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17
CERTIFICATE OF ANALYSIS Date: 09.12.2008 CA No.: 1613/1 Product name: Hyperoside Basic Data Denotation: Hyperoside Chem. 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.: 08120119 CAS-No.: [482-36-0] Formula: C 21 H 20 O 12 Molecular weight: 464.38 Storage conditions: 4 °C Source: Hypericum perforatum Stable until: December 2013 Last purity control: December 2008 Date of production: December 2008 Article No.: A2062 Molecular formula O O OH 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.); conforms 1 H-NMR consitent with structure, data in literature conforms 13 C-NMR consitent with structure, data in literature conforms IR-Spectrum consitent with structure conforms UV-Spectrum consitent with structure, data in literature λ max [nm] = 256, 360 ± 2 log ε λmax = 4.38, 4.30 ± 0.05 conforms λ max [nm] = 258; 360 log ε λmax = 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

Transcript of CERTIFICATE OF ANALYSIS - applichem.com · Appearance fine yellow needles conforms ... Analytical...

Page 1: CERTIFICATE OF ANALYSIS - applichem.com · Appearance fine yellow needles conforms ... Analytical Report to the Certificate of Analysis (CA) CA No.:1613/1 Date: ... Sample Solvent:

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

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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

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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

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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

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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

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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

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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

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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

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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]

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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'

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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]

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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'

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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]

.

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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

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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.

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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]-.

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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