Mass Directed Fraction Collection of Natural Products: Examples from Turmeric & Green Tea Extract
Natural products have been a source of inspiration for pre-clinical drug discovery both by exploring traditional medicines and to discover new spaces in pharmacology. Isolation and characterization of natural products remains a major barrier in drug discovery. Isolation is generally done on an analytical scale and then compounds are characterized fully before any scaleup purifications are attempted. The ability to purify compounds in complex natural product mixtures by highly specific MS data allows for the simplification of the purification and characterization steps of the process. Here, flash and prep chromatography are coupled with MS detection to purify natural products in green tea and turmeric extracts.
Isolation of the major catechins in green tea and the major curcuminoids in turmeric was completed via extractions. Green tea leaves were extracted, and the crude material was analyzed using UPLC-MS and analytical standards to identify the catechins of interest and develop a suitable prep-LC method for isolation. Turmeric powder was extracted and then analyzed by TLC-MS using Advion’s Plate Express™ to identify the compounds of interest and develop a suitable flash chromatography method for isolation. Both extracts were purified using mass-directed fraction collection using Interchim’s puriFlash 5.250 flash/prep chromatography system connected to Advion’s expression® single quadrupole mass spectrometer. Target compounds were detected using XIC MS channels. Purity of the isolated compounds was then determined using HPLC-MS.
We were able to successfully isolate the 3 major curcuminoids (curcumin, demethoxycurcumin, and bisdemethoxycurcumin) in turmeric and the 5 major catechins in green tea ((-)-epigallocatechin (EGC), (-)-epicatechin (EC), (-)-epigallocatechin-3-gallate (EGCG), (-)-epicatechin-3-gallate (ECG), and (-)-gallocatechin gallate (GCG) with high purity (≥95%).
An isocratic flash chromatography method (97:3 DCM:MeOH) was developed using TLC (97:3 DCM:MeOH) to purify the curcuminoids. The TLC plate was analyzed by APCI– MS using the Plate Express which extracts spots directly from the plate with no need for sample preparation. Fractions were collected using extracted ion chromatogram (XIC) channels with APCI– MS. Fractions were then characterized by ASAP– MS and purity for each fraction was determined using HPLC-MS.
A preparative LC method was developed for the catechins in green tea using HPLC-MS and reference standards to identify each compound of interest. Using a water methanol gradient and collecting fractions using XIC channels set for the compounds of interest. Fractions were characterized and their purity determined by HPLC-MS. EGC, EGCG, GCG, and ECG fractions were determined to have purities of 100 %, 99.8 %, 98.8, and 100 % respectively.