Targeted Metabolite Profiling: Sample Preparation Techniques for GC-MSBased Steroid Analysis

Sadanala, Krishna Chaitanya; Lee, Jeongae; Chung, Bong Chul; Choi, Man Ho

doi:10.5478/msl.2012.3.1.004

Cited by 6 publications

(7 citation statements)

References 62 publications

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“…Metabolomics comprises qualitative and quantitative analysis of intracellular and intercellular metabolites using two distinct analytical approaches: (1) non-targeted metabolite profiling, i.e. identification and characterization of a large number of metabolites and their precursors; and (2) targeted metabolite profiling, with an emphasis on quantitative variations in metabolites of interest (e.g., amino acids, carbohydrates, lipids and nucleotides) based on a prior understanding of their biological roles in metabolic pathways ( Sadanala et al, 2012 ).…”

Section: Introductionmentioning

confidence: 99%

A Metabolomic Approach to Understanding the Metabolic Link between Obesity and Diabetes

Park¹,

Sadanala²,

Kim³

2015

Molecules and Cells

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132

117

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Obesity and diabetes arise from an intricate interplay between both genetic and environmental factors. It is well recognized that obesity plays an important role in the development of insulin resistance and diabetes. Yet, the exact mechanism of the connection between obesity and diabetes is still not completely understood. Metabolomics is an analytical approach that aims to detect and quantify small metabolites. Recently, there has been an increased interest in the application of metabolomics to the identification of disease biomarkers, with a number of well-known biomarkers identified. Metabolomics is a potent approach to unravel the intricate relationships between metabolism, obesity and progression to diabetes and, at the same time, has potential as a clinical tool for risk evaluation and monitoring of disease. Moreover, metabolomics applications have revealed alterations in the levels of metabolites related to obesity-associated diabetes. This review focuses on the part that metabolomics has played in elucidating the roles of metabolites in the regulation of systemic metabolism relevant to obesity and diabetes. It also explains the possible metabolic relation and association between the two diseases. The metabolites with altered profiles in individual disorders and those that are specifically and similarly altered in both disorders are classified, categorized and summarized.

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Section: Introductionmentioning

confidence: 99%

A Metabolomic Approach to Understanding the Metabolic Link between Obesity and Diabetes

Park¹,

Sadanala²,

Kim³

2015

Molecules and Cells

Self Cite

132

117

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“…It also allows the separation and detection of regioisomeric conjugates. However, hydrolysis of conjugated androgen using enzymatic digestion is still commonly performed in the laboratories equipped with GC–MS/MS because of the low volatility and low thermal stability of the conjugated analytes [46]. In addition, ion suppression due to matrix effects affects the direct quantitation of steroid conjugates (e.g.…”

Section: Discussionmentioning

confidence: 99%

Simultaneous quantitation of nine hydroxy-androgens and their conjugates in human serum by stable isotope dilution liquid chromatography electrospray ionization tandem mass spectrometry

Zang

Tamae

Mesaros

et al. 2017

The Journal of Steroid Biochemistry and Molecular Biology

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Castration resistant prostate cancer (CRPC), the fatal form of prostate cancer, remains androgen dependent despite castrate levels of circulating testosterone (T) and 5α-dihydrotestosterone (DHT). To investigate mechanisms by which the tumor can synthesize its own androgens and develop resistance to abiraterone acetate and enzalutamide, methods to measure a complete androgen profile are imperative. Here, we report the development and validation of a stable isotope dilution liquid chromatography electrospray ionization tandem mass spectrometric (SID-LC-ESI-MS/MS) method to quantify nine human hydroxy-androgens as picolinates, simultaneously with requisite specificity and sensitivity. In the established method, the fragmentation patterns of all nine hydroxy-androgen picolinates were identified, and [ 13 C 3 ]-5α-androstane-3α, 17β-diol and [ 13 C 3 ]-5α-androstane-3β, 17β-diol used as internal standards were synthesized enzymatically. Intra-day and inter-day precision and accuracy corresponds to the U.S. Food and Drug Administration Criteria for Bioanalytical Method Validation. The lower limit of quantitation (LLOQ) of nine hydroxy-androgens is 1.0 pg to 2.5 pg on column. Diols which have been infrequently measured: 5-androstene-3β, 17β-diol and 5α-androstane-3α, 17β-diol can be determined in serum at values as low as 1.0 pg on column. The method also permits the quantitation of conjugated hydroxy-androgens following enzymatic digestion. While direct detection of steroid conjugates by electrospray-ionization tandem mass spectrometry has advantages the detection of unconjugated and conjugated steroids would require separate methods for each set of analytes. Our method was applied to pooled serum from male and female donors to provide reference values for both unconjugated and conjugated hydroxy-androgens. This method will allow us to interrogate the involvement of the conversion of 5-androstene-3β, 17β-diol to T, the backdoor pathway involving the conversion of 5α-androstane-3α, 17β-diol to DHT and the inactivation of DHT to 5α-androstane-3β, 17β-diol in advanced prostate cancer. HHS Public Access

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“…In addition, the chemical derivatization‐induced formation of a steroid with unclear fragmentation may lead to a product whose mass spectrum shows better diagnostic patterns and expected fragments. Silylation is the most widely used derivatization reaction in steroid analysis; active hydrogens from acid, alcohol, thiol, amine, and amide groups can be protected, mainly with TMS groups (Poole & Zlatkis, ; Choi et al, , ; Sadanala et al, ). In general, silylation reagents are moisture‐sensitive, but an excess of the silylating agent can be used to minimize problematic moisture.…”

Section: Biological Sample Preparationmentioning

confidence: 99%

Bringing GC–MS profiling of steroids into clinical applications

Choi

Chung

2014

Mass Spectrometry Reviews

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Abnormalities of steroid biosynthesis and excretion are responsible for the development and prevention of endocrine disorders, such as metabolic syndromes, cancers, and neurodegenerative diseases. Due to their biochemical roles in endocrine system, qualitative and quantitative analysis of steroid hormones in various biological specimens is needed to elucidate their altered expression. Mass spectrometry (MS)-based steroid profiling can reveal the states of metabolites in biological systems and provide comprehensive insights by allowing comparisons between metabolites present in cells, tissues, or organisms. In addition, the activities of many enzymes related to steroid metabolism often lead to hormonal imbalances that have serious consequences, and which are responsible for the progress of hormone-dependent diseases. In contrast to immunoaffinity-based enzyme assays, MS-based methods are more reproducible in quantification. In particular, high-resolution gas chromatographic (GC) separation of steroids with similar chemical structures can be achieved to provide rapid and reproducible results with excellent purification. GC-MS profiling therefore has been widely used for steroid analysis, and offers the basis for techniques that can be applied to large-scale clinical studies. Recent advances in analytical technologies combined with inter-disciplinary strategies, such as physiology and bioinformatics, will help in understanding the biochemical roles of steroid hormones. Therefore, comprehensive analytical protocols in steroid analysis for different research purposes may contribute to the elucidation of complex metabolic processes relevant to steroid function in many endocrine disorders, and in the identification of diagnostic biomarkers.

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Targeted Metabolite Profiling: Sample Preparation Techniques for GC-MSBased Steroid Analysis

Cited by 6 publications

References 62 publications

A Metabolomic Approach to Understanding the Metabolic Link between Obesity and Diabetes

A Metabolomic Approach to Understanding the Metabolic Link between Obesity and Diabetes

Simultaneous quantitation of nine hydroxy-androgens and their conjugates in human serum by stable isotope dilution liquid chromatography electrospray ionization tandem mass spectrometry

Bringing GC–MS profiling of steroids into clinical applications

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