Determination of oxytetracycline, tetracycline, chlortetracycline and doxycycline residues in honey by liquid chromatography-ultraviolet detection

12.
2.
2.
1 Scope of application

Determination of oxytetracycline , tetracycline , chlortetracycline , and doxycycline residues in honey
.

Detection limit of the method: oxytetracycline, tetracycline, chlortetracycline, and doxycycline are all 0.

12.
2.
2.
2 Principle of the method

The tetracycline antibiotic residues in the honey sample are extracted with 0.
1mol/L Na ₂ EDTA-Mcllvaine (pH=4.
0±0.
05) buffer solution.
After centrifugation, the supernatant is purified with OasisHLB or equivalent solid phase extraction column and anion exchange column , UV detector high performance liquid chromatograph determination, external standard method for quantification
.

12.
2.
2.
3 Reagents and materials

Methanol , acetonitrile : primary chromatographic purity; disodium hydrogen phosphate (Na ₂ HPO ₄ ·2H ₂ O): superior grade; ethyl acetate : analytical grade, redistilled; citric acid (C ₆ H ₈ O ₇ ·H ₂ O), disodium ethylenediaminetetraacetic acid (Na ₂ EDTA·2H ₂ O), oxalic acid: analytically pure
.

Disodium hydrogen phosphate solution: 0.
2mol/L
.

Weigh 28.

Oxytetracycline, tetracycline, chlortetracycline, doxycycline standard materials: purity ≥95%
.

Standard solution of oxytetracycline, tetracycline, chlortetracycline and doxycycline: 0.
1mg/mL
.

Accurately weigh appropriate amounts of oxytetracycline, tetracycline, chlortetracycline, and doxycycline standard materials, and mix them with methanol to prepare a standard stock solution of 0.

OasisHLB solid phase extraction column or equivalent: 500mg, 6mL
.

Before use, pretreated with 5mL methanol and 10mL water respectively to keep the column moist

Anion exchange column: carboxylic acid type, 500mg, 3mL
.

Pre-treatment with 5mL ethyl acetate before use, keep the column moist

12.
2.
2.
4 Instruments and equipment

High performance liquid chromatograph: equipped with ultraviolet detector; analytical balance: sensing volume 0.
1mg, 0.
01g; liquid mixer; solid phase extraction vacuum device; liquid reservoir: 50mL; micro syringe: 25μL, 100μL; graduated sample tube : 5mL, accuracy of 0.
1mL; vacuum pump: vacuum degree should reach 80kPa; centrifuge tube: 50mL; flat bottom flask: 100mL; pH meter: measurement accuracy of ±0.
02
.

12.
2.
2.
5 Sample preparation

(1) Sample preparation

For laboratory samples without crystals, stir them evenly
.

For samples with crystals, place them in a water bath not exceeding 60°C under airtight conditions to warm them, shake them, stir them after all the samples are melted, and quickly cool to room temperature

(2) Extraction

Weigh 6g sample, accurate to 0.
01g
.

Place it in a 150mL Erlenmeyer flask, add 30mL Na ₂ EDTA-Mcllvaine buffer solution, and mix quickly on a liquid mixer for 1 min to completely dissolve the sample

(3) Purification

Pour the supernatant into a reservoir connected to the OasisHLB column or equivalent, and pass the supernatant through the Oasis or equivalent solid phase extraction column at a flow rate of ≤3mL/min.
After the supernatant has completely flowed out, use 5mL methanol -Wash the column with water and discard all effluent
.

Under a negative pressure of 65kPa, vacuum pump for 20min, and finally eluted with 15mL ethyl acetate .

According to the above method, let the eluent pass through the anion exchange column at a flow rate of ≤3mL/min under reduced pressure.
After all the eluent has flowed out, wash the column with 5mL methanol and discard all the effluent
.
Under 65kPa negative pressure, drain under reduced pressure for 5 minutes, and then eluted with 4mL mobile phase.
Collect the eluent in a 5mL sample tube and dilute to 4mL for measurement by liquid chromatograph
.

12.
2.
2.
6 Determination

(1) Liquid chromatography conditions

Column: μBondapakC ₁₈ , 10μ, 300mm×3.
9mm or equivalent; mobile phase: acetonitrile+methanol+0.
01mol/L oxalic acid solution (20+10+70, v/v/v); flow rate: 1.
5mL/min; Column temperature: 25℃; detection wavelength: 350nm; injection volume: 100μL
.

(2) Liquid chromatography determination

According to the content of oxytetracycline, tetracycline, chlortetracycline, and doxycycline in the sample solution, select a standard working solution with similar peak heights, oxytetracycline, tetracycline, chlortetracycline, and doxycycline in the standard working solution and the sample solution The response value of the element should be within the linear range determined by the instrument
.
Measure the difference in volume of the standard working solution and the sample solution
.
Under the above chromatographic conditions, the resolution of the chromatographic peaks of oxytetracycline, tetracycline, chlortetracycline, and doxycycline should be greater than 1.
2, and their reference retention times are 4.
0min, 4.
8min, 9.
6min, and 14.
0min, respectively
.
The chromatograms of oxytetracycline, tetracycline, chlortetracycline, and doxycycline standard substances are shown in Figure 12-4
.

Figure 12-4 Oxytetracycline, tetracycline, chlortetracycline, doxycycline standard substance chromatogram.

4.
027min, oxytetracycline: 4.
757min, tetracycline; 9.
578min, aureomycin: 14.
015min, doxycycline