Commission Regulation (EC) No 152/2009 of 27 January 2009 laying down the methods of sampling and analysis for the official control of feed (Text with EEA relevance)

Where titration of the ammonia content of the distillate is performed manually, the procedure mentioned below applies. Where the distillation unit is fully automated to include titration of the ammonia content of the distillate, follow the manufacturer’s instructions for operation of the distillation unit.

Place a collecting flask containing 25 ml to 30 ml of the boric acid solution (point 3.18) under the outlet of the condenser in such a way that the delivery tube is below the surface of the excess boric acid solution. Adjust the distillation unit to dispense 50 ml of sodium hydroxide solution (point 3.9). Operate the distillation unit in accordance with the manufacturer’s instructions and distil off the ammonia liberated by the addition of the sodium hydroxide solution. Collect distillate in the boric acid receiving solution. The amount of distillate (time of steam distillation) depends on the amount of nitrogen in the sample. Follow the instructions of the manufacturer.

• Note: In a semi-automatic distillation unit, the addition of excess sodium hydroxide and the steam distillation are performed automatically.

5.3.   Titration

Proceed according to point 5.3.1 or 5.3.2.

5.3.1.   Sulphuric acid

Titrate the excess sulphuric acid in the collecting flask with sodium hydroxide solution (point 3.10 or 3.11) depending on the concentration of the sulphuric acid used, until the end point is reached.

5.3.2.   Boric acid

Titrate the contents of the collecting flask with the hydrochloric acid standard volumetric solution (point 3.19) or with the sulphuric acid standard volumetric solution (point 3.6) using a burette and read the amount of titrant used.

When colorimetric end-point detection is applied, the end-point is reached at the first trace of pink colour in the contents. Estimate the burette reading to the nearest 0,05 ml. An illuminated magnetic stirrer plate or a photometric detector may aid visualisation of the end-point.

This can be done automatically using a steam distiller with automatic titration.

Follow the manufacturers’ instructions for operation of the specific distiller or distiller/titrator.

• Note: When an automatic titration system is used, titration begins immediately after distillation starts and the 1 % boric acid solution (point 3.18) is used.

Where a fully automatic distillation unit is employed, the automatic titration of the ammonia can also be carried out with end-point detection using a potentiometric pH system.

In this case an automatic titrator with a pH-meter is used. The pH-meter shall be calibrated properly in the range of pH 4 to pH 7 following normal laboratory pH-calibration procedures.

The pH end-point of the titration is reached at pH 4,6, being the steepest point in the titration curve (inflection point).

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 (point 3.14) in place of the sample.

6. Calculation of results

Calculations are performed according to point 6.1 or 6.2.

6.1.   Calculation for titration according to point 5.3.1

The content of crude protein, expressed as a percentage by weight, is calculated according to the following formula:

Image omitted; see official source

where:

V0	=	is the volume (ml) of NaOH (point 3.10 or 3.11) used in the blank test
---	---	---
V1	=	is the volume (ml) of NaOH (point 3.10 or 3.11) used in the sample titration
---	---	---
c	=	is the concentration (mol/l) of sodium hydroxide (point 3.10 or 3.11)
---	---	---
m	=	is the weight (g) of sample.

6.2.   Calculation for titration according to point 5.3.2

6.2.1.   Titration with hydrochloric acid

The content of crude protein, expressed as a percentage by weight, is calculated according to the following formula:

Image omitted; see official source

where:

m	=	is the weight (g) of the test portion
---	---	---
c	=	is the concentration (mol/l) of the standard volumetric solution of the hydrochloric acid (point 3.19)
---	---	---
V0	=	is the volume (in ml) of hydrochloric acid used for the blank test
---	---	---
V1	=	is the volume (in ml) of hydrochloric acid used for the test portion.

6.2.2.   Titration with sulphuric acid

The content of crude protein, expressed as a percentage by weight, is calculated according to the following formula:

Image omitted; see official source

where:

m	=	is the weight (g) of the test portion
---	---	---
c	=	is the concentration (mol/l) of the standard volumetric solution of sulphuric acid (point 3.6)
---	---	---
V0	=	is the volume (in ml) of sulphuric acid (point 3.6) used for the blank test
---	---	---
V1	=	is the volume (in ml) of sulphuric acid (point 3.6) used for test portion.

7. Verification of the method

7.1.   Repeatability

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

• — 0,4 % in absolute value, for crude protein contents of less than 20 %,

• — 2,0 % relative to the higher value, for crude protein contents from 20 % to 40 %,

• — 0,8 % in absolute value, for crude protein contents of more than 40 %.

7.2.   Reproducibility

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

• — 1,8 % in absolute value, for crude protein contents of less than 20 %,

• — 9,0 % relative to the higher value, for crude protein contents from 20 % to 40 %,

• — 3,6 % in absolute value, for crude protein contents of more than 40 %.

7.3.   Accuracy

Carry out the analysis (digestion, distillation and titration) on an appropriate quantity of acetanilide (point 3.13) (e.g. 0,2 to 0,3 g) in the presence of 1 g of sucrose (point 3.14); 1 g acetanilide consumes 14,80 ml of sulphuric acid (point 3.5). Recovery must be at least 99 %.

8. Observations

• 8.1.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.

• 8.2.If the digest solidifies, recommence the determination using a larger amount of sulphuric acid (point 3.4) than that specified in point 5.1.

• 8.3.For products with a low nitrogen content, the volume of sulphuric acid (point 3.7) 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.

• 8.4.For routine analysis, alternative methods of analysis can be applied for the determination of crude protein but the Kjeldahl method described in this Part C is the reference method. The equivalence of the results obtained with the alternative method (e.g. DUMAS) compared to the reference method must be demonstrated for each matrix individually. As the results obtained with an alternative method, even after having verified the equivalency, might deviate slightly from the results obtained with the reference method, it is necessary to mention in the analytical report the method of analysis used for the determination of crude protein.

D. DETERMINATION OF UREA

1. Purpose and scope

This method makes it possible to determine the level of urea used as feed additive in ruminant feed.

2. Principle

The sample is suspended in water with a clarifying agent. The suspension is filtered. The urea content of the filtrate is determined after the addition of 4-dimethylaminobenzaldehyde (4-DMAB) by measuring the optical density at a wavelength of 420 nm.

3. Reagents

• 3.1.Solution of 4-dimethylaminobenzaldehyde: dissolve 1,6 g of 4-DMAB in 100 ml of 96 % ethanol and add 10 ml of hydrochloric acid (ρ20 1,19 g/ml). This reagent keeps for a maximum period of two weeks.

• 3.2.Carrez solution I: dissolve in water 21,9 g of zinc acetate, Zn(CH3COO)2 2H2O and 3 g of glacial acetic acid. Make up to 100 ml with water.

• 3.3.Carrez solution II: dissolve in water 10,6 g of potassium ferrocyanide, K4Fe(CN)6 3H2O. Make up to 100 ml with water.

• 3.4.Active carbon which does not absorb urea (to be checked).

• 3.5.Urea, 0,1 % solution (w/v).

4. Apparatus

• 4.1.Mixer (tumbler): approximately 35 to 40 rpm.

• 4.2.Test tubes: 160 × 16 mm with ground-glass stoppers.

• 4.3.Spectrophotometer.

5. Procedure

5.1.   Analysis of sample

Weigh out 2 g of the sample to the nearest mg and place with 1 g of active carbon (point 3.4) in a 500 ml volumetric flask. Add 400 ml of water and 5 ml of Carrez solution I (point 3.2), mix for approximately 30 seconds and add 5 ml of Carrez solution II (point 3.3). Mix for thirty minutes in the tumbler. Make up to volume with water, shake and filter.

Remove 5 ml of the transparent colourless filtrates, place in test tubes with ground-glass stoppers, add 5 ml of 4-DMAB solution (point 3.1) and mix. Place the tubes in a water bath at 20 °C (+/– 4 °C). After fifteen minutes measure the optical density of the sample solution with the spectrophotometer at 420 nm. Compare with the blank test solution of the reagents.

5.2.   Calibration curve

Remove volumes of 1, 2, 4, 5 and 10 ml of the urea solution (point 3.5), place in 100 ml volumetric flasks and make up the volume with water. Remove 5 ml from each solution, add 5 ml of 4-DMAB solution (point 3.1) to each of them, homogenise and measure the optical density as shown above in comparison with a control solution containing 5 ml of 4-DMAB and 5 ml of water free from urea. Plot the calibration curve.

6. Calculation of results

Determine the amount of urea in the sample using the calibration curve.

Express the result in mg urea per kg sample.

7. Evaluation of the method

7.1.   Repeatability

The difference between the results of two determinations carried out on the same sample in the same laboratory and by the same operator must not exceed:

• — At 420 nm: — 50 % relative to the higher value, for urea contents from 3 000  mg/kg to lower than 5 000  mg/kg, — 25 % relative to the higher value, for urea contents from 5 000  mg/kg to lower than 7 000  mg/kg, — 20 % relative to the higher value, for urea contents of 7 000  mg/kg or more.

• — At 435 nm: — 40 % relative to the higher value, for urea contents from 3 000  mg/kg to lower than 5 000  mg/kg, — 25 % relative to the higher value, for urea contents from 5 000  mg/kg to lower than 9 000  mg/kg, — 5 % relative to the higher value, for urea contents of 9 000  mg/kg or more.

7.2.   Reproducibility

The difference between the results of two determinations carried out on the same sample in different laboratories and/or by different operators must not exceed:

• — At 420 nm: — 3 000  mg/kg, in absolute value, for urea contents from 3 000  mg/kg to lower than 12 000  mg/kg, — 4 500  mg/kg, in absolute value, for urea contents of 12 000  mg/kg or more.

• — At 435 nm — 50 % relative to the higher value, for urea contents from 3 000  mg/kg to lower than 8 000  mg/kg, — 25 % relative to the higher value, for urea contents of 8 000  mg/kg or more.

8. Results of a collaborative study

An EU interlaboratory comparison exercise was organised in which 18 laboratories took part. Five positive ruminant compound feed samples (in Tables 1 and 2 referred to as MAT) were analysed (1 analysis) in blind duplicates while one blank compound ruminant feed was analysed once.

Calculations for repeatability (r) and reproducibility (R) limits as defined by international guidelines were carried out after the removal of outliers using Analysis of Variance of the valid values.

The calculated method performance figures (repeatability, reproducibility) are presented in the following tables. Over all tested samples including the blank material, no false positives or false negatives were found.

Table 1

Method performance characteristics for urea measured at λ = 420 nm in all materials

	MAT 2	MAT 5	MAT 3	MAT 4	MAT 6
	Sheep	Cattle	Sheep	Sheep	Cattle
Target mass fraction (mg kg-1)	3 000	5 000	7 001	9 036	11 000
Average mass fraction. (mg kg-1)	4 241	6 993	7 830	9 962	12 071
Reproducibility standard deviation sR (mg kg-1)	1 141	1 303	985	994	1 711
Repeatability standard deviation sr (mg kg-1)	723	601	549	712	737
Reproducibility relative standard deviation RSDR (%)	27	19	13	10	14
Repeatability relative standard deviation RSDr (%)	17	9	7	7	6
Limit of reproducibility, R [R = 2,8 × sR]	3 195	3 649	2 759	2 784	4 790
Limit of repeatability, r [r = 2,8 × sr]	2 024	1 684	1 536	1 994	2 064

Table 2

Method performance characteristics for urea measured at λ = 435 nm in all materials

	MAT 2	MAT 5	MAT 3	MAT 4	MAT 6
	Sheep	Cattle	Sheep	Sheep	Cattle
Target mass fraction (mg kg-1)	3 000	5 000	7 001	9 036	11 000
Average mass fraction. (mg kg-1)	4 101	6 467	7 890	10 062	11 642
Reproducibility standard deviation sR (mg kg-1)	706	1 194	675	745	1 378
Repeatability standard deviation sr (mg kg-1)	570	628	613	196	167
Reproducibility relative standard deviation RSDR (%)	17	18	9	7	12
Repeatability relative standard deviation RSDr (%)	14	10	8	2	1
Limit of reproducibility, R [R = 2,8 × sR]	1 977	3 344	1 889	2 087	3 859
Limit of repeatability, r [r = 2,8 × sr]	1 596	1 759	1 715	549	467

9. Observations

• 9.1.In the case of contents of urea exceeding 3 %, reduce the sample to 1 g or dilute the original solution so that there are not more than 50 mg of urea in 500 ml.

• 9.2.In the case of low contents of urea, increase the sample as long as the filtrate remains transparent and colourless.

• 9.3.The above results from collaborative trials do not indicate a significant difference in precision between urea measured at 420 nm or at 435 nm.

E. DETERMINATION OF AMINO ACIDS (EXCEPT TRYPTOPHAN)

The methods of analysis to be used for the determination of amino acids (except tryptophan) are:

• — EN ISO 13903 Animal feeding stuffs – Determination of amino acids content,

• — EN ISO 17180 Animal feeding stuffs – Determination of lysine, methionine, and threonine in commercial amino acid products and premixtures ([^E0030]),

• — the method of analysis as described in points 1 to 10 hereafter.

1. Purpose and scope

This method makes the determination possible of free (synthetic and natural) and total (peptide bound and free) amino acids in feed materials, compound feeds and premixtures containing less than 10 % ([^E0031]) of each amino acid, using an amino acid analyser. It is applicable to the following amino acids: cyst(e)ine, methionine, lysine, threonine, alanine, arginine, aspartic acid, glutamic acid, glycine, histidine, isoleucine, leucine, phenylalanine, proline, serine, tyrosine and valine.

The method does not distinguish between the various salts of amino acids and it cannot differentiate between D and L forms of amino acids. It is not valid for the determination of tryptophan or hydroxy analogues of amino acids.

2. Principle

2.1.   Free amino acids

The free amino acids are extracted with diluted hydrochloric acid. Co-extracted nitrogenous macromolecules are precipitated with sulfosalicylic acid and removed by filtration. The filtered solution is adjusted to pH 2,20. The amino acids are separated by ion exchange chromatography and determined by reaction with ninhydrin with photometric detection at 570 nm.

2.2.   Total amino acids

The procedure chosen depends on the amino acids under investigation. Cyst(e)ine and methionine must be oxidised to cysteic acid and methionine sulphone respectively prior to hydrolysis. Tyrosine must be determined in hydrolysates of unoxidised samples. All the other amino acids listed in point 1 (Purpose and scope) can be determined in either the oxidised or unoxidised sample.

Oxidation is performed at 0 °C with a performic acid/phenol mixture. Excess oxidation reagent is decomposed with sodium disulphite. The oxidised or unoxidised sample is hydrolysed with hydrochloric acid (point 3.20) for 23 hours. The hydrolysate is adjusted to pH 2,20. The amino acids are separated by ion exchange chromatography and determined by reaction with ninhydrin using photometric detection at 570 nm (440 nm for proline).

3. Reagents

Double distilled water or water of equivalent quality must be used (conductivity < 10 μS).

• 3.1.Hydrogen peroxide, w (w/w) = 30 %.

• 3.2.Formic acid, w (w/w) = 98–100 %.

• 3.3.Phenol.

• 3.4.Sodium disulphite.

• 3.5.Sodium hydroxide.

• 3.6.5-Sulfosalicylic acid dihydrate.

• 3.7.Hydrochloric acid, density approximately 1,18 g/ml.

• 3.8.tri-Sodium citrate dihydrate.

• 3.9.2,2'-Thiodiethanol (thiodiglycol).

• 3.10.Sodium chloride.

• 3.11.Ninhydrin.

• 3.12.Light petroleum, boiling range 40–60 °C.

• 3.13.Norleucine, or other compound suitable for use as internal standard.

• 3.14.Nitrogen gas (< 10 ppm oxygen).

• 3.15.1-Octanol.

• 3.16.Amino acids.

• 3.16.1.Standard substances of the amino acids listed under point 1 (Purpose and scope). Pure compounds containing no water of crystallisation. Dry under vacuum over P2O5 or H2SO4 for 1 week prior to use.

• 3.16.2.Cysteic acid.

• 3.16.3.Methionine sulphone.

• 3.17.Sodium hydroxide solution, c = 7,5 mol/l:

• Dissolve 300 g NaOH (point 3.5) in water and make up to 1 litre.

• 3.18.Sodium hydroxide solution, c = 1 mol/l:

• Dissolve 40 g NaOH (point 3.5) in water and make up to 1 litre.

• 3.19.Formic acid – phenol solution:

• Mix 889 g formic acid (point 3.2) with 111 g water and add 4,73 g phenol (point 3.3).

• 3.20.Hydrolysis mixture, c = 6 mol HCl/l containing 1 g phenol/l:

• Add 1 g phenol (point 3.3) to 492 ml HCl (point 3.7) and make up to 1 litre with water.

• 3.21.Extraction mixture, c = 0,1 mol HCl/l containing 2 % thiodiglycol: Take 8,2 ml HCl (point 3.7), dilute with approximately 900 ml water, add 20 ml thiodiglycol (point 3.9) and make up to 1 litre with water (do not mix points 3.7 and 3.9 directly).

• 3.22.5-Sulfosalicylic acid, ß = 6 %:

• Dissolve 60 g 5-sulfosalicylic acid (point 3.6) in water and make up to 1 l with water.

• 3.23.Oxidation mixture (Performic acid – phenol):

• Mix 0,5 ml hydrogen peroxide (point 3.1) with 4,5 ml formic acid-phenol solution (point 3.19) in a small beaker. Incubate at 20–30 °C for 1 hour in order to form performic acid, then cool on an ice-water bath (15 min) before adding to the sample.

• Caution: Avoid contact with skin and wear protective clothing.

• 3.24.Citrate buffer, c = 0,2 mol Na⁺/l, pH 2,20:

• Dissolve 19,61 g sodium citrate (point 3.8), 5 ml thiodiglycol (point 3.9), 1 g phenol (point 3.3) and 16,50 ml HCl (point 3.7) in approximately 800 ml water. Adjust pH to 2,20. Make up to 1 litre with water.

• 3.25.Elution buffers, prepared according to conditions for the analyser used (point 4.9).

• 3.26.Ninhydrin reagent, prepared according to conditions for the analyser used (point 4.9).

• 3.27.Standard solutions of amino acids. These solutions shall be stored below 5 ^oC.

• 3.27.1.Stock standard solution of amino acids (point 3.16.1).

• c = 2,5 μmol/ml of each in hydrochloric acid.

• May be obtained commercially.

• 3.27.2.Stock standard solution of cysteic acid and methionine sulphone, c = 1,25 μmol/ml.

• Dissolve 0,2115 g cysteic acid (point 3.16.2) and 0,2265 g methionine sulphone (point 3.16.3) in citrate buffer (point 3.24) in a 1 litre graduated flask and make up to mark with citrate buffer. Store below 5 °C for not more than 12 months. This solution is not used if the stock standard solution (point 3.27.1) contains cysteic acid and methionine sulphone.

• 3.27.3.Stock standard solution of the internal standard e.g. norleucine, c = 20 μmol/ml.

• Dissolve 0,6560 g norleucine (point 3.13) in citrate buffer (point 3.24) in a graduated flask and make up to 250 ml with citrate buffer. Store below 5 °C for no more than 6 months.

• 3.27.4.Calibration solution of standard amino acids for use with hydrolysates, c = 5 nmol/50 μl of cysteic acid and methionine sulphone and c = 10 nmol/50 μl of the other amino acids. Dissolve 2,2 g sodium chloride (point 3.10) in 100 ml beaker with 30 ml citrate buffer (point 3.24). Add 4,00 ml stock standard solution of amino acids (point 3.27.1), 4,00 ml stock standard solution of cysteic acid and methionine sulphone (point 3.27.2) and 0,50 ml stock standard solution of internal standard (point 3.27.3) if used. Adjust pH to 2,20 with sodium hydroxide (point 3.18).

• Transfer quantitatively to a 50 ml graduated flask and make up to the mark with citrate buffer (point 3.24) and mix.

• Store below 5 °C for not more than 3 months.

• See also observations point 9.1.

• 3.27.5.Calibration solution of standard amino acids for use with hydrolysates prepared according to point 5.3.3.1 and for use with extracts (point 5.2). The calibration solution is prepared according to point 3.27.4 but omitting sodium chloride.

• Store below 5 °C for not more than 3 months.

4. Apparatus

• 4.1.100 or 250 ml round-bottomed flask fitted with a reflux condenser.

• 4.2.100 ml borosilicate glass bottle with screw cap with rubber/teflon liner (e.g. Duran, Schott) for use in the oven.

• 4.3.Oven with forced ventilation and a temperature regulator with an accuracy better than ± 2 ^oC.

• 4.4.pH-meter (three decimal places).

• 4.5.Membrane filter (0,22 μm).

• 4.6.Centrifuge.

• 4.7.Rotary vacuum evaporator.

• 4.8.Mechanical shaker or magnetic stirrer.

• 4.9.Amino acid analyser or HPLC equipment with ion exchange column, device for ninhydrin, post column derivatisation and photometric detector.

• The column is filled with sulfonated polystyrene resins capable of separating the amino acids from each other and from other ninhydrin-positive materials. The flow in the buffer and ninhydrin lines is provided by pumps having a flow stability of ± 0,5 % in the period covering both the standard calibration run and the analysis of the sample.

• With some amino acid analysers hydrolysis procedures can be used in which the hydrolysate has a sodium concentration of c = 0,8 mol/l and contains all the residual formic acid from the oxidation step. Others do not give a satisfactory separation of certain amino acids if the hydrolysate contains excess formic acid and/or high sodium ion concentrations. In this case the volume of acid is reduced by evaporation to approx. 5 ml after the hydrolysis and prior to pH adjustment. The evaporation shall be performed under vacuum at 40 °C maximum.

5. Procedure

5.1.   Preparation of the sample

The sample is ground to pass through a 0,5 mm sieve. Samples high in moisture must be either air-dried at a temperature not exceeding 50 °C or freeze dried prior to grinding. Samples with a high fat content shall be extracted with light petroleum (point 3.12) prior to grinding.

5.2.   Determination of free amino acids

Weigh to the nearest 0,2 mg an appropriate amount (1-5 g) of the prepared sample (point 5.1), into a conical flask and add 100,0 ml of extraction mixture (point 3.21). Shake the mixture for 60 min using a mechanical shaker or a magnetic stirrer (point 4.8). Allow the sediment to settle and pipette 10,0 ml of the supernatant solution into a 100 ml beaker.

Add 5,0 ml of sulfosalicylic acid solution (point 3.22), with stirring and continue to stir with the aid of magnetic stirrer for 5 min. Filter or centrifuge the supernatant in order to remove any precipitate. Place 10,0 ml of the resulting solution into a 100 ml beaker and adjust the pH to 2,20 using sodium hydroxide solution (point 3.18), transfer to a volumetric flask of appropriate volume using citrate buffer (point 3.24), and make up to the mark with the buffer solution (point 3.24).

If an internal standard is being used add 1,00 ml of internal standard (point 3.27.3) for each 100 ml final solution and make up to the mark with the buffer solution (point 3.24).

Proceed to the chromatography step according to point 5.4.

If the extracts are not being examined the same day, they must be stored below 5 °C.

5.3.   Determination of total amino acids

5.3.1.   Oxidation

Weigh to the nearest 0,2 mg from 0,1 to 1 g of the prepared sample (point 5.1) into:

• — a 100 ml round-bottomed flask (point 4.1) for open hydrolysis (point 5.3.2.3), or

• — a 250 ml round-bottomed flask (point 4.1) if a low sodium concentration is required (point 5.3.3.1), or

• — a 100 ml bottle fitted with a screw cap (point 4.2) (for closed hydrolysis point 5.3.2.4).

The weighed sample portion must have a nitrogen content of about 10 mg and a moisture content not exceeding 100 mg.

Place the flask/bottle in an ice-water bath and cool to 0 °C, add 5 ml of oxidation mixture (point 3.23) and mix using a glass spatula with a bent tip. Seal the flask/bottle containing the spatula with an air-tight film, place the ice-water bath containing the sealed container in a refrigerator at 0 °C and leave for 16 hours. After 16 hours remove from the refrigerator and decompose the excess oxidation reagent by the addition of 0,84 g of sodium disulphite (point 3.4).

Proceed to point 5.3.2.1.

5.3.2.   Hydrolysis

• 5.3.2.1.Hydrolysis of oxidised samples

• To the oxidised sample prepared according to point 5.3.1 add 25 ml of hydrolysis mixture (point 3.20) taking care to wash down any sample residue adhering to the sides of the vessel and the spatula.

• Depending on the hydrolysis procedure being used, proceed according to point 5.3.2.3 or 5.3.2.4.

• 5.3.2.2.Hydrolysis of unoxidised samples

• Weigh into either a 100 ml or a 250 ml round-bottomed flask (point 4.1) or a 100 ml bottle fitted with a screw cap (point 4.2), to the nearest 0,2 mg, from 0,1 to 1 g of the prepared sample (point 5.1). The weighed sample portion must have a nitrogen content of about 10 mg. Add carefully 25 ml of hydrolysis mixture (point 3.20) and mix with the sample. Proceed according to either point 5.3.2.3 or point 5.3.2.4.

• 5.3.2.3.Open hydrolysis

• Add 3 glass beads to the mixture in the flask (prepared in accordance with point 5.3.2.1 or 5.3.2.2) and boil with continuous bubbling under reflux for 23 hours. On completion of hydrolysis, wash the condenser down with 5 ml of citrate buffer (point 3.24). Disconnect the flask and cool it in an ice bath.

• Proceed according to point 5.3.3.

• 5.3.2.4.Closed hydrolysis

• Place the bottle containing the mixture prepared in accordance with point 5.3.2.1 or 5.3.2.2 in an oven (point 4.3) at 110 °C. During the first hour in order to prevent a build up of pressure (due to the evolution of gaseous substances) and to avoid explosion, place the screw cap over the top of the vessel. Do not close the vessel with the cap. After one hour close the vessel with the cap and leave in the oven (point 4.3) for 23 hours. On completion of hydrolysis, remove the bottle from the oven, carefully open the cap of the bottle and place the bottle in an ice-water bath. Leave to cool.

• Depending on the procedure for pH adjustment (point 5.3.3), quantitatively transfer the contents of the bottle to a 250 ml beaker or a 250 ml round-bottomed flask, using citrate buffer (point 3.24).

• Proceed according to point 5.3.3.

5.3.3.   Adjustment of pH

Depending on the sodium tolerance of the amino acid analyser (point 4.9) proceed according to point 5.3.3.1 or 5.3.3.2 for the pH adjustment.

• 5.3.3.1.For chromatographic systems (point 4.9) requiring a low sodium concentration.

• It is advisable to use an internal stock standard solution (point 3.27.3) when amino acid analysers requiring a low sodium concentration are employed (when the acid volume has to be reduced).

• In this case add 2,00 ml of the internal stock standard solution (point 3.27.3) to the hydrolysate before the evaporation.

• Add 2 drops of 1-octanol (point 3.15) to the hydrolysate obtained in accordance with point 5.3.2.3 or 5.3.2.4.

• Using a rotary evaporator (point 4.7) reduce the volume to 5-10 ml under vacuum at 40 °C. If the volume is accidentally reduced to less than 5 ml the hydrolysate must be discarded and the analysis recommenced.

• Adjust the pH to 2,20 with sodium hydroxide solution (point 3.18) and proceed to point 5.3.4.

• 5.3.3.2.For all other amino acid analysers (point 4.9)

• Take the hydrolysates obtained in accordance with point 5.3.2.3 or 5.3.2.4 and partly neutralise them by carefully adding with stirring, 17 ml of sodium hydroxide solution (point 3.17), ensuring that the temperature is kept below 40 °C.

• Adjust the pH to 2,20 at room temperature using sodium hydroxide solution (point 3.17) and finally sodium hydroxide solution (point 3.18). Proceed to point 5.3.4.

5.3.4.   Sample solution for chromatography

Quantitatively transfer the pH adjusted hydrolysate (point 5.3.3.1 or 5.3.3.2) with citrate buffer (point 3.24) to a 200 ml graduated flask, and make up to the mark with buffer (point 3.24).

If an internal standard has not already been used, add 2,00 ml of internal standard (point 3.27.3) and make up to the mark with citrate buffer (point 3.24). Mix thoroughly.

Proceed to the chromatography step (point 5.4).

If the sample solutions are not being examined the same day they must be stored below 5 °C.

5.4.   Chromatography

Before chromatography bring the extract (point 5.2) or hydrolysate (point 5.3.4) to room temperature. Shake the mixture and filter a suitable amount through a 0,22 μm membrane filter (point 4.5). The resulting clear solution is subjected to ion exchange chromatography, using an amino acid analyser (point 4.9).

The injection may be performed manually or automatically. It is important that the same quantity of solution ± 0,5 % is added to the column for the analysis of standards and samples except when an internal standard is used, and that the sodium:amino acid ratios in the standard and sample solutions are as similar as is practicable.

In general, the frequency of calibration runs depends on the stability of the ninhydrin reagent and the analytical system. The standard or sample is diluted with citrate buffer (point 3.24) to give a peak area of the standard of 30–200 % of the sample amino acid peak area.

The chromatography of amino acids will vary slightly according to the type of analyser employed and resin used. The chosen system must be capable of separating the amino acids from each other and from the ninhydrin-positive materials. In the range of operation the chromatographic system must give a linear response to changes in the amounts of amino acids added to the column.

During the chromatography step the valley:peak height ratios mentioned below apply, when an equimolar solution (of the amino acids being determined) is analysed. This equimolar solution must contain at least 30 % of the maximum load of each amino acid which can be accurately measured with the amino acid analyser system (point 4.9).

For separation of threonine-serine the valley:peak height ratio of the lower of the two overlapping amino acids on the chromatogram must not exceed 2:10 (if only cyst(e)ine, methionine, threonine and lysine are determined, insufficient separation from adjoining peaks will adversely influence the determination). For all other amino acids the separation must be better than 1:10.

The system must ensure that lysine is separated from ‘lysine artefacts’ and ornithine.

6. Calculation of results

The area of the sample and standard peaks is measured for each individual amino acid and the amount (X), in g amino acid per kg sample, is calculated.

Image omitted; see official source

If an internal standard is used multiply by:

Image omitted; see official source

A	=	peak area, hydrolysate or extract
---	---	---
B	=	peak area, calibration standard solution
---	---	---
C	=	peak area, internal standard in hydrolysate or extract
---	---	---
D	=	peak area, internal standard, calibration standard solution
---	---	---
M	=	molar weight of the amino acid being determined
---	---	---
c	=	concentration of standard in μmol/ml
---	---	---
m	=	sample weight (g) (corrected to original weight if dried or defatted)
---	---	---
V	=	ml total hydrolysate (point 5.3.4) or ml calculated total dilution volume of extract (point 6.1).

Cystine and cysteine are both determined as cysteic acid in hydrolysates of oxidised sample, but calculated as cystine (C6H12N2O4S2, M 240,30 g/mol) by using M 120,15 g/mol (= 0,5 × 240,30 g/mol).

Methionine is determined as methionine sulphone in hydrolysates of oxidised sample, but calculated as methionine by using M of methionine: 149,21 g/mol.

Added free methionine is determined after extraction as methionine, for the calculation the same M is used.

• 6.1.The total dilution volume of extracts (F) for determination of free amino acids (point 5.2) is calculated as following:

Image omitted; see official source

V	=	Volume of final extract.

7. Evaluation of the method

The method has been tested in an intercomparison made at international level in 1990 using four different feeds (mixed pig feed, broiler compound, protein concentrate, premixture).

• Note: The method has been tested during a second international intercomparison study in 2003 by using blind duplicate pairs of broiler finisher feed, broiler starter feed, corn, fishmeal and poultry meal samples. For details see EN ISO 13903.

The results of 1990 intercomparison, after elimination of outliers, of mean and standard deviation are given in the tables in this point:

Means in g/kg

Reference Material	Amino Acid	Amino Acid	Amino Acid	Amino Acid
Reference Material	Threonine	Cyst(e)ine	Methionine	Lysine
Mixed Pig Feed	6,94 n = 15	3,01 n = 17	3,27 n = 17	9,55 n = 13
Broiler Compound Feed	9,31 n = 16	3,92 n = 18	5,08 n = 18	13,93 n = 16
Protein Concentrate	22,32 n = 16	5,06 n = 17	12,01 n = 17	47,74 n = 15
Premixture	58,42 n = 16	—	90,21 n = 16	98,03 n = 16
n = Number of participating laboratories.	n = Number of participating laboratories.	n = Number of participating laboratories.	n = Number of participating laboratories.	n = Number of participating laboratories.

7.1.   Repeatability

The repeatability expressed as ‘within laboratory standard deviation’ of the intercomparison of the previous table is given in the following table:

Coefficient of variation (%) for repeatability (CVr)

Reference Material	Amino Acid	Amino Acid	Amino Acid	Amino Acid
Reference Material	Threonine	Cyst(e)ine	Methionine	Lysine
Mixed Pig Feed	1,9 n = 15	3,3 n = 17	3,4 n = 17	2,8 n = 13
Broiler Compound Feed	2,1 n = 16	2,8 n = 18	3,1 n = 18	2,1 n = 16
Protein Concentrate	2,7 n = 16	2,6 n = 17	2,2 n = 17	2,4 n = 15
Premixture	2,2 n = 16	—	2,4 n = 16	2,1 n = 16
n = Number of participating laboratories.	n = Number of participating laboratories.	n = Number of participating laboratories.	n = Number of participating laboratories.	n = Number of participating laboratories.

7.2.   Reproducibility

The results for between laboratory standard deviation by the above mentioned intercomparison are given in the table below:

Coefficient of variation (%) for reproducibility (CVR)

Reference Material	Amino Acid	Amino Acid	Amino Acid	Amino Acid
Reference Material	Threonine	Cyst(e)ine	Methionine	Lysine
Mixed Pig Feed	4,1 n = 15	9,9 n = 17	7,0 n = 17	3,2 n = 13
Broiler Compound feed	5,2 n = 16	8,8 n = 18	10,9 n = 18	5,4 n = 16
Protein Concentrate	3,8 n = 16	12,3 n = 17	13,0 n = 17	3,0 n = 15
Premixture	4,3 n = 16	—	6,9 n = 16	6,7 n = 16
n = Number of participating laboratories.	n = Number of participating laboratories.	n = Number of participating laboratories.	n = Number of participating laboratories.	n = Number of participating laboratories.

8. Use of Reference Materials

The correct application of the method shall be verified by making replicate measurements of certified reference materials when available. Calibration with certified amino acid calibration solution is recommended.

9. Observations

• 9.1.Because of differences between amino acid analysers the final concentrations of the calibration solutions of standard amino acids (see points 3.27.4 and 3.27.5) and of the hydrolysate (see point 5.3.4) shall be taken as a guideline.

• The range of linear response of the apparatus has to be checked for all amino acids.

• The standard solution is diluted with citrate buffer to give peak areas in the middle of the range.

• 9.2.Where high performance liquid chromatographic equipment is used to analyse the hydrolysates, the experimental conditions must be optimised in accordance with the manufacturer’s recommendations.

• 9.3.By applying the method to compound feed or premixtures containing more than 1 % chloride (concentrate, mineral feeds, complementary feeds) underestimation of methionine could occur and special treatment shall be done.

10. Performance criteria

Compilation of the results (except for tyrosine) coming from the 2 collaborative studies (from 1990 reported in point 7 above and from 2005 reported in EN/ISO 13903) gives the following criteria for repetability and reproducibility. The values derived from these 2 interlaboratory tests may not be applicable to concentration ranges and matrices other than those given.

10.1.   Repeatability

The difference between the results of two determinations carried out on the same sample in the same laboratory and by the same operator must not exceed:

• — 6 % relative to the higher value, for total amino acids in case of glycine, alanine, lysine, proline, glutamic acid, isoleucine and histidine,

• — 8 % relative to the higher value, for total amino acids in case of threonine, phenylalanine, methionine, aspartic acid and leucine,

• — 10 % relative to the higher value, for total amino acids in case of arginine and valine,

• — 12 % relative to the higher value, for total serine amino acid,

• — 15 % relative to the higher value, for total cyst(e)ine amino acid.

10.2.   Reproducibility

The difference between the results of two determinations carried out on the same sample in different laboratories and/or by different operators must not exceed:

• — 15 % relative to the higher value, for total amino acids in case of glycine, alanine and threonine,

• — 20 % relative to the higher value, for total amino acids in case of lysine, proline, phenylalanine, methionine and aspartic acid,

• — 22 % relative to the higher value, for total amino acids in case of glutamic acid and leucine,

• — 27 % relative to the higher value for total arginine amino acid,

• — 32 % relative to the higher value, for total isoleucine amino acid,

• — 35 % relative to the higher value, for total amino acids in case of valine and serine,

• — 40 % relative to the higher value, for total histidine amino acid,

• — 50 % relative to the higher value, for total cyst(e)ine amino acid.

F. DETERMINATION OF TRYPTOPHAN

The methods of analysis to be used for the determination of tryptophan are:

• — EN ISO 13904 Animal feeding stuffs – Determination of tryptophan content,

• — the method of analysis as described in points 1 to 9 hereafter.

1. Purpose and scope

The method makes the determination possible of the total and free tryptophan in feed. It does not distinguish between D- and L- forms.

2. Principle

For the determination of the total tryptophan, the sample is hydrolysed under alkaline conditions with saturated barium hydroxide solution and heated to 110 °C for 20 hours. After hydrolysis internal standard is added.

For the determination of free tryptophan, the sample is extracted under mild acidic conditions in the presence of internal standard.

The tryptophan and the internal standard in the hydrolysate or in the extract are determined by HPLC with fluorescence detection.

3. Reagents

• 3.1.Double distilled water or water of equivalent quality must be used (conductivity < 10 μS/cm).

• 3.2.Standard substance: tryptophan (purity/content ≥ 99 %) dried under vacuum over phosphorous pentoxide.

• 3.3.Internal standard substance: α-methyl-tryptophan (purity/content ≥ 99 %), dried under vacuum over phosphorous pentoxide.

• 3.4.Barium hydroxide octa-hydrate (care shall be taken not to expose the Ba(OH)2 .8 H2O excessively to air in order to avoid formation of BaCO3, which could disturb the determination) (see observation point 9.3).

• 3.5.Sodium hydroxide.

• 3.6.Ortho-phosphoric acid, w (w/w) = 85 %.

• 3.7.Hydrochloric acid, ρ20 1,19 g/ml.

• 3.8.Methanol, equivalent to HPLC grade.

• 3.9.Light petroleum, boiling range 40–60 °C.

• 3.10.Sodium hydroxide solution, c = 1 mol/l:

• Dissolve 40,0 g NaOH (point 3.5) in water and make up to 1 litre with water (point 3.1).

• 3.11.Hydrochloric acid, c = 6 mol/l:

• Take 492 ml HCl (point 3.7) and make up to 1 litre with water.

• 3.12.Hydrochloric acid, c = 1 mol/l:

• Take 82 ml HCl (point 3.7) and make up to 1 litre with water.

• 3.13.Hydrochloric acid, c = 0,1 mol/l:

• Take 8,2 ml HCl (point 3.7) and make up to 1 litre with water.

• 3.14.Ortho-phosphoric acid, c = 0,5 mol/l:

• Take 34 ml ortho-phosphoric acid (point 3.6) and make up to 1 litre with water (point 3.1).

• 3.15.Concentrated solution of tryptophan (point 3.2), c = 2,50 μmol/ml:

• In a 500 ml volumetric flask dissolve 0,2553 g tryptophan (point 3.2) in hydrochloric acid (point 3.13) and make up to the mark with hydrochloric acid (point 3.13). Store at – 18 °C for a maximum of 4 weeks.

• 3.16.Concentrated internal standard solution, c = 2,50 μmol/ml:

• In a 500 ml volumetric flask dissolve 0,2728 g α-methyl-tryptophan (point 3.3) in hydrochloric acid (point 3.13) and make up to the mark with hydrochloric acid (point 3.13). Store at – 18 °C for a maximum of 4 weeks.

• 3.17.Calibration standard solution of tryptophan and internal standard:

• Take 2,00 ml concentrated solution of tryptophan (point 3.15), and 2,00 ml of concentrated internal standard (α-methyl-tryptophan) solution (point 3.16). Dilute with water (point 3.1) and methanol (point 3.8) to approximately the same volume and to approximately the same concentration of methanol (10-30 %) as the finished hydrolysate.

• This solution must be prepared freshly before use.

• Protect from direct sunlight during preparation.

• 3.18.Acetic acid.

• 3.19.1,1,1-trichloro-2-methyl-2-propanol.

• 3.20.Ethanolamine w (w/w) > 98 %.

• 3.21.Solution of 1 g 1,1,1-trichloro-2-methyl-2-propanol (point 3.19) in 100 ml methanol (point 3.8).

• 3.22.Mobile phase for HPLC: 3,00 g acetic acid (point 3.18) + 900 ml water (point 3.1) + 50,0 ml solution (point 3.21) of 1,1,1-trichloro-2-methyl-2-propanol (point 3.19) in methanol (point 3.8) (1 g/100 ml). Adjust pH to 5,00 using ethanolamine (point 3.20). Make up to 1 000  ml with water (point 3.1).

4. Apparatus

• 4.1.HPLC equipment with a spectrofluorometric detector.

• 4.2.Liquid chromatographic column, 125 mm × 4 mm, C18, 3 μm packing, or equivalent.

• 4.3.pH-meter.

• 4.4.Polypropylene flask, capacity 125 ml, with wide neck and screw cap.

• 4.5.Membrane filter, 0,45 μm.

• 4.6.Autoclave, 110 (± 2) °C, 1,4 (± 0,1) bar.

• 4.7.Mechanical shaker or magnetic stirrer.

• 4.8.Vortex mixer.

5. Procedure

5.1.   Preparation of samples

The sample is ground to pass through a 0,5 mm sieve. Samples high in moisture must be either air-dried at a temperature not exceeding 50 °C or freeze dried prior to grinding. Samples with high fat content shall be extracted with light petroleum (point 3.9) prior to grinding.

5.2.   Determination of free tryptophan (extract)

Weigh to the nearest 1 mg an appropriate amount (1-5 g) of the prepared sample (point 5.1), into a conical flask. Add 100,0 ml hydrochloric acid (point 3.13) and 5,00 ml concentrated internal standard solution (point 3.16). Shake or mix for 60 min. using a mechanical shaker or a magnetic stirrer (point 4.7). Allow the sediment to settle and pipette 10,0 ml of the supernatant solution into a beaker. Add 5 ml ortho-phosphoric acid (point 3.14). Adjust the pH to 3 using sodium hydroxide (point 3.10). Add sufficient methanol (point 3.8) to give a concentration of between 10 and 30 % of methanol in the final volume. Transfer to a volumetric flask of appropriate volume and dilute with water to a volume necessary for the chromatography (approx. the same volume as the calibration standard solution (point 3.17)).

Filter a few ml of the solution through a 0,45 μm membrane filter (point 4.5) before injection on the HPLC column. Proceed to the chromatography step according to point 5.4.

Protect standard solution and extracts against direct sunlight. If it is not possible to analyse the extracts the same day, the extracts may be stored at 5 °C for a maximum of 3 days.

5.3.   Determination of total tryptophan (hydrolysate)

Weigh to the nearest 0,2 mg from 0,1 to 1 g of the prepared sample (point 5.1) into the polypropylene flask (point 4.4). The weighed sample portion shall have a nitrogen content of about 10 mg. Add 8,4 g barium hydroxide octa-hydrate (point 3.4) and 10 ml water. Mix on a vortex mixer (point 4.8) or magnetic stirrer (point 4.7) Leave the teflon-coated magnet in the mixture. Wash down the walls of the vessel with 4 ml water. Put on the screw cap and close the flask loosely. Transfer to an autoclave (point 4.6) with boiling water and let it steam for 30-60 minutes. Close the autoclave and autoclave at 110 (± 2) °C for 20 hours.

Before opening the autoclave reduce the temperature to just under 100 °C. In order to avoid crystallisation of Ba(OH)2. 8 H2O, add to the warm mixture 30 ml water which is at room temperature. Shake or stir gently. Add 2,00 ml concentrated internal standard (α-methyl-tryptophan) solution (point 3.16). Cool the vessels on water/ice bath for 15 minutes.

Then, add 5 ml ortho-phosphoric acid (point 3.14). Keep the vessel in the cooling bath and neutralise with HCl (point 3.11) whilst stirring and adjust the pH to 3,0 using HCl (point 3.12). Add sufficient methanol to give a concentration of between 10 and 30 % of methanol in the final volume. Transfer to a volumetric flask of appropriate volume and dilute with water to the defined volume necessary for the chromatography (for example 100 ml). The addition of methanol shall not cause precipitation.

Filter a few ml of the solution through a 0,45 μm membrane filter (point 4.5) before injection on the HPLC column. Proceed to the chromatography step according to point 5.4.

Protect standard solution and hydrolysates against direct sunlight. If it is not possible to analyse the hydrolysates the same day, they may be stored at 5 °C for a maximum of 3 days.

5.4.   HPLC determination

The following conditions for isocratic elution are offered for guidance; other conditions may be used, provided they yield equivalent results (see also observations point 9.1 and 9.2):

Liquid chromatographic column (point 4.2):	125 mm × 4 mm, C18, 3 μm packing or equivalent
Column temperature:	Room temperature
Mobile phase (point 3.22):	3,00 g acetic acid (point 3.18) + 900 ml water (point 3.1) + 50,0 ml solution (point 3.21) of 1,1,1-trichloro-2-methyl-2-propanol (point 3.19) in methanol (point 3.8) (1 g/100 ml). Adjust pH to 5,00 using ethanolamine (point 3.20). Make up to 1 000  ml with water (point 3.1)
Flow rate:	1 ml/min
Total run time:	approx. 34 min
Detection wavelength:	excitation: 280 nm, emission: 356 nm.
Injection volume	20 μl.

6. Calculation of results

The amount of tryptophane (X), in g per 100g sample, is calculated.

Image omitted; see official source

A	=	peak area of internal standard, calibration standard solution (point 3.17)
---	---	---
B	=	peak area of tryptophan, extract (point 5.2) or hydrolysate (point 5.3)
---	---	---
V1	=	volume in ml (2 ml) of concentrated tryptophan solution (point 3.15) added to the calibration solution (point 3.17)
---	---	---
c	=	concentration in μmol/ml (= 2,50) of concentrated tryptophan solution (point 3.15) added to calibration solution (point 3.17)
---	---	---
V2	=	volume in ml of concentrated internal standard solution (point 3.16) added at the extraction (point 5.2) (= 5,00 ml) or to the hydrolysate (point 5.3) (= 2,00 ml)
---	---	---
C	=	peak area of internal standard, extract (point 5.2) or hydrolysate (point 5.3)
---	---	---
D	=	peak area of tryptophan, calibration standard solution (point 3.17)
---	---	---
V3	=	volume in ml (= 2,00 ml) of concentrated internal standard solution (point 3.16) added to calibration standard solution (point 3.17)
---	---	---
m	=	sample weight in g (corrected to original weight if dried and/or defatted)
---	---	---
M	=	molar weight of tryptophan (= 204,23 g/mol).

7. Repeatability

The difference between the results of two parallel determinations carried out on the same sample must not exceed 10 % relative to the highest result.

8. Results of a collaborative study

An EU collaborative study (4th intercomparison) was arranged in which three samples were analysed by up to 12 laboratories to certify the method for hydrolysis. Replicate (5) analyses were performed on each sample. The results are given in the following table:

	Sample 1 Pig feed	Sample 2 Pig feed supplemented with L-tryptophan	Sample 3 Feed concentrate for pigs
L	12	12	12
n Mean [g/kg]	50 2,42	55 3,40	50 4,22
sr [g/kg]	0,05	0,05	0,08
r [g/kg]	0,14	0,14	0,22
CVr [%]	1,9	1,6	1,9
SR [g/kg]	0,15	0,20	0,09
R [g/kg]	0,42	0,56	0,25
CVR [%]	6,3	6,0	2,2
L =  number of laboratories submitting results n =  number of single results retained after eliminating outliers (identified by Cochran, Dixon outlier test) sr =  standard deviation of repeatability SR =  standard deviation of reproducibility r =  repeatability R =  reproducibility CVr =  coefficient of variation of repeatability, % CVR =  coefficient of variation of reproducibility, %.	L =  number of laboratories submitting results n =  number of single results retained after eliminating outliers (identified by Cochran, Dixon outlier test) sr =  standard deviation of repeatability SR =  standard deviation of reproducibility r =  repeatability R =  reproducibility CVr =  coefficient of variation of repeatability, % CVR =  coefficient of variation of reproducibility, %.	L =  number of laboratories submitting results n =  number of single results retained after eliminating outliers (identified by Cochran, Dixon outlier test) sr =  standard deviation of repeatability SR =  standard deviation of reproducibility r =  repeatability R =  reproducibility CVr =  coefficient of variation of repeatability, % CVR =  coefficient of variation of reproducibility, %.	L =  number of laboratories submitting results n =  number of single results retained after eliminating outliers (identified by Cochran, Dixon outlier test) sr =  standard deviation of repeatability SR =  standard deviation of reproducibility r =  repeatability R =  reproducibility CVr =  coefficient of variation of repeatability, % CVR =  coefficient of variation of reproducibility, %.

Another EU collaborative study (3rd intercomparison) was arranged in which two samples were analysed by up to 13 laboratories to certify the method for extraction of free tryptophan. Replicate (5) analyses were performed on each sample. The results are given in the following table:

	Sample 4 Wheat and soya mixture	Sample 5 Wheat and soya mixture (= sample 4) with added tryptophan (0,457 g/kg)
L n	12 55	12 60
Mean [g/kg]	0,391	0,931
sr [g/kg]	0,005	0,012
r [g/kg]	0,014	0,034
CVr [%]	1,34	1,34
SR [g/kg]	0,018	0,048
R [g/kg]	0,050	0,134
CVR [%]	4,71	5,11
L =  number of laboratories submitting results n =  number of single results retained after eliminating outliers (identified by Cochran, Dixon outlier test) sr =  standard deviation of repeatability SR =  standard deviation of reproducibility r =  repeatability R =  reproducibility CVr =  coefficient of variation of repeatability, % CVR =  coefficient of variation of reproducibility, %.	L =  number of laboratories submitting results n =  number of single results retained after eliminating outliers (identified by Cochran, Dixon outlier test) sr =  standard deviation of repeatability SR =  standard deviation of reproducibility r =  repeatability R =  reproducibility CVr =  coefficient of variation of repeatability, % CVR =  coefficient of variation of reproducibility, %.	L =  number of laboratories submitting results n =  number of single results retained after eliminating outliers (identified by Cochran, Dixon outlier test) sr =  standard deviation of repeatability SR =  standard deviation of reproducibility r =  repeatability R =  reproducibility CVr =  coefficient of variation of repeatability, % CVR =  coefficient of variation of reproducibility, %.

Another EU intercomparison study was arranged in which four samples were analysed by up to 7 laboratories with the aim of a tryptophan certification for hydrolysis. The results are given below Replicate (5) analyses were performed on each sample.

	Sample 1 Mixed pig feed(CRM 117)	Sample 2 Low fat fish meal (CRM 118)	Sample 3 Soybean meal (CRM 119)	Sample 4 Skimmed milk powder (CRM 120)
L	7	7	7	7
n	25	30	30	30
Mean [g/kg]	2,064	8,801	6,882	5,236
sr [g/kg]	0,021	0,101	0,089	0,040
r [g/kg]	0,059	0,283	0,249	0,112
CVr [%]	1,04	1,15	1,30	0,76
SR [g/kg]	0,031	0,413	0,283	0,221
R [g/kg]	0,087	1,156	0,792	0,619
CVR [%]	1,48	4,69	4,11	4,22
L =  number of laboratories submitting results n =  number of single results retained after eliminating outliers (identified by Cochran, Dixon outlier test) sr =  standard deviation of repeatability SR =  standard deviation of reproducibility r =  repeatability R =  reproducibility CVr =  coefficient of variation of repeatability, % CVR =  coefficient of variation of reproducibility, %.	L =  number of laboratories submitting results n =  number of single results retained after eliminating outliers (identified by Cochran, Dixon outlier test) sr =  standard deviation of repeatability SR =  standard deviation of reproducibility r =  repeatability R =  reproducibility CVr =  coefficient of variation of repeatability, % CVR =  coefficient of variation of reproducibility, %.	L =  number of laboratories submitting results n =  number of single results retained after eliminating outliers (identified by Cochran, Dixon outlier test) sr =  standard deviation of repeatability SR =  standard deviation of reproducibility r =  repeatability R =  reproducibility CVr =  coefficient of variation of repeatability, % CVR =  coefficient of variation of reproducibility, %.	L =  number of laboratories submitting results n =  number of single results retained after eliminating outliers (identified by Cochran, Dixon outlier test) sr =  standard deviation of repeatability SR =  standard deviation of reproducibility r =  repeatability R =  reproducibility CVr =  coefficient of variation of repeatability, % CVR =  coefficient of variation of reproducibility, %.	L =  number of laboratories submitting results n =  number of single results retained after eliminating outliers (identified by Cochran, Dixon outlier test) sr =  standard deviation of repeatability SR =  standard deviation of reproducibility r =  repeatability R =  reproducibility CVr =  coefficient of variation of repeatability, % CVR =  coefficient of variation of reproducibility, %.

9. Observations

• 9.1.Following special chromatographic conditions may give better separation between tryptophan and α-methyl-tryptophan.

• Isocratic elution followed by gradient column cleaning:

Liquid chromatographic column:	125 mm × 4 mm, C18, 5 μm packing or equivalent	125 mm × 4 mm, C18, 5 μm packing or equivalent
Column temperature:	32 °C	32 °C
Mobile phase:	A: 0,01 mol/l KH2PO4/Methanol, 95 + 5 (V + V)	A: 0,01 mol/l KH2PO4/Methanol, 95 + 5 (V + V)
Mobile phase:	B: Methanol	B: Methanol
Gradient programme:	0 min 100 % A	0 % B
Gradient programme:	15 min 100 % A	0 % B
Gradient programme:	17 min 60 % A	40 % B
Gradient programme:	19 min 60 % A	40 % B
Gradient programme:	21 min 100 % A	0 % B
Gradient programme:	33 min 100 % A	0 % B
Flow rate:	1,2 ml/min	1,2 ml/min
Total run time:	approx. 33 min.	approx. 33 min.

• 9.2.The chromatography will vary according to the type of HPLC and column packing material used. The chosen system must be capable of giving baseline separation between the tryptophan and the internal standard. Moreover, it is important that degradation products are well separated from the tryptophan and the internal standard. Hydrolysates without internal standard shall be run in order to check the base line under the internal standard for impurities. It is important that the run time is sufficiently long for the elution of all the degradation products, otherwise late eluting peaks may interfere with subsequent chromatographic runs.

• In the range of operation, the chromatographic system shall give linear response. The linear response shall be measured with a constant (the normal) concentration of the internal standard and varying concentrations of tryptophan. It is of importance that the size of both the tryptophan and internal standard peaks are within the linear range of the HPLC/fluorescence system. If either the tryptophan and/or the internal standard peak(s) is (are) too small or too high the analysis shall be repeated with another sample size and/or a changed final volume.

9.3.   Barium hydroxide

With age barium hydroxide becomes more difficult to dissolve. This results in an unclear solution for the HPLC determination, which may produce low results for tryptophan.

G. DETERMINATION OF CRUDE OILS AND FATS

1. Purpose and scope

This method is for the determination of crude oils and fats in feed.

The use of the two procedures described below depends on the nature and composition of the feed and the reason for carrying out the analysis.

For the determination of crude oils and fats in oil seeds and oleaginous fruit as well in feed in which the crude oil/fat content is higher than 15 %, the extraction should be performed by Procedure A and re-extraction by Procedure B (point 5.3).

1.1.   Procedure A – *Directly extractable crude oils and fats*

This method is applicable to feed materials of plant origin, except those included within the scope of Procedure B.

1.2.   Procedure B – *Total crude oils and fats*

This method is applicable to feed materials of animal origin and to all compound feeds. It is to be used for all materials from which the oils and fats cannot be completely extracted without prior hydrolysis (e.g. gluten, yeast, potato proteins and products subjected to processes such as extrusion, flaking and heating).

1.3.   Interpretation of results

In all cases where a higher result is obtained by using Procedure B than by Procedure A, the result obtained by Procedure B shall be accepted as the true value.

2. Principle

2.1.   Procedure A

The sample is extracted with light petroleum. The solvent is distilled off and the residue dried and weighed.

2.2.   Procedure B

The sample is treated under heating with hydrochloric acid. The mixture is cooled and filtered. The residue is washed and dried and submitted to the determination according to Procedure A.

3. Reagents

• 3.1.Light petroleum, boiling range: 40 to 60 °C. The bromine value must be less than 1 and the residue on evaporation less than 2 mg/100 ml.

• 3.2.Sodium sulfate, anhydrous.

• 3.3.Hydrochloric acid, c = 3 mol/l.

• 3.4.Filtration aid, e.g. Kieselguhr, Hyflo-supercel.

4. Apparatus

• 4.1.Extraction apparatus. If fitted with a siphon (Soxhlet apparatus), the reflux rate shall be such as to produce about 10 cycles per hour; if of the non-siphoning type, the reflux rate shall be about 10 ml per minute.

• 4.2.Extraction thimbles, free of matter soluble in light petroleum and having a porosity consistent with the requirements of point 4.1.

• 4.3.Drying oven, either a vacuum oven set at 75 ± 3 °C or an air-oven set at 100 ± 3 °C.

5. Procedure

5.1.   Procedure A (see point 8.1)

Weigh 5 g of the sample to the nearest 1 mg, transfer it to an extraction thimble (point 4.2) and cover with a fat-free wad of cotton wool.

Place the thimble in an extractor (point 4.1) and extract for six hours with light petroleum (point 3.1). Collect the light petroleum extract in a dry, weighed flask containing fragments of pumice stone ([^E0032]).

Distil off the solvent. Dry the residue maintaining the flask for one and a half hours in the drying oven (point 4.3). Leave to cool in a desiccator and weigh. Dry again for 30 minutes to ensure that the weight of the oils and fats remains constant (loss in weight between two successive weighings must be less than or equal to 1 mg).

5.2.   Procedure B

Weigh 2,5 g of the sample to the nearest 1 mg (see point 8.2), place in a 400 ml beaker or a 300 ml conical flask and add 100 ml of hydrochloric acid (point 3.3) and fragments of pumice stone. Cover the beaker with a watch glass or fit the conical flask with a reflux condenser. Bring the mixture to a gentle boil over a low flame or a hot-plate and keep it there for one hour. Do not allow the product to stick to the sides of the container.

Cool and add a quantity of filtration aid (point 3.4) sufficient to prevent any loss of oil and fat during filtration. Filter through a moistened, fat-free, double filter paper. Wash the residue in cold water until a neutral filtrate is obtained. Check that the filtrate does not contain any oil or fats. Their presence indicates that the sample must be extracted with light petroleum, using Procedure A, before hydrolysis.

Place the double filter paper containing the residue on a watch glass and dry for one and a half hours in the air oven (point 4.3) at 100 ± 3 °C.

Place the double filter paper containing the dry residue in an extraction thimble (point 4.2) and cover with a fat-free wad of cotton wool. Place the thimble in an extractor (point 4.1) and proceed as indicated in the second and third paragraph of point 5.1.

5.3.   Procedure A and re-extraction by Procedure B

For the determination of crude oils and fats in oil seeds and oleaginous fruit as well in feed in which the crude oil/fat content is higher than 15 % the extraction should be performed by Procedure A and re-extraction by Procedure B.

This means after the extraction with light petroleum (procedure A), the residue or a portion of the residue is re-extracted with hydrochloric acid (procedure B). The crude oil and fat content is the sum of the result of procedure A and B.

6. Expression of result

Express the weight of the residue as a percentage of the sample.

7. Repeatability

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

• — 0,2 %, in absolute value, for contents of crude oils and fats lower than 5 %,

• — 4,0 % relative to the highest result for contents of 5 to 10 %,

• — 0,4 % in absolute value, for contents above 10 %.

8. Observations

• 8.1.For products with a high content of oils and fats, which are difficult to crush or unsuitable for drawing a homogeneous reduced test sample, proceed as follows.

• Weigh 20 g of the sample to the nearest 1 mg and mix with 10 g or more of anhydrous sodium sulfate (point 3.2). Extract with light petroleum (point 3.1) as indicated in point 5.1. Make up the extract obtained to 500 ml with light petroleum (point 3.1) and mix. Take 50 ml of the solution and place in a small, dry, weighed flask containing fragments of pumice stone. Distil off the solvent, dry and proceed as indicated in the last paragraph of point 5.1.

• Eliminate the solvent from the extraction residue left in the thimble, crush the residue to a fineness of 1 mm, return it to the extraction thimble (do not add sodium sulfate) and proceed as indicated in the second and third paragraphs of point 5.1.

• Calculate the content of oils and fats as a percentage of the sample by using the following formula:

Image omitted; see official source

• where:

m1	=	weight in grams of the residue after the first extraction (aliquot part of the extract),
---	---	---
m2	=	weight in grams of the residue after the second extraction.

• 8.2.For some products (e.g. low in oils and fats) the test sample may be increased.

• 8.3.Pet foods containing a high content of water may need to be mixed with anhydrous sodium sulfate prior to hydrolysis and extraction as per Procedure B.

• 8.4.In point 5.2 it may be more effective to use hot water in place of cold water to wash the residue after filtration.

• 8.5.The drying time of 1,5 h may need to be extended for some feed. Excessive drying shall be avoided as this can lead to low results. A microwave oven can also be used.

H. DETERMINATION OF CRUDE FIBRE

1. Purpose and scope

This method makes it possible to determine fat-free organic substances in feed which are insoluble in acid and alkaline media and are conventionally described as crude fibre.

The method is not applicable in the case of lignocellulose and vegetable carbon (particles too fine).

2. Principle

The sample, defatted where necessary, is treated successively with boiling solutions of sulphuric 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 crude fibre present in the test sample.

3. Reagents

• 3.1.Sulphuric acid, c = 0,13 mol/l.

• 3.2.Anti-foaming agent (e.g. n-octanol).

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

• 3.4.Acetone.

• 3.5.Light petroleum boiling-range 40 to 60 °C.

• 3.6.Hydrochloric acid, c = 0,5 mol/l.

• 3.7.Potassium hydroxide solution, c = 0,23 mol/l.

4. Apparatus

• 4.1.Heating unit for digestion with sulphuric acid and potassium hydroxide solution, equipped with a support for the filter crucible (point 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.

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

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

• 4.4.Drying oven with thermostat.

• 4.5.Muffle furnace with thermostat.

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

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

5. Procedure

Weigh out 1 g of the prepared sample to the nearest 1 mg and place it in the crucible (point 4.2), (see observations points 9.1, 9.2 and 9.3) and add 1 g of filter aid (point 3.3).