Matrix effect in F₂-isoprostanes quantification by HPLC-MS/MS: a validated method for analysis of iPF₂α-III and iPF₂α-VI in human urine

tLiquid chromatography coupled with tandem mass spectrometry (HPLC–MS/MS) has become the methodof choice for analysis in biological matrices, because of its high specificity and sensitivity. However, itshould be taken into account that the presence of matrix components coeluting with analytes mightinterfere with the ionization process and affect the accuracy and precision of the assay. For this reason,the presence of a “matrix effect” should always be evaluated during method development, above allin complex matrix such as urine. In the present work, a HPLC–MS/MS method was developed for thequantification of urinary iPF2-III and iPF2-VI. A careful assessment of matrix effect and an accuratevalidation were carried out, in order to verify the reliability of quantitative data obtained. Ion suppression,due to the matrix components, was reduced through optimization of both chromatographic method andsample extraction procedure. Urine samples were purified by solid phase extraction (SPE) and the extractsinjected into the HPLC–MS/MS system, equipped with a TurboIonSpray ionization source operated innegative ion mode (ESI−). Stable isotope-labeled analogues (iPF2-III-d4and iPF2-VI-d4) were used asinternal standards, and quantification was performed in multiple reaction monitoring (MRM) mode bymonitoring the following mass transitions: m/z 353.4 → 193.2 for iPF2-III, m/z 357.2 → 197.0 for iPF2-III-d4, m/z 353.4 → 115.1 for iPF2-VI, and m/z 357.4 → 115.1 for iPF2-VI-d4. The validated assay, appliedto the analysis of urinary samples coming from healthy and overweight subjects, resulted suitable for anaccurate quantification of iPF2-III and iPF2-VI in human urine