Tentative Translation

JAS
0016

JAPANESE AGRICULTURAL
STANDARD
Determination of the ornithine in mushroom (Hypsizygus marmoreus)― High-performance liquid chromatographic method

Date of Establishment: 2021 – 3 – 31

Ministry of Agriculture, Forestry and Fisheries

0016：2021

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)

0016：2021

Contents

Page

1

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

2

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

3

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

4

Principle .............................................................................................................................................................1

5

Reagents .............................................................................................................................................................1

6

Apparatus ..........................................................................................................................................................27

Preparation of test samples ......................................................................................................................3

8

Procedure ..........................................................................................................................................................3

8.1

Extraction ..........................................................................................................................................................3

8.2

Determination .................................................................................................................................................39

Calculation.........................................................................................................................................................4

9.1

Quantitation .....................................................................................................................................................4

9.2

Expression of results ....................................................................................................................................5

10

Precision ............................................................................................................................................................5

10.1

Interlaboratory test ......................................................................................................................................5

10.2

Repeatability ....................................................................................................................................................5

10.3

Reproducibility ...............................................................................................................................................5

11

Quality control.................................................................................................................................................5

12

Test report ........................................................................................................................................................5

Annex A (informative)

Results of interlaboratory tests..........................................................................6

Bibliography.....................................................................................................................................................................8(3)

0016：2021

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, based on theprovision of Article 4, paragraph (1) of the Act on Japanese Agricultural Standards as applied mutatis
mutandis pursuant to the provision of Article 5 of the Act.
Attention is drawn to the possibility that some parts of this Standard may conflict with patent rights,
applications for a patent after opening to the public or utility model rights. The Minister of Agriculture,
Forestry and Fisheries and the Council for the Japanese Agricultural Standards are not responsible for
identifying any of such patent rights, applications for a patent after opening to the public or utility modelrights.

(4)

JAPANESE AGRICULTURAL STANDARD(Tentative Translation)

JAS0016：2021

Determination of the ornithine in mushroom (Hypsizygus
marmoreus)― 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 high-performance liquid chromatographic test method for the determination ofornithine in mushroom (Hypsizygus marmoreus) (fresh).

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 editions of the referenced documents (including any
amendments) apply.
ISO 1042, Laboratory glassware — One-mark volumetric flasks
JIS K 0557, Water used for industrial water and wastewater analysis
JIS K 8180, Hydrochloric acid (Reagent)

3 Terms and definitions
No terms and definitions are listed in this document.

4 Principle
Ornithine is extracted from a test portion of finely chopped mushroom with dilute hydrochloric acid. The
ornithine in the extract is determined with high-performance liquid chromatograph (HPLC) system thatmeets the specified criteria.

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 Standard reagent, either of the following:

20016：2021a) Ornithine monohydrochloride, of minimum mass fraction, ≥ 97,0%; orb) Commercially available amino acid standard solution prepared with ornithine at a concentration
suitable for HPLC system and operating conditions.
5.3

Hydrochloric acid, of minimum mass fraction, 35,0 %, according to JIS K 8180.

WARNING — Since it is a deleterious substance, avoid contact with eyes, mucous membranes and skin.

5.4
Dilute hydrochloric acid, prepared by adding 0,86 mL of hydrochloric acid to water andobtaining a constant volume of 1,0 L.
5.5Internal standard, necessary when using the internal standard method for the measurement(see 8.2), and confirmed to be neither included in Hypsizygus marmoreus, nor overlapping otheringredient peaks therein. Suitable for HPLC system and operating conditions.
NOTE
Norvaline and theanine were used on the HPLC system equipped with a tandem mass spectrometer in theinterlaboratory tests described in Annex A.

5.6
Reagents for HPLC analysis, necessary for HPLC system and operating conditions, withsuitable quality.
5.7

A series of standard solutions,

made by dissolving the standard reagent in dilute hydrochloric acid and preparing it to a concentration of
3 or more stepwise concentrations suitable for HPLC system and the operating conditions to be used. The
minimum concentration of the standard solution shall be set at or above the minimum limit ofquantitation of HPLC system (see 8.2.1 a)).
NOTE
In the interlaboratory tests described in Annex A, a series of standard solutions was prepared with anornithine concentration of 400 mg/kg to 2 500 mg/kg for the test samples.

6 Apparatus
Usual laboratory apparatus and, in particular, the following.
6.1
Electronic analytical balance, capable of weighing to a readability of ±0,1 mg, capable ofweighing more than 200 g.
6.2
Extraction container, made of glass or resin that is resistant to acidic solutions, with the lid, ofapproximately 50 mL capacity, capable of maintaining necessary space for enough shaking.
6.3

Shaker, capable of shaking an extraction container, back and forth, at 100 r/min or more.

6.4
One-mark volumetric flasks, capacity suitable for the preparation of standard solutions (see5.7) and extraction procedures (see 8.1.2 and 8.1.3), of ISO 1042, class A.
6.5
Centrifuge tubes, made of resin that is resistant to acidic solutions, with the lid. They shall beable to withstand centrifugation at 13 000 ×g.
6.6

Centrifuge, capable of carrying out centrifugation at 13 000 ×g with centrifuge tubes.

WARNING — To prevent accidents, operate the centrifuge according to the instruction manual of the equipment.

6.7
Membrane filters, made of hydrophilic polytetrafluoroethylene (PTFE), suitable for acidic
solutions, with a nominal pore size of 0,2 µm. The filter and the housing shall be unitary, and the materialof the housing shall be resistant to acidic solutions.
6.8
Vials, suitable for HPLC system to be used, made of deactivated glass or high purity
polypropylene. The septum of the lid shall be made of or coated with PTFE.
6.9

HPLC system,

30016：2021

equipped with HPLC on the separation part, and either a fluorescence detector, a visible absorption
detector (both detectors shall carry out the measurement by the post-column derivatization method), or atandem mass spectrometer on the detection part. They shall be able to set the operating conditions as
specified in 8.2.1.

7 Preparation of test samples
After removing the hard tip of the sample, finely chop it to make it visually homogeneous with a foodprocessor or the like. Use this as a test sample. Immediately carry out the extraction specified in 8.1, or
keep the test sample frozen. To keep it frozen, transfer the whole test sample, or a portion thereof whichis stirred to be homogeneous, into the container that is capable of being tightly sealed, as soon as the test
sample is prepared. Take the test sample out of the freezer before use, allow it to return to room
temperature and mix well.
NOTE

It has been confirmed that the test sample will remain stable for at least 20 weeks when kept frozen at -30 °C to -20 °C.

8 Procedure
8.1 Extraction
8.1.1 Weigh, to the nearest 10 mg, approximately 2 g of the test sample (see Clause 7) into an extraction
container, add 15 mL of dilute hydrochloric acid and shake it with a shaker for 15 min.
8.1.2 Transfer the mixture in the extraction container (see 8.1.1) to a one-mark volumetric flask. Add
dilute hydrochloric acid to the residue in the extraction container, and transfer the entire residue to theflask.
NOTE

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

8.1.3 Fill the flask (see 8.1.2) up to the mark with dilute hydrochloric acid and mix it. Use this as a mixed
solution. When adopting the internal standard method on the measurement (see 8.2), add the internal
standard before filling the flask or when diluting the measurement solution (see 8.1.5) in 8.2.2.
8.1.4 Transfer some or all of the mixed solution (see 8.1.3) to a centrifuge tube, centrifuge it for 10 min
at 13 000 ×g.
8.1.5 Filter supernatant liquid through a membrane filter. Use this as a measurement solution. Performthe measurement (see 8.2) on the day of preparation or keep it frozen.
NOTE
It has been confirmed that the measurement solution will remain stable for at least 18 weeks when kept
frozen at -30 °C to -20 °C.

Take the measurement solution stored at -30 °C to -20 °C out of the freezer before use and mix it well.

8.2 Determination
8.2.1

Setting of operating conditions for HPLC system and confirmation of extraction conditions

The measurement shall be performed by the absolute calibration curve method or the internal standard
method. Operate HPLC system according to the instruction manual, and set the operating conditions tomeet the following criteria:a) The minimum limit of quantitation shall be 50 mg/kg or less as test sample (see Clause 7)
concentration;b) The ornithine peak shall be separated from the peaks that precede and succeed it, so as not to interferewith the measurement of the standard solution and the measurement solution (or its dilution);c) The correlation coefficient of the linear calibration curve shall be 0,995 or more;40016：2021

d) Perform a recovery test by adding approximately the same amount of ornithine[1] as contained in arandom test sample (see Clause 7) to the sample, and the recovery shall be within the range shown in
Table 1[2];Table 1— Recovery range
Added concentration

Recovery（%）≧1 000 mg/kg

95 to 105

≧100 mg/kg

90 to 107

≧10 mg/kg

80 to 110e) Determine random test samples (see Clause 7) 6 times or more[3] under repeatability condition, andthe standard deviation (mass fraction) (Srl) shall meet the following condition[4]:

Srl
≦1,3
0,02w 0,85

where
w
8.2.2

is the concentration of ornithine in the test sample (mass fraction)
Measurement with HPLC system

Confirm that the fluctuation of base line gives no hindrance for determination of ornithine by a blank run
under the condition of above-mentioned setting, and inject a series of standard solutions and the sameamount of the measurement solution (see 8.1.5) into HPLC system. Confirm the fluctuation of sensitivity
during measurement.
NOTE
In the interlaboratory tests described in Annex A, an intermediate concentration solution of the series ofstandard solutions was measured at 1 intervals for 5 measurement solutions, and it was confirmed that the fluctuation
was within ±10 %.

Obtain the output signal of ornithine for each injected standard solution and injected measurement solution.
Prior to measurement, the measurement solution may be diluted to an ornithine concentration suitable for
the HPLC system.

9 Calculation
9.1 Quantitation
Construct linear calibration curve for each standard solution from the ornithine concentrations against theoutput signal intensity. Confirm that the following conditions are met:a) The correlation coefficient of the linear calibration curve shall be 0,995 or more;b) The ornithine concentration of the measurement solution (or its dilution) shall be within thecalibration curve range.
Calculate the ornithine concentration (µg/mL) in the measurement solution from the output signal
intensity of each measurement solution (or its dilution) by the calibration curve. The ornithine content in
the test sample, wi (mg/kg), is given by the following formula:

wi =

where

C ×V
W

5
0016：2021

C

is the ornithine concentration in the measurement solution, in µg/mL;V

is the constant volume (mL) at filling up (see 8.1.3);

W
NOTE

is the mass (g) of the test sample (see 8.1.1);
In the interlaboratory tests described in Annex A, V was 50.

9.2 Expression of results
Express the results to two significant figures.

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 (6,7×102 mg/kg to 1,9×103 mg/kg) nor the matrices other than those given.

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 on averageas long as the specified operation is correctly done[6].

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

12 Test report
The test report shall include at least the following information:a)the title of this document or the reference number;b)

every detail to identify the test sample;c)the date of the test;d)the results of the test.

60016：2021

Annex A
(informative)
Results of interlaboratory tests
Interlaboratory tests were carried out in accordance with IUPAC protocol[7] in 2020 in Japan, and gavethe statistical results shown in Table A.1. Commercially available Hypsizygus marmoreus, from which
the hard tips were removed, were finely chopped for 5 min at 3 600 r/min by food processor. After the
homogeneity [8] was confirmed, finely chopped samples were used as a test sample. The experimental
protocol, the ornithine standard reagent and test samples were supplied to the participating
laboratories by the Food and Agricultural Materials Inspection Center (FAMIC), the organizer of theinterlaboratory tests. Each laboratory tested a total of 10 test samples (5 pairs of blind duplicates)
according to the experimental protocol.

Table A.1 — Precision data
Sample identification

Sample 1

Sample 2

Sample 3

Sample 4

Sample 5

Number of participating laboratories

10

10

10

10

10

Number of accepted test results

10

8

10

9

8

Mean ornithine content, mg/kg

671

895

1 216

1 538

1 853

Repeatability standard deviation sr, mg/kg

12

12

44

40

57

Repeatability relative standard deviation, %

1,9

1,4

3,7

2,6

3,1

Repeatability limit r (r = 2,8 sr), mg/kg

35

34

124

111

161

Reproducibility standard deviation sR, mg/kg

46

31

86

59

60

Reproducibility relative standard deviation, %

6,9

3,5

7,1

3,8

3,2

Reproducibility limit R (R = 2,8 sR), mg/kg

129

88

240

164

167

NOTE 1 Considering the usage situation in Japan, the HPLC system equipped with HPLC on the separation part, andeither a fluorescence detector, a visible absorption detector or a tandem mass spectrometer on the detection part werechosen for the interlaboratory tests. The details of the HPLC system used are as follows: the fluorescence detectors(post-column derivatization method) were used in 2 laboratories, the visible absorption detectors (post-column
derivatization method) were used in 3 laboratories, and the tandem mass spectrometers were used in 5 laboratories.
NOTE 2 Examples of the operating conditions adopted in the interlaboratory tests are shown below. This
information is given for the convenience of users of this document and does not constitute an endorsement of the
products by Ministry of Agriculture, Forestry and Fisheries, Japan.
EXAMPLE 1

Detector: tandem mass spectrometer;
Column: ACQUITY UPLC BEH Amide (150×2.1 mm i.d., Waters);
Mobile phase A: water-formic acid (99,7/0,3, v/v);
Mobile phase B: acetonitrile-water-formic acid (95/4,7/0,3, v/v);
Flow rate: 0,3 mL/min;
Injection volume: 10 µL;
Column temperature: 30 °C;
Ionization mode: ESI Positive;
Precursor ion (m/z): 133,00;
Product ions (m/z): 69,80 and 115,92;70016：2021

Run time: 18 min
EXAMPLE 2

Detector: fluorescence detector (post-column derivatization method);
Column: Shim-pack Amino-Na (100×6,0 mm i.d., Shimadzu);
Mobile phase: Mobile phase kit for amino acid analysis (Na Type);
Flow rate: 0,3 mL/min to 0,4 mL/min;
Injection volume: 10 µL;
Column temperature: 60 °C;
Derivatization reagent: o-phthalaldehyde;
Excitation wavelength: 350 nm;
Emission wavelength: 450 nm;
Run time: 53 min

EXAMPLE 3

Detector: visible absorption detector (post-column derivatization method);
Column: Packed column for bioanalysis＃2622 (60×4,6 mm i.d., Hitachi);Mobile phase: Buffer for biological fluid PF-SET;
Flow rate: 0,35 mL/min;
Injection volume: 20 µL;
Column temperature: 30 °C to 70 °C;
Derivatization reagent: ninhydrin;
Wavelength: 570 nm;
Run time: 125 min80016：2021

Bibliography[1]

Official Methods of Analysis (2019) 21st Edition, AOAC INTERNATIONALNOTE“Appendix D: Guidelines for Collaborative Study Procedures To Validate Characteristics of a
Method of Analysis” of the referenced document was referred to for the amount of ornithine to be added in
the recovery test.

[2]

Codex Alimentarius Commission Procedural Manual (2019) Twenty-seventh edition, World
Health Organization, Food and Agricultural Organization of the United Nations
NOTE“Table 1: Guidelines for establishing numeric values for the criteria” in “Principles for the
Establishment of Codex Methods of Analysis” of the referenced document was referred to for the recovery
range.

[3]

Validation of Analytical Procedures: Text and Methodology Q2(R1) (2005), ICH Harmonised
Tripartite Guideline
NOTE

[4]

“5.1. Repeatability b)” of the referenced document was referred to for the repeatability.

Codex, Guidelines on Analytical Terminology, CAC/GL 72-2009
NOTE
The formula was derived from the range of RSD (r) / PRSD (R) shown in "HorRat (Horwitz ratio)"of the referenced document.

[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 calculationof 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]

Thompson, M., et al., The International Harmonized Protocol for the Proficiency Testing ofAnalytical Chemistry Laboratories, Pure Appl. Chem., 2006, 78(1), pp. 145-196
NOTE
Section 3.11 “Testing for sufficient homogeneity and stability” of the referenced document wasreferred to for the method to confirm the homogeneity.

History of Enactment
Enactment

Public Notice of the Ministry of Agriculture, Forestry and Fisheries No. 445 of March
31, 2021

Enactment Provisions, Amended Provisions and Supplementary Provisions (Extract)— Public Notice of the Ministry of Agriculture, Forestry and Fisheries No. 445 of March 31, 2021
This public notice comes into effect on April 30, 2021.