Quantification of Endogenous Steroids and Hormonal Contraceptives in Human Plasma via Surrogate Calibration and UHPLC-MS/MS

Abstract

Quantifying endogenous and exogenous steroids at low concentrations in biological matrices remains a major analytical challenge. Immunoassay-based diagnostics are limited by cross-reactivity, particularly at low levels, prompting a shift toward (ultra)high-performance liquid chromatography-tandem mass spectrometry ((U)HPLC-MS/MS) for clinical applications. A key limitation for endogenous hormone quantification is the absence of a true blank matrix for external calibration. To address this, we developed a surrogate calibration method employing 1,2-dimethylimidazole-5-sulfonyl chloride (DMIS) derivatization for estrogens, enabling sensitive and selective quantification alongside nonderivatized steroids. Stable isotope-labeled surrogate calibrants and internal standards were used to achieve matrix-matched quantification within a clinically relevant range. Parallelism between analytes and surrogate calibrants was systematically verified in plasma across multiple calibration levels. The method was further optimized through the use of narrow-bore UHPLC columns and refined chromatographic conditions to enhance sensitivity and resolution for a broad analyte panel. Combined with efficient protein precipitation and 96-well plate-based solid-phase extraction, the developed assay achieves pg/mL-level quantification in human plasma with high precision and accuracy. This integrated approach uniquely combines surrogate calibration for endogenous steroids with external calibration for exogenous contraceptives, including sensitive DMIS-based derivatization for estrogens, enabling comprehensive hormonal profiling in a single run. Beyond its analytical scope, the method outlines a structured validation strategy, which is aligned with regulatory principles, and may therefore serve as a practical reference for future LC-MS/MS assays employing surrogate calibration.

1

Overlay of normalized chromatograms (quantifier mass transition) of steroids in human plasma. Target analytes are shown in black, surrogate calibrants are marked in blue, and internal standards are highlighted in red. 1: cortisol-d ₄; 2: cortisol; 3: cortisone-d ₈; 4: cortisone; 5: cortisone-¹³C₃; 6: CORT-d ₈; 7: CORT; 8: E3-d ₃ DMIS; 9: E3 DMIS; 10: dienogest-d ₈; 11: dienogest; 12: T; 13: T-¹³C₃; 14: 17OHP-d ₈; 15: 17OHP–¹³C₃; 16: 17OHP; 17: E2 DMIS; 18: E2-¹³C₃ DMIS; 19: DHT-d ₃; 20: DHT; 21: LNG-d ₆; 22: LNG; 23: EE2-d ₄ DMIS; 24: EE2 DMIS; 25: E1-¹³C₆ DMIS; 26: E1-¹³C₃ DMIS; 27: E1 DMIS; 28: NoAc; 29: Preg; 30: Preg-¹³C₂d₂; 31: P-d ₉; 32: P–¹³C₃; 33: P; 34: ChAc-d ₆; 35: ChAc; 36: ALLO-d ₅; 37: ALLO.

2

Boxplot distribution of endogenous and exogenous steroid concentrations in samples collected from females with a natural menstrual cycle (NC), using intrauterine devices (IUD), and using oral contraceptives (OC).