Ultra high-performance supercritical fluid chromatography-mass spectrometry emerging as a complementary approach for targeted steroid analysis.

BACKGROUND: Steroid analysis plays a crucial role in biomedical research, yet the simultaneous quantification of multiple steroid classes in biological fluids remains challenging because of their high structural similarity and the resulting crosstalk. Ultra-high-performance supercritical fluid chromatography (UHPSFC) has emerged as a promising alternative to traditional liquid and gas chromatography approaches, offering improved separation of isomers. However, the limited ionization efficiency of 3-hydroxy steroids constrains MS sensitivity not only in UHPSFC-MS/MS. UniSpray (US) represents an alternative ionization source with the potential to enhance method sensitivity compared to a common electrospray ionization (ESI) source, given the improved droplet dispersion and desolvation mechanism of US. Yet, the implementation of US in UHPSFC-MS/MS in steroidomics has not been systematically explored.

RESULTS: A UHPSFC-US-MS/MS method was developed as a complementary approach for profiling and quantification of 36 steroids, comprising a complex mixture of 15 stereoisomers, 17 positional isomers, and 4 additional non-interfering compounds. Despite the high level of isomer coverage, the UHPSFC exhibited excellent chromatographic performance with separation scores higher by 13-17% over UHPLC, observed already during the column screening. Using an optimized CO2/methanol gradient on a Torus 1-AA column, a complete separation of critical isomers was achieved in 14 min, reducing the analysis time by 25% compared to published UHPLC-MS/MS methods. Compared to ESI, US resulted in a 5-12.3× increase in S/N-related MS response for both 3-keto-4-ene and poorly ionizing 3-hydroxy steroids, and exhibited lower matrix effects in human plasma, especially at low concentration levels. The validated UHPSFC-US-MS/MS method matched or exceeded the performance and limits of quantification of published UHPSFC-MS/MS and UHPLC-MS/MS studies while simultaneously resolving a uniquely broad panel of steroid isomers.

SIGNIFICANCE: This study presents a comprehensive UHPSFC-MS/MS method for steroid profiling, integrating the complete separation of an extensive number of steroid isomers, achieved in a shorter analysis time compared to UHPLC, and the first systematic characterization of US in this field. Enhanced method sensitivity, improved limits of quantification, and reduced matrix effects reinforced the potential of US as a complementary ionization source to ESI and provided a valuable reference point for future UHPSFC-US-MS/MS studies. The viability of UHPSFC-MS/MS approaches in biomedical applications has been further supported by successful method validation.