Liquid chromatography (HPLC), liquid-mass spectrometry (LC-MS or LC-MS/MS) and gas chromatography (GC-MS) are the main methods used for the detection of glucocorticoid drugs in different matrices, such as food of animal origin.

Operation method

High performance liquid chromatography-tandem mass spectrometry

Principle

Glucocorticoids are a class of steroid hormones secreted by the adrenal cortex that regulate the biosynthesis and metabolism of sugars, fats, and proteins, as well as having anti-inflammatory effects, and are called "glucocorticoids" because of their earliest recognized activity in regulating glucose metabolism. The basic structural features of glucocorticoids include the carbonyl group of C3, the Δ4 and 17 β-ketol side chains of adrenocorticoids, and the 17 α-OH and 11 β-OH unique to glucocorticoids.

Materials and Instruments

Foods of Animal Origin
Standard Prednisone Prednisolone Dexamethasone Betamethasone Fludrocortisone Methylprednisone Beclomethasone Hydrocortisone
TSQ Quantum Triple Quadrupole Tandem Mass Spectrometer Electrospray Ionization Source Surveyor AS Autosampler

Move

I. Sample Preparation

1. Extraction

At room temperature, weigh (2 ± 0.05) g of tissue samples in a 50 mL centrifuge tube, add ethyl acetate 15 mL, vortex mixing extraction for 3 min, centrifugation for 10 min (4000 rpm), transfer the ethyl acetate layer to a 50 ml pear-shaped bottle. To the residue, add 10 mL of 0.1 mol/L NaOH solution, mix well, add 15 mL of ethyl acetate to extract and remove the ethyl acetate layer. Combine the two extracts, concentrated under reduced pressure at 40 ℃ nearly dry, add ethyl acetate 1.0 mL and hexane 5 mL, so that it is fully dissolved, to be purified.

2. Purification

SPE was carried out using a Silica solid phase extraction column. After activating the SPE column with 5 mL of hexane, the sample was washed with 5 mL of hexane, dried, and then eluted with 5 mL of hexane-acetone (6:4, v/v). The eluate was blown dry at 40 ℃ under N2, and the residue was dissolved in 20% aqueous acetonitrile 1.0 mL, and then passed through a 0.45 μm filter membrane for the determination of liquid chromatography and mass spectrometry conditions by LC/MS/MS method.

Chromatographic conditions

1. Chromatographic column: Thermo Hypersil Gold (150 × 2.1 mm, 5 μm); see Table 1 for gradient elution, in which A: water (containing 0.01% formic acid); B: acetonitrile; flow rate: 250 μL/min; injection volume: 10 μL.

2. Mass spectrometry conditions

Electrospray ionization source (ESI), negative ion mode; selective reaction monitoring (SRM) scanning mode, spray voltage: 3500 V; ion transfer tube temperature: 350 ℃; nitrogen was used as the sheath gas and auxiliary gas, of which 40 arb for the sheath gas and 5 arb for the auxiliary gas, and argon was used as the collision gas, with a collision pressure of 1.5 mTorr; selective reaction monitoring (SRM).

Common Problems

The recoveries of glucocorticoids in liver and muscle of pig, cow and sheep, chicken, egg and milk were 60 - 110 %, which meet the minimum detection limits at home and abroad, and the quantitative and qualitative results were accurate and reproducible.

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