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WO2002099419A1 - Composition et procede pour detecter un adulterant dans un echantillon aqueux - Google Patents

Composition et procede pour detecter un adulterant dans un echantillon aqueux Download PDF

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Publication number
WO2002099419A1
WO2002099419A1 PCT/US2001/018560 US0118560W WO02099419A1 WO 2002099419 A1 WO2002099419 A1 WO 2002099419A1 US 0118560 W US0118560 W US 0118560W WO 02099419 A1 WO02099419 A1 WO 02099419A1
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WIPO (PCT)
Prior art keywords
composition
oxidizing agent
stabilizer
amine
creatinine
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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PCT/US2001/018560
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English (en)
Inventor
John Novinski
Barry Sample
Richard L. Hilderbrand
Susan Mills
Victoria Johnson
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Quest Diagnostics Inc
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Quest Diagnostics Inc
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Publication date
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Priority to PCT/US2001/018560 priority Critical patent/WO2002099419A1/fr
Publication of WO2002099419A1 publication Critical patent/WO2002099419A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/52—Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
    • C12Q1/28—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving peroxidase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/483—Physical analysis of biological material
    • G01N33/487—Physical analysis of biological material of liquid biological material
    • G01N33/493—Physical analysis of biological material of liquid biological material urine

Definitions

  • Urine-based testing is a widely practiced way of detecting the use of controlled substances or substances of abuse.
  • those who have found themselves placed in the predicament of having been exposed to certain drugs, either purposefully or inadvertently, and receiving a request for drug testing have identified and begun using chemicals which mask or interfere with the chemistries used to detect certain drugs. These chemicals are called adulterants.
  • These adulterants can be readily obtained by consumers, being that they have a number of uses and are readily available through many consumer or retail channels. For example, adulterants now showing up with increasing frequency are certain oxidizing agents, particularly nitrite salts, Cr(VI) salts and the alkali metal hypochlorites, e.g., sodium hypochlorite or common bleach.
  • compositions for use in detecting the presence of at least one adulterant in an aqueous sample are featured.
  • the composition comprises at least one amine and at least one stabilizer.
  • An aqueous sample may be a body fluid, for example blood or urine.
  • an adulterant according to this invention comprises an oxidizing agent.
  • oxidizing agents include, without limitation, chromates, nitrogen heterocyclic salts of oxidizing agents, peroxides, hypohalites, halite, halates, perhalate, periodide, oxone, permanganate, N- chlorosulfonamides, peracids, oxidative enzymes, nitrites and the like and mixtures thereof.
  • the presence of an oxidizing agent is identified by a presence of a range of broad band at about 470 nm to about 604 nm.
  • the presence of an oxidizing agent such as a nitrite is identified by a presence of a band at about 408 nm to about 410 nm.
  • the amine comprises a primary aromatic amine.
  • the amine comprises N,N-diethylphenylene diamine.
  • the stabilizer comprises a creatinine and/or citrate. Still further in accordance with the present invention, the composition further comprises an iodide.
  • the composition may comprise N,N-diethylphenylene diamine /Iodide/Creatinine/Citrate/Creatinine.
  • a method for detecting at least one adulterant comprises the step of combining a composition of the present invention with a urine sample and making a reading of a band which is associated with an adulterant.
  • the step of combining the composition and the urine sample, and the step of reading the absorbance may be achieved manually or by an automated spectrophotometer.
  • Fig. 1 is a tracing of the visible absorption spectra of a sample treated with DPD and a stabilizing agent to which sodium nitrite has been added.
  • Fig. 2 is a tracing of the visible absorption spectra from a sample containing DPD and a stabilizing agent to which sodium hypochlorite has been added.
  • Fig. 3 is a tracing of a visible absorption spectra from a sample containing DPD and a stabilizing agent to which pyridinium chlorochromate has been added.
  • Fig. 4 is a graphical representation of measured concentration vs. spiked concentration data from an evaluation study involving solutions of sodium nitrite treated with DPD and creatinine.
  • Fig. 5 is a graphical representation of measured concentration vs. spiked concentration data from an evaluation study involving solutions of pyridinium chlorochromate treated with DPD and creatinine.
  • Fig. 6 is a graphical representation of measured concentration vs. spiked concentration data from an evaluation study involving solutions of sodium hypochlorite treated with DPD and creatinine.
  • Fig. 7 is a tracing of a visible absorption spectra showing a "cherry red” band and a "purple" band.
  • a composition comprising at least one amine and at least one stabilizer may be useful in detecting the presence of at least one adulterant in an aqueous sample.
  • an aqueous sample is combined, or added, to a composition to form a mixture, and an adulterant is detected from the mixture.
  • the detection of an adulterant in the mixture is achieved through an observation of absorption band(s) which is/are associated with the respective adulterants.
  • the observations of the absorption bands are made with a spectrophotometer.
  • a band may be a simple band of one peak or a complex band of more than one peak.
  • an aqueous sample where an adulterant may be detected may include a body fluid.
  • a body fluid includes a blood sample, a tear sample and/or, preferably, a urine sample.
  • an adulterant is any chemical, composition or complex which may interfere with the detection of a test compound. These adulterants may be added to the aqueous sample to be tested. For example, a person may add an adulterant to his or her urine sample to prevent the detection of the presence of a test compound.
  • a test compound includes a drug precursor, a drug, a drug metabolite and/or any molecule suggesting that there is a presence of a drug in the test urine.
  • a drug includes opioids (e.g.
  • an adulterant comprises any molecule or complex which is capable of preventing the detection of another chemical entity.
  • an adulterant comprises an oxidizing agent.
  • An oxidizing agent as used herein may be any material which is capable of directly or indirectly facilitating the oxidation of another chemical entity, preferably a chemical molecule, more preferably a test compound, even more preferably a drug or a drug metabolite.
  • the oxidizing agent prevents the detection of a presence of a drug in an aqueous sample, for example a urine sample.
  • One type of oxidizing agent comprises a transition metal.
  • the transition metal has a high oxidation state.
  • An example of such an oxidizing agent includes a chelate complex. See, for example, Collins et al. U.S. Patent No. 6,100,394, the disclosure of which is inco ⁇ orated in its entirety herein by reference.
  • Another example of such oxidizing agent includes a transitional metal salt.
  • an oxidizing agent comprises chromium compounds.
  • these oxidizing agents comprise Chromium (VI) compounds, for example, chromate salts, pyridinium chlorochromate, pyridinium fluorochromate, chromium oxide, dichromate and the like and mixtures thereof.
  • an oxidizing agent comprises vanadium compounds, for example sodium meta vandate (V).
  • these oxidizing agents comprise Cerium (IV) compounds, for example ammonium hexanitrato cerate (IV).
  • oxidizing agent comprises a nitrite, alkyl nitrite, arylnitrite, nitrous acid, the like and mixtures thereof.
  • oxidizing agents comprises nitrogen heterocyclic salts of oxidizing agents, for example, pyridinium hydrogen perbromide, quinolinium dichromate, the like and mixtures thereof.
  • oxidizing agents include peroxides (e.g. hydrogen peroxide), hypohalites (e.g., hypochlorite, hypobromite), ferricyanide, halites (e.g., chlorite), halates (e.g., chlorate), perhalate (e.g., perchlorate), periodide, oxone, permanganate, N- chlorosulfonamides (e.g., chloroamine-T, chloroamine-B), peracids (e.g., perselenic acid, 3-chloroperbenzoic acid), oxidative enzymes (e.g., catalase, peroxidase, microperoxidase), the like and mixtures thereof.
  • the oxidizing agent as used herein does not include a nitrite, alkyl nitrite, arylnitrite, nitrous acid, the like and mixtures thereof.
  • the composition comprises a nitrogen containing molecule, for example an azine or a diphenylazine.
  • the composition comprises an amine.
  • the amine comprises an aromatic amine. More preferably, the amine comprises a primary aromatic amine, for example a primary aromatic diamine.
  • the aromatic group of the primary aromatic amine may have about 4 to about 10 carbons, preferably 6 carbons. Additionally, the aromatic group of the primary aromatic amine may be a heterocyclic molecule.
  • Chart 1 shows non-limiting examples of amines that may be used in accordance with the present invention.
  • R 2 , R 3 , R 4 , and R 5 may each individually be an alkyl (e.g., methyl, ethyl, isopropyl, t-butyl and n-butyl), aryl, a hydrogen, a halogen, hydrocarboxy, carboxylic acid ester, sulfonic acid ester or amino.
  • the alkyl comprises about 2 to about 10 carbons, more preferably about 2 to about 5 carbons.
  • the amino may be R ⁇ -N-R 7 , where R ⁇ and R may each individually be an alkyl, aryl group, hydrogen, halogen, hydrocarboxy, carboxylic acid ester or sulfonic acid ester.
  • R ls R 2 , R 3 , R 4 , and R 5 , or R ⁇ and R 7 may bridge with each other.
  • Compound II is an example of a bridging between R 2 and R 3 .
  • Compound III is an example of a bridging between Ri and R 2 .
  • the amine is an o, m or -N,N-diethylphenylene diamine (hereinafter "DPD"), compounds IV, V and VI, respectively (Available from Aldrich catalog no. 26151-3).
  • the amine is a ?-DPD (compound VI).
  • amines may include anilines (e.g., para-ethoxyaniline, ethylaniline, diethylaniline, toluidines, xylidenes), 2-methyl-4-amino- 5-formylamino pyridimidine, 2-methyl-4-amino-5-pyrimidinecarboxylic acid, 1,5 dimethyl-6-aminonaphthaline, the like and mixtures thereof.
  • the amine may be a secondary aromatic amine, for example anilinopyridine.
  • an amine in accordance with the present invention may be a
  • the conjugated-system may be a carbon-chain.
  • a carbon conjugated system may comprise 3 to 15 carbons, preferably 4 to 8 carbons.
  • the conjugated system may also include a heteroatom.
  • the amount of amine, for example aromatic amine, preferably primary aromatic amine, used will be some concentration sufficient to give a useful visible abso ⁇ tion spectra.
  • the amine is at a concentration of about 0.1 g/L to about 1.2 g/L, preferably about 0.3 g/L to about 0.9 g/L, more preferably about 0.6 to about 0.8 g/L of the composition.
  • the concentration of the composition be diluted, for example with water. In one embodiment, the composition is diluted to about half the original concentration, at the time an abso ⁇ tion reading is made.
  • the dilution is especially advantageous when the aqueous sample is suspected of containing a transition metal oxidizing agent.
  • the amine is dissolved in a suitable solvent prior to being added to the test sample.
  • a suitable solvent Various solvents can be used so long as the compound dissolves in it.
  • organic type or aprotic solvents are used.
  • DPD when DPD is used, it is convenient to first dissolve it in a short-chain organic acid such as formic acid, glacial acidic or acetic acid before diluting it further with water.
  • the composition comprises a stabilizer.
  • the stabilizer stabilizes a species which gives rise to a band associated with at least one adulterant.
  • the stabilized species is an adulterant-stabilizer intermediate, for example an (oxidizing agent)-DPD intermediate.
  • the stabilized species gives rise to a more persistent band.
  • the stabilizer may facilitate an increase in the life-time of a band associated with the oxidizing agent.
  • the stabilizer increases the life-time of a band associated with the oxidizing agent, wherein the band is not defined by the pu ⁇ le spectra region, see figure 7.
  • the stabilizer helps maintain a band associated with an adulterant for a period of time long enough for adequate measurement of a band. In a more referred embodiment, the stabilizer helps maintain a band associated with an adulterant for at least about 30 seconds.
  • a stabilizer useful in this invention includes any molecule or chemical entity having at least one non-bonding pair of electrons.
  • a stabilizer may be any nucleophile.
  • a stabilizer is an oxygen or nitrogen containing molecule.
  • Non-limiting examples of stabilizers include sodium ethoxide, sodium hydroxide, creatinine, citrate salt and mixtures thereof.
  • a stabilizer is a creatinine (available from Sigma Catalog No. C4255).
  • a stabilizer is a citrate.
  • the composition of the present invention comprises at least two stabilizers, for example a creatinine and a citrate.
  • a stabilizer according to the present invention is not used as an acid.
  • An effective concentration of the stabilizer in the composition is about 1 g and 6 g, preferably about 2 g and about 5 g, more preferably about 4 g to about 4.5 g/L.
  • concentration of the stabilizer in the composition be diluted, for example with water.
  • the original concentration of the stabilizer in the composition is diluted to about half at the time an abso ⁇ tion reading is made.
  • the composition is effective in aiding in the detection of at least one adulterant in an aqueous sample when it is added or combined with the aqueous sample.
  • a small volume of an aqueous sample may be added to a composition of the present invention to form a mixture.
  • about 1 to about 5 uL of an aqueous sample may be added to about 100 to about 300 uL of the composition, or similar ratios.
  • about 3 uL of an aqueous sample may be added to 250 uL of the composition, or similar ratios.
  • the aqueous samples above are urine samples.
  • a mixture containing an adulterant for example an oxidizing agent, will have an abso ⁇ tion band or set of bands characteristic of that type of adulterants. These particular bands may be determined empirically through commonly known methods by one of ordinary skill in the art.
  • the various types of oxidizing agents identified above may be generally categorized into two classes.
  • the first class of oxidizing agents, class I may be identified by an abso ⁇ tion of a broad complex band at about 470 nm to about 604 nm, or as is substantially described in figures 2 and 3.
  • Examples of the class I oxidizing agents include, without limitation, the chromium compounds and hypochlorites.
  • the second class of oxidizing agents, class II may be identified by a strong abso ⁇ tion band at about 408 nm to about 420 nm, preferably at about 410 nm, and a weak abso ⁇ tion band at about 470 nm to about 620 nm, preferably at 511 nm.
  • Figure 1 substantially describes the abso ⁇ tion characteristics of class II oxidizing agents.
  • a non-limiting example of a class II oxidizing agent includes a nitrite.
  • the abso ⁇ tion band at about 470 nm to about 604 nm may sometimes be referred to herein as the "cherry red" band. In most cases, this band is relatively short-lived and is usually not amenable for detection and measurement.
  • this band exists for less than a second.
  • certain class II oxidizing agents for example chromium compounds
  • the unique elements of the present composition allow for this cherry red band to be persistent enough to be detected and measured.
  • the composition of the present invention enhances the lifetime of the cherry red band in a urine sample. The exact chemical basis behind this phenomenal is presently unknown. Without wishing to limit this invention to any mechanism or theory of operation, it is believed that the cherry red band reflects an intermediate which is formed when an oxidizing agent is placed in the present composition.
  • the cherry red band persists for at least about 15 seconds to about 10 minutes, preferably 5 minutes. In a preferred embodiment, the cherry red band persists for at least about 30 seconds to about 3 minutes. In a more preferred embodiment, the cherry red band persists for at least about 40 seconds to about 2 minutes.
  • the composition further comprises an iodide. Without wishing to limit the invention to any mechanism or theory of operation, it is believed that the inclusion of an iodide in the composition increases the lifetime of the cherry red band. This increased time allows for a better detection and measurement of the band.
  • the molar ratio of the iodide to the amine is about two to about ten, preferably about three to about seven, more preferably about five.
  • a preferred composition comprises iodide in about five molar excess of DPD.
  • the composition comprises about 2 g to about 8 g, preferably 4.5 g of iodide salt, for example sodium iodide, per lOOOmL.
  • concentration of the iodide be diluted, for example with water.
  • the iodide concentration is diluted to about half at the time an abso ⁇ tion reading is made.
  • the composition is effective in facilitating the detection of a single adulterant, preferably an oxidizing agent.
  • the composition may facilitate in the detection of class II oxidizing agents (such as a nitrite), which is characterized by a strong abso ⁇ tion band at about 408 nm to about 420 nm, preferably about 410 nm, and a weak abso ⁇ tion band at about 470 nm to about 604 nm, preferably 570 nm; or the composition may facilitate in the detection of class I oxidizing agents (such as a hypochlorite), which is primarily characterized by the abso ⁇ tion band at about 408 nm to about 604 nm, preferably 570 nm.
  • class II oxidizing agents such as a nitrite
  • class I oxidizing agents such as a hypochlorite
  • the composition is effective in facilitating the detection of more than one adulterant, preferably more than one oxidizing agent.
  • a composition of the present invention may be useful in facilitating a simultaneous detection of both class I and class II oxidizing agents. See Example 5 below.
  • the composition of the present invention may be useful in facilitating the detection of one or more types of oxidizing agents within the same class.
  • the cherry red complex band may reflect a detection of a single oxidizing agent, for example hypochlorite, but the cherry red complex band may also reflect a measurement of more than one type of oxidizing agents belonging to the same class, such as a hypochlorite and a pyridinium chlorochromate.
  • the cherry red complex band may reflect a summation of an abso ⁇ tion from a species indicative of a hypochlorite and an abso ⁇ tion from a species indicative of chlorochromate.
  • the detection of an oxidizing agent is not by the detection of a band associated with the pu ⁇ le region of the visible spectrum. That is, in one embodiment, the oxidizing agent is not detected by an observation of a band having wavelengths of about 670 nm.
  • compositions of the present invention may be appropriate for use in a spot test. For example, a person may add an effective quantity of the present composition to a test tube containing a suspect urine sample. The person may then observe for a transitional cherry red color from the composition/urine mixture with the naked eye. An identification of a transitional cherry red color in the test tube is indicative that the urine may be adulterated with at least one oxidizing agent, for example a chromium compound.
  • the detection of an adulterant using the present compositions is facilitated by a spectrophotometer.
  • a spectrophotometer instead of observing for the transitional presence of the cherry red color with a naked eye, it is preferred that the composition/urine mixture be placed in a spectrophotometer for a detection of a band at about 470 nm to about 604 nm. The detection of such a band is indicative that the urine may contain an adulterant, for example a hypochlorite.
  • the spectrophotometer may detect for abso ⁇ tions at other wavelengths, for example at about 408 nm to about 420 nm, preferably 410 nm.
  • a detection of a strong an absorbance at about 408 nm to about 420 nm, and a weak absorbance at about 470 nm to about 604 nm suggests that the urine may be adulterated with another type of oxidizing agents, for example nitrites.
  • the detection of an adulterant using the present composition is facilitated by an automated spectrophotometer.
  • an automated spectrophotometer may be employed for the pu ⁇ ose of this invention, provided that it is capable of making a reading relatively quickly (e.g., less than a few minutes, preferably less than a few seconds) after the composition and the aqueous sample is mixed.
  • Non-limiting examples of automated spectrophotometers include the Olympus AU-800, AU-5000, AU-5200, AU-5400, Hatatchi 717, Hatatchi 747 and Beckman DU70 can be programmed to make abso ⁇ tion measurements within less than 30 seconds of mixing the composition and the aqueous sample.
  • the observation of bands associated with certain adulterants may be qualitative.
  • the abso ⁇ tion bands may yield quantitative data by preparing a calibration curve within the specified wavelengths.
  • the calibration curve may be prepared by methods commonly known in the art.
  • the composition comprises an N,N-diethylphenylene diamine and a creatinine.
  • This composition may be employed in accordance with this invention to detect the presence of one or more types of oxidizing agents, for example a hypochlorite, a pyridinium chlorochromate, and a hydrogen peroxide.
  • the composition may additionally be employed in accordance with this invention to detect one or more class of oxidizing agents, for example class I comprising chromium compounds and class II comprising nitrites.
  • the composition may be employed to detect the presence of more than one class of oxidizing agents simultaneously.
  • the concentration of the N,N-diethylphenylene diamine and a creatinine in this composition may be about 0.4 g/L and 2 g/L of the final composition/aqueous sample mixture volume, respectively.
  • the composition is employed to detect for these adulterants in a urine sample.
  • the concentrations of the amine and stabilizer in the composition prior to adding the aqueous sample to the composition and prior to abso ⁇ tion reading, may be twice the final composition/aqueous sample mixture concentrations.
  • a dilution of the composition may be achieved with adding water immediately prior to adding the aqueous sample, for example urine sample, or immediately prior to abso ⁇ tion reading.
  • the composition comprises (a) more than one amine, for example an N,N-diethylphenylene diamine and an aniline, and (b) one or more stabilizer, for example citrate and/or creatinine.
  • This composition may be employed in accordance with this invention to detect adulterants, such as oxidizing agents.
  • the concentration of the N,N-diethylphenylene diamine and aniline here may be about 0.2 g to about 0.4 g and about 0.1 g to about 0.4 g per the composition/urine sample mixture, respectively. If only one stabilizer is used, the citrate or creatinine concentrations may be about 2 g or about 2.5 g per the final volume of the composition urine sample mixture, respectively.
  • the citrate and creatinine concentration may be 2.25 g and 1.15 g per the final volume of the composition/urine sample mixture, respectively.
  • the composition is employed to detect for these adulterants in a urine sample.
  • the concentrations of the amine and stabilizer(s) in the composition may be twice the final composition/aqueous sample mixture concentrations.
  • a dilution of the composition may be achieved with adding water immediately prior to adding the aqueous sample, for example urine sample, or immediately prior to abso ⁇ tion reading.
  • the composition comprises an N,N-diethylphenylene diamine, iodide, citrate and creatinine.
  • This composition may be employed in accordance with this invention to detect the presence of one or more class of oxidizing agents, for example a hypochlorite, a pyridinium chlorochromate, and a hydrogen peroxide.
  • the composition may additionally be employed in accordance with this invention to detect one or more groups of oxidizing agents, for example the group comprising chromium compounds and the group comprising nitrites. Even more preferably, the composition may be employed to detect the presence of more than one group of oxidizing agents simultaneously.
  • the concentration of the N,N- diethylphenylene diamine, iodide, citrate and a creatinine in this composition may be about 0.1-0.5 g, about 1.0-3.5 g, about 1.0-2.0 g and about 1.5-3.0 g per the 1 liter volume of the final composition/urine sample mixture, respectively.
  • the concentration of the N,N-diethylphenylene diamine, iodide, citrate and a creatinine in this composition may be about 0.3 g, about 2.25 g, about 1.25 g and about 2.25 g per the 1 liter volume of the final composition/urine sample mixture, respectively.
  • the concentrations of the N,N-diethylphenylene diamine, iodide, citrate and a creatinine in the composition may be twice the final composition/aqueous sample mixture concentrations.
  • a dilution of the composition may be achieved with adding water immediately prior to adding the urine sample, or immediately prior to abso ⁇ tion reading.
  • the composition has a pH of about 5 to about 8, preferably about 6 to about 7.5, more preferably about 6.5 to about 7.5. Without wishing to limit the invention to any particular theory or mechanism of operation, it is believed that the present invention may function more properly at a neutral pH, not an acidic pH. In a preferred embodiment, the composition/aqueous sample has a neutral pH at the time an abso ⁇ tion reading is made. In one embodiment, a composition of the present invention may be used to detect oxidizing agents other than nitrite and the like.
  • a composition according to the present invention is preferably a solution
  • the composition may be also a gel or a solid.
  • a solid composition may be formed by dissolving DPD in a polymer that has the characteristics of a stabilizer.
  • Such stabilizer may comprise nucleophilic groups attached to a polymer backbone, for example polyvinylalcohol.
  • a solid composition may be formed by dissolving DPD in a polymer such as gelatin. The DPD/gelatin may then be sandwiched between two layers of gelatin in which the stabilizer has been dissolved.
  • the layers are thin, for example a micron or less in thickness.
  • both the DPD and the stabilizer may be dissolved in a polymer.
  • such polymer allows for the DPD and the stabilizer to substantially interact as though they are in solution.
  • Example 1 DPD/Creatinine Composition A composition for detecting the presence of oxidizing agents such as nitrites, chlorochromates and/or hypochlorites is prepared as follows:
  • Creatinine (about 3 g to about 5 g, preferably 4 g) is dissolved in about 1000 mL of deionized water. Then dissolve about 0.5 g to about 1 g, preferably about 0.8 g of N,N diethyl- 1,4-phenylene diamine in 60 mL of glacial acetic acid. To this latter solution, add enough of the creatinine solution prepared above to give a volume of 1000 mL. This creatining/diamine solution is then ready for use in a colorometric assay for detecting a nitrite and/or an oxidizing agent. Preferably this composition is used in conjunction with a urine sample. In one embodiment, the composition has a neutral pH.
  • the urine does not contain an additive and is maintained at room temperature, for example about 25°C to about 30 °C, preferably about 27°C. Furthermore, it is preferable that the urine be analyzed within about 20 hours, more preferably within about 10 hours, even more preferably within about 6 hours after it is extracted from a person or animal.
  • the step of adding the composition, water and urine sample to the cuvette, and the step of determining the presence of certain bands may be conducted manually.
  • these steps may be achieved by an automated spectrophotometer, provided that the automated machine is able to make a reading of the absorbance in about less than 40 seconds after the urine is added to the composition. Readings of the absorbance are made at about 25 °C to about 30 °C, preferably about 27 °C.
  • Example 3 DPD/Iodide/Creatinine/Citrate/Creatinine Composition A composition for detecting the presence of oxidizing agents such as nitrites, chlorochromates and/or hypochlorites is prepared as follows:
  • Dissolve Iodide (about 3 g to about 6 g, preferably about 4.5 g), creatinine (about 3 g to about 6 g, preferably about 4.5 g) and citrate (about 1 g to about 4 g, preferably about 2.5 g) in about 1000 mL deionized water. Then dissolve about 0.6 g of N,N diethyl- 1,4-phenylene diamine in about 80 mL of glacial acetic acid. To this latter solution, add enough of the iodide/creatinine/citrate solution prepared above to the give a volume of 1000 mL.
  • the iodide/creatinine/citrate/diamine solution is then ready for use in a colorometric assay for detecting a nitrite and/or an oxidizing agent.
  • this composition is used in conjunction with a urine sample.
  • the composition has a neutral pH.
  • Example 4 Method of Detecting an Adulterant in Urine Prepare a composition according to Example 3. Place about 250 uL of the composition into a cuvette. Add to the cuvette containing the composition about 250 uL of water and 3 uL of a test urine.
  • a presence of a former band is indicative of a nitrite adulterant or the like in the urine; a presence of a latter complex band is indicative of an oxidizing agent, for example hypochlorite and/or pyridinium chlorochromate, in the urine.
  • the step of adding the composition, water and urine sample to the cuvette, and the step of determining the presence of certain bands may be conducted manually.
  • these steps may be achieved by an automated spectrophotometer, provided that the automated machine is able to make a reading of the absorbance in about less than 40 seconds after the urine is added to the composition. Readings of the absorbance are made at about 25 °C to about 30 °C, preferably about 27 °C.
  • a mixture containing a nitrite shows a sha ⁇ abso ⁇ tion band at about 408nm to about 420 nm, and a small complex band at about 470 nm to about 604 nm.
  • a mixture containing the other group of oxidizing agents, for example hypochlorite also shows an abso ⁇ tion of a complex band at about 470 nm to about 604 nm.
  • a positive number may be indicative that there is a higher concentration of nitrite than non- nitrite oxidizing agent; and a negative number may be indicative that there is a higher concentration of oxidizing agent than nitrite.
  • the instrument used may detect only one band. In another embodiment, the instrument used may simultaneously detect more than one band, for example two bands. The use of a particular instrument is readily known to one of ordinary skill in the art.
  • Example 6 Figures 1 and 2 set out the visible spectra for the DPD composition prepared in Example 1 when sodium nitrite, sodium hypochlorite and pyridinium chlorochromate are added.
  • the visible spectra associated with the sodium hypochlorite are identical to the visible spectra associated with the pyridinium chlorochromate. It can easily be seen that the sodium nitrite produces an intense abso ⁇ tion at about 411 nm and a weak abso ⁇ tion at around 540 nm. Sodium hypochlorite generates an intense abso ⁇ tion only at 540 nm as does pyridinium chlorochromate. The method is useful for concentrations of sodium nitrite up to two thousand ug/ml. A high concentration of sodium hypochlorite (20% solutions) increases the lifetime of the cherry-red band. Results are shown in Table 1. These results are based on readings taken with the spectrophotometer set to record at 410 nm and 540nm.
  • Nitrite Specimens were tested for nitrite using a current nitrite reagent and DPD and creatinine as prepared in Example 1.
  • Pyridinium Chlorochromate Specimens tested for pyridinium chlorochromate and found to be positive were also evaluated by gas chromatography/mass spectrometry to confirm the presence of the adulterant.
  • Sodium hypochlorite (bleach) Specimens were tested for bleach and found to be positive were also evaluated using an AquaCheck dipstick to confirm the presence of chlorine. Carryover - High concentrations of nitrite, pyridinium chlorochromate and bleach were evaluated along with negative controls to determine the level at which carryover occurs in the testing process.
  • the target adulterant was spiked into deionized water for nitrite and urine for pyridinium chlorochromate and bleach and then DPD/creatinine composition prepared as per Example 1 was added as described below.
  • Table 3 sets outs results observed when solutions containing increasing concentrations of sodium nitrite were treated with DPD/creatinine composition described in Example 1.
  • Water was spiked with sodium nitrite to give different concentrations of nitrite as the starting point for generating an absorbance curve.
  • Spiked samples were processed through an Olympus AU800 autoanalyzer which sampled a 3 ⁇ l aliquot of the spiked specimen, mixed it with 250 ml of the DPD/creatinine composition described in Example 1 and 250 ml of deionized water.
  • Readings up to 200 ⁇ g/ml are considered to reflect unadulterated samples. Samples with readings between 201 and 499 ⁇ g/ml are flagged as being unacceptable and samples with readings of 500 ⁇ g/ml or higher are retested for nitrites using a second colorometric assay.
  • Urine was spiked with various amounts of commercial bleach comprising about 5.25% of sodium hypochlorite as the starting point for generating an absorbance curve.
  • Spiked samples were processed through an Olympus AU800 autoanalyzer which sampled a 3 ⁇ l aliquot of the spiked specimen, mixed it with 250 ml of the DPD/creatinine composition described in Example 1 and 250 ml of deionized water.

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Abstract

L'invention concerne une composition qui comprend au moins une amine et au moins un stabilisant, laquelle peut être utile pour détecter la présence d'un adultérant dans un échantillon d'urine. Un tel adultérant peut être un agent oxydant.
PCT/US2001/018560 2001-06-06 2001-06-06 Composition et procede pour detecter un adulterant dans un echantillon aqueux Ceased WO2002099419A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ITCS20080020A1 (it) * 2008-11-20 2010-05-21 Garofalo Alessandro Metodo e kit per la determinazione della concentrazione di perossidi in un liquido organico

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5464775A (en) * 1991-09-16 1995-11-07 Chimera Research And Chemical, Inc. Method of detecting adulterant in urine
US5801060A (en) * 1990-10-10 1998-09-01 Chimera Research & Chemical, Inc. Method of using automated analyzer testing of urine for presence of a pH abnormality with single reagent indicator
US5955370A (en) * 1993-11-02 1999-09-21 U.D. Testing, Inc. Urine adulteration test method
WO2000052464A1 (fr) * 1999-03-04 2000-09-08 Quest Diagnostics Investments Incorporated Reactif et procede utilises pour detecter un adulterant dans un echantillon aqueux

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5801060A (en) * 1990-10-10 1998-09-01 Chimera Research & Chemical, Inc. Method of using automated analyzer testing of urine for presence of a pH abnormality with single reagent indicator
US5464775A (en) * 1991-09-16 1995-11-07 Chimera Research And Chemical, Inc. Method of detecting adulterant in urine
US5955370A (en) * 1993-11-02 1999-09-21 U.D. Testing, Inc. Urine adulteration test method
WO2000052464A1 (fr) * 1999-03-04 2000-09-08 Quest Diagnostics Investments Incorporated Reactif et procede utilises pour detecter un adulterant dans un echantillon aqueux

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ITCS20080020A1 (it) * 2008-11-20 2010-05-21 Garofalo Alessandro Metodo e kit per la determinazione della concentrazione di perossidi in un liquido organico

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