Composition for assaying for nitrites

A composition for assaying nitrites comprising a diazotizable amine, an acid component and a coupling component, wherein the coupling component is a compound of the following formula I: ##STR1## wherein R represents a hydrogen atom or a straight-chain alkyl group having 1 to 6 carbon atoms, such as a methyl, ethyl, n-propyl, n-butyl, n-pentyl or n-hexyl group and n represents an integer of 1 to 6, with the proviso that the sulfoalkyl group may be substituted with at least 1 hydroxyl group, or a water-soluble salt thereof as well as test devices carrying the composition. This composition is far more sensitive than conventional compositions for assaying nitrites. With this composition, for example, a trace amount of as small as 0.02 mg/100 ml of a nitrite to be detected in urine in the diagnosis of a bacterial infection such as urinary tract infection can be detected surely.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
The present invention relates to a composition for detecting nitrites or 
nitrite-forming bacteria in foods and body fluids, particularly urine. 
2. Description of the Prior Art 
The frequency of occurrence of urinary tract infection is high. This 
disease is asymptomatic and the patients are women in most cases. When 
bacteriuria patients are left untreated, they suffer from manifest urinary 
tract infection frequently to cause various problems. However, an early 
diagnosis of the infection is possible by detecting bacteria excreted in 
the urine. When the diagnosis is to be effected biologically, urine is 
taken in such a manner that it is not contaminated with persistent 
bacteria. After a quantitative culture, at least 10.sup.5 /ml of the 
bacteria should be detected. However, the quantitative culture requires a 
complicated technique and a long time. Therefore, a chemical method has 
been employed for the rough quantitative determination of the bacteria 
more easily and rapidly. 
Bacteria such as E. coli (the most common bacterium causing urinary tract 
infection), Proteus, Klebsiella, Staphylococcus and Enterococcus contain 
in their microbial bodies an enzyme which reduces nitrates into nitrites. 
Vesical urine of a healthy person is free from bacteria and, therefore, 
when a nitrite is detected in urine, it may be considered that a urinary 
tract such as renal pelvis or bladder is infected with one or more of the 
above-mentioned bacteria and that a nitrate in urine was reduced into a 
corresponding nitrite in the infected part. 
A known, highly sensitive method of detecting bacteria in the urinary tract 
is based on the determination of the nitrite in excreted urine by a Griess 
test. A typical reagent for determining the nitrite by this test comprises 
an acidic solution of sulfanilic acid and 1-naphthylamine. In the Griess 
test, a nitrite contained in a sample diazotizes sulfanilic acid and a 
resulting diazonium salt is coupled with 1-naphthylamine to form a red 
dye. The concentration of this dye is proportional to the concentration of 
the nitrite. Therefore, the concentration of the nitrite can be determined 
by, for example, colorimetric analysis using a previously prepared 
calibration curve. This method had demerits in that the reagent solution 
is unstable and the operation is troublesome. To overcome these demerits, 
a method has been proposed wherein the Griess test is conducted on a 
carrier on which the reagent composition has been fixed. 
In the field of so-called "dry chemistry" in which a reagent composition 
used for the assay of a body fluid composition is fixed on a carrier, a 
reagent composition comprising sulfanilic acid, 1-naphthylamine and a 
solid organic acid has been known (see Fischl & Pinto "Clin. Chim. Acta" 
2, 527-533 (1957). Such a reagent mixture in dry state is far more stable 
than the solution so that it can be stored for a long time. Another 
example of the assaying agents is a reagent mixture comprising a 
diazotizable amine, an N,N-dialkyl-2-naphthylamine and a solid organic 
acid as disclosed in the specification of U.S. Pat. No. 3,415,717. 
However, these known test pieces have a minimum detection limit of 0.1 to 
1 mg/100 ml in terms of sodium nitrite. Such a low sensitivity is 
insufficient for detecting only a trace of the nitrite to be detected in 
the examination of bacterial infection. Under these circumstances, the 
development of highly sensitive and stable test pieces has been demanded. 
After intensive investigations made for the purpose of overcoming the 
above-mentioned defects of the conventional methods, the inventors have 
completed the present invention. 
SUMMARY OF THE INVENTION 
The present invention aims at solving the problems of the prior art. An 
object of the invention is to provide a stable composition for assaying 
nitrites which has a sensitivity sufficient for assaying nitrites to be 
detected in patients with bacterial infections, and also test devices 
carrying said composition. 
The composition of the present invention for assaying for nitrites 
comprises a diazotizable amine, an acid component and a coupling 
component, wherein said coupling component is a compound of the following 
general formula I: 
##STR2## 
wherein R represents a hydrogen atom or a straight-chain alkyl group 
having 1 to 6 carbon atoms, such as a methyl, ethyl, n-propyl, n-butyl, 
n-pentyl or n-hexyl group and n represents an integer of 1 to 6 with the 
proviso that the sulfoalkyl group may be substituted with at least 1 
hydroxyl group, 
or a water-soluble salt thereof.

DETAILED DESCRIPTION OF THE INVENTION 
The inventors have found that when the compound of the above general 
formula (I) or a water-soluble salt thereof is used as the coupling 
component, nitrites to be detected in the bacterial infections (in only a 
trace amount, i.e., 0.02 mg/100 ml of the nitrites) can be assayed 
quantitatively in a highly sensitive manner. 
The compounds preferably used as the coupling component in the present 
invention include, for example, the following compounds, though they are 
not limited thereto: 
N-(1-naphthyl)-3-aminopropanesulfonic acid, 
N-(2-methyl-1-naphthyl)-3-aminopropanesulfonic acid, 
N-(2-ethyl-1-naphthyl)-3-aminopropanesulfonic acid, 
N-(1-naphthyl)-4-aminobutanesulfonic acid, 
N-(1-naphthyl)-2-aminoethanesulfonic acid, and 
N-(1-naphthyl)-3-amino-2-hydroxypropanesulfonic acid. 
Though most of these compounds have not been disclosed in literature as 
yet, they can be prepared easily by known processes. 
For example, these compounds can be obtained by reacting 1-naphthylamine 
with a corresponding sultone (see Oda et al. "Kogyo Kagaku Zasshi" 59, No. 
9, 1028 to 1030 (1956)) or by reacting 1-naphthylamine with an 
alkylsulfonic acid or hydroxyalkylsulfonic acid having a halogen atom at 
its terminal (see N. E. Good et al., "Analytical Biochemistry", 104, 300 
to 310 (1980)). 
The composition of the present invention for assaying for nitrites is 
obtained by mixing the obtained coupling component with a diazotizable 
amine and an acid component. When the composition of the present invention 
is incorporated in an absorptive carrier and the carrier is applied to a 
support made of, for example, plastic so as to use it conveniently, a test 
device having an excellent storage stability and being usable easily is 
obtained. The most frequently used absorptive carrier is filter paper. In 
addition, non-woven fabrics, cotton and wood pieces may also be used. It 
is also possible to apply the assaying composition to a support such as a 
sheet of plastics, e.g., polyvinyl chloride, polystyrene, or polyester, 
together with a suitable adhesive such as gelatin or a synthetic resin, 
e.g. polyvinyl alcohol. After drying, the composition is fixed on the 
support. It will be understood that various modifications can be readily 
obtained. 
The diazotizable amines include, for example, sulfanilic acid, arsanilic 
acid, p-aminobenzoic acid and particularly sulfanilamide. 
The acid components include solid organic acids such as tartaric, citric, 
oxalic, malic, malonic, succinic and glutaric acids. Among them, tartaric, 
citric and oxalic acids are preferred. 
The test devices may be prepared by, for example, the following process: 
the diazotizable amine, coupling component and solid organic acid are 
dissolved in water, an organic solvent, or a mixture of water and organic 
solvent to attain given concentrations. An absorptive carrier, preferably 
a filter paper, is immersed in the resulting solution and then the carrier 
is taken out and dried to obtain a test paper. The test paper is cut into 
pieces having a desired size and a desired shape and then applied to a 
support such as a plastic sheet to obtain the test pieces carrying the 
nitrite-assaying composition of the present invention. 
The amount of the diazotizable amine is 0.5 to 10 mM, preferably 1 to 7 mM 
and more preferably 1.5 to 5 mM, per 100 ml of the solution. The amount of 
the coupling component is 0.1 to 5 mM, preferably 0.2 to 3 mM and more 
preferably 0.5 to 2 mM. The molar ratio of the diazotizable amine to the 
coupling component is in the range of 1/2 to 7/1, preferably 3/1 to 6/1 
(practically, the reaction ratio is 1/1). 
The concentration of the solid organic acid is 2 to 15 wt. %, preferably 
2.5 to 12 wt. % and more preferably 3 to 8 wt. % based on the amount of 
the composition solution. 
Suitable organic solvents for the above-mentioned components are lower 
aliphatic alcohols, particularly methanol. As a matter of course, other 
easily evaporable solvents in which the respective components are soluble 
may also be used. 
The test devices are not only suitable for detecting nitrite-forming 
bacteria in urine but are also usable generally for detecting nitrities or 
nitrite-forming bacteria such as those in drinking water or foods. 
DESCRIPTION OF THE PREFERRED EMBODIMENTS 
The following examples will further illustrate the present invention, which 
by no means limit the invention. 
Preparation of coupling components 
Preparation Example 1 
Synthesis of sodium N-(1-naphthyl)-3-aminopropanesulfonate: 
11.8 g of 1-naphthylamine was dissolved in 300 ml of 1-propanol. A solution 
of 10.1 g of 1,3-propanesultone in 20 ml of methanol was added to the 
above solution and the mixture was heated under reflux for about 3 h. 
After cooling to room temperature, the mixture was neutralized with a 1N 
aqueous NaOH solution and then the solvent was evaporated to dryness. 
After recrystallization from a solvent mixture of methanol and benzene, 
7.8 g of white crystals were obtained (yield: 33%). 
Physical properties: 
Thin layer chromatography (silica gel plate (a product of Merck Co.); 
developer: chloroform/methanol/water=8/5/2) 
UV absorption observed 
ninhydrin reaction (+) 
Rf=0.14 
Infrared absorption spectrum (KBr tablet, .nu..sub.max (cm.sup.-1)) 
.nu..sub.SO.sbsb.3 1200, 1050 cm.sup.-1 ; .nu..sub.NH 3400 cm.sup.-1 
.nu..sub.C.dbd.C 1620 cm.sup.-1 ; .nu..sub.CH.sbsb.2 2850 cm.sup.-1 
Melting point: 208.degree. to 209.degree. C. (decomposition) 
Preparation Example 2 
Synthesis of sodium N-(2-methyl-1-naphthyl)-3-aminopropanesulfonate: 
12.6 g of 2-methyl-1-naphthylamine was dissolved in 300 ml of 1-propanol. A 
solution of 9.8 g of 1,3-propanesultone in 20 ml of methanol was added to 
the above solution and the mixture was heated under reflux for about 3 h. 
After cooling to room temperature, the mixture was neutralized with a 1N 
aqueous NaOH solution and then the solvent was evaporated to dryness. 
After recrystallization from a solvent mixture of methanol and benzene, 
4.4 g of white crystals were obtained (yield: 18%). 
Physical properties: 
Thin layer chromatography (silica gel plate (a product of Merck Co.); 
developer: chloroform/methanol/water=8/5/2) 
UV absorption observed 
ninhydrin reaction (+) 
Rf=0.33 
Infrared absorption spectrum (KBr tablet, .nu..sub.max (cm.sup.-1)) 
.nu..sub.SO.sbsb.3 1200, 1050 cm.sup.-1 ; .nu..sub.NH 3400 cm.sup.-1 
.nu..sub.C.dbd.C 1620 cm.sup.-1 ; .nu..sub.CH.sbsb.2 2930 cm.sup.-1 
.nu..sub.CH.sbsb.3 2950 cm.sup.-1 
Melting point: 228.degree. to 229.degree. C. (decomposition) 
Preparation of the composition for assaying for nitrites 
EXAMPLE 1 
0.3 g of sulfanilamide, 0.2 g of sodium 
N-(1-naphthyl)-3-aminopropanesulfonate and 3.0 g of tartaric acid were 
dissolved in a suitable amount of methanol. Additional methanol was added 
to the solution to prepare 100 ml of a solution of the composition of the 
present invention for assaying for nitrites in methanol. 
EXAMPLES 2 to 9 
Compositions of the present invention were prepared in the same manner as 
in Example 1 using various diazotizable amines, coupling components and 
acid components. 
Comparative Example 
A conventional composition for assaying for nitrites was prepared in the 
same manner as in Example 1 except that sodium 
N-(1-naphthyl)-3-aminopropanesulfonate was replaced with 1-naphthylamine. 
The compositions for assaying nitrites prepared in the above examples and 
comparative example are summarized in Table 1. 
TABLE 1 
__________________________________________________________________________ 
Composition for assaying for nitrites 
No. Diazotizable amine 
Coupling component 
Acid component 
Solvent 
__________________________________________________________________________ 
Ex. 1 
sulfanilamide 
sodium N--(1-naphthyl)-3-amino- 
tartaric acid 
methanol 
(0.3 g, 1.7 mM) 
propanesulfonate (0.2 g, 0.7 mM) 
(3.0 g, 20 mM) 
(total 100 ml) 
Ex. 2 
sulfanilic acid 
sodium N--(1-naphthyl)-3-amino- 
citric acid 
ethanol 
(2 mM) propanesulfonate (0.2 g, 0.7 mM) 
(5.0 g, 26 mM) 
(total 100 ml) 
Ex. 3 
arsanilic acid 
sodium N--(1-naphthyl)-3-amino- 
citric acid 
ethanol 
(2 mM) propanesulfonate (0.2 g, 0.7 mM) 
(5.0 g, 26 mM) 
(total 100 ml) 
Ex. 4 
p-aminobenzoic 
sodium N--(1-naphthyl)-3-amino- 
citric acid 
ethanol 
acid (2 mM) 
propanesulfonate (0.2 g, 0.7 mM) 
(5.0 g, 26 mM) 
(total 100 ml) 
Ex. 5 
p-aminobenzamide 
sodium N--(1-naphthyl)-3-amino- 
citric acid 
ethanol 
(2 mM) propanesulfonate (0.2 g, 0.7 mM) 
(5.0 g, 26 mM) 
(total 100 ml) 
Ex. 6 
sulfanilamide 
N--(2-methyl-1-naphthyl)-3- 
citric acid 
ethanol 
(0.3 g, 1.7 mM) 
aminopropanesulfonic acid (2 mM) 
(5.0 g, 26 mM) 
(total 100 ml) 
Ex. 7 
sulfanilamide 
N--(2-ethyl-1-naphthyl)-3-amino- 
citric acid 
ethanol 
(0.3 g, 1.7 mM) 
propanesulfonic acid (2 mM) 
(5.0 g, 26 mM) 
(total 100 ml) 
Ex. 8 
sulfanilamide 
N--(1-naphthyl)-4-aminobutane- 
citric acid 
ethanol 
(0.3 g, 1.7 mM) 
sulfonic acid (2 mM) 
(5.0 g, 26 mM) 
(total 100 ml) 
Ex. 9 
sulfanilamide 
N--(1-naphthyl)-2-aminoethane- 
citric acid 
ethanol 
(0.3 g, 1.7 mM) 
sulfonic acid (2 mM) 
(5.0 g, 26 mM) 
(total 100 ml) 
Comp. 
sulfanilamide 
1-naphthylamine (0.1 g, 0.7 mM) 
tartaric acid 
methanol 
Ex. 1 
(0.3 g, 1.7 mM) (3.0 g, 20 mM) 
(total 100 ml) 
__________________________________________________________________________ 
Preparation of test pieces for assaying for nitrites 
EXAMPLE 10 
A filter paper (No. 2316; a product of Schleicher & Schull Co.) was 
immersed in the composition prepared in Example 1 for a given time and 
then taken out and dried. The resulting test paper was cut into pieces of 
5 mm.times.5 mm and applied to an end of a polystyrene sheet of 5 
mm.times.80 mm by means of a double-coated adhesive tape (thickness: 0.35 
mm) to obtain a test piece carrying the composition of the present 
invention for assaying nitrite concentration as shown in the FIGURE, 
wherein 1 indicates the test paper and 2 indicates a support, e.g., 
polystyrene sheet. The support may have numerals, letters, symbols, 
standard color tone, etc. applied to the surface thereof or printed 
thereon, if desired. 
EXAMPLES 11 TO 18 
Test pieces were prepared in the same manner as in Example 10 except that 
the composition prepared in Example 1 was replaced with a composition 
prepared in each of Examples 2 to 9. 
Comparative Example 2 
Test pieces were prepared in the same manner as in Example 10 except that 
the composition prepared in Example 1 was replaced with the composition 
prepared in Comparative Example 1. 
Performance comparison test 
The test pieces for assaying nitrite concentration prepared in Examples 10 
to 18 and Comparative Example 2 were immersed in urine containing a 
nitrite of various concentrations to estimate their analytical accuracy. 
The results of the performance comparison tests of the test pieces for 
assaying nitrite concentration prepared in Examples 10 to 18 and 
Comparative Example 2 are summarized in Table 2. 
TABLE 2 
__________________________________________________________________________ 
Results of the performance comparison tests 
Supported Lower limit of 
No. composition 
Developed color tone.sup.1 
detection (mg/100 ml) 
__________________________________________________________________________ 
Ex. 10 
Ex. 1 light red to reddish purple 
0.02-0.03 
Ex. 11 
Ex. 2 light reddish purple to purple 
0.02-0.03 
Ex. 12 
Ex. 3 rose pink to red 
0.02-0.03 
Ex. 13 
Ex. 4 light red to reddish purple 
0.02-0.03 
Ex. 14 
Ex. 5 light red to reddish purple 
0.02-0.03 
Ex. 15 
Ex. 6 rose pink to red 
0.02-0.04 
Ex. 16 
Ex. 7 rose pink to red 
0.02-0.04 
Ex. 17 
Ex. 8 light reddish purple to purple 
0.02-0.04 
Ex. 18 
Ex. 9 light red to reddish purple 
0.02-0.04 
Comp. 
Comp. rose pink to red 
0.1-0.12 
Ex. 2 
Ex. 1 
__________________________________________________________________________ 
.sup.1 The color tone was fixed after about 10 to 20 sec. 
It is apparent from the results shown in Table 2 that the test pieces 
carrying the composition of the present invention for assaying for 
nitrites had a lower limit of detection of 1/3 to 1/5 of that of the 
conventional test pieces. This fact suggests that the detection 
sensitivity of the test pieces according to the present invention has been 
improved remarkably. 
As described above in detail, the composition for assaying nitrites and 
test devices carrying the composition of the present invention are far 
more sensitive than the conventional composition for assaying nitrites and 
test devices carrying the composition. According to the present invention, 
a trace amount of as small as 0.02 mg/100 ml of a nitrite to be detected 
in urine in the diagnosis of a bacterial infection such as urinary tract 
infection can be detected surely. 
Thus, the composition of the present invention and test pieces carrying the 
composition exhibit excellent effects in the highly sensitive detection of 
nitrites in various samples as well as in the easy and rapid assay 
thereof.