P–006 Simultaneous determination of bisphenol A and S in the samples of human seminal fluid
Authors: Fialková S , Král T , Kohoutek J , Franzová K , Ješeta M , Navrátilová J 
Published: August 2021

Abstract

Study question
Can we quantitatively determine concentrations of endocrine disruptors namely bisphenol A and S in seminal fluid?

Summary answer
We developed selective analytical method to simultaneously screen for the presence of bisphenol A (BPA) and S (BPS).

What is known already
The male reproductive system involves processes, which may be influenced by the disruption of the endocrine system by chemicals called endocrine disruptors (EDs). There is a growing evidence that EDs such as bisphenol A and S may be responsible for the decline in male reproductive health. To date, the claimed adverse effects on male fertility are largely based on the results from studies assessing the relationship between urinary BPA and BPS concentration and semen parameters. The best evidence of an adverse effect of BPA and BPS directly on spermatozoa could be provided by measuring bisphenols concentration directly in seminal fluid.

Study design, size, duration
To selectively and quantitatively analyzed bisphenols in any biological matrix advanced analytical tools and selective sample preparation protocols must be employed. In this study we developed targeted analytical method based on liquid chromatography tandem mass (LC-MS/MS) detection to measure bisphenol A and S in seminal fluid samples obtained from IVF clinic. A total of 140 samples were analysed.

Participants/materials, setting, methods
BPA and BPS was extracted from 140 seminal fluid samples using solvent extraction followed by preconcentration step. Samples were analyzed on Agilent 6495 Triple Quadrupole (Agilent Technologies, Santa Clara, CA) operating in the ESI-negative mode. Two MS/MS transitions were used for quantitative LC-MS/MS analyses. Chromatographic separation was achieved on Waters™ ACQUITY™ UPLC™BEH C18 (100 × 2.1 mm, 1.7 µm) column using gradient elution with a mixture of 0.1mM ammonium fluoride and methanol as mobile phases.

Main results and the role of chance
We developed selective sample preparation method for detection of BPA and BPS in seminal fluid followed by LC-MS/MS detection. The method validation was performed based on FDA guidelines. Validation criteria included limit of detection (LOD), limit of quantitation (LOQ), accuracy and precision. Due to the lack of the certified reference material the validation criteria of the method were assessed in pool of spiked seminal samples. The accuracy of the LC-MS/MS method was evaluated as a percent recovery of the amount of target analyte added into the sample. Recovery rates were above 80% for both analytes. LOD was 0.04 ng/mL for BPA and 0.01 ng/mL for BPS. LOQ was 0.14 ng/mL and 0.02 ng/mL for BPS. Measured BPA concentration ranged from 0.04 ng/mL to 1.62 ng/mL. For BPS, the concentration ranged from 0.01 ng/mL to 0.47 ng/mL. BPA and BPS were detected in 64% and 81% of samples, respectively. Interestingly, BPA showed lower detection frequency compared to BPS. These results are consistent with other studies performed on urine samples.

Limitations, reasons for caution
The limitation of the developed method is the time-consuming sample preparation and analysis cost.
Wider implications of the findings: These results document for the first time the presence of BPS in seminal fluid. Knowing the concentration of BPA and BPS in seminal fluid is crucial for mitigating the associated health risks and initiating intervention and prevention strategies. Our future work will evaluate the influence of BPS concentration on spermatozoa.

Trial registration number
AZV NV18–01–00544; CZ.02.2.69/0.0/0.0/19_074/0012727