Estimation of Steroid Hormones in Biological Samples Using Micro Extraction and Advanced Chromatography Techniques

Review Article

Austin J Anal Pharm Chem. 2022; 9(3): 1150.

Estimation of Steroid Hormones in Biological Samples Using Micro Extraction and Advanced Chromatography Techniques

Anshuman Srivastava¹, Madan Madhav Godbole² and Ashutosh Shrivastava¹*

1Center for Advanced Research, King George’s Medical University, Lucknow, India

2Jawaharlal Nehru Medical College, KLE University, Belagavi, Karnataka, India

*Corresponding author: Dr. Ashutosh Shrivastava, Center for Advanced Research, Faculty of Medicine, King George’s Medical University, Lucknow, India

Received: September 27, 2022; Accepted: November 08, 2022; Published: November 15, 2022

Abstract

Steroid profiling plays an important role in the clinical setting and also in the diagnosis of various physiological disorders. Steroids in the research laboratory and patient care has been routinely measured by immunoassay. Limitations of immunoassay in quantifying steroids have been well documented and the advent of advanced mass spectrometry is offering a viable alternative to measure multiple steroids in single reaction. Analytes ranging from the steroid and their metabolites that are present in the body carry out various important functions and are relevant to maintaining homeostasis. Blood plasma and urine samples are the clinical material for such analysis, however, due to the difference in polarity of the target steroid, a major challenge exists as to how faithfully analyze different steroids in the given sample type. These steroids are usually present at very low concentrations in the body and are present in several forms leading to increased complexity. Achieving an excellent chromatographic separation for the analysis of steroids requires an effective sample preparation and analysis procedure. Hence various methods are developed to analyze the presence of steroids and to know their exact concentration in the body. This review covers various analytical methodologies such as chromatographic procedures and downstream mass spectrometry for the identification and measurement of the levels of steroid hormones. At the same time, this review also covers the most recent developments that have taken place in the recent past to cover the enhanced understanding of steroid hormone analysis.

Keywords: Steroid Hormone; Gas Chromatography-Mass Spectrometry; Liquid Chromatography-Mass Spectrometry; Sample extraction procedure

Abbreviations

BSTFA, N, O-Bis(Trimethylsilyl)Trifluoroacetamide; DBS: Dried Blood Spot; DHEA: Dehydroepiandrosterone; DHT: Dihydrotestosterone; GC-MS/MS: Gas Chromatography-Mass Spectrometry; LOD: Limit of Detection; LOQ: Limit of Quantification; LC-MS/MS: Liquid Chromatography-Mass Spectrometry; LLE: Liquid-Liquid Extraction; MALDI: Matrix-Assisted Laser Desorption Ionization; MSTFA: N-Methyl-N-(trimethylsilyl)trifluoroacetamide: 17-OHP: 17 a-Hydroxyprogesterone; SPE: Solid Phase Extraction; UPLC: Ultra Performance Liquid Chromatography; UPLC (QTOF) MS: Ultra Performance Liquid Chromatography Quadrupole Timeof- Flight (QTOF) Mass Spectrometers; UHPSFC-MS/MS: Ultra- High Performance Supercritical Fluid Chromatography-Tandem Mass Spectrometry

Introduction

Steroid hormones play a significant role in regulating different body functions, including salt and water balance, immune response, ability to endure illness and injury, and development of secondary sexual characteristics [1-3]. Steroid hormones are a biologically active group of molecules derived from cholesterol [4]. Any dysregulation in the biosynthesis, metabolism and excretion of steroids could lead to an endocrine disorder. Endocrine disease which is associated with the alteration of steroids can be diagnosed by the quantification of steroids and the ratio analysis of their specific pathway products.

The hyphenated chromatography technique, coupled with mass spectrometry, is gradually becoming a significant approach for the quantification and identification of steroid hormones in biological samples [5,6]. Fast analysis, improved selectivity, and the need for only a small sample volume make this type of technique superior in comparison to traditional and routinely used immunoassay methods [7]. Immunoassay methods are selective and helpful in the detection of a single steroid hormone. However, the robustness of the data, accuracy, and reproducibility of the immunoassay process is not high due to a lot of matrix interference and batch-to-batch variation in the antibodies [8,9].

Liquid Chromatography-Mass Spectrometry (LC-MS/MS) and Gas Chromatography-Mass Spectrometry (GC-MS/MS) are advanced chromatography approaches in the field of steroid hormone analysis [10]. Liquid chromatography-mass spectrometry is an excellent tool in comparison to GC-MS/MS in respect of the determination of individual steroids, but in the case of metabolite identification, GCMS/ MS shows a better response [11,12]. A lot of research studies have been undertaken on the identification and quantification of steroids by using the above chromatography techniques, but only few of those studies are robust and reliable [10,13]. The additional recent analytical approach that has become more prevalent in the analysis of steroids over the past few years is Ultra-High Performance Supercritical Fluid Chromatography-Tandem Mass Spectrometry (UHPSFC-MS/MS) and Matrix-Assisted Laser Desorption Ionization (MALDI-MS). These chromatography instruments are designed to resolve numerous challenging retention/separation problems.

Biological samples are complex and are not directly compatible with mass spectrometry. Therefore, a fast, selective, effective, and robust analytical sample extraction procedure is always important before the chromatographic separation and analysis of biological samples [14]. Generally, sample extraction is a time-consuming procedure and needs to be highly accurate. Sample extraction has conventionally been executed using Liquid-Liquid Extraction (LLE) [15,16], Solid-Phase Extraction (SPE) [17,18], and Protein Precipitation (PP) [9]. The manual operations associated with these techniques are labor-intensive and time-consuming. Modern trends in sample extraction procedures include Supported Liquid Extraction (SLE), Dried Blood Spot (DBS) [20,21], and Dispersive Liquid-Liquid microextraction (DLLME).

The measurement of steroid hormones is generally performed for public health assessment, patient care, diagnosis and clinical research. All steroid hormones are derivatives of cholesterol which is generally a waxy substance present in blood and animal tissue (Figure 1). The transportation of steroid hormones in bloodstream takes place with carrier globulin proteins in a bound form. Abnormalities in the production and metabolism of steroid hormones could potentially lead to various endocrine disorders. For the diagnosis, and treatment of endocrine-related diseases, the absolute quantification of steroid hormones and their metabolites is necessary. In this review, contemporary development in conventional and modern approaches for sample extraction for the analysis of steroids using chromatographic separation and mass spectrometry analysis have been drawn and reviewed. Attention has also been placed on different modern mass spectrometry instruments for the quantification of steroids in different biological samples.