Testing Method of 10-Hydroxy-2-Decenoic Acid

-----Stipulated by GB9697-2008/China

5.3 Testing Method of 10-Hydroxy--2- Decenoic Acid

5.3.1 Reagent
The water used in this test is re-distilled water.
a) Methanol: Spectral pure or Analytical reagent(AR) with a transmittance more than 30% at the
detection wavelength.
b) Anhydrous Ethanol: Guaranteed Reagent (GR) grade
c) Internal Standard : Methyl 4-hydroxybenzoate, content 99.0%.
d)10-HDA Standard : More than 99.0%. Before using, putting into the apparatus with concentrated
sulfuric acid to decompress and dry for 24 hours.
e) 10-HDA Standard Solution: Accurately weigh 25mg dried 10-HDA standard, dissolve with
anhydrous ethanol, transfer to 25mL volumetric flask , dilute with anhydrous ethanol to the scale
of 25mL, shaking well to even quality. It is 1mg/mL 10HDA in solution.
f) Internal standard solution: Accurate weigh 650mg dried Methyl 4-hydroxybenzoate, dissolve
with anhydrous ethanol, transfer to 1000mL volumetric flask , dilute with anhydrous ethanol to the
scale of 1000mL，shaking well to even quality. It is 0.65mg/mL internal standard in solution.
g) Chlorhydric acid (c=0.03mol/L): take 100mL of 0.1mol/L chlorhydric acid, adding into 200mL
re-distilled water.
h) Mobile phase Ø (CH3OH+0.03mol/L HCL+H2O)=55+10+35

5.3.2 Instrument
a) HPLC: Collocate UV detector, recording instrument or processor.
b) Chromatographic Column: 4.6mm*250mm column, packing silica gel C18 bonded phase,5μm
or 10μm
c) Ultrasonic washing instrument
d) Vortex mixing instrument
e) Analytical balance: Inductance 0.0001g

5.3.3 Test Steps

5.3.3.1 Test Specimen Arrangement
After thawing the sample to room temperature, mix it with a glass stick, take about 0.5g, put it into
a weighed 50mL volumetric flask, accurately weigh it, add 1mL of 0.03mol/L hydrochloric acid
and 2mL water, mix it on a vortex mixer to make the sample dissolve, add 30mL of anhydrous
ethanol, shake it gently while adding , then accurately add 10mL of internal standard solution,
dilute it to the scale with anhydrous ethanol, shake it well to even quality, and immediately put it
in an ultrasonic bath for 15minutes, or put it on a vortex mixer to oscillate for 15min, take it out,
centrifuge at 3000r/min for 10minutes, and then measure it immediately. If it cannot be measured
in time, it must be placed in the refrigerator for being measured.

5.3.3.2 Chromatographic Conditions

Detector Wavelength: 210nm; Column temperature: 35C°; Mobile phase flow rate: 1mL/min.

5.3.3.3 Determination of Correction Factor

Accurately transfer 0.5, 1, 2, 3, 4, 5mL of 10HDA standard solution to 10mL volumetric flask
separately , accurately add 2mL of internal standard solution to each volumetric flask, dilute to the
scale of 10mL with anhydrous ethanol each, and shake well to even quality. Take 2μL of these 6
solutions respectively, inject them respectively into the chromatograph, to calculate with the peak
area ratios, which should form linear and calculate the correction factor F.

5.3.3.4 Sample Determination

Transfer sample solution 4μL, inject into the Chromatograph and quantity it according to the
“Internal Standard Method”.

5.3.4 Calculation
The content of 10-Hydroxy-2-Decenoic Acid in royal jelly is calculated according to formula (2):

In the formula:
X2------------------------------- Content of 10-Hydroxy-2-Decenoic Acid in royal Jelly, %;
F--------------------------------- Correction Factor;
Ai-------------------------------- Peak Area of the tested Component in the sample;
As-------------------------------- Peak Area of Internal Standard in sample;
ms--------------------------------Mass of Internal Standard, in grams (g);
Mi--------------------------------Mass of Sample, in grams (g).

5.3.5 Relative Deviation of Parallel Test

The relative deviation of parallel test should not exceed 2.0%.