Salting-Out Assisted Liquid-Liquid Extraction for UPLC-MS/MS Determination of Thyroxine and Steroid Hormones in Human Serum and Fish Plasma

Abstract

Measuring the level of steroid and thyroxine hormones is key to understanding organism health conditions. Liquid chromatography coupled with tandem mass spectrometry has become the method of choice for such hormone analyses in clinical laboratories. Detection of hormones at low levels typically requires a time-consuming sample preparation, such as liquid-liquid extraction followed by solvent evaporation and re-solubilization of the sample extract. Instead, we applied salting-out assisted liquid-liquid extraction (SALLE) for the extraction of thyroxine, testosterone, cortisone, and cortisol from human serum and fish plasma samples. SALLE allowed direct injection of sample extracts. Sodium chloride and ammonium sulfate were evaluated as salting-out reagents together with four different organic solvents. High extraction recovery and reduced matrix interference were achieved by using ammonium sulfate together with 10% methanol in acetonitrile. Limits of quantification were in the range of 0.1–0.2 ng/mL and signal responses were linear (R2 > 0.997) up to at least 100 ng/mL for all hormones. The method was applied for hormone measurements in fish plasma. In conclusion, SALLE combines the simplicity of crude protein precipitation with the high analyte enrichment of a liquid-liquid extraction. Here we have presented it as a novel sample preparation method for clinical laboratories where mass spectrometry is utilized in the field of endocrinology.

Sensitivity Enhancement for Separation-Based Analytical Techniques Utilizing Solid-Phase Enrichment Approaches and Analyte Derivatization for Trace Analysis in Various Matrices

“…Furthermore, it is broadly applicable for the enrichment of pharmaceuticals and extraction of biological interferences with high removal effectiveness. SPE is seen as a good substitute for LLE because it moves past many of the shortcomings of LLE [16][17][18]. Moreover, the entire procedure can be automated.…”

“…Additionally, the offline sample preparation approach is anticipated to cause more than one-third of the analytical errors [15]. Conventional sample work-up processes in bio-analysis frequently entail a variety of pretreatment stages to purify the protein-rich samples before they are loaded into the LC, but three of the most frequent are solid-phase extraction (SPE) [3,4], liquid-liquid extraction (LLE) [16][17][18], and protein precipitation [19].…”

Method Development for Aminopyralid Residues Determination by Ultra-High-Performance Liquid Chromatography Coupled to Tandem Mass Spectrometry in Straw

“…However, the QuEChERS method, including its modifications, is primarily intended for the purposes of multi-residue analyses involving a wide range of different, but mainly less polar, analytes [25]. Thus, for aminopyralid, as a polar acidic compound easily soluble in water, the salting-out assisted liquid-liquid extraction (SALLE) may be a better alternative [26]. According to European Union Reference Laboratories (EURLs), QuEChERS-based methodology provides a 0.05 mg/kg limit of quantitation (LOQ) for the majority of routinely analyzed matrices, which is sufficient efficiency for all maximum residue levels (MRLs) recommended by EFSA in common crop grains (with exception of rape seeds) [2].…”