Supplementary Materials

Tracing the thermal history of products through lyso-phospholipid analysis by hydrophilic interaction liquid chromatography-electrospray ionization Fourier- transform mass spectrometry

Ilario Losito1,2,*, Laura Facchini1, Rosa Catucci1, Cosima Damiana Calvano1,2, Tommaso R.I. Cataldi1,2, Francesco Palmisano1,2

1 Dipartimento di Chimica, Università degli Studi di Bari “Aldo Moro”, Via E. Orabona 4, 70126 Bari, Italy; [email protected] 2 Centro Interdipartimentale SMART, Università degli Studi di Bari “Aldo Moro”, Via E. Orabona 4, 70126 Bari, Italy * Correspondence: [email protected]; Tel.: +39-080-544-2506

Titles of supplementary figures, as reported at the end of the main manuscript:

Figure S1. Comparison of typical ESI(+)-FTMS spectra obtained for PC in fresh seafood products

Figure S2. Comparison of typical ESI(+)-FTMS spectra obtained for LPC in fresh seafood products

Figure S3. Comparison of typical ESI(+)-FTMS spectra obtained for PE in fresh seafood products

Figure S4. Comparison of typical ESI(+)-FTMS spectra obtained for LPE in fresh seafood products

Figure S5. Comparison of LPE/PE and LPC/PC ESI(+)-FTMS response ratios obtained for fresh or refrigerated European flat ( Edulis)

Figure S6. Comparison of typical ESI(+)-FTMS spectra obtained for LPC in seafood products after thermal treatments leading to the maximum accumulation of LPC

Figure S7. Comparison of typical ESI(+)-FTMS spectra obtained for LPE in seafood products after thermal treatments leading to the maximum accumulation of LPE

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38:7 804.5537 34:2 36:5 38:6 758.5694 780.5531 806.5682 40:7 100 832.5836 80 o-36:5 o-38:6 Ostrea edulis 60 40:6 42:7 40 30:0 32:1 34:4 p-36:4 p-38:5 39:5 834.5987 (flat ) 754.5382 860.6146 20 706.5373732.5531 766.5738 792.5881 822.5995 0 36:5 34:1 780.5539 o-38:6 38:6 40:7 100 760.5843 36:6 p-38:5 806.5689 39:6 832.5840 778.5381 80 792.5886 820.5843 gigas 60 o-36:5 40:6 34:4 39:5 834.5996 (Japanese oyster) 40 30:0 32:1 p-36:4 37:5 42:7 754.5385 794.5690 822.5999 20 706.5380 732.5536 766.5745 860.6154 0 34:1 38:6 o-38:6 806.5689 38:0 760.5840 818.6639 100 34:2 p-38:5 80 758.5694 o-36:5 Ruditapes philippinarum 60 32:1 36:5 792.5889 34:4 p-36:4 39:5 40:6 (Manila ) 40 30:0 31:0 732.5533 780.5542 42:7 754.5382 822.5995 834.5996 860.6162 20 706.5377 720.5532 766.5745 0 o-36:5 36:4 38:6 p-36:4 782.5696 o-38:6 806.5685 100 40:7 vulgaris 766.5737 p-38:5 832.5835 80 () 60 o-34:1 34:1 36:5 792.5883 40:6 760.5836 780.5541 42:7 tentacle 40 30:0 32:1 p-34:0 834.5990 20 706.5736 732.5533 746.6047 860.6142 Relative Relative Abundance 0 o-36:5 o-36:4 36:4 38:6 p-36:4 p-36:3 782.5696 766.5737 768.5896 806.5685 Octopus vulgaris 100 o-38:6 40:7 80 36:5 p-38:5 832.5836 (common octopus) 60 <<< <<< 34:1 780.5540 792.5883 39:6 40:6 mantle 40 32:1 760.5837 834.5990 20 732.5533 820.5838 0 34:1 760.5837 100 36:2 38:5 32:1 786.5996 808.5854 Parapaeneus longirostris 80 732.5530 38:6 60 36:5 (pink shrimp) 33:1 806.5684 40 780.5533 40:7 746.5689 36:1 40:6 body 20 788.6153 832.5838 834.5991 0 700 710 720 730 740 750 760 770 780 790 800 810 820 830 840 850 860 870 m/z

Figure S1. Comparison between typical ESI(+)-FTMS spectra obtained for the PC class during the HILIC-ESI(+)- FTMS analysis of lipid extracts obtained from different fresh seafood products. Spectra were averaged in the retention time interval 7.3 - 11.6 min (see Figure 1 in the paper).

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16:0 496.3396 100 80 18:1 60 522.3549 Ostrea edulis (flat oyster) 40 20:1 14:0 o-16:0 17:0 18:0 524.3711 550.3861 20 468.3082 482.3601 510.3550 0 16:0 496.3395 100 80 16:1 18:1 Crassostrea gigas 60 494.3246 522.3553 20:1 550.3865 (Japanese oyster) 40 14:0 o-16:0 17:0 18:0 468.3083 482.3604 510.3554 524.3713 20 0 16:0 496.3393 100 80 Ruditapes philippinarum 60 14:0 o-16:0 482.3602 (Manila clam) 468.3081 17:0 40 510.3554 20 0 16:0 496.3392 100 o-16:0 80 482.3599 Octopus vulgaris 60 (common octopus) Relative Abundance o-18:0 18:0 tentacle 40 o-15:0 510.3913 524.3709 20:1 20 468.3444 550.3862 0 16:0 496.3392 100 <<< 80 o-16:0 Octopus vulgaris 60 482.3600 (common octopus) 40 o-18:0 18:1 mantle 522.3551 20 510.3913 0 16:0 496.3394 100 80 o-16:1 16:1 18:1 18:0 Parapaeneus longirostris 480.3447 494.3241 60 522.3551 524.3709 (pink shrimp) 40 o-15:0 o-16:0 17:0 20:1 22:6 body 482.3603 510.3554 20 468.3082 550.3865 568.3393 0 450 460 470 480 490 500 510 520 530 540 550 560 570 580 590 m/z

Figure S2. Comparison between typical ESI(+)-FTMS spectra obtained for the LPC class during the HILIC-ESI(+)- FTMS analysis of lipid extracts obtained from different fresh seafood products. Spectra were averaged in the retention time interval 14.6 - 16.7 min (see Figure 1 in the paper).

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o-40:7/p-40:6 776.5584 100 o-38:6 o-40:3 80 p-38:5 p-40:2 Ostrea edulis 60 750.5427 784.6210 (flat oyster) o-39:7 40 p-39:6 38:5 766.5379 20 762.5430 0 o-40:7/p-40:6 776.5587 100 o-39:7 o-38:6 o-40:3 80 p-38:5 p-39:6 Crassostrea gigas 762.5430 p-40:2 60 o-35:2 750.5429 38:5 o-41:7 36:5 o-39:3 784.6215 (Japanese oyster) p-35:1 o-38:3 766.5376 p-41:6 40 738.5064 p-39:2 716.5586 p-38:2 790.5738 20 756.5899 770.6060 0 o-40:7/p-40:6 o-40:3 776.5583 100 p-40:2 o-35:2 o-39:3 784.6213 80 o-38:6 o-38:3 o-39:7 Ruditapes philippinarum o-34:2 p-35:1 o-37:2 p-38:5 p-38:2 p-39:6 p-39:2 60 o-36:2 (Manila clam) p-34:1 770.6056 716.5582 p-36:1 p-37:1 750.5425 756.5897 762.5426 40:6 40 702.5426 744.5898 730.5739 792.5532 20 0 o-38:6 o-38:5/p-38:4 o-40:7 p-38:5 752.5588 p-40:6 100 36:5 38:6 750.5430 776.5587 738.5062 764.5216 Octopus vulgaris 80 38:5 o-36:5 o-40:6 (common octopus) Relative Abundance 60 o-37:6 766.5375 o-36:6 p-36:4 o-39:7 p-40:5 40:6 tentacle 40 p-37:5 36:4 p-36:5 p-39:6 778.5735 724.5274 736.5272 740.5221 792.5534 20 722.5115 762.5429 0 36:5 o-38:6/p-38:5 738.5063 750.5430 o-38:5/p- 38:4 100 38:5 36:4 752.5584 38:6 40:7 80 740.5222 766.5380 Octopus vulgaris o-36:6 o-36:5 764.5219 o-40:7 790.5372 60 o-37:6 o-38:7 (common octopus) p-36:5 p-36:4 38:4 p-40:6 p- 37:5 p- 38:6 768.5528 40:6 mantle 40 776.5591 722.5118 724.5277 748.5273 792.5531 20 736.5276 0 764.5215 o-36:6 o-37:6 o-38:7 38:6 40:10 100 o-40:7 p- 36:5 p- 37:5 p- 38:6 784.4908 80 o-36:5 p-40:6 Parapaeneus longirostris 736.5278 748.5270 o-38:6 40:7 722.5117 p- 36:4 776.5587 60 p- 38:5 790.5376 (pink shrimp) 724.5274 36:5 750.5428 body 40 34:1 39:6 718.5383 738.5065 20 778.5376 0 700 705 710 715 720 725 730 735 740 745 750 755 760 765 770 775 780 785 790 795 m/z

Figure S3. Comparison between typical ESI(+)-FTMS spectra obtained for the PE class during the HILIC-ESI(+)- FTMS analysis of lipid extracts obtained from different fresh seafood products. Spectra were averaged in the retention time interval 2.3 – 3.4 min (see Figure 1 in the paper).

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466.3285 100 80 Ostrea edulis 60 o-17:1 o-20:2 (flat oyster) 40 p-17:0 18:0 p-20:1 20:5 482.3235 20 452.3128 492.3440 500.2764 0 466.3289 100 o-20:2 o-17:1 80 o-19:1 p-20:1 Crassostrea gigas 60 p-17:0 p-19:0 492.3445 20:1 (Japanese oyster) 452.3131 40 18:0 508.3395 22:6 480.3445 20:5 526.2926 20 482.3237 500.2768 0 22:6 526.2925 100 80 o-18:1 20:5 Ruditapes philippinarum 60 o-17:1 p-18:0 o-17:2 17:0 500.2766 (Manila clam) 40 p-17:1 p-17:0 466.3287 468.3081 20 450.2977 452.3130 0 p-18:0/ o-18:1 100 466.3285 80 Octopus vulgaris (common octopus) 60 o-16:1 o-17:1 o-18:2 18:0 tentacle

Relative Abundance 20:1 40 p-16:0 p-17:0 p-18:1 482.3234 20:5 22:6 508.3392 526.2922 20 438.2972 452.3129 464.3131 500.2764 0

466.3287 100 18:1 18:0 20:4 22:6 502.2925 526.2923 80 480.3081 482.3236 Octopus vulgaris 20:5 20:1 60 o-16:1 o-17:1 o-18:2 o-19:3 508.3393 (common octopus) p-16:0 500.2766 40 p-17:0 p-18:1 p-19:2 mantle 438.2973 20 452.3129 464.3130 476.3115 0 22:6 526.2923 100 o-16:1 o-18:1/p-18:0 18:1 80 p-16:0 480.3080 20:5 466.3287 438.2974 o-17:1 16:0 500.2766 60 o-18:2 454.2922 18:0 p-17:0 p-18:1 20:1 40 482.3236 20:4 452.3131 464.3132 502.2925 508.3394 20 0 440 445 450 455 460 465 470 475 480 485 490 495 500 505 510 515 520 525 530 m/z

Figure S4. Comparison between typical ESI(+)-FTMS spectra obtained for the LPE class during the HILIC-ESI(+)- FTMS analysis of lipid extracts obtained from different fresh seafood products. Spectra were averaged in the retention time interval 3.4 – 4.3 min (see Figure 1 in the paper).

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Ostrea edulis, LPE/PE a 0.12 a 0.10 a 0.08 0.06 0.04 0.02 666 0.00 Fresh Refrigerated (4°C) Refrigerated (4°C) 4 days 7 days

Ostrea edulis, LPC/PC 0.06 a a 0.05 0.04 a 0.03 0.02 0.01 666 0 Fresh Refrigerated (4°C) Refrigerated (4°C) 4 days 7 days

Figure S5. Comparison between LPE/PE and LPC/PC ratios of ESI-(+)-FTMS responses obtained from the HILIC- ESI(+)-FTMS analysis of lipid extracts of European flat oysters (O. edulis) subjected to lipid extraction soon after purchase (as fresh product) or after refrigeration at 4°C for 4 or 7 days in a laboratory refrigerator. Mean values and standard deviations (indicated as error bars) are reported, referred to the number of replicates indicated for each type of sample. The results of a Tukey-Kramer test are reported in the form of sample type grouping, emphasized by letters. See the main text for details on the calculation of the LPE/PE and LPC/PC ratios from XIC chromatograms.

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16:0 496.3396 17:0 18:1 100 510.3553 522.3552 80 18:2 Ostrea edulis 60 20:1 17:1 520.3397 20:5 (flat oyster) 40 o-16:0 550.3867 22:6 508.3403 18:0 542.3241 refrigerated 7 days 482.3605 524.3710 568.3397 20 0 16:0 496.3396 100 Crassostrea gigas 80 18:1 (Japanese oyster) 60 o-16:0 refrigerated 7 days 522.3552 18:0 482.3605 20:1 40 17:0 524.3718 22:6 550.3865 20 510.3553 568.3393 0 568.3396 22:6 100 18:3 20:4 518.3239 544.3395 80 Ruditapes philippinarum o-16:0 (Manila clam) 60 482.3603 refrigerated 7 days 16:0 18:1 20:5 40 496.3396 542.3240 (open valves) 522.3552 20 0

Relative Abundance Relative 18:1 22:6 522.3560 20:5 568.3402 100 16:0 542.3247 o-16:0 22:5 Octopus vulgaris 496.3401 18:0 80 482.3608 18:2 570.3558 524.3722 (common octopus) 60 520.3401 o-18:0 20:4 tentacle 40 544.3402 defrozen/refrigerated 7 days 510.3923 22:4 20 572.3711 608.3852610.1843 0 600 610 16:0 496.3398 18:1 100 522.3560 17:0 Parapaeneus longirostris 80 18:0 (pink shrimp) 16:1 510.3558 60 524.3720 20:5 22:6 body 494.3244 40 17:1 542.3247 20:4 568.3404 defrozen/refrigerated 7 days 20 508.3400 544.3398 0 450 460 470 480 490 500 510 520 530 540 550 560 570 580 590 m/z

Figure S6. Comparison between ESI(+)-FTMS spectra obtained for the LPC class during the HILIC-ESI(+)-FTMS analysis of lipid extracts obtained from seafood products after thermal treatments leading to the maximum accumulation of LPC species. Red labels indicate LPC species that exhibited a significant variation (increase or decrease) in treated products with respect to fresh ones. Spectra were averaged in the retention time interval 14.6 - 16.7 min (see Figure 1 in the paper).

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o-18:1/p-18:0 466.3289 o-20:2 100 p-20:1 Ostrea edulis 80 492.3445 (flat oyster) 60 o-17:1 refrigerated 7 days 40 p-17:0 18:0 20:5 482.3239 20 452.3132 500.2769 0 o-18:1/p-18:0 466.3291 100 20:2 Crassostrea gigas 80 506.3241 18:0 60 o-17:1 o-19:1 (Japanese oyster) 482.3240 o-20:2 p-17:0 p-19:0 20:1 refrigerated 7 days 40 p-20:1 20:5 452.3135 480.3447 508.3398 20 492.3448 500.2769

0 530 o-18:1/p-18:0 466.3288 100 Ruditapes philippinarum 80 (Manila clam) o-17:1 refrigerated 7 days 60 o-20:2 o-16:1 p-17:0 (open valves) 40 p-20:1 22:6 p-16:0 452.3133 492.3448 20 438.2977 526.2924 0

Relative Abundance Relative o-18:1/p-18:0 467.3286 100 Octopus vulgaris 80 (common octopus) tentacle 60 o-17:1 o-19:1 20:5 20:4 22:6 p-19:0 500.2766 defrozen/refrigerated 7 days 40 p-17:0 502.2922 526.2925 20 452.3131 480.3445 0 22:6 526.2924 100 16:0 o-18:1/p-18:0 20:4 Parapaeneus longirostris 80 o-16:1 454.2923 466.3288 18:1 18:0 502.2923 482.3235 (pink shrimp) p-16:0 480.3081 20:5 60 o-17:1 o-18:2 body 500.2767 40 438.2973 o-16:0 p-17:0 p-18:1 17:0 20:1 defrozen/refrigerated 7 days 452.3131 464.3132 468.3088 496.4210 20 440.3131 508.3393 0 435 440 445 450 455 460 465 470 475 480 485 490 495 500 505 510 515 520 525 530 m/z

Figure S7. Comparison between ESI(+)-FTMS spectra obtained for the LPE class during the HILIC-ESI(+)-FTMS analysis of lipid extracts obtained from seafood products after thermal treatments leading to the maximum accumulation of LPE species. Red compositional labels indicate LPE species that exhibited a significant variation (increase or decrease) in treated products with respect to fresh ones. Spectra were averaged in the retention time interval 3.4 – 4.3 min (see Figure 1 in the paper).

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