The Feedingstuffs (Sampling and Analysis) (Amendment) Regulations 1994

• Citation: S.I. 1994/1610
• Sections: 139

Section 1 Title, commencement and interpretation

(1) The Regulations may be cited as the Feedingstuffs (Sampling and Analysis) (Amendment) Regulations 1994 and shall come into force on 11th July 1994.

(2) In these Regulations “the principal Regulations ” means the Feedingstuffs (Sampling and Analysis) Regulations 1982 .

Section 2 Amendment of the principal Regulations

The principal Regulations are hereby further amended in accordance with regulation 3 below.

Section 3 Amendment of the principal Regulations

In Schedule 2 ( METHODS OF ANALYSIS )—

(a) in paragraph 3 (Methods of Analysis)—

(i) item 16 shall be deleted,

(ii) for item 26a there shall be substituted the following item:

(26a) Aflatoxin B 1 — one-dimensional thin layer chromatographic method

(iii) for item 26b there shall be substituted the following item:

(26b) Aflatoxin B 1 — high performance liquid chromatographic method

(b) for the provisions relating to method 4: Protein, there shall be substituted the provisions set out in Schedule 1 to these Regulations;

(c) for the provisions relating to method 9: Fibre, there shall be substituted the provisions set out in Schedule 2 to these Regulations;

(d) the provisions contained in method 16: Copper — Diethyldithiocarbamate spectrophotometric method, shall be deleted;

(e) in method 26a: Aflatoxin B 1 — for certain straight feedingstuffs—

(i) for the heading and paragraph 1 there shall be substituted the following provisions:

26a. AFLATOXIN B 1 — ONE-DIMENSIONAL THIN LAYER CHROMATOGRAPHIC METHOD

Scope and field of application

(1) This method is for the determination of aflatoxin B 1 in raw materials and straight feedingstuffs. This method is not applicable to raw materials and straight feedingstuffs containing citrus pulp. The lower limit of determination is 0.01 mg/ kg (10 ppb). In the presence of interfering substances it is necessary to repeat the analysis using method 26b (high performance liquid chromatography).

(ii) in paragraph 5.0 (Defatting) for the word “before” there shall be substituted the word “after”;

(f) for the provisions relating to method 26b: Aflatoxin B 1 — for all other feedingstuffs, there shall be substituted the provisions set out in Schedule 3 to these Regulations.

Section 1 Scope and Field of Application

This method is for the determination of the protein content of feedingstuffs on the basis of the nitrogen content, determined according to the Kjeldahl method.

Section 2 Principle

The sample is digested by sulfuric acid in the presence of a catalyst. The acidic solution is made alkaline with sodium hydroxide solution. The ammonia is distilled and collected in a measured quantity of sulfuric acid, the excess of which is titrated with a standard solution of sodium hydroxide.

Section 3.1 Reagents

Potassium sulfate.

Section 3.2 Reagents

Catalyst: copper ( II ) oxide CuO or copper (II) sulfate pentahydrate, CuSO 4 — 5H 2 O.

Section 3.3 Reagents

Granulated zinc.

Section 3.4 Reagents

Sulfuric acid, ρ 20 = 1.34 g/ml.

Section 3.5 Reagents

Sulfuric acid c(½H 2 SO 4 ) = 0.5 mol/l.

Section 3.6 Reagents

Sulfuric acid c(½H 2 SO 4 ) = 0.1 mol/l.

Section 3.7 Reagents

Methyl red indicator; dissolve 300 mg of methyl red in 100 ml of ethanol, σ = 95-96% (v/v).

Section 3.8 Reagents

Sodium hydroxide solution (Technical grade may be used) β = 40 g/100 ml (m/v: 40%).

Section 3.9 Reagents

Sodium hydroxide solution c = 0.25 mol/l.

Section 3.10 Reagents

Sodium hydroxide solution c = 0.1 mol/l.

Section 3.11 Reagents

Granulated pumice stone, washed in hydrochloric acid and ignited.

Section 3.12 Reagents

Acetanilide (m.p. = 114deg;C, N = 10.36%).

Section 3.13 Reagents

Sucrose (nitrogen free).

Section 4 Apparatus

Apparatus suitable for performing digestion, distillation and titration according to the Kjeldahl procedure.

Section 5.1 Digestion.

Weigh 1 g of the sample to the nearest 0.001 g and transfer the sample to the flask of the digestion apparatus. Add 15 g of potassium sulfate (3.1.), an appropriate quantity of catalyst (3.2.) (0.3 to 0.4 g of copper (II) oxide or 0.9 to 1.2 g of copper (II) sulfate pentahydrate), 25 ml of sulfuric acid (3.4.) and a few granules of pumice stone (3.11.) and mix. Heat the flask moderately at first, swirling from time to time if necessary until the mass has carbonized and the foam has disappeared; then heat more intensively until the liquid is boiling steadil y. Heating is adequate if the boiling acid condenses on the wall of the flask. Prevent the sides from becoming overheated and organic particles from sticking to them. When the solution becomes clear and light green continue to boil for another two hours, then leave to cool.

Section 5.2 Distillation.

Add carefully enough water to ensure complete dissolution of the sulfates. Allow to cool and then add a few granules of zinc (3.3.).

Place in the collecting flask of the distillation apparatus an exactly measured quantity of 25 ml of sulfuric acid (3.5.) or (3.6.) depending on the presumed nitrogen content. Add a few drops of methyl red indicator (3.7.).

Connect the digestion flask to the condenser of the distillation apparatus and immerse the end of the condenser in the liquid contained in the collecting flask to a depth of at least 1 cm (see observation 8.3.). Slowly pour 100 ml of sodium hydroxide solution (3.8.) into the digestion flask without loss of ammonia (see observation 8.1.).

Heat the flask until the ammonia has distilled over.

Section 5.3 Titration.

Titrate the excess sulfuric acid in the collecting flask with sodium hydroxide solution (3.9.) or (3.10.) depending on the concentration of the sulfuric acid used, until the end point is reached.

Section 5.4 Blank test.

To confirm that the reagents are free from nitrogen, carry out a blank test (digestion, distillation and titration) using 1 g of sucrose (3.13.) in place of the sample.

Section 6 Calculation of results

The content of protein is calculated according to the following formula:

Where,

V 0 = Volume (ml) of NaOH (3.9. or 3.10.)

V 1 = Volume (ml) of NaOH (3.9. or 3.10.) used in the sample titration.

c = Concentration (mol/l) of sodiumm hydroxide (3.9 or 3.10).

m = Mass (g) of sample.

Section 7.1 Repeatability.

The difference between the results of two parallel determinations carried out on the same sample must not exceed:

0.2% in absolute value, for protein contents of less than 20%;

1.0% relative to the higher value, for protein contents from 20% to 40%;

0.4% in absolute value, for protein contents of more than 40%.

Section 7.2 Accuracy.

Carry out the analysis (digestion, distillation and titration) on 1.5 to 2.0 g of acetanilide (3.12.) in the presence of 1 g of sucrose (3.13.); 1 g acetanilide consumes 14.80 ml of sulfuric acid (3.5.). Recovery must be at least 99%.

Section 8.1 Observations

Apparatus may be of the manual, semi-automatic or automatic type. If the apparatus requires transference between the digestion and distillation steps, this transfer must be carried out without loss. If the flask of the distillation apparatus is not fitted with a dropping funnel, add the sodium hydroxide immediately before connecting the flask to the condenser, pouring the liquid slowly down the side.

Section 8.2 Observations

If the digest solidifies, recommence the determination using a larger amount of sulfuric acid (3.4.) than that specified above.

Section 8.3 Observations

For products with a low nitrogen content, the volume of sulfuric acid (3.6.) to be placed in the collecting flask may be reduced, if necessary, to 10 or 15 ml and made up to 25 ml with water.

Section 1 Scope and Field of Application

This method is for the determination of the content in feedingstuffs of fat-free organic substances which are insoluble in acid and alkaline media and are conventionally described as fibre.

Section 2 Principle

The sample, defatted where necessary, is treated successively with boiling solutions of sulfuric acid and potassium hydroxide of specified concentrations. The residue is separated by filtration on a sintered-glass filter washed, dried, weighed and ashed within a range of 475 to 500° C. The loss of weight resulting from ashing corresponds to the fibre present in the test sample.

Section 3.1 Reagents

Sulfuric acid, (c(½H 2 SO 4 ) = 0.13 mol/l).

Section 3.2 Reagents

Anti-foaming agent (e.g. n—octanol).

Section 3.3 Reagents

Filter aid (Celite 545 or equivalent), heated at 500° C for four hours (8.6.).

Section 3.4 Reagents

Acetone.

Section 3.5 Reagents

Light petroleum, boiling-range 40 to 60° C.

Section 3.6 Reagents

Hydrochloric acid, (c = 0.5 mol/l).

Section 3.7 Reagents

Potassium hydroxide solution, (c equals; 0.23 mol/l).

Section 4.1 Apparatus

Heating unit for digestion with sulfuric acid or potassium hydroxide solution, equipped with a support for the filter crucible (4.2.) and provided with an outlet tube with a tap to the liquid outlet and vacuum, possibly with compressed air. Before use each day preheat the unit with boiling water for five minutes.

Section 4.2 Apparatus

Glass filter crucible with fused sintered-glass filter plate (pore size 40-90μm). Before first use, heat to 500° C for a few minutes and cool (8.6.).

Section 4.3 Apparatus

Cylinder of at least 270 ml with a reflux condenser, suitable for boiling.

Section 4.4 Apparatus

Drying oven with thermostat, to a temperature of 130 ± 5° C.

Section 4.5 Apparatus

Muffle furnace with thermostat, to a temperature in the range 475 to 500° C.

Section 4.6 Apparatus

Extraction unit consisting of a support plate for the filter crucible (4.2.) and with a discharge pipe with a tap to the vacuum and liquid outlet.

Section 4.7 Apparatus

Connecting rings to assemble the heating unit (4.1.), crucible (4.2.) and cylinder (4.3.) and to connect the cold extraction unit (4.6.) and crucible.

Section 5 Procedure

Weigh out to the nearest 0.001 g, 1 g of the prepared sample and place in the crucible (4.2.), (see observations 8.1., 8.2. and 8.3.) and add 1 g of filter aid (3.3.).

Assemble the heating unit (4.1.) and the filter crucible (4.2.), then attach the cylinder (4.3.) to the crucible. Pour 150 ml of boiling sulfuric acid (3.1.) into the assembled cylinder and crucible and if necessary add a few drops of anti-foaming agent (3.2.).

Bring the liquid to the boil within 52 minutes and boil vigorously for exactly 30 minutes.

Open the tap to the discharge pipe (4.1.) and, under vacuum, filter the sulfuric acid through the filter crucible and wash the residue with three consecutive 30 ml portions of boiling water, ensuring that the residue is filtered dry after each washing.

Close the outlet tap and pour 150 ml of boiling potassium hydroxide solution (3.7.) into the assembled cylinder and crucible and add a few drops of anti-foaming agent (3.2.). Bring the liquid to boiling point within 5 ± 2 minutes and boil vigorously for exactly 30 minutes. Filter and repeat the washing procedure used for the sulfuric acid step.

After the final washing and drying, disconnect the crucible and its contents and reconnect it to the cold extraction unit (4.6.). Apply the vacuum and wash the residue in the crucible with three consecutive 25 ml portions of acetone (3.4.) ensuring that the residue is filtered dry after each washing.

Dry the crucible to constant weight in the oven at 130° C. After each drying cool in the desiccator and weigh rapidly. Place the crucible in a muffle furnace and ash to constant weight at 475C to 500° C for at least 30 minutes.

After each heating cool first in the furnace and then in the desiccator before weighing.

Carry out a blank test without the sample. Loss of weight resulting from ashing must not exceed 4 mg.

Section 6 Calculation of results

The fibre content as a percentage of the sample is given by the expression:

where

a = mass of sample in g;

b = loss of mass after ashing during the determination, in g;

c = loss of mass after ashing during the blank test, in g.

Section 7 Repeatability

The difference between two parallel determinations carried out on the same sample must not exceed:

0.3 in absolute value for fibre contents lower than 10%,

3% relative to the higher result, for fibre contents equal to or greater than 10%.

Section 8.1 Observations

Feedingstuffs containing more than 10% fat must be defatted prior to analysis with light -petroleum (3.5.). Connect the filter crucible (4.2.) and its contents to the cold extraction unit (4.6.) and apply vacuum and wash the residue with three consecutive 30 ml portions of light petroleum, ensuring that the residue is dry. Connect the crucible and its contents to the heating unit (4.1.) and continue as described under 5.

Section 8.2 Observations

Feedingstuffs containing more than 10% fat after extraction with light petroleum (3.5.) must be defatted as shown in 8.1. and then defatted once more after boiling with acid.

After boiling with acid and the subsequent washing connect the crucible and its contents to the cold extraction unit (4.6.) and wash three times with 30 ml acetone followed by three further washings with 30 ml portions of light petroleum. Filter under vacuum until dry and continue the analysis as described under 5, beginning with potassium hydroxide treatment.