Supporting Information for Publication - page S-1. Comparative analysis of sample preparation methods to handle the complexity of

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1 Supporting Information for Publication - page S-1 Comparative analysis of sample preparation methods to handle the complexity of the blood fluid metabolome: when less is more Sara Tulipani, Rafael Llorach, Mireia Urpi-Sarda, Cristina Andrés-Lacueva* Biomarkers and Nutritional & Food Metabolomics Research Group, Department of Nutrition and Food Science, XaRTA, INSA, Faculty of Pharmacy, University of Barcelona, Av. Joan XXIII s/n, 08028, Barcelona, Spain. INGENIO-CONSOLIDER Program, Fun-C-Food CSD , Ministry of Science and Innovation, Barcelona, Spain *Corresponding Author candres@ub.edu. Phone: Fax: Abstract In the present Supporting Information for Publication file, the authors present additional details potentially useful for a deeper understanding of the analytical workflow applied, as cited in the main manuscript. Details include additional information about the experimental section (LC-MS data acquisition phase) and the Results and Discussion section (data quality assurance and comparative chemometric data analysis). Five Supplementary Figures and four Supplementary Tables are included in this SI file, together with their corresponding legends and descriptive text, and presented in the order of their mention within the main manuscript.

2 Figure S-1. Sample collection and preparation (A). Overview of the metabolomics workflow applied for comparative analysis (B). The workflow consists of five stages: LC-ESI-q-ToF-MS data acquisition, data conversion and preprocessing, data quality assurance, chemometric data analysis and result browsing and interpretation.

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4 Experimental Section. Supplemetal details Aqueous 16-Component Metabolite Mix. An aqueous 16-component metabolite standard mixture was prepared at two final concentrations (5 and 25 µg/ml) from individual standard solutions of the following metabolites (individual stock solutions 1000 µg/ml): gallic acid, syringic acid, (-)-epicatechin, naringenin, 4-hydroxyhippuric acid, D-L-carnitine, L-phenylalanine, L-tryptophan, leucine, isoleucine, acetylcholine chloride, acetyl-l-carnitine hydrochloride, 1-O-palmitoyl-sn-glycero-3-phosphocholine, 1-O-stearoyl-sn-glycero-3-phosphocholine, α-hydroxybutyric acid, glycochenodeoxycholic acid and palmitic acid. All individual standard were dissolved in methanol/water 80/20, v/v, except for palmitic acid (13% tertbutylmethylether in methanol). The standard components were chosen to represent both endogenous and exogenous metabolites, and to approximately cover the retention time and mass range of the biological samples. Two lysophosphatidylcholines were included in the mix to check for the extraction of phospholipids from the samples, through the different sample preparation techniques. Raw and Spiked Biological Samples. Biological samples were collected at the beginning of each study, after an overnight fast, following a low-in-polyphenols washout diet for at least 48 h (baseline, T0), and after the dietary intervention (T1). Post-intervention samples of plasma and serum were respectively collected 2 h after receiving a serving of 40 g of cocoa powder dissolved in 250 ml of water, and 12 h after receiving the last serving of red wine (30 g alcohol/d) of a 4 wk intervention, as detailed in the references suggested within the manuscript (Llorach et al. 2009; Rotches- Ribalta et al. 2012). All samples were stored at -80ºC until analysis.working material for both biological matrices was prepared by pooling aliquots of T0 and T1 samples coming from the distinct volunteers. Samples were subjected to the five metabolite extraction procedures, and extraction carried out in triplicate, to provide technical replicates. Besides on biological samples, the five preparation techniques were also tested on the aqueous 16-component metabolite standard mix (5 µg/ml), in triplicate, to evaluate the procedure effects (solvent-based effects, eventual extraction of plastic liners and plasticizers such as phthalate esters) on metabolite coverage separately from the biological matrix effects. Optimization of LC-MS Analytical Conditions. Different combinations of MS spray parameters were tested in consecutive chromatographic runs, such as IonSpray voltage (from 2500 to 5000 V in both ion modes), declustering potential (from 60 to 90 V in both ion modes) and focusing potential (from 200 to 380 V in both ion modes), and compared for the best peak resolution and metabolic profiling analysis. Other spray parameters were kept as follows: declustering potential 2, -10 V; ion release decay, 6 microseconds; ion release width, 5 microseconds; temperature, 400 ºC; N2 as curtain (50 arbitrary units) and nebulizer (60 arbitrary units) gases. The ToF was calibrated with taurocholic acid (ions at m/z and m/z ) (1 pmol/µl) and reserpine (ions at m/z and m/z ) (1 pmol/µl) for negative and positive mode calibration, respectively. The LC gradient elution program and MS operating conditions (in ES+ and ES modes) optimized for plasma and serum metabolomics analysis are presented below. A range of

5 different RP gradients were tested in the present study, so to obtain the elution of all the standard compounds monitored within the runtime, extend the gradient wash time and avoid the progressive increase in the background signal noise in the lipophilic part of the chromatogram. HPLC gradient elution program and MS optimized conditions for plasma and serum metabolomics analysis (ES + and ES modes): HPLC gradient elution (ES+ and ES-) Flow rate Mobile Mobile Time (min) (ml min 1 ) phase A (%) phase B (%) Initial 0, , ,5 0, , , ,1 0, ,5 0, MS conditions ES+ mode ES- mode IS (V) DP (V) FP (V) DP2 (V) IRD 6 6 IRW 5 5 TEM (ºC) CUR (L/min) GAS (L/min) GAS 2 (L/min) 60 60

6 Figure S-2. Overview of the data quality was obtained through PCA of the global data sets (biological and QC samples), based on the hypothesis that the closer the samples of each category appear on the scores plot the more reproducible the performance of the LC MS system should be. (A) PCA scores plot of all the QC and biological samples injected (plasma, ES- ion mode). Colors indicate different batches. (B) (C-D) PCA scores plot of the biological samples injected, with colors and ellipses indicating QC3 (plasma, ES+ ion mode). Colors indicate different batches.pca scores plots of all the plasma samples injected, excluding the aqueous QC samples (C, ES- ion mode; D, ES+ ion mode). Colors indicate different extraction methods, regardless of the types of samples (raw T0, spiked T0, T1). Sample positioning in the 2D scores plot space appears to depend more on the extraction than on the sample type. On the other hand, samples extracted by methods A, B and C positioned more closely among them, indicating more similar profiles for samples subjected to conventional solvent extraction.

7 Results and discussion Table S-1. Variation in mass accuracy, retention time and response for selected ions in aqueous QC2 samples injected along the doubly randomized plasma samples analysis Detected mass (m/z) Ret time (min) Peak Area Analyte Name mean Error (mda) mean CV (%) mean CV (%) Negative mode α-hydroxyisobutyric acid Gallic acid Hydroxyhippuric acid Siryngic acid (-)-Epicatechin Naringenin Glycochenodeoxycholic acid Palmitic acid Positive mode D-L-carnitine Acetylcholine Acetyl-L-carnitine Leucine/Isoleucine L-Phenylalanine L-Triptophan Palmitoyl-sn-glycero-3-phosphocholine Steroyl-sn-glycero-3-phosphocholine

8 Table S-2. Mean values and percentage variation coefficients (CV) for retention times and peak areas of the eighteen standard compounds (eight compounds in negative mode, eight in positive mode) spiked in aqueous, plasma and serum samples (5 µg/ml), for each of the extraction conditions tested (at least 6 datapoints each). Negative mode H2O - 5ppm PLASMA 0h_5ppm SERUM 0h_5ppm RT Peak Area RT Peak Area RT Peak Area Analyte Name Method mean CV(%) mean CV(%) mean CV(%) mean CV(%) mean CV(%) mean CV(%) α-hydroxyisobutyric acid A B C D E Gallic acid A B C D E Hydroxyhippuric acid A B C D E Siryngic acid A B

9 C D E (-)-Epicatechin A B C D E Naringenin A B C D E Glycochenodeoxycholic acid A B C D E Palmitic acid A B C D E Positive mode H2O - 5ppm PLASMA 0h_5ppm SERUM 0h_5ppm RT Peak Area RT Peak Area RT Peak Area Analyte Name Method mean CV(%) mean CV(%) mean CV(%) mean CV(%) mean CV(%) mean CV(%)

10 D-L-carnitine A B C D E Acetylcholine A B C D E Acetyl-L-carnitine A B C D E Leucine/Isoleucine A B C D E L-Phenylalanine A B C D E L-Triptophan A

11 Palmitoyl-sn-glycero-3- phosphocholine B C D E A B C D E Steroyl-sn-glycero-3-phosphocholine A B C D E Note. A. solvent extraction with acetonitrile; B. solvent extraction with methanol; C. solvent extraction with methanol/ethanol (50:50 v/v); D. ultrafiltration; E. Ostro 96-well plates technology.

12 Table S-3. Evaluation of the sample preparation techniques according to the ability to significantly differentiate raw and iked samples, and to give the highest fold change in the mass signal intensities 1. Metabolites RT Ion mode Monoisotopic mass Raw versus spiked (1 µg/ml) samples p < Highest Fold Change Raw versus spiked (5 µg/ml) samples p < Highest Fold Change D-L-carnitine 0.27 ESI A A A, B, C, D, E D acetylcholine 0.3 ESI D, E E A, B, D B acetyl-l-carnitine 0.33 ESI B,C, D, E E A, C, D, E B (iso)leucine 0.57 ESI E E A, C, D, E B L-phenylalanine 1.14 ESI D, (E) E A, B, C, D, E A L-tryptophan 2.58 ESI (A), B, D A A, B, C, D, E B α-hydroxyisobutyric acid 0.8 ESI A A Gallic acid 1.01 ESI A, B, C, D, E E A, B, C, D, E F 4-Hydroxyhippuric acid 2.65 ESI A, B, C, D, E B A, B, C, D, E B Syringic acid 4.22 ESI A, B, C, E A A, B, C, D, E D (-)-Epicatechin 4.28 ESI A, B, C, D, E C A, B, C, D, E C Naringenin 5.09 ESI A, B, C, D, F C A, B, C, D, E C Glycochenodeoxycholic acid 5.42 ESI A, B, E E A, B, C, E B Palmitoyl-sn-glycero ESI phosphocholine - - (B) - Steroyl-sn-glycero-3-phosphocholine 6.43 ESI (B) B Palmitic acid 7.32 ESI (B) - B, C B 1 As results of paired t-test (data not adjusted with Bonferroni correction). 2 Methods within parentheses enable to detect differences although with lower significance (p < 0.05)

13 Figure S-3. Methods comparison (ES+ ion mode). Signal intensities trend plots of common metabolites monitored in raw plasma samples subjected to the five sample treatments (Ext A Ext E). Samples subjected to the different preparation procedures are distinguished by colors at the top of the intensity bars.

14 Figure S-4. (A) PCA-DA scores plot of the raw plasma samples injected (ES+ ion mode), classified according to the different sample preparation methods (Ext A Ect E). As observed in the corresponding loading plot (B), a series of mass features (within the red square) contributed to the classes separation. (C) Superimposed signal intensity trend plot of the main discriminative mass features with higher intensity in samples subjected to extraction methods A, B, C. (D) Signal intensity trend plots of lysophosphatidilcholines detected among the discriminative metabolites.

15 Figure S-5. Venn diagrams obtained by second-order analysis showing the overlap of the five sample treatments in the detection of significant plasma mass features following a dietary intervention (metaxcms outputs).

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17 As major drawback, the SPE-mediated removal of phospholipids was again associated with lower feature coverage of other compounds of unpolar nature and late elution time (RT > 4.9 min), namely free fatty acids. In contrast, the conventional solvent extraction procedures were able to detect a significant reduction in their plasma concentration following cocoa consumption, which could be a reasonable cause of the decrease in circulating acylcarnitines observed concomitantly, and would be consistent with the increased fat storage previously argued as a consequence of coffee consumption