# Systematic Review and Meta-Analysis of Melatonin Quantification in Wine
## 3 Results and Discussion
### The Methodology of Melatonin Quantification in Wines  
The ranked order of methods used for quantification in studies included in the review is as follows: HPLC-MS/MS (60%) > HPLC-FL (25%) > HPLC-DAD (5%) > CEC (5%) > ELISA (5%). Different extraction and cleanup procedures were used in the sample preparation step, and the overall rank order is as follows: SPE (35%) > DLLME (25%) > just filtered (20%) > concentration (10%) > MEPS (5%) > liquid extraction (LE) (5%). Two major challenges are generally present in melatonin quantification: basal melatonin levels and the desired sensitivity. HPLC is the most commonly used method under different conditions with an isocratic reversed mobile phase and several types of detectors. The DAD detector is useful in different matrices, but in the analysis of wine samples, it was not frequently used due to detection/quantification limits and the concentration level of this analyte. The detection limit (LoD) and the quantification limit (LoQ) of the HPLC-DAD technique are in the \(\mathrm{\SIUnitSymbolMicro m}^{-1}\) domain. For Brazilian and Argentinian wine sample analysis, prior to determination, an ultrasound-dispersive liquid-liquid microextraction (US-DLLME) method for the preconcentration of melatonin was developed, optimized, and validated [27]. HPLC-MS/MS is a powerful and robust analytical technique for precise and sensitive measurement, even at low concentrations with a shorter run time. Due to its capability of selectively detecting and quantifying active principles in complex matrices, it is often used in melatonin research in wine samples. Of all the developed methods for the analysis of melatonin in wines, HPLC-MS/MS methods are the most widely used. The application of HPLC-MS/MS in melatonin quantification has enabled tremendous quantitative capabilities in the MRM mode with high sensitivity, specificity, and selectivity [47]. One of the many advantages of HPLC-MS/MS is its ability to determine melatonin isomers, which show cytoprotective and antioxidant activity depending on the changing position of two side chains in the indole ring [48]. Although melatonin and its identified isomer had the same fragment ions, melatonin's most stable abundant fragment is 174. The most abundant fragment for the isomer found in wine is 216, and the ratios of these ions were different for melatonin and melatonin isomers. The relative abundance of the fragment ion 216 was higher than 174 for melatonin isomers [37; 38; 39]. Another advantage is that the wine samples can be analyzed without the involvement of sample pretreatments, which can only be filtered [9; 40; 42]. HPLC-FL is a simple, fast and sensitive detection technique. In addition to the most common HPLC-MS methods, considering melatonin has strong native fluorescence, a HPLC-FL method was applied for analysis of food with simple matrix [49]. The LoD and LoQ of this technique are in pg mL\({}^{-1}\) domain and are suitable for the determination of melatonin in wines [8; 14; 28; 30; 41]. Compared with HPLC, several unique characteristics of CEC, such as minimal reagent consumption, high resolving power, low cost, and rapid analyses, make it particularly attractive for the analysis of compounds in wine [50]. As a result, the high electro-chromotography robustness, high resolution, and high sensitivity, accompanied by the straightforwardness and reproducibility of the extraction procedure, results in a useful tool for determination of melatonin in wine samples [25]. Today, techniques used to detect and quantify melatonin include immunological assays, such as ELISA. The results obtained using this method are not considered accurate, since the various compounds present in grapes can cross-react with antibodies and enzymes, overestimating the real values of melatonin [51]. The main drawbacks of the ELISA test are a lack of linearity, false positive and false negative results, and the relatively high price of the kit.