The Application of QuEChERS in the Extraction of Anabolic Steroids in Whole Blood

This application utilizes the original, non-buffered QuEChERS (acronym for quick, easy, cheap, effective, rugged, and safe) technique to quantify anabolic steroids in human whole blood. Previous extraction techniques typically involve a protein precipitation step followed by a liquid-liquid extraction (LLE) or solid phase extraction (SPE). Target analytes are extracted from human whole blood by combining 1 mL of sample with acetonitrile (MECN), magnesium sulfate (MgSO₄), and sodium chloride (NaCl) to enhance phase separation and partioning of the anabolic steroids into the organic layer. Following adequate shaking and centrifugation, further dispersive SPE is conducted on the sample to absorb residual water and remove matrix co-extractives. This ultimately results in a clean, final extract for LC–MS-MS analysis.

Extraction/Analytical Materials

QuEChERS Procedure

1) Add 2 mL of MECN to 15-mL centrifuge tube containing 400 MgSO₄ and 100 mg NaCl (ECQUUS1015CT).

2) Add internal standards (IS), and appropriate amounts of steroids spiking solution to fortified samples.

3) Add 1 mL of negative whole blood into the 15-mL tubes.

4) Cap and shake for 1 min at 1000 strokes per min using a Spex 2010 Geno-Grinder.

5) Centrifuge at 3000 g for 5 min.

dSPE Cleanup

6) Transfer 1 mL of the supernatant to a 2-mL dSPE tube containing 150 mg MgSO₄ and 50 mg of PSA (CUMPS2CT).

7) Shake 1 min at 1000 strokes/min using the Spex 2010 Geno-Grinder.

8) Centrifuge at 3000 g for 5 min.

9) Transfer 0.4 mL of the cleaned extract into a 2-mL auto-sampler vial; add 0.4 mL of reagent water, and vortex for 30 s.

10) The samples are ready for LC–MS-MS analysis.

Instrumental

LC–MS-MS: Agilent 1200 Binary Pump SL with AB Sciex API 4000 QTrap MS/MS
Column: UCT Selectra® C18 LC column, 100 × 2.1 mm, 3 μm
Guard Column: UCT Selectra® C18 guard column, 10 × 2.1 mm, 3 μm
Injection Vol.: 10 μL
Mobile Phase A: 0.1% formic acid in water
Mobile Phase B: 0.1% formic acid in methanol
Column Flow rate: 0.30 mL/min

Results

Conclusion

Excellent recoveries ranging from 81.4% to 101.6% were obtained using UCT's QuEChERS extraction kit (ECQUUS1015CT) and dispersive clean-up products (CUMPS2CT) in conjunction with the Selectra® C18 HPLC column (SLC-18100ID21-3UM) for the extraction and analysis of anabolic steroids in human whole blood.

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