Selection of analysis conditions for determination of zearalenol, zearalenone, diethylstilbestrol, hexestrol, and diethylstilbestrol in liver, kidney and muscle tissues of bovine pigs

9.
2.
1.
7 Selection of analysis conditions

(1) Extraction of the test substance

This method tested the extraction with ethyl acetate and tert-butyl methyl ether (MTBE), and the results are shown in Table 9-5
.
Can be seen from Table 9-5 ether and MTBE extract zeranol efficiency and corn gibberellic one equivalent for the stilbene hormone MTBE as an extraction solvent recovery is better, and ultimately the selection MTBE as an extraction solvent

Table 9-5 The influence of different solvent extraction on recovery rate

Zearalenol and stilbene hormones in animal-derived samples exist in two forms, namely the free state and the protein-bound state.
The addition of β-glucuronidase can hydrolyze the bound zearalenol and stilbene hormones Into a free state
.
In this experiment, the standard substance of diethylstilbestrol combined with monoglucuronic acid was added to the matrix to simulate and compare the recovery rate of diethylstilbestrol before and after enzymatic hydrolysis.

(2) Solid phase extraction purification

LC-MS/MS has high detection sensitivity, but the complex matrix background will inhibit the ionization efficiency of the target compound, greatly reduce the detection sensitivity of the measured component, and the degree of sample purification also has a great impact on the reproducibility of the results
.
This method uses a silica gel column for SPE purification and compared different grades of silica gel columns.

The five types of estrogen and metabolites detected by this method require low limits and complex substrates.
Therefore, the application of automatic solid phase extraction (AS PE) is considered to reduce human error and improve reproducibility
.
The parameters of eluent ratio, eluent ratio, and eluent dosage are optimized, and the results are shown in Table 9-6 to Table 9-8

Table 9-6 The influence of the eluent ratio on the recovery rate (the amount of eluent is 6mL)

Table 9-7 The effect of eluent ratio on recovery rate (first elution with ethyl acetate-n-hexane [6+94], then elution)

Table 9-8 The influence of the amount of eluent on the recovery rate (three times elution, 3mL each time)

The targets tested in the experiment are two types, zearalenol and its metabolites, and stilbene hormones
.
There are differences in the properties of the two types of substances, so it is necessary to take into account the two types of substances when determining the final experimental conditions

(3) Optimization of mass spectrometry conditions

A syringe pump was used for direct sample injection, and standard solutions of zearalenol, zearalenone, diethylstilbestrol, diethylstilbestrol, and diethylstilbestrol were injected into the ion source of the tandem mass spectrometer at 5 μL/min
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The primary mass spectrometry analysis (Q1, precursor ion scan) compares the positive and negative methods of electrospray ionization (ESI).

Figure 9-3 The secondary mass spectra of five compounds

The collision gas energy (CE) is optimized for the selected product ions for each analyte
.
After connecting the liquid chromatograph, I tried to use the atmospheric pressure chemical ionization (APCI) negative method for measurement, and found that the APCI(-) ionization method had higher detection sensitivity

(4) Optimization of HPLC conditions

This method has tried several C18 and C8 columns of Waters, Agilent, Merck and other brands.
After comparing Agilent’s ZORBAX SB C8 (150mm×4.
6mm, 3.
5μm) on zearalenol and stilbene hormones The analytical column efficiency is better
.

When using APCI ionization, acetonitrile is the preferred mobile phase with a flow rate of 1 mL/min
.
Under this premise, different flow ratios and gradient programs were tested