**2.10. HPLC analysis**

Three grams of fresh frozen strawberry from different production systems was mixed with 15 mL acetone and homogenized using a Polytron® homogenizer (Brinkmann Instruments, Inc., Westbury, NY, USA) for 1 min. The extracts were then filtrated through no. 1 Whatman filter paper and concentrated under vacuum in a water bath at 35°C with a Heidolph Laborota 4000 rotary evaporator (Elk Grove village, IL, USA). The concentrated sample was dissolved in 15 mL of acidified water (3 g/100g formic acid) and then passed through a C18 Sep-Pak cartridge (Waters Ltd., Mississauga, ON, Canada), which was previously activated with methanol followed by water, and then 3 g/100 g aqueous formic acid. The anthocyanins and other phenolics were then recovered with 3 mL of acidified methanol containing 3 g/100 g formic acid. The methanol extract was passed through a 0.45-μm Acrodisc syringe filter (Sigma Chemical Co., Oakville, ON, Canada), and 10 μL of each sample was injected for the HPLC analysis. All standards except for anthocyanins were dissolved in methanol. Anthocyanins were dissolved in 1 g/100g HCl in methanol [30].

The individual phenolic compounds of berries were analysed by HPLC, using a Varian (Varian Inc., Palo Alto, CA, USA) HPLC system. A photodiode array detector (Model 335) and a quaternary pump (Model 240) were equipped in the HPLC system. Samples were injected at 40°C, and the separation was performed with a reverse phase 4.6-mm Polaris C18-A column coupled with a 4.6-mm MetaGuard Polaris C18-A guard column (Varian Inc., Palo Alto, CA, USA). The mobile phase was acidified water containing 2.5 g/100g formic acid (A) and acidified methanol containing 2.5 g/100 g formic acid (B). The flow rate was 1.0 ml/min, with a gradient profile consisting of B with the following proportions (v/v) of A: 0–15 min, 15–30% B; 15–20 min, 30% B; 20–25 min, 30–80% B; 25–42 min, 80% B; 42–42.5 min, 80–100% B; 42.5–50.25 min, 100% B; and 50.25–51.25 min, 0% B. The detector was simultaneously monitoring the different groups of phenolics at 254, 280, 320, 350 and 510 nm. Equilibration time between samples was 15 min. Data were collected and analysed by Varian Workstation Star version 6.41. Total phenolic compounds were divided into six groups and quantified as described in our previous work [30].
