# SPME Method Optimized by Box-Behnken Design for Impact Odorants in Reduced Alcohol Wines
## 3 Results and Discussion
### Optimization of SPME Factors
Temperature has a significant impact upon the vapor pressures of analytes and, therefore, upon the headspace concentrations in which the SPME fiber is immersed during sampling [29]. Increasing the sample temperature will inevitably increase the headspace concentrations of the higher boiling point analytes, leading to improved analytical sensitivities for these compounds and increasing the competitive binding for SPME fiber space with lower boiling point analytes [13]. Increased competition between high and low boiling point compounds for fiber space explains the negative \(b\)-coefficients for early eluting compounds. SPME fiber exposure time is important for the quantification of compounds. Negative \(b\)-coefficient for linear terms are associated with early eluting compounds and positive coefficients for late eluting and high boiling point compounds, which is evident at all ethanol concentrations. The partitioning of volatile compounds into the headspace during fiber exposure is a dynamic phenomenon as the compounds absorb onto the SPME fiber from the headspace [30]. As semivolatile compound concentrations in the headspace are relatively low compared to their liquid concentration, the overall mass transfer rates are low and the longer extraction times lead to the increased mass transfer of these compounds from the vapor onto the fiber. Interestingly, the sample volume in the headspace vial was insignificant for all compounds at 5% \(v/v\) ethanol (Figure 1), but became a significant factor for some compounds as the ethanol concentration increased (Figures 2 and 3). This observation should be considered in light of the relative concentration and partition coefficients of ethanol in the base wines compared to the analytes of interest. As the ratio of sample to headspace volume increases, compounds with high partition coefficients--i.e., compounds with relatively lower headspace concentrations--partition less into the headspace relative to compounds with low partition values. Ethanol has a relatively high partition coefficient but is present in high concentrations relative to other volatile compounds. Competition between ethanol and other volatile compounds for SPME fiber binding sites will arise, which would lower sampling sensitivity. Larger sample volumes will increase the quantity of some analytes present in the headspace vial relative to ethanol, thereby, increasing the sensitivity of the SPME sampling for these compounds at higher ethanol concentrations. The calibration parameters, including analyte quantification and qualification ions, elution times and method performance characteristics, are presented in Supplementary Table S6. The linearity, limits of quantification, and signal to noise ratios are within the acceptable thresholds for compound quantification [21]. Figure 1: The \(b\)-coefficients (absolute units) for solid phase microextraction (SPME) optimization parameters for 23 compounds with an ethanol concentration of 5% \(v/v\). Significant coefficients for specific compounds are indicated with an asterisk (*). Compound identification is cross-referenced to the numbered compounds in Table 1.  
Figure 2: The \(b\)-coefficients (absolute units) for SPME optimization parameters for 23 compounds with an ethanol concentration of \(8\%\,v/v\). Significant coefficients for compounds are indicated with an asterisk (*). Compound identification is cross-referenced to the numbered compounds in Table 1.