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BOEHRINGER MANNHEIM / R-BIOPHARM
Enzymatic BioAnalysis / Food Analysis
UV-method
for the determination of ammonia in foodstuffs and other
materials and for the determination of nitrogen after
Store at 2-8°C
Kjeldahl-digestion (see pt. 12.2)
Cat. No. 11 112 732 035
For recommendations for methods and standardized procedures see Test-Combination for approx. 50 determinations Principle (Ref. 1)
If the absorbance A2 decreases constantly, extrapolate the absorbance to In the presence of glutamate dehydrogenase (GIDH) and reduced nicotin- the time of the addition of solution 3 (GlDH).
amide-adenine dinucleotide (NADH), ammonia reacts with 2-oxoglutarate to L-glutamate, whereby NADH is oxidized.
Subtract the absorbance difference of the blank from the absorbance differ- The amount of NADH oxidized in the above reaction is stoichiometric to the amount of ammonia. NADH is determined by means of its light absorbance The measured absorbance differences should, as a rule, be at least 0.100 absorbance units to achieve sufficiently precise results (see "Instructions forperformance of assay" and “Sensitivity and detection limit”, pt. 4).
The Test-Combination contains
If the absorbance differences of the samples (⌬A 1. Bottle 1 with approx. 60 ml solution, consisting of: 1.000 (measured at 340 nm or Hg 334 nm respectively) or 0.500 (measured triethanolamine buffer, pH approx. 8.0; 2-oxoglutarate, 150 mg at 365 nm), the concentration of ammonia in the sample solution is too high.
2. Bottle 2 with approx. 50 tablets; each tablet contains: The sample is to be diluted according to the dilution table in that case.
3. Bottle 3 with approx. 1.2 ml glutamate dehydrogenase solution, approx.
Calculation
According to the general equation for calculating the concentration: 4. Bottle 4 with ammonia assay control solution for assay control purposes (measurement of the assay control solution is not necessary for calculat- ing the results.) Use the assay control solution undiluted. (Expiry date: Preparation of solutions
1. Use contents of bottle 1 undiluted.
2. Dissolve one tablet of bottle 2 with one ml solution of bottle 1 in a beaker
MW = molecular weight of the substance to be assayed [g/mol] or in a reagent tube for each assay (blank and samples) depending on the number of determinations. Use forceps for taking the tablets out of bottle 2. This results in reaction mixture 2*.
3. Use contents of bottle 3 undiluted.
Stability of reagents
Solution 1 is stable at 2-8°C (see pack label).
Bring solution 1 to 20-25°C before use.
Tablets 2 are stable at 2-8°C (see pack label).
Reaction mixture 2 is stable for 3 days at 2-8°C.
Bring reaction mixture 2 to 20-25°C before use.
If the sample has been diluted on preparation, the result must be multiplied The contents of bottle 3 are stable at 2-8°C (see pack label).
Procedure
When analyzing solid and semi-solid samples which are weighed out for sample preparation, the result is to be calculated from the amount weighed: Read against air without a cuvette in the light path) or against waterSample solution: 0.2-8 µg ammonia/assay3 (in 0.100-2.000 ml sample Instructions for performance of assay
The amount of ammonia present in the assay has to be between 0.2 ␮g and 8 ␮g. In order to get a sufficient absorbance difference, the sample solutionis diluted to yield an ammonia concentration between 0.01 and 0.08 g/l.
Dilution table
Mix***, and read absorbances of the solutions (A1) after approx. 5 min at 20- Mix***, wait for completion of the reaction (approx. 20 min) and read absor- If the reaction has not stopped after 20 min, continue to read absorbances in If the measured absorbance difference (⌬A) is too low (e.g. Ͻ 0.100), the 2 min intervals until the absorbances increase constantly over 2 min.
sample solution should be prepared again (weigh out more sample or diluteless strongly) or the sample volume to be pipetted into the cuvette can be For simplification of the assay performance it is also possible to pipette directly 1.000 ml of increased up to 2.000 ml. The volume of water added must then be reduced solution 1 into the cuvette and add 1 tablet from bottle 2. After dissolution of the tablet with to obtain the same final volume in the assays for sample and blank. The new the aid of a spatula continue working as described in the procedure. The difference in sample volume v must be taken into account in the calculation.
volume of ca. 1% (increase of volume by 1 tablet per 3.020 ml assay volume) has to be takeninto account in the calculation by multiplication of the result with 1.01.
** Rinse the enzyme pipette or the pipette tip of the piston pipette with sample solution before The absorption maximum of NADH is at 340 nm. On spectrophotometers, measurements are taken at the absorption maximum; if spectralline photometers equipped with a mercury *** For example, with a plastic spatula or by gentle swirling after closing the cuvette with vapor lamp are used, measurements are taken at a wavelength of 365 nm or 334 nm.
Parafilm (trademark of the American Can Company, Greenwich, Ct., USA) If desired, disposable cuvettes may be used instead of glass cuvettes.
See instructions for performance of assay 0503.11748 220②
Technical information
The absorbance differences measured and the weights of sample used 2.1 Use only freshly distilled water for the assay.
should be proportional for identical sample volumes.
2.2 Work in an atmosphere free from ammonia (ban smoking in the 8.4 Possible interference caused by substances contained in the sample can be recognized by using an internal standard as a control: in additionto the sample, blank and standard determinations, a further determination Specificity (Ref. 1)
should be carried out with sample and assay control solution in the
same assay. The recovery can then be calculated from the absorbancedifferences measured.
In the analysis of commercial ammonium sulfate results of approx. 100%have to be expected.
8.5 Possible losses during the determination can be recognized by carrying out recovery tests: the sample should be prepared and analyzed with 4. Sensitivity and detection limit (Ref. 1.2)
and without added standard material. The additive should be recovered The smallest differentiating absorbance for the procedure is 0.005 absorb- quantitatively within the error range of the method.
ance units. This corresponds to a maximum sample volume v = 2.000 ml and Reagent hazard
measurement at 340 of an ammonia concentration of 0.02 mg/l sample solu-tion (if v = 0.100 ml, this corresponds to 0.4 mg/l sample solution).
The reagents used in the determination of ammonia are not hazardousmaterials in the sense of the Hazardous Substances Regulations, the The detection limit of 0.08 mg/l is derived from the absorbance difference of Chemicals Law or EC Regulation 67/548/EEC and subsequent alteration, 0.020 (as measured at 340 nm) and a maximum sample volume v = 2.000 ml.
supplementation and adaptation guidelines. However, the general safety 5. Linearity
measures that apply to all chemical substances should be adhered to.
Linearity of the determination exists from approx. 0.2 ␮g ammonia/assay After use, the reagents can be disposed of with laboratory waste, but local (0.08 mg ammonia/l sample solution; sample volume v = 2.000 ml) to 8 ␮g regulations must always be observed. Packaging material can be disposed ammonia/assay (0.08 g ammonia/l sample solution; sample volume v = 0.100 10. General information on sample preparation
6. Precision
In a double determination using one sample solution, a difference of 0.005 to Use clear, colorless and practically neutral liquid samples directly, or
0.010 absorbance units may occur. With a sample volume of v = 0.100 ml after dilution according to the dilution table, and of a volume up to 2.000 ml; and measurement at 340 nm, this corresponds to an ammonia concentration Filter turbid solutions;
of approx. 0.4-0.8 mg/l. (If the sample is diluted during sample preparation, Degas samples containing carbon dioxide (e.g. by filtration);
the result has to be multiplied by the dilution factor F. If the sample is Adjust acid samples to pH 7-8 by adding sodium or potassium hydroxide
weighed in for sample preparation, e.g. using 1 g sample/100 ml = 10 g/l, a difference of 0.004-0.01 g/100 g can be expected.) Adjust acid and weakly colored samples to pH 7-8 by adding sodium or
potassium hydroxide solution and incubate for approx. 15 min;
The following data have been published in the literature: Treat “strongly colored” samples that are used undiluted or with a higher
sample volume with polyvinylpolypyrrolidone (PVPP) - (e.g. 2.5-5 g/100 ml); CV = 0.88-1.16 % (ammonium chloride solutions) Crush or homogenize solid or semi-solid samples, extract with water or
dissolve in water and filter if necessary; Deproteinize samples containing protein with perchloric acid or with
trichloroacetic acid;
Interference/sources of error
Extract samples containing fat with hot water (extraction temperature
During protein precipitation with perchloric acid which is to be carried out in should be above the melting point of the fat involved). Cool to allow the fat foodstuffs protein fragments are occasionally obtained. These protein to separate, make up to the mark, place the volumetric flask in an ice bath fragments are kept in solution and may gradually form ammonia in alkaline buffer systems leading to creep reactions. This formation of ammonia is very Break emulsions with trichloroacetic acid.
low and can be differentiated and calculated from the ammonia content ofthe sample by extrapolation of the absorbance A Important note
The Carrez-clarification should not be used in the sample preparation
for ammonia determination due to a too low recovery rate
The common ingredients of foodstuffs do not interfere with the determina- (adsorption of ammonia).
tion of ammonia. Only high concentrations of tannins in fruit juices maycause an inhibiton of the GIDH reaction. Fruit juices should therefore always 11. Application examples
Determination of ammonia in fruit juices
As high concentrations of heavy metals cause turbidity, they also make a Add 0.5-1.0 g (!) wet polyvinylpolypyrrolidone (PVPP) to 10 ml fruit juice reliable determination of ammonia difficult. In most cases high concen- (clear, turbid or colored juices) - when the sample volume is increased, trations of metal ions can be removed as hydroxides by alkalization of the neutralize, if necessary, and fill up to 20 ml with water - in a beaker and stir for 1 min (magnetic stirrer). Filter sample solution immediately and use it forthe assay. Sodium thiosulfate, occasionally added to samples of swimming-pool water, Determination of ammonia in water (swimming-pool water) (Ref. 3.1,
does not interfere with the assay up to 1 mg per assay.
Recognizing interference during the assay procedure
Filter the water sample if necessary, and use the clear sample for the assay.
Dilute sample solution according to the dilution table or use up to v = 8.1 If the conversion of ammonia has been completed according to the time 2.000 ml sample volume for the assay.
given under “Procedure”, it can be concluded in general that no inter-ference has occurred.
Determination of ammonia in milk
Mix 1 ml milk with 4 ml trichloroacetic acid (0.3 M). After approx. 5 min
8.2 On completion of the reaction, the determination can be restarted by centrifuge for separation of the precipitate. Decant the supernatant and adding ammonia (ammonium chloride, ammonium sulfate; qualitative neutralize with KOH (10 M) (dilution factor can be neglected due to the high or quantitative): if the absorbance is altered subsequent to the addition concentration of KOH), filter and use 1.000-2.000 ml sample solution for the of the standard material, this is also an indication that no interference Determination of ammonia in bakery products
8.3 Operator error or interference of the determination through the Accurately weigh approx. 10 g of the minced sample into a homogenizer presence of substances contained in the sample can be recognized by beaker, add approx. 20 ml perchloric acid (1 M) and homogenize for approx.
carrying out a double determination using two different sample 2 min. Proceed as stated under “meat and meat products”. Use at most volumes (e.g. 0.100 ml and 0.200 ml): the measured differences in absorbance should be proportional to the sample volumes used.
When analyzing solid samples, it is recommended that differentquantities (e.g. 1 g and 2 g) be weighed into 100 ml volumetric flasks.
Determination of ammonia in meat, meat products (Ref. 3.2, 3.3) and
12.3 Determination of ammonia in fermentation samples and cell
culture media
Accurately weigh approx. 5 g of the homogenized sample (from a sample of Place the sample (after centrifugation, if necessary) in a water-bath at 80°C 100 g, that has been ground and homogeneously mixed in a mixer) into a for 15 min to stop enzymatic reactions. Centrifuge and use the supernatant homogenizer beaker, add approx. 20 ml perchloric acid (1 M) and homo- (diluted according to the dilution table, if necessary) for the assay.
genize for approx. 2 min. Transfer the contents quantitatively with approx.
Alternatively, deproteinization can be carried out with perchloric acid. See 40 ml water into a beaker. Adjust to pH 7.0 (Ͻ 7.5) first with potassium hydroxide (5 M) and then exactly with potassium hydroxide (2 M). Transfer Homogenize gelatinous agar media with water and treat further as the contents quantitatively with water into a 100 ml volumetric flask, fill up to the mark with water, whereby it must be taken care that the fatty layer is
above the mark and the aqueous layer is at the mark.
For separation of fat and for precipitation of the potassium perchloraterefrigerate for 20 min. Afterwards filter. Discard the first few ml. Use theclear, possibly slightly turbid solution for the assay, if necessary after dilutionacc. to the dilution table.
Calculate the amount of ammonia according to the afore mentioned calcula-tion formula, whereby it must be multiplied with the volume displacementfactor K = 0.98.
Determination of ammonia in licorice products, sal ammoniac
pastilles
Accurately weigh out approx. 1 g minced and homogenized sample into a
100 ml volumetric flask, add about 60 ml water of 60 to 70°C and stir with a
magnetic stirrer about 10 min till the material is completely dissolved. Adjust
to 20-25°C, fill up with water to the mark, mix and filter.
Discard the first ml, use the (almost) clear filtrate for the assay, after dilutionaccording to the dilution table if necessary.
12. Further applications
The method may also be used in the examination of fertilizers, pharmaceuticals,cosmetics, paper (Ref. 2.1) and in research when analyzing biologicalsamples. For details of sampling, treatment and stability of the sample seeRef. 1.1-1.2.
Examples:
12.1 Determination of ammonia in fertilizers
Grind approx. 10 g of the sample and mix thoroughly. Accurately weigh
approx. 100 mg of the homogeneous material into a 100 ml beaker and add
approx. 50 to 60 ml water. Adjust to pH 7-8 with diluted hydrochloric acid
(1 M) or in the case of acidic fertilizer with diluted sodium hydroxide (1 M).
Warm to 60-70°C on a heatable magnetic stirrer for approx. 10 min. Allow to
cool, transfer quantitatively into a 100 ml volumetric flask and fill up to the
mark with water. Mix the solution and filter, if necessary.
Use 0.100 ml of the clear solution diluted, if necessary, for the assay.
12.2 Determination of nitrogen after Kjeldahl-digestion
The determination of total nitrogen can be obtained via the ammonia
determination in a sample mineralized according to the Kjeldahl-method.
References
Normally, the samples are to be incinerated wet (sulfuric acid). The ammonia, da Fonseca-Wollheim, F., Bergmeyer, H. U. & Gutmann, I. (1974) in Methoden der enzy- formed from nitrogen, is determined according to the procedure as follows: matischen Analyse (Bergmeyer, H. U. Hrsg.) 3. Aufl., Bd. 2, S. 1850-1853, Verlag Chemie,Weinheim and (1974) in Methods of Enzymatic Analysis (Bergmeyer, H. U. ed.) 2nd ed., Accurately weigh approx. 2 g of the ground and homogenized sample into a vol. 4, pp. 1802-1806, Verlag Chemie, Weinheim/Academic Press, Inc., New York and 100 ml Kjeldahl-flask, add 20 ml sulfuric acid (specific gravity = 1.84 g/ml) and approx. 30 mg catalyst mixture (e.g. acc. to Wieninger) or one Kjeldahl- Bergmeyer, H. U. & Beutler, H.-O. (1985) in Methods of Enzymatic Analysis (Bergmeyer, tablet, heat for approx. 2-3 h until the sample is disintegrated (yellowish or H. U., ed.) 3rd ed., vol. VIII, pp. 454-461, Verlag Chemie, Weinheim, Deerfield Beach/ blue-greenish solution). Allow the sample to cool and carefully (protective glasses), transfer the material quantitatively into a beaker filled with 600 ml Untersuchung von Papieren, Kartons und Pappen für die Lebensmittelverpackungen ice-cold water, stirring all the time (magnetic stirrer, icebath). Neutralize with (gem. Empfehlungen XXXVI der Kunststoffkommission des Bundesgesundheitsamtes) approx. 60 ml KOH (10 M) (pH 6-8). Transfer the neutralized solution quanti- Kapitel 8 (Methoden) Pkt. 3.4.2 (Ammoniak) tatively into a 1 l volumetric flask, fill up to the mark with water and mix. If Brautechnische Analysenmethoden, Band III, S. 597-599 (1982), Methodensammlung necessary, filter the mixture (sometimes necessary after disintegration with der MitteleuropÑischen Brautechnischen Analysenkommission (MEBAK), herausgege- Kjeldahl-tablets); discard the first few ml. Use the solution diluted, if neces- ben von F. Drawert im Selbstverlag der MEBAK, Freising Höpner, Th. (1977) Enzymatische Methoden in der Wasseranalytik - Mîglichkeiten und
Grenzen, Vom Wasser 49, 173-182
Gerhardt, U. & Quang, T. D. (1979) Methoden zur Ammoniakbestimmung in Fleisch und Fleischerzeugnissen, Fleischwirtschaft 59, 946-948
Barchietto, G., Cantoni, C., Frigerio, R. & Provera, D. (1984) Esame comparativo dei prodotti di autolisi nella carne di maiale (Azoto non proteico, Urea, Ammoniaca), ε × d × v × 1000 × amount weighed [g] Conservazione degli Alimenti 3, 12-17
Bartels, U. (1991) Die enzymatische Bestimmung von Ammonium im Niederschlags- wasser, CLB Chemie in Labor und Biotechnik 42, 377-382
ε × 1000 × 0.100 × 1000 × amount weighed [g] Cheuk, W. L. & Finne, G. (1984) Enzymatic determination of urea and ammonia in refrig-
erated seafood products, J.Agric.Food Chem. 32, 14-18
Ammonia assay control solution (Bottle 4)
Concentration: see bottle label
Ammonia assay control solution is a stabilized aqueous solution of ammo- The assay control solution can be used as an internal standard in order to nia. It serves as an assay control solution for the enzymatic determination of check the determination for correct performance (gross errors) and to see ammonia in foodstuffs and other materials.
whether the sample solution is free from interfering substances: Application:
1. Addition of ammonia assay control solution to the assay mixture: Instead of sample solution the assay control solution is used for the assay. 2. Restart of the reaction, quantitatively: After completion of the reaction with sample solution and measuring of A2, add 0.050 ml assay control solution to the assay mixture. Read absorbance A3 after the end of the reaction (approx. 20 min). Calculate the concentration Mix, and read absorbances of the solutions (A 2-A3) according to the general equation for calculating the concentration. The altered total volume must be taken into Continue as described in the pipetting scheme under "Procedure". Follow account. Because of the dilution of the assay mixture by addition of the the instructions given under "Instructions for performance of assay" and assay control solution, the result differs insignificantly from the data stated The recovery of the standard is calculated according to the followingformula: R-BIOPHARM AGLandwehrstr. 54D-64293 DarmstadtTelefon + 49 61 51 / 81 02-0Fax

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