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The Minister for Agriculture, Food
and Forestry Policies
CENTRAL INSPECTORATE DIVISION FOR THE PROTECTION OF QUALITY AND FOR
THE REPRESSION OF FRAUD CONCERNING AGRO-FOOD PRODUCTS
DIRECTORATE GENERAL FOR FRAUD PREVENTION AND FOR THE REPRESSION
OF FRAUD CONCERNING AGRO-FOOD PRODUCTS
1. ------IND- 2013 0578 I-- EN- ------ 20131105 --- --- PROJET
Decree No _______
Approval of the official analysis method for determining free amino acid content in cheese
THE DIRECTOR-GENERAL
IN AGREEMENT WITH
THE DIRECTOR-GENERAL
OF ANIMAL HEALTH AND VETERINARY MEDICINE
OF THE MINISTRY OF HEALTH
THE DIRECTOR
OF THE CUSTOMS AND MONOPOLIES AGENCY
AND
THE DIRECTOR-GENERAL
FOR THE INDUSTRIAL AND COMPETITION POLICY
OF THE MINISTRY OF ECONOMIC DEVELOPMENT
HAVING CONSIDERED Article 43 of Royal Decree 2033 of 15 October 1925, amended
by Law 562 of 18 March 1926, laying down provisions on the "Repression of fraud in the
preparation and sale of substances for agricultural use and farming products" and Article 108 of
Royal Decree 1361 of 1 July 1926, laying down rules for the implementation of the aforementioned Royal Decree 2033/1925 which state that tests on the products and substances to which
the decree-law refers must be conducted by laboratories entrusted with official methods set forth
and adopted by this Ministry in concert with the Ministries of Finance, Health and of Industry,
Commerce and Artisan Crafts;
HAVING CONSIDERED Legislative Decree 282 of 18 June 1986 converted with
amendments to Law 462 of 7 August 1986, Article 10 of which establishes the foundation of a
Central Inspectorate for the Repression of Fraud within the Ministry of Agriculture and Forestry for
the performance, among other things, of duties inherent to the prevention and repression of
violations in the preparation and trade of agro-food products and substances for agrarian and
forestry use;
HAVING CONSIDERED Presidential Decree 41 of 14 February 2012 laying down the
“Reorganisation of the Ministry of Agricultural, Food and Forestry Policies (omitted)”, Article 4 of
which establishes for the Central Inspectorate, the title “Dipartimento dell’Ispettorato centrale della
tutela della qualità e repressione frodi dei prodotti agro-alimentari” (Central Inspectorate
Department for the Protection of Quality and for the Repression of Fraud concerning Agro-Food
The Minister for Agriculture, Food
and Forestry Policies
CENTRAL INSPECTORATE DIVISION FOR THE PROTECTION OF QUALITY AND FOR
THE REPRESSION OF FRAUD CONCERNING AGRO-FOOD PRODUCTS
DIRECTORATE GENERAL FOR FRAUD PREVENTION AND FOR THE REPRESSION
OF FRAUD CONCERNING AGRO-FOOD PRODUCTS
Products) with the acronym ICQRF, confirming among its responsibilities, the updating of official
methods of analysis of agro-food products and substances for agrarian and forestry use;
HAVING CONSIDERED Article 2(1) of Legislative Decree 300 of 30 July 1999, amended
by Decree-Law 85 of 16 May 2008, converted with amendments into Law 121 of 14 July 2008
laying down "Urgent provisions for the adaptation of government structures in application of
Article 1(376) and (377) of Law 244 of 24 December 2007" which establishes the number and the
name of Ministries;
HAVING CONSIDERED Articles 57, 63 and 68(1) of the afore-mentioned Legislative
Decree 300/1999, in regard to the institution of revenue agencies, including the "Customs and
Monopolies Agency" and, for the latter, the assignment of duties and the appointment of the role of
Director who is entrusted to represent and manage the Customs and Monopolies Agency;
HAVING CONSIDERED Law 172 of 13 November 2009, laying down the institution of the
"Ministry of Health" which took effect on 13 December 2009;
HAVING CONSIDERED Legislative Decree No 165 of 30 March 2001 and subsequent
amendments;
HAVING CONSIDERED Ministerial Decree of 05 May 2006 of the pro tempore, Ministry
of Agricultural and Forestry Policies concerning the institution, in accordance with Article 44 of
Law No 82 of 20 February 2006, of the Consultative Commission for the update of the official
analysis method of agro-food products and substances for agrarian and forestry use, structured into
10 sub-commissions with sector-specific competences, including the Sub-commission for milk and
derivatives, instituted and appointed by Ministerial Decree No 1795 of 19 December 2008 as
subsequently amended in its composition by Ministerial Decree of 16 December 2011;
HAVING CONSIDERED the Directive of the President of the Council of Ministers of
04 August 2010 in relation to interpretation guidelines on the re-order of collegial organisations and
the reduction of the costs of administrative equipment, which ordered the operation, under an
extended regime, of the above-mentioned Consultative Commission and the related Subcommissions, until 28 June 2012;
HAVING CONSIDERED the Commission Implementing Regulation (EU) No 584/2011 of
17 June 2011, setting out approval of the non-secondary amendments to the regulation of the use of
the denomination "Grana Padano (DOP)", registered on the register of protected denominations of
origin and protected geographical indications, in which reference is made to the specific amino acid
composition, to be determined by means of the use of ion exchange chromatography with postcolumn photometric detection with ninhydrin;
The Minister for Agriculture, Food
and Forestry Policies
CENTRAL INSPECTORATE DIVISION FOR THE PROTECTION OF QUALITY AND FOR
THE REPRESSION OF FRAUD CONCERNING AGRO-FOOD PRODUCTS
DIRECTORATE GENERAL FOR FRAUD PREVENTION AND FOR THE REPRESSION
OF FRAUD CONCERNING AGRO-FOOD PRODUCTS
HAVING CONSIDERED Commission Regulation (EU) No 794/2011 of 08 August 2011,
setting out the approval of the amendments to the regulation of the denomination "Parmigiano
Reggiano", registered on the register of protected denominations of origin and protected
geographical indications, in which it is established that the "grated" type must, among other
requirements, have an amino acid composition that is specific to "Parmigiano Reggiano";
HAVING CONSIDERED the Decrees of the President of the Council of Ministers of
04 November 1991, whereby the denomination of origin of the cheese "Parmigiano Reggiano" and
the cheese "Grana Padano" was extended to the respective "grated" types, with the expressed
requirement of the respective specific amino acid composition;
HAVING CONSIDERED European Parliament and Council Regulation (EC) No 882/2004,
on official controls in regard to feed and food, in which Chapter III, Article 11 :"Methods of
Sampling and Analysis" states that "analysis methods used in the context of official controls shall
comply with relevant Community rules or, (a) if no such rules exist, with internationally recognised
rules or protocols (omitted) or those agreed in national legislation;" and that "Wherever possible,
methods of analysis shall be characterised by the appropriate criteria";
CONSIDERING it necessary to establish an analysis method for the control of free amino
acid content of the afore-mentioned Protected Designation of Origin cheeses, in order to strengthen
the instruments protecting and safeguarding products registered as protected denomination of origin
and protected geographical indications;
WITH REGARD to the opinion given in the meeting of the Consultative Commission held
on 15 July 2011 for the update of the official methods of analysis of agro-food products and
substances for agrarian and forestry use - Sub-commission for milk and derivatives, as mentioned
previously;
HAVING CONSIDERED the results, documented by note prot. DGPREF No 4953 of
4 April 2013 of ICQRF of the inter-laboratory circuit instituted by note prot. EX DG PREF No 9553
of 05 July 2012 of ICQRF, aimed at ensuring the validation of a method of analysis for determining
the free amino acid content of cheese by using ion exchange chromatography with post-column
photometric detection with ninhydrin, in compliance with the Commission opinion pursuant to the
point above, which showed the suitability of the method to be used for application to the official
control;
HAVING CONSIDERED Directive 98/34/EC and subsequent amendments laying down a
procedure for the provision of information in the field of technical standards and regulations;
The Minister for Agriculture, Food
and Forestry Policies
CENTRAL INSPECTORATE DIVISION FOR THE PROTECTION OF QUALITY AND FOR
THE REPRESSION OF FRAUD CONCERNING AGRO-FOOD PRODUCTS
DIRECTORATE GENERAL FOR FRAUD PREVENTION AND FOR THE REPRESSION
OF FRAUD CONCERNING AGRO-FOOD PRODUCTS
HEREBY DECREES
Article 1
1. The official method of analysis for determining the free amino acid content of cheeses
described in an attachment to this decree is hereby approved.
2. The scope of application of the method pursuant to Paragraph 1 is defined in the method
itself.
Article 2
The method of analysis described in this decree applies to the control of domestic products.
This decree, which shall be sent to the relevant supervisory body, shall take effect on the day
after its publication in the Official Journal of the Italian Republic.
on behalf of THE MINISTRY OF AGRICULTURAL, FOOD AND FORESTRY POLICIES
The Director-General of the Directorate General for the Prevention and for the
Repression of Frauds concerning Agro-Food Products
on behalf of THE MINISTRY OF HEALTH
on behalf of THE CUSTOMS AND MONOPOLIES AGENCY
The Director
on behalf of THE MINISTRY OF ECONOMIC DEVELOPMENT
Rome,
The Minister for Agriculture, Food
and Forestry Policies
CENTRAL INSPECTORATE DIVISION FOR THE PROTECTION OF QUALITY AND FOR
THE REPRESSION OF FRAUD CONCERNING AGRO-FOOD PRODUCTS
DIRECTORATE GENERAL FOR FRAUD PREVENTION AND FOR THE REPRESSION
OF FRAUD CONCERNING AGRO-FOOD PRODUCTS
Decree _______
ATTACHMENT: METHOD OF ANALYSIS
CHEESE - DETERMINATION OF THE FREE AMINO ACID CONTENT
1. SCOPE AND AREA OF APPLICATION
This standard describes the determination of the free amino acid content of cheese, with the
use of the ion exchange amino acid analyser with reference to the amino acids given in
Table 1 below:
Table 1 – Amino acids and respective codes
Amino acid
Code
Aspartic Acid
γ-aminobutyric acid
Glutamic acid
Alanine
Arginine
Asparagine
Citrulline
Phenylalanine
Glycine
Glutamine
Isoleucine
Histidine
Leucine
Lysine
Methionine
Ornithine
Proline
Serine
Tyrosine
Threonine
Valine
ASP
GABA
GLU
ALA
ARG
ASN
CIT
PHE
GLY
GLN
ILE
HIS
LEU
LYS
MET
ORN
PRO
SER
TYR
THR
VAL
The Minister for Agriculture, Food
and Forestry Policies
CENTRAL INSPECTORATE DIVISION FOR THE PROTECTION OF QUALITY AND FOR
THE REPRESSION OF FRAUD CONCERNING AGRO-FOOD PRODUCTS
DIRECTORATE GENERAL FOR FRAUD PREVENTION AND FOR THE REPRESSION
OF FRAUD CONCERNING AGRO-FOOD PRODUCTS
WARNING: Use of this method may require the use of dangerous substances or the
implementation of operations that entail a certain risk. This method does not aim to deal
with all safety problems connected with its use; the user is responsible for defining
appropriate safety procedures and for compliance with current legislation.
2. REFERENCE LEGISLATION
UNI EN ISO 3696:1996 – Water for analytical laboratory use. Specifications and test
methods.
ISO 707:2008 (IDF 50:2008) – Milk and milk products. Guidance on sampling
UNI ISO 5725-2:2004 - Accuracy (trueness and precision) of measurement methods and
results - Part 2: Basic method for the determination of repeatability and reproducibility of a
standard measurement method
3. TERMS AND DEFINITIONS
3.1. XXX (code) amino acid content: quantity of XXX amino acid in the sample, expressed in
mg/kg
3.2. Total free amino acids: sum of the content of the amino acids pursuant to Table 1,
expressed in mg/kg.
4. PRINCIPLE
The cheese is solubilised with sodium citrate buffer; the macro molecules in the solution are
precipitated with sulphosalicylic acid and removed for filtration.
The filtered solution containing the free amino acids is suitably diluted and the amino acids
are separated by ion exchange chromatography and determined by post-column reaction with
ninhydrin and photometric detection at 570 and 440 nm.
5. REAGENTS
5.1. Water, ultra-pure Type I, according to standard UNI ISO 3696
5.2. Chloric acid, smoking 37 %
5.3. 2.2’-Thiodiethanol (thiodiglycol), aqueous solution 25 % for the preparation of elution
buffers
The Minister for Agriculture, Food
and Forestry Policies
CENTRAL INSPECTORATE DIVISION FOR THE PROTECTION OF QUALITY AND FOR
THE REPRESSION OF FRAUD CONCERNING AGRO-FOOD PRODUCTS
DIRECTORATE GENERAL FOR FRAUD PREVENTION AND FOR THE REPRESSION
OF FRAUD CONCERNING AGRO-FOOD PRODUCTS
5.4. 2-Propanol, for the preparation of elution buffers
5.5. Standard amino acids, listed in paragraph 1
5.6. Sodium hydroxide solution, 12 mol/L.
Dissolve 480 g of NaOH in water and make up to 1 L
5.7. Sodium citrate buffer, 0.2 mol/L, pH 2.20 for the extraction of the free amino acids.
Dissolve 19.61 g of sodium citrate dihydrate, 400 mg of EDTA disodium salt,
100 mL of pentachlorophenol (50 mg of pentachlorophenol dissolved in 10 mL of
ethyl alcohol) and 16 mL of chloric acid in approx. 800 mL of water. Leave
shaking for 24 hours, adjust pH to 2.20 using chloric acid, checking with a pH
metre (6.2), make up to 1 L with water and then filter under a vacuum through a
cellulose acetate membrane with pores of 0.2 μm.
5.8. 5-sulphosalicylic acid, β = 7.5 %, deproteinising. Dissolve 75 g of 5-sulphosalicylic acid
dihydrate in approx. 800 mL of citrate buffer (5.7). Adjust the pH to 1.7-1.8 with a
sodium hydroxide solution (5.6), make up to 1 L with citrate buffer (5.7) and then
filter under a vacuum through a cellulose acetate membrane with pores of 0.45 μm
(6.6)
5.9. Lithium citrate buffer, for the dilution of samples and reference standards. Dissolve
8.4 g of lithium hydroxide monohydrate, 9.6 g of citric acid, 100 mL of
pentachlorophenol (50 mg of pentachlorophenol dissolved in 10 mL of ethyl
alcohol) and 8 mL of 2.2' - Thiodiethanol, aqueous solution 25 % in approx.
800 mL of water.
Leave shaking for 24 hours, adjust pH to 2.20 using chloric acid (5.2), checking
with a pH metre (6.2), make up to 1 L with water and then filter under a vacuum
through a cellulose acetate membrane with pores of 0.2 μm (6.6)
5.10.
Norleucine solution at approximately 600 mg/L, as per internal standard. In a
100 mL calibrated beaker, use analytical scales (6.1) to weigh out approximately
60 mg of norleucine and make up to volume with citrate lithium buffer (5.9). The
solution should be kept in the refrigerator and has a shelf life of six months.
5.11.
Ninhydrin, prepared according to the manufacturer's instructions
5.12.
Elution buffers, acquired ready or prepared in the laboratory, in accordance with
the instructions given in Table 1.
The buffers are prepared with lithium salts needed to separate the asparagine and
glutamine. The method needs five elution buffers and a regenerating solution.
Their composition is given in Table 2 (weighed for 1 L of solution):
The Minister for Agriculture, Food
and Forestry Policies
CENTRAL INSPECTORATE DIVISION FOR THE PROTECTION OF QUALITY AND FOR
THE REPRESSION OF FRAUD CONCERNING AGRO-FOOD PRODUCTS
DIRECTORATE GENERAL FOR FRAUD PREVENTION AND FOR THE REPRESSION
OF FRAUD CONCERNING AGRO-FOOD PRODUCTS
Table 2 – Elution buffer composition
BUFFER 1
BUFFER 2
BUFFER 3
BUFFER 4
BUFFER 5
BUFFER 6
DILUTION
BUFFER
2.84
3.00
3.15
3.50
3.58
----
2.20
0.2
0.3
0.5
0.9
1.62
0.3
0.2
8.4 g
8.4 g
8.4 g
4.2 g
7.0 g
12.59 g
8.4 g
CITRIC ACID
9.6 g
9.6 g
9.6 g
9.6 g
21.0 g
----
9.6 g
LITHIUM
CHLORIDE
----
4.25 g
12.72 g
34.0 g
61.5 g
----
----
0.1 mL
0.1 mL
0.1 mL
0.1 mL
0.1 mL
0.1 mL
0.1 mL
8 mL
8 mL
8 mL
----
----
----
8 mL
15 mL
15 mL
----
----
----
----
----
14 mL
13 mL
12 mL
3 mL
----
----
14 mL
pH
MOLARITY
(LI+)
LITHIUM
HYDROXIDE
MONOHYDRAT
E
PHENOL
PENTACHLORIDE
THIODIGLYCOL
(25 %)
2-PROPANOL
CHLORIC
37 %
ACID
6. EQUIPMENT
Standard laboratory equipment and, in particular:
6.1. Analytical scales, with sensitivity 0.1 mg
6.2. pH metre, with sensitivity 0.01 unit pH
6.3. Magnetic shaker
6.4. Homogeniser Ultra-Turrax type
6.5. Electronic burette, or calibrated glass pipette
6.6. Syringe inert membrane filters with pores of 0.2 μm and 0.45 μm
6.7. Paper filters, rapid (type S&S 589/1, Whatman Grade 41 or equivalent)
6.8. Paper filters, slow (type S&S 589/3, Whatman Grade 42 or equivalent)
6.9. Ion exchange chromatograph, complete with ion exchange column, thermostat reaction
bath for post-column derivitisation with ninhydrin and photometric detector able to
read at wavelengths of 570 and 440 nm.
The Minister for Agriculture, Food
and Forestry Policies
CENTRAL INSPECTORATE DIVISION FOR THE PROTECTION OF QUALITY AND FOR
THE REPRESSION OF FRAUD CONCERNING AGRO-FOOD PRODUCTS
DIRECTORATE GENERAL FOR FRAUD PREVENTION AND FOR THE REPRESSION
OF FRAUD CONCERNING AGRO-FOOD PRODUCTS
7. SAMPLING
Sampling is not part of the method specified in this regulation. ISO707/IDF50 gives a
recommended sampling method.
A representative sample must be sent to the laboratory. It must not have been damaged or
modified during transport or storage.
The laboratory keeps the sample in order to avoid any deterioration or alteration to its
composition.
8. PREPARATION OF THE SAMPLE
8.1. Preparation of the sample
Before extracting the free amino acids, the cheese sample must be finely ground.
8.2. Extraction of the free amino acids
The procedure for the extraction of free amino acids is carried out at room temperature,
which, as a general rule, should not exceed 25 °C.
Use analytical scales (6.1) to weigh 1.5 g of finely ground cheese in a 100 mL beaker, then
use an electronic burette or calibrated glass pipette to add 40 mL of sodium citrate buffer 0.2
N pH 2.20 (5.7). Place the beaker onto the magnetic shaker with a small magnetic anchor and
leave to shake for 15 minutes. Place the solution under the homogeniser (6.4) at minimum
speed for five minutes, and then filter through a quick paper filter (6.7) into a 100 mL flask.
Use a calibrated glass pipette to collect 10 mL of the filtrate and transfer it into a calibrated
25 mL beaker. Therefore, with a small magnetic anchor, place on the shaker (6.3) and add,
drop by drop, 10 mL of sulphosalicylic acid solution 7.5 %, pH 1.7-1.8 (5.8). Leave to shake
for five minutes and make up to volume with citrate buffer 0.2 N at pH 2.2 (5.7); shake, then
filter through slow paper filter (6.8) into a test tube. Use a calibrated glass pipette to collect
10 mL of the filtrate and transfer it into a calibrated 100 mL beaker. Add 2 mL of norleucine
solution to approximately 600 mg/L (5.10) and make up to volume with lithium citrate buffer
(5.9). Filter the solution thus obtained using a syringe filter through a regenerated cellulose
(RC) membrane with pores of 0.2 μm, before proceeding with instrumental analysis.
8.3. Preparation of the standard reference solution
8.3.1.
Concentrated reference mix of amino acids to approximately:
 300 mg/L for: γ-Aminobutyric, Citrulline, Glycine, Glutamine;
 400 mg/L for: Alanine, Arginine, Asparagine, Methionine, Ornithine, Tyrosine,
Threonine;
 600 mg/L for: Aspartic acid, Phenylalanine, Isoleucine, Histidine, Serine;
 800 mg/L for: Leucine, Proline, Valine;
The Minister for Agriculture, Food
and Forestry Policies
CENTRAL INSPECTORATE DIVISION FOR THE PROTECTION OF QUALITY AND FOR
THE REPRESSION OF FRAUD CONCERNING AGRO-FOOD PRODUCTS
DIRECTORATE GENERAL FOR FRAUD PREVENTION AND FOR THE REPRESSION
OF FRAUD CONCERNING AGRO-FOOD PRODUCTS
 900 mg/L for: Glutamic acid, Lysine.
In a calibrated 100 mL flask, use analytical scales to weigh out approximately 30 mg of γAminobutyric, Citrulline, Glycine, Glutamine, approximately 40 mg of Alanine, Arginine,
Asparagine, Methionine, Ornithine, Tyrosine, Threonine, approximately 60 mg of Aspartic
acid, Phenylalanine, Isoleucine, Histidine, Serine, approximately 80 mg of Leucine, Proline,
Valine and approximately 90 mg of Glutamic acid and Lysine. Make up to volume with
lithium citrate buffer (3.9). In calculating the concentrations, consider the purity of each of
the amino acids weighed.
The solution should be kept in the freezer and has a shelf life of six months.
8.3.2.
Intermediate reference mix of amino acids:
Use a calibrated glass pipette to transfer 50 mL of the concentrated mix into a 100 mL
calibrated flask and make up to volume with citrate lithium buffer (3.9).
The solution should be kept in the freezer and has a shelf life of six months.
8.3.3.
Diluted reference mix "A" of amino acids to approximately:
 0.75 mg/L for: γ-Aminobutyric, Citrulline, Glycine, Glutamine;
 1.0 mg/L for: Alanine, Arginine, Asparagine, Methionine, Ornithine, Tyrosine,
Threonine;
 1.5 mg/L for: Aspartic acid, Phenylalanine, Isoleucine, Histidine, Serine;
 2.0 mg/L for: Leucine, Proline, Valine;
 2.25 mg/L for: Glutamic acid, Lysine.
Use a calibrated glass pipette to transfer 0.5 mL of the intermediate reference mix of amino
acids and 2 mL of the norleucine solution to approximately 600 mg/L into a calibrated
100 mL flask (3.10). Make up to volume with lithium citrate buffer (3.9).
The solution should be kept in the freezer and has a shelf life of two months.
8.3.4.
Diluted reference mix "B" of amino acids to approximately:
 1.5 mg/L for: γ-Aminobutyric, Citrulline, Glycine, Glutamine;
 2.0 mg/L for: Alanine, Arginine, Asparagine, Methionine, Ornithine, Tyrosine,
Threonine;
 3.0 mg/L for: Aspartic acid, Phenylalanine, Isoleucine, Histidine, Serine;
 4.0 mg/L for: Leucine, Proline, Valine;
 4.5 mg/L for: Glutamic acid, Lysine.
The Minister for Agriculture, Food
and Forestry Policies
CENTRAL INSPECTORATE DIVISION FOR THE PROTECTION OF QUALITY AND FOR
THE REPRESSION OF FRAUD CONCERNING AGRO-FOOD PRODUCTS
DIRECTORATE GENERAL FOR FRAUD PREVENTION AND FOR THE REPRESSION
OF FRAUD CONCERNING AGRO-FOOD PRODUCTS
Use a calibrated glass pipette to transfer 1 mL of the intermediate reference mix of amino
acids and 2 mL of the norleucine solution to approximately 600 mg/L into a calibrated
100 mL flask (3.10). Make up to volume with lithium citrate buffer (3.9).
The solution should be kept in the freezer and has a shelf life of two months.
8.3.5.
Diluted reference mix "C" of amino acids to approximately:
 3.0 mg/L for: γ-Aminobutyric, Citrulline, Glycine, Glutamine;
 4.0 mg/L for: Alanine, Arginine, Asparagine, Methionine, Ornithine, Tyrosine,
Threonine;
 6.0 mg/L for: Aspartic acid, Phenylalanine, Isoleucine, Histidine, Serine;
 8.0 mg/L for: Leucine, Proline, Valine;
 9.0 mg/L for: Glutamic acid, Lysine.
Use a calibrated glass pipette to transfer 2 mL of the intermediate reference mix of amino
acids and 2 mL of the norleucine solution to approximately 600 mg/L into a calibrated
100 mL flask (3.10). Make up to volume with lithium citrate buffer (3.9).
The solution should be kept in the freezer and has a shelf life of two months.
8.3.6.
Diluted reference mix "D" of amino acids to approximately:
 7.5 mg/L for: γ-Aminobutyric, Citrulline, Glycine, Glutamine;
 10.0 mg/L for: Alanine, Arginine, Asparagine, Methionine, Ornithine, Tyrosine,
Threonine;
 15.0 mg/L for: Aspartic acid, Phenylalanine, Isoleucine, Histidine, Serine;
 20.0 mg/L for: Leucine, Proline, Valine;
 22.5 mg/L for: Glutamic acid, Lysine.
Use a calibrated glass pipette to transfer 5 mL of the intermediate reference mix of amino
acids and 2 mL of the norleucine solution to approximately 600 mg/L into a calibrated
100 mL flask (3.10). Make up to volume with lithium citrate buffer (3.9).
The solution should be kept in the freezer and has a shelf life of two months.
9. PROCEDURE
9.1. Separation of the free amino acids
9.1.1.
Principle of the method
The analytical principle is based on the elution of the sample by means of an ion exchange
chromatograph column followed by post-column derivitisation with ninhydrin, detection
The Minister for Agriculture, Food
and Forestry Policies
CENTRAL INSPECTORATE DIVISION FOR THE PROTECTION OF QUALITY AND FOR
THE REPRESSION OF FRAUD CONCERNING AGRO-FOOD PRODUCTS
DIRECTORATE GENERAL FOR FRAUD PREVENTION AND FOR THE REPRESSION
OF FRAUD CONCERNING AGRO-FOOD PRODUCTS
using a colour metre, acquisition and subsequent data processing using a computer with
specific software.
The comparison of the different retention times with those of a standard amino acid solution
(6.1-6.6) enables the identification of the individual amino acids in the sample.
The laboratory verifies its capacity to apply the method, with specific reference to the
separation capacity (above all with regards to the trio ASN/GLU/GLN) and the linearity of
the response.
9.1.2.
Chromatographic conditions
Set the instrument according to the following conditions:
 injected volume: 100 l sample previously filtered through cellulose acetate 0.2
m
 buffer flow velocity: 20 mL/h
 ninhydrin flow velocity: 20 mL/h
 duration of data acquisition: approx. 160 min.
 regeneration time: 47 min.
 reaction bath temperature: 135 °C
 elution gradient: see Table 3
Table 3 - elution gradient
No
Time
Temp.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
End
01:00
00:00
01:00
05:30
38:00
17:00
10:00
34:00
50:00
06:00
10:00
01:00
30:00
06:00
31 oC
31 oC
31 oC
31 oC
31 oC
40 oC
64 oC
64 oC
76 oC
76 oC
31 oC
31 oC
31 oC
31 oC
Buffer (see
Table 1)
1
1
1
1
2
3
3
4
5
6
1
1
1
1
Flow
Ninhydrin
20 mL/h
20 mL/h
20 mL/h
20 mL/h
20 mL/h
20 mL/h
20 mL/h
20 mL/h
20 mL/h
20 mL/h
20 mL/h
OFF
25 mL/h
20 mL/h
ON
ON
ON
ON
ON
ON
ON
ON
ON
ON
ON
OFF
OFF
ON
Commands
Reset
Load
The Minister for Agriculture, Food
and Forestry Policies
CENTRAL INSPECTORATE DIVISION FOR THE PROTECTION OF QUALITY AND FOR
THE REPRESSION OF FRAUD CONCERNING AGRO-FOOD PRODUCTS
DIRECTORATE GENERAL FOR FRAUD PREVENTION AND FOR THE REPRESSION
OF FRAUD CONCERNING AGRO-FOOD PRODUCTS
Figure 1 gives an example of chromatographic separation of the amino acids. The abovespecified separation conditions can be modified, in any case assuring a separation capacity of
the amino acids that is at least equal to that stated in Figure 1.
Figure 1 - Separation of the free amino acids of a standard solution (with specific reference
to the critical trio ASN-GLU-GLN and GLY-ALA-CIT)
Name
Minutes
10. CALCULATIONS AND EXPRESSION OF RESULTS
10.1.
Calculations
Starting from the values obtained for the four standard solutions of amino acids (8.3.3-8.3.6),
the calibration straight lines are constructed for the individual amino acids. In determining the
relevant equations, the calibration straight lines are forced to go through the axis origin.
By linear interpolation on the respective calibration straight lines, the concentrations of the
individual amino acids present in the sample extraction solutions (8.2) are derived.
The result is given at the initial sample quantity.
10.2.
Expression of results
The result is expressed for each amino acid in mg amino acid per kg cheese sample, with no
decimal figures.
The Minister for Agriculture, Food
and Forestry Policies
CENTRAL INSPECTORATE DIVISION FOR THE PROTECTION OF QUALITY AND FOR
THE REPRESSION OF FRAUD CONCERNING AGRO-FOOD PRODUCTS
DIRECTORATE GENERAL FOR FRAUD PREVENTION AND FOR THE REPRESSION
OF FRAUD CONCERNING AGRO-FOOD PRODUCTS
11. EXPLANATION
The repeatability and reproducibility values for each amino acid have been obtained in an
inter-laboratory circuit, carried out in 2012 (see Attachment A). Tests were carried out on
samples with total free amino acid content that is variable between approximately
57 000 mg/kg and 80 000 mg/kg and with content of the individual amino acid that varies
from approximately 100 mg/kg (GABA) to approximately 13 000 mg/kg (GLU).
11.1.
Repeatability
Refer to the data given in Table 4.
11.2.
Reproducibility
Refer to the data given in Table 4.
Table 4 – Repeatability type rejection values and reproducibility type rejection for each
individual amino acid, obtained
Repeatability (σr)
ASP
Reproducibility (σR)
y = mx + b1
Mean
y = mx + b1
Mean
σr
M
B
σR
M
B
23
0.017
-13.437
165
0.055
47.353
GABA
10
0.08
8.605
42
0.123
23.704
GLU
129
0.006
56.718
389
0.026
85.965
ALA
27
0.043
-49.995
132
0.042
57.752
ARG
28
0.011
13.396
96
0.033
49.429
ASN
42
0.002
35.914
136
0.122
-237.218
CIT
37
0.022
-4.933
113
0.040
35.943
PHE
46
0.002
40.108
167
0.088
-137.105
108
0.041
39.611
GLY
21
0.008
7.391
GLN
45
0.025
27.105
ILE
97
0.082
-261.360
201
89
0.070
39.611
0.102
-244.161
HIS
37
0.016
3.253
154
0.034
81.302
LEU
139
0.091
-430.871
370
-0.017
474.065
LYS
76
0.004
43.866
377
-0.007
433.832
MET
42
0.084
-91.746
110
0.143
-119.131
ORN
7
0.013
3.911
28
0.045
19.295
PRO
134
0.051
-187.572
349
-0.117
1094.494
SER
52
0.013
2.414
234
0.043
65.764
TYR
111
0.484
-766.276
195
0.282
-316.060
THR
25
0.010
1.490
106
0.003
99.774
VAL
54
0.017
-29.961
216
0.040
20.369
TOT. AA
596
0.022
-925.668
2907
0.051
-610.841
In the equation given (y = mx + b), y corresponds to σr/R and x corresponds to the concentration in
mg/kg of the individual amino acid
1
The Minister for Agriculture, Food
and Forestry Policies
CENTRAL INSPECTORATE DIVISION FOR THE PROTECTION OF QUALITY AND FOR
THE REPRESSION OF FRAUD CONCERNING AGRO-FOOD PRODUCTS
DIRECTORATE GENERAL FOR FRAUD PREVENTION AND FOR THE REPRESSION
OF FRAUD CONCERNING AGRO-FOOD PRODUCTS
12. BIBLIOGRAPHY
 D.H. Spackman, W.H. Stein, e S. Moore, Anal. Chem., 30, 1190 (1958);
 J.V. Benson, Jr, M.J. Gordon, e J.A. Patterson, Anal. Biochem., 18, 228 (1967);
 Final report of the inter-laboratory circuit CI 02.01.2012, sent by the Central
Inspectorate Department for the Protection of Quality and for the Repression of
Frauds concerning Agro-Food Products (ICQRF) with note DG PREF - Central
Laboratory of Rome - prot. no 0004953 of 04 April 2013.
13. ATTACHMENT A (INFORMATION) - RESULTS OF THE INTER-LABORATORY
CIRCUIT
The inter-laboratory circuit organised to validate this method was carried out in 2012. The
results obtained in the two tests were statistically analysed in accordance with ISO 57252:2004 to provide the precision data given in Table A.1 below.
The circuit was carried out on four samples with a variable content of the individual amino
acids (from approximately 100 mg/kg to approximately 13 000 mg/kg) and total free amino
acids (from approximately 57 000 mg/kg to 80 000 mg/kg).
Seven expert laboratories participated in the circuit. No laboratory was excluded from the
test. Some results were excluded from statistical processing following the application of the
Cochran and Grubbs tests. Please refer to the circuit report (see bibliography) for more
details.
Below is a summary table with the most important data obtained from the circuit.
The Minister for Agriculture, Food
and Forestry Policies
CENTRAL INSPECTORATE DIVISION FOR THE PROTECTION OF QUALITY AND FOR
THE REPRESSION OF FRAUD CONCERNING AGRO-FOOD PRODUCTS
DIRECTORATE GENERAL FOR FRAUD PREVENTION AND FOR THE REPRESSION
OF FRAUD CONCERNING AGRO-FOOD PRODUCTS
Table A.1 - Inter-laboratory circuit results of free amino acids
Field 1
ASP
GABA
GLU
ALA
ARG
ASN
CIT
PHE
GLY
GLN
ILE
mean (mg/kg)
2651
139
13816
1994
242
3387
2989
3913
2386
609
4957
sr
24
214
13
45
110
443
49
140
16
47
49
236
91
203
44
202
26
138
39
95
127
284
0.9 %
8.1 %
9.6 %
32.5 %
0.8 %
3.2 %
2.5 %
7.0 %
6.4 %
19.3 %
1.5 %
7.0 %
3.0 %
6.8 %
1.1 %
5.2 %
1.1 %
5.8 %
6.3 %
15.7 %
2.6 %
5.7 %
2523
210
12261
1908
2188
2891
1205
3512
1557
350
4807
37
173
1.5 %
6.6 %
9
48
4.2 %
22.8 %
168
423
1.4 %
3.5 %
24
158
1.3 %
8.3 %
55
216
2.5 %
9.9 %
38
182
1.3 %
6.3 %
14
94
1.2 %
7.8 %
56
184
1.6 %
5.2 %
24
109
1.5 %
7.0 %
38
57
10.9 %
16.3 %
174
293
3.6 %
6.1 %
2077
100
11958
1776
328
3213
2872
3615
1550
494
4236
25
139
1.2 %
6.7 %
7
33
7.0 %
33.1 %
144
370
1.2 %
3.2 %
16
105
0.9 %
5.9 %
15
60
4.7 %
18.1 %
36
51
1.1 %
1.6 %
30
99
1.0 %
3.5 %
40
172
1.1 %
4.8 %
18
99
1.1 %
6.4 %
38
72
7.6 %
14.5 %
49
54
1.2 %
1.3 %
1390
8920
1417
2459
2752
578
2877
1155
1346
3493
8
134
0.6 %
9.7 %
94
321
1.1 %
3.6 %
16
124
1.2 %
8.7 %
26
48
1.1 %
2.0 %
47
74
1.7 %
2.7 %
15
54
2.6 %
9.4 %
42
11
1.5 %
3.9 %
15
87
1.3 %
7.5 %
62
132
4.6 %
9.8 %
39
171
1.1 %
4.9 %
sR
CV%r
CV%R
mean (mg/kg)
sr
Field 2
sR
CV %r
CV %R
mean (mg/kg)
sr
Field 3
sR
CV %r
CV %R
mean (mg/kg)
sr
Field 4
Field 1
Field 2
Field 3
Field 4
sR
CV %r
CV %R
mean (mg/kg)
sr
sR
CV %r
CV %R
mean (mg/kg)
sr
sR
CV %r
CV %R
mean (mg/kg)
sr
sR
CV%r
CV%R
HIS
2184
56
153
2.5 %
7.0 %
LEU
6688
198
478
3.0 %
7.2 %
LYS
9346
67
421
0.7 %
4.5 %
MET
1925
67
152
3.5 %
7.9 %
ORN
382
10
36
2.6 %
9.4 %
PRO
7060
200
260
2.8 %
3.7 %
SER
4540
44
287
1.0 %
6.3 %
TYR
1858
165
266
8.9 %
14.3 %
THR
1849
22
108
1.2 %
5.8 %
VAL
5640
62
321
1.1 %
5.7 %
TOT. AA
79059
864
4251
1.1 %
5.4 %
1980
37
168
1.9 %
8.5 %
2317
31
166
1.3 %
7.2 %
6226
264
375
4.2 %
6.0 %
6616
64
240
1.0 %
3.6 %
8180
100
342
1.2 %
4.2 %
7803
77
308
1.0 %
4.0 %
1722
64
167
3.7 %
9.7 %
1519
19
45
1.3 %
3.0 %
102
9
26
8.9 %
25.8 %
253
5
31
1.9 %
12.2 %
6377
139
373
2.2 %
5.8 %
6628
102
306
1.5 %
4.6 %
4651
69
250
1.5 %
5.4 %
3708
63
212
1.7 %
5.7 %
1682
63
141
3.8 %
8.4 %
2000
188
216
9.4 %
10.8 %
2620
23
148
0.9 %
5.7 %
2450
36
47
1.5 %
1.9 %
4988
74
84
1.5 %
1.7 %
4880
44
235
0.9 %
4.8 %
72008
828
3338
1.2 %
4.6 %
69364
267
944
0.4 %
1.4 %
mean (mg/kg)
sr
sR
CV %r
CV %R
2043
23
129
1.1 %
6.3 %
5650
29
388
0.5 %
6.9 %
6340
60
437
0.9 %
6.9 %
1226
16
76
1.3 %
6.2 %
80
2
20
3.1 %
25.6 %
5376
94
457
1.7 %
8.5 %
2722
31
189
1.1 %
6.9 %
1710
30
156
1.7 %
9.1 %
2091
18
120
0.9 %
5.7 %
3928
36
222
0.9 %
5.6 %
57682
426
3093
0.7 %
5.4 %