# Quantitative Analysis of Caffe in Roasted Coffee: A Comparison of Brewing Methods
## 3 Results and Discussion
### Caffee Determination Methods
The Diode Array Detector (DAD) was used in more than a third of the studies (34.5%) in which the HPLC method was used, the Ultraviolet Detector in 27.6%, the Photodiode Array Detector (PDA) in 20.7% and the Variable Wavelength Detector in 10.4%. Two studies used the more advanced liquid chromatography method coupled with mass spectrometry [52; 58]. DAD and PDA detectors were the most commonly employed due to their broad availability, cost-effectiveness, and the absence of ion suppression or significant matrix effects. Furthermore, these detectors allow for the simultaneous quantification of multiple compounds, although their analyte coverage remains more limited than that of mass spectrometers. The studies used a reversed-phase system with a C18 column (stationary phase of n-octadecylsilane molecules), except the [PERSON] et al. [52], which used a Kinetex PFP (pentafluorophenyl stationary phase) column. Many studies (27.4%) used a classical column configuration (250 \(\times\) 4.6 mm) to determine substances of medium polarity, such as caffeine. The remaining studies used shorter columns (150, 125, 100 mm) with different diameters (5.0, 4.0, 3.0. 2.7, 2.1 \(\upmu\)m). The particle size of the stationary phase inside the column in 62.1% of the studies was 5 \(\upmu\)m; in others, it was 2.6-4.0 \(\upmu\)m. Only a few studies (20.7%) used a precolumn [30; 31; 32; 51; 53]. Caffe was determined using a mobile phase containing different amounts of water and methanol. This method was used in most studies (69.0%), with modifications regarding the proportion between water and methanol. Only a few papers (31.0%) described HPLC methods using a water-acetonitrile mobile phase [30; 31; 32; 41; 45; 47; 49; 58; 59]. In almost all the studies, the mobile phase was modified by the addition of acids such as formic, acetic, phosphoric, and citric acids, except in the studies by [PERSON] et al. [50], [PERSON] et al. [42], and [PERSON] et al. [46]. Gradient elution was used in 62.1% of the studies with varying gradient profiles. Sample injection volumes in the analyses ranged from 1 to 100 \(\upmu\)L, with 10 \(\upmu\)L (37.9%) and 20 \(\upmu\)L (31.0%) being the most commonly used volumes. In most studies, the flow rate was 1 mL/min (41.4%), while 20.7% of studies used a flow rate of 0.4 mL/min. The remaining studies' flow rate ranged from 0.2 to 1.5 mL/min. In 37.9% of the studies, no information was available about the column temperature used; in other studies, the authors mostly used the temperature 25 \({}^{\circ}\)C (24.1%) [29; 36; 37; 38; 42; 44; 56], and in others room temperature [30; 31; 32] or in the range (30-40 \({}^{\circ}\)C) [52; 57; 58; 59]. Table 2 also includes the sample preparation step; in some studies, samples were filtered (65.5% of studies), centrifuged (37.9%), and diluted (34.5%) before HPLC analysis. Different wavelengths were used in the studies, as shown in Figure 3.  
Table 3 shows the analytical method validation parameters such as coefficient of determination (R\({}^{2}\)), limit of detection, limit of quantification, and relative standard deviations. Figure 3: Wavelengths used in the HPLC method to detect caffeine.