AGROSLUŽBY OLOMOUC, s.r.o.AGROSLUŽBY OLOMOUC, s.r.o.

Výzkumný ústav pro chov skotu Rapotín spol. s r.o.,
oddělení biotechnologických laboratoří, 788 13 Rapotín.
(The Research Institution for the horned cattle-breeding Rapotín, Ltd.
biotechnological laboratories division, 788 13 Rapotín.)

 

The analytical parameters of the automatic analyzer 

"ureakvant"

 evaluating the urea content  in a cow's milk

Vation of the method

In Rapotín: 29.05.1997

 

We carried out the validation of the method intended for the evaluation of the urea concentration in a cow's milk by an electro-chemical sensor, installed in the analyzer UREAKVANT

 

The description of the analyzer.

 

            The automatic analyzer UREAKVANT is intended for the quantitative evaluation of the urea in a cow's milk. It is designed for the routine processing of the higher number of analyses. It consists of electromechanical units, keyboard, display and printer. Analyzer is controlled by a microcomputer available to be attached to the other equipment through the communication lines.

 

Analytical principal

            The UREAKVANT analyzer function is based on the monitoring of the change rate in conductance of the sample during the decomposition of urea by the urease enzyme. The reaction proceeds in a tempered measuring cell. The basic component of the cell is a replaceable enzymic reactor.

 

The description of a function

            After the switch on the analyzer sets to the operating mode, carries out the autotest of all servo components and emerges itself in the dialog box of the display. After the temperature being stable (app. 10 minutes) it permits the further procedures.

            During the first putting into operation the calibration should be carried out by using five standard samples. The results of the calibration are presented to the user together with the final conclusion (System OK, resp. faults notifications). If the system is in good conditions the analyzer will permit the analysis of the milk samples.

            The correctness of the analysis is tested by the compulsory analysis of the check sample in which the urea concentration is already known. If the result of the check sample test does not meet the required range the further analysis of next samples will not be allowed.

            60 milk samples can be analysed in one series. Before the proper analysis the milk is diluted into an one-shot plastic micro test tubes. This action should be carried out on the removable circle carousel. The analyses are carried out only from the engaged positions on the carousel.

            The process of the analysis is indicated on the display. The printing of the results is provided by the external printer. After the series analysis of the samples on one carousel being finished the apparatus is automatically set to the stand-by mode and is ready for another analysis command.

 

Maintenance

            No special maintenance is required. The brief cleaning routine should be carried out after the whole-day operation. The replacement of the enzymic reactor (the wearable components) should be preceded by the utter-cleaning routine of the fluid and measuring system.

            The milk clotted samples or the samples containing mechanical impurities are impossible to be analysed.

 

The basic analytical parameters specified by the manufacturer.

Output .................................................              90 samples an hour

Reproducibility ....................................              up to 3 % in the whole linearity range

Standard deviation ...............................              ± 0.5 mg/dl

Guaranteed linearity for 3 % ................              6 - 60 mg/dl

Measurement range ..............................              1 - 120 mg/dl


 

The used material: - A set of calibration solutions supplied by the manufacturer in the concentration sequences: 12, 24, 36, 48 and 60 mg/dl, resp.:  2, 4, 6, 8 and 10 mmol/l.

Diluting solution supplied by the manufacturer.

 

 

i - calibration functions

 

          The calibration solutions has been diluted according to the instructions for operating the analyzer by a buffer solution. The calibration function is defined as a dependence of the measured relative conductance quantity "H" on the urea concentration in the diluted standard.

 

Urea concentration (mmol/l)

"H"

 0.2

 7746

0.4
0.6
0.8
1.0

17671
27057
33594
45398

 

Linear regression ...........                        H = 46915 [ mmol/l ] - 1335

Regression index ...........                        r = 0.997

SD   instruction B .........                        SB = 587, i.e. 1.25 % of the "H" value

 

 

ii - repeatability

 

          The repeatability was evaluated on a set of 20 samples on levels of: 14, 24, 47 mg/dl

 

 

average

mg/dl

SD

mg/dl

VK

%

R

mg/dl

R/average

%

low level

14.13

0.32

2.2

1.2

8.5

medium level

23.74

0.34

1.4

1.3

5.5

high level

47.44

0.68

1.4

2.9

6.1

 

 

iii - standard deviation of the method

 

17 parallel evaluations of different milk samples was carried out on levels 14 - 60 mg/dl.

 

the standard deviation of the method was stated according to the relation as:

 

S = 0.886 * 1/k S(Rimax  - Ri min)

 

k = 17

S(Rimax  - Ri min) = 9.4

S = 0.49 [ mg/dl ]


 

iv - impact of the preservation by bronopole

 18 random samples of the native breast milk was analysed. Each sample was then eked out with bronopol in concentration: 1 pastille for 25 ml of the solution. The urea concentration was evaluated again in these modified samples.

 

 

average

mg/dl

SD

mg/dl

VK

%

before the preservation

41.3

4.8

11.7

after the preservation

40.9

5.0

12.2

difference of averages

-0.4

2.3

5.6

 

Since the difference of averages before and after the preservation is considerably smaller than 2 SD, it could be regard as the insignificant one as to a statistics.

 

v - recovery

 The set formed by  9 native breast milk and 9 preserved milk random samples was tested. Each sample was analysed in doublets before and after the addition of 11 and 33 mg/dl of urea.

 

free of preservation

average

mg/dl

 

41.9

difference between the estimated and the actual value

recovery

%

with addition of 11 mg/dl

52.9

0

99.8

with addition of 33 mg/dl

74.9

0

99.9

 

 

 

 

 

preserved

average

mg/dl

 

28.7

difference between the estimated and the actual value

recovery

%

with addition of 11 mg/dl

39.7

0

100.6

with addition of 33 mg/dl

61.4

-0.3

99.3

 

 v - transfer  between samples

 In the first series a group of five samples with low concentration and then a group of five samples with high concentration were analysed. In the second series the same samples were analysed so that the high concentration sample followed the one with low concentration and vice versa. The transfer was stated as a quotient of the difference of averages from 1. and 2. series to the average from 1. series.

 

 

average from 1.series

mg/dl

average from 2.series mg/dl

transfer

%

low concentration

13.8

14.1

+ 2.2

high concentration

47.5

47.5

0


 

vii - comparison with the state-of-art method

 The results of evaluation of the urea in 17 native breast milk samples by a photometric method with Erlich´s reagent (the reference method) and by the UREAKVANT analyzer method (the present method) were rated according to a linear regression.

 

linear regression equation                        Y = 1.028 * X + 6.4 [ mg/dl ],

 

                        where

 

                        X = the reference method

                        Y = the present method

 

regression index                        r = 0.986

 

          The present method has been proved to correlate the reference method. The systematic shift had to be done due to the essential impossibility of using the same standards.

 

 

viii - conclusion

 

          The urea analysis carried out by the UREAKVANT apparatus in whole range of the observed parameters meets the standards required for the routine analyses in laboratories for its observation. It is an advisable method for a milk sample analysis. A possibility to apply the method to other biological fluids has not been tested. The distinguished aspect of the method can also be found in a possibility to analyse the milk preserved by bronopol with no impact on the results validity. The present method does not require any pre-modifications to the samples being analysed. We have detected the fact that the samples with milk clotted or the samples containing the mechanical impurities are impossible to be analysed. The method is fast and does not require any special training of the operating staff. At present the material costs are lower than those needed to the other state-of-art validated methods. The UREAKVANT apparatus enables to evaluate up to 900 samples a day. On basis of the results mentioned above and of our own experiences

 

we recommend the analyzer UREAKVANT for the routine evaluation of the urea in milk

 

The tests were carried out in two blocks: from 28.04. till 03.05.1997 and from 26.05. till 27.05.1997

 

 

In Rapotín 28.05.1997                        prepared by: Ing. Jaroslav FINCAR

 

 

Tests checked out by:

 

on behalf of the Milk Analysis Laboratory in Prerov                        Nìmèáková Hana

on behalf of VUCHS Rapotín                        Ing. Oto Hanuš

on behalf of the manufacturer                        RNDr. Milan Jílek, CSc.

 

Note: The original test reports are filed in VUSCH Rapotín, a copy is held by the manufacturer.


 

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