Critical parameters affecting analytical performance in the determination of environmental relevant steroids using HPLC-MS/MS

MARINO, D.J.; WUNDERLIN, D.A.; RONCO, A.E.; CARRIQUIRIBORDE, P.

Portland

Congreso; SETAC- North America 31th Annual Meeting; 2010

Society of Environmental Toxicology and Chemistry

In the present study some critical parameters on the analytical determination of the environmental relevant steroids E1, E2, EE2 and DHT by HPLC-MS/MS were assessed. Binary (ACN:H2O and MeOH:H2O) and ternary (ACN:MeOH:H2O) mobile phases were evaluated in the liquid separations system (HPLC). Several concentrations of formic acid and ammonium acetate were tested as additives in the aqueous components. Two alternative stationary phases were evaluated using Gemini C18 (Phenomenex) and Kinetex PFP (Phenomenex) columns. The instrumental variables of mass spectrometer studied were: ionization sources APCI and ESI, positive and negative mode, needle voltage, capillary energy (CID), temperature of drying gas and your influences on type and relative ions abundances. The [M-OH]+, [M+H]+ and [M-H]- were identified as monitoring ions of the studied steroids in positive and negative mode respectively. The most abundant total ion count (TIC) was obtained with ESI source in positive mode. Under this instrumental condition, the [M-OH]+/ [M+H]+ ratio was favored (in both HPLC columns) by the increases of H+ in the mobile phase for the estrogens (aromatic ring) but not for the androgen. The Signal/Noise ratio was improved 10 times by increasing the drying gas temperature (DGT) from 50 to 300¡ãC. The optimum CID conditions for the [M+H]+ ion were 20, 45 and 55eV for E2, EE2 and E1, respectively. The number of counts was increased at higher CID for DHT, but no changes were observed in the type of ion. Under same conditions, the formation of [M-OH]+ ion was 20% higher in the polar (PFP) than in the non-polar (C18) stationary phase. Under the ESI negative mode, only ammonium acetate produced the [M-H]- ion. No negative ions were formed by DHT due to the absence of the aromatic ¨COH. The study showed the relevance of considering the carbocation deshidratation product stability (EE2¡Ö E2> E1>DHT), the mobile phase composition and the nature of the stationary phase, together with the instrument set up, in order of improving the performance of the ion monitoring for steroid analysis in environmental samples using HPLC-MS/MS.