Tentative Translation

JAS0024

JAPANESE AGRICULTURAL
STANDARD

Determination of the procyanidins in apple juice—High performance liquid chromatographic method

Date of Establishment: 2022 - 3 - 31

Ministry of Agriculture, Forestry and Fisheries

0024：2022

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 the Food and Agricultural Materials Inspection Center (FAMIC), Incorporated
Administrative Agency, with permission of the publisher of the original text (Ministry of Agriculture, Forestry andFisheries).
The translation is made in consideration of technical contents, but it is aimed to provide information when using JASoriginal 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)0024：2022

ContentsPage

1

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

2

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

3Terms and definitions..................................................................................................................................14Principle .............................................................................................................................................................1

5

Reagents .............................................................................................................................................................26Apparatus ..........................................................................................................................................................2

7Test samples.....................................................................................................................................................38Procedure ..........................................................................................................................................................3

8.1

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

8.2

Measurement ...................................................................................................................................................39Calculation.........................................................................................................................................................49.1

General................................................................................................................................................................49.2

Quantitation .....................................................................................................................................................49.3

Expression of results ....................................................................................................................................510Precision ............................................................................................................................................................510.1

Interlaboratory test ......................................................................................................................................510.2

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

10.3

Reproducibility ...............................................................................................................................................511

Quality control.................................................................................................................................................512Test report ........................................................................................................................................................5

Annex A (informative)

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

Annex B (informative)

Typical HPLC chromatograms...............................................................................7

Bibliography.................................................................................................................................................................. 10(3)0024：2022

Foreword
This Japanese Agricultural Standard has been established by the Minister of Agriculture, Forestry andFisheries through deliberations at the Council for the Japanese Agricultural Standards, as the result ofproposal for establishment of Japanese Agricultural Standard submitted by Food and Agricultural Materials
Inspection Center, Incorporated Administrative Agency with the original bill being attached, based on theprovision of Article 4, paragraph (1) of the Act on Japanese Agricultural Standards.
Being in conformance with this standard may come under the use of the patent rights held by the following:—Name: National Agriculture and Food Research Organization—Address: 3-1-1 Kannondai, Tsukuba, Ibaraki, Japan—Patent number: Japanese Patent No, 6508741—Title of invention: Analysis method and analysis system of procyanidins
The relevant holder of the above-mentioned patent right has indicated an intention of granting license toanyone under the nondiscriminatory and reasonable conditions, except to the other holders of the patentrights related to this standard who will not grant its licenses under the same conditions.
It should be noted that following this standard does not always refer to granting a free license.
Attention is drawn to the possibility that some parts of this standard may conflict with patent rights as otherthan mentioned above. The Minister of Agriculture, Forestry and Fisheries and Council for the Japanese
Agricultural Standards are not responsible for identifying such patent rights.
The "patent rights" as mentioned here include patent rights, published patent application or utility modelrights.

(4)

JAPANESE AGRICULTURAL STANDARD(Tentative Translation)

JAS0024：2022

Determination of the procyanidins in apple juice—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 theresponsibility 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 method for the determination ofprocyanidins in apple juice (limited to not from-concentrate juice, the same applies hereinafter).

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.
ISO 648, Laboratory glassware — Single-volume pipettes
ISO 1042, Laboratory glassware — One-mark volumetric flasks
JIS K 0124, General rules for high performance liquid chromatography
JIS K 0557, Water used for industrial water and wastewater analysis
JIS K 8034, Acetone
JIS K 8355, Acetic acid (Reagent)
JIS K 9502, L(+)-Ascorbic acid (Reagent)

3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
3.1
procyanidins
polymers formed from catechin or epicatechin
Note 1 to entry: Procyanidins are, for example, procyanidin B2.

4 Principle
Procyanidins are extracted from apple juice with acetone/water mixture. The procyanidins in the extract
are determined with high performance liquid chromatograph (HPLC) system with a fluorescence detector.
The concentration of procyanidins is calculated from peak area ratio of procyanidins in the extract tostandard substance, procyanidin B2.

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5 ReagentsUse 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 Procyanidin B2, confirmed purity.
5.3 Acetone, of minimum mass fraction, 99,5 %, according to JIS K 8034.
5.4 Acetic acid, of minimum mass fraction, 99,7 %, according to JIS K 8355.
5.5 Acetonitrile, HPLC grade.
5.6 Methanol, HPLC grade.
5.7 L(+)-ascorbic acid, of minimum mass fraction, 99,6 %, according to JIS K 9502.
5.8 L(+)-ascorbic acid-containing acetone/acetic acid/water mixture, mix 70 parts per volume ofacetone with 0,5 parts per volume of acetic acid and 29,5 parts per volume of water, and dissolve 0,5 g ofL(+)-ascorbic acid per 1,0 L of the mixture.
5.9 L(+)-ascorbic acid-containing acetic acid solution, dissolve 50 mL of acetic acid and 5 g of L(+)ascorbic acid per 1,0 L of water.
5.10

HPLC mobile phase5.10.1 Mobile phase A, acetonitrile/acetic acid mixture,mix 98 parts per volume of acetonitrile with 2 parts per volume of acetic acid. Degas the mixture before use.
NOTE

Degassing prevents troubles of bubbles, then gives stable flow and background.

5.10.2 Mobile phase B, methanol/acetic acid/water mixture,mix 95 parts per volume of methanol with 2 parts per volume of acetic acid and 3 parts per volume of water.
Degas the mixture before use.
NOTE

5.11

Degassing prevents troubles of bubbles, then gives stable flow and background.

A series of standard solutions,dissolve procyanidin B2 in L(+)-ascorbic acid-containing acetone/acetic acid/water mixture and prepare it
to a concentration of 3 or more stepwise concentrations suitable for testing samples. Concentrations ofstandard solutions shall be corrected by purity of procyanidin B2.
NOTE1 In the interlaboratory tests described in Annex A, standard solutions were prepared to 10 μg/mL, 20 μg/mL,
40 µg/mL, 60 μg/mL, 80 μg/mL and 100 μg/mL of concentrations.
NOTE2–20 °C.

It has been confirmed that 100 μg/mL standard solution remains stable for at least 28 days when kept at

6 Apparatus
Usual laboratory apparatus and, in particular, the following.
6.1 Electronic analytical balance, capable of weighing to an accuracy of ±0,1 mg, capable of weighing
more than 200 g.
6.2 One-mark volumetric flasks, capacity suitable for the extraction procedure (see 8.1), of ISO 1042,
class A.
6.3 Single volume pipettes, capacity suitable for the extraction procedure (see 8.1), of ISO 648, class A.

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6.4 Membrane filters, made of polytetrafluoroethylene (PTFE), suitable for organic solvents, with a poresize of 0,45 μm or less. The filter and the housing shall be unitary, and the material of the housing shall beresistant to organic solvents.
6.5 HPLC system
6.5.1 HPLC, equipped with a degasser, a liquid-sending pump that can perform binary gradient elution,a sample injector with cooling function, a column compartment with thermostatically controlling function,a fluorescent detector that allowed to set a measurement wavelength described in 8.2.1 and datacollection/integration system, prescribed in JIS K 0124.
6.5.2 Chromatographic column for HPLC, normal-phase columns, with the following characteristics:—stationary phase: diol phase;—capable to separate a procyanidins peak and a catechin or epicatechin peak enough.
NOTEIn the interlaboratory tests described in Annex A, the columns were used, that were 4,6 mm internal diameterand 250 mm length stainless pipes packed with 5 μm spherical particle size silica gel bonded with dihydroxypropylgroup chemically.

7 Test samples
Use ordinary temperature apple juice as test samples.

8 Procedure
8.1 Extraction
8.1.1 Measure 2 mL of the well-shaking test sample (see Clause 7) into 10 mL one-mark volumetric flaskwith a single volume pipette, and add 7 mL of acetone.
8.1.2 Fill the flask (see 8.1.1) up to the mark with L(+)-ascorbic acid-containing acetic acid solution andmix it. Filter the mixed solution through membrane filter, and use it as the measurement solution.
8.1.3 Perform the measurement in 8.2.1 and 8.2.2 on the day of preparation.

8.2 Measurement
8.2.1 Setting of HPLC system conditions
Set up the HPLC system conditions in accordance with the manufacturer's instructions and adjust it asfollows. In this conditions, peaks of catechin or epicatechin and peaks of procyanidins shall be separated.
a)Column temperature: 30 °C to 35 °C;b)

Measurement wavelength (excitation and emission): 230 nm and 321 nm;

c)Volume injected: 1 μL to 10 μL;d)Sample cooler temperature: 4 °C;e)

Binary gradient conditions: for 1 min to 2 min after injecting, mix and develop 91 % to 95 % mobile
phase A and 5 % to 9 % mobile phase B, then increase the volume ratio of mobile phase B to 90 % to98 % quickly in order to elute other substances from the column.

NOTE

In the interlaboratory tests described in Annex A, the conditions described in Table 1 were used.

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Table 1 — HPLC system conditions in the interlaboratory tests
Flow rate of the mobile phase

1,0 mL/min

Temperature of the column

35 °C

Temperature of the detector

40 °C

Excitation wavelength

230 nmEmission wavelength

321 nmVolume injected

5 μL

Temperature of the sample cooler

4 °C

Mixing volume

0,5 mL

Binary gradient conditions

Time (min)Mobile phase A(%, v/v)

Mobile phase
B (%, v/v)

0 to 1,593

7

1,5 to 10298

10 to 2093

7

NOTE The values given are examples.

8.2.2 Measurement with HPLC system
Run the entire HPLC system to stabilize it. Confirm that the fluctuation of base line gives no hindrance fordetermination of procyanidins by a blank run under the specified conditions (see 8.2.1). Inject a series ofstandard solutions and the same amount of the measurement solutions (see 8.1) 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 every 7 measurement of the measurement solutions, and it was confirmed that thefluctuation was within ±10 %.

8.2.3 Identification
Identify the individual procyanidins peak in the sample chromatogram by comparing the retention times
with those of procyanidin B2 obtained from the standard solutions under the same chromatographicconditions. Include the peak immediately following procyanidin B2 peak in the procyanidins peak.
NOTE

Typical HPLC chromatogram of apple juice is given in Annex B.

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.

9.2 Quantitation
Obtain the areas of procyanidin B2 in each of a series of standard solutions. Construct linear calibrationgraphs for each standard of the procyanidin B2 concentrations against the peak areas obtained from thedata collection/integration system. The correlation coefficient of the linear calibration is required to be0,995 or more.
Calculate the concentration of procyanidins from the area of each measurement solution by the calibration
curve. The procyanidins content in test sample, ρ, is given by the formula:50024：2022

where=×ρ

is the procyanidins content in the test sample (mg/L)

ρS

is the concentration of procyanidins in the measurement solution (μg/mL)

VS

is the volume (mL) of the test sample (see in 8.1.1)

Vis the constant volume (mL) at filling up (see in 8.1.2)

9.3 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 aresummarized in Annex A. The values derived from this interlaboratory test can be inapplicable to the content
ranges other than the given one (8,9×10 mg/L to 2,5×102 mg/L) 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 [1] given in Table A.1 on average as long as the specified operation is correctly done [2].

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 [1] given in Table A.1 onaverage as long as the specified operation is correctly done [2].

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

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

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Annex A(informative)
Results of interlaboratory tests
Interlaboratory tests were carried out in accordance with IUPAC protocol [3] in 2021 in Japan, and gave thestatistical results given in Table A.1. After the homogeneity [4] was confirmed, commercially available apple
juice, well shaken at ordinary temperature, was used as a test sample. The experimental protocol, 100μg/mL procyanidin B2 solution and test samples were supplied to the participating laboratories by theFood and Agricultural Materials Inspection Center (FAMIC), the organizer of the interlaboratory tests. Eachlaboratory 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

Number of participating laboratories

11111111

Number of accepted test results

991010Mean procyanidins content, mg/L88,9171,4193,9253,3Repeatability standard deviation sr, mg/L

2,62,92,1

3,4

Repeatability relative standard deviation RSDr, %2,91,71,11,3Repeatability limit r (r = 2,8 sr), mg/L

7,28,1

5,8

9,5

Reproducibility standard deviation sR, mg/L

8,921,323,631,5

Reproducibility relative standard deviation RSDR, %10,012,412,212,4

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

24,959,766,088,1NOTEAlthough 5 pairs of test samples were supplied according to IUPAC protocol in the interlaboratory tests, it
was found that one pair of the sample was short of the specified volume in some laboratories. The data of this sample
was not adopted for Table A.1 because participating laboratories were not able to test these samples according to thespecified procedure.

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Annex B(informative)
Typical HPLC chromatograms
Y1XKeyX

Retention time, minY

Response, mV

1

Procyanidin B2

NOTE
HPLC system conditions were in accordance with Table 1 and Inertsil® WP300 Diol 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 B.1 — 100 μg/mL standard solution

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Y21XKeyX

Retention time, minYResponse, mV

1Catechin or epicatechin

2Procyanidins

NOTE
HPLC system conditions were in accordance with Table 1 and Inertsil® WP300 Diol 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 B.2 — Apple juice extract

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Bibliography[1]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.

[2]

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.

[3]

Horwitz, W., Protocol for the design, conduct and interpretation of method-performance studies,Pure Appl. Chem., 1995, 67(2), pp. 331-343[4]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.