## 3 Results and Discussion
### Quantification of Aromatic Compounds and Monosaccharides by HILIC-UV/ELSD  
As pointed out in the Introduction, our aim was to develop a simple and fast screening method for simultaneous quantification of the main aromatic compounds and monosaccharides from willow bark extract. Since monosaccharides do not contain chromophores, ELSD was added to the detector train, in addition to UV (Figure 3). ELSD, a quasi-universal detector, has several advantages over the more commonly used refractive index detector. To name a few, ELSD has fairly similar response factors for structurally similar compounds, it is compatible with most of the solvents and modifiers, it can be used with solvent gradients, and it is not sensitive to pulsation of the HPLC pump [24]. One disadvantage of ELSD is that, in the case of most applications, the detector response is non-linear. The peak area (\(A\)) correlates with the mass of the analyte (\(m\)) as follows:  
\[A=a\times m^{b} \tag{1}\]  
where \(a\) and \(b\) are coefficients depending the ELSD instrumentation. Similar behavior was also observed here; exponential behavior was evident both for aromatic compounds and for monosaccharides (examples of ELSD calibration curves are presented in Figure S1). Aromatic compounds can also be detected with UV and, as shown in Table 1, the limit of quantification (LOQ) decreased a hundredfold if UV was used instead of ELSD. The sensitivity of ELSD was, however, better compared with the RI detector for which the LOQ was in the order of few hundred mg/L (150 for fructose and 300 mg/L for glucose with a 10 uL injection) (personal communication with [PERSON]).  
As shown in Figure 3, picein, triandrin, fructose, and glucose are the main components in willow bark extract. Other components, such as (+)-catechin, are also present. The combination of both a UV detector and ELSD is powerful, especially in cases in which the amount of aromatic compounds is very low (the sensitivity of UV significantly higher than ELSD). In this study, the standard 10 mm flow cell was used, but sensitivity can be increased if needed by changing the 10 mm flow cell to a longer 60 mm flow cell.  
The quantified amounts for picein, triandrin, fructose, and glucose for willow bark extract (calculated using ELSD signals) are presented in Table 2. The most prevalent component in the extract was glucose, followed by fructose and picein. The results for the UV-absorbing components picein and triandrin, obtained with ELSD and a UV detector, agreed well with each other, being within 5%. The results obtained with the HILIC method were validated using GC-FID for aromatic compounds and HPAEC-PAD for monosaccharides (Table S1). The HILIC results are in accordance with the results obtained with these established methods. Day-to-day variation in the ELSD response was observed, and thus the addition of a suitable internal standard would be feasible if the method were to be used routinely for quantification.  
\begin{table}
\begin{tabular}{c c c c c} \hline \hline  & **ELSD** & & **UV** & \\  & **LOD** & **LOQ** & **LOD** & **LOQ** \\ \hline Picein & 5 & 10 & 0.01 & 0.05 \\ Triandrin & 5 & 10 & 0.01 & 0.05 \\ Fructose & 10 & 50 & n.a. & n.a. \\ Glucose & 25 & 125 & n.a. & n.a. \\ \hline \hline \end{tabular}  
* LOD = 2 \(\times\) detector noise, LOQ = 5 \(\times\) detector noise for UV (260 mm), LOQ for ELSD determined experimentally (please note the non-linear response of ELSD); 5 \(\mu\)L injections were used in systems containing UV and ELDS; Phenomenex Luna® Omega Sugar columns were used in all analyses; n.a. = not applicable.  
\end{table}
Table 1: Limit of detection (LOD) and limit of quantification (LOQ) presented as mg/1 for main compounds in willow bark extract.  
Figure 3: Chromatogram (UV at 260 nm and ELSD signals) of willow bark extract. Eluent used was A = 30% MeOH in water, B = ACN (A/B = 20/80).