Method for determination of avermectin drug residues (2)

(3) High performance liquid chromatography (HPLC)

AVMs are low-polarity compounds, and the use of reversed-phase-high performance liquid chromatography (RP-HPLC) can obtain better selectivity
.

There are many reports on the use of HPLC in the analysis of AVMs residues, mainly using ultraviolet detector (UVD), diode array detector (PDADAD) and fluorescence detector (FLD)

1) Ultraviolet detector (UVD)

The conjugated diene structure of AVMs has strong ultraviolet absorption at 240～250nm.
Using this feature, ultraviolet detection can be established, but there are many endogenous substances such as corticosteroids, vitamins, lipids, nucleic acids, etc.
in this spectral region , Will seriously interfere with the detection
.

In addition, the minimum effective concentration of AVMs in the body is very low.

Cao Hong et al.
established an HPLC-UVD method for the determination of AVM residues in sheep plasma
.

The samples were extracted with ethyl acetate and directly analyzed by HPLC

2) Diode array detector (PDA/DAD)

The Diode Array Detector (PDADAD) can realize instantaneous full-spectrum scanning of the effluent chromatographic peak, and at the same time obtain information on the retention value, purity and absorption spectrum of the chromatographic peak
.

Since AVMs residue confirmation methods are generally more complicated, PDADAD can be an option when the drug residue level in the sample is high

3) Fluorescence detector (FLD)

Since AVMs have no symmetrical conjugated structure, they cannot be directly detected by FLD.
Only after derivatization can generate a symmetrically conjugated benzene ring structure can they emit fluorescence.
Therefore, it is very important to choose appropriate fluorescence derivatization reagents and reaction conditions
.

The fluorescence derivatization of AVMs generally follows the mechanism of acylation, dehydration and then aromatic ring formation (Figure 21-1), but the instability of fluorescent derivatives remains to be resolved

Figure 21-1 The fluorescence derivation mechanism of AVM

Tolan et al.
established an analysis method for IVM in plasma
.

The dried extract uses pyridine as a catalyst, acetic anhydride as a dehydrating agent, and reacts at 105-110°C for 22-24 hours.

Payne et al.
used HPLC-FLD to determine EPR in cattle tissues
.

Because the fluorescence derivatization reagent of AVMs is not stable, it will lose 50% within 2h.

Rupp et al.
determined the IVM and DOR in the muscle tissue of Atlantic salmon.
The extract was purified with C8 and silica gel column, and then dehydrated with TFAA and N-methylimidazole to derive the fluorescent product, which was then dissolved and converted with ammonium acetate-methanol solution.
A stable alcohol form is formed, and the reaction takes 15 min at 50-55°C
.

Then use HPLC-FLD for detection, the chromatographic column is a Hypersil C ₁₈ column, the mobile phase is acetonitrile-water (90+10, v/v), the column temperature is 65°C, the excitation wavelength is 272nm, and the emission wavelength is 465nm

When Ali et al.
used HPLC-FLD to determine the residues of AVM, IVM, DOR, EPR, and MOX in cattle liver, the derivatization yields of AVM, IVM, DOR and MOX can reach the peak instantaneously, while the yield of EPR takes 7h to reach Peak
.
When heated to 65°C, the EPR reaction speed is fast, and the yield is the highest at 90 minutes, and the yield of other drugs also increases by 10% to 15%
.
The method recovery rate is greater than 70%, and the CV is less than 20%
.
Hou Xiaolin and others used HPLC separation and FLD detection to establish a SPE method for analyzing EPR, AVM, DOR and IVM residues in cattle tissues
.
The sample was extracted with acetonitrile, purified with basic alumina and C18 column, and acetonitrile solution of acetic anhydride and 1-methylimidazole was used as the derivatization reagent.
At 96℃, the complete derivatization required 100min
.
The average recovery rate of the four drugs was 70.
02%-88.
75%, the intraday CV was less than 8.
52%, and the intraday CV was less than 7.
13%
.
The LOD of EPR, AVM, DOR and IVM is 0.
4～0.
5μg/kg, and the LOQ is 2ug/kg
.
Roudaut et al.
reported the HPLC method for detecting AVM, IVM, DOR and MOX in liver tissue
.
The sample was extracted with acetonitrile, cleaned with a C18 column, derivatized with TFAA, and detected by HPLC-FLD.
The excitation wavelength was 361nm and the emission wavelength was 465nm
.
The method LOQ meets the EU MRLs requirements, the recovery rate is between 77.
5% and 90.
8%, and the RSD is between 2.
7% and 7.
7%
.
Hou et al.
detected EPR, AVM, DOR and IVM residues in beef liver and beef
.
The sample was extracted with acetonitrile, cleaned up with C18 column, and after derivatization reaction, it was measured by HPLC-FLD
.
The recovery rate in beef liver is 70.
31%～87.
11%, beef is 79.
57%～93.
65%, RSD is less than 17.
84% and 14.
68% respectively; method LOD is 0.
5～1.
0ng/g, LOQ is 1～2ng/g
.