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Abstract Introduction
Physiological processes naturally produce Volatile Organic Compounds (VOCs). In a pathological condition, different processes occur, causing the generation of VOCs which are not usually produced and/or changing their concentration. Thus, VOCs can be analyzed in biological samples to identify potential disease biomarkers, notably cancer.
One of the techniques used for VOCs analysis is the Static Headspace Gas Chromatography-Mass Spectrometry (SHS-GC-MS). This technique depends on numerous factors, such as incubation time, temperature, salt addition, agitation, and sample volume. Here, we study and optimize the significant factors that affect VOCs analysis in oral fluid (OF) and urine by SHS-GC-MS, using Plackett-Burman (PB) and Response Surface Methodology (RSM).
Objectives
To study and optimize the parameters for VOCs analysis in OF and urine by SHS-GC-MS.
Methods
Pooled OF and urine were analyzed in different conditions by SHS-GC-MS. The PB12 factorial design was used to evaluate how incubation time (tinc, min), temperature (T, °C), agitation (rpm), sample volume (SV, mL), pH, and salt addition (NaCl, g), affected the total response area. The significant variables obtained via PB were optimized by central composite design (CCD) and RSM. All statistical analyses were performed with Statistica 7.0.
Results and Discussion
Statistical analyses of the PB12 showed that OF significant variables were T, salt addition and SV (p < 0.05). The other factors were not statistically significant (p > 0.05). Regarding urine, only salt addition was not significant. The main reasons why the significant factors differ between OF and urine are the SV and pH. The OF SV is limited by the amount obtained in one sample collection (about 1mL), while in urine higher volumes may be analyzed. Therefore, a higher tinc and agitation may be required to increase the amount VOCs in urine headspace. Regarding the pH, several compounds are eliminated in urine as conjugates. An acidic environment may enhance the quantity of compounds in their non-conjugated form, resulting in a significant higher response area.
For OF CCD evaluation, SV was fixed at 1 mL, agitation at 250 rpm, pH was neutral (since it avoided an additional sample preparation step), and the other factors were optimized. Although tinc was not statistically significant, it was evaluated in CCD to ensure that the best results were obtained within the shortest time. The analysis of the RSM provided the best conditions: T = 95 °C, NaCl = 0.8 g, and tinc = 5 min.
Conclusion
The PB factorial design allowed us to study and differentiate the statistical significant variables in OF and urine VOCs analysis by SHS-GC-MS, and the RSM provided the OF optimized conditions. The urine optimization will be included at the poster presentation. These parameters will be used to analyze the OF and urine of healthy people and individuals with head and neck Cancers, to determine possible biomarkers of this disease. |
= Discovery stage.
= Translation stage.
= Clinically available.
