Selection of analytical methods for determination of residues of carbachol, olaquindox and metabolites in liver and muscle of cattle and pigs

15.
2.
1.
7 Choice of analysis method

(1) Sample processing method

1) Extraction method of carbachol

Carbachol has poor solubility in polar solvents such as water and acetone.
In the literature, methanol-water solution is usually selected as the tissue extract.
The solution inevitably contains a large amount of impurities such as water-soluble proteins and pigments.
These impurities are complex and have color.
Make the extract turbid, seriously affect the qualitative and quantitative effects, and contaminate the chromatographic column at the same time
.

2) Extraction methods of metabolites DCBX, QCA and MQCA

Metabolites such as DCBX, QCA and MQCA exist in the animal's body in a combined state with protein.
Usually, the target compound can be separated from the protein tissue by acid hydrolysis, alkali hydrolysis or enzymatic hydrolysis.
Among them, the effect of enzymatic hydrolysis is the best, not only the reaction conditions The most gentle, and the most thorough separation from animal tissues
.

Add 0.
3mol/L HCI to the sample solution to acidify the enzymatic hydrolysate, and then use anion exchange solid phase extraction column for purification.
This study compared the effects of Bond Elute Certify II and Oasis MAX commercial SPE columns.
The results show that Oasis The purification effect and recovery rate of MAX are better than the former.
This study optimizes the conditions of solution, flow rate control, elution and elution of the analyte used in solid phase extraction.
According to the nature of the compound, DCBX, QCA and MQCA need to be divided into two steps.
For elution, DCBX was eluted with dichloromethane in the first step , and QCA and MQCA were eluted with ethyl acetate containing 2% formic acid in the second step
.

(2) Liquid chromatography conditions

1) Selection of chromatographic column

The separation effect of Phenomenex Luna C ₁₈ , Waters Atlantis dC ₁₈ and Inertsil ODS-3 with different packing chromatographic columns was compared.
The results showed that when using Inertsil ODS-33um, 2.
1×100mm chromatographic column, the peak time and resolution of each compound And the peak shape is better
.

2) Selection of mobile phase

When mass spectrometry adopts positive ion mode for detection, mobile phases with a certain acidity are usually used, so that the analyte is protonated and positively charged, thereby improving the sensitivity of detection.
This experiment compares 0.
05% to 0.
3% formic acid solutions.
The results showed that the components of the mobile phase using 0.
1% formic acid solution were better separated, and there was a strong response on the mass spectrum, so the experiment chose 0.
1% formic acid solution as the mobile phase
.

(3) Mass spectrometry conditions

The composition of animal tissues is complex, and the sample matrix is ​​likely to interfere with the target analyte.
In this study, electrospray ionization source (ESI) and positive ion scanning were used for LC-MS/MS detection.
Multiple reaction monitoring mode (MRM) can effectively eliminate samples The interference of the matrix greatly improves the sensitivity of the method
.

Related Links: Procedures for the determination of carbachol, olaquindox and metabolite residues in cattle and pig liver and muscle