Effect of kinds of DES on recovery
Three kinds of DES were prepared by choline chloride with ethylene glycol (DES1), glycerol (DES2) and urea (DES3), respectively. The effects of three kinds of DES as extractant on the recovery of γ-nonolactone were compared.The extraction recoveries of γ-nonolactone by DES1, DES2 and DES3 were 85%, 63% and 52% respectively. The extraction efficiency of γ-nonolactone by DES1 was significantly higher than the other kinds of DES. At the same time, the viscosity of EDS1 in ethylene glycol system was significantly lower than the other kinds of DES, Therefore, ethylene glycol system is selected for subsequent experiments.
Effect of solvent ratio on recovery
According to the literature and the material characteristics of eutectic solvents, the molar ratio of choline chloride and ethylene glycol was selected as 1:3 and stirred at 60 ℃ for 30 min, to make uniform transparent liquid. 0, 20, 50 and 100 μL of eutectic solvents were added respectively to investigate the effects of different proportions of eutectic solvents on recovery. The results are shown in Figure 1.
It can be seen from the experimental results that without adding eutectic solvent, the recovery of γ-nonolactone is only 0.04%, adding 20 μL is 45.2%, adding 50 μL is 95.4%, and the recovery of adding 100 μL is 91.8%. The experimental results are shown in Fig.1. With the increase of EDS volume, the recovery increases gradually, reaches the maximum at 50 μL, continue to increase the volume and the recovery rate begins to decline. In the extraction process, after extracting γ-nonolactone fully, increasing the volume of DES1 has no obvious effect on the recovery of γ-nonolactone. Instead, the adsorbed γ-nonolactone is dislodged from the surface and dispersed into coconut juice, this leads to a decrease in recovery. Therefore, the volume of DES1 added in this experiment is 50 μL.
Effect of salt dosage on recovery
The influence of inorganic salt used for salting out on recovery and adding 0.2, 0.5 and 1.0 g of sodium chloride on the test results were investigated. After 10 min of ultrasound, clear two-phase distribution can be seen after centrifugation at 4℃, 10000 r/min for 5 min. The results are shown in Fig.2.
Matrix effect (ME) is one of the key factors affecting the quantitative accuracy, which will have a great impact on the accurate quantification of the target compound. Before established method, the matrix effect of the tested substance must be evaluated, and effective measures must be taken to eliminate or compensate, so as to improve the reliability of the analytical method. At present, the evaluation method of ME is often evaluated by the ratio of the slope of the matrix standard curve to the slope of the solvent standard curve. The ME the following formula can be calculated.
ME = Ka / Kb ----------------------------- -(1)
-Ka refers to the slope of the calibration curve of the sample matrix,
-KB refers to the slope of the solvent calibration curve.
The established method, Kb is 8.25, Ka is 2.16(0.2 g NaCl), Ka is 7.97 (0.5 g NaCl), and Ka is 8.99 (1.0 g NaCl). When the matrix effect is closer to 1, it indicates that the matrix has less obvious influence on the results. Inorganic salt addition and recovery are satisfactory at this time. Considering the influence of ME, 0.5 g sodium chloride is selected as the inorganic salt used in this experiment (See Fig. 2).
Effects of centrifugal temperature on test results
The effects of different centrifugation temperatures on the test results were investigated. Under the conditions of 0, 4 and 20 ℃, 10000 r/min centrifugation for 5 min, the two-phase distribution was not obvious under the condition of 20 ℃, the acetonitrile phase had white flake precipitation, the two-phase stratification was obvious under the condition of 4 ℃, the acetonitrile phase was clear and transparent, and the results at 0 ℃ were similar to those at 4 ℃. Considering the test cost, 4 ℃ was selected as the centrifugation temperature in this experiment
Verification of the Methodology
Standard curve, linear range and detection limit
In this study, external standard method was used for quantification, concentration of 0.1, 0.5, 1.0, 2.0, 5.0, 10.0 and 20.0 μg/mL γ-nonolactone standard solution were prepared, respectively. According to peak area (Y) and concentration of standard solution (X, μg/mL) drawing standard curve the linear relation between peak area and concentration of γ-nonolactone was good in the range of 0.1~20.0 μg/mL with the correlation coefficient r is 0.99999. The limits of detection (LOD) and the limits of quantification (LOQ) were obtained by the linear equations LOD=3 s/N and LOQ=10 s/N. See Fig. 3 and Fig. 4. The results show that LOD of γ-nonolactone is 0.2 mg/kg and LOQ is 0.5 mg/kg. The developed method has good sensitivity.
Accuracy and precision
To determine the accuracy, recovery experiments were performed using three different concentrations of real coconut juice samples spiked with known amounts of γ-nonolactone standard. γ-nonolactone spiked levels were 0.2, 0.5, and 10 mg/kg, respectively. The samples were measured using the optimized procedures established above. The experiment was performed in six repeated tests at each concentration level. The results showed that the recoveries of the γ-nonolactone are 89.4% ~ 105.0% within the range of spiked concentration. The relative standard deviation (RSD) is 0.6% ~ 5.8% (Table 2), It shows that the method has good stability and accuracy, and can meet the detection requirements of γ-nonolactone compounds in coconut juice.
Real samples testing
The quantitative analysis of γ-nonolactone in 10 purchased coconut juice samples from supermarkets was carried out by using the established method. The results show that, 3 purchased coconut juice samples were detected to contain γ-nonolactone , with the contents of 15.4 mg/kg, 35.8 mg/kg and 41.6 mg/kg respectively. γ-nonolactone was not detected in the samples of the other seven brands. Explain that γ-nonolactone is used in some coconut juice.