Determination of phenolic derivatives of antipyrine in plasma with HPLC-Tandem MS using ESI and Turbo ion spray as interfaces

Free radical damage plays an important role in biology and medicine, especially in the aging process of human beings. In the field of free radical products, determining the degree of this free radical damage is still a major problem. Several determination methods exist, most of which involve the use of a so-called endogenous marker. One of the disadvantages of endogenous markers is that they are involved in biochemical pathways. An exogenous marker with a well-known metabolic pathway can distinguish between naturally formed metabolites and free radical products. Whereas these radical products are formed only via competitive reactions, their concentration in biological fluids will be low. In this work, antipyrine was used as a potential exogenous marker for oxidative stress. A method to determine antipyrine and its phenolic derivatives in plasma by means of reversed-phase high performance liquid chromatography combined with a mass spectrometer (RP-HPLC-MS) was developed. Electrospray ionization (ESI) and turbo ion spray ionization were used as interfaces between the HPLC and the mass spectrometer. To optimize the ESI measurements and the turbo ion spray measurements, a -column (1 mm I.D.) was used for the LC separation. The mass spectrometer was operated in the multiple reaction mode (MRM). For the different phenolic derivatives of antipyrine, different target ions were used and optimized. A m/z=205/120 (mother/daughter) resulted in the best signal to noise ratio. Using -HPLC instead of conventional HPLC combined with ESI resulted in an increase of the signal to noise ratio by a factor 15. The turbo ion spray interface combined with -HPLC increased this factor by 1.4. The detection limit, determined for antipyrine because no standards for the phenolic derivatives of antipyrine are available, for this method (MS/MS mother/daughter m/z 188.9/104.0) was 1.25 pg in 20-L water.