Salting-Out Assisted Liquid-Liquid Extraction for Simultaneous Determination of Steroid Hormones and Thyroxine by Using LC-MS/MS: Application in Fish Plasma

Most biological fluids contain hormones, sometimes in small amounts, whose detection and quantification as biological markers can be crucial. Measuring the level of steroid and thyroxine hormones is a key step to underfunding the overall fish physiology. Liquid chromatography with tandem mass spectrometry (LC-MS/MS) combines the high sensitivity of MS with the significant physical separation capacity of LC, becoming the method of choice for hormone analyses. However, a throughput, fast, reliable, simple, and environmentally friendly sample preparation method is still missing, and research is needed to carefully extract those hormones from the biological sample. This study aimed to develop a novel salting-out assisted liquid-liquid extraction (SALLE), a method to simultaneously extract L-thyroxine, testosterone, cortisone, and cortisol from human and fish plasma samples. The analysis was performed by using reversed-phase Acquity ultra-high-performance liquid chromatography with the column BEH C182.1x 50mm ID with 1.7 µm particle size (Waters) connected to a triple quadrupole Quattro Premier XE mass spectrometer (Waters) with ESI+ mode and MRM channel for ion monitoring. To achieve high extraction recovery, the performance of four different organic solvents and NaCl and (NH4)2SO4, as salting-out reagents, was evaluated. The obtained result shows that SALLE with acetonitrile, as an organic solvent, and ammonium sulfate, as a salting-out reagent, was effective both in terms of reducing matrix interference and increasing extraction recovery. Additionally, acetonitrile with 10% methanol and saturated ammonium sulfate was selected for the current SALLE experiment, as they yield high analyte recovery and less ion suppression. Finally, the optimized SALLE method was validated according to the Eurachem guide. The method showed high linearity (R2 > 0.997) for all hormones. The limit of detection (LOD) and limit of quantifications (LOQ) ranged between 0.01 to 0.18 ng/mL for all hormones. Furthermore, the recoveries of the method were 76% to 95% for total thyroxine (TT4), 55 % to 74% for cortisol, 81 % to 96 % for cortisone, and 90% to 102% for testosterone, respectively. Moreover, the accuracy of the method was calculated as the bias in (RSD%) and found to be < 8% for TT4 and testosterone and <10 % for cortisone. Similarly, both repeatability and reproducibility of the method were found to be (RSD%) <15% across all hormones. Finally, the developed and validated method was applied to the fish plasma samples and the obtained results were good for all hormones. This thesis finding provides a significant contribution for FishLab AS and other laboratories aiming to identify biomarkers of stress, smoltification, and maturity in fish plasma samples.