LegalizeCommission 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)
— 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.
5.3.2.1.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.
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.
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.
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.
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:
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:
| Reference Material | Amino Acid | |||
|---|---|---|---|---|
| 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. |
The repeatability expressed as ‘within laboratory standard deviation’ of the intercomparison of the previous table is given in the following table:
| Reference Material | Amino Acid | |||
|---|---|---|---|---|
| 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. |
The results for between laboratory standard deviation by the above mentioned intercomparison are given in the table below:
| Reference Material | Amino Acid | |||
|---|---|---|---|---|
| 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. |
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.
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.
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
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.
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.
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.
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.
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, %. |
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, %. |
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, %. |
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 | |
|---|---|---|
| Column temperature: | 32 °C | |
| Mobile phase: | A: 0,01 mol/l KH2PO4/Methanol, 95 + 5 (V + V) | |
| B: Methanol | ||
| Gradient programme: | 0 min 100 % A | 0 % B |
| 15 min 100 % A | 0 % B | |
| 17 min 60 % A | 40 % B | |
| 19 min 60 % A | 40 % B | |
| 21 min 100 % A | 0 % B | |
| 33 min 100 % A | 0 % B | |
| Flow rate: | 1,2 ml/min | |
| Total run time: | 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.
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).
This method is applicable to feed materials of plant origin, except those included within the scope of Procedure B.
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).
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
The sample is extracted with light petroleum. The solvent is distilled off and the residue dried and weighed.
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
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 (17).
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).
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.
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:
where:
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).
Assemble the heating unit (point 4.1) and the filter crucible (point 4.2), then attach the cylinder (point 4.3) to the crucible. Pour 150 ml of boiling sulphuric acid (point 3.1) into the assembled cylinder and crucible and if necessary add a few drops of antifoaming agent (point 3.2).
Bring the liquid to the boil within 5 ± 2 minutes and boil vigorously for exactly 30 minutes.
Open the tap to the discharge pipe (point 4.1) and, under vacuum, filter the sulphuric 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 boiling potassium hydroxide solution (point 3.7) to the assembled cylinder and crucible and add a few drops of antifoaming agent (point 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 sulphuric acid step.
After the final washing and drying, disconnect the crucible and its contents and reconnect it to the cold extraction unit (point 4.6). Apply the vacuum and wash the residue in the crucible with three consecutive 25 ml portions of acetone (point 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 (loss in weight between two successive weighings must be less than or equal to 2 mg) at 475 °C 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.
6. Calculation of results
The crude fibre content as a percentage of the sample is given by the expression:
where:
7. Repeatability
The difference between two parallel determinations carried out on the same sample must not exceed:
— 0,6 % in absolute value for crude fibre contents lower than 10 %,
— 6 % relative to the higher result, for crude fibre contents equal to or greater than 10 %.
8. Reproducibility
The difference between the results of two determinations carried out on the same sample in different laboratories must not exceed:
— 1,0 % in absolute value for crude fibre contents lower than 10 %,
— 10 % relative to the higher result, for crude fibre contents equal to or greater than 10 %.
9. Observations
9.1.Feed containing more than 10 % crude fat must be defatted prior to analysis with light petroleum (point 3.5). Connect the filter crucible (point 4.2) and its contents to the cold extraction unit (point 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 (point 4.1) and continue as described under point 5.
9.2.Feed containing fats which cannot be extracted directly with light petroleum (point 3.5) must be defatted as shown in point 8.1 and 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 (point 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 point 5, beginning with potassium hydroxide treatment.
9.3.If the feed contain over 5 % of carbonates, expressed as calcium carbonate, connect the crucible (point 4.2) with the weighed sample to the heating unit (point 4.1). Wash the sample three times with 30 ml hydrochloric acid (point 3.6). After each addition let the sample stand for about one minute before filtering. Wash once with 30 ml water and then continue as described under point 5.
9.4.If an apparatus in the form of a stand is used (several crucibles attached to the same heating unit) no two individual determinations on the same sample for analysis may be carried out in the same series.
9.5.If after boiling it is difficult to filter the acidic and basic solutions, use compressed air through the discharge pipe of the heating unit and then continue filtering.
9.6.The temperature for ashing shall not be higher than 500 °C in order to extend the lifetime of the glass filter crucibles. Care must be taken to avoid excessive thermal shock during heating and cooling cycles.
I. DETERMINATION OF SUGAR
1. Purpose and scope
This method makes it possible to determine the amount of reducing sugars and total sugars after inversion, expressed as glucose or where appropriate as sucrose, converting by the factor 0,95. It is applicable to compound feed. Special methods are provided for other feed. Where necessary, lactose shall be measured separately and taken into account when calculating the results.
This method is to be used for the determination of the sugar content for use in energy value calculation of the feed.
In case the sugar content is to be determined for other purposes, other methods of analysis can be used.
2. Principle
The sugars are extracted in dilute ethanol; the solution is clarified with Carrez solutions I and II. After eliminating the ethanol, the quantities before and after inversion are determined by the Luff-Schoorl method.
3. Reagents
3.1.Ethanol solution 40 % (v/v) density: 0,948 g/ml at 20 °C, neutralised to phenolphthalein.
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.Methyl orange, solution 0,1 % (w/v).
3.5.Hydrochloric acid 4 mol/litre.
3.6.Hydrochloric acid 0,1 mol/litre.
3.7.Sodium hydroxide solution 0,1 mol/litre.
3.8.Luff-Schoorl reagent:
Stirring carefully, pour the citric acid solution (point 3.8.2) into the sodium carbonate solution (point 3.8.3). Add the copper sulphate solution (point 3.8.1) and make up to 1 litre with water. Leave to settle overnight and filter.
Check the concentration of the reagent thus obtained (Cu 0,05 mol/litre; Na2 CO3 1 mol/litre), see point 5.4, last paragraph. The solution’s pH shall be approximately 9,4.
3.8.1.Copper sulphate solution: dissolve 25 g of copper sulphate, Cu SO4 5H2O, free from iron, in 100 ml of water.
3.8.2.Citric acid solution: dissolve 50 g of citric acid, C6H8O7•H2O in 50 ml of water.
3.8.3.Sodium carbonate solution: dissolve 143,8 g of anhydrous sodium carbonate in approximately 300 ml of warm water. Leave to cool.
3.9.Sodium thiosulphate solution 0,1 mol/litre.
3.10.Starch solution: add a mixture of 5 g of soluble starch in 30 ml of water to 1 litre of boiling water. Boil for three minutes, leave to cool and if necessary add 10 mg of mercuric iodide as a preservative.
3.11.Sulphuric acid 3 mol/litre.
3.12.Potassium iodide, solution 30 % (w/v).
3.13.Granulated pumice stone boiled in hydrochloric acid, washed in water and dried.
3.14.3-methylbutan-1-ol.
4. Apparatus
Mixer (tumbler): approximately 35 to 40 rpm.
5. Procedure
Weigh 2,5 g of the sample to the nearest mg and place in a 250 ml volumetric flask. Add 200 ml of ethanol (point 3.1) and mix in the tumbler for one hour. Add 5 ml of Carrez solution I (point 3.2) and stir for approximately 30 seconds. Add 5 ml of Carrez solution II (point 3.3) and again stir for one minute. Make up to volume with ethanol (point 3.1), homogenise and filter. Remove 200 ml of the filtrate and evaporate to approximately half volume in order to eliminate most of the ethanol. Transfer the evaporation residue quantitatively to a 200 ml volumetric flask using warm water, cool, bring up to volume with water, homogenise and filter if necessary. This solution will be used to determine the amount of reducing sugars and, after inversion, of total sugars.
Using a pipette, remove not more than 25 ml of the solution containing less than 60 mg of reducing sugars expressed as glucose. If necessary, make up to 25 ml with distilled water and determine the content of reducing sugars by the Luff-Schoorl method. The result is expressed as the percentage content of glucose in the sample.
Using a pipette take 50 ml of the solution and transfer to a 100 ml volumetric flask. Add a few drops of methyl orange solution (point 3.4) then, carefully and stirring continuously, add hydrochloric acid (point 3.5) until the liquid turns a definite red. Add 15 ml of hydrochloric acid (point 3.6), immerse the flask in a fast boiling water bath and keep there for thirty minutes. Cool rapidly to approximately 20 °C and add 15 ml of sodium hydroxide solution (point 3.7). Make up to 100 ml with water and homogenise. Remove not more than 25 ml containing less than 60 mg of reducing sugars expressed as glucose. If necessary, make up to 25 ml with distilled water and determine the content of reducing sugars by the Luff-Schoorl method. The result is expressed as the percentage of glucose or, where appropriate, sucrose, by multiplying by the factor 0,95.
Using a pipette, take 25 ml of Luff-Schoorl reagent (point 3.8) and transfer to a 300 ml Erlenmeyer flask; add exactly 25 ml of the clarified sugar solution. Add 2 granules of pumice stone (point 3.13), heat, stirring by hand, over a free flame of medium height and bring the liquid to the boil in approximately two minutes. Place the Erlenmeyer immediately on an asbestos-coated wire gauze with a hole approximately 6 cm in diameter under which a flame has been lit. The flame shall be regulated in such a way that only the base of the Erlenmeyer is heated. Fit a reflux condenser to the Erlenmeyer flask. Boil for exactly ten minutes. Cool immediately in cold water and after approximately five minutes titrate as follows:
Add 10 ml of potassium iodide solution (point 3.12) and immediately afterwards (carefully, because of the risk of abundant foaming), add 25 ml of sulphuric acid (point 3.11). Titrate with sodium thiosulphate solution (point 3.9) until a dull yellow colour appears, add the starch indicator (point 3.10) and complete titration.
Carry out the same titration on an accurately measured mixture of 25 ml of Luff-Schoorl reagent (point 3.8) and 25 ml of water, after adding 10 ml of potassium iodide solution (point 3.12) and 25 ml of sulphuric acid (point 3.11) without boiling.
6. Calculation of results
Using the table establish the amount of glucose in mg which corresponds to the difference between the values of the two titrations, expressed in ml of sodium thiosulphate 0,1 mol/litre. Express the result as a percentage of the sample.
7. Special procedures
7.1.In the case of feed which are rich in molasses and other feed which are not particularly homogeneous, weigh out 20 g and place with 500 ml of water in a 1 litre volumetric flask. Mix for one hour in the tumbler. Clarify using Carrez solutions I (point 3.2) and II (point 3.3) reagents as described under point 5.1, this time however using four times the quantities of each reagent. Bring up to volume with 80 % ethanol (v/v).
Homogenise and filter. Eliminate the ethanol as described under point 5.1. If there is no dextrinised starch, bring up to volume with distilled water.
7.2.In the case of molasses and feed materials which are rich in sugar and almost starch-free (carobs, dried beetroot cossettes, etc.), weigh out 5 g, place in a 250 ml volumetric flask, add 200 ml of distilled water and mix in the tumbler for one hour, or more if necessary. Clarify using Carrez solutions I (point 3.2) and II (point 3.3) reagents as described under point 5.1. Bring up to volume with cold water, homogenise and filter. In order to determine the amount of total sugars, continue as described under point 5.3.
8. Observations
8.1.In order to prevent foaming it is advisable to add (irrespective of the volume) approximately 1 ml of 3-methylbutan-1-ol (point 3.14) before boiling with Luff-Schoorl reagent.
8.2.The difference between the content of total sugars after inversion, expressed as glucose, and the content of reducing sugars, expressed as glucose, multiplied by 0,95, gives the percentage content of sucrose.
8.3.In order to determine the content of reducing sugars, excluding lactose, two methods may be adopted:
8.3.1.For an approximate calculation, multiply by 0,675 the lactose content established by a different method of analysis and subtract the result obtained from the content of reducing sugars.
8.3.2.For an accurate calculation of reducing sugars, excluding lactose, the same sample must be used for the two final determinations. One of the analyses is carried out on part of the solution obtained under point 5.1, the other on part of the solution obtained during the determination of lactose by the method laid down for that purpose (after fermenting the other types of sugar and clarifying).
In both cases the amount of sugar present is determined by the Luff-Schoorl method and calculated in mg of glucose. One of the values is subtracted from the other and the difference is expressed as a percentage of the sample.
Example
The two volumes taken correspond, for each determination, to a sample of 250 mg.
In the first case 17 ml of sodium thiosulphate solution 0,1 mol/litre corresponding to 44,2 mg of glucose is consumed; in the second, 11 ml, corresponding to 27,6 mg of glucose.
The difference is 16,6 mg of glucose.
The content of reducing sugars (excluding lactose), calculated as glucose, is therefore:
| Na2 S2 O3 0,1 mol/litre | Glucose, fructose invert sugars C6 H12 O6 | Lactose C12 H22 O11 | Na2 S2 O3 0,1 mol/litre | ||
|---|---|---|---|---|---|
| ml | mg | difference | mg | difference | ml |
| 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 | 2,4 4,8 7,2 9,7 12,2 14,7 17,2 19,8 22,4 25,0 27,6 30,3 33,0 35,7 38,5 41,3 44,2 47,1 50,0 53,0 56,0 59,1 62,2 | 2,4 2,4 2,5 2,5 2,5 2,5 2,6 2,6 2,6 2,6 2,7 2,7 2,7 2,8 2,8 2,9 2,9 2,9 3,0 3,0 3,1 3,1 | 3,6 7,3 11,0 14,7 18,4 22,1 25,8 29,5 33,2 37,0 40,8 44,6 48,4 52,2 56,0 59,9 63,8 67,7 71,7 75,7 79,8 83,9 88,0 | 3,7 3,7 3,7 3,7 3,7 3,7 3,7 3,7 3,8 3,8 3,8 3,8 3,8 3,8 3,9 3,9 3,9 4,0 4,0 4,1 4,1 4,1 | 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 |
J. DETERMINATION OF LACTOSE
1. Purpose and scope
This method makes it possible to determine the level of lactose in feed containing more than 0,5 % of lactose.
2. Principle
The sugars are dissolved in water. The solution is subjected to fermentation by the yeast Saccharomyces cerevisiae which leaves the lactose intact. After clarification and filtration the lactose content of the filtrate is determined by the Luff-Schoorl method.
3. Reagents
3.1.Suspension of Saccharomyces cerevisiae: suspend 25 g of fresh yeast in 100 ml of water. The suspension will keep for a maximum period of one week in a refrigerator.
3.2.Carrez solution I: dissolve in water 21,9 g of zinc acetate, Zn (CH3 COO)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.Luff-Schoorl reagent:
Stirring carefully, pour the citric acid solution (point 3.4.2) into the sodium carbonate solution (point 3.4.3). Add the copper sulphate solution (point 3.4.1) and make up to 1 litre with water. Leave to settle overnight and filter. Check the concentration of the reagent thus obtained (Cu 0,05 mol/litre; Na2 CO3 1 mol/litre). The solution’s pH shall be approximately 9,4.
3.4.1.Copper sulphate solution: dissolve 25 g of copper sulphate Cu SO4 5H2O, free from iron, in 100 ml of water.
3.4.2.Citric acid solution: dissolve 50 g of citric acid C6H8O7 • H2O in 50 ml of water.
3.4.3.Sodium carbonate solution: dissolve 143,8 g of anhydrous sodium carbonate in approximately 300 ml of warm water. Leave to cool.
3.5.Granulated pumice stone boiled in hydrochloric acid, washed in water and dried.
3.6.Potassium iodide, solution 30 % (w/v).
3.7.Sulphuric acid 3 mol/litre.
3.8.Solution of sodium thiosulphate 0,1 mol/litre.
3.9.Starch solution: add a mixture of 5 g of soluble starch in 30 ml of water to 1 litre of boiling water. Boil for three minutes, leave to cool, and if necessary add 10 mg of mercuric iodide as a preservative.
4. Apparatus
Water bath with thermostat set at 38–40 °C.
5. Procedure
Weigh 1 g of the sample to the nearest mg and place this portion of the sample in a 100 ml volumetric flask. Add 25 to 30 ml of water. Place the flask in a boiling water bath for thirty minutes and then cool to approximately 35 °C. Add 5 ml of yeast suspension (point 3.1) and homogenise. Leave the flask to stand for two hours in a water bath, at a temperature of 38–40 °C. Cool to approximately 20 °C.
Add 2,5 ml of Carrez solution I (point 3.2) and stir for thirty seconds, then add 2,5 ml of Carrez solution II (point 3.3) and again stir for thirty seconds. Make up to 100 ml with water, mix and filter. Using a pipette, remove an amount of filtrate which does not exceed 25 ml and which preferably contains from 40 to 80 mg of lactose and transfer it to a 300 ml Erlenmeyer flask. If necessary, make up to 25 ml with water.
Carry out a blank test in the same way with 5 ml of yeast suspension (point 3.1). Determine the lactose content according to Luff-Schoorl, as follows: add exactly 25 ml of Luff-Schoorl reagent (point 3.4) and two granules of pumice stone (point 3.5). Stir by hand while heating over a free flame of medium height and bring the liquid to the boil in approximately two minutes. Place the Erlenmeyer immediately on an asbestos-coated wire gauze with a hole approximately 6 cm in diameter under which a flame has been lit. The flame shall be regulated in such a way that only the base of the Erlenmeyer is heated. Fit a reflux condenser to the Erlenmeyer flask. Boil for exactly ten minutes. Cool immediately in cold water and after approximately five minutes titrate as follows:
Add 10 ml of potassium iodide solution (point 3.6) and immediately afterwards (carefully, because of the risk of abundant foaming) add 25 ml of sulphuric acid (point 3.7). Titrate with sodium thiosulphate solution (point 3.8) until a dull yellow colour appears, add the starch indicator (point 3.9) and complete titration.
Carry out the same titration on an accurately measured mixture of 25 ml of Luff-Schoorl reagent (point 3.4) and 25 ml of water, after adding 10 ml of potassium iodide solution (point 3.6) and 25 ml of sulphuric acid (point 3.7) without boiling.
6. Calculation of results
Using the attached table, establish the amount of lactose in mg which corresponds to the difference between the results of the two titrations, expressed in ml of sodium thiosulphate 0,1 mol/litre.
Express the result of anhydrous lactose as a percentage of the sample.
7. Observation
1.For products containing more than 40 % of fermentable sugar, use more than 5 ml of yeast suspension (point 3.1).
2.In ‘lactose reduced’ feed (e.g. cat milk), lactose is converted into fructose, which is not completely fermented within 2 hours resulting in higher or false positive results (because residues of fructose remain in the extract).
| Na2 S2 O3 0,1 mol/litre | Glucose, fructose invert sugars C6 H12 O6 | Lactose C12 H22 O11 | Na2 S2 O3 0,1 mol/litre | ||
|---|---|---|---|---|---|
| ml | mg | difference | mg | difference | ml |
| 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 | 2,4 4,8 7,2 9,7 12,2 14,7 17,2 19,8 22,4 25,0 27,6 30,3 33,0 35,7 38,5 41,3 44,2 47,1 50,0 53,0 56,0 59,1 62,2 | 2,4 2,4 2,5 2,5 2,5 2,5 2,6 2,6 2,6 2,6 2,7 2,7 2,7 2,8 2,8 2,9 2,9 2,9 3,0 3,0 3,1 3,1 | 3,6 7,3 11,0 14,7 18,4 22,1 25,8 29,5 33,2 37,0 40,8 44,6 48,4 52,2 56,0 59,9 63,8 67,7 71,7 75,7 79,8 83,9 88,0 | 3,7 3,7 3,7 3,7 3,7 3,7 3,7 3,7 3,8 3,8 3,8 3,8 3,8 3,8 3,9 3,9 3,9 4,0 4,0 4,1 4,1 4,1 | 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 |
K. DETERMINATION OF STARCH
POLARIMETRIC METHOD
This method makes it possible to determine the levels of starch and of high molecular weight starch degradation products in feed for the purpose of checking compliance with the declared energy value (provisions in Annex VII) and Regulation (EC) No 767/2009.
This method is to be used for the determination of the starch content for use in energy value calculation of the feed.
In case the starch content is to be determined for other purposes, other methods of analysis can be used.
The method comprises of two determinations. In the first one, the sample is treated with dilute hydrochloric acid. After clarification and filtration, the optical rotation of the solution is measured by polarimetry.
In the second one, the sample is extracted with 40 % ethanol. After acidifying the filtrate with hydrochloric acid, clarifying and filtering, the optical rotation is measured as in the first determination.
The difference between the two measurements, multiplied by a known factor, gives the starch content of the sample.
3.1.Hydrochloric acid, solution 25 % (w/w) density: 1,126 g/ml.
3.2.Hydrochloric acid. solution 1,13 % (w/v)
The concentration must be checked by titration using a sodium hydroxide solution 0,1 mol/litre in the presence of 0,1 % (w/v) methyl red in 94 % (v/v) ethanol. For the neutralisation of 10 ml, 30,94 ml of NaOH 0,1 mol/litre is needed.
3.3.Carrez solution I: dissolve 21,9 g of zinc acetate Zn(CH3COO)2 2H2O and 3 g of glacial acetic acid in water. Make up to 100 ml with water.
3.4.Carrez solution II: dissolve 10,6 g of potassium ferrocyanide K4 Fe(CN)6 3H2O in water. Make up to 100 ml with water.
3.5.Ethanol, solution 40 % (v/v), density: 0,948 g/ml at 20 °C.
4.1.250 ml Erlenmeyer flask with standard ground-glass joint and with reflux condenser.
4.2.Polarimeter or saccharimeter.
Crush the sample until it is fine enough for all of it to pass through a 0,5 mm round-meshed sieve.
Weigh 2,5 g of the crushed sample to the nearest mg and place in a 100 ml graduated flask. Add 25 ml of hydrochloric acid (point 3.2), shake to obtain even distribution of the test sample and add a further 25 ml of hydrochloric acid (point 3.2). Immerse the flask in a boiling water bath shaking vigorously and steadily for the first three minutes to prevent the formation of agglomerates. The quantity of water in the water bath must be sufficient for the bath to remain at boiling point when the flask is introduced into it. The flask must not be taken out of the bath whilst being shaken. After exactly 15 minutes, remove from the bath, add 30 ml of cold water and cool immediately to 20 °C.
Add 5 ml of Carrez solution I (point 3.3) and shake for approximately 30 seconds. Then add 5 ml of Carrez solution II (point 3.4) and shake again for approximately 30 seconds. Make up to volume with water, mix and filter. If the filtrate is not perfectly clear (which is rare), repeat the determination using a larger quantity of Carrez solutions I and II, for example 10 ml.
Measure the optical rotation of the solution in a 200 mm tube with the polarimeter or saccharimeter.
Weigh 5 g of the sample to the nearest mg, place in a 100 ml graduated flask and add about 80 ml of ethanol (point 3.5) (see observation point 7.2). Leave the flask to stand for 1 hour at room temperature; during this time, shake vigorously on six occasions so that the test sample is thoroughly mixed with the ethanol. Make up to volume with ethanol (point 3.5), mix and filter.
Pipette 50 ml of the filtrate (corresponds to 2,5 g of the sample) into a 250 ml erlenmeyer flask, add 2,1 ml of hydrochloric acid (point 3.1) and shake vigorously. Fit a reflux condenser to the erlenmeyer flask and immerse the latter in a boiling water bath. After exactly 15 minutes, remove the erlenmeyer flask from the bath, transfer the contents to a 100 ml graduated flask, rinsing with a little cold water, and cool to 20 °C.
Clarify using Carrez solutions I (point 3.3) and II (point 3.4), make up to volume with water, mix, filter and measure the optical rotation as indicated in point 5.2, second and third paragraphs.
The starch content (%) is calculated as follows:
The difference between the results of two parallel determinations carried out on the same sample must not exceed 0,4 in absolute value for a starch content lower than 40 % and 1 % relative for starch contents equal to or greater than 40 %.
7.1.If the sample contains more than 6 % of carbonates, calculated in terms of calcium carbonate, they must be destroyed by treatment with an exactly appropriate quantity of dilute sulphuric acid before determination of the total optical rotation.
7.2.In the case of products with a high lactose content, such as powdered milk serum or skimmed milk powder, proceed as follows after adding 80 ml of ethanol (point 3.5). Fit a reflux condenser to the flask and immerse the latter in a water bath at 50 °C for 30 minutes. Leave to cool and continue the analysis as indicated in point 5.3.
7.3.The following feed materials, where they are present in significant amounts in feed, are known to give rise to interferences when determining the starch content by the polarimetric method and thereby incorrect results could be yielded:
— (sugar) beet products such as (sugar)beet pulp, (sugar) beet molasses, (sugar) beet pulp – molassed, (sugar) beet vinasse, (beet) sugar,
— citrus pulp,
— linseed; linseed expeller; linseed extracted,
— rape seed; rape seed expeller; rape seed extracted; rape seed hulls,
— sunflower seed; sunflower seed extracted; sunflower seed, partially decorticated, extracted,
— copra expeller; copra extracted,
— potato pulp,
— dehydrated yeast,
— products rich in inulin (e.g. Chips and meal of Jerusalem artichokes),
— greaves,
— soybean products. In these cases the method of analysis as provided by Commission Regulation (EC) No 121/2008 (18) can be applied. This method can also be used for feed containing less than 1 % starch.
L. DETERMINATION OF CRUDE ASH
1. Purpose and scope
This method makes it possible to determine the crude ash content of feed.
2. Principle
The sample is ashed at 550 °C; the residue is weighed.
3. Reagents
Ammonium nitrate, solution 20 % (w/v).
4. Apparatus
4.1.Hot-plate.
4.2.Electric muffle-furnace with thermostat.
4.3.Crucibles for ashing made of silica, porcelain or platinum either rectangular (approx. 60 × 40 × 25 mm) or circular (diameter: 60 to 75 mm, height: 20 to 40 mm).
5. Procedure
Weigh out to the nearest mg approximately 5 g of the sample (2,5 in the case of products which have a tendency to swell) and place in a crucible for ashing which has first been heated at 550 °C, cooled down and tared. Place the crucible on the hot-plate and heat gradually until the substance carbonises. Ash according to point 5.1 or 5.2.
5.1.Put the crucible into the calibrated muffle furnace set at 550 °C. Keep at this temperature until white, light grey or reddish ash is obtained which appears to be free from carbonaceous particles. Place the crucible in a desiccator, leave to cool and weigh immediately.
5.2.Put the crucible into the calibrated muffle-furnace set at 550 °C. Ash for 3 hours. Place the crucible in a desiccator, leave to cool and weigh immediately. Ash again for 30 minutes to ensure that the weight of the ash remains constant (loss in weight between two successive weighings must be less than or equal to 1 mg).
6. Calculation of results
Calculate the weight of the residue by deducting the tare.
Express the result as a percentage of the sample.
7. Observations
7.1.The ash of substances which are difficult to ash must be subjected to an initial ashing of at least three hours, cooled and then a few drops of 20 % solution of ammonium nitrate or water added to it (carefully, to avoid dispersal of the ash or the formation of lumps). Continue calcining after drying in the oven. Repeat the operation as necessary until ashing is complete.
7.2.In the case of substances resistant to the treatment described under point 7.1, proceed as follows: after ashing for three hours, place the ash in warm water and filter through a small, ash-free filter. Ash the filter and its contents in the original crucible. Place the filtrate in the cooled crucible, evaporate until dry, ash and weigh.
7.3.In the case of oils and fats, weigh accurately a sample of 25 g in a suitably sized crucible. Carbonise by setting light to the substance with a strip of ash-free filter paper. After combustion, moisten with as little water as possible. Dry and ash as described under point 5.
M. DETERMINATION OF ASH WHICH IS INSOLUBLE IN HYDROCHLORIC ACID
1. Purpose and scope
This method makes it possible to determine the level in feed of mineral substances which are insoluble in hydrochloric acid. Two methods can be used, depending on the nature of the sample.
1.1. Method A: applicable to organic feed materials and to most compound feed;
1.2. Method B: applicable to mineral compounds and mixtures and to compound feed, whose content in substances insoluble in hydrochloric acid, as determined by Method A, is greater than 1 %.
2. Principle
2.1. Method A: the sample is ashed, the ash boiled in hydrochloric acid and the insoluble residue filtered and weighed.
2.2. Method B: the sample is treated with hydrochloric acid. The solution is filtered, the residue ashed and the ash thus obtained treated in accordance with Method A.
3. Reagents
3.1.Hydrochloric acid 3 mol/litre.
3.2.Trichloroacetic acid, solution 20 % (w/v).
3.3.Trichloroacetic acid, solution 1 % (w/v).
4. Apparatus
4.1.Hot plate.
4.2.Electric muffle-furnace with thermostat.
4.3.Crucibles for ashing made of silica, porcelain or platinum, either rectangular (approx. 60 × 40 × 25 mm) or circular (diameter: 60 to 75 mm, height: 20 to 40 mm).
4.4.Ash free filters
5. Procedure
Ash the sample using the method described for the determination of crude ash. Ash obtained from that analysis may also be used.
Place the ash in a 250 to 400 ml beaker using 75 ml of hydrochloric acid (point 3.1). Bring slowly to the boil and boil gently for fifteen minutes. Filter the warm solution through an ash-free filter paper and wash the residue with warm water until the acid reaction is no longer visible. Dry the filter containing the residue and ash in a tared crucible at a temperature of not less than 550 °C and not more than 700 °C. Cool in a desiccator and weigh.
Weigh 5 g of the sample to the nearest mg and place in a 250 to 400 ml beaker. Add 25 ml of water and 25 ml of hydrochloric acid (point 3.1) successively, mix and wait for effervescence to cease. Add a further 50 ml of hydrochloric acid (point 3.1). Wait for any release of gas to cease then place the beaker in a boiling water bath and keep it there for thirty minutes or longer, if necessary, in order to hydrolyse thoroughly any starch which may be present. Filter while warm through an ash-free filter and wash the filter in 50 ml of warm water (see observation point 7). Place the filter containing the residue in a crucible for ashing, dry and ash at a temperature of not less than 550 °C and not more than 700 °C. Place the ash in a 250 to 400 ml beaker using 75 ml of hydrochloric acid (point 3.1); continue as described in the second subparagraph of point 5.1.
6. Calculation of results
Calculate the weight of the residue by deducting the tare. Express the result as a percentage of the sample.
7. Observation
If filtration proves difficult recommence the analysis, replacing the 50 ml of hydrochloric acid (point 3.1) by 50 ml of trichloroacetic acid, solution 20 % (w/v) (point 3.2) and washing the filter in a warm solution of 1 % trichloroacetic acid (point 3.3).
N. DETERMINATION OF TOTAL PHOSPHORUS
The total phosphorus is to be determined by
— the method of analysis provided for by EN 15510 Animal feeding stuffs: Methods of sampling and analysis – Determination of calcium, sodium, phosphorus, magnesium, potassium, iron, zinc, copper, manganese, cobalt, molybdenum and lead by ICP-AES, or
— the method of analysis provided for by EN 15621 Animal feeding stuffs: Methods of sampling and analysis – Determination of calcium, sodium, phosphorus, magnesium, potassium, sulphur, iron, zinc, copper, manganese and cobalt after pressure digestion by ICP-AES, or
— the photometric method, as described hereafter.
PHOTOMETRIC METHOD
This method makes it possible to determine the content of total phosphorus in feed. It is particularly appropriate for the analysis of products low in phosphorus. In certain cases (product rich in phosphorus), a gravimetric method may be used.
The sample is mineralised, either by dry combustion (in the case of organic feed) or by acid digestion (in the case of mineral compounds and liquid feed), and placed in an acid solution. The solution is treated with molybdovanadate reagent. The optical density of the yellow solution thus formed is measured in a spectrophotometer at 430 nm.
3.1.Calcium carbonate.
3.2.Hydrochloric acid, ρ20 = 1,10 g/ml (approx 6 mol/litre).
3.3.Nitric acid, ρ20 = 1,045 g/ml.
3.4.Nitric acid, ρ20 = 1,38 to 1,42 g/ml.
3.5.Sulphuric acid, ρ20 = 1,84 g/ml.
3.6.Molybdovanadate reagent: mix 200 ml of ammonium heptamolybdate solution (point 3.6.1), 200 ml of ammonium monovanadate solution (point 3.6.2) and 134 ml of nitric acid (point 3.4) in a 1 litre graduated flask. Make up to volume with water.
3.6.1.Ammonium heptamolybdate solution: dissolve in hot water 100 g of ammonium heptamolybdate (NH4) 6Mo7O24.4H2O. Add 10 ml of ammonia (density 0,91 g/ml) and make up to 1 litre with water.
3.6.2.Ammonium monovanadate solution: dissolve 2,35 g of ammonium monovanadate NH4VO3 in 400 ml of hot water. Stirring constantly, slowly add 20 ml of dilute nitric acid (7 ml of HNO3 (point 3.4) + 13 ml of H2O and make up to 1 litre with water.
3.7.Standard solution of 1 mg phosphorus per ml: dissolve 4,387 g of potassium dihydrogen phosphate KH2PO4 in water. Make up to 1 litre with water.
4.1.Silica, porcelain or platinum ashing crucibles.
4.2.Electric muffle-furnace with thermostat set at 550 °C.
4.3.250 ml Kjeldahl flask.
4.4.Graduated flasks and precision pipettes.
4.5.Spectrophotometer.
4.6.Test tubes about 16 mm in diameter, with stoppers graded to a diameter of 14,5 mm; capacity: 25 to 30 ml.
According to the nature of the sample, prepare a solution as indicated in point 5.1.1 or 5.1.2.
Weigh 1 g or more of the sample to the nearest 1 mg. Place the test sample in a Kjeldahl flask, add 20 ml of sulphuric acid (point 3.5), shake to impregnate the substance completely with acid and to prevent it from sticking to the sides of the flask, heat and keep at boiling point for 10 minutes. Leave to cool slightly, add 2 ml of nitric acid (point 3.4), heat gently, leave to cool slightly, add a little more nitric acid (point 3.4) and bring back to boiling point. Repeat this procedure until a colourless solution is obtained. Cool, add a little water, decant the liquid into a 500 ml graduated flask, rinsing the Kjeldahl flask with hot water. Leave to cool, make up to volume with water, homogenise and filter.
Weigh about 2,5 g of the sample to the nearest 1 mg in an ashing crucible. Mix the test sample until completely merged with 1 g of calcium carbonate (point 3.1). Ash in the oven at 550 °C until white or grey ash is obtained (a little charcoal does not matter). Transfer the ash into a 250 ml beaker. Add 20 ml of water and hydrochloric acid (point 3.2) until effervescence ceases. Add a further 10 ml of hydrochloric acid (point 3.2). Place the beaker on a sand bath and evaporate until dry to make the silica insoluble. Redissolve the residue in 10 ml of nitric acid (point 3.3) and boil on the sand bath or hot plate for 5 minutes without evaporating until dry. Decant the liquid into a 500 ml graduated flask, rinsing the beaker several times with hot water. Leave to cool, make up to volume with water, homogenise and filter.
Dilute an aliquot part of the filtrate obtained by point 5.1.1 or 5.1.2 to obtain a phosphorus concentration of not more than 40 μg/ml. Place 10 ml of this solution in a test tube (point 4.6) and add 10 ml of molybdovanadate reagent (point 3.6). Homogenise and leave to stand for at least 10 minutes at 20 °C. Measure the optical density in a spectrophotometer at 430 nm against a solution obtained by adding 10 ml of the molybdovanadate reagent (point 3.6) to 10 ml of water.
From the standard solution (point 3.7) prepare solutions containing respectively 5, 10, 20, 30 and 40 μg of phosphorus per ml. Take 10 ml of each of these solutions and add thereto 10 ml of molybdovanadate reagent (point 3.6). Homogenise and leave to stand for at least 10 minutes at 20 °C. Measure the optical density as indicated in point 5.2. Trace the calibration curve by plotting the optical densities against the corresponding quantities of phosphorus. For concentrations between 0 and 40 μg/ml, the curve will be linear.
Determine the amount of phosphorus in the test sample by using the calibration curve.
Express the result as a percentage of the sample.
The difference between the results of two parallel determinations carried out on the same sample shall not exceed:
— 3 %, relative to the higher result, for phosphorus contents of less than 5 %,
— 0,15 % in absolute value, for phosphorus contents of 5 % or more.
O. DETERMINATION OF CHLORINE FROM CHLORIDES
1. Purpose and scope
This method makes it possible to determine the amount of chlorine in chlorides which are soluble in water, conventionally expressed as sodium chloride. It is applicable to all feed.
2. Principle
The chlorides are dissolved in water. If the product contains organic matter it is clarified. The solution is slightly acidified with nitric acid and the chlorides precipitated in the form of silver chloride by means of a solution of silver nitrate. The excess silver nitrate is titrated with a solution of ammonium thiocyanate, by Volhard’s method.
3. Reagents
3.1.Solution of ammonium thiocyanate 0,1 mol/litre.
3.2.Solution of silver nitrate 0,1 mol/litre.
3.3.Saturated solution of ammonium ferric sulphate (NH4)Fe(SO4)2.
3.4.Nitric acid, density: 1,38 g/ml.
3.5.Diethyl ether.
3.6.Acetone.
3.7.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.
3.8.Carrez solution II: dissolve in water 10,6 g of potassium ferrocyanide K4Fe(CN)6·3H2O. Make up to 100 ml with water.
3.9.Active carbon, free from chlorides and not absorbing them.
4. Apparatus
Mixer (tumbler): approximately 35 to 40 rpm.
5. Procedure
According to the nature of the sample, prepare a solution as shown under point 5.1.1, 5.1.2 or 5.1.3.
At the same time carry out a blank test omitting the sample to be analysed.
Weigh to the nearest mg a sample of not more than 10 g and containing not more than 3 g of chlorine in the form of chlorides. Place with 400 ml of water in a 500 ml volumetric flask at approximately 20 °C. Mix for thirty minutes in the tumbler, bring up to volume, homogenise and filter.
Weigh approximately 5 g of the sample to the nearest mg and place with 1 g of active carbon in a 500 ml volumetric flask. Add 400 ml of water at approximately 20 °C and 5 ml of Carrez solution I (point 3.7), stir for 30 seconds then add 5 ml of Carrez solution II (point 3.8). Mix for thirty minutes in the tumbler, bring up to volume, homogenise and filter.
Prepare the solution as described under point 5.1.2 but do not filter. Decant (if necessary centrifuge), remove 100 ml of the supernatant liquid and transfer to a 200 ml measuring flask. Mix with acetone (point 3.6) and bring up to volume with this solvent, homogenise and filter.
Using a pipette, transfer to an Erlenmeyer flask from 25 ml to 100 ml of the filtrate (according to the assumed chlorine content) obtained as described under point 5.1.1, 5.1.2 or 5.1.3. The aliquot portion must not contain more than 150 mg of chlorine (Cl). Dilute if necessary to not less than 50 ml with water, add 5 ml of nitric acid (point 3.4), 2 ml of saturated solution of ammonium ferric sulphate (point 3.3) and two drops of ammonium thiocyanate solution (point 3.1) transferred by means of a burette filled up to the zero mark. Using a burette, transfer the silver nitrate solution (point 3.2) in such a way that an excess of 5 ml is obtained. Add 5 ml of diethyl ether (point 3.5) and shake hard to coagulate the precipitate. Titrate the excess silver nitrate with the ammonium thiocyanate solution (point 3.1) until the reddish-brown tint has lasted for one minute.
6. Calculation of results
The amount of chlorine (X), expressed as % sodium chloride is calculated by using the following formula:
where:
If the blank test indicates that silver nitrate solution 0,1 mol/l has been consumed deduct this value from the volume (V1 – V2).
7. Observations
7.1.Titration may also be carried out by potentiometry or amperometry.
7.2.In the case of products which are very rich in oils and fats, first de-fat with diethyl ether or light petroleum.
7.3.In the case of fish-meal, titration may be carried out by Mohr’s method.
P. DETERMINATION OF CARBONATES
1. Purpose and Scope
This method makes it possible to determine the amount of carbonates, conventionally expressed as calcium carbonate, in feed with the exception of feed where iron carbnate is present.
2. Principle
The carbonates are decomposed in hydrochloric acid; the carbon dioxide released is collected in a graduated tube, and its volume compared with that released under the same conditions by a known quantity of calcium carbonate.
3. Reagents
3.1.Hydrochloric acid, density 1,10 g/ml.
3.2.Pure calcium carbonate.
3.3.Sulphuric acid, approximately 0,05 mol/litre, coloured with methyl red.
4. Apparatus
Scheibler-Dietrich apparatus (see diagram in the Appendix) or equivalent apparatus (calcimeter).
5. Procedure
Depending on the sample’s carbonate content, weigh a portion of the sample as shown below:
(a) 0,5 g for products containing from 50 % to 100 % of carbonates, expressed as calcium carbonate;
(b) 1 g for products containing from 40 % to 50 % of carbonates, expressed as calcium carbonate;
(c) 2 g to 3 g for other products.
Hydrochloric acid (point 3.1, above) is added to the portion of the sample to decompose any carbonates present. The volume of carbon dioxide is measured by using a Scheibler-Dietrich apparatus or equivalent apparatus (calcimeter) and is compared with the volume of carbon dioxide produced by 0,5 g pure calcium carbonate (point 3.2, above).
All determinations shall be carried out under the same conditions in order to avoid making corrections for differences in temperature and pressure. The determination should be preferably carried out in a temperature-controlled room.
The procedure making use of the Scheibler-Dietrich apparatus is described in detail in the Appendix.
6. Calculation
The content of carbonates, expressed as pure calcium carbonate, is calculated by using the formula:
| X = | V ×100 |
|---|---|
| V1 × 2m |
where:
7. Observations
7.1.If the Scheibler-Dietrich apparatus is used and the sample weighs more than 2 g, first place 15 ml of distilled water in the flask (item 4 in the diagram in the Appendix, below) and mix before beginning the test by adding hydrochloric acid (point 3.1, above). Use the same volume of distilled water for the control test.
7.2.If the apparatus used has a different volume from that of the Scheibler-Dietrich apparatus, the portions taken from the sample and from the control substance and the calculation must be adapted accordingly.
Appendix
Detailed procedure making use of the Scheibler-Dietrich apparatus
Place the portion of the sample in the special flask (item 4 in the diagram) of the apparatus, fitted with a small tube of unbreakable material containing 10 ml of hydrochloric acid (point 3.1, above), and connect the flask to the apparatus. Turn the three-way cock (item 5 in the diagram) so that the graduated tube (item 1 in the diagram) connects with the outside. Using the mobile tube (item 2 in the diagram), which is filled with coloured sulphuric acid (point 3.3, above) and is connected to the graduated tube (item 1 in the diagram), bring the level of the liquid up to the zero mark. Turn the three-way cock (item 5 in the diagram) in order to connect up the tubes (items 1 and 3 in the diagram) and check that the level is at zero.
Run the hydrochloric acid (point 3.1, above) slowly over the portion of the sample, tilting the special flask (item 4 in the diagram). Make the pressure equal by lowering the mobile tube (item 2 in the diagram). Shake the special flask (item 4 in the diagram) until the release of carbon dioxide has stopped completely.
Restore the pressure by bringing the liquid back to the same level in the tubes (items 1 and 2 in the diagram). After a few minutes, when the volume of gas has become constant, take the reading.
Carry out a control test in the same conditions on 0,5 g of calcium carbonate (point 3.2, above).
SCHEIBLER-DIETRICH APPARATUS FOR THE DETERMINATION OF CO2
(measured in mm)
ANNEX IV
A. DETERMINATION OF VITAMIN A
The vitamin A is to be determined by:
— the method of analysis provided for by EN 17547 Animal feeding stuffs: Methods of sampling and analysis – Determination of vitamin A, E and D (19) content – Method using solid phase extraction (SPE) clean-up and high-performance liquid chromatography (HPLC), or
— by reversed phase high performance liquid chromatography (RP-HPLC) using a UV or a fluorescence detector, as described in the points 1 to 9 hereafter.
1. Purpose and scope
This method makes it possible to determine the level of vitamin A (retinol) in feed. Vitamin A includes all-trans-retinyl alcohol and its cis-isomers which are determined by this method. The content of vitamin A is expressed in International Units (IU) per kg. One IU corresponds to the activity of 0,300 μg all-trans-vitamin A alcohol or 0,344 μg all-trans-vitamin A acetate or 0,550 μg all-trans-vitamin A palmitate.
The limit of quantification is 2 000 IU vitamin A/kg.
2. Principle
The sample is hydrolysed with ethanolic potassium hydroxide solution and the vitamin A is extracted into light petroleum. The solvent is removed by evaporation and the residue is dissolved in methanol and, if necessary, diluted to the required concentration. The content of vitamin A is determined by reversed phase high performance liquid chromatography (RP-HPLC) using a UV or a fluorescence detector. The chromatographic parameters are chosen so that there is no separation between the all-trans- vitamin A alcohol and its cis isomers.
3. Reagents
3.1.Ethanol, σ = 96 %.
3.2.Light petroleum, boiling range 40 °C-60 °C.
3.3.Methanol.
3.4.Potassium hydroxide solution, c = 50 g/100 ml.
3.5.Sodium ascorbate solution, c = 10 g/100 ml (see point 7.7 observations).
3.6.Sodium sulphide, Na2S · x H2O (x = 7 – 9).
3.6.1.Sodium sulphide solution, c = 0,5 mol/l in glycerol, ß = 120 g/l (for x = 9) (see point 7.8 observations).
3.7.Phenolphthalein solution, c = 2 g/100 ml in ethanol (point 3.1).
3.8.2-Propanol.
3.9.Mobile phase for HPLC: mixture of methanol (point 3.3) and water, e.g. 980 + 20 (v + v). The exact ratio will be determined by the characteristics of the column employed.
3.10.Nitrogen, oxygen free.
3.11.All-trans-vitamin A acetate, extra pure, of certified activity, e.g. 2,80 × 106 IU/g.
3.11.1.Stock solution of all-trans-vitamin A acetate: Weigh to the nearest 0,1 mg, 50 mg of vitamin A acetate (point 3.11) into a 100 ml graduated flask. Dissolve in 2-propanol (point 3.8) and make up to the mark with the same solvent. The nominal concentration of this solution is 1 400 IU vitamin A per ml. The exact content shall be determined according to point 5.6.3.1.
3.12.All-trans-vitamin A palmitate, extra pure, of certified activity, e.g. 1,80 × 106 IU/g.
3.12.1.Stock solution of all-trans-vitamin A palmitate: Weigh to the nearest 0,1 mg, 80 mg of vitamin A palmitate (point 3.12) into a 100 ml graduated flask. Dissolve in 2-propanol (point 3.8) and make up to the mark with the same solvent. The nominal concentration of this solution is 1 400 IU vitamin A per ml. The exact content shall be determined according to point 5.6.3.2.
3.13.2,6-Di-tert-butyl-4-methylphenol (BHT) (see point 7.5 observations).
4. Apparatus
4.1.Vacuum rotary evaporator.
4.2.Amber glassware.
4.2.1.Flat bottom or conical flasks, 500 ml, with ground-glass socket.
4.2.2.Graduated flasks with ground-glass stoppers, narrow-necked, 10, 25, 100 and 500 ml.
4.2.3.Separating funnels, conical, 1 000 ml, with ground-glass stoppers.
4.2.4.Pear-shaped flasks, 250 ml, with ground-glass sockets.
4.3.Allihn condenser, jacket length 300 mm, with ground-glass joint, with adapter for gas feed pipe.
4.4.Pleated filter paper for phase separation, diameter 185 mm (e.g. Schleicher & Schuell 597 HY 1/2).
4.5.HPLC equipment with injection system.
4.5.1.Liquid chromatographic column, 250 mm × 4 mm, C18, 5 or 10 μm packing, or equivalent (performance criterion: only a single peak for all retinol isomers under the HPLC-conditions).
4.5.2.UV or fluorescence detector, with variable wavelength adjustment.
4.6.Spectrophotometer with 10 mm quartz cells.
4.7.Water-bath with magnetic stirrer.
4.8.Extraction apparatus (see Figure 1) consisting of:
4.8.1.Glass cylinder of 1 l capacity fitted with a ground glass neck and stopper.
4.8.2.Ground glass insert equipped with a side-arm and an adjustable tube passing through the centre. The adjustable tube shall have a U-shaped lower end and a jet at the opposite end so that the upper liquid layer in the cylinder may be transferred into a separating funnel.
5. Procedure
Note: Vitamin A is sensitive to (UV-) light and to oxidation. All operations shall be carried out in the absence of light (using amber glassware, or glassware protected with aluminium foil) and oxygen (flush with nitrogen). During extraction air above the liquid shall be replaced by nitrogen (avoid excess pressure by loosening the stopper from time to time).
Grind the sample so that it passes a 1 mm mesh sieve, taking care to avoid generation of heat. Grinding must be carried out immediately before weighing and saponification otherwise there may be losses of vitamin A. Do not grind the sample(s) if the particle size distribution is adequate (e.g. premixtures and feed additives).
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