GB2030292A - Substituted indoles as urobilinogen controls - Google Patents

Abstract

A urobilinogen control composition, control preparation a device and a method for preparing a liquid control in which the presence of urobilinogen is simulated are disclosed. A method for simulating the presence of urobilinogen in a liquid and a liquid control in which the presence of urobilinogen is simulated are also disclosed. The control composition incorporates compounds reactable with p- dimethylaminobenzaldehyde to produce a color change such that the presence of urobilinogen is simulated. Indoles which are selectively substituted at the 1, 2 and/or 5 positions are used. The control may be in the form of a solution, but is advantageously incorporated with a carrier, such as a matrix or tablet, to provide a device. <IMAGE>

Description

SPECIFICATION Substituted indoles as urobilinogen controls The present invention relates generally to the field of control preparations and, more particularly, to means for preparation of urobilinogen controls and demonstration samples.

The use of physiological fluid controls in laboratory practice is widely understood and utilized as it relates to clinical chemistry laboratory routine. For example, controls have recently played an increasingly important role in urinalysis, and their potential contribution to upgrading the quality of body fluid testing is only beginning to be realized. In the United States, the Clinical Laboratories Improvement Act of 1967 (42 U.S.C. 263a) and regulations thereunder indicate that laboratories which are to be accredited by the Department of Health, Education and Welfare must use urine controls daily in qualitative urinalysis. Also, the use of control or standard solutions has found applicability in training and evaluation of laboratory technicians and others.

Thus, convenient preparation of reliably uniform control and demonstration solutions has become increasingly important. These solutions are useful in the frequent comparison required to insure proper technique, reagent quality, and the like.

Various naturai and simulated body fluids and control compositions have been developed, including control standards provided for the field of urine chemistry. Such include TEK-CHEK 1-4 controls (combinations of natural and artificial ingredients in lyophilized human urine specimens manufactured by Ames Company, Division of Miles Laboratories, Inc., Elkhart, Indiana 46515). Also, Shukla, U.S. Patent No. 3,901,655, discloses a lyophilized human urine control prepared from pooled normal human urine.

As can be seen, these urine controls have principally been preparations of actual urine samples, thus requiring the collection and processing of large volumes of donor urine. None has provided artificial simulation of urobilinogen.

The detection of urobilinogen has for many years been undertaken by the Ehrlich reaction using pdimethylaminobenzaldehyde to produce a color reaction with the urobilinogen. Other substances known to react with Ehrlich's reagent in a liquid system and the colors produced are as follows: porphobilinogen, opsopyrroledicarboxylic acid, opsopyrrole carboxylic acid, melanogen (5,6-dihydroxyindole), melagin, excretion products of allylisopropylacetamide or SedormidB (Roche Inc., Nutley, N.J.), indole-3-acetic acid, phylloerythrinogen, indole, and the indole derivatives urorosein and indirubin give a red color; urea and indican give a yellow color; tryptophan gives a orange color; indoxyl sulfate an orange-brown color; bilirubin gives green; and skatole produces a blue color.Sulfonamides, phenothiazine and meprobamate metabolites, procaine, indoleacetic acid, and 5-hydroxyindoleacetic acid are also said to react with Ehrlich's reagent.

Thus, in summary, prior art controls which reflect the presence of urobilinogen have been limited to those using urobilinogen, itself. Tests for the detection of urobilinogen in solution have used pdimethylaminobenzaldehyde which has been observed to react with various other compounds.

OBJECTS OF THE INVENTION It is therefore an object of the present invention to provide a method for simulating the presence ol urobilinogen and a urobilinogen control composition.

A further object of the invention is to provide a control which simulates urobilinogen and which is stable for a prolonged period, even when in the form of a solution.

A still further object is to provide a control which simulates urobilinogen and which is particularly suited for reaction with p-dimethylaminobenzaldehyde incorporated in commercially available dip-andread test devices to give a high degree of color coincidence with the color reaction produced by urobilinogen itself.

Other objects and a fuller understanding of the invention will be had by referring to the following description and claims drawn to preferred embodiments thereof.

BRIEF DESCRIPTION OF THE DRAWINGS Figure lisa graphical representation of the data reported in Example II for compositions according the invention in comparison with urobilinogen solutions of 2 Ehrlich units, color standards for 2 Ehrlich units and other substances reactive with Ehrlich's reagent.

Figure 2 is a graphical representation of the data reported in Example II for compositions according to the invention in comparison with urobilinogen solutions of 4 Ehrlich units, color standards for 4 Ehrlich units and other substances reactive with Ehrlich's reagent.

Figure 3 is a graphical representation of the data reported in Example II for compositions according to the invention in comparison with urobiljnogen solutions of 8 Ehrlich units, color standards for 8 Ehrlich units and other substances reactive with Ehrlich's reagent.

Figure 4 is a graphical representation of the data reported in Example II for compositions according to the invention in comparison with urobilinogen solutions of 12 Ehrlich units, color standards for 12 Ehrlich units and other substances reactive with Ehrlich's reagent.

SUMMARY OF THE INVENTION In accordance with the present invention a urobilinogen control composition, control preparation device and a method for preparing a liquid control in which the presence of urobilinogen is simulated are provided. A method for simulating the presence of urobilinogen in a liquid and a liquid control in which the presence of urobilinogen is simulated are also provided.The urobilinogen control composition includes at least one compound reactable with p-dimethylaminobenzaldehyde to produce a color change which simulates the presence of urobilinogen and which has the formula:

wherein R1 and R2 are the same or different and are H or a substituted or unsubstituted C1-C4aIkyI, and R3 is H, a substituted or unsubstituted C1-C4 alkyl, alkoxy, or halogen, with the proviso that R1, R2 and R3 cannot simultaneously be hydrogen. Preferred among these substituted indoles are 5methoxyindole and 2-methyl-5-methoxyindole. The control composition is advantageously incorporated with a carrier, such as a matrix or tablet, to provide a control device.The method comprises adding to a predetermined amount of a suitable solvent, such as a physiological or body fluid, a predetermined amount of the composition according to the invention.

The compounds defined above are incorporated in the composition in an amount sufficient to give a control concentration range of from about 0.001 grams/deciliter (g/dl) to about 0.1 g/dl and to simulate, accordingly, a urobilinogen concentration range of from about 2 to about 12 Ehrlich units.

The use of these substituted indoles has been found to have a number of unexpected, advantageous characteristics. These include stability in solution as compared to urobilinogen itself, which is highly labile. Additionally, controls prepared according to the invention react with commercially available test devices for the detection of urobilinogen to yield superior coincidence of color with that produced by reaction of urobilinogen itself.

As a further aspect of the invention there is also provided a method for preparing the control preparation device which comprises releasably incorporating a carrier with a predetermined concentration of the control composition.

As is evident, the present invention is used in the preparation of a control rather than the testing of a sample. The control is not contacted or otherwise associated with a sample to be tested.

DESCRIPTION OF THE PREFERRED EMBODIMENTS Specific terms used in the following description are intended to refer only to the particular embodiment of the invention selected for illustration and are not intended to limit the scope of the invention.

The reagents used are commercially available from Eastman Kodak Co., Organic Chemicals Dept., Rochester, New York 14650 or others.

The solution itself containing the compositions according to the invention can be used to simulate a body fluid specimen, such as urine, which contains urobilinogen. Formation of a chromophoric complex with resultant color change is effected upon testing thereof. However, the composition is advantageously incorporated in a solid preparation for later preparation of liquid controls.

Solid preparations, as described below, are preferably incorporated with a carrier matrix in strip format. The term carrier matrix can be envisioned to refer to bibulous and nonbibulous matrices which are insoluble in and maintain their structural integrity when exposed to water or physiological fluids.

Suitable bibulous matrices which can be used include paper, cellulose, wood, synthetic resin fleeces, woven and nonwoven fabrics and the like. Nonbibulous matrices include organoplastic materials, such as polystyrene, polypropylene or the like. When a bibulous matrix is employed, the matrix is advantageously affixed, such as by double-faced adhesive tape, to an insoluble support member, such as an organoplastic strip, for ease of use.

Alternatively, the compositions of the invention can be embodied in a carrier taking the form of a pressed or molded tablet containing conventional carrier material.

Also provided is a method of making such devices which comprises contacting a carrier, such as a matrix ortableting agent. When this contacting is by impregnation with a solution of the composition according to the invention, the carrier so contacted is then dried. In addition to impregnation, the devices of the present invention can be made by other suitable techniques such as printing or spraying the composition onto a substrate or matrix. The solvent used in preparing solutions for the method can be water, physiological solutions, organic solvents or mixtures thereof.

As an additional aspect of the invention there is provided a method for simulating the presence of urobilinogen in a fluid control sample which comprises contacting a solvent liquid of a predetermined volume with a carrier releasably incorporated, as described above, with a predetermined amount of a control composition according to the invention and allowing said control composition to be released into said solvent in amounts sufficient to provide a control solution of desired concentration. The amounts of solvent and the control composition can be selected such that when the control composition has been released from the carrier matrix into the solvent to the extent that equilibrium concentrations thereof are obtained in the liquid and the matrix, the resulting concentration provides the desired control solution.

In contrast to prior art control preparations, which required use of urobilinogen itself, the devices of the present invention have improved stability. This stability can be even further improved by separating components of a control composition of a substrate and by suitably packaging the finished product in foil, a desiccant-containing container or the like. The physical separation of components can be accomplished by printing, by encapsulation of one or more components, by use of a separate matrix or substrate for different components, by placement of incompatible components on opposite sides of a substrate, or the like.

For any color within the gamut of the tristimulus color system the amounts of the three stimuli required to produce it (red, green and blue) serve as a measure of the color produced. The amounts are called tristimulus values and the measurement is done on any commercially available tristimulus colorimeter. The tristimulus values for each of red, green and blue are observed. Thus, in essence, two colors differ by the relative overall difference in their tristimulus values. This difference (Ac) is expressed as the extent to which they are separated in color space.

The laws of color-matching by additive mixture of color stimuli can be expressed by simple algebraic equations and illustrated geometrically in a three-dimensional space, the tristimulus color space. The calculations by which the color space illustrations, used in Example II, are derived are known and have been aptly described in Judd et al, Color in Business, Science and Industry, 3rd Ed. John Wiley s Sons N.Y., N.Y. (1975).

The following examples are illustrative of the invention defined by the claims. One skilled in the art will be able to make such variations, substitutions and changes in the ingredients and parameters as may seem desirable.

EXAMPLE I A comparison was made of the ability of certain compounds to simulate the presence of urobilinogen in a liquid sample.

The various compounds identified in Table I were dissolved in donor urines pre-tested for freedom from urobilinogen or bilirubin in amounts sufficient to give the indicated concentrations. The solutions so prepared were tested by momentary immersion of a UROBILISTIXB reagent test strip (Ames Company, a Division of Miles Laboratories, Inc., Elkhart, Indiana 46515) therein. The results reported indicate the color observed on the reagent test strip when read at 60 seconds and compared to the standard color chart accompanying the test strips.

TABLE I UROBI LISTI Xi Compound Test Strip (Ehrlich Units) 5-chloroindole (0.1 g/dl) 4 2,5-dimethylindole (0.1 g/dl) 12 2,5-dimethylindole (0.002 g/dl! 2 2-methylindole (0.1 g/dl! 12 2-methylindole (0.01 g/dl) 8 2-methylindole (0.001 g/dl! 2 5-methoxyindole (0.1 g/di) 12 1,2 dimethylindole (0.1 g/dl) 12 2-methyl-5- methoxyindole (0.t g/dl) 12 As can be seen from the results reported, the control compositions are effective to simulate a broad range of clinically significant urobilinogen values.

EXAMPLE II Color coincidence of the control compositions according to the invention with urobilinogen and recognized color standards was measured. The inventive composition was also compared with other compounds, known to react with Ehrlich's reagent, for coincidence of color production with that of bilirubin and the color standards.

Tristimulus values and coordinates in color space were calculated for printed urobilinogen color standards, urobilinogen in solution reacted with the urobilinogen portion of a UROBILISTEX# test strip, control compositions according to the invention reacted with UROBILISTIX test strips and other substances reacted with UROBILISTIX test strips. The materials tested, their concentrations, in grams/deciliter (g/dl) where applicable, and the color space coordinates for each are reported in Table II.

TABLE IT Test Subject Color Space Coordinates L* a* b* Printed Color Standard 2 Ehrlich Units 76.15 4.81 50.97 4. Ehrlich Units 65.49 10.91 52.91 8 Ehrlich Units 62.62 22.33 39.48 12 Ehrlich Units 54.42 31.87 34.80 Urobilinogen in Urine 2 Ehrlich Units 70.32 3.12 52.93 4 Ehrlich Units 64.61 14.74 43.64 8 Ehrlich Units 56.39 26.26 35.66 12 Ehrlich Units 52.48 32.11 31.36 Control Compositions 5-methoxy indole (0.01 give) 63.34 8.22 41.97 2-methyl indole (0.01 g/dl) 59.16 37.49 40.92 1,2-dimethyl indole (0.001 gIdI) 73.93 2.81 55.46 2,5-dimethyl indole (0.01 g/dl) 56.29 41.38 22.59 2,5-dimethyl indole (0.002 g/dl) 75.11 4.59 48.06 methoxy indole (0.02 g/dl) 59.88 12.39 36.52 Other Substances indole 3-acetic acid (0.1 g/dl) 86.90 -20.01 76.03 3-methyl indole (0.1' g/dl) 87.44 -22.80 68.19 bilirubin (0.1- g/dl) 76.50 11.12 79.36 sulfathizole (0.1 g/dl) 87.17 -7.32 96.66 indoxyl sulfate (0.1 g/dl) 88.3 -22.5 76.1 The color difference units (E) between the printed urobilinogen color standards and urobilinogen, control compositions and other substances dissolved in negative urines is reported in Table Ill.

TABLE Ill Test Subject Color Difference Units (##) 2 Ehrlich Unit Printed Standard Urobilinogen 2 Ehrlich Units 6.38 2,5-dimethyl indole (0.002 g/dl) indole-3-acetic acid (0.1 g/dl) 36.86 indole (0.01 g/dl) 31.75 bilirubin (0.1 g/dl) 44.84 4 Ehrlich Units Printed Standard Urobilinogen 4 Ehrlich Units 10.21 5-methoxy indole (0.01 g/dl) 11.47 indole-3-acetic acid (0.1 g/dl) 44.13 bilirubin (0.1 g/dl) 43.04 8 Ehrlich Unit Printed Standard Urobilinogen 8 Ehrlich Units 8.30 Methoxy indole (0.02 g/dl) 10.72 indole-3-acetic acid (0.1 g/dl) 60.96 12 Ehrlich Units Printed Standard Urobilinogen 12 Ehrlich Units 3.96 2-methyl indole (0.1 gSdl) 9.57 indoxyl sulfate (0.1 g/dl) 76.25 The results set out in Table Ill are graphically illustrated in Figs. 14 for simulation of various Ehrlich unit levels. The printed color standard of a UROBILISTIX test strip (A), and a solution of the indicated Ehrlich Unit concentration of actual urobilinogen (C) are compared with various embodiments of the control composition and with other substances known to react with Ehrlich's reagent.

In Figure 1 printed color standards for 2 Ehrlich units and urobilinogen solutions of 2 Ehrlich units concentration are compared with a control composition according to the invention formulated to include 0.002 g/dl 2,5-dimethylindole (B) and other substances, in this case 0.1 g/dl indole-3-acetic acid(1),0.01 g/dl indole (2) and 0.1 g/dlbilirubin(3).

In Figure 2 printed color standards for 4 Ehrlich units and urobilinogen solutions of 4 Ehrlich units concentration are compared with a control composition according to the invention formulated to include 0.1 g/dl 5-methoxy indole (B) and other substances, in this case 0.1 gdl indole-3 -acetic acid (1) and 0.1 g/dl bilirubin (3).

In Figure 3 printed color standards for 8 Ehrlich units and urobilinogen solutions of 8 Ehrlich units concentration are compared with a control composition according to the invention formulated to include 0.02 g/dl 5-methoxy indole (B) and other substances, in this case 0.1 g/dl indole-3-acetic acid (1).

In Figure 4 printed color standards for 12 Ehrlich units and urobilinogen solutions of 12 Ehrlich units concentration are compared with a control composition according to the invention formulated to include 0.1 g/dl 2-methyl indole (B) and another substance, in this case 0.1 g/dl indoxyl sulfate (4).

As can be seen from the Color Difference Units reported and the graphic illustrations the control composition according to the invention shows excellent coincidence of color when compared with the other substances.

Claims (14)

1. A method for preparing a liquid control in which the presence of urobilinogen is simulated, comprising incorporating with a suitable liquid medium at least one compound of the formula:

wherein R1 and R2 are the same or different and are H or a substituted or unsubstituted C1-C4 alkyl, and R3 is H, a substituted or unsubstituted C1-C4 alkyl, substituted or unsubstituted C1-C4 alkoxy, or halogen, with the proviso that R1, R2 and R3 cannot simultaneously be hydrogen.

2. The method of Claim 1 wherein the said compound is 2-methylindole.

3. The method of Claim 1 wherein the said compound is 1,2-dimethylindole.

4. The method of Claim 1 wherein the said compound is 2,5-dimethylindole.

5. The method of Claim 1 wherein the said compound is 2-methyl-5-methoxyindole.

6. The method of Claim 1 wherein the said compound is 5-methoxyindole.

7. The method of any of Claims 1 to 6 wherein the liquid medium is urine.

8. A method for simulating the presence of urobilinogen in a liquid which comprises adding to a predetermined amount of said liquid a predetermined amount of a composition including at least one compound of formula:

wherein R1 and R2 are the same or different and are H or a substituted or unsubstituted C1-C4alkyl, and R3 is H, a substituted or unsubstituted C1-C4 alkyl, substituted or unsubstituted C1-C4 alkoxy, or halogen, with the proviso that R1, R2 and R3 cannot simultaneously be hydrogen.

9. A urobilinogen control composition which comprises at least one compound of formula:

where R1 and R2 are the same or different and are H or a substituted or unsubstituted C1-C4 alkyl, and R3 is H, a substituted or unsubstituted C1-C4 alkyl, substituted or unsubstituted C1-C4 alkoxy, or halogen, with the proviso that R1, R2 and R3 cannot simultaneously be hydrogen.

10. A urobilinogen control preparation device which comprises a carrier and, incorporated therewith, a predetermined amount of the composition of Claim 9.

11. A method for making a urobilinogen control preparation device which comprises incorporating a carrier with a predetermined amount of the composition of Claim 9.

1 2. A liquid control in which the presence of urobilinogen is simulated, comprising a suitable liquid medium having incorporated therein the composition of claim 9.

1 3. A liquid control according to claim 12 in which the said liquid medium is urine.

14. A method of testing a composition for the determination of urobilinogen which comprises reacting the said composition with a compound of the formula defined in claim 1 and observing the colour produced.

1 5. A method according to claim 14 wherein the said composition comprises pdimethylaminobenzaldehyde.

1 6. A method according to claim 14 substantially as hereinbefore described.