Determination of 16 sulfonamide residues in milk and milk powder. Liquid chromatography-tandem mass spectrometry (3)

2.
2.
5.
7 Selection of analysis conditions

(1) Optimization of extraction and purification methods

Because the structure of trimethoprim and other sulfonamides are different, and they have different pK and solvent affinity, it is difficult to extract simultaneously in common organic solvents
.

Taking into account the subsequent purification process, 0.
33mol/L perchloric acid and water are mixed in three different ratios (1+4, 2+3, 3+2), and 16 substances to be tested are added to 5mL solution.
After passing the MCX column, eluting with 5mL water, 5mL methanol , and eluting with 5mL 25% ammonia water methanol, after concentration, instrumental analysis revealed that the recovery rate of 16 substances was above 95% by simply adding reagents
.

In the elution process, make an elution curve, it can be known that the first 2 mL of methanol will elute all the analytes, so 3 mL of methanol is used for elution
.

(2) Establishment of chromatographic conditions

In the experiment, 0.
1% acetic acid-methanol was used as the mobile phase, and time gradient elution was used.
The components to be tested were separated on a C ₁₈ column of 150mm×2.
1mmi.
d, particle size 5um, and the analytes were separated to the greatest extent.
The peak symmetry and sharpness, coupled with the high selectivity of mass spectrometry, can qualitatively quantify 16 analytes through MRM chromatograms, and no endogenous interferences affect the determination of components
.

(3) Optimization of mass spectrometry conditions

A peristaltic pump was used to inject 1ug/mL of 16 sulfonamide mixed standard solutions at 10uL/min to determine the best mass spectrometry conditions for each compound, including selection of characteristic ion pairs, optimization of electrospray voltage, sheath gas, auxiliary gas, collision energy and other mass spectrometry analysis Condition
.

2.
2.
5.
8 Linearity range and lower limit of determination

According to the sensitivity of each sulfa drug, a series of matrix standard working solutions were prepared with blank solutions of milk and milk powder samples, and measured under selected chromatographic conditions and mass spectrometry conditions.
The injection volume was 10 μL, and the peak area was used to work with the matrix standard.
The concentration of the tested component in the solution is plotted.
The linear range, linear equation and linear correlation coefficient are shown in Table 2-26
.

Table 2-26 Linear ranges, linear equations and correlation coefficients of 16 sulfonamides

It can be seen that under the operating conditions of this method, sulfacetamide, sulfamethazine, sulfapyridine, sulfamethoxidazine, sulfa-p-methoxypyrimidine, sulfachloropyridazine, sulfamethoxazole, sulfamethoxazole 16 kinds of oxygen pyrimidine, sulfa-6-methoxypyrimidine, sulfamethazine, sulfaquinoxaline, sulfadiazine, sulfathiazole, sulfamethizole, trimethoprim and sulfamethoxazole Sulfonamide is 1-50ng/mL (corresponding to the sulfa drug residue in milk in the range of 0.
5-25.
0μg/kg, corresponding to the sulfonamide drug residue in the milk powder in the range of 2.
0-100.
0ug/kg), the response values ​​are all within the linear range of the instrument Within
.

Related Links: Determination of 16 Sulfonamide Residues in Milk and Milk Powder Liquid Chromatography-Tandem Mass Spectrometry (2)