focus on Chromatography Establishment of the Saliva Volatomic Profi le as an Exploratory and
Non-invasive Strategy to Find Potential Breast Cancer Biomarkers Carina Cavaco1
, Rosa Perestrelo1 , Catarina Silva1 , Fernando Aveiro2 , Jorge Pereira1 , José S. Câmara1,3
1) CQM/UMa, Centro de Química da Madeira, Universidade da Madeira, Campus Universitário da Penteada, 9000-390 Funchal, Portugal 2) Hospital Dr Nélio Mendonça. Haematooncology Service, Dr. Nélio Mendonça Hospital, Avda Luis de Camões, Funchal 3) Universidade da Madeira; Centro de Ciências Exatas e da Engenharia da Universidade da Madeira, Campus Universitário da Penteada, 9000-390 Funchal, Portugal
Breast cancer (BC) is a major health problem worldwide. It is the most common malignancy in women and the second cause of cancer- related mortality. BC late diagnosis is a major cause of BC morbidity and improved diagnosis tools are needed. Here we present a sensitive assay aiming to identify volatile organic metabolites (VOMs) as biomarkers of BC development using saliva samples. Saliva is a promising source of VOMs that can be used as potential cancer biomarkers. The extraction of saliva VOMs was performed using dynamic headspace solid phase microextraction (dHS-SPME). Several parameters that infl uence the extraction were tested. These included the nature of the adsorptive phase, sampling temperature, extraction time, volume, pH, salt concentration and agitation.
Introduction
Cancer is characterised by abnormal growth and development of normal cells beyond their natural boundaries. In the last 50 years, and despite the global efforts to decrease the incidence of this disease, cancer has become the leading cause of death. Particularly, breast cancer (BC) is the most common malignancy and the second most common cause of cancer-related mortality in women [1].
The diagnosis at an early stage allows the management of high-risk cancers and a much better prognosis, but to allow this it is necessary to fi nd specifi c and sensitive biomarkers. In certain circumstances some molecules are differentially expressed in cancer cells relative to their normal counterparts, and their altered levels can be measured to establish a correlation with the diseased state [2,3]. A promising class of biomarkers for cancer early diagnosis are the volatile organic metabolites (VOMs) [4]. Saliva contains VOMs that can be used as potential cancer biomarkers and its sampling is easy and non-invasive. Moreover, when compared with blood, saliva contains less proteins, thus decreasing any potential risk of non-specifi c interference and hydrostatic interactions [5]. Therefore, the aim of this study was to establish the saliva volatomic profi le between BC patients and healthy individuals, and to explore VOMs as potential biomarkers in BC diagnosis at early stage. Dynamic headspace solid-phase microextraction (dHS-SPME) using Carboxen®
/polydimethylsiloxane
(CAR/PDMS) sorbent was selected as a powerful and highly sensitive strategy to isolate VOMs from saliva. Combined with GC-qMS, it was used to obtain metabolomic information patterns of BC patients and healthy individuals. Additionally, several parameters that infl uence the extraction such as the nature of the adsorptive phase, sampling temperature, extraction time, volume, pH, salt concentration and agitation were tested.
Methods dHS-SPME Procedure
The saliva samples used in the optimisation of the dHS-SPME parameters were from normal subjects and kept at -80°C, being thawed at room temperature before use. For the selection of the best fi bre, fi ve SPME fi bres (Supelco) with different stationary phases and fi lm thicknesses were tested. These include polydimethylsiloxane (PDMS) (100 µm), polyacrylate (PA) (85 µm), divinylbenzene/Carboxen® (50/30 µm), Carboxen®
/polydimethylsiloxane (DVB/CAR/PDMS) /polydimethylsiloxane (CAR/PDMS) (75 µm),
and polydimethylsiloxane/divinylbenzene (PDMS/DVB) (65 µm). In the dHS-SPME optimisation, 2 ml saliva aliquots were placed in 4-ml headspace glass vials, adjusted to pH 1–2 with 0.25 ml of 5.0 M HCl, 0.2 g of NaCl added and stirred at 800 rpm. The sample vial was placed in a thermostat bath adjusted to 38.0 ± 0.1°C and the SPME fi bre was introduced into the headspace for 45 min. After sampling, the SPME fi bre was withdrawn into the needle, removed from the vial and inserted into the injector port (250°C) of the GC-qMS system for 6 min. The metabolites were thermally desorbed and transported directly to the analytical column. Each sample was analysed in duplicate.
A CAR/PDMS fi bre was used to study the temperature and the time of extraction parameters. Extraction was performed at temperatures of 28, 38 and 48°C for 30, 45, and 60 min with a stirring rate of 800 rpm. The optimum conditions were determined by the totality of the peak areas obtained in each parameter, number of compounds extracted and reproducibility. The experimental design is represented in Figure 1.
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