New Frontiers in the Quality of Medicines€¦ · New Frontiers in the Quality of Medicines Workshop Herbals: Traditional Chinese Medicines Moderators: Prof. Dr Rudolf Bauer Prof

New Frontiers in theQuality of Medicines

WorkshopHerbals: Traditional Chinese Medicines

Moderators:Prof. Dr Rudolf BauerProf. Dr Gerhard Franz

EDQM International Conference13-15 June 2007

Strasbourg, France

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Thank you for your attention

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ELABORATING AN EUROPEANMONOGRAPH FROM A CHINESE

MONOGRAPH

DR KEITH HELLIWELL

Senior Technical Advisor – Natural Products

William Ransom & Son plc

13 – 15 JUNE 2007

HERBAL DRUGS AND HERBAL DRUG PREPARATIONS

Defined as: Herbal drugs are mainly whole, fragmented or cut plants, parts of

plants, algae, fungi, lichen in an unprocessed state, usually in dried form but

sometimes fresh.

Herbal Drug (European Pharmacopoeia)

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Defined as: Herbal Drug Preparations are obtained by subjecting

herbal drugs to treatments such as extraction, distillation,

expression, fractionation, purification, concentration or

fermentation. These include comminuted or powdered herbal

drugs, tinctures extracts, essential oils, expressed juices and

processed exudates.

Herbal Drug Preparation (European Pharmacopoeia)



Herbal Drugs used in Traditional Chinese Medicine

Majority appear to be processed.

Examples of types of processing in the Chinese Pharmacopoeia which

produce a Herbal Drug Preparation:

Boiling

Calcining

Stir-baking

Addition of other ingredients

- honey, vinegar, wine

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Borderline types of processing:

Softening of dried herbal drugs by:

Soaking in water

Spraying with water

Covering with damp cloths

prior to slicing and re-drying

Statement in the Chinese Pharmacopoeia 2005, Appendix IID

‘2. CUTTING: unless cutted in fresh or dry form, the crude drugs should bemoistened to soft for cutting, it is better to keep moisten than to soak inwater to prevent elimination of active principles……….’


Cutting of Herbal Drugs to produce Herbal Drug Preparations

European Pharmacopoeia

Cutting for specific applications (e.g. Herbal Teas)

Chinese Pharmacopoeia 2005 Appendix IID:

‘Processing of crude drugs [Note: this is equivalent in meaning to herbal drugs in the

European Pharmacopoeia] is to make the crude drugs into small processed

pieces through processing procedures such as cleaning, cutting, stir-baking,

so as to obtain the processed drugs fulfilling the requirements of therapy, dispensing

and making preparations, thus assuring the safety and efficacy of the drugs.’

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EUROPEAN REGULATORY CONSIDERATIONS

Herbal Drug

Cut/Powdered Extracted Processed (Herbal Extracts)

Herbal Drug Preparation

Teas Solid Dosage Forms Solid Dosage forms (tablets/capsules)

(tablets/capsules) Oral Liquid ProductsTraditional Chinese Medicines (tincture/mixtures/etc)

Ointments/Creams

Herbal Medicinal Product

Herbal Medicinal Products and their ingredients

SAMPLES

Availability of samples for monograph elaboration.

Commercial samples:

5-8 samples as a minimum for herbal drugs/herbal drug preparations.

Samples should be typical of material supplied to market where the monographs

are to be legally binding.

Samples are used for confirmation of identification parameters and determining

numerical values to be given in tests and assays.

Reference Samples

Authentic reference samples: used to confirm that commercial samples are both

genuine and of an acceptable quality.

Reference samples of substitute/adulterant herbal drugs/herbal drug preparations

for exclusion tests.

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SAMPLES

Present situation for those herbal drugs/herbal drug preparations used in TraditionalChinese Medicine and on the high priority list of the European Pharmacopoeia.

Commercial Samples:

Difficult to obtain sufficient samples typical of material available in the European market.

Are there differences in quality between material in European market and those used in China?

Reference samples:

Herbal Drugs: difficult to obtain.

Herbal Drug Preparations: virtually impossible to obtain.

Substitutes/Adulterants: difficult to obtain

Both types of samples are essential to ensure applicability of published EuropeanPharmacopoeia monographs.

ELABORATED MONOGRAPHS – EUROPEAN PHARMACOPOEIA FORMAT

Requirement for the elaboration of an European Pharmacopoeia monograph from a ChinesePharmacopoeia monograph:

Style and quality parameters of the European Pharmacopoeia.

Based on current requirements for a Herbal Drug monograph.

Production section to be included if the herbal drug is processed.

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Quality parameters to be applied selected from:

Identification: macroscopy, microscopy, chemical (usually chromatographic)

Quantification:

Assay (eg. titrametric, spectrophotometric, chromatographic)

Application of minimum numerical values (eg, content of essential oil, extractable

matter, swelling index, bitterness value, colour intensity).

Absence of adulterants. Other quality parameters (eg. total ash, ash insoluble in hydrochloric acid, foreign matter, loss on drying, water content). General quality criteria (eg. heavy metals, pesticides, aflatoxins, microbial contamination). Additional requirements (eg. absence of rancidity for materials processed by stir-baking with oil).

Tests


Chinese Pharmacopoeia 2005 – Appendices II A-C detail a similarapproach

For a European Pharmacopoeia monograph elaborated from aChinese Pharmacopoeia monograph there may be a difference inemphasis as to the quality parameters which should be included inthe monograph.

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Radix Arnebiae [Zicao] Arnebia Root

The dried root of Arnebia euchroma (Royle) Johnst. or Arnebia guttata Bunge

Draft European Pharmacopoeia monograph [published in Pharmeuropa

(2007)19.1]

All samples obtained from European market were of processed herbal

drugs.

Any adulteration would be from other pigment containing herbal drugs or

synthetic dyes.

Arnebia root is only for external use based on the presence of

hydroxynaphthaquinone pigments.

A spectrophotometric assay was considered adequate.

Content of hydroxynaphthaquinone pigments: not less than 1.0%w/w

(all samples examined in range 1.2 – 5.0%w/w).


Radix Arnebiae [Zicao] Arnebia RootThe dried root of Arnebia euchroma (Royle) Johnst. or Arnebia guttata Bunge

Xnlt 0.30%nlt 0.30%Hplc assay for dimethylacrylalkanin

nlt 1.0%nlt 0.80%nlt 0.80%Spectrophotometric assay forhydroxynaphthaquinone pigments

√XXAbsence of methanol soluble dyes

√XXAbsence of water soluble dyes

nmt 14.0%XXTotal Ash

nmt 2.0%XXForeign matter

nmt 12.0%XXLoss on drying

XXnmt 15.0%Water Content

√X√Thin-layer chromatographicidentification

XX√Chemical reaction to heating

√√√Macroscopy

Processed RootProcessed RootUnprocessedroot

Quality Parameter

Chinese Pharmacopoeia2005 monograph

Draft EuropeanPharmacopoeia monograph[Pharmeuropa (2007) 19.1]

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Cortex Acanthopanacis [Wujiapi] Slenderstyle Acanthopanax Bark

The dried root bark of Acanthopanax gracilistylus W.W. Smith

Challenges:

Different materials traded on the market as Wujiapi


Thin-layer Chromatogram

1 2 3 4 5 6 7 8 9 10 11Track:1. Wujiapi (ex shop in Hangzhu – Republic of China) [Ref. 25813]2. Acanthopanacis gracilis radius cortex [Ref. 284778]3. Acanthopanacis radius cortex [Ref. 28541].4. Acanthopanacis cortex INA p123-1380 23.11.055. Acanthopanacis giroldii cortex Taiwan 036. Acanthopanacis cortex (ex TCM shop in UK)7. Acanthopanacis senticosi Taiwan 038. Periplocae cortex9. Acanthopanacis henryi cortex10. Periploca septum11. Acanthopanacis senticosus

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Information suggests that material being trade as Cortex Acanthopanacis is

80% Periploca species 10% other Acanthopanax species 10% Acanthopanax gracilistylus



The dried root bark of Acanthopanax gracilistylus W.W. Smith

Challenges:

Different materials traded on the market as Wujiapi



The dried root bark of Acanthopanax gracilistylus W.W.Smith.

nmt 12.0%w/wXXTotal Ash

nmt 12.0%w/wXXLoss on Drying

nmt 2.0%w/wXXForeign Matter

To be decidedXXQuantification

√XXAbsence of Periploca species

√XXThin-layer chromatographic identification

√X√Microscopy

√X√Macroscopy

Processed RootProcessed RootUnprocessedroot

Quality Parameter

Chinese Pharmacopoeia2005 monograph

Proposed draft EuropeanPharmacopoeia monograph

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FINAL COMMENTS

[1] Many challenges lie ahead in the elaboration of European

monographs from Chinese monographs.

[2] European Pharmacopoeia Groups of Experts have only been

engaged in this work for the last 12-18 months.

[3] Key requirement is the availability of samples, both commercial and reference samples.

[4] Core group of European Pharmacopoeia Experts.

[5] Draft monographs published in Pharmeuropa should have an

accompanying detailed commentary.

[6] We must not forget the reason for undertaking this work:

‘……. assuring the safety and efficacy of the drugs’ [Chinese

Pharmacopoeia 2005, Appendix II D].

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Status of Quality Control of Raw Chinese Materia Medica andProcessed Chinese Materia Medica (slice) in China

Ji ShenShanghai Institute for Food and Drug Control

Address: 1500 Zhang Heng Road Shanghai China Post Code: 201203

Email: [email protected]

Shanghai Institute for Food and Drug Control

The presentation is focused on:

Ⅰ.Profile of quality control standard of raw Chinese Materia Medica(CMM) and theprocessed CMM in China

Ⅱ. Relation between raw CMM and processed CMM(slice)

Ⅲ. Profile of Shanghai Municipal Standard for Processed CMM

Ⅳ. Characteristics of quality standard for raw CMM and the quality standard forprocessed CMM and difference between them

Ⅴ. Profile of study on quality control of Processed CMM

Ⅵ. The trend of the quality control of raw CMM and processed CMM

Ⅶ. The work we are engaged in


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Ⅰ

Profile of quality control standard of raw Chinese Materia

Medica (CMM) and the processed CMM in China


Standards for Raw Chinese Materia Medica

State standards China Pharmacopoeia (PartⅠ)

Health Administration Standard for raw Chinese Materia Medica

State Food and Drug Administration Standard for Imported raw Chinese Materia Medica.

Local standards

Provincial /regional standards for raw Chinese Materia Medica


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Standards for Processed Chinese Materia Medica

State standard China Pharmacopoeia (PartⅠ) (Edition 2005)

Local standards Provincial standards/regional standards for processed CMM.

China Pharmacopoeia (PartⅠ) (Edition 2005) collects 21 monographs of processedCMM and 47 monographs whose processing items contain characteristics, generalrequirements or assay. But China Pharmacopoeia collects only some of the specificationsof processed CMM used in China.

So nowadays the main standards for processed CMM are provincial standards/regionalstandard for raw CMM.


Ⅱ Relation between raw CMM and processed CMM(slice)

Processed CMM is product of raw CMM, the procedure of the producing iscalled processing

The processed CMM is one of basic parts of Traditional ChineseMedicine’s (TCM) formula and is the material of Chinese Patent Medicine.

Export of TCM includes raw CMM and processed CMM. Processed CMMaccounts for most of the total in China. The most of export Processed CMMis only treated simply with procedure of cleaning and slicing into certainsize.


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Shanghai Municipal Standard for Processed CMM (Edition 1994) 877 monographs (including different specification of the same raw CMM) is collected.

Shanghai Municipal Standard for Processed CMM (Edition 2007) 952 monographs (including different specification of the same raw CMM is collected . 87 CMM is supplemented. 4 items on different specification of the same raw CMM are upgraded to monographs. 19 monographs are put under the other monograph as items. 3 monographs is deleted. The edition will enact by the end of July.

ⅢProfile of Shanghai Municipal Standard for Processed CMM


ⅣCharacteristics of quality standard for raw CMM and the quality

standard for processed CMM and difference between them

Commonness: Both include description, identification, general requirement, content of extract, assay and tolerance limit on safety.

Difference: Characteristics of quality standard for processed CMM

Procedure of different processing specification is described

Tolerance limit of some items, general requirement, content of extract and assay of processed

CMM are different from that of raw CMM because the indice above are affected by theprocedure of processing.


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Contrast between standard for raw CMM and standard for processed CMM

1.01.39.010.05.010.010.0Xu Chang Qing

CA: 1.5 LG: 0.10CA: 1.5 LG: 0.102.09.010.0Jin Yin Hua

0.9★1.86.06.0Zhi Zi

TⅡA: 0.16SA B：2.0

TⅡA: 0.20SA B：3.0

WS: 30.0ES: 12.0

WS: 35.0ES: 15.0

2.53.09.010.0Dan Shen

2.53.012.014.08.08.0Huang Bai

8.5*9.035.540.06.06.0Huang Qin

1.21.525.00.80.810.010.0Da Huang

SSPCMMChpSSPCMMChpSSPCMMChpSSPCMMChp

AssayContent of extractAcid soluble ashTotal ash

Minimum content (%) Tolerance Limit (%)

*: BothHuangqing and stir-fried Huang Qin ★: Zhi Zi Tan Chp: China Pharmacopoeia SSPCMM : Shanghai Municipal Standard for Processed CMMDa Huang: Radix et Rhizoma Rhei Huang Qin: Radix Scutellariae Huang Bai: Cortex Phellodendri Dan Shen: Radix Salviae MiltiorrhizaeZhi zi: Fructus Gardeniae Jin Yin Hua: Flos Lonicerae Xu Chang Qing: Radix Cynanchi paniculati Zhi Zi Tan: processed Fructus Gardeniae (Carbonized)WS: Water soluble ES: Ethanol soluble TⅡA: TanshinoneⅡA SA B: Sahianolic acid B CA: Chlorogenic acid LG: Luteolin-7-O-glucoside


Example one: Radix Scutellariae Radix Scutellariae is the dried root of Scutellaria baicalensis Georgi (Fam. Labiatae)

Purpose of processing

Processed Radix Scutellariae (simply) : washed clean, cut into slices anddried for dispenser’s preparation, pharmaceutical preparation and storage.

Radix Scutellariae (stir-fried): weaken the cool action.

Research on processing procedure of processed Radix Scutellariae(simply) and processed Radix Scutellariae (stir-fried)


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Processing procedure of processed Radix Scutellariae (simply):

Raw CMM is got rid of impurity, quick washed, taken out. steamed for half anhour, cut into thin slices while hot and dried at 70℃ for 2 hours.

Procedure of processed Radix Scutellariae (stir-fried):

Raw CMM is stir-fried at 320℃ for 4 minutes, and got rid of impurity.


The result of content of baicalin of 10 batches of Radix Scutellariae, processedRadix Scutellariae (simply) and processed Radix Scutellariae(stir-fried)

14.313.314.4050509

14.913.313.7050419

14.113.313.0050411

15.113.112.5050328

8.513.014.8050323

13.713.116.1050314

8.513.014.5050301

15.013.516.9050216

10.711.610.2050110

13.812.410.7041129

contents of baicalin of processedRadix Scutellariae (stir-fried)

content of baicalin of processedRadix Scutellariae (simply)

content of baicalin ofRadix Scutellariae

Item No.


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Minimum content of Baicalin Minimum content of baicalin of Radix Scutellariae set by Chp is 9.0%.

The average content of baicalin of Radix Scutellariae is 13.68% The average content of baicalin of processed Radix Scutellariae (simply) is 12.96%, which is 95% of average content of Radix Scutellariae. According to the contrast of data available, the minimum content of baicalin of processed Radix Scutellariae (simply) is 94% of Radix Scutellariae’s minimum content, which is 8.5%.

The average content of baicalin of processed Radix Scutellariae (stir-fried) is 12.86%, which is 94% of content of Radix Scutellariae.

According to the contrast of data available, the minimum content of baicalin of processed Radix Scutellariae (stir-fried) is 95% of Radix Scutellariae’s minimum content, which is 8.5%.


Example two: Fructus Gardeniae

Fructus Gardeniae is the dried ripe fruit of Gardenia jasminoidesEllis (Fam. Rubiaceae).

Processing can weaken the cool action of Fructus Gardeniae

Procedure of ProcessingFructus Gardeniae (carbonized): place the clean raw CMM in a hot potand stir-fried at a high temperature until the surface of CMM turn dark-brown but keep the nature of CMM, take them out and stand cool.


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Research on processing procedure The processing procedure determined on the basis of study

Choose and clean Stir-fried at 200℃

Fructus Gardeniae clean Fructus Gardeniae until darkness For 10 minutes

spray a little water

take out and stand cool stir-fried for another 1 minutes until dryness


The result of gardenoside content in 10 batchs of Fructus Gardeniae and FructusGardeniae （carbonized）

55.11.853.36Guangdong provinceZZ-42005111410

53.61.803.36Guangdong provinceZZ-42005111409

51.41.793.48Yujiang, Jiangxi provinceZZ-112005111408

51.41.793.48Yujiang, Jiangxi provinceZZ-112005111407

50.51.913.78Hunan provinceZZ-52005111406

50.31.903.78Hunan provinceZZ-52005111405

50.01.853.70Jiangxi provinceZZ-62005111404

56.22.133.79Zhejiang provinceZZ-32005111403



Transfer rate ofprocessing

Content of BContent of AOrigin PlaceLot:No.

A: gardenoside of Fructus GardeniaeB: gardenoside of processed Fructus Gardeniae（carbonized）(calculated as dry CMM)


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Minimum content of gardenosidecontents

Chinese Pharmacopoeia part I prescribes that gardenoside contents of Fructus Gardeniae is not less than 1.8%.

According to the above results, calculated by the transfer rate of 50%, minimum gardenoside content of processed Fructus Gardeniae（carbonized）, calculated as dry CMM is 0.9%.


Study on contrast of fingerprinting chromatogram of FructusGardeniaes from different origin places

The HPLC fingerprinting method has been established for Fructus Gardeniae to study on contrast of Fructus Gardeniaes from different origin places

Mark of peaks in common in the fingerprinting chromatogram of Fructus Gardeniae

14 peaks in common are marked and gardenoside peak is marked with S

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ⅥThe trend of the quality control of raw Chinese Materia Medica

and processed Chinese Materia Medica

Under the guidance of the principle of scientification, practicabilityand specialization, modern scientific research achievements havebeen absorbed as many as possible. Supplementing of new assayindexes and new reference substances has upgraded the overallquality control.

The usage of reference Chinese Materia Medica, referencechemicals and reference extracts shows a rising trend. Referenceextracts were listed as the part of standard substances in ChinaPharmacopeia for the first time, which indicates the developingdirection that determining a group of components would graduallytake the place of determining a single index component.


282 reference chemicals are collected in China Pharmacopoeia, in which 90 arecollected for the first time.

218 reference Chinese Materica Medica are collected, in which 69 are collectedfor the first time.

11 reference extracts are collected, in which 6 are collected for the first time.

Now, reference substances, including 477 reference chemicals from naturalmedicines and 594 reference Chinese Materia Medica, are exclusively providedby NICPBP (National Institute for the Control of Pharmaceutical and BiologicalProducts) in China.

But obtaining reference chemicals from other provider is permitted if NICPBPis short of it but the certificate is necessary.


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Monography revised and supplemented mainly includes as follows.

1. The monographs of Chinese materia medica from many origins whosechemicals are obviously different from each other, are gradually listed asnew monographs according to the principle of one name to one drug.

2. According to the situation of commodities of Chinese material medica,revision is made to the medicinal part and resource.

3. The control indexes of safety are supplemented.

4. The specificity of testing methods are strengthened.


5. Scientification, advancement and practicability of the testing methodsare strengthened.

① Many modern analytical methods are adopted.

② The indexes of assay become more rational.

③ The concept of the Traditional Chinese Medicine’s theory on whole isemphasized on. The mode of quality control with a single chemical is brokenthrough. The method of quality control with multi-chemicals or characteristicchromatogram peak group is introduced.

④ The microscopic identification has the character of convenience, and speedand visibility


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6. The quality control of both toxic and active medicinal materials iscontrolled strictly.

The species, in which toxic constituents found out, are controlled strictly. For example, pyrrolizidine alkaloids exist in partial plants of Asteraceae. These compounds show no toxicity themselves. But they will produce toxic pyrrole through internal metabolism, as a metabolite in vivo. Therefore, they must be controlled strictly.


ⅦThe work we are engaged in

1. To propose China Pharmacopoeia committee to revise and supplement tolerance limitof aflatoxin in some Chinese materia medica and Chinese patent medicine in ChinesePharmacopoeia(Edition 2010).

2. To propose China Pharmacopoeia committee to increase the number of both pesticideresidues and Chinese materia medicas containing pesticide residue in item of method ofmonitoring of pesticide residue in Chinese Pharmacopoeia 2010.

3. To propose China Pharmacopoeia Committee to revise and supplement the number ofboth harmful elements and Chinese materia medica and Chinese patent medicinecontaminated with harmful elements in Chp (Edition 2010).

4. We propose China Pharmacopoeia Committee to use HPLC coupled to ICP-MS foranalysis of valence configuration of element in Chinese Pharmacopoeia 2010.


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THANK YOU!


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Traditional Chinese Medicines

A practical example of existingcontrol methods: identity andassay of Aristolochic acid

lögdInstitute of Public HealthNRW, Germany

Official Medicines Control Laboratory (OMCL)Dr. M. Heuermann

[email protected]

Dr. M. Heuermann OMCL Annual Meeting May 2006 Folia 2

OMCL Münster´s TCM History

• TCM are an important part of our work

• About 40 samples last year

• Sample origin: - wholesalers- pharmacies- customs

• TCM labeled as - food supplements- cosmetics- seldom as medicines

• Readable declaration often missing

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TCM Pharmaceutical formulations

• herbal drugs• tea (mixtures)• tablets• capsules• ointments• liquids• pills• globuli• powder


Summary of analytical results of TCM

• Formulations often exist of an unidentifiable herbalmatrix

• Formulations often contain no herbal material

• Often powerful enrichment with chemical APIs– Glucocoticoids - Sibutramin - Phenformin– Minoxidil - Finasterid - Glibenclamid– Sildenafil - Homosildenafil– Cinnabaris - Arsenoxid - Antrachinone

• Mixup with Aristolochic acid containing herbals

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Analytical strategy

1. Literature– Traditional Chinese monographs– Special literature dealing with TCM medicinal herbal drugs– “Special” Pharmacognosy

2. Internet recherche– Often the only way to find readable product information– Information about product composition– Information about the indications

3. Markroscopic and microscopic analysis– Herbal drugs with “added value”– Crystals of chemical APIs


Analytical strategy

4. Analytical methods for screening– HPLC/DAD UV-spectra database– GC/MS (MS spectra database)– TLC (aristolochic acid, antrachinoids)– HPLC/MS (MS spectra database)

5. Heavy metal analysis– ICP-MS– AAS

6. Assay and identity confirmation– HPLC/DAD and HPLC/MS for API and impurities– GC for pesticides

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Publication 9.2000

stemMutongLardizabalaceaeAkebia quinata

stemGuanmutongAristolochiaceaeAristolochiamanshuriensis

radixGuangfangjiAristolochiaceaeAristolochiafangchi

radixFangji, Fenfangji,Hanfangji

MenispermaceaeStephaniatetrandra

DrugChinese NameFamilySpecies

Chinese herbal drugs Fangji and Mutong: common mistakes / impurities

TCM with nephro-toxic substances caused several death casesAristoloclochic acid I and II caused the deathNon reliable certificates of the suppliers/brokers


Stephania Tetrandra: 1. level = “optical test”

• original sample of Stephania tetrandra

Chinese name: Fangji

• herbal drug, mistaken for Stephaniatetrandra probablyAristolochiae fangji radix

Chinese name: Guangfangji

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TLC: Monograph Radix Aristolochiae Fangji

Identity test 3, Chinese Pharmacopoeia

TLC: Camag, Horizontal Developing ChamberTLC-plates: Merck, HPTLC-plates, Kieselgel 60 F254, 10x20cmUV-Cabinet: Desaga, CabUVIS with Hitachi CCD-Camera HP-C550Mobile phase: 20% Toluol 10% Acetyl ethyl ester

1% water 1% Ameisensäure.Detection: UV-Light, 366 nm and 254 nm

Sample preparation3 g powdered drug bowled under reflux in 50 ml ethanol, 1h.Filtration and evaporation under vacuum.Residue solved in 5 ml ethanol.References: Radix Aristolochiae Fangji and

Aristolochic acid standard, 0.5 mg/ml.3 µl sample or reference solution.


TLC on Aristolochic acid, 254 nm

1 2 3 4 5 6 7 8 9

4, 6, 7: samples of Stephaniae tetrandrae radix 2 and 91 and 3: Aristolochiae spez. suspicious5 and 8: Reference substance: Aristolochic acid sample

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TLC on Aristolochic acid, 366 nm

1 2 3 4 5 6 7 8 9

4, 6, 7: samples of Stephaniae tetrandrae radix 2 and 91 and 3: Aristolochiae spez. suspicious5 and 8: Reference substance: Aristolochic acid sample


HPLC-System:Hewlett Packard Serie 1100diodenarray-detectormass-selective detector (MSD)software ChemStation A.06.04.

Reference material:Aristolochic Acid, Sigma, Lot 36H1311Tetrandrine, Aldrich, Lot 09704TN

main alkaloid of Stephaniae Tetrandrae

HPLC DAD/MSD Method: Identity and Assay 1/4

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Sample preparation:• 2 g powdered drug• 50 ml methanol/formic acid –solution, pH 2-3• 30 min shaking of the suspension• suspension 4 min centrifugation 4000 rpm• Upper phase membrane filtered


HPLC-Parameter:Mobile phase A: 0.1%ige formic acid pH 2.7Mobile phase B: methanol

0 -21 min: Isocratic 40% A and 60% B21- 29 min: 5% A and 95% B

Flow: 0.2 ml/minTemperature: 35 °CInjection volume: 5 µlColumn: Phenomenex Prodigy ODS(3) 5µm, 100A, 150 x 2.0mm

DAD:220 -500 nm


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MSD-Parameter:Ionisation mode: API-ESPolarity: PositiveScan-mode: 90 - 600 amuSIM-mode: 294.0 , 334.0 amu ( 0 -15 min)

324.0 , 364.0 amu (15 -21 min)Gas temperature (N2): 350 °CDry gas: 10.0 l/minNebuliser-pressure: 20 psigVoltage: 4500 V

Calibration range:Aristolochic acid I: 0.7 – 18.9 ppm (correlation: 0.99954)Aristolochic acid II: 0.2 – 5.2 ppm (correlation: 0.99969)



Suspicious sample: Stephania Tetrandra DAD

Identification and confirmation– HPLC-DAD on reversed phase with library search– GC-MS with library search– LC-MS

UV-spectrum of aristolochic acid I

UV-spectrum of aristolochic acid II

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Chromatogram Stephaniae Tetrandrae Radix(suspicious sample)

• Aristolochic Acid II, DAD = LOQ


Aristolochic Acid I: HPLC MSD

adduct MW 324 364

MW 341

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Aristolochic Acid II: HPLC MSD

adduct MW 294 334

MW 311


SIM Chromatogramm Reference Standards

Aristolochic Acid IMW 341adduct MW 364

Aristolochic Acid IIMW 311adduct MW 334

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Summary

• TCM are quite popular in Germany– But: no benefit without risk

• Lack of “official monographs” with European standard

• Required analytical techniques:– Information recherche: internet– Organoleptic (macrosopic and microscopic) (don´t taste)– TLC, AAS, ICP-MS– HPLC/DAD screening for chemical APIs– HPLC/MS and or GC/MS screening and confirmation


Acknowledgments

Tanks to all colleagues in MünsterDr. Petra Schmoltzi

Dipl. Ing. Michael Scherges

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New Frontiers in the Quality of Medicines€¦ · New Frontiers in the Quality of Medicines Workshop Herbals: Traditional Chinese Medicines Moderators: Prof. Dr Rudolf Bauer Prof