Mass Spectrometry Reference Standard 1-Heptadecanoyl-2-(5Z,8Z,11Z,14Z-Eicosatetraenoyl)-sn-Glycero-3-Phospho- (1 -Myo-Inositol)(Ammonium Salt)

Transcription

1 Systematic name: Common name(s): Chemical Molecular weight: Exact Mass Molecular Weight: Product# / Lot #: 1-Heptadecanoyl-2-(5Z,8Z,11Z,14Z-Eicosatetraenoyl)-sn- Glycero-3-Phospho- GPIns(17:0-20:4(5Z,8Z,11Z,14Z)) (Proton Form) (Ammonium Salt Form) (Proton Form) (Ammonium Salt Form) LMGP / LM7-082A Manufacture date: August 15, 2006 Storage Conditions: Concentration: Volume: Total Amount/Ampoule: Solvent: -16 to -24ºC μm 1.0 ml μg methanol Structural Information: H P H H H H H H C 46 H P Exact Mass: Molecular Weight: Reviewed: Signature on file Approved: Signature on file Date: 9/26/06 Date: 9/26/06 Page 1 of 13

2 TLC: Conditions: Plate A Mobile Phase: 65:25:4 CHCl 3 :MeH:H 2 Data: Plate A 1 2 Lane 1: Suitability (18:1 PE, 18:1 LPE + oleic acid) Lane 2: GPIns(17:0-20:4(5Z,8Z,11Z,14Z)) Interpretation: Ninhydrin (-), Iodine 1 spot, Phosphorus (+), Char (+), Wet Dip 1 spot Flame Atomic Absorption (Calcium): Interpretation: GPIns(17:0-20:4(5Z,8Z,11Z,14Z))< 10 ppm Ca Page 2 of 13

3 Proton Nuclear Magnetic Resonance ( 1 H-NMR): Conditions: Sample conc.: 6.67 mg/ml in 8:2 CDCl 3 : CD 3 D Temp.: 300 K Field Strength: MHz Pulse: 30 for 2.62 sec. Relaxation time: 2 sec. # of scans: 48 Line Broadening: 0.20 Hz Data: Interpretation: Shift (ppm) Actual Theoretical protons Protons Assigment not detected 0 4 NH4 group not detected 0 5 H groups Chloroform peak oleifinic protons proton on the 2nd carbon of the glycero backbone proton on the 1st carbon of the glycero backbone + water peak proton on the 1st carbon of the glycero backbone + ring proton protons on the 3rd carbon of the glycero backbone ring proton ring proton ring proton ring proton methanol peak ring proton + CH2 group of the ethanol peak "interior" allylic protons alpha protons allylic protons on both ends beta protons remaining -CH2- on the fatty acid chains + CH3 group of the ethanol peak methyls on the fatty acid chains Actual Theoretical Sum Protons Sum Protons Note: sum excludes chloroform & methanol peaks Page 3 of 13

4 Phosphorus Nuclear Magnetic Resonance ( 31 P-NMR): Conditions: Sample conc.: 6.67 mg/ml in 8:2 CDCl 3 : CD 3 D Temp.: 300 K Field Strength: MHz Pulse: 30 for 2.62 sec. Relaxation time: 2 sec. # of scans: 230 Line Broadening: 1.0 Hz Data: 5e+8 4e e+8 2e+8 1e+8 0-1e Interpretation: ne shift under described conditions Page 4 of 13

5 Normal Phase HPLC with Evaporative Light Scattering Detection (HPLC/ELSD): Conditions: Mobile Phase A: 80:14:6 MeH:CHCl 3 :H2 + 15mM NH 4 AC + 0.1% NH 4 H Gradient: Isocratic Column: Agilent Eclipse XDB C8, 150 X 4.6mm, 5u Flow rate: 0.5 ml/min ambient Detector: Sedex 75, 40 degrees C, 3.5 bar, gain of 7 Injection Volume: µg/µl Data: mau Time (min) Interpretation: Purity = 97.9% (AUC) Page 5 of 13

6 Fatty Acid Methyl Ester by Capillary Gas Chromatography (GC-FAME): Conditions: Column: DB-225, 30M X 0.22 mm, i.d., ser. # US H Temperatures: ven = 220 C isocratic, Injection = 250 C, Detector = 250 C Carrier: Helium at 1 ml/min Injection: 1 µl at 1:20 split Sample: 5 mg/ml in hexane after reaction with 0.2N sodium methoxide in methanol at room temperature. Data: : : Time (min) Interpretation: Purity = 46.6% 17:0, 53.4% 20:4, 100% total Page 6 of 13

7 UV/VIS xidation: Conditions: Agilent Model 8453 UV Visible Detector Sample:1.0 mg/ml in ethanol Data: Absorbance (AU) Wavelength (nm) Interpretation: 234nm = 1.40% Conjugated 268nm = 0.19% Conjugated 278nm = 0.23% Total = 1.82% Page 7 of 13

8 Mass Spectroscopy: Qualitative Conditions: Period 1 Experiment 1: Scan Type: Q3 MS (Q3) Polarity: Negative Scan Mode: Profile Ion Source: Turbo Spray Resolution Q3: Unit Intensity Thres.: 0.00 cps Settling Time: msec MR Pause: msec MCA: Yes Center/Width: No Step Size: 0.10 amu Start (amu) Stop (amu) Time (sec) Parameter Table(Period 1 Experiment 1) CUR: IS: TEM: GS1: GS2: 0.00 ihe: N DP EP CXP Page 8 of 13

9 Data: LIPID MAPS Certificate f Analysis -Q3: 30 MCA scans from Sample 3 (10 ug.ml -Q3 FI-BS DP=-170) of PI LM7-082 LM-1502.wiff (Turbo Spray) 9.0e7 8.5e Max. 9.0e7 cps. 8.0e7 7.5e7 7.0e7 6.5e7 6.0e7 5.5e7 Intensity, cps 5.0e7 4.5e7 4.0e7 3.5e7 3.0e7 2.5e7 2.0e7 1.5e7 1.0e7 5.0e m/z, amu Interpretation: -Q3: [M-H] - = u, [M-H + CH 3 CNa] - = u Page 9 of 13

10 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: pen Scan Rate: 250 amu/s Intensity Thres.: 0.00 cps Settling Time: msec MR Pause: msec Q0 trapping: No MCA: Yes Center/Width: Yes LIT fill time: 5.00 msec Q3 Entry Barrier: 8.00 V Step Size: 0.03 amu Start (amu) Stop (amu) Time (sec) Parameter Table(Period 1 Experiment 1) CUR: IS: TEM: GS1: GS2: 0.00 ihe: N CAD: High DP CES 0.00 CE Page 10 of 13

11 Data: LIPID MAPS Certificate f Analysis -ER: 60 MCA scans from Sample 10 (10 ug.ml -ER FI-BS DP=-180) of PI LM7-082 LM-1502.wiff (Turbo Spray) 3.2e Max. 3.3e8 cps. 3.0e8 2.8e8 2.6e8 2.4e8 2.2e8 2.0e Intensity, cps 1.8e8 1.6e8 1.4e8 1.2e8 1.0e8 8.0e7 6.0e e7 2.0e m/z, amu Interpretation: -ER: [M-H] - = u Page 11 of 13

12 Quantitative Conditions: Column = Luna C18 2x20mm Mobile Phase A = 720:207:62:10, 20 mm (NH4)2C3 in H2 ph adj. to 9.6 w/ NH4H: IPA: THF: ACN, Mobile Phase B = 741:222:37, IPA: THF: ACN, A:B 70:30, flow = 0.4 ml/min, temp = 20 C Column = Luna C18 2x20mm Mobile Phase A = 720:207:62:10, 20 mm (NH4)2C3 in H2 ph adj. to 9.6 w/ NH4H: IPA: THF: ACN, Mobile Phase B = 741:222:37, IPA: THF: ACN, A:B 70:30, flow = 0.4 ml/min, temp = 20 C Step Table: 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: Page 12 of 13

13 Data: LIPID MAPS Certificate f Analysis ihe: N CAD: Medium DP EP CE CXP XIC of -MRM (1 pair): 871.6/269.5 amu from Sample 9 (Ampoule 2 Inj 2) of PI LM-1502 LM7-082A Stability wif e5 1.8e Max. 1.9e5 cps. 1.7e5 1.6e5 1.5e5 1.4e5 1.3e5 1.2e5 1.1e5 Intensity, cps 1.0e5 9.0e4 8.0e4 7.0e4 6.0e4 5.0e4 4.0e4 3.0e4 2.0e4 1.0e Time, min Interpretation: GPIns(17:0-20:4(5Z,8Z,11Z,14Z)) = µ Page 13 of 13