Prominence 1 lc2030c plus system

Prior to analysis, all samples were filtered through a PTFE filter (VWR, Leuven, Belgium). Naringenin and p-coumaric acid were quantified using a Waters Acquity UPLC H-Class system connected to an ACQUITY TUV-detector operating at 30 °C and 290 nm. A Kinetex^® 2.6 µm Polar C18 100 Å column (Phenomenex, Utrecht, The Netherlands) was used to separate metabolites using the following method, at a flow rate of 0.6 mL/min:

Time (min)	Eluent A: 0.1% TFA in water (%)	Eluent B: 100% acetonitrile (%)
0	90	10
0.5	75	25
5	75	25
7	30	70
8.5	30	70
10	90	10

Glycerol was quantified on a Shimadzu Prominence-I LC2030c Plus system connected to an RID-20A (Shimadzu) detector operating at 40 °C. A Rezex ROA-Organic Acid H + (8%) – 150 × 7,8 mm column (Phenomenex, Utrecht, The Netherlands) at 60 °C was used to separate metabolites using an isocratic method with a flow rate of 0.6 mL/min and 0.005 N H₂SO₄ in water as eluens.

An HPLC Prominence-i LC-2030C Plus system with UV–vis and a RID-20A refractometric detector operated with LabSolutions DB—GPC software v.6.87 (all from Shimadzu, Kyoto, Japan) were used for stability analysis of HA solutions and prepared eye drops. Aqueous gel filtration chromatography (GFC) with UV at 230 nm [47 ] and RI detection (RID) was performed in a Yarra^TM SEC-4000 column (300 mm × 7.8 mm, 3 µm ultra-pure silica particle size, 500 Å pore size) with a SecurityGuard^TM Cartridge GFC-4000 (4 mm × 3.0 mm) System (all from Phenomenex^®, Torrance, CA, USA), at 30 °C. The running buffer was a mixture of 0.2 M NaNO₃ and 0.01 M NaH₂PO₄ (pH 7.0) at a flow rate of 1.0 mL/min [48 ]. The mobile phase was prefiltered through an OlimPeak^TM 0.2 μm hydrophilic PTFE filter (Teknokroma, Barcelona, Spain) and degassed on an ultrasonic degasser. Injections of 20 µL were made. The temperature of both detectors was 40 °C.