Sample pretreatment of 8 kinds of macrolides and lincosamide drug residues in puffer fish and eels

6.
2.
4.
5 Sample pretreatment

(1) Sample preparation

Take out a representative sample of about 1 kg from all the samples, fully mash, mix, and divide them into two parts, and put them into clean containers
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(2) Extraction

Weigh 5g sample, accurate to 0.
01g, place it in a 50mL centrifuge tube, add 20.
0uL internal standard working solution and 10.
0mL tris solution, and shake vigorously on a shaker for 10 minutes
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(3) Purification

After all the sample solution flows out, wash the column with 10 mL water and 10 mL methanol solution successively , discard all the effluent, and drain the solid phase extraction column with a vacuum pump for 1 hour
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According to the above operation steps, prepare the blank sample extract solution used to prepare the series of matrix standard working solutions
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6.
2.
4.
6 Determination

(1) Liquid chromatography conditions

Column: Atlantis C ₁₈ , inner diameter 3μm, 150mm×2.
1mm (inner diameter) or equivalent: mobile phase A: acetonitrile ; mobile phase B: 0.
1% formic acid aqueous solution; mobile phase C: methanol; see Table 6 for gradient elution conditions 15; Flow rate: 0.
2mL/min; Column temperature: 30°C; Injection volume: 20μL
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(2) Mass spectrometry conditions

Ion source: electrospray ion source; scanning method: positive ion scan; detection method: multi-reaction monitoring: electrospray voltage: 5500V; atomizing gas pressure: 0.
24MPa; auxiliary gas flow rate: 0.
4L/min; ion source temperature: 550 ℃; Collision cell outlet voltage: 2.
0V; Qualitative ion pair, quantitative ion pair, collision gas energy and declustering voltage, see Table 6-4
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(3) Qualitative determination

Select 1 parent ion and 2 or more product ions for the component to be tested.
Under the same test conditions, the retention time of the substance to be tested in the sample is within ±2.
5% of the corresponding retention time in the standard solution; and the sample to be tested In the solution, the relative abundance of each qualifier ion in the test substance is close to the concentration of the matrix standard working solution.
The ratio of the relative abundance of each qualitative ion in the test substance is k, and the deviation does not exceed the range specified in Table 1-5, then it can be judged The corresponding analyte is present in the sample
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(4) Quantitative determination

Under the best working conditions of the instrument, use the matrix standard mixed working solution to inject samples separately, and take the ratio of the peak area of ​​the measured component in each standard solution to the peak area of ​​the internal standard substance as the ordinate, and take the measured component of each standard solution The ratio of the concentration (ng/mL) to the concentration of the internal standard substance is the abscissa, draw a standard working curve, and use the standard working curve to quantify the sample
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The multiple reaction monitoring (MRM) chromatograms of the standard substances of lincomycin, elixomycin, erythromycin, tilmicosin, tylosin, spiramycin, kitasamycin and josamycin are shown in Figure 6.