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Microsoft word - sfe535

Determination of Steroids in
A solution to these problems is to use an SFE instrument and method that simplifies the Animal Tissues by
separation and recovery of trace level drug residues from an analyte/fat matrix. This Supercritical Fluid
application describes a procedure for coupling SFE Extraction and Inline
technology with an inline trapping technique to quickly and easily extract residue levels of steroids Trapping
from animal tissue samples without co-extracting Introduction
Equipment
Applied Separations’ Spe-ed™ SFE-4 Teflon Inline Cartridge Holder- Cat. #7923 Materials
HPLC. Unfortunately, these methods are time consuming, require large volumes of organic solvent, and must be optimized for every new Spe-ed Polypropylene Wool (Cat. #7952) SFE is an alternative technique using supercritical Carbon dioxide –SFC/SFE grade without carbon dioxide to extract analytes from a variety of matrices. SFE significantly reduces the use, exposure to, and disposal of hazardous solvents, while providing comparable extraction results to There have been some problems identified with using typical SFE methods to isolate drug residue from tissue matrices. One of the main difficulties is that when trace levels of residues are isolated from fat tissue by SFE using CO2, fat is co- extracted. If a modifier is used with CO2, the N-Methyl-N- (trimethylsilyl) resultant extract becomes more complex and the desired analyte is more difficult to recover from www.appliedseparations.com
Homogenize 5.0 g of tissue and freeze dry. When Incubate this mixture for 30 minutes at 37 °C. Add ready for extraction, blend freeze dried tissue with 1.0 ml of acidic buffer. (The acidic buffer is 3.5 g of Spe-ed Matrix. Add internal standard and prepared by mixing 1.7 ml of hydrochloric acid 1 mL of water. Remove the end flanges of a 3 ml (37%) with 98.3 ml of acetate buffer (2 mol 1-1). SPE alumina cartridge and insert into an Extract the mixture twice with 6 ml of TBME and appropriately sized teflon holder. Close one end of evaporate the combined extract to dryness under a an extraction vessel, and place the cartridge/holder gentle stream of nitrogen. Transfer the residue to a into the vessel. Set the cartridge luer into the derivatisation vial filled with 0.5 ml of ethanol. outlet of the vessel. Next, place a plug of Spe-ed Evaporate the ethanol and add 0.05 ml of HFBA- Wool on top of cartridge/holder and compress with acetone (1/4; v/v). Vortex mix the vial and a stainless steel tamping rod. Pour the sample incubate at 60 °C for 1 hr. Once incubation is mixture of tissue and Spe-ed Matrix into the complete, evaporate the reaction mixture to extraction vessel and tamp down with rod. Fill the dryness under a gentle stream of nitrogen at 50 °C. remaining void in the extraction vessel with Next, dissolve the derivatised residue in 0.025 ml another plug of Spe-ed Wool, and then use a of isooctane and transfer into a GC injection vial stainless steel tamping rod to tightly pack the with a micro-insert. Derivatisation method I can vessel. Close vessel with an end-cap and perform Extraction Conditions
Ethynylestradiol Ethynylestradiol-d Analyte Recovery
When the extraction sequence is complete, remove the SPE column and elute with 6 ml of methanol- Derivatisation Method II:
Evaporate the second portion of the eluate under a gentle stream of nitrogen in a water-bath at 50 °C. Post-SFE Analysis
Add 2 mLs of water and mix in vortex for 30 s. Divide the eluate into two equal portions. Next, extract mixture twice with 6 ml of TBME. Evaporate TBME and transfer residue into a Derivatisation Method I:
derivatisation vial with 0.5 ml of ethanol. Evaporate the solvent of the first portion and Evaporate ethanol and add 25 µl of MSTFA- dissolve the residue in 0.2 ml of alkaline ammonium iodide-dithioerythritol (1000+2+4, v/w/w). Mix vial for 30 s and incubate for 1 hour (Prepare alkaline solution by dissolving 5.6 g of potassium hydroxide in 100 ml of methanol). www.appliedseparations.com
When incubation is complete, evaporate reaction References
mixture to dryness under a gentle stream of Stolker, A.; Zoontjes, P.; and van Ginkel, L. “The nitrogen at 50 °C. Dissolve derivatised residue in use of Supercritical fluid extraction for the 0.025 ml of iscoctane. Derivatisation method two determination of steroids in animal tissues.” The Analyst. 1998, 123, 2671-2676.
Nortestosterone
Nortestosterone-d3
Methylboldenone
Methylboldenone-d3
Norethandrolone
Norgestrel
Chlorotestosterone acetate
Chlorotestosterone acetate-d3
Analysis
GC-MS
Results
Validation results of SFE-GC-MS of Steroids
from Fortified Bovine Muscle*
Analyte
Repeatability,
laboratory
Reproducibility
RSD (%) (n=3)

*(MSTFA Derivative)

Conclusion
The supercritical carbon dioxide extraction of
steroids form animal tissue samples offers a viable
alternative to solvent-based procedures. The
accuracy and precision of the results were
comparable to the standard method while
extraction times were reduced. In addition, levels
of detection were 2 ug/k for melengestrol acetate.
www.appliedseparations.com

Source: http://www.cspl.in/application/pdfs/extraction/sfe535.pdf

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