The principle of the experimental method:
High performance liquid chromatography-tandem mass spectrometry(HPLC-MSIMS) using high performance liquid chromatography as the separation method, mass spectrometry as the identification and determination method, and connecting the two into a complete instrument through an appropriate interface. The sample is injected through the liquid chromatography system, separated by the chromatographic column, and then enters the interface. In the interface, the sample is converted from ions or molecules in the liquid phase to ions in the gas phase, which are then focused in the mass analyzer and separated according to the mass-to-charge ratio. Finally, the ion signal is converted into an electrical signal, which is detected by an electron multiplier, and the detected signal is amplified and transmitted to a computer data processing system.
Experimental Materials:
food of animal origin
Reagents, kits:
Standard Testosterone, Methyl Testosterone, Progesterone, Trenbolone
Instruments and consumables:
TSQ Quantum triple quadrupole tandem mass spectrometer, electrospray ionization source, Surveyor AS autosampler
Experimental steps:
1. Sampling: Weigh about 500 g of animal muscle and liver, completely chop them, and homogenize them for later use; take 10 fresh eggs, peel them, and homogenize them for later use; take 500 mL of fresh milk and mix them for later use.
2. Extraction: 5 g sample was placed in a 50 mL centrifuge tube, 3 mL of 10% sodium carbonate solution and 10 mL of ethyl acetate were added, homogenized for 30 s, shaken for 10 min (4°C), and centrifuged at 6000 r/min 10 min, transfer the upper organic phase to a pear-shaped bottle. The extraction was repeated once more with 10 mL of ethyl acetate, and the upper organic phases were combined. Rotary evaporate to dryness at 40°C. The residue was dissolved in 1 mL of 50% acetonitrile aqueous solution, the solution was frozen for 30 min, centrifuged at 16,000 r/min for 5 min, and an appropriate amount of solution was taken through a 0.22 μm filter membrane for LC/MS/MS determination.
3. Injection Analysis
Precautions:
1. The ion source, mass analyzer, and detector of the mass spectrometer must all work under high vacuum to reduce background interference and avoid unnecessary ion-molecule reactions. Insufficient vacuum will result in insufficient sensitivity.
2. The LC-MS interface should avoid entering non-volatile buffers, avoid buffers containing phosphorus and chlorine, and the components containing potassium and potassium must be <1mmol/1. Too high salt will inhibit the signal of the ion source, block the spray needle and contaminate it. instrument.
3. Choose the appropriate voltage: When the DP voltage (declustering voltage) is high, the sample will decompose or fragment in the source, which will also make the ratio of multiply charged ions low and multimers reduced.
other:
1. The selection of the ion source is very important for the qualitative and quantitative analysis and detection of the compounds to be tested. Generally speaking, electrospray ionization sources are used for medium-polarity or large-polarity compounds, while atmospheric pressure chemical ionization is used for small-polarity compounds. Compounds also tend to choose atmospheric pressure chemical ionization, and electrospray is beneficial for the analysis of biological macromolecules or other compounds with large molecular weights.
2. Selection of positive and negative ion modes: The general principle is that positive ions are suitable for alkaline samples, and can be used to acidify samples with acetic acid or formic acid. When the samples contain secondary or tertiary ammonia, the positive ion mode can be preferred. Negative ions are suitable for acidic samples, and ammonia water can be used to alkalize the samples.
