Method for the determination of chlorpyrifos residues in exported honey - Master's thesis - Dissertation
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Honey
Medium insecticidal residue test method
Chromatography
1
Subject content and scope of application
This standard specifies the sampling, sample preparation, and gas chromatographic determination of pesticide residues in exported honey. It applies to the testing of insecticide residues in exported honey.
2
Sampling and sample preparation
2.1 Inspection lot
A batch of no more than 1000 pieces is considered an inspection lot. All goods in the same lot should have similar characteristics such as packaging, labeling, origin, specifications, and grade.
2.2 Number of samples
The minimum number of samples required depends on the batch size:
- For 50 pieces or less: 5 samples
- For 50–100 pieces: 10 samples
- For 101–500 pieces: Add 5 samples for every 100 pieces
- For over 500 pieces: Add 2 samples for every additional 100 pieces
2.3 Sampling tools
2.3.1 Sampling tube: Stainless steel, 115 cm long, 2.5 cm in diameter.
2.3.2 Mixer: Enamel bucket or cup.
2.3.3 Rods: Stainless steel set.
2.3.4 Vial bottle: 500 mL frosted glass bottle with wide mouth.
2.4 Sampling method
Randomly select the specified number of samples from the batch. Open each one and insert the sampling tube slowly to collect at least 100 g per sample. Transfer the sample into a container, mix thoroughly, and seal it. Label and send it to the lab promptly.
2.5 Sample preparation
Stir uncrystallized samples evenly. If crystallization occurs, warm the sample in a water bath at no more than 60°C until fully melted. Cool quickly and avoid moisture evaporation. Store the prepared sample in a sealed vial and label it.
2.6 Sample storage
Keep the sample at room temperature. During all operations, ensure no contamination or changes in residue levels occur.
3
Test methods
3.1 Method summary
Residual insecticides in honey are extracted using acetone and sodium hydroxide solution with n-hexane. After purification through liquid-liquid partitioning, the compound is analyzed using gas chromatography with a nitrogen-phosphorus detector. Diphenylamine is used as an internal standard for quantification.
3.2 Reagents and materials
All reagents are analytical grade unless otherwise stated. Water is distilled or deionized.
3.2.1 Acetone: Heavy distillation.
3.2.2 N-hexane: Heavily distilled with alkali.
3.2.3 Sodium hydroxide solution: 1 mol/L.
3.2.4 Sodium hydroxide solution: 0.5 mol/L.
3.2.5 Hydrochloric acid solution: 0.1 mol/L.
3.2.6 Internal standard solution: 0.500 μg/mL diphenylamine in n-hexane.
3.2.7 Insecticide standard: Purity > 99%.
3.2.8 Standard solution: Prepare 40.0 mg insecticide in 100 mL n-hexane to get 0.400 mg/mL. Dilute further with n-hexane and internal standard to prepare working solutions. Freshly prepared.
3.3 Instruments and equipment
3.3.1 Gas chromatograph with nitrogen-phosphorus detector.
3.3.2 Integrator or recorder.
3.3.3 Rotatory evaporator.
3.3.4 Micro-syringe: 10 μL.
3.4 Measuring step
3.4.1 Extraction
Weigh 20 g sample, add 30 mL 1 mol/L NaOH, dissolve, add 50 mL acetone, transfer to a separatory funnel. Add 50 mL n-hexane, shake for 2 minutes, separate and retain the upper layer.
3.4.2 Purification
Wash n-hexane layer with 30 mL 0.5 mol/L NaOH, then with 20 mL 0.1 mol/L HCl. Repeat washing steps, combine HCl layers, neutralize with 30 mL 1 mol/L NaOH, add 60 mL n-hexane, shake, and concentrate under reduced pressure.
3.4.3 Determination
3.4.3.1 Chromatographic conditions:
a. Column: Glass column (2 m × 3 mm), filled with 6% SE-54 or 15% SE-30 on Chromosorb WHP with 0.6% KOH.
b. Nitrogen: ≥99.99%, 60 mL/min.
c. Hydrogen: 2.5 mL/min.
d. Air: 170 mL/min.
e. Column temperature: 198°C.
f. Injection temperature: 250°C.
g. Detector temperature: 250°C.
h. Bead voltage: 6.8×2V.
3.4.3.2 Chromatographic determination
Inject 2 μL of both standard and sample solution. Retention time for diphenylamine is about 3.8 min, and for the insecticide is 4.4 min.
3.4.4 Blank test
Perform the procedure without adding any sample.
3.5 Calculation and representation of results
Use the internal standard method for calculation. Formula:
X = (Cs × A × Asi × mi) / (Csi × Ai × As × m)
Where:
- X = Insecticide content in honey (mg/kg)
- Cs = Insecticide concentration in standard solution (μg/mL)
- Csi = Diphenylamine concentration in standard solution (μg/mL)
- A = Peak area of insecticide in sample
- Ai = Peak area of diphenylamine in sample
- As = Peak area of insecticide in standard
- Asi = Peak area of diphenylamine in standard
- mi = Amount of diphenylamine added
- m = Total sample weight (g)
Note: Subtract blank value from the final result.
4
Method for determining lower limit and recovery rate
4.1 Lower limit
The lower limit of detection is 0.005 mg/kg.
4.2 Recovery rate
Recovery rate ranges between 89.8% and 100.5% for concentrations from 0.005 to 0.100 mg/kg.
Additional information:
This standard was proposed by the State Administration of Import and Export Commodity Inspection of the People’s Republic of China.
It was drafted by the Shanghai Import and Export Commodity Inspection Bureau of the People’s Republic of China.
Main drafters: Ge Xiuli and Cai Zeci.
Reference: Dr. Wiertz, Kurzer Abri β der Methode – Chlordimeformin Honig.