Systematic name: Common name(s):, GPA(17:0/14:1(9Z)) Chemical Molecular weight: Exact Mass Molecular Weight: Product# / Lot #: 632.85 (proton form) 649.88 632.44 (proton form) 649.47 LMGP10010006 / LM1-057A Manufacture date: September 28, 2005 Storage Conditions: -16 to -24 C Concentration: Volume: Total Amount/Ampoule: 17.937 μm 0.988 ml 11.215 µg (based on proton form) Solvent: Structural Information: methanol H P H H Chemical Formula: C 34 H 65 8 P Exact Mass: 632.44 Molecular Weight: 632.85 Reviewed: Date: Signature on file 10/19/05 www.lipidmaps.org Page 1 of 9 www.avantilipids.com
TLC: Conditions: Plate A Mobile Phase: Plate B Mobile Phase: 65:25:4 CHCl 3 :MeH:H 2 65:25:4 CHCl 3 :MeH:NH 4 H Plate A Plate B 1 2 1 2 Lane 1: Suitability (18:1 PE + oleic acid and 18:1 LPE) Lane 2 GPA(17:0/14:1(9Z)) ne spot in iodine and water, ninhydrin (-), phosphorus (+), char (+) Flame Atomic Absorption (Calcium): GPA(17:0/14:1(9Z)) = 19 ppm Ca www.lipidmaps.org Page 2 of 9 www.avantilipids.com
Proton Nuclear Magnetic Resonance ( 1 H-NMR): Conditions: Sample conc.: 25 mg/ml in 8:2 CDCl 3 : CD 3 D Temp.: 300 K Field Strength: 300 MHz Pulse: 30 for 2.70 sec. Relaxation time: 1 sec. # of scans: 16 Line Broadening: 0.20 Hz Shift standard: TMS = -0.0002 PPM 8 7 6 5 4 3 2 1 0-1 Chemical Shift PPM Shift (PPM) Actual Protons Theoretical Protons Assignment not detected 0 2 H 7.4009 0.000 0 chloroform peak 5.22548-5.38158 2.765 3 oleifinic protons & proton oncarbon 2 of the glycero backbone 4.41246-4.42188 1.029 1 proton on carbon 1 of the glycero backbone 4.34093-4.38105 4.828 0 H 2 peak 4.14987-4.21268 1.393 1 proton on carbon 1 of the glycero backbone & H 2 peak 3.9795-3.9970 1.840 2 protons on carbon 3 of the glycero backbone ~3.35 0.181 0 Methanol peak 2.2827-2.3522 3.767 4 alpha protons 1.9915-2.0316 3.775 4 allylic protons 1.5967 3.943 4 beta protons 1.2608-1.3397 36.507 38 remaining -CH 2 - on the fatty acid chains 0.8595-0.9223 6.000 6 methyls on the fatty acid chains Actual Theoretical Sum Protons Sum Protons 61.018 65 www.lipidmaps.org Page 3 of 9 www.avantilipids.com
Phosphorus Nuclear Magnetic Resonance ( 31 P-NMR): Conditions: Sample conc.: 25 mg/ml in 8:2 CDCl 3 : CD 3 D Temp.: 300 K Field Strength: 121.5 MHz Pulse: 30 for 0.786 sec. Relaxation time: 2 sec. # of scans: 256 Line Broadening: 1.0 Hz 10 5 0-5 -10 Chemical Shift PPM ne shift under described conditions www.lipidmaps.org Page 4 of 9 www.avantilipids.com
Reversed Phase HPLC with Evaporative Light Scattering Detection (HPLC/ELSD): Conditions: Mobile Phase A: 1:1 MeH:H 2 + 20 mm NH 4 AC Mobile Phase B: 100 % MeH + 20 mm NH 4 AC 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: 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 450 400 350 13.326 300 250 mv 200 150 100 50 0 0 5 10 15 20 25 30 Time (min) Purity = 100% AUC www.lipidmaps.org Page 5 of 9 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 Injection = 250 C, Detector = 250 C Carrier: Grade 5 Helium at 1 ml/min Injection: Sample: 1 µl @ 1:20 split 5 mg/ml in hexane after reaction with 0.2N sodium methoxide in methanol at room temperature for 5 minutes. 110000 100000 90000 3.237 (14:1) 4.336 (17:0) 80000 70000 Counts 60000 50000 40000 30000 20000 10000 0 0 5 10 15 20 25 Time (min) Purity = 100% (AUC) 14:1 and 17:0 FAMEs www.lipidmaps.org Page 6 of 9 www.avantilipids.com
Mass Spectroscopy: Qualitative Conditions: Flow infusion scan Period 1 Experiment 1: Instrument: Applied Biosystems 4000 Q Trap Scan Type: Enhanced MS (EMS) Polarity: Negative Scan Mode: Profile Ion Source: Turbo Spray Start (amu) Stop (amu) Time (sec) Param Start Stop 431.00 831.00 0.4001 AF3 0.55 0.76 EXB 150.71 146.83 1.2E+07 1.0E+07 631.68 [M-H] - 8.0E+06 Intensity, cps 6.0E+06 4.0E+06 2.0E+06 699.52 [M-H+ Na + HC] - 713.52 [M-H+ Na +CH 3 C] 0.0E+00 431 471 511 551 591 631 671 711 751 791 831 m/z, amu -EMS: [M-H] - = 631.68 u, [M-H+Na+HC] - = 699.52 u, [M-H+Na+CH 3 C] - = 713.52 u. www.lipidmaps.org Page 7 of 9 www.avantilipids.com
Qualitative Conditions: Flow infusion QTrap Period 1 Experiment 2: Instrument: Applied Biosystems 4000 Q Trap Scan Type: Enhanced Resolution (ER) Polarity: Negative Scan Mode: Profile Ion Source: Turbo Spray Start (amu) Stop (amu) Time (sec) Param Start Stop 616.43 646.43 0.1201 AF3 0.25 0.25 EXB 151.75 151.61 3.5E+08 3.0E+08 631.44 [M-H] - 2.5E+08 Intensity, cps 2.0E+08 1.5E+08 632.43 1.0E+08 5.0E+07 633.42 634.41 0.0E+00 613 618 623 628 633 638 643 m/z, amu -ER: [M-H] - = 631.44 u www.lipidmaps.org Page 8 of 9 www.avantilipids.com
Quantitative Conditions: HPLC/MS/MS Comment: Luna C18 2x20mm, 3µ Mobile Phase A: 7.2:2.0:6.2:1.0, 20 mm (NH 4 ) 2 C 3 in H 2 ph adj. to 9.6 w/ NH 4 H: IPA: THF: ACN Mobile Phase B: 7.4:2.2:3.7, IPA: THF: ACN MRM Transition Q1 Mass (amu) Q3 Mass (amu) Dwell(msec) 631.56 269.10 1000.00 Step Table Step Total Time Flow Rate A (%) B (%) (min) (µl/min) 0 0.00 450 60.0 40.0 1 3.00 450 60.0 40.0 2.5E+05 0.91 2.0E+05 Intensity, cps 1.5E+05 1.0E+05 5.0E+04 0.0E+00 0 0.5 1 1.5 2 2.5 3 Time (min) Total GPA(17:0/14:1(9Z)) = 17.937 µm www.lipidmaps.org Page 9 of 9 www.avantilipids.com