1. Field of the Invention
The present invention relates to elements for the quantitative or semi-quantitative analysis of liquids which may contain gentisic acid as an interferent.
2. Description of the Related Art
It is well known in the art to perform a quantitative or semi-quantitative analysis of a liquid by contacting that liquid with an analytical element containing reagents capable of yielding a detectable product in proportion to the concentration of a predetermined analyte in the liquid. One particularly useful method involves an enzymatic assay wherein the predetermined analyte, upon contact with the analytical element, is oxidized in the presence of an enzyme contained therein to produce a peroxide in proportion to the concentration of the predetermined analyte in the liquid undergoing analysis. A detectable product is then yielded by the reaction of the peroxide with an indicator composition in the presence of a substance having peroxidative activity. This detectable product should be formed in direct proportion to the peroxide present and thus also in proportion to the concentration of the predetermined analyte. Elements and analyses of this type are described in U.S. Pat. No. 3,992,158 and in a copending U.S. application by B. J. Bruschi, Ser. No. 712,972, filed Aug. 9, 1976, both of which are incorporated herein by reference.
Methods of analysis employing reaction mechanisms other than the above-described peroxide mechanism to produce a detectable product are also known. For example, U.S. Pat. No. 3,711,252, describes a method for the quantitative analysis of uric acid in aqueous liquids wherein the aqueous liquid is contacted with a carrier element containing a ferric salt and either 2,4,6-tri(2-pyridyl)-1,3,5-triazine of 2,2':6',2"-terpyride, in a buffered acidic medium. A color change is produced which is directly proportional to the concentration of uric acid in the aqueous liquid.
Additional methods are described in U.S. Pat. No. 3,801,466.
In all of the above-cited references to elements and methods for their use, it is also recognized that substances present in the liquid undergoing analysis other than the predetermined analyte may interfere with or bias the analytical reactions such that the detectable product is not formed in direct proportion to the predetermined analyte alone. This is particularly true for relatively low concentration analytes. For example, in analyses for uric acid or lactic acid in aqueous liquids such as serum or urine, it is recognized that gentisic acid can interfere with the reactions used to indicate the concentrations of uric acid or lactic acid. This is a significant problem, because it is well known that gentisic acid may often be present in liquids such as serum or urine.
Gentisic acid is a metabolic product of acetylsalicylic acid (aspirin) and would be expected to be found in the body fluids of anyone who has recently ingested common aspirin. Its presence has been recognized and methods are available for its analysis in liquids. Since uric acid and lactic acid are normally present in body fluids in relatively small concentrations, the recent ingestion of one or two doses of aspirin can effectively destroy the accuracy of a lactic acid or uric acid analysis.
In some of the analytical methods described above, such as those discussed in U.S. Pat. Nos. 3,711,252 and 3,801,466, gentisic acid is falsely detected as more of the predetermined analyte, because it reacts with the analytical reagents to form the detectable product in the same way that the predetermined analyte does. The result is a false indication that there is a higher concentration of predetermined analyte than is actually present.
Methods are available and known to avoid interferences of this type. For example, U.S. Pat. No. 3,711,252, suggests prevention of gentisic acid interference by incorporation of persulfate in the analytical element. U.S. Pat. No. 3,801,466 suggests a multi-step method of avoidance involving preparation of comparative test samples in one of which the predetermined analyte is totally eliminated by a pre-analysis reaction. The two samples are then analyzed for predetermined analyte, and the difference in results between the two indicates the concentration of interferents such as gentisic acid that may be present. While such methods of avoidance are useful, they are either inconvenient to use (involving multiple steps) or are applicable only to one method of analysis. For example, the use of persulfate suggested by U.S. Pat. No. 3,711,252, would not be successful in avoiding gentisic acid interference with the proxide-linked analyses described previously.
The mechanism of interference of gentisic acid with a peroxide-mechanism-type analysis is quite different from the interferences described above, wherein gentisic acid is falsely detected as predetermined analyte. In a peroxide-mechanism-type analysis, gentisic acid interference produces the opposite effect. It causes a false indication that there is a lower concentration of predetermined analyte than is actually present. Unlike the other analytical methods wherein gentisic acid reacts to form the detectable product just as the predetermined analyte does, in the peroxide-linked analyses gentisic acid competes with the indicator composition in the presence of a substance having peroxidative activity in order to react with the peroxide formed by the interaction of predetermined analyte and enzyme. Thus, less peroxide is available to react with the indicator composition to produce the detectable product, and the concentration of predetermined analyte indicated is falsely low.
Although the mechanism of the competition between the indicator composition and gentisic acid for peroxide is not definitely known, the following hypothesis is presented as a possible explanation of the interference.
In the peroxide-linked analyses which use a analytical element as a test-reagent carrier, all of the test reagents except the liquid being analyzed are usually incorporated into the element itself. The indicator composition may be dispersed or dissolved in a suitable organic solvent within the element. When the liquid to be analyzed is contacted with the analytical element, some of the liquid is imbibed into the element. Any predetermined analyte present in the imbibed liquid then reacts with oxygen in the presence of the enzyme incorporated in the element to produce a peroxide. The peroxide is formed within the element itself and is situated in close proximity to, or interspersed with, the organic solvent containing indicator composition and a substance having peroxidative activity. It is desirable at this point that all of the peroxide formed in the element should act in the presence of the substance having peroxidative activity to oxidize some of the proximately located indicator composition. This oxidation of indicator composition produces a detectable product whose relative concentration is then determined by measuring its optical density spectrophotometrically or otherwise to indicate the concentration of predetermined analyte in the liquid undergoing analysis. It is apparent that any gentisic acid which is situated in similar proximity to the peroxide as is the indicator composition may itself be oxidized by the peroxide. Any peroxide undergoing such reaction is thus made unavailable for oxidation of indicator composition. What is not so apparent is the reason why significant amounts of gentisic acid come to be as well situated for this reaction as is the indicator composition which is dissolved in the organic solvent. Since gentisic acid is originally dispersed throughout the body of the liquid being analyzed, while indicator composition is in organic solvent within the element itself where the peroxide is first formed, one would expect that most peroxide formed would react with indicator composition before it had a chance to come into contact with significant amounts of gentisic acid. However, this is not the case, and it is accordingly hypothesized that the organic solvents previously chosen to facilitate dispersion of the indicator composition within the element, e.g., a solvent such as N,N-diethyl lauramide, which is used in the prior art (see, for example, U.S. Ser. No. 712,972 referred to above), act also to preferentially partition gentisic acid into the organic solvent from the aqueous liquid. This means that a much higher concentration of gentisic acid may be found in the organic solvent than in the aqueous liquid and thus is just as well situated to react with any peroxide being formed as is the indicator composition itself.
Accordingly, it would be desirable to provide an analytical element using the peroxide-linked assay mechanism wherein gentisic acid is not preferentially partitioned into the organic solvent containing indicator composition and is thus not as well situated for reaction with peroxide as is the indicator composition and, therefore, does not interfere with the formation of detectable product to such a significant extent as it does in the analytical elements of the prior art.