Picogram-level quantification of some growth hormones in bovine urine using mixed-solvent bubble-in-drop single drop micro-extraction.

Growth hormones are important biologically active compounds. However, they can cause deleterious effects if not used with care and their use in farmed animals is banned in the European Union. This study presents the development and application of a mixed-solvent "bubble-in-drop single drop micro-extraction" (BID-SDME) method for enrichment of stilbene hormones in bovine urine samples. The hormones are quantified using GC-MS showing good linearity with the coefficient of determination (R(2)) of 0.999 and 0.999 for hexestrol and diethylstilbestrol, respectively, in the concentration range 0.05-10 ng mL(-1). Excellent precision (RSD<10%) and accuracy (using a bovine urine certified reference material) were obtained. The observed detection capability (CCα, or LOD) values were 0.01 ng mL(-1) (hexestrol), 0.03 ng mL(-1) (cis-diethylstilbestrol) and 0.02 ng mL(-1) (trans-diethylstilbestrol), respectively, while the decision limit (CCβ, or LOQ) values were 0.03 ng mL(-1), 0.08 ng mL(-1) and 0.07 ng mL(-1), which are comparable to or better than those reported in literature. Importantly, sample handling is significantly simplified by our method and enrichment values are greatly enhanced. The results show that a 3:1 chloroform/toluene mixture gave the highest extraction efficiency with a drop-bubble ratio of 2:1. We highlight the importance of solvent density on the success of the BID-SDME method.