First results

Results of target analysis - LC-Qtrap-MS

Digestion and fecal fermentation

DIGESTION AND FECAL FERMENTATION - WORKFLOW

Method development and validation for determination of 36 neuroactive compounds in tomato prior and after digestion and fecal extracts - Target analysis using LC-Qtrap-MS

Tomato prior and after digestion

•100 mg lyophilized tomato/digested pallet + ISTD mix, ultrasound extraction with methanol

•Calibration curve standards (16 calibration points)

•Sample dilution 0x (aspartame, bisphenols, guanine)x, 100x (chlorogenic, pantothenic acid, tryptophane, phenylalanine), 1000x (citric, glutamic acid) dilution

•2-3 concentration level

•LOQ: 0.0004 (p-coumaric acid, tomatine) – 20 µg/g dry tomato (linoleic acid)

•Linearity R2 > 0.82

•Range 0.0004 – 80 µg/g dry tomato (16 calibration points)

•Precision RSD < 15%

•Matrix effect ± 0.5–96%

Recovery 48.5 – 163.5 %

Fecal extracts

•100µl of fecal sample à 5x dilution with methanol + ISTD mix

•2 concentration level

•Matrix matched calibration (17 calibration points), subtraction of blank

•LOQ: 0.0005 (aspartame, acetaminophen) – 5 ppm (phenylalanine, linoleic acid)

•Linearity R2 > 0.85

•Range 0.0005 – 200 ppm

•Precision RSD < 15%

Distribution of neuroactive compounds after diogestino of tomato

Percentage of compounds after tomato digestion remaining in pallet (dark blue), in liquid phase (light blue) and percentage of biotransformation (light green) and formation (dark green) during digestion. The comopunds in pallet have the potential to reach the colon while will the ones in liquide most probably be extracted with urine.

Fecal batch culture fermentation model

Kinetics of neuroactive compounds during fecal fermentation

No biological variability
No difference between tomato and toxic tomato
Similar trend but higher presence in the presence of digested tomato: pyridoxic acid, eriodyctiol, caffeic acid, isomers of chlorogenic acid, glutamine, linoleic acid, quinic acid, quercetin, coumaric acid, serotonin, tomatine, rutin, tryptophane
Increasing concentration during fermentation: serotonin, phenylalanine

Batch repeatability
Biotransformation
- Fast (< LOQ, 3-6h): citric acid, chlorogenic acid isomers, quinic acid, rutin, tomatine, aspartame, guanine
- Moderate (< LOQ, 24h): pantothenic acid, eriodyctiol, glutamic acid
- Slow (< 50%, 24h): BPS, difenoconazole, pyridoxic acid, L-dopa, linoleic acid, tyramine, tyrosine

ROS analysis of all fecal samples - DPPH assay

Donor 1

No difference of antioxidant activity between different coniditon, time of fermentation nor different donor.

Donor 2

Donor 3

Results of non-target analysis - LC-QTOF-MS

Digestion and fecal fermentation

Pre-processing of non target data using mzMine4

all samples anlaysed in full scan and data dependent acquisition
after pre-processing, including imputation, normalization, transformation - generation of mass feature list (> 10,000 mass features) for both polarities
the highest variance - time of fermentation

Objectives and question to be answered

Does the digested tomato change the fermentation when added prior fermentation (control = digested inulin versus samples with digested tomato)
Is there an effect of toxic compounds added to tomato prior digestion which were also later fermented (tomato without toxic compounds versus tomato with toxic compounds)
What is the metabolism of compounds that were added before fermentation (samples with added compounds versus blank samples)

Reduction of mass feature list - in house develop scritps in R

Statistical analysis: significance: abs(ES) > 1 & p-value < 0.05

Relevant mass features selected = both criteria are true for at least two consecutive
- time points comparing the value of mass feature in two groups (control versus treatment)
- kinetic curve behavior
Possible relevant mass features ~700 per polarity
Presence in media
All time points significant: source of digestion mixture
RT cut
~300 per polarity: checking the MS2, peak shape, isotopic pattern, difference treatment versus
- controlling with annotated target compound for which the trend is known

Final list of mass features for identification

Identification of unknown mass features using in-house and online data bases and sirius

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