Tentative Translation

JAS
0023

JAPANESE AGRICULTURAL
STANDARD
Testing method of K-value as a freshness index for fish
－High performance liquid chromatographic method

Date of Establishment: 2022 – 3 – 31

Ministry of Agriculture, Forestry and Fisheries

0023：2022

Precautions for using English version of JAS
This English translation has been made by the drafting party etc., based on the original text (Japanese version), and has
been posted on the website of Food and Agricultural Materials Inspection Center (FAMIC), Incorporated Administrative
Agency, with permission of the publisher of the original text (Ministry of Agriculture, Forestry and Fisheries).
The translation is made in consideration of technical contents, but it is aimed to provide information when using JAS
original text, and is not recognized as having the same effects as the original text.
If there is any doubt in the translation, please follow the original.
FAMIC is not responsible for inconvenience by using only the translation.
Food and Agricultural Materials Inspection Center, Incorporated Administrative Agency(2)

0023：2022

Contents

Page

1

Scope ....................................................................................................................................................................1

2

Normative references ..................................................................................................................................1

3

Terms and definitions..................................................................................................................................1

4

Principle .............................................................................................................................................................2

5

Reagents .............................................................................................................................................................26

Apparatus ..........................................................................................................................................................57

Preparation of test samples ......................................................................................................................6

8

Procedure ..........................................................................................................................................................6

8.1

General................................................................................................................................................................6

8.2

Extraction ..........................................................................................................................................................6

8.3

pH adjustment ..............................................................................................................................................6

8.4

Protein removal..............................................................................................................................................7

8.5

Preparation of sample solution ...............................................................................................................7

8.6

Measurement ...................................................................................................................................................79

Calculation.........................................................................................................................................................8

9.1

General................................................................................................................................................................8

9.2

Calculation of K-value ..................................................................................................................................8

9.3

Expression of results ....................................................................................................................................9

10

Precision ............................................................................................................................................................9

10.1

Interlaboratory test ......................................................................................................................................9

10.2

Repeatability ....................................................................................................................................................9

10.3

Reproducibility ...............................................................................................................................................9

11

Quality control.................................................................................................................................................9

12

Test report ..................................................................................................................................................... 10

Annex A (informative)

Results of interlaboratory tests....................................................................... 11

Annex B (informative)

Results of studies on the pH of HxR standard solutions forabsorbance measurement ...................................................................................................................... 12
Annex C (informative)

Typical HPLC chromatograms .......................................................................... 13(3)

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Bibliography.................................................................................................................................................................. 15(4)

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Foreword
This Japanese Agricultural Standard has been established by the Minister of Agriculture, Forestry and
Fisheries through deliberations at the Council for the Japanese Agricultural Standards as the result of
proposal for establishment of Japanese Agricultural Standard submitted by the Hakodate Regional Industry
Promotion Organization, Public Interest Incorporated Foundation with the original bill being attached,based on the provision of Article 4, paragraph (1) of the Act on Japanese Agricultural Standards.
Attention is drawn to the possibility that some parts of this Standard may conflict with patent rights,
published patent application or utility model rights. The Minister of Agriculture, Forestry and Fisheries andthe Council for the Japanese Agricultural Standards are not responsible for identifying any of such patentrights, published patent application or utility model rights.

(5)

JAPANESE AGRICULTURAL STANDARD(Tentative Translation)

JAS0023：2022

Testing method of K-value as a freshness index for fish
－High performance liquid chromatographic method
WARNING — The user of this document should be familiar with normal laboratory practice. This
document does not purport to address all the safety problems associated with its use. It is the
responsibility of the user of this document to establish appropriate safety and health practices andto ensure compliance with any national regulatory conditions.

1 ScopeThis document specifies a test method for calculating the K-value, which is a freshness index, from thecontent of ATP-related compounds in fresh fish (limited to Osteichthyes, and excluding thawed fish)measured by high performance liquid chromatography.

2 Normative references
The following documents are referred to in the text in such a way that some or all of their content constitutes
requirements of this document. The latest edition of the referenced document (including any amendments)
applies.
JIS K 0115, General rules for molecular absorptiometric analysis
JIS K 0124, General rules for high performance liquid chromatography
JIS K 0557, Water used for industrial water and wastewater analysis
JIS K 8223, Perchloric acid (Reagent)
JIS K 9005, Phosphoric acid
JIS K 9009, Sodium dihydrogenphosphate dihydrate (Reagent)
JIS K 9020, Disodium hydrogenphosphate (Reagent)
JIS Z 8802, Methods for determination of pH of aqueous solutions
ISO 648, Laboratory glassware — Single-volume pipettes
ISO 1042, Laboratory glassware — One-mark volumetric flasks
ISO 8655-2, Piston-operated volumetric apparatus — Part2:Piston pipettes

3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
3.1
ATP-related compound
adenosine 5’-triphosphate (ATP), adenosine 5’-diphosphate (ADP), adenosine 5’-monophosphate (AMP),
inosine 5’-monophosphate (IMP), inosine (HxR) and hypoxanthine (Hx)3.2

20023：2022

K-valueratio of the sum of the contents of HxR and Hx to the sum of the contents of the ATP-related compounds intest samples expressed as a percentage [1]Note 1 to entry: See 9.2.3, Formula [3]4 Principle
The endogenous enzymes that decompose ATP-related compounds are inactivated and ATP-related
compounds are extracted with dilute perchloric acid. The contents of the ATP-related compounds in thesample solution are measured using a high performance liquid chromatograph (hereinafter referred to asHPLC) equipped with an ultraviolet-visible absorbance detector. The K-value is calculated from thosecontents [1], [2].

5 Reagents
Use only reagents of recognized analytical grade, unless otherwise specified.
WARNING — It is the responsibility of users of this document to comply with legal regulations
regarding the use of reagents.
5.1 Water, conforming to grade A3 or A4 of JIS K 0557.
5.2 Perchloric acid, of mass fraction 60,0 % to 62,0 %, specified in JIS K 8223.
5.3 Phosphoric acid, of minimum mass fraction, 85,0 %, specified in JIS K 9005.
5.4 Sodium dihydrogenphosphate dihydrate, of mass fraction 99,0 % to 102,0 %, specified in JIS K
9009.
5.5 Disodium hydrogenphosphate, of minimum mass fraction, 99,0 %, specified in JIS K 9020.
5.6 Standards of ATP-related compounds, are given in Table 1. Those purity shall be confirmed byseparating impurities (including other ATP-related compounds) with HPLC. The purity may be confirmedby the supplier of the standards or the user of this document.
Table 1— Standards of ATP-related compounds
ATP-related
compoundName of standards

CAS number

purityATP

Adenosine 5’-triphosphate disodium salt hydrate

34369-07-8

98 % or more

ADP

Adenosine 5’-diphosphate monopotassium salt dihydrate

72696-48-1

95 % or more

AMP

Adenosine 5’-monophosphate sodium salt hydrate

149022-20-8

98 % or more

IMP

Inosine 5’-monophosphate disodium salt hydrate

352195-40-5

98 % or more

HxR

Inosine58-63-998 % or more

Hx

Hypoxanthine

68-94-0

98 % or more

NOTE In the interlaboratory tests described in Annex A, adenosine 5’-triphosphate disodium salt hydrate of formulaweight 551,1(anhydrous basis), adenosine 5’-diphosphate monopotassium salt dihydrate of formula weight 501,3,
adenosine 5’-monophosphate sodium salt hydrate of formula weight 347,2(anhydrous free acid basis), inosine 5’monophosphate disodium salt hydrate of formula weight 392,2(anhydrous basis), inosine of formula weight 268,2and hypoxanthine of formula weight 136,1 were used.

5.7 Dilute perchloric acid, prepared by adding 40,0 g of perchloric acid to 440 mL of water.
5.8 Sodium hydroxide (water) solution, prepared to a concentration of about 1 mol/L to 5 mol/L inwater. A commercially available sodium hydroxide solution of the same concentration may be used.

30023：2022

5.9 Dilute hydrochloric acid, prepared to a concentration of about 1 mol/L in water. A commerciallyavailable dilute hydrochloric acid of the same concentration may be used.
5.10 Phosphate buffer solutions
5.10.1 0,25 mol/L Phosphate buffer solution (pH7,0)
Prepare 0,25 mol/L disodium hydrogenphosphate solution by diluting disodium hydrogenphosphate inwater. Prepare 0,25 mol/L sodium dihydrogenphosphate solution by diluting sodium
dihydrogenphosphate dihydrate in water. Add 0,25 mol/L sodium dihydrogenphosphate solution to 0,25
mol/L disodium hydrogenphosphate solution until pH of the mixture reaches 7,0.
NOTEThis buffer is used in the case using a silica gel-based column specified in 6.1.2 a).

5.10.2 0,05 mol/L Phosphate buffer solution (pH7,0)
Prepare 0,05 mol/L disodium hydrogenphosphate solution by diluting disodium hydrogenphosphate inwater. Prepare 0,05 mol/L sodium dihydrogenphosphate solution by diluting sodium
dihydrogenphosphate dihydrate in water. Add 0,05 mol/L sodium dihydrogenphosphate solution to 0,05
mol/L disodium hydrogenphosphate solution until pH of the mixture reaches 7,0.
5.10.3 1 mol/L Phosphate buffer solution (pH2,9)Prepare 1 mol/L sodium dihydrogenphosphate solution by diluting sodium dihydrogenphosphate
dihydrate in water. Prepare 1 mol/L phosphoric acid solution by diluting phosphoric acid in water. Add 1
mol/L phosphoric acid solution to 1 mol/L sodium dihydrogenphosphate solution until pH of the mixture
reaches 2,9.
NOTE This buffer is used in the case using a polymer-based column specified in 6.1.2b).

5.10.4 0.2 mol/L Phosphate buffer solution (pH2,9)Prepare 0,2 mol/L sodium dihydrogenphosphate solution by diluting sodium dihydrogenphosphate
dihydrate in water. Prepare 0,2 mol/L phosphoric acid solution by diluting phosphoric acid in water. Add
0,2 mol/L phosphoric acid solution to 0,2 mol/L sodium dihydrogenphosphate solution until pH of the
mixture reaches 2,9.
NOTE This buffer is used in the case using a polymer-based column specified in 6.1.2 b).

5.11 Stock standard solutions of ATP-related compounds
Prepare ATP stock standard solution, ADP stock standard solution, AMP stock standard solution, IMP stock
standard solution, HxR stock standard solution and Hx stock standard solution respectively, by adding eachstandard of ATP-related compounds specified in 5.6 to water to a concentration of about 5 mmol/L. Among
those, Hx stock standard solution may be prepared by adding hypoxanthine to water heated about 70 ℃
and allowing it to cool down.
NOTE
It has been confirmed that hypoxanthine is difficult to dissolve in cool water at the concentration given inthis subclause.

5.12 Standard solutions
5.12.1 Standard solutions for absorbance measurement
Prepare ATP standard solution for absorbance measurement, ADP standard solution for absorbancemeasurement, AMP standard solution for absorbance measurement, IMP standard solution for absorbancemeasurement, HxR standard solution for absorbance measurement and Hx standard solution forabsorbance measurement respectively, by diluting each stock standard solution of the ATP-related
compounds 100-fold with 0,05 mol/L phosphate buffer solution (pH7,0) (see 5.10.2), using single volume
pipettes or piston pipettes, and one-mark volumetric flasks.

40023：2022

NOTE
In the interlaboratory tests described in Annex A, 1 mL of the stock standard solutions were poured into 100
mL one-mark volumetric flasks with single volume pipettes, and 0,05 mol/L phosphate buffer solution (pH7,0) was
added to the mark.

Set up and operate the spectrophotometer (see 6.2) in accordance with the manufacturer’s instructions.
Measure the absorbance of standard solutions for absorbance measurement at wavelengths given in Table
2 with 0,05 mol/L phosphate buffer solution (pH7,0) as reference. The concentration of the ATP-related
compound X in each stock standard solution, cX, std, is given by the following formula:

where

,

=

(

×

)

× 10 ×(1)

cX, std is the concentration of the ATP-related compound X in the stock standard solution (µmol/L);
A(λX) is the absorbance of the ATP-related compound X standard solution for absorbance measurement
determined at measurement wavelengths λX given in Table 2;εX

is the molar absorption coefficient of the ATP-related compound X (mol-1 L cm-1) (see Table 2);lis the optical path length (cm) of the absorption cell (see 6.3);

V1

is used volumetric flask’s capacity (mL) ;V2

is the volume (mL) of the stock standard solution used.

Table 2—Measurement wavelengths and molar absorption coefficients of ATP-related
compounds [3]ATP-related compound

NOTE

Measurement wavelength (nm)Molar absorption coefficient (mol-1
L cm-1)

ATP259

15 400ADP

259

15 400AMP

259

15 400IMP

249

12 700HxR

249

12 300Hx

250

10 700Results of studies on the molar absorption coefficient of HxR at pH7,0 are shown in Annex B.

5.12.2 A series of standard working solutions
Using single volume pipettes or piston pipettes, pour ATP stock standard solution, ADP stock standard
solution, AMP stock standard solution, IMP stock standard solution, HxR stock standard solution and Hx
stock standard solution into a one-mark volumetric flask, and water is added to the mark. It is used as themixed stock standard working solution. Depending on the type of column specified in 6.1.2, prepare a series

50023：2022of standard working solutions with a concentration of 4 or more stepwise from the mixed stock working
standard solution by one of the following steps.
a) For using a silica gel-based column: Dilute the mixed stock standard working solution with water using
a single volume pipette or a piston pipette. Mix 4 parts per volume of this diluted solution with 1 partper volume of 0,25 mol/L phosphate buffer solution (pH7,0) (see 5.10.1) to prepare the standard
working solutions.
b) For using a polymer-based column: Dilute the mixed stock standard working solution with water using
a single volume pipette or a piston pipette. Mix 4 parts per volume of this diluted solution with 1 partper volume of 1 mol/L phosphate buffer solution (pH2,9) (see 5.10.3) to prepare the standard working
solutions.
NOTE 1 In the interlaboratory tests described in Annex A, 1,25 mL of each standard working solution was prepared.
Their concentrations were about 0,8 µmol/L, about 3,2 µmol/L, about 8 µmol/L, about 32 µmol/L, about 160 µmol/L,
about 320 µmol/L and about 480 µmol/L.
NOTE 2 It has been confirmed that the standard working solutions will remain stable for at least 7 days when kept
at 10 °C.

6 Apparatus
Usual laboratory apparatus and, in particular, the following.
6.1 HPLC system
6.1.1
High performance liquid chromatograph, equipped with a liquid feeding pump, athermostatically controlled column compartment (column oven), an ultraviolet-visible detector set at 260
nm, and data collection/integration system specified in JIS K 0124. It should have a degas chamber at a
mobile phase feeding unit, and a cooling system at a sample injector.
6.1.2 Column, silica gel-based or polymer-based and with characteristics that the peaks of the ATPrelated compounds are detected within 60 minutes and that the resolution specified in JIS K 0124 is 1,5 orhigher when the minimum concentration of a series of standard working solutions is measured according
to 8.6. When using a guard column, select the one with the same packing material as the column used forthe measurement. The specifications given in a) and b) are available.
a) Silica gel-based column:― packing material: adamantyl group introduced silica gel;― material of chromatographic tube: stainless steel;― length: 250 mm;― internal diameter: 4,6 mm;― spherical particle size: 5 μm.
NOTESome of octadecyl group introduced silica gel columns have been confirmed that the peaks of HxR or Hx
overlap with the peaks of impurities when the sample solution is measured, and the K-value is calculated higher thanactual value.

b) Polymer-based column:― packing material: polyvinyl alcohol-based gel with a pore size of 40 nm;― material of chromatographic tube: stainless steel;― length: 300 mm;― internal diameter: 7,5 mm;

60023：2022― spherical particle size: 6 μm.
6.2 Spectrophotometer, capable of measuring at wavelengths of 249 nm, 250 nm and 259 nm, capableof holding cells of optical path length 1 cm, specified in JIS K 0115.
6.3 Cells, quartz glass, of optical path length 1 cm.
6.4 One-mark volumetric flasks, with the precision equivalent or higher than class A of ISO 1042,
covering the volume range of solution preparation.
6.5 Single volume pipettes, with the precision equivalent or higher than class A of ISO 648.
6.6 Piston pipettes, suitable for the dilution of standard solutions, etc., of ISO 8655-2.
6.7 Extraction container, capable of being used in the extraction (see 8.2) process, and resistant tosolutions to be used.
6.8 Membrane filters, made of hydrophilic polytetrafluoroethylene (PTFE) suitable for acidic solutions,pore size of 0,45 µm or less. The filter and housing shall be unitary, and the housing material shall be
resistant to acidic solutions, Capable of being used with a syringe attached.
6.9 Electronic analytical balance, capable of weighing to an accuracy of ±10 mg, and capable ofweighing more than 200 g.
6.10 Homogenizer, capable of stirring test samples and reagents, and suspending test samples in reagents,
in the extraction (see 8.2) process.
6.11 pH meter, specified in JIS Z 8802.
6.12 pH test paper, capable of identifying pH2,5 to pH3,5.

7 Preparation of test samples
In the case that heads, bones, viscera, epidermis, fins, etc. are found, remove them and obtain the lateralmuscle. Remove dark meat, collect ordinary meat and homogenize it with a food processor, etc. Use it as atest sample. Immediately perform the operation in 8.1. For frozen samples, perform the operation in thisclause immediately after thawing or partial thawing.

8 Procedure
8.1 General
In order to avoid decomposition of ATP-related compounds, cool samples and extracts with ice and keepthem at low temperature until the operation in 8.4 is performed, except during the experimental operation.

8.2 Extraction
Weigh, to the nearest 10 mg, approximately 2,0 g of the test sample (see Clause 7) into an extraction
container, add ice-cooled dilute perchloric acid. Using homogenizer, stir and suspend the sample in the
dilute perchloric acid. In the case the suspension adheres to the homogenizer, rinse it off with the ice-cooled
dilute perchloric acid and mix both well to make a suspended extract. The total amount of the diluteperchloric acid to be used is about 30 mL.
NOTE
In the interlaboratory tests described in Annex A, 20 mL of ice-cooled dilute perchloric acid was added to thesample in a 50 mL polypropylene tube and was stirred with a rotor-stator type homogenizer at a rotation speed of 10000 min-1 for 30 seconds. Subsequently, the homogenizer shaft was washed with 10 mL of ice-cooled dilute perchloric
acid, and both were mixed to obtain a suspended extract.

8.3 pH adjustment [4]8.3.1 Add sodium hydroxide solution to the suspended extract (see 8.2) and stir it to adjust the pH to 2,570023：2022

to 3,5. Check the pH with a pH meter or pH test paper. In the case the pH exceeds 3.5, add dilute hydrochloric
acid to adjust the pH.
NOTE
In the interlaboratory tests described in Annex A, 2,7 mL of 5 mol/L sodium hydroxide solution was added
to a suspended extract and stirred with a glass rod. A small portion of this solution was collected and the pH waschecked with pH test paper. In the case the pH was 2,5 or less, 100 µL of 1 mol/L sodium hydroxide solution was added,
stirred, and the pH was checked again. In the case the pH exceeds 3,5, 100 µL of dilute hydrochloric acid was added,
and the pH was checked. This process was repeated until the pH reached 2,5 to 3,5.

8.3.2 Rinse the residue with water, transfer the entire pH-confirmed suspension extract (see 8.3.1) to a
one-mark volumetric flask. Add water to the capacity mark with water and shake to mix it.
NOTE

In the interlaboratory tests described in Annex A, 50 mL one-mark volumetric flasks were used.

8.3.3 Collect all or a portion of the content of the one-mark volumetric flask (see 8.3.2) and cool thecollection with ice for at least 30 minutes. Use this as a pH adjusted extract.

8.4 Removal of protein
Filter a portion of the supernatant from the pH adjusted extract (see 8.3.3) through a membrane filter toobtain a filtrate.

8.5 Preparation of sample solution
Mix 4 parts per volume of the filtrate (see 8.4) with 1 part per volume of the phosphate buffer solution withsingle volume pipettes or piston pipettes. Use this as a sample solution. Use one of the following as thephosphate buffer solution:a) For using a silica gel-based column: 0,25 mol/L phosphate buffer solution (pH7,0) (see 5.10.1);b)

For using a polymer-based column: 1 mol/L phosphate buffer solution (pH2,9) (see 5.10.3).

NOTE

It has been confirmed that sample solutions will remain stable for at least 3 days when kept at 10 °C.

8.6 Measurement
8.6.1 Setting of operating conditions
In accordance with the manufacturer's instructions, set the HPLC conditions as follows:
NOTE 1 In the case the mobile phase contains a large amount of potassium ions, it reacts with perchlorate ionscontained in the sample solution and forms a precipitate, which interferes with the measurement by HPLC. Accordingly,
in this document, sodium salts are used to prepare the mobile phase.

a) For using a silica gel-based column:
1)

Mobile phase: 0,05 mol/L phosphate buffer solution (pH7,0) (see 5.10.2);

2)

Column temperature: 40 °C;

3)Measurement wavelength: 260 nm;

4)Injection volume: 10 µL.

NOTE 2 In the interlaboratory tests described in Annex A, flow rate was set to 1,0 mL/min.

b) For using a polymer-based column:
1)

Mobile phase: 0,2 mol/L phosphate buffer solution (pH2,9) (see 5.10.4);

2)

Column temperature: 40 °C;

3)Measurement wavelength: 260 nm;

4)Injection volume: 20 µl.

NOTE 3 In the interlaboratory tests described in Annex A, flow rate was set to 0,6 mL/min.

80023：2022

8.6.2 HPLC analysis
Stabilize the entire system appropriately. Confirm that the fluctuation of base line gives no hindrance formeasurement of the ATP-related compounds by a blank run under the specified condition (see 8.6.1). Then
inject a series of standard working solutions and the sample solutions (see 8.5) onto the column. The orderof injections should be random.
8.6.3 Identification
Identify the individual ATP-related compounds peaks in the sample chromatograph by comparing retention
times with those obtained from the standard solutions under the same chromatographic conditions (see8.6.1).
NOTE

Typical HPLC chromatograms of ATP related compounds are given in Annex C.

9 Calculation
9.1 General
Quantitative determination is performed by the external standard method with integration of the peak area,
which is then related to the corresponding value for the standard substance. For the peaks of impurities,
take appropriate measures according to the perpendicular or tangent method specified in JIS K 0124.

9.2 Calculation of K-value
9.2.1 Obtain the areas of the ATP-related compounds in each of a series of standard working solutions.
Construct linear calibration graphs of each ATP-related compound, for each standard concentration
against the peak area of each ATP-related compound obtained from the data collection/integration
system. The correlation coefficients of the linear calibration are required to be 0,998 or more.
NOTE
In the interlaboratory tests described in Annex A, with reference to the content of ATP-related compounds
in general Osteichthyes, the standard working solutions with the following concentrations were used for each ATPrelated compound:—for ATP: about 0,8 µmol/L, about 3,2 µmol/L, about 8 µmol/L, about 32 µmol/L, about 160 µmol/L and about
320 µmol/L;—

for ADP: about 0,8 µmol/L, about 3,2 µmol/L, about 8 µmol/L, about 32 µmol/L, and about 160 µmol/L;—

for AMP: about 0,8 µmol/L, about 3,2 µmol/L, about 8 µmol/L, and about 32 µmol/L;—

for IMP: about 0,8 µmol/L, about 3,2 µmol/L, about 8 µmol/L, about 32 µmol/L, about 160 µmol/L, about 320µmol/L, and about 480 µmol/L;—

for HxR: about 0,8 µmol/L, about 3,2 µmol/L, about 8 µmol/L, about 32 µmol/L, about 160 µmol/L and about
320 µmol/L;—

for Hx: about 0,8 µmol/L, about 3,2 µmol/L, about 8 µmol/L, about 32 µmol/L, about 160 µmol/L and about 320µmol/L.

9.2.2 Calculate the concentrations of the ATP-related compounds from the peak area of each sample
solution by the calibration curves. The content of ATP-related compound X in the test sample, bX, is givenby the following formula:
＝

where
bX

,

×

×

－

×

is the content of ATP related compound X in the test sample (µmol/g);(2)

90023：2022

cX,sp

is the concentration of X in the sample solution (µmol/L);

V3

is the constant volume (mL) at filling up in 8.3.2;

mis the mass (g) of the test sample;

V4

is the volume (µL) of the filtrate in 8.5;

V5

is the volume (µL) of the phosphate buffer in 8.5.

NOTE

In the interlaboratory tests described in Annex A, V3 was 50 mL, V4 was 1000 µL, and V5 was 250 µL.

9.2.3 Calculate the K-value from the contents of ATP-related compounds in the test sample by thefollowing formula:
K-value =

where

× 100

bA

is the ATP content in the test sample (µmol/g);bBis the ADP content in the test sample (µmol/g);

bC

is the AMP content in the test sample (µmol/g);

bDis the IMP content in the test sample (µmol/g);bE

is the HxR content in the test sample (µmol/g);

bF

is the Hx content in the test sample (µmol/g);(3)

9.3 Expression of results
Express the results to two significant figures. Describe the unit (%) with them, as needed.

10 Precision
10.1 Interlaboratory test
The interlaboratory test was carried out to determine the precision of the test method, and the results are
summarized in Annex A. The values derived from this interlaboratory test can be inapplicable to the content
ranges other than the given one (6,12 % to 83,4 %).

10.2 Repeatability
The absolute difference between two independent single test results, obtained using the same method on
identical test material in the same laboratory by the same operator using the same equipment within ashort interval of time, is expected in not more than 5 % of cases to be greater than the repeatability limit (r)values [5] shown in Table A.1 on average as long as the specified operation is correctly done [6].

10.3 Reproducibility
The absolute difference between two single test results, obtained using the same method on identical test
material in different laboratories with different operators using different equipment, is expected in not
more than 5 % of cases to be greater than the reproducibility limit (R) values [5] given in Table A.1 onaverage as long as the specified operation is correctly done [6].

11 Quality control
The laboratory shall have internal quality control procedures for tests.

100023：2022

12 Test report
The test report shall include at least the following information:a)the title or the reference number of this document;b)every detail to identify the test sample;c)the date of the test;d)the results of the test.

110023：2022

Annex A
(informative)
Results of interlaboratory tests
Interlaboratory tests were carried out in accordance with IUPAC protocol [7] in 2021 in Japan, and gave thestatistical results shown in Table A.1. The ordinary meats of lateral muscles of commercially available fresh
Osteichthyes were collected, chopped, frozen in liquid nitrogen to suppress deterioration, and crushed
while frozen. The crushed materials were subdivided into about 2,0 g, which is the amount to be used for
one test, and were used as a test sample, after the homogeneity [8] was confirmed. The test samples werestored at -80 °C until sent to the participating laboratories. The experimental protocol, the stock standard
solutions of ATP-related compounds and test samples were supplied to the participating laboratories by the
Hakodate Regional Industry Promotion Organization, Public Interest Incorporated Foundation, theorganizer of the interlaboratory tests, and the mass of the test sample were notified. The temperature of thetest samples was maintained below -20 °C during transportation to each laboratory and until the start of
the test. Within 4 days of receipt, each laboratory tested a total of 10 test samples (5 pairs of blind
duplicates) according to the experimental protocol. In order to avoid deterioration of the samples due tothawing, each laboratory conducted the test using the entire amount of the test samples in the frozen state,
and used the mass of the samples notified by the organizer to calculate the K-value.
Table A.1 — Precision data
Sample identification

1

2

3

4

5

Number of participating laboratories

11

11

11

11

11

Number of accepted test results

11

10

9

10

11

Mean value, %(K-value)

6,12

10,08

26,78

39,25

83,4

value)

0,10

0,11

0,05

0,11

0,2

Repeatability relative standard deviation

1,6

1,1

0,18

0,28

0,2

RSDr, %

0,27

0,31

0,14

0,31

0,6

value)

0,21

0,23

0,45

0,50

1,2

Reproducibility relative standard deviation

3,5

2,3

1,7

1,3

1,4

RSDR, %

0,60

0,65

1,3

1,4

3,3

Repeatability standard deviation sr, %(K-

Repeatability limit r (r = 2,8 sr), %(K-value)
Reproducibility standard deviation sR, %(K-

Reproducibility limit R (R = 2,8 sR), %(K-value)
Key to samples:
1 Japanese flounder(Paralichthys olivaceus), wild caught.
2 Yellowtail(Seriola quinqueradiata) , wild caught.
3 Yellowtail(Seriola quinqueradiata), farmed.
4 Pacific mackerel(Scomber japonicus), wild caught.

5 Atlantic salmon(Salmo salar), farmed.

120023：2022

Annex B(informative)
Results of studies on the pH of HxR standard solutions for absorbancemeasurement
Regarding the molar absorption coefficient of ATP-related compounds, the values in pH7,0 phosphatebuffer solution are used for ATP, ADP, AMP, IMP, and Hx, and the value in pH6,0 phosphate buffer solution
is used for HxR [3]. Studies to change the pH of absorbance measurement solution of HxR to pH7,0 as wellas other ATP-related compounds, were conducted to reduce workload for user of this document.
The evaluation results of the absorbance of the HxR solution due to the difference between pH6,0 and pH7,0of the solvent are shown in Table B.1.
Table B.1 — Absorbance of HxR solutions at pH6,0 and pH7,0
Replicate

Absorbance
pH6,0

pH7,0

1

0,617

0,619

2

0,617

0,616

3

0,617

0,617

4

0,617

0,619

5

0,618

0,617

Mean value

0,617

0,618

NOTE Significant difference could not found in the absorbance between at pH6,0 and at pH7,0
by Welch's t-test (two-tail)(significance level 5 %).

130023：2022

Annex C
(informative)
Typical HPLC chromatogramsb) Sample solution[Bluefin tuna(Thunnus orientalis)]a) Standard working solution(ATP-related
compounds, each with a concentration of about 160µmol/L)Key
X

Retention time, min

3

ADP

Y

Response, mV

4

AMP

1

IMP

5

Hx

2

ATP

6

HxR

NOTE HPLC operating conditions are in accordance with 8.6.1a) and CAPCELL PAK ADME-HRTM was used as thecolumn. This information is given for the convenience of users of this document and does not constitute an
endorsement of this product.

Figure C.1 — Examples of the chromatograms using the silica gel-based column

140023：2022a) Standard working solution(ATP-related
compounds, each with a concentration of about 160µmol/L)b) Sample solution[Japanese flounder(Paralichthys
olivaceus)]

Key
X

Retention time, min

3

AMP

Y

Response, mV

4

IMP

1

ATP

5

HxR

2

ADP

6

Hx

NOTE HPLC operating conditions are in accordance with 8.6.1b) and Asahipak® GS-320 HQ was used as the column.
This information is given for the convenience of users of this document and does not constitute an endorsement of thisproduct.

Figure C.2 — Examples of the chromatograms using the polymer-based column

150023：2022

Bibliography[1]

Saito, T., et al., A new method for estimating the freshness of fish. Nippon Suisan Gakkaishi, 1959,
24(9), pp. 749-750
NOTE The K-value calculation formula in the referenced document was referred to for calculating the Kvalue.

[2]

Lee, EH., et al., High performance liquid chromatographic determination of K value as an index of
freshness of fish. Nippon Suisan Gakkaishi, 1982, 48(2), pp. 255
NOTE The analysis method in the referenced document was referred to for the method of calculating Kvalue using the measured value of HPLC.

[3]National Academy of Science, Specifications and Criteria for Biochemical Compounds. 3rd Edition.,
Washington D.C..1972
NOTE Molar absorption coefficient of the ATP-related compounds in referenced document was referred tofor calculating the concentration of the ATP-related compounds standard solution using the measured valueof spectrophotometer.

[4]

HU, Y., et al., Development of simplified method for extracting ATP-related compounds from fishmeat. Nippon Suisan Gakkaishi, 2013, 79(2), pp. 219-225
NOTE “Results, and the neutralization conditions of the extract” of the referenced document was referred
to for the pH adjustment of the suspended extract.

[5]

ISO 5725-6:1994, Accuracy (trueness and precision) of measurement methods and results — Part 6:
Use in practice of accuracy values
NOTE Section 4 “Determination of limits” of the referenced document was referred to for the calculation of
the repeatability limit and the reproducibility limit.

[6]

ISO 5725-1:1994, Accuracy (trueness and precision) of measurement methods and results — Part 1:
General principles and definitions
NOTE Section 7.1.5 of the referenced document was referred to for the expression of the repeatability limit
and the reproducibility limit.

[7]

Horwitz, W., Protocol for the design, conduct and interpretation of method-performance studies,
Pure Appl. Chem., 1995, 67(2), pp. 331-343[8]

ISO 13528:2005, Statistical methods for use in proficiency testing by interlaboratory comparisons
NOTE Appendix B of the referenced document was referred to for the method to confirm the homogeneity.