US 3409405 A
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United States Patent O 7 3,409,405 DIAGNOSTIC PREPARATION FOR THE DETECTION OF FORMALDEHYDE Raam R. Mohan, 1 Irish Lane, Randolph Township, Dover, NJ. 07801; Frank J. Turner, 2 Brent Place, Succasunna, NJ. 07876; and Stephen J. Schulte, 13 Timber Hill Drive, Livingston, NJ. 07039 No Drawing. Filed Feb. 17, 1965, Ser. No. 433,476 3 Claims. (Cl. 23-230) ABSTRACT OF THE DISCLOSURE A diagnostic preparation for the detection of formaldehyde comprising a bibulous material impregnated with a specific solution consisting of phenylhydrazine hydrochloride, ferric ammonium citrate and tribasic potassium phosphate. In use the impregnated material is allowed to come incontact with a sample and hydrochloric acid. The presence of formaldehyde is indicated by a red color.
This invention relates to a novel composition for the rapid detection of formaldehyde in body fluids and relates more particularly to a composition for the rapid and semi-quantitative determination of formaldehyde in urine.
The bactericidal effects of form-aldehyde are well known. In treating urinary tract infections, for example, use has been made of a complex of formaldehyde with ammonia to form an amine such as methenamine which in acidic urine will release free formaldehyde. Methenamine complexed with mandelic acid to yield methenamine mandelate is available under the trademark Mandelamine. When a patient suffering from urinary tract infections takes this drug in suitable dosage form, the metabolim of the drug in the body is such that the patient gets the benefit of the bactericidal effects of the formaldehyde which is released. On contact with urine, the methen-amine mandela-te releases formaldehyde which acts directly on invading micro-organisms in the unitary tract. As a routine procedure, urine from patients receiveing this therapy is frequently assayed from time to time to ensure not only that formaldehyde is present but that it is present in adequate concentration for the desired bactericidal effect. However, in available analytical procedures for determining the presence of formaldehyde in urine, prolonged and cumbersome manipulative steps are required.
Thus, in a preferred method, for example, a sample of the patients urine is reacted with phenylhydrazine and the corresponding phenylhydrazone of formaldehyde which forms is then colorimetrically estimated.
'When this test is utilized, a positive result will also be observed when other :CHO groups are present since phenylhydrazine will react with all free aldehyde groups. This test is, therefore, non-specific. In another method, urine containing formaldehyde is acidified and the form aldehyde is then colorimetrically assayed by reacting with a suitable oxidizing agent such as Schiffs base and then measuring photometrically the depth of color of the resulting characteristically colored complex. There are many disadvantages to this method since it involves the use of a spectrophotometer which requires skilled technicians and expensive equipment. On the other hand, a quantitative test utilizing the formation of a phenylhydrazone is too complex to be caried out as a regular procedure in the usual doctor's office or even in small clinics where skilled chemical technicians are not readily available.
Accordingly, a primary object of this invention is to provide a novel composition for the detection of formaldehyde in urine.
Another object of this invention is to provide a composition for the rapid and semi-quantitative detection of formaldehyde in body fluids.
A further object of this invention is to provide a novel composition useful for detecting formaldehyde in body fluids and which has enhanced stability and shelf life.
Yet another object of this invention is to provide a composition which is specific for the detection of formaldehyde in body fluids.
Another object of this invention is to provide a highly sensitive test for detecting the presence or absence of formaldehyde.
Other objects and advantages will become more apparent from the following detailed description.
We have now found that the aforementioned and other objects are fulfilled by providing a strip of a bibulous material which is impregnated over at least a portion of its area with a reagent system comprising the combination of a salt of phenylhydrazine and ferric ammonium citrate with tribasic potassium or sodium phosphate. The concentration of ferric ammonium citrate and potassium phosphate tribasic in the composition applied to the bibulous strip and in the final product is critical. The impregnating solution should contain these components in the ratio of about 0.25% by weight of ferric ammonium citrate to 0.2% by weight of potassium phosphate tribasic. The phenylhydrazine salt present may vary in amount from 0.2 to 0.375% by weight. The reagent solution is prepared by dissolving the respective active ingredients in water in the amounts given. Conveniently, when impregnating a bibulous material such as filter paper the proper amount can be applied by employing phenylhydrazine hydrochloride as a 7.5% by weight aqueous solution ferric ammonium citrate as a 5.0% by weight aqueous solution and potassium phosphate tribasic as a 20% by weight aqueous solution and combining them in a volume ratio of 5 :5 :1. The resulting aqueous mixture containing the respective reagents is then applied to a suitable bibulous material and allowed to dry. The amount of the reagents deposited on the carrier material varies with the nature of the material, but generally an increase in dry weight of about 10 to 30% is desirable to give optimum results.
In place of ferric ammonium citrate, other ferric salts such as ferric chloride, ferric sulfate and the like may also be advantageously employed.
Bibulous materials which can be employed as the carrier are those materials which by means of capillary action are able to draw a liquid upward. Suitable carrier materials are, for example, filter paper, felt, porous ceramic strips, woven or matted glass fibers and the like. The bibulous material is normally cut into narrow strips after being impregnated with the reagents to facilitate packaging in small bottles or other containers from which they may be dispensed as needed.
A suitable barrier zone may be applied to the strips to prevent the migration of the reagents beyond any specific chosen area. Hydrophobic materials such as lacquer or a chrome complex of the formula:
CnHas with the reagent system is allowed to come in contact with urine or other body fluid being tested for the presence of formaldehyde. To this wetted reagent strip is added the composition of this invention in detecting the presence of free formaldehyde in urine:
TABLE I.REACTIVITY OF \ARIO US COMBINATIONS OF REAGENTS IN THE DEVELOPMENT OF A DIAGNOSTIC PAPER STRIP FOR THE DETECTION OF FREE CI'IgO IN URINE HCHO, meg/ml. of Urine Comments ReegentsRatio (v./v.)
phenylhydrazinezl Fe Am Citratezl K PO4. ND=N0 Differentiation. 5 plieuylhydrazinezl Fe Am Citratezl K3PO-l Do. 5 phenylhydrazinezb Fe Am Citrate-.1 K PO Do. 5 phenylhydrazineA Fe Am Citratezl K3PO D Differentiation Fair. 5 phenylhydrazinezo Fe Am Citratczl K3POl =D fic e eti0fl Good and Rapid. 5 phenylhydrazinezfi Fe Am Citratezl K3PO4 D=Difierentiation Fair. 5 phenylhydrazinefl Fe Am Citratezl K PO4" ND Differentiation Poor. 5 phenylhydrazinezB Fe Am Citratezl KJPOL. Do. 5 phenylhydrazinezt) Fe Am Citratezl K lo n Do. 5 pheuylhydrazinezlO Fe Am Citratezl K POi... Do.
+= Color change; =No color change.
one or two drops of 6 N hydrochloric acid. A red color 20 From the above table, it is evident that a reagent sysdevelops within 5 to 10 seconds if formaldehyde is present. The depth of this red coloration is proportional to the concentration of formaldehyde present.
It is believed that the reaction proceeds according to the following equations:
HCl [C H5NIIN CH K3PO4] Fe+++ Red Color In order to further illustrate this invention, the following examples are given:
Example 1.-Preparation of reagent system A. 7.5% w./v. solution of .phenylhydrazine hydrochloride is prepared by dissolving about 7.5 grams phenylhydrazine hydrochloride in 100 ml. of distilled water with some heating;
B. 5.0% w./v. of aqueous ferric ammonium citrate solution is prepared by dissolving about 5.0 grams ferric ammonium citrate in 100 ml. of distilled water; and
C. 20.0% aqueous potassium phosphate tribasic is prepared by dissolving about grams of potassium phosphate tribasic in 100 ml. of distilled water.
Five parts by volume of solution A, five parts by volume of solution B and one part by volume of solution C are then mixed together in a suitable container.
Example 2.-Preparation of test strip Bibulous material such as Whatmans No. 3 filter paper is impregnated with a 4 millimeter wide band by applying a solution of lacquer. The lacquer band is placed such that it is 12 millimeters from one edge along the length of the paper. To the dried paper is then applied an aqueous reagent mixture prepared in accordance with Example 1 such that it is 12 millimeters from the edge of the paper and just overlapping the lacquer band. The paper is then allowed to dry at room temperature. The impregnated paper is then cut perpendicular to the reagent band into 6 millimeters wide by 82 millimeters long strips.
Example 3.-Use of the reagent strips The test strip prepared in accordance with Example 2 is allowed to be wetted with the urine of a patient who has been on Mandelamine therapy. A few drops of 6 N hydrochloric acid are added to the wetted area. A red color develops within five to 10 seconds. As a reference standard urines containing only free formaldehyde which has been added to give a known concentration are prepared and subjected to the same test. The color produced by the urine of unknown formaldehyde concentration is then compared with the reference standard and the relative depth of color gives a semi-quantitative estimation of the concentration of formaldehyde.
Example 4 The following test results indicated the sensitivity of tem comprising a solution containing 5 parts by volume of 7.5% of phenylhydrazine to 5 parts by volume of 5% ferric ammonium citrate and one part by volume of a 20% by weight solution of tribasic potasium phosphate will quantitatively detect meg/ml. of free formaldehyde and allow for sem-quantitative estimation of levels of formaldehyde greater than 100 meg/m1. in urine.
Example 5 When the test strip is allowed to come in contact with urines containing the following added compounds: benzaldehyde, acetaldehyde, furfuraldehyde, salicylaldehyde, and then a few drops of hydrochloric acid are added to the same area, no red color forms on any of the test strips which indicates the remarkable specificity of the reagent system.
It is understood that the foregoing detailed description is given merely by way of illustration and that many variations may be made therein without departing from the spirit of our invention.
Having described our invention, what we desired to secure by Letters Patent is:
1. A diagnostic preparation for the detection of formaldehyde which comprises a strip of a bibulous material impregnated with a solution consisting of a combination of 7.5 grams phenylhydrazine hydrochloride in 100 ml. of water, 5 .0 grams of ferric ammonium citrate in 100 ml. of water, and 20 grams of tribasic potassium phosphate in 100 ml. of water in a ratio by volume ranging from 5:411 to 5:621.
2. Process for the detection of formaldehyde in body fluids which comprises allowing the test strip as defined in claim 1 to come in contact with said body fluid and then allowing the test strip to come in contact with 6 N hydrochloric acid.
3. A process for the semi-quantitative determination of formaldehyde in body fluids which comprises allowing a test strip as defined in claim 1 to come in contact with said body fluid then allowing said test strip to come in contact with 6 N hydrochloric acid and determining the color intensity thus resulted.
References Cited UNITED STATES PATENTS 2,314,548 3/1943 McClintlock 23553 OTHER REFERENCES Tanenbaum, M. and Bricker, C. E., Analytical Chemistry, vol. 23, pp. 345357 (1951).
MORRIS O. WOLK, Primary Examiner.
J. SCOVRONEK, Examiner.
E. A. KATZ, Assistant Examiner.