Abstract
BackgroundVolatile organic compounds (VOCs) can be intermediates of metabolic pathways and their levels in biological samples may provide a better understanding about diseases in addition to potential methods for diagnosis. Headspace analysis of VOCs in urine samples using solid phase micro extraction (SPME) coupled to gas chromatography - mass spectrometry (GC-MS) is one of the most used techniques. However, it generally produces a limited profile of VOCs if applied to fresh urine. Sample preparation methods, such as addition of salt, base or acid, have been developed to improve the headspace-SPME-GC-MS analysis of VOCs in urine samples. These methods result in a richer profile of VOCs, however, they may also add potential contaminants to the urine samples, result in increased variability introduced by manually processing the samples and promote degradation of metabolites due to extreme pH levels. Here, we evaluated if freeze-drying can be considered an alternative sample preparation method for headspace-SPME-GC-MS analysis of urine samples.ResultsWe collected urine from three volunteers and compared the performances of freeze-drying, addition of acid (HCl), addition of base (NaOH), addition of salt (NaCl), fresh urine and frozen urine when identifying and quantifying metabolites in 4 ml samples. Freeze-drying and addition of acid produced a significantly higher number of VOCs identified than any other method, with freeze-drying covering a slightly higher number of chemical classes, showing an improved repeatability and reducing siloxane impurities.ConclusionIn this work we compared the performance of sample preparation methods for the SPME-GC-MS analysis of urine samples. To the best of our knowledge, this is the first study evaluating the potential of freeze-dry as an alternative sample preparation method. Our results indicate that freeze-drying has potential to be used as an alternative method for the SPME-GC-MS analysis of urine samples. Additional studies using internal standard, synthetic urine and calibration curves will allow a more precise quantification of metabolites and additional comparisons between methods.Graphical abstractEnhancing VOC profiling from urine samples.Electronic supplementary materialThe online version of this article (doi10.1186/s13065-016-0155-2) contains supplementary material, which is avaialble to authorize users.
Highlights
Volatile organic compounds (VOCs) can be intermediates of metabolic pathways and their levels in biological samples may provide a better understanding about diseases in addition to potential methods for diagnosis
The urine samples were all randomly analysed by headspace-solid phase micro extraction (SPME)-gas chromatography - mass spectrometry (GC-MS)
Experimental conditions The following treatments or sample preparation methods were applied to the urine samples collected from each volunteer prior to analysis by headspace-SPME-GC-MS: Fresh, where five aliquots were kept at room temperature and quickly analysed after collection; Frozen, where five aliquots were frozen at −80 °C and defrosted; Freeze-dry, where five aliquots were frozen at −80 °C and freezedried for 18 h; NaCl, where five aliquots were frozen at −80 °C, defrosted and treated with 1 ml of salt solution; HCl, where five aliquots were frozen at −80 °C, defrosted and treated with 1 ml of acid solution (i.e. HCl 5M); and NaOH, where five aliquots were frozen at −80 °C, defrosted and treated with 1 ml of base solution (i.e. NaOH 5M)
Summary
Freeze‐drying: an alternative method for the analysis of volatile organic compounds in the headspace of urine samples using solid phase micro‐extraction coupled to gas chromatography ‐ mass spectrometry. M. Aggio1* , Arno Mayor, Séamus Coyle, Sophie Reade, Tanzeela Khalid, Norman M.
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