LIPID MAPS Certificate of Analysis
Product Number / Lot Number: LMST01010004 / LM6-034A
Mass Spectrometry Reference Standard
5,6α-epoxy-5α-cholestan-3β-ol(d7)
Systematic name:
5,6α-epoxy-5α-cholestan-3β-ol(d7)
Common name(s):
5,6-alpha-epoxy-cholesterol(d7)
Chemical
Molecular weight:
409.70
Exact Mass
Molecular Weight:
409.39
Product# / Lot #:
LMST01010004 / LM6-034A
Manufacture date:
December 16, 2004
Storage Conditions:
-16 to -24°C
Concentration:
Volume:
Total Amount/Ampoule:
127.52 μM
1.00 ml
52.24 µg
Solvent:
methanol
H
CD3
Structural Information:
H
D
CD3
H
H
HO
H
O
Chemical Formula: C27H39D7O2
Exact Mass: 409.39
Molecular Weight: 409.70
Reviewed:
Date:
Signature on file
2/25/06
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Page 1 of 7
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LIPID MAPS Certificate of Analysis
Product Number / Lot Number: LMST01010004 / LM6-034A
Mass Spectrometry Reference Standard
5,6α-epoxy-5α-cholestan-3β-ol(d7)
TLC:
Conditions:
Mobile Phase:
3:2 cyclohexane : ethylacetate
Data:
1
2
Lane 1: Suitability (18:1 PE + oleic acid)
Lane 2 (5,6alpha-epoxy-cholesterol(d7))
Interpretation:
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One spot in iodine and water, ninhydrin (-), phosphorus (-), char (+)
Page 2 of 7
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LIPID MAPS Certificate of Analysis
Product Number / Lot Number: LMST01010004 / LM6-034A
Mass Spectrometry Reference Standard
5,6α-epoxy-5α-cholestan-3β-ol(d7)
Proton Nuclear Magnetic Resonance (1H-NMR):
Conditions:
Sample conc.:
Temp.:
Field Strength:
Pulse:
Relaxation time:
# of scans:
Line Broadening:
7.5 mg/ml in 8:2 CDCl3 : CD3OD
300 K
300 MHz
30° for 5.24 sec.
1 sec.
32
0.20 Hz
Data:
-1
0
1
2
3
4
5
6
7
8
Chemical Shift PPM
Interpretation:
Shift (PPM)
4.47
3.8
3.36
2.95
1.08
0.89
0.63
Assignment
H2O
CH-3
CH3OH
CH-6
CH3-19
CH3-21
CH3-18
The above peaks match those of the literature values.
Wilson, W.K.; Swaminathan, S.; Pinkerton, F.D.; Gerst, N.; and Schroepfer, G.J. Jr. Steroids, 1994, vol. 59, page 310-317.
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Page 3 of 7
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LIPID MAPS Certificate of Analysis
Product Number / Lot Number: LMST01010004 / LM6-034A
Mass Spectrometry Reference Standard
5,6α-epoxy-5α-cholestan-3β-ol(d7)
Reversed Phase HPLC with Evaporative Light Scattering Detection (HPLC/ELSD):
Conditions:
Mobile Phase :
Gradient:
Column:
Flow rate:
Detector:
Injection Volume:
75:25 (v/v) ACN : IPA
Isocratic
Luna C-18, 250 mm X 4.5 mm i.d., 5 µ
1 ml/min
Sedex 75, 40 degrees C, 3.5 bar, gain of 7
20µl @ 1.0 mg/ml
Data:
Interpretation:
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Purity = 100% AUC
Page 4 of 7
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LIPID MAPS Certificate of Analysis
Product Number / Lot Number: LMST01010004 / LM6-034A
Mass Spectrometry Reference Standard
5,6α-epoxy-5α-cholestan-3β-ol(d7)
Mass Spectroscopy:
Qualitative
Conditions:
Flow infusion scan
Period 1 Experiment 1:
Instrument:
Applied Biosystems 4000 Q Trap
Scan Type:
Q1 MS (Q1)
Polarity:
Positive
Scan Mode: Profile
Ion Source:
Turbo Spray
Start (amu)
Stop (amu)
Time (sec)
227.00
627.00
1.00
Data:
+Q1: 0.619 to 0.988 min from Sample 3 (1 ug.mL +Q1 INF FIA) of 5-alpha-6-alpha-Epoxy-Cholesterol-d7 LM6-014 LM410...
Max. 1.7e6 cps.
427.6
1.7e6
1.6e6
1.5e6
1.4e6
1.3e6
1.2e6
1.1e6
Intensity, cps
1.0e6
9.0e5
8.0e5
7.0e5
6.0e5
392.5
5.0e5
410.4
4.0e5
3.0e5
2.0e5
1.0e5
429.8
374.4
240
260
280
300
320
340
360
380
400
420
440
m/z, amu
460
480
500
520
540
560
580
600
Interpretation: +Q1: [M-2H2O+H]+ = 374.4 u, [M-H2O+H]+ = 392.5 u, [M+H]+ = 410.4 u,
[M+NH4]+ = 427.6 u
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Page 5 of 7
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620
LIPID MAPS Certificate of Analysis
Product Number / Lot Number: LMST01010004 / LM6-034A
Mass Spectrometry Reference Standard
5,6α-epoxy-5α-cholestan-3β-ol(d7)
Qualitative
Conditions:
Flow infusion QTrap
Period 1 Experiment 2:
Instrument:
Applied Biosystems 4000 Q Trap
Scan Type:
Enhanced Resolution (ER)
Polarity:
Positive
Scan Mode:
Profile
Ion Source:
Turbo Spray
Start (amu)
395.60
Stop (amu)
442.60
Time (sec)
0.1201
Param
AF3
EXP
Start
0.20
-153.53
Stop
0.22
-153.29
Data:
+ER: 30 MCA scans from Sample 9 (1.0 ug.mL +ER INF MCA) of 5-alpha-6-alpha-Epoxy-Cholesterol-d7 LM6-014 LM410...
Max. 4.8e7 cps.
410.3
4.8e7
4.6e7
4.4e7
4.2e7
4.0e7
3.8e7
3.6e7
3.4e7
3.2e7
3.0e7
Intensity, cps
2.8e7
2.6e7
2.4e7
2.2e7
411.3
2.0e7
1.8e7
413.2
1.6e7
1.4e7
432.3
1.2e7
427.4
1.0e7
8.0e6
6.0e6
412.3
4.0e6
409.3
2.0e6
396
Interpretation:
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398
400
402
404
406
408
416.3
410
412
425.3 428.3
414.2
414
415.2
416
423.3 426.2
418 420
m/z, amu
422
424
426
433.3
434.3
431.3
428
430
432
434
436
438
+ER: [M+H]+ = 410.3 u, [M+NH4]+ = 427.4 u, [M+Na]+ = 432.3 u
Page 6 of 7
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440
442
LIPID MAPS Certificate of Analysis
Product Number / Lot Number: LMST01010004 / LM6-034A
Mass Spectrometry Reference Standard
5,6α-epoxy-5α-cholestan-3β-ol(d7)
Quantitative
Conditions:
HPLC/MS/MS
Comment:
Mobile Phase A:
Column: Mercury 20 x 2 mm, C8
6:2:2 methanol : water : acetonitrile + 0.1%/v HCOOH
and 10 mM NH4OAC
2:3:1 chloroform : methanol :water + 0.1%/v HCOOH
and 10 mM NH4OAC
Mobile Phase B:
MRM Transition
Q1 Mass (amu) Q3 Mass (amu)
427.47
392.40
Dwell(msec)
1000
Step Table
Step
Total Time
(min)
0.00
3.00
0
1
Flow Rate
(µl/min)
500
500
A (%)
B (%)
80
80
20
20
XIC of +MRM (1 pair): 427.5/392.4 amu from Sample 9 (Ampoule 2 Inj. 2) of 5a-6a Epoxycholesterol D7 Stability 020606.w...
Data:
Max. 2.1e4 cps.
1.5
2.1e4
2.0e4
1.9e4
1.8e4
1.7e4
1.6e4
1.5e4
1.4e4
Intensity, cps
1.3e4
1.2e4
1.1e4
1.0e4
9000.0
8000.0
7000.0
6000.0
5000.0
4000.0
3000.0
2000.0
1000.0
0.0
Interpretation:
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0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
Time, min
1.8
2.0
2.2
2.4
2.6
5,6-alpha-epoxy-cholesterol(d7) = 127.52 µM (52.24 µg/ml)
Page 7 of 7
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2.8