BS ISO 4134:2021 pdf download – Meat and meat products — Determination of L-(+)- glutamic acid content — Reference method
7.5 Precision
7.5.1 Interlaboratory test
The precision of the method was established by an interlaboratory test carried out in accordance with ISO 5725-1 and ISO 5725-2. The results of these tests have been published (see Reference [4]). The values derived from these tests may not be applicable to concentration ranges and matrices types other than those given.
7.5.2 Repeatability
The value less than or equal to which the absolute difference between two test results obtained under repeatability conditions can be expected to be 0,55 % with a probability of 95 % of cases exceeding 0,159 2 g/100 g up to 0,305 0 g/100 g for L-(+)-glutamic acid contents.
7.5.3 Reproducibility
The value less than or equal to which the absolute difference between two test results obtained under reproducibility conditions can be expected to be 0,82 % with a probability of 95 % of cases exceeding 0,159 2 g/100 g up to 0,305 0 g/100 g for L-(+)-glutamic acid contents.
7.6 Detection limit
When W m (the moisture content of test sample in 7.3.4.4) is 70 and V 1 /50 (the dilution factor of filtrate taken in 7.3.3.6) is 1/15, the detection limit is 0,02 g/100 g of L-(+)-glutamic acid contents.
8 Test method of light absorption microplate reader
8.1 Reagents
The reagents are the same as those used in 7.1.
8.2 Apparatus
In addition to the equipment from 7.2.1 to 7.2.9, the following apparatus should be also included.
8.2.1 Single channel or multi-channel transferring pipettes and tips, of capacities 1 000 μl, 200 μl,100 μl and 20 μl conforming to ISO 8655-2.
8.2.2 Light absorption microplate reader, of wavelength 490 nm.
8.2.3 Microplate, of 96 holes (recommended), flat and transparent.
8.2.4 Brown glass vial, with caps, of volume not less than 1,5 ml.
8.3 Procedure
8.3.1 General
If it is required to check whether the repeatability requirement is achieved, two separate determinations
should be carried out.
8.3.2 Extraction of L-(+)-glutamic acid in the test portion
The extraction method is the same as 7.3.2 and 7.3.3.
8.3.3 Determination
8.3.3.1 Preparation of detection instrument
Set up the light absorption microplate reader (8.2.2), preheat the instrument according to the instrument specification until equilibrium conditions achieved. Set the detection parameters: the detection wavelength of 490 nm, the microplate reading mode by row and by hole, and the reading time interval of approximately 0,5 s per hole.
8.3.3.2 Absorbance determination
8.3.3.2.1 Maintain the temperature of solution R123 (7.1.6), water, the L-(+)-glutamic acid series standard solution (7.1.10), the L-(+)-glutamic acid test solution (8.3.2) between 20 °C and 25 °C. Pipette 300 μl of solution R123 (7.1.6) and 600 μl of water into a brown glass vial with caps (8.2.4). Mix by inverting or shaking the vial slowly. The solution obtained is the blank solution. Pipette 300 μl of solution R123 (7.1.6), 540 μl of water and 60 μl of the L-(+)-glutamic acid test solution (8.3.2) into a brown glass vial (8.2.4). Mix by inverting or shaking the vial slowly. The solution obtained is the test solution. Pipette 300 μl of solution R123 (7.1.6), 540 μl of water and 60 μl of the L-(+)-glutamic acid series standard solution (7.1.10) into a brown glass vial (8.2.4). Mix by inverting or shaking the vial slowly. The solution obtained is the standard solution.
8.3.3.2.2 Pipette 200 μl of the blank solution, the standard solution and the test solution (8.3.3.2.1) into different holes of the same microplate (8.2.3). Put the microplate into the light absorption microplate reader and swing for 5 s to 10 s. Read the absorbance A b3 of the blank solution, A s2 of the L-(+)-glutamic acid series standard solution and A 2 of the L-(+)-glutamic acid test solution at a wavelength of 490 nm.
8.3.3.2.3 Pipette 10 μl of the GLDH solution (7.1.7) into each of the brown glass vials in 8.3.3.2.1. Mix by inverting or shaking the vial slowly.
Take the microplate out and pipette 200 μl of the solution from each brown glass vial into different holes. Each vial is recommended to be determined three times and the numerical average can be used as the test result.