Selection and optimization of analytical conditions for determination of nitrofuran metabolite residues in foods of animal origin

7.
2.
1.
7 Selection and optimization of analysis conditions

(1) Selection of derivatization conditions

This method uses two derivatization agents, 2-NBA and 4-NBA, to compare the derivatization effects of AOZ, AMOZ, SEM and AHD residues in positive samples.
It is found that 2-NBA is more sensitive than 4-NBA
.
Therefore, 2-NBA was chosen as the derivatizing agent

Table 7-6 The influence of the amount of derivatizing agent on the measurement result (response signal peak area)

It can be seen from Table 7-5 that the amount of derivatizing agent added is less than 1.
0mg, which may cause incomplete derivatization of the test substance.
If the amount is more than 1.
0mg, there will be crystals of derivatizing agent when drying with nitrogen, which is not easy to be The mobile phase dissolves and interferes with the measurement
.
Choose the amount of derivatizing agent to be 1.

Table 7-7 Response signal (peak area) of four nitrofuran metabolites at different pH

(2) Selection of purification conditions

Two sample purification methods were tested.
One was to adjust the pH of the derivatized solution and extract it twice with ethyl acetate, centrifuge and dry the ethyl acetate layer with nitrogen to a constant volume
.
This method has better extraction efficiency, but the operation steps are cumbersome, and the ethyl acetate layer cannot be completely taken out

Table 7-8 Response signals (peak areas) of four nitrofuran metabolites in different extraction columns

(3) Selection of liquid chromatography conditions

1) Pork, beef, chicken, pork liver, fish, shrimp, crab and shellfish samples

Compare Pinnacle II C ₁₈ , 5um, 150mm×2.
1mm; Inertsil ODS-3, 5um, 150mm×2.
1mm, ZORBAX SB-C ₁₈ , 3.
5μm, 150mm×2.
1mm and Atlantis-C ₁₈ , 3μm, 150mm×2.
1 The separation effect of the four liquid phase analytical columns of mm on the four nitrofuran metabolites, the results found that the Atlantis-C ₁₈ analytical column has ideal sensitivity and resolution
.
Therefore, this column was selected as the analytical column

In order to optimize the mobile phase, the chromatographic effects of methanol + acetic acid aqueous solution and acetonitrile + acetic acid aqueous solution as the mobile phase were compared.
Experiments show that when acetonitrile + 0.
3% acetic acid aqueous solution is used as the mobile phase and gradient elution is adopted, the derivatives of the four metabolites are separated Degree and sensitivity are high
.
See Table 7-2 for mobile phase gradient elution conditions

2) Milk or milk powder sample

Compare Inertsil ODS-3, 5um, 150mm×2.
1mm, ZORBAXSB-C ₁₈ , 3.
5um , 150mm×2.
1mm and Atlantis-C ₁₈ , 3um, 150mm×2.
1mm three liquid phase analysis columns to four nitrofurans The separation effect of metabolites, the results found that the Atlantis-C ₁₈ analytical column, sensitivity and resolution are ideal
.
Therefore, this column was selected as the analytical column

In order to optimize the mobile phase, the chromatographic effects of methanol + formic acid aqueous solution and acetonitrile + formic acid aqueous solution as mobile phase were compared.
Experiments show that when acetonitrile + 0.
1% acetic acid aqueous solution is used as the mobile phase and gradient elution is adopted, the derivatives of the four metabolites are separated Degree and sensitivity are high
.
See Table 7-3 for mobile phase gradient elution conditions

3) Honey sample

Compare Pinnacle II C ₁₈ , 5um, 150mm×2.
1mm; Hypersil C ₁₈ 5um, 150mm×4.
6mm; Inertsil ODS-3, 5μm, 150mm×2.
1mm, ZORBAXSB-C ₁₈ , 3.
5um , 150mm×2.
1mm, four The separation effect of a liquid phase analysis column on four nitrofuran metabolites, the results found that the ZORBAXSB-C ₁₈ , 3.
5um , 150mm×2.
1mm analytical column, the sensitivity and resolution are ideal
.
Therefore, this column was selected as the analytical column

In order to optimize the mobile phase, the chromatographic effects of methanol + ammonium acetate aqueous solution and acetonitrile + acetic acid aqueous solution as the mobile phase were compared.
Experiments show that when acetonitrile + 0.
4% acetic acid aqueous solution is used as the mobile phase and gradient elution is adopted, the derivatives of the four metabolites The resolution and sensitivity are high
.
See Table 7-4 for mobile phase gradient elution conditions
.

(4) Selection of mass spectrometry conditions

Take 20 μg/mL standard solutions of four nitrofuran metabolites and derivatize them with 2-NBA to obtain 2-NBA-AOZ, 2-NBA-AM0Z, 2-NBA-SEM, 2-NBA-AHD standard derivatization solutions, respectively
.
The derivatives of the four metabolite standard solutions were injected into the ion source at a flow rate of 5 μL/min
.
Under the positive ion detection mode, the first-stage mass spectrometry analysis (Q1 scan) was performed to obtain the molecular ion peaks
.
Then perform the secondary mass spectrometry analysis (product ion scan) on the molecular ions to obtain the secondary mass spectrum, as shown in Figure 7-4
.
Then optimize the parameters such as declustering voltage (DP), focusing voltage (FP), collision gas energy (CE) and collision cell exit voltage (CXP) to maximize the molecular ion and characteristic fragment ion intensity, and determine the best mass spectrum Parameters
.
The liquid chromatography and mass spectrometry are connected online to further optimize the parameters such as the atomizing gas flow rate, the temperature of the atomizing chamber, and the position of the spray needle, so as to achieve the best various parameters
.

Figure 7-4 The secondary mass spectra of four nitrofuran metabolites

(5) Selection of quantitative methods

Two quantitative methods, the external standard method and the internal standard method, were tested
.
When the standard solution and sample solution have the same matrix, there is little difference between the two
.
When the matrix of the standard solution and the sample solution are different, the ionization efficiency of the nitrofuran metabolite derivatives is different, and the internal standard method is more accurate for quantification
.

Related Links: Determination of Nitrofuran Metabolite Residues in Food of Animal Origin