Abstract
Urinary volatile compounds (VCs) have been recently assessed for disease diagnoses. They belong to very diverse chemical classes, and they are characterized by different volatilities, polarities and concentrations, complicating their analysis via a single analytical procedure. There remains a need for better, lower-cost methods for VC biomarker discovery. Thus, there is a strong need for alternative methods, enabling the detection of a broader range of VCs. Therefore, the main aim of this study was to optimize a simple and reliable liquid–liquid extraction (LLE) procedure for the analysis of VCs in urine using gas chromatography-mass spectrometry (GC-MS), in order to obtain the maximum number of responses. Extraction parameters such as pH, type of solvent and ionic strength were optimized. Moreover, the same extracts were analyzed using Proton Nuclear Magnetic Resonance Spectroscopy (1H-NMR), to evaluate the applicability of a single urine extraction for multiplatform purposes. After the evaluation of experimental conditions, an LLE protocol using 2 mL of urine in the presence of 2 mL of 1 M sulfuric acid and sodium sulphate extracted with dichloromethane was found to be optimal. The optimized method was validated with the external standards and was found to be precise and linear, and allowed for detection of >400 peaks in a single run present in at least 50% of six samples—considerably more than the number of peaks detected by solid-phase microextracton fiber pre-concentration-GC-MS (328 ± 6 vs. 234 ± 4). 1H-NMR spectroscopy of the polar and non-polar extracts extended the range to >40 more (mainly low volatility compounds) metabolites (non-destructively), the majority of which were different from GC-MS. The more peaks detectable, the greater the opportunity of assessing a fingerprint of several compounds to aid biomarker discovery. In summary, we have successfully demonstrated the potential of LLE as a cheap and simple alternative for the analysis of VCs in urine, and for the first time the applicability of a single urine solvent extraction procedure for detecting a wide range of analytes using both GC-MS and 1H-NMR analysis to enhance putative biomarker detection. The proposed method will simplify the transport between laboratories and storage of samples, as compared to intact urine samples.
Highlights
Modern metabolomics is a high-throughput approach for the monitoring of metabolites in biological tissue or fluid in a defined time point
A specific class of metabolomics focused on the profile of volatile compounds (VCs) is termed volatolomics, the applications of which for diagnostic purposes is growing [8,9,10,11,12,13,14,15]
We successfully demonstrated for the first time the applicability of single urine solvent extracts for both Gas Chromatography-Mass Spectrometry (GC-MS) as well as 1 H-Nuclear MagneticResonance Spectroscopy (NMR) analysis, for volatile and semi-volatile compound analysis
Summary
Modern metabolomics is a high-throughput approach for the monitoring of metabolites in biological tissue or fluid in a defined time point. The profile of metabolites may vary during pathological states, hormonal changes, exposure to environmental pollutants, diet, etc., and the changes can be determined in different biological specimens, such as urine, saliva, blood, skin, feces, breath and sweat [1,2]. Because of the non-invasive methods of collection and the richness of metabolites, is a commonly used fluid in metabolite profiling [3,4], potentially giving a large amount of information about the metabolic state of the body. Urine is frequently analyzed using Liquid Chromatography-Mass. Spectrometry (LC-MS), Gas Chromatography-Mass Spectrometry (GC-MS) and Nuclear Magnetic. VCs are secreted by cells of the human body, as a result of their metabolism
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