# SPME Method Optimized by Box-Behnken Design for Impact Odorants in Reduced Alcohol Wines
## 3 Results and Discussion  
### Optimization of SPME Factors  
Optimization of SPME sample experimental conditions is a crucial analytical step to ensure the accuracy and sensitivity of the method for headspace analysis [18]. In the present study, a quantitation method for a range of wine impact odorants was optimized by a three-level Box-Behnken design for three different ethanol content wine-model solutions (i.e., 5, 8 and 13% \(v/v\)). This multivariate statistical model minimized the number of experiments required to determine the significant coefficients for linear interactions and quadratic terms for SPME fiber exposure temperature, time, and wine sample volume for each analyte responses. A global optimized condition for SPME sampling for the suite of analytes, at each ethanol concentration was then developed by using a desirability function that accounts for a low signal response for compounds. This approach has been previously employed for determining the optimized conditions for the quantification of fungal off-flavors [22]. Global optimized SPME sampling conditions (at each ethanol concentration) were determined from the predicted maximum response for each analyte and associated sampling conditions(Equation (2) and Tables S3-S5) using the \(b\)-coefficients, which were determined using the design matrix (Supplementary Materials) and measured responses. A global and weighted mean SPME fiber extraction temperature, time, and sample volume are determined from the relative size of the inverse maximum predicted response. This approach increases the importance of maximizing the SPME sampling conditions for compounds with relatively low detector responses, whilst down weighting the influence of compounds with large detector responses. Ethanol is the most abundant volatile compound in wine and will compete with other compounds of interest for binding sites on the SPME fiber [26]. As wine ethanol concentrations impact the partitioning of volatile compounds from liquid to the headspace, optimized sample analysis conditions were determined at wine ethanol levels to avoid confounding analytical results associated with sample dilution. This approach ensures that the headspace concentrations for each compound of interest at each ethanol concentration are accurately determined and allow for comparisons between wines at differing ethanol levels to be made. The results regarding the combination of optimized fiber exposure time, temperature, and sample volume at different ethanol contents are summarized in Supplementary Materials (Tables S3-S5 for 5, 8 and 13% \(v/v\) ethanol content model wine solutions, respectively). Inspection of the \(b\)-coefficients for each compound at each ethanol concentration (Figures 1-3 for 5, 8, and 13% \(v/v\) ethanol content model wine solutions) reveals the impact of each experimental factor upon the analyte behavior for SPME. The modulus of each \(b\)-coefficient is determined by the relative detector response for each analyte and the sign infers either a positive or negative impact upon the analytical sensitivity. The SPME temperature of fiber exposure is the most critical factor for analyte quantification in agreement with the previous findings for different matrices other than wine [27; 28]. This is indicated by the number of analytes with a significant \(b\)-coefficient for the linear and second-order interaction terms of temperature, and this trend is evident at all ethanol concentrations. Of interest is the association of the negative \(b\)-coefficients of temperature with early eluting compounds (1-10) and the positive \(b\)-coefficients of late eluting compounds (15-23), and this can be correlated with the boiling point and the elution order of the analytes of interest (Supplementary Table S6). The overall SPME fiber binding of analytes from the vial headspace is determined by mass transfer of the analytes from the liquid to volatile phase followed by mass transfer onto the fiber.