SPLASH Booklet

Avanti’s high-purity lipids are transformed into high-accuracy tools for quantitation and identification of lipids by the research and pharma markets. These products support clinical biomarker discovery and have brought Lipidomics Analysis to the forefront of personalized medicine research and the study of disease mechanisms. Lipidomic Standards

UltimateSPLASH ™ ONE ONE Internal Standard for Lipidomic Analysis Product Number 330820L-1EA Lipids comprise an amazingly complex and diverse set of molecules that are involved in numerous biological processes including energy storage/release, signaling, growth, disease, and death. Over the last 20 years, use of soft ionization techniques in conjunction with single and multistage mass spectrometry both with and without prior separations have enabled unprecedented levels of sensitivity, speed, and specificity for identification, structure determination, and quantitation of these highly diverse ensemble of molecules. However, one of the primary challenges that remained was the development of a comprehensive set of internal standards which will allow accurate quantitation of numerous individual molecular species. UltimateSPLASH™ONEwas created to address the need for a comprehensive set of internal standards and greatly expands the number of chain length and unsaturation variants within a given class, which may be used to control for differences in ionization and fragmentation efficiency. These internal standards are intentionally designed to appear in the spectral gaps between naturally occurring lipid species, and their relative abundances have been varied to reflect the patterns found in nature. Additionally, UltimateSPLASH™ incorporates these features into more lipid classes than ever before including: PI, PS, PG, LPI, LPS, and LPG are now included for even broader lipidomics applications. The resulting final mixture contains a total of 69 unique and highly pure lipids across 15 lipid classes, which have been meticulously characterized, quantitated, and blended for accurate quantitation using multiple lipidomics techniques.

Inaddition toour easy tousemixtureof all 69 internal standards inonevial, UltimateSPLASH™ internal standards are available in class specific mixtures, as well as individual internal standards. Details on ordering individual internal standards can be found on our website, along with the 15 separate class mixtures. One vial containing 69 high-purity deuterium labeled internal standards covering 15 lipid classes. Each vial contains 1.2mL of 1:1 DCM:MeOH solution that has been meticulously characterized, quantitated, and blended for accurate quantitation using multiple lipidomics techniques.

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Ultimate SPLASH™ ONE | Product Number 330820L-1EA

Product Number 860906 860907 860908 860910 860909 860911 860914 860912 860913 700220 700221 700222 700223 700226 860748 860747 860746 860745 860679 860741 860740 860742 860743 860744 855683 855682 855681 855680 855678 856721 856720 856719 856718 856717 858135 858134 858133 858132 858131 858153 858152

-

(M+H) +

(M-NH4) -

(M+NH4) +

(M-Na) -

(M+Na) +

(M+AcO) -

Internal Standard Component

Formula

Exact Mass Conc µg/mL

(M-H)

14:0-13:0-14:0 TG-d5 14:0-15:1-14:0 TG-d5 14:0-17:1-14:0 TG-d5 16:0-15:1-16:0 TG-d5 16:0-17:1-16:0 TG-d5 16:0-19:2-16:0 TG-d5 18:1-17:1-18:1 TG-d5 18:1-19:2-18:1 TG-d5

C 44 H 79 D 5 O 6 C 46 H 81 D 5 O 6 C 48 H 85 D 5 O 6 C 50 H 89 D 5 O 6 C 52 H 93 D 5 O 6 C 54 H 95 D 5 O 6 C 56 H 97 D 5 O 6 C 58 H 99 D 5 O 6 C 60 H 103 D 5 O 6 C 41 H 63 D 7 O 2 C 43 H 67 D 7 O 2 C 45 H 71 D 7 O 2 C 47 H 71 D 7 O 2 C 49 H 73 D 7 O 2 C 34 H 58 D 7 NO 3 C 36 H 62 D 7 NO 3 C 38 H 66 D 7 NO 3 C 40 H 70 D 7 NO 3 C 42 H 74 D 7 NO 3

731.692

713.6582 739.6738 767.7051 795.7364 823.7677 849.7834

25 50 75

757.7077

785.739

100 125 100

813.7703 841.8016 867.8172 893.8329 919.8485 947.8798 619.6153 647.6466 675.6779 699.6779

875.799

75 50 25 25 50 75 50 25 75 50 25 50 75 75 50 25 50 75 50

901.8147

18:1-21:2-18:1 TG-d5

929.846

14:1 cholesteryl-d7 ester 16:1 cholesteryl-d7 ester 18:1 cholesteryl-d7 ester 20:3 cholesteryl-d7 ester

601.5815 629.6128 657.6441 681.6441 707.6598 542.5404 570.5717

624.5707

652.602

680.6333 704.6333

22:4 cholesteryl-d7 ester

725.6936

730.649

C16:1 Ceramide-d7 C18:1 Ceramide-d7 C20:1 Ceramide-d7 C22:1 Ceramide-d7 C24:1 Ceramide-d7

541.5331 569.5644 597.5957

543.5477

565.5296 593.5609 621.5922 649.6235

601.5542 629.5855 657.6168 685.6481 713.6794 768.6223 796.6536 824.6849 852.7162 880.7475 781.5761 809.6074 837.6387 861.6387

571.579

598.603

599.6103 627.6416 655.6729 710.6157

626.6343

625.627

654.6656

653.6583

677.6548

16:1 SM (d18:1/16:1)-d9 18:1 SM (d18:1/18:1)-d9 20:1 SM (d18:1/20:1)-d9 22:1 SM (d18:1/22:1)-d9 24:1 SM (d18:1/24:1)-d9

C 39 H 68 D 9 N 2 O 6 P 709.6084 C 41 H 72 D 9 N 2 O 6 P 737.6397 C 43 H 76 D 9 N 2 O 6 P 765.671 C 45 H 80 D 9 N 2 O 6 P 793.7023 C 47 H 84 D 9 N 2 O 6 P 821.7336 C 39 H 71 D 5 NO 8 P 722.5622 C 41 H 75 D 5 NO 8 P 750.5935 C 43 H 79 D 5 NO 8 P 778.6248 C 45 H 79 D 5 NO 8 P 802.6248 C 47 H 81 D 5 NO 8 P 828.6405 C 36 H 65 D 5 NO 8 P 680.5153 C 38 H 69 D 5 NO 8 P 708.5466 C 40 H 73 D 5 NO 8 P 736.5779 C 42 H 73 D 5 NO 8 P 760.5779 C 44 H 75 D 5 NO 8 P 786.5935 C 37 H 65 D 5 NaO 10 P 733.4918 C 39 H 69 D 5 NaO 10 P 761.5231 C 41 H 73 D 5 NaO 10 P 789.5544 C 43 H 73 D 5 NaO 10 P 813.5544 C 45 H 75 D 5 NaO 10 P 839.5701 C 37 H 64 D 5 NNaO 10 P 746.4871 C H D NNaO P 774.5184

738.647

766.6783 794.7096 822.7409 723.5695 751.6008 779.6321 803.6321 829.6478 681.5226 709.5539 737.5852 761.5852 787.6008 734.4991 762.5304 790.5617 814.5617 840.5773 747.4943 775.5256

17:0-14:1 PC-d5 17:0-16:1 PC-d5 17:0-18:1 PC-d5 17:0-20:3 PC-d5 17:0-22:4 PC-d5 17:0-14:1 PE-d5 17:0-16:1 PE-d5 17:0-18:1 PE-d5 17:0-20:3 PE-d5 17:0-22:4 PE-d5 17:0-14:1 PG-d5 17:0-16:1 PG-d5 17:0-18:1 PG-d5

100 150 100

887.6543

50 25 50 75 50 25 25 50 75 50 25 25 50

679.508

707.5393 735.5706 759.5706

785.5863

751.5256 779.5569 807.5882 831.5882

710.5026 738.5339 766.5652 790.5652 816.5808 723.4978 751.5291

17:0-20:3 PG-d5

3

17:0-22:4 PG-d5 17:0-14:1 PS-d5 17:0-16:1 PS-d5

857.6039

745.4798 773.5111

17:0-14:1 PE-d5

C 36 H 65 D 5 NO 8 P 680.5153 C 38 H 69 D 5 NO 8 P 708.5466 C 40 H 73 D 5 NO 8 P 736.5779 C 42 H 73 D 5 NO 8 P 760.5779 C 44 H 75 D 5 NO 8 P 786.5935 C 37 H 65 D 5 NaO 10 P 733.4918 C 39 H 69 D 5 NaO 10 P 761.5231 C 41 H 73 D 5 NaO 10 P 789.5544 C 43 H 73 D 5 NaO 10 P 813.5544 C 45 H 75 D 5 NaO 10 P 839.5701 C 37 H 64 D 5 NNaO 10 P 746.4871 C 39 H 68 D 5 NNaO 10 P 774.5184 C 41 H 72 D 5 NNaO 10 P 802.5497 C 43 H 72 D 5 NNaO 10 P 826.5497 C 45 H 74 D 5 NNaO 10 P 852.5653 C 58 H 99 D 5 6 901.8147 Formula C 44 H 79 D 5 6 C 46 H 81 D 5 6 C 48 H 85 D 5 6 C 50 H 89 D 5 6 C 52 H 93 D 5 6 C 54 H 95 D 5 6 C 56 H 97 D 5 6 13 6 82 739 6738 767.705 95.7364 823.7677 49.7834 875.799

25 50 75 50 25 25 50 75 2 50

856721 856720 856719 856718 856717 858135 Product Number 858134 858133 60906 858132 60907 858131 858153 60910 858152 858151 6091 858150 60914 860912 858149 800827 6 913 800826 800824 7 2 1 800825 7 2 2 700223 800823 850109 70 226 850110 850111 6 747 850112 6 746 860745 850118 850107 6 679 850108 6 741 860740 850106 858146 860742 858148 860743 860744 858147 858123 855683 858130 855682 855681 858129 870309 855680 855679 855678 856721 855778 856709 856720 856710 856719 856718 856716 856717 858135 858134 858133 858132 858131 858153 858152 60908 60909 7 2 0 6 748

679.508

681.5226 709.5539 737.5852 761.5852 787.6008 734.4991 762.5304 790.5617 814.5617 840.5773 747.4943 775.5256 803.5569 827.5569

17:0-16:1 PE-d5 17:0-18:1 PE-d5 17:0-20:3 PE-d5 17:0-22:4 PE-d5

707.5393 735.5706 759.5706

Continued

Ultimate SPLASH™ ONE | Product Number 330820L-1EA

785.5863

17:0-14:1 PG-d5 17:0-16:1 PG-d5 Internal Standard Component

751.5256 779.5569 807.5882 831.5882 757.7077 857.6039 785.739 813.7703 841.8016 867.8172 731.692 893.8329 919.8485 575.5406 603.5719 19.6153 631.6032 655.6032 7 . 779 681.6188 699.6779 725.6936 947.8798 47. 466

710.5026 738.5339 (M-Na) - 766.5652 790.5652 816.5808 723.4978 751.5291 779.5604 803.5604

-

(M+H) +

(M-NH4) -

(M+NH4) +

(M+Na) +

(M+AcO) -

Exact Mass Conc µg/mL

(M-H)

17:0-18:1 PG-d5 4 3 0-14:0 TG-d5 14:0-15:1-14:0 TG-d5 17:0-20:3 PG-d5 17:0-22:4 PG-d5 17:0-14:1 PS-d5 16:0-15:1-16:0 TG-d5 17:0-16:1 PS-d5 17:0-18:1 PS-d5 16:0-19:2-16:0 TG-d5 17:0-20:3 PS-d5 17:0-22:4 PS-d5 18:1-19:2-18:1 TG-d5 17:0-14:1 DG-d5 17:0-16:1 DG-d5 4:1 cholesteryl-d7 ester 17:0-18:1 DG-d5 6:1 cholesteryl-d7 ester 18:1 cholesteryl-d7 ester 17:0-20:3 DG-d5 17:0-22:4 DG-d5 20:3 cholesteryl-d7 ester 22:4 cholesteryl-d7 ester 17:0-14:1 PI-d5 17:0-16:1 PI-d5 C16: Ceramide-d7 17:0-18:1 PI-d5 C18: Ceramide-d7 C20:1 Ceramide-d7 17:0-20:3 PI-d5 17:0-22:4 PI-d5 C22:1 Ceramide-d7 C24:1 Ceramide-d7 15:0 Lyso PI-d5 17:0 Lyso PI-d5 6:1 SM (d18:1/16:1)-d9 19:0 Lyso PI-d5 15:0 Lyso PS-d5 20:1 SM (d18:1/20:1)-d9 17:0 Lyso PS-d5 22 1 SM (d18:1/22:1)-d9 24:1 SM (d18:1/24:1)-d9 19:0 Lyso PS-d5 15:0 Lyso PG-d5 17:0-14:1 C-d5 14:0-17:1-14:0 TG-d5 16:0-17:1-16:0 TG-d5 18:1-17:1-18:1 TG-d5 8:1-21:2-18:1 TG-d5 18:1 SM (d18:1/18:1)-d9

25 25 100 50 75 100 7 125

745.4798 773.5111 801.5424 825.5424

50 75 50 25 25 50 75 50 50 75 50 25 25 50 75 50 50 25 50 25 25 7 50 75 50 25 25 25 50 50 75 25 25 50 25 75

851.558

853.5726

829.5761

557.5068 585.5381 601. 815 613.5694 637.5694 5 .6441 663.585 681.6441 707.6 98 5 2. 404 5 0.5717 929.846 29.6128

C 34 H 59 D 5 O 5 C 36 H 63 D 5 O 5 C 38 H 67 D 5 O 5 C 40 H 67 D 5 O 5 45 71 7 2 C 42 H 69 D 5 O 5 47 71 7 2 C 49 H 73 D 7 O 2 C 34 H 58 D 7 NO 3 C 36 H 62 D 7 NO 3 60 103 5 6 41 63 7 2 43 67 7 2 C 38 H 66 D 7 NO 3 C 40 H 70 D 7 NO 3 C 42 H 74 D 7 NO 3

580.496 24. 707 608.5273 636.5586 652.602 660.5586 8 .6333 704.6333 686.5742 730.649 565.5296 593.5609 621.5922 649.6235

C 40 H 73 D 5 NO 13 P 816.5525 C 42 H 77 D 5 NO 13 P 844.5838 C 44 H 81 D 5 NO 13 P 872.6151 C 46 H 81 D 5 NO 13 P 896.6151 C 48 H 83 D 5 NO 13 P 922.6307 C 24 H 45 D 5 NO 12 P 580.3384 C 26 H 49 D 5 NO 12 P 608.3697 39 68 9 2 O 6 7 9.6084 C 41 H 72 D 9 N 2 O 6 P 737.6397 C 28 H 53 D 5 NO 12 P 636.401 C 21 H 36 D 5 NNaO 9 P 510.273 C 43 H 76 D 9 N 2 6 P 765.671 C 23 H 40 D 5 NNaO 9 P 538.3043 C 25 H 44 D 5 NNaO 9 P 566.3356 C 21 H 37 D 5 NaO 9 P 497.2778 C 23 H 41 D 5 NaO 9 P 525.3091 C 41 H 75 D 5 N 8 P 750.5935 C 25 H 45 D 5 NaO 9 P 553.3404 C 23 H 43 D 5 NO 7 P 486.3482 C 45 H 79 D 5 NO 8 P 802.6248 C 25 H 47 D 5 NO 7 P 514.3795 C 27 H 51 D 5 NO 7 P 542.4108 C 36 H 65 D 5 NO 8 P 680.5153 C 20 H 37 D 5 NO 7 P 444.3013 C 22 H 41 D 5 NO 7 P 472.3326 C 40 H 73 D 5 NO 8 P 736.5779 C 24 H 45 D 5 NO 7 P 500.3639 C 42 H 73 D 5 NO 8 P 760.5779 C 44 H 75 D 5 NO 8 P 786.5935 C 37 H 65 D 5 NaO 10 P 733.4918 C 39 H 69 D 5 NaO 10 P 761.5231 C 47 H 81 D 5 NO 8 P 828.6405 C 38 H 69 D 5 NO 8 P 708.5466 C 41 H 73 D 5 NaO 10 P 789.5544 C 43 H 73 D 5 NaO 10 P 813.5544 C 45 H 75 D 5 NaO 10 P 839.5701 C 37 H 64 D 5 NNaO 10 P 746.4871 C H D NNaO P 774.5184 598.603 626.6343 654.6656 C 45 H 80 D 9 N 2 O 6 P 793.7023 C 47 H 84 D 9 N 2 6 821.7336 C 39 H 71 D 5 N 8 P 722.5622 C 43 H 79 D 5 NO 8 P 778.6248

798.5186 826.5499 854.5812 878.5812 904.5969 562.3046 590.3359

541.5331 569.5644 597.5957

543.5477

545.3621 573.3934 887.6543 809.6074 837.6387 880.7475 781.5761 861.6387 601.5542 629.5855 657.6168 685.6481 713.6794 768.6223 796.6536 824.6849 852.7162

571.579

599.6103 627.6416 655.6729 710.6157

625.627

940.6645 598.3723 626.4036

653.6583

677.6548

738.647

618.3672

654.4349

487.3555 515.3868 829.6478 543.4181 445.3086 709.5539 473.3399 737.5852 761.5852 501.3712 787.6008 766.6783 794.7096 822.7409 723.5695 751.6008 779.6321 803.6321 681.5226 734.4991 762.5304 790.5617 814.5617 840.5773 747.4943 775.5256

509.2658 537.2971

487.2838 515.3151 543.3464 474.2886 502.3199

565.3284

17:0 Lyso PG-d5 17:0-16:1 C-d5 19:0 Lyso PG-d5 15:0 Lyso PC-d5 7: -20:3 - 17:0 Lyso PC-d5 19:0 Lyso PC-d5 17:0-14:1 PE-d5 15:0 Lyso PE-d5 17:0 Lyso PE-d5 : -18:1 - 19:0 Lyso PE-d5 17:0-20:3 PE-d5 17:0-22:4 PE-d5 17:0-14:1 PG-d5 17:0-16:1 PG-d5 17:0-18:1 PG-d5 7: -18:1 C- 17:0-22:4 -d5 7: -16:1 -

50 100 150 25 25 100

530.3512

485.341

50 50 25 25 25 50 50 75 50 25 25 25 50 75 50 25 25 50

513.3723 679.508 541.4036 707.5393 443.294 735.5706 471.3253 759.5706 499.3566 785.5863

601.4247

751.5256 779.5569 807.5882 831.5882

710.5026 738.5339 766.5652 790.5652 816.5808 723.4978 751.5291

4

17:0-20:3 PG-d5

17:0-22:4 PG-d5 17:0-14:1 PS-d5 17:0-16:1 PS-d5

857.6039

745.4798 773.5111

Best Seller Spotlight SPLASH ® LIPIDOMIX ® Mass Spec Standard Product Number 330707-1EA The original SPLASH internal standard mixture has been a best-seller since it was first developed in 2016. It contains one deuterium labeled internal standard for each of 14 lipid classes at ratios relative to human plasma lipids. Each vial contains 1mL of internal standard mixture in methanol solution with a suggested sample spiking ratio of 10µL SPLASH to 10µL human plasma, allowing for up to 100 samples to be analyzed with one vial. Concentrations are verified for use as a quantitative standard.

EquiSPLASH ™ LIPIDOMIX ® Mass Spec Standard | Product Number 330731-1EA

EquiSPLASH is popular for its higher concentrations of internal standard for each lipid class, allowing users to apply to multiple sample types (tissues, cell extracts, plasma). Each vial contains 1mL of internal standard mixture in methanol solution, 13 lipid classes supplied at 100µ g/mL each. Concentrations are verified for use as a quantitative standard. This standard is also ideal for use in method development and system verification experiments to determine linearity and sensitivity prior to analyzing samples.

Target Conc. µM 213 48 8

Target Conc. µg/mL 160.7 25.5

Target Conc. µM

Target Conc. µg/mL

Exact Mass

Chemical Formula

Exact Mass

Chemical Formula

Internal Standard 15:0-18:1(d7) PC 18:1(d7) Lyso PC 15:0-18:1(d7) PE 18:1(d7) Lyso PE 15:0-18:1(d7) PG 15:0-18:1(d7) PI 15:0-18:1(d7) PS 15:0-18:1(d7)-15:0 TAG 15:0-18:1(d7) DAG 18:1(d7) MAG

Internal Standard 15:0-18:1(d7) PC 18:1(d7) Lyso PC 15:0-18:1(d7) PE 18:1(d7) Lyso PE 15:0-18:1(d7) PG 15:0-18:1(d7) PI 15:0-18:1(d7) PS 15:0-18:1(d7)-15:0 TAG 15:0-18:1(d7) DAG 18:1(d7) MAG

752.6061 528.3921 710.5591 486.3451 741.5537 829.5698 754.5490 811.7646 587.5506 363.3366 657.6441 737.6397 689.4989

C 41 H 73 D 7 NO 8 P C 26 H 45 D 7 NO 7 P C 38 H 67 D 7 NO 8 P C 23 H 39 D 7 NO 7 P C 39 H 68 D 7 O 10 P C 42 H 72 D 7 O 13 P C 39 H 67 D 7 NO 10 P

133 189 141 205 131 118 129 123 170 275 152 135 188

752.6061 528.3921 710.5591 486.3451 741.5537 829.5698 754.5490 811.7646 587.5506 363.3366 657.6441 737.6397 530.5404

C 41 H 73 D 7 NO 8 P C 26 H 45 D 7 NO 7 P C 38 H 67 D 7 NO 8 P C 23 H 39 D 7 NO 7 P C 39 H 68 D 7 O 10 P C 42 H 72 D 7 O 13 P C 39 H 67 D 7 NO 10 P C 51 H 89 D 7 O 6 C 36 H 61 D 7 O 5 C 21 H 33 D 7 O 4 C 45 H 71 D 7 O 2 C 41 H 72 D 9 N 2 O 6 P C 33 H 58 D 7 NO 3

100 100 100 100 100 100 100 100 100 100 100 100 100

5.7 5.3 29.1

11 38 11 5

9.1 4.2 57.3 9.4 2 356.1 30.9 7.4

C 51 H 89 D 7 O 6 C 36 H 61 D 7 O 5 C 21 H 33 D 7 O 4 C 45 H 71 D 7 O 2

71 16 6 541

18:1(d7) Chol Ester d18:1-18:1(d9) SM 15:0-18:1(d7) PA

18:1(d7) Chol Ester d18:1-18:1(d9) SM C15 Ceramide-d7

C 41 H 72 D 9 N 2 O 6 P C 36 H 61 D 7 NaO 8 P

42 11

Cholesterol-d7

248 393.3988

C 27 H 39 D 7 O

98.4

5

Continued

Ceramide LIPIDOMIX Ceramides have significant clinical implications with regards to inflammation, immune response, and a variety of diseases. Ceramide LIPIDOMIX was developed to provide a clinically relevant ceramide mixture, and as such it contains several key ceramides shown to be correlated to cardiovascular disease 1 .

SPLASH II SPLASH II is a variation on the original SPLASH designed for plasma lipidomics. Modifications include the addition of plasmalogens and the removal of cholesterol, PG, PA, and MAG.

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New Splash Mixtures

MALDI SPLASH for Mass Spec Imaging As the field of Mass Spectrometry Imaging grows, there is a need to increase standardization and quantitation across samples and between labs. MALDI SPLASH is designed to be sprayed onto tissue samples similarly to how matrix is applied prior to MALDI imaging. As a result, multiple internal standards on the surface of the tissue sample can be used for accurate spatial lipid quantitation. The concentrations of the mixture are based on lipid ratios present in brain tissue.

SphingoSPLASH I Sphingolipids are increasingly recognized as a fascinating class of lipids involved in a variety of molecular functions from membrane composition to cellular signaling. As such, it is vital that sphingolipids can be accurately quantitated- that is why we developed SphingoSPLASH I. This mix is composed of equimolar concentrations of lipids representative of the sphingolipid class.

7

Mouse SPLASH Mouse SPLASH is similar to the original SPLASH, but with concentrations based on the lipid composition of murine samples.

8

Frequently Asked Questions How do I handle Avanti Mass Spec Standards properly?

Can I get a Custom Mass Spec Standard? Yes, we custom formulate mass spec standards using the lipids and solvents of your choice. Weworkwith you to design your custommass spec standard and canmake recommendations if needed. Let us know how we can help by emailing lipidomics@avantilipids.com. Avanti’s Lipidomics Division has been making Custom Quantitative Mass Spec Standards for the Lipidomics community for over 15 years. We specialize in high-accuracy quantitative packaging of lipids for use as mass spec standards, robotic assisted packaging of solutions, accurate quantitation of lipid concentrations, and rigorous quality control testing. Who do I contact if I have additional questions? Please e-mail us at lipidomics@avantilipids.com if you have any additional questions. Custom Synthesis Avanti prides itself on being innovative by discovering new lipids for our customers. We are constantly adding to our extensive list of products for research. However, if you cannot find the perfect lipid to make your project a success, or you have a suggestion for a new product that is needed in the field, our synthesis team is ready to take on a new challenge. With over 150 years of combined lipid synthesis experience, our team can solve most synthesis problems and deliver custom lipids with Avanti’s signature quality. Please contact our synthesis team at customsynthesis@avantilipids.com to discuss the amazing things we can do for you.

Your Avanti Mass Spec Standard should be stored in a -10°C to -25°C freezer until ready for use. It is designed to be a one-time use ampule, and we do not recommend extended storage after opening. Always make sure to warm bath sonicate the unopened ampoule for approximately 2 minutes prior to opening the ampoule. Lipids in solution may precipitate during shipping and storage conditions, and it may not be visible with a solution at extremely low concentrations such as this. Directly transfer from ampule to sample preparation glass vial for immediate use. General handling guidelines for lipids should be followed as outlined on our website. How do I order Avanti Mass Spec Standards? Please visit the Avanti website. Customers in the United States can order directly from Avanti, and customers outside the United States will be directed to our worldwide distribution partner for country specific ordering information and pricing. Can I use my Avanti Mass Spec Standard more than once? Avanti quantitative mass spectrometry standards are designed to be “one-time use” items. It is important to directly transfer from the ampule to the experiment or prepare as a dilution for immediate use. Avanti cannot guarantee product purity and subsequent performance if used outside these guidelines. Howdo I reference anAvanti MassSpecStandard inmypublication? Please list the full name and product number of the Avanti product in your materials and methods sections, along with “Avanti Polar Lipids, Alabaster, AL” as the source. If you publish using an Avanti product we would love to hear about it! Please drop us an email and share your success stories. How do I open the sealed glass ampule? Ampules are pre-scored for easy opening. With the ampule upright, hold the ampule in a gloved hand and firmly push the top section away from you with even pressure. The top should cleanly snap off, but proceed with care, as it will leave a sharp glass edge.

9

Suggested Extraction Procedures For Research Use Only. Not for use in diagnostic procedures. Extraction Protocol for Plasma 1. Use 13 x 100 mm new glass screw capped tubes. Do not use washed tubes as you may extract detergent residue. 2. Add 990 µl water to 10 µl plasma, then let sit on ice for 10 minutes. 3. Add 2.0 mL methanol. 4. Add 0.9 mL dichloromethane. 5. Vortex. 6. A singe phase should appear. If there are two distinct phases, add 50 µl methanol and vortex. If solution is still not a single phase, repeat addition of 50 µl methanol and vortex. 7. Add 10 µl UltimateSPLASH™ ONE Internal Standard, vortex, and let mixture sit for 30 minutes at room temperature. 8. Add 1 mL water. 9. Add 0.9 mL dichloromethane.

Continued

3. Add 2.0 mL methanol.Add 990 µl water to 10 µl plasma, then let sit on ice for 10 minutes. 4. Add 0.9 mL of dichloromethane. 5. Vortex. 6. Repeat steps 6-16 from Extraction Protocol for Plasma. Extraction Protocol for Solid Tissue 1. Weigh tissue to be extracted. 50-100 mg is sufficient. Calculate water content. Expected values are as follows: • Adipose 18% • Brain 60% • Bone 44% • Heart, kidney, liver, lung, intestines, spleen, and stomach 65% • Testes 18% 2. Add water to tissue so that total volume is 1 mL. Example: 100 mg of brain tissue corresponds to 60 µL water. Add 940 µL water. 3. Disperse tissue. I. Grind tissue frozen in liquid nitrogen using cold mortar and pestle. II. Blend using a homogenizer. 4. Sonicate for 30 seconds with 5 seconds bursts and 20 second rest time. Perform sonication steps on ice. 5. Add 2.0 mL of methanol. 6. Add 0.9 mL dichloromethane. 7. Vortex. 8. Repeat steps 6-16 from Extraction Protocol for Plasma.

10. Invert tube 10 times. DO NOT VORTEX. 11. Centrifuge at 1200 rpm for 10 minutes. 12. Collect lower layer and put into a new glass tube. 13. Add 2 mL dichloromethane to remains in extraction tube. 14. Mix, centrifuge, and collect lower layer. Add to first extract. 15. Evaporate solvent under a stream of nitrogen. 16. Re-suspend lipids in injection solvent.

Extraction Protocol for Cell Culture 1. Use 13 x 100 mm new glass screw capped tubes. Do not use washed tubes as you may extract detergent residue. 2. Collect cells: I. Wash cells with non-buffered saline to remove cell culture medium. II. For cells in suspension: centrifuge, discard saline, and add 1 mL water. Vortex and transfer to glass tube for extraction. Rest on ice for 10 minutes. Ensure final volume is 1 mL and adjust if necessary. III. For adhered cells: wash cells with non-buffered saline. Add 1 mL water to lyse cells and scrap. Collect cell lysate and transfer to glass tube for extraction. Rest on ice for 10 minutes. Ensure final volume is 1 mL and adjust if necessary.

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New Products Bile Acids

Bile acids are potent digestive surfactants which promote the absorption of lipids. They also represent the primary pathway for cholesterol catabolism and account for approximately 50% of the daily turnover of cholesterol [2]. However, bile acids are no longer limited to solely that. Bile acids have now been recognized as hormones involved in the regulation of various metabolic processes. Bile acids regulate not only their own synthesis and enterohepatic circulation, but also triglyceride, cholesterol, glucose and energy homeostasis. Current metabolomic and lipidomic efforts aim to provide new insights into the mechanism of bile acid signaling in the regulation of lipid metabolism and the maintenance of lipid homeostasis. [1,2] Avanti’s comprehensive line of pure bile acid primary standards and internal standards are the perfect complement to primary, secondary, and/or conjugated bile acid research in any clinical, toxicology, or research laboratory. [1] Qi Y, Jiang C, Cheng J, Krausz KW, Li T, Ferrell JM, Gonzalez FJ, Chiang JY. Bile acid signaling in lipid metabolism: metabolomic and lipidomic analysis of lipid and bile acid markers linked to anti-obesity and anti-diabetes in mice. Biochim Biophys Acta. 2015 Jan;1851(1):19-29. PMID: 24796972 [2] Staels B, Fonseca VA. Bile acids and metabolic regulation: mechanisms and clinical responses to bile acid sequestration. Diabetes Care. 2009 Nov;32 Suppl 2(Suppl 2):S237- 45. PMID: 19875558

Bile Acid Primary Standards Now Available from Avanti: https://avantilipids.com/product-category/sterols/bile-acids

Bile Acid Internal Standards Now Available from Avanti: **weblink when available**

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IsoPure Phospholipids

Mixed-acyl glycerophospholipids are among the most abundant lipids in nature. Famously, 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC) is the most abundant phospholipid in most eukaryotic cells and tissues. The molecular structure of POPC follows the textbook consensus that unsaturated fatty acyl chains occupy the sn-2 position of the glycerol backbone while saturated fatty acyl chains are relegated to the sn-1 position. As our analytical technologies advance, this conventional wisdom is being turned inside out with the isomer 1-oleoyl-2-palmitoyl phosphatidylcholine (OPPC) being identified at high abundance in samples ranging from plasma to tissue [1,2,3]. Adding to the complexity, both isomers are present in many instances in ratios ranging from 1:100 to 1:1. Resolving the puzzle of lipid isomers requires advanced analytical tools in conjunction with structurally defined reference materials. Until recently, commercially available synthetic lipids were also comprised of mixtures, as transacylation occurring in the round-bottom flask always led to 10-20% OPPC in any preparation of POPC. Moreover, the regiopurity of the product was difficult to define. Avanti has now introduced the IsoPure line wherein innovative synthetic procedures produce mixed-acyl glycerophospholipids with >99% regiopurity. These next- generation standards will be pivotal to isomer-resolved identification of glycerophospholipids in biological samples. [1] Ekroos K, Ejsing CS, Bahr U, Karas M, Simons K, Shevchenko A. Charting molecular composition of phosphatidylcholines by fatty acid scanning and ion trap MS3 fragmentation. J. Lipid Res. 2003, 44, 2181. PMID: 12923235 [2] Zacek P, Bukowski M, Rosenberger TA, Picklo M. Quantitation of isobaric phosphatidylcholine species in human plasma using a hybrid quadrupole linear ion-trap mass spectrometer. J. Lipid Res. 2016, 57, 2225. PMID: 27688258 [3] Maccarone AT, Duldig J, Mitchell TW, Blanksby SJ, Duchoslav E, Campbell JL. Characterization of acyl chain position in unsaturated phosphatidylcholines using differential mobility-mass spectrometry.J. Lipid Res. 2014, 55, 1668. PMID: 24939921

Dive Into Lipidomics with UltimateSPLASH ™ ONE Complete Internal Standard Solution for Quantitative Lipidomics Now available from:

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IsoPure Phospholipids Now Available from Avanti: https://avantilipids.com/product-category/phospholipids/isopure

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