Non-steroidal anabolic hormone drug residue analysis pretreatment method-purification (1)

(3) Purification method

The classic purification treatment methods mainly include liquid-liquid distribution (LLP).
With the development of science and technology and the continuous improvement of veterinary drug residue detection requirements, various new technologies and methods have also been used as non-steroidal anabolic hormones in biological samples.
sample processing method comprising solid phase extraction (the SPE), molecular imprinting (the MIT), immunoaffinity chromatography (IAC), solid phase microextraction (the SPME), matrix solid phase dispersion (MSPD) and the like
.

1) Liquid liquid partition (LLP)

LLP is a purification method that uses the difference in partition coefficients of two immiscible solvents (solvent pairs) in the sample to separate the analyte and the interfering substance.
It usually uses a polar solvent that is compatible with water and A non-polar solvent that is not compatible with water is paired for distribution.
After repeated distributions, the analyte is separated from impurities
.

LLP is the most commonly used purification method, and is often used in combination with other methods for degreasing the extract

Shin et al.
used the LLP method to extract and purify ZON in mouse serum
.

Add tert-butyl methyl ether to the rat serum, vortex to mix, centrifuge, dry the organic layer with nitrogen, add acetonitrile-0.

2) Solid phase extraction (SPE)

SPE uses an adsorbent to adsorb the target compound in the liquid sample to separate it from the sample matrix and interfering substances, and then eluate or heat to desorb the target compound by elution or heating
.

Compared with traditional LLP, SPE does not require a large amount of organic solvents, does not produce emulsification, and can purify small-volume samples.

A.
Stilbene

In the analysis method of stilbene compounds, silica gel column, HLB column, graphitized carbon black (GCB) column, amino column (NH2) and diatomaceous earth column are all used for the purification of stilbene in animal matrix.

.

Xu Hong et al.
used a silica gel SPE column to purify the residues of DES, HES, and DIS in food of animal origin
.

Pre-wash the silica gel column twice with 6 mL n-hexane at a flow rate of 4 mL/min; load the sample at a flow rate of 2 mL/min.

Wu Yinliang and others also used silica gel SPE columns to purify DES, DIS and HES in animal tissues
.

Extracted with ethyl acetate under alkaline conditions, the extract was passed through a silica gel SPE column (5mL n-hexane, 5mL ethyl acetate activation), followed by 2mL n-hexane, 2mL n-hexane-ethyl acetate (85+15, v/ v) Rinse with mixed solvent and drain it, eluting with 4mL n-hexane-ethyl acetate (80+20, v/v) mixed solvent, collect the eluate, and analyze by GC-MS

Xu Yingjiang and others used HLB column to purify DES in grass carp blood, muscle and liver
.

It was extracted with 10% sodium carbonate and ethyl acetate, and the extract was dissolved with 1 mL of methanol to dissolve the residue, then diluted with 9 mL of water, and passed through an SPE column

Ding Yayun et al.
used matrix solid phase dispersion (MSPD) to extract and purify DES in animal liver, and the eluate was purified by alkaline diatomaceous earth and silica gel SPE column
.

1.

Yang et al.
reported the residual analysis methods of 50 anabolic hormones (including DIS, HES and DES) in pork, beef, shrimp, milk and liver
.

The sample was cleaned up with a GCB series NH2SPE column

B.
RALs

Strong anion exchange columns (MAX), NH2 columns, etc.
are often used to purify RALs in animal matrices
.

Xia et al.
used MAX columns to purify ZER, TAL, ZAN.
ZON, a-ZOL and β-ZOL in milk
.
Add 5mL acetonitrile to 5mL milk, vortex for 30s, centrifuge at 9000r/min for 5min (4℃), take the supernatant, add 1.
0mL ammonia (2.
5mol/L) and 10mL water, mix well and pass through the MAX column (2mL acetonitrile) Equilibrate with 2mL water), wash with 2mL 5% ammonia water and 0.
5mL acetonitrile, vacuum dry for 2min, 3mL acetic acid-acetonitrile solution (2+98, v/v) elution, dry with nitrogen in a water bath at 50C, 0.
5mL water-acetonitrile (50 +50, v/v) reconstituted, after passing through 0.
22um microporous membrane, LC-MS/MS analysis
.
The average recovery rate of this method is 92.
6%～112.
5%, CV is lower than 11.
4%, LOD and LOQ are 0.
01～0.
05μg/L and 0.
05～0.
2μg/L, respectively
.
Qian Zhuozhen and others used NH ₂ column to purify ZER, TAL, ZAN, ZON, a-ZOL and B-ZOL in aquatic products
.
The sample was extracted with acetonitrile, the n-hexane was degreased, the lower acetonitrile solution was rotary evaporated to dryness, and the acetonitrile saturated n-hexane was dissolved for later use
.
Equilibrate the NH2 column with 5 mL ethyl acetate and 5 mL n-hexane
.
Take the extract through the column at a flow rate not exceeding 1 mL/min, and then wash with 5 mL n-hexane, 5 mL n-hexane-ethyl acetate (60+40, v/v), and 4 mL n-hexane-ethyl acetate (20 +80, v/v) and 4mL ethyl acetate elution
.
The eluent was blown dry under nitrogen at 50C, and the volume of 1 mL of acetonitrile-water solution (20+80, v/v) was constant.
After passing through a 0.
22μum microporous membrane, LC-MS/MS analysis
.
The LOQ of this method is 1.
0lg/kg, the recovery rate is 75.
9%-103.
8%, and the RSD is 3.
90%-13.
5%
.

C.
Simultaneous purification of stilbene and RALs

Stilbene compounds and RALs in animal-derived matrices can also be purified by SPE at the same time
.

Rubies et al.
established the SPE-LC-MS method to detect TAL, ZER, HES, DES and DIS in animal urine
.
After the urine is enzymatically digested, it is purified with an HLB solid phase extraction column and detected by LC-MS/MS
.
The recovery rate of this method is 71.
4%-106.
4%; CCa is 0.
2-0.
9ug/L, and CCβ is 0.
3-1.
0ug/L
.
Kathrin et al.
2 established a method to simultaneously purify stilbene compounds (DES, DIS, HES) and RALs (ZER, TAL, a-ZOL, β-ZOL and ZON) in cow urine using SPE
.
Add 2mL sodium acetate buffer (2mol/L, pH5.
2) to 5mL urine sample, add 100μL snail juice (Helixpomatiajuice), and place at 37°C in the dark for 16h or 50°C for 3h.
After hydrolysis is complete, use 2mol/L hydroxide Adjust the pH of the sodium solution to pH 9.
0+0.
1, extract twice with 5 mL of ether, add 1.
5 mL of methanol and 3 mL of water, wash twice with 2 mL of n-hexane, discard the n-hexane layer, and use 5 mL of methanol and 5 mL of water.
Equilibrated HLB column (6cm3, 200mg), washed with 5mL methanol and 5mL methanol-water (55+45, v/v), eluted with 5mL acetone, and the eluate is then passed through an aminopropyl column (500mg) equilibrated with 5mL methanol and 5mL acetone , 3mL).
After the effluent was evaporated to dryness, it was reconstituted with 100μL of acetonitrile-water (1+1, v/v) and analyzed by LC-MS/MS
.
The LODs of stilbene compounds and RALs detected by this method were lower than 1μg/L and 1.
5μg/L, respectively, and the recoveries were 99.
2%-106.
0% and 99.
4%-107.
9%, respectively
.