5 cqa

The analytical standard 5-CQA (CAS Number: 327-97-9) was provided by Carl Roth GmBH (purity: >97%, Karlsruhe, Germany). 3-CQA (CAS Number: 906-33-2, purity > 95%), 4-CQA (CAS Number: 905-99-7, >95%), caffeine (CAS Number: 58-05-2, purity > 99%), glacial acetic acid, and potassium phosphate monobasic were purchased from Sigma-Aldrich (St. Louis, MO, USA). 3-FQA (CAS Number: 1899-29-2, purity 98%) was supplied by Chem Faces (Hubei, China). 3,4-DiCQA (CAS Number: 14534-61-3, purity > 95.0%), 3,5-diCQA (CAS Number: 2450-53-5, purity > 95%), and 4,5-diCQA (CAS Number: 32451-88-0, purity > 95%) were obtained from Biopurity Phytochemicals (Sichuan, China). HPLC-grade acetonitrile and methanol were provided by J.T. Baker (Griesheim, Germany). A Barnstead Nanopure Diamond (Dubuque, IA, USA) was used to make refined deionized water.

High-performance liquid chromatography (HPLC) grade acetonitrile, HPLC grade water, ethyl acetate and caffeic acid (CA) were from Merck (Darmstadt, Germany). Quercetin, kaempferol and isorhamnetin were provided from Sigma-Adrich. 5-caffeoylquinic acid (5-CQA) was purchased from Carl Roth (Karlsruhe, Germany).
Among the isomers, only 5-CQA is available commercially. 5-CQA was isomerized in a saturated aqueous sodium hydrogen carbonate (NaHCO3) solution at 60 °C (28 ) or in 0.2% aqueous sodium carbonate (29 (link)). The 5-CQA yields a mixture of 3-caffeoylquinic acid (3-CQA), 4-caffeoylquinic acid (4-CQA) and 5-CQA by isomerization. To obtain 3-CQA and 4-CQA from 5-CQA, isomerization was performed according to the procedure reported in the literature (34 ). Twenty-five milligrams of 5-CQA was dissolved in 25 ml of distilled water and 25 ml of 0.2% aqueous sodium carbonate was added. After 30 min, 1 ml of 10% (v/v) acetic acid and 5 ml of methanol were added to 4 ml of the reaction solution.