# The Feeding Stuffs (Sampling and Analysis) Regulations 1999, Paragraph 5: Procedure


Hydrolysis
5.1 Weigh to the nearest 0.001 g, between 3 and 7 g of the prepared sample containing about 0.2 g of isobutylidenediurea into a concical flask (4.1). Add 100 ml buffer solution (3.4) and 20.0 ml toluene (3.1) to the sample and place in the flask a magnetic bar. Stopper firmly to ensure that the flask remains tightly closed during the hydrolysis.
Place the flask in the water bath (4.5) and stir vigorously for 20 minutes. Remove the flask and immerse in an ice-water bath for 5 minutes. Add 15 g sodium sulphate (3.2) and 5.0 ml internal standard solution (3.6) to the contents of the flask. Stopper the flask again, shake, return to the water bath (4.5) and warm for 3 minutes with stirring. Cool in the ice-water bath for 5 minutes. Transfer slowly between 15 and 25 ml of the mixture to the centrifuge tube (4.2), stopper, and centrifuge for 5 minutes to separate the layers. (Repeat the transfer if insufficient toluene is decanted). Transfer a portion of the upper (toluene) layer to a test tube with a pasteur pipette.
Determination
5.2 Inject between 0.5 and 1.0 μl of the toluene solution (5.1) into the gas chromatograph (4.3).
Suggested conditions:
Column
70iC
Nitrogen
40 ml per minute
Injection
150iC
Hydrogen
30 ml per minute
Detector
150iC
Air
370 ml per minute
Approximate retention times:
Isobutyraldehyde
1 min.
Internal standard
1.5 min.
Toluene
3 min.
Measure the peak heights of the isobutyraldehyde and internal standard. Calculate the peak height ratio, isobutyraldehyde/internal standard, and from this value determine the quantity of isobutylidenediurea present by reference to the calibration curve (5.3).
Calibration curve
5.3 Weigh to the nearest mg, 100, 200 and 300 mg isobutylidenediurea (3.5) into three conical flasks (4.1). Add 100 ml buffer solution (3.3), 20.0 ml toluene (3.1) and a magnetic bar to each. Stopper the flasks firmly. Continue as in 5.1 from “… Place the flask in the water bath …”. Inject the toluene solutions into the gas chromatograph (4.3), and measure the peak heights. Calculate the peak height ratios, isobutyraldehyde/internal standard, and plot the calibration curve using peak height ratios as the ordinates and the corresponding weights or isobutylidenediurea as the abseissae.

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Source: https://www.legislation.gov.uk/uksi/1999/1663/schedule/2/part/5/paragraph/5 (the official version)
Page: https://openlegislation.uk/feeding-stuffs-sampling-and-analysis-regulations-1999/schedule-2/part-5/paragraph-5
Contains public sector information licensed under the Open Government Licence v3.0. Source: legislation.gov.uk, The National Archives.