Salt of 6-carboxy-3-methylbenzothiazolone hydrazone hydrate in colorimetric determination of hydrogen peroxide

Water soluble salts of hydrazone compounds react quickly with aniline compounds in the presence of a peroxidase and hydrogen peroxide to provide a color which can be measured spectrophotometrically or visually, either in solution or when incorporated in a device for the measurement of analytes of interest. These water-soluble hydrazone salts are useful in determining hydrogen peroxide or an analyte which reacts to produce hydrogen peroxide in an aqueous liquid, by, in the presence of a substance having peroxidative activity, physically contacting a sample of a liquid with a dye-forming composition comprising a water-soluble salt of a hydrazone compound and a substituted or unsubstituted aniline compound to produce a colored dye; and detecting the dye formed as a result of the presence of hydrogen peroxide or an analyte which reacts to produce hydrogen peroxide.

FIELD OF THE INVENTION 
The present invention is directed to a reagent, method and apparatus for 
detection of hydrogen peroxide. 
BACKGROUND OF THE INVENTION 
Medical science has an increasing need for quick, accurate determination of 
analytes in blood or other body fluids. Traditionally, assays for analytes 
have been performed by laboratories and required skilled technicians, 
requiring complex apparatus and reagents, as well as considerable time in 
order to obtain accurate results. Numerous qualitative and some 
quantitative devices and methods have been developed which eliminate or 
decrease the need for laboratory diagnostic services. Many of these 
devices and methods include test strips or dip sticks which can be exposed 
to blood or another body fluid in order to obtain a diagnostic result. 
Common examples of this technology include the various test products for 
determining the concentration of glucose in diabetics, and determining 
cholesterol levels in blood. 
Many clinical assays depend upon a detection and/or quantitative 
determination of hydrogen peroxide and compounds yielding hydrogen 
peroxide as a result of chemical or enzymatic reactions. For example, 
hydrogen peroxide is produced in the enzymatic assay of analytes such as 
glucose, cholesterol, uric acid, lipase, triglycerides, creatine kinase, 
etc. in the presence of oxygen. The quantity Of analyte present in a test 
sample is determinable from the amount of hydrogen peroxide produced and 
detected. 
Known compositions for detecting or quantifying hydrogen peroxide in such 
assays generally comprise a substance having peroxidative activity, e.g., 
peroxidase, and a material which undergoes a detectable change (e.g., 
oxidation to a colored dye) in the presence of hydrogen peroxide and the 
peroxidative substance. Various materials which undergo such a detectable 
change include monoamines, diamines, phenols, leuco dyes and other known 
dyes or dye formers. 
Hydrogen peroxide detection has also been accomplished by the reaction of a 
color-forming coupler and an oxidizable color developing compound, e.g,. 
4-amninoantipyrine, in the presence of peroxidase. Color-forming couplers 
which have been used for this purpose include N-substituted anilines, such 
as those described in U.S. Pat. No. 4,251,629, Yamanisi et al.; U.S. Pat. 
No. 4,260,679, Tsuda et al.; and U.S. Pat. No. 4,396,714, Maeda et al. 
Some of the anilines described in these references have solubilizing 
groups, such as hydroxy or sulfo groups, attached to the nitrogen atom. 
Although the dye-producing materials which have been conventionally used 
are 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 dye-forming materials. However, where the analyte concentration 
is initially low, or where high dilution of the test sample is required, 
such material may still provide insufficient detectable color in such 
instances. 
The problem of low analyte concentration is particularly acute when analyte 
determination is attempted with a dry analytical element, such as 
described in U.S. Pat. No. 3,992,158, Przybylowicz et al.; U.S. Pat. No. 
5,087,556, to Ertingshausen; and U.S. Pat. No. 5,234,813, McGeehan et al. 
In many of these types of devices, the indicator or reagent layer present 
in the device is very thin, and the concentration of the dye-forming 
material is necessarily quite low. Hence, the density of the color formed 
from low level analytes, or even from abnormally low concentrations of 
high level analytes, can be rather low. 
For accurate quantitative determinations, the color formed from reaction of 
the color former should be an intense, easily discernible color, i.e., to 
permit easier reading, which is of some consequence for devices to be used 
by patients for self-monitoring, e.g., of blood glucose or cholesterol. 
Moreover, the reaction of hydrogen peroxide with the dye must be very 
rapid so that the color formed can be read by the user without delay. 
Since the ideal pH for effecting many enzymatic reaction is approximately 
7, it is important that the compounds used be soluble in aqueous solution 
at a pH around 7. 
Making quantitative determinations of analytes in body fluids for 
diagnostic purposes requires formation of a color and reading the 
endpoint. Greater color depth (higher molar absorptivity) of the color 
formed from the reaction of the indicator with a dye permits easier 
reading. Therefore, it has been desirable to develop dye systems that have 
very high molar absorptivity. Additionally, many of these devices are 
stored at room temperature over a period of months. Some of the currently 
used indicators have been found to degrade under long-term storage 
conditions, particularly in the presence of functional groups from other 
components of the device. High heat stability is necessary for long-term 
shelf life at 60.degree. C. in dry form. One of the most commonly used 
indicators, 3-methyl benzothiazolone-2-hydrazone HCL, commonly known as 
MBTH.HCl, is insufficiently stable under such high heat conditions, as 
noted in U.S. Pat. No. 4,101,381. 
Another problem with the conventionally used indicator MBTH is the 
relatively poor water solubility of the dye formed in the color reactions. 
This poor water solubility makes it difficult to remove all of the dye 
from the containers in an automatic analyzer, so that the method is poorly 
suited for use in continuous flow automatic analyzers. This effect is 
amplified in the oxidation procedure necessary for the development of the 
color, wherein secondary reactions occur in which an insoluble product is 
formed. In automatic analyzers, this product is deposited in the tubes and 
glass spirals, and thus further increases the carry over. Furthermore, 
this leads to an unstable, i.e., drifting, base line. In order to 
compensate for these disadvantages of MBTH as an indicator for automatic 
analyzers, the analysis time must be lengthened considerably, and the 
apparatus must be washed frequently. Lastly, allowances must be made for 
drifting of the base line. 
Prior workers in the field have developed reagents for clinical assays 
which are based upon compounds related to MBTH, but none of these 
compounds has proved to be superior to MBTH. For example, Klose et al., in 
U.S. Pat. No. 4,101,381, disclose a reagent for detecting substances 
forming hydrogen peroxide using 
3-methyl-2-sulfonyl-benzothiazolononhydrazone. Unfortunately, this 
compound has a much poorer solubility in water than MBTH, and thus cannot 
successfully be used in liquid assay systems. 
Another disadvantage of using MBTH or its 2'sulfonated analog is that these 
dyes can only be used in systems at a pH below 7. Otherwise, an 
autoxidative color reaction can occur even in the absence of hydrogen 
peroxide, resulting in a relatively rapid coloration and thus of 
distinctly limited storage stability for a ready-to-use reagent. 
Bomer et al., in U.S. Pat. No. 4,800,169, disclose decolorants for use in 
reagents for tests which contain single-component oxidation indicators. In 
this case, the decolorant prevents the appearance of a blank color value 
of the test aid. The decolorants do not couple with themselves, and they 
are not used with any coupling component. Esters of hydrozones are 
disclosed in Wei, U.S. Pat. Nos. 3,694,450 and 3,859,280. These compounds, 
however, are said to be central nervous system depressants, and there is 
no indication that these esters can be used as dyes for assays, either 
coupled or uncoupled. 
SUMMARY OF THE INVENTION 
Water soluble salts of 6-carboxy-3-methylbenzothiazolone hydrazone, or 
carboxy MBTH compounds have been found to react quickly with an 
aniline-type dye in the presence of a peroxidase and hydrogen peroxide to 
provide a color which can be measured spectrophotometrically or visually, 
either in solution or when incorporated in a device for the measurement of 
analytes of interest. The soluble salts of 
6-carboxy-3-methylbenzothiazolone hydrazone hydrate, such as sodium 
potassium, or ammonium 6-carboxy-3-methylbenzothiazolone hydrazone 
hydrate, (NaCMBTH) have been found to be superior to MBTH.HCl in the speed 
of reactivity, long-term stability, solubility characteristics, and the 
molar absorptivity of the color these new compounds form with aniline-type 
dyes.

DETAILED DESCRIPTION OF THE INVENTION 
The soluble hydrazone compounds of the present invention, in the presence 
of hydrogen peroxide, react rapidly with an aniline compound to form a 
deeply-colored compound. The dyes thus formed are useful in detecting 
compounds which can react to form hydrogen peroxide, particularly those 
compounds which react to form hydrogen peroxide as a result of the action 
of an enzyme, such as glucose in the presence of glucose peroxidase, or 
cholesterol in the presence of cholesterol oxidase. In the dyes formed 
from these soluble hydrazone compounds, the reaction to form a 
deeply-colored dye proceeds rapidly either on a substrate impregnated with 
the reactants or in solution. 
The soluble salts of carboxy MBTH preferably have a molar extinction 
coefficient of at least 13,000 and preferably at least 15,000. The salts 
are soluble in cold water and rapidly form strongly colored dyes with 
aniline compounds in the presence of hydrogen peroxide. Dye formation with 
the compounds of the present invention occurs over a wide pH range, 
generally from about 4 to about 11. 
Assays 
The compounds of the present invention can be used in both solution and dry 
element assays. The compounds can be formulated with optional substances 
having peroxidative activity or a buffer which maintains the pH of the 
composition in an aqueous environment at a pH of from about 4 to 11. 
Substances having peroxidative activity useful in the practice of the 
present invention are also known as peroxidative substances, and are 
capable of catalyzing the oxidation of another substance by means of 
hydrogen peroxide or another peroxide. Such substances include natural and 
synthetic peroxidases, cytochromes, hemin, forms of hemoglobin, alkaline 
hematin, iron sulfocyanate, iron tannate, tungstic acid and its salts, 
molybdic acid and its salts, chromic salts and the like. Peroxidase is a 
particularly useful peroxidative substance. A catalytic amount of the 
peroxidative substance can be used in a reagent formulation, as is well 
known to those skilled in the art. 
Substantially any buffer can be used in the composition of this invention 
which does not interfere with the assay and which maintains the 
composition at a pH which is conducive to dye formation as well as to the 
reactions required for a given assay. Generally, the pH is maintained 
within the range of from about 4 to about 11, but a specific pH depends 
upon the particular analyte being assayed and the reagents used therein. 
An advantage of the instant dye is that it can be used in systems at a pH 
of 7 or above. For example, when a fluid is assayed for uric acid using 
uricase, the pH of the composition is preferably maintained between about 
8 and 9. However, the instant dye may be used in a acidic system (pH&lt;7) as 
well. For example when a sample is assayed for glucose using glucose 
oxidase, the pH of the composition is generally maintained between about 4 
and about 7. Useful buffers for various assays include carbonates, 
borates, phosphates, maleates, glutarates, the tris materials, such as 
tris(hydroxy-methyl)aminomethane, and others known in the art. One of the 
advantages of the compounds of the present invention is that they are 
readily soluble and rapidly couple with aniline compounds at a pH of 
between about 4 and 11, which is the pH range at which most clinical 
assays are conducted. 
Compositions for assays can be prepared for use in a solution assay by 
mixing the soluble hydrazone compound with an oxidizable color developing 
compound such as an aniline dye. Additional materials can be mixed in as 
needed. 
The molar ratios of soluble hydrazone compound to aniline compound 
preferably range from about 20:1 to about 1:20, with more nearly equimolar 
ratios being more preferred for the optimum combination of detection 
sensitivity and interference resistance. When the compounds of the present 
invention are used in solution assays, the soluble hydrazone compound is 
preferably present in a concentration of up to about 10.sup.-3 molar, and 
more preferably from about 5.times.10.sup.-5 to about 5.times.10.sup.-4 
molar. The aniline compound is preferably present in an amount sufficient 
to react with the soluble hydrazone compound, such as a molar ratio of 
from about 20:1 to about 1:20, and more preferably about equimolar. For 
example, the aniline compound is preferably present in an amount of up to 
about 10 millimolar, and preferably from about 0.5 to about 2 millimolar. 
The amounts of the optional components of the composition, e.g., buffer, 
surfactant, peroxidative substances, etc., are within the skill of a 
worker in the art. Cf. U.S. Pat. Nos. 4,492,754; 4,672,029; 5,179,005; 
5,043,269, all of which disclose clinical assays reciting varying amounts 
of such optional components as required by the individual assay. 
The soluble hydrazone compounds of the present invention can be used to 
determine an analyte which is capable of producing hydrogen peroxide 
(i.e., analyte which can participate in a reaction or series of reactions 
which produce hydrogen peroxide) in an fluid sample. 
The sample may be a biological fluid or a non-biological fluid. 
Non-limiting examples of biological fluids obtained in vivo include whole 
blood, a separated blood fraction, urine, semen, saliva, cerebrospinal 
fluid, amniotic fluid, ascites fluid, pleural effusion, cyst fluid, pus, 
tissue extracts, etc. Non-limiting examples of biological fluids obtained 
in vitro include tissue culture supernatant, such as that of hybridoma 
cells, or microbial fermentation medium. 
The sample may also be a non-biological fluid such as drinking water, 
wastewater, groundwater, or a nonaqueous fluid. 
This composition can be used with the appropriate interactive reagent or 
combination of reagents which produces hydrogen peroxide upon interaction 
with the analyte during the assay. Analytes which can be determined in 
this matter include glucose, triglycerides, uric acid, lipase, 
cholesterol, galactose, amino acids, creatine kinase, and others known to 
those skilled in the clinical chemistry art. For example, to determine 
uric acid, the composition is used with uricase. To determine cholesterol, 
the composition is used with cholesterol oxidase and cholesterol ester 
hydrolase. Other interactive compositions can be fashioned for a given 
analyte by one skilled in the art. The amounts of the reagents suitable 
for a given assay are known to one skilled in the art of clinical 
chemistry. In the context of the present disclosure, determination means 
either qualitative (i.e., merely detection), semi-quantitative, or 
quantitative analysis unless otherwise specified. 
Aniline Dyes 
The soluble hydrazones of the present invention can be coupled with any 
type of aniline dye, i.e., any dye which includes an amino group bonded to 
a phenyl group. The substituents on the phenyl or amino group can be 
chosen to change the .lambda..sub.max of the dye or to change the visible 
color for optical or spectrophotometric determinations. 
Preferably, the soluble hydrazone is coupled with an aniline having the 
formula given in FIG. 1, where R.sup.1 and/or R.sup.2 can be H; C.sub.1 
-C.sub.9 alkyl; C.sub.1 -C.sub.9 alkoxy; NR.sup.3 R.sup.4 where R.sup.3 
and/or R.sup.4 are H, C.sub.1 -C.sub.9 alkyl, aryl or heteroaryl; F; Cl; 
Br; I; COOR.sup.5 where R.sup.5 is H, C.sub.1 -C.sub.9 alkyl, aryl or 
heteroaryl; CN; CONR.sup.6 R.sup.7 where R.sup.6 and/or R.sup.7 are H, 
C.sub.1 -C.sub.9 alkyl, aryl or heteroaryl; aryl; aryloxy; heteroaryl, 
heteroaryloxy; and 
R.sup.11, R.sup.12, R.sup.13 and/or R.sup.14 are H; C.sub.1 -C.sub.9 alkyl; 
C.sub.1 -C.sub.9 alkoxy; NR.sup.15 R.sup.16 where R.sup.1 5 and/or R.sup.1 
6 are H, C.sub.1 -C.sub.9 alkyl, aryl or heteroaryl; F; Cl; Br; I; 
COOR.sup.17 where R.sup.17 is H, C.sub.1 -C.sub.9 alkyl, aryl or 
heteroaryl; CN; CONR.sup.18 R.sup.19 where R.sup.1 8 and/or R.sup.1 9 are 
H, C.sub.1 -C.sub.9 alkyl, aryl or heteroaryl; aryl; aryloxy; heteroaryl, 
heteroaryloxy; or any other group that does not interfere with the 
coupling reaction; and 
n is an integer of from 0 to 10; and 
Z and/or Y is H; OH; SH; COOR.sup.8 where R.sup.8 is H, C.sub.1 -C.sub.9 
alkyl, aryl or heteroaryl; CN; CONR.sup.9 R.sup.10 where R.sup.9 and/or 
R.sup.10 are H, C.sub.1 -C.sub.9 alkyl, aryl or heteroaryl; NR.sup.20 
NHR.sup.21 where R.sup.20 and/or R.sup.21 are H, C.sub.1 -C.sub.9 alkyl, 
aryl or heteroaryl; and at least one of Z and Y is COOR.sup.8, CN.sub.1 
NR.sup.9 R.sup.10 or NR.sup.20 NHR.sup.21. 
The term "aryl" includes substituted and unsubstituted single, plural and 
fused ring groups which have aromatic bonding, including but not limited 
to phenyl, naphthyl, biphenyl, fluoryl, pyryl, and the like. Substituents 
on the aryl ring may be any substituents which do not interfere with 
coupling to the soluble hydrazones of the present invention, including but 
not limited to C.sub.1 -C.sub.5 alkyl, C.sub.1 -C.sub.5 alkenyl, F, Cl, 
Br, I, hydroxyl, C.sub.1 -C.sub.5 alkoxy, CN, COOR.sup.1, CONR.sup.6 
R.sup.7, and any combinations thereof. Likewise, the alkenyl and alkyl 
groups may be substituted by F, Cl, Br, I, hydroxyl, CN, and other groups 
which do not interfere with the coupling. 
The term "heteroaryl" encompasses rings having aromatic bonds having at 
least one heteroatom in the ring. The heteroatoms may be N, S, or O, and 
any combination thereof. Non-limiting examples of the heteroaryl groups 
which can be included in the amines which can be coupled to the soluble 
hydrazones of the present invention are pyridine, quinoline, isoquinoline, 
pyrazine, pyrimidine, purine, oxathialone, oxazole, dithiazine, indole 
xanthene, acridine and the like. These heteroaryl groups may likewise be 
substituted at one or more positions by at least one substituent selected 
from the group consisting of C.sub.1 -C.sub.5 alkyl, C.sub.1 -C.sub.5 
alkenyl, C.sub.1 -C.sub.5 alkoxy, CN, COOR.sup.1, CONR.sup.6 R.sup.7, and 
the like. 
The above aniline compounds are disclosed and described in more detail in 
application Ser. No. 08/111,490, filed Aug. 25, 1993, now U.S. Pat. No. 
5,518,891 the entire contents of which are hereby incorporated by 
reference. 
Among the aniline dyes that can be used to form dyes according to the 
present invention are the following: 
N,N-(biscarboxymethyl)aniline (PAGA) 
N,N-(biscarboxymethyl)-4-methyoxyaniline (MOPAGA) 
N,N-(bis-.beta.-carboxyethyl)aniline (PAPA) 
N-ethyl-N-phenylglycine (EPG) 
N-ethyl-N-carboxyethylaniline (NENCEA) 
N-phenylpiperidinyl succinate (PPS) 
N-ethylanilinopropaneamine (NEAP) 
N-methylanilinopropaneamine (NMAP) 
N-methyl-N-carboxyethylanilin (NMNCEA) 
N,N-(bis-.beta.-carboxyethyl )-2,5-dimethylaniline (BCEDMA) 
N-.beta.-carboxyethylaminobenzoic acid (NCEABA) 
Compounds of the formula: 
##STR1## 
wherein n and m are individually selected from whole numbers 1 to 4, X and 
Y, which can be the same or different, represent a valence bond or a 
phenylene radical, 
R.sub.1 and R.sub.2 are individually selected from carboxyl or sulfonic 
acid groups and one of R.sub.1 and R.sub.2 can also be hydrogen or lower 
alkyl, and 
R.sub.3 and R.sub.4 are individually selected from hydrogen and alkyl 
radicals of up to 6 carbon atoms. 
Test means prepared with dyes made according to the present invention, and 
test systems employing these test means, are preferably used in a 
generally neutral or slightly acid pH range, although the dyes remain 
operative even at a somewhat higher pH up to almost pH11. The maintenance 
of a generally neutral or acid pH provides improved reactivity in terms of 
speed and resistance to interference. The soluble hydrazones of the 
present invention are particularly well suited to clinical assays because 
they react quickly with aniline compounds at approximately neutral pH to 
produce dyes having high molar absorptivity. 
The soluble hydrazones of the present invention react with aniline 
compounds in the presence of hydrogen peroxide. Many analytes, including 
glucose, cholesterol, uric acid, etc. produce hydrogen peroxide when acted 
upon by a peroxidase enzyme, i.e., an enzyme which will catalyze a 
reaction wherein hydrogen peroxide oxidizes another substance. The 
peroxidases are generally conjugated proteins containing iron porphyrin. 
Peroxidase occurs in horseradish, potatoes, figtree sap and turnips (plant 
peroxidase); in milk (lacto peroxidase); and in white blood corpuscles 
(verdo peroxidase). Peroxidase also occurs in microorganisms and may be 
produced by fermentation. Certain synthetic peroxidases, such as those 
disclosed by Theorell and Maehly in Acta. Chem. Scand. Vol. 4, pages 
422-434 (1950), are also satisfactory for use in hydrogen peroxide 
detection systems. Less satisfactory but also useful are substances such 
as hemin, methemoglobin, oxyhemoglobin, hemoglobin, hemochromogen, 
alkaline hematin, hemin derivatives, and certain other compounds which 
demonstrate peroxidative or peroxidase-like activity. 
Other substances which are not enzymes but which demonstrate peroxidative 
activity and could be used as oxidizers are iron sulfocyanate, iron 
tannate, ferrous ferrocyanide, chromic salts (such as potassium chromic 
sulfate) absorbed in silica gel, etc. 
Among the analytes that can be determined by detecting hydrogen peroxide 
produced by action of an oxidizing agent on the analyte are glucose, 
cholesterol, uric acid, cholesterase, phospholipids, creatine or 
creatinine. All of these assays can readily be conducted using the dye 
formed according to the present invention. 
In the present invention, the term "reagent layer" is used to refer to a 
layer in which an analyte is converted into a visually detectable species 
in the presence of a dye composition according to the present invention. 
This basically comprises a substance having peroxidase activity and a 
substance capable of causing a detectable change in the presence of 
hydrogen peroxide and the substance having peroxidase activity. In this 
case, the substance capable of causing a detectable change in the presence 
of hydrogen peroxide and the substance having peroxidase activity is the 
combination of the soluble hydrazone compound of the present invention and 
an aniline compound. 
In use, the reagent, i.e., the substance which reacts with the analyte of 
interest to form hydrogen peroxide, can be incorporated in an assay device 
with the dye-forming substance, or can be in a layer separate from the 
dye-forming substance. The formation of a colored dye with the soluble 
hydrazone compound and the aniline compound indicates the presence and/or 
concentration of a desired analyte, or a reaction or decomposition product 
of the analyte. 
The term "substance having peroxidase activity" is used herein to mean a 
substance which catalyzes oxidation of a hydrogen donor with hydrogen 
peroxide (as a substrate) and is well recognized in the art. Examples of 
substances having peroxidase activity include peroxidases extracted from 
various organisms, synthetic peroxidase and other chemical substances 
extracted from organisms which exhibit an activity similar to that of 
peroxidase. Of these, horseradish peroxidase is preferred. 
In a test device, a color-forming reaction layer can contain an analyte 
component which differs from hydrogen peroxide, which is hereinafter 
referred to as an "analyte precursor component", and a reagent composition 
system capable of forming hydrogen peroxide through chemical reaction. 
Alternatively, a reagent layer containing the reagent system for forming 
hydrogen peroxide can also be provided separately from the reagent layer 
or any other layer in the test device. 
The hydrogen peroxide forming reagent layer can be any reagent composition 
system in which hydrogen peroxide is produced from the analyte precursor 
component through chemical reaction in one step or a reagent composition 
system in which hydrogen peroxide is produced from the analyte precursor 
components through chemical reaction comprising continuous enzyme 
reactions. One example of such a reaction is, for example: 
##STR2## 
Depending upon the hydrogen peroxide-forming reagent system, the reagent 
system for forming hydrogen peroxide can be incorporated into the reagent 
layer, the color-forming reaction layer or the dye-fixing layer, or a 
single layer or a plurality of layers different from the aforesaid layer 
can be provided as the hydrogen-peroxide forming layer. 
The dye-forming formulation of the present invention can be incorporated in 
a dry analytical element such as a multilayer assay device, which 
generally comprises an absorbent carrier material, i.e., a self-supporting 
absorbent sheet or pressed material, such as filter paper or strips, which 
contains the analytical composition and, optionally, any other desired 
reagents such as the peroxidative substances. 
When used in a dry multilayer assay device, the dye-forming composition can 
be incorporated into a suitable carrier material by imbibition, 
impregnation, coating, or by immobilization onto an insoluble matrix. 
Useful carrier materials are those which are insoluble and maintain their 
structural integrity when exposed to water or physiological fluids such as 
urine or serum. Useful elements can be prepared from paper, porous 
particulate structures, cellulose, wood, glass fibers, woven and nonwoven 
fabrics (both synthetic and nonsynthetic) and the like. A useful dry 
analytical device is made by imbibing a solution of the analytical 
composition into the material and drying. 
The components of the analytical composition, as well as the peroxidative 
substance, interactive component, etc., can be incorporated in any of the 
element zones. The location of individual components is well within the 
skill of a worker in the clinical chemistry art. 
The composition and method of the present invention can also be used with a 
dry analytical element which contains an absorbent carrier material, i.e., 
a thin sheet of a self-supporting, absorbent or bibulous material, such as 
filter paper or strips, which contains the composition of the present 
invention. Preferably, these elements also contain a peroxidative 
substance. These elements, which generally provide qualitative assays, are 
known in the art as test strips, dip sticks, and the like. 
When employed in dry chemistry elements, the composition can be 
incorporated into a suitable absorbent carrier material by imbibition, 
impregnation, coating or others suitable technique. Useful absorbent 
materials are insoluble and maintain their structural integrity when 
exposed to water or physiological fluids such as urine or serum. Useful 
elements can be prepared from paper, porous particulate structures, porous 
polymeric films, cellulose, wood, glass fiber, woven and nonwoven fabrics 
(synthetic and natural) and the like. Useful materials and procedures for 
making such elements are well known in the art, and many are described in 
the following U.S. Pat. Nos.: 3,092,465; 3,802,842; 3,915,647; 3,917,453; 
3,936,357; 4,248,829; 4,255,384; 4,270,920; 4,312,834; 5,087,556; and 
5,234,813; the entire contents of which are hereby incorporated by 
reference. 
Preferably, the dry analytical elements of the present invention have at 
least one porous spreading zone as a carrier material. This zone can be 
self-supporting, i.e., composed of a material rigid enough to maintain its 
integrity, but preferably it is carried on a separate supporting 
substrate, commonly called a support. This support can be any suitable 
dimensionally stable, and preferably radiation transmissive, material 
which transmits electromagnetic radiation of a wavelength between about 
200 and about 900 nm. A support of choice for a particular element should 
be compatible with the intended mode of detection (reflection or 
transmission spectroscopy). Useful support materials include paper, metal 
foils, polystyrene, polyesters such as polyethylene terephthalate, 
polycarbonate, cellulose esters, and the like. 
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 
analyzed. 
Useful spreading zones can be prepared using fibrous materials, either 
mixed with a suitable binder material or woven into a fabric, as described 
by Kitajima et al. in U.S. Pat. No. 4,292,272. Alternatively, the 
spreading zone is prepared from polymeric compositions such as blush 
polymers or particulate material, with or without binding adhesives. Other 
useful spreading zone materials are described in German OLS No. 3,150,102 
and Japanese Patent Publication No. 57-101760. It is desirable that the 
spreading zone be isotopically porous, meaning that the porosity is the 
same in each direction in the zone as created by interconnected spaces or 
pores between particles, fibers, polymeric strands, etc. 
The elements can have more than one zone, e.g., reagent zones, spreading 
zones, registration zones, mordant zones, radiation-blocking or filter 
zones, subbing zones, barrier zones, buffer zones, etc. The zones are 
generally in fluid contact with each other, meaning that fluids, 
interactive reagents and reaction products such as colored dyes can pass 
between superposed regions of adjacent zones. Stated in another manner, 
fluid contact refers to the ability to transport components of a fluid 
between the zones in fluid contact. Preferably, the zones are separately 
coated layers, although two or more zones can be part of a single layer, 
or a zone can contain two or more separate layers. The compounds of the 
present invention can be incorporated in any of the zones of the elements 
that would be suitable for the particular analysis. The location of other 
reagents or addenda can be any suitable zone known by a worker skilled in 
the art of clinical chemistry. 
In the elements of the present invention, the amounts of the soluble 
hydrazone compound and other reagents can be varied widely depending upon 
the analyte to be determined. Preferably, the soluble hydrazone compound 
is present to provide a coverage of from about 0.2 to about 3 
gram/m.sup.2. The peroxidative substance can be present in a coverage 
within the skill of a worker in the art. For peroxidase, for example, the 
coverage is preferably up to about 150,000 and more preferably from about 
40,000 to about 60,000 IU/m.sup.2. A variety of other desirable, but 
optional, reagents and addenda can be present in the element in amounts 
known to one skilled in the art. These materials include surfactants, 
buffers, binders, pigments, activators, interactive reagents, etc. In the 
context of this application, IU represents the International Unit for 
enzyme activity required to catalyze the conversion of 1 .mu.mole of 
substrate per minute under standard pH and temperature conditions for the 
given enzyme. 
In one embodiment of an apparatus for use with the soluble carboxy MBTH 
compounds of the present invention, a self-contained chromatic 
quantitative analyzer is used for quantitatively detecting an analyte in a 
biological fluid. The device includes a base having a first open reservoir 
for receiving the bio-logical fluid. A means for separating solids from 
the bio-logical fluid is provided in the first open reservoir. The second 
open reservoir draws the biological fluid from the channel by capillary 
and/or wicking action and, when the second open reservoir is filled with 
the biological fluid, the capillary and/or wicking action terminates. A 
membrane is provided in the channel which is permeable to the biological 
fluid. There is at least one chromatic chemical indicator immobilized in 
the membrane in a predetermined concentration. The membrane enables 
biological fluid to interact with the chromatic chemical indicator. Such a 
device is described in detail in U.S. Pat. No. 5,087,556, the entire 
contents of which are hereby incorporated by reference. 
In a preferred apparatus for use in the present invention, the analytical 
device used includes a fluid sample well means connected to a sample 
initiation area in such a fashion that the assay will not commence unless 
sufficient sample is introduced into the sample well means to conduct the 
assay. Once sufficient sample has been deposited into the sample well 
means, the sample flows into an initiation area and the assay commences. 
Such a device is described in U.S. Pat. No. 5,234,813, the entire contents 
of which are hereby incorporated by reference. 
In one embodiment of a device which can be used with the solubly hydrazone 
compounds of the present invention, a fluid sample measuring device 
comprises three distinct parts: 
(a) a sample well into which sample is introduced so as to meter the sample 
to ensure that sufficient sample is present to conduct an assay; 
(2) an assay initiation area located at a level above the sample well; and 
(3) siphon means for connecting the sample well and the assay initiation 
area by which liquid can readily flow, resulting in a siphoning action 
from the sample well to the assay initiation area. 
The assay initiation area is connected to a detection zone where the actual 
assay occurs. This detection zone includes an indicator means, including 
at least one of the soluble hydrazone compounds of the present invention, 
which develops a detectable signal such as a color. The detectable portion 
of the detection zone caused by reaction of the indicator means with the 
analyte, or a derivative thereof, as observed after the capillary action 
is terminated, corresponds to the concentration of the analyte in the 
fluid sample. A scale is provided along the length of the detection zone 
channel to readily equate the detectable portion of the channel to the 
concentration of analyte. 
The amount of the dye-forming components can be varied widely. Preferably, 
the soluble hydrazone is present in an amount of coverage of at least 
about 100, and more preferably from about 300 to about 5000 mg/m.sup.2. 
The aniline compound is preferably present in an amount of coverage of at 
least about 100, and more preferably from about 1000 to about 5000 
mg/m.sup.2. The peroxidative substance can be present in a coverage 
preferably of at least about 25,000, and more preferably from about 50,000 
to about 100,000 I.U./m2 for peroxidase. A variety of other desirable but 
optional reagents and additives can be present in the elements in amounts 
known to one skilled in the art. Such materials include surfactants, 
buffers, binder, pigments, activators, reagents of interactive 
compositions, etc. 
To quantify an analyte using such a device, a fluid sample is deposited 
into the sample well means. If there is sufficient volume of sample to 
conduct an assay, the sample is drawn up into the assay initiation area 
through the siphon means. There may be a separation zone below the assay 
initiation area to remove any solids suspended in the fluid sample. The 
fluid sample is then drawn through the detection zone by capillary and/or 
wicking action, preferably to a reservoir means, which contains an 
absorbent. The reservoir means draws the fluid sample through the 
detection zone and, when the reservoir is filled with the fluid sample, 
the capillary and/or wicking action is terminated. While the fluid sample 
is being drawn through the detection zone, the indictor means is permeated 
with the fluid sample. The detection zone includes a suitable indicator 
immobilized therein in a predetermined concentration to react with the 
analyte. Thus, the analyte in the fluid sample is completely reacted in a 
single step or a series of chemical reactions with the indicator means. 
Determination of hydrogen peroxide or an analyte is achieved when the 
soluble hydrazone compound and the aniline compound react to form a dye. 
This dye can be detected with the unaided eye or with suitable 
spectrophotometric means and procedures, preferably at a wavelength 
greater than or equal to 600 nm. The substituents on the soluble hydrazone 
as were as on the aniline compound can be chosen to provide a dye with an 
absorption maximum at any desired wavelength. One skilled in the art of 
dye chemistry can readily determine which substituents should be present 
on the soluble hydrazone compound and/or aniline compound to achieve a dye 
with the desired maximum absorbance. 
The composition and method of the present invention are adaptable to 
solution as well as to dry element assays. In a solution assay, generally 
the chromogenic composition (optionally containing interactive reagents) 
is physically contacted and mixed with a liquid test sample in a suitable 
container, such as a test tube, Petri dish, beaker, cuvette, etc. The 
resulting solution is incubated for a relatively short time, generally 
less than about five minutes, at a suitable temperature, such as 
37.degree. C. The sample is then evaluated by measuring the amount of dye 
provided upon reaction of the color-forming coupler with color developing 
compound in the presence of hydrogen peroxide. 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 analyte. Such 
an evaluation can be effected visually or with suitable colorimetric 
detection equipment and procedures. 
The color-forming coupler and color developing compound can be provided as 
part of a diagnostic test kit for either dry or solution assays. For 
solution assays, the kit components can be supplied as lyophilized 
reagents in individual packets having predetermined amounts. 
Alternatively, they can be provided in bottled or otherwise packaged 
solutions sufficient in size for one or more assays. Other reagents or 
non-reactive addenda can also be supplied in the kit along with suitable 
assay utensils or containers for performing the assay, if desired. A dry 
analytical element, such as one of those described below, containing one 
or more reagents necessary for an assay can also be included as part of a 
diagnostic test kit. 
When the kit is to be employed in an analysis, the components are mixed 
together, dissolved in or diluted with water as necessary, and employed to 
effect the intended analysis by generally known techniques. That is, the 
various ingredients are mixed with the sample to be analyzed, and the 
resulting mixture is held at a predetermined temperature to permit the dye 
of the present invention to form. The concentration of the dye is then 
determined, as by conventional photometric analysis. These analyses may be 
performed manually or automatically using equipment and techniques already 
well known to the art. 
Assays for analytes using the dye composition of the present invention can 
be manual or automated. In general, using dry elements containing reagents 
including the soluble hydrazone compounds of the present invention, 
hydrogen peroxide or analyte 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. This contact can be 
accomplished in any suitable manner, including dipping or immersing the 
element into the sample or, preferably, spotting the element by hand or 
machine With a drop of the sample using a suitable dispensing means. After 
sample application, the element is left for a period of generally less 
than five minutes while any hydrogen peroxide formed from the analyte in 
the sample causes the soluble hydrazone to couple with the aniline 
compound to form a dye. This dye can be detected with the unaided eye or 
with suitable spectrophotometric means and procedures. Alternatively, for 
a quantitative assay, the element can be designed so that a color bar is 
formed which is proportional to the amount of hydrogen peroxide in the 
sample or formed by the analyte in the sample with the peroxidative agent. 
The following examples are given for purposes of illustration only, and are 
not meant to be limiting of the scope of the invention. Although the 
examples given here are for the preparation of 
3-methylbenzothiazolone-2-hydrazone-6-carboxylic acid, sodium salt, other 
soluble salts according to the present invention can be prepared using 
processes known to a synthetic organic chemist. 
Example 1 
Preparation of 2-aminobenzothiazole-6-carboxylic acid 
As shown in FIG. 2, 2-aminobenzo-thiazole-6-carboxylic acid, 2, is prepared 
from 4-aminobenzoic acid and ammonium thiocyanate in the presence of 
bromine and glacial acetic acid. One hundred thirty seven grams of 
4-aminobenzoic acid was slurried with 70 mL of acetic acid in a 500 mL RB 
flask which was cooled in a water bath to 5.degree.-10.degree. C. Then, 
30.4 grams of ammonium thiocyanate was added to the slurry, followed by an 
additional 70 mL of glacial acetic acid. Next, 11.2 mL of bromine diluted 
in 30 mL of glacial acetic acid was added dropwise over a 30-minute 
period. After the bromine was completely added, the slurry was allowed to 
warm to room temperature and was stirred at room temperature for 30 
minutes. After stirring at room temperature, the reaction mixture was 
transferred to a 1-L Erlenmeyer flask, and 400 mL of water was added. 
The aqueous mixture was heated to boiling (95.degree.-100.degree. C.) and 
allowed to stir at boiling for 15 minutes. The reaction mixture was 
filtered while hot, and the residue discarded. The clear-orange-colored 
filtrate was stored at 4.degree. C. in a refrigerator until cool and a 
precipitate had formed. The precipitate was slurried in 800 mL water and 
neutralized with 190 mL ammonium hydroxide to pH 6 as measured by litmus. 
The precipitate formed was collected in a Buchner funnel, and when sucked 
dry gave a wet weight of about 290-300 grams of crude product. The crude 
product was slurried in 400 mL water and the pH of the slurry was adjusted 
to 10 by the addition of 30 mL of 30% sodium hydroxide. The resulting 
turbid solution was filtered and the residue discarded. 
The filtrate was cooled to room temperature and neutralized while cooling 
in a ice/water bath to pH 6 by the addition of 17-20mL acetic acid. The 
resulting aqueous suspension of precipitate was cooled at 4.degree. C. 
overnight. After fully cooling and settling, the precipitate produced was 
collected in a Buchner funnel and sucked dry. The purified product was 
dried in air for 24 hours. The yield was 34.3 grams (88%) of a yellow, 
grainy solid. 
Elemental analysis: 
Calcd: C, 49.48%; H, 3.09%; N, 14.43%; S, 16.49% 
Found: C, 49.26%; H, 3.09%; N, 14.01%; S, 16.22% 
HPLC C.sub.18 column, 250 mm.times.5 .mu.m; phosphate buffer/acetonitrile 
mobile phase. RT=9.75 min. 
Preparation of 2-amino-3-methyl-6-carboxybenzothiazolium iodide 
As shown in FIG. 3, 2-amino-3-methyl-6-carboxybenzothiazolium iodide, 3, is 
produced from compound 2 above. Thirty four grams of 
2-amino-6-carboxybenzothiazole was placed in a 2-L RB flask and slurried 
with 400 mL of methoxyethanol. Methyl iodide was added all at once, and 
425 mL more methoxyethanol was added. The flask was fitted with a 
condenser and the reaction mixture was brought to reflux. The mixture was 
refluxed for fourteen hours. The resulting dark amber solution was cooled 
to room temperature and poured into one liter of diethyl ether, out of 
which a precipitate formed. The precipitate was collected in a Buchner 
funnel and washed with 800mL ether. It was dried in the air with gentle 
warming. The yield was 36.5 grams (62%) of a pale yellow solid with purple 
tinges. 
Elemental analysis: 
Calcd.: C, 32.14; H, 2.68; N, 8.33; S, 9.52; I, 37.80 
Found: C, 32.17; H, 2.64; N, 8.17; S, 9.43; I, 37.68 
HPLC C.sub.18 column, 250 mm.times.5 .mu.m; phosphate buffer/acetonitrile 
mobile phase. RT=2.24 min. 
Preparation of 3-methylbenzothiazolone-2-hydrazone-6-carboxylic 
As shown in FIG. 4, CMBTH, 4 is prepared from compound 3. 
2-Amino-3-methyl-6-carboxybenzothiazolium iodide (36.5 g) was placed into a 
500-mL RB flask and slurried in 170 mL water. The slurry was dark purple. 
Hydrazine hydrate (26.5 mL) was added all at once, and the slurry 
decolorized immediately. One hundred mL additional water was added and the 
mixture was brought to reflux. The reaction mixture was refluxed with 
stirring for one hour and filtered while hot. The residue was discarded 
and the filtrate neutralized to pH 6 with 27 mL glacial acetic acid. The 
aqueous suspension was refrigerated overnight. The product was collected 
in a Buchner funnel and sucked dry. The crude wet weight was about 135-140 
grams. 
Elemental analysis 
Calcd.: C, 45.00; H, 4.17; N, 17.50; S, 13.33 
Found: C, 44.81; H, 4.11; N, 18.80; S, 13.79 
HPLC C.sub.18 column, 250 mm.times.5 .mu.m; phosphate buffer/acetonitrile 
mobile phase. RT=2.24 min. 
NMR 
(D.sub.2 O) 8.10, s, 1H, aryl H; 7.97, d, 1H, aryl H; 3.6, s, 3H, N-Me 
Preparation of 3-methylbenzothiazolone-2-hydrazone-6-carboxylic acid, 
sodium salt (NaCMBTH) 
As shown in FIG. 5, NaCMBTH, 5, is produced from CMBTH, 4. 
One hundred eighteen grams of wet crude 3- 
methylbenzothiazolone-2-hydrazone-6 carboxylic acid was slurried in 
sufficient water to allow stirring in a 600-mL beaker. The mixture was 
heated to 70.degree. C. Then, 15.5 mL sodium hydroxide solution was added 
by pipetful until the pH was 10. Insoluble matter was filtered out and the 
clear filtrate was cooled to room temperature and transferred to a 500-mL 
Erlenmeyer flask. Isopropanol was carefully pipetted onto the caustic 
solution so that there was a two phase system, after which the flask was 
cooled quickly in an ice-salt bath. The contents of the flask were swirled 
to mix the two phases and to allow crystallization to begin. Cooling 
continued as more isopropanol was pipetted into the flask to complete 
precipitation of the product. The resulting precipitate was collected in a 
Buchner funnel. The yield of the sodium salt was 75.3 grams wet. The crude 
product was slurried in 250 mL isopropanol, collected on a Buchner funnel, 
then resuspended in 250 mL ether and recollected on a Buchner funnel. The 
clean product was dried in air and stored in the dark in an airtight 
container. 
Elemental analysis: 
Calcd.: C, 35.06; H, 4.87; N, 13.64; S, 10.39 
Found: C, 35.12; H, 4.77; N, 13.80; S, 10.06 
HPLC C.sub.18 column, 250 mm.times.5 .mu.m; phosphate buffer/acetonitrile 
mobile phase. RT=2.25 min. 
TGA 
% volatiles ranges from around 5% to around 19%, depending upon degree of 
hydration. 
Example 2 
Relative Molar Extinction Coefficients 
The relative absorbance of MBTH and sodium carboxy MBTH, an illustrative 
soluble hydrazone compound, was measured and compared. Since each compound 
was measured at the same molar concentration, the molar extinction was 
proportional to the concentration. 
Each sample contained the following: 
1 mL phosphate buffer, 0.035M, pH 6.5 
10 .mu.L R-MBTH solution, 10 mM, where R=H or COONa 
100 .mu.L N-ethylanilinopropanamine hydrochloride, 10 mM 
10 .mu.L POD solution, .about.120 IU/mL 
50 .mu.L H.sub.2 O.sub.2, 20 mM 
A 1-mL aliquot of each solution was transferred to a cuvette and diluted 
with an addition 2 mL of phosphate buffer solution. Absorbance at 600 nm 
was read: 
______________________________________ 
MBTH.HCl 0.1513 absorbance units 
CMBTH 0.2115 absorbance units 
______________________________________ 
Example 3 
Heat Stability of CMBTH 
In order to evaluate the heat stability of sodium carboxy MBTH, 38 mg. of 
either MBTH.HCl or NaCMBTH was added to 1.2 mL water to dissolve. Then, 
0.75 mL of this solution was added to 21 mg Hydrisel SX 660, a commercial 
surfactant and 1015 mL 0.1% ACP polymer (ISP Technologies), prepared in 
70% isopropanol solution. Each solution was coated onto a 4-inch wide 
polyester fabric in a thin layer and dried at 40.degree. C. for fifteen 
minutes. The coated fabric was sealed in an airtight foil bag and stored 
at 4.degree. C. 
Small samples (2 cm.times.5 cm) of each coated fabric were placed into a 
120.degree. C. oven and removed at appropriate intervals. The 
heat-stressed fabric samples were assayed spectrophotometrically. 
The fabric samples were placed into a tared test tube and weighed. Ten mL 
of 20 mM phosphate buffer, pH 7.0, was added. The tubes were capped and 
agitated for ten minutes at room temperature. Absorbance at 310 nm was 
read for each sample and the values were compared against a standard 
curve. Table 1 shows the comparative densities for the two compounds after 
being held at 120.degree. C. for periods. 
TABLE 1 
______________________________________ 
Time Density of NaCMBTH 
@120.degree. C. 
(mg/m.sup.2) Density of MBTH.HCl (mg/m.sup.2) 
______________________________________ 
0 75.4 114.7 
4 50.4 10.2 
6 51.2 
23 50.3 
______________________________________ 
MBTH.HCl is effectively decomposed after only two hours at 120.degree. C., 
while NaCMBTH is still viable after six hours at the same temperature, and 
even retains most of its activity after 23 hours. 
Example 4 
Solubility Comparison 
The solubility of MBTH.HCl was compared with that of NaCMBTH. One hundred 
mg samples of each of MBTH.HCl (Aldrich) and NaCMBTH were placed into test 
tubes. Water was added in 50 .mu.-L aliquots until the sample completely 
dissolved. The solubility of the compounds is shown in Table 2. 
TABLE 2 
______________________________________ 
Compound Water added 
Solubility (mg/mL) 
______________________________________ 
MBTH.HCl 2900 .mu.L 
34 
NaCMBTH 1150 .mu.L 
87 
______________________________________ 
An identical experiment was performed using pH 7 buffer (phosphate, 0.1M) 
instead of water. The results are shown in Table 3. 
TABLE 3 
______________________________________ 
Compound Water added 
Solubility (mg/mL) 
______________________________________ 
MBTH.HCl &gt;9600 .mu.L 
&lt;10 
NaCMBTH 575 .mu.L 
174 
______________________________________ 
Example 5 
Device for Quantitatively Determining Hydrogen Peroxide 
An oven dried, three neck, 250-mL, round bottom flask was fitted with a 
stoppered addition funnel, condenser with tubing adapter to a nitrogen 
bubbler, and a stopper. A stirbar was placed into the flask. Under 
constant nitrogen purge, the cool, dry flask was placed into an ice/water 
bath. Twenty five grams of isocyanatopropyltriethoxysilane were introduced 
into the flask while the flask was flushed with nitrogen. The addition 
funnel was filled with twenty grams of an amine-functional aniline-type 
dye as the purge continued. The setup was then switched from nitrogen 
purge to nitrogen bubbler. The amine was added dropwise, one drop per 
second, via the addition funnel, while the contents of the flask were 
stirred. When addition was complete, the mixture was allowed to stir for 
ten minutes. The ice/water bath was removed and the contents were warmed 
to room temperature with stirring. The product should be used immediately, 
and must remain under nitrogen until used in the next step of the process. 
The product is a viscous, clear liquid. 
Five hundred grams of silica gel and six liters of toluene were slurried in 
a ten liter, three neck, round bottom flask equipped with a reflux 
condenser and a stirring shaft. The amine-silane mixture just prepared was 
transferred all at once to the large flask containing the slurry. The 
small flask was rinsed with 500 mL of toluene and the rinse was added to 
the silica/silane slurry. The mixture was brought to reflux and refluxed 
with stirring for two hours. The slurry was cooled to room temperature. 
The product was collected in a Buchner funnel. The product was placed into 
a large vessel and covered with four liters of toluene and stirred for 30 
minutes. The finished product was air-dried. 
Preparation of Analytical Film 
A 12" by 5" swatch of polyester fabric (PeCap.RTM. from Tetko, Briarcliffe 
Manor, N.Y.) is attached to a glass plate using double-stick tape. A paste 
containing 0.25 gram of the dyed matrix as prepared above, 125 .mu.L of 25 
mg/mL NaCMBTH in methanol, 100 .mu.L of 18 mg/1.5 mL water, 200 .mu.L of 
anhydrous methanol and 400 .mu.L of polyvinyl acetate, medium MW, 12% 
methanolic solution, a film-forming polymer, was placed at one end of the 
swatch and a film is drawn down onto the fabric using a film-casting knife 
(Paul N. Gardner Company, Pompano Beach, Fla.) set at 1 mil (0.001 inch). 
This film is dried at 40.degree. C. for ten minutes, and then 5 mm wide 
strips are cut and heat sealed with polyester top and bottom films to form 
very precise flow channels. 
Determination of Hydrogen Peroxide 
Plasma with varying concentrations of hydrogen peroxide is introduced into 
the channels prepared as above. As the sample flows through the channels, 
dark blue color bars form with sharp color fronts. The length of the color 
bars is proportional to the concentration of hydrogen peroxide in each 
sample. 
Any recitation of a preferred range is to be deemed to include a 
description of the included subranges. Any recitation of a multimember 
class of elements is to be deemed to include a description of the possible 
subclasses. Any recitation of individual embodiments is to be deemed to 
also include a description of all possible combinations of said 
embodiments. All references, including prior patent applications cited (if 
any), are to be deemed incorporated in their entirety by reference. 
The foregoing description of the specific embodiments will so fully reveal 
the general nature of the invention that others can, by applying current 
knowledge, readily modify and/or adapt for various applications such 
specific embodiments without departing from the generic concept, and, 
therefore, such adaptations and modifications should and are intended to 
be comprehended within the meaning and range of equivalents of the 
disclosed embodiments. It is to be understood that the phraseology or 
terminology employed herein is for the purpose of description and not of 
limitation.