CA1312536C - Method and device for ketone measurement - Google Patents

Abstract

ABSTRACT

The present invention relates to methods and materials for the detection of ketone and aldehyde analytes in fluid samples by means of reacting analyte containing samples with a first solid matrix material to which a nitroprusside salt is coupled and a second solid matrix material to which an amine is covalently coupled. Methods and devices are also provided for ascertaining the fat catabolism effects of a weight loss dietary regimen comprising determining the breath acetone concentration of the subject.

Description

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BACKGROUND

The present invention relates generally to methods and materials for the detection of ketones and aldehydes in fluid (liquid or vapor) samples. The invention is particularly directed to the quantitatîve determination of ketone and aldehyde concentrations in physiological fluids including blood, urine and breath samples. The invention eu~ther relates to methods and ma~erlals for monltorlng the ef~ect~ of die~, exerclse and diabetlc condltions through the quantitative measurement o~ breath acetone levels.
It i known that "ketone bodies" by which term is generally meant acetone, acetoacetic acid and B-hydroxybutyric acid, tend to accumulate in the blood stream during periods of relative or absolute carbo-hydrate deprivation due to the breakdown of storagetriglycerides. The process through which overproduction of ketone bodies occurs is not well defined but is related to increased oxidation of long chain fatty acids by the liver. Specifically, acetoacetic acid and B-hydroxybutyric acid are formed by the liver as inter-mediates during the oxidation of fatty acid molecules by acetoacetyl coenzyme A. Acetone is formed from the spontaneous decarboxylation of ace~oacetic acid. Under normal conditions the intermediate products are eurther degraded to carbon dioxide and water and the ketone products do not appear at significant concentrations in the bloodstream. Nevertheless, certain metabolic and disease states interfere with the normal degradation of these intermediates which then accumulate in the bloodstream as a result.

~3~2~36 The quantitative measurement of ketone concentrations in blood serum is important because of the relationship between elevated serum ketone levels and clinical conditions such as diabetes, disorders of the digestive organs, renal insufficiency, uremia and malignant carcinoma. In the course of these disorders, ketone bodies pass into the blood stream and a state of metabolic acidosis (ketosis) occurs. Monitorin~ for the onset Oe keto~is i~ of particular importance in ~he 1~ maintenance of diabetics becau~e the occurrence Oe keto~is may indicate ~he need ~or modiication Oe insulin dosage or other disease management.
The concentration and identity o various ketone and aldehyde components present i.n the serum may lS be determined by direct chemical or chromatographic analysis. While such direct analysis provides the most accurate determination of serum ketone and aldehyde concentrations it suffers from numerous deficiencies includin~ the requirement that blood be drawn to provide serum for analysis. Moreover, the analysis must be carried out promptly due to decomposition Oe acetoacetic acid to acetone during storage. In addition, the analysis of blood serum for ketones and aldehydes by chemical means requires the use of various reagents and procedures which can be complex and inconvenient for consumer use. Further, the use of certain chromato-graphic techniques such as gas chromatography is often impractical for consumer and many types of professional use.
As a consequence of the limitations of measuring serum ketone levels directly, a large body o~
art has developed directed to the testing of urine for the presence of ketone bodies. It is known that the concentration of ketone bodles in urine bears an imper-fect relationship to serum ketone concentrations. While urine ketone concentrations depend on numerous factors ~, ~3-~536 and are not always directly proportionaL to serum ketone concentrations, testing of urine for ketones is a simple and relatively inexpensive means of monitoring serum ketone concentrations. Such methods are in widespread use by diabetics in both home and clinical settings.
A number of test devices and methods for the detqrmination o~ urine ketone concentrations are Icnown to ~he art. Some assays utillze the reaction of ~ce~ne with sallcylaldehyde in ~lkalin~ solution ~o giv~ the deeply colo~ed or~nge ~o red compound salicylalace-tone. Any acetoacetic acid in such solutions is con-verted by the alkali to acetone which further contributes to the color reaction.
Kamlet, U.S. Patent ~o. 2,283,262 discloses lS compositions for the detection of acetone and aceto~
acetic acid in solutions such as urine. The materials comprise a dry mixture of a member of the group con-sisting of the alkali metal and alkali-earth metal bi3ulfite addition products of salicylaldehyde and a member o~ the group consisting of the alkali metal and a~kali-earth metal oxides and hydroxides.
Many assays take advantage of the "Legal"
method which utilizes the reaction of a carbonyl group containing compound such as a ketone or an aldehyde with a nitroprusside (nitroferricyanide) salt in the presence of an amine to form a colored complex. While acetone will react, albeit slowly, with nitroprusside under aqueous conditions, the reaction of acetoacetic acid is some lO0 to 200 times faster with the result that "Legal" reactions under aqueous conditions whether detecting "acetone," "acetone bodies" or "ketone bodies"
primarily detect acetoacetic acid. The color reaction is believed to occur as a result of a coupling reaction through the nitroso group of the nitroprusside with the analyte to form an intermediate which then complexes with the amine to produce a color characteristic of the 1 3 11 ~

specific amine. In forming the complex, the trivalent iron of the nitroprusside is reduced to its divalent state. The color complex, however, is unstable because nitroprusside decomposes rapidly in alkaline solu-tions. Further, nitroprusside salts are subject todecomposition in the presence of moisture and high pH.
Frequently during storage, a brown decomposition product is formed which can inter~ere with sensitive detection during assay~. These limitations have led to numerous att~mp~s to ~tabilixe ~he colo~ colnplex by utilizin~
mixtures o~ nitroprussides and amines or amino acl~s in combination with a variety of buffers, metal salts, -- organic salts, organic stabilizers and polymers.
Numerous combinations of reagents have been shown to be suitable for detection of a variety of ketone bodies in liquid samples although the analyte predominantly detected in physiological fluids is acetoacetic acid.
Fortune, U.S. Patent No. 2,186,902 discloses the use of soluble nitroprusside chromogens in the presence of ammonia and soluble carbonates for the detection of what was termed "acetone" (actuall~ aceto-acetic acid) in urine samples. ~arying colorations are observable for the ~uantitative determination of "aceton~" levels.
Galat, U.S. Patent No. 2,362,478 discloses a solid reagent for the detection of "acetone" (actually acetoacetic acid) in liquid samples. The reagent comprises a dry mixture of a powdered anhydrous soluble nitroprusside, granular anhydrous soluble nitroprusside and granular anhydrous ammonium sulfate. The reagent signals the presence of "acetone" by producing a color reaction when a drop of sample is added thereto.
Free, U.S. Patent No. 21509r,140 discloses improvements on the materials of Fortune comprising solid dry formulations which may be in the form of tablets for the detection of "acetone bodies" oe "ketone 1312~3~

bodies" in liquids. The materials comprise a nitro-prusside salt, glycine and an alkaline salt.
Nicholls, et al., U.S. Patent No. 2,577,97~
discloses improvements on the dry formulations of Free for the detection of "acetone bodies" or "ketone bodies"
in bodily fluids. Such compositions comprise alkali metal nitroprussides and alkali metal glycinates combined with sugars such as lactose, dextrose and ~ucrose.
Whlle many assay device~ oE the prior a~t utilize dr~ t~blets or powder~ in performing an a~say, other assay devices utilize adsorbant carriers upon which some or all of the reagents have been dried. The adsorbant carriers may be in the form of strips which can be immersed in a sample of the liquid to be analyæed with the color reaction taking place in solution on the carrier. These assay devices, like those utilizing tablets or powders, suffer from decomposition of the nitroprusside indicator. In addition, indicator materials which are merely adsorbed onto the adsorbant carriers tend to suffer from diffusion of reagents away from the strip which affects the strength o~ the color signals. Further, the strips exhibit a certain amount of "bleeding" of color product in the aqueous environ-ment which limits the stability of the color indicatorsignal of the reacted device.
Magers, et al., U.S. Patent 4,147,514 discloses test strips for the detection of ketone bodies such as acetoacetic acid in bodily fluids utilizing a 3~ solution comprisin~ nitroprusside in combination with at least one inorganic metal salt where the metal is selected from the group of magnesium and calcium. The solution optionally comprises at least one primary amine combined therewith. Test strips are dipped in the solu-tion and are dried. They may be immersed in fluidsamples and the occurrence of a color reaction observed.

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U.K. Patent No. 1,012,542 discloses methods for the detection of ketone bodies in bodily fluids wherein alkaline components, in an a~ueous solution are impregnated onto a carrier to which, soclium nitroprus-side salt in an organic carrier also containing largeamounts of an organic film-forming polymer is later applied. The carrier material is said to be very stable and is used eor the detection oE ketone bodies ~acetoacetic acld) in liquid sample~.
~0 U.K. Patent No. 1,369,13~ disclo~e~ imp~oved methods ~or the detection oE ket~nes in bodily Eluld~
wherein an absorbant carrier is first impregnated with a solution consisting of an amino acid, tetrasodium ethylenediamine-tetraacetate buffer and water which is then dried. The carrier is then impregnated with a solution of sodium nitroprusside in dimethyl formalde-hyde and optionally an alcohol containing one to four carbons and is dried.
Smeby, U.S. Patent No. 2,990,253 discloses a device Eor the detection of ketone bodies in fluid samples comprising a bibulous carrier onto which nitro prusside is first applied in an aqueous acidic media and to which is subsequently applied a non-aqueous solution of organic bases such as amines or amino alcohols to achieve the alkalinity necessar~ for the assay reaction.
Mast, et al., U.S. Patent No. 3,212,855 discloses an improved method for the production of a "dipstick" device for the detection of ketone bodies in fluids in which a bibulous carrier is first impregnated with an aqueous svlution comprising an alkaline buffer and a water soluble amino acid. The carrier is then dried and impregnated with a solution in an organic solvent comprising an alkali metal nitroprusside and an organic film producing polymer.

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Takasaka, Japanese Patent Application No.
1980-45270 discloses methods for the detection of ketones in body fluids utilizing test strips impregnated with alkali metal salts of nitroprusside and yttrium metal salts. The strips indicate a color reaction in acidic pHs in the presence of acetoacetic acid.
Federal Republic of Germany Patent No.
3,029,865 discloses improved test strips for the detection of ketones in bodily Eluids comprisin~
ab~o~bant carriers impregnated with sodlum nitropru~slde, ~ water-soluble amino acid, an alkalin~
bu~er compound and phosphoric acid trlmorpholide ag a stabili2er.
Kikuchi, Japanese Patent Application No. 19~32-10208 discloses test strips for the detection of ketones in bodily fluids which are produced by immersion of absorbant carrier material in a solution comprising an amino acid, sodium triphosphate and sodium hydroxide and distilled water. The carrier strips are then dried and are immersed in a solution comprising a nitroprussidesalt dissolved in dimethylformamide. They are then dried again and are ready for use.
Hirsch, U.S. Patent No. 4,097,240 discloses a process for the production of dipstick devices for the detection of ketones in fluids such as urineO The process comprises the impregnation of an absorbant carrier with sodium nitroprusside, an alkaline buffer substance and a water soluble amino acid. The carrier is first impregnated with an aqueous solution of amino acid and tetrasodium ethylenediamine tetraacetate buffer and dried. It îs then impregnated with a solution of sodium nitroprusside in a solvent mixture consisting of methanol and an organic solvent miscible with methanol such as a linea~ or branched aliphatic alcohol with two to six carbon atoms.

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Habenstein, U.S. Patent No. 4,184,850 discloses a dipstick device for the detection of ketone bodies in fluids comprising an absorbant carrier medium impregnated with sodium nitroprusside, a water-soluble lower amino acid, an alkaline buffer substance, and at least one organic acid which serves to form a stabilizing environment around the nitroprusside salt.
Kohl, U~S. Patent No. 4,405,721 disc10ses devices for the detection of ketone bodies in bodily ~lulds compri~ing a carrier impregnaked with A bu~er, an amino acid, sodium nitroprusside and a hetero~yclic stabilizing compound.
Tabb, et al., U.S. Patent No. 4,440,724 discloses devices for the detection of ketone bodies in bodily fluids and methods for their preparation. The devices may be constructed according to steps com-prising; impregnating a carrier with an aqueous solution of a soluble nitroprusside chromogen, drying the carrier, impregnating the carrier with an aqueous solution including a metal salt, a primary amine, TAPS
(N-Tris (hydroxymethyl) 3-aminopropane sulfonic acid) and TRI5 (tris-hydroxymethyl aminomethane) and drying the carrier, the pH of the Einished test device being no greater than 7Ø
Of interest to the present application is the disclosure of Ogawa, et al., U.S. Patent No. 3,880,590 which discloses a dipstick device for the semiquantitative detection of acetoacetic acid in liquids such as urine. The Ogawa, et al. strip is said to be incapable of detecting other ketone bodies, such as acetone and ~-hydroxybutyric acid. The device comprises an absorbant material, a nitroprusside salt and a heavy metal salt such as nickel or ferric chloride. The absorbant materials include silica gel paper, diethylaminoethyl (DEAE) cellulose paper and amino ethyl cellulose paper with which the nitroprusside ~ ~253~

salt is associated. The absorbant strips are impregnated with a solution of a nitroprusside salt and a heavy metal salt in water or organic solvents including dimethyl formamide, dimethyl sulfonate methanol and ethanol or mixtures thereof. Solvents disclosed to be useful in forming the devices include dimethylformamide, dimethylsulfoxide, methanol and ethanol and mixtures thereo~. ~ccording to one example, dil~ethyl orm~mide solu~on i~ ed to impregnate VEA~
cellulose paper along wlth nlc~cel chloride and sod.~
nitroprusside. The s~rips were dried and later used to detect the presence of acetoacetic acid in urine. It is disclosed that the impregnating solution itself may be useful for the detection of ketone bodies but that the dried test strips are preferred in view of preservation, ; stability and handling considerations.
While references variously refer to the use of nitroprusside and amine compositions for the detection of "acetone", "acetone bodies" and "ketone bodies", the assays primarily detect acetoacetic acid and are generally incapable of distinguishing between reaction products formed from reaction of acetone and reaction products formed from reaction vf other ketone bodies including acetoacetic acid. Other assays, such as those of Ogawa, et al. are disclosed to be incapable of detecting acetone at all. While numerous advances have been made with respect to "Legal" assays for the detection of ketones and aldehydes, such assays are still limited by the instability of nitroprusside at pHs greater than 7. Finally, such assays still measure only the concentrations of ketone bodies in urine and fail to necessarily provide accurate measurements of ketone bodies present in the blood serum.
~ It is well known in the art that breath samples may be assayed for the presence of acetone in order to determine serum acetone levels. Acetone is a ~311 2~

relatively volatile compound having a partition coefficient of approximately 330. It readily diffuses from the blood into the alveolar air of the lungs according to an equilibrium relationship. As a consequence of this equilibrium state, concentrations of acetone in alveolar air are directly proportional to those in the blood and measurements of acetone in alveolar air can be used to determine the concentration Oe acetone in the serum. Croford, et al., Trans. Arner.
Clin. Climatol. A.~soc. 88, 128 ~1977). CroEfordt et al.
al90 di~close~ th~ use Oe head ~pace analysi~ to determine the ketone concentration of liquid samples.
Current methods for the measurement of breath acetone levels include the use of gas chromatography.
Rooth, et al., The Lancet, 1102 (1966) discloses the use of a gas chromatograph capable of detecting acetone at concentrations of 2 to 4 nM of air with 18 nM being the concentration for breath of normal individuals.
Subjects breathe directly into the device and the acetone peak i5 read after 40 seconds. Reichard, et al., J. Clin. Invest. 63, 619 (1979) discloses gas chromatographic techniques for the determination of breath acetone concentrations wherein breath samples are collected through the use of a calibrated suction flask into which the test subject breathes through a glass inlet tube. These methods and the instruments required for their use are complicated and expensive and tend to be impractical for use by consumers.
Other methods for the measurement of breath acetone levels involve the use of mass spectrographic equipment. Krotosynski, J. Chrom. Sci., 15, 239 (1977) discloses the use of mass spectrographic equipment in evaluating the ketone content of alveolar air. Twelve ke~one components of breath were identified with acetone comprising the major component. Mass spectrographic methods suffer from the same limitations, however, as relate to gas chromatographic techniques.

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Methods utilizing color reactions for the detection of acetone in liquid or air have also been reported in the art. Greenberg, et al., J. Biol. Chem.
Vol. 154-155, 177 ~1944) discloses methods for the determination of acetone levels apart from those of other ketone bodies in solution. The methods involve reaction of acetone and other ketones with 2,4-dinitrophenylh~draz~ne, to form hy~razone product~ which may then be ~eparat~d and i~olated owing to di~Eering ~olubilitles.
Peden, ~. Lab. Clin. Med. 63,332 (196~) discloses improvements over the methods of Greenberg, et al. utilizing salicylaldehyde as a coloring reagent.
According to this method, 3-hydroxybutyric acid is converted to acetone by oxidation with the amount of acetone formed measured by reaction with salicylaldehyde. Preformed acetone and acetoacetic acid are removed prior to the conversion of the ~-hydroxybutyric acid by heating in the presence of acid. While these methods are useful for the determination of acetone concentrations apart from those of other ketone bodies they are complex and time consuming.
These various colorime~ric methods known for detection of acetone in biological fluids are complex, time consuming and necessitate the use of a spectrophotometer or color charts. Moreover, the methods often require the use of high concentrations of alkali or acids. Alternative methods for the detection of acetone often require the use of complex and expensive apparatus. There thus continues to exist a need for methods for the quantitative determination of fluid acetone concentrations which are simple, accurate, inexpensive and do not require the use of high concentrations of hazardous reagents.

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There exists a desire for methods for the measurement of the rate of fat catabolism. It is a particular problem that many individuals undergoing diets are unable to determine their rate of fat-loss because of daily variation in their body fluid content. Significantly, it is known that ea~ly in a diet individuals lose high proportions of fluid as compared to fat. Later in their diets, when individuals may still be catabolizing fat at a constant rate they may cea~e to lose Eluids at the previous high rate or may, i~ only temporarlly, regain Eluid weight. q'h~
experience of hitting a plateau in weight loss or even regaining weight is psychologically damaging and weakens the subject's resolve to continue with the diet often with the effect that the subject discontinues the diet.
Recently, a method has been disclosed for the determination of daily rate of fat loss. Wynn, et al., Lancet, 482 (1985) discloses that daily fat-loss may be calculated by subtracting daily fluid and protein mass changes from daily weight changes. Changes in body water are estimated from the sum of external sodium and potassium balances and protein mass changes are calculated from nitrogen balances. Such a method is complex and time consuming thus making it inconvenient for the consumer.
One set of methods for measuring body fat is by quantitating total body water (T~W). A number of methods are available for determining TBW. These include isotopic dilution procedures using deuturiated water, tritiated water and l3O-labelled water. Urine, blood serum or saliva samples are collected after a 2 to 4 hour equilibration. The fluid samples are then vacuum sublimed and the concentration of tracer in the sublimate is determined by mass spectrometer, gas chromatography, or infrared or nuclear magnetic ~3~2~

resonance spectroscopy. Body composition can also be measured by a bioelectrical impedence method using a body composition analyzer. These methods are well known in the literature and are readily performed by those of skill in the art. Equipment for performing such measurements is available commercially from medical instrument manufacturers such as R~L Systems, Inc.
(Detroit, MI).
Hydrostatic weighing method is a well known method wherein the su~ject is completely submerged in a tank of water and the body fat is calculated by taking into acco-lnt the average densit~ of fat and the amount o~ water displaced, This method i~ inconvenient and is still not completely accurate because assumptions must be made relating to nonfat density, lung capacity and other factors. Another method for calculating the percentage of body fat utilizes skin calipers to measure the thickness of fat deposited directly beneath the skin. Pincers are used to measure the thickness of folds of skin and fat at various locations on the body. The results of these measurements are compared with standardi~ed tables to arrive at a figure for percentage of body fat. This method, while more convenient than the use of hydrostatic weighing is less accurate. All methods for determination of body fat content suffer from the fact that they do not reveal the rate of fat loss but only the at content of the body at a particular time. Because means for determining body fat content are of limited accuracy, means for the determination of the rate of fat loss are similarly limitedO Nevertheless it is desired that a simple and convenient method be developed for the determination of the rate of fat-loss wherein such a method is capable of distinguishing weight loss due to loss of fat as opposed to weight loss from the elimination of bodily fluids.

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of interest to the present invention are observations that ketosis occurs in non-diabetic individuals undergoing weight loss through diet, fasting or exercise. Freund, Metabolism 14, 985-990 ~1965) observes that breath acetone concentration increases on l'fasting." It is disclosed that breath acetone concentrations increased gradually from the end of the first day of the fast to approximately 50 hours into the fast a~ which time the concentration ~ose sharply in a linear ~ashlon and reached a plateau on the ourth day. The acetone concentratlon oE the plateau was approximately 300 ~g/liter (5,000 nM) a hundred-fold increase over the normal value of 3 ~g/liter (50 nM).
When, lnstead of fasting, the subiect was placed on a "ketogenic" diet with a minimum of 92% of calories derived from fat, the subject suffered a lesser degree of ketosis wherein the plateau had an acetone concen-tration of approximately 150 ~g/liter (2,500 nM).
Rooth, et al., The ~ancet, 1102-1105 ~1966) discloses studies relating to the breath acetone concentrations of a number o obese and diabetic subjects. When the caloric intake of three non-diabetic obese subjects was reduced, their breath acetone concentrations as measured by a gas chromatograph increased approximately three-fold. On fasting, the subjects' breath acetone concentrations increased to one hundred times normal. Within 16 hours after a heavy meal the subjects' breath acetone concentrations dropped almost to normal. In a study of obese diabetic patients, the authors disclosed evidence that those obese patients who had lost weight in the last three months had higher breath acetone concentrations than those patients who had gained weight.
Walther, et al., ~cta ~iol. Med. Germ. 22, 117-121 ~1969) discloses the results of a study on the effects of continued exercise of a well-trained ~31~3~

cyclist. The authors disclose that breath acetone concentration, increases prior to, during and after the cessation of the physical load and reached a maximum 15 to 20 minutes after cessation of the physical load.
Breath acetone concentrations approach a normal level one to two hours after the cessation of the physical load. It is suggested that the increased production of acetone is due to the increased utilization oE plasma ~ree ~atty acids ln liver and reduced utilization in peripheral tis~ue.
More recent ~tu~les have shown a correlation between fasting in normal and obese patients and increased blood acetone levels. Rooth, et al., Acta Med. Scand. 187, 455-463 (1970); Goschke, et al., Res.
Exp. Med. 165, 233--244 (1975); and ~eichard et al., J.
Clin. Invest. 63, 619-626 (1979) all show the development of ketosis in both overweight and normal individuals during fasting. Rooth, et al., (1970) suggests the use of breath ketone measurements as a motivational tool to enforce against dietary cheating.
The studies disclose that development of ketosis is slower in overweight than in normal weight individuals. Reichard, et al., discloses that there is a better correlation between breath acetone and plasma ketone concentrations than between urine ketone and plasma ketone concentrations. In addition, Rooth, et al., (1970) discloses that certain urine ketone tests which detect the presence of acetoacetic acid are not entirely reliable because some individuals do not excrete acetoacetic acid in the urine despite increased blood serum concentrations.
Crofford, et al., tl977) discloses the use of breath acetone monitoring for monitoring of diabetic conditions and as a motivational tool in following patients on long-term weight reduction programs. Such monitoring i5 said to be particularly effective as ~3~25~

normalization of the breath acetone is disclosed to occur upon significant dietary indiscretion. Patients' breath samples were monitored using a gas chromatograph and it is suggested that patients be instructed to restrict their caloric input to that which will maintain breath acetone concentrations of approxi.mately 500 nM.
It is further suggested that if breath acetone is controlled at this level and the proper balance oE car-~ohydrate, protein and eat are maintained in the diet that weigh~ 10~9 will occur at a rate o~ approximatel~
one-half pound per week.

SUMMARY OF THE INVENTION

The present invention relates to methods and materials for the detection of ketones and aldehydes in fluid ~liquid or vapor) samples. The invention is particularly directed to the quantitative determination of ketone and aldehyde concentrations in physiological fluids including serum, urine and breath samples. The invention is particularly suited for the determination of acetone concentrations. According to one aspect of the invention, methods are disclosed for the quantitative determination of serum acetone concentra-tions through the measurement of breath acetoneconcentrations. The method of breath acetone measurement utilizing the methods and materials of this invention is also adoptable for monitoring the insulin dose requirement for Type l insulin-dependent diabetic patients and to distinguish between Type 1 (ketotic) and Type 2 (non~ketotic) diabetic patients. Alternatively, concentrations of acetone or other ketones or aldehydes in serum, urine or other liquids may be determined by head space analysis of vapors in equilibrium with a liquid sample. According to further aspects of the present invention, liquid samples may be analyæed 1312~3~

quantitatively in a liquid phase reaction for the presence of aldehydes or ketone bodies such as acetoacetic acid. According to still further aspects of the invention, methods are disclosed for ascertaining the fat catabolism effects of a weight loss dietary regimen comprising diet, fasting or exercise through the quantitative determination of serum or alveolar air ~breath) acetone concentrations. Preferred methods for de~erminatio~ of the rate o eat catabolism comprise measurement o bre~th acetone concentrations and may b~
readily determ~ned b~ utili~ing the devices o~ th~
inventlon. The present Lnvention also provides kit~ Eor the determination of fluid ketone and aldehyde concentrations and for the determination oE the rate of fat catabolism.
The present invention comprises methods and materials for the determination of fluid ketone and aldehyde analyte concentrations through the reaction of analytes present in the sample fluid with a nitroprusside compound in the presence of an amine and a solvent to produce a colored reaction product. Devices according to the invention comprise a first solid matrix material to which a nitroprusside salt such as sodium nitroprusside is coupled. The devices further comprise a second solid matrix material to which is covalently bound an amine. ~ccording to one aspect of the . invention, the nitroprusside salt and the amine may be coupled with and covalently bound, respectively, to the same solid matrix material. Preferably, however, the first and second solid matrix materials are in the form of discrete particles which are treated accordingly with a nitroprusside salt or an amine and are intermixed so as to place nitroprusside and amine moieties in intimate contact with one another. The solid matrix materials may be selected from a variety of materials including cellulose and silica gel which present suitable coupling . .

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moieties or are susceptible to reaction with suitable coupling moieties.
While the various methods of th~ present invention vary according to their specifics, they share S the common aspect wherein ketone or aldehyde analytes present in a sample are contacted in the presence of a solvent with a nitroprusside salt coupled to a first solid matrix material and an amine covalently bound to a second solid matrix material. These materials together react to form detectable reaction products of characteristic colors which may then be observed ~or a ~ualitative or quantitatlve dete~mination o~ the pre~ence o~ ketone or aldehyde analytes.
SpeclEic method~ and confiquratlons o~ deviceg ~or carrying out tho~e methods are known accordlng to the identity of the analyte of interest and the nature of the sample material to be assayed. When the sample material is a vapor, a fixed quantity of the vapor may be collected by suitable means and the ketone or aldehyde analyte component preconcentrated on a preadsorbent material. The adsorbant for preconcentration of such analytes may be a material such as Tenax TA~(a 2, 6-diphenyl-p-phenylene oxide polymer) or activated silica which may be maintained in the device in a preadsorbent zone separate from the first and second solid matrix materials associated with the nitroprusside salt and amine which are located in a reaction æone. After preconcentration, the ketone or aldehyde analytes are desorbed from the adsorbant by means of a solvent and contacted with the first and second solid matrix materials for reaction with the nitroprusside and amine reagents. According to a preferred embodiment, ketone and aldehyde components present in a vapor sample may be preconcentrated on the first and second solid matrix materials themselves. The analytes may then be solubilized by addition of a 5 3 ~

solvent to react with the nitroprusside and amine moieties present on the solid matrix mat:erials. Where the analyte of interest is acetone methods for the assay of vapor samples, particularly breath samples, preferably utilize a desiccating bed for the preadsorption of water which can interfere with the quantitative detection of acetone.
In vapor ~ample devices wherein ketone~ and aldeh~des are preconcentra~ed by adsorptlon onto the ~ir~t an~l ~econd solld matrix ma~erial~, ~ "linear reading sys~em" for de~erminatio~ oE ~nalyte concentration may be utilized. The system provides a visual indication, in the form of a color bar of colored reaction products, indicating the quantity of ketone or aldehyde analytes adsorbed onto the first and second solid matrix materials. ~ecause the adsorption sites on the matrix materials are limited, the extent to which ketone and aldehyde analytes will be adsorbed is dependent upon the quantity of analytes in the vapor sample. Analyte vapors will initially be adsorbed onto the solid matrix materials at the first portion of the reaction zone. As adsorption sites on those materials are saturated analyte vapors are adsorbed at more distant points within the reaction zone. Where the volume of the vapor sample is fixed, the distance from the first end of the reaction zone at which the analyte vapors are finally adsorbed is dependent upon the concentration of the analytes in the vapor sample. The extent to which the analytes are adsorbed, and hence the ketone or aldehyde concentration of the sample, is indicated by the extent of formation of colored reaction products.
When the sample material is a liquid such as urine or serum, head-space vapor assays may be carried out by analysis of vapor in equilibrium with the liquid for the presence of acetone and other ~olatile ketone ~3~ 2~6 and aldehyde components. After collection of a known volume of vapor in equili~rium with the liquid sample, the vapor is analyzed in the same way as breath and other vapor samples. Such head-space analysis is particularly suitable for analysis of the more volatile ketone and aldehyde fractions of samples as "lighter"
analytes such as acetone will be present in the heacl space vapor in higher proportions than other less volatile "heavy" analyte components.
Quantl~ative liqu~d phase colorlmetric assays m~y also be conducted on ~amples such a~ serum or urine according to the tnethods of the present invention.
Liquid assays are useful for detection of most ketones and aldehydes but are particularly useful for quantitative detection of less volatile analytes such as acetoacetic acid. According to such methods, liquid samples are applied to microcolumns packed with or dipsticks coated with the solid matrix materials of the invention. The presence of ketones or aldehydes in such samples produces a color reaction. Quantitative results can be obtained through use of ascending chromatography methods in microcolumns comprising the solid matrices of the invention. The concentration of ketone and aldehyde analytes present in the sample may be determined by the height of color bar produced in the tube. Where a dipstick coated with the solid matrix materials of the invention is used, analyte concentrations may be determined by visual or spectrophotometric evaluation of the color signal.
These liquid phase methods for analysis of liquid samples are not particularly suitable for detection of acetone in aqueous solutions such as bodily fluids, however, because the presence of water in these solutions retards the reaction rate of acetone to less than one one-hundredth the reaction rate of acetoacetic acid. Nevertheless, if the concentration of acetone in ~12~3~

an aqueous solution is sufficiently high, the liquid phase methods may be adopted.
One preferred device of the present invention utilizes a breath collection device into which a subject breaths and which can collect a selected amount of alveolar air. The breath sample is then passed through the analyzer device wherein an anhydrous calcium chloride desiccant bed removes water vapor ~rom the breath ~ample. The ~ample iq t~en pa3sed through a bed L0 eilled wl~h a mlx~ure Oe ~irst and ~econd solid ~atrix materials comprising nitroprus~lde-DEAE silica gel and aminopropyl silica gel where acetone contained within the breath is adsorbed into the matrix. The distance to which the acetone is adsorbed is dependent upon the total amount of acetone present in the sample. A
solvent mixture containing either methanol or methanol and dimethyl sulfoxide (DMSO) is then added to the matrix to activate the color reaction and form a blue color bar. The length of the color bar is proportional to the concentration of acetone in the fixed breath sample volume and may be compared with a table or calibration marks on the side oE the matrix bed to determine the breath and serum acetone concentrations.
The methods and materials of the present invention may be utilized to monitor diabetic patients, to analyze for various metabolic abnormalitieq or may be utilized according to one aspect of the present invention for the monitoring of the rate of fat catabolism. It has been found that serum acetone concentrations and hence breath acetone concentrations which can be measured by the methods and devices of the present invention may be correlated directly with the rate of fat catabolism and fat-loss experienced by a subject undergoing a weight loss dietary regimen comprising fasting, dieting, exercise or a combination of the three. Serum and breath acetone concentrations 1 3~2~3~

may be determined by a variety of means and the rate of fat-loss calculated therefrom according to the invention. The methods and devices of the invention, however, are extremely convenient, are accurate within about 10~ in determining serum acetone levels and are therefore particularly suitable for measuring fat catabolism and the rate of actual fat-loss as opposed to determining welght loss which is variable and often ~e~lects variatiQns in eluid losses. B~ measurement o~
breath acetone levels, a sub~ect will be abl~ to estimate with a hlgh degree of accuracy his rate of fat-loss, the water-loss/fat-loss ratio and be able to adjust his diet and amount of exercise according to his desired weight loss goals.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a view of a vapor test device of the present invention.
FIG. 2 is a view of an alternative vapor test device of the present invention.
FIG. 3 is a graph illustrating the relationship between the height of the color bar in a vapor test device of the present invention and the concentration of acetone present in a vapor sample.
FIG. 4 is a view of a liquid test device of the present invention.
FIG. 5 is a graph illustrating the relationship between breath acetone concentrations and the rate of fat loss corresponding thereto.
FIG. 6 is a graph illustrating the degree of water and fat loss for dieters 0 to 10 pounds overweight over a period of days.
FIG. 7 is a graph illustrating the degree of water and fat loss for dieters lO to 20 pounds overweight over a period o~ days.

13~ 2~3~

FIG. 8 is a graph illustrating the degree of water and fat loss for dieters 20 to 40 pounds overweight over a period of days.
FIG. 9 is a graph illustrating the degree of water and fat loss for dieters 40 to 100 pounds overweight over a period of days.
FIG. 10 is a graph illustrating the relationship between breath ace~one concentration~ and blood head-space aaetone concentrations measu~ed by ga~
10 chromatograph~
FIG. 11 is a graph illustrat1ng the relationship between breath acetone concentrations as measured by a gas chromatograph and by devices according to the invention.
FIG. 12 is a graph illustrating the average morning breath acetone concentration for a dieting and a non-dieting population.
FIG, 13 is a graph illustrating the average height of an indicator color bar in breath acetone measurement devices according to the invention for a dieting and for a non-dieting population.
FIG. 14 is a graph illustrating the average cumulative fat loss for a dieting and for a non-dieting population.
YIG. lS is a graph illustrating the cumulative fat loss for a first individual dieter.
FIG. 16 is a graph illustrating the daily height of an indicator color bar in breath acetone measurement devices according to the invention for the first individual dieter monitored in FIG. 15.
FIG. 17 is a graph illustrating the cumulative fat loss for a second individual dieter.
FIG. 18 is a graph illustrating the daily height of an indicator color bar in breath acetone measuring devices according to the invention for the second individual dieter monitored in FIG. 17.

~3~2~3~

FIG. 19 is a graph illustrating the relationship between the concentration of breath acetone and the height of an indicator color bar in breath acetone measuring devices according to the invention.
FIG. 20 is a graph illustrating the relationship between breath acetone concentrations and the rate of fat loss.
FIG. 21a depicts a diet progress chart for use in monitoring a diet program in con~unction with the p~en~ inven~ion. ~IG. 21b depicts the diet progr~
chart o FIG. 21a Eilled out to monitor a dietary welght loss program.
FIG. 22 is a perspective view of a breath-sampling kit of the present invention.
FIG. 23 is an exploded perspective view of the kit shown in FIG. 22.
FIG. 24 is an exploded perspective view of the outer tubular member of blow tube of the kit shown in FIGS. 22 and 23.
FIG. 25 is an enlarged fragmentary longitudinal sectional view taken generally along line 25-25 of FIG. 24.
FIG. 26 is a perspective view of a disposable analyzer column which is usable with the breath-sampling kit of FIGS. 22 and 23.
FIG. 27 is a side elevational view of the breath-sampling assembly of the present invention with the housing portions of the kit removed and showing the position of the parts and the flow of air when a user is blowing into the mouth piece to expand the inflatable bag and provide a known-volume of breath to be analyzed.
FIG. 28 is an end elevational view, partially in transverse section, of the assembly taken generally along the line 28-28 of FIG. 27.
FIG. 29 is a side elevational view of the assembly of FIG. 27 showing the position of the parts 1312~3~

and the flow of air when the collected sample of alr is being discharged from the bag through the analyzer column.
FIG. 30 is an end elevational view, partially in transverse section, of the assembly taken generally along the line 30-30 of FIG. 29.
FIG. 31 is a transverse sectional view taken generally along line 31-31 of FIG. 29.
~ G. 32 ls a ~ragmentary longitudlnal sectional view taken gener~lly along line 32-32 Oe FIG. 29 showing ~he unbroken ampule o~ reactant in th~
disposable analyzer column.
FIG. 33 is a fragmentary longitudinal sectional view similar to FIG. 32 but showing the ampule of reactant after same has been broken.
FIG. 34 is a transverse sectional view taken generally along line 34-34 of FIG. 27 with the analyzer column in its first rotary and axial position (breath-receiving mode) in the blow tube.
FIG. 35 is a transverse sectional view taken generally along line 35-35 of FIG. 29 with the analyzer column in its second rotary and axial position (breath-discharging mode~ in the blow tube.
FIG. 36 is a transverse sectional view taken generally along line 36-36 of FIG. 27.
FIG. 37 is an enlarged fragmentary longitudinal sectional view showing the valve operation of the breath-sampling assembly when a user is blowing into the mouth piece as shown in FIG. 27.
FIG. 38 is an enlarged fragmentary longitudinal sectional view similar to FIG. 37 that is showing the valve operation after the user has completely filled the breath collection bag, a static condition during which the breath sample is retained in the collection bag.

~312~

FIG. 39 is an enlarged ~ragmentary longitudinal sectional view similar to FIGS. 37 and 3 but showing the valve operation after the analyzer column has been rotated 90 degrees from its initial position and then pushed further inwardly into the blow tube to the position shown in FIGS. 29 and 39 whereby the collected sample of ~reath is forced through the analyzer column and out through the mouth piece as a result oE bia~e~ d~flation of the collection bag.
DETAlL~ DES~IPTION

The present invention comprises methods and materials for the determination of fluid ketone and aldehyde concentrations through the reaction of such carbonyl group containing compounds with a nitroprusside compound in the presence of an amine and a suitable solvent to produce a color reaction. Devices according to the invention comprise a first solid matrix material to which a nitroprusside salt is coupled and a second solid matrix material to which an amine is covalently bound. The addition of magnesium or calcium salts in the test composition promotes chelate formation thus stabili~ing the color product and enhancing the kinetics of the reaction between the carbonyl compound, the amine and the nitroprusside.
Specifically, the first solid matrix ~aterial may be coupled to the nitroprusside salt by means of a suitable secondary or tertiary amine compound. The secondary or tertiary amine compound is itself coupled either directly to the first solid matrix material or to a coupling agent or coupling moiety which is attached to the first solid matrix material. Such coupling moieties include silane epoxides such as 3'-glycidoxy-propyltrimethoxysilane having a first functionalltyreactive with materials such as silica gel and a second ~3~2~3~

epoxide functionality reactive with an aspect of a secondary or tertiary amine compound. ~latrix material~
presenting suitable coupling moieties include 9el~t ion exchange resins, glasses and ~ellulosic materials which may be o~tained commercially. Such matrix materials include diethylaminoethyl lDEAE) silica gel~, DEAE
cellulose, diethylamino (DEA) ilica gel, aminoethyl (AB) silica gel, quarternary aminoethyl (QAE) silica qel as well as other weakly or strongly baslc ion exchange materials.
M~trix materials compr1~ing suitable coupl1n~
~oiet~e for coupllng o ni~roprusslde alts n~ed no~ be obtalned colNmerc1ally, but may be produced according to known procedures in the art. In Kundu, et al., J. ~ipid Res. 19, pp ~90-394 (1978) applicant discloses methods for the preparation of DEAE-silica gel. In Rundu, et al., J. Chrom. 170, pp. 65-72 (1979) applicant dlscloses methods for the preparation of DEAE-silica gel as well as DEAE-controlled porous glass.
The second solid matrix ~aterial is covalently bound to an amine by means of a coupling moiety which may be lnitially coupled to eithe~ the solid mattix material or to the amine. Illustrative of suitable chemistry is the reaction between 3'-aminopropyl-trimethoxysilane and silica gel to produce a~inopropyl silica gel. Lower alkyl amine silica gels such as aminopropyl silica gel are available commercially but may readily produced according to methods known to the art. In Kundu, et al., J Lipid Res., 20, pp. 825-833 ~1979~ applicant discloses suitable methods for the preparation of aminopropyl silica gel.

~' :

Nitroprusside Sa}ts Nitroprusside sal~s suitable ~or coupling with the first solid matrix material of the present invention include those salts capable of reacting with ketone and aldehyde analytes in the presence of an amine and a solvent to produce a detectable color complex. Suitable nitroprusside salts include elemental metals and preferably alkali metal and alkali earth metal salts of nitroprusside. Preferred alkali metal salts of nitroprusside include sodium nitroprusside, while pre~erred alkali earth metal salts include salts of magnesium and calcium.

Seconda~and ~ V mines S~condary and tertlary amines suitable for coupling the nitroprusslde salt to the first solid matrix material include those amines capable of forming an ionic complex with the nitroprusside salt and immobilizing it on the first solid matrix material. A
pre~erred material is N,N-diethylethanolamine the hydroxy group of which can react with the epoxide moiety of a silane epoxide such as 3'-glycidoxypropyl-trimethoxysilane to form diethylaminoethyl substituted materials such as DEAE silica gel and DEAE cellulose.
Couplinq Agents Coupling agents suitable for use with the present invention include those agents having a first group reactive to form a bond with the first matrix material and a second group reactive to form a bond to a secondary or tertiary amine compound. Particularly preferred ic the use of silane coupling agents having an alkoxy silane qroup. Preferred coupling agents include those such as Y-aminopropyltriethoxy silane, N-B-Y
~aminoethyl)-y-aminopropyl-trimethoxy silane and chloropropyl triethoxy silane. Particularly preferred c .~

~3~ 253~

are silane coupling agents such as 3'-glycidoxypropyl trimethoxy silane having a first alkoxy silane group and a second epoxide group.

Amines Amines suitable for covalent binding to the second solid matrix materials of the present invention include those amines capable o~ reactin~ with ketones or aldeh~d~ and nitropru~de materials in the pr~s~nce Oe a ~olvent to pro~uce a detectable color complex.
Suitable amines include primary and secondary pol~amines and primary and secondary lower alkyl amines with from 1 to 10 carbons. Primary amines are preferred although secondary amines are also suitable for methods and procedures of the present invention. Amines are coupled to the second solid matrix materials of the invention by means o~ coupling moieties. $ypically the matrix materials are reacted with silane substituted amine-coupling agent conjugates such as 3'-aminopropyltrimethoxysilane. ~his material will reactwith a suitable matrix material such as silica gel or cellulose to prQduce aminopropyl silica gel or aminopropyl cellulose although the invention is not limited to aminopropyl moieties and other materials are e~ually suitable.

Solid Matrix Materials Suitable solid matrix materials for coupling with nitroprusside salts and for covalent binding to amines include high surface area materials such as silica gels, glass materials such as controlled porous glass, granular cellulosic or agarose based materials, cross-linked dextran polymers, inorganic or organic ion exchanger materials, kieselsur and other silicate materials. Preferred first and second solid matrix materials for the vapor phase devices of the present invention are the high surface area gel materials such as silica gels which are characterized by their high surface area, high flow properties and exceptional dimensional stability. While silica gels of varying sizes and porosities may be used, materials with pore diameters between about 60 and about 1000 angstroms and particle sizes between about 40 and about 400 microns are preferred. Particularly preeerred are silica gel particles with pore diameters between about 100 and about 200 ~ngstrom~ and part.icle ~izes ranging bet:ween about 200 and about ~00 microns. Mo~t preEerred f~or uge as the first solid matrix materials for coupling with nitroprusside salts are diethyl amino (DEA) silica gel particles obtained from Diagnostic Specialties/Separa-tion Industries, (Metuchen, N.J.). The particles are characterized by having particle sizes ranging from about 250 to about 400 microns, mean pore diameters of 130 angstroms; mean surface area of 194 m2/g; mean settle volumes of 1.9 cc/g; and an elemental composition 20 comprising 10.90% C, 0.85% N, and 2.17~ ~. Other suitable first solid matrix materials include diethylaminoethyl (DEAE), aminoethyl ~E), quaternary aminoethyl (QAE) and other weakly or strongly basic ion exchangers on different organic or inorganic supports.
First and second solid matrix materials suitable for the liquid phase detection devices of the present invention include those gel materials generally suitable for the vapor phase detection devices, although preferably with smaller diameters. A preferred DEA-silica gel for use as the first and second matrix materials in the liquid phase assays according to the invention may be obtained from Diagnostic Specialties/Separation Industries (Metuchen, N~). The material is characterized by having particle sizes ranging from about 40 to about 60 microns in diameter, mean pore diameters of 200 angstroms, mean surface area ~3~2~3~

of 180 m2/g, mean settle volume of 1.8 cc/g; and an elemental composition of 10.57% C, 0.82% N and 2.10~ X. Suitable materials additionally include a number of materials less suited for the vapor phase devices of the present invention such as cellulosic materials. Preferred celluosic materials to be coupled with a nitroprusside salt include diethylaminoethyl (DEAE) cellulose and diethylamino (DEA) cellulose. A
pr~erre~ material Eor the second matrix material is amlnopropyl cellul4~e.
~ umerou~ other appropriately substituted materials are suitable as the matrix materials of the present invention. These include:
(A) Natural polymeric carbohydrates and their synthetically modified, cross-linked or substituted derivatives, such as agar, agarose and cross-linked dextran polymers.
(B) Synthetic polymers which can be prepared with suitably porous structures, such as (a) vinyl polymers, such as polyethylene, polypropylene, polystyrene, polyvinylchloride, polyvinylacetate and its partially hydrolysed derivatives, polyacrylates, polyacrylamides, polymethacrylates; (b) copolymers and terpolymers of the above vinyl monomers among themselves and with other monomers; (c) polycondensates, such as polyesters, polyamides and (d) addition polymers, such as polyurethanes or polyepoxides.
(C) Inorganic materials which can be prepared in a suitably porous form, such as sulfates or carbonates of alkaline earth metals and magnesium, e.g., barium sulfate, calcium sulfate, calcium carbonate, magnesium carbonate, or silicates of alkali and alkaline earth metals and/or aluminum and/or magnesium, and aluminum or silicon oxides or hydrates, such as clays, alumina, talc, kaolin, zeolite, silica gels and glass such as controlled porous glass. These materials can be ~3~ 2~3~

used as such or as fillers in one of the above polymeric materials.
(D) Mixtures or copolymers of the above classes, such as graft copolymers obtained by initiating polymerization of synthetic polymers on a pre-existing natural polymer.
The following examples disclose methods for the production of the solid matrix materials of the invention.

In thl~ example, DEA~ silica gel was prepared according to the procedure described by Kundu, et al., J. Lipid Res., 19, 390-395 (1978). According to this procedure 100 grams of silica gel which was obtained from Diagnostic Specialties/Separation Industries (Metuchen, NJ) was deareated under vacuum for 30 min.
and then heated at 45C for 20 hours with a mixture containing 1000 ml of 10% 3'-glycidoxypropyltrimethoxysilane (Polyscience, Inc.,Warrington, PA) and 100 ml of N,N-diethanolamine (Aldrich Chemical Co., Milwaukeel WI). The reaction mixture was allowed to cool to room temperature. It was filtered through a coarse-porosity aintered glass funnel and washed with 2 liters of-methanol to remove unbound rea~tants and by-products. The silica matrix was then converted to the chloride form by treatment with hydrochloric acid until the pH became 4.5.
According to an alternative procedure described by Roy and Kundu, Anal. Biochem., 98, 238-241 (1979), 100 grams of silica gel is heated with 1000 ml of 10% 3'-glycidoxypropyltriethoxysilane in toluene at 60C for 15 hours. After cooling to room temperature, the reaction mixture is ~iltered and washed with 2 liter of acetone and dried under vacuum to yield epoxy silica gel. The epoxy silica gel (100 g) is heated with 1000 ~3~ ~3~

ml of 10% diethylamine (Sigma Chemical Company, St.
Louis, MO) in toluene at 50C for 20 hours.
Diethylamino (DEA) silica gel thus obtained is processed to the chloride form as described above for DEAE-silica gel~.
The DEA-silica or DEAE-silica gel prepared according to the above procedure is then treated with sodium nitropru~slde alone or sodium nitroprusside mixed with magnesl~m or c~lcium sul~ate to orm nitroprusside D~ or DEAE-~illc~. ~ccording to one procedure, one hundred gram aliquots o DE~ silica gel were then t~lcen in dark bottles and each mixed with one liter of aqueous solution of sodium nitroprusside at concentrations of 2 g/liter, 4 g/liter, 5 g liter, 6 g liter, 8 g/liter and lO g/liter. The mixtures were rotated in the dark for 5 minutes, filtered on coarse-porosity sintered glass funnels and dried thoroughly under vacuum.
Alternatively, the DEA or DEAE-silica materials were additionally treated with nitroprusside at concentrations of 2 g/liter, 4 g/liter, 5 g/liter, 6 g/liter, 8 g/liter and 10 g/liter mixed with equimolar amounts of magnesium sulfate. The mixtures were then rotated in the dark for 5 minutes, filtered on coarse-porosity sintered glass funnels and dried thoroughly under vacuum.
The total binding capacity of this preferred DEA silica gel matrix was lO0 mg nitroprusside per gram of matrix. The binding efficiency of sodium nitroprusside alone or when mixed with equimolar amounts of magnesium sulfate was 100% for materials treated with nitroprusside at concentrations of 2 to 5 g/liter, 98%
for materials treated with 6 g/liter, 96% for 8 g/liter and 90% for 10 g/liter. Because the nitroprusside-DEA
or DEAE silica gel matrix is sensitive to light these operations were performed so as to avoid direct exposure to light. Nevertheless, the matrix is stable at room ~3~2~3~

temperature for extended periods if protected from the light.

In this example, aminopropyl silica gel was prepared according to the procedure described by Kundu, et al., J. Lipid, Res., 20, 825-833 (1979). In this method, lO0 grams of silica gel was deareated under vacuum for 30 min. and then shaken at 50C ~or 20 hours with 600 ml o a solutlon comprising 10~ ~by weight) 3'-aminopropyltriekhoxy~ilane (Poly~alence, Inc., Warrington, PA) in toluene. ~he reaction mixture wa~
allowed to cool to room temperature and filtered through a coarse-porosity sintered glass funnel. The gel was washed with 2 liters of methanol to remove unreacted materials and other by-products and then with water.
The material was then vacuum dried and stored at room temperature. The aminopropyl silica in the basic form is stable for a extended periods at room temperature.
Similar procedures may be carried with other silanes containing an amino function. It could be a short chain (Cl-Cl~) or polymeric type amine containing a silane function.
The aminopropyl silica matrix most preferred for use as the second solid matrix material for vapor phase detection devices according to the invention may be obtained from Diagnostic Specialties/Separation Industries (Metuchen, NJ) and is characterized by having a mean particle size ranging from about 250 to about 400 microns; mean pore diameter of 130 angstroms; mean surface area of 194 m2/g; mean pore volume of 0.63 m3/g;
and an elemental composition comprising 6.67% C, 2.42% N
and lo 64% H.

~2~3~

In this example, aminobutyl silica gel was prepared utilizing epoxy silica gel prepared according to Example 2. One hundred grams of epoxy silica gel was deareated under vacuum for 30 min. and then shaken at 50C for 20 hours with 600 ml of a solution comprising 10~ (by weight) 1,4-diaminobutane (Aldrich Chemical Company, Milwaukee, WI) in toluene. The reaction mixture wa~ allowed to cool to room temperature, wa~
ilt~red through a coar~e-poroslty ~intered glass ~unnel and washed successively wi~h 1 liter of toluene, 2 liters of methanol and 2 liters of water. The mixture was then vacuum dried and was stored at room temperature.
Similar procedures comprising opening of an epoxy silica matrix with diamines may be carried out with any short chain (Cl-C10~ or polymeric amines. The opening of epoxy silica can also be extended by ammoniacal toluene or aqueous ammonia to generate a primary amine. In addition, the epoxy silica gel may be used as an intermediate to form secondary amine function with any short chain or polymeric secondary amine.
Similarly, short chain or polymeric compounds containing tertiary amine functionalities may be used to produce a silica matrix with a tertiary amine structure.

In this example, test matrices were formed by mixing varying amounts of nitroprusside-DEAE silica produced according to the methods of Example 1 with aminopropyl silica produced according to the methods of Example 2. The matrices comprised varying amounts of nitroprusside ranging from 20 to 90 mg nitropru~side per gram of matrix. The matrices were prepared using DEA-silica, characterized by particle sizes ranging from 200to 400 microns and average pore diameters of 130 , . .

131~3S

angstroms obtained from Diagnostic Specialties/
Separation Industries as described in Example 1.
Numerous materials were evaluated inclucling those with varying ratios of nitroprusside-DEA silica as described in Example 1 as well as a preferred aminopropyl silica material with particle sizes from 200-400 microns and average pore diameters of 130 A (angstroms) obtained from Diagnostic ~pecialties/Separation as described in ~xa~ple 2.
L0 Makerials ~uitable ~or a vapor as~a~ d~v1ces according to the invention include various commercially available DEA or DEAE silica matrices of particle sizes ranging from 40 to 60, 60 to 100 and 100 to 200 microns with average pore diameters of 200 angstroms (Diagnostic Specialties/Separation Industries, Metuchen, NJ) as well as those prepared as described in Example 1. These materials were evaluated by mixing with different ratios with aminopropyl silica matrices of the same particle size, i.e., 40 to 60, 60 to 100 and 100 to 200 microns and same pore diameter, 200 angstroms obtained from the same commercial source as well as alkyl silica matrices prepared as according to Examples 2 and 3. Evaluation of materials with varying matrix ratios of DEA or DEAE-silica and aminoalkyl silica showed that the most preferred composition for detection of acetone vapor samples comprised a first solid matrix material of porous DEA-silica gel particles with diameters ranging from 250 to 400 microns and average pore diameters of 130 angstroms. The preferred matrix comprised DEA
silica particles containing 50 mg nitroprusside per gram of matrix (optionally associated with magnesium) and aminopropyl silica at a ratio of 1:2 (by weight).
The preferred composition for detection of ketones and aldehydes in a~ueous media utilizes porous DEA- or DEAE-silica gel particles with diameters ranging from about 40 to about 100 microns with average pore ~2~36 diameters of about 200 angstroms. The pre~erred first solid matrix ma~erial utilizes DEA-silica materials with particle diameters of from about 40 to about 60 microns and average pore diameters of about 200 angstroms and may be obtained from Diagnostic Specialties/Separation Industries (Metuchen, NJ). Aminopropyl silica particles with diameters ranging from about 40 to about 60 microns and having average pore diameters of about 2Q0 angstroms may be obtained ~rom Diagnostic Specialties/Separation Indu~trie~ ~nd are the mo~t preeerred second ~olld matrl~ materlals. The most p~e~erred reactlon matrix for detection of acetoacetic acid in aqueous solution~
comprises the above described nitroprusside DEA silica and aminopropyl silica materials at weight ratios of l:l with the DEA silica containing 20 mg nitroprusside per gram of matrix.

In this example, nitroprusside-DEAE cellulose was formed according to the following procedure. Ten grams of DEAE cellulose powder (Sigma Chemical Company, St. Louis, M0, Cat. # D-8632) was reacted with 100 ml o~
aqueous sodium nitroprusside solution at a concentration of lO g/liter. After mixing for 10 minutes in the dark at room temperature, the mixture was filtered on a sintered glass funnel, washed with 500 ml of water and dried thoroughly under vacuum. The nitroprusside-DEAE-cellulose powder was then stored at room temperature, protected from light and was stable for an extended period of time.

In this example, aminopropyl cellulose was formed according to the general procedure described in Example 2. Ten grams of cellulose powder (~hatman Chemical Separation Ltd., U.K.; Microgranular CC 41, 13~ 2~

Cat. # 4061-050) was deareated under vacuum for 30 minutes. The material was shaken at 50~ for 20 hours with 100 ml of a solution comprising 10% 3'-aminopropyltriethoxy~ilane (Polyscience Inc., ~arrington, PA) in toluene. The reaction mixture was then allowed to cool to room temperature, was filtered, washed successively with 400 ml of methanol and water and was vacuum dried. It was ~tored at room temperature and i~ ~table ~or an extended period of ~ime.
EX~MPLE 7 In this example, test matrices were formed by mixing varying amounts of nitroprusside-DEAE cellulose produced according to the method of Example 5 with aminopropyl cellulose produced according to the method of Example 6 or aminopropyl silica produced according to the method of Example 2. The matrices so formed were tested by treatment with urine samples to which specific concentrations of acetoacetic acid had been added.
Acetoacetic acid present in the test samples reacted with the nitroprusside salt and the amine present on the solid matrix materials to form a color product. The results ind~cating the sensitivity of the various materials are shown in Table 1 below.

; 30 3 ~

a~ ~
Q~ra E~
O _ J~ ~ _ ~ ~ ~ ~ ._ ~
C: S S ~ r,,, S
~U ~~ ~ 0 0 0 4 E~- r E~ r ~ m o ~: ~ s s ~ s s s ~ o r~
r-l ~ U ~ .J.~ ~ ~ ~ tP 0 r~r--l .rl C aJ (~ ~1 rl ~1 .-1 ~1 ~1 U~ U3 ~ ~ U~
E~~It r-l ~ r-~ ~ ~ ~ ~ ~ ~ ~ ~ ~: ~ O
r~ O ~ ra rc~ U CO 1~3 4 ~, ~0 c o o 8 o o o 0 c~ r; 111 ~ ~ ~ ~ C!) ~ p, pl ~ h ~ , o ,~ 0 p~ .U
~ 0 I E3 C.
~ ~ ~ O
~ Uo~ ~,w,~o ,~

.rl :~ U ~ (~ (~ O U- ~
Hr~U r ~ Z ~3 U rX0 4 ) U~ ~ r~ ~U ~1) ~1~1 r~lI ~ r~l ~ O ~ ~ C

~- 0 r--l ~ r ~ ~¢ rl ~; DX C 3 r-t r; r-l ~1 H r; r;~ ~H r~P Ul E~ ~ r H ~z ra ~J ~ ~ 11~ ~1 ~ ~r Z ~~ (~1 O (~1 r-l ra U t) ~rl U C rl .~i t~5 ro ra 3 0 rl r~l X ~ -~C a o u ~.q .~ ~ ~,) u~ ra ~ IC C r-l U rl U~ D ~S 3 H ~ ie r ~ ¢ ~ a.
a ~ o a 3 U~ 3 0 ~: 3 ~ .. ~a .. ,~ o ~ ~ u O o H U -K U ~ Ul V O C ~ ~ O U~ r 1 P~ U r~l ~c r l lt H3 ~ .L~ . r~ ra U Ql r-l a~
H rl ~.~ a~ ~C U~ V C r~
1 1 a) a~ 0~ U ro r-1 ~-r~ 3 r~
V~ r1 ~ r--lr--l r--l r-l r-lU~ 0 0 D --1 a) ~
1> 0 ~ I ~ Ql r~U Pl a I or-l O r l O a~ ~ O O ro r-l ~ ) Z t~ ~) rl O ~1 ¢ Q~
v ~ 10 Ul U~ O O O r-l ~ ~-r1 Cl C
1~ rl3 rl 3 -- u~ c 0 o v a ~ a~ a ~ ¢ u ~ o ~ ~ z z z z ~ ~ ~ c K ~ ~ ~c ~3~ 2~6 Analysis of the test results indicates that the combination of nitroprusside DEA silica with aminopropyl silica at a weight ratio of 1:2 provides the - best sensitivity. The combination of nitroprusside-DEAE-cellulose and aminopropyl silica provided good results. It was observed, however, that the mixture of nitroprusside--DEAE cellulose and aminopropyl cellulose had relakively poor sen~itivity, pos~ibly due to lower reactlviky o aminopropyl cellulo~e. With Le~ than a 5 mg/dl urine acetoacetlc acid concentration, it wa~l ~ound dif f icult to read the color.
Sensitivity of the matrix can be enhanced by increasing the number of amino groups present. This may be accomplished by reaction of cellulose based matrix with epichlorohydrin to form the intermediate "epoxy cellulose." The epoxy ring present in the intermediate may then be opened with alkyl diamino compounds as disclosed in Example 3, with the result that the sensitivity can be enhanced. To illustrate this, nitroprusside-DEAE cellulose matrix was mixed with ; aminopropyl silica (the number of amino groups in aminopropyl silica was at least 5 times less compared to aminopropyl cellulose) and S mg/dl of urine acetoacetic acid could be easily detected, the sensitivity being as 25- good as that of the silica matrices. The preferred composition for detection of urine acetoacetic acid comprised nitroprusside-DEAE cellulose and aminopropyl silica at a weight ratio of 1:4 with DEAE cellulose containing approximately 5 mg nitroprusside per gram of matrix as shown in Table 1.

In this example a nitroprusside-DEAE-aminopropylsilica bifunctional gel matrix was prepared wherein both the nitroprusside and amine functiona:Lities were coupled to a single solid matrix material. Epoxy 13~2~3~

silica gel was prepared by reaction of 3l-glycidoxy-propyl triethoxy silane according to the method of Example 1. The epoxy silica gel was then reacted with a mixture comprising 1,4-diaminobutane and diethylamine at S a molar ratio of 20:1, in toluene at 50C ~or 20 hours. The reaction mixture was then cooled to room temperature and filtered on a coarse-porosity glass funnel, e~hau~tively wa~hed with ~oluene and methanol and dried under ~acuum ~or ~ hours.
Xt w~ then necesqary ~b~cause ni~ropru~ld~
reacts with Eree amine) to protect the amino group by reaction with trifluroacetic anhydride and ethylacetate at a 1:1 volume ratio at room temperature for 24 hours. The matrix was washed exhaustively with lS ethylacetate and methanol and dried in air.
Nitroprusside was then incorporated onto the DEAE
functionality by treatment of 10 grams of matrix with 100 ml of 10 g/liter sodium nitroprusside in water in the dark for 5 minutes as described according to Example 1.
The matrix was then filtered, washed thorouyhly with water and dried under vacuum for 4 hours. The trifluroacetyl group was removed from the amino function by reaction with solid anhydrous potassium carbonate suspended in dry methanol ~-hile the pH of the reaction medium was maintained between 8.0 and 8.5 to prevent cleavage of the nit.oprusside group from the DEAE functionality. The reaction mixture was mixed at room temperature in the dark for 8 hours with the pH
monitored each hour and potassium carbonate added as needed to maintain the pH. The matrix was then filtered on a coarse porosity glass funnel in the dark, washed exhaustively with water and dried under vacuum for 12 hours.
The bifunctional gel demonstrated a sensitivity limit of 10 ng o~ liquid acetone or ~ 3 ~

- ~2 -0.17 nM. Because the partition coefficient of liquid and vapor state of acetone is approximately 330, 10 ng of liquid acetone would be equivalent to 3300 ng ~58 nM) of acetone vapor. While the bifunctional matrix may not be suitable for detection of very low levels of acetone, it can be used to measure acetone vapor concentration of highly ketotic individuals (dieting or fasting) or monitoring breath acetone of insulin dependent type 1 diabetic pa~ients whose breath acetone concentrations can b~ a~ high a~ 100-200 n~.

V~POR TEST DEVICES

The devices of the present invention are suitable for the detection of ketone and aldehyde analytes in both liquid and gaseous (vapor) forms.
Vapors which may be analyzed for the presence of such analyes include atmospheric air, laboratory and industrial vapors, breath and other vapors for which the quantitative analysis of ketone or aldehyde content i9 desired. A particular aspect of the present invention comprises methods and devices for the quantitative detection of acetone in human breath. Such detection is useful for the monitoring of serum acetone levels whlch is of value to diabetics in monitoring the onset of ketosis. Such detection is also useful according to a further aspect of the present invention relating to the quantitative monitoring of fat catabolism.
Referring to the drawing, Figure 1 depicts a vapor test device (10) for the detection of ketone or aldehyde analytes present in a vapor sample comprising a length of inert cylinder (11) having a first end ~12) at which a vapor sample may be introduced to the device by a sample means and a second end (13) at which vapor is exhausted ~rom the device. Within the inert cylinder (11) and progressing from the first end (12) to the 13~2~6 second end (13) are a first porous barrier (14), a second porous barrier (15), a third porous barrier (16) and a fourth porous barrier (17). The first porous barrier (14) and the second porous barrier (15) define a pretreatment zone (18) filled with desiccant means. I'he second (15) a~d third (16) porous barriers define a reaction zone (19) filled with a first solid matrix material to which a nitroprusside salt is coupled and a second solid matrlx ma~erl~l to which an amine is covalently bound. ~h~ reaction zone ~19) may also comprise an axially aligned ille~ rod (22) which fi~ls space within the reaction zone (19) and increases the length of the device over which a fixed volume of the first and second solid matrix materials are spread.
Said third porous barrier (]6) and said fourth porous barrier (17) define a solvent zone (20) in which is located a solvent ampule (21).
According to a procedure for use of the device, a fixed volume of sample vapor is introduced to the first end (12) of the device by suitable sample means and is allowed to flow through the length of the device before it is exhausted from the second end (13). As sample vapor flows through the device, water vapor present in the sample is adsorbed by desiccant means present in the pretreatment zone (18). Dehydrated vapor then flows through the reaction zone (19) where ketones and aldehydes present in the vapor are adsorbed by the first and second solid matrix materials.
Analytes are first adsorbed at the end of the reaction zone (19) adjacent to the first porous barrier (14) closest the first end (12) but as the volume of vapor sample is passed through the device the solid matrix materials closest the first end become saturated and additional analytes are adsorbed onto the solid matrix materials progressively farther from the first end (12). Where the volume of the sample is fixed the ~3~3~

distance to which the analytes are adsorbed into the reaction zone (19) may be directly correlated with the concentration of analytes present in the sample.
When the volume of sample vapor has been passed through the device, the solvent ampule t21) is broken allowing a volume of solvent to plass downward through the third porous barrier (15) into the reaction zone (19) where the first and second solid matrix materials are thoroughly wetted with the solvent. The nitroprusside salt which has been coupled to the first solid matrix material and the amine which has been covalently bound to the second solid matrix material then react ln the presence o~ solvent with ketones anct aldehyde~ adsorb~d onto the ~olid matrix mate~ials to lS ~orm color~d ~eactlon products which form ~ colored bar in the device. These reaction products p~ovide a visual signal indicating the areas of the reaction zone where vapor ketones were adsorbed and thus the concentration of analytes present in the sample.
~eferring to the drawing, Figure 2 depicts an alternative vapor test device ~30) for the detection of ketones and aldehydes in a vapor sample comprising a length of inert cylinder (31) having a first end (32) at which a vapor sample may be introduced and a second end 2S ~33) at which vapor is exhausted from the device.
Within the inert cylinder (31) and progressing ~rom the first end (32~ to the second end (33) are a first porous barrier (34~, a second porous barrier (35) and a third porous barrier (36). The eirst porous barrier (34) and the second porous barrier (35) define a preadsorbent zone (37) which is filled with an adsorbent matexial capable of selectively adsorbing ketone and aldehyde analytes. The second porous barrier (35) and third porous barrier ~36) deeine a reaction zone (38) Eilled with a first solid matrix material to which a nitroprusside salt is coupled and a second solid matrix material to which an amine is covalently bound.

. ~ ., ~3~3~

According to a procedure for use of the device, a fixed volume of sample vapor is introduced to the first end t32) of the device by suitable sample means and is allowed to flow through the length of the device before it is exhausted from the second end ~33~. Ketones and aldehydes present in the sample vapor are selectively adsorbed on the adsorbent material present in the preadsor~ent zone (37). When the volume of sample vapor has been passed through the device a quantity of solvent is introduced to the first end ~32 of the device which then desorbs analytes adsorbed in the preadsorben~ zone (37) and transports them to the reaction zone (38). ~here the analytes react in the presence o~ the solvent wlth the nit~oprusslde ~alt3 coupled to the ~irst ~olld matrix material and the ~mlne covalently bound to the second solid matrlx mater~al to form a colored reaction product.

Analyzer Column The analyzer column comprises an inert cylinder fabricated from a material which will neither react with nor adsorb ketones or aldehydes and which is nonreactive with the reagents utilized in the assay.
The column material~ are preferably transparent in order that the presence of a color reaction product may be detected and evaluated. Preferred materials include transparent plastics such as polystyrene and polyethylene terephthalate. Glass tubes are acceptable but columns fabricated from polyethylene terephthalate are particularly preferred. Where the device is one according to Figure 1, the cylinder is preferably somewhat flexible in order that pressure may be applied to rupture the solvent ampule. Alternatively, where the solvent ampule comprises a readily puncturable end such as one of metal foil, means such as a plunger means may be incorporated with the device to free solvent rom the A

1312~3~

ampule. The analyzer column may~be of generally any size and geometry selected to contain sufficient reagents to analy~e a sample of a selected size.
According to one embodiment the column comprises a polyethylene terephthalate cylinder 13 cm long with an inner diameter of 0.8 cm. The cylinder is divided by means of three 0.5 cm porous barriers defining pretreatment zone 1.3 cm long, a reaction zone, 3.0 crn long and a 4.Q cm long solvent zone containing a solvent ampule. Optionally pre~ent in the reaction zone i~
iller means whlch may be an a~ially cent~red ro~ which can be used to eill a portion o~ the volume oE the reaction zone and thereby elongate the length of the zone filled by a Eixed volume of the first and second solid matrix materials. The rod itself may be transparent or opaque and should be inert to the analytes and reagents of the invention. It is preferably fabricated from polystyrene, being polyethylene terephthalate, or polypropylene with polypropylene being particularly preferred.
!

Porous Barriers Porous barrier materials suitable for use with devices of the present invention include those materials which~are inert to and nonreactive with the analytes and reagents of the invention and are porous with respect to the passage of vapor samples and solvents utilized in such devices. Suitable materials include various porous materials such as nylon fabric, glass wool, sponge, styrofoam and other ceramic and plastic materials.
Preferred materials for use with the vapor phase devices of the present invention are porous polyethylene frits with a pore size of 100 microns (Porex Technologies, Fairburn, GA).

~3~2~

Preadsorbent Materials Preadsorbent materials suitabLe for use with the present invention include those materials which are capable of selectively adsorbing ketone and aldehyde analytes from vapor samples. Such materials should also readily and completely desorb such analytes in the presence of preferred solvents of the invention such as methanol and methanol with dimethylsulfoxide ~DMSO).
Suitable materials include activated silica gel. A
particularly preferred material is Tenax TA (a trademark of Enka N.V., Arnham, Netherlands) a 2,6-diphenyl-p-phenylene oxide polymer.

Deslccant Means Vapor analyzer devlces according to the present invention which are designed foc the analysis of vapors containing water vapor, particularly breath, require means for the removal o~ water from the vapor sample prior to reaction with the assay matrix. This is particularly so where the material analyzed for is acetone, as the presence of water substantially reduces the rate of reaction of acetone with the nitroprusside and amine reagents of the invention. While a variety of desiccant materials are available which are capable of pre-drying vapor samples, it is desired to utilize a material which is inexpensive, safe and will not adsorb or react adversely with the ketone or aldehyde components Oe the vapor samples. Materials such as granular silica, anhydrous caicium sulfate, molecular sieve type 3A or 4A (W.R. Grace, aaltimore~ MD), magnesium perchlorate, activated charcoal, Bio-beads SM-2 and SM-4 (styrene-divinyl copolymers obtained from ~io-Rad Laboratories, Richmond, CA) are generally undesirable materials becaus~ of their tendency to adsorb acetone. Ascarite II~16-20 mesh) (obtained from Thomas Scientific Swedesboro, NJ~ is an excellent ~, ~.3~2~3~

desiccant and does not adsorb acetone but comprises sodium hydroxide silica particles and is corrosive and unsuitable from a handling standpoint.
A preferred material is anhydrous calcium chloride in pellet form. Various commercially available materials are suitable, although a particularly preferred material may be obtained in pellet form from Dow Chemicals, Ludington, Michigan as type 94 XFS~
43284. The material may be fractured using a Fritzmill size reduction instrument (Fritzpatrick Company, Elmhurst, IL) and sized to between 16 and 24 mesh. The pre~erred calcium chloride granules are then heated at 200C for 20 hours and are stor~d in a closed bottle.
The amount o~ mois~ur~ permitted eor the pr~cred calcium chlo~ide i~ between 0-0.5~. The amount o~
calcium chloride needed ~or removal of molsture from vapor samples may readily be determined by one oE skill in the art. The amount of calcium chloride required for removal of moisture from a 450 cc breath sample is between 120 and 180 mg.
It has been established that 150 mg of calcium chloride (Dow 94 XFS, 16-24 mesh, 0-0.4~ moisture) adsorbs 100~ of moisture from a 450 cc breath sample.
The amount of moisture in a 450 cc breath sample is 4.90 ~ 0.99 (SD, n = 56). The recovery of acetone vapor from 450 cc breath samples (n = 20) using same amount of calcium chloride is also 100~. It is important to note that if the amount of anhydrous calcium chloride exceeds about 200 mg, there is a tendency to adsorb acetone from 450 cc breath samples. Other types of calcium chloride such as Type P 90 (Dow Chemical) obtained in pellet form and fractured to the desired 16-24 mesh size can also be used to remove moisture from breath and other vapor samples. To reduce the moisture content of P 90 calcium chloride to less than 0.5%, it must be heated at 200C
for at least 48 hours. The material may then be stored in a closed container until needed.
jA
t ~

13~ 253~
~ 49 -S~mple Means The devices of the present invention for the quantitative detection of ketones and aldehydes in vapor samples require means for the introduction of a fixed quantity of vapor sample to the detectic,n column.
Suitable means are those which comprise materials which are inert with respect to the ketone ~amples and are capable of reproducibly delivering a fixed volume of sample vapor to the device. It is desired that vapor samples be introduced to the devices of the invention at a relatlvely steady rate in order that analytes present ~n the sample be ~eproduclbly ad~orbed on the Eir~t and second solld ~atrix materials o~ the reactlon zone.
Unst~ady vapor flQw ~nto the devices may cause analytes to be unevenly adsorbed onto the matrix materials o~ the device with the consequence that inconsistent and unreproducible analyte concentrations would be indicated by the assay devices.
Balloons and ba~s are particularly suitable for such applications although it is necessary that the material from which the bag or balloon is constructed be inert to the ketone and aldehyde materials of the ~ample. It was found that rubberized films and polyvinyl films adsorbed greater than 25% of acetone present in a breath sample ln ten minutes. Fi1ms found to be suitable included those fashioned frorn nylon, teflon, very low density density polyethylene, and a copolymer of polyester with polyvinyl chloride/vinylidene chloride (Saran). Bolton, et al., U.S. Patent No. 4,579,826 describes methods and devices for sampling of predominantly alveolar breath.
Bolton, et al. specifically discloses one device comprising a non-self-supporting polymeric tu~e and a spring means effective to roll the tube upon itself in spirai fashion toward the mouthpiece unit.

~.., ~ .

1~1%~3~

Particularly preferred due to high permeability of wa~er vapor and its durability and 1QW
cost is the use of bags of 1 mil thick nylon. According to one embodiment, a nylon bag with a capacity of 450 - 5 cubic centimeters iB attached to a valve device comprising a column, a mouthpiece, and a plunger. With the plunger set in one position the test subject takes a deep breath, holds it for five seconds and blows a breath sample into the device at a steady rate until th~
sample bay i~ completely in~lated. The plunger i~ th~n pu~hed down to an altern~te position and the ~ample bag i~ steadily deElated by a ~pring means blowing the sample vapor through the device and contacting analytes with either the preadsorbant bed or the nitroprusside and amine treated first and second matrix materials.
Where the material to be sampled is atmospheric air or an industrial or laboratory vapor sample, a sample port may be substituted for the mouthpiece. Vapor samples may be collected by a bellows or other suitable means and appropriate volumes of material introduced to the device.
With reference now to ~igures 22-39, one embodiment of a breath sampling kit for practicing a method of the present invention will now be described.
25 As best illustrated in Figures 22, 23 and 26, a breath-sampling kit (50) includes a portable housing (52) characterized by: an elongated base (54) having an inverted U-shaped portion (56) open at its right end ~as viewed in Figure 23) and having an integral depending but upwardly opening U-shaped portion (58) at its left end, defining a vertically disposed compartment (60) open at its right end but including an end wall (62) at its left end having a circular opening (64) provided therein; a breath-sampling assembly (66) which is fitted in the base (54) and retained therein by a cover (68) which is characterized by an elongated U-shaped portion lL3~L2~3~

~70) having a vertically disposed end wall (72) at its left end (as viewed in Figure 23) which is adapted to overlay the end wall ~62) of the base (54); and an end cap (74) which fits over the closes the right end of the housing (52) in a suitable manner. The meeting edges (76) and (78) of the U-shaped portions (56) and (70) of the base (54~ and the cover (68), respectively, are designed for interfitting engagement in a known manner~ The kit (50) also includes a breaker button (8~) which is di~posed in an opening ~2) provided in a ~ide wa.ll oF the hou~lng base (5~) Eor a purpo~e wh.ich will be described in detail hereina~ter. A slightly raised marker (84) depends from the bottom oE an opening (64) on the outer surface of the end wall (62) of the compartment (60) for a purpose which will also be described hereinafter.
As best illustrated in Figures 23, 24, 27 and 28, the breath-sampling assembly (66) is characterized by: an elongated outer tubular member or blow tube (86) which is formed of a transparent inert plastic material and which has an opening (88) formed in the side wall thereof toward the bottom of the tube (86); an inflatable/deflatable air or breath collecting plastic bag (90) having a specific volumetric capacity and having an open end secured to the outer surface of the tube (86) and in communication with the opening ~88) provided therein, the opposite end of the bag (90) being closed and secured to a rigid support member ~92) which is disposed generally parallel to the tube (86); a valve housing (94) which is secured in a valve housing recess (96) provided in the lower end of the tube (86); a ball valve (97) which may be formed of polypropylene and which is disposed in an axial recess (98) provided in the inner end of the valve housing (94~ and; an O-ring (lO0) which is retained against downwardly facing O-ring seating means (102) provided in the tube (86) by the .

~3~253~

inner end of the valve housing (94). As best illustrated in Figures 37, 38 and 39, the inner end of the valve housing (94), which is reduced in diameter relative to the outer end, i5 provided w.ith a diametrically extending bore tlO4) which is axially aligned with the opening (88) in the blow tube (86) to the bag (90) and with a pair of diametrically opposite vertically extending slots (106) adjacent to but ~paced ~rom the inner end of the valve housing (94) with a lower valve ~eat ~108) for the ball valve (97) be.in~
defined in the rece~s (98) above the lower end oE the slots (106) for a reason which will be made clear hereinafter. It is also noted that the diameter of the ball valve (97) is somewhat greater than the inner diameter of the O-ring (100).
With reference to Figures 24, 25, 35, 36, 37, 38 and 39, it is noted that the inner surface of the blow tube (86) is provided with four equidistantly spaced, longitudinally extending ribs (110) and (112).
Three of the ribs (110) terrninate short of the upper end of the blow tube ~86) by a distance approximately equal to 1/5 of the overall length of the tube (86). The fourth rib (112) terminates almost at the upper end of the tube (86). All four of the ribs (110) and (112) converge inwardly at their lower ends to define the O ring seating means (102). As shown in Figure 25, the upper end of the rib (112) is provided with a stepped configuration to define a first holding surface (114) for the analyzer column and a second positive stop surface (116). An external annular rib (118) at the upper end of the tube (86) limits insertion of the blow tube (86) into the opening (64) provided in the end wall (52) of the housing compartment (60). The blow tube (86) i9 also provided with a button hole (120) which is adapted to be radially aligned with the breaker button opening (82) in the housing base (54) when the breath-~3~2~

sampling assembly (66) is fitted in the housing base (54).
.~ disposable analyzer column (122) is provided for use with the breath-sampling assembly (66) described herein. As best illustrated in Figures 26, 27, 29 and 32-39, the disposable analyzer column (122), which is formed of a transparent inert plastic having a degree of flexibility as previously noted herein, is characterlzed by a tubular member (12~) having a series o~ axially ~paced zone~ which will be deined hereinafter. ~'he tubular member (12~) ha~ an upper mouth piece po~tion (126) o a diameter which i~ receivable in the open end of the blow tube (86) seated in the opening (64) in the housing base ~54), which opening is exposed upon the removal of the cover ~68). The tubular member also has ; a first reduced-diameter portion (128), which comprises a major portion of the length of a column (122), and a second further reduced-diameter end portion (130) of a relatively short length. An annular shoulder (132) is deEined between the two reduced-diameter portions (128) and (130) and an inclined or frusto-conical wall segment (134) is defined between the mouth piece portion (126) and the first reduced-diameter portion ~128). For a purpose which will be discussed hereinafter, a series of air or breath openings (136) are provided in the inclined wall segment (134). The openings (136) preferably are equidistantly, circumferentially spaced around the wall segment (134) as illustrated in Figure 34. Further, as illustrated in Figures 26, 27, 34 and 35, a pair of diametrically opposite longitudinally extending flats ~138) are provided on the outer surface of the mouth piece portion (126), which flats (138) extend from the inclined wall segment (134) for a distance approximately equal to 1/2 of the length of the mouth piece portion (126), at which point inwardly facing shoulders (I40) are defined.

13~2:~G

Within the analyzer column (122), from the lower end to the upper end as viewed in :Figure 26, there is provided a first porous barrier or frit filter (142) which is secured in the end of the second reduced-diameter end portion (130); a second porous barrier orfrit filter (144) which is seated against the annular shoulder (132) defined between the two reduced-diameter portions (lZ3) and (130); an inert Eiller rod ~146) havlng a longl~udinal slot (148) formed therein which i~
~ea~ed agalnst the ~ilter (1~) in the eir~t red~lced-diameter portion (128); a thlrd porous barrier or ~rit filter (150) which is seated against the opposite upper end of the filler rod (146); a breakable ampule (152) containing liquid solvent or reactant disposed in the first reduced-diameter portion (128) above the third filter (150); and a fourth porous barrier or frit filter t154) which is provided in the first reduced-dia~eter portion (128) above the ampule (152). Each of the porous filters or frits is held in place by friction or raised projections on the inner surface of the analyzer column (122).
With reference again to the axially spaced zones of the analyzer column (122), a pretreatment zone (156) is defined in the second reduced diameter end portion t130) between the first and second porous barriers or filters ~142) and ~143). The pretreatment zone (156) is filled with a suitable desiccant means (158) such as CaC12. A reaction zone (160) is defined between the second and third filters (144) and (150) in that the longitudinal slot (148) in the filler rod ~146) is filled with one or more solid reactive materials (162) as described elsewhere. A solvent zone (164) in which the ampule (152) is disposed is defined between the third and fourth filters (150) and (154). As previously noted, herein the filler rod (146) fills a substantial portion of the space withln the reaction ~ 3~2~3~

zone (160) and thus increases the length of the zone (160) over which a fixed volume of a solid reactive material or materials (162) is spread. .~s best shown in Figures 27, ~9, 32 and 33, indicia markings (166) may be provided on the filler rod (146) adjacent to slot (148).
Beeore describing how the breath-sampling kit (50) is used to collect and test a sample of a person's breath, reerence is first made to Flgures 22, 23, 24 and 31, showing th~ breaker button (80), and to Flgure~ 32 and 33. ~he breaker button (80) ls characteri2ed by: a manually depre~sable head portion (168) which is freely receivable in the opening (82) provided in the housing base (54); an inwardly projecting stem portion (170) which extends through the opening (120) provided in the blow tube (86) and which is retained therein by an enlarged head (172) on the end thereof; and a coil spring (174) which is disposed about the stem portion (170) between the underside of the breaker button head (168) and the outer surface of the blow tube (86), whereby the breaker button (80) is normally biased away from the blow tube (86). As shown in the drawings, the breaker button (80) is longitudinally positioned on the blow tube (86) such that the stem head (172) i5 aligned with the solvent zone (164~ of the analyzer column (122) when the analyzer column (122) is inserted into the blow tube (86) during a collection and testing procedure. Thus, sufficient inward pressure on the breaker button ~80) forces the stem head (172) against the flexible solvent zone portion (164) of the analyzer column (122) with sufficient pressure to break the ampule (152) and permit the reactant solvent to flow downwardly through the "exposed" reactive material or materials (162) in the longitudinal slot (148) of the filler rod (146).
Obviously, the ampule (152) should preferably be broken at the end most closely adjacent to the reaction zone ~ 3 ~

~160) as shown in the drawings, to ensure a maximum flow of the reactant/solvent. If the ampule (152) were broken at an upper end, it is possible that part of the reactant/solvent would be retained in the unbroken lower portion of the ampule (152) with unreliable test results possibly resulting therefrom.
To collect and test a sample of a person's breath using the breath-sampling kit disclo~ed herein, the cover ~68) is eir~t removed from the portable hou~ing (52) to provide access to the open end o~ the blow tube (86) and also to permit inflation of the collection bag (90). When one o~ the disposable analyzer columns (122) is first inserted into the blow tube (86), it is important that one of the flats (138) on the mouth piece portion (126) be aligned with the raised marker (84) provided on the end wall (72) of the housing base (54). This ensures that neither of the flats (138) will be in initial alignment with the fourth rib (112) which, as can ~e seen in Figure 23, is positioned 90 from the raised marker (84). Therefore, the inclined wall segment (134) engages the second or positive stop surface (116) on the rib (112) to limit further inward movement of the analyzer column (122).
In this rotational and axial position of the analyzer ; - 25 column (122), the breath-sampling assembly (66) is in : its breath-collecting mode wherein the parts are positioned as shown in Figures 27, 28, 34 and 37. In this mode, as best illustrated by the arrows in Figures 27, 28 and 37, the person whose breath is to be tested or monitored blows into the mouth piece (126) with the blown breath passing through the mouth piece (126), through the air openings (136) in the inclined wall segment (134) and longitudinally through the space defined between the inner surface of the blow tube (86) and the outer surface of the first reduced-diameter portion (128) of the analyzer column (122). This is the ~ 3~2~3~

path of least resistance due to the frit filters (154, 150, 144 and 142) disposed within the analyzer column (122). The blown breath proceeds through the O-ring (100) and into the recess (98) provided in the valve housing (94), through the slots (106) around the ball valve (97), through the aligned valve housing bore (104) and the blow tube opening ( sa ) into the inflatable/
deflatable collection bag (90). As illustrated in Figures 27 and 28, the bag (90) 1~ ully lnElated a~ain~t the ~orce of a ~lat coil spring (176) which 1~
adhesively secured to the outer surace of the bag (90) approximately midway between the opposite side edges thereof to provide a known volume of a user's breath.
The spring (176) normally biases the bag (90) into the deflated rolled-up condition as shown in Figure 23.
Once the bag (90) has béen filled to its capacity, the user stops blowing and the bag (90) remains in its fully inflated condition inasmuch as the back pressure of the filled bag (90) forces the ball valve (97) into sealing engagement against the underside of the O ring (100), as is clearly illustrated in the static mode of Figure 38.
To discharge the collected sample of breath or air past the reactive material (162), the analyzer 25 column (122) is first rotated 90 (in either direction) to align one of the flats (138) with the longest rib (112) (see Figure 35) whereby the analyzer column (122) may now be advanced further into the blow tube (86) to its second rotational and axial position, best illustrated in Figures 29 and 39. In this deflation mode, the second reduced-diameter end portion (130) of the analyæer column (122~ passes through and engages the O-ring (100) to seal off the external passage of lesser resistance, as well as forcing the ball valve (97) downwardly against its lower valve seat (108). This action permits the bag ~90) to deflate, at a ~2~3g substantially constant rate due to the spring (176) with the discharged air passing from the bag (90~ through the blow tube opening (88), the aligned valve housing bore (104), the slots (106) around the seated ball valve (97), and finally through the first frit filter (142) into the analyzer column (122). It then passes through the pretreatment zone (156), the reactive zone (}60), the solvent zone (164), and out through the mouth piece (126)l since the les~er resistance pa~sage is bloclced.
~h~ ampule (152) may then be broken by ~ppl~ing lnw~rd pressure on the breaker button (80) as previously described herein.
It is noted that the ball check valve 197) described herein coulcl be replaced by other known type valve arrangements such as a flapper valve. To provide for ai~ flow through the analyzer column (122) in the bag deflation mode when a flapper valve is used, transverse notches are provided in the inner end of the analyæer column (122).
The testing procedure has been thoroughly discussed previously herein in connection with the testing device of Figure 1 and therefore will not be repeated herein.
After the breath-sampling test has been completed, the used analyzer column (122) is disposed of and a new analyzer column (122) can be used with the same kit (50) for a further test.

Solvents Suitable solvents must provide an environment in which ketone and aldehyde analytes may react with the nitroprusside and amine reagents of the invention. In embodiments of the invention where analytes are adsorbed onto preadsorbant materials suitable solvents must be capable of desorbing the analytes and transporting them to the reaction zone. Such solvents inclucle methanol ~3~2~3~

although a preferred solvent mixture comprises dimethyl sulfoxide (DMSO~ and methanol a~ a 1:3 ratio by volume containing 30 mg/ml TRISMA-base. A particularly preferred solvent mixture i5 that compri~ing DMSO and methanol at a 1:3 ratio by volume containing 5 ~l/ml N,N-diethanolamine (DEA~.

In this example, the composition of the color developing solvent was optimized to provide for a dark blue ~for acetone) color signal havlng a high background cQntrast. Accocding to this example, vapor te~t devices wer~ constructed acco~dlng to the gene~al detail~ of Figure 1 comprising te~t matrlce~ according to Example 4. The matrices comprlsed nitroprusside-DEA and aminopropyl silica particles 250 to 400 microns in diameter with average pore diameters of 130 angstroms wherein the particles were present at a weight ratio of 1:2 and wherein the DEA-silica contained 50 mg of nitroprusside per gram of matrix. The devices were tested by administration of a test vapor comprising a 20 nM concentration of acetone in air. Various solvents were tested each comprising DMSO and methanol at a 1:3 ~v/v~ ratio in the presence of optionally substituted ingredients. Two-tenths of a milliliter o~ each of the test solvents was applied to the reaction zone and after five minutes, the height, color and degree of contrast of the color bar was judged with the results presented in Table 2 below. Use of the DMSO and methanol solvent alone gives generally poor results while incorporation of diethyl amine (Et2NH) and triethylamine (Et3N) improves the color and degree of background contrast.
Incorporation of TRIS buffer also results in improved color and background contrast with a preferred solvent mixture comprising DMSO and methanol at a 1:3 (v/v) ratio containing 30 mg/ml TRISMA-base. Most preferably, ~L3~

the solvent mixture comprises DMSO and methanol at a 1:3 (v/v) ratio containing 5 ~l/ml DEA.

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According to this example, vapor test devices were constructed according to the general details of Figure 1 comprising test matrices according to Example 9. Alternatively, vapor detection devices were formecl wherein DEA-silica contained 50 mg of nitroprusside per gram of matrix and was additionally treated with a solution of magnesium sulfate such that the matrix con~alned 2Q mg Oe magn~ium sula~e per gram of matrix.
The devices were then tested utilizing vapor samples containing calibrated concentrations of acetone. Acetone vapor samples ranging in concentration from 15 nM to 500 nM were tested in the devices of the invention as well as against a gas chromatograph ~Shimadzu Model GC-8A, equipped with a heated gas sampler HGS-2 with a flame ionization detector and a chromosorb 102 3% 80-100 mesh). Results of the tests are shown on Table 3 below which confirm the existence of a relationship between acetone concentrations and the ~engths of color bars. The results are highly reproducible and the height of the color bar may be read by an untrained consumer within an accuracy of 1 to 2 millimeters. At lower acetone concentrations, the results show production of darker, easier to read color with the magnesium treated matrices. At acetone concentrations of 200 nM or greater, however, treated and untreated reaction matrices provide similar results.

~31 2~3~

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According to this example, vapor test devices were constructed according to the general details of Figure 1 comprising various combinations of nitroprusside and amine-treated solid matrix materials. Concentrations of nitroprusside associated with the first solid matrix material were varied Erom 30 to 100 mg/gram oE material while the welght ratio oE
nitropru~ide-DEA silica to aminopropyl ~lllca wa~
varied.
According to the results (shown in Table 4, below), optimum results occurred with nitroprusside concentrations of 40 to 50 mg per gram of matrix. A
particularly preferred combination was nitroprusside at a concentration of 50 mg/g with a nitroprusside DEA to aminopropyl silica ratio of 1 to 2 by weight.

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Ketones and aldehydes other than acetone will react with the materials of the present invention.
While acetone reacts with the nitroprusside and aminopropyl silica materials in the present of suitable solvents to produce a blue reaction product, other ketones and aldehydes in the presence of these materials and in the presence of alternative amine materials will react to produce reaction products with di~fering colors. The color of a reaction product thus procluced i~ therefor~ indicatlve of the type of ketone or aldehyde present whlle the length o~ the color bar produced provides a quantitative de~ermination of the concentration of the ketone or acetone.

Head Space Analysis Concentrations of ketones and aldehydes present in liquid samples may be determined utilizing the same methods and materials of the invention used for analysis of vapor. According to well known procedures, however, head space vapor in equilibrium with the liquid sample to be analyzed is collected and analyzed according to procedures for analyzing vapor samples.
Ketone and aldehyde vapor concentrations may be related to liquid sample concentrations through use of known vapor pressure and partition coefficient relationships. Head space analysis is useful for the determination of concentrations of more volatile ketone and aldehyde sample components and is particularly useful for the determination of acetone concentrations in aqueous samples. Detection of acetone in such aqueous samples is otherwise hampered by the interference of water with the nitroprusside/amine color reaction. Vapor collected by head-space analysis may be desiccated according to the methods disclosed above in order to prevent the adverse effects of water on the color reaction.

3 ~

LIQUID ASSA~ l:)EVICES

The present invention also provides methods and devices for the direct quantitative and semiquantitative analysis of ketones and aldehydes present in liquid samples. Assay devices suitable for direct analysis o liquid samples are particularly useeul as he~d space vapor analysis according to the nv~ntion tends ~o be primarily ~uitable eO~ anal~si~ Oe more volatile analytes. Dir~ct li~uid assay devlc:ea according to the invention include microcolumns for capillary adsorption of liquid samples and dipst~cks for dipping into liquid samples. Suitable sample materials for testing according to the present invention include various laboratory and industrial reagents as well as physiologi~al fluids including urine, serum and other materials. Devices according to the invention include a first solid matrix material to which a nitroprusside salt has been coupled and a second solid matrix material to which an amine is covalently bound. This allows the stable formation of color complexes after reaction of the matrix with analytes such as acetoacetic acid. Such stable color formation in turn facilitates the semiquantitative analysis of ketones and aldehydes through colorimetric methods including comparisons with color charts and use of spectrophotometers.
Figure 4 depicts a "microcolumn" assay device (40) suitable for ascending chromatographic analysis of liquid samples according to the invention. The de~ice comprises an inert microcylinder (41) having a first end ~42) and a second end (43). Flush with the first end (42) is a first porous barrier (44). A second porous barrier (45) is flush with or spaced from the second end (43) of the device, while an optional third porous barrier (46) is located in the interior of the device 2 ~ 3 ~

spaced from the first end (42). The first porous barrier (44) and third porous barrier (46) define a diffusion zone, the purpose of which is to slow down the rapid infusion of sample material. The diffusion zone is filled with an inert substance such as cellulose powder which is capable of promoting liquid diffusion.
The second (45) and third ~46) porous barriers define a reaction zone (~8) which comprlses a mixture of ~he first ~olid matrix material to which a nitroprusside ~alt ls coupled an~ a s~cond solid m~trix material to which an amine 1~ co~alentl~ bound.
According to a procedure for use of devi.ce (40) of Figure ~, the device is dipped at its first end (44) in a sample of a liquid to be assayed. The liquid rises through capillary action through the first porous barrier (44) into the diffusion zone (47) and then continues through the third porous barrier (46) into the reaction zone (48). Ketones and aldehydes present in the sample liquid there react with the nitroprusside salt and the amine presented by the first and second solid matrix materials to form a colored reaction product. The sample liquid continues to diffuse into the device (40) and into the reaction zone (48) until the liquid reaches the second porous barrier (45) and capillary transport ceases. After a suitable waiting period for formation of color, the colored reaction product is observed and the concentration of analytes present in the sample liquid is determined spectrophotometrically or by comparison with color charts.

Microcylinders The microcylinders from which the microcolumn devices of the invention are fabricated preferably comprise a material which will neither adsorb nor react with the analytes or reagents of the assay. The ~3~2~3~

microcylinder materials are preferably transparent in order that the presence of a color reaction product may be detected and evaluated. Preferred materials include transparent plastics such as polystyrene and polyethylene terephthalate. Glass tubes are acceptable but columns fabricated from polyethylene terephthalate are particularly preferred.
The microcylinders may generally be of any size and geom~try selected to contain sufficient reagent~ ~o analyze a ~ample of a ~elected size. Column diame~er~ pre~erably range erom about 1.0 mm to about 3.0 mm with preEerred column lengths ranging from about 10 mm to about 40 mm.

Porous Barriers -Porous barriers suitable for the liquid assay devices of the present invention include those porous barrier material suitable for conducting vapor assays including mateEials such as nylon fabric, glass wool, sponge and styrofoam. A particularly preferred porous barrier material for use with the liquid assa~ devices of the present invention is nylon fabric.

EXAMPLÆ 12 According to this example, microcolumn devices of the invention were fabricated by covering microcylinders (1.5 mm by 50 mm) with a piece of nylon fabric at one end. The cylinders were each then filled with an amount of cellulose powder sufficient to fill a 5 mm length of the tube (Whatman Chemical Separation, Ltd., U.K.; microgranular cc 41, Cat. # 4061-050). The columns were then filled to a height of 40 mm with a mixture of nitroprusside-DEA and aminopropyl silica (1:2 by weight), with a pore diameter of 200A (angstroms), and average particle size of 40 to 60 microns as described in Example 4. The top of the silica matrix ~3~3~

was then covered with 1 mm thick nylon fabric as before. A vibrator (Vibrograver, Supelco, Inc.
Bellefonte, PA) was used to pack each of the microcolumns uniformly. After packing, the columns were stored in a dark bottle at room temperature. The devices may not only be used for semiquantitative analysis through spectrophotometric techniques or reerence to color charts, but 1~ particularl~ useful or the ~uan~itatlve detectlon oE ketone and aLd~hydes through the u~e o~ asc~nding chLomatographic techni~ue~.

According to another aspect of the invention, "dipstick" devices may be fashioned wherein the solid matrix material of the invention is used in suitable shapes such as films, strips or sheets. The materials may also be coated onto, or bonded or laminated to appropriate inert carriers by using glue or adhesive by mixing with binders or by heat treatment and the compression of the particles onto plastic surfaces.
Suitable materials include paper, glass, plastic, metal or fabrics. The matrix material i9 preferably in the form of strips of thickness in the range from about 0.10 mm to about l mm, and most preferably of about 0.25 mm. The strips preferably range from 2 mm to 4 mm wide and from 2 mm to 4 mm long, but may be virtually any dimension consistent with economy and sample size.
According to one procedure, polystyrene sheets, 0.025 mm thick, were obtained (Vinyl Plastics, Inc.l Milwaukee, WI, Cat. ~ 1045), and were sprayed uniformly with an adhesive (Scotch 3M Spray Mount Adhesive, Cat. # 6065). The sheets were then sprayed with a mixture of nitroprusside-DEA-silica and aminopropyl silica (1:2 by weight) with a pore diameter of 200A and particle size of 40-60 microns.
Alternatively, the sheets were treated with a mi~ture of 3 ~

nitroprusside-DEAE-cellulose and aminopropyl silica (1:4 and 2:1 by weight) as described in Example 7. Excess particles which did not stick to the glued surface were removed by tapping.
The coated strips were then cut in small pieces (4 mm x 2 mm size) attached to double-faced adhesive tape and used to measure acetoacetic acid concentration in urine samples. According to an aIternative procedure, dipsticks were eashioned erom (,el Bond Strips ~FMC Corp., Rockland, M~ine). According to thi~ method, 100 mg oE nitroprus~ide-DEA silica and aminopropyl silica at a 1:2 ratio by weight and 100 mg nitroprusside-DEAE cellulose and aminopropyl silica at 1:4 and 2:1 ratios by weight were each mixed with 80 ~1 hydroxyethyl cellulose agarose (5%) ~FMC Corp., Rockland, Maine) and 50 microliters hydroxy propyl cellulose (0.5~) obtained from Hercules, Hercules, Del.
taken in 470 microliters of water at 50C. Twenty microliters of the resulting slurry was pipètted onto each Gel Bond Strip (4 mm x 2 mm size). After allowing the strip to air-dry in the dark at room temperature for roughly two to three hours, the strips were used to measure the acetoacetic acid concentration in urine samples.

In this example, a solid matrix comprising nitroprusside-DEA silica according to Example 1 and aminopropyl silica according to Example 2 in a weight ratio of 1:1 or 1:2 was incorporated in a microcolumn as described in Example 12. In order to test the devices, known amounts of acetoacetic acid were added to various buffers and to fresh urine samples from normal subjects whose acetoacetic concentrations were determined to be less than 1 mg/dl. Urine solutions were discarded aEter being stored for 2 hours at 4C.

~ ~.2~36 SampleConcentration of Acet:oacetic Acid (mg/dl) Urine pH 5.0 - 7.0lO0 50 25 :L0 5 Acetate buffer pH 4.0 " " " " " "
Borate buffer pH 8.0 " " " " " "
Tris buffer pH 9.5 " " " " " "

The devices gave posi~lve color results for th~ urine samples withln one mlnute. The colors varied accordlng to the concentrakion o the samp1e ~nd ranged from dark purple/magenta for the lO0 mg/dl sample to light pink for the 1 mg/dl sample. Urine samples not spiked with acetoacetic acid gave a slightly yellowish-tan color. The sensitivity threshold in all cases was 1 mg/dl. The colors were stable for a period of 72 hours and the amount of acetoacetic acid could be accurately determined over that period by comparison with a color chart.

In this example, the devices of Example 12 were tested against buffer solutions at pHs 4.0, 7.3, 8.0 and 9.5 to which varying amounts of acetoacetic acid has been added as shown in Example 14, Table 5. The test columns gave positive colors within one minute with - colors being very similar to those observed with the urine samples as in Example 14. Control buffers not spiked with acetoacetic acid gave a slightly yellowish color. The sensitivity threshold in all cases was 1 mg/dl. The colors were stable for a period of 72 hours and the amount of acetoacetic acid could accurately be determined over that period by comparison with a color chart.

3 ~

In this example, a solid matri~ comprising nitroprusside-DEAE-silica according to Example 1 and aminopropylsilica according to Example 2 in a 1:2 (by weight) ratio was incorporated onto a polystyrene sheet or Gel Bond Strip as described in Example 13. The device was tested against the urine samples of 'rable 5 and ~ave positive color resul~ eor all samples within one minute. 'rhe color~ varied a~co~dlng to the concentration of the sample and ranged from dark purple/magenta for the 100 mg/dl sample to light pink for the 1 mg/dl sample. Urine samples not spiked with acetoacetic acid gave a slightly yellowish-tan calor.
The sensitivity threshold in all cases was 1 mg/dl. The colors were stable for a period of 72 hours and the amount of acetoacetic acid in the samples could accurately be determined over that period by comparison with a color chart.

In this example, the dipsticlc devices of Example 13 were tested against the buffer solutions of Table 5 at pHs 4.0, 7.3, 8.0 and 9.5 to which varying amounts of acetoacetic acid had been added according to Example 15 ~Table V). The test strips gave positive colors within 1 minute with the colors very similar to those observed with the urine samples of Examples 14 and 16. Control buffers not spiked with acetoacetic acid gave a slightly yellowish color. The sensitivlty threshold in all cases was 1 mg/dl. The colors were stable for a period of 72 hours and the amount of acetoacetic acid in the samples could accurately be determined by comparison with a color chart.

3 ~

In this example, dipstick devices according to Example 13 were constructed wherein the solid matrix comprised nitroprusside-DEAE-cellulose and aminopropylsilica in weight ratios of 1:2 and 1:4.
These devices were tested against the urine and buffer solutions (at pHs 4.0, 7.3, 8.0 and 9.5) to which var~ing amounts of acetoacetLc acid had been added acco~ding to Table 5. The~e dRvlc~s gave the same re3ults as the de~lce~ Oe ~xampLes l~, 15, 16 and 17.

In this example, dipstick devices according to Example 12 were constructed wherein the solid matrix comprised nitroprusside-DEAE-cellulose and aminopropylcellulose in weight ratios of 1:4 and 1:8.
The dipsticks were constructed of polystyrene sheets or of Gel Bond and were tested against the urine and buffer solutions (at p~s 4.0, 7.3, 8.0 and 9.5) of '~able 5.
The devices were sensitive to acetoacetic acid and gave colors ranging from light purple to faint purple which were visible for 48 hours. The sensitivity limit ranged from between 5 to 10 mg/dl.

In this example, microcolumn chromatography devices were constructed according to Example 12. The devices were packed with solid matrix material comprising nitroprusside-DEAE-silica and aminopropyl silica at a ratio of 1:2 by weight. The devices were immersed in 300 ul of a standard solution comprising ~cetoacetic acid in 0.2 molar phosphate buffer (pH 6.8) containing 0.9~ sodium chloride which rose through the columns by capillary action. In rising through the device, acetoacetic acid within solution reacted with the matrix materials to produce a purple reaction ~3~3~

product which was stable for 24 hours. l'he amount of acetoacetic acid could therefore be determined 'oy measuring the length of the color bar. The elution took approximately ten minutes but it would be possible to shorten this time as may be desired. Table 6 below shows the relationship between acetoacetic acid concentration and the height of the color bar.

T~hE 6 ~Acetoacetic Acld ~Ieight o Concentration Color ~ar~
(mg/dl) (Millimeter) Color 100 30 Dark Violet 22 Dark Violet 15 25 16 Dark Violet 7 Medium Violet 4 Medium Violet ; 1 2 Light Violet * Average of two experiments.

In this example, the devices of Example 20 were used to detect the presence of acetoacetic acid in urine samples to which acetoacetic acid had been added. Seven samples were tested with linear color bars detected in four of the samples. The color bar heights in four samples were very similar to those depicted in Example 20, Table 6. The other three samples did not produce linear color bars but rather produced color bars which were more elongated and diffused. It is believed that the failure of certain of the samples to produce elongated color bars is a consequence of the prese'nce of high salt concentrations in the urine.

In this example, urine samples were diluted to one-tenth their original concentration with water in ~3~2~
- 7g -order to prevent interference of the salt solutions with the chromatography. Various samples were tested with the micro column capillary devices of Example 14. The results are shown in Table 7 below.

RELATIONSHIP BETWEEN HEIGHT OF COLOR
~AR AND TEN-FOLD DILUTED URINE SAMPLES

Acetoacetic Acid Height of 1~Concentration Color Bar*
(MG/DL~ (Millimeter) 100 11 ~ 3 g ~ 2 7 i 2 ~Average of seven experiment~.

In this example, the bifunctional solid matrix material of Example 8 comprising both nitroprusside and aminopropyl functions coupled and bound to the same matrix was tested according to the procedure of Example 16 for the detection of acetoacetic acid in aqueous solutions. The materials were only slightly sensitive to acetoacetic acid in buffers (p~ 6.8). Only a faint purple color was detected at 100 mg/dl of acetoacetic acid. The sensitivity was increased by addition of Trisma base (pH 10.0) to about 20 mg/ml of acetoacetic acid. When aminopropyl silica was added to the matrix material, this increased sensitivity to about 2a mg/ml of acetoacetic acid. The device of Example 16 was also tested for sensitivity to acetone in the presence of dimethylsulfoxide and methanol (v/v, 1:3) containing 30 m~/ml Trisma base. The sensitivity limit was approximately 7 nM acetone in solution. A faint blue was detected which could be slightly enhanced by addition of aminopropyl silica to the mixed matrix material.

131~3~

MONITORING OF ~IEIGHT LOSS
.
In the course of development of the devices of the present invention it was discovered that serum acetone concentrations and hence breath acetone concentrations as measured by the methods and devices of the present invention may be correlated directly with the ~ate o~ a~-metabolism (~a~-10~9) experienced by a su~ject und~rgolng a w~l~h~ los~ dlet~ry regimen comprisillg ~asting, dletlng, ~x~rcise or a combinatio of the a~oresaid.
The invention comprises methods for ascertaining the fat catabolism effects of a weight loss dietary regimen comprising (a) periodically assaying breath for acetone content and (b) correlating breath acetone to a standard reflecting the effect on breath acetone of fixed rates of fat catabolism. A direct correlation between alveolar air (breath) concentrations and the rate of fat-loss has been established. Because breath acetone concentrations are directly proportional to serum acetone concentrations, the correlation between acetone and the rate of fat-loss also holds for serum acetone. References to breath acetone concentrations 2S will therefore, unless otherwise stated, also refer to the serum acetone concentrations which are speciEically associated therewith.
Methods for determining the fat catabolism effects of a weight loss dietary program involve the collection of alveolar air (breath) samples and assaying for acetone content. Various methods may be utilized for determination of sample acetone concentrations including mass spectrometry and gas chromatography with preferred methods utilizing the ketone assay devices of the invention. Such assay devices may be provided in which tabular color charts are calibrated to indicate a ~3~3~

rate of fat catabolism expressed in suitable units such as pounds of fat catabolized per week. Assay devices comprising a linear reading system may comprise a graphic adjunct such that a color bar sciale may be calibrated to indicate a rate of fat catabolism expressed in units such as pounds of fat catabolized per week. Breath may be sampled on a periodic basis such as once daily with samples preferably collected before breakfast in the morning. Breath samples may be taker, more frequently than once daily, although samples taken soon after consumption of a meal or after the completion of exercise may indicate lower or higher rates of fat catabolism, re~pectively, than would be expected to be maintained over a 24 hour period.

In this example, breath acetone concentrations were measured for a group of dieting individuals and controls utilizing a Shimadzu gas chromatograph (Model GC-8A, Columbia, MD) equipped with a heated gas sampler HGS 2 and a flame ionization detector. The chromatographic column consisted of a 2 meter stainless steel coil, 1/8 inch OD packed with chromosorb 102 3~
80-100 mesh (Supelco, Inc.). The column temperature was maintained at 120C with ultrapure helium as a carrier gas (5 kg/cm2 pressure). Hydrogen and air pressures were 0.5 kg/cm2 and 0.2 kg/cm2, respectively. The retention time of acetone was 4.2 minutes and the acetone peak was well separated from the methanol, ethanol, isopropanol and acetaldehyde peaks.
Calibrations were made by preparing acetone vapor in glass gas jars or from commercially available cylinders containing a compressed air-acetone mixture ~Linde Div., Union Carbide). Calibration standards ranging from 4-1000 nM were used to demonstrate a linearrelationship between the height of the acetone peak and ~ 3 ~

the concentration of acetone in a sample. A Shimadzu C-~RA integrator was used for calibration purposes.
In order that breath samples taken from different individuals at different times provide accurate and reproducible results, several types of expired breath specimens were tested for acetone concentration. Several types of expired breath samples are suitable for chemical analysis including (1) expired alveolar air: (2) end-tidal air; (3) end-expiratory air and (4) re-breathed air. Mixed expired air is not suitable for breath analysis because it contains variable proportions o~ alveolac air and dead-space air.
Varlous types o~ breath ~amples were collected rom a number o~ volunteers by methods includlng ~a) end-tidal alr by collectlon of the last part of a big breath, (b) end-expiratory breath specimen by means of a device ~Intoximeter, Inc., St. Louis, MO) according to the method of Dubowski, Clin. Chem. 20, 966 (1974) and (c) equilibrated vital capacity air by holding a deep breath for 5 seconds and expelling various fractions of the breath according to the method of Erikson, New Scientist, 381, 608 (lg64) The acetone content of all collected specimens showed differences of less than 2~ between the various methods. It was thus concluded that diagnostically useful samples could be obtained simply by holding a deep breath for 5 seconds and expelling the entire breath to obtain a sample of equilibrated vital capacity air. For analysis of breath acetone by gas chromatography, volunteers were asked to take a deep breath, hold for 5 seconds and blow into a silicone coated balloon (1 liter capacity~ via a one-way valve and T-connection connected to the gas inlet of a gas chromatograph. After the gas-loop was purged for 10 seconds with a breath sample, a constant volume of 1 cc was allowed to be swept into the chromatographic column for analysis.
,~ ~

~2~

In this example, a diet study was conducted with 170 normal volunteers who were between 0 and 100 pounds above desirable body weight for height according to the Metropolitan Life Height/Weight table. The criteria for selection of volunteers were that they were normal in other respect~, had completed a physic~l examina~ion within the previou~ 12 mon~hs and did not ~all lnto one or more Oe the eollowlng categorie~
pregnant women; (2~ individuals taklng lithium sa:Lt5 ~or depression; (3) individuals with renal or hepatic disease requiring protein restriction; (4) individuals with arteriosclerotic heart disease; (5) diabetics receiving insulin or oral hypoglycimic agents; and (6) individuals with cardiac arrythmias.
The diet program continued for two weeks and the diet included fish, poultry, lean beef, eggs, vegetables, salad, cottage cheese, coffee, tea, sugar free gelatin, and not more than 2 cans o diet soda.
Each volunteer was allowed to plan his own daily diet plan, none of which exceeded the limits of 1200 calorie, 40 grams of fat and 40 grams of carbohydrate on any day. Each volunteer also took one multivitamin plus mineral tablet and at least lSQ0 ml fluid per day.
Breath acetone concentrations of each individual were measured early in the morning before breakfast by gas chromatography according to the procedure of Example 24 as well as by the colorimetric method according to Example 11. Urine concentrations of acetoacetic acid were measured by Ketostix (Miles Laboratories, Elkhart, Indiana~ as well as by the method according to Example 14. The body weight of each volunteer was also recorded prior to breakfast.
All subjects participating in the program lost between five and ten pounds of body weight in the first ~2~

week of the diet. The specific amount of weight loss depended on the obesity, gender and leve:L of physical activity of the individual. While it is generally accepted that women in general have lower metabolic rates than men, Wynn, et al., Lancet, 482 (}985), this was confirmed by the study. It was also found that the rate of fat-loss, and hence development of ketosis is depend~nt on the extent of obesity of an individual, with ~everely obese individuals losing Eat and becomin~
1~ ketotlc at a s~ower rake than ;le~s obese individu~
It was noted ~hat the ~ate oE ~t-loss and increase in breath acetone also depends on individual's physical activity, e.g., a person on a diet and additionally performing physical activity such as aerobics, bicycling or jogging, has a higher breath acetone concentration and rate of fat-loss than one who is on a diet only and not doing any physical exercise.
The relationship between rate of fat-loss and serum/breath acetone concentration was determined by analysis of the subjects of the example during their second week of dieting. More than 50% of the weight lost in the first week of the diet was due to water loss. By comparison, in the second week of dieting, the amount of water loss for those subjects between 0 and 20 pounds overweight became minimal, approaching 10 to 15%
of weight loss, and the loss in body weight was primarily due to fat catabolism. Figure S illustrates the data from individuals between 0 and 20 pounds overweight during the second week of the diet. The "straight line", calculated by linear regression, gives the statistical value of the relation between breath acetone concentration and rate of fat-loss in pounds per week. The relation between breath acetone, fat-loss and calories burned is shown in Table 8 below.

~3~

RELATIONSHIP BETWEEN BREAT$ ACETONE
CONCENTRATION, FAT LOSS
AND CALORIE BURNED DURING DIETING
Breath Acetonea Fat-Lossb Calories BurnedC
Concn. (NM) Per Day Per Week Per Day (lbs) (lbs) 8-30 - _ _ 0~07 0.5 ~6 67 0.1~ 1.0 57 120 0. 2a 2.0 11 l0 212 o,~ 3,0 1757 330 ~,5 3,5 20~3 a Breath acetone concentration was calculated by gas chromatography and colorimetric method of Example 10.
b Fat-loss was calculated from the slope of the straight line (shown in Fig. 5) c Calorie burned was devised from the relationship between calories and fat consumption: l g fat burned =
9 calorie.

The weight, water and fat-loss profiles of dieters are shown in Figures 6 through 9. The values for fat-loss were calculated from the breath acetone measurement and the standard obtained by determination oE the slope of the straight line in Figure 5. The values for water-loss were calculated by subtracting from the actual body weight. It should also be noted that in the first week of dieting, the fat-loss figure accounts for the loss of glycogen carbohydrate stores in addition to loss of body fat.
It was found that urine acetoacetic acid has no direct relationship with fat-loss. Although an increase was clearly noted with all dieters after 2 to 3 days of dieting, the increase was not quantitatively related to breath acetone concentrations or to the rate of fat-loss. The blood sugar levels of the dieters did not change during the dieting period.

~3~2~

In this example, a diet program was conducted for one month with 30 otherwise normal 40-100 pound overweight volunteers. This established that the direct linear relationship between breath acetone and fat-loss exists beyond two weeks using the same low-fat/low carbohydrate diet. The selection oE the subjects was the same as in the two-week program except all the subject~ had to undergo complet~ ph~lcal examinationv laboratory tests including complete blood count, serum chemistries (SMCC 12 or 20) and urinalysis before participation. Breath acetone, urine ketone and body weight of each individual were measured daily and blood sugar level determined weekly. It was noted that volunteers in this group tend to lose water for a longer period of time than less obese people.
It was found that for this group, the water-loss becomes minimum (10-15%) in the third week (~igure 9). It was also found that breath acetone concentrations of subjects in this group were directly proportional to their fat-loss in the third and ~ourth week as well as in the second week. Although urine acetoacetic acid concentrations of each individual were elevated, there was no direct relationship to the rate of fat loss. No changes in blood sugar levels were noted.
It is interesting to note that more obese ; people tend to lose water for a longer period of time.
For the group who are between 0-lO pounds overweight, ~ the water-loss becomes minimal ~<15%) on day 8, for lO-; 20 pounds overweight the day shifts to day 9 and for 20-40 pounds overweight it shifts to day lO. People who are between 40-100 pounds overweight, the water-loss continues in the second week of dieting and becomes minimal (<15%) on day 14.

~3~3~

The program also indicated that water-loss in four very obese subjects (100-200 pounds overweight) continues for a much longer time and fluctuates even in the week 4. This group developed ketosis at a slower rate than the other less obese groups and also experienced a lower rate of fat loss.

EX~MP~E 27 ` In ~his example, ~ group of sub~cts entlanced th~ ex~en~ o th~ir k~kosl~ by par~iclpatln~ in pt~ysical exercise without decreasing their daily calorîe intake. An increase of 20 40~ in breath acetone was observed after burning 400-500 calories by physical exercise tbicycle or jogging). Immediately after physical exercise, there was a drop in breath acetone level which then slowly rose after 1 hour and plateaued after 4 to 5 hours.
It was found that ketosis (breath acetone) drops considerably for volunteers when they don't perform exercise on any given day. As a typical illustration, a male subject with a daily intake of 1000 calorie, 30 to 40 grams of fat, and 30 to 40 grams of carbohydrate plateaued at a breath acetone level of 100 nM from the 8th day onwards. He did not perform any rigorous physical exercise. On day 11, he rode on a bicycle for 10 miles at a rate of 10 miles/hr. (500 calorie burnedO) It was observed that his breath acetone increased to 200 nM on the next day (day 12).
It was found that his breath acetone dropped again to 100 nM when he stopped his physical exercise. This increase in breath acetone in conjunction with exercise suggests that it may be possible to correlate the number of calories burned by exercise with increased breath acetone levels. It has been found that excessive coffee or tea intake also enhances breath acetone production during dieting.

~3~3$

In this example, the antiketotic effect of dietary "cheating" was measured It was observed that dieters consuming a high carbohydrate meal by mistake lowered their breath acetone levels appreciably within a few hours. Subjects participating on the diet of Example 27 for 2 weeks or fasting eor 12 hours consumed an 8 ounce can of ENSVR~(Ross Laboratories, Columbus, OH) containing 250 calories and 36 grams of carbohydrate. ~he breath acetone of those consuming the product dropped by about 20% a~ter one hour and by al~out 30% a~ter 3 hour~. Slmilarly, when the test sub~ect~
discontlnued the dlet program and ate d high calorie diet (800 caloeie, 100 grams Oe carbohydrate and 20 to 40 grams fat), a drop of approximately 40~ in breath acetone was observed in 5 hours. Within 24 hours, the breath acetone concentration dropped to the pre-diet level.

In this example, the relationship between development of Itetosis (breath acetone) and caloric intake was studied. The results are shown in Table 9 below. As may be observed, the increase in breath acetone is directly proportional to the intake of calorie.

~0 1312~36 EFFECT OF CALORIE INTAKE
ON ~ETOSIS DEVELOPMENT
Calorie Intakea Breath Acetone Level (times normal x) Day 1 Day 2 Day 3 0 4x 16x 600-700 l.5x Gx 13x 1100-1300 1.5x~ ~x 8-10x 2000 l.lx2.~x ~x a Diet comprised of hlgh-protein and le~s than ~0 gm carbohydrates/day wa~ used in thi~ study.

In this example, a diet study was conducted wherein breath acetone concentrations were measured for a group of dieting individuals utilizing the devlces produced according to the methods of Examples 10 and 11 as well as by a gas chromatograph. A gas chromatograph was also utilized to measure blood head-space acetone concentrations. Weight, total body water and total body fat were periodically determined for the subjects of the study.
The study was limited to normal, healthy male and female subjects, between the ages of 24 and 54, with no chronic medical disorder except obesity. Fifty-eight volunteers, (20 male and 38 female) participated in the diet study in three groups. Each study period was for 30 consecutive days, excluding weekends and holidays.
Twenty volunteers, ~10 male and 10 female) were included in the non-dieting control group. This study continued or 19 days excluding weekends and holidays. The participants were between 10% and 30% above their ideal body weight as determincd-by age/sex/frame/height/weight and Metropolitan Life Insurance Company tables. The participants had complete physical examinations including blood and urine analysis before entering the ~3~ 2~

study, in the middle of and at the end of the study period.
Two diet plans, one providing 1,000 calories per day and another providing 1,200 calories per day were developed for this study by a physician and a consulting dietician. The 1,000 calorie diet included 60-80 gm protein, 90-130 gm carbohydrate and 22-24 gm fat per day. The 1,200 calorie diet included 80-110 gm protein, 113-147 gm carbohydrate and 25-47 gm Eat per day. The H~rri~-Benedlct equatlon was employed to determine the basal en~rgy expenditure (BEE) of ~ach participant before entering the study and the selection of the diet plan was made by the resident dietician according to their BEE requirements. The volunteers refrained from any strenuous physical activity during the entire study period and pedometer was provided to each dieter to record the daily number of steps taken.
Data collected during the diet study allowed correlation of blood and breath acetone concentrations. In addition, the data allowed comparison of breath acetone concentrations as measured by a gas chromatograph and by devices prepared according to the methods of Examples 10 and 11.
According to the procedure, blood acetone measurements of the dieting population were performed on days 1, 2, 16, 23 and 30, and for the non-die~ing group, the measurements were made on days 3, 10 and 17. Gas chromatographic head-space analysis was carried out according to the method of Van Stekelenburg and Koorevaar, Clin. Chim. Acta, 34, 305-310, 1971 to measure blood acetone concentrations. Breath acetone concentrations were determined by using a gas chromatograph and by means of breath acetone devices produced according to the methods of Examples 10 and 11.
Figure 10 shows the comparison of breath acetone concentrations (y-axis) to acetone 3 ~

concentrations of blood head-space (x-axis) in dieting and non-dieting groups. Analysis of t~lis data by linear regression techniques provides a formu]a of y = 1.45 (x) + 0.954, where x = blood acetone head-space S concentration (nm) and y = breath acetone concentrations (nM). The data has a correlation coefficient Ir) of 0.92.
Figure 11 shows the comparison Oe breath acetone concentratlon of dleting and non-dietlng volunteers determlne~ by ga~ chromatography (x-axi~) and by devices prepared according to Examples 10 and 11 (y-axis). The column heights obtained from the breath analyzer devices of the invention were converted to nM
acetone concentrations using the standard curve specific for that lot of analyzer columns. Analysis of the data by linear regression techniques provides a formula of y = 1.173 (x) + 8.65, where x = breath acetone concentration (nM) as calculated by gas chromatography and y = breath acetone concentration (nM~ as calculated by devices prepared according to Examples 10 and 11.
The data has a correlation coefficient (r) of 0.91.
Data collected during the diet study also allowed improved correlations to be made between the rate of fat loss and breath acetone concentrations.
According to this example, total body water and body fat determinations were made by means of bioelectrical impedance instrumentation. According to the experimental procedure, five breath samples were collected from each experimental subject each day immediately upon awakening. Three of the samples were promptly assayed for breath acetone by each volunteer using a breath acetone analyzer prepared according to the methods of Examples 10 and 11. The remaining two samples were analyzed by trained personnel. One was used to measure breath acetone concentration with a breath acetone analyzer device of the invention 13~2~

;

performed by a trained technician and the other assayed by gas chromatography. ~hole body weights were measured by the volunteers daily immediately upom awakening, after defecation and urination. For ea~h determination, the volunteers weighed themselves in their own home with ` a precision scale Eive consecutive times. The scales were calibrated at the beginning and end of each study period. Total body water and body fat determinations were performed on each volunteer, five d~ys a week ~Mond~y-Friday) in the morning beEore bre~kEa~t, with a Bi~elect~ical Impedan~e ~nalyzer Model ~ lOl (R~L
Systems, Inc., De~roit, Ml).
During ~he course o this study, the breath acetone concentration of all subjects increased during the first few days of the diet, reached a plateau after approximately seven days and remained elevated during the course of the 30 day study. Figures 12 and 13, respectively, show the average cumulative breath acetone concentration and column color bar height during the 30 day study for the 58 dieting volunteers and the 20 non-dieting (control) volunteers. The average column height of the dieting population ranged between about 22 to 24 mm (275-300 nM acetone concentration), compared to the non-dieting population average which ranged between 25 about 5 to lO mm (17-27 nM acetone concentration).
The study also demonstrated that all subjects lost fat while on the study diet. The total fat loss was determined by body composition analysis using electrical impedance. Figure 14 shows the average cumulative fat loss during the course of the 30 day study. The total average cumulative fat loss was approxima~ely 150 ounces for the 30 day period, i.e., approximately 5 ounces fat loss per day, per volunteer.
It is important to note that the rate of fat loss was not the same for all participant dieters. To illustrate this, the individual profiles of two dieters i 13t2~3~

are shown in Figures 15 and 16, together and 17 and 18 together. In Figure 16, (Dieter 1), the column heights, as determined by the breath acetone analyzer devices of the inventlon, rose progressively from day 1 and reached a plateau on day 8. The column heights of this dieter remained elevated over 22 mm, corresponding to a breath acetone concentration approximately of 250 nM
(Figure 19). The total loss of body fat was 165 ozs.
(10.3 lbs) (Figure 15) or approximately 5.5 ounces per day. In F~gure 16, ~Dieter 2), the column height~ rose progre~ively rom day ~ and re~ched a plateau on day ~. In contrast to Dieter 1, the average column height was reduced to approximately 13 mm. A reduced cumulative fat loss of 86 ounces, or approximately 3 ounces per day, corresponding to a breath concentration of approximately 70 nM (Figure 19), was also observed for Dieter 2 ~Figure 17).
Individuals in a normal, healthy and non-dieting population have a breath acetone concentration of 15 nM with S.D. Oe 11 (N = 78). This value was calculated from the day 0 baseline level for the dieting (N = 58) and non-dieting groups. A threshold level of 37 nM acetone (above which indicates that the patient is losing fat) was calculated on the basis of the average breath acetone concentration of non-dieting subjects plus 2 S.D. The threshold level is indicated on the Diet Progress Chart.
The column height corresponding to the 37 nM
acetone threshold level will vary slightly for each lot of columns and this lot specific adjustment is incorporated into a lot specific Diet Progress Chart, an example of which is illustrated in Figure 21a.
The average daily breath acetone concentration during the "plateau" phase of the study period (approximately days 8-30) was calculated for each volunteer (dieter and non-dieter) from daily ~3~2~3~

determinations using devices produced according to the procedure of Examples 10 and 11 and a gas chromatograph. These values are shown in Table ~ along with the calculated average daily rate of fat loss determined by impedance over the same time period.
correlation of breath a~etone concentrations using the Breath Acetone Analyzer and rate of fat loss is shown in Figure 20. Analysis of the data by linear regression techniques provides a formula o: Rate of fat loss (o~/day) = (breath acetone conc. (nM) - 15.3)/52.2.
The ~ormul~ has a correlation coe~icient (r) o~ 0.~.
The resultq demon~tra~e that the level oE hreath a~one measured by the devices of the invention is indicative of the relative rate of fat loss in the patient.
According to a method for use of the devices of the present invention, the acetone concentration of a breath sample may be determined by matching the observed column color zone height with a scale on the left side of a "~iet Progress Chart" shown in Figure 21a. The scale is correlated to breath acetone concentration, which i9 itself correlated to the rate of fat loss. The correlation between column color zone height and breath acetone may be adjusted from lot to lot of the test device according to quality control techniques. In the case of the "Diet Progress Chart" of Figures 21a and Zlb, quality control considerations indicated that a column height of 9.5 mm correlated to an acetone concentration of 120 nM, a column height of 7 mm correlated to an acetone concentration of 220 nM and a column height of 30 mm correlated to an acetone concentration of 330 nM. The subject plots the nM
acetone concentration reading on the graph daily. All normal subjects should be below the threshold acetone concentration while not dieting. As the diet continues, the breath acetone concentration will rise for several days and then plateau. The rate of fat loss can be estimated by a scale on the right side of the graph.
Non-dieting normal healthy people should have readings in the zone which is below the threshold line. The 0 zone indicates less than 2 ounces of fat loss/day. The + zone estimates a rate of fat loss of 2-4 ounces/day and the ++ zone estimates a rate of fat loss of 4-6 ounces/day.
Figure 21b illustrates the actual data o an individual who was on a 1,000 caloric diet program Eor 30 day~, The acetone concent~ation as determinecl ~rom the measured column color zone height wa~ plotted daily on the graph. As can be seen from the graph, the column heights (nM acetone concentrations) rise progressively from day 1 and exceeded the threshold mark on day 3.
The nM acetone concentrations remained elevated in most of the days and fluctuated within the ++ zone. The graph indicates that this dieter remained above the threshold zone (4-6 ounces of fat loss per day). One can estimate the amount of at loss during the 30 day 20 period to be approximately between 120-180 ounces (4 6 ounces per day). The actual amount of at loss over the 30 day period calculated using impedance measurements was 165 ounces.
Normal, healthy and non-dieting individuals have an average breath acetone concentration of 15 nM
with a S.D. of 11 (N = 78). The range varies between 6 to 30 nM in this population. The "threshold level" was calculated to be 37 nM acetone concentration of the basis of average breath acetone concentration plus 2 S.D. Subjects whose breath acetone concentration is below 37 nM are expected not to lose fat (less than 2 ounces/day). Individuals who consume a mixed 1,000-1~200 caloric diet can expect breath acetone concentrations above the threshold level corresponding to a fat loss rate of greater than two ounces per day.
The rate of fat loss may not be the same for each '~ 13~2~

individual. Those with a higher metabolic rate can expect greater fat loss.

Average Breath Acetone Concentration and Fat Loss (Day 8-3Q) I.D.Fat I.D. Fat Number Acetone Loss Number Acetone ~088 (Patient/ Conc. Imp. (Patient/ Conc. Imp.
_~_~ ~L ~ ~L
lCl 1~3 S.2 lC3 1~1 2.1 2C1 120 3.~2 2C3 279 3.69 3C1 61 2.28 3C3 144 2.72 4C1 264 4.17 4C3 275 3.05 SCl 137 4.72 5C3 380 2.76 6C1 302 5.61 6C3 242 4.97 7C1 293 4.4 7C3 223 4.09 8C1 70 2.8 8C3 416 6.32 9C1 177 3.79 9C3 159 3.92 lOCl 188 4.17 lOC3 249 4.89 llCl 121 2.73 llC3 259 2.75 12C1 110 2.49 12C3 263 3.53 13C1 62 2.27 13C3 317 6.09 14C1 84 3.13 14C3 290 3.98 15C1 162 3.53 15C3 290 4.01 16C1 232 3.31 16C3 257 3.72 17C1 153 2.81 17C3 324 3.64 18C1 105 2.54 18C3 ~83 2.7 lC2 185 2.36 l9C3 281 5.08 2C2 347 3.9 20C3 200 5.18 3C2 339 3.31 lC4 44 0 4C2 276 3.6 2C4 70 0.16 5C2 177 3.5 3C4 7 0.8 6C2 343 6.62 4C4 9 0 7C2 97 1.85 5C4 7 0.16 8C2 68 2.1 6C4 51 1.12 9C2 164 3.58 7C4 7 0.16 13~2~3~

I.D. Fat I.D. ~ Fat Number Acetone Loss Number Acetone Loss (Patient/ Conc. Imp. (Patient/ Conc. Imp.
Group)_ (nM) Oz/Day Group) (nM) Oz/D~y 10C2 294 2.76 8C4 39 0.16 llC2 202 4.8 9C4 4 0.64 12C2 322 4.03 10C4 17 0.:L6 13C2 233 4.27 llC~ 14 0.:32 l~C2 ~8 3.05 12C~ 18 1.2 15C2 3~2 ~.52 13C~ 18 1.
16C2 105 3.53 l~C~ 68 0.~6 17C2 176 3.5S 15C4 6 0 18C2 239 4.84 16C4 4 0.16 l9C2 295 5.32 17C4 32 0.48 20C2 190 4.6 18C4 44 0.16 l9C4 5 0.48 20C4 48 0.16 Numerous modifications and variations in practice of the invention are expected to occur to those skilled in the art upon consideration of the foregoing descriptions of preferred embodiments thereof.
Consequently, only such limitations should be placed on the invention as appear in the following claims.

Claims (43)

1. A method for the detection of a ketone or aldehyde analyte in a fluid sample comprising:
(a) contacting the analyte present in the sample with a first solid matrix material to which a nitroprusside salt is coupled and a second solid matrix material to which a primary or secondary amine is covalently coupled, (b) reacting the analyte with the nitroprusside and the amine to form a detectable reaction product, and (c) detecting the reaction product.

2. The method according to claim 1 wherein the nitroprusside salt is sodium nitroprusside.

3. The method according to claim 1 wherein the amine is a primary amine.

4. The method according to claim 1 wherein the fluid sample is a vapor.

5. The method according to claim 1 wherein the analyte is acetone.

6. The method according to claim 1 wherein analytes are adsorbed onto an adsorbent material and are desorbed from the adsorbent material and contacted with the first and second solid matrix materials by means of a solvent.

7. The method according to claim 6 wherein the solvent comprises methanol and dimethylsulfoxide.

8. The method according to claim 4 wherein analytes present in the vapor sample are adsorbed onto the first and second solid matrix materials.

9. The method according to claim 1 wherein the analyte concentration may be determined by detecting the extent to which analytes present in the sample fluid are linearly adsorbed onto the first and second matrix materials.

10. A device for the detection of ketones and aldehydes in a fluid sample comprising a first solid matrix material to which a nitroprusside salt is coupled and a second solid matrix material to which a primary or secondary amine compound is covalently bound.

11. The device according to claim 10 wherein said first and second solid matrix materials are immobilized on a strip.

12. The device according to claim 10 wherein said first and second solid matrix materials are immobilized in a column.

13. The device according to claim 10 wherein the nitroprusside salt is sodium nitroprusside.

14. The device according to claim 10 wherein the amine is a primary amine.

15. The device according to claim 10 wherein said first and second matrix materials are selected from the group consisting of silica gel materials and cellulosic materials.

16. The device according to claim 10 further comprising a preadsorbent zone.

17. The device according to claim 10 comprising means for isolation of a fixed volume of vapor in equilibrium with the liquid sample and means for contacting said fixed volume of vapor with the first and second solid matrix materials.

18. The device according to claim 10 wherein the nitroprusside salt and the amine are coupled to the same solid matrix material.

19. The device according to claim 10 comprising amino propyl silica gel and sodium nitroprusside coupled to diethylamino silica gel.

20. A method for ascertaining the fat catabolism effects of a weight loss dietary regimen, said method comprising:
(a) periodically assaying breath for acetone content, and (b) correlating breath acetone content to a standard reflecting the effect on breath acetone of fixed rates of fat catabolism.

21. The method according to claim 20 wherein the period is 24 hours and wherein the fixed rate is expressed in ounces of fat catabolized per day.

22. The method according to claim 20 wherein the standard is in the form of an equation.

23. The method according to claim 20 wherein the standard is in tabular form.

24. The method according to claim 20 wherein the standard is a graphic adjunct to a breath acetone assaying device.

25. The method for ascertaining the fat catabolism effect of a weight loss dietary regimen according to claim 20 wherein the breath is assayed for acetone content by the steps of: (i) contacting a breath sample with a first solid matrix material to which a nitroprusside salt is coupled and a second solid matrix material to which a primary or secondary amine is covalently coupled, (ii) reacting the acetone with the nitroprusside and amine to form a detectable reaction product, and (iii) detecting the reaction product

26. A kit for the determination of ketone and aldehyde concentrations in a vapor sample comprising:
(a) a first solid matrix material to which a nitroprusside salt is coupled, (b) a second solid matrix material to which a primary or secondary amine is covalently bound, (c) a solvent, and (d) means for collecting a fixed volume of sample vapor and contacting it with the first solid matrix material and the second solid matrix material.

27. A kit for the determination of ketone and aldehyde concentrations in a liquid sample comprising:
(a) a first solid matrix material to which a nitroprusside salt is coupled, (b) a second solid matrix material to which a primary or-secondary amine is covalently bound, and (c) means for collecting a fixed volume of the liquid sample and contacting it with the first solid matrix material and the second solid matrix material.

28. A kit for ascertaining the fat catabolism effects of a weight loss dietary regimen comprising:
(a) a first solid matrix material to which a nitroprusside salt is coupled, (b) a second solid matrix material to which a primary or secondary amine is covalently bound, (c) a solvent, (d) means for collecting a fixed volume of breath and contacting it with the first solid matrix material and second solid matrix material, and (e) a standard reflecting the effect on breath acetone of fixed rates of fat catabolism.

29, A device for monitoring levels of various components in the breath of a user, comprising:
a) an outer tubular member having an open first end and a second end;
b) valve means disposed toward the second end of said outer tubular member;
c) a breath sample collection means having one end mounted on said tubular member and having the other end closed, and having an interior in communication with the inside of said outer tubular member through said valve means; and d) a disposable tubular analyzer column having a mouthpiece and containing material reactive to the presence of said breath components, wherein said column is insertable into said outer tubular member and co-operates with said valve means to permit breath blown by a user to pass through the outer tubular member into said sample collection means and to permit said collected breath sample to discharge through the reactive material of said analyzer column.

30, The monitoring device as recited in claim 29 wherein said analyzer column is insertable into said outer tubular member to a first rotational and axial position for breath collecting in which breath blown into the mouthpiece bypasses said reactive material and passes through said valve means into said collecting means, said analyzer column being rotatable and further insertable to a second rotational and axial position for breath sample discharging in which a collected breath sample is discharged through said valve means into said reactive material from said collecting means.

31. The monitoring device as recited in claim 29 wherein the analyzer column further comprises a reduced-diameter central portion creating a passageway between the inside of said outer tubular member and the outside of said analyzer column, said central portion being connected to said mouthpiece by an inclined wall segment having at least one orifice therein such that, when said valve means is in a first position, the breath of a user passes from said mouthpiece through said passageway to said sample collecting means.

32. The monitoring device as recited in claim 29 wherein longitudinally extending rib means are provided on the inner surface of said outer tubular member for guiding said analyzer column when inserted therein.

33. The monitoring device as recited in claim 32 wherein positioning flats are provided on said analyzer column for cooperation with said rib means on said outer tubular member.

34. The monitoring device as recited in claim 32 wherein stop means are provided on at least one of said rib means for engagement by said analyzer column to determine a first axial position of said column.

35. The monitoring device as recited in claim 30 wherein said valve means comprises a housing member, a ball member disposed therein, an O-ring, and a further reduced-diameter end portion of said analyzer column, said further reduced-diameter end portion of said column being axially spaced from said O-ring in said first position of the column and, in said second position of the column, passing through and sealingly engaging said O-ring and abutting against said ball.

36, The monitoring device as recited in claim 30 wherein said sample collection means comprises an inflatable/deflatable bag and a flat coil spring is provided on the outer surface of said bag so that, after said bag is inflated and said analyzer column is moved into said second position, said spring coils said bag into its deflated condition with the fixed volume of air being discharged at a substantially constant rate through said reactive material in the analyzer column.

37, The monitoring device as recited in claim 29 wherein said reactive material includes at least one fluid reactant, said tubular analyzer column has a flexible portion adjacent said reactive material and said outer tubular member includes means for squeezing said flexible portion thereby to break an ampule of said fluid reactant disposed in said flexible portion.

38. The monitoring device as recited in claim 37 wherein said means for squeezing comprises an outwardly biased but inwardly movable breaker button mounted in a portable housing surrounding said outer tubular member, said breaker button being in alignment with said flexible portion of the disposable analyzer column and having a stem and head for transferring pressure to said flexible portion.

39. The monitoring device as recited in claim 37 wherein said means for squeezing is axially positioned to break the end of said ampule most closely adjacent said reactive material.

40. A kit for the determination of levels of various components in a user's breath, said kit comprising:
a) a portable housing adapted to receive a disposable analyzer column containing a mouthpiece and reactive materials;
b) bag means in communication with said housing for collecting a fixed volume of a user's breath blown through the mouthpiece and discharging it at a substantially constant rate through the reactive materials; and c) valve means disposed in said housing which, in a first position, prevents said fixed volume of breath from contacting said reactive materials and, in a second position, permits contact of said breath with said reactive materials, said valve means being operable by insertion of the column into the housing.

41. A disposable, tubular analyzer column for use in a kit for monitoring levels of various components in the breath of a user, said analyzer column comprising:
a hollow mouthpiece portion of a diameter permitting partial insertion of said analyzer column into a blow tube of said kit;

a central portion of reduced-diameter having an inclined wall segment interconnecting said mouthpiece and said reduced-diameter central portions, said central portion housing a volume of reactive material, and said inclined wall segment defining at least one breath passage opening connecting the hollow interior of the mouthpiece with the exterior of the central portion.

42. The analyzer column as recited in claim 41 wherein said central portion also houses a breakable ampule of liquid reactant adjacent said reactive material, said central portion being flexible enough to permit breaking of said ampule by applying pressure to said central portion.

43. The analyzer column as recited in claim 41 wherein said tubular member is further characterized by an end portion opposite said mouthpiece portion, said end portion having a further reduced-diameter which is operably engageable with valve means of said kit in at least one position of said analyzer column in the blow tube of said kit.

0432q