APPAREIL ET SUBSTANCE POUR LE DOSAGE DE SUBSTANCES A ACTIVITE PEROXYDATIVE

TEST COMPOSITION AND DEVICE FOR DETERMINING PEROXIDATIVELY ACTIVE SUBSTANCES

Abrégé anglais

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
<IMG> , <IMG> ,
<IMG> , 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
<IMG>
in which m, n and p, same or different, are integers of 1 to
about 4.

Revendications

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
<IMG> , <IMG> ,
<IMG> , 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
<IMG>
in which m, n and p, same or different, are integers of 1 to
about 4.
- 26 -

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.
- 27 -

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.
- 28 -

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
<IMG> , <IMG>,
<IMG> , 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
<IMG>
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.
- 29 -

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.
- 30 -

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.
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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
<IMG> , <IMG> ,
<IMG> , 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
<IMG>
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.
- 32 -

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 -

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.
- 34 -

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.
- 35 -

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.
- 36 -

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.
--2-

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.
- 7

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.
- 8

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
- 10 -
'
.. .... .. .. . ..

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~
-11-

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.
- 12 -
, ~.

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~
-13

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
-14-

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.
-16-

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.
-18-
.

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.
-19-

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~
4 5
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-
.