Patent Application: US-55624709-A

Abstract:
a rapid method of volatile analysis and interpretation is taught enabling inferences on the surrounding environment as sophisticated as commonly achieved by dogs via olfaction . the method is based on rapid analysis of vapors released by persons or other organisms into a gas , correction of said analysis due to competing ambient volatiles , extraction of abundance patterns of certain preselected metabolites present in said vapor analysis , and classification of said persons or organisms by comparison of said abundance patterns with preestablished standard metabolite patterns . a preferred approach for rapid analysis involves an atmospheric pressure ionization , such as an electrospray cloud , followed by a mass spectrometer with an atmospheric pressure source . a preferred method for background correction is subtraction of the background signal from the sample signal when both are ionized at similar humidity levels . a preferred comparison pattern involves the abundance of fatty acids and other common metabolites . preferred classification criteria include recognition of individuals , or species , or health state .

Description:
a first embodiment of the invention uses a sesi charger ( also referred to as an ionizer ) interfaced to an api - ms ( api mass spectrometer ) for breath analysis . this is shown schematically in fig1 , which improves upon the arrangement of u . s . patent application ser . no . 11 / 732 , 770 in two key respects : ( i ) the charger operates now in negative polarity to promote ionization of fatty acids and related vapors , and ( ii ) the background spectrum ( to be subtracted from the breath spectrum in order to provide a signal more directly representative of breath ) is obtained from ambient air after bringing it to near 100 % relative humidity . we have used a variety of api mass spectrometers for rapid analysis of breath , including sciex &# 39 ; s api 365 triple quadrupole , and sciex &# 39 ; s qstar time of flight mass spectrometer . the use of these and other api mass spectrometers with similar sesi sources also in negative mode for explosive detection yields comparable results martinez - lozano et al . ( 2009 ). many other api ms systems are commercially available and many others have been fabricated ad hoc by those familiar with the field . most of them can be readily coupled to a sesi ionizer for practicing the subject invention . the ionization source of fig1 includes an electrospray ( es ) capillary 1 facing the ms ( mass spectrometer ) sampling orifice 2 . neutral vapors enter the chamber through an inlet tube 3 and are forced to pass through a negative electrospray ( es ) plume 4 . a preferred electrospraying solution to ionize weak acids in negative mode uses 0 . 1 % nh 4 oh in 1 : 1 methanol / water ( v / v ), obtaining deprotonated vapors ( molecular weight minus 1 da ). a variety of other buffers can be used to produce negative ions . a sample flow rate in the range from 2 - 8 l / min with a preferred value of 6 l / min is driven in and out of the charging chamber with a source of negative pressure 14 , such as a suctioning pump , connected to a second outlet tube 5 . this flow is a mixture of a small flow ( typically 0 . 5 l / min ) of co 2 ( which helps avoiding electrical discharges with negative es , but is not strictly necessary ) entering through inlet 6 and 1 . 5 - 7 . 5 l / min of air entering through branch 7 , which is either humidified ambient laboratory air coming through branch 8 , or breath coming through branch 9 . some of the vapor molecules carried by the flow entering through line 3 are ionized by the charged drops ( or by solution ions released from the drops ) in spray 4 , are driven by the electric field against a stream of dry counterflow gas fed through line 10 and exiting into the electrospray chamber through orifice 11 , and sucked together with approximately 0 . 5 l / min of dry counterflow gas into the ms 12 through sampling orifice 2 . the electric field driving the ions through 11 against the drying gas may be simply created by the electrospray needle , but also with the help of auxiliary electrodes 13 . these ions are finally “ weighed ” in the ms , which determines a signal intensity and an ion mass for each ion or for a desired subgroup of selected ions . in one approach the breath sample is taken by sealing the sampling tube 9 with the lips and letting the suctioning pump 14 connected to the outlet tube 3 sample the gas from the lungs without opposing any resistance or forcing it in , so that the pressure is almost identical to atmospheric pressure , and the flow rates of sample and background are also almost the same ( as seen directly in flowmeter 15 ). in another approach , the sampling tube 9 is open to the atmosphere in the vicinity of the mouth , and breath is directed into the tube inlet during exhalation . care must be exerted when handling the sampling tube 9 to avoid contamination coming from the skin . although wearing gloves is a common approach to control this contamination , note that many plastic gloves are themselves a source of contaminating volatiles . a preferable approach is to fix the sampling tube 9 and avoiding contact with it , or at least restricting contact to portions of the sampling tube 9 sufficiently separated from its sampling end . preferably the breathing subject fasts overnight to minimize interferences coming from the mouth and obtain a repeatable breath pattern . samplings without this precaution are also useful to observe the interferences associated to eating . exhaled breath consists of vapors previously inhaled from the ambient , with some additions and subtractions made in the lung . accordingly , one must discriminate between endogenous vapors coming from the subject and exogenous vapors originally in the atmosphere . it is important to note that simple subtraction of the mass spectra obtained from either breath or ambient air is often inappropriate to correct for the background , because humidity from breath drastically affects the ionization efficiency ( hence the output signal ) of many vapors . fig2 represents the ratio of ion signals obtained for saturated fatty acid vapors charged in the setup of fig1 in either dry or humid air , as a function of the number of carbon atoms in the fatty acid molecule . note the almost tenfold increase in the charging probability of large chains in humid air . as a result , the large fatty acids produced by human skin and contained in the laboratory background are more highly charged when first humidified by passing them through the lung than when drawing them directly from the dry ambient . simple background subtraction therefore gives the incorrect appearance that breath contains large fatty acids . consequently , our blank is preferably based on humidified room air and is taken every time immediately before the breath sample . in this way , by simply substituting humid room air spectra from breath spectra , we preserve the detection probability of background contaminants as well as breath metabolites . in the present embodiment of the invention , ambient air entering through line 16 is humidified in chamber 17 prior to entering into the ionization chamber . humidification can be achieved in a number of well known ways . in one embodiment the humidification chamber 17 is a passive device containing distilled water at 37 ° c ., while ambient air passes through the headspace above the water pool . in another common embodiment , a well controlled flow of distilled water is injected with a syringe pump through port 18 into humidification chamber 17 . in this approach one must ensure that the humidification chamber 17 provides complete evaporation of the water fed through 18 , and effective mixing of the resulting water vapor with the gas flow coming through 16 . this has been previously achieved in work by zhan and fenn ( 2002 ) by spraying the liquid into very small drops which evaporate immediately into the heated gas . similar results are reported by sgro and de la mora ( 2004 ) by heating sufficiently the injected liquid and the gas . many other variants have been reported , including that used by martinez - lozano et al . ( 2009 ), who inject controlled quantities of explosive vapor from a solution into a gas , where the injected liquid solution was electrosprayed into particularly small drops , and the gas was also heated to promote complete evaporation . as already noted , the mass spectrometer uses a dry stream of curtain gas flow , entering through line 10 and meeting the electrospray plume frontally after passing through orifice 11 . this dry stream precludes penetration of neutral vapors into the analyzer , and handles the high humidity in these samples with no apparent interference of hydrated peaks . note that this technique is online and requires no sample preparation . the sampling tube 9 is continuously drawing room air at a fixed flow rate , and the subject needs only to exhale in it . selection between sampling from either the subject &# 39 ; s breath ( or dry ambient air ) through line 9 or sampling humidified ambient air through line 16 is accomplished by a two - way valve 19 . a background - corrected mass spectrum obtained by taking the difference between sample and humidified laboratory air is shown in fig3 . among the dominant peaks in the spectrum one sees a series spaced by 14 da , clearly associated to ch 2 addition in a hydrocarbon chain . the series starts with a weak peak at 73 da , corresponding to deprotonated propionic acid ( c3 ). its structure was confirmed by its exact mass combined with collision induced dissociation ( cid ) of the parent ion into several characteristic product peaks . the series continues uninterrupted with well - defined peaks up to deprotonated tetradecanoic acid ( myristic ; c14 ). larger fatty acid chains are contained in the background at concentrations larger than in breath as a result of contamination from skin vapors of persons in the laboratory . this competition makes it difficult to determine small breath concentrations of larger fatty acid by the approach described . however , larger fatty acids can still be quantified if the subject inhales clean bottled air instead of atmospheric air . when applicable , this alternative method of reducing the background is also useful , an is included within the invention . along with the fatty acids , the high resolution of the time of flight ( tof ) ms instrument used cleanly resolves another series of peaks slightly lighter , starting with pyruvic acid and ending at 185 da . this series most likely corresponds to ketomonocarboxylic acids , according to their exact mass ( besides the positive identification by cid of pyruvic and 4 - ketohexanoic acid ). note that the breath signal exceeds the background only from 129 da ( 4 - ketohexanoic acid ) to 185 da ( 4 - ketodecanoic acid ). the particular position of the keto group has not been confirmed by alternative procedures and could be different . interestingly , 2 - ketohexanoic acid is a known potent insulin secretagogue ( lenzen et al . 1982 ). benzoic acid ( 121 da ) is also present , as confirmed by cid . 3 - methylbutanal ( 85 da ) has been tentatively identified , and some other minor peaks may be also aldehydes according to their exact mass . for instance , butanal ( 71 da ); 3 - methylbut - 2 - enal ( 83 da ); 3 - hexenal ( 97 da ; related to r - linolenic acid metabolism ); 4 - methylpentanal ( 99 da ); and heptanal ( 113 da ). we also observe a series of peaks displaced 2 da to the left of the main saturated fatty acid series . on the basis of their exact mass , they correspond most probably to singly unsaturated fatty acids : c7 : 1 ( 127 da ), c8 : 1 ( 141 da ), c9 : 1 ( 155 da ), and c10 : 1 ( 169 da ). longer chains up to c18 : 1 are observed in the background at concentrations higher than in breath . the dominant background peak at 89 da corresponds to 2 - hydroxypropanoic acid ( lactic acid ), secreted in bulk quantities by the skin . however , we observe 2 - hydroxyhexanoic acid ( 131 da ), 2 - hydroxyheptanoic acid ( 145 da ), and 2 - hydroxyoctanoic acid ( 159 da ) clearly above the background level ( assigned only by their exact mass ). in conclusion , the method of analysis described yields a large number of peaks in breath , many of which are associated to known human metabolites , and can be differentiated from the background . unlike prior art , the approach therefore has all the outstanding characteristics of biological detectors . a preferred method for classification of persons would therefore extract from a spectrum such as that shown in fig1 a list of the n abundances of certain pre - established metabolite ions . the associated n - dimensional vector characteristic of that person can be compared with a reference vector to achieve a match or a mismatch . many techniques exist to perform the comparison between a pattern and one or many reference standards , some of which account through training for the variability of the fingerprint . this variability could in our case be due to environmental conditions such as cleanness , eating history , etc . there is similarly a variability in the mass spectra produced by electron fragmentation of given molecules , but this does not preclude their identification in existing algorithms ( i . e . the nist ms search software ), once the recognition software is suitably trained by being fed the range of mass spectra characteristic of this natural variability . cristoni et al . ( 2009 ) have illustrated the capabilities of this nist software in the recognition of cancerous vs . healthy mass spectra , showing a better performance than traditional multivariate analysis ( clustering and principal component analysis ). we have so far discussed the analysis of human breath . but the invention includes also probing volatiles from other organisms , with small variants that can be readily implemented by those familiar with the field of breath analysis . in one such variation applicable also to humans , a breathing mask 9 a may be used to connect the mouth of an animal to sampling line 9 for greater convenience . in this case the two - way valve 19 is removed so that the gas continuously ingested through inlet 3 comes automatically through the path of least resistance , namely , from the animal breath when it exhales , and otherwise from the background line 16 . when the animal inhales , a valve in the breathing mask opens widely such that the flow rate of gas entering through 16 , requiring efficient humidification , does not increase substantially above the flow sample through line 3 when the animal is not breathing . depending on the size of the animal , the volume sampled through inlet line 3 would have to be adjusted . the term breath should in some cases be understood broadly . for instance , metabolytes released by a colony of organisms , such as a cellular colony , may be evaluated . for example , when analyzing the metabolytes released by a colony of organisms residing ( for instance ) on a fermenting piece of cheese , the distinction between vapors released from either breath and skin ( or membrane ) is lost , but the method can still be used with small modifications . in this case , the colony may be enclosed in a container 9 b having an inlet 9 c and an outlet 9 d , while purified gas ( also humidified if appropriate for that organism ) would enter through this inlet 9 c , circulate through the interior of the container 9 b , and carry the vapors released by the colony to the outlet tube 9 d connecting to 9 and into the analyzer . naturally , for a small volume chamber ( relative to the volume of the human lung ), a flow rate substantially smaller than 6 l / min should be used . for other small animals having distinct breathing organs , it may be difficult or impractical to distinguish between vapors released from the lungs or from other organs , in which case , placing the animal in the container 9 b ( similarly to the case of the colony just discussed ) and sampling the vapors released from organs other than the lungs ( perhaps including also urine and faeces ) is equally appropriate and is included within the invention . metabolytes originating from a fish tank ( or a cell culture ) residing in water can similarly be sampled and analyzed by passing the sampling gas through the headspace above this water . in a second embodiment of the invention using the same instrument as in the prior embodiment , cutaneous vapors are analyzed . the sample is now drawn by locating the open end of line 9 in the vicinity of the human skin ( for instance the palm of the hand ). this embodiment can also be used with other organisms via slight variations , as just discussed for breath , whereby the term skin is also used broadly to include various organisms , for instance the cellular membrane or the leaf of a plant . in one embodiment the background air is drawn also from the atmosphere through the open end of line 9 , placing it at a safe distance from any individual . in this case , the level of humidity in the vicinity of the hand is not greatly affected by the hand , so , humidification of the background is not essential . however , humidification of both the sample and the background is a useful possibility that not only provides a better humidity control , but also enhances considerably the sensitivity towards long chain fatty acids ( fig2 ). to achieve this benefit , both the sample and the room background are drawn in sequence ( first one , then the other ) into the mass spectrometer through line 16 . for this measurement valve 19 would only admit gas coming through 8 . a background - corrected mass spectrum obtained for human skin in negative mode is shown in fig4 , with more details provided by martinez - lozano and fernandez de la mora 2009 . fig4 shows that such skin spectra are as well suited for classification or recognition purposes as that shown in fig3 for breath . some particularly notable features of human skin spectra are the presence of long chain fatty acids up to at least oleic acid ( c18 : 1 ), and the singular intensity of lactic acid , and to a lesser extent pyruvic acid ( notice the break in the scale introduced in fig4 to enable representing these high abundances ). the extraordinary signal obtained for lactic acid suggests its use for simple detection of the presence of humans in closed controlled areas , or for related search purposes . note also that pyruvic acid is known to be involved in type ii diabetes mellitus metabolism . the skin spectrum of fig4 includes the complete series of saturated fatty acids from c12 : 0 to c16 : 0 , as well as unsaturated fatty acids from c12 : 1 to c18 : 1 . other abundant metabolites are pyruvaldehyde , glyoxylic acid , 4 - hydroxybutanoic acid ; 3 - methyl - 2 - oxobutanoic acid ; 5 - hydroxypentanoic acid and 4 - methyl - 2oxopentanoic acid . fatty acids smaller than c12 surely originate also from the skin , but their presence is difficult to measure in the open atmosphere method described due to competition from more abundant light fatty acids originating from the breath of persons in the laboratory . this is the converse problem to the one previously described in relation to the detection of heavy fatty acids from breath . a variety of cleaning strategies can similarly be adopted here to avoid or reduce background contamination from breath and enable determination of lighter skin fatty acids . the most informative strategy involves obtaining separate mass spectra for both the skin and breath , since the corresponding metabolytes have different origins and therefore provide independent pieces of intelligence . breath provides a window into the composition of volatile species dissolved in the bloodstream , while many skin volatiles are also influenced by the activity of skin bacteria . several hundred distinct skin fatty acids exist in the human skin ( nicolaides 1974 ) offering an incredibly rich fingerprint . many of these species are isomers having exactly the same mass . but even isomers may often be distinguished by suitable analytical techniques , including the combination between an ion mobility spectrometer ( for instance , a differential mobility analyzer , dma ) and a mass spectrometer . fig5 shows a schematic of such an embodiment . target vapors enter into an ionization chamber 20 through inlet tube 3 . said vapors are sampled into the chamber by applying suction through a small pump connected to the exit port 5 . the sampled gas gets in contact with the electrospray cloud 4 , some of its vapor components become ionized and are then driven by the prevailing electric field against a clean curtain gas flow coming from the dma through orifice ( slit ) 21 located at the left dma electrode 22 . these ions are classified according to their electrical mobility within the dma , with the help of an auxiliary flow of clean gas moving vertically . as a result , only those ionized vapors within a narrow range of mobilities proceed into the exit dma sample orifice leading directly to the inlet orifice 13 to the mass spectrometer 12 . because the various fatty acid isomers have different electrical mobilities , the dma - ms combination can distinguish a larger number of such vapors than the ms alone . a more detailed description of the coupling of a dma and a ms is given in u . s . patent application ser . no . 11 / 786 , 688 by juan rus et al . u . s . patent application ser . no . 11 / 786 , 688 is incorporated by reference herein in its entirety . the work described has demonstrated the ability to detect vapor chains including up to 18 carbon atoms . even heavier vapors are known to be part of the skin spectrum and may be similarly detected in more sensitive mass spectrometers , such as modern triple quadrupoles . also included in the invention are alternative sampling methods previously used in skin analysis via gc - ms . in one approach ( slightly slower than direct sampling from the atmosphere , but must faster than gc - ms ) a cotton with a solvent such as ethanol briefly touches the skin . this cotton is inserted into a small chamber and heated above body temperature to release low volatility vapors conveyed through heated lines to the same sesi - dma - apims device previously described . the methods described admit many variants considered also part of this invention . for instance , clean , rich and readily interpretable spectra containing numerous metabolites ( such as amines ) are obtained in the embodiment of figure with positive electrospray ionization , as discussed in more detail by martinez - lozano ( 2009 ) in the case of cutaneous vapors . volatiles released from the headspace above other body fluids can similarly be analyzed to provide complementary classification criteria . for instance , martinez - lozano ( 2008 ) has examined the volatiles from urine and found a number of metabolytes different from those produced by breath or skin . we also note that recent nuclear magnetic resonance ( nmr ) metabolic studies on urine have shown that , in spite of temporal variabilities ( e . g . biochemical cycles , diet , etc . ), a stable metabolic fingerprint strongly individual - specific exists ( assfalg et al . 2008 ; holmes et al . 2008 ), even at timescales as long as 3 years ( bernini et al . 2009 ). the electrospray ionizer described here has numerous advantages , but useful spectra can also be obtained with alternative chargers . for instance , passing the sample gas through the interior of a metal tube coated with 10 millicurie of ni 63 produces a spectrum from breath or skin containing a similar number of fatty acids as the sesi ionizer . other alternative geometries and radioactive source intensities would also be useful , as would other chargers commonly used for atmospheric pressure ionization . the tests described have involved persons , but other individual organisms or colonies of organisms can be similarly probed and classified . some comment is finally appropriate regarding the immense range of different criteria or purposes for which one would wish to analyze and classify different organisms according to the present invention . nature provides numerous examples of the practical benefits that an improved sense of smell would bring to our own species . some additional particularly interesting examples are provided by the interaction between the dog and his human master ( besides the dog &# 39 ; s capacity to distinguish different concrete persons by their scent ). for instance , the dog has demonstrated an ability to sniff various human cancers , often with great accuracy , sometimes at an early stage at which no other detection means are known ( church and williams 2001 ; welsh et al . 2008 ). also , anecdotal reports suggest that some dogs can provide early warning of hypoglycemia episodes by ‘ sniffing ’ their owner &# 39 ; s dropping blood sugar levels . these observations show that not only individual persons , but also their state of health often have distinct volatile signatures . these states of health can therefore be classified according to the present invention . another important fact to be noted is that dogs are often claimed to be sensitive to parts per trillion ( ppt ) concentrations of certain vapors ( walker et al ., 2006 ), while sesi - 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