Patent Publication Number: US-9429564-B2

Title: System and method for eluting and testing substance from exhaled aerosol sample

Description:
FIELD OF THE INVENTION 
     The present invention relates to methods for eluting nonvolatile compounds from a solid matrix. In particular, the invention is a method for eluting and optionally analyzing nonvolatile compounds collected on a membrane from exhaled air. 
     BACKGROUND OF THE INVENTION 
     Exhaled breath is commonly used in alcohol testing and today&#39;s technology makes it possible to perform on-site breath testing with legally defensible results using infrared spectroscopy. 
     Testing DNA and testing for drugs, whether abused or used therapeutically, generally requires the taking of blood or urine samples for testing. Hair, sweat, and oral fluid can also used for drug and DNA testing. Blood sampling is invasive and requires medically trained personnel, and the test subject is often transported to a hospital or other specialized facility for sampling. These procedures are time and effort consuming. The collection of urine samples may also be considered intruding on personal integrity. Other problematic issues related to samples and specimens taken from a subject include risk of disease transmission or infection and the risk of the subject exchanging samples or using a clean sample from another subject to avoid detection of illicit drugs. 
     The need to provide a non-invasive, not-specimen based apparatus, system and/or method for detecting the presence (i.e. qualitative) or determining the quantitative amount of a substance in a subject is at least partially met by the invention disclosed in PCT Application No. PCT/EP2012054180, the contents of which are incorporated herein by reference for all purposes. PCT/EP2012054180 discloses an apparatus, system and method for sampling the exhaled breath of a subject for a drug substance and/or a biomarker. Aerosols from the lungs of the subject are collected on a sampling membrane and then analyzed for the presence and optionally the amount of one or more nonvolatile compounds. 
     In DE19718924, breath is in a specific exhalation device conveyed through a fiber fleece. Large aerosol particles are caught in the fiber fleece by condensation. The device of DE19718924 comprises several valves which increase the resistance to blow through the device. The fiber fleece is removed from the housing, folded into the upper part of a centrifugal tube. The fiber fleece can be contaminated by this procedure. A standard elution solution, such as ELISA buffer or salt solutions, is added to the tube. A total amount of lung substances from the caught aerosol is determined from the eluate obtained. The determined total amount of lung substances allows for diagnosis and therapy of diseases, such as asthma. However, the disclosure of DE19718924 is not very detailed. The total amount of lung substances obtained as a result is not very specific and allows only coarse diagnostics and doubtful therapy based on such diagnostics. Sampling times in a range of 5-15 minutes are rather long and consistency of results over the range is not demonstrated. The method and device of DE19718924 do not appear to be very efficient and have a limited applicational range. Despite DE19718924 being published in 1998, no major improvement has been disclosed during 15 years. Hence a long felt need exists for improvement. 
     The present invention provides a method for eluting nonvolatile compounds from a sampling membrane in such a way that the eluted compounds are easily assayed. The present method improves the yield and consistency of recovery of nonvolatile compounds from sampling membranes used to collect aerosols from exhaled breath. 
     SUMMARY OF THE INVENTION 
     Accordingly, embodiments of the present invention preferably seek to mitigate, alleviate or eliminate one or more deficiencies, disadvantages or issues in the art, such as the above-identified, singly or in any combination by providing a method, according to the appended patent claims. 
     According to one aspect of the invention, a method for recovery is provided for collecting nonvolatile compounds from a sample taken from the exhaled breath of a subject for further analysis. The method may comprise removing a sampling membrane from a housing of a breath sampling device. Alternatively, the sampling membrane may be in place in a housing of a breath sampling device. Nonvolatile compounds may be eluted by passing a polar solvent through an inlet and an outlet in an adapted housing. By leaving the sampling membrane in place and eluting through the housing lowers the risk of contamination of the membrane. It also provides a more convenient and fast way of handling a collected breath sample. 
     A sampling membrane comprising collected from exhaled breath and aerosols containing nonvolatile compounds of interest is eluted by passing a polar organic solvent through the membrane using gravity alone or under pressure or centrifugal force. 
     In some examples of the invention, the sampling membrane is a filter membrane, preferably an electrostatic filter membrane, and most preferably an electrostatic membrane comprising nonwoven acrylic and polyethylene fibers. 
     The sampling membrane may be a filter membrane comprising at least one layer of non-woven filtration media with a specific weight in the range of 23 g/m3 to 500 g/m3, preferably in the range of 150 up to 300 g/m3, and even more preferably in the range of 200 up to 280 g/m3 and may comprise at least one further layer that is a spunbonded carrier with a scrim weight of 10 to 20 g/m3. 
     The polar solvents suitable for use in the method are miscible in water, are capable of dissolving water soluble and lipid soluble components of the exhaled aerosol, and are capable of eluting the nonvolatile compounds of interest. Polar solvents meeting these criteria include methanol, acetonitrile, isopropanol, and acetone. 
     The nonvolatile compounds of interest may originate from blood by a mechanism of producing a gas phase in the alveoli or from other parts of the airways and are transferred from the lungs carried by an aerosol. The nonvolatile compounds may be medical drugs, drugs of abuse, metabolites of drugs, markers indicative of drug use, or naturally occurring markers of disease states or metabolic conditions of subjects. Analysis of nonvolatile compounds from sample membranes may include mass-spectroscopy (MS) and Surface Enhanced Raman Spectroscopy (SERS) and other sensitive analytical methods known in the art. 
     In some examples of the invention, the aerosol on the sampling membrane may contain nonvolatile compounds of at least one drug substance in the exhaled breath. Nonlimiting examples of drug substances include amphetamines, ecstasy, Cannabis, THC, cannabinoids, opiates, heroin, morphine, 6-AM, cocaine, benzodiazepines, Propoxyphene, Methadone, Buprenorphine, Tramadol, LSD, Designer/Internet drugs, Kathinon, GHB, Meprobamat, Z-drugs, tryptamines, and anabolic steroids. 
     MS and SERS are the preferred analyzing methods with liquid chromatography/MS (LC/MS) being preferred over gas chromatography/MS (GC/MS). 
     Further examples of the invention are defined in the dependent claims, wherein features for the second and subsequent aspects of the invention are as for the first aspect mutatis mutandis. 
     Some known nonvolatile biomarker that may be transported by aerosols in exhaled breath is comprised in the list comprising lipids, peptides, nucleotides, prostanoids, proteins, DNA or RNA. These biomarkers may be used for diagnosis of diseases or illnesses, such as cancer (such as lung cancer), asthma, inflammation, infection (such as tuberculosis) and/or oxidative stress or other medical conditions. 
     The term “comprises/comprising” when used in this specification is taken to specify the presence of stated features, integers, steps or components but does not preclude the presence or addition of one or more other features, integers, steps, components or groups thereof. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       These and other aspects, features and advantages of which examples of the invention are capable of will be apparent and elucidated from the following description of examples of the present invention, reference being made to the accompanying drawings, in which 
         FIG. 1  is a schematic illustration illustrating an example of a portable device configured to collect a sample from exhaled breath of a subject; 
         FIG. 2  is a flow-chart illustrating a method for using a portable device configured to collect a sample from exhaled breath of a subject; 
         FIG. 3  is a flow chart of method steps for a recovery method according to the invention; 
         FIG. 4  illustrates a system for detecting the presence or determining the quantitative amount of at least one drug compound in exhaled breath; and 
         FIG. 5  illustrates typical chromatograms for four different drugs. 
     
    
    
     DESCRIPTION OF EMBODIMENTS 
     Specific embodiments of the invention will now be described with reference to the accompanying drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art. The terminology used in the detailed description of the embodiments illustrated in the accompanying drawings is not intended to be limiting of the invention. In the drawings, like numbers refer to like elements. 
     In  FIG. 1  is a schematic illustration showing an example of a portable system configured to collect a sample from exhaled breath of a subject. Sampling system  20  is configured to collect a sample from exhaled breath of a subject  201 . The subject exhales through a mouthpiece and a tubular element  202  being in flow communication with a housing  204  via at least one inlet  203 . The mouthpiece and tubular element  202  may be of same size or type as conventionally used for alcohol-testing. Preferably, the mouthpiece and tubular element  202  are configured to mainly permit aerosols to pass through. 
     The housing  204  can be made of any suitably material or combinations thereof such as, metal, plastic, glass or ceramics and comprises a sampling membrane  205 , being a fibre membrane, for collecting aerosols comprising non-volatile drug substances from the exhaled breath. The exhaled breath exits the housing through at least one outlet  206 . The portable system may be sealed after being used and sent to a laboratory  207  to be analyzed. Analysis of the amount of illicit drugs may be performed using any suitable method with Surface Enhanced Raman Spectroscopy (SERS) and mass-spectroscopy (MS), for example Liquid chromatography MS (LCMS), being most preferred. 
     The sampling membrane can be placed anywhere in the housing  204 . Preferably the sampling membrane  205  is fastened to the inner walls of the housing  204  and stretched over the cross-section of the housing. The housing  204  is preferably constructed out of two or more parts, a first part with an inlet  203  and one part with at least one outlet  206 . 
     The mouthpiece and tubular element  202  are detachable and in fluid communication with the housing  204  via the inlet  203 . The tubular element  202  functions as a pre-filter for filtering out saliva and/or mucus and/or large particles and/or aggregates from the exhaled breath. Thus a cleaner sampling membrane, such as a filter membrane, to be analyzed is obtained, which may result in better and more accurate analysis. Another advantage of using a tubular element  202  is to prevent contamination between subjects and samples. 
     The exhaled breath will, after flowing through the tubular element  202 , travel into the housing  204  and be brought into contact with the sampling membrane  205 , preferably a filter membrane. 
     The drug substances, being non-volatile compounds conveyed by aerosols in the exhaled breath, are collected by the sampling membrane  205  as the exhaled breath is permeated through the sampling membrane  205 . 
     The sampling membrane  205 , being a filter membrane is not to be confused with an electronic or chemical sampling unit and/or trap. The sampling element  205  is a physical entity on which the drug substance is collected. Collection may, in different embodiments, be based on various principles, singly or in combination, comprising depositing, catching, fastening, and condensing of non-volatile constituents on the sampling element  205 . 
     Using a filter membrane allows for a low pressure drop through the portable sampling system, making it easy and comfortable to exhale through the device. This is important for subjects experiencing problems with their lungs. 
     In an example of a portable sampling system, the filter membrane is placed to block the path for the exhaled breath through the portable system. 
     There are many possibilities for fastening the filter membrane, for example, using a separate support structure to retain the filter, which may be an element that is either fastened to the inside walls of the housing  204  or a segment being slid onto a first part of the housing during the assembly before the second part of the housing is mounted. 
     Alternatively and/or additionally, the filter membrane may be fastened direct onto the inside walls of the housing  204 , either by glue or by heat and thereby melting a small part of the edge of the filter membrane. 
     The second part of the housing is either screwed or slid onto the first part of the housing. The second part comprises at least one outlet  206 . Alternatively and/or additionally, the housing  204  may comprise at least two outlets  206 . This will aid to avoid a subject being tested to block the outlet and thereby blowing up the volume measuring element with minimum exhaled breath being permeated through the filter membrane 
     The sampling element  205  is preferably a filter membrane made of synthetic and/or half-synthetic fibers for the exhaled breath to diffuse through. The filter membrane has a structure that catches the aerosols and thereby collects the drug substances being exhaled while letting gas pass through. The aerosol particles comprise the non-volatile drug compounds. Preferably, the filter membrane is operable to collect the aerosols from the air with a high volumetric capacity while maintaining a low pressure drop across the filter substrate. Alternatively and/or additionally, in some examples the filter membrane may be an electrostatic filter made of fibers. Alternatively and/or additionally, the filter membrane may be of a non-woven polymeric fibrous that may be an electret. 
     Alternatively and/or additionally, the filter membrane may be an electrostatic filter being preferably a non-woven filter membrane comprising a blend of acrylic fibers and polypropylene fibers. The filter membrane may have a density (sometimes called grade or weight) in the range 23 up to 500 g/m2. But it has been shown that the range 130 up to 300 g/m2 is preferred. Even more preferably is the range 200 up to 280 g/m2. 
     Alternatively and/or additionally, the non-woven layer may be attached to at least one further layer. Further layers can be used to enhance a filter membrane&#39;s physical properties or improve filtration performance. The extra layer can be a carrier, preferably a polypropylene spunbonded carrier. The spunbond carrier may have a scrim weight in the range 10 up to 20 g/m2, preferably 15 g/m2. 
     For example a three layered filter membrane comprising one non-woven layer with a density of 250 g/m2 and two layers being carriers each with a scrim weight of 15 g/m2 will have an air flow resistance of about 36 Pa at 9.5 m/min media velocity. Alternatively and/or additionally, the filter membrane may be corrugated to enhance the filtering area within a given housing volume. 
     The portable system combined with the method of eluting the content of the filter membrane is configured and adapted to have a sensitivity for illicit drugs high enough to obtain results of a standard to be used as proof in a court of law. 
     The collected aerosols may comprise nonvolatile compounds of at least one drug substance in the exhaled breath. The drug substance may, by way of example, be an amphetamine, ecstasy, Cannabis, THC, a cannabinoid, an opiate, heroin, morphine, 6-AM, cocaine, a benzodiazepine, Propoxyphene, Methadone, Buprenorphine, Tramadol, LSD, a designer/Internet drug, Kathinon, GHB, Meprobamat, a Z-drug, a tryptamine, or an anabolic steroid. Other illicit drugs (drugs of abuse) and therapeutic drugs not included in the list may also be detectable due to similar interchanges with the human body as the above mentioned illicit drug substances. 
     Alternatively and/or additionally, non-volatile compounds may include biomarkers transported by aerosols in exhaled breath. These biomarkers may comprise lipids, peptides, nucleotides, prostanoids, proteins, DNA or RNA and may be used for diagnosis of diseases or illnesses, such as cancer (such as lung cancer), asthma, inflammation, infection (such as tuberculosis) and/or oxidative stress or other medical conditions. These biomarkers may be collected and recovered using the system and methods described herein. 
       FIG. 2  is a flow-chart illustrating a method  50  for using a portable system configured for collecting a sample  502  from exhaled breath from a subject. Briefly,  FIG. 2  illustrates detecting the presence and/or determining the quantitative amount  503  of at least one illicit drug substance or compound in a collected sample. The illicit drug compounds are collected as aerosols, being non-volatile particles and/or compounds, from the exhaled breath. The method comprises: a subject exhaling  501  into the invented portable system; a sampling unit collects a sample  502  comprising drug substances or compounds; the collected sample is extracted from the sampling membrane and analyzed  503  using MS or SERS. 
     With reference to  FIG. 2 , a subject exhales  501  in and out; preferably the subject exhales into the portable system until a specific volume of exhaled breath has been passed through the sampling membrane, preferably a filter membrane. 
     Alternatively and/or additionally, the subject may exhales either for a certain time or for a fixed number of times, such as 1 to 10 times, into a portable system. When breathing a fixed number of times, each exhalation may be set to last for a fixed time. 
     To measure a specific volume, one preferred method is to use a port located on the mouthpiece or between the mouthpiece and the inlet of the housing. A portion of the exhaled breath is extracted  504  through the port and inflates an element, such as a non-elastic bag. Hence when the bag is full, the volume of said bag will be proportional to the volume passed through the sampling membrane. 
     Alternatively and/or additionally, a bag with elastic properties may be used. 
     A deep breath is preferred to obtain exhaled breath from deep lying lung portions, such as the central or peripheral lung regions. 
     The exhaled breath is then collected  502  by the sampling element, i.e. an easy to breathe through filter membrane, suitable for collecting drug substances before it exits the system. The filter membrane is preferably made of synthetics and/or half synthetic fibers. Preferably, the filter membrane has electrostatic-properties. Using a filter membrane creates a small, light-weight and easy to use method that can be used everywhere by anyone to detect if a subject is under influence of an illicit drug. The sampling method is of such high quality that the obtained results are of a court approved standard. These results are obtained using a method that is neither invasive, e.g. Blood sampling, nor intruding on personal integrity, e.g. urine sampling. Hence the known drawbacks with these methods are prevented. 
     After being used, the housing of the sampling system is sealed off by sealing the inlet and the outlet and the device may be sent to a laboratory, where the collected compounds in the filter are recovered an analyzed. 
       FIG. 3  illustrates a method  30  for recovering one or more nonvolatile compounds from a sampling filter membrane. The fibers are preferably nonwoven and filter is most preferably a felt or a matted material. The filter is preferably an electrostatic filter and most preferably comprises acrylic fibers and polypropylene fibers. The method comprises contacting  301  the filter with an amount of a polar solvent capable of neutralizing the electrostatic charge on the filter. The contacting of the filter with the polar solvent elutes the nonvolatile compounds from the sampling filter to form an eluate. Preferred polar solvents include methanol, acetonitrile, isopropanol, acetone, and combinations thereof, with methanol and acetonitrile being most preferred for their relative ease of handling. The polar solvents are miscible in water, are capable of dissolving water soluble and lipid soluble components of the aerosol, and thereby eluting the nonvolatile compounds of interest from the filter membrane. Polar solvents meeting these criteria include methanol, acetonitrile, isopropanol, and acetone. Methanol and acetonitrile are preferred polar solvents in combination with filter membranes comprising acrylic and polyethylene fibers. Preferably, the polar solvents are used in pure form, i.e. in no mixture with other compounds or solvents. Pure solvents are e.g. pure methanol, or pure acetonitrile. 
     The amount of solvent used depends on the size of the filter and should be enough to elute the nonvolatile compounds but should not unnecessarily dilute the compounds. By way of example, the amount of solvent may be between 0.02 ml and 20 ml. Contacting may be accomplished by washing the filter with the solvent, for example by passing an amount of the solvent through the filter. The filter  205  is preferably in the housing  204  of the sampling device with the solvent being passed though the device from one of the inlet  203  or outlet  206  to the other of the inlet  203  or outlet  206  of the device. Alternatively, the filter  205  may be removed from the device and placed in a column for elution with the solvent by gravity or with pressure. The filter may alternatively be soaked in the solvent before being placed in a centrifuge tube or column. 
     After the solvent is contacted with the filter membrane, the eluate is collected  302  in a suitable container. Collection may be accomplished, for example, by gravity flow from a filter in a device or, alternatively, saturating the filter with solvent and placing the device in a centrifuge with a collecting vessel positioned to receive the eluate during centrifugation. The eluate may also be collected by applying solvent to a sampling device and applying pressure to force the solvent through the filter and into a collecting vessel. Collection may also be accomplished by gravity, pressure, or centrifugation from a filter that has been removed from a sampling device and placed in a column or tube for elution by gravity, pressure, or centrifugation. 
     The collected eluate may be stored or optionally delivered  303  to an assay system capable of detecting (qualitative analysis) and/or measuring (quantitative analysis) one or more nonvolatile compounds in the eluate. Any suitable assay system may be used, with LC/MS, GC/MS, and SERS being most preferred. MS may include, for example, time-of-flight MS, quadrupole ion trap MS, tandem MS, electrospray ionization MS, and matrix-assisted laser desorption/ionization MS. Depending on the assay system being used, the eluate may be pretreated or concentrated before delivery to the assay system. For LC/MS, the eluate may be loaded into the liquid chromatography column, optionally after reducing the volume of the eluate by evaporation of the solvent with or without partial vacuum. For SERS, the eluate may be placed on a SERS substrate comprising, for example, gold or silver colloids, optionally after reducing the volume of the eluate. The eluate may alternatively be evaporated to dryness to form a dry residue that is resuspended in the same or a different solvent or combination of solvents before application to an assay system. 
       FIG. 4  illustrates a system  60  for detecting the presence or determining the quantitative amount of at least one drug compound in exhaled breath. The system comprising a portable drug testing device  602  for handheld collection of a sample from exhaled breathe from a subject  601 . The device  602  could be according to any embodiment described herein. Further, the system  60  comprises a sensor unit  603  for analyzing the collected compounds in the testing device  602 . The sensor could be any know type of sensor but preferably mass spectroscopy or SERS. To be able to analyze the collected sample the content of the filter membrane in the device  602  is extracted. This could be done for example by using a solvent to be analyzed using the sensor unit  603 . 
     An advantage is that the test may be performed almost anywhere at a low cost and short lead time before obtaining a result. 
     Example 1 
     Table 1 shows the results of recovering a variety of nonvolatile compounds from an electrostatic filter membrane comprising acrylic fibers and polyethylene fibers by elution with methanol. The eluates were analyzed using LC-MS/MS with reference solution. Recovery and assay for each nonvolatile compound analyte was performed in triplicate. Typical chromatograms for some drugs when analyzing the eluates using LC-MS/MS equipment are illustrated in  FIG. 5 . From the top to the bottom the chromatogram shows peaks for, amphetamine 70, 75; Morphine 80, 85; Diazepam, 90, 95; and THC, 100, 105. As there is a linear relation between concentration (pg/filter) and the area ratio of analyte to internal standard for all compounds the amount of drug in the filter may be determined based on calibration curves from reference solutions. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Recovery of nonvolatile compounds by methanol elution 
               
            
           
           
               
               
               
               
            
               
                 Analyte 
                 Recovery (%) 
                 Analyte 
                 Recovery (%) 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 Amphetamine 
                 101 
                 Methamphetamine 
                 107 
               
               
                 THC 
                 95 
                 Morphine 
                 98 
               
               
                 Codeine 
                 97 
                 MDMA 
                 93 
               
               
                 Benzoylecgonine 
                 95 
                 Cocaine 
                 95 
               
               
                 Buprenorphine 
                 116 
                 Methadone 
                 115 
               
               
                 Oxazepam 
                 106 
                 Flunitrazepam 
                 107 
               
               
                 6-Acetylmorphine 
                 97 
                 Diazepam 
                 105 
               
               
                   
               
            
           
         
       
     
     The present invention has been described above with reference to specific embodiments. However, other alternatively and/or additionally embodiments than the above described are equally possible within the scope of the invention. Different method steps than those described above, performing the method by hardware or software, may be provided within the scope of the invention. The different features and steps of the invention may be combined in other combinations than those described. The scope of the invention is only limited by the appended patent claims.