Summary
This post is going to introduce the technology of GC-MS to detect the β2- Stimulant Content in animal Tissues. The theory is that mix animal tissue with 1% solution of perchloric acid. And then extracting it with the help of ethyl acetate: isopropanol(6:4) and purifying it with solid-phase extraction (SPE) and derivatizating it with BSTFA and 1% TMCS to process the GC-MS detection. Minimum detectable concentration in the sample were terbutaline 0.5μg / kg, clenbuterol 110 μg / kg, salbutamol 0.2 μg / kg.
Chromatographic conditions
Column: HP-5MS 5% phenyl methyl silicone fused silica capillary column (30m × 0.25mm × 0.25 μm); inlet temperature: 300 ℃; Column temperature program: initial temperature is 150℃for 3 min, and then rising from 10℃/ min to 240℃ and then 20℃/ min to 280 ℃ for 3 min; carrier gas: high-purity helium (99.999%), a flow rate of 110 ml / min, no split injection.
MS conditions
EI source temperature is 230 ℃; electron energy is 70eV; interface temperature is 280 ℃, electron multiplier voltage is 1388 V, mass scan range is 50 ~ 550amu; Solvent delay for 8 min.
Sample Preparation
Weighing 5g minced sample with adding 1% perchloric acid solution 30 ml. And then homogenizing it in a high speed tissue homogenizer for 1 min and for 15-minute ultrasound at 80℃. After those steps, sample will be centrifuged at 5000 r / min for 10 min. Its supernatant, which should be regulated to alkaline with 50% sodium hydroxide, was transferred to a separatory funnel. And adding 30ml organic solvent for extraction and centrifugation. The organic phase was transferred to a stoppered flask with grinding mouth and extracted again. And then combining organic phase and drying over with anhydrous sodium sulfate at 50℃. Firstly, we should use 3 ml of acetonitrile and 3 ml of n-hexane to dissolve it and put it into a test tube, and then separating the exane phase. After evaporation of acetonitrile phase, we should use 3% ethanol / ethyl acetate to dissolve the volume to 2 ml. Rinsing Cle-SLX column with 5ml 3% ethanol / ethyl acetate. And then adding 1 ml sample extraction and 5 ml 5% methanol / ethyl to eluent for getting component 1. In order to get component 2 we should use 10 ml 50% methanol / ethyl acetate to eluent. Eluent should be blown to dry with nitrogen. Adding 100 μl BSTFA and 1% TMCS for derivating at 60℃ for 30 min. And then drying it with nitrogen and dissolving with 200μl toluene. After that we can process the GC-MS technology to analyze it.
This post is going to introduce the technology of GC-MS to detect the β2- Stimulant Content in animal Tissues. The theory is that mix animal tissue with 1% solution of perchloric acid. And then extracting it with the help of ethyl acetate: isopropanol(6:4) and purifying it with solid-phase extraction (SPE) and derivatizating it with BSTFA and 1% TMCS to process the GC-MS detection. Minimum detectable concentration in the sample were terbutaline 0.5μg / kg, clenbuterol 110 μg / kg, salbutamol 0.2 μg / kg.
Chromatographic conditions
Column: HP-5MS 5% phenyl methyl silicone fused silica capillary column (30m × 0.25mm × 0.25 μm); inlet temperature: 300 ℃; Column temperature program: initial temperature is 150℃for 3 min, and then rising from 10℃/ min to 240℃ and then 20℃/ min to 280 ℃ for 3 min; carrier gas: high-purity helium (99.999%), a flow rate of 110 ml / min, no split injection.
MS conditions
EI source temperature is 230 ℃; electron energy is 70eV; interface temperature is 280 ℃, electron multiplier voltage is 1388 V, mass scan range is 50 ~ 550amu; Solvent delay for 8 min.
Sample Preparation
Weighing 5g minced sample with adding 1% perchloric acid solution 30 ml. And then homogenizing it in a high speed tissue homogenizer for 1 min and for 15-minute ultrasound at 80℃. After those steps, sample will be centrifuged at 5000 r / min for 10 min. Its supernatant, which should be regulated to alkaline with 50% sodium hydroxide, was transferred to a separatory funnel. And adding 30ml organic solvent for extraction and centrifugation. The organic phase was transferred to a stoppered flask with grinding mouth and extracted again. And then combining organic phase and drying over with anhydrous sodium sulfate at 50℃. Firstly, we should use 3 ml of acetonitrile and 3 ml of n-hexane to dissolve it and put it into a test tube, and then separating the exane phase. After evaporation of acetonitrile phase, we should use 3% ethanol / ethyl acetate to dissolve the volume to 2 ml. Rinsing Cle-SLX column with 5ml 3% ethanol / ethyl acetate. And then adding 1 ml sample extraction and 5 ml 5% methanol / ethyl to eluent for getting component 1. In order to get component 2 we should use 10 ml 50% methanol / ethyl acetate to eluent. Eluent should be blown to dry with nitrogen. Adding 100 μl BSTFA and 1% TMCS for derivating at 60℃ for 30 min. And then drying it with nitrogen and dissolving with 200μl toluene. After that we can process the GC-MS technology to analyze it.