PI(17:0/14:1(9Z)) (Proton Form) (Ammonium salt Form) (Proton Form) (Ammonium salt Form) LMGP / LM7-112B

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1 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)) (Proton Form) (Ammonium salt Form) (Proton Form) (Ammonium salt Form) LMGP / LM7-112B Manufacture date: June 24, 2008 Storage Conditions: Concentration: Volume: Total Amount/Ampoule: Solvent: -16 to -24 C μm 1.0 ml µg methanol Structural Information: Reviewed: Signature on file Approved: Signature on file Date: Date: Page 1 of 12

2 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 Page 2 of 12

3 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: ppm Shift (ppm) Actual protons Theoretical Protons Assignment not detected 0 4 NH4 group not detected 0 5 OH groups ~ oleifinic protons ~ proton on the 2nd carbon of the glycero backbone ~ proton on the 1st carbon of the glycero backbone ~ proton on the 1st carbon of the glycero backbone + ring proton + water ~ protons on the 3rd carbon of the glycero backbone ~ ring proton ~ ring proton ~ ring proton ~ ring proton ~ methanol peak ~ ring proton ~ alpha protons ~ allylic protons ~ beta protons ~ remaining -CH2- on the fatty acid chains ~ methyls on the fatty acid chains Actual Theoretical Sum Protons Sum Protons Note: sum excludes chloroform & methanol peaks Page 3 of 12

4 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: MHz Pulse: 30 for sec. Relaxation time: 2 sec. # of scans: 64 Line Broadening: 0.0 Hz ppm -15 Interpretation: One shift under described conditions Page 4 of 12

5 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 1 µg/µl 140 mv minutes Interpretation: Purity = 100% AUC Page 5 of 12

6 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 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 counts : : minutes 30 Interpretation: Purity = 42.5% 14:1 and 57.0% 17:0, 99.5% total Page 6 of 12

7 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: msec MR Pause: msec Q0 trapping: No MCA: No Center/Width: No LIT fill time: msec Start (amu) Stop (amu) Time (sec) Parameter Table(Period 1 Experiment 1) CUR: IS: TEM: GS1: GS2: 0.00 ihe: ON CAD: High DP 0.00 CES 0.00 CE Page 7 of 12

8 -EMS: to min from Sample 1 (10 µg/ml -EMS FI) of PI LM-1504 LM7-112.wiff (Turbo Spray), subtracted (0.346 to... Max. 1.5e7 cps. 1.5e e7 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 1.0e m/z, amu Interpretation: -EMS: [M-H] - = u Page 8 of 12

9 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: msec MR Pause: msec Q0 trapping: No MCA: No Center/Width: Yes LIT fill time: msec Start (amu) Stop (amu) Time (sec) Parameter Table(Period 1 Experiment 1) CUR: IS: TEM: GS1: GS2: 0.00 ihe: ON CAD: High DP 0.00 CES 0.00 CE Page 9 of 12

10 -ER: to min from Sample 2 (10 µg/ml -ER FI) of PI LM-1504 LM7-112.wiff (Turbo Spray), subtracted ( 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 5.00e m/z, amu Interpretation: -ER: [M-H] - = u. Page 10 of 12

11 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 (%) 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: msec MR Pause: msec MCA: No Step Size: 0.00 amu Q1 Mass (amu) Q3 Mass (amu) Dwell(msec) Parameter Table(Period 1 Experiment 1) CUR: IS: TEM: GS1: GS2: ihe: ON CAD: Medium DP EP CE CXP CE CXP Page 11 of 12

12 XIC of -MRM (1 pair): 793.6/224.9 amu from Sample 9 (Ampoule 2 Inj 2) of PI LM-1504 LM7-112B Stability wiff (Turbo... Max. 1.2e5 cps. 1.25e 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 1.00e Time, min Interpretation: PI(17:0/14:1(9Z)) = µm Page 12 of 12