Aggio Raphael B M, Mayor Arno, Coyle Séamus, Reade Sophie, Khalid Tanzeela, Ratcliffe Norman M, Probert Chris S J
Department of Cellular and Molecular Physiology, Institute of Translational Medicine, University of Liverpool, Crown Street, L693BX Liverpool, UK.
Marie Curie Palliative Care Institute Liverpool, University of Liverpool, London Road, L39TA Liverpool, UK.
Chem Cent J. 2016 Mar 1;10:9. doi: 10.1186/s13065-016-0155-2. eCollection 2016.
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. 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.
We 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.
In 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.
挥发性有机化合物（VOCs）可能是代谢途径的中间体，其在生物样品中的水平除了可作为潜在的诊断方法外，还能让我们更好地了解疾病。使用固相微萃取（SPME）结合气相色谱-质谱联用（GC-MS）对尿液样本中的VOCs进行顶空分析是最常用的技术之一。然而，如果应用于新鲜尿液，它通常只能检测到有限的VOCs种类。已开发出如添加盐、碱或酸等样品制备方法，以改进尿液样本中VOCs的顶空-SPME-GC-MS分析。这些方法能检测到更丰富的VOCs种类，但也可能会向尿液样本中添加潜在污染物，因人工处理样本而导致变异性增加，且极端pH值会促使代谢物降解。在此，我们评估了冷冻干燥是否可被视为尿液样本顶空-SPME-GC-MS分析的另一种样品制备方法。
我们收集了三名志愿者的尿液，并比较了冷冻干燥、添加酸（HCl）、添加碱（NaOH）、添加盐（NaCl）、新鲜尿液和冷冻尿液在鉴定和定量4毫升样本中代谢物时的性能。冷冻干燥和添加酸所鉴定出的VOCs数量明显多于其他任何方法，冷冻干燥涵盖的化学类别略多，显示出更好的重复性并减少了硅氧烷杂质。
在这项工作中，我们比较了尿液样本SPME-GC-MS分析的样品制备方法的性能。据我们所知，这是第一项评估冷冻干燥作为替代样品制备方法潜力的研究。我们的结果表明，冷冻干燥有潜力用作尿液样本SPME-GC-MS分析的替代方法。使用内标、合成尿液和校准曲线的进一步研究将使代谢物的定量更精确，并能对不同方法进行更多比较。
增强尿液样本中的VOC分析图谱
