*2.5. Phytochemical Analysis by LC-MS*/*MS*

For the description of bark extracts' phytochemical composition a validated analytical method of liquid chromatography tandem mass spectrometry was employed (LC-MS/MS). The analysis was performed using an Agilent 1100 HPLC Series system (Agilent, Santa Clara, CA, USA) which was equipped with binary gradient pump, degasser, auto sampler, column thermostat, and UV detector. The LC system was coupled with an Agilent Ion Trap 1100 SL mass spectrometer (LC/MSD Ion Trap VL).

The LC-MS/MS analytical method was previously developed and validated [15,16] and was used for the identification of 18 polyphenols in BBE samples: caftaric acid, gentisic acid, caffeic acid, chlorogenic acid, *p*-coumaric acid, ferulic acid, sinapic acid, hyperoside, isoquercitrin, rutin, myricetol, fisetin, quercitrin, quercetin, patuletin, luteolin, kaempferol, and apigenin. For the chromatographic separation of polyphenols, a reverse-phase analytical column was used (Zorbax SB-C18, 100 mm × 3.0 mm i.d., 3.5 μm). The mobile phase was a mixture of methanol: acetic acid 0.1% (v/v) and a binary gradient was used. The elution started with a linear gradient, beginning with 5% methanol and ending at 42% methanol for 35 minutes; then isocratic elution followed for 3 min with 42% methanol. For the chromatographic data processing, the ChemStation and DataAnalysis software (Agilent, Santa Clara, CA, USA) were used.

Moreover, in order to identify other six polyphenols in BBE samples (epicatechin, catechin, syringic acid, gallic acid, protocatechuic acid, and vanillic acid) a second LC-MS method was employed [15]. For the separation of the aforementioned compounds, the same analytical column as previously specified was used. Likewise, the mobile phase consisted of a mixture of methanol: acetic acid 0.1% (v/v) and a binary gradient was used. The elution started with 3% methanol for 3 min, followed by 8% methanol until 8.5 min when 20% methanol was used and kept for the next 10 min, and then the column was rebalanced with 3% methanol. The flow rate was set at 1 mL/min and the sample injection volume was 5 μL. The MS detection mode was selected for detection of the polyphenolic compounds. The ionization on the MS system was in negative mode using an electrospray ion source (capillary +3000 V, nebulizer 60 psi (nitrogen), dry gas nitrogen at 12 L/min, dry gas temperature 360 ◦C). Each identified polyphenol was further quantified in BBE extracts. The results were expressed as milligrams of phenolic compound per gram of herbal material.
