Minor phenolic compounds with polar characteristics were extracted and subsequently quantified using high-pressure liquid chromatography (HPLC) according to the standard method of the IOC [58 ]. Some modifications were made based on Türkay et al. [16 (link)] to improve accuracy. HPLC (Agilent 1260 model infinity II, Santa Clara, CA, USA) was equipped with a Spherisorb ODS-2 C18 reverse-phase column (4.6 mm × 25 cm), a 100 A° spectrophotometric UV detector, and an integrator at 280 nm. Two grams of the sample were carefully weighed and transferred into glass tubes, after which 1 mL of a solution containing syringic acid was introduced as an internal standard. The resulting mixture underwent vortexing for 30 s, and then 5 mL of a methanol and water solution in an 80/20 (v/v) ratio was added. Subsequently, the samples underwent a 15 min ultrasonic treatment in a bath, followed by centrifugation at 5000 × rpm for 25 min. The injection volume was 40 μL. The mobile phases were water (0.2% H3PO4 v/v) (A), methanol (B), and acetonitrile (C), and the flow rate was 0.8 mL/min with a gradient flow composition. The gradient elution was as follows, starting from 96% A, 2% B, and 2% C; 50% A, 25% B, and 25% C at 40 min; 40%, 30%, and 30% at 5 min; 50% B and 50% C at 15 min with a 10 min standby; and 96% A, 2% B, and 2% C at 2 min with 10 min standby. The peaks were identified according to the relative retention time of the internal standard peak (syringic acid) described in the standard method. To quantify and express the identified phenolics as tyrosol, the respective response factor (RRF value) was found to be 4.7 (the ratio of the response factor of syringic acid to tyrosol), as described in the standard method. The peaks were identified using the RRT of phenolic compounds against syringic acid, which served as the internal standard of the method.
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