Tentative Translation

JAS
0003

JAPANESE AGRICULTURAL
STANDARD

Determination of the β-cryptoxanthin in Satsuma Mandarin― High performance liquid chromatographic method

Date of Establishment: 2018-3-29
Date of Revision: 2023-6-28

Ministry of Agriculture, Forestry and Fisheries

0003：2023

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 publisherof 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 theoriginal 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)

0003：2023

Contents

Page

1

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

2

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

3

Terms and definitions.......................................................................................................................................... 2

4

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

5

Reagents ..................................................................................................................................................................... 2

6

Apparatus .................................................................................................................................................................. 4

7

Preparation of test samples .............................................................................................................................. 5

8

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

9

Calculation................................................................................................................................................................. 7

10

Precision .................................................................................................................................................................... 8

10.1

Interlaboratory test .............................................................................................................................................. 8

10.2

Repeatability ............................................................................................................................................................ 8

10.3

Reproducibility ....................................................................................................................................................... 8

11

Quality control......................................................................................................................................................... 8

12

Test report ................................................................................................................................................................ 8

Annex A (informative) Results of interlaboratory test ....................................................................................... 9Annex B (informative) Typical HPLC chromatograms ......................................................................................10
Annex C (informative) Shoulder peaks derived from isomers of BCR.......................................................12
Bibliography...........................................................................................................................................................................13(3)

0003：2023

Foreword
This Japanese Agricultural Standard has been revised by the Minister of Agriculture, Forestry and Fisheries
through deliberations at the Council for the Japanese Agricultural Standards as a result of proposal for therevision of Japanese Agricultural Standard submitted by Food and Agricultural Materials Inspection Center,
Incorporated Administrative Agency with the original bill being attached, based on the provisions of Article
4, paragraph (1) of the Act on Japanese Agricultural Standards as applied mutatis mutandis pursuant to
Article 5 of the same Act. This edition replaces the previous edition (JAS 0003:2019), which has beentechnically revised.
Attention is drawn to the possibility that some parts of this document may conflict with patent rights,
published patent applications 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 such patentrights, published patent applications or utility model rights.

(4)

JAPANESE AGRICULTURAL STANDARD(Tentative Translation)

JAS
0003：2023

Determination of the β-cryptoxanthin in Satsuma Mandarin― 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
and to ensure compliance with any national regulatory conditions.

1 ScopeThis document specifies a high performance liquid chromatographic method for the determination of βcryptoxanthin (BCR) in the edible part of satsuma mandarin (Citrus unshiu Marc.) (fresh fruits).

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 8101, Ethanol (99,5) (Reagent)
JIS K 8150, Sodium chloride (Reagent)
JIS K 8355, Acetic acid (Reagent)
JIS K 8361, Ethyl acetate (Reagent)
JIS K 8574, Potassium hydroxide (Reagent)
JIS K 8593, Petroleum ether (Reagent)
JIS K 8780, Pyrogallol (Reagent)
JIS K 8839, 2-Propanol (Reagent)
JIS K 8848, Hexane (Reagent)
JIS K 8987, Sodium sulfate (Reagent)
JIS K 9705, Tetrahydrofuran (Reagent)20003：2023

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

4 Principle
BCR is extracted from the test sample using ethanol. The extract is saponified with potassium hydroxide.
BCR is collected from the saponified sample using hexane and ethyl acetate mixture to obtain themeasurement solution. The BCR in the measurement solution is determined by high performance liquid
chromatograph (HPLC) system with UV-visible absorbance detector.

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 BCR, of minimum mass fraction, 99 % (HPLC).
5.3 Ethanol, of minimum mass fraction, 99,5 %, according to JIS K 8101.
5.4 Pyrogallol, of minimum mass fraction, 99,5 %, according to JIS K 8780.
5.5 Sodium sulfate, of minimum mass fraction, 99,0 %, according to JIS K 8987.
5.6 Potassium hydroxide, of minimum mass fraction, 85,0 %, according to JIS K 8574.
5.7 Sodium chloride, of minimum mass fraction, 99,5 %, according to JIS K 8150.
5.8 Hexane, of minimum mass fraction, 96,0 %, according to JIS K 8848.
5.9 Ethyl acetate, of minimum mass fraction, 99,5 %, according to JIS K 8361.
5.10

2-Propanol, of minimum mass fraction, 99,7 %, according to JIS K8839.

5.11

Acetonitrile, HPLC grade.

5.12

Methanol, HPLC grade.

5.13 Tetrahydrofuran, with or without antioxidants, of minimum mass fraction, 99,5 %, according to JIS
K 9705.
5.14

Acetic acid, of minimum mass fraction, 99,7 %, according to JIS K 8355.

5.15dl-α-tocopherol, of minimum mass fraction, 97,0 %.

5.16

Petroleum ether, special grade stipulated in JIS K 8593 or those of quality equivalent or superior.

5.17

Nitrogen, of minimum volume fraction, 99,5 %.

5.18

β–carotene, of minimum mass fraction, 90 %.

5.19 Pyrogallol ethanol, dissolve 30 g of pyrogallol per 1,0 l of ethanol. Do not use if the color changes
to brown.
5.20

Potassium hydroxide solution, dissolve 60 g of potassium hydroxide per 100 ml of water.

WARNING ― Since irritating gas is generated, work shall be done in a place with good ventilation
inside a fume cupboard etc.
5.21

Sodium chloride solution, dissolve 10 g of sodium chloride per 1,0 l of water.

5.22

Hexane/ethyl acetate mixture, mix 9 parts per volume of hexane with 1 part per volume of ethyl

30003：2023

acetate.
5.23 HPLC mobile phase, mix 55 parts per volume of acetonitrile with 40 parts per volume of methanol,
5 parts per volume of tetrahydrofuran and 0,1 parts per volume of acetic acid. Dissolve 0,05 g of dl-αtocopherol per 1,0 l of this mixture. Degas before use.
5.24 BCR stock standard solution, prepare a BCR stock standard solution in petroleum ether
containing approximately 10 μg/ml BCR. Transfer this solution into several bottles with screw cap, seal andstore.
NOTE
In the interlaboratory test described in Annex A, BCR stock standard solutions were stored at –30 °C to–20 °C. It has been confirmed that the BCR stock standard solutions remain stable for at least half a year when stored
at –30 °C to –20 °C.

Return the BCR stock standard solution to room temperature and shake to mix before use. Remove
undissolved matter with a membrane filter.
5.25

Standard solutions

5.25.1 General
A standard solution for absorbance measurement (see 5.25.2) and a series of the standard solutionsprescribed in 5.25.3 shall be prepared from the same BCR stock standard solution each time. Once the BCR
stock standard solution is returned to room temperature, the standard solution for absorbancemeasurement shall be prepared and measured the concentration of that on the same day. Any remaining
BCR stock standard solution shall not be stored.
5.25.2 Standard solution for absorbance measurement, dilute the BCR stock standard solution 5times with petroleum ether using a single volume pipette and a volumetric flask.
NOTE
In the interlaboratory test described in Annex A, 2 ml of BCR stock standard solution was transferred into a
10 ml volumetric flask and filled up to the mark.

Set up and operate the spectrometer in accordance with the manufacturer’s instructions. Measure theconcentration of standard solution for absorbance measurement by using a spectrometer at 452 nm withpetroleum ether as reference. The BCR concentration of the stock standard solution, ρ0, is given by theformula:
ρ0 =

where

A × V2 × 10 000
ε × V1

ρ0is the concentration of BCR in the stock standard solution (μg/ml);

A

is the absorbance of the standard solution for absorbance measurement determined at 452 nm(petroleum ether, optical path length 1 cm);εis the absorption coefficient of BCR in concentration 1 % and optical path length 1 cm, 2 386 [7]
[8];

V1

is the nominal volume (ml) of the single volume pipette used;V2
is the nominal volume (ml) of the one-mark volumetric flask used.
Perform the procedure in 5.25.3 on the same day.
5.25.3 A series of standard solutions, using a single volume pipette, transfer the BCR stock standard
solution into a round-bottomed flask. Gently blow nitrogen on this solution to evaporate the petroleum
ether. Dissolve the contents in the round-bottomed flask completely with ethanol. Ultrasound may be used
for about 10 seconds as a means of dissolution. Rinsing this flask several times with ethanol, transfer thecontents completely into a one-mark volumetric flask. Add ethanol up to the mark of the volumetric flask
and shake to mix. Filter the mixed solution with a membrane filter and use as the standard solution. Use the40003：2023

same procedure to prepare standard solutions with a total of 4 or more concentration levels to create aseries of standard solutions. An example of the prepared series of standard solutions is given in Table 1. The
BCR concentration of the dilute standard solution, ρi, is given by the formula:
ρi =

where

ρ0 × V3
V4

ρiis the concentration of BCR in ith levels of standard solution (μg/ml);

ρ0

is the concentration of BCR in the stock standard solution (μg/ml) obtained in 5.25.2;

V3

is the nominal volume (ml) of the single volume pipette used;V4

is the nominal volume (ml) of the one-mark volumetric flask used.
Table 1 ― An example of the prepared series of standard solutions
Standard

The nominal volume of the

The nominal volume of the

The concentration of BCR

solutions

single volume pipette (ml)one-mark volumetric flask

in

(ml)

(equivalent to μg/ml)

standard

A

1

5

2,0

B

1,5

10

1,5

C

1

10

1,0

D

0,5

10

0,50

E

0,5

20

0,25

solutions

Perform the procedure in 8.5.2 on the day of preparation, or store the standard solutions at –30 °C to –20 °C.
A series of standard solutions stored at –30 °C to –20 °C shall be returned to room temperature before HPLC
measurement (see 8.5.2). After mixing thoroughly and using ultrasound for about 10 seconds as necessary
to dissolve the undissolved matter, pass through a membrane filter.
NOTE 1 Between 0,25 μg/ml and 2,0 μg/ml of the calibration curve, it has been confirmed that the coefficient ofdetermination is more than 0,990 and the origin is within the 95 % confidence interval of the y-intercept.
NOTE 2 It has been confirmed that the standard solutions remain stable for at least a week when stored at –30 °C to–20 °C.

5.26

β-carotene solution

Dissolve β-carotene in ethanol, and prepare a solution of a certain concentration that is within the BCR
concentration range of the series of standard solutions prepared in 5.25.3.
NOTE
When preparing a high concentration β-carotene solution, the β-carotene may not completely dissolve in theethanol. In such a case, dissolve the β-carotene in hexane, and take a fraction. Blow nitrogen gently to evaporate thehexane so that it can dissolve in ethanol to prepare a solution with the targeted concentration.

6 Apparatus
The usual laboratory apparatus and the following shall be used.
6.1 Electronic analytical balances, capable of weighing to an accuracy of ±0,1mg, and more than 200 g.
6.2 Centrifuge tubes, made of glass with a round-bottom and around 50 ml in capacity, with a cap,
keeping the space for shaking, and suitable for centrifugation at 400×g. The cap shall be either a stopper orscrew type that is resistant to organic solvents. Furthermore, the cap for saponification (see 8.2) shall be50003：2023

resistant to strong alkaline solutions.
6.3 Shaker, capable of mixing the centrifuge tube by shaking vertically.
6.4 Centrifuge, capable of 400×g.
WARNING ― To prevent accidents, operate the centrifuge according to the instruction manual of theequipment.
6.5 Single volume pipettes, to cover the volume range for dilution of standard solutions (see 5.25) andsaponification (see 8.2), of ISO648, class A.
6.6 One-mark volumetric flasks, to cover the volume range for dilution of standard solutions (see 5.25),
extraction (see 8.1) and saponification (see 8.2), of ISO1042, class A.
6.7 Water bath, capable of being maintained at (70±3) °C and size for centrifuge tube rack.
6.8 Round-bottomed flasks, of 100 ml capacity, with ground neck, usable for evaporation.
6.9 Rotary evaporator, with water bath and vacuum control, for evaporation of solvent, for example
hexane, ethyl acetate and ethanol.
6.10 Membrane filters, made of polytetrafluoroethylene (PTFE), suitable for organic solvents, with apore size of 0,2 μm or less. The filter and the housing shall be unitary, and the housing material shall be
resistant to organic solvents.
6.11 Vials, suitable for HPLC to be used, made of glass, deactivated, or other glass vials which have beenchecked for no influence on the measurement. Septum of the cap shall be made of PTFE or coated with
PTFE.
6.12

Spectrometer, capable of measuring wavelengths 452 nm and holding cells.

6.13 Cells, made of quartz glass or glass, with optical path length of 1 cm, and should have stoppers. Whenusing multiple cells, optical characteristics shall be equivalent.
6.14

HPLC system

6.14.1 HPLC, equipped with a mobile phase delivery system, a column oven with temperature control
function, a UV-visible absorbance detector able to measure absorbance at 455 nm and a data processing
unit, prescribed in JIS K 0124. A mobile phase delivery system should have a degassing device.
6.14.2 Chromatographic column for HPLC, reverse-phase C18 (ODS) columns, with the following
characteristics:—length: 150 mm—internal diameter: 4,6 mm—spherical particle size: 3 μm to 5 μm
—β-carotene shall be eluted within 20 min. Confirm that elution time of β-carotene and its peak do not
overlap with BCR peak in accordance with 8.5.
If a guard column is used, select the one matching the C18 (ODS) column.

7 Preparation of test samples
After removing only the outer peel of the sample, pulverize it using a homogenizer or the like to obtain thetest sample.
Immediately perform the procedure in 8.1, or freeze the test sample to store. When storing the test sample
frozen, transfer into a sealable glass container soon after pulverized. Return it to room temperature and mix
well before use.

60003：2023

NOTE 1 It has been confirmed that the samples remain stable for 2 weeks when stored fresh and refrigerated [4].
NOTE 2 It has been confirmed that the test samples remain stable for at least 2 months when stored frozen at –20 °C
or below.

8 Procedure
8.1

Extraction

8.1.1 Weigh, to the nearest 10 mg, approximately 2 g of the test sample into a centrifuge tube. Add 15 mlof pyrogallol ethanol and 10 g of sodium sulfate to the tube.
8.1.2 Shake vigorously for 5 min by shaker. Separate the contents by the centrifuge at 400×g for 5 min.
Transfer the supernatant into a 50 ml one-mark volumetric flask.
8.1.3 Add 15 ml of pyrogallol ethanol to the residue in the tube and repeat the procedure in 8.1.2. Transfer
the supernatant into the same volumetric flask as in 8.1.2.
8.1.4 Repeat the procedure in 8.1.3.
8.1.5 Add pyrogallol ethanol up to the mark of the volumetric flask containing the supernatant, shake tomix, and use as the extract.

8.2

Saponification

Using a single volume pipette, transfer 10 ml of the extract (8.1.5) into a centrifuge tube and add 1 ml ofpotassium hydroxide solution to the tube. Shake gently. Place the tube in a constant temperature water bathset at 70 °C, heat the tube for 30 min shaking every 5 min. Then cool the tube to room temperature and use
as the saponified sample.

8.3

Collection of BCR

8.3.1 Add 20 ml of sodium chloride solution, 5 ml of 2-propanol and 12 ml of hexane/ethyl acetate
mixture to the saponified sample (8.2) and shake to mix.
8.3.2 Shake the tube vigorously for 5 min by shaker. Separate the contents by the centrifuge at 400×g for
5 min. Transfer the supernatant into a round-bottomed flask.
8.3.3 Add 12 ml of hexane/ethyl acetate mixture to the liquid left in the tube. Repeat the procedure in
8.3.2. Transfer the supernatant into the same round-bottomed flask as in 8.3.2.
8.3.4 Repeat the procedure in 8.3.3.
8.3.5 Evaporate almost the solvent in the round-bottomed flask (8.3.4) at 40 °C or below by rotary
evaporator. Then gently blow nitrogen on the residue to dryness, and use as the dry residue.

8.4

Dissolution

Dissolve the dry residue in the round-bottomed flask (8.3.5) completely with ethanol. Ultrasound may be
used for about 10 seconds as a means of dissolution. Rinsing the flask several times with ethanol, andtransfer the solution completely to a one-mark volumetric flask.
NOTE 1 In the interlaboratory test described in Annex A, 5 ml one-mark volumetric flasks were used.

Add ethanol up to the mark of the volumetric flask and shake to mix. Pass through a membrane filter into avial, and use as the measurement solution.
Perform HPLC measurement (see 8.5.2) on the same day, or store the measurement solution at –30 °C to–20 °C.
NOTE 2 It has been confirmed that the measurement solution remains stable for at least one week when stored at–30 °C to –20 °C.

70003：2023

The measurement solution stored at –30 °C to –20 °C shall be returned to room temperature on the day ofmeasurement. Mix using a test tube mixer and use ultrasound for about 10 seconds as necessary to dissolve
the undissolved matter well, then pass through a membrane filter.

8.5

Determination

8.5.1 HPLC operating conditions
Set up the HPLC system in accordance with the manufacturer's instructions and adjust it as follows.
a)

Flow rate of the mobile phase: 1,5 ml/min.

b) Column Temperature: 40 °C.
c)

Detection wavelength: 455 nm.

d) Volume injected: 20 μl.
e)Measurement time: 25 min. If, by injecting β-carotene, it is confirmed that the peak of β-carotene does
not affect the subsequent measurement, the measurement time may be shortened.

8.5.2 HPLC analysis
Allow the entire system to run for a while to stabilize it. Confirm that the fluctuation of the base line gives
no hindrance for the determination of BCR by a blank run under the specified conditions (see 8.5.1). Then
inject the series of standard solutions into the column, followed by equal volumes of the measurement
solutions.
8.5.3 Identification
Identify the individual BCR peak in the sample chromatogram by comparing retention times with thoseobtained from the standard solutions under the same HPLC conditions (see 8.5.1).
NOTE

Typical HPLC chromatogram of a satsuma mandarin extract are given in Annex B.

9 Calculation
9.1

GeneralQuantitative 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 prescribed in JIS K 0124. For
the shoulder peaks derived from cis isomers, their peak areas are summed up as the peak area of BCR.
NOTE
Typical shoulder peaks derived from cis isomers are given in Annex C. Generally with isomers of BCR, alltrans isomer is the most stable, and partial isomerization to cis isomer has been reported. Under the HPLC operating
conditions described in 8.5.1, cis isomers peaks are detected immediately after the all-trans isomer peak. (see Figure
C.1 a)). Furthermore, it has been reported that the main absorption spectrum of cis isomer shifts at 2 nm to 5 nm
towards the short wavelengths compared to the all-trans isomer, and an characteristic peak appears in the near-UV
region that is not present in the all-trans isomer [9] (see Figure C.1 b)).

9.2

Quantitation

Obtain the BCR peak areas for each of the series of standard solutions. Perform a linear regression of peakareas for each standard solution against the BCR concentrations of the respective standard solutions tocreate a calibration curve.
Calculate the concentration of BCR in each measurement solution by using the calibration curve. The BCR
content in the test sample, wi, is given by the formula:wi =C × V5 × d1
W × d280003：2023

where

9.3

wi

is the content of BCR in the test sample (mg/kg);C

is the concentration of BCR in the measurement solution (μg/ml);

V5

is the constant volume (ml) in preparation of the measurement solution (see 8.4);

d1

is the constant volume (ml) in preparation of the extract (see 8.1), typically 50;

d2

is the volume of fraction (ml) in saponification of the extract (see 8.2), typically 10;

W

is the mass (g) of the test sample.

Expression of results

Express the results to two significant figures.

10 Precision
10.1 Interlaboratory test
An 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 (4,7 mg/kg to 23 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 [1] given in Table A.1 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 as longas the specified operation is correctly done [2].

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 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.

90003：2023

Annex A
(informative)

Results of interlaboratory test
An interlaboratory test was carried out in accordance with IUPAC protocol [3] in 2015 in Japan, and gavethe statistical results given in Table A.1 [5]. Some commercially available satsuma mandarins, with the outer
peel removed, were weighed 150 g to 200 g. Pyrogallol was added as an antioxidant at 10 % of the sample
mass and pulverized at 12 000 r/min for 10 min by a pulverizing device.
NOTE 1 Since carotenoids can be decomposed by light, oxygen and enzymes in the sample, the antioxidant was added.

After the homogeneity [6] was confirmed, the pulverized material was used as a test sample. The
experimental protocol and test samples were supplied to the participating laboratories by the Food andAgricultural Materials Inspection Center (FAMIC), the organizer of this interlaboratory test. Each laboratory
tested a total of 10 test samples (5 pairs of blind duplicates) according to the experimental protocol.
NOTE 2 In this interlaboratory test, instead of the HPLC mobile phase prescribed in 5.23, a mixture of 24 parts pervolume of methanol and 1 part per volume of chloroform, containing 0.05 g per 1.0 l of ascorbyl palmitate was used.
When FAMIC examined the difference in quantitative value from using these mobile phases in 2022, no significant
difference was found (significance level: 5 %).

Table A.1 — Precision data
Sample identification

Sample 1

Sample 2

Sample 3

Sample 4

Sample 5

Number of participating laboratories

11

11

11

11

11

Number of accepted test results

10

9

9

9

10

Mean BCR content, mg/kg (mass fraction),

4,73

6,75

10,2

13,7

23,4

Repeatability standard deviation sr mg/kg

0,12

0,13

0,32

0,57

0,50

Repeatability relative standard deviation, RSDr, %

2,6

2,0

3,1

4,2

2,1

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

0,34

0,36

0,90

1,6

1,4

Reproducibility standard deviation sR mg/kg

0,67

0,61

1,0

1,3

2,6

Reproducibility relative standard deviation, RSDR, %

14

9,0

9,9

9,6

11

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

1,9

1,7

2,8

3,6

7,3

10
0003：2023

Annex B(informative)

Typical HPLC chromatograms

Y
1

X

Key
X

retention time, minYresponse, mAU

1

BCR

NOTE
For the HPLC operating conditions, in addition to 8.5.1, Inertsil® ODS-3 was used as the column. This
information is given for the convenience of the users of this document and does not constitute an endorsement of the
product.

Figure B.1 — BCR standard solution (equivalent to 1 μg/ml)

110003：2023

Y
1

2

X

Key
X

retention time, minYresponse, mAU

1

BCR

2

β-carotene

NOTE
For the HPLC operating conditions, in addition to 8.5.1, Inertsil® ODS-3 was used as the column. This
information is given for the convenience of the users of this document and does not constitute an endorsement of the
product.

Figure B.2 — Measurement solution

120003：2023

Annex C
(informative)

Shoulder peaks derived from isomers of BCR

Y

Y

20
20

1
10

4

2

10

0
0
1

2

3

4

5

X

a) Chromatogram of BCR standard solution

3
350

400

450

500

550

Z

b) Comparison of isomeric absorption spectraKeyX

retention time, minYresponse, mAU

Z

wavelength, nm

1

peak of all-trans isomer of BCR (X=3,7 min)2

shoulder peak of cis isomers of BCR (X=3,9 min)3

absorption spectrum of all-trans isomer4absorption spectrum of cis isomers (distinct at 325 nm to 350 nm)

NOTE
For the HPLC operating conditions, in addition to 8.5.1, TSKgel® ODS-120A was used as the column. This
information is given for the convenience of the users of this document and does not constitute an endorsement of the
product.

Figure C — Examples of detected isomers

130003：2023

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]

Matsumoto, H., et al., Effect of Postharvest Temperature and Ethylene on Carotenoid Accumulationin the Flavedo and Juice Sacs of Satsuma Mandarin (Citrus unshiu Marc.) Fruit, J. Agric. Food Chem.,
2009, 57(11), pp. 4724-4732.

[5]

Kumagai, M., et al., Validation of a Method for Determination of β-cryptoxanthin in Satsuma
Mandarin (Citrus unshiu Marc.) by Interlaboratory Study , Nippon Shokuhin Kagaku Kogaku Kaishi.
2016, 63(10), pp. 450-454.

[6]

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.

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