DE1295887C2 - REAGENT COMPOSITION FOR THE COLORIMETRIC ANALYSIS OF AMMONIA NITROGEN - Google Patents

Description

isiengen water, ζ. B. 0.1 ml of water per 5 g of phenol, contained, but this water is physically occluded by the solidified phenol and can therefore with do not come into contact with nitroprusside. The reagent is therefore practically anhydrous.

When using the composition of reagents according to the invention for the determination of urea, first the urease reaction is used to form ammonia accomplished. Then first the phenol / nitroprusside salt reagent and then the alkaline hypochlorite solution was added, whereby the color developed. Will this order of If the addition is complied with, it is not necessary that the reagents are added immediately one after the other will. When the alkaline hypochlorite was added after 2 hours and longer, there was practically none Change in the intensity or the position of the tip vk-r optical density of the color was observed. Through this, that the point in time at which the reagents are added is not of critical importance, becomes practically one Considerable improvement has been achieved as it is now possible to identify determinations with a large number of samples to perform without constantly having one of the reagents immediately after the previous one must be admitted.

Another advantage of the reagent assembly according to the invention is that the same Devices, e.g. B. pipettes, can be used for various of the reagents. The color reaction will does not change if the same pipette is used, for example, for the phenol nitroprusside reagent and for the alkaline hypochlorite reagent is used and only rinsed with water in between. Further the concentrations of the reagents can fluctuate within wide limits without any intensity or the absorption maximum of the color can be influenced if the colorimetrically to be examined Solutions are diluted to the same final volume.

The measurement of the color intensity is carried out in the usual way, e.g. B. with a spectrophotometer 640 millimicrons or a filter photometer with a green filter.

The following example explainsp ·· '. uie invention without to restrict them.

EXAMPLES Preparation of the reagents To prepare the phenol nitroprusside color reagent, 37.5 g of sodium nitroprusside were dissolved in distilled water, whereupon the volume was made up to 150 ml with distilled water. In each case 0.1 ml of this nitroprusside solution was placed in 1250 flasks with a capacity of 10 ml each. Then 6,500 g of phenol was melted by heating the crystals in a container in a water bath at 80 ° C. ^{5.0 ml of the liquefied phenol at 65 °} C. were then introduced into each of the small flasks described above. After the phenol had cooled and solidified, the vials were closed.

To prepare the alkaline hypochlorite reagent, 5120 ml of a 5.25% strength aqueous Sodium hypochlorite solution is given to 13,000 ml of a 25% strength aqueous sodium hydroxide solution. Respectively ml of this solution were then placed in 1250 flasks with a capacity of 20 ml, which were then locked.

Diagnostic test

Ammonia-free water was used to prepare the reagents:

a) Buffered urease:

1. D.; The contents of the vials described above with buffered urease were added by adding 20 ml of distilled water made ready for use again.

2. The ready-to-use urease is stable for 1 month if it is stored ^{at 2 to 10 ° C.}

35

b) Phenol nitroprusside color reagent:

To produce buffered urease, 250 g of ethylenediaminetetraacetic acid were first dissolved in 3500 ml of water. The pH of the ethylenediaminetetraacetic acid solution was adjusted to 6.5 with 54.6 ml of 40% sodium hydroxide solution. 50 g sodium azide was added with stirring until the product went into solution. Then 37.5 g of urease was dissolved in 1000 ml of distilled water. The urease had an activity of 1000 Sumner-Einheitcn per gram, measured at 30 ⁰ C. The solution was added to the above-described EDTA buffer. The volume of the combined solutions was brought to 5000 ml. After filtering, 4 ml of the solution were placed in 1250 vials with a capacity of 25 ml each. The material in the flasks was subjected to freeze drying, whereupon the flasks were sealed.

1. One of the flasks described above was filled with water and left to stand for 5 minutes, to liquefy the phenol.

2. The contents of the vial were transferred to a 500 ml flask, the vial was rinsed with distilled water to completely capture its contents, and that Volume was brought to 500 ml with distilled water.

3. The solution obtained in this way is stable for at least 1 month if it is stored in glass or at 2 to 10 ° C Plastic containers is stored.

^ ₀ c) Alkaline hypochlorite reagent:

1. The concentrated alkaline hypochlorite solution from one of the flasks described above was transferred to a container with a capacity of 500 ml, and that The flask was rinsed with distilled water so that its contents were fully recorded. The volume was brought to 500 ml with distilled water.

2. This solution is stable for at least 6 months when stored in glass or plastic containers at 2 to 10 ° C is stored.

d) To produce a nitrogen standard with a content of 1.5 ml nitrogen per 100 ml 70.7 mg of analytically pure ammonium sulfate dissolved in 100 ml of water.

The test was carried out with Seruni. It can also be performed with plasma or atomized urine

will. A urine dilution of 1:50 will generally suffice. Stronger or weaker dilutions may be required to determine the concentration in a for colorimetric determination bring suitable area.

1. In each case 0.2 ml of the buffered urea solution was poured into a series of test tubes Testing of a blank sample, a standard sample and a sample with an unknown content were introduced.

2. With the exception of those intended for the blank sample, the test tubes were put into the respective test tubes 0.02 ml of a standard solution or 0.02 ml of a serum sample whose urea content is not was known (this sample is called "Unknown") using a hemoglobin pipette and introduced using the auto wash procedure.

3. These test tubes were kept in an incubator at 37 ^C C for 15 minutes to convert the urea to ammonia. (Instead, the test tubes can be left at room temperature for 30 minutes at this stage and the following stage 5.)

Absorption of the sample of unknown content - absorption of the blank sample

4. In each test tube (including the rind sample) were in the order given

a) 5 ml of phenol color reagent,

b) 5 ml alkaline hypochlorite reagent,

given, whereupon the contents of each test tube immediately mixed and an inert material as Lid was used for the test tubes. to prevent contamination.

5. The test tubes were again incubated for 15 minutes to develop the color held at 37 ° C.

6. The absorbance was read using a spectrophotometer at 640 millimicrons.

calculation

Measured with a spectrophotometer is the absorbance corrected by the blank for the concentration of ammonia nitrogen directly proportional. The values obtained in the analyzes described above were used to calculate urea nitrogen of samples of unknown content is used according to the following equation:

Absorption of the standard
- Absorption of the blank sample

Concentration of the standard (mg / 100 ml)

= Urinary nitrogen of the sample of unknown content (mg / 100 ml).

The urinary stalk nitrogen content of the sample is unknown According to the procedure described in this example, the content was 15 mg per 100 ml Serum determined.

The urea starch content was used as a control of the sample of unknown content used in this example according to that in the Manual of Clinical Laboratory Methods described standard method determined with Neßler's reagent. According to this method, it was believed the urea nitrogen content of the sample of unknown content is also 15 ml per 100 ml of serum.

It should be noted that this colorimetric reaction can also be used to determine the ammonia nitrogen can serve in various other liquids or solutions, provided that after Addition of the phenol color reagent, an alkali hypochlorite reagent is added in such an amount that any initially present acidity of the solution is neutralized and a sufficient excess of it Alkali is present to convert the phenol to the alkali salt. For example, when determining of nitrogen in a so-called Kjeldahl digestion by the presence of larger amounts of protein or salts, e.g. B. sodium chlorite or sulfate, does not noticeably affect the color reaction.

Claims (1)

1 2 components phenol, a nitroprusside saiz, a hypo patent claim: chlorite, characterized by ]. Phenol in a practically anhydrous mixture mil _{Set of} reagents for the colorimemem Nitroprussidsaiz and bag analysis. Ammonia nitrogen, and in particular 5 ^ _eme alkaline aqueous solution of an alkali Sondere for use in the enzymatic "'jjvp _O chlorite
Urea determination by means of urease containing effective reaction components are phenol, one side by side that cannot be mixed before use Nitroprussidsaiz, a hypochlorite, known state. characterized by 'io The reagent combination according to the invention:>, ·; - _{Λ D,.} · ....,. ... luno enables a very precise determination of jn 1. Phenol m a practically anhydrous mixture _Am ^o _m01liak ^D _{sückst0ff in} biological fluids, with a nitroprusside _{alkali salt and z} . _{claimed and} a minimum au 2. eme alkaline aqueous solution of an alkali _{sample liquid} ; _{igkelt and} laboratory equipment requires, hypocnlonts ^ _wu persist in the reagents used: g next to each other are immiscible before use and the time of addition of the reagents τλ <η Status. is critical. In addition this has reagent addition, menstelluna the advantage that they eii: .s Effective waste easily and safely transported wl - 20 den, whereby analytical determinations under external circumstances are relieved that would otherwise For the determination of urea in biological such determinations make or considerably more difficult Liquids, such as serum, plasma or urine, would make standing completely impossible several known methods are available. In the determination of urea in biological for example, urea with the aid of the enzyme as liquids with the aid of the invention Urease in the presence of a buffer solution for ammo reagent composition, the urea is added nium carbonate is hydrolyzed, the ammonia is hydrolyzed by urease and the formed Addition of sodium borate released, debuted and ammonia with the practically anhydrous mixture is collected in 0.05 η hydrochloric acid. a nitropri- "idall: alisalzes *" it phenol and the The nitrogen determination is then carried out in a colorimetric, aqueous, alkaline solution of an alkali hypo- after adding Neßler's reagent (vgi. Opal. hlorits reacted to form a blue color, H e ρ 1 e r, Manual of Clinical Laboratory Method, whereby the one present in the liquid sample 4th edition, Charles C. Thomas, Springfield, Illinois). Urea quantitatively by colorimetric means You can also use the yellow pigment that can be detected in the. Be before using them Condensation of diacetyl with urea in a 35 the individual reagents of the invention protein-free filtrate of the biological fluid - composition dissolved and diluted, is used to determine. Bish ^p r were used to analyze ammonia nitrogen These known colorimetric procedures, solutions of sodium phenolate and nitroprusside, were used by Fawcett and Scott by applying sodium, the effectiveness of which diminishes considerably in a relatively short time which tends to form a blue color. In the reaction of ammonia with sodium phenolate and in contrast to nitroprusside, the sL'u improves in the presence of hypochlorite (Fawcett and Scott, free water with formation of colored substances, J. din. Path., 1960, vol. 13, p. 156). This method, which disrupts the colorimetric determination, decomposes, is more precise and works with smaller amounts of the reagent contained in the inventive method, but has certain disadvantages, as there is no free water. Solid phenol has been introduced by heating to its top reason, which it does not position itself for routine analysis. So the concentration of the liquid and then ™ \ t nitroprusside is combined. The reagents critical. Nitroprusside can also be used as a single crystal or in particle probes reagents. The diversity form present. Instead, the phenol of reagents is important because time at this 50 and the nitroprusside also combine in that mode of operation is a critical factor. If the dyeing one does not divide a physical mixture of the solid constituent reagents according to Fawcett and Scott into parts, e.g. B. in particulate! State, can be added in short succession of water, then creates and stores free conditions, which means that the optical density is noticeably reduced in the end. Reagent does not degrade even with longer storage. Such a random occurrence suffers. tion makes the simultaneous analysis of a large The nitroprusside acts as a catalyst for the Number of samples impossible and continues the prak- color reaction, with the reaction rate the applicability of the method has narrow limits. is influenced by the amount of catalyst. Optimal Also, some of the reagents are subject to conditions if the concentrated Fawcett and Scott unstable, making the custom 60 reagent about 324 mg phenol and 25 mg nitro availability of the method is further limited. It contains prvssid sodium. This concentrated Reageaz The task is therefore to identify the disadvantages of the practical implementation of the analysis th method to eliminate. dissolved in water. The amount of nitroprusside in de: .i invented The subject of the invention is a reagent composition according to the invention can considerably- Composition for colorimetric analysis 65 Hch fluctuates and only needs to be so large that of ammonia nitrogen, especially for using the catalytic effect of nitroprusside, the practical in the enzymatic urinary streak determination. Application needs are sufficient, by means of urease, containing as an effective reaction- This concentrated reagent may be small