Tetracycline residue analysis technology-pretreatment method (2)

(2) Purification method

At present, the sample purification methods for TCs residues in biological samples at home and abroad mainly include solid phase extraction (SPE), magnetic solid phase extraction (MSPE), solid phase microextraction (SPME), matrix solid phase dispersion (MSPD), molecular imprinting technology ( MIT), limited access medium (RAM), metal chelating affinity chromatography (MCAC) and other technologies
.

1) Solid phase extraction (solidphase extraction, SPE)

SPE does not require a large amount of organic solvents, does not produce emulsification, can purify a small volume of samples, and is widely used to separate and purify TCs in samples
.
SPE columns usually choose HLB, C ₁₈ , Strata-X purification based on reversed-phase retention mechanism , and SAX, MAX based on anion exchange mechanism , etc.

, and can also be combined with columns to improve the purification effect

Peres et al.
used a C ₁₈ SPE column to purify OTC, TC and CTC in honey
.
Add 15mL 0.

1mol/L Na2EDTA-Mcllvaine buffer solution (pH4.

Pang Guofang et al.
compared the effects of single solid phase extraction column purification (OasisHLB) and double column purification (HLB and Carboxylic acid anion exchange columns) of OTC, TC, CTC, and DC in poultry meat
.
The poultry meat sample was extracted with 0.

1mol/L Na2EDTA-Mcllvaine buffer solution (pH4), and the extract was passed through the HLB solid phase extraction column at a flow rate of no more than 3mL/min, washed with 5mL methanol-water (5+95, v/v), Drain under reduced pressure for 20 min, eluted with 15 mL of ethyl acetate, and pass the eluent through the Car-boxylicacid anion exchange column at a flow rate of no more than 3 mL/min under reduced pressure.

2) Magnetic solid phase extraction (MSPE)

MSPE is a solid phase extraction technology that uses magnetic or magnetizable materials as the adsorbent matrix
.
In the MSPE process, the magnetic adsorbent is not directly filled into the adsorption column, but is added to the sample solution or suspension to adsorb the target analyte on the surface of the dispersed magnetic adsorbent, which can be made under the action of an external magnetic field The target analyte is separated from the sample matrix

.

Rodriguez et al.
used the MSPE method to purify the CTC, TC, and OTC in the milk.
The milk sample was directly purified by MSPE and eluted with acidified methanol
.
Add 0.

3g of silanized magnetic SPE filler to 50mL of milk, stir for 20min for uniform dispersion, let stand for 5min, separate the silanized magnetic filler with a magnet, wash with 10mL of EDTA (1mmol/L), 3×1.

3) Solid phase microextraction (SPME)

The selectivity of SPME is based on the principle of "similar compatibility", combined with the polarity, boiling point and distribution coefficient of the test substance, and achieved by selecting fiber extraction heads with different coating materials
.
SPME-HPLC technology has achieved great development in both coating materials and operating methods.

In terms of operating methods, in addition to the initial manual SPME-HPLC continued to develop, in-tube SPME-HPLC (in-tube-SPME-HPLC ) And other newly developed operating methods have also made rapid development

Wen et al.
used a biocompatible polymer monolithic capillary column as the adsorption medium, and used in-line SPME-HPLC to analyze and detect CTC, TC, OTC and DC in fish meat
.
Take 1.

0g crucian carp muscle sample, add 10mL 0.

4) Metal chelate affinity chromatographic (MCAC)

MCAC is to treat the adsorbent material with CuSO4 solution, and the extract containing TCs is purified by MCAC.
The TCs are integrated with Cu bound on the agar gel column to be exclusively adsorbed on the column packing, and then EDTA-Mcllvaine buffer is used.
Elution, to achieve the purpose of separation from other impurities
.

Stubbings et al.
used online MCAC-HPLC to detect TCs residues in animal tissues
.
25uL CuSO ₄ solution (50g/L) and 500μL pure water activate the MCAC column, the extract is loaded at a flow rate of 0.

36mL/min, 500uL pure water, 500μL methanol, 500μL pure water are washed separately, KH ₂ PO ₄ -EDTA-Mcllvaine buffer Solution elution, KH ₂ PO ₄ -EDTA-Mcllvaine buffer-methanol-acetonitrile and KH ₂ PO-EDTA-Mllvaine buffer were respectively equilibrated to regenerate the MCAC column

5) Matrix solid phase dispersion (MSPD)

MSPD mixes and grinds the sample with adsorbents such as C ₁₈ , alumina, etc.
, so that the sample is evenly dispersed on the surface of the stationary phase particles, made into a semi-solid state and packed into a column, and then a suitable eluent is selected for elution
.
The MSPD technology condenses the homogenization, extraction, and purification processes in the traditional sample pretreatment, avoids the loss of the analyte in these processes, and has the advantages of high extraction and purification efficiency, solvent savings, and low sample consumption
.

Zhang Suxia and others established a method for the extraction and purification of MSPD of CTC, TC, OTC and DC residues in milk
.
Weigh 22g of _C18 filler, put it into a 50mL glass syringe, wash with two column volumes of n-hexane, dichloromethane and methanol, and vacuum dry; weigh 2g of dried C18 filler and place it in a glass mortar, respectively Add 0.
05g Na ₂ EDTA and oxalic acid, add 0.
5mL milk sample to the C18 filler, gently grind with a glass pestle for 30s to mix the sample and the filler evenly, take a 5mL glass syringe, pad a piece of filter paper at the bottom, and add anhydrous sodium sulfate To the volume of 0.
2mL, load the sample, put a piece of filter paper on it, press down to the volume of 4.
2mL; wash with 8mL n-hexane, vacuum dry, and elute with ethyl acetate-acetonitrile (1+3, v/v) Add 0.
1 mL of ethylene glycol in a chicken heart bottle, remove the solvent with a rotary evaporator, and dissolve the residue with 0.
4 mL of methanol-acetonitrile-0.
01mol/L oxalic acid (2+3+5, v/v), vortex and mix for 15s , Transfer to a glass centrifuge tube, centrifuge at 2000g for 10 min; take the supernatant for injection, HPLC determination
.
The average recovery of the method is 78.
7%-90.
2%, the CV is between 2.
4% and 1.
18%, and the LOD is 0.
02-0.
05μg/mL
.
Mu, respectively, using magnesium silicate (Florisil), and C18 silica gel as adsorbent MSPD established milk Residues TC, OTC and DC
.
Mix the adsorbent and the milk sample in a 4:1 ratio and put it into the syringe, wash with 6mL n-hexane for degreasing, 6mL 0.
1mol/L citric acid aqueous solution-methanol (1+9, v/v) elution, and the eluent is blown with nitrogen.
Dry, reconstituted with 0.
5mL methanol, filtered with 0.
45um filter membrane, CE analysis
.
The results showed that the recovery rate of C18 was the highest, the average recovery rate was 93.
4%～102%, and the LOD was 0.
0745～0.
0808μg/mL
.

6) Molecular imprinting technology (MIT)

MIT is a polymer bionic material artificially synthesized by chemical means
.
In the presence of the target molecule (template molecule), it is cross-linked and polymerized, and then the template molecule is eluted and removed.
It has a complementary structure to the template molecule in the three-dimensional cavity and the action site, and has the advantage of recyclable and reused template molecules.
There is a special selectivity in molecular recognition
.

Li Qian et al.
used the method of precipitation polymerization to synthesize MIPs that specifically recognize TC, OTC, and CTC using DC as a template molecule
.
Mix 1mmoL doxycycline hydrochloride, 60mL acetonitrile, 8mmoL methacrylic acid (MAA), 16mmoL trimethylolpropane trimethacrylate and 45mg azobisisobutyronitrile (ABIN).
After deoxygenating with nitrogen, 60℃ magnetic force Stir the reaction for 24 hours to obtain MIPs
.
The verification of recognition performance and physical characteristics showed that the MIPs had a specific adsorption effect on TCs and had good stability
.
The SPE cartridge was prepared with the MIPs as the filler, and the purification conditions were: 5mL methanol activation, 10mL aqueous solution loading, 3mL 5% formic acid water solvent elution, and 6mL 1% formic acid-methanol solution for elution
.
The maximum sample load of the cartridge is 0.
2mg, the average recovery is 72.
31%-90.
85%, and the CV is less than 10%
.
Sun et al.
used TC as the template, MAA as the functional monomer, ethylene glycol dimethacrylate (EGDMA) as the cross-linking agent, methanol as the solvent, and cyclohexanol and dodecanol as the mixed porogenic solvent, and used in-situ for the first time MIT technology prepared the TC imprinted monolithic column, and optimized and evaluated the synthesis conditions and performance
.
The monolithic column is connected with the C18 SPE column and is used for the purification of TCs in milk and honey.
The method recovery rate is between 73.
3%～90.
6% (milk) and 62.
6%～82.
3% (honey)
.
Hu et al.
used TC as a template and acrylamide as a functional monomer to synthesize MIPs through trimethylolpropane trimethacrylate, coated with SPME fiber, and used online HPLC to detect trace amounts of TC, OTC, DC, and CTC in chicken and milk.

.
The recovery rates of the methods all exceeded 71.
6%
.
The experiment was compared with the recovery rate obtained with ordinary coating, and the results showed that the extraction effect of MIPs coating on TCs was satisfactory
.

7) Restricted access materials (RAM)

RAM, also known as restricted access stationary phase, is a new type of online sample pretreatment filler, which has both separation and enrichment functions.
By controlling the pore size of the filler and modifying its outer surface hydrophilically, it makes the outer surface hydrophilic.
There is no irreversible denaturation and adsorption with the macromolecular substances in the biological sample, and then it is eluted and removed in the dead volume or close to the dead volume
.
It has the advantages of avoiding the loss of the analyte due to pre-processing, reducing the tedious steps in traditional biological sample preparation, saving manpower and material resources, and being easy to realize automation
.
Chico et al.
bonded the diol group to the outer surface of the porous silica gel pores, and the Cs alkyl chain was bonded to the inner surface as a restrictive medium material.
On-line RAM-HPLC was used to detect TCs in milk
.
Using Mg(NO ₃ ) ₂ ·6H ₂ O as the post-column derivatization reagent, the measured CV of milk is 6% to 9.
2%; the method improves the detectability of the analyte, but the recovery rate of the measured OTC and TC is only 50% and 67%
.

Related links: Tetracycline residue analysis technology-pretreatment method (1)