Trace analysis of steroid hormones in tear films via liquid chromatography-high resolution mass spectrometry

In this study, we developed an ultra-performance liquid chromatography-quadrupole-Orbitrap mass spectrometry (UPLC-Q-Orbitrap-MS) method for the analysis of seven steroid hormones in human tears. Tear samples were collected using Schirmer strips and extracted with methanol. The analytes were then subjected to a "one-step" clean-up process using solid phase extraction, and subsequently separated on a C₁₈ column by UPLC. Detection was performed using an Orbitrap MS detector, operated at a resolution of 17 500 FWHM in parallel reaction monitoring mode with an HESI ion source under positive ionization. Our data showed the sensitivity with limits of detection for steroid hormones in tears ranging from 0.12 to 0.86 pg μL^-1, and high correlation coefficients in the corresponding concentration range exceeding 0.99. The results also had high accuracy with spiking recoveries for spiked tear samples ranging from 78.2% to 96.7% and relative deviations of less than 15%. Furthermore, we successfully applied our method to detect the pg μL^-1 level of steroid hormones in real human tear samples. Our findings showed the potential of this UPLC-Q-Orbitrap-MS method for the accurate and sensitive determination of steroid hormones in human tears, providing a valuable tool for ophthalmic and endocrine research.