CA1223512A

CA1223512A - Compositions and elements containing triarylmethane leuco dyes and methods using same

Info

Publication number: CA1223512A
Application number: CA000458834A
Authority: CA
Inventors: Bruce E. Babb; Daniel S. Daniel
Original assignee: Eastman Kodak Co
Current assignee: Eastman Kodak Co
Priority date: 1984-05-21
Filing date: 1984-07-13
Publication date: 1987-06-30
Also published as: JPS60256056A; EP0162685A2; EP0162685A3; EP0162685B1; US4670385A; DE3586336T2; DE3586336D1

Abstract

COMPOSITIONS AND ELEMENTS CONTAINING
TRIARYLMETHANE LEUCO DYES AND METHODS USING SAME

Abstract of the Disclosure A particular class of triarylmethane leuco dyes are useful in analytical compositions, elements and methods. These leuco dyes are triarylmethane compounds having an aromatic heterocyclic moiety attached to the central methane carbon and which, upon interaction with hydrogen peroxide, provide dyes having a maximum absorption at a wavelength equal to or greater than about 600 nm. Particularly useful leuco dyes have the structure:

Description

Jo COMPOSITIONS AND ELECTS CONTAINING
TRIARYLMETHANE LUKE DYES AND METHODS USING SAME
Field ox the Invention This invention relate to a novel composition, element and method using particular Luke dyes to detect hydrogen peroxide or Another analyze which reacts to produce hydrogen peroxide in the analysis of aqueous liquids, e.g. biological fluids. This invention is particularly useful in clinical chemistry.
Background of the Invention The detection and quantitative determination of hydrogen peroxide and compounds yielding hydrogen peroxide as a result of chemical or enzymatic reactions are of importance in many areas. For example they are important in the detection of hydrogen peroxide produced in the enzymatic assay of ` chemical or biological substances (sometimes called annihilates such as glucose, cholesterol, uric acid, triglycerides, creative Cannes, etc. in the presence of oxygen. The quantity of analyze present in a specimen sample is determinable from the amount of " hydrogen peroxide produced and detected.
Known compositions for detecting or ` 25 quantifying hydrogen peroxide in such assays I; generally comprise a substance having peroxidative activity, e.g. peroxides, and a material which undergoes a detectable change (e.g. a color change) in the presence of hydrogen peroxide and tune peroxidative substance. Various materials which undergo such a detectable change include monoamine, dominoes, phenols, Luke dyes and other known dyes or dye former. Dye-providing materials also useful in such assays include triarylimidazoles 88 described, for example, in U. S. Patent 4,089,747 (icily My 16, 1978 to Brush).

` to

-2-Triar~lmethane dyes and their Luke precursors are also known a commerc-L~lly useful compounds. Triarylmethane Luke dyes, for example, are known as useful indicators of hydrogen peroxide.
S Examples of such Luke dyes include the following compounds:

No I N
Jo I /.
Luke malachite green;
H
No I N
I
! I!

ON\
SHEA SHEA
Luke crystal violet;
H

No I No .

SUE SUE
Luke naphthalene green V; and H
No I No I-I! i SUE
Luke form of Acid Dye #3040.

.
I, ..

3-However, it has been observed that such Luke dyes readily oxidize in elf or in aqueous solutions containing a substance having p0roxidative activity (see e.g. Example 1 hereinbelow). Hence, this instability renders them unsuitable for analytical determinations, and especially for dry assays where the analytical composition is stored for a period of time prior to use.
Although other dye-providing materials have the desired stability and are, in general, useful as indicators for hydrogen peroxide determination, there are instances when the concentration of hydrogen peroxide to be analyzed is too low to produce sufficient detectable color from such indicators. In some instances, this shortcoming can be overcome by ; using increased amounts of indicator. However, where ` the analyze concentration is initially low or high dilution of the test sample it required, such indicators may still be deficient because they still provide insufficient detectable color in such instances.
Jo Such problems of instability and low analyze concentration are particularly acute when analyze Jo determination is attempted with a dry analytical element, e.g. with the commercially fiuccessful elements described in U. S. Patent 33992,158 (issued November 16, 1976 to Przybylowicz et at). In such instances, the indicator or reagent layer present in such elements is necessarily very thin, and the dye concentration is relatively low. Hence, the density of the color formed can be rather low even with high analyze concentrations. However, it would be desirable to use such elements for very low analyze concentrations.
Hence, there is a continuing need in the art for dye-providing materials which are stable to 51~

oxidation and which can be used to detect low concentrations of hydrogen peroxide or of annihilate which react to produce same.
Summary of the Invention The present invention utilizes a particular class of Luke dyes which are unexpectedly table to oxidation in air and in the presence of a peroxidative substance, but which can be advantageously used to detect low levels of hydrogen peroxide or analyzes which react to produce hydrogen peroxide in both solution and dry assays.
Advantageously, dyes obtained from these compounds have a maximum absorption at or above 600 no whereby avoiding potential spectral interferents commonly found in biological fluids. These compounds are particularly useful for the determination of hydrogen peroxide generated by one or more (i.e. coupled or uncoupled) enzymatic reactions in response to an analyze such as glucose galactose, amino acids, uric acid, triglycerides, creative Cannes, cholesterol and the like.
The Luke dyes useful in the practice of this invention are triarylmethane Luke dyes having an aromatic he~erocyclic moiety attached to the central methane carbon atom.
Therefore, in accordance with this invention, a composition for determination of hydrogen peroxide in an aqueous liquid comprises a substance having peroxidative activity, and a triarylmethane Luke dye having an aromatic heterocyclic moiety attached to the central methane carbon atom which provides a dye having a maximum absorption at a wavelength equal to or greater than about 600 no upon interaction with hydrogen peroxide. Such a compulsion it particularly useful for the determination of an analyze which react to .
.
.~.
.

,, ,., I

provide hydrogen peroxide, and con include an interactive composition which produces hydrogen peroxide upon interaction with the analyze, Still another feature of this invention is a dry analytical element for determination ox hydrogen peroxide or an analyze which reacts to produce hydrogen peroxide. Such an element comprises a composition described in the preceding paragraph.
According to yet another feature of this lo invention, a method for determining hydrogen peroxide or an analyze which reacts to produce hydrogen peroxide in an aqueous liquid comprises the steps of: A. physically contacting a sample of the liquid with a composition comprising a substance having peroxidative activity and the Luke dye described hereinabove; and B. detecting the resulting dye at a wavelength equal to or greater than about 600 no.
Detailed Description of the Invention The Luke dyes useful in the practice of this invention are triarylmethane compounds which have an aromatic heterocyclic moiety attached to the central methane carbon. These Luke dyes provide dyes, in the presence of hydrogen peroxide, which have a maximum absorption at a wavelength equal to or greater than about 600 no. The Luke dyes also have two carbocyclic aureole groups attached to the central methane carbon. Generally, these carbocyclic aureole groups are the same although they can be different, if desired, and have from 6 to 14 carbon atoms (e.g.
phenol, naphthyl or anthryl). Preferably, each aureole group is phenol. usher each aureole group has an amino group (primary, secondary or tertiary) which is attached to the aureole ring in such a position as to allow dye formation, e.g. in the position of a phenol group. Preferably, the amino group is twirler.

Particularly useful Luke dyes con be represented by the 8 structure (I):
H

I C _ /

wherein R is a S- to 15-membered aromatic heterocyclic moiety, and generally a moo- or poly-heterocyclic moiety containing carbon and one or more nitrogen, sulfur, oxygen or selenium atoms.
Examples of R groups are illustrated hereinbelow. R' And R" are independently open chain (primary, secondary or tertiary) or cyclic amine. Preferably, R' and R" are independently tertiary open chain or cyclic amine. As used herein, a cyclic amine it represented by the structure -N
. z"
wherein Z represents the carbon, nitrogen, oxygen, sulfur or selenium atoms necessary to complete, with the illustrated nitrogen atom, a 5- to 15-membered mono-heterocyclic ring, as further identified hereinbelow for Al, R2, R3 and K4.
The Luke dyes preferred in the practice of this invention have the structure (II):
H
No I
R
wherein R is a 5- to 15-membered aromatic heterocyclic moiety which can be unsubstituted or substituted with one or more substituents such as alkyd preferably of 1 to 18 carbon atoms), aureole (preferably of 6 Jo 14 carbon atoms, e.g. phenol, xylyl, etc.); alkoxy (preferably of I to 18 carbon :

.
'.'' .

:~2;~5~
` -7-ohms eye. methoxy, propoxy, n-pen~oxy, eke; halo (e.g. flyer, sheller, broom end loo); Nero Sweeney;
` amino US described hereinabove for R' Hod R";
car boxy; sulfa; c~rboxyester~; c~rbonyl~lkyl (preferably of 2 to 20 carbon atoms, ~ub~tltuted or unsubstituted); 8ul phony; ~ulfon~mide~; end the like. The heterocycllc moiety con be a moo- or polyheterocyclic ring and con Conklin carbon, nitrogen, sulfur, oxygen end ~elenlum Atoms in any combln~tion. Examples of useful heterocyclic moieties include fury, thinly, selenophenyl, ` pyridyl, pyrlndinyl, pyrlmidyl, tri~zinyl, thi~zolyl, ox~zolyl, selenazolyl, perineal, personnel, quinollnyl, lsoqulnollnyl, tetr~zlnyl, pyrld~zinyl, quin~zolinyl, ~cridlnyl, benzothiszolyl, qulnox~linyl, cinnollr~yl, phthsl~zlnyl, pterldinyl, phen~zinyl, N-methylpyrrole, N-methylindole, N-methylimid~zole, ~-methylbenzimld~zole, etc.
Heterocycl1c moieties which sure not useful ore those containing an -NH group in the rung, e.g. those derived from purl, lndole, lmidazole, benzimld~zole, etc. Preferred heterocycllc Mattel ore those which ore deficient, including pyrldyl, pyrldazinyl, pyrlmidyl, pyr~zlnyl, trl~zlnyl, tetr~zinyl, quinolinyl, i~oqulnolinyl, quln~zollnyl, scrldinyl, quinoxsllnyl, cinnollnyl, phth~l~zinyl, pteridinyl, phen~zlnyl, pyrindlnyl sod the like.
Partlculsrly preferred R groups are 3-pyridyl end

4-pyrldyl. For B definition of "deflations", see the book by A. Albert, HeterocYcllc Chemistry end Ed., Univ. of London, Athlone Preys, Chapter 4 (particularly pp. 67-70), 1968.
In the ~bove-ldentlfied structure, R , R , R end R Are independently sub~tltuted or unsubs~ltuted ~lkyl, preferably of 1 to 14 carbon ,` ...
`' .
:

stows (e.g. methyl, chloromethyl, ethyl, n-propyl, i80propyl Huxley, d~cyl, ~etr~decyl, etc.);
~ubstltuted or un~ubstituted ~lkaryl or ~r~lXyl, preferably of 7 to 14 carbon atom ego. bouncily, 2-ethylphenyl, p-methylbenzyl, me~hyln~phkhyls, etc.); ~ub~t$tuted or un~ubstituted uryl, preferably of 6 to 14 carbon Atoms in the rheumatic nucleus (e.g.
phenol, p-nltrophenyl, xylyl, n~phthyl, ~nthryl, etc.); or on ~rom~tlc or non~rom~tlc monoh~terocycllc moiety, preferably of 5 to 15 atoms. Ex~mpleq of ~romstic heterocycllc moletleq ore given above in the definition of R. Examples of non~rom~tic heterocyclic moieties include piperldlnyl, pyrrolidinyl, morphollnyl and the like.
Alternatively, R end R token together, and R
end R token together can represent, with the nltrs~en Tom to which they ore itched the atoms needed to complete substituted or unsubstltuted heterocyclic ring, preferably of 5 to 15 Tom (i.e.
carbon, oxygen, sulfur, nitrogen, selenium Tom), e.g. piperidinyl, morpholinyl, Julolidinyl, piperszinyl and the like.
Preferably, R , R , R end R ore independently substituted or un~ubstituted lower ~lkyls of 1 to 6 carbon Tom (e.g. methyl, chloromethyl, ethyl, Huxley, etc.). More pre~ersbly R it the sue a R end R it the me I
R . Most preferably, each of these group it the same.
The ~rylenP rings ox the Luke dye illustrated hereinabove can be further sub tituted, if desired, in the positions met to the central methane carbon, with one or more (up to 2 etch) lower ~lkyl (1 to 4 carbon Tom, substituted or unsub~tituted), lower nlkoxy (l to 4 carbon Tom, ~ubstltuted or un~ub~tltuted, e.g. methoxy, ethics, .
I: `

l 1.,'~3 I
I-;.
etc.), halo (e.g. sheller, broom, etc.), ~nlno~ or other groups known in the art. Electron donating groups, such as lower alkyd and lower alkoxy Are preferred.
Upon oxidation by hydrogen peroxide and ` substance having peroxidative activity (e.g.
peroxides), the Luke dyes are converted to detectable dyes which have a maximum absorption at or above 600 no. This lecture is advantageous in what dye detection is not hindered by certain spectral interferents (e.g. hemoglobin, bilirubin, etc.) commonly found in biological fluids. The following equation illustrates the conversion of Luke dye to positively-charged detectable dye:
lo H
No C Jo N/RHydrogen peroxide I \.~./ I \-=-/ ~R4Peroxidase R

R

No I ON/ I
I R
wherein I is a suitable monovalent anion (e.g.
halide, per chlorate, Tulane sulfonate, etc.).
Representative Luke dyes are listed in Table I hereinbelow in reference to the compound ; structure (II) given hereinabove.
.

., : ' I

I

- - - - - - y , c~~1 . I - - - - - - I
. En . I

- - - - - -I` I\
I . - .
Jo T, I
;` . . Z . .=. .=. . Jo .=. .=.
'! ' '' o/ I ",/ I z;// I ./ \. / \
I, / / \ I \ I // \ I Jo // //
.` , , ,= ,= a\ ! = - - - -I, o . I H H H H H H
. En H H H H H

I' .

35~
-11~

\
, . 0 I/ 0 I, so . a) o a o 'U .' \. ./ \.~, ,,~, . I I I q) I I I

; o V

. J

.
. Jo // \ En 1 0 to . , C V

. H Pi SO to 0 Ill a I 'a) /-\ a 0 . I oily owe 1 I ' Al Jo \ / \ / V I:
. I; Z U 5 ZZ O
Jo ; I V I
. IO O
. I
.
I\ ') Jo I\ zoo / / Jo; O Jo l l lo . a) b so Luke dyes I and II are preferred.
The Luke dyes useful in the practice of this invention can be readily prepared using commercially available or readily prepared starting materials. They can be prepared using synthetic procedures well known in the art as illustrated, for example, in U.S. Patent 3,995,088 (issued November 30, 1976 to Garner et at). In general, the Luke dyes of structure (II) are prepared by reacting a mole of an alluded of the formula RCH0 with two moles of an amine or a mixture of amine (i.e. 2 moles of \.=./ N\R.2 if Rl=R3 and R2-R4, or a 1:1 molar mixture of I No and N/

(if R', R2, R3 and R4 are different) in the presence of a suitable catalyst (e.g. ZnCl2).
The analytical composition of this invention can be used in both solution and dry element assays, and comprises a Luke dye as described hereinabove, a substance having peroxidative activity, and optionally but preferably, a buffer which maintains a pi of from about 4 to about 9 in the composition when it is used in either solution or dry assay.
Substances having peroxidative activity useful in the practice of this invention are also known as peroxidative substances and are capable of catalyzing the oxidation of another substance by means of hydrogen peroxide and other peroxides. Such substances include natural and synthetic pcroxidnses, cytochromes, hymen, forms of hemoglobin, alkaline Hampton, iron sulfocyanate, iron twenty, chromlc salts and the like. Peroxldase it a particularly useful peroxidative substance.
Substantially any buffer is useful in the composition of this invention. Useful buffers provide a pi in the composition which it conducive to dye formation. Generally, the pi is within the range of from about 4 to about 9, but a specific pi will depend to some extent on the particular analyze being assayed. For example, when used to detect uric acid using unease, it is preferred to buffer the composition at a pi between about 8 and about 9.
Useful buffers include carbonates, borate, phosphates, glutarates and the iris materials, e.g.
tris(hydroxymethyl)aminomethane.
The compositions of this invention can be prepared for use in a solution assay by mixing the peroxidative substance (generally in an aqueous solution) with the Luke dye. Since the Luke dyes have limited volubility in water, they can be dissolved in a water-miscible solvent, such as an alcohol or N,N-dimethylformamide, prior to mixing with the peroxidative substance. The details of preparing a representative hydrogen peroxide determining composition are given in Example 2 hereinbelow.
When the compositions of this invention are used in solution assay, generally the Luke dye is present in a concentration of up to about 0.1, and preferably from about 0.02 to about 0.05, mg/mL of solution. Similarly, the peroxidative substance is present in an amount sufficient to catalyze the Luke dye-dye reaction. For example, peroxides it prevent in Run amount up to 1, and preferably from bout 0.1 to about 0.5, U/mL. the alienate of the optional composition components (e.g. buffer, ~urfactnnt, 351~

etc.) and of the interactive composition (described hereinbelow) are within the skill of a wormer in the art.
The compositions of this invention can be used to determine an analyze which it capable of producing hydrogen peroxide, i.e. it can participate in a reaction or series of reactions which produce hydrogen peroxide, in an aqueous liquid by -including in such compositions an interactive composition which produces hydrogen peroxide upon interaction with the analyze. Analyzes which can be determined in this manner include glucose, triglycerides, uric acid, cholesterol, galactose, amino acids, creative Cannes, and others known to one skilled in the clinical lo chemistry art. For example, an interactive composition for determining uric acid includes unease, and an interactive composition for determining cholesterol includes cholesterol oxidize and cholesterol ester hydrolyze. Further, an interactive composition for determining creative Cannes includes glycerol Cannes, adenosine triphosphate and ~-glycerophofiphate oxidize. Other interactive compositions can be fashioned for a given analyze by those skilled in the art.
The compositions and method of this invention are adaptable to both solution and dry element assays. In a solution assay, generally the Luke dye, peroxidative substance and interactive composition, if included, are physically contacted and mixed with a liquid test sample in a suitable container (e.g. test tube, metric dish, beaker, cuvette, etc.). The resulting solution it incubated for a relatively short time (i.e. bout 5 minutes) at a temperature of up to about 25C. The sample is then evaluated by measuring the amount of dye provided upon interaction with hydrogen peroxide.

it The amount of dye can then be correlated to the amount of hydrogen peroxide either initially present in the sample, or produced as a result of the presence of an analyze. Such an evaluation can be done visually or with suitable calorimetric detection equipment and procedures.
Alternatively, the composition and method of this invention can be utilized with a dry analytical element which can be a simple carrier matrix, -Lye.
thin sheet or self-supporting absorbent or bibulous material, such as filter paper or strip, which contain the Luke dye with or without the peroxidative substance. Preferably, such elements also contain the peroxidative substance. Such elements are known in the art as text trips, diagnostic elements, dip sticks, diagnostic agents and the like.
When employed in "dry chemistry" elements, the composition of this invention can be incorporated into a suitable carrier matrix by imbibi~ion, impregnation, coating or another suitable technique.
Useful carrier matrices are infallibly and maintain their structural integrity when exposed to water or physiological fluids such as urine or serum. Useful carrier matrices can be prepared from paper, porous particulate structures, cellulose, wood, glass fiber, woven and non woven fabrics (synthetic and non synthetic) and the like. A useful dry analytical element is made by imbibing a solution of the reagent composition into the matrix and drying. Useful materials and procedures for making such elements are well known in the art as exemplified in U.S. Patents 3,092,465 (issued June 4, 1963 to Adonis et at);
3,802,842 (issued April 9, 1974 to Lange et By);
3,915,647 (issued October 28, 1975 to Wright);
3,917,453 (issued November 4, 1975 to Mulligan

5 ~'~

et at); 3,936,357 (issued February 3, 1976 to Mulligan et at), 4,248,829 (issued February 3, lg81 to Kitajima et at); 4,255,384 (issued March 10, 1981 Kitajima et at); and 4,270,920 (issued June 2, 1981 to Rondo et at); and U.S. Patent 2,052,057 published January 21, 1981) .
Preferably, the dry analytical elements of this invention have at least one porous spreading zone. This zone can be a self-3upporting carrier matrix (i.e. composed of a material rigid enough to maintain its integrity), but preferably it I carried on a separate supporting substrate (commonly called a support). Such a support can be any suitable dimensionally stable, and preferably, transparent (i.e. radiation transmisslve) material which transmits electromagnetic radiation of a wavelength between stout 200 And about 900 no. A support of choice for a particular element should be compatible with the intended mode of detection (reflection or transmission 6pectroscopy). Useful support materials include paper, metal foils, polystyrene, polyesters [e.g. polyethylene terephthalate)], polycarbonates, cellulose esters (e.g. cellulose acetate), etc.
The porous spreading zone can be prepared from any suitable fibrous or non-fibrous material or mixtures of either or both. The void volume and average pore size of this zone can be varied depending upon the use intended. For example, if whole blood or other liquid samples containing high molecular weight materials are to be assayed, the void volume and average pore size are generally greater than if serum or urine is to be assayed.
Useful spreading Zion can be prepared using fibrous materials, either mixed with a suitable binder material or woven into A fabric, AS de8Crlbed in U. S. Patent 4,292,272 (issued September 29, 1981 to K~t~1im~ et I the declare of which it incorporated herein by reference in its entirety.
Alternatively, end preferably, the spreading zone it prepared from polymeric compositions (e.g. blush polymer) or p~rtlcul~te m~terisls, with or without binding sdheslves, I described in U. S. Patents 3t992,158 (issued November 16, 1976 to Przybylowicz et I Rod 4,258,001 (issued March 24, 1981 to Pierce et I Other u eful spreading zone materiels are lo described in W. German OWLS 3,150,102 (published July 29, 1982) and Japanese Potent Public~tlon 57(1982)-101760 (published June 24, l9B2), both Resigned to Koni3hlroku Photo. It I desirable that the prodding zone be isotroplc~lly porous, meaning that the porosity it the me in etch direction in the zone crested by interconnected spouse or pores between particles, fiber, polymeric strands, etc.
P~rtlcul~rly useful spre~dln8 zones ore those hsv1ng a partlcul~te structure formed by org~no-polymeric particles end polymeric Adhesive for these particle go described in the Pierce et Al patent noted hereinabove.
The element can hove more than one zone, e.g. one or more reagent zones, spreading zones, regi~tr~tlon zone, mordant zone, radiAtion-blocking or filter zone, tubbing zone, barrier zone, buffer zone, etc. The zones sure generally in fluid contact with etch other me~nlng that fluids, reuniter end reaction products can pays between superposed regions of decent zones. Stated in another manner, fluid eont~ct refers to the Ability to transport components of fluid between the zones in fluid contact.
Preferably, the zones ore separately costed layers, although two or more zones con be single layer, or I

8 zone can contain two or mows apricot Lowry.
Buds the reruns noted hereinabove, ~ulta~le element format end components ore de3crlbed, for ample in U. S. Patents 4,042,335 sued Await 16, 1977 to Clement); 4,132,528 (Lydia January 2, 1979 to Elkenberry et at); end 4,144,30~ sued March 13, 1979 to Figures.
The component of the compo~ltlons of this invention, i.e. peroxid~tlve sub~t~nce, Luke dye, o interactive composition (if prevent), buffer (if pronto, etc. con be lncorporsted in Any of the zone of the element that would be suitable for the particular ~n~lyqls. The location of indivldu~l components is within the skill of worker in the olinic~l chemistry Art.
In the element of they'll invention, the mount ox the Luke dye con be varied widely, but it it enroll present in a coverage of up to bout 5, end preferably from about 0.01 to stout US gym .
The Luke dyes can be bull mulled directly into the ~preadlng or regent layer mRterlsl, or dlsRolved in A high boiling coupler solvent and dispersed in the layer. Useful coupler solvents include trl-m-cresyl phosphate and trl-o-tolyl fifty. The peroxid~tive substance it prevent in Q coverage within the skill of a worker in the sot. For peroxldRse, for example, the coverage is up to bout 150,000, end preferably from stout 50,000 to about Lowe U/m . A variety of other deslrsble, but option81 reagent end addenda can be prevent in the element in mounts known to one skilled in the art.
Such materiels include surf~ctant~, buffers, binders, pigments, activators, reQBent~ for the lntar~ctlve compassion, etc.

so One embodiment of this invention is a multi layer dry analytical element for determining an analyze. This element comprises a support having thereon, in order and in fluid contact, a hydrophilic layer containing a hydrophilic binder material (natural or synthetic), such as gelatin or polyacrylamide, and a spreadlng/reagent layer containing: 1) an interactive composition which produces hydrogen peroxide upon interaction with the analyze, 2) a substance having peroxidative activity, and 3) a Luke dye as described hereinabove.
A variety of different elements, depending on the method of assay, can be prepared in accordance with the present invention. Elements can be configured in a variety of forms, including elongated tapes of any desired width, sheets, slides or chips.
The assay of this invention can be manual or automated. In general, in using the dry elements, hydrogen peroxide or analyze determination is made by taking the element from a supply roll, chip packet or other source and physically contacting it with a sample of the liquid to be tested. Such contact can be accomplished in any suitable manner, e.g. dipping or immersing the element into the sample or, I preferably, by spotting the element by hand or machine with a drop of the sample with a suitable dispensing means.
After sample application, the element is exposed to any conditioning, such as incubation, heating or the like, that may be desirable to quicken or otherwise facilitate obtaining any test result.
Determination of hydrogen peroxide or an analyze is achieved when the Luke dye is oxidized to provide a detectable dye. This dye can be detected with the unaided eye or with suitable spectrophotometric means and procedures. Generally, -20~
the dyes formed in the practice of this invention have a a or an absorption maximum, equal to or greater than 600 no.
The following preparations and examples are provided to illustrate the practice of the invention. In those examples; Estanel~ was obtained from B. F. Goodrich (Cleveland, Ohio); Alkanol'~ XC
was obtained from ELI. Dupont (Wilmington, Delaware);
Surfactant log was obtained from Olin Mathewson Co. (Stamford. Connecticut); and Briton X-102, Briton X-100 and Briton X-200 were obtained from Room & Hays (Philadelphia, Pennsylvania).
Peroxides was obtained from Miles Laboratories, Elk hart, Indiana. Glycerol Cannes was obtained from Beckman Clinical Diagnostics Division (Carlsbad, California All other reagents (including enzymes) were obtained from Aldrich Chemicals Co. (Milwaukee, Wisconsin), Sigma Chemical Co. sty. Louis, Missouri) or Eastman Kodak Company (Rochester, New York).
Air stability of both the Luke dyes useful in the practice of this invention and those known in the art was demonstrated by allowing a sample of each Luke dye to be exposed to air for several weeks.
Only slight oxidation, as evidenced by a slight color formation, was observed in the case of the Luke dyes useful in this invention. The Luke dyes tested in the following examples were stable in the analytical solutions and elements prepared until oxidized by hydrogen peroxide in the presence of peroxides.
Luke dyes outside the scope of this invention, e.g.
Luke malachite green and Luke crystal violet, were readily oxidized in air.
The maximum absorption of the dyes formed by the Luke dyes useful in this invention were measured in the following manner: a stock solution of 1-5 my of the Luke dye in 100 my of methanol way prepared.

US

Ton my of the stock solution was mixed with I my of an aqueous sDlutlon of peroxide ~100 unlit per my Do water) end 0.1 my of 0.1 M hydrogen peroxide ~oiution. The resulting solution we diluted to 50 my with methanol and water, end after 2 hour, the re~ultlng spectra were determined in conventional Carey spectrophotometer.
Preparation of Luke Dye I (Table I) A mixture of 4-pyridinecarboxaldehyde ~11 8), N,N-dimethylsnlllne (25 g) and An hydrous zinc chloride (5 g) was heated under a nitrogen atmosphere sty 150C for two hour. The resulting hot melt Wag poured unto water and the mixture wag mode strongly alkaline by the addition of dilute sodium hydroxide.
This mixture was then team distilled to remove excess dimethylaniline. After cooling, the water war decanted from the residue yielding a sticky solid.
Thin solid was dissolved in ethanol and filtered to remove zinc oxide. The filtrate wow cooled, diluted with water and allowed to stand until cryat~llization of the product wan complete. The product was collected and recrystallized from aceton~trlle to yield 10 g of Luke dye I. Maws spectral snails showed a parent ion it m/e 331 sod no recognlzsble impuritle8. The Luke dye wow very table in air and the maximum ab~orptlon of the corresponding dye way at 630 no.
Preparation of Luke Dye II (Table I) This compound WAS prepared in a manner similar to the preparation of Luke dye I, but using 3-pyridinecarboxsldehyde. The resulting product had mop. of 137-189C and my g spectral annul showed a parent ion at m/e 331 and no recognizable impurities. The Luke dye was very stable in sir sod the corresponding dye had maximum ~bsorptlon at 632 no.

l'h~35~

Preparation of Luke Dye III table I) A mixture of 2-furaldehyde (10 g), N,N-dimethylaniline (30 g) and an hydrous zinc chloride (14 g) was heated together at 100C under a nitrogen atmosphere until essentially all of the alluded was consumed, as indicated on thin layer chromatography (silica, 4:1 toluene/ethyl acetate).
The reaction lime was about 2 hours. Dilute potassium hydroxide solution was then added, and the resulting solution was steam distilled to remove excess N,N-dimethylaniline. The residue Way extracted with ethyl acetate, filtered and the organic layer separated. After drying over magnesium sulfate, the solvent was concentrated under reduced pressure. The residue crystallized on cooling, and was recrystallized from ethyl alcohol. The yield was 15 g and TLC analysis showed one major spot. Mass spectral analysis showed a parent ion at m/e 336 and no recognizable impurities. This Luke dye was very stable to air. The maximum absorption of the corresponding dye was at 628 no.
Preparation of Luke Dye V (Table I) Acetone (58 g) was brominated according to the procedure described by C. Rapper in Archive For Kemp, 21(46), 512 (1963) to provide 153 g of Intermediate A, o Br2CHCCH2Br.
This compound (145 g) was reacted with Jo ~--C-NH2 = -(69 g) according to the procedure described by B~ganz and Ruler in Chum. Ben., 101, pp. 3872-3882 (1968) to provide 203 g of Intermediate B

OH
I Bra Brush-; OH
US/ \.=./
Intermediate B (44 g) was converted to 29 g of Intermediate C
By 2 Ho US/ \-'-/
in the presence of sulfuric acid as described by Bygones et at, upper. Intermediate C (29 g) was when converted to 14 g of Intermediate D, o lo HO

S \ . . /
according to Bygones et at, swooper. Thin layer chromatography (silica, 4:1 toluene/ethyl acetate) showed one product.
A mixture of Intermediate D (13 g), N,N-dimethylaniline (20 g) and an hydrous zinc chloride (10 g) was heated to 120C. A vigorous reaction commenced and water was expelled from the mixture. After the reaction had subsided, the mixture was heated to 150C for a few minutes. The resulting viscous syrup was poured into a dilute potassium hydroxide solution with stirring. The resulting mixture was extracted with ethyl acetate, 3 the solvent was dried and concentrated under reduced pressure. The residue solidified on addition of a small amount of ethyl alcohol. Recrystallization from ace~onitrile gave 15 g of Luke dye V. TLC
(silica, 4:1 toluene/ethyl acetate) showed one product. Mass spectral analysis showed one parent 3'J~'~

, ion at m/e 413 end no recognizable impurltie~ e Luke dye was very stable to air. The maximum absorption of the corresponding dye we at 635 no.
Example 1 Comparison of Luke Dyes This is a comparison of the stability of a Luke dye useful in the practice of this inven~Lon to a known Luke dye, Luke naphthalene green V which has the 6 structure:
H

C~3 C -N/

H03~ \S03H
A test solution was prepared from 0.1 my of a 5 my solution of Luke naphthalene Green V and 0.8 my of 50 my phosphate buffer (pi 7). Upon addition of 0.01 my peroxides (2 gel dye formation was observed within 5 minutes, indicating instability of the Luke dye in the presence of a peroxidative material. In contrast, Luke dye II which it useful in this invention, when treated in a similar manner, did not produce dye after one hour.
Example 2 Solution Assay of Hvdro en Peroxide _ . Y
A Luke dye within the scope of this invention was used to determine hydrogen peroxide in solution in the following manner. A 0.3 my Alcott of a stock methanol solution of Luke dye II (about 5mM) was diluted 10:1 with aqueous buffer solution (pi 7), then 0.1 my of an aqueous peroxides solution (100 units of peroxides per my of water), and 0.1 my of a 0.1 M hydrogen peroxide solution were added.
Dye formation was observed after about 5 minutes with this composition. In contrast, no dye was observed after 60 minutes when the dye wins mixed with only the buffer or peroxides or both.

ho Example 3 Solution Assay for Glucose A Luke dye useful in this invention was used to assay for glucose in solution by the following procedure. Two stock solutions were prepared: stock solution A contained 50 my of potassium phosphate buffer (pi 7) and 10 my of a methanol solution of Luke dye II (21.9 my of Luke dye/100 my of methanol; stock solution B contained 5 my of potassium phosphate buffer (pi 7), peroxides (120 U/mL) and glucose oxide (100 U/mL).
A control solution way prepared prom 1 my ox solution A, 1 my of solution B and 1.4 my of distilled water. A test solution was prepared from 1 my of solution A, 1 my of solution B, 1 my of distilled water and 0.4 my of glucose solution (10 Mel). The optical density of the control and test solutions were then measured at 25C at 632 no in a conventional Carey spectrophotometer. The difference in density between the two solutions after one minute was 0.12 optical units indicating that the test solution of this invention was useful for determination of glucose.
Example 4 Multi layer Elements Using Luke Dyes I-VI for Determining hydrogen Peroxide Luke dyes I-VI were used to determine hydrogen peroxide with individual multi layer analytical elements by incorporating each Luke dye into an individual element, contacting those elements with solutions containing hydrogen peroxide and measuring the reflection density of the resulting dye.
ash analytical element had the following format:

oh __ _ Barium sulfate 5-5~ gym Cellulose acetate 0~4-4 g/m2 Estate elastomers 0.05-1 g/mZ
Briton X-102 eurfactant 0.06-0.25 g/m2 5 Spreading/ Potassium phosphate buffer Reagent (pi 7) 0.04-0.2 g/m2 Layer Sodium phosphate buffer (pi 7) 0.02-0.1 g/m2 Dime done 0.01-0.05 gym 4-Hydroxypyr-lmidine 0.001-0.01 g/mZ
Luke dye 0.06-0.5 g/m2 Peroxides U/m 2 Hydrophilic Gelatin g/m' Layer Surfactant lug g/m2 . .
// polyethylene terephthalat // / / /
/ / / Support The elements were evaluated using two hydrogen peroxide test solutions: test solution A
(pi 5) contained 5 x 10-2M dimethylglutaric acid and 10-2M hydrogen peroxide; test solution B (pi 7) contained 5 x 10-2M sodium dihydrogen phosphate, 5 x 10-2M potassium hydrogen phosphate and 10-2M
hydrogen peroxide. Water was used as a control liquid. Ten AL of each solution were spotted onto individual samples of the elements containing the respective Luke dyes. After 5 minutes incubation at 37C, the reflection density was measured in a modified conventional reflectometer at 630 no.
Table II hereinbelow shows the change in reflection density (DRY) between the water control and each test solution with each Luke dye.
It is apparent from these data that each of the Luke dyes is effective in determining hydrogen peroxide at both pi 5 and pi 7 in a dry element.

-27~
Table II
ad at 630 nod at 630 no, Luke Dye _ Solution A Solution B
I 0.89 1.1 5II 0.41 1.0 III 0.25 0.29 IV 0.56 0.23 V 0.78 0.~2 VI 0.86 0.37 example 5 Multi layer Element for Determination of Triglycerides An analytical element was prepared AS shown in the following format.

Lopez M ~400 Elm Triton'n X-100 2.7-11 g/m2 Spreading Titanium dioxide 25-200 g/m2 Layer Cellulose acetate 3_30 g/m2 Estate parathion resin 0.5-5 g/m2 Subbing PoIy(~-isopropylacryl-Layer aside 0.1-1.0 g/m2 Gelatin 2-20 g/m' BVSME hardener g/m2 Hydrophilic Boric Acadia g/m2 Layer Briton X-1000.03-0.12 g/m2 Ascorbic acid oxidize 1000-5000 U/m2 elation 2-15 gym Poly(methylacrylate-co-2-acrylamido-2-methy propane sulfonic acid-co-2-acetoacetoxy-methacrylate)2-15 g/m2 Luke dye I0.2-2.5 g/m2 Tricresyl phosphate g/m2 Reagent Alkanol'~ XC0.1-0.4 g/m2 Lowry Magnesium chloride g/m2 Boric Acadia g/m2 Adenosine triphosphate (AT) 0.6-2.4 g/m2 Briton X-2000.02-0.1 my Glycolic Acadia g/m2 BVSME h~rdener0.02-0.08 g/m2 Peroxides Us Glycerol Cannes U/m2 Glycerophosph~te oxidize U/m2 . j -- / / / /

/ Polyethylene terephthalate)/ / /
/ / / Support / / /
_ _ .

So I
To evaluate this coated eletnent~ a series of triglyceride calibrator fluids, varying in analyze concentration from 17.4 mg/mL to 565.8 mg/dL, were prepared and samples of the element were spotted S with 10 AL drops of each calibrator fluid. After 5 minutes incubation at 37C, the reflection density (DRY) was measured at 650 no in a modified conventional re1ectometer. The results are listed in Table III hereinbelow. These data indicate that lo the element of this invention illustrated in this example is suitable for determining triglycerides.
Table III
Calibrator Fluid-Triglyceride Concentration at 650 no 15(mg/dL) 17.4 0.020 94~4 0.106 250.0 0.120 400.6 0.126 20565.8 0.141 The invention hue been described in detail with particular reference to preferred embodiments thereof, but it will be understood that variations and modifications can be effected within the spirit and scope of the invention.

Claims

We claim:

1. A composition for the determination of hydrogen peroxide in an aqueous liquid, said composition comprising a substance having peroxidative activity and a triarylmethane leuco dye having an aromatic heterocyclic moiety attached to the central methane carbon atom which provides a dye having a maximum absorption at a wavelength equal to or greater than about 600 nm upon interation with hydrogen peroxide.

2. The composition of claim 1 wherein said leuco dye has the structure:

wherein R is a 5- to 15-membered aromatic heterocyclic moiety; and R' and R" are independently open chain or cyclic amines.

3. The composition of claim 2 wherein said leuco dye has the structure:

wherein R is a 5- to 15-membered aromatic heterocyclic moiety; and R1, R2, R3 and R4 are independently alkyl, alkaryl, aralkyl, aryl or a heterocyclic moiety, or R1 and R2 or R3 and R4 independently taken together with the respective nitrogen atom, represent the atoms necessary to complete a heterocyclic ring.

4. The composition of claim 3 wherein R1=R3 and R2=R4,

5. The composition of claim 4 wherein R1, R2, R3 and R4 are the same.

6. The composition of claim 3 wherein each of R1, R2, R3 and R4 is a lower alkyl of 1 to 6 carbon atoms.

7. The composition of claim 3 wherein said leuco dye is selected from the group consisting of:
I. ;

II. ;
III. ;

IV. ;

V. ;

VI. ;

VII. ;

VIII. ;

IX. ;

X. XI. XII.

8. The composition of claim 3 wherein R is a .pi.-deficient heterocyclic moiety.

9. The composition of claim 7 wherein said leuco dye is compound I or II.

10. The composition of claim 1 wherein said substance having peroxidative activity is peroxidase.

11. A composition for the determination of an analyte in an aqueous liquid, said composition comprising: 1) an interactive composition which produces hydrogen peroxide upon interaction with said analyte, 2) a substance having peroxidative activity and 3) a triarylmethane leuco dye having an aromatic heterocyclic moiety attached to the central carbon atom which provides a dye having a maximum absorption at a wavelength equal to or greater than about 600 nm upon interaction with hydrogen peroxide.

12. The composition of claim 11 wherein said leuco dye has the structure:

wherein R is a 5- to 15-membered aromatic heterocyclic moiety; and R' and R" are independently open chain or cyclic amines.

13. The composition of claim 12 wherein said leuco dye has the structure:

wherein R is a 5- to 15-membered aromatic heterocyclic moiety; and R1, R2, R3 and R4 are independently alkyl, alkaryl, aralkyl, aryl or heterocyclic moiety, or R1 and R2 or R3 and R4 independently taken together with the nitrogen atom, represent the atoms necessary to complete a heterocyclic ring.

14. A dry analytical element for the determination of hydrogen peroxide in an aqueous liquid, said element comprising: 1) a substance having peroxidative activity, and 2) a triarylmethane leuco dye having an aromatic heterocyclic moiety attached to the central carbon atom which provides dye having a maximum absorption at a wavelength equal to or greater than about 600 nm upon interaction with hydrogen peroxide.

15. The element of claim 14 wherein said leuco dye has the structure:

wherein R is a 5- to 15-membered aromatic heterocyclic moiety; and R' and R" are independently open chain or cyclic amines.

16. The element of claim 15 wherein said leuco dye has the structure:

wherein R is a 5- to 15-membered aromatic heterocyclic moiety; and R1, R2, R3 and R4 are independently alkyl, alkaryl, aralkyl, aryl or a heterocyclic moiety, or R1 and R2 or R3 and R4 independently taken together with the respective nitrogen atom, represent the atoms necessary to complete a heterocyclic ring.

17. The element of claim 14 comprising an interactive composition which produces hydrogen peroxide upon interaction with an analyte.

18. A dry analytical element for the determination of an analyte in an aqueous liquid, said element comprising:
a support having thereon a porous spreading zone and 1) an interactive composition which produces hydrogen peroxide upon interaction with said analyte, 2) a substance having peroxidative activity and 3) a leuco dye which provides a dye having maximum absorption at a wavelength equal to or greater than about 600 nm upon interaction with hydrogen peroxide, said leuco dye having the structure:

wherein R is a 5- to 15-membered aromatic heterocyclic moiety; and R1, R2, R3 and R4 are independently alkyl, alkaryl, aralkyl, aryl or heterocyclic moiety, or R1 and R2 or R3 and R4 independently taken together with the respective nitrogen atom, represent the atoms necessary to complete a heterocyclic ring.

19. The element of claim 18 wherein R1=R3 and R2=R4.

20. The element of claim 18 wherein R1, R2, R3 and R4 are the same.

21. The element of claim 20 wherein each of R1, R2, R3 and R4 is a lower alkyl of 1 to 6 carbon atoms.

22. The element of claim 18 wherein said leuco dye is selected from the group consisting of:

I. ;

II. ;

III. ;

IV. ;

V. ;

VI. ;

VII. ;

VIII. ;

IX. ;

X. XI. ;and XII. .

23. The element of claim 22 wherein said leuco dye is compound I or II.

24. The element of claim 18 wherein said substance having peroxidative activity is peroxidase.

25. A method for determining hydrogen peroxide or an analyte which reacts to produce hydrogen peroxide in an aqueous liquid, said method comprising the steps of:
A. physically contacting a sample of said liquid with a composition comprising a substance having peroxidative activity and a triarylmethane leuco dye having an aromatic heterocyclic moiety attached to the central methane carbon atom which provides a dye having a maximum absorption at a wavelength equal to or greater than about 600 nm upon interaction with hydrogen peroxide; and B. detecting said dye at a wavelength equal to or greater than about 600 nm.

26. The method of claim 25 wherein said leuco dye has the structure:

wherein R is a 5- to 15-membered aromatic heterocyclic moiety; and R1, R2, R3 and R4 are independently alkyl, alkaryl, aralkyl, aryl or a heterocyclic moiety, or R1 and R2 or R3 and R4 independently taken together with the respective nitrogen atom, represent the atoms necessary to complete a heterocyclic ring.

27. The method of claim 25 wherein said leuco dye is in a dry analytical element.

28. The method of claim 25 wherein said contacting step a occurs in the presence of an interactive composition which reacts with said analyte to provide hydrogen peroxide.