Systematic name: Common name(s): Chemical Molecular weight: Exact Mass Molecular Weight: Product# / Lot #: 1-heptadecanoyl-2-(9Z-tetradecenoyl)-sn-glycero-3-phospho-(1'- myo-inositol)(ammonium Salt) PI(17:0/14:1(9Z)) 794.99 (Proton Form) 812.02 (Ammonium salt Form) 794.49 (Proton Form) 811.52 (Ammonium salt Form) LMGP06010006 / LM7-112B Manufacture date: June 24, 2008 Storage Conditions: Concentration: Volume: Total Amount/Ampoule: Solvent: -16 to -24 C 12.72 μm 1.0 ml 10.11 µg methanol Structural Information: Reviewed: Signature on file Approved: Signature on file Date: Date: www.lipidmaps.org Page 1 of 12 www.avantilipids.com
TLC: Conditions: Plate A Mobile Phase: 65:25:4 CHCl 3 :MeOH:H 2 O Plate A 1 2 Lane 1: Suitability (18:1 PE, 18:1 LPE + oleic acid) Lane 2: PI(17:0/14:1(9Z)) Interpretation: Ninhydrin (-), Iodine 1 spot, Phosphorus (+), Char (+), Wet Dip 1 spot Flame Atomic Absorption (Calcium): Interpretation: PI(17:0/14:1(9Z)) < 10 ppm Ca www.lipidmaps.org Page 2 of 12 www.avantilipids.com
Proton Nuclear Magnetic Resonance ( 1 H-NMR): Conditions: Sample conc.: 7.5 mg/ml in 8:2 CDCl 3 : CD 3 OD Temp.: 300 K Field Strength: 400 MHz Pulse: 30 for 3.96 sec. Relaxation time: 1 sec. # of scans: 16 Line Broadening: 0.0 Hz Interpretation: 8 7 6 5 4 3 2 1 ppm Shift (ppm) Actual protons Theoretical Protons Assignment not detected 0 4 NH4 group not detected 0 5 OH groups ~5.3 1.986 2 oleifinic protons ~5.26 0.934 1 proton on the 2nd carbon of the glycero backbone ~4.4 1.031 1 proton on the 1st carbon of the glycero backbone ~4.2 15.692 2 proton on the 1st carbon of the glycero backbone + ring proton + water ~4.0 2.228 2 protons on the 3rd carbon of the glycero backbone ~3.91 1.010 1 ring proton ~3.78 1.064 1 ring proton ~3.65 0.961 1 ring proton ~3.44 1.000 1 ring proton ~3.3 1.083 0 methanol peak ~3.28 0.909 1 ring proton ~2.3 3.905 4 alpha protons ~2.0 3.897 4 allylic protons ~1.6 4.012 4 beta protons ~1.2 38.553 38 remaining -CH2- on the fatty acid chains ~0.89 6.000 6 methyls on the fatty acid chains Actual Theoretical Sum Protons Sum Protons Note: sum excludes chloroform & methanol peaks 67.490 78 0 www.lipidmaps.org Page 3 of 12 www.avantilipids.com
Phosphorus Nuclear Magnetic Resonance ( 31 P-NMR): Conditions: Sample conc.: 7.5 mg/ml in 8:2 CDCl 3 : CD 3 OD Temp.: 300 K Field Strength: 162.0 MHz Pulse: 30 for 0.505 sec. Relaxation time: 2 sec. # of scans: 64 Line Broadening: 0.0 Hz -3.1188 10 5 0-5 -10 ppm -15 Interpretation: One shift under described conditions www.lipidmaps.org Page 4 of 12 www.avantilipids.com
Reversed Phase HPLC with Evaporative Light Scattering Detection (HPLC/ELSD): Conditions: Mobile Phase A: 1:1 MeOH:H 2 O + 20 mm NH 4 OAC Mobile Phase B: 100 % MeOH + 20 mm NH 4 OAC Gradient: Mobile Phase A Mobile Phase B 0 min 25% 75% 5 min 25% 75% 10 min 10% 90% 20 min 0% 100% 30 min 0% 100% Column: Zorbax C8, 4.6 mm X 150 mm i.d,5µm Flow rate: 1 ml/min Detector: Sedex 75, 40 degrees C, 3.5 bar, gain of 5 Injection Volume: 10 µl @ 1 µg/µl 140 mv 13.413 120 100 80 60 40 minutes 0 5 10 15 20 25 30 Interpretation: Purity = 100% AUC www.lipidmaps.org Page 5 of 12 www.avantilipids.com
Fatty Acid Methyl Esters by Capillary Gas Chromatography (GC-FAME): Conditions: Column: DB-225, 30M X 0.25 mm, i.d. Temperatures: Initial Temp. = 220 C, 25 min. Injection = 250 C Detector = 250 C Carrier: Grade 5 Helium at 1 ml/min Injection: 1 µl @ 1:20 split Sample: 5.0 mg/ml in hexane after reaction with 0.2N sodium methoxide in methanol at room temperature for 5 minutes. 110000 counts 100000 90000 80000 70000 60000 50000 40000 30000 20000 10000 0 3.613-14:1 5.202-17:0 0 5 10 15 20 25 minutes 30 Interpretation: Purity = 42.5% 14:1 and 57.0% 17:0, 99.5% total www.lipidmaps.org Page 6 of 12 www.avantilipids.com
Mass Spectroscopy: API 4000 QTRAP Qualitative Conditions: Period 1 Experiment 1: ---------------------------- Scan Type: Enhanced MS (EMS) Polarity: Negative Scan Mode: Profile Ion Source: Turbo Spray # Scans to Sum: 2 Scan Rate: 1000 amu/s Intensity Thres.: 0.00 cps Settling Time: 0.0000 msec MR Pause: 5.0070 msec Q0 trapping: No MCA: No Center/Width: No LIT fill time: 20.00 msec Start (amu) Stop (amu) Time (sec) 594.00 994.00 0.4001 Parameter Table(Period 1 Experiment 1) CUR: 10.00 IS: -4500.00 TEM: 100.00 GS1: 20.00 GS2: 0.00 ihe: ON CAD: High DP 0.00 CES 0.00 CE -10.00 www.lipidmaps.org Page 7 of 12 www.avantilipids.com
-EMS: 3.431 to 4.332 min from Sample 1 (10 µg/ml -EMS FI) of 17.0-14.1 PI LM-1504 LM7-112.wiff (Turbo Spray), subtracted (0.346 to... Max. 1.5e7 cps. 1.5e7 793.5 1.4e7 1.3e7 1.2e7 1.1e7 1.0e7 9.0e6 Intensity, cps 8.0e6 7.0e6 6.0e6 5.0e6 4.0e6 3.0e6 2.0e6 1.0e6 796.6 600 620 640 660 680 700 720 740 760 780 800 820 840 860 880 900 920 940 960 980 m/z, amu Interpretation: -EMS: [M-H] - = 793.5 u www.lipidmaps.org Page 8 of 12 www.avantilipids.com
Qualitative Conditions: (cont d) Period 1 Experiment 1: ---------------------------- Scan Type: Enhanced Resolution (ER) Polarity: Negative Scan Mode: Profile Ion Source: Turbo Spray # Scans to Sum: 2 Resolution Q1: Open Scan Rate: 250 amu/s Intensity Thres.: 0.00 cps Settling Time: 0.0000 msec MR Pause: 5.0070 msec Q0 trapping: No MCA: No Center/Width: Yes LIT fill time: 20.00 msec Start (amu) Stop (amu) Time (sec) 778.52 808.52 0.1201 Parameter Table(Period 1 Experiment 1) CUR: 10.00 IS: -4500.00 TEM: 100.00 GS1: 20.00 GS2: 0.00 ihe: ON CAD: High DP 0.00 CES 0.00 CE -10.00 www.lipidmaps.org Page 9 of 12 www.avantilipids.com
-ER: 0.009 to 0.967 min from Sample 2 (10 µg/ml -ER 793.52 FI) of 17.0-14.1 PI LM-1504 LM7-112.wiff (Turbo Spray), subtracted (0.21... Max. 1.2e7 cps. Intensity, cps 1.19e7 1.15e7 1.10e7 1.05e7 1.00e7 9.50e6 9.00e6 8.50e6 8.00e6 7.50e6 7.00e6 6.50e6 6.00e6 5.50e6 5.00e6 4.50e6 4.00e6 3.50e6 3.00e6 2.50e6 2.00e6 1.50e6 1.00e6 5.00e5 793.5 794.5 795.5 796.6 780 782 784 786 788 790 792 794 796 798 800 802 804 806 808 m/z, amu Interpretation: -ER: [M-H] - = 793.5 u. www.lipidmaps.org Page 10 of 12 www.avantilipids.com
Quantitative Conditions: Column = Luna C18 2x20mm Mobile Phase A = 720:207:62:10, 20 mm (NH4)2CO3 in H2O ph adj. to 9.6 w/ NH4OH: IPA: THF: ACN, Mobile Phase B = 741:222:37, IPA: THF: ACN, A:B 70:30, Flow = 0.4 ml/min, temp = 20c Step Total Time(min) Flow Rate(µl/min)A (%) B (%) 0 0.00 500 50.0 50.0 1 2.50 500 50.0 50.0 Period 1 Experiment 1: ---------------------------- Scan Type: MRM (MRM) Polarity: Negative Scan Mode: N/A Ion Source: Turbo Spray Resolution Q1: Unit Resolution Q3: Unit Intensity Thres.: 0.00 cps Settling Time: 4997.1200 msec MR Pause: 5.0070 msec MCA: No Step Size: 0.00 amu Q1 Mass (amu) Q3 Mass (amu) Dwell(msec) 793.58 224.90 800.00 Parameter Table(Period 1 Experiment 1) CUR: 10.00 IS: -4500.00 TEM: 550.00 GS1: 50.00 GS2: 50.00 ihe: ON CAD: Medium DP -115.00 EP -10.00 CE -64.00 CXP -11.00 CE -64.00 CXP -11.00 www.lipidmaps.org Page 11 of 12 www.avantilipids.com
XIC of -MRM (1 pair): 793.6/224.9 amu from Sample 9 (Ampoule 2 Inj 2) of 17.0-14.1 PI LM-1504 LM7-112B Stability 063008.wiff (Turbo... Max. 1.2e5 cps. 1.25e5 1.16 Intensity, cps 1.20e5 1.15e5 1.10e5 1.05e5 1.00e5 9.50e4 9.00e4 8.50e4 8.00e4 7.50e4 7.00e4 6.50e4 6.00e4 5.50e4 5.00e4 4.50e4 4.00e4 3.50e4 3.00e4 2.50e4 2.00e4 1.50e4 1.00e4 5000.00 0.00 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2.0 2.1 2.2 2.3 2.4 Time, min Interpretation: PI(17:0/14:1(9Z)) = 12.716 µm www.lipidmaps.org Page 12 of 12 www.avantilipids.com