Methods for the Detection of Oxidative Adulterants in Urine Sample

Abstract

The problem of confirming the presence of an adulterant in a urine sample is solved by the use of a reagent capable of reacting with uric acid and non-urate markers in a urine sample. In one embodiment, a phosphotungtate reagent is used to react with the urine sample to create a blue coloration in the presence of uric acid or uric acid equivalents. A reduction or elimination of the blue coloration, resulting in a reduction in the light absorbance, of the urine sample can be used as an indicator of the historical presence of an adulterant. An Oxidant History test can also be generated using the phosphostungtate reagent, wherein the light absorbance resulting from the blue coloration is measured over time, with a measured reduction in the absorbance being an indication that an adulterant is or has been present in the urine sample and is oxidizing the uric acid and non-urate markers over time.

Claims

1. An assay method that measures the disappearance of a reducing constituent normally present in human urine, wherein the disappearance of such reducing constituent indicates adulteration of the urine.

2. An assay method that measures Uric Acid Equivalents in urine, the method comprising: reacting the biological sample with a reagent that is oxidized by Uric Acid Equivalents in the sample; exposing the reacted biological sample to a light source; obtaining a light absorbance measurement of the reacted biological sample; and calculating a Uric Acid Equivalents value utilizing the light absorbance measurement.

3. The assay method according to claim 2, wherein the light absorbance is measured at between 600-700 nm.

4. The assay method according to claim 1, wherein the Uric Acid Equivalents value determines the historical presence of an oxidative-adulterant in the biological sample.

5. The assay method according to claim 4, wherein the reagent comprises phosphotungstate.

6. The assay method according to claim 5, wherein the phosphotungstate reagent is adapted to be utilized in an automated clinical analyzer suitable for analyzing biological samples.

7. The assay method according to claim 2, wherein the oxidant to be detected is selected from the group consisting of: potassium nitrite, potassium chlorochromate, hydrogen peroxide/peroxidase, and sodium hypochlorite.

8. An assay method that determines the historical presence of an oxidative-adulterant in a biological sample, the method comprising: reacting the biological sample with a reagent comprising phosphotungstate; analyzing the reacted sample using spectrophotometry to obtain a first light absorbance measurement of the reacted biological sample; calculating a first Uric Acid Equivalents value utilizing the first light absorbance measurement; analyzing the reacted sample again by spectrophotometry, after a pre-determined amount of time; obtaining an additional light absorbance measure of the reacted biological sample; calculating an additional Uric Acid Equivalents value utilizing the additional light absorbance measurement; and utilizing the first Uric Acid Equivalents value and additional Uric Acid Equivalents value to obtain an Oxidant History for the sample.

9. The method according to claim 8, further comprising analyzing the Oxidant History to determine if the biological sample was treated with an oxidizing-adulterant.

10. The method according to claim 8, wherein the Oxidant History is obtained utilizing an automated system for analyzing biological samples.

11. The assay method according to claim 10, wherein the oxidizing-adulterant to be detected is selected from the group consisting of: potassium nitrite, potassium chlorochromate, hydrogen peroxide/peroxidase, and sodium hypochlorite.

12. An assay kit that detects oxidation of Uric Acid Equivalents in urine, the kit comprising: a first reagent comprising phosphotungstate that reacts with Uric Acid Equivalents in urine; a second reagent comprising carbonate that is used to buffer the reacted first reagent; and a third reagent comprising uric acid that can be used as a calibrator.

13. The kit according to claim 11, wherein the first reagent, the second reagent, and the third reagent are adapted to be used with an automated analyzer.

14. The kit according to claim 13, wherein the amount of oxidized Uric Acid Equivalents is determined colorimetrically.

15. A method that determines the Uric Acid Equivalents in a sample of a biological fluid, the method comprising: (a) reacting the sample with a first reagent that is oxidized by Uric Acid Equivalents in the sample, such that the sample changes color; (b) analyzing the reacted sample with a spectrophotometer to obtain a first measure of the absorbance of the sample; (c) calculating a Uric Acid Equivalents value utilizing the first absorbance measure; (d) reanalyzing the reacted sample with a spectrophotometer, after a predetermined amount of time, to obtain a second measure of the absorbance of the sample; and (e) calculating a Uric Acid Equivalents value utilizing the second absorbance measure.

16. The method according to claim 15, further comprising comparing the first absorbance measure to the second absorbance measure, wherein a second Uric Acid Equivalents value that is less than the first Uric Acid Equivalents value indicates that the sample was adulterated with an oxidant.

17. The method according to claim 15, wherein steps (b) through (e) are repeated to obtain additional Uric Acid Equivalent values, such that a decrease in the Uric Acid Equivalent values over time indicates that the sample was adulterated with an oxidant.

18. The method according to claim 15, wherein the Uric Acid Equivalents in the sample reacted with the phosphotungstate in the first reagent to change the sample to blue.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0030] In order that a more precise understanding of the above recited invention can be obtained, a more particular description of the invention briefly described above will be rendered by reference to specific embodiments thereof that are illustrated in the appended drawings. The drawings presented herein may not be drawn to scale and any reference to dimensions in the drawings or the following description is specific to the embodiments disclosed. Any variations of these dimensions that will allow the subject invention to function for its intended purpose are considered to be within the scope of the subject invention. Thus, understanding that these drawings depict only typical embodiments of the invention and are not therefore to be considered as limiting in scope, the invention will be described and explained with additional specificity and detail through the use of the accompanying drawings in which:

[0031] FIG. 1 is a graph of an Oxidant History of Uric Acid Equivalents in a urine sample that has been adulterated with the STEALTH, a commercially-available adulterant.

[0032] FIG. 2 is a graph of an Oxidant History of Uric Acid Equivalents in a urine sample that has been adulterated with a 10% potassium nitrate.

[0033] FIG. 3 is a graph of an Oxidant History of Uric Acid Equivalents in a urine sample that has been adulterated with 2% bleach.

[0034] FIG. 4 is a graph that indicates the results obtained with a prior art General Oxidant Test reagent used on a sample adulterated with a Stealth-like adulterant. It can be seen that the nitrite equivalents fell below the accepted absorbance cut-off of 500 μg/mL within a few hours.

[0035] FIG. 5 is a graph that indicates the results obtained with a prior art General Oxidant Test reagent used on a sample adulterated with bleach.

[0036] FIG. 6 is a graph that indicates the results obtained with a prior art General Oxidant Test reagent used on a sample adulterated with pyridum chlorochromate (PCC).

DETAILED DISCLOSURE

[0037] The subject invention pertains to an assay reagent and methods of use for detecting a decrease in specific markers present in a urine sample. In specific embodiments, the subject invention provides an assay reagent that reacts with uric acid and non-urate constituents found normally in urine. The methods described herein can be useful for determining whether a urine sample has been treated with an oxidative-adulterant. The methods and reagents of the subject invention can provide a visually detectable and quantifiable result. Advantageously, the embodiments of the subject invention can also be incorporated with standard automated laboratory equipment, such as clinical analyzers, utilized to conduct automated testing of urine samples for Drugs of Abuse (DOA).

[0038] The subject invention is particularly useful in the field of laboratory screening and testing of urine samples, more particularly, the procedures used to test for the presence of an adulterant used to mask the presence of DOA. While the subject application describes, and many of the terms herein relate to, a use for detection of urine sample adulterants that mask DOA, other modifications apparent to a person with skill in the art and having benefit of the subject disclosure are contemplated to be within the scope of the present invention.

[0039] Currently, both Immunoassay Laboratories and Confirmatory Labs using GC/MS can report contradictory results, usually due to a positive DOA test being reported by the Immunoassay Laboratories followed by a negative DOA test in the Confirmatory Lab. This result is usually attributed to greater specificity for DOA assay by GC/MS and known cross-reactants causing false positives in the initial immunoassay tests. In some cases, this may be the cause; however, it has been shown that this may also be due to slow oxidation of uric acid and non-urate constituents in the sample by an oxidative adulterant. By using the methods and reagents of the subject invention, such results can be used to indicate that (a) the sample was adulterated and (b) that the first immunoassay result was more likely to be correct.

[0040] In certain embodiments, the subject invention provides a phosphotungstate reagent and method that can be more accurate than the current assay reagents and methods used to detect DOA in urine samples. The phosphotungstate reagent operates by reacting with the normally-occurring uric acid and non-urate markers. In the presence of these markers, the phosphotungstate reagent is reduced, which creates a blue coloration in the urine sample. It has been found that these markers are also affected by the presence of adulterants in the urine, which also oxidize these markers. As such, these markers can be used in the practice of the subject invention. When oxidized, the markers are inhibited from reacting with the phosphotungstate reagent thereby decreasing or preventing the blue coloration. These markers are generally stable in urine and will not oxidize under normal conditions. Thus, a reduction or lack of blue coloration in a urine sample treated with the phosphotungstate reagent of the subject invention is an indication of the presence of an oxidant destroying or inactivating the markers, which could indicate use of an adulterant.

[0041] The blue coloration, when analyzed spectroscopically, can indicate the concentration of markers present in the urine sample. In one embodiment, the blue coloration affects light absorbance in the range of from between approximately 580 to approximately 800 nm. In a more particular embodiment, the blue coloration affects light absorbance in the range of from between approximately 600 to approximately 700 nm. In a specific embodiment, the blue coloration affects light absorbance in the range of from between approximately 650 nm to approximately 700 nm.

[0042] An initial test result where the amount of available marker is below the established cut-off levels can be a definitive indication that an oxidative-adulterant is present or was historically present in the sample. Advantageously, the effect of an oxidant-adulterant on a urine sample can be enhanced or can continue over time. Thus, the presence of an oxidative-adulterant in a urine sample can further oxidize these markers over time. Thus, when subject to confirmatory testing, if the test results obtained for a sample show that the available markers are less than those from earlier test results obtained from the initial screening test, i.e., the blue coloration is further reduced in the sample, the presence of an oxidant adulterant in the sample is definitively indicated.

[0043] In a further embodiment, an Oxidant History (OHist) can also be obtained for samples, allowing an initial test result to be compared to later test results. The results of the OHist can be used as effective evidence of the presence of an adulterant in a urine sample. A further embodiment provides a method by which an OHist can be established if a second test result is below the established cut-off value for the markers in a urine sample.

[0044] The level of uric acid can vary between samples. However, the embodiments of the subject invention can still be effective because the effects of the oxidative-adulterants can still reduce the blue color in a sample to levels that are far lower than the lowest range possible of even pathologically lowered human urine. Diseases that lower uric acid levels are rare. Friedman & Young report only 5 relatively-uncommon diseases capable of causing lowered uric acid levels while reporting 40 more common diseases that are capable of elevating uric acid levels. [Friedman, R. B. and Young, D. S.; Effects of Disease on Clinical Laboratory Tests, Second Edition, pp. 3-(232-232); AACC Press, 2029 K Street, NW, Seventh Floor, Washington, D.C. 20006.] The effectiveness of oxidative-adulterants is such that they can significantly reduce the uric acid and non-urate markers in a sample. Advantageously, with the embodiments of the subject invention, the reduction in these markers, absent the presence of an adulterant, can still be significantly greater than that seen in adulterated samples and can still reduce the problem of presenting a false positive result. Table 1 illustrates certain drugs and the prevalence of false negative results caused by using oxidant-adulterants.

TABLE-US-00001 TABLE 1 Effects of Oxidant Adulterants on Initial and Confirmatory Drug Testing Initial Drug Testing Adulterant Amphetamines Cocaine Metabolites Opiates Phencylidines Cannabinoid Metabolites Whizzies, Klear (Nitrite) − − − − − Urine Luck (PCC) − + ++ + +++ Stealth (H2O2/Peroxidase) + + ++ + +++ Confirmation Drug Testing 6-Acetyl Carboxy Adulterant Methamphetamine Benzoyecgonine Morphine Morphine Phencylidines THC Whizzies, Klear (Nitrite) − − +++ − − +/− Urine Luck (PCC) − − +++ ++ − +++ Stealth (H2O2/Peroxidase) − − +++ + − +++ Drug Testing Suppression: − (<20%); + (>20% but <50%); ++ (>50% but <80%); +++ (>80%) and +/− (Variable).

[0045] Certain embodiments of the subject invention described herein utilize a sodium phosphotungstate reagent for reacting with a urine sample. There are several types phosphotungstate that can be utilized with the embodiments of the subject invention. For example, lithium phosphotungstate could also be used in the embodiments of the subject invention. Preferably, the selected phosphotungstate that is utilized is molybdate-free. A person with skill in the art, having benefit of the subject disclosure, would be able to determine any one of several types of phosphotungstate that could be used in the reagent of the subject invention.

[0046] The effects of phosphotungstate can be enhanced by the addition of cyanide to the reagent. While, in one embodiment, the subject invention utilizes a phosphotungstate reagent, other uric acid or non-urate-oxidants could also be used. Non-limiting examples include arsenotungstic acid, arsenophosphotungstic acid, potassium ferricyanide, uranyl acetate, iodine, dichromate, permanganate, and chloramine. A person with skill in the art would be able to determine other indicators that are capable of quantifying the presence of uric acid or non-urate markers by oxidation/reduction, which are capable of being used to practice the subject invention.

[0047] A phosphotungstate reagent, according to the subject invention and a General Oxidant Test (DRI® General Oxidant-Detect® Test, ThermoScientific) known in the art were tested on urine samples treated with potential adulterants. Table 2 lists the ingredients of the adulterants tested and adulterant percentage addition to the urine samples.

TABLE-US-00002 TABLE 2 Adulterants Used to Characterize Detection ADULTERANT INGREDIENTS USED TO % TYPE PREPARE ADULTERANT AND ADDED TO (TRIVIAL NAME) CONCENTRATIONS USED SAMPLE Stealth 3% Hydrogen Peroxide + 1,000 Units 2% Peroxidase Liquid Laundry 8.25% Sodium Hypochlorite 2% Bleach Klear, Whizzies 12.2 g/dL Potassium Nitrite 10% Urine Luck pyridium chlorochromate (PCC) 10% 4.31 g/L

[0048] The General Oxidant Test reagent was used in accordance with the manufacturer's instructions. The product insert states the intended use: “The Test is intended for the detection of urine adulteration by oxidizing compounds” and claims in the product insert that the “test can be performed on any automated clinical chemistry analyzer to detect oxidants”. The test utilizes Tetramethybezidine (TMB) as a chromogen in acid solution. The insert further states that “Marijuana samples adulterated with oxidants can produce a positive result, during initial screening by immunoassay, notably the marijuana metabolite (THC). However, the results cannot be confirmed by GC/MS”. The product insert does not mention specifically the disappearance of the oxidant over time or indicate what other drugs might be irregular between immunoassay and the confirmatory GC/MS testing.

[0049] The General Oxidant Test Reagent insert further states that “Fresh urine specimens should be used” and that the “Mandatory Guidelines for Federal Workplace Drug Testing Programs: Final Guidelines” notice recommends that specimens that do not receive an initial test within 7 days of arrival at the laboratory should be placed into secure refrigeration units.

[0050] FIGS. 4, 5, and 6 show an OHist of the absorbance levels obtained over a span of 24 hours on urine samples adulterated with a Stealth-type, bleach, and pyridium chlorochromate adulterants. The data is plotted as mg/dal Nitrite Equivalents. The tests were calibrated with a 500 μg/mL potassium nitrite solution.

[0051] For this test, either a 500 μg/mL or 200 μg/mL upper cut-off level is used for positive General Oxidant tests. This is because patients taking certain medications, such as, for example, cardiac drugs, or patients with urinary tract infections, often have nitrite in their urine. Federal Guidelines mandate an oxidant cutoff level at 500 μg/mL. Thus, any samples that exhibit nitrite levels above the cut-off are considered to be potentially adulterated. The insert that is provided with the prior art General Oxidant test states that “urinary nitrite concentrations of 200 μg/mL or greater is . . . proof of adulteration of the specimen.”

[0052] The OHist determination of the present invention can be calibrated with a reagent containing uric acid. The results can then be reported as Uric Acid Equivalents because the urine-reducing substance acts on both uric acid and non-urate constituents.

[0053] Several oxidative-adulterants were tested and an OHist prepared for each sample, according to the subject invention. Pyridium chlorochromate (PCC) did not produce a positive oxidative-adulterant result in a 24 hour period of measurement. While PCC did lower the measurement of Uric Acid Equivalent values, it did not lower values to the accepted cutoff level in the same amount of time as other oxidative adulterants. It is theorized that the PCC may have actually reacted with and destroyed some of the non-urate markers in the urine. This caused a reduction in the concentration of PCC as well as the markers available for measurement, but sufficiently to reach cut-off levels in the given time perior. The operation of PCC in the urine over time could result in further destruction of PCC and markers, thus reducing the levels of oxidized markers over time. Consequently, the OHist test method of the subject invention may be require additional time to detect adulteration with PCC. However, a noticable drop in marker levels over time could warrant further investigation, even if they do not reach cut-off levels.

[0054] In contrast to the General Oxidant tests, which, as shown in FIGS. 4-6, are reported as positive for adulteration when results are above a Nitrite Equivalent cutoff, the OHist test of the present invention is positive when the Uric Acid Equivalents are below a Uric Acid Equivalent cutoff level. Although laboratories may choose the cutoff level for the OHist test of the present invention, 20 mg/dL has been chosen for these studies. A cutoff value of, for example, 10 mg/dL of Uric Acid Equivalents could also be used.

[0055] To establish a cut-off level, one hundred or more non-adulterated urine samples could be assayed with the phosphotungstate reagent of the subject invention, using a uric acid calibrator to determine the Uric Acid Equivalents of the samples. A mean and a standard deviation of the results can then be calculated. The cutoff value could be, for example, less than 3 standard deviations from the mean. A lower value can be used. The values for males and females should also be calculated separately to determine if there is a gender difference. Also, any sample having a very low value that appears not to be a part of the population may be an adulterated sample and should be excluded from the calculations. Known methods for excluding data such as the 4D Method, the use of t-values, or the Q-Test can be used.

[0056] The graph in FIG. 4 indicates that the results obtained with the General Oxidant Test reagent fell below the Nitrite Equivalent cut-off absorbance of 500 μg/mL just a few hours after the sample was treated. Thus, the laboratory results would likely not indicate that this sample was adulterated. Thus, the false negative result produced by the tests for drugs of abuse would be considered accurate. Automated testing is seldom performed in less than an hour and is often performed at least one day following collection of the sample. Thus, the sample would likely be tested well after the adulterant had affected the DOA in the sample. Further, the accepted cut-off levels for nitrite would prevent the sample from being flagged for use of an adulterant. Thus, this sample would not have been subject to confirmatory testing.

[0057] Other General Oxidants were tested and the results are shown in FIG. 5 and FIG. 6. FIG. 5 shows the results obtained from a urine sample adulterated with commercial bleach. While common bleach products may not have the notoriety of Stealth, they can be just as effective and can also disappear from the sample as effectively as Stealth. Additionally, a bleach adulterant does not contain peroxidase. Therefore, it is likely that tests used to detect peroxidase-based adulterants will not be effective for detecting bleach.

[0058] FIG. 6 shows results obtained from a sample adulterated with pyridium chlorochromate (PCC). PCC disappears, or falls below, the accepted cut-off levels, slower than either Stealth or bleach. However, as mentioned previously, many DOA tests are performed at least 24 hours or longer following sample collection. Thus, even the slow-acting PCC can have sufficient time to destroy the existence of DOA in the sample. Even if the initial test showed positive results, by the time the sample was tested by a confirmatory laboratory, the presence of the PCC would result in a false negative test result and would be accepted as the final result, despite the positive results obtained from the initial screening test.

[0059] The current screening tests and procedures utilized to analyze urine samples for DOA can be inaccurate for the detection of adulterants in the sample. Samples that exhibit positive test results can be subject to confirmatory testing, whose results are considered conclusive. However, the confirmatory tests conducted on these samples can also provide inaccurate results because of the time delays often involved before testing samples. Adulterated samples may not be tested before the adulterant in the sample, detected during the initial screening test, has sufficient time to further degrade the evidence of DOA in the urine sample.

[0060] The embodiments of the subject invention provide a phosphotungstate reagent and a method of use that can be more accurate than the current reagents and methods used to detect DOA in urine samples. The phosphotungstate reagent can operate by reacting with normally occurring constituents in urine to impart a blue coloration to the sample. Thus, a reduction or lack of blue coloration in a urine sample treated with the phosphotungstate reagent of the subject invention can be an indication of the presence of an oxidative adulterant affecting or inactivating the markers, thus lowering the amount of activation of the chromogen. Further, an OHist can be obtained for samples allowing an initial test result, which may not have been indicative for DOA, to be compared to later test results, which may be indicative of DOA. The results of the OHist can be used as effective evidence of the presence of an oxidative adulterant in a urine sample.

[0061] In one embodiment of the method of the subject invention, a urine sample is initially tested by the screening laboratory for DOA, which can include obtaining and recording an initial measurement of the markers to obtain a Uric Acid Equivalents value for the sample utilizing the reagent of the subject invention. However, if the test results show a Uric Acid Equivalents value that is not initially indicative of the presence of an oxidative-adulterant, other tests conducted on the sample may warrant confirmatory testing. In that situation, the sample can be sent to a confirmatory laboratory for further testing. One embodiment of the method of the subject invention includes the confirmatory laboratory conducting further tests of the markers to obtain additional Uric Acid Equivalents values. If the test results of a confirmatory laboratory do not indicate or corroborate the use of DOA for that sample, the one or more Uric Acid Equivalents values can then be compared to the initial Uric Acid Equivalents value obtained by the screening laboratory. All of the Uric Acid Equivalents values can be plotted to create an OHist. If the OHist confirms that the levels of Uric Acid Equivalents values decreased during the time window of the sample, which is from the time the sample was obtained to the time of the confirmatory testing, then the presence or historical presence of an oxidative-adulterant can be definitively confirmed. Consequently, the confirmation of the use of an oxidative-adulterant on the sample can be indicative of the presence or the historical presence of DOA. Procedures dictated by state or federal law can then be initiated.

[0062] The scope of the invention is not limited by the specific examples and suggested procedures and uses related herein since modifications can be made within such scope from the information provided by this specification to those skilled in the art.

[0063] Following are examples that illustrate procedures for practicing the subject invention. These examples are provided for the purpose of illustration only and should not be construed as limiting. Thus, any and all variations that become evident as a result of the teachings herein or from the following examples are contemplated to be within the scope of the present invention.

EXAMPLE 1

Reagents and Methods of use, Including Obtaining an Oxidant History (OHIST)

A. Reagent Preparation:

[0064] Reagent 1: Phosphotungstate Reagent—1 Liter: Add 27 grams of molybdenum free sodium tungstate to approximately 333 mL deionized water in a reflux vessel. Add 30.9 grams of phosphoric acid. Add boiling chips and reflux gently for 2 hours. Cool to room temperature and then dilute to 1 Liter with deionized water. Add 21.0 grams of Lithium Sulfate and mix.

[0065] All ingredients are ACS grade. Molybdinum free Sodium Tungstate was obtained from GFS Chemicals, Powell Ohio 43065. The reagent so prepared is stable for at least 1 year refrigerated at 2-8 degrees Celsius.

[0066] Reagent 2: Carbonate Buffer Reagent—1 Liter. Dissolve 119 grams of ACS grade sodium carbonate to 800 mL of deionized water. Add 7.5 grams sodium hydroxide. Add deionized water to bring the volume to 1 Liter. This reagent is used to buffer the test sample and blank.

[0067] The reagent is stable for at least 1 year at ambient temperature 20-30 degrees Celsius.

[0068] Reagent 1 and 2 are stable for at least 1 year when packaged together and stored at 2-8 degrees Celsius.

[0069] Reagent 3: Uric acid 40 mg/dL Calibrator: 1 Liter—Dissolve 0.63 grams of ACS grade lithium carbonate in 500 mL of Deionized water. Warm to about 45 degrees Celsius. The mixture should not be warmer than 50 degrees Celsius for the next step. Add 400 mg of ACS grade uric acid and mix until dissolved. Dissolve 0.5 grams of sodium azide and then dilute to 1 liter with deionized water.

[0070] This calibrator is stable for at least 1 year refrigerated at 2-8 degrees Celsius.

B. Oxidative-Adulterant Assay Method:

[0071] Manual Method: Test—Add 100 μL of Urine Sample to 2 mL of Phosphotungstate Test Reagent 1 and mix. [0072] Reagent Blank—Add 100 μL of Deionized water to 2 mL of Phosphotungstate Reagent 1 and mix. [0073] Add 1 mL of Carbonate Buffer Reagent 2 to Reagent Blank and Test Reagent and mix each one separately. [0074] Incubate for 5 minutes at 37 degrees Celsius. [0075] Measure absorbance at 700 nm of Reagent Blank and Test Reagent. [0076] Calibrator absorbance is developed by using the calibrator as a Test. [0077] The Test Net absorbance (Test Absorbance—Blank Absorbance) is used to calculate the Uric Acid Equivalent result as follows: [0078] (Test Net Absorbance/Calibrator Net Absorbance)×Calibrator Absorbance=Value of Calibrator

[0079] The absorbance measurement may be made at 600-700 nm. No secondary bichromatic wavelength should be used.

C: Automated Measurement Parameters:

[0080] 1. Mindray BS200 Clinical Analyzer Parameters: [0081] Test: OX Hist [0082] No. User Defined [0083] Full Name: Oxidant History [0084] Reaction Type: Endpoint [0085] Pri. Wave 670 nm [0086] Sec Wave none [0087] Direction: Increase [0088] Reac. Time: 0 and 11 [0089] Incubation Time: 3 [0090] Unit: Mg/dL UAEq [0091] Precision: Integer [0092] R1: 180 [0093] R2: 90 [0094] Sample Volume; 10 [0095] Mixed Reagent Blank: Optional [0096] Compensate: Slope:1 Intercept: 0 [0097] 2. AU 400, AU 400e, AU 480, AU 640, AU640e and AU680 Series Clinical Analyzers Parameters. [0098] Reagent ID: User defined [0099] Test Name: Oxidant History [0100] Sample Volume: 10 [0101] R1 Volume: 180 [0102] R2 Volume: 90 [0103] Wavelength: Pri: 700 Sec. None [0104] Method: END [0105] Reaction Slope: POS [0106] Measuring Point 1: First 0 Last 27 [0107] Measuring Point 1: (Not Applicable) [0108] Calibration Type: AB Formula: Y=AX+B

EXAMPLE 2

Procedures for Obtaining an Oxidant History (OHIST)

[0109] The reagents and methods of automation described in Example 1, above, can be used to exploit the kinetics of Uric Acid Equivalents measurement by performing a screening test on a urine sample soon after collection and then repeating the screening test hours or even days following the first test. For example, the collecting site or first immunoassay screening laboratory can test the sample and obtain an initial Uric Acid Equivalents value for the sample. If the initial value concludes the possible presence of an adulterant, the results can be reported to the confirmatory laboratory. The confirmatory laboratory can also obtain one or more Uric Acid Equivalent values on the same sample. The results of the initial Uric Acid Equivalents value can be compared with the results of the one or more Uric Acid Equivalents values obtained by the confirmatory laboratory. If analysis of the Uric Acid Equivalents values shows a reduction in the values over time, the presence of an oxidative-adulterant can be considered definitive proof of the use of an oxidative-adulterant in the sample. Even if the analysis of the Uric Acid Equivalents values do not fall below the mandated cutoff level indicative for positive adulteration, adulteration can still be definitively proven if the Uric Acid Equivalents values obtained from confirmatory laboratory tests have fallen significantly during the time window of the sample.

[0110] Any reference in this specification to “one embodiment,” “an embodiment,” “example embodiment,” “further embodiment,” “alternative embodiment,” etc., is for literary convenience. The implication is that any particular feature, structure, or characteristic described in connection with such an embodiment is included in at least one embodiment of the invention. The appearance of such phrases in various places in the specification does not necessarily refer to the same embodiment. In addition, any elements or limitations of any invention or embodiment thereof disclosed herein can be combined with any and/or all other elements or limitations (individually or in any combination) or any other invention or embodiment thereof disclosed herein, and all such combinations are contemplated with the scope of the invention without limitation thereto.

[0111] The invention has been described herein in considerable detail, in order to comply with the Patent Statutes and to provide those skilled in the art with information needed to apply the novel principles, and to construct and use such specialized components as are required. However, the invention can be carried out by specifically different equipment and devices, and that various modifications, both as to equipment details and operating procedures can be effected without departing from the scope of the invention itself. Further, although the present invention has been described with reference to specific details of certain embodiments thereof and by examples disclosed herein, it is not intended that such details should be regarded as limitations upon the scope of the invention except as and to the extent that they are included in the accompanying claims.