Purification of urine samples to improve detection limit of anabolic agents

Objective: To investigate recovery percentage of clenbuterol, nandrolone, stanozolol, and epimetendiol by two different solid phase extraction procedures viz. XAD2 (polystyrene divinylbenzene) and SPE columns (C18, Samprep, Rankem) so as to improve detection limit of anabolic steroids.
Materials and Methods: The urine samples were spiked with different concentrations of drugs. The preliminary work was done with six samples, each of clenbuterol, nandrolone, and epimetendiol at 1, 2, 5, and 10 ng/mL and of stanozolol at 5,10, 20, and 40 ng/mL that were processed and injected into high resolution mass spectrometer. Later the study was limited to clenbuterol and epimetendiol at 2, 5, and 10 ng/mL concentrations. The data were analysed by comparing XAD2 and SPE column values.
Results: The results show that the recovery percentage of nandrolone and stanozolol at various concentrations did not signify any difference between the two columns. However, there was a significant increase in the recovery of clenbuterol at 2 ng/mL ( P < 0.002), 5 ng/mL ( P < 0.001), and 10 ng/mL ( P < 0.001) concentrations, where as for epimetendiol there was significant increase in the recovery at 2, 5, and 10 ng/mL ( P < 0.01) with SPE column compared to XAD2 column.
Conclusion: It is possible to enhance the detection limit of clenbuterol and epimetendiol by SPE column compared to XAD2 column. This procedure may be used for confirmation of suspicious samples found in routine testing.

Keywords
Anabolic steroid, detection limit, mass spectrometry, SPE column, urine test, XAD2 extraction.