FI98864B - Method and test arrangement for determination of acetone - Google Patents

Description

98864

Method and test apparatus for the determination of acetone

FIELD OF THE INVENTION z f

The invention relates to a method and a test device for determining the position per ton by a gas diffusion method, in which acetone, which evaporates from a sample, is introduced into an absorption liquid, where it causes a color change which is detected.

Technology background

Acetone is a metabolite with an increase in ‘10 concentration in the presence of metabolic imbalance, | such as as a result of diabetes or hunger, in which case the amount of acetone increases, e.g., in body fluids and exhalation.

In ruminants, changes in feeding are also reflected in the body's acetone content, and the determination of acetone is a valuable tool in monitoring ruminant feeding and preventing ketosis. Proper feeding of the ruminant is also of great economic importance, as it can both increase milk production and shorten the calving interval. Ketosis is generally defined as a metabolic disorder due to a lack of energy intake in which body fluids. . the concentration of ketones has risen to abnormally high levels.

• ♦ j In addition to acetone, ketones include B-hydroxybutyric acid and acetic acid. The importance of acetone is emphasized when ..7 when the disease state is severe.

• 25 The amount of ketones in cow's milk is: only about half the amount in the blood. Correspondingly, 7: the amount of ketones in the urine is about four times the concentration of ketones in the blood. A healthy cow has a weapon ·. less than 2.5 mg / iooml in milk, while 5 mg / 100 • · ... '30 ml is considered the lower limit for clear acetone disease. Concentration range *. 2.5-5.0 mg / 100 ml refers to latent acetone disease (Se- ***, J., Suomen Eläinlääkärilehti, ^ 5 (1989) 3, 115-117).

'**: Acetone can be determined in many ways and the following articles describe, for example, the gas chromatographic determination of dairy products • · 35 and milk acetone by the head- • e 2 98864 - m space method: Hild, J. Determination of some aroma Γ! * compounds in dairy products by headspace analysis. Appl. ’--35 ... ....... .rjr; r

Headspace Gas Chromatogr., GC Headspace Symp., Meeting Date 1978, 89-94? and Urbach, G., Journal of Chromatography, 404 (1987) 163-174. Gas chromatographic determination of acetone is reliable, but it is slow and requires expensive and complex equipment.

Acetone can also be determined by the so-called by a microdiffusion technique based on the selective transfer of a volatile compound, generally via the gas phase, to an absorption solution from which it is determined, e.g. colorimetrically or volumetrically. Such methods are described, e.g., in Lapland, G. R. & Clark, L., Anal. Chem., 23 (1951) 541, in which acetone is absorbed into 2,4-dinitrophenylhydrate-15. Kobayashi, Y. J., Chem. Soc. Jpn. Ind. Chem.

Sect., 56 (1953) 174 and Chem. Abstr., 48 (1954) 9271 describe an acetone assay in which acetone is absorbed into dichromate-sulfuric acid and Anger, V. & Ofri, S. Fresenius, Z. Anal. Chem., 206 (1969) 186 describe a process in which acetone absorption utilizes salicylaldehyde. . chloride. In Rahim, S. A. & Bashir, W. A., Microchem.

| J., 26 (1981) 329 describes a method for the determination of acetone using diazotized sulphanilic acid.

.. *! * Acetone can also be selectively absorbed • * .ί from 25 samples into a hydroxylamine solution where it reacts with: hydroxylamine to form an acetooxime ··· ...

• · ·. .

• · · • ......

(CH 2) 2 CO + NH 3 OH - * (CH 3) 2 C = NOH + H 2 O, «· • · • ·· 30 where the hydroxylammonium / hydroxylamine balance • · · '. changes • * / NH 3 OH + - NH 3 OH + H +.

···:: • · s. * ·. The pH change can be measured with an acid / base indicator. Ai- · * · 35 times the microdiffusion method was carried out in the so-called Conway’n • ·· ...

• · 98864; 3 chambers. It is a circular 10-15 mm high glass vessel 5 to 10 cm in diameter covered with a lid. In the middle of the base, separated by a low wall, is another circular space in which the absorption liquid is placed. The sample and other necessary reagents are placed in the space surrounding the absorption liquid, whereby the sample and the absorption solution are in the same horizontal state. Conway’s method is not suitable for modern mass analytics and requires a lot of sample. On this basis, it has not been possible to develop any YK-10 simple serial measurement equipment.

The current application of the microdiffusion method in the determination of volatile compounds is flow injection analysis, or FIA. In it, diffusion does not take place through the gas phase but through a semipermeable membrane, which at the same time prevents the mixing of the 15 samples and the absorption liquid (Marstorp, P., Anfält, T. & Andersson, L., Anal. Chim.

Acta, 149 (1983) 283-289). The disadvantages of the FIA method are that it requires special equipment and that it is relatively slow. In the case of milk samples, another problem is that the milk easily clogs the hose systems of the device. . and in practice the membrane has to be replaced and the caloric • · - -: ·: · broinite replaced at very short intervals.

* '”· Summary of the invention .. *! * A simple, sensitive and fast method for the determination of e e ------- • 25 acetone has now been invented, which is suitable for both serial analyzes and the so-called cow-site ana- eee e ** · * · lysis. The method is based on gas diffusion, in which e the volatile acetone from the sample is passed to the absorption liquid, where it causes a color change which is observed.

* ... 30 The method according to the invention is characterized in that · # · *. the sample and the absorption liquid are juxtaposed so that they are separated by a common closed gas space *: ***: The invention further relates to a test device for determining acetone, characterized in that it comprises • · e Γ *. 35 container in which the absorption liquid is located so that the absorption liquid and the sample can be placed so that they are separated by a common closed gas space. Opposite microtiter plates form a particularly suitable container. ---- 5 Detailed Description of the Invention

The method according to the invention can be carried out as follows: A sample and an absorption liquid to which the reagents necessary for the color change, e.g. a pH indicator, have been added, are placed opposite each other in the vessel separated by the gas phase. By juxtaposing, for example, two microtiter plates, a series-measured Convay of 96 samples is thus obtained in a space of less than 1 dm2. Preferably, the absorption solution is in the wells of the bottom plate and the samples are placed in the wells of the top plate. This is possible, for example, if the samples are tightly gelled in the wells of the top plate or if the wells of the top plate have a porous absorbent for binding the liquid or the bottom surface of the top plate is chamfered with a semipermeable film permeable to acetone.

The sample can be gelled with e.g. polyacrylate, 20 starch or CMC. If the sample is bound to a carrier,. . Thus, for example, glass wool, cellulose, polyacrylate, etc. can be used as a carrier. As the sample separating semi-permeable film, various types of films which are permeable to high molecular weight compounds such as acetone can be used. The semipermeable membrane can be, for example, Teflon (so-called Teflon tape or Teflon filter paper Millipore · *: *; HVHP09050, pore size 0.45 μη) or cheaper so-called "diffusion tape" (Micropore).

; ·. The • * · * ... '30 microtiter plate loaded with the samples by the above procedures can be inverted on the bottom plate • · 9 *. as a lid. Between the plates, the following is suitably placed: V a routed silicone rubber or similar seal to provide a common sealed gas space for each pair of wells. Sheets of extruded. ) ·. lightly put together.

• e 9 • 99 9 9 9 9 9 9 99 9 9 5 98864

The reaction is preferably carried out at room temperature. Raising the temperature may speed up the reaction, but the temperature must not rise above 40 eC, because then the hydroxylammonium chloride begins to decompose. After an absorption time of 5 (usually about 3-5 hours), the plates can be separated and the absorption solution of the bottom plate analyzed in the usual manner colorimetrically, e.g. spectrophotometrically with an automatic plate reader or visually. For visual reading, it is more practical to use 10 discs with 1 to 20 sample slots. In the visual flash method, it may also be more advantageous to use an inverse system in which the sample is in the lower state and the reagents are attached to the upper state.

The method according to the invention is preferably suitable for the determination of acetone in milk. Instead of milk, the sample may also be other biological fluids, body fluids such as blood or urine, or products of the food industry, such as the dairy industry, or other technical preparations. The shelf life of the sample can be improved by the addition of some conventional preservatives such as pronopo-. . sodium azide or dichromate.

• * | As acetone absorption liquids, e.g.

** *: 2,4-dinitrophenylhydrazine, dichromate sulfuric acid, salicylaldehyde or diazotized sulphanilic acid containing; ’, · * 25 added any other lines required for the color reaction; reagents. For the most suitable concentration ranges for milk samples s *: *; however, is obtained by using hydroxylamine as the absorption liquid, preferably in the form of hydroxylammonium chloride (NH 2 OH x HCl). For milk samples, the concentration of the absorption liquid is preferably about 0.005 to 0.05 M, and in particular 0.01 to 0.03 M, which is practical to dilute. from about 1-5% stock solution.

s] ”: The pH change of the acetone in the absorption solution. ] ·. to the hydroxylamine, a pH indicator • · 0 V 'is added. 35 ri. Suitable indicators or combinations of indicators • ·· ...........

• ·

98864 I

6 are those that change color in the pH range of 2.8 to 5.4, especially in the range of 3.8 to 4.6. Examples are “ΐ

Indicators: pH range Color readings 5 acidic base

Methyl yellow 2.8 to 4.0 red yellow

Bromophenolsin 3.0 to 4.6 yellow red

Methyl orange 3.1 to 4.4 red yellow

Bromocresol green 3.8 - 5.4 yellow blue - 10 ..................

Preferably bromophenol is used. The concentration range of the indicator depends on the amount of other reactants in the microtiter plate, and a suitable amount is one in which the absorbance range does not exceed 1000 units.

The ratio of indicator to hydroxylammonium chloride affects the determination of the acetone content and the lower the concentration of hydroxylammonium chloride the more sensitive the method of determination.

Preferred embodiments of the invention are also set out in the dependent claims. The following examples illuminate. . invention.

• »: Example l 0 .....

* * The following equipment was used in the assays: ..T Hamilton Microlab 2200 dispensers and 900 additional dispensers; And 25 Hamilton HR 7000 microtiter plate readers. The microtiter plates were Hunclon / Greiner 96-well, flat-bottomed non-sterile plates.

• .....

Reagent solutions were prepared as follows: 100 mg; ·, the Na salt of bromophenolsine (Sigma B-6131) was dissolved in 100 4 · · * ···. To 30 ml of deionized water. 2 g of hydroxylammonium chloride (Merck 4616) was dissolved in 100 ml of deionized water.

; '/ A hydroxylammonium chloride / indicator, ϊ.,. Ϊ mixture was then prepared by taking 7.5 ml of the above 0.1% bromide. * ·. nolsin solution and 5 ml of the abovementioned 2% hydroxy e · e V ': 35 cyclammonium chloride per 100 ml distilled or deionized * ·· • ·. S3 ~ · - - · ---- · ...............-. .. - *: "· '- ..............” "T" “- rr * r-r ~" “* m ^ 1¾¾ 98864 7 water. This reagent solution was dispensed into 50-200 μΐ 96-well microtiter plates. A silicone gasket with corresponding holes was placed on top of the plate. m. ... - ··· *. · - · - ·· ·· ':' '·': '. R'.!

For another 96-well microtiter plate, 50-200 μl of standard or milk samples were loaded with an-5. 26% fat milk powder 10 g / 100 ml of water was added as standards, to which acetone 0.5, 1.0 and 2.0 mg / 100 ml were added. The plate was coated with a semipermeable film, i.e. a so-called with "diffusion tape" (Micropore; 75 mm x 9.1 m; 3M 10 1530-3) and placed upside down on a plate and seal containing the reagent mixture. The plates were incubated for 3-6 hours. The color change in the indicator mixture was measured spectrophotometrically with a microtiter reader at 590 nm or visually.

15 Example 2

Acetone concentrations were determined from 166 milk samples by the method of Example 1. 100 μΐ of hydroxylammonium chloride / indicator mixture was added to the microtiter plate and 80 μΐ of milk was added to the other plate. The incubation time 20 was 3-5 hours. Gas was used as the reference method. chromatographic (GC) head-space method I (Urbach, G., Journal of Chromatography, 404 (1987) 163- .....

174). The limits of determination ranged from 0.5 to 2.0 mg acetone / 100 ml * ·· * · · milk. The results are shown in Table 1.

* V 25!, ·: Table 1. Distribution of milk samples (pcs) in different ί acetone content categories

Acetone <0.5 0.5 - 1.0 1.0 - 2.0> 2.0 Γ · .. mg / 100 mi_________ UK: 30 <0.5 144 - -; ·. ·, 0.5 - 1.0 2 6 1. ·· «. 1.0 - 2.0 - 1 3> 2.0_. - __1_8_ ***** Ref method Λ.Ϊ 35 units / each 146 7 5 8 166 8 98864

The method according to the invention evaluated 97% of the samples in the correct category compared to the reference method.

Example 3: ...... ..........

The method according to the invention was compared with both the GC reference method used in Example 2 and the "." PIA method (Marstorp, P., Anfält, T. & Andersson, L.,

Anal. Chim. Acta, 149 (1983) 281-289). The samples were 166 cow-specific raw milk samples. In Table 2, samples-10 are classified into five different categories using the GC method as a reference.

Table 2 · Distribution of milk samples (pieces) into different acetone content classes 15 Microtiter plate Fia

Category acetone 123 4 5123 4 5 mg / 100 ml 1> 2.0 81 9 2 1.0-2.0 31 31 •. *. 3 0.5 - 1.0 1 6 2 1 6 2 eeer ··· · 20 4 0.25 - 0.5 35 17 37 is 5 <0.25 30 61 17 74 • e · 9 m · • · · ...

• · .....

• ·:. *. According to the method of the invention, the samples fell into the correct class ee "e! *: ·!! As follows: Class 1, 89%; Class 2, 75 25%; Class 3, 67%; Class 4, 67%; and Class 5, 67%. - .. for practical applications, the samples were divided into three levels, ie • · "level I with an acetone content> 1 mg / 100 ml; level II, V: 0.5-1.0 mg / 100 ml; and level III, <0.5 mg / 100 ml. In this case 92 · * · ’:% of level I samples; 67% of the level II samples and 100% e e of the level III samples fell into the correct category according to the invention. * For livestock monitoring samples which • e · .....

are milk samples representative of the whole herd, operating limit op 1.0 mg / 100 ml acetone / 100 ml milk. If the acetone content _ _. , rv .. - | Ί, - -, Γ ^ ιρττ / —i-fj-t J · * -T - ^ -. > iiWS ι ϊΐίίΓΐ <Α £ ί ·· * ί'ΐ "'·" ί: - **' ^ ί * '98864 9 exceeds this, individual milk samples per cow shall be taken from the herd, giving a concentration range of 2,5 to 5,0 mg / 100 ml indicates latent acetone disease. . __

The results were also statistically treated with the following 5 results:

Repeatability and recovery XäSaJL Level 2 Iasg-3 X 0.52 1.22 1.98 diff. 0.097 0.080 0.093 10 CV% 18.5 6.6 4.7 N 8 8 8 range 0.4-0.6 1.0-1.4 1.8-2.1

Regression 15 GC / Fia GC / microtiter

Correlation coefficient 0.96 0.92

The equation of the line y = 1.009x-0.03 y = 0.923x + 0.02

x = * GC

y = Fia / microtiter 20 • e e · | In time, the method according to the invention was the most advantageous. The GC method was able to analyze about 90 samples / day, the FIA method about 100 samples / day and the microtiter application about 300 samples / day.

: 25 Example 4 ··· · · * · *; The effect of the indicator and hydroxylammonium chloride on the limits of determination of the acetone content was investigated.

; ·. The indicator strain as a solution was 0.01% bromophenolsin • ·· '... t ...... and the absorbent 1 solution was 2% hydroxylammonium chloride.

*. 30 The results show e.g. that the lower the hydroxylammonium chloride concentration, the more sensitive the method of determination and ...; is. This is shown in Table 3.

e ..... .....

e e e • e 9 • ee • e • · · • · «• e 10 98864

Table 3. Effect of indicator and hydroxyl iononium chloride subtex on specific limits of acetone concentration

Hydroxylammonium chloride concentration 51 0.007M 7.5 ml indicator + 2.5 ml hydroxylammonium chloride / 100 ml HjOj limit of determination> 0.3 mg / 100 ml acetone II 0.014K 7.5 ml indicator + 5.0 ml hydroxyl sylammonium chloride / 100 ml Hp; assay and> 0.5 mg / 100 ml acetone III 0.029M 7.5 ml indicator + 10 ml hydroxylammonium chloride / 100 mlHp; limit of determination> 0.8 mg / 100 ml acetone IV 0.043M 7.5 ml indicator + IS ml hydroxyethylammonium chloride / 100 ml Hp; limit of determination> 1,5 mg / 100 ml of acetone m m - e · • e e> ·· e e e e e · »» • e e "'• e ......

m * e .....

• e • · • '· m e e • · e ··· e ···· "·.

• e e • · e • »e • e • # · ·· ♦ · · · s e e; ·. ··· • ·· · • e · • e. · E ·» · • · · .....

• * · • · · ...... - - -.....

··· .....

e e • · e • e · e · il

J

Claims (10)

A method for determining acetone down the gas diffusion method, wherein the acetone is evaporated from a sample conducted in an absorption liquid, where it causes a color change to be observed, characterized in that the sample and the absorption liquid are placed roughly each other. that they are separated by a gently closed gas effluent. 10 2. A method according to claim 1, characterized in that the sample to be examined is milk. 3. A method according to claim 1 or 2, characterized in that the sample is transferred into the gel furnace, bonded to a carrier or separated by an acetone-nonsurable substance. 4. A process according to claim 1, characterized in that, by means of a pH indicator, the change of the pH as the acetone causes the absorption liquid to determine. •. ·. 5. A process as claimed in any one of claims 1-4, characterized in that said nanosorption liquid uses a hydroxylanine solution in which said nanophenolphenolate sonic pH indicator is added. : 25 6. A method according to any one of claims 1 to 5, characterized in that the sample and the absorbent liquid are placed in the nitrite plates, so that each other is inserted into each other and to each other which puts an end perforated seal. 30 7, A method according to claim 6, characterized in that the absorbent liquid and the pH-indi- cator son are added therein placed in the lower plate and the sample in the upper plate. IN : Eighth A method according to claim 7, characterized in that the color change of the color indicator in the s 98864 absorbent liquid is read with an automatic plate reader. Test apparatus for determining acetone by the gas-diffusion method, wherein the acetone evaporating from a sample is passed into an absorption liquid, where it causes a color change to be detected, characterized in that it comprises a vessel in which the absorption liquid is located. say, that the absorbent liquid and sample may be placed Λ against each other such that they are separated by a jointly closed gas space. 10. Test apparatus according to claim 9, characterized in that the vessel consists of microtiter plates which are set against each other and an appropriately perforated seal which is inserted between them. • »» e • «· * ·· ·. • «•• e ·· e m. ·. : • e »......... ·» ·. • · .... * · E • m • · • · • ♦ e * · · ·· * ♦ ··· • * e • · · • · • # ·. 9 ··· • · · · · · ··· • · '* · e ·· ··· ... ...... • · · · · «« e ·% · · • ·· • ·