BACKGROUND OF INVENTION

Drug abuse is a critical problem throughout the world. Commonly abused drugs include cocaine, cannabinoids, amphetamines, and phencyclidine (PCP), as well as designer drugs such as 3,4-methelenedioxyamphetamine (“meth”) and 3,4-methelenedioxy-methamphetamine (“Ecstasy”).

On Sep. 15, 1986, President Ronald Reagan issued Executive Order No. 12564 directing federal agencies to achieve a drug-free work environment. The Department of Health and Human Services (HHS) has developed guidelines and protocols for drugs-of-abuse testing. It is estimated that approximately 20 million employees are screened each year for illicit drug use. Drug testing programs in the United States can be classified as mandatory or non-mandatory. In the mandatory programs (e.g., the Department of Transportation), a regulated employer is required by federal regulation to test employees. In the non-mandatory programs, employers choose to test for reasons other than federal regulations.

Persons using drugs may attempt to adulterate urine specimens to avoid detection. Adulterants are substances that can be added to, for example, a urine sample to change the integrity of the sample. Some other types of adulterants can be used to mask that a sample has been diluted and can cause a laboratory test to fail in detecting that the sample was diluted. These adulterants are often oxidants that have been shown to destroy, or to partially destroy, evidence of drugs of abuse (DOA) in a urine sample. Thus, both screening and confirmatory tests can be rendered negative.

Oxidant adulterants include potassium nitrite (e.g., “Klear” and “Whizzies”), potassium chlorochromate (e.g., “Urine Luck™”) and hydrogen peroxide/peroxidase (e.g., “Stealth”) and are readily available, usually at low cost. Household bleach is also an effective adulterant that can cause negative DOA results. Adulterants can degrade and disappear in a sample within hours after being added, making them difficult or impossible to detect. Further, specific tests have to be conducted to detect each type of adulterant.

DOA testing often involves several steps, which can include: (a) sample collection, (b) sample integrity testing, (c) DOA screening, and (d) confirmatory testing. These steps may: (a) occur at different locations, (b) be conducted by personnel with varying qualifications, (c) be conducted by different institutions, and/or (d) be conducted sequentially at different times. For example, confirmatory tests are conducted after the screening tests and often require that the sample be transported from one institution or laboratory to another. The steps leading to the confirmatory testing step may occur over one or more days.

Laboratories typically screen samples using commercially available Enzyme Immunoassays (EIA) and related methods and reagents. These screening methods are not always specific, as there can be cross-reacting substances that cause false positive results. Consequently, cut-off values or minimum concentrations for each drug have been established, whereby assay values below the cut-off value are considered negative results. If a positive result is obtained by the EIA screen, the sample can be sent to a confirmatory testing laboratory where Gas Chromatography/Mass Spectrometry equipment is often utilized for the confirmatory test.

Generally, the cut-off values used for the confirmatory test are lower than the values used for the initial screening (e.g., the EIA). In general, only initially positive screening tests are submitted for confirmatory testing and the confirmatory test results are considered conclusive. Because of the cut-off value criteria used to establish a positive result, adulterants only need to lower the tested value of the urine sample below the cut-off level to produce a negative result.

Clinical laboratory professional organizations have called attention to the problems with adulterated urine samples for illicit drug testing, but detecting an adulterated urine sample is difficult. The American Journal of Clinical Pathology concluded that new adulterants can produce false negative results for DOA. The Substance Abuse and Mental Health Services Administration (SAMHSA) reports that of 6,320,000 samples tested in 2013 one of every 300 was invalid. If the adulterant testing procedures failed to detect “disappearing” adulterants, that number may have been even higher. The invalid tests may also be much higher in non-mandated labs or other labs not performing adequate tests for adulterants.

Manual spot tests have become available to test for the presence of adulterants in a sample. Although a step in the right direction in detecting adulterants, these tests can be laborious and expensive to perform and primarily provide only qualitative results, which may still result in under-detection or false positives. The high cost of these manual tests may lead non-mandated laboratories to take short cuts in adulterant testing, partially because reimbursement for adulterant testing has at times been insufficient. Adulterant testing products and labor can be costly, and the cost is escalating, causing non-mandated laboratories to forego adulterant testing. Ironically, clinical laboratory testing costs in general are one of the few areas in health care where costs of an individual diagnostic test have declined since the 1950's. That reduction in cost has been brought about by laboratory automation and the integration of the automation with laboratory/hospital information systems. The use of automated systems and methods, and the use of reagents, combined with automated sample identification and documentation by an integrated Laboratory Information System computer would enable the costs to be reduced to a fraction of the costs of manual adulteration spot test, if a reagent were available.

Several sample integrity tests are recommended by laboratory professional organizations and government oversight groups. These tests are intended to detect adulteration that destroys drugs, adulteration that interferes with testing, and adulteration by dilution or sample substitution. The principal tests recommended are: temperature measurement—intended to detect sample substitution; pH measurement—intended to detect substances that interfere with testing; specific gravity—intended to detect sample dilution and substances that interfere with testing; and creatinine assay—intended to detect dilution. Additionally, oxidant and aldehyde tests are intended to detect substances known to destroy drugs of abuse.

All of these tests have specific purposes in detecting adulteration of samples. The purpose of the specific gravity test is typically to corroborate the results of prior tests that indicate one of many substances may have been used to adulterate the sample. One of the most common tests for specific gravity involves measuring weight per unit volume (w/v), usually using a mechanical hydrometer placed in an aliquot of the sample. Refractive Index based Specific gravity detectors are also available; however, both of these methods tend to be cumbersome and labor intensive. None of these methods can be used with standard automated laboratory equipment or clinical analyzers. Important record keeping is also laborious, prone to errors, and expensive.

Water is the most common adulterant or dilution agent used to lower levels of drugs in a sample, thereby causing a false negative drug test. Water may be added at the time of collection or by ingestion of a liter or more of water prior to sample collection. The components in urine found in the highest concentrations and that contribute most to the specific gravity of urine are sodium chloride and potassium chloride salts. Urea is also a major contributor to the specific gravity of urine. Specific gravity testing, which relies on one or more of these components, has been shown to be a poor test for detecting sample dilution, particularly in comparison to methods that utilize creatinine measurement. This is because the addition of simple table salt to a urine sample can sufficiently mask the effects of dilution.

In addition, confirmatory laboratory tests are often conducted one or more days following the initial screening test. Urea is known to degrade over a period of one or more days. This degradation involves converting one molecule of urea into two molecules of ammonia and one molecule of carbon dioxide. This degradation can increase the specific gravity of a urine sample, which can also mask any prior dilution of the sample with water.

Recently, reagent strips have become available that can provide an index of specific gravity by measuring the sodium content of the urine. These strips may be less sensitive to ammonia from urea degradation; however, the sodium-detecting reagent strips typically employ a pH indicator dye, which is affected significantly by change in pH resulting from the levels of ammonia. While the strips do not measure the ammonia released by urea degradation, the pH change associated with the urea degradation can affect results, particularly in tests conducted later by confirmatory laboratories. Further, tests performed with reagent strips must be done manually, not with automated laboratory equipment, and the strips do little to solve the testing accuracy and data management costs of conducting tests.

There are commercially available reagents that can be used in automation equipment or clinical analyzers for conducting specific gravity testing on urine samples. These reagents usually depend upon the detection of chloride to derive an index of specific gravity, or a specific gravity derived from just the chloride constituent in a urine sample. Although chloride detection can provide a reasonably accurate index of specific gravity on normal or unadulterated samples, the use of chloride detection to derive specific gravity can be a poor indicator of adulterated samples. This is because some of the most common adulterants contain no chloride and produce no change in the chloride concentration in the sample. Therefore, while the specific gravity of the sample may change, the change will not be detected by these types of chloride-detecting reagents.

Some of the principal adulterants known to affect specific gravity are baking soda (sodium bicarbonate/sodium carbonate), liquid drain openers (sodium hydroxide), Klear (potassium nitrite), and Urine Luck™ (potassium chlorochromate), none of which contain chloride.

Other reagents used in automated analyzers to determine a specific gravity index contain ferric perchlorate or mercury, which is highly toxic. Perchlorate can also be combustible. These issues raise serious questions as to the safety and effectiveness of using these reagents in automated analyzers for the detection of adulteration.

The use of DOA continues to be a problem around the world. The ability to determine whether an individual is using or has used a DOA can be a critical safety issue. Acquiring a urine sample from individuals to be tested is the most expedient and least invasive way to obtain a testable bodily fluid. While there are a variety of screening tests that can be conducted to try to detect DOA in a urine sample, many are inaccurate or are not sufficiently reliable to be considered undeniable confirmation. Confirmatory testing can be used to try to corroborate samples with a positive screening test. One of the most common tests conducted by confirmatory laboratories is measuring specific gravity or, more specifically, an index of specific gravity based on one or more constituents in the urine. Unfortunately, these tests have not been entirely reliable and changes in the urine sample over time can affect the results of testing for an index of specific gravity.

Compounding the historical reliability questions surrounding the use of prior specific gravity tests is the fact that drug users wanting to subvert DOA sample integrity testing receive advice from internet sites. Most DOA testing labs currently perform creatinine assays as a means of detection invalid sample integrity due to dilution. Commonly, specific gravity tests are only performed if the creatinine concentration of a sample is below 20 mg/dL. Although creatinine has historically been the gold standard for the detection of dilution, new subversion techniques, such as in vivo dilution, are now being used to subvert detection. The technique of in vivo dilution was first discovered in studies athletes and body builders who often take, a.k.a. “load”, supplemental creatine to increase muscle mass. Creatine is converted in muscle to creatinine, which is excreted in the urine. [Schedel J, Tanaka M, Tanaka H, Kiyonaga M. et al. Consequences of one-week creatine supplementation on creatinine levels in athletes' serum and urine, Schweizerische Zeitschrift für <<Sportmedizin and Sporttraumatologie>> 2000; 48: 111-116.] Information can also be obtained from the World Wide Web that provides advice about how to “load” with creatine and protein, consume large amounts of water prior to the test, and perform exercise to obtain both a normal urine creatinine and specific gravity while still diluting DOA below detection cut off levels. There remains a need for safe, effective reagents for measuring specific gravity of a urine sample. Furthermore, the ability to use such reagents in automated clinical analyzers would provide a quicker and more cost effective method for conducting confirmatory urine tests to detect DOA.

BRIEF SUMMARY

The subject invention provides methods and reagents useful for analysis and measurement of specific constituents in a urine sample that can be used to derive a Specific Gravity Index (SGI) for a urine sample. When the SGI of a given urine sample is compared to the SGI of known normal urine samples, results can be used to determine whether the given sample was adulterated, typically, in an effort to mask use of Drugs-of-Abuse (DOA). In particular, the subject invention provides reagents and methods for the measurement of specific markers normally present in a urine sample, where such markers can be useful indicators for detecting adulteration of the sample.

Beneficially, the reagents and methods of the subject invention can be used with standard laboratory automation equipment or clinical analyzers to measure the markers in the given sample, thereby facilitating automated urine sample analysis for detection of adulteration.

Specifically, spectrophotometry analysis of a sample treated with a reagent of the subject invention can provide a quantitative measurement of specific markers that can be used to derive a SGI for the sample. Abnormal SGI measurements can be an indication that the sample has been adulterated by dilution or addition of another substance, such as salt, which can alter the specific gravity of the sample. More specifically, spectrophotometry analysis of a sample treated with a reagent of the subject invention can be used to determine a SGI for the sample, which, if outside of an accepted normal range for the SGI, would indicate that the integrity of the sample was compromised.

Specific gravity is a measure of the weight of a liquid divided by the weight of water. The constituents found in the highest concentrations in urine are sodium chloride and potassium chloride and, as such, contribute most to the specific gravity measurement for a urine sample. However, there are other constituents, such as urea, which also contribute to the overall specific gravity of a urine sample. A Specific Gravity Index, (SGI), according to the subject invention, is a measurement obtained by utilizing a subset of the constituents found in a urine sample. More specifically, the SGI is a measurement of the weight of the non-aqueous constituents of the sample per unit volume. Advantageously, the subset of constituents can also be used as markers, according to the subject invention, for automating analysis of a urine sample, so as to obtain a SGI.

One embodiment of the subject invention utilizes the sodium (Na+) and potassium (K+), naturally found in a urine sample, as markers. In a further embodiment, these same markers are used to obtain a SGI for the sample. In a specific embodiment, a sodium-potassium dependent β-Galactosidase is utilized along with an indicator chromogen of o-nitrophenylgalactoside (o-NPG). In one embodiment, the method of the subject invention results in the formation of a yellow color due to cleavage of the o-NPG into o-nitrophenol, a molecule that can be analyzed by the spectrophotometry methods utilized in most clinical analyzers to obtain a SGI for the given sample. The chromogen can be cleaved to o-nitrophenol by using the sodium or potassium activated β-Galactosidase. Thus, the amount of sodium and/or potassium in a given urine sample can dictate the amount of cleaved o-NPG created by the reaction. Magnesium, Mg++ has also been known to activate β-galactosidase; however, the amount of Mg++ in a urine sample is significantly less than the amount of Na+ or K+.

The amount of yellow o-nitrophenol that is produced from the colorless o-NPG can be measured spectrophotometrically at a primary wavelength of 405 nm-410 nm. The rate of increase in absorbance at 405 nm-410 nm is proportional to total sodium and potassium concentration in the sample. Light absorbance measurements outside a known normal range of a SGI for urine can be an indication of abnormal levels of sodium and/or potassium in the sample. This can be an indication that the sample integrity has been compromised.

Many of the current methodologies measure the amount of chloride in a urine sample, where the amount of chloride detected can be used as an indication of adulteration; however, many of the products used to adulterate urine samples do not contain chloride. Thus, their addition to a urine sample may have no effect on the total chloride amounts. Advantageously, by utilizing the total amount of sodium and potassium, to obtain a SGI, instead of attempting to directly detect the amount of chloride in the sample, the reagent and methods of the present invention can be used to indicate more generally whether the sample has been adulterated.

If it can be determined initially that the sample has been adulterated in a fashion that has affected the SGI, remaining automated tests can be halted and another sample obtained as soon as possible. This can save time and resources, and may aid in obtaining an unadulterated sample in time to determine whether DOA have been used.

Specific gravity measurement of urine samples is used by testing laboratories. Currently, the adulteration of samples capable of being detected by a Specific Gravity measurement can be divided into two types: (a) sample dilution, which can cause a low sample specific gravity; and (b) sample adulteration, such as, for example, by addition of salt, which can cause a higher than normal specific gravity. Of these two types, adulteration by addition of substances to the sample that causes a higher than normal specific gravity can be the most important. This is because it has been determined that detection of sample dilution is more accurately detected by methods that measure the amount of urine creatinine. Current methodology requires that a specific gravity measurement to detect dilution of a sample need only be performed if the creatinine level is below a certain threshold, such that measurement of specific gravity on such samples can provide a corroborative indication of dilution. Among routine integrity tests, certain adulterants can only be detected by a specific gravity measurement.

The present invention employs a unique and advantageous departure from currently known technologies for the measurement of sodium. Further, according to the novel concepts of the present invention, both sodium and potassium, which are normally present in urine, can be used in a single test yielding a total sodium and potassium value. The total sodium and potassium value can then be used to calculate a SGI, utilizing an automated analysis system.

In one embodiment of the subject invention, a given urine sample, or portion thereof, is combined with a pre-determined amount of o-nitrophenylgalactoside (o-NPG), which is normally colorless. The treated sample can be reacted with β-galactosidase, which, in the presence of the sodium and potassium in the urine, will cleave the o-NPG into o-nitrophenol. The amount of cleavage is dependent upon the amount of sodium and potassium in the urine sample. The resulting O-nitrophenol has a yellow color that intensifies with concentration. Analysis of the reacted sample by spectrophotometry can yield a measurement of the mEq/L of total sodium and potassium in the urine sample, which can be converted to provide an SGI for the sample. If the calculated SGI is outside the normal range for urine, then it can be presumed that the sample was subject to tampering.

Specific gravity testing is used as one of the principal means, along with pH and creatinine measurement, for detecting tampering or adulteration of samples submitted for DOA testing. However, the process of obtaining specific gravity is time-consuming, expensive, and prone to error. It is a goal of the present invention to provide reagents and methods for obtaining instead a Specific gravity Index (SGI), which can be optimized for the detection of sample tampering. It is also a goal of the subject invention to utilize automated testing devices and techniques for the analysis of urine by measuring the normally occurring substituents of sodium (Na+) and potassium (K+) as indicators of adulteration or tampering with a urine sample.

Magnesium (Mg++) is also known to activate β-galactosidase. Human urine contains approximately 1 mMol/L of Mg++. This is significantly less than the amount of sodium and potassium normally present in urine, though still likely to be sufficient to activate the β-Galactosidase in the reagent of the subject invention. However, as with the sodium and potassium, dilution of a urine sample will also equally dilute the magnesium and thus reduce activation of β-galactosidase. Thus, the overall effect of magnesium on the SGI obtained by the methods of the subject invention is minimal. However, it has been observed that inclusion of magnesium in the reagent and calibrators, in a ratio normal to the sodium and potassium in urine, can improve sensitivity to effects of urine dilution.

