## 2 Results and Discussion
### Validation of the Quantitative Method
#### 2.2.1 Linearity of Calibration Curve in Different Wine Matrix
  
* Three “dry” style Pinot Gris wines, 1 Four “medium dry” style Pinot Gris wines; 4 One “dry” style Riesling wine, 4 Four “medium dry” style Riesling wine, 5 Seven “medium sweet” style Riesling wine, 6 Seven “sweet” style Riesling wine, 72 2011 dry Riesling, 10.8% alcohol content (_viv_), 0.58 g/L residual sugar, 2013 Pinot Gris, 11.8% alcohol content (_viv_), 16. One of the differences observed between the L and H calibration curves was the slope. A lower slope for 2 and 4 (limonene isomers) and higher slope for 5, 6, 9 and 10 (rose oxide isomers) was found in the H base calibration curve compared with the L base calibration curve. Rose oxide isomers seemed more easily released from the diluted wine solution with higher residual sugar content, while limonene isomers were retained longer in the same wine base. Other work has shown that high concentrations of fructose in model wine solutions have strong odorant retaining effects in headspace extraction [24]. However, another study showed the extraction of esters was not affected by sugar content in a varying saccharose content (0-200 g/L) synthetic wine using HS-SPME-HRGC [25], suggesting that the impact of sugar concentration on extraction for HS-SPME measurements varies depending on the volatile compound being measured. Our results showed that the measurement for concentration of chiral monoterpenes in wine with a high residual sugar would not be accurate if a calibration curve from a dry wine were utilized to calculate the concentration of terpen, especially for limonene and rose oxide isomers. This suggested a strong matrix effect on the investigated compounds. Several other method factors were investigated to ensure accurate and repeatable measurements of the chosen chiral monoterpenes. HS-SPME was used as it is an inexpensive and reproducible method to measure volatile compounds [26; 27; 28]. In our study, single fiber repeatability was investigated in both wine bases. Calibration curves were divided into three parts, based on the age (or usage) of a new fiber; the beginning (first 8-10 days of fiber life), middle use (within 8-13 days of fiber life) and at the last life of fiber (last 10 days of fiber life). In total, six calibration curves were plotted in terms of wine matrix and fiber age over 300 injections; L first, L middle, L last, H first, H middle and H last. A significant change in slope for compounds 2, 4, 5, 6, 9 and 10 was noted from first to last intervals for both wine bases (Table 1). Interestingly, limonene isomers (2 and 4) had decreasing affinity for the SPME fiber in single fiber durability. Rose oxide isomers (5, 6, 9 and 10) showed a greater affinity for the fiber during the middle interval, in comparison with the decreasing affinity noted for other compounds. 7, 8, 11, 12, 14, 16, 18, 20 and 21 were not affected by the life of the SPME fiber. The reason for the poor repeatability of single fiber was not clear. It was reported that SPME fibers suffer from some weakness, such as limited lifetime and sample carryover [29]. The carry over effect had been eliminated in our study by re-desorption in the heater port for 10 min, lengthening this time did not improve compound affinity over time. One report mentioned poor reproducibility from one fiber to another, and suggested a new calibratation each time the fiber was changed [30]. To eliminate the error due to single fiber variance, chemical composition was determined using calibration curves from the same time period. 2.2 Limit of Detection (LOD), Limit of Quantitation (LOQ), Wine Reproducibility and Internal Standards Stability  
LOD and LOQ data are reported in Table 2. The LOQ ranges from 1 ng/L to 1.11 ug/L and these limits are lower than the known olfactory thresholds for the measured compound found in Section 3.1, Table 5. The detected LOQ (ug/L) values are also lower than previous methods measuring chiral terpenes [2,31,32]. Wine reproducibility values varied around 15% (RSD %) for all compounds in Riesling and Pinot Gris wines.