17 GC-qMS Analysis
The gas chromatograph (GC) system was composed of an Agilent Technologies 6890N Network (Palo Alto, CA, USA) equipped with a 30.0 m×250 µm ID×0.25 µm film thickness BP-20 (SGE, Dortmund, Germany) fused silica capillary column and interfaced with an Agilent 5975 quadrupole inert mass selective detector (MS). The oven temperature program was as follows: initial 60°C held for 5min then ramped 2°C/min up to 150°C, held for 5 min, again ramped 15°C/min to 220°C, and held for 1 min. The total GC run time was 60 min. The column flow was maintained constant at 1 ml min-1
using He (Helium N60, Air Liquide) as the carrier gas.
The injection port was operated in the splitless mode and held at 250°C. For the MS system, the operating temperatures of the transfer line, quadrupole and ionisation source were 270, 150 and 230°C, respectively. Electron impact mass spectra were recorded at a 70 eV ionisation voltage and the ionisation current was 10 µA. The acquisitions were performed in Scan mode (30–300 m/z), and the electron multiplier was set to the auto tune procedure. Metabolite identification was accomplished through manual interpretation of spectra and matching against the Agilent MS ChemStation Software (Palo Alto, CA, USA). This software was equipped with an NIST05 mass spectral library.
Extraction Temperature and Time
Using an extraction time of 45 min with a 2 mL saliva sample, the effect of extraction temperature on SPME extraction efficiency was investigated at 28, 38 and 48°C. The best results were obtained at 38°C since the number of identified metabolites was higher.
To evaluate the influence of extraction time on the extraction capacity, different extraction times (30, 45 and 60 min) were tested using the previously selected fibre (CAR/PDMS) and 38°C. Of the three extraction times tested, the one with higher extraction efficiency was 45 min (Figure 2).
Fibre Selection
To select the most appropriate fibre to isolate volatile metabolites from saliva, five SPME fibres were tested. By comparing all of the tested fibres in terms of peak areas and the number of identified metabolites, the best efficiency was obtained using a CAR/PDMS coating as observed in Figure 2, whereas the lowest efficiency was obtained using the PDMS and PA fibres. Thus, the CAR/PDMS fibre was chosen as the SPME fibre for the following optimisation studies.
Figure 2. Influence of fibre coating, extraction time and temperature on the extraction efficiency of VOMs
Ionic Strength and pH Figure 1. Experimental design Results and Discussion
This study involved the optimisation of the best experimental conditions to extract volatile metabolites from the saliva samples using HS-SPME. This was followed by a comparison of the metabolomic patterns from saliva of BC patients and healthy volunteers. This was achieved by the identification of the metabolites and through peak area differences.
Optimisation of the dHS-SPME Parameters
The optimisation of the parameters involved in HS-SPME was executed by selecting the conditions that achieved the maximum response in terms of metabolite peak area, number of metabolites detected and reproducibility.
Several sodium chloride concentrations were assayed (0, 5, 10, 15 and 20% (w / v)), the best results obtained were with a concentration of 10%. In order to study the influence of the sample pH on the efficiency of the HS-SPME extraction, samples were prepared at neutral, acidic (5M HCl) and basic (1M NaOH) conditions. As observed in Figure 3 acidic pH facilitates the extraction of volatile compounds.
Volume Sample and Stirring
Three sample volumes (1, 2 and 4 mL) were analysed and it was found that the 2 mL samples allowed a more efficient extraction. Finally, two more assays were performed, extraction with and without agitation. It was verified that the agitation facilitated the extraction of the volatile compounds present in saliva (Figure 3). In summary, the best extraction conditions for the determination of the volatile profile of the saliva from BC patients and healthy volunteers were 2 mL saliva samples containing 10% of NaCl (w/v) and acidified to pH 1, extracted at a 38°C during 45 min, using a CAR/ PDMS fibre and 800 rpm stirring.
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