# Systematic Review and Meta-Analysis of Melatonin Quantification in Wine
## 3 Results and Discussion
### The Methodology of Melatonin Quantification in Wines
In an attempt to determine melatonin in wines using this test, a 25.0% false positive rate and a 28.6% false negative rate were obtained. While the ELISA kit could not be validated for accurate melatonin quantification in wines [26], it could still be useful for screening if validated for matrix effects. Differences in the methodology employed for analyzing melatonin in wine can indeed lead to variations in results. Variances in sample preparation/extraction techniques, quantification techniques, and detection methods may influence the reported levels of melatonin in different studies. Sample pretreatment, purification, separation, and concentration usually require more than 60% of the analysis time and mainly determine the accuracy of the analysis. It is difficult to use appropriate sampling methods to achieve satisfactory recovery. A variety of pretreatment methods have been used for purifying and extracting melatonin in wine samples. Their characteristics and superiorities are worth considering in order to achieve a level of recovery that is appropriate for analytical methods. LE is a general extraction technology known for its simple operation and ease-of-use. Among the different pretreatment methods for melatonin, SPE has replaced LE due to its advantages of extraction efficiency, timesaving capability, low levels of consumption, user-friendly control, and so on. To further achieve miniaturization and high-throughput development requirements, numerous technologies like microextraction technologies such as MEPS and DLLME were also used. These methods have excellent extraction efficiency that can achieve a-hundred-times-higher enrichment and are quicker and less expensive than standard SPE [52]. A variety of pretreatment and analytical methods used for identifying melatonin in wines are summarized in Table 2. \begin{table}
\begin{tabular}{c c c c c c} \hline \hline
**Sample** & **Cartridge/Sorbent** & \begin{tabular}{c} **Elution/Extraction** \\ **Solvent** \\ \end{tabular} & \begin{tabular}{c} **Analytical** \\ **Method** \\ \end{tabular} & \
\begin{tabular}{c} **LoD and LoQ** \\ **ng mL-1** \\ \end{tabular} & **References** \\ \hline SPE & C8 cartridges & methanol & HPLC-MS/MS & 0.12 and 0.42 & [38] \\ \hline SPE & Bond Elut C18 & methanol & HPLC-MS/MS & 0.6 & [32] \\ \hline \multirow{3}{*}{SPE} &
\begin{tabular}{c} Strata \\ X-Polymeric \\ Reversed \\ Phase \\ \end{tabular} & methanol & HPLC-MS/MS & 2.3 and 18 & [29] \\ \hline SPE & ProElut C18 & methanol & HPLC-MS/MS & - & [17] \\ \hline SPE & HLB Oasys & methanol & HPLC-MS/MS & - & [43] \\ \hline SPE & C18 & methanol & ELISA & - & [26] \\ \hline SPE & BondElut C18 & methanol & HPLC-FL & 0.03 and 0.01 & [14] \\ \hline MEPS & C8 & methanol & HPLC-FL & 0.05 and 0.02 & [28] \\ \hline DLLME & - & chloroform & HPLC-FL & 0.01 and 0.05 & [41] \\ \hline DLLME & & chloroform & HPLC-FL & 0.01 and 0.05 & [30] \\ \hline DLLME & - & chloroform & HPLC-FL & 0.07 and 0.24 & [8] \\ \hline US-DLLME & - & dichloromethane & HPLC-DAD & 230 and 700 & [27] \\ \hline only filtered & - & - & HPLC-MS/MS & 0.059 and 0.12 & [40] \\ \hline only filtered & - & - & HPLC-MS/MS & - & [9] \\ \hline only filtered & - & - & HPLC-MS/MS & - & [42] \\ \hline LE & - & methanol & CEC & 0.01 and 0.03 & [25] \\ \hline concentration & 3:1 (v:v) & methanol & HPLC-MS/MS & - & [39] \\ \hline - & - & - & HPLC-MS/MS & 0.033 and 0.112 & [37] \\ \hline \hline \end{tabular}
\end{table} 
Table 2: Methods that have been used in published research for melatonin quantification. The results of meta-analyses of studies, in which the HPLC-MS or HPLC-FL methods were used to quantify melatonin in wines, are reported in Table 3.  
The meta-analysis was performed on the results obtained from a total of 100 wine samples quantified with HPLC-MS across nine studies. The highest total samples used in one study was 35 [9]. The MRAW was 4.3, with a 95% confidence interval of [2.59; 6].