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INTRODUCTION

Consumption of Kombucha, a fermented tea product

containing a variety of live microorganisms, is

associated with improved gastrointestinal health and

general improvement of wellbeing. Of particular interest

is the modification of the microbiome, or host of

microbial entities that populate the human gut as a result

of consumption.1,2

In addition, the tea contains a number

of polyphenols and other antioxidants that are also

associated with improved health.3

This mixture of polyphenols from tea and metabolites

from the bacteria and yeast co-culture represent a

complex mixture that requires high resolution methods

to profile. Multiple chromatographic methods are

required to provide coverage over the breadth of diverse

analytes.

UNTARGETED LC/IMS/MS PROFILING OF POLYPHENOLS AND BACTERIA/YEAST COCULTURE METABOLITES IN KOMBUCHA AND TEAS

Melissa L Caldwell1; ANDREW BAKER2

1University of California, Santa Cruz, Santa Cruz, CA; 2Waters, Inc., Pleasanton, CA

METHODS

Samples of commercial kombucha and home-brewed teas

were centrifuged at 14,000 x g for 10 minutes. Five replicate

1 µLinjections of each sample were analyzed in random order

by RP or HILIC chromatography (Waters HSS T3 2.1 x

100mm or BEH Amide, 2.1x100 mm) coupled to an IMS-

enabled Q-TOF instrument (Waters VION ) operated in

positive and negative ion ESI mode. Nitrogen was used as

the mobility gas at a pressure of 7.5x10-3 mbar). A data-

independent approach was used to generate comprehensive

fragmentation profiles for the mobility and chomatographically

separated components. Resulting LC/IMS profiles were

processed with Progenesis QI to identify differences in the

polyphenol profiles for several kombucha

preparations. Collision cross section (CCS) values were

measured for a number of polyphenols and related species.

References

1. Kinross, J.m., von Roon, A. C., Holmes, E., Darzi, A., and Nicholson, J.K., Curr. Gastroenterol Rep.

2008, 10, 396.

2. Kozyrovska, N.O., Reva, O. M., Goginyan, V., and de Vera, J.P., Biopolymers and Cell. 2012, 28. 83.

3. Yao, L.H., Jiang, Y. M., Shi, J., Tomas-Barberan, F/ A., Datta, N., Singanusong, R., Chen, S,S., Plant

Foods for Human Nutrition. 2004, 59, 113.

4. Ge, Y, Tang, Y., Guo, S., Liu, X, Zhu, Z., Liu, P., and Duan, J., Int. J. Anal. Chem. 2015,

http://dx.doi.org/10.1155/2015/394526

CONCLUSION

Increased analytical peak capacity by incorporating IMS separation.

Rapid, confident characterization of polyphenolic components from teas and identification additives.

Use of Progenesis QI for multivariate classification of starting teas and finished product as well as characterization of mobility resolved isomeric species.

VION has excellent mass and CCS measurement reproducibility across wide dynamic range.

Kombucha is an acquired taste.

RP Method BEH HSS T3, 2.1x100 mm 1.8 µm dp

Mobile Phase A 0.1% Aqueous Formic Acid Mobile Phase B 0.1% Formic Acid in Acetonitrile Gradient Hold 99% A for 1 minutes Ramp from 1 to 65% A in 5 minutes Ramp to 1% A in 0.5 minute Hold 2 minutes Return to Initial Conditions HILIC Method

4 BEH Amide, 2.1x100 mm 1.7 µm dp

Mobile Phase A 5mM NH4OAc, 5 mM NH4For, 0.2% HFor in Water Mobile Phase B 1mM NH4OAc, 1 mM NH4For, 0.2% HFor in Acetonitrile Gradient Hold 2% A for 1 minutes Ramp from 2 to 18% A in 5 minutes Ramp from 18 to 20% A in 6 minutes Ramp to 65% A in 2 minutes Hold 2 minutes Return to Initial Conditions

Figure 1. Kombucha production

Figure 2. VION IMS Q-Tof

UNTARGETED SCREENING REVERSED PHASE ES-

UNTARGETED SCREENING REVERSED PHASE ES+

Figure 3. UNIFI Discovery tool used for automated compound

identification. The workflow consists of an elemental composition

determination, ChemSpider search, and then automated matching of

theoretical fragment ions to the experimental data.

Figure 4. Left Panel: Raw low energy and high energy spectra.

Right Panel: IMS filtered low and high energy spectra. Fragment ions

are matched to theoretical (in silico) or authentic spectra.

Figure 8. Results of targeted analysis of Black Tea. Left Panel: Reconstructed total ion chromatogram of all

found components and IMS-Filtered XICs for Caffeine from low and high energy functions.

Middle Panel Annotated low and high energy spectra along with UV spectrum. Right Panel: Trend plot for

caffeine across multiple replicate analyses of each tea or Kombucha sample. Caffeine is not observed in

Rooibos tea or Kombucha made from Rooibos tea.

Figure 6. PCA Bi-Plot and trend Plot for ESI– analysis of tea and Kombuchas. Panel A top, Black tea; bottom, Kombucha made with

Balck tea. Panel B top, Green tea; bottom, Kombucha made with Green tea. Panel C top, White tea; bottom, Kombucha made with

White tea. Panel D top, Rooibos tea; bottom, Kombucha made with Rooibos tea.

Figure 7. PCA Bi-Plot and trend Plot for ESI+ analysis of tea and Kombuchas. Panel A top, Black tea; bottom, Kombucha made with

Balck tea. Panel B top, Green tea; bottom, Kombucha made with Green tea. Panel C top, White tea; bottom, Kombucha made with

White tea. Panel D top, Rooibos tea; bottom, Kombucha made with Rooibos tea.

Figure 5. Figures of merit for Epicatechin 3 gallate.

Panel A, Observed CCS 200.31 Å2, 0.21% RSD.

Panel B, mass measurement accuracy –0.12 mDa

Standard deviation 0.25 mDa.

Panel C, Detector counts.

A

B

C

Figure 9. Representative data for HILIC separation of

Kombucha. While retention times are greatly

different between RP and HILIC methods, good

agreement is observed for CCS; typically less than

1%.

Figure 10. Representative plots from Multivariate analysis of teas and

kombuchas. Panel A 3d scores plot. Panel B O-PLS-DA S-Plot and

variable trend plots for species high in White tea (mild or non-

fermented). Panel C. Mobility resolved plots for a isomers of a

compound observed in Green (compact form) or Rooibos (larger CCS)

teas.

A

B

C

A B C D

A B C D

Ionization Mode ESI +ve or –ve MS Resolution 53,000 fwhm @ m/z 556 Acquisition Rate 10 spectra/sec HDMSE Method Low Energy 4 V High Energy Ramp 20 to 45V Mobility Gas N2 at 25 mL/min

LC METHODS

MS PARAMETERS