Optimisation of Urine Sample Preparation for Headspace-Solid Phase Microextraction Gas Chromatography-Mass Spectrometry: Altering Sample pH, Sulphuric Acid Concentration and Phase Ratio.

Prashant Aggarwal,Chris Probert,Mark T Boyd,James Baker,Séamus Coyle,Elinor A Chapman

doi:10.3390/metabo10120482

Abstract

Headspace-solid phase microextraction gas chromatography-mass spectrometry (HS-SPME-GC-MS) can be used to measure volatile organic compounds (VOCs) in human urine. However, there is no widely adopted standardised protocol for the preparation of urine samples for analysis resulting in an inability to compare studies reliably between laboratories. This paper investigated the effect of altering urine sample pH, volume, and vial size for optimising detection of VOCs when using HS-SPME-GC-MS. This is the first, direct comparison of H2SO4, HCl, and NaOH as treatment techniques prior to HS-SPME-GC-MS analysis. Altering urine sample pH indicates that H2SO4 is more effective at optimising detection of VOCs than HCl or NaOH. H2SO4 resulted in a significantly larger mean number of VOCs being identified per sample (on average, 33.5 VOCs to 24.3 in HCl or 12.2 in NaOH treated urine) and more unique VOCs, produced a more diverse range of classes of VOCs, and led to less HS-SPME-GC-MS degradation. We propose that adding 0.2 mL of 2.5 M H2SO4 to 1 mL of urine within a 10 mL headspace vial is the optimal sample preparation prior to HS-SPME-GC-MS analysis. We hope the use of our optimised method for urinary HS-SPME-GC-MS analysis will enhance our understanding of human disease and bolster metabolic biomarker identification.

Highlights

This paper investigates the effect of altering urine sample volume, vial size, and pH for optimising detection of volatile organic compounds (VOCs) when using HS-solid phase microextraction (SPME)-gas chromatography-mass spectrometry (GC-MS)
1 M H2 SO4 has been used; we found that both 2.5 M and 5 M H2 SO4 were significantly more effective at producing VOCs detected using our HS-SPME-GC-MS method [8]
We optimised the detection of VOCs in urine using HS-SPME-GC-MS by altering pH, H2 SO4 concentration, and phase ratio

Summary

Introduction

Metabolomics identifies de novo or changing metabolites, often in the form of volatile organic compounds (VOCs) from biological samples [1]. VOCs usually have fewer than 12 carbon atoms, a boiling point of less than 300 ◦ C, and are biological or synthetic in origin [3,5,6,7]. They have a high vapour pressure and low molecular weight enabling them to enter the gas phase at ambient temperatures; they may have an odour and emanate from urine, faeces, saliva, breath, and other bodily products [5,8]. VOCs are produced by a variety of processes including degradation from metabolic pathway intermediates, and, their concentration in samples can give an insight into biochemical activity upstream, with potential to be biomarkers of disease [2,6]

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Conclusion