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) (c. 152)

ANNEX IV U.K. METHODS OF ANALYSIS TO CONTROL THE LEVEL OF AUTHORISED ADDITIVES IN FEED

G. DETERMINATION OF LASALOCID SODIUM U.K.

Sodium salt of a polyether monocarboxylic acid produced by Streptomyces lasaliensis

1. Purpose and scope U.K.

The method makes it possible to determine the level of lasalocid sodium in feed and premixtures. The limit of detection is 5 mg/kg, the limit of quantification is 10 mg/kg.

2. Principle U.K.

Lasalocid sodium is extracted from the sample into acidified methanol and determined by reversed-phase high performance liquid chromatography (HPLC) using a spectrofluorometric detector.

3. Reagents U.K.

3.1. Potassium dihydrogen phosphate (KH₂PO₄). U.K.

3.2. Orthophosphoric acid, w (w/w) = 85 %. U.K.

3.3. Orthophosphoric acid solution, c = 20 %. U.K.

Dilute 23,5 ml of orthophosphoric acid (3.2) to 100 ml with water.

3.4. 6-Methyl-2-heptylamine (1,5-dimethylhexylamine), w (w/w) = 99 %. U.K.

3.5. Methanol, equivalent to HPLC grade. U.K.

3.6. Hydrochloric acid, density = 1,19 g/ml. U.K.

3.7. Phosphate buffer solution, c = 0,01 mol/l. U.K.

Dissolve 1,36 g of KH₂PO₄ (3.1) in 500 ml of water (3.11), add 3,5 ml of orthophosphoric acid (3.2) and 10,0 ml of 6-methyl-2-heptylamine (3.4). Adjust the pH to 4,0 with orthophosphoric acid solution (3.3) and dilute to 1 000 ml with water (3.11).

3.8. Acidified methanol. U.K.

Transfer 5,0 ml of hydrochloric acid (3.6) into a 1 000 ml graduated flask, make up to the mark with methanol (3.5) and mix. This solution must be prepared freshly before use.

3.9. HPLC mobile phase, phosphate buffer-methanol solution 5 + 95 (V + V). U.K.

Mix 5 ml of phosphate buffer solution (3.7) with 95 ml of methanol (3.5).

3.10. Lasalocid sodium standard substance with guaranteed purity, C₃₄H₅₃O₈Na (sodium salt of a polyether monocarboxylic acid produced by Streptomyces lasaliensis), E763. U.K.

3.10.1. Lasalocid sodium stock standard solution, 500 μg/ml U.K.

Weigh to the nearest 0,1 mg, 50 mg of lasalocid sodium (3.10) into a 100 ml graduated flask, dissolve in acidified methanol (3.8), make up to the mark with the same solvent and mix. This solution must be freshly prepared before use.

3.10.2. Lasalocid sodium intermediate standard solution, 50 μg/ml U.K.

Pipette 10,0 ml of stock standard solution (3.10.1) into a 100 ml graduated flask, make up to the mark with acidified methanol (3.8) and mix. This solution must be prepared freshly before use.

3.10.3. Calibration solutions U.K.

Into a series of 50 ml graduated flasks transfer 1,0, 2,0, 4,0, 5,0 and 10,0 ml of the intermediate standard solution (3.10.2). Make up to the mark with acidified methanol (3.8) and mix. These solutions correspond to 1,0, 2,0, 4,0, 5,0 and 10,0 μg of lasalocid sodium per ml respectively. These solutions must be prepared freshly before use.

3.11. Water, equivalent to HPLC grade. U.K.

4. Apparatus U.K.

4.1. Ultrasonic bath (or shaking water-bath) with temperature control. U.K.

4.2. Membrane filters, 0,45 μm. U.K.

4.3. HPLC equipment with injection system, suitable for injecting volumes of 20 μl. U.K.

4.3.1. Liquid chromatographic column 125 mm x 4 mm, reversed-phase C₁₈, 5 μm packing or equivalent. U.K.

4.3.2. Spectrofluorometer with variable wavelength adjustment of excitation and emission wavelengths. U.K.

5. Procedure U.K.

5.1. General U.K.

5.1.1. Blank feed U.K.

For the performance of the recovery test (5.1.2) a blank feed shall be analysed to check that neither lasalocid sodium nor interfering substances are present. The blank feed shall be similar in type to that of the sample and lasalocid sodium or interfering substances shall not be detected.

5.1.2. Recovery test U.K.

A recovery test shall be carried out by analysing the blank feed which has been fortified by addition of a quantity of lasalocid sodium, similar to that present in the sample. To fortify at a level of 100 mg/kg, transfer 10,0 ml of the stock standard (3.10.1) to a 250 ml conical flask and evaporate the solution to approximately 0,5 ml. Add 50 g of the blank feed, mix thoroughly and leave for 10 minutes mixing again several times before proceeding with the extraction step (5.2).

Alternatively, if a blank feed similar in type to that of the sample is not available (see 5.1.1), a recovery test can be performed by means of the standard addition method. In this case the sample to be analysed is fortified with a quantity of lasalocid sodium similar to that already present in the sample. This sample is analysed together with the unfortified sample and the recovery calculated by subtraction.

5.2. Extraction U.K.

5.2.1. Feed U.K.

Weigh to the nearest 0,01 g, from 5 g to 10 g of the sample into a 250 ml conical flask with stopper. Add 100,0 ml of acidified methanol (3.8) by pipette. Stopper loosely and swirl to disperse. Place the flask in an ultrasonic bath (4.1) at approximately 40 ^oC for 20 minutes, then remove and cool to room temperature. Allow to stand for about 1 hour until the suspended matter has settled, then filter an aliquot portion through a 0,45 μm membrane filter (4.2) into a suitable vessel. Proceed to the HPLC determination (5.3).

5.2.2. Premixtures U.K.

Weigh to the nearest 0,001 g about 2 g of the unground premix into a 250 ml graduated flask. Add 100,0 ml of acidified methanol (3.8) and swirl to disperse. Place the flask and contents in an ultrasonic bath (4.1) at approximately 40 ^oC for 20 minutes, then remove and cool to room temperature. Dilute to the mark with acidified methanol (3.8) and mix thoroughly. Allow to stand for 1 hour until the suspended matter has settled, then filter an aliquot portion through a 0,45 μm membrane filter (4.2). Dilute an appropriate volume of the clear filtrate with acidified methanol (3.8) to produce a final test solution containing about 4 μg/ml of lasalocid sodium. Proceed to the HPLC determination (5.3).

5.3. HPLC determination U.K.

5.3.1. Parameters U.K.

The following conditions are offered for guidance; other conditions may be used, provided they yield equivalent results:

Liquid chromatographic column (4.3.1):	125 mm × 4 mm, reversed-phase C18, 5 μm packing or equivalent
Mobile phase (3.9):	Mixture of phosphate buffer solution (3.7) and methanol (3.5), 5+95 (V+V)
Flow rate:	1,2 ml/min.
Detection wavelengths:
Excitation:	310 nm
Emission:	419 nm
Injection volume:	20 μl

Check the stability of the chromatographic system, injecting the calibration solution (3.10.3) containing 4,0 μg/ml several times, until constant peak heights (or areas) and retention times are achieved.

5.3.2. Calibration graph U.K.

Inject each calibration solution (3.10.3) several times and determine the mean peak heights (areas) for each concentration. Plot a calibration graph using the mean peak heights (areas) as the ordinates and the corresponding concentrations in μg/ml as the abscissae.

5.3.3. Sample solution U.K.

Inject the sample extracts obtained in 5.2.1 or 5.2.2 several times, using the same volume as taken for the calibration solution and determine the mean peak heights (areas) of the lasalocid sodium peaks.

6. Calculation of results U.K.

From the mean peak height (area) produced by injection of the sample solution (5.3.3) determine the concentration of lasalocid sodium (μg/ml) by reference to the calibration graph.

6.1. Feed U.K.

The lasalocid sodium content, w (mg/kg) in the sample is given by the following formula:

[mg/kg]

where:

c

=

lasalocid sodium concentration of the sample solution (5.2.1) in μg/ml

V₁

=

volume of the sample extract according to 5.2.1 in ml (i.e. 100)

m

=

weight of the test portion in g

6.2. Premixtures U.K.

The lasalocid sodium content, w (mg/kg) in the sample is given by the following formula:

[mg/kg]

where:

c

=

lasalocid sodium concentration of the sample solution (5.2.2) in μg/ml

V₂

=

volume of the sample extract according to 5.2.2 in ml (i.e. 250)

f

=

dilution factor according to 5.2.2

m

=

weight of the test portion in g

7. Validation of the results U.K.

7.1. Identity U.K.

Methods based on spectrofluorometry are less subject to interference than those in which UV detection is used. The identity of the analyte can be confirmed by co-chromatography.

7.1.1. Co-chromatography U.K.

A sample extract (5.2.1 or 5.2.2) is fortified by the addition of an appropriate amount of a calibration solution (3.10.3). The amount of added lasalocid sodium must be similar to the amount of lasalocid sodium found in the sample extract. Only the height of the lasalocid sodium peak shall be enhanced after taking into account the amount of lasalocid sodium added and the dilution of the extract. The peak width, at half height, must be within ± 10 % of the original peak width produced by the unfortified sample extract.

7.2. Repeatability U.K.

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

• 15 % relative to the higher value for lasalocid sodium contents from 30 mg/kg to 100 mg/kg,

• 15 mg/kg for lasalocid sodium contents from 100 mg/kg to 200 mg/kg,

• 7,5 % relative to the higher value for lasalocid sodium contents of more than 200 mg/kg.

7.3. Recovery U.K.

For the fortified (blank) feed sample, the recovery shall be at least 80 %. For the fortified premixture samples, the recovery shall be at least 90 %.

8. Results of a collaborative study U.K.

A collaborative study(1) was arranged in which 2 premixtures (samples 1 and 2) and 5 feeds (samples 3-7) were analysed by 12 laboratories. Duplicate analyses were performed on each sample. The results are given in the following table:

a Content declared by manufacturer.
b Feed prepared in the laboratory.
	Sample 1Chicken premix	Sample 2Turkey premix	Sample 3Turkey pellets	Sample 4Chicken crumbs	Sample 5Turkey Feed	Sample 6Poultry Feed A	Sample 7Poultry Feed B
L	12	12	12	12	12	12	12
n	23	23	23	23	23	23	23
Mean [mg/kg]	5 050	16 200	76,5	78,4	92,9	48,3	32,6
sr [mg/kg]	107	408	1,71	2,23	2,27	1,93	1,75
CVr [%]	2,12	2,52	2,24	2,84	2,44	4,0	5,37
sR [mg/kg]	286	883	3,85	7,32	5,29	3,47	3,49
CVR [%]	5,66	5,45	5,03	9,34	5,69	7,18	10,7
Nominal content [mg/kg]	5 000a	16 000a	80a	105a	120a	50b	35b

L

=

number of laboratories

n

=

number of single results

s_r

=

standard deviation of repeatability

s_R

=

standard deviation of reproducibility

CV_r

=

coefficient of variation of repeatability, %

CV_R

=

coefficient of variation of reproducibility, %.

(1)

Analyst, 1995, 120, 2175-2180.