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Selection of analysis conditions for determination of α-Trenbolone and β-Trenbolone residues in cattle muscle, liver and kidney

 Last Update: 2021-09-20
 Source: Internet
 Author: User

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8.
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7 Selection of analysis conditions

(1) Enzymatic hydrolysis

Trenbolone is often stably combined with proteins and other substances in samples, and the literature mostly uses β-glucuronidase /arylsulfatase hydrolysis
.

However, the enzymolysis conditions are different (including the amount of enzyme, enzymolysis temperature and time, different buffer systems and pH, etc.

(2) Extraction and purification

Trenbolone is an ester-soluble compound.
Experiments have found that ethyl acetate can effectively extract the test substance from the enzymatic hydrolysis solution
.

Solid phase extraction technology is a relatively effective purification method

Figure 8-14 Trenbolone standard, GPC chromatogram of liver, kidney, and muscle blank samples (UV: 250nm)
(a) a, β-Trenbolone mixed standard chromatogram: (b) Kidney blank sample chromatogram: ( c) Chromatogram of blank liver sample: (d) Chromatogram of blank muscle sample

Considering that trenbolone is a weakly polar compound, the purification conditions of the silica gel solid phase extraction column were investigated
.

Both the eluent and the eluent were used to test the recovery rate under different ratios of acetone and n-hexane with 5 mL.
The experimental results are shown in Table 8-27 and Table 8-28

Table 8-27 The influence of eluent ratio on recovery rate

Table 8-28 The influence of eluent ratio on recovery rate

According to the experimental results in Table 8-27, 5 mL of acetone - n-hexane (10+90, v/v) eluent has the best impurity removal effect, and the test substance is not eluted
.

According to the test results in Table 8-28, when the volume ratio of acetone-hexane is greater than 3:7, 5 mL of eluent can completely desorb the analyte

(3) Selection of instrument conditions

1) Selection of HPLC instrument conditions

According to reports in various literatures, Cloversil ODS-U 5um 4.

6mm×250mm, ZORBAX Eclipse XDB-C ₁₈ 4.
6mm×150mm 5um, and Diamonsil (TM) diamond C ₁₈ 5um 150mm×4.

2) Selection of HPLC-MS instrument conditions (determination of monitoring ions)

According to the general requirements of mass spectrometry, first perform a full scan for each compound, and then determine the monitoring ion for each compound, and use the selected ion mode to improve the detection sensitivity
.

The determination of the monitored ions is selected according to the mass spectrum and structural characteristics of each compound

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