Hormone residues in foods of animal origin may jeopardize the health of consumers, with sex and growth-promoting hormones, which are currently the most used, posing the greatest risk to human health. Source: Food Safety Monitoring Technology (Chemical Industry Press)
Operation method
HPLC-MS/MS method
Materials and Instruments
Animal tissue Move 1. Pre-treatment Weigh 10 g of sample, accurately add 0.5 mL of internal standard solution (13C-hexenestrol, 13C-testosterone, 13C-estradiol) (20 ng/mL), add 10 mL of sodium acetate-acetic acid buffer solution at pH 5.2, homogenize, and then add 10 uL of β-glucosidase-sulfate esterase solution, and then perform enzyme digestion by oscillation at (37±1)°C overnight. After removal and cooling, 36 mL of methanol was added and extracted by shaking for 30 min, then transferred to a centrifuge tube and centrifuged at 2000 g for 10 min. 40 mL of hexane was added to the supernatant and extracted twice, and the hexane layer was discarded. In the water-methanol layer, 0.5 mL of n-propanol was added, and the methanol was removed by rotary evaporation at 50 ℃. The HLB solid phase extraction column (6 mL, 500 mg) was activated with 6 mL methanol, 6 mL water. 50 mL of water was added to the solution after rotary evaporation and passed through the column at 2-3 mL/min. After washing Pan with 6 mL of water, the extraction column was blown dry with nitrogen. The column was eluted with 10 mL of 5 mmol/L triethylamine methanol solution, the eluate was blown dry with nitrogen, 0.3 mL of trichloromethane was added to dissolve the residue, and 3 mL of n-hexane was added for the next purification step. The cinnamon gum solid phase extraction column (6 mL, 500 mg) was activated with 6 mL of hexane, the solution was passed through the column and washed with 5 mL of hexane. The column was eluted with 6 mL of water-saturated ethyl acetate, and the eluate was nitrogen-dried and dissolved in 2 mL of methanol-ethyl acetate (40 + 60) solution for passing through the amino column. Amino column (6 mL, 500 mg) was washed with 3 mL methanol-ethyl acetate (40 + 60) solution, 3 mL water-saturated ethyl acetate activation, the previous step solution over the column and receive, then add 3 mL methanol - ethyl acetate (40 + 60) solution elution and receive, nitrogen gas blowing dry, add 0.5 mL of mobile phase dissolved on the machine. 2. Determination (1) androgen determination of liquid chromatographic conditions column: phenyl column (2.0 × 250 mm, 5 um); mobile phase: methanol - water, gradient elution, the initial conditions of 65% water - 35% methanol, maintained for 3 min, 27 min linear gradient so that the proportion of methanol 100%, maintained for 5 min, 1 min the proportion of methanol down to 35%, maintained for 20 min to the next injection. Flow rate: 0.2 mL/min. (2) Mass spectrometry reference conditions for androgen determination Ionization source: electrospray positive ion mode; capillary voltage: 3.5 KV; conical hole voltage: 70 V; RF lens 1 voltage: 30 V; RF lens 2 voltage: 0.5 V; source temperature: 100 ℃; desolvent gas temperature: 300 ℃; desolvent gas flow rate: 400 L/h; electron multiplication voltage: 650 V; injection chamber pressure: 2.8 × 10-3 mbar; androgen determination mass spectrometry reference conditions. 10-3 mbar; (3) Estrogen determination of liquid chromatographic conditions column: phenyl column (2.0 mm × 250 mm, 5 um); mobile phase: acetonitrile - water, gradient elution, the initial conditions of 65% water - 35% acetonitrile, keep 3 min, 27 min linear gradient so that the proportion of acetonitrile 100%, keep 5 min, 1 min the proportion of acetonitrile down to 35%, keep 20 min to the next injection. Flow rate: 0.2 mL/min (4) Mass spectrometry conditions for estrogen determination Ionization source: electrospray negative ion mode; capillary voltage: 3.1 KV; dimensional pore voltage: 80 V; RF lens 1 voltage: 40 V; RF lens 2 voltage: 0.5 V; source temperature: 100 ℃; desolvation gas temperature: 300 ℃; desolvation gas flow rate: 400 L/h; electron multiplication voltage: 650 V; injection chamber pressure: 2.8 × 10 -3 mbar. For more product details, please visit Aladdin Scientific website.
Sodium acetate-acetic acid buffer solution β-glucosidase-sulfatase solution Methanol Hexane Water saturated ethyl acetate
Centrifuge tube HLB solid phase extraction column Liquid chromatography conditioned columns