Maximum allowable residue limits and analytical techniques of nitrofuran metabolites-pretreatment methods (2)

(3) Extraction and purification

Commonly used sample extraction and purification methods in the detection process of NFs metabolites include liquid-liquid partitioning (LLP), solid phase extraction (SPE) and precipitation, etc.
In recent years, supercritical fluid extraction (SFE) and matrix solid phase dispersion technology ( MSPD) etc.
also have applications
.

1) Precipitation

For high-protein and high-fat samples such as milk powder and egg powder, after hydrolyzing and derivatizing protein-bound nitrofuran metabolites, protein precipitation agents ZnSO ₄ and K ₄ Fe(CN) ₆ can be added to effectively remove milk powder and egg powder To avoid emulsification and gelation in the process of extraction and purification
.

For royal jelly samples, trichloroacetic acid is a good protein precipitation agent, and its strong acidity can provide a suitable acidic reaction environment
.

2) Liquid liquid partition (liquidliquidpartition, LLP)

The LLP of NFs metabolites is generally divided into two phases with ethyl acetate in a pH 7 aqueous solution
.

Lopez et al.
used a 10% sodium chloride solution to dissolve the honey.
After centrifugation, the supernatant was passed through an OasisHLB solid phase extraction column, and the eluent was used for hydrolysis and derivatization
.

At the same time, the pH of the sample solution has a great influence on the extraction efficiency
.

3) Solid phase extraction (solidphase extraction, SPE)

SPE is widely used in the purification of NFs metabolites in animal tissue samples
.

Peng Tao et al.
compared ordinary C18SPE column and OasisHLBSPE column with vinylpyrrolidone-divinylbenzene copolymer as adsorbent, and found that the latter has strong selective retention of analytes, while most matrix interferences are retained.
Weaker
.

4) Supercritical fluid extraction (SFE)

SFE has the advantages of high efficiency, speed, and solvent saving, but it needs to be implemented in a special instrument
.

5) Accelerated solvent extraction (ASE)

An Qiang et al.
established a method for the rapid determination of NFs metabolites in animal-derived foods using ASE-HPLC
.
Weigh 10.
0 g of the mashed sample, mix it with 20 g of diatomaceous earth, and fill it into a 33 mL extraction cell.
The extraction cell is placed in the heating furnace cavity of the ASE instrument, and the extraction process is completed under the set extraction conditions
.
The author optimized the extraction solvent, extraction temperature, extraction pressure, extraction time and other conditions
.
The best extraction solvent is methanol-trichloroacetic acid (0.
68mol/L) with a volume ratio of 1:1, the best extraction temperature is 100℃, the best extraction pressure is 1.
0×10 ⁷ Pa, and the best extraction time is 10min×3 times
.
After the extract was cooled and filtered, it was derivatized with o-chlorobenzaldehyde, adjusted to pH 7.
0 with NaOH, LLP with ethyl acetate, purified by a C ₁₈ -CN mixed SPE column, and detected by HPLC-UVD
.
The method LOD (S/N=3) is: SEM 0.
005μg/mL, AHD 0.
005μg/mL, AMOZ 0.
005μg/mL, AOZ 0.
005μg/mL; the recovery rate when adding a concentration of 5.
0mg/kg is: SEM88.
6%±3.
8%, AHD82.
9%±4.
9%, AMOZ89.
3%±3.
9%, AOZ93.
7%±3.
8%
.
Tao et al.
established a method for the determination of four NFs metabolites in carp and rice field eel using ASE and ultrasonic accelerated derivation technology
.
ASE was used to extract the analytes, the derivatization reaction was performed in ultrasound for 1 h, and then SPE was used for purification
.
The CCa of the method is between 0.
07～0.
13μg/kg, the CCβ is between 0.
31～0.
49μg/kg, and the method recovery rate is 77.
2%～97.
4%
.
Compared with the traditional method, this method greatly shortens the pre-processing time
.

6) Solid-supported-liquid-liquidextraction (solid-supported-liquid-liquidextraction, SSLLE)

Zhu Weixia et al.
used diatomaceous earth SSLLE and parallel evaporation combined pretreatment technology for purification and enrichment, and established furazolidone metabolites (AOZ), furantoin metabolites (AMOZ), and nitrofurantoin metabolites (AHD) in chicken, honey, and milk.
) And nitrofural metabolites (SEM) ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) confirmation method
.
After derivatization of the NFs metabolites, the ultra-efficient 1.
7μm C ₁₈ column was used to separate 4 kinds of analytes, and they were quantified by the isotope internal standard method
.
The minimum detection limit of AOZ and AMOZ is 0.
1μg/kg, AHD and SEM are 0.
25μg/kg; the recovery rate of this method is 85.
6%-104.
3% at 3 different addition levels, and the RSD is 3.
2%-95%
.
The equilibration time of the sample solution in the column is an important parameter that affects the recovery rate.
The study separately measured the effects of different equilibration times on the extraction efficiency.
The results show that the solution can form a firm support with the diatomite filler at 30 minutes
.
At the same time, the extraction efficiency of different volumes of ethyl acetate was optimized.
The recovery rate of 20 mL of ethyl acetate was 85%.
40 mL of ethyl acetate can completely extract 4 NFs metabolites in the solid phase extraction column under the action of gravity
.

7) Matrix solid-phase dispersion (MSPD)

Cao Wenqing et al.
used MSPD technology, purified by LLP, and quantified isotope internal standards to establish an LC-MS/MS method for the determination of NFs metabolite residues in egg yolk powder, such as furantrone, nitrofurazone, nitrofurantoin, and furazolidone
.
Use diatomaceous earth as the matrix dispersant to disperse the egg yolk powder, the trichloroacetic acid solution precipitates the protein and provides a hydrolysis environment, 2-nitrobenzaldehyde derivatization, ethyl acetate LLP and other pretreatment methods to metabolize the NFs in the egg yolk powder The product was extracted and purified
.
The LOQ of this method is 0.
5μg/kg, the linear range is 0.
5～6.
0μg/kg, the indoor verification recovery rate is 90.
06%～109.
8%, and the RSD is 2.
0%～7.
7%
.
This method is suitable for the monitoring and detection of NFs metabolites in egg yolk powder matrix by residue testing laboratories
.