A Preparative-HPLC Method Development and Validation for the Estimation and Identification of Laccaic Acid-A, Laccaic Acid-B, and Laccaic Acid-D

Lac is derived from Laccifer lacca Kerr and has historically been used as a dye due to its coloring qualities. It is now widely utilised in the food and colouring industries. Lac dye was isolated for the first time with the help of preparative HPLC. Further characterization was also performed using NMR and MS spectroscopy techniques.

The column used for the isolation was the Inertsil ODS-3 reverse-phase C18 column. The wavelength of the PDA detector was set between 275 nm and 410 nm. Data acquisition was carried out for Empower 2.0 (Waters Ltd., United States). For the mobile phase, Methanol: acetonitrile (85:15) is the ideal solvent system A, while 0.15% perchloric acid in water is the ideal solvent system B. Ultimately, a 0.22 µm membrane filter was used to filter the mobile phase. We conducted an effective gradient run after the runtime of 33 minutes, setting the flow rate at 15 mL/min. We set up the fraction collector to operate on a time basis, with a maximum collection volume of 5 ml per vial and a period of 0.41 min for each vial.

After the chromatographic run, three single elution peaks were observed with retention times (Rt) of 15.19, 25.09, and 30.54 min, respectively. The compounds associated with Rt 15.19 were effectively collected in vials nos. 13, 22, 31, and 40, respectively, in the fraction collector. The compound with a retention time of 25.09 was collected in vials no. 24, 33, 42, and 51, respectively, whereas the compound with a retention time of 30.54 was collected in vials no. 52, 61, 70, and 79, respectively, in the fraction collector.

A sufficient amount of the samples was collected, and the Rota evaporator (Buchi, Germany) was used to evaporate the solvents. After lyophilization, the dried product was stored at 4°C for further analysis. The compound having an Rt of 15.19 min revealed that it is laccaic acid A. The compound having an Rt of 25.09 indicated that it is laccaic acid B. Finally, the compound having an an Rt of 30.54 min indicated that the compound is laccaic acid D.