composition from the protective atmosphere and introducing it to the reservoir concurrently with a provision of a sample to the reservoir.
  • the sample vessel comprises means for removing the composition from the protective atmosphere and introducing it to the reservoir prior to the provision of the sample to the reservoir.
  • the sample vessel of the invention may comprise any of the features as defined above in relation to the apparatus of the invention.
  • the sample may be provided to the reservoir by using a sampler, or the sample may be provided to the reservoir by using the lid comprising a rod member, as defined above.
  • the sample vessel may be a sample vessel as defined above.
  • the sample and the composition are introduced into the sample vessel at substantially the same time.
  • the composition may be introduced into the reservoir within one minute of the sample, such as within 30, 20 or 10 seconds, for example within 9, 8, 7, 6, 5, 4, 3, 2, or 1 second of the sample.
  • the composition may be introduced into the sample vessel prior to the introduction of the sample into the sample vessel, as defined above.
  • the composition may be introduced into the reservoir and the sampler containing the sample may be introduced within 120 seconds of the composition being introduced into the reservoir, such as within 110, 100, 90, 80, 70, 60, 50, 40, 30, 20, 15, or 10 seconds before the sample.
  • the sample vessel of the invention may be comprised in a kit-of-parts, which may contain any of the further features detailed above in respect of the kit-of-parts containing the end cap.
  • the kit-of-parts may comprise a plurality of sample vessels, such as at least 10, for example at least 20, 30, 40, 50 or 100 sample vessels.
  • the apparatus of the invention and the sample vessel of the invention may both be used for sampling a fluid, e.g. bodily fluid, such as blood, urine, or saliva.
  • a fluid e.g. bodily fluid, such as blood, urine, or saliva.
  • Such a use can be to sample a target compound in the fluid for future quantification and qualification.
  • a target compound may be a biomarker, a pharmaceutically active compound, endogenous or exogenous toxins, illegal drugs, veterinary drugs or other chemicals that are valuable to measure in bodily fluids, preferably in blood.
  • chemicals, compounds and biomarkers that may be present in one or several body fluid sample(s) may be referred to as "target chemicals/compounds" or "analytes”.
  • the apparatus/sample vessel is envisaged to be supplied to end users with the compound for analysis having been predetermined and, therefore, the Internal Standard included in the composition may have been preselected as a suitable Internal Standard for future analysis, quantification and/or qualification of the target compound.
  • the composition comprising the Internal Standard being stored in an atmosphere that protects it from degradation, at the time of supplying the apparatus to the user the amount of non-degraded Internal Standard will be known.
  • the composition comprising the Internal Standard and target compound will degrade at the same rate, meaning that on future analysis the concentration of the target compound in the fluid at the time of sampling can be reliably determined based on the relative concentration of the Internal Standard on the basis that the Internal Standard and target compound degrade at the same rate.
  • samples may be taken by the user (e.g., patient) themselves rather than needing a medical professional. Therefore, the patient may sample the bodily fluid at home rather than in a hospital setting.
  • Another advantage of the present invention is that samples do not need to be analysed immediately on being taken to reliably calculate concentrations of target compounds in the fluid sample, which means that delays in laboratory analysis will not impact the reliability of the analysis. This means that the technology is suitable for regular mail transport to the laboratory without this impacting the reliability of the analysis due to variations in time from sampling, through to delivery and then to analysis.
  • kit-of-parts comprising multiple apparatuses allowing for the sampling of bodily fluids over a period of time. Therefore, such apparatuses can be useful in, for example, precision medicine settings by sampling blood serum levels of an active pharmaceutical ingredient over time after taking a drug composition and, following analysis of target compound levels in the sample, the drug dose may be tailored to that particular patient. Following from this, such kits may also find use in clinical trials where rather than keeping trial candidates in hospital settings for analysing blood serum levels they can take samples themselves and send the samples on for further analysis in a laboratory setting.
  • the apparatus may, therefore, be for use in sampling blood serum levels of pharmaceutically active compounds that have been administered to a subject.
  • Suitable target compounds that may been sampled by the present invention may be compounds that have a narrow therapeutic index, such as those selected from the list consisting of Abemaciclib, Acalabrutinib, Acenocoumarol, Alatrofloxacin, Aldesleukin, Alectinib, Alemtuzumab, Alpelisib, Altretamine, Amikacin, Amineptine, Aminoglutethimide, Aminophylline, Amiodarone, Amitriptyline, Amitriptylinoxide, Amoxapine, Amphotericin B, Amsacrine, Anagrelide, Arbekacin, Argatroban, Arsenic trioxide, Asparaginase Erwinia chrysanthemi, Asparaginase Escherichia coli, Astemizole, Atezolizumab, Avelumab, Ax
  • the substance suitable for use as an Internal Standard may be a derivative of the target compound that has been predetermined for analysis, which Internal Standard is isotopically substituted (e.g., labelled).
  • the substitution e.g., labelling
  • the Internal Standard is substituted (e.g., labelled) with a stable isotope.
  • the substance suitable for use as an Internal Standard may be a compound that is structurally different to the target compound, but is known to degrade at the same rate as the target compound.
  • Such Internal Standards may be isotope substituted compounds as defined above.
  • the isotope with which the compound is isotopically substituted may be selected from the list consisting of deuterium ( 2 H, d), carbon-13 ( 13 C), nitrogen-15 ( 15 N), oxygen-18 ( 18 O) and mixtures thereof.
  • the compound suitable for use as an Internal Standard may be substituted either at a single location or multiple locations.
  • a preferred substance for use as an Internal Standard is a derivative of the target compound being analysed, comprised of isotopically substituted variants of the compound being analysed.
  • Stable isotopes such as deuterium ( 2 H), carbon-13 ( 13 C), or nitrogen- 15 ( 15 N)
  • a suitable Internal Standard can be a structurally similar compound to that being analysed, but that does not naturally occur in the samples.
  • using stereoisomers like doxorubicin as an Internal Standard for measuring epirubicin ensures identical chemical stability, despite differing chromatographic properties.
  • the Internal Standard degrades at the same rate as the target compound that is being analysed.
  • the composition may comprise multiple substances suitable for use as Internal Standard, such that the apparatus is able to be used to sample multiple target compounds for onward analysis, quantification and/or qualification.
  • the present invention may also be used to quantify bodily fluid levels (e.g., blood levels) of antifungal drugs.
  • Such antifungal drugs may be selected from the list presented in the table 1. below, with this table showing suitable Internal Standards that may be incorporated into the composition for analysing the drugs. Table 1. Examples of antifungal drugs and used Internal Standards.
  • the present invention may also be used to quantify bodily fluid levels (e.g., blood levels) of antiviral drugs.
  • antiviral drugs may be selected from the list presented in Table 2. below, with this table showing suitable Internal Standards that may be incorporated into the composition for analysing the drugs.
  • the present invention may also be used to quantify bodily fluid levels (e.g., blood levels) of anticonvulsants.
  • anticonvulsants may be selected from the list presented in the Table 3. below, with this table showing suitable Internal Standards that may be incorporated into the composition for analysing the anticonvulsant.
  • the present invention may also be used to quantify bodily fluid levels (e.g., blood levels) of antidepressants.
  • antidepressants may be selected from the list presented in Table 4. below, with this table showing suitable Internal Standards that may be incorporated into the composition for analysing the antidepressant.
  • the present invention may also be used to quantify bodily fluid levels (e.g., blood levels) of anticancer drugs.
  • anticancer drugs may be selected from the list presented in Table 5. below, with this table showing suitable Internal Standards that may be incorporated into the composition for analysing the anticancer drug.
  • Common pharmaceuticals and drugs of abuse that may be analysed using the apparatus/vessel of the present invention may be selected from the list consisting of Alprazolam (Diazepam-D5), Amitriptyline (Mianserin-D3), Amphetamine (Amphetamine- D5), Benzoylecgonine (Amphetamine-D5), Bromazepam (Diazepam-D5), Buprenorphine (Methadone-D3), Chlordiazepoxide (Diazepam-D5), Chlorprothixene (Mianserin-D3), Citalopram (Mianserin-D3).
  • Clonazepam (Diazepam-D5), 7-Aminoclonazepam (Diazepam- 05), Clozapine (Mianserin-D3), Cocaine (Methadone-D3), Codeine (Methadone-D3), Diazepam (Diazepam-D5), Flunitrazepam (Diazepam-D5), 7-Aminoflunitrazepam (Diazepam-D5), Fluoxetine (Mianserin-D3), Ketamine (Amphetamine-D5), Ketobemidone (Amphetamine-D5), Lamotrigine (Mianserin-D3), Levomepromazine (Mianserin-D3), Lidocaine (Mianserin-D3), Lorazepam (Diazepam-D5), 6-MAM (Amphetamine-D5), MDA (Amphetamine-D5), MDEA (Amphetamine-D5), MDMA (Amphetamine-D
  • the apparatus/vessel of the present invention may also be used to sample bodily fluid levels of controlled substances such as narcotics and stimulants, such as controlled substances selected from the list consisting of heroin, marijuana, LSD, codeine, fentanyl, hydrocodone, hydrocodone combinations (e.g., with acetaminophen), hydromorphone, morphine, methadone, oxycodone, oxycodone combinations (e.g., with acetaminophen) tapentadol, amphetamine, methamphetamine and methylphenidate, cocaine, pentobarbital, secobarbital, buprenorphine, dronabinol, ketamine, tramadol, alprazolam, diazepam, clonazepam, lorazepam, midazolam, and combinations thereof.
  • controlled substances such as narcotics and stimulants, such as controlled substances selected from the list consisting of heroin, marijuana, LSD
  • the apparatus/vessel of the invention may also be for use in the diagnosis of diseases or conditions by, for example, measuring certain target compounds in bodily fluid samples indicative of those diseases.
  • the apparatus/vessel of the invention may be for use in diagnosing a disease or condition selected from the list consisting of addiction (for example drug addiction or alcoholism), cancer (such as breast cancer or prostate cancer), cardiovascular diseases, high blood pressure, viral infections, fungal infections, bacterial infections, epilepsy, depression, pain, vitamin deficiency, immune-deficiency and steroid deficiency.
  • addiction for example drug addiction or alcoholism
  • cancer such as breast cancer or prostate cancer
  • cardiovascular diseases high blood pressure
  • viral infections for example drug addiction or alcoholism
  • fungal infections bacterial infections
  • epilepsy bacterial infections
  • depression depression
  • pain vitamin deficiency
  • immune-deficiency immune-deficiency
  • steroid deficiency steroid deficiency
  • the sample vessel may contain an analysis solution, which may also be referred to as an extraction fluid or solution.
  • an analysis/extraction solution may comprise chemicals that increases the stability of the target molecules enabling cold chain free transports. Chemicals may also be added to pre-process the sample by enriching target molecules or to further lower the time of handling at the lab.
  • Known methods to preserve analytes in samples are precipitation with water miscible solvents such as acetone, 1-butanol, 2- butanol, 2-metyl-l-propanol, 2-metyl-2-propanol, dimethylformamide, dimethyl sulfoxide, 1,4-dioxane, dimethylpropyleneurea, sulfonate.
  • Bases such as ammonia, tris(hydroxymethyl)aminomethane, triethylamine and non-nucleophilic amines e.g., N,N-Diisopropylethylamine, l,8-Diazabicycloundec-7-ene, 1,5- Diazabicyclo(4.3.0)non-5-ene, 2,6-Di-tert-butylpyridine, and buffers e.g., the amines exemplified above being 10-90% protonated with e.g., hydrochloric acid, Ammonium acetate, Phosphate-buffered saline, tris EDTA buffer, Britton-Robinson buffer, 2,2'-[(2- amino-2-oxoethyl)azanediyl]diacetic acid, N-(2-Acetamido)-2-aminoethanesulfonic acid, HEPES, HEPBS and HEPP
  • the use of the apparatus or sample vessel according to the invention may follow a method comprising the steps of: a. providing a sampler comprising a fluid sample; and b. introducing the sampler into the interior of the sample vessel so as to introduce the sample to the interior of the sample vessel concurrently with the composition comprising a substance suitable for use as an Internal Standard.
  • a method of using the apparatus, sample vessel or kit-of-parts comprising the steps of: a. providing a sampler comprising a fluid sample, a sample vessel comprising a composition positioned within an aperture and, optionally, a lid comprising a rod member; and either b. introducing the sampler into the interior of the sample vessel so as to contact the fluid sample with the analysis/extraction solution and concurrently releasing the composition into the interior of the sample vessel, or, provided that when the lid comprising a rod member is present, c. introducing the rod member through the aperture so as to release the composition into the interior of the sample vessel; and d. introducing the sampler into the interior of the sample vessel so as to contact the fluid sample with the analysis/extraction solution.
  • the method may further comprise the step of coupling a lid to the end cap to seal the interior of the apparatus.
  • the rod member of the lid when used to introduce the composition into the interior of the sample vessel the rod member is removed from the aperture before the sampler is introduced.
  • a method for analysing, quantifying and/or qualifying the presence and/or concentration of a target compound in a fluid sample from a subject comprises the steps of: providing a kit-of-parts for sampling a fluid as defined above; distributing the kit-of-parts to a location of the subject; receiving a sample vessel comprising a fluid sample with the end cap coupled to the sample vessel; processing and analysing the sample to determine the concentration of the target compound and the Internal Standard; and calculating the concentration of the target compound in the fluid sample at the time of sampling.
  • the method of using the apparatus, sample vessel or kit-of-parts, and/or the method for analysing, quantifying and/or qualifying the presence and/or concentration of a target compound in a fluid sample from a subject may include a step of shaking the sample vessel for a period of from about 1 second to about 10 minutes, such as from about 10 seconds to about 1 minute, for example greater than 30 seconds, such as from about 30 seconds to about 2 minutes, such as from about 30 seconds to 60 seconds.
  • the shaking may be done manually or mechanically, such as through use of a homogeniser.
  • the processing step may include a step of centrifuging the sample in order to remove proteins and/or polar chemicals in the sample.
  • the sample may be analysed by any means known in the art that would be suitable to calculate the concentration of the target compound in the sample.
  • the sample may be analysed using liquid chromatography single or tandem mass spectrometry such as (LC-MS/MS), mass spectrometry (MS/MS), or gas chromatography (GC-MS/MS).
  • the apparatus may be produced according to regulatory guidelines.
  • the apparatus can be sold directly to the customer online or in pharmacies or given to the end customer at a healthcare facility with instructions on how to use it.
  • the customer/patient may be provided with the apparatus in a box containing instructions for the sampling procedure, including the following items which may be included in the kit-of-parts as defined herein and used according to the following method:
  • a lancet to puncture the skin e.g., fingertip;
  • a capillary to collect a specified exact amount of blood preferably 20-50 uL;
  • the device may be marked with a specific and unique barcode and a digital or analogue solution to mark the date and time of sampling;
  • the results will be returned to the customer registered for that unique barcode (the patient or a health care institution).
  • the apparatus of the invention may be identified with a unique marking, e.g., barcode, which identifies the sample for onward analysis.
  • a unique marking e.g., barcode
  • Figure 1 shows a side cross-sectional view of an end cap according to the invention.
  • Figure 2 shows an aerial view of the end cap of figure 1.
  • Figure 3 shows a side, non-cross-sectional view of the end cap of figures 1 and 2.
  • Figure 4A to 4C shows a series of depictions of an embodiment of how the device according to the invention is used, with figure 4A showing the sampler and the sample vessel separate to one another, figure 4B showing the sampler extending through the aperture of the end cap, and figure 4C showing the sample holder coupled to the end cap.
  • Figure 5A to 5E shows a series of depictions of another embodiment of how the device according to the invention is used, with figures 5A to 5C showing how a syringe and plunger sampler is used with a sample vessel coupled to an end cap according to the invention, and figures 5D and 5E showing how the end cap can be coupled to a lid after sample introduction.
  • Figure 6A to 6H shows a series of depictions of another embodiment of how the device according to the invention is used, with figure 6A showing the sample vessel, figure 6B showing the lid comprising the rod member and sample vessel separate to one another, figure 6C showing the lid comprising the rod member extending through the aperture of the end cap, figure 6D showing the lid comprising the rod member and sample vessel separate to one another and the composition in the interior of the sample vessel, figure 6E showing the sampler and sample vessel separate to one another, figure 6F showing the sampler extending through the aperture of the end cap, figure 6G showing the lid comprising the rod member and sample vessel separate to one another and the composition and fluid sample in the interior of the sample vessel and figure 6H showing how the end cap can be coupled to the lid comprising a rod member after sample introduction.
  • Figure 7 shows the shows the dose-response curves (0-1000 nM epirubicin) obtained by the method using the apparatus according to the invention and a Traditional method at different time points (direct to 7 days).
  • Figure 8 shows the low impact of haematocrit concentration on the signal of epirubicin when analysed with the method utilising the apparatus according to the invention.
  • Figure 1 shows a side profile cross-sectional view of an end cap (100) according to the present invention.
  • the end cap (100) comprises an aperture (102) in which a dry pellet (104) is positioned.
  • the dry pellet contains a substance suitable for use as an Internal Standard.
  • the dry pellet (104) is sealed on both sides of the aperture (102) by penetrable films (106).
  • the end cap (100) has two sets of threads, one at the lower portion (108) for coupling to a sample vessel and another set at the upper portion (110) for coupling to a lid or sampler handle.
  • Figure 2 shows an upper profile view of the end cap (100) of Figure 1 with the center showing the aperture (102).
  • the outer section (112) has a series of ridges for ease of user handling.
  • Figure 3 shows a side profile non-cross-sectional view of the end cap of figure 1. Here the upper threading portion (110) can be more clearly seen.
  • FIGS 4A, 4B and 4C shows an embodiment of the invention.
  • the fluid sample may be provided by any means, such as by use of a lancet for drawing blood via a finger prick.
  • a lancet for drawing blood via a finger prick.
  • the user grasps the sampler (200) by the handle portion (202) and dips the exposed end of the sampler (204), in this case a capillary tube, into the blood sample. On doing so the capillary draws up a defined amount of blood sample.
  • the user takes a sample vessel (300), which has been coupled to the end cap (100) and advances the capillary tube (204) through the aperture thus penetrating the upper film and pushing the dry pellet (104) through the lower film and exposing the dry pellet to the interior of the sample vessel (300).
  • the sample vessel (300) contains an analysis/extraction solution (302), which is shown in Figure 4B, and on penetrating the lower film (106) the dry pellet (104) drops into the analysis/extraction solution (302) and releases the substance suitable for use as an Internal Standard into the solution.
  • the user advances the capillary tube (204) fully into the sample vessel (300) and contacts the end of the capillary tube with the analysis/extraction solution (302).
  • the blood sample is contacted with the analysis/extraction solution concurrently with the dry pellet (104).
  • the handle portion (202) of the sampler (200) is then screw fit onto the top of the end cap for storage.
  • FIGS 5A, 5B, 5C, 5D and 5E show an alternative embodiment of the invention.
  • the user when in use the user provides a fluid sample for analysis.
  • the fluid sample may be provided by any means, such as by use of a lancet for drawing blood via a finger prick.
  • the user grasps the sampler (400), which in this embodiment is in the form of a syringe (402) with a plunger (404), and draws up the blood sample into the syringe (402) by withdrawing the plunger (404).
  • the syringe may comprise volume markings so that the user can draw up a determined amount of fluid (e.g., blood).
  • the user takes a sample vessel (300), which has been coupled to the end cap (100) and advances the end of the syringe (402) through the aperture (102) thus penetrating the upper film and pushing the dry pellet (104) through the lower film and exposing the dry pellet to the interior of the sample vessel (300).
  • the sample vessel (300) contains an analysis/extraction solution (302), which is shown in Figure 4B, and on penetrating the lower film (106) the dry pellet (104) drops into the analysis/extraction solution (302) and releases the substance suitable for use as an Internal Standard into the solution. Simultaneously the user presses the plunger (404) on the syringe (402) to expel the fluid sample into the interior of the sample vessel (300).
  • the user may withdraw the syringe out of the end cap aperture (102) and fit a lid (500) onto the top of the end cap (100) to close the apparatus.
  • FIGS 6A, 6B, 6C, 6D, 6E, 6F, 6G and 6H show an alternative embodiment of the invention.
  • the user When in use, the user takes a sample vessel (300) which has been coupled to the end cap (100).
  • the user grasps the lid (600) comprising a rod member (602) and advances the end of the rod member (602) through the aperture (102) thus penetrating the upper film and pushing the dry pellet (104) through the lower film and exposing the dry pellet to the interior of the sample vessel (300).
  • the sample vessel (300) contains an analysis/extraction solution (302), which is shown in Figure 6C, and on penetrating the lower film (106) the dry pellet (104) drops into the analysis/extraction solution (302) and releases the substance suitable for use as an Internal Standard into the solution.
  • the user withdraws the lid (600) comprising the rod member (602).
  • the user provides a fluid sample for analysis.
  • the fluid sample may be provided by any means, such as by use of a lancet for drawing blood via a finger prick.
  • the user grasps the sampler (400), which in this embodiment is in the form of a syringe (402) with a plunger (404), and draws up the blood sample into the syringe (402) by withdrawing the plunger (404).
  • the syringe may comprise volume markings so that the user can draw up a determined amount of fluid (e.g., blood).
  • the user advances the end of the syringe (402) through the aperture (102) and presses the plunger (404) on the syringe (402) to expel the fluid sample into the interior of the sample vessel (300).
  • the user may withdraw the syringe out of the end cap aperture (102) and fit the lid (600) comprising the rod member onto the top of the end cap (100) to close the apparatus.
  • the apparatus may then be shaken to achieve mixing of the sample, suitable substance and analysis/extraction solution. In this way, a homogeneous protein precipitation and quantitative release of the internal standard from the composition may be achieved.
  • analysis of the target compounds can be performed by any means known in the art, such as liquid chromatography mass/mass spectrometry (LC-MS/MS).
  • LC-MS/MS liquid chromatography mass/mass spectrometry
  • the apparatus of the present invention provides a number of advantages, including:
  • Example 1 Use of a Sample Apparatus for Determining the Concentration of Epirubicin using Doxorubicin as an Internal Standard
  • the method using the apparatus according to the invention quantifies drugs, i.e., epirubicin in biological samples, contrasting its performance with a typical "Traditional" analytical method.
  • the apparatus according to the invention facilitates convenient, timely sample collection, which can be mailed to a laboratory for precise analysis using liquid chromatography-tandem mass spectrometry (LC-MS/MS).
  • Epirubicin-HCI (Cat 992, Batch 3559), doxorubicin-HCI (Cat 990, Batch 3285) and daunorubicin-HCI (Code D0125000, Batch 5.0) were obtained from British Pharmacopoeia Reference Chemical Standards. Acetone (99.5 %), acetonitrile (HPLC grade), methanol (>99.9%,), formic acid (95%), methylene blue (M4159-25G), 2-propanol (99.9%) and zinc sulphate heptahydrate (Essential-i- grade), were all obtained from Sigma-Aldrich. Lutrol micro 127 came from BASF.
  • Non-woven polypropylene (PP) (Milisten, 172 x 150 x 0.2 cm; 800 g, part no. 75925CNDVBCYJ04IRK2PZQW) and was obtained from amazon.se. Sealing of the caps was done by placing a heat-sealing foil between the bottom cap and a heat-sealing machine (Vevor , Wt-90DS230421320820010) for 3s at 170 °C. Stainless-steel staples (24/6, Rapid) were obtained from 123ink.se.
  • PP Non-woven polypropylene
  • venous blood samples (EDTA, 3 x 10 mL) from subjects.
  • Stock solutions were prepared from epirubicin, doxorubicin, and daunorubicin at (2000 pM) in methanol. Stocks were diluted to 100 pM for calibration series preparation.
  • Calibration samples were prepared by spiking fresh blood with varying concentrations of epirubicin (0-1000 nM).
  • the device/apparatus used in this study equates to the device depicted in Figures 6a to 6h.
  • this device and the method of using it, as the "apparatus according to the invention” or the “method using an apparatus according to the invention”, or the like.
  • the PP matrix (8.0 mm id.) was inserted into the device cap and sealed with pierceable alumina heat sealing foil (170 C, 3 S). Insert the PP-matrix to the bottom sealed hole, add internal standard (10 uL) consisting of: Doxorubicin, daunorubicin (400 nM, respectively), and agents that encapsules the internal standard (IS) on the matrix, e.g., polyethylene glycols (PEG, Lutrol Micro 127), being non-reactive and soluble in the solvent for protein precipitation. Dyes and antioxidants such as methylene blue (1.0 mM) was added to the PP-matrix being inserted to the hole of a bottom-sealed cap.
  • internal standard 10 uL
  • Doxorubicin Doxorubicin, daunorubicin (400 nM, respectively
  • agents that encapsules the internal standard (IS) on the matrix e.g., polyethylene glycols (P
  • Stainless steel grinding balls (6 x 2 mm i.d.) and the precipitation solution (500 uL IPA: MeOH, 1: 1, 0,1% formic acid (FA)) were added to a 1.5 mL vial. If caps were not used directly, they were top sealed within Ih after the addition of the IS.
  • Doxorubicin (10 uL of 400 nM) is added to the precipitation solution (500 uL IPA: MeOH, 1: 1, 0,1% FA) in a 1.5 mL vial with a screw cap.
  • Blood or plasma (20 uL or 50 uL) is added by (same procedure):
  • a blood sampling capillary (Sarstedt POCT 20 uL or 50 uL)
  • Apparatus according to the invention After adding the sample 20 uL of blood (or plasma), the apparatus is closed and shaken for a minimum of 30 s. The grinding balls in the tube enhance the protein precipitation.
  • Protein precipitation experiments were conducted using six different solvent mixtures, as shown in Table 6. For each solvent, the precipitation volume was fixed at 500 pl. Blood samples incubated with epirubicin (111 nM) and control samples without epirubicin were prepared for each shaking technique for the respective protein precipitation solutions.
  • the shaking techniques employed included handshake for 30 seconds, vortex mixing for 10 seconds (Vortex 1x10 sec), vortex mixing for 60 seconds (Vortex 6x10 sec), shaking with a Thermomix at 1200 rpm for 20 minutes, and handshake for 30 seconds with 3 steel balls (2.8 mm diameter).
  • the precipitates were visually inspected, and the quality of the precipitation was rated based on the relative grade of fine grinding and homogeneity. Following precipitation, all samples were stored at room temperature for 3 days prior to analysis by LC-MS/MS.
  • Precipitation solutions 1. IPA: MeOH (1: !, 0.1% FA), 2. IPA;MeOH (1: 1, 0.2% FA), 3. IPA: MeOH (90: 10, 0.1% FA), 4. ACN : MeOH (90: 10, 0.1% FA), 5. ZnSO 4 :water (10:90, 0.1% FA), 6. ACN :acetone (8:2, 0.1% FA).
  • Mixing method H30s (handshake 30 seconds), VlOs (vortex 1x10 seconds), V60s (vortex 6x10 seconds), T20m (thermomix, 1200 rpm, 20 minutes), H30s+B handshake with 3 x balls.
  • a comparative validation study was conducted comparing the method using an apparatus according to the invention with a traditional method for analysis the traditional approach to measure Traditional method to validate the inventive method and apparatus.
  • Blood samples incubated with epirubicin were measured using the method utilising the apparatus according to the invention and the Traditional Method over a time span from immediately after preparation to; 18h, 3d, 7d up to 14d delay until sonication, centrifugation and analysis.
  • Figure 7 shows the dose-response curves (0-1000 nM epirubicin) obtained by the method using the apparatus according to the invention and Traditional methods at different time points (direct to 7d).
  • the Y-axis shows the ratio of signal intensity for epirubicin (Epi) to doxorubicion (Dox).
  • the X-axis shows the concentration of epirubicin (nM9 in incubated blood samples.
  • the method using the apparatus according to the invention also measures daunorubicin as a second internal standard. While the traditional method requires samples to be processed immediately, the inventive method and apparatus allows for processing samples stored for days up to two weeks before analysis. As shown in Table 7, the highest AUC of epirubicin was observed after 7 days with both methods, indicating a consistent performance over time.
  • the comparison in Figures 7 consists of eight plots, each representing the linear regression analysis of epirubicin concentration versus the signal ratio of epirubicin/doxorubicin at four different time points for both methods.
  • the linear regression equations and R 2 values are indicated in each subplot. The comparison was made at the highest concentration of epirubicin (1000 nM). However, the same pattern of higher variability was observed when analysing samples immediately (direct) and at 18 hours at lower concentrations of epirubicin.
  • the data compares the performance of two methods— Traditional (Trad) and inventive (Inv)— in measuring the concentration of epirubicin and doxorubicin in blood samples over different time intervals (Oh, 18h, 3d, 7d, and 14d).
  • the inventive method using the apparatus according to the invention measures also daunorubicin.
  • the "Method Ratio (Trad/Inv)" column represents the ratio of the Epirubicin/Doxorubicin ratio for the Traditional method to that of the method using the apparatus according to the invention at each time point. This ratio indicates how well the two methods correlate by comparing the AUC Epi/AUC Dox ratios.
  • Figure 8 shows the low impact of haematocrit concentration on the signal of epirubicin when analysed with the method using the apparatus according to the invention.
  • the data collected shows that the method using the device according to the invention performed comparably to the Traditional method.
  • the apparatus according to the invention designed for self-sampling and delayed analysis, was compared with the Traditional method, which requires samples to be processed immediately. That is, in the Traditional method traditional sample analysis is done in the laboratory with a minimum delay between the protein precipitation step (with the IS being added in this step) and the sample sonication done prior the centrifugation and separation of the clear eluate being analysed by LC-MS/MS. Results from both methods were evaluated over various time intervals (Oh, 18h, 3d, 7d, and 14d) to assess the impact of storage time on analytical measurements. Surprisingly, the inventive method provided consistent results, with better performance observed after 3, 7, and even 14 days of room temperature storage compared to samples processed directly and after 18 hours. The data shows that the highest yield of epirubicin was obtained on days 3 and 7 for both methods. This suggests that prolonged storage facilitates slow equilibrium processes, releasing epirubicin from pockets that are difficult to extract, thus levelling out variations in blood precipitation.
  • the highest AUC of epirubicin was observed after 7 days for both methods, indicating the stability and robustness of the inventive apparatus and method over time.
  • the introduction of stainless-steel balls improved the inventive method by grinding the blood samples during protein precipitation, although full precipitation was still possible in the absence of the steel balls. Additionally, including an antioxidant to preserve the internal standard proved effective.
  • the robust data from these studies allowed progress to the clinical validation phase, where early measurements are promising.
  • the inventive apparatus allows sampling several days before laboratory processing and analysis. Data indicated that extended storage times (3d, 7d, and 14d) improved dose-response relationships and reduced variability for all measured analytes compared to samples processed immediately by both methods. This unexpected positive effect suggests that delaying analysis may facilitate in a more homogeneous drug extraction process that improves the analytical outcome.
  • the True Dose method showed stable performance across different time points, with the ratio of Epirubicin/Doxorubicin remaining consistent.
  • the method ratio (Trad/Inventive) demonstrated high agreement, ranging from 84.6% to 102.3%, indicating minimal variability between methods.
  • haematocrit study indicated that varying haematocrit levels (7 to 18 g/dL) did not significantly affect the signal of epirubicin, confirming the reliability of the True Dose method in different haematocrit conditions.
  • the method using the apparatus according to the invention shows significant promise for reliable and consistent quantification of epirubicin in blood samples, even with delays in processing from the time that the sample is taken. Its stability over time supports its use for home-based self-sampling, enhancing patient compliance and comfort while maintaining the accuracy required for effective therapeutic monitoring. Future studies should explore the application of the inventive method and apparatus across different drugs and clinical scenarios to validate its utility in personalized medicine further.
  • the inventive method and apparatus provide a reliable alternative to the Traditional method for measuring drug concentrations in blood samples. Its ability to maintain accuracy and precision over extended storage times makes it highly suitable for home-based self-sampling, improving patient compliance and convenience.
  • the strong correlation between the inventive method and Traditional methods across various conditions and time points supports its potential for widespread clinical application, particularly in therapeutic drug monitoring and personalized medicine.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Hematology (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Heart & Thoracic Surgery (AREA)
  • Veterinary Medicine (AREA)
  • Physics & Mathematics (AREA)
  • Public Health (AREA)
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  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Medical Informatics (AREA)
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  • Chemical & Material Sciences (AREA)
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  • Chemical Kinetics & Catalysis (AREA)
  • Dermatology (AREA)
  • Sampling And Sample Adjustment (AREA)

Abstract

La présente invention concerne un appareil de collecte d'échantillon de fluide comprenant un capuchon d'extrémité comprenant une substance appropriée pour être utilisée en tant que norme interne, ladite composition étant scellée à l'intérieur du capuchon d'extrémité dans une atmosphère qui protège la substance destinée à être utilisée en tant que norme interne contre la dégradation et le capuchon d'extrémité étant conçu pour pouvoir être accouplé à un récipient d'échantillon et pour libérer de manière contrôlée la composition à l'intérieur du récipient d'échantillon. La présente invention concerne également un récipient d'échantillon pour la collecte d'échantillon de fluide, qui comprend une substance appropriée pour être utilisée en tant que norme interne qui est scellée dans une atmosphère qui protège la substance destinée à être utilisée en tant que norme interne contre la dégradation et le récipient d'échantillon comprenant des moyens pour éliminer la composition de l'atmosphère protectrice et l'introduire dans le récipient d'échantillon simultanément avec la fourniture d'un échantillon ou le récipient d'échantillon comprenant des moyens pour éliminer la composition de l'atmosphère protectrice et l'introduire dans le récipient d'échantillon avant la fourniture d'un échantillon. La présente invention concerne également un kit de pièces comprenant l'appareil de collecte d'échantillon de fluide et/ou le récipient d'échantillon, ainsi que des procédés et des utilisations de ceux-ci.
PCT/EP2024/073021 2023-08-15 2024-08-15 Dispositif Pending WO2025036981A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
GB2312444.9 2023-08-15
GB2312444.9A GB2633004B (en) 2023-08-15 2023-08-15 Sample collection apparatus
EP24179708 2024-06-03
EP24179708.3 2024-06-03

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WO2025036981A1 true WO2025036981A1 (fr) 2025-02-20

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Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090216213A1 (en) * 2005-12-09 2009-08-27 Dna Genotek Inc. Container system for releasably storing a substance
CN203958963U (zh) * 2014-07-04 2014-11-26 三诺生物传感股份有限公司 一种试剂盒组件
US20170087547A1 (en) * 2014-05-21 2017-03-30 Orion Diagnostica Oy Sampling and assay kit, sample holder and method
US20200047183A1 (en) * 2016-08-11 2020-02-13 Instrumentation Laboratory Company Reagent component dispensing caps for reagent containers used in automated clinical analyzers
WO2020156849A1 (fr) 2019-01-28 2020-08-06 Redhotdiagnostics Ab Méthode de mesure d'une concentration d'un biomarqueur/analyte dans le sang de mammifères
US20200256767A1 (en) * 2017-10-24 2020-08-13 Bioneer Corporation Bio sample collection device
US11592368B2 (en) * 2011-06-19 2023-02-28 DNA Genotek, Inc. Method for collecting and preserving a biological sample

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090216213A1 (en) * 2005-12-09 2009-08-27 Dna Genotek Inc. Container system for releasably storing a substance
US11592368B2 (en) * 2011-06-19 2023-02-28 DNA Genotek, Inc. Method for collecting and preserving a biological sample
US20170087547A1 (en) * 2014-05-21 2017-03-30 Orion Diagnostica Oy Sampling and assay kit, sample holder and method
CN203958963U (zh) * 2014-07-04 2014-11-26 三诺生物传感股份有限公司 一种试剂盒组件
US20200047183A1 (en) * 2016-08-11 2020-02-13 Instrumentation Laboratory Company Reagent component dispensing caps for reagent containers used in automated clinical analyzers
US20200256767A1 (en) * 2017-10-24 2020-08-13 Bioneer Corporation Bio sample collection device
WO2020156849A1 (fr) 2019-01-28 2020-08-06 Redhotdiagnostics Ab Méthode de mesure d'une concentration d'un biomarqueur/analyte dans le sang de mammifères

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