Hieracium, L.

Milutinović, Violeta, Niketić, Marjan, Krunić, Aleksej, Nikolić, Dejan, Petković, Miloš, Ušjak, Ljuboš & Petrović, Silvana, 2018, Sesquiterpene lactones from the methanol extracts of twenty-eight Hieracium species from the Balkan Peninsula and their chemosystematic significance, Phytochemistry 154, pp. 19-30 : 29

4.4. LC-MS analysis of dry MeOH extracts of flowering aerial parts of 28 Hieracium species

Dried and powdered flowering aerial parts of 28 Hieracium species were pre-extracted with CH 2 Cl 2 and then bimacerated with MeOH in the same manner as in the case of the flowering heads of H. calophyllum . The number of individual plants from each species used for the extraction, the total masses of plant material, the masses of the obtained methanol extracts, and extraction yields are presented in Table 4 . The obtained dried MeOH extracts were dissolved in MeOH (8.5 mg /ml), filtered through a membrane filter (0.22 μm) and subjected to qualitative and quantitative LC/SQ-MS analysis. For this purpose, Zorbax SBAq column (150 × 3.0 mm; 3.5 μm particle size, Agilent Technologies), operating at 25 ̊C, was used. The mobile phases consisted of 5 mM HCO 2 NH 4 in H 2 O (A), and MeOH (B). The gradient program was as follows: 5–10% B (8 min), 10–30% B (5 min), isocratic 30% B (2 min), 30-20% B (2 min), isocratic 20% B (2 min), 20–30% B (4 min), 30–40% B (5 min), 40–95% B (2 min), with the total run of 30 min, post run of 5 min, flow rate of 0.3 ml/min, and Vinj of 3 μl. DAD was operating at 210 and 254 nm, while MSD data were acquired in the positive ion mode: in the full-scan mode in the range of m/z 80–700, and in the Selected Ion Monitoring (SIM) mode at m/z 540.20 (1 and 3), 378.10 (2) of [M+H] +, and 442.20 (4) of [M+NH 4] +. Ion source parameters were the same as for the preparative LC-MS analysis. The MSD (in SIM mode) peak areas were used for the quantitative analysis by the external standard method. Isolated compounds (1–4) were used as the reference substances and dissolved in MeOH to obtain stock solutions (0.2 mg /ml). The calibration curves were constructed using ten different concentrations of 1–4. For each concentration, the data were recorded three times. Linearity, linear ranges, LODs and LOQs of the compounds were determined according to International Conference on Harmonization guidelines (ICH, 2005). Resulting regression equations were used to test the linearity by calculating the correlation coefficients (r 2), as well as to determine LODs and LOQs using the standard deviations of the response (σ) and the calibration curve slopes (a), in the following way:

LOD=3.3 × σ / a

LOQ =10 × σ / a

(1)

(2)