Abstract
A sensitive and rapid high performance liquid chromatography–mass spectrometry (HPLC–MS) method was developed and validated for simultaneous quantification of ten steroid hormones, including estrogens, androgens, progesterones, and corticosteroids four classes of steroids. The following ten steroid hormones were analyzed: progesterone, 21-deoxycortisol, estrone, 4-androstenedione, testosterone, dihydro-testosterone, androstenone, dehydroepiandrosterone, corticosterone and cortisone. Stable deuterated isotopes were used as internal standards for quantification. Sample preparation with and without derivatization were performed after liquid–liquid extraction, and the corresponding results were compared according to sensitivity and selectivity. Hydroxylamine derivatization was found to improve the ionization efficiency of the analytes for electrospray ionization MS analysis. The gradient of mobile phase and experimental parameters for HPLC separation were optimized. The lower limits of quantification were in the range of 0.05–5 ng mL−1 with wide linear range for the ten steroid hormones. The intra-day precision < 11.1% and recovery of 84.5–120% with negligible matrix effect were achieved, where within the acceptance limits of the FDA guideline. Total HPLC–MS analysis time was 6 min. This method enables simultaneous quantification of steroids in human serum. It will be helpful for the serum steroid profiling in order to understand various endocrinology diseases.Graphical
Highlights
Steroid hormones are a class of tetracyclic aliphatic hydrocarbons with cyclopentane polyhydrophenanthrene nuclei, which play an important regulatory role in various physiological activities of human body and are indispensable hormones for maintaining life [1]
Because steroid hormones present in very low concentration in organisms, the structures of different steroid hormones are very similar and their polarity is relatively small, it is of challenge to detect and quantify steroids with high precision and accuracy
A sensitive method for the simultaneous determination of ten steroid hormones in human serum was established by liquid–liquid extraction (LLE), derivatization, stable isotope labeling and HPLC–electrospray ionization (ESI)–Q-TOF–MS
Summary
Steroid hormones are a class of tetracyclic aliphatic hydrocarbons with cyclopentane polyhydrophenanthrene nuclei, which play an important regulatory role in various physiological activities of human body and are indispensable hormones for maintaining life [1]. Immunoassay is the mainstream method for steroid analysis in clinic in the past few decades, which has the advantages of high throughput and fast speed This method is vulnerable to interference by body fluid matrix or structural analogues, resulting in the lack of specificity and accuracy [2, 3]. Some trace and non-ionizable hormones cannot be detected by conventional methods, but with the continuous improvement of ionization technology, chemical derivatization has become a mature strategy in improving the sensitivity and detection limit of steroid hormones [11, 12] It improves the ionization efficiency during electrospray ionization (ESI) process by adding ionizable groups to the analyte which makes more analytes charged into mass spectrometry, and changes the structure of the analyte and chromatographic separation behavior, and so more structural analogues or interferences can be separated. The method has three advantages that are small sample consumption, short detection time and more hormones detected at one injection
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