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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 AND HERBAL DRUG PREPARATIONS
HERBAL DRUGS AND HERBAL DRUG PREPARATIONS
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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HERBAL DRUGS AND HERBAL DRUG PREPARATIONS
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……….’
HERBAL DRUGS AND HERBAL DRUG PREPARATIONS
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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ELABORATED MONOGRAPHS – EUROPEAN PHARMACOPOEIA FORMAT
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
ELABORATED MONOGRAPHS – EUROPEAN PHARMACOPOEIA FORMAT
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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ELABORATED MONOGRAPHS – EUROPEAN PHARMACOPOEIA FORMAT
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).
ELABORATED MONOGRAPHS – EUROPEAN PHARMACOPOEIA FORMAT
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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ELABORATED MONOGRAPHS – EUROPEAN PHARMACOPOEIA FORMAT
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
ELABORATED MONOGRAPHS – EUROPEAN PHARMACOPOEIA FORMAT
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
ELABORATED MONOGRAPHS – EUROPEAN PHARMACOPOEIA FORMAT
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
ELABORATED MONOGRAPHS – EUROPEAN PHARMACOPOEIA FORMAT
Cortex Acanthopanacis [Wujiapi] Slenderstyle Acanthopanax Bark
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
Shanghai Institute for Food and Drug Control
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Ⅰ
Profile of quality control standard of raw Chinese Materia
Medica (CMM) and the processed CMM in China
Shanghai Institute for Food and Drug Control
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
Shanghai Institute for Food and Drug Control
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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.
Shanghai Institute for Food and Drug Control
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.
Shanghai Institute for Food and Drug Control
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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
Shanghai Institute for Food and Drug Control
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.
Shanghai Institute for Food and Drug Control
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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
Shanghai Institute for Food and Drug Control
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)
Shanghai Institute for Food and Drug Control
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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.
Shanghai Institute for Food and Drug Control
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.
Shanghai Institute for Food and Drug Control
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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%.
Shanghai Institute for Food and Drug Control
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.
Shanghai Institute for Food and Drug Control
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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
Shanghai Institute for Food and Drug Control
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
55.72.113.79Zhejiang provinceZZ-32005111402
54.92.083.79Zhejiang provinceZZ-32005111401
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)
Shanghai Institute for Food and Drug Control
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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%.
Shanghai Institute for Food and Drug Control
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.
Shanghai Institute for Food and Drug Control
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.
Shanghai Institute for Food and Drug Control
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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.
Shanghai Institute for Food and Drug Control
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
Shanghai Institute for Food and Drug Control
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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.
Shanghai Institute for Food and Drug Control
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.
Shanghai Institute for Food and Drug Control
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THANK YOU!
Shanghai Institute for Food and Drug Control
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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
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
2
Dr. M. Heuermann OMCL Annual Meeting May 2006 Folia 3
TCM Pharmaceutical formulations
• herbal drugs• tea (mixtures)• tablets• capsules• ointments• liquids• pills• globuli• powder
Dr. M. Heuermann OMCL Annual Meeting May 2006 Folia 4
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
3
Dr. M. Heuermann OMCL Annual Meeting May 2006 Folia 5
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
Dr. M. Heuermann OMCL Annual Meeting May 2006 Folia 6
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
4
Dr. M. Heuermann OMCL Annual Meeting May 2006 Folia 7
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
Dr. M. Heuermann OMCL Annual Meeting May 2006 Folia 8
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
5
Dr. M. Heuermann OMCL Annual Meeting May 2006 Folia 9
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.
Dr. M. Heuermann OMCL Annual Meeting May 2006 Folia 10
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
6
Dr. M. Heuermann OMCL Annual Meeting May 2006 Folia 11
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
Dr. M. Heuermann OMCL Annual Meeting May 2006 Folia 12
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
7
Dr. M. Heuermann OMCL Annual Meeting May 2006 Folia 13
HPLC DAD/MSD Method: Identity and Assay 2/4
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
Dr. M. Heuermann OMCL Annual Meeting May 2006 Folia 14
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
HPLC DAD/MSD Method: Identity and Assay 3/4
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Dr. M. Heuermann OMCL Annual Meeting May 2006 Folia 15
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)
HPLC DAD/MSD Method: Identity and Assay 4/4
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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
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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
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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
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Acknowledgments
Tanks to all colleagues in MünsterDr. Petra Schmoltzi
Dipl. Ing. Michael Scherges
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Your questions