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8.
2.
1.
7 Selection of analysis conditions
(1) Selection of purification and enrichment conditions
1) Selection of sample extraction method
The steroidal anabolic hormones in animal tissues will form glucuronide conjugates.
When extracting samples, β-glucase/sulfatase must be added for hydrolysis before extraction
.
Steroid anabolic hormones are fat-soluble substances and have high solubility in polar organic solvents.
2) Selection of purification conditions
The residual level of steroidal anabolic hormones in animals is low, and there are many endogenous interfering substances.
It is difficult to achieve the ideal purification effect only through pure liquid-liquid extraction or simple solid-phase extraction purification.
In the experiment, the lower polarity is used.
The C 18 SPE column removes the interference impurities with greater polarity, and then uses the silica gel SPE cartridge with greater polarity to remove the interferences with weaker polarity.
From the test results, this uses two solid phases with opposite polarities.
The method of extracting material for sample purification and enrichment can effectively remove the interference caused by the sample matrix
.
Compared with the Canadian Food Inspection Agency method, the use of this bipolar solid-phase extraction purification method also reduces the cumbersomeness of multiple liquid-liquid extractions, shortens the extraction time, and improves work efficiency
(2) Selection of liquid chromatography conditions
When the liquid chromatography is combined with the mass spectrometer, because the ion source of the mass spectrometer has certain restrictions on the mobile phase composition of the liquid chromatography, in order to ensure the maximum ionization efficiency, the mobile phase uses the acetonitrile- ammonium formate system
.
The composition of animal tissues is complex.
(3) Selection of mass spectrometry conditions
Use a peristaltic pump to continuously inject 1 mg/L of three steroid hormones and two isotope internal standard solutions into the mass spectrometer at a flow rate of 10uL/min, and perform primary mass spectrometry (Q1 scan) on five test substances to obtain molecular ions Peak (M+1) + , optimize the gas path parameters such as spray gas (NEB), curtain gas (CUR), auxiliary heating gas, optimize the declustering voltage (DP) of the molecular ion peak of each component to be measured, and focus voltage (FP) ), the entrance voltage (EP), the molecular ion peak (M+1) is subjected to secondary mass spectrometry (product ion scan) to obtain testosterone, epitestosterone, progesterone, deuterated testosterone (d 5 -testosterone), deuterated progesterone (d 9 -Progesterone) the secondary mass spectrum
.
Select the ion pair with higher intensity and less interference as the monitoring ion pair
Two kinds of stable isotope internal standards are used in the quantitative method, deuterated testosterone (d 5 -testosterone) is used as the quantitative reference substance of trineurone and epitestosterone, and deuterated progesterone (d 9 -progesterone) is used as progesterone.
The quantitative reference substance reduces the quantitative error caused by the sample processing process and the mass spectrometry ionization process to the substance to be tested, and greatly improves the accuracy of the quantification
.
8.
2.
1.
8 Linearity range and lower limit of determination
Concentrations of testosterone and epitestosterone prepared with methanol-water are 0, 0.
25μg/L, 0.
5μg/L, 1.
0μg/L, 2.
5μg/L, and progesterone concentrations are 0, 1.
25μg/L, 2.
5μg/L , 5.
0μg/L, 12.
5μg/L mixed standard working solution, each milliliter of the mixed standard working solution contains 5ng deuterated testosterone (d 5 -testosterone) and deuterated progesterone (d 9 -progesterone)
.
Measure under the selected chromatographic and mass spectrometry conditions, and use the ratio of the peak area of the measured component of the standard working solution to the peak area of the internal standard substance of the standard working solution to the concentration of the measured component of the standard solution working solution and the internal standard substance of the standard working solution The ratio of the concentration is used as a linear regression curve
Table 8-9 Quantitative transitions, linear equations, and correlation coefficients of testosterone, epitestosterone, and progesterone
Considering the signal-to-noise ratio of the mass spectrum of each component to be tested and the actual needs of the detection work, the determination of the lower limit (LOQ) of testosterone and epitestosterone in animal tissues (muscles) is determined to be 0.
Related Links: Determination of Testosterone, Epitestosterone, and Progesterone Residues in Beef Liver and Beef