Customs Standard Methods CLASS-04-04-00 : 0061 Korea Customs Service

Customs Standard Methods

CUSTOMS LABORATORY AND SCIENTIFIC SERVICE STANDARD METHODS

CLASS 04-04-00 : 0061

Quantitative analysis of royal jelly inside natural honey

1. Scope 4.2 0.02M Ammonium monohydrogen

phosphate [0.02M (NH4)2HPO4]

This method can be used for analysis After dissolving 2.6412g of

procedures to confirm the classification (NH4)2HPO4 into 200ml of distilled

requirements regulated by Article 26 of water, and add distilled water until the

Notice of Goods Classification standard solution reaches to 1,000ml.

pursuant to Article 85 of Customs 4.3 Methanol: HPLC grade

Act(CLASS-99-01-00), Natural honey

augmented with royal jelly.
5. Methods

2. Principle 5.1 10-HDA Standard Solution

After drying 10-HAD standard

Using HPLC, conduct quantitative analysis product at sulfuric acid desiccator,

on “10-HDA(10-Hydroxy decanoic acid)", then dissolve into methanol so that

the special component for natural royal the concentration reaches 0.1mg/ml.

jelly. 5.2 producing the specimen

Take a specimen which is 10-HAD and
reaches the amount of 0.1 mg/ml. Put
3. Equipment
them in a 100ml flask, add 5ml of
water, then heat it with 50℃ while
HPLC system
shaken and blended. Then, add 35ml
of methanol, then run the ultrasound
4. Reagent
shaking extractor for thirty minutes,
then to match 100ml with adding, or
4.1 10-Hydroxy decanoic acid, 10-HAD
deducting ethanol.
Standard.

CUSTOMS LABORATORY AND SCIENTIFIC SERVICE STANDARD METHODS

 Customs Standard Methods CLASS-04-04-00 : 0061 Korea Customs Service

5.3 Machine analysis

5.3.1 HPLC Analysis Condition
- Column: μ-Bondapak C18 or with a 8. Notes
similar function
- Moving phase: 0.02 M (NH4) 2HPO4:
Notice of product classification
Methanol (7:3) (Add
standards pursuant to Article 85
phosphoric acid, to
make pH 7.0.) of Customs Act
- Detector: UV 214 nm Article 26 (Natural honey augmented

- Fluid velocity: 1.4 ml/min (Appropriately by royal jelly) ① Natural honey

adjust so that 10-HAD augmented with royal jelly, shall be

major peak maintains 7 classified as No.2106, in case of meeting

to 8 minutes) all following criteria.

- Injection: 20㎕ 1. Concentration of royal jelly is 5% or

5.3.2 Conduct analysis pursuant to CLASS higher. (concentration of 10-HDA is 0.08%

00-02-00: HPLC General Testing, then or higher of comprehensive weight).

quantify. 2. It is Irreversible to natural honey.

② In case of honey that does not meet
the criteria prescribed by paragraph 1
shall be classified as No. 0409.
6. Methods of Calculation Not
Applicable

7. Reference and Resources

7.1 Korea food code 29-7 Honey (2)

Processed product, royal jelly, “①
10-Hydroxy decanoic acid” [Attached]
7.2 CLASS 99-01-00: Notice of product
classification standards pursuant to
Article 85 of Customs Act.
7.3 CLASS 99-01-23: product classification
standards of natural honey augmented
with royal jelly.
7.4 Analysis guide to determine HS
(2004.10.)

CUSTOMS LABORATORY AND SCIENTIFIC SERVICE STANDARD METHODS

 Customs Standard Methods CLASS-04-04-00 : 0061 Korea Customs Service

[Attached] Korea Food Code 29-7 Honey

6) Testing methods
(2) Royal jelly processed product
① 10-Hydroxy-2-Decaonic Acid(10-HDA)
ⓐ Equipment
High speed liquid chromatograph
ⓑ Producing standard solution
After precisely taking 50 mg of 10-HDA standard product to dry in sulfuric acid
desiccator, dissolve it to methanol to obtain 50mL of standard agent (1,000 ppm).
Use with dilution when necessary.
ⓒ Producing test agent
Take the specimen which reaches the equivalent amount of 48 mg of 10-HAD. (If
it is soft capsule, contents and capsule should be taken together) Put the
specimen in a flask of 500 mL in volume, heat it with 50℃ then shake and mix.
In case of soft capsule, create even mixture of capsule and contents, then run the
process of 30-minute ultrasound extraction by shaking after adding 350 mL
methanol. Then add more methanol to make the solution 500mL. filter this
solution to use as a test solution. In case of capsule products, take more 20
capsules or more, excise them and measure precise weight of contents to obtain
the mean value of the weight of capsules, then take the value as an average
weight per one capsule.
ⓓ Test Operation
Ⓐ Measurement condition for high speed liquid chromatography
ⓐ Column∶μ—Bondapak C18 or similar
ⓑ Moving phase∶ 0.02 M (NH4)2HPO4 : Methanol (7:3) (Add phosphoric acid,
to make pH 7.0..)
ⓒ Extractor∶ UV 214 nm
ⓓ Fluid velocity∶1.4 mL/min (Appropriately adjust so that 10-HAD major peak
maintains 7 to 8 minutes)
Ⓑ Quantitative testing
Inject 10μL of test and standard solutions to liquid chromatograph to obtain a
graphical representation of 10-HDA. Compare the graph with the standard
graph to confirm the peak that corresponds to 10-HDA. Concentration of
10-HDA shall be derived from area or height of such peaks and calculated as
below:
<Calculation>

CUSTOMS LABORATORY AND SCIENTIFIC SERVICE STANDARD METHODS

 Customs Standard Methods CLASS-04-04-00 : 0061 Korea Customs Service

Peak area of 10-HAD in Taken from the

10-HDA test solution standard product(mg)
× ×100
Concentration(%) ＝ Peak area of 10-HAD in Collected from the
standard solution test sample(mg)

CUSTOMS LABORATORY AND SCIENTIFIC SERVICE STANDARD METHODS