Innovative molecular networking analysis of steroids and characterisation of the urinary steroidome.

Steroids are cholesterol-derived biomolecules that play an essential role in biological processes. These substances used as growth promoters in animals are strictly regulated worldwide. Targeted assays are the conventional methods of monitoring steroid abuse, with limitations: only detect known metabolites. Metabolism leads to many potential compounds (isomers), which complicates the analysis. Thus, to overcome these limitations, non-targeted analysis offers new opportunities for a deeper understanding of metabolites related to steroid metabolism. Molecular networking (MN) appears to be an innovative strategy combining high-resolution mass spectrometry and specific data processing to study metabolic pathways. In the present study, two databases and networks of steroids were constructed to lay the foundations for the implementation of the GNPS-MN approach. Steroids of the same family were grouped together, nandrolone and testosterone were linked to other analogues. This network and associated database were then applied to a few urine samples in order to demonstrate the annotation capacity in steroidome study. The results show that MN strategy could be used to study steroid metabolism and highlight biomarkers.

Cyanoacetohydrazide as a Novel Derivatization Agent for the Determination of UHPLC-HRMS Steroids in Urine.

The possibility of cyanoacetohydrazide usage as a novel derivatizing agent is demonstrated in the presented article, and a comparison with hydroxylamine as the most commonly used reagent is provided. Optimal conditions for steroid derivatization with cyanoacetohydrazide are provided. According to the collected data, the maximum yield of derivatives was observed at pH 2.8 within 70 min at 40 °C with 5 ng/mL limit of detection for all investigated analytes. It was shown that cyanoacetohydrazide derivatives produces both syn- and anti-forms as well as hydroxylamine, and their ratios were evaluated and shown in presented work. An efficiency enchantment from two to up to five times was achieved with a novel derivatization reagent. Its applicability for qualitative analysis of steroids in urine was presented at real samples. Additionally, the reproducible fragmentation of the derivatizing agent in collision-induced dissociation offers opportunities for simplified non-targeted steroidomic screening. Furthermore, cyanoacetohydrazide increases ionization efficiency in positive mode, which can eliminate the need for redundant high-resolution instrument runs required for both positive and negative mode analyses.

Urinary steroid profiling using liquid chromatography-tandem mass spectrometry for the diagnosis of canine Cushing's syndrome.

Serum cortisol measurements by chemiluminescence enzyme immunoassay (CLEIA) are widely used to diagnose hypercortisolism (HC) or Cushing's syndrome in dogs. However, they are associated with problems such as the need for multiple blood collections under stressful conditions or cross-reactivity between hormones. Therefore, a less invasive and more accurate diagnostic method is required. This study aimed to develop a urinary steroid profile analysis method using liquid chromatography-tandem mass spectrometry (LC/MS/MS) and to evaluate its clinical usefulness. Sixty-five healthy dogs and 38 dogs with suspected HC were included in the study. Using LC/MS/MS, the levels of 11 steroid hormones in the urine were determined. We established the upper limit of the reference interval for each urinary steroid-to-creatinine ratio and evaluated their diagnostic performances. The levels of the five steroid hormones were significantly higher in the 14 dogs with HC than in the 24 dogs with mimicking HC and 65 healthy dogs. The urinary corticosterone-to-creatinine ratio showed the highest diagnostic accuracy (area under the curve, 0.96). A significant correlation was seen between urinary cortisol concentrations measured by LC/MS/MS and CLEIA (rs = 0.88, P <0.001), although the CLEIA measurements were significantly higher than the LC/MS/MS measurements (P <0.001). LC/MS/MS-based urinary steroid profiles are a promising tool for diagnosing canine HC.

Profiling of urinary steroids aided by lithium ion adduction-based ultrahigh-performance liquid chromatography-tandem mass spectrometry.

RATIONALE: As 3-OH-containing steroids are prone to dehydration by conventional electrospray ionization, reducing detection sensitivity, Li ion adduction-based ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC/MS/MS), developed to prevent dehydration and effectively detect 3-OH steroids, was applied for profiling total and free steroids in urine. METHODS: Free urinary steroids were isolated directly from urine by solid-phase extraction (SPE) with 80% acetonitrile. The total steroids were prepared by enzymatic treatment of urine with a cocktail of sulfatase and glucronidase, protein precipitation, and separation with the above SPE. In order to detect as many steroid types as possible, UHPLC/MS/MS (Li method) with Li+ solution added after the column was used for analysis in addition to the conventional method of detecting protonated ions (H method). The 13 3-OH steroids and the remaining 16 steroids were quantified by standard curves prepared using product ion transitions derived from [M + Li]+ and MH+ , respectively. RESULTS: Two groups of human urine, male and female urine, were analyzed. 3-OH steroids could be detected with greater sensitivity using the Li method than the conventional method. The absolute amounts of each steroid were normalized based on creatinine levels. The difference between the male and female groups are clearly attributable to sex steroids. CONCLUSIONS: Twenty-nine total steroids and 19 free steroids were identified in a limited volume (240 mL) of urine. Of these, 13 3-OH steroids were better detected by Li+ adduction-based UHPLC/MS/MS.

Urinary 17-ketosteroids and ketogenic steroids in a mixed Jamaican population

The mean daily 17-ketosteroid excretion for a group of coloured Jamaicans was not found to be significantly different from that of Europeans living under the same conditions. Mean 17-ketogenic steroid excretion was significantly lower in the Jamaicans (Summary)