CA1098808A

CA1098808A - Test composition and device for determining peroxidatively active substances

Info

Publication number: CA1098808A
Application number: CA295,641A
Authority: CA
Inventors: Charles T. W. Lam
Original assignee: Miles Laboratories Inc
Current assignee: Bayer Corp
Priority date: 1977-03-14
Filing date: 1978-01-25
Publication date: 1981-04-07
Also published as: DE2857298C2; IT7847859A0; AR220329A1; SE7801164L; AU3279478A; JPS53115288A; JPS6122780B2; IN148528B; DE2803955C3; FR2384261B1; HU177004B; DE2803955B2; IT1104148B; DE2803955A1; BR7800603A; GB1552747A; SE430104B; FR2384261A1

Abstract

Docket No. 11709 TEST COMPOSITION, DEVICE AN METHOD FOR DETERMINING PEROXIDATIVELY ACTIVE SUBSTANCES ABSTRACT OF THE DISCLOSURE A test composition, device, and method for determining the presence of a peroxidatively active substance in a sample are disclosed. The composition comprises an organic hydroperoxide, an indicator capable of producing a detect-able response in the presence of a hydroperoxide and a peroxidatively active substance, and one or both of (a) a diluent compound having the structure , , , or mixtures thereof, in which R1 and R2, same or different, are hydrogen, alkyl or alkoxy having 1 to about 6 carbon atoms, or aryl, and (b) a borate ester having the structure in which m, n and p, same or different, are integers of 1 to about 4.

Description

8 ~ ~

The device comprises a carrier matrix incorporated with the composition, and the m~thod comprises contacting a sample9 suspected of containing a peroxidatively active substance, wi~h said composition.

BACKG~OUND OF TH~ I~VE~TIO~

FieZd of the Inuention The present invention relates to the detection of con-stituents in a test sample. More particularly, the invention relates to the quali~ative and semi-quantitative analysis of a sample for constituents which possess peroxidative activity.

- Description af the Prior Art ''', ~:
Many analytical methods are presently available for detecting the presence of peroxidatively active substances in samples such as urine, fecal suspensions, and gastro-intestinal cQntonts. Hemoglobin and its derivati~es are typical o such "peroxidatively active" substances because they behave in a manner similar to the behavior of the enzyme peroxidase. Such substances have also been referred to as pseudope~oxidases. Peroxidatively active substances are enzyme-like in that they catalyze the redox reaction between peroxides and benzidine, o-tolidine, 3,3',5,5'-tetramethyl-benzidine, 2,7-diaminofluorene or similar indicator sub-stances, thereby producing a detectable response such as a - cDlor change. Hence, most methods for determining the presence of occult blood in test samples rely on this pseudoperoxidase activity.

8 ~ 8 Several methods have evolved over the years which rely on enzyme-like catalysis of the peroxidic oxidation of color-orming indica~ors. Primarily these include wet chemical procedures and "dip-and-read" ~ype reagent-bearlng strips. Of the former, a typical example is set forth in Richard M, Henry) et aZ., CZ~n~caZ Chsmistry Pr~noipZes and Techniques (Hagerstown, Maryland: Harper and Row, 1974), pp. 1124-1125. This procedure in~olves the use of glacial acetic acid tbufer)~ diphenylamine (indicator), and hydro- ~ -gen peroxide. While such wet methods have proven analytical ability, t~ey are nevertheless fraught with ob~ious short-comin~s, not the least of which are poor reagent stability and inadequate sensltivity. Inherent to such reagent solu-tions is a decline in stability ~ergo sensiti~ity) so rapid that fresh rea~ent solutions must be prepared after several ;~: days of storage, a necessity resulting in both excessi~e ti~e ~equired of analytical personnel, and poor economy , because of having to waste costly reagents ~:~ A second m~thod for the determination of peroxidatively active subs~ances, and the one presently Rreferred by clinical assayists and analysts, is the use of so-called "dip-and-read" reagent strips. Typical of such devices is a reagent strip manufac~ured by the Ames Company Division of rqiles oratorles 9 Inc. and sold under the name HEMASTIX~. This

2$ reagent strip comprises, in essenc~, a porous paper matrix afixed to a plastic strip or handle. The paper is impreg-nated with a buffered mix~ure of an organic hydroperoxide and o-tolidine. Upon immersion in a liquid containing ,

-3-hemoglobin, myoglobin, erythrocytes or other pseudoperoxidases, a blue color de~elops in the paper, the intensity of which is proportional to the concentration of the peroxidatively active substance in the sample. Thus, by comparing the color developed in the reagent strip to a standard color chart, the assayist can determine, on a semi-quantitative basis, the amount o~ unknown prese~t in the sample.
.
~ Henceg the advanta~es o reagen~ strips over wet ~
. .
chemistry methods are predominantly two~old: strips are i 10 easier to use because neither the preparation of reagents -` nor the attendant apparatus are required; and grea~er stability of reagents is afforded~ resulting in greater : . , accuracy, sensitivity and economy.
; .
But the inherent advantages of strips o~er wet chemistry notwithstanding, ~he characteristics of stability and sensi-tivity are in need of still further improvement. Whereas these properties in cuTrent state-of-the-art strips or ;~
determining pseudoperoxidases are greatly enhanced over ` those of wet chemical methods; th~re would nevertheless accrue a great advanc~ in the art i~ such strips ~ould be made even more stable during storage and even more sensitive to peroxidatively active substances. It was towards achieving these improvements that the research acti~ities resulting in the present invention were directed.
At least three attempts at achieving the above-mentioned ; goals are recorded in the prior art. A r~cltation in ChemiceI ABstracts Volume 85, ,age 186 (1976~ describes a two-dip method for preparing reagent strips containing ~ -;
o-tolidine and phenylisopropyl hydroperoxide. In this method a solution is made o-f the indicator ~o-tolidine ~ 2HCl) and polyvinylpyrrolidone in ethanol. To this solution were added a small amount of sur~actant and enough citrate bu~fer to provide a pH of 3.7. Filter paper strips impreg-nated with ethyl cellulose were dipped in this solution and dri~d. The thus-impregnated filter paper was subsequently -~ 10 dipped into a second solution containing 1,4-diazabicyclo [2,2,2]oc~aney phenylisopropyl hydroperoxide and polyvinyl-pyrrolldone dissolved in an ethanol-toluene mixture. The thrust of this experiment was to s~abilize the peroxide and indicator combination through the use of the bicyclooctane derivati~e and the polyvinylpyrrolidone.
A second such met~od is disclosed in U.S. Patent 3,853,471. This paten~ ~eaches the use of phosphoric or phosphonic acid amides where the substituent amido groups are primarily N morpholine radicals.
Besides these attempts, there also exists the dis-closure of U.S. Patent 3,252,762 wherein the organic hydro-peroxide is physically encapsulated within a colloidal material such as gelatin. Thus, when such a test strip is utilized, ~he aqueous ~es~ sample dissolves ~he gelatin capsules, thereby freeing the hydroperoxide ~or further reaction with the indicator in ~he presence of a per-oxidatively active substance.

8 ~

Each of these prior attem~ts was aimed at 3tabiZiz~ng the reagents so that the potentially incompatible reactive i~gredients (hydroperoxide and indicator) would not pre-maturely combine and thereby render the test strips less ~ 5 sensitive. Hence, it can be said that the prior art methods .~ were not directed towards the combined objectives of simul-taneously enhancing stability and sensitivity, but rather they attemp~ed to preserve existing sensiti~ity by preventing ~ reagent decomposition during storage.
`~ 10 Another prior art reference which is of interest is U.S.
Patent 3,236,850. This patent is directed towards stabilizing ~ organic hydroperoxides used as catalysts and oxidizing ;~ agents, The patentees in this reference disclose the use of primary) secondary, or tertiary amine salts with organic ;~ 15 peroxides. This reference is in no way directed toward reagent test strips.
Upon realizing that none of the above-described methods would achiçve the kind of stability and sensitivity desired in a test strip for detecting peroxidatively active substances, the present inventor decided to take a completely dif-ferent tack. This different approach was discovered during the re-search which led to the present invention, and resulted in a composition and device which completely fulfilled the desired objectives of increased stability and sensitivity.
But, even more surprisingly, yet another advantage re-sulted from this work - an improved method for preparing the device presently disclosed whereby the manufacture is dramatically simpler than processes enumerated in the fore-going prior art references - a one-dip method.

-G-;
SUMMARY OF THE INVENTIOlll :

Briefly stated, the present invention relates to a test composition and devîce for determining the presence of a psuedoperoxidase in a test sample. The test composition :
S comprises an organic hydroperoxide; an indicator capable of producin~ a detectable response in the presence of a hydro-peroxide and a peroxidatively active substance; and one or both of ; (i) a diluent having the structure O
:

R ~ ' Rl ~ ~ ~ R2 o ~0 Rl--C~
NRlR2 , or mixtures thereof, in which Rl and R2, same or dif~erent, are alkyl or alkoxy having 1 to abou~ 6 carbon atoms~ or aryl~ and : tli) a borate ester having the structure /(CH2)m--\
N -(CH~)n - O - B

in which m~ n and p~ same or different, are integers of 1 to about 4, The device of the present invention comprises the above composition incorporated with a carrier matrix. The process comprises ~he steps of preparing the test composition and incorporating it with the matrix.

DETAILED DESCRIPTION OF THE INVENTION

The organic hydroperoxide cont~mplated for use in the ;~
test composition can be selected from many well-known organic hydroperoxides. It must, however, be capable o reacting with a peroxidatively active substance in the presence of a peroxide-sensitive indicator to produce a detectable response such as a color change o~ change in the ~ amount of light absorbed or reflected by the test compo- ~;~
`~ sition Among hydroperoxides which have been found suitable are t-butyl hydroperoxide~ cumene hydroperoxide, diisopropyl-~ benzene hydroperoxide, 2,5-dimethylhexane-2~5-dihydroperoxide, paramenthane hydroperoxide or mixtures thereo. 0 these, cumene hydroperoxide has been ~ound to be most preerable.
There exist many indicators which are capable of pro-ducing a detectable response in the presence of a hydro-peroxide and a pseudoperoxidase, and which are, therefore, ~-suitable ~or use~ in the present invention. These include the~so-called '1benzidine-type" compounds, typical o which are ben7idine, o-tolidine, 3,3',595'-tetramethylbenzidine, 2,7-diaminofluorene OI' mixtures of these in varying pro-portions.
The borate esters which are presently believed to contribute to increased stability and sensitivity have the structure depicted above. Of the compounds included in this generic structure, it has been found that trimethanolamine borate, triethanolamine borate and tri(n-propanol)amine borate are particularly suitable. These compounds corre-spond to the above generic formula where m, n and p are the same interger and are 1, 2, and 3, respectively.

9~ ~ ~ 8 The amount o-f the bora~e ester useful in the present composition and device can vary broadly. This is reflected in the Examples, i~fra. Thus, in Example I the molar ratio of triethanolamine borate equivalents to cumene hydro-peroxide equivalents is 4.71. Conversely 9 Example III
evidences a 2.83 molar ratio of horate to hydroperoxide ~1,42 normality ratio if the difunctionality of the peroxide is considered).
But the equivalents ratio range of about 1.4 to about 5, as shown in the Examples, is by no means limiting with respect to the amount of borate useful in the present in-vention. Any amount sufficient to achieve the desired degree of test composition stability and sensitivity can be employed, and this amount is easily determinable at the laboratory bench, given the present invention disclosure, In a preferred embodiment of the present invention, the composition comprises cumene hydroperoxide, o-tolidine, a mixture of dimethyl sulfone and dimethyl sulfoxide 9 and triethanolamine borate.
The test composition is typically prepared by dissolv-ing or suspending portions of each ingredient to be used in water or other sui~able suspending medium or solvent. Such solvent or medium includes chloroform, methanol, ethanol, methylene chloride~ cyclohexane, etc.
The test device can be prepared from a one-dip process.
Accordingly, a portion of carrier matrix material is im-mersed in the solution or suspension and subsequently dried.
Test devices thus prepared exhibit little loss in reactivity even after storage under stress conditions such as about 60 .

to ab~ut 70C for 1 to 3 days and longer. By way of com-parison, test devices were simil.~ily prepared, but without the presence of the borate ester and/or tha diluent. When these strips were stored under substantially identical S stress conditions, a dramatic loss in reactivity and sensi-tivity was observed The carrier matrix utilized in forming the test device can take on a multitude of forms. Thus, U.S. Pa~ent 3,846,247 teaches the use of felt, porous ceramic strips, and woven or matted glass fibers. Additionally, U S. Patent 3,552,938 teaches the use of wood sticks~ cloth, sponge material~ and argillaceous substances. The use of synthetic resin fleeces and glass fiber felts as a carrier matrix is suggested in British Patent 1,369,139. Another British Patent, 1,349,623, proposes the use of light-permeable mesh^
work o thin filaments as a cover for an underlying paper matrix. Polyamide fibers are taught in French Patent 2~170,397. These suggestions notwithstanding, howe~er, the material predominantly used in the art as a carrier matrix, and that which is especially suitable for the present invention, is a bibulous paper such as filter paper. It can thus be seen that there is a great deal of leeway in se-lecting an appropriate material for use as a carrier matrix, and the matrix can take on various physical forms. All of these ~ypes are intended as being within the scope of the present invention The mechanism whereby the present approach to enhanced stability and sensitivity is realized is not known with cer-tain~y. Ho~ever the unique chemical characteristics of the ' .. .... .. .. . ..

8~i~

bora~e e~ter described and claim~d in ~he instant inventio~
give rise to reasonable grounds for spe~ula~ion. It is known that peroxides ~re generally unstable co~pounds, or at least less stable than most compounds occu~ring in ~a~ure~
s Some are explos~ve, Oth~rs, such as organie hydropero~ide~
(vi~. cumene hydr~p~roxide) are relatively stable~ but are believed to ~asily dis~ociate in the presence of aclds such as thos~ commonly employed in occult ~l~od-sensitiYe tes~
de~ices, Whon this d~composition Qccurs in the pres~nce o~
an oxidiæ~ble indi~ator ~such as ~hose described herein~ a r~dox re~ction takes place, It is belle~ed that ~hls pre~ ::
m~ture $n~e~a~ion i9 th~ ~ause of decreased sensi~ivity in peroxidati~oly s~sitive reag~en~ strips.
On th~ o~her hand, bo~ate esters such as those describcd lS ~qrein ~re unique in the geom~ry o~ ths nitrog~n and boro~
a~om~ în ~he bicyclia ~tructure, each comprlsing a separ~e bridgeh~ad atomO The nitrogen atom a~ one b~idgeh~ad is e~ectron-rich, containing an unsh~r~d pair of el~ctrons pr~cting Qut~ards rom th~ molecula~ axisO The o~her bridg~h~ad a~om, the boron atom~ situated at the other axial ~nd o~ th~ m~lecule, is electron de~icient and tends to coordlna~ly ~ond with elec~ron-rich anions.
Hen~e, becau$~ o its electron richness, the nitrogen end of ~h~ mol~cul~ could quite cono~irably tie up a pro-ton, whe~eas the ele~tron-d~icient boron brid~ehead atom could coordinately couple with an aniQni~ pero~ide residu~.
: Thus, it is b~lie~ed ~hat the uni~u~ electran di~ributi~

in the presently discussed bicyclic molecules stabillzes the organic peroxide in the present ~est composition by chemi-cally inserting itself between the peroxidic proton and oxygen atom, forming a coordinately coupled ion pair.
Thus, in the present test composition, the surprisingly increased stability is believed to arise from the organic hydroperoxide being precluded rom ionically interacting with the indicator until the solvating power of the test sample destroys the peroxide-borate complex, and frees the peroxide ~o oxidize the indicator in the presence of a peroxidatively active substance.
But even though marked increases in s~ability and sensitivity result from the use of borate esters, it has surprisingly been found that even greater stability and sensitivity can ensue when the borate ester and the above-described diluent are both included in the test composition.
The diluen~ compounds which are presently believed to contribute to the increased stability and sensitivity of the present invention have the structures depicted above. Of the compounds included in these generic structures, it has been found that N,N-dimsthyl formamide, dimethyl sulfoxide, dimethyl sulfone, or their mixtures are especially suitable.
Other diluents found to b0 operable are benzyl sulfoxide, 4-chlorophenyl sulfone 9 4-fluoro-3-nitrophenyl sulfone and others. These compounds correspond to the above structural formulas where Rl and R2 are each msthyl.

, ~.

The scope of Rl and R2 encompassed by the presently disclosed inventive concep~s is ~road. Hence ! by Rl and R2 are meant s~bstltuted or unsubstitu~ed alkyl of 1 to about 6 carbon atoms. Illus~r~tive of alkyl groups are methyl, ethyl, propyl t isopropyl, n-butyl, isobutyl, sec-butyl, n-pentyl, isopentyl, neopentyl, t-pentyl and other isomers, including those o~ haxane. Thsse alkyl groups are also illus~rati~e of th~ alko~y groups intended as satisfyîng the foregoin~ definîtian of Rl a~d R2.
When Rl and R2 ara aryl, the scope is likewise broad and the term aryl, as used herein, includes substituted and unsubsti~uted aryl groups such as phenyl, benzyl, tolyl, anilino~ napht~yl, e~c.
Typical af groups with which Rl and R~ may be sub-stitut~d are ami~o, nitro 9 amido, nitrilo, hydroxyl, alkyloxy, haloge~, phenyl, sulfonic acid, carbo~ylic acid, etc.
The amount o diluent u~ ed in ~he presently dis~losed compo ition, device and proces~ can rary wldel~S and can easlly b~ determined at ~he laboratory ben~h. Thus, when the diluen~ i~ a liquid such as dime~hyl sulfoxide, ~se~
Table II, ~n~Pa) the amaunt can vary from about 10% to about 100% bas~d on the volume o~ diluent added ~o ~he composltion, comp~red with the volume of solvent or suspending agent (in the ea e o Example XII~ water). A preferred range is about 25% to about 100%~
Likewise, when the diluent is a mixture of more than one o~ ~he diluent compounds disclosed herein, the ratio 3f ~he amounts of the sev~ral compounds used in the dilue~t can vary broadly, and these ~ also, can readily be determined in the labora~ory~

9 ~

In order to more clearl~ t ach how to make and use the present in~ntion, and to illus~rate presently pr~ferred em~odimen~s thereof, the fQllowing examples are provided.

A, PREPAR~TION OF ~H~ TEST COMPOSITION AND DEVrCE

~ampZe I The ~est Compo~t~on ;::

A test compositio~ was prepared by di~solving ~he fallowing ingredi~nts in 150 ml ~millillters) of deionized w~e~. The ingredient~ were added ~o the water in descending order as lis~ed~

Trisodium ~itra~e 3.~ g ~grams) Citric Acid 2.2 g Ethylenediamine~etraacetic : acid, tetrasodlum salt 0,1 g Dime~hylsulfone 10 . O g Sodium lauryl sulfate 1.0 g 6~Me~hoxyquinoline 0.5 g Dimethylsulfoxide25,0 ml (milliliters) Ace~one 25.0 ml Cu~ene hydroperoxide 2.0 g Trieth~nolamine borate ~Aldrich Chemical Co., Inct) 10.0 g o-tolidine 0.4 g E~camp l e II - The Tes t Dev~oe Strips o~ Wha~mann 3MM f~lter pape~ were immersed in the test composition of Example I, The immersed skrips were dried at 70C ~or abou~ lS minutes to form tes~ compositlon^
impregnated carrier matrices. The dried carrier ma~rices were than attached to plastic (polystyrene) strips or handles by means of double-faced adhesive ~ape purçhased from 3M Com-pany. The resul~ing test de~ices can bs us~d ~o de~eçt peroxi-: da~ively active sub~t~nces by immersion in a test sample~
such as urine ? suspeGted o containing such substance, and observin~ any de~elopment q~ color in ~he carrier ma~rix.

B. STABILITY TESTINC~ OF VA~IOUS ES~ COMPOSI~IO~S

E$amp~ The E~at o~ the Bor~b~ Es~er ~' The purpose o ~hls experimen~ was to demons~rate ~he e~fect of triethanolamine borate on occul~ blood test compo-sitiQ~s as compar~d wi~h o~her known additives~ ~he followlng solution was prepared, chloroo~m 100 ml o-tolidine 0~5 g 2l5-dimethylh~xan~-Z,5~dlhydroperoxide2.0 ~
poly~N-vinylpyrrolid~ne) lO.q g To aliquot por~ions o~ this solution (20 ml each~ w~re added 1 gram of the additives in ~he fallawing table. These -15~

test composition solutions were ~rmitted to stand at room ;~
temperature for about 5 hours and the results observed are tabulated below in Table I, TABLE I

S ¦ SAMPLB NO. ADDITIVE
,, - ~ .. . ~ ., .. ~ _ 1 hexamethylene- Very dark 3 almost black solu-tetramine tion. Poor sensiti~ity.
2 1,4-dia~abicyclo- Dark green/brown discoloration.
[2.2~2~octane Minimally sensitive to occult blood in urine.
lQ 3 triethanolamine Slight browning.
borate High sensiti~ity to occult blood in urine

4 control ~no Turned black in abou~ 2 hours.
additi~e) , Unusable in occult blood analy-! ' sis, _ E~campZe IV - The Ef~ect of the ~iZuent To a 500 ml beaker were added the following components:
chloroform 100 ml cumene hydroperoxide 4.0 g ~grams) o-tolidine 0,4 g dimethyl sulfoxide 25.0 ml The resultant test compositlon, which was slightly yellow upon combining the abo~e ingredients, was transferred to' ~n Erlenmeyer flask which was stoppered and left on the laboratory bench at room temperature ~about 18.3C) o~ernight. After one day the ~est composition had grown only slightly darker than when it was prepared.

ExampZe V - Comparison wi~h E~YmpZe IV

A tes~ composition for con~rol purposes~ i.e., for comparison with the present invention, was prepared by following the procedure of Example IV, except that ~he di-methyl suloxide was substituted by 25 ml additional chloro- :
form, This ~est composition, wi~hout dimethyl sulfoxide, was stoppered in an Erlenmeyer flask and left on the laboratory bench as in Example IV (i.~., at 18.3C or one day~
Contrary to the composition o~ Example IV, the control compo-si~ion da~kened to almost black, thus indicating the im-proved stability impar~ed to the test composition by the diluen~.

C. ~H~ STABILIZATrON OF TEST DEYICES BY T~E BORATE ESTEF~

~amp ?,e VI - Cont~o ~ .
~' A solution of ~he following tes~ composition was pre-: pared for preparation of a reagent strip sensiti~e to ~seudoperoxidases. This formulation does not con~ain the bora~e ester component of the present invention.

H2O 150 ml Trisodium Citrate 3.2 g Citric Acid 2.2 g Ethylenediamlnetetra-acetic acid, tekrasodium salt 0.1 g g~

Dimethylsulfone 10. 0 g Sodium lauryl sulfate 1.0 g 6-methoxyquinoline0.5 g Dimethylsulfoxide25.0 ml S Acetone 25.0 ml Cumene hydroperoxide 2,0 g o-tolidine 0.4 g :;:
A section o~ Whatmann 3~M filter paper was immersed i~ the above test composition solution and dried at 70C. The dried paper was cut into squares of about 5 mm, and these ~ere attached to plastic handles using double-faced adhesive tape (available from 3M Company) ~hereby orming test devices.

ExampZe VII - Pr~esont Inven~iorL

A solution was prepared as in Examplç VI, above, ex-cept that lOg triethanolamine borat~ was added prior to the addition of the cumene hydroperoxide. Test devices were prepared from this solu~ion in identical fashion as in Example VI.

E~ampZe VIII ~ di,azabicycZo~2,2.2JootaMe :

A solution was prepared as in Example VI, supr~, except that lOg 1,4 diazabicyclo[2.2.2]octane was added prior to the addition of cumene hydroperoxide. This solu-tion was used to prepare test devices in identical fashion as in Example ~I.

.

i8 E~empZe IX - Hexamet~y~ene~etr~ ~ne A solution was prepared as in Example VI, supra~ ex-cepk that 10g hexame~hylenetetramine was added prior to the addition o cum~ne hydrope~oxide. Test devices were prepared from this solution as in Example VI, supra.

E~mp~e X - Stab~ty and Se~s~tivity Compari~on The ~st devices of Examples VI - IX were placed ~:
under stressed conditions to determîne their relative stabilities and sensitivi~ies. Devices from F~xamples VI - VIII were stored or three days at about 60C~ and those of ~xample IX for one day at about 70C. These stressed t~st devices were then ~ested for sensitivi~y by immersion in urine ~on~aining one par~ per million blood. Only ~he device containing triethanolamine borate (Example VII) produced a c.hange in color at this occult blood concen-tra~ion (v~z., 0.015 milligrams per deciliter). The re-maining devices were insensiti~e to this level o occult blood.

~. T~E STABILIZATIOP~ OF TEST DEVICES BY THE DILUENT

E~amp~e XI

Six test devices were prepared from six test composi-tion solutions contai~ing varying amoun~s o dimethyl sulfoxide in accordance with Table II below.

The six test composition solutions were all prepared :
with the following formula:
water 75 ml trisodium citrate 3.2 g S citric a~id 4.5 g triethanolamine borate 10.0 g e~hylenediamine tetra acetic acid, te~rasodium salt 0.1 g dimethyl sulfo~e 5.0 g sodium lauryl sulfate 1.0 g . 6-methoxyquinoline 0.5 g cumene hydroperoxide 2.0 g o-tolidine 0.4 g ;~
To ~ach of ~he six test composition solutions were added, ~:
respectively, the following amounts of dimethyl sulfoxide ~ ; ~ a~d methanol:
:; `;
TABLE II
----Test Dimethyl Me~hanol Composi~ion Sul.oxide ~ml) No (ml) _ ~5 1~ ~ ~ J

., ~

~ -20-~ ' Test devic~s were then pre,ared from each of the above test composition solutions by immersing a piece of Whatmann 3MM ilter paper into each solution. The immersed strips of filter paper were withdrawn from their respective solutions~
and dried and stress~d in an oven at 70C for 18 hours.
After stressing, each strip was tested in a test solu-tion ~omprising a 1:1,000,000 dilutlon of resh whole blood in urine. The intensity of color de~eloping in each strip provided a comparison means. The results are tabulated in Table III.
Table III
__ __ Tes~ Device Color Rating**
\~
1 0 ~no color~
3 1 ~trace~

6 7 ~almost ~o loss in rea~ti~ity3 -*Test device numbers corr~spond to ~he test composition numbers in Table II.
**The ~olor rating was based on a scale of 0 to 8, 8 being the cqlor produced by a reshly pr~pared, unstressed test device prepared ~rom test composition no. 6 ~Table II)o A rating of 0 indlcates no color $ormation, a rating of - 7 inticates almost no loss of reactivity, and a rating o 1 indicates only tra~e amounts of color.

It is dramati~ally apparent from the data in Tabls III, that sensitivity increasss with the amount sf dilu~n~
(here, dimethyl suloxide) used in the test omposition ~ ~ formulation.

.: :
. .
., - ~1 -. ~ A . .

~9~8 . INCREASED STABI~ITY O~ THE -EST COMPOSITION f WITH
BORA~B ESTER~ IMPARTED BY T~ARIOUS DI~UENTS

Exam p 1, ~ XI I

Eight test compositions were prepared as in Example XI, 7 of which contained different diluents and one (control) having no diIuent. Th~ purpose o ~his e~periment was to assess ~he ef1cacy of v~rious diluen~s in increasin~ the s~ability o~ test compositions sensi~ive to peroxida~ively active substances where the amount of bora~e es~er in ~he composi~ion ls h~ld constant.
A solutio~ was prepared containing the following ingredi~nts:

watsr S0 ml ~risodiu~ citrate 3.2 g citric acid 2.2 g :~ sodium lauryl sulfate 1.~ g 6-met~xyquinoline 1.0 g methanol 50.0 ml thanolamine borate 5.0 g ~: 20 cumen~ hydroperoxide 2.0 g o-tolidine 0,4 g `
: A small aliquot of this solution was added to each of 8 tes~ tubes. There~f~er small ~mounts of the diiuents listed in Table I~ were added to each test ~ube, the tes~ tubes stoppered, and allowed to stand at room temperature on the laboratory b~nch for one week. At the ~nd o the week, -.: -22-... ... . . . .. . .. .... . . .. . . . ...

~ ~9 ~

the test tubes were examined ~o relative darkening in color, the darker the color, ~he less stable the test compo-sition. The results are gi~en in Table I~ in order o~
decr~asi~g s~abili~y (i.e., test composition 1 being most s~able and tes~ composi~ion 8 being least stable).

TABLE IV
. ~ ~
Tes~ Composition Diluent No.
_______ 1 dimt~thyl suloxide 2 N9N-dime~hyl ~or~amide 3 dim~thyl sul~one 4 benzyl sul~oxide 4-chlo~ophenyl sulfone 6 4-fluoro-3-ni~rophenyl sul~one 7 2 imidazolidolle 8 (control~ none In Table IV, samples 1 and 2 exhibited excellent stability;
scIution 3 less so, bu~ nevertheless having good s~ability;
solution~ 4-7 were moderat~ly stable; and solution 8~ the contrQl, was ~ar les~ s~able than solu~ions 1-7.

F. PRE~EP~RED }~MBOpIMENTS
' ' Exemp Z e XIII
t ' , The following ingredienks are mixed in 150 ml wa~er:
i ,-;
-` ~ Trisodium citsa~e 3~2 g .~ 25 Citric Acid 2.2 g : ~thylenediaminetetraacetic acid 9 ` : ~etrasodium sal~ 0.1 g ,, ,- ,~
: -23-.

~ ~ :

Dim~hyl sulone 10.0 g Sodium lauryl sulfate 1.0 g 6-mothoxrquinoline 005 ~
Dimethylsulfoxide 2S.0 ml Acetone 25.0 ml Triethanolamine borate 10.0 g cumene hydroperoxide 2.0 g 4 -tolidine0.4 g S~rips o~ Wha~mann 3MM ilter paper were immersed in the ~.
i abo~e 501ution and dried at 70C. When dried, the ~ er paper was ~ut i~to squ~res measuring 4 mm on a side. These squ~r~s were then mounted on polyst~rene handl0s ~easuring . about 8 x 15 mm, b~ use o~ double-a~d adhesire tape ~3M
; ~ompany).
Tes~ devi~es prepared in accordance with the abo~e ~ eXpe~iment were st~es ed at 60C or three days and w~re found : ~ s~nsi~ive to:occult blood in u~ine a~ con~entr~tions at le.ast :~ as low as O.Oli milligram pescent, ':
E~amp Ze ~fIV
' :~

This e~ample illustrat~s a second preerred embodiment o~ the pre~ently disclosed ~8S~ de~ice. -:
A test composition is prepared by mixing the following ::
ingredients, in the following order, in a b~aker wlth stirring:

2~-~3~
`
water 75 ml trisodium citrate 3,2 g citric acid 4.15 g triethanolamine borate 10.0 g ethylenediaminetetraacetic acid 0.1 g sodium lauryl sulfa~e 1~5 g dime~hyl suIone 10.0 g 6-me~hoxyquinoline 0.6 g N,N-dimathyl ~ormamide 75 ml cumene hydroperoxide 3.0 g o-tolid.in~ 0.8 g S~rips o~ Wha~mann 3P~ filter paper were immersed in th~ above co~position and dried for about 11 minutes at about 90 to 92C. The dried strips were ~hen moun~ed on plastic ~andles using double adhesi~e tape.
~, S~me of the strips were stressed by storage at 70C
f~r one ~ay, others ~y storing at 40C for 12 weeks. Both se~s of strips developed a blue/green color when contacted by a 1:1 X 106 dilution of fresh whole blood in urine.
;- In summary, the preceding Bxamples point out how to make ~nd use the compositlon and ~est device of the present inventlon (Examples I and II), present comparative results :: of s~abili~y and sensi~ivity (Examples III to XII) J and des-crib~ presently preferred embodiments ~xamples XIII and XIV). Th~ ~ffects o the borate ester and the diluent, both .~ singly and in conc~rt, on the test composition are portrayed -~ in ~xamples III to V~ wher~as the effects on the ~es~ device . ,, ~-- are shown in Examples VI to XI. The use o~ various diluents is o~bodied in Exanlple XII.

~25-.

Claims

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A test composition for determining the presence of a peroxidatively active substance in a test sample, said composition comprising an organic hydroperoxide, an indi-cator capable of producing a detectable response in the presence of said hydroperoxide and a peroxidatively active substance, and one or both of (a) a diluent having the structure , , , or mixtures thereof, in which R1 and R2, same or different, are hydrogen, alkyl or alkoxy having 1 to about 6 carbon atoms, or aryl, and (b) a borate ester having the structure in which m, n and p, same or different, are integers of 1 to about 4.

2. The composition of Claim 1 in which said diluent is present, the diluent being dimethyl sulfoxide, dimethyl sulfone, N,N-dimethyl formamide, benzyl sulfoxide, 4-chlorophenyl sulfone, 4-fluoro-3-nitrophenyl sulfone or mixtures thereof.

3. The composition of Claim 1 in which said diluent is present, the diluent being a mixture of dimethyl sulfone and N,N-dimethyl formamide.

4 The composition of Claim 1 in which the borate ester is present and is trimethanolamine borate, triethanol-amine borate, tri(n-propanol)amine borate or mixtures thereof.

5. The composition of Claim 1 in which the borate ester is present and is triethanolamine borate.

6. The composition of Claim 1 in which both the diluent and borate ester are present, the diluent being dimethyl sulfone, N,N-dimethyl formamide or mixtures there-of, and the borate ester being trimethanolamine borate, triethanolamine borate, tri(n-propanol)amille borate or mixtures thereof.

7. The composition of Claim 6 in which the diluent is a mixture of dimethyl sulfone and N,N-dimethyl formamide, and the borate ester is triethanolamine borate.

8. The composition of Claim 1 in which the organic hydroperoxide is t-butyl hydroperoxide, cumene hydroperoxide, diisopropylbenzene hydroperoxide, 2,5-dimetylhexane-2, 5-dihydroperoxide, paramenthane hydroperoxide or mixtures thereof.

9. The composition of Claim 1 in which the organic hydroperoxide is cumene hydroperoxide.

10. The composition of Claim 1 in which the indicator is benzidine, o-tolidine, 3,3',5,5'-tetramethylbenzidine, 2,7-diaminofluorene, or mixtures thereof.

11. The composition of Claim 1 in which the indicator is o-tolidine.

12. A test composition for determining the presence of a peroxidatively active substance in a test sample, said composition comprising (a) cumene hydroperoxide, (b) an indicator selected from benzidine, o-tolidine, 3,3',5,5'-tetramethylbenzidine, 2,7-diaminofluorene and mixtures thereof, (c) a mixture of dimethyl sulfone and N,N-dimethyl formamide, and (d) triethanolamine borate.

13. A test device for determining the presence of a peroxidatively active substance in a test sample, said device having a carrier matrix incorporated with a test composition comprising an organic hydroperoxide, an indi-cator capable of producing a detectable response in the presence of said hydroperoxide and a peroxidatively active substance, and one or both of (a) a diluent having the structure , , , or mixtures thereof, in which R1 and R2, same or different, are hydrogen, alkyl or alkoxy having 1 to about 6 carbon atoms, or aryl, and (b) A borate ester having the structure in which m, n and p, same or different, are integers of 1 to about 4.

14. The test device of Claim 13 in which the diluent is present and is dimethyl sulfoxide, dimethyl sulfone, N,N-dimethyl formamide, benzyl sulfoxide, 4-chlorophenyl sulfone, 4-fluoro-3-nitrophenyl sulfone or mixtures thereof.

15. The test device of Claim 13 in which the diluent is present and is a mixture of dimethyl sulfone and N,N-dimethyl formamide

16. The test device of Claim 13 in which the borate ester is present and is trimethanolamine borate, triethanol-amine borate, tri(n-propanol)amine borate or mixtures thereof

17. The test device of Claim 13 in which the borate ester is present and is triethanolamine borate.

18. The test device of Claim 13 in which both the diluent and borate ester are present; the diluent being dimethyl sulfoxide, dimethyl sulfone, N,N-dimethyl formamide or mixtures thereof, and the borate ester being trimethanol-amine borate, triethanolamine borate, tri(n-propanol)amine borate or mixtures thereof.

19. The test device of Claim 18 in which the diluent is a mixture of dimethyl sulfone and N,N-dimethyl formamide, and the borate ester is triethanolamine borate.

20. The test device of Claim 13 in which the organic hydroperoxide is t-butyl hydroperoxide, cumene hydroperoxide, diisopropylbenzene hydroperoxide, 2,5-dimethylhexane-2,5-dihydroperoxide, paramenthane hydroperoxide or mixtures thereof.

21. The test device of Claim 13 in which the organic hydroperoxide is cumene hydroperoxide.

22. The test device of Claim 13 in which the indicator is benzidine, o-tolidine, 3,3',5,5'-tetramethylbenzidine, 2,7-diaminofluorene, and mixtures thereof.

23. The test device of Claim 13 in which the indicator is o-tolidine.

24. A test device for determining the presence of a peroxidatively active substance in a test sample, said device having a carrier matrix incorporated with a test composition comprising (a) cumene hydroperoxide, (b) an indicator selected from benzidine, o-tolidine, 3,3',5,5'-tetramethylbenzidine, 2,7-diaminofluorene and mixtures thereof, (c) a mixture of dimethyl sulfone and N,N-dimethyl formamide, and (d) triethanolamine borate.

25. The test device of Claim 24 in which said carrier matrix is bibulous paper.

26. A process for preparing a test device for deter-mining the presence of a peroxidatively active substance in a test sample, said process comprising the steps of (a) preparing a test composition by combining an organic hydroperoxide, an indicator capable of producing a detectable response in the presence of said hydroperoxide and a peroxidatively active substance, and one or both of (i) a diluent having the structure , , , or mixtures thereof, in which R1 and R2, same or different, are hydrogen, alkyl or alkoxy having 1 to about 6 carbon atoms, or aryl, and (ii) a borate ester having the structure in which m, n and p, same or different, are integers to 1 to about 4; and (b) incorporating said test composition with a carrier matrix.

27. The process of Claim 26 in which the diluent is present and is dimethyl sulfoxide, dimethyl sulfone, N,N-dimethyl formamide, benzyl sulfoxide, 4-chlorophenyl sulfone 9 4-fluoro-3-nitrophenyl sulfone or mixtures thereof.

28. The process of Claim 26 in which the diluent is present and is a mixture of dimethyl sulfone and N,N-dimethyl formamide

29. The process of Claim 26 in which the borate ester is present and is trimethanolamine borate, triethanolamine borate, tri(n-propanol)amine borate or mixtures thereof.

30. The process of Claim 29 in which the borate ester is triethanolamine borate,

31. The process of Claim 26 in which both the diluent and borate ester are present, the diluent being dimethyl sulfoxide, dimethyl sulfone, N,N-dimethyl formamide or mixtures thereof, and the borate ester being trimethanolamine borate, triethanolamine borate, tri(n-propanol)amine borate or mixtures thereof.

32. The process of Claim 31 in which the diluent is a mixture of dimethyl sulfone and N,N-dimethyl formamide, and the borate ester is triethanolamine borate.

33 The process of Claim 26 in which the organic hydroperoxide is t-butyl hydroperoxide, cumene hydro-peroxide, diisopropylbenzene hydroperoxide, 2,5-dimethylhexane-2,5-dihydroperoxide, paramenthane hydroperoxide or mixtures thereof.

34. The process of Claim 26 in which the organic hydroperoxide is cumene hydroperoxide.

35. The process of Claim 26 in which the indicator is benzidine, o-tolidine, 3,3',5,5'-tetramethylbenzidine, 2,7-diaminofluorene, or mixtures thereof.

36. The process of Claim 26 in which the indicator is o-tolidine.

37. A one-dip process for preparing a test device for determining the presence of a peroxidatively active sub-stance in a test sample, said process comprising the steps of (a) preparing a test composition comprising cumene hydroperoxide, o-tolidine, a mixture of dimethyl sulfone and N,N-dimethyl formamide, and triethanolamine borate; and (b) incorporating said composition with a carrier matrix.

38. The process of Claim 37 in which the carrier matrix is bibulous paper.

39. A method for determining the presence of a peroxi-datively active substance in a test sample, said method comprising contacting said sample with the composition of Claim 1, and observing a detectable response.

40. The method of Claim 39 in which the diluent is present and is dimethyl sulfoxide, dimethyl sulfone, N,N-dimethyl formamide, benzyl sulfoxide, 4-chlorophenyl sulfone, 4-fluoro-3-nitrophenyl sulfone or mixtures thereof.

41. The method of Claim 39 in which the diluent is present and is a mixture of dimethyl sulfone and N,N-dimethyl formamide.

42. The method of Claim 39 in which the borate ester is present and is trimethanolamine borate, triethanolamine borate, tri(n-propanol)amine borate or mixtures thereof.

43. The method of Claim 42 in which the borate ester is triethanolamine borate.

44. The method of Claim 39 in which both the diluent and borate ester are present, the diluent being dimethyl sulfoxide, dimethyl sulfone, N,N-dimethyl formamide or mixtures thereof, and the borate ester being trimethanol-amine borate, triethanolamine borate, tri(n-propanol)amine borate or mixtures thereof.

45. The method of Claim 44 in which the diluent is a mixture of dimethyl sulfone and N,N-dimethyl formamide, and the borate ester is triethanolamine borate.

46. The method of Claim 39 in which the organic hydro-peroxide is t-butyl hydroperoxide, cumene hydroperoxide, diisopropylbenzene hydroperoxide, 2,5-dimethylhexne-2, 5-dihydroperoxide, paramenthane hydroperoxide or mixtures thereof.

47 The method of Claim 39 in which the organic hydro-peroxide is cumene hydroperoxide.

48. The method of Claim 39 in which the indicator is benzidine, o-tolidine, 3,3',5,5'-tetramethylbenzidine, 2,7-diaminofluorene, or mixtures thereof.

49. The method of Claim 39 in which the indicator is o-tolidine.

50. A method for determining the presence of a peroxi-datively active substance in a test sample said method comprising contacting said sample with the composition of Claim 12, and observing a detectable response.