View
254
Download
0
Category
Preview:
Citation preview
CERTIFICATE OF ANALYSIS Date: 09.12.2008
CA No.: 1613/1
Product name: Hyperoside
Basic DataDenotation: HyperosideChem. name (CAS): 2-(3,4-Dihydroxyphenyl)-3-(β-D-
galactopyranosyloxy)-5,7-di-hydroxy-4H-1-benzopyran-4-one
Synonyms: Hyperin, Quercetin-3-β-D-galactoside
Batch No.: 08120119CAS-No.: [482-36-0]Formula: C21H20O12
Molecular weight: 464.38Storage conditions: 4 °CSource: Hypericum perforatumStable until: December 2013Last purity control: December 2008Date of production: December 2008Article No.: A2062
Molecular formula
O
OOH
HO
OH
OH
OR
R = β-D-Galactopyranosyl
DETERMINATION SPECIFICATION RESULT
Properties
Appearance fine yellow needles conforms
Odour odourless conforms
Solubility soluble in Methanol, aq. Ethanol
low soluble in Water
conforms
Identity
Melting point 220 – 240 °C (decomp.) (Methanol 90 %) 223 – 224 °C (decomp.); conforms1H-NMR consitent with structure, data in literature conforms
13C-NMR consitent with structure, data in literature conforms
IR-Spectrum consitent with structure conforms
UV-Spectrum consitent with structure, data in literature
λmax [nm] = 256, 360 ± 2
log ελmax = 4.38, 4.30 ± 0.05
conforms
λmax [nm] = 258; 360
log ελmax = 4.37, 4.30
Mass-Spectrum
hr-MS
molecular ion peak at m/z 463 [M-H]-
measured mass tolerance < +/-10 ppm
Peak at m/z 463; conforms
0.4 ppm; conforms
Purity
TLC 1 band 1 band; conforms
HPLC 1. Method
2. Method
Sum of impurities at 254 nm and 355 nm:<1.0 %
0.51 %, 0.58 %; conforms
0.57 %, 0.70 %; conforms
Assay
HPLC 1. Method >99.0 % at 254 nm, Spectrum Max Plot 99.49 %, 99.45 %; conforms
Result: The product meets the requirements
Analytical Report to the Certificate of Analysis (CA) CA No.: 1613/1Date: 09.12.2008
Hyperoside Page: 1 of 16
Batch No.: 08120119
Table of Contents
Page
1. Manufacturing Procedure ....................................................................................................................................2
2. Characteristics .....................................................................................................................................................2
3. Melting Point ........................................................................................................................................................2
4. TLC-Analysis........................................................................................................................................................3
5. HPLC-Analysis.....................................................................................................................................................4
6. 1H-NMR-Spectrum...............................................................................................................................................8
7. 13
C-NMR-Spectrum............................................................................................................................................10
8. IR-Spectrum.......................................................................................................................................................12
9. UV-VIS-Spectrum ..............................................................................................................................................13
10. Mass-Spectrum................................................................................................................................................14
11. Instrumentation ................................................................................................................................................16
12. References ......................................................................................................................................................16
Analytical Report to the Certificate of Analysis (CA) CA No.: 1613/1Date: 09.12.2008
Hyperoside Page: 2 of 16
Batch No.: 08120119
1. Manufacturing Procedure
Crude Hyperoside was isolated from Hypericum perforatum by an extraction process using a mixture ofmethanol and water and extraction of the obtained concentrate with ethyl acetate. The pure product was ob-tained by several recrystallisations from methanol/water mixtures. The substance was dried at 45 °C / 0.1 mbarover a period of 48 hours.
2. Characteristics
The crystallization of Hyperoside from 85% methanol yields the product as light yellow microcrystalline powder.When dissolved in methanol/water under heating a slight yellow coloured solution is obtained. In the solid statethe substance is stable and shows no markedly sensibility to air and moisture. In methanolic solutions it is stablefor some days. In order to prevent any decompositon the substance should be stored at a dry place at 4° C.
3. Melting Point
Found: 223 - 224 °C (from Methanol 85%), not corr.
Ref.[1]
: 226 - 229 °C (Methanol)Ref.
[2]: 236 °C
Analytical Report to the Certificate of Analysis (CA) CA No.: 1613/1Date: 09.12.2008
Hyperoside Page: 3 of 16
Batch No.: 08120119
4. TLC-Analysis
Stationary phase: Silica gel 60 F254, 0.20 mm thickness (Art.-No. 1.05554, Merck, Darmstadt, Ger.)Mobile Phase: Ethyl acetate / Formic acid / Water (20/2/3; v/v/v)Sample Solvent: MethanolDevelopment length: 10 cmRetention factor: Rf = 0.43 (chamber saturation)Applied quantities: 20, 10, 5 µgDetection: a) UV254: 1 band
b) Diphenylboryloxyethylamine (Naturstoffreagenz A), 10 % in ethanol, afterspraying with Macrogel 400 / 10 min. at 110 °C; detected at UV365: 1 band
TLC-Chromatogram (1:1)
Thinlayer Chromatogram
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
Rf/dm
1 2 3
TLC of Hyperoside Batch No.: 08120119 afterspraying with Naturstoffreagenz A;Trace 1: 20µg, Trace 2: 10 µg, Trace 3: 5 µg
Analytical Report to the Certificate of Analysis (CA) CA No.: 1613/1Date: 09.12.2008
Hyperoside Page: 4 of 16
Batch No.: 08120119
5. HPLC-Analysis
1. MethodColumn type: Hypersil Gold 175-5µ, C18, 250 x 4.6 mmMobile Phase: Methanol/Acetonitrile/Orthophosphoric acid pH 2.5 (18/12/70, v/v/v)Sample solvent: Methanol/Water 3:1Injection vol.: 5 µLFlow rate: 1.00 mL/minDetection: DAD, 210-450 nmColumn temp.: 20 °C
2: 254 nm, 4 nmPk # Retention Time Area Area Percent Capacity factor Lambda Max
1 4.917 272 0.01 0.70 2562 5.547 250 0.01 0.92 2133 6.240 2486 0.07 1.16 2174 9.291 3796614 99.49 2.21 2565 12.928 15784 0.41 3.47 2656 14.219 679 0.02 3.92 214
Totals3816085 100.00
Minutes
0 5 10 15 20
0
100
200
300
400
500
600
4.8
85
6.2
40
9.2
91
12
.939
Spectrum Max PlotHyperosid Ch.-B.: 08120119Hyperosid 08120119 RPmet7-043.dat
Retention Time
Minutes
0 10 20
mA
U
0
50
100
150
200
250
300
350
4.9
17
5.5
47
6.2
40
9.2
91
12
.92
8
14.2
19
2: 254 nm, 4 nmHyperosid Ch.-B.: 08120119Hyperosid 08120119 RPmet7-043.dat
Retention Time
Analytical Report to the Certificate of Analysis (CA) CA No.: 1613/1Date: 09.12.2008
Hyperoside Page: 5 of 16
Batch No.: 08120119
HPLC-Analysis
Spectrum Max Plot*)
Pk # Retention Time Area Area Percent Capacity factor Lambda Max
1 4.885 700 0.01 0.69 3392 6.240 6150 0.10 1.16 2233 9.291 6264800 99.45 2.21 2564 12.939 27812 0.44 3.48 267
Totals6299462 100.00
*)A Spectrum Max Plot is a chromatogram with each point plotted at ist maximum absorbance within the detection range.This plot gives an indication of the appearance of the chromatogram when the wavelengths are opimized for each peak.
4: 355 nm, 4 nmPk # Retention Time Area Area Percent Capacity factor Lambda Max
1 6.229 2566 0.08 1.16 2122 9.291 3171998 99.42 2.21 2563 10.229 300 0.01 2.54 2124 12.928 15712 0.49 3.47 265
Totals3190576 100.00
DAD UV-Spectrum
Spectrum at time 9.29 min.
nm
250 300 350 400 450
mA
U
0
100
200
300
400
500
600m
AU
0
100
200
300
400
500
600
9.29 minLambda max : 256 355Lambda min : 237 282
Analytical Report to the Certificate of Analysis (CA) CA No.: 1613/1Date: 09.12.2008
Hyperoside Page: 6 of 16
Batch No.: 08120119
HPLC-Analysis
2. MethodColumn type: Reprosil-Pur CN, 5µ, 250 x 4mmMobile Phase: A: Acetonitrile/Orthophosphoric acid pH 2.5 10/90 (v/v)
B: Acetonitrile/Orthophosphoric acid pH 2.5 90/10 (v/v)0-20 min.: linear gradient 0%B ->25%B20-30 min.: linear gradient 25%B ->75%B
Sample solvent: Methanol/Water 3:1Injection vol.: 5 µLFlow rate: 1.00 mL/minDetection: DAD, 210-450 nmColumn temp.: 20 °C
2: 254 nm, 4 nmPk # Retention Time Area Area Percent Capacity factor Lambda Max
1 11.840 1981 0.04 4.95 2172 13.557 4652084 99.43 5.81 2553 14.869 21569 0.46 6.47 2654 15.147 1716 0.04 6.61 2115 16.395 544 0.01 7.24 2146 22.731 836 0.02 10.42 211
Totals4678730 100.00
Minutes
0 10 20 30
mA
U
0
100
200
300
400
500
11.8
51
13.5
57
14
.86
915
.16
816
.384
22
.752
4: 355 nm, 4 nmHyperosid Ch.-B.: 08120119Hyperosid 08120119 CNmet2-009.dat
Retention Time
Minutes
0 10 20 30
mA
U
0
100
200
300
400
500
600
11.8
40
13
.557
14.8
69
15.1
47
16
.39
5
22.7
31
2: 254 nm, 4 nmHyperosid Ch.-B.: 08120119Hyperosid 08120119 CNmet2-009.dat
Retention Time
Analytical Report to the Certificate of Analysis (CA) CA No.: 1613/1Date: 09.12.2008
Hyperoside Page: 7 of 16
Batch No.: 08120119
HPLC-Analysis
4: 355 nm, 4 nmPk # Retention Time Area Area Percent Capacity factor Lambda Max
1 11.851 2270 0.06 4.96 2212 13.557 3885884 99.30 5.81 2553 14.869 21437 0.55 6.47 2654 15.168 1322 0.03 6.62 2115 16.384 1109 0.03 7.23 2176 22.752 1178 0.03 10.43 363
Totals3913200 100.00
Analytical Report to the Certificate of Analysis (CA) CA No.: 1613/1Date: 09.12.2008
Hyperoside Page: 8 of 16
Batch No.: 08120119
6. 1H-NMR-Spectrum
[300 MHz, 300 K, Sovent: DMSO-d6]
Analytical Report to the Certificate of Analysis (CA) CA No.: 1613/1Date: 09.12.2008
Hyperoside Page: 9 of 16
Batch No.: 08120119
1H-NMR-Spectrum
Assignment of the signals
[500 MHz, 303 K, Solvent: DMSO-d6]
Proton(s) Chemical shifts [ppm] Data in literature[3]
[ppm]Solvent signalsHydroxyl signals [ppm]
H-6
H-8
H-2'
H-5'
H-6'
H-1''
H-2’’
H-3’’
H-4’’
H-5"
H-6”a
H-6”b
6.20 (d; 2.0 Hz) [1H]
6.41 (d; 2.0 Hz) [1H]
7.53 (d; 2.2 Hz) [1H]
6.82 (d; 8.5 Hz) [1H]
7.67 (dd; 8.5, 2.2 Hz) [1H]
5.38 (d; 7.7 Hz) ], [1H]
3.56 (m) [1H]
3.36 (m) [1H]
3.65 (‘t’; 3.7 Hz) [1H]
3.33 (m) [1H]
3.33 (m) [1H]
3.45 (m) [1H]
6.18 d (1.9 Hz)
6.38 d (1.9 Hz)
7.52 d (2.2 Hz)
6.80 d (8.3 Hz)
7.65 dd (8.3, 2.2 Hz)
5.35 d (7.6 Hz)
3.56 dd (9.4, 7.6 Hz)
3.36 dd (9.4, 3.2 Hz)
3.65 br d (3.2 Hz)
3.33 m
3.33 m
3.46 dd (9.5, 5.1 Hz)
12.60 (OH-5)
DMSO-d6: 2.50
Water signal: ~ 3.37 (unspecific)
Ar-OH: 12.64 (OH-5), 10.85, 9.72,
9.14 [4H]
(shift unspecific)
Glu-OH: 4.43, 4.84, 5.12 [4H] (shift
unspecific)
Solvent residue:
Methanol: 3.17 (br d), 4.08 (OH)
(≈ 1.65 w%)
O
O
OH
OH
O
OH
OH
O
OH
OH
OHOH
2
3
46
7
9
10
8
5
1'
2'
4'5'
6'
1''
2''3''
4''
5''
6''
3'
Analytical Report to the Certificate of Analysis (CA) CA No.: 1613/1Date: 09.12.2008
Hyperoside Page: 10 of 16
Batch No.: 08120119
7. 13
C-NMR-Spectrum
[75 MHz, 300 K, Solvent: DMSO-d6]
Analytical Report to the Certificate of Analysis (CA) CA No.: 1613/1Date: 09.12.2008
Hyperoside Page: 11 of 16
Batch No.: 08120119
13C-NMR-Spectrum
Assignment of the signals
[75 MHz, 300 K, Solvent: DMSO-d6]
C-Atom Chemical shift [ppm] Data in Ref[3]
[ppm] Solvent signals [ppm]
2
3
4
5
6
7
8
9
10
1'
2'
3'
4'
5'
6'
1"
2"
3"
4"
5"
6"
156.2
133.5
177.5
161.2
98.6
164.1
93.5
156.3
103.9
121.1
115.2
144.8
148.4
115.9
122.0
101.8
71.2
73.2
67.9
75.8
60.1
156.2
133.4
177.4
161.1
98.9
164.2
93.4
156.2
103.8
121.0
115.1
144.7
148.4
115.9
121.9
101.8
71.2
73.2
67.9
75.7
60.1
DMSO-d6: 39.0 - 40.0
Solvent residue:
Methanol: 48.6
O
O
OH
OH
O
OH
OH
O
OH
OH
OHOH
2
3
46
7
9
10
8
5
1'
2'
4'5'
6'
1''
2''3''
4''
5''
6''
3'
Analytical Report to the Certificate of Analysis (CA) CA No.: 1613/1Date: 09.12.2008
Hyperoside Page: 12 of 16
Batch No.: 08120119
8. IR-Spectrum
KBr-Pellet
Hyp08120119KBr-Pressling
3410
1654
1608
1576
1559
1506
1446
1363
1303
1205
1171
1137
1116
1089
1022
999
826
790
654
599
5001000150020002500300035004000
Wavenumber cm-1
20
30
40
50
60
70
80
90
100
Tra
nsm
itta
nce [
%]
Assignment: 3410 cm-1
: OH-Stretch vib 1654 cm-1
: C=O-Stretch vib1608 cm
-1: C=C-Stretch vib
For reference see Ref[1]
.
Analytical Report to the Certificate of Analysis (CA) CA No.: 1613/1Date: 09.12.2008
Hyperoside Page: 13 of 16
Batch No.: 08120119
9. UV-VIS-Spectrum
Solvent: MethanolConcentration: 5.0x10
-5 mol/l
Temperature: 20 °C
Result
Maxima: λmax [nm] εmax log εmax Minima: λmin [nm] εmin log εmin
258 23412 4.37 238 13101 4.12
270 sh 17995 4.26 284 7745 3.89
308 sh 9675 3.99
360 20123 4.30
Ref[4]
: λmax = 257 nm, 269sh, 299sh, 262 nm
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
210 260 310 360 410 460
Wellenlänge
Ex
tin
kti
on
Analytical Report to the Certificate of Analysis (CA) CA No.: 1613/1Date: 09.12.2008
Hyperoside Page: 14 of 16
Batch No.: 08120119
10. Mass-Spectrum
Ionization mode: ESI negative
The ESI-negative Mass spectrum shows the peak of the molecular ion at m/z 463 ([M-H]-). At m/z 927 the dimer
[2M-H]- and at m/z 499 the adduct of the molecular ion with chloride is detected.
Analytical Report to the Certificate of Analysis (CA) CA No.: 1613/1Date: 09.12.2008
Hyperoside Page: 15 of 16
Batch No.: 08120119
Mass-Spectrum
ESI-negative Mass spectrum, peak list
ESI-negative Mass spectrum, High Resolution
The found value of the high resolution of the peak at m/z 463 agrees within the mass tolerance with the
proposed molecular composition of the molecular ion C21H19O12 [M-H]-.
Analytical Report to the Certificate of Analysis (CA) CA No.: 1613/1Date: 09.12.2008
Hyperoside Page: 16 of 16
Batch No.: 08120119
11. Instrumentation
Determination Apparatus
Melting Point MEL-TEMPII, Laboratory Devices, USA
HPLC-Analysis Pump: Shimadzu LC-10ADvp
Detector (PDA): Shimadzu SPD-M10Avp
Injector: Rheodyne 7725i, 5, 10 µL loop
1H-NMR Spectrum Bruker AM 300
13C-NMR Spectrum Bruker AM 300
UV-VIS Spectrum Agilent 8452A-DAD
FT-IR Spectrum Bruker Vektor 22
FAB-Mass Spectrum ICR Apex-Qe
12. References
[1] N. R. Farnsworth, H. Wagner, L. Hörhammer, H.-P. Hörhammer,J. Pharm. Sciences 1968, 57(6), 1059-1061.
[2] U. Fiedler, Naturwiss. 1953, 40(7), 226.
[3] B. Bennini, A. J. Chulia, M. Kaouadji, F. Thomasson, Phytochemistry, 1992, 31(7), 2483-2486.
[4] T. J. Marby, K. R. Markham, M. B. Thomas "The Systematic Identification of Flavonoids”,Springer Verlag Berlin-Heidelberg-New York, 1970
Recommended