1. Fingerprinting of Herbal Drugs using
Advanced Bioanalytical Techniques
Dr. Bhaswat S. Chakraborty
10/12/12 1

2. 10/12/12 2

3. Herbal Drugs as Traditional Medicine
“…the medicine that refers to health practices,
approaches, knowledge and beliefs
incorporating plant, animal and mineral based
medicines, spiritual therapies, manual
techniques and exercises, applied singularly or
in combination to treat, diagnose and prevent
illnesses or maintain well-being …”
WHO
10/12/12 3

4. Some Indian Medicinal Plants
10/12/12 4

5. Use of Traditional Medicine
• Worldwide 40-70% people use traditional medicine (of herbal,
animal or mineral origin)
– Exclusively, alternately or terminally
• The quality of herbal medicines is often poor
– Production of herbals is not controlled or regulated
• Herbal medicines usually contain a range of pharmacologically
active compounds
– Often not known which of these constituents produces the therapeutic
effect.
– Testing for efficacy is more complex than with synthetic drugs
• Complex safety issues
– Toxicity of natural herbal constituents
– Presence of contaminants or adulterants
– Herb-herb or herb-prescription drug interactions
10/12/12 5

6. Renewed Interest in Herbal Medicine
• The effectiveness of plant medicines.
• The preference of consumers for natural therapies, a greater interest in
alternative medicines and a commonly held erroneous belief that herbal
products are superior to manufactured products.
• A dissatisfaction with the results from synthetic drugs and the belief that
herbal medicines might be effective in the treatment of certain diseases
where conventional therapies and medicines have proven to be inadequate.
• The high cost and side effects of most modern drugs.
• Improvements in the quality, efficacy, and safety of herbal medicines with
the development of science and technology.
• Patients’ belief that their physicians have not properly identified the
problem; hence they feel that herbal remedies are another option.
• A movement towards self-medication
10/12/12 6

7. But the Problem is
• Herbal drugs are usually mixtures of many constituents.
• The active principle(s) is (are), in most cases unknown.
• Selective analytical methods or reference compounds may not be
available commercially.
• Plant materials are chemically and naturally variable.
• The same species varies in various drug contents depending on
locality, cultivation process, etc.
• The source and quality of the raw material are variable.
• The methods of harvesting, drying, storage, transportation, and
processing (for example, mode of extraction and polarity of the
extracting solvent, instability of constituents, etc.) have an effect.
10/12/12 7

8. QC of Herbal Drugs
• Identity
• Purity
• Content or assay – very challenging for herbal drugs
• Parameters for Quality Control of Herbal Drugs
– Microscopy
– Foreign matter determination
– Determination of ash
– Determination of heavy metals
– Determination of Microbial Contaminants and Aflatoxins
– Determination of Pesticide Residues
– Determination of Radioactive Contamination
– Analytical method and its validation
10/12/12 8

9. Why QC Herbal Drugs?
• Accurate identity and specifications
• Content uniformity
• Reduced batch to batch variability
• Efficacy related to quality
• Safety
– Some sub-species can be very toxic
• Raw drug and dosage form stability
• Control of shelf life decreasing contaminations
10/12/12 9

10. Sample Preparation for
Chromatographic Fingerprinting
• Follows the same priciples as those for any HPLC (or
other chromatographic) analysis
• Herbs may require very specific additional steps prior
to chromatographic analysis
– Grinding and pulverizing
– Extraction
• Organic solvent
• Organic solvent free
• Super-critical fluid
• Co-solvent super-critical
– Hydro-distillation of essential oils
10/12/12 10

11. Instead of chemical solvent, compressed CO2 is used. It is a fine
extracting solvent and doesn’t contaminate the herb, environment
or our body.
10/12/12 11

12. LC-MS-MS
10/12/12 12

13. MS vs. MS/MS
Mass
HPLC Inlet Ionize Detect
Analyze
MS
Separation Identification
Mass Mass
Inlet Ionize Fragment Detect
Analyze Analyze
MS1 Collision MS2
Cell
MS/MS
10/12/12 13

14. Chromatographic Fingerprinting of Herbs
• These are chromatograms representing characteristics of the
herbs
– Identity, consistency and authenticity of samples
• Hyphenated techniques (GC/MS, LC/MS and CE/MS) show
greatly improved performances
– retention time shift, selectivity, chromatographic separation abilities
and measurement precision
• Chromatographic fingerprinting is a highly accurate,
meaningful quality control method of herbal medicines
• Since it is usually difficult to distinguish herbal drugs by
different manufactures visually, principal components analysis
(PCA) is used.
10/12/12 14

15. Curcumin
+ 1 0 1 m fr m a p 1( 1 4 9 P S IV ( 1_ U C M ) o Q .w ( ub S r y, C n o e
Q : .0 7 in o S mle 0 0 0 _ O IT EQ ) C R U IN f 1 iff T r o pa ) e tr id d Mx 2 e c s
a . .3 7 p .
23
2 .1
2 e
.3 7
2 e
.2 7
2 e
.1 7
2 e
.0 7
1 e
.9 7
1 e
.8 7
1 e
.7 7
1 e
.6 7
1 e
.5 7 22
1 .0
1 e
.4 7
Intensity, cps
1 e
.3 7 27
1 .0
1 e
.2 7
1 e
.1 7
1 e
.0 7 31
9 .4
29
7 .2
9 e
.0 6
24
4 .9
8 e
.0 6
26
9 .2
7 e
.0 6 44
6 .5
39
6 .1
6 e
.0 6 26
5 .3 34
3 .6
22
3 .4
22
0 .1
5 e
.0 6 29
4 .2 21
6 .1 35
0 .2
23
1 .9 26 30
6 .2 0 .5 32
5 .3 48 43
0 .5 1 .4
4 e
.0 6 3 4 1 .0
1 .33 9 42
9 .6
2 .1 2 0 2 5
21 3 .1 3 .1 25 21
7 .0 9 .1 38
5 .3 38
8 .4
41 48
1 .4 2 .5 45
6 .4
3 e
.0 6 21
5 .4 33
6 .2 45
5 .3
33
0 .2 34
4 .3 40
0 .6
22
7 .3 36
2 .8 37
7 .2 42
3 .4 48 44
5 .4 7 .6 55 53
2 .0 3 .0 5 0
5 .8
2 e 27 25 28
.0 6 0 .1 1 .3 3 .9 23
6 .2
35 39
6 .2 7 .1
57
0 .3 58
7 .9
32
4 .4 45
2 .3 56 51
1 .9 4 .0 55
6 .3
34
7 .4 47
4 .3 40
7 .5 4 8 54
4 .9 52
9 .7
1 e
.0 6 7 .9
43
2 .4 57
8 .2
20
0 20
2 20
4 20
6 20
8 30
0 30
2 30
4 30
6 30
8 40
0 40
2 40
4 40
6 40
8 50
0 50
2 50
4 50
6 50
8 60
0
m ,a u
/z m
10/12/12 15

16. Total Ion Current of Parent Ion
Parent Ion of Curcumin

17. HPLC Fingerprint of Ashwagandha
10/12/12 Dr. Quazi et al (downloaded), CSIR 17

18. Glycowithanolides and Withanolides
from Ashwagandha
10/12/12 Dr. Quazi et al (downloaded), CSIR 18

19. LC-MS Fingerprint of Ashwagandha
10/12/12 Dr. Quazi et al (downloaded), CSIR 19

20. Fingerprint of Formulations
10/12/12 20
Dr. Quazi et al (downloaded), CSIR

21. Total Ion Chromatogram of Ginseng Sample
10/12/12 21

22. 10/12/12
Chromatograms of Different Batches of
Ginseng in Shenmai injection
22

23. Similarity of Fingerprints
• Equivalence of herbal drugs of different sources –
“Phytoequivalence”
– Very good, reproducible and accurate chromatograms are needed
• For good comparison, correction of retention time shifts of the
component drugs is necessary
• So many components from so many sources
– Visual comparison is extremely difficult
– May even be misleading
• Chemometric techniques,e.g., Principal Component
Analysis (PCA) are used to confirm similarity or dis-
similarity
10/12/12 23

24. Phytoequivalence
TIC of essential oils of Cortex
cinnamomi from four different
sources
10/12/12 Liang et al (2004), J Chromat B, 812, 53-70 24

25. Correction for RT Shift
Chromatograms of Ginko biloba
samples by two different HPLC
instruments
10/12/12 Liang et al (2004), J Chromat B, 812, 53-70 25

26. Principal Component Analysis
HPLC UV of G. biloba extracts from 17 different sources
10/12/12 Liang et al (2004), J Chromat B, 812, 53-70 26

27. Principal Component Analysis
HPLC UV of G. biloba extracts from 17 different sources
Different fingerprint
from those below
10/12/12 Liang et al (2004), J Chromat B, 812, 53-70 27

28. Principal Component Analysis
HPLC UV of G. biloba extracts from 17 different sources
10/12/12 Liang et al (2004), J Chromat B, 812, 53-70 28

29. On-Line Chemometric Comparison
HPLC-MS TIC of Schisandra extracts by 6 different methods
Liang et al (2004),
10/12/12
J Chromat B, 812, 53-70 29

30. DNA Microarray of Herbal Drugs
10/12/12 Wang M et al (2005), Phytotherap Res 19, 173-182 30

31. Thank You
10/12/12 31