Determination method of non-steroidal anabolic hormone drug residues (2)

(5) High performance liquid chromatography (HPLC)

HPLC is an internationally recognized method for residue analysis, with strong selectivity and high sensitivity
.

However, because there are many sample pre-processing steps, cumbersome operation and long analysis time, it is not suitable for rapid screening of residue detection

1) UVD, DAD/PDA

UVD is a commonly used detector in HPLC methods
.

The molecular structure of non-steroidal anabolic hormones contains a long conjugated system composed of phenolic hydroxyl group and carbon-carbon double bond, ketone group and phenolic hydroxyl group, which can be dissolved in dilute alkali solution and has strong UV absorption at 240-300nm

Liu Hongcheng and others established an HPLC-UVD method for the analysis of DES, HES and DIS in milk
.

Use MSPD technology to extract and purify, use 20mmol/L phosphoric acid- acetonitrile (42+58, v/v) as mobile phase, and separate on Xterra C18 (250mm×4.

Zhang Qingjie and others used HPLC-UVD method to detect ZER, TAL, ZAN, ZON, ax-ZOL and β-ZOL in milk and milk powder
.

The sample was extracted with acetonitrile, purified by IAC column, and detected by HPLC-UVD

Koole et al.
reported the simultaneous determination of 20 anabolic hormones (including stilbenes and RALs) in cow urine using HPLC-DAD
.

The RP-Select B chromatographic column was used for separation, with acetonitrile-water as the mobile phase and gradient elution.

2) FLD

FLD has strong selectivity and high sensitivity, and is a detector often used in residue analysis
.

Stilbenes such as DES need to be derivatized before they can be detected by FLD, and there are few reports in the literature.

Chen et al.
used 4-(4,5-diphenyl-1Himidazol-2-yl)benzoyl chloride (DIB-Cl) as a derivatization reagent to establish an HPLC-FLD detection method for DES in urine
.

Add 0.

Shin et al.
established an HPLC-FLD method to detect ZON in mouse serum
.

Serum samples were extracted with tert-butyl methyl ether and analyzed by HPLC-FLD.

3) ELSD

ELSD is a general-purpose detector that can detect any sample with a lower volatility than the mobile phase without the need for a chromophore
.
The response value of ELSD is directly proportional to the quality of the sample, so it can be used to determine the purity of the sample or detect unknowns
.
ELSD has higher sensitivity than refractive index detectors, is insensitive to temperature changes, has a stable baseline, and is suitable for HPLC gradient elution
.

Jiang Yong et al.
established a HPLC-ELSD analysis method for the detection of DES and other drugs in meat products
.
Weigh 5g of the sample into a cork with a stopper, add 10.
0 mL of methanol, sonicate for 10 min, take the supernatant, centrifuge and analyze it by HPLC-ELSD
.
The conditions of HPLC and ELSD are determined by experiment: Chromatographic column Diamonsil TM C ₁₈ ODS (250mm×4.
6mm.
id, 5um), mobile phase is 5% acetic acid and acetonitrile, gradient elution, flow rate 1.
0mL/min, injection volume 10μL , Column temperature is 30℃; ELSD gain value is 7, temperature is 40℃, carrier gas is air, pressure is 3.
4bar
.
Calculated with a signal-to-noise ratio (S/N) of 3, the LOD of this method DES is 0.
0058mg/kg, the recovery rate is 85.
2%, and the CV is 3.
18%
.

4) ECD

ECD is the measurement of changes in the electrical signal of a substance
.
Compounds with redox properties, such as organic compounds containing nitro and amino groups, as well as inorganic anions and cations, can be detected by ECD
.
ECD also includes polarography, coulomb, amperometric and conductivity detectors.
The first three are collectively called voltammetric detectors, which are used for the detection of compounds with redox properties, while conductivity detectors are mainly used for ion detection
.

Reuvers et al.
used HPLC-ECD to detect DES residues in edible animal tissues
.
The tissue samples were first extracted with tert-butyl methyl ether, and then extracted again with 1mol/L sodium hydroxide, purified with a C18 solid phase extraction column, with methanol-0.
05mol/L phosphate buffer (pH3.
5) (67+33, v/v) As the mobile phase, it was separated on a Nucleosil C ₁₈ chromatographic column, and the ECD was detected at +0.
90V
.
The LOD of this method is 0.
5～2.
0μg/kg, and the recovery rate is 66%
.
Wan Meimei and others established an HPLC-ECD detection method for DES in animal tissues
.
Chromatographic column is Hypersil ODS C18 column (5um, 4.
6mm×250mm), mobile phase is acetonitrile-0.
007mol/L ammonium dihydrogen phosphate (48+52, v/v, pH3.
5), column temperature is 32℃, flow rate It is 1.
0mL/min
.
ECD parameter setting: mode is pretreat, working potential is 0.
9V, pretreatment control is set to stop, zero point control is set to preparation, post-time is 30min, pre-treatment cycles 8 times, pre-treatment potential 1 is 1.
4V, pre-treatment The potential 2 is -0.
4V, the pretreatment time 1 is 200ms, the pretreatment time 2 is 300ms, the pretreatment time 3 is 500ms, the peak width is 0.
10min, the electrode is an oxidation electrode, and the instrument sensitivity is 0.
5μA
.
The DES concentration has a good linear relationship in the range of 0.
2-100ng/g, and the correlation coefficient is 0.
9995; the average recovery rate of different additive concentrations in each tissue is 80%, the intraday CV is less than 3.
60%, the intraday CV is less than 3.
72%, and the LOD is 0.
2 ng/g
.