U.S. patent application number 10/912528 was filed with the patent office on 2005-03-03 for composition and method for detecting an adulterant in an aqueous sample.
This patent application is currently assigned to Quest Diagnostics Investments Incorporated. Invention is credited to Hilderbrand, Richard L., Johnson, Victoria, Mills, Susan, Novinski, John, Sample, Barry.
Application Number | 20050048663 10/912528 |
Document ID | / |
Family ID | 24061649 |
Filed Date | 2005-03-03 |
United States Patent
Application |
20050048663 |
Kind Code |
A1 |
Novinski, John ; et
al. |
March 3, 2005 |
Composition and method for detecting an adulterant in an aqueous
sample
Abstract
It is discovered that a composition comprising at least one
amine and at least one stabilizer may be useful in detecting the
presence of an adulterant in a urine sample. Such adulterant
includes an oxidizing agent.
Inventors: |
Novinski, John; (Leesburg,
FL) ; Sample, Barry; (Alpharetta, GA) ;
Hilderbrand, Richard L.; (Lady Lake, FL) ; Mills,
Susan; (Ambler, PA) ; Johnson, Victoria;
(Orlando, FL) |
Correspondence
Address: |
STOUT, UXA, BUYAN & MULLINS LLP
4 VENTURE, SUITE 300
IRVINE
CA
92618
US
|
Assignee: |
Quest Diagnostics Investments
Incorporated
Wilmington
DE
|
Family ID: |
24061649 |
Appl. No.: |
10/912528 |
Filed: |
August 4, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10912528 |
Aug 4, 2004 |
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09876716 |
Jun 6, 2001 |
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09876716 |
Jun 6, 2001 |
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09517891 |
Mar 3, 2000 |
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6303384 |
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60123299 |
Mar 4, 1999 |
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Current U.S.
Class: |
436/164 |
Current CPC
Class: |
Y10T 436/112499
20150115; Y10T 436/179228 20150115; G01N 33/52 20130101; Y10T
436/141111 20150115; Y10T 436/212 20150115; G01N 33/70 20130101;
G01N 33/84 20130101; Y10T 436/173845 20150115; G01N 33/493
20130101; G01N 31/22 20130101; Y10T 436/173076 20150115 |
Class at
Publication: |
436/164 |
International
Class: |
G01N 033/48 |
Claims
1-64. (cancelled)
65. A method for determining whether a urine sample has been
adulterated by an oxidizing agent which interferes with detection
of a drug or metabolite thereof present in the urine sample,
comprising contacting a urine sample with a reagent composition,
the reagent composition comprising an amine reagent which reacts
with an oxidizing agent which interferes with detection of a drug
or metabolite thereof in the urine sample; and
spectrophotometrically detecting a color signal produced by the
amine reagent upon reaction with the oxidizing agent if the
oxidizing agent is present in the urine sample, wherein a
detectable signal indicates the urine sample has been adulterated
with the oxidizing agent.
66. The method of claim 65, wherein the urine sample is from a
person undergoing a drug screening test.
67. The method of claim 65, wherein the amine reagent comprises an
aromatic ring.
68. The method of claim 67, wherein the aromatic ring is a phenyl
ring.
69. The method of claim 68, wherein said phenyl ring is a
substituted phenyl ring.
70. The method of claim 65, wherein the amine reagent is
N,N-diethylphenylene diamine.
71. The method of claim 65, wherein the oxidizing agent is an agent
selected from the group consisting of transition metals, nitrites,
peroxides, halites, hypohalites, salts thereof, and combinations
thereof.
72. The method of claim 66, wherein the oxidizing agent interferes
with the detection of an illegal drug used by the person.
73. The method of claim 65, wherein the reagent composition further
comprises a stabilizer which enhances a time the color signal is
detectable relative to a substantially identical reagent
composition without the stabilizer.
74. The method of claim 65, wherein the detecting comprises
detecting a signal at a wavelength from about 470 nm to about 604
nm.
75. The method of claim 65, wherein the detecting comprises
detecting a signal at a wavelength from about 408 nm to about 420
nm.
76. A method for detecting the presence of an oxidizing agent
adulterant in a urine sample, the method comprising: combining a
urine sample with a reagent composition, the reagent composition
comprising an amine reagent which reacts with an oxidizing agent
which interferes with detection of a drug or metabolite thereof in
the urine sample, and detecting a signal produced by interaction of
the amine reagent with an oxidizing agent adulterant which was
added to the urine sample before the urine sample was combined with
the reagent composition.
77. A reagent composition for determining whether a urine sample
has been adulterated by an oxidizing agent which interferes with
detection of a drug or metabolite thereof present in the urine
sample, comprising an amine reagent which reacts with an oxidizing
agent present in the urine sample to produce a
spectrophotometrically detectable color signal if present in the
urine sample, wherein said oxidizing agent interferes with
detection of a drug or metabolite thereof in the urine sample.
78. The composition of claim 77, further comprising a stabilizer
which enhances a time the color signal is detectable relative to a
substantially identical reagent composition without the
stabilizer.
79. The composition of claim 77, wherein the amine reagent
comprises an aromatic ring.
80. The composition of claim 79, wherein the aromatic ring is a
phenyl ring.
81. The composition of claim 80, wherein the phenyl ring is a
substituted phenyl ring.
82. The composition of claim 77, wherein the amine reagent is
N,N-diethylphenylene diamine.
83. The composition of claim 77, wherein the amine reagent reacts
with an oxidizing agent selected from the group consisting of
transition metals, nitrites, peroxides, halites, hypohalites, salts
thereof, and combinations thereof.
84. The composition of claim 77, wherein the amine reagent reacts
with an oxidizing agent effective in interfering with the detection
of an illegal drug.
85. The composition of claim 77, wherein the amine reagent produces
a detectable signal at a wavelength from about 470 nm to about 604
nm when reacted with the oxidizing agent present in the urine
sample.
86. The composition of claim 77, wherein the amine reagent produces
a detectable signal at a wavelength from about 408 nm to about 420
nm when reacted with the oxidizing agent present in the urine
sample.
87. The composition of claim 77 in combination with a urine sample
obtained from a person undergoing a drug screening test.
Description
RELATED APPLICATION
[0001] The present application is a continuation-in-part of
co-pending application Ser. No. 09/517,891, filed Mar. 03, 2000,
the disclosure of which is hereby incorporated in its entirety,
herein by reference.
BACKGROUND OF THE INVENTION
[0002] Drug testing is mandated or strongly supported by many
political and regulatory groups and private industry. Hiring or
continued employment may depend on a drug-free test. Urine-based
testing is a widely practiced way of detecting the use of
controlled substances or substances of abuse. Not too surprisingly,
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.
[0003] Various compositions and methods are presently available for
detecting an adulterant in urine samples. For example, Kell in U.S.
Pat. No. 5,955,370 disclosed a method for detecting a diuretic in a
urine sample. However, there continues to be a need for a better
composition and method for detecting the presence of adulterants
such as oxidizing agents in urine samples.
SUMMARY OF THE INVENTION
[0004] This invention provides for that need. In accordance with
the present invention, a composition for use in detecting the
presence of at least one adulterant in an aqueous sample is
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.
[0005] Further in accordance with the present invention, an
adulterant according to this invention comprises an oxidizing
agent. Examples of 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.
[0006] Still further in accordance with the present invention, 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.
[0007] Still further in accordance with the present invention, 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.
[0008] Still further in accordance with the present invention, the
amine comprises a primary aromatic amine. For example, the amine
comprises N,N-diethylphenylene diamine.
[0009] Still further in accordance with the present invention, the
stabilizer comprises a creatinine and/or citrate.
[0010] Still further in accordance with the present invention, the
composition further comprises an iodide. For example, the
composition may comprise N,N-diethylphenylene diamine
/Iodide/Creatinine/Citrate/Creatini- ne.
[0011] Still further in accordance with the present invention, a
method for detecting at least one adulterant is provided. The
method 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.
[0012] Any feature or combination of features described herein are
included within the scope of the present invention provided that
the features included in any such combination are not mutually
inconsistent as will be apparent from the context, this
specification, and the knowledge of one of ordinary skill in the
art.
[0013] Additional advantages and aspects of the present invention
are apparent in the following detailed description and claims.
DESCRIPTION OF THE FIGURES
[0014] 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.
[0015] 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.
[0016] 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.
[0017] 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.
[0018] 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.
[0019] 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.
[0020] FIG. 7 is a tracing of a visible absorption spectra showing
a "cherry red" band and a "purple" band.
DETAILED DESCRIPTION OF THE INVENTION
[0021] The present invention is, in part, based upon the discovery
that, 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. In one embodiment, 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. Preferably the observations of the absorption bands
are made with a spectrophotometer. As used herein, a band may be a
simple band of one peak or a complex band of more than one
peak.
[0022] An aqueous sample where an adulterant may be detected may
include a body fluid. In one embodiment, a body fluid includes a
blood sample, a tear sample and/or, preferably, a urine sample.
[0023] As used herein, 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. Non-limiting examples of a drug
includes opioids (e.g. Heroin, morphine, methadone, codeine), CNS
depressants (e.g. barbiturates, phenobarbital, benzodiazephines
valium, Librium), psychostimulants (e.g. cocaine, amphetamines,
methamphetamines, "crack"), cannabinoids (e.g. marihuana, hashish,
tetrahydrocannabinol, cannabinol, cannabidiol), psychedelics (e.g.
hallucinogens, psychotomimetics, psychotogens, lysergic acid
diethylamide, mescaline, peyote, psilocybin, "magic" mushrooms),
arylcyclohexylamines (e.g. phencyclidine, "angel dust", "crystal",
PCP).
[0024] In one embodiment, an adulterant comprises any molecule or
complex which is capable of preventing the detection of another
chemical entity. In a preferred embodiment, 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. Preferably, the oxidizing agent prevents
the detection of a presence of a drug in an aqueous sample, for
example a urine sample.
[0025] One type of oxidizing agent comprises a transition metal.
Preferably, 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. Pat. No. 6,100,394, the disclosure
of which is incorporated in its entirety herein by reference.
Another example of such oxidizing agent includes a transitional
metal salt. In one embodiment, an oxidizing agent comprises
chromium compounds. Preferably, these oxidizing agents comprise
Chromium (VI) compounds, for example, chromate salts, pyridinium
chlorochromate, pyridinium fluorochromate, chromium oxide,
dichromate and the like and mixtures thereof. In another preferred
embodiment, an oxidizing agent comprises vanadium compounds, for
example sodium meta vandate (V). In yet another preferred
embodiment, these oxidizing agents comprise Cerium (IV) compounds,
for example ammonium hexanitrato cerate (IV).
[0026] Another type of oxidizing agent comprises a nitrite, alkyl
nitrite, arylnitrite, nitrous acid, the like and mixtures thereof.
Yet another type of oxidizing agents comprises nitrogen
heterocyclic salts of oxidizing agents, for example, pyridinium
hydrogen perbromide, quinolinium dichromate, the like and mixtures
thereof.
[0027] Other types of 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.
[0028] In one embodiment, the oxidizing agent as used herein does
not include a nitrite, alkyl nitrite, arylnitrite, nitrous acid,
the like and mixtures thereof.
[0029] In one embodiment, the composition comprises a nitrogen
containing molecule, for example an azine or a diphenylazine.
Preferably, the composition comprises an amine. In a preferred
embodiment, 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.
[0030] Chart 1 shows non-limiting examples of amines that may be
used in accordance with the present invention. 1
[0031] With respect to compound I, R.sub.1, R.sub.2, R.sub.3,
R.sub.4, and R.sub.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. In a preferred embodiment, the alkyl comprises about 2 to
about 10 carbons, more preferably about 2 to about 5 carbons. The
amino may be R.sub.6--N--R.sub.7, where R6 and R.sub.7 may each
individually be an alkyl, aryl group, hydrogen, halogen,
hydrocarboxy, carboxylic acid ester or sulfonic acid ester.
[0032] Furthermore, R.sub.1, R.sub.2, R.sub.3, R.sub.4, and
R.sub.5, or R.sub.6 and R.sub.7 may bridge with each other.
Compound II is an example of a bridging between R.sub.2 and
R.sub.3. Compound III is an example of a bridging between
R.sub.1and R.sub.2.
[0033] In a preferred embodiment, the amine is an o, m or
p-N,N-diethylphenylene diamine (hereinafter "DPD"), compounds IV, V
and VI, respectively (Available from Aldrich catalog no. 26151-3).
In a more preferred embodiment, the amine is a p-DPD (compound
VI).
[0034] Other amines, or preferably primary aromatic 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-aminonaphtha- line, the like and mixtures thereof. In
one embodiment, the amine may be a secondary aromatic amine, for
example anilinopyridine.
[0035] In one embodiment, an amine in accordance with the present
invention may be a NH.sub.2 covalently linked to a conjugated
system, forming a conjugated-system-amine. The conjugated-system
may be a carbon-chain. A carbon conjugated system may comprise 3 to
15 carbons, preferably 4 to 8 carbons. For example, an amine in
accordance with the present invention may be
NH.sub.2CH.dbd.CH--CH.dbd.CH.sub.2. The conjugated system may also
include a heteroatom. For example, an amine in accordance with the
present invention may be NH.sub.2C.dbd.N--N.dbd.C.
[0036] The amount of amine, for example aromatic amine, preferably
primary aromatic amine, used will be some concentration sufficient
to give a useful visible absorption spectra. Generally, 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. At the time an absorption reading is
made, it is preferable that 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
absorption reading is made. The dilution is especially advantageous
when the aqueous sample is suspected of containing a transition
metal oxidizing-agent. Furthermore, it is preferable that the amine
is dissolved in a suitable solvent prior to being added to the test
sample. Various solvents can be used so long as the compound
dissolves in it. In one embodiment, organic type or aprotic
solvents are used. For example, 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.
[0037] In one embodiment, the composition comprises a stabilizer.
Without wishing to limit the invention to any particular mechanism
or theory of operation, it is believed that the stabilizer
stabilizes a species which gives rise to a band associated with at
least one adulterant. In some situations, it is further believed
that the stabilized species is an adulterant-stabilizer
intermediate, for example an (oxidizing agent)-DPD intermediate. In
any case, the stabilized species gives rise to a more persistent
band. For example, the stabilizer may facilitate an increase in the
life-time of a band associated with the oxidizing agent. In one
embodiment, the stabilizer increases the lifetime of a band
associated with the oxidizing agent, wherein the band is not
defined by the purple spectra region, see FIG. 7.
[0038] In a preferred embodiment, 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. For example, a stabilizer may be
any nucleophile. Preferably, 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. In a preferred embodiment, a stabilizer is a
creatinine (available from Sigma Catalog No. C4255). In another
preferred embodiment, a stabilizer is a citrate. In yet another
embodiment, the composition of the present invention comprises at
least two stabilizers, for example a creatinine and a citrate.
[0039] In one embodiment, a stabilizer according to the present
invention is not used as an acid.
[0040] 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. At the time an
absorption reading is made, it is preferable that the concentration
of the stabilizer in the composition be diluted, for example with
water. In one embodiment, the original concentration of the
stabilizer in the composition is diluted to about half at the time
an absorption reading is made.
[0041] 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. In one embodiment, a small volume
of an aqueous sample may be added to a composition of the present
invention to form a mixture. For example, 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. In a preferred embodiment, about 3
uL of an aqueous sample may be added to 250 uL of the composition,
or similar ratios. Preferably, the aqueous samples above are urine
samples.
[0042] A mixture containing an adulterant, for example an oxidizing
agent, will have an absorption 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.
[0043] In one embodiment, 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
absorption of a broad complex band at about 470 nm to about 604 nm,
or as is substantially described in FIGS. 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 absorption band at about
408 nm to about 420 nm, preferably at about 410 nm, and a weak
absorption band at about 470 nm to about 620 nm, preferably at 511
nm. FIG. 1 substantially describes the absorption characteristics
of class II oxidizing agents. A non-limiting example of a class II
oxidizing agent includes a nitrite.
[0044] The absorption 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. For example, in urine
samples, this band exists for less than a second. As such, the
detection of certain class II oxidizing agents, for example
chromium compounds, in urine would be difficult or nearly
impossible. However, it has been surprisingly discovered that the
unique elements of the present composition allow for this cherry
red band to be persistent enough to be detected and measured.
Preferably, 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.
[0045] In one embodiment, 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. In one embodiment, 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. Preferably, 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. For
example, a preferred composition comprises iodide in about five
molar excess of DPD. In one embodiment, the composition comprises
about 2 g to about 8 g, preferably 4.5 g of iodide salt, for
example sodium iodide, per 1000 mL. At the time an absorption
reading is made, it is preferable that the concentration of the
iodide be diluted, for example with water. In one embodiment, the
iodide concentration is diluted to about half at the time an
absorption reading is made.
[0046] In one embodiment, the composition is effective in
facilitating the detection of a single adulterant, preferably an
oxidizing agent. For example, the composition may facilitate in the
detection of class II oxidizing agents (such as a nitrite), which
is characterized by a strong absorption band at about 408 nm to
about 420 nm, preferably about 410 nm, and a weak absorption 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 absorption band at about 408 nm to about 604 nm, preferably
570 nm.
[0047] In a preferred embodiment, the composition is effective in
facilitating the detection of more than one adulterant, preferably
more than one oxidizing agent. For example, 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.
[0048] Furthermore, 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. For example, 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. In the latter case, the cherry red
complex band may reflect a summation of an absorption from a
species indicative of a hypochlorite and an absorption from a
species indicative of chlorochromate.
[0049] In one embodiment, the detection of an oxidizing agent is
not by the detection of a band associated with the purple 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.
[0050] 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.
[0051] Preferably, the detection of an adulterant using the present
compositions is facilitated by a spectrophotometer. For example,
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. Additionally, the
spectrophotometer may detect for absorptions 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.
[0052] More preferably, the detection of an adulterant using the
present composition is facilitated by an automated
spectrophotometer. In some situations, certain transitional bands
appear within seconds after the composition is mixed with the
suspect urine. For example, a detection of the absorption bands
associated with DPD stabilized products must be made within seconds
after the mixing of the composition and the urine. Any automated
spectrophotometer may be employed for the purpose 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
absorption measurements within less than 30 seconds of mixing the
composition and the aqueous sample.
[0053] In one embodiment, the observation of bands associated with
certain adulterants may be qualitative. However, the absorption
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.
[0054] In one embodiment, 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.
Preferably, 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. Even more preferably, 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. Preferably, the composition is
employed to detect for these adulterants in a urine sample. In one
embodiment, prior to adding the aqueous sample to the composition
and prior to absorption reading, the concentrations of the amine
and stabilizer 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 absorption reading.
[0055] In one embodiment, 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. If a combination of
stabilizer is used, 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. Preferably, the composition is
employed to detect for these adulterants in a urine sample. In one
embodiment, prior to adding the aqueous sample to the composition
and prior to absorption reading, 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 absorption reading.
[0056] In a preferred embodiment, 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. Preferably, 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. Preferably, 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. In one
embodiment, prior to adding the aqueous sample to the composition
and prior to absorption reading, 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 absorption reading.
[0057] In one embodiment, 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
absorption reading is made.
[0058] In one embodiment, a composition of the present invention
may be used to detect oxidizing agents other than nitrite and the
like.
[0059] Although a composition according to the present invention is
preferably a solution, the composition may be also a gel or a
solid. In one embodiment, 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. In
another embodiment, 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. Preferably, the layers are thin, for example a
micron or less in thickness. In yet another embodiment, both the
DPD and the stabilizer may be dissolved in a polymer. Preferably,
such polymer allows for the DPD and the stabilizer to substantially
interact as though they are in solution.
[0060] The following examples are provided to illustrate the
invention but are not intended to limit it in any fashion or to any
degree.
EXAMPLE 1
DPD/Creatinine Composition
[0061] A composition for detecting the presence of oxidizing agents
such as nitrites, chlorochromates and/or hypochlorites is prepared
as follows:
[0062] 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.
EXAMPLE 2
Method of Detecting an Adulterant in Urine
[0063] Obtain urine sample from a patient. In one embodiment, the
urine does not contain an additive and is maintained at room
temperature, for example about 25.degree. C. to about 30.degree.
C., preferably about 27.degree. 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.
[0064] Prepare a composition according to Example 1. Place about
250 uL of the composition into a cuvette. Preferably the
composition is maintained at 25-30.degree. C., more preferably
27.degree. C., at all times. Add to the cuvette containing the
composition about 250 uL of deionized water and about 3 uL of a
test urine. Immediately after the addition of water and urine,
determine the presence of a band at about 408 nm to about 420 nm,
preferably 410 nm, and a complex band at about 470 nm to about 604
nm, preferably 540 nm. A presence of a former band is indicative of
a nitrite adulterant in the urine; a presence of a latter complex
band is indicative of an oxidizing agent in the urine.
[0065] 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. Preferably, 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.degree. C. to about 30.degree. C., preferably about 27.degree.
C.
EXAMPLE 3
DPD/Iodide/Creatinine/Citrate/Creatinine Composition
[0066] A composition for detecting the presence of oxidizing agents
such as nitrites, chlorochromates and/or hypochlorites is prepared
as follows:
[0067] 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. Preferably, this composition is
used in conjunction with a urine sample. In one embodiment, the
composition has a neutral pH.
EXAMPLE 4
Method of Detecting an Adulterant in Urine
[0068] 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. Immediately after the addition of the water and urine,
determine the presence of a band at about 408 nm to about 420 nm,
preferably 410 nm, and a complex band at about 470 nm to about 604
nm, preferably 540 nm. 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.
[0069] 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. Preferably, 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.degree. C. to about 30.degree. C., preferably about 27.degree.
C.
EXAMPLE 5
Distinguishing Between a Nitrite and Other Oxidizing Agents in a
Single Assay
[0070] A mixture containing a nitrite shows a sharp absorption band
at about 408 nm 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
absorption of a complex band at about 470 nm to about 604 nm.
[0071] Thus, to determine the presence of non-nitrite oxidizing
agents, subtract the absorption in the range of the broad band (470
nm to 604 nm) for the non-nitrite oxidizing agent from the
absorption in the sharp absorption (408 nm to 420 nm) of the
nitrite. (The absorption is quantified either by the area of the
band or the height of the band.) A positive number may be
indicative of the presence of nitrite and a negative number may be
indicative of the presence of a halogen containing bleach. In one
embodiment, 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.
[0072] In one embodiment, 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
[0073] FIGS. 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
absorption at about 411 nm and a weak absorption at around 540 nm.
Sodium hypochlorite generates an intense absorption 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 540 nm.
1TABLE 1 Absorbances for NaNo.sub.2, NaOCl, and Pyridinium
Chlorochromate NaNo.sub.2 Pyridinium Conc. Absorption Chloromate
Absorb. NaOCl Absorb. 200 .mu.g/ml 1647 125 ppm -79.25 0.05% -742.2
400 .mu.g/ml 3230 250 ppm -159.6 0.21% -1724.8 500 .mu.g/ml 4158
500 ppm -320.2 0.32% -2212.4 1000 .mu.g/ml 9280 1000 ppm -601.4
0.52% Flagged as high 2000 .mu.g/ml 1.58% Flagged as high
[0074]
2TABLE 2 Spectrophotometer Parameters - Olympus AU800 Description
Setting Sample vol.: 3 uL Composition vol. 250 uL Diluent vol.: 250
uL Wavelength 1: 410 nm Wavelength 2: 540 nm Method End Reaction
slope + Measure pt S.sub.1 = 0 E.sub.1 = 1
EXAMPLE 7
Validation Studies
[0075] Validation Studies were Performed to Evaluate the Following
Parameters for Nitrite, Pyridinium Chlorochromate and Sodium
Hypochloritc:
[0076] Linearity--The linear range at multiple concentration ranges
above and below the cutoff were evaluated.
[0077] Precision--Intra-run precision was evaluated at the
concentration ranges used for linearity evaluation. Inter-run
precision was evaluated on quality control samples spiked at +25%
and -25% of cutoff.
[0078] Nitrite: Specimens were tested for nitrite using a current
nitrite reagent and DPD and creatinine as prepared in Example
1.
[0079] 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.
[0080] 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.
[0081] 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.
[0082] Olympus AU 5061 and AU800 chemistry analyzers were used for
recording absorbance spectra.
[0083] In each of these assays 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.
EXAMPLE 7(a)
Sodium Nitrite Evaluation with DPD/Creatinine Composition
[0084] 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 ul 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. The analyzer control software was set to S1=0
and E1=2 and a reading was taken at 410 nm. These settings allow
for an absorption reading at about 30 seconds after the composition
is mixed with the urine.
[0085] Readings up to 200 ug/ml are considered to reflect
unadulterated samples. Samples with readings between 201 and 499
ug/ml are flagged as being unacceptable and samples with readings
of 500 ug/ml or higher are retested for nitrites using a second
colorometric assay.
3TABLE 3 Nitrite Nitrite Evaluation with DPD/Creatinine Conc.
Composition .mu.g/ml Assayed Values (concentration units) Average
50 54 56 56 55 50 54 100 106 112 108 108 106 108 250 246 267 260
263 256 258 375 373 398 383 379 372 381 500 479 504 501 491 487 492
625 597 645 619 629 611 620 750 690 737 729 733 700 718 1000 885
961 930 905 905 917 2000 1498 1553 1561 1580 1475 1533 3000 1887
1997 1928 1931 1881 1925 Series 1 Series 2 Series 3 Series 4 Series
5 Series 6
EXAMPLE 7(b)
Pyridium Chlorochromate Evaluation with DPD/Creatinine
Composition
[0086] Urine was spiked with various amounts of pyridinium
chlorochromate as the starting point for generating an absorbance
curve. Spiked samples were processed through an Olympus AU800
autoanalyzer which sampled a 3 .mu.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. The analyzer
control software was set to S1=0 and E1=2 and a reading was taken
at 540 nm. Table 4 contains the data from five runs and FIG. 4 is a
graph of these results.
[0087] Based on the instrument printouts in concentration mode,
readings less than -50 .mu.g/ml are considered to reflect
adulterated samples are subjected to alternative testing to confirm
the presence or absence of pyridinium chlorochromate.
4TABLE 4 Evaluation of Pyridinium Chlorochromate with
DPD/creatinine Composition Conc. Assayed Values .mu.g/ml
(concentration units) Average 50 40 41 42 36 42 40 75 60 61 63 62
63 62 112.5 85 89 92 86 87 88 125 101 104 99 103 102 150 116 123
123 118 121 120 187.5 147 153 154 154 154 152 225 176 186 175 172
177 500 372 403 403 407 390 395 1000 757 802 786 779 750 775 2000
1399 1472 1460 1410 1416 1431 3000 2017 2092 2110 2068 2045 2066
4000 2596 2724 2687 2665 2644 2663 Series 1 Series 2 Series 3
Series 4 Series 5 Series 6
EXAMPLE 7(c)
Evaluation of DPD/Creatinine as a Test for Na Hypochlorite
[0088] 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 .mu.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. The analyzer control software was set to S1=0 and
E1=2 and a reading was taken at 540 nm, 30 seconds after mixing.
Results are given in Table 5 and in graphic form in FIG. 5.
[0089] Based on the instrument printout in concentration mode,
readings of greater than -50 .mu.g/ml are considered to represent
normal unadulterated samples and readings equal or less than -50
.mu.g/ml or higher are confirmed by a second test for chlorine.
5TABLE 5 Evaluation of Sodium Hypochlorite with DPD/Creatinine
Conc. Composition .mu.g/ml Assayed Values (Absorbance units)
Average 0.03 61 9 36 42 9 37 0.05 101 25 71 124 29 80 0.13 256 158
231 183 103 184 0.20 122 303 234 147 202 0.26 304 151 289 211 135
218 0.33 267 143 275 191 131 201 0.38 256 130 268 173 113 188 0.53
278 269 747 390 349 407 1.05 1734 Abs Error Abs Error Abs Error Abs
Error 1.58 Abs Abs Error Abs Error Abs Error Abs Error Error Series
1 Series 2 Series 3 Series 4 Series 5 Series 6
[0090] While this invention has been described with respect to
various specific examples and embodiments, it is to be understood
that the invention is not limited thereto and that it can be
variously practiced with the scope of the following claims.
* * * * *