Determination of Dehydroacetic acid in Bread using HPLC with UV detection following the Chinese National Standard (GB) Method

Dean Duan, Application Scientist


Dehydroacetic acid is an organic compound which has several industrial applications. It can be used as a plasticizer in synthetic resins; as a fungicide or bactericide; but also as a food preservative (E265).

This application focus on testing Dehydroacetic acid in bread using a Discovery® HS C18 HPLC column, following the current Chinese national standard method (GB 5009.121-2016).

Obtained results shows satisfying chromatographic resolution of dehydroacetic acid from the bread sample matrix, and the method linearity, Limit of Detection (LOD) and Limit of Quantitation (LOQ) meet set testing requirements.
Dehydroacetic acid
Dehydroacetic acid



Experimental Conditions
column Discovery® HS C18 250 x 4.6 mm,5um (568523-U)
mobile phase [A] 20 mM ammonium acetate, pH 3.5 with acetic acid; [B] methanol; (70 : 30, A : B)
flow rate 1.0 mL/min
detector UV 293 nm
injection 10 µL
column temp 30 °C
sample Standard stock solution: Weigh accurately 0.1 g of dehydroacetic acid to a 100-mL volumetric flask, add into 10 mL of 20 g/L of sodium hydroxide solution, add  water to volume to obtain a 1.0 mg/mL of stock solution. standard solution: Dilute the stock solution with water to obtain a 50 µg/mL of solution.
Sample: Homogenize bread sample, weigh accurately 2 - 5 g of homogenized sample to a 25-mL centrifuge tube. Add into 10 mL of water, 5 mL of 120 g / L of ZnSO4 aqueous solution, adjust pH value to pH 7.0 with 20 g/L of NaOH aqueous solution. add water to volume, shake well. sonicate for 10 minutes. transfer the supernatant to separating funnel, add into 10 mL of hexane, shake for 1 minute, stand for separating, remove the hexane layer, repeat the procedure twice, pool the aqueous layer to centrifuge, centrifuge at 4000 r/min for 1o minutes. filter the supernatant with 0.45 µm filter membrane for HPLC analysis.


Standard stock solution


No. Compound RT (min) Resolution Plates (USP) Tailing factor
1 Dehydroacetic acid 19.5 -- 22832 1.0

Specificity and repeatability

1. Specificity: Inject Standard Solution and Determine the Retention Time and Monitor the Peak Purity

No. Compound RT (min) Plates (USP) Tailing factor Peak purity
1 Dehydroacetic acid 19.5 22832 1.0 1.0000


2. Standard Repeatability (Dehydroacetic acid, 10 ppm)

Measurements Mean area
STD 1 283.7
STD 2 284.8
STD 3 284.2
STD 4 284.8
STD 5 283.2
Mean 284.1
Standard Deviation     0.7
RSD (%)     0.2


Specificity and repeatability


3. Linearity

Concentration (μg/mL) Mean area
    0.20    5.7
    0.50  14.8
    1.00  28.8
  10.0 284
  50.0 1439
100.0 2875
200.0 5783


4. LOD & LOQ

Concentration (μg/mL) Mean area
0.20   5.73
0.50 14.79
1.00 28.76
STEYEX   0.35
Slope 28.70
LOD (ppm)   0.04
LOQ (ppm)   0.12