Optimization of ICP-MS/MS-based methodology for multielemental analysis in follicular fluid: new approaches in female infertility research

Introduction: Heavy metals are recognized as potential endocrine disruptors associated with female infertility. Their determination in follicular fluid (FF), a biologically relevant matrix reflecting the ovarian microenvironment, may improve the assessment of environmental exposure and support studies on reproductive toxicity as it offers valuable insights into its multielemental composition. However, FF remains poorly characterized, and standardized analytical methodologies are lacking. This study aimed to optimize ICP-MS/MS conditions for multielemental determination in FF, providing a robust analytical basis for future toxicological and risk assessment studies.

Methods: Five FF samples from patients undergoing in vitro fertilization were pooled, aliquoted, and processed according to each type of analytical test. Different dilution factors were evaluated, and half of the aliquots were spiked for recovery assessment. Continuous ICP-MS/MS acquisition was performed using helium (He) as collision gas. Additionally, strategies to reduce spectral interferences for arsenic (⁷⁵As) and selenium (⁷⁴Se) were assessed using oxygen (O) as reaction gas.

Results: Continuous analysis in He mode achieved recoveries within the optimal range (100–120%) for all aliquots, including undiluted samples, supporting reliable multielemental determination. In continuous O mode, spectral interferences persisted under most conditions. The best performance for ⁷⁵As and ⁷⁴Se was obtained using a 1:2 dilution with 5–10% oxygen, yielding recoveries close to 100%. The optimized method enabled the determination of heavy metals and rare earth elements relevant to environmental exposure assessment and reproductive toxicology.

Conclusions: Continuous ICP-MS/MS analysis in He mode was identified as the most suitable approach for multielemental analysis of FF, while O reaction mode may improve the determination of selected elements after further optimization. Although biological validation requires larger cohorts, this standardized analytical workflow provides a reproducible platform for high-quality exposure assessment, facilitating future integration with multivariate statistical analyses, mechanistic studies on endocrine disruption, and next-generation risk assessment within NAM-based reproductive toxicology.