Click to view or download PDF versionJANUARY, 2015
Skullcap Adulteration Laboratory Guidance Document
By Stefan Gafner, PhD
Chief Science Officer, American Botanical Council
Technical Director, ABC-AHP-NCNPR Botanical Adulterants Program
Keywords:
Adulterant, adulteration, germander, Scutellaria lateriflora, Scutellaria spp., scullcap, skullcap, skullcap adulteration, Teucrium canadense, Teucrium chamaedrys
1. Purpose
Skullcap (Scutellaria lateriflora, family
Lamiaceae) herb has a long history of adulteration, evidenced in comments from
over 100 years ago by Felter and Lloyd that “Scutellaria versicolor Nuttall and Scutellaria canescens Nuttall are the species generally collected
by herbalists and substituted for Scutellaria
lateriflora.” [1] Besides the substitutions with other species from the genus Scutellaria, adulteration with germander
(Teucrium) species containing
hepatotoxic furano neo-clerodane diterpenes has been reported in the early 1990s
and seems to persist in the herb trade in North America and possibly elsewhere.
[2] This Laboratory Guidance Document presents a review of the various publicly-available
analytical technologies and methods used to differentiate between authentic S. lateriflora and its potentially
adulterating species, listed in Table 1.
2. Scope
The various analytical methods described below were reviewed with the specific purpose of
identifying strengths and limitations of existing methods for differentiating S. lateriflora from its potentially
adulterating species. Analysts can use this review to help guide the
appropriate choice of techniques for their specific skullcap products for
qualitative purposes. The positive evaluation of a specific method for testing S. lateriflora material in the products’
particular matrix in this Laboratory
Guidance Document does not reduce or remove the responsibility of laboratory personnel
to demonstrate adequate method performance in their own laboratory using
accepted protocols outlined in the US Food and Drug Administration’s Final Rule
for Current Good Manufacturing Practices for Dietary Supplements (as published
in 21 CFR Part 111) and by AOAC (Association of Official Analytical Chemists) International,
International Organization for Standardization (ISO), World Health Organization
(WHO), and International Conference on Harmonisation (ICH).
3. Common and scientific names
3.1 Common Name: skullcap
[3]
3.2 Other Common Names
English: blue skullcap, helmet flower, hoodwort,
European or greater skullcap, Quaker bonnet, mad-dog skullcap, mad weed, scullcap,
Virginia skullcap
French: scutellaire, scutellaire latériflore,
scutellaire de Virginie, toque, toque bleue, toque casquée, toque des marais
German: Helmkraut, Fieberkraut, Fleckenkraut,
Blaues Gnadenkraut, Kappenhelmkraut,
Gemeines Schildkraut
Italian: scutellaria
Spanish: escutelaria, escutelaria de Virginia
3.3 Latin Binomial: Scutellaria lateriflora L. [4,5]
3.4 Synonyms: Cassida lateriflora (L.) Moench; Scutellaria polybotrya Bernh. [4,5]
3.5 Botanical Family: Lamiaceae
4. Botanical Description
Botanical
descriptions for Scutellaria and Teucrium species are provided in local, national,
and international floras, including Flora
of North America, Flora Europaea,
and Flora of China. Additionally, the
genus Scutellaria (including tables
distinguishing them from Teucrium species), is described in the skullcap monograph of the American Herbal
Pharmacopoeia (AHP), accompanied by illustrations and images. [6] Morphological
identification to the species level requires personnel trained in botany as
well as authenticated materials with intact and characteristic botanical features.
Table 1. Scientific names, family, and common names of known* skullcap (Scutellaria
lateriflora) adulterants
Speciesa |
Synonymsb |
Family |
Common namec |
Other common namesd |
Scutellaria alpina L. |
Cassida alpina (L.) Moench |
Lamiaceae |
|
Alpine skullcap |
Scutellaria baicalensis Georgi |
S. lanceolaria Miq.; S. macrantha Fisch. |
Lamiaceae |
Chinese skullcap |
Baikal skullcap, scute |
Scutellaria galericulata L. |
Cassida galericulata (L.) Scop. |
Lamiaceae |
|
Marsh skullcap, marsh
skullwort |
Scutellaria incana Biehler |
S. canescens Nutt. |
Lamiaceae |
|
Hoary skullcap, downy
skullcap |
Scutellaria ovata Hill |
S. versicolor Nutt. |
Lamiaceae |
|
Heartleaf skullcap |
Teucriume canadense L. |
T. boreale E.P. Bicknell;
T. bracteosum Raf.;
T. menthifolium E.P. Bicknell;
T. mexicanum Sessé & Moc.;
T. nashii Kearney;
T. occidentale A. Gray;
T. roseum E.P. Bicknell;
T. virginicum L. |
Lamiaceae |
|
Canada germander, American
germander, wood sage |
Teucriume chamaedrys L. |
T. stevenianum Klokov |
Lamiaceae |
Germander |
Wall germanderc |
aAccording to The Plant List and the Tropicos
database. [4,5]
bAccording to The Plant List and the Tropicos
database. [4,5] A comprehensive list of synonyms can be accessed through The
Plant List website. [4]
cAccording to the American Herbal Products
Association’s Herbs of Commerce, 2nd ed. (2000). [3]
dAccording to The Plant List, the Tropicos
database, Herbs of Commerce, 2nd ed., and the USDA PLANTS Database. [3,4,5,7]
eTeucrium species have also been referred to as “pink skullcap” which contributes to the
nomenclatural confusion.
*Note: The list of known adulterants is based on published
data, e.g., references 1, 2, and 6. Some of the listed species may represent
incidences of historical significance but occurrence may be rare or absent in
the current marketplace.
Sections 5-8 of the present document discuss macroscopic,
microscopic, genetic, and chemical authentication methods for S. lateriflora. A comparison among the
various approaches is presented in Table 3 at the end of section 8.
5. Identification and Distinction using Macroanatomical Characteristics
Macroscopic identification criteria for S. lateriflora have been published in the AHP monograph by Upton et
al.,[6] in the Ph.D. thesis by Brock, [8] and in Applequist.[9] Descriptions in
the AHP are more detailed and include the potential adulterants T. chamaedrys, T. canadense, S.
galericulata, and S. incana in a table format. High-quality
drawings illustrate the text in the table and make it more easily
understandable. The text by Brock contains details on S. lateriflora, T. chamaedrys, T. canadense, S. galericulata, and S. ovata, and many of her comments are
based on the 2009 AHP monograph. In addition to a table listing the main
features of each plant, Brock also provides helpful guidance outlining the main
distinctive features between the different Scutellaria species and Teucrium.
6. Identification and Distinction using Microanatomical Characteristics
Detailed microscopic descriptions of S. lateriflora, and the germander species T. canadense and T.
chamaedrys, are found in a number of references. [6,10,11] The textbook by
Upton et al. [10] also contains a section on the roots of S. baicalensis. However, there are no microscopic descriptions in
the recent literature for other Scutellaria species listed in Table 1.
Comments: While
outside the scope of this document, botanical microscopy is one of the easiest
ways to detect adulteration with inert materials and undisclosed fillers (e.g.,
cellulose, starch, sand). However, it is unclear if a microscopic distinction
of powdered aerial material of S. lateriflora and closely related Scutellaria species can be achieved. The
use of microscopy for the authentication of S.
lateriflora, in addition to a macroscopic assessment, may be adequate for
companies that grow their own plant material, or when whole plant material is
purchased. In all other cases, a microscopic examination should be combined
with other appropriate methods (genetic or chemical) for authentication or
detection of adulteration. The identification of S. lateriflora extracts cannot be achieved using microanatomical
characteristics.
7. Genetic Identification and Distinction
Methods described in the following literature were evaluated
in this review: Hosokawa et al. [12] and Hosokawa et al. [13]
Comments: The
approach using direct sequencing [13] has been tested successfully with a
number of closely related skullcap species and is considered the more reliable
of the two genetic methods to authenticate S.
lateriflora. However, genetic assessment will not identify the plant part,
which is a legal requirement of dietary supplement ingredient identification.
Thus, genetic testing must typically be coupled with another appropriate test
for distinguishing the plant parts. As DNA methods are usually inappropriate and
unreliable for identity testing of extracts or certain other processed material
(see Table 3), the authentication and detection of adulteration has to be done
by chemical means in these cases.
8. Chemical Identification and Distinction
There are numerous analytical methods available for authenticating S. lateriflora and differentiating it
from other Scutellaria species as
well as potential adulterants, such as Teucrium species. These methods are cited in the Laboratory Methods section below.
Distinction based on the phytochemical profile requires a detailed knowledge of
the constituents of S. lateriflora and its adulterants. Below is a summary of the phytochemical composition of
skullcap and its known adulterants, including chemical structures of the
principal flavonoids occurring in S.
lateriflora (Figure 1) and phenylpropanoid glycosides in Teucrium species (Figure 2).
8.1 Chemistry of Scutellaria
lateriflora and Potential Adulterants
Scutellaria
lateriflora: According to a review of the analytical literature, the main
flavonoid in dried S. lateriflora aerial
parts is baicalein-7-O-glucuronide (syn: baicalin, 1). Other important
flavonoids are dihydrobaicalin (2), lateriflorein-7-O-glucuronide (syn:
lateriflorin, 3), wogonoside (4),
ikonnikoside I (5), and oroxylin A-7-O-glucuronide
(6). [11, 14-18] Larger amounts of the aglycone, baicalein, point to a cleavage
of the glucuronic acid moiety in 1 and are often indicative of stability
issues. Since most of the flavone-glucuronides (1, 4, 6, and scutellarein-7-O-glucuronide
[syn: scutellarin, 7]) are found in many species of the genus Scutellaria, methods for authentication
of S. lateriflora relying on its
major constituents must be based on the totality of compounds present (phytochemical
fingerprint) in regard to both the composition and the relative amounts.
|
|
1: R1 = R2 =
R4 = H, R3 = OH
3: R1 = R4 =
H, R2 = OCH3, R3 = OH
4: R1 = R2 =
R3 = H, R4 = OCH3
5: R1 = R4 = H, R2 = R3 = OH
6: R1 = R2 = R4 = H, R3 = OCH3
7: R1 = R3 = OH, R2 = R4 = H |
2 |
Figure 1: Chemical structures of principal flavonoids found in aerial parts of S. lateriflora
Other compounds reported
from S. lateriflora aerial parts include
waxes, essential oil,
neo-clerodane diterpenes, amino acids, coumarins, and stilbenes. [15,16,19-22] According to several authors who used
authenticated samples in their analysis, neither verbascoside (8) nor teucrioside (9)(Figure 2; see also Teucrium species below) occur in S. lateriflora [11,17,23,24]; therefore,
the phenylpropanoid glycosides were proposed as markers to detect adulteration
of skullcap with germander. The findings of two studies [18,25] that reported 8 from skullcap materials remain controversial,
since the analysis was performed on unauthenticated commercial products [18] or
showed a very untypical chemical composition [25].
8: R = H
9: R = lyxose
Figure 2: Chemical structures of
phenylpropanoid glycosides from Teucrium species
Scutellaria alpina: Seven flavonoids are known from S. alpina leaves [26]: chrysin,
2’-methoxychrysin, apigenin, scutellarein, 7,
chrysin-7-O-glucuronide (10), and apigenin-7-O-glucuronide (11). While the lack of 1, 2, 4, and 6 is easily detected
in a substitution, a mixture of S. alpina with S. lateriflora may be difficult
to detect based on flavonoids only. The presence of the scutalpins, neo-clerodane
diterpenes characteristic to S. alpina,
can be used to unequivocally identify this species. [27,28]
10: R1 = R2 = H
11: R1 = H, R2 = OCH3
12: R1 = OH, R2 = H
Figure 3: Chemical structures of important flavone-glucuronides found in Scutellaria species
Scutellaria galericulata: The flavonoid compositions of aerial
parts of S. galericulata and S. lateriflora grown in North America are
very similar, the only difference being the presence of 10 and the absence of 6 in S. galericulata. [29] Phenylpropanoid glycosides [2-(4-hydroxyphenyl)-ethyl-(6-O-caffeoyl)-β-D-glucopyranoside, calceolarioside B, osmanthuside E, and martynoside] have been reported from the
aerial parts of S. galericulata material from Turkey. [30] These four phenylpropanoid glycosides represent appropriate marker compounds to distinguish S.
galericulata from S. lateriflora.
Additionally, the scutegalins, neo-clerodane diterpenes characteristic of S. galericulata, can be used for
authentication. [31,32]
Scutellaria incana: The only quantitative phytochemical
characterization of aerial parts of S.
incana available is summarized in Table 2, indicating large amounts of 4 and 7, but no 1-3, 5,
or 6. [29] Qualitatively, a total of
40 flavonoids have been reported from S.
incana, including a number of flavone C-glycosyl compounds, 1, 4,
and 6, 7, and 10. [33] In addition, the authors
detected 8 in the plant material.
The identification was mainly based on HPLC-MS, a sensitive technique that may
have allowed detecting very low amounts of 1 and 6. Structure assignments were
tentative in some cases, but the presence of flavone C-glycosides and 8 should allow a distinction between S. incana and other species.
Scutellaria ovata: The major flavone-glucuronide from the
aerial parts of S. ovata is 6, with smaller amounts of 1 and 10. [29] In addition, oroxylin A-7-O-glucoside and ovatin (5,6-dimethoxyflavone-7-O-glucoside) were also reported from the species. [34] Presence of
oroxylin A-7-O-glucoside and absence
of 2, 3, and 5 differentiates S. ovata and S. lateriflora.
Table 2. Comparison of contents (in % [w/w]) of flavone- and
flavanone-glucuronides in dried hydroethanolic (70% ethanol) extracts of S. lateriflora, S. alpina, S. galericulata, S. incana, and S. ovata [29]
Speciesa |
1 |
2 |
3 |
4 |
5 |
6 |
7 |
10 |
12 |
S. lateriflora |
7.0-17.2 |
3.2-14.6 |
0.8-2.3 |
0.6-0.9 |
0.3-1.1 |
0.7-0.9 |
0.5-0.7 |
0 |
0-0.3 |
S. alpinab |
* |
0 |
0 |
0.5 |
0 |
0 |
6.0 |
0.9 |
* |
S. galericulata |
1.6-16.7 |
9.2-24.2 |
0.2-0.6 |
0-0.1 |
0-0.8 |
0 |
0.8-3.4 |
0.5-8.9 |
0-9.1 |
S. incana |
0 |
0 |
0 |
1.4-3.9 |
0 |
0 |
6.4-8.7 |
0.1-0.3 |
0 |
S. ovata |
1.1 |
0 |
0 |
0 |
0 |
9.9 |
0.1 |
0.9 |
0 |
1: Baicalein-7-O-glucuronide; 2: Dihydrobaicalin; 3: Lateriflorein-7-O-glucuronide; 4: Wogonoside; 5: Ikonnikoside I; 6:
Oroxylin A-7-O-glucuronide; 7: Scutellarein-7-O-glucuronide; 10: Chrysin-7-O-glucuronide; 12:
2’-Methoxychrysin-7-O-glucuronide
aThere are no
comparative results for S. baicalensis available.
b1 and 12 were
present at low levels in one of two voucher samples of S. alpina. The material with 1 and 12 was not quantitatively analyzed.
[Gafner, unpublished]
Scutellaria
baicalensis: The composition of Baikal skullcap has been extensively studied, and a large
number of chemical structures have been reported from this plant. [35] The main
flavonoids in the roots have been identified as 1, 4, 6, baicalein, and wogonin, [36-38] and
the contents of 1 have to be no less
than 10% in the crude root drug according to the Japanese Pharmacopoeia. [39] The aerial parts contain mainly 1, 4,
and 7. [25] It is distinct from S. lateriflora by the absence of 3 and 5 in aerial parts and roots, and by the presence of large amounts
of 4 in root material.
Teucrium
canadense: The aerial parts are
characterized by the presence of 8 as
the major component, with smaller amounts of flavone-glycosides (e.g., 11), but not 1-3, 5, or 7. [11,17] The lipophilic fraction is
dominated by the neo-clerodane diterpenes teuflin and teucvidin. [17,40]
Teucrium
chamaedrys: The major compound in T. chamaedrys aerial parts is 9, with other
phenylpropanoid glycosides (8, teucrioside
3’’’’-O-methyl ether, and teucrioside-3’’’’, 4’’’’-O-dimethyl ether [41]) as
minor compounds. Also present are flavonoids, mainly glycosides of apigenin,
diosmetin, and luteolin, [17] and the reportedly characteristic hypolaetin- and isoscutellarein-7-O-(6'''-O-acetyl)allosyl-(1→2)-O-glucosides. [42] In the lipophilic
fraction, the predominant neo-clerodane diterpene is teucrin A. [17,40]
A complicating factor with regard to
establishing a phytochemical profile is the known instability of some of the
major components in S. lateriflora.
This is of particular concern if fresh material is used for extraction, or if
the material is improperly dried, since the high amount of water will expose
the flavonoids to oxidative degradation. Degradation has also been observed in
tinctures with low amounts of ethanol, leading to a complete absence of known
skullcap metabolites in certain products. [43]
8.2 Laboratory
Methods
Note:
Unless otherwise noted, all methods summarized below use only aerial parts of Scutellaria spp. and/or Teucrium spp.
8.2.1 HPTLC
Methods from the
following sources were evaluated in this review: Upton et al., [6] Gafner et
al., [11] and Hong et al. [44]
Figure 4: HPTLC analysis of S. lateriflora and its adulterants
according to [6]. For
lanes 1-16, the identities of the materials are indicated above the lane. Lane
17: mixture of Scutellaria lateriflora: Teucrium canadense (80:20); Lane 18:
mixture of Scutellaria lateriflora: Teucrium chamaedrys (80:20). Detection:
Natural products/polyethylene glycol (NP/PEG) reagent, UV at 366 nm. Image
provided by Camag AG; Switzerland.
Comments: The conditions described in references
11 and 44 are the same with the exception of the extraction process. Using
methanol [44] as the extraction solvent will shorten the application time
(methanol dries easier than a 70% aqueous ethanol solution) and may lead to
more uniform bands.
Hong et al. have
been able to detect as little as 0.5% T.
chamaedrys in S. baicalensis using the HPTLC conditions initially published by Gafner et al. [11] Upton et
al. [6] have modified the solvent system to include suitable conditions for the
less polar components, in particular 1.
The modification comes at the expense of a lower resolution between the
abundant flavone-glucuronides (e.g., 1, 7) in S. baicalensis and the phenylpropanoid glycosides (8, 9)
in Teucrium.
Both systems
are suitable for authentication of S.
lateriflora. For dried skullcap raw material where the flavone-glucuronides
are predominant, the mobile phase developed by Gafner et al. [11] and Hong et
al. [44] may be preferred. System suitability parameters have not been
published for any of the methods and will need to be included in the validation
process.
8.2.2 HPLC and UPLC
Methods
described in the following literature were evaluated in this review: Upton et
al., [6] Bergeron et al., [15] Li et al., [16] Lin et al., [17] Zhang et al.,
[18] Sun and Chen, [24] Islam et al., [25] Gao et al., [43] Makino et al., [45]
Parajuli et al., [46] Tascan et al., [47] Cole et al., [48] and Brock et al. [49]
A comparison among the various HPLC and UPLC methods are given in Appendix 1,
Table 4. Specific comments on strengths and weaknesses of each of the methods
are listed in Appendix 1, Table 5.
Figure 5: Typical HPLC-UV trace for S. lateriflora. Conditions as outlined in [15]. 1:
Scutellarin; 2: Ikonnikoside I; 3: Baicalin; 4: Lateriflorin; 5:
Dihydrobaicalin; 6: Oroxylin A-7-O-glucuronide;
7: Baicalein
Comments: In most cases, sample preparation is
the most time-consuming part of an analysis. For routine quality control, a
quick and easy method is helpful, e.g., the sample preparation outlined by Lin
et al. [17] The solvent of choice in most cases is a mixture of MeOH-water
(between 6:4 and 8:2, v/v) or EtOH-water (6:4 or 7:3, v/v), which will give
adequate extraction of the flavone-glucuronides. All published methods will be
able to detect adulteration with Teucrium species, if the adulterant is present in a sufficient quantity. The methods
which have been the most thoroughly validated [6,15] should be preferred. If
the run time is of essence, the conditions developed by Sun and Chen [24] are
the best option. System suitability parameters (e.g., tailing factor,
resolution) have not been published for any of the methods and will need to be
included in the validation process.
8.2.3 MS-Fingerprinting
Chen et al.
[23]:
Comments: A statistics-based authentication
method is state of the art for analytical technologies. The analysis is very
short and environmentally friendly due to low solvent use. The method will
provide a “yes” or “no” answer without relying on the interpretation of an
expert after constructing an appropriate library of authenticated materials;
however, an expert analyst is required to setup the parameters for the instrument
and the statistical evaluation, and to run the instrument. Initial costs for
the instrumentation are high. A sonication time of 1 hour and small sample
volume may lead to a high temperature extraction and increase the risk of
degradation. Very small sample amounts are used for extraction. The method has
not been validated and no system suitability parameters were described.
8.2.4. NMR
Colson et al.
[50]:
Comments: A statistics-based authentication
method is state of the art for analytical technologies. The results show that
this approach is able to clearly distinguish S. lateriflora from Teucrium samples and the instrument will provide a “yes” or “no” answer without relying
on expert interpretation. As with other statistics-based evaluations, added
materials (e.g., carriers, processing aids) will modify the outcome of the PCA
and thus may cluster the material outside the acceptable range. Therefore, the
construction of a library containing authenticated materials of the same
composition as the analyte is necessary. Expert analysts are required to setup
the appropriate parameters and run the instrument. The analysis time is short
and ecologically responsible due to the low amount of solvent used. As a result
of the reproducibility using NMR, new samples can be directly compared to
samples run earlier without having to rerun the whole series. System
suitability for any botanical analysis is the same: the 1H line
shape and the 1H sensitivity have to comply with the probe
specifications. In addition, the temperature must be stable to 0.1°C. However,
the sample preparation is very time-consuming due to the need to freeze-dry the
extract before analysis in order to avoid a large signal from residual water. Data
on method validation are not available.
8.2.5 NIR Hyperspectral
Imaging
Sandasi et
al. [51]:
Comments: Near-infrared hyperspectral imaging
(NIR-HSI) is a fast, non-destructive,
environmentally friendly, and affordable method. Samples of raw material,
extracts, or even ingredients in heterogeneous matrices can be analyzed with
little or no pre-processing. Sandasi et al. subjected the hyperspectral images to
multivariate statistical analysis in order to differentiate the three species
analyzed (S. lateriflora, T. canadense, and T. chamaedrys). Using the statistical approach, a “yes” or “no”
answer can be obtained without the need to rely on the interpretation of an
expert. HSI also enables large quantities of material to be analyzed thus
avoiding sampling problems. The technique has good selectivity; nevertheless,
the sensitivity is lower compared to conventional chromatographic techniques.
In the case of skullcap, the method could detect only admixtures with Teucrium spp. at levels ≥ 40%, as the
error of detection (deviation between the exact and predicted values) becomes
larger with decrease in percentage adulteration. Sandasi et al. have used only one
authenticated sample of each species to create their library, so it needs to be
seen how differences due to agricultural or processing variations impact the
results, and how well the method can distinguish the various known adulterants
from within the Scutellaria genus.
[51]
Table 3.
Comparison among the different techniques to authenticate S. lateriflora
Method |
Applicable to |
Pro |
Con |
Macroscopic |
Raw materiala |
Quick
Inexpensive
No solvents required |
No
automation/statistics
Outcome relies on
analysts’ expertise
Difficult or
impossible for c/s material |
Microscopic |
Raw material |
Quick
Inexpensive
Can readily detect
adulterating Teucrium species
Few solvents
required |
No
automation/statistics
Outcome relies on
analysts expertise
Challenge to
distinguish closely related Scutellaria species |
Genetic |
Raw material |
Able to distinguish
closely related species
Reliable
Able to detect small
amounts of adulterants |
Labor-intensive
sample preparation and analysis
Expensive equipment
Cannot distinguish
between plant parts |
HPTLC |
Raw material,
extracts |
Quick
Basic systems affordable
for smaller labs
Able to detect small
amounts of adulterants |
No statistics
High-end equipment
expensive
Need for standard
compoundsc |
HPLC-UV |
Raw material,
extracts |
Standard equipment
in many laboratories
Able to detect small
amounts of adulterants
Mostly quantitative
(less specific than HPLC-UV/MS) |
Equipment expensive
Often no statistics
applied (although software is available)
Need for standard
compoundsc |
HPLC-UV/MS |
Raw material,
extracts |
Standard equipment
in many laboratories
Able to detect small
amounts of adulterants
Qualitative and
quantitative |
Equipment very expensive
Often no statistics
applied (although software is available)
Quality of data
depends on ability to ionize analyte
Need for standard
compoundsc |
Standalone MS (no
prior separation) |
Raw material,
extracts |
Short analysis time
Reliable
State-of-the art
statistical evaluation
Independent of
analyst’s expertise after method is set up
Qualitative and
quantitative |
Equipment very expensiveb
Initial setup of
parameters complex
Quality of data
depends on ability to ionize analyte |
NMR |
Raw material,
extracts |
Short analysis time
Reliable and highly
reproducible
State-of-the art
statistical evaluation
Independent of
analysts expertise after method is set up
Qualitative and
quantitative |
Equipment and
maintenance very expensiveb
Initial setup of
parameters complex
Labor- and
time-intensive sample preparation
Needs at least 4’ x
7’ floor space |
aOnly whole and cut and sifted (c/s)
bSome useful standard compounds (dihydrobaicalin, ikonnikoside I, 2’methoxychrysin-7-O-glucuronide, teucrioside, teuflin) are
not commercially available.
cCosts for high-resolution mass
spectrometers and NMR instruments are generally above $250,000. A low-cost NMR
for natural products analysis can be obtained for ca. $150,000.
9. Conclusion
There are
numerous techniques and analytical methods that readily allow for the
differentiation of Scutellaria lateriflora from the potentially toxic
adulterant Teucrium species even when
admixtures occur at small concentrations. Differentiating among closely related
species of Scutellaria is more
challenging. Authentication of Scutellaria species solely based on the presence of marker compounds (e.g., baicalein-7-O-glucuronide,
scutellarein-7-O-glucuronide, wogonoside, or baicalein) is insufficient unless
a thorough statistical evaluation (e.g., with HPLC, direct MS, [23] or NMR [50])
is performed.
For species authentication
of commercially available whole plant material, confirmation of species
identity and purity may be achieved by organoleptic methods, if conducted by
qualified personnel (e.g., a botanist). For cut or powdered raw material, a
combination of a physical assessment test (e.g., macroscopic or microscopic)
and/or a genetic approach [e.g., 13] combined with chemical identification
methods is recommended. HPTLC and HPLC methods can be used for chemical
characterization of raw material and extracts. Suggested HPTLC methods include
the methods described in references 6, 11, and 44. For laboratories with an
HPLC, the suggested methods are detailed in 6 and 15; for laboratories with
UPLC equipment, the method of choice is presented in 24. It should be noted
that with all the available methods, data on method validation is limited, and
system suitability data are lacking.
Note: A
number of identity tests for skullcap materials are offered by third-party
analytical laboratories. According to input from five contract laboratories,
the testing methods include microscopy, HPTLC, and HPLC-UV. Additional testing
methods (HPLC-MS or near-infrared [NIR] methods) can be developed upon request.
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Appendix 1
Table 4. Comparison among different published HPLC methods for S. lateriflora
Reference |
Number of samplesa |
Origin of samples (aerial parts when not
specified) |
Sample preparation:
handlingb / duration [min]c |
Column type |
Run time
[min]d |
Detection wavelength (UV) or ion mode
(MS) |
[6] |
1e |
AHP |
6
/ 1460 |
C18 |
46 |
UV:
280 |
[15] |
1e |
Commercial
raw material |
9f / 2460f |
C18 |
46 |
UV:
280 |
[16] |
1 |
Commercial
raw material |
15
/ 200 |
C18 |
75 |
MS
(negative) |
[17] |
9 |
AHP |
5
/ 90 |
C18 |
75 |
UV:
280, 310, 330, 350
MS
(pos/neg) |
[18] |
10 |
Commercial
products off shelf |
ASEg |
C18 |
85 |
UV:
278 |
[24] |
17 |
AHP
& Internet |
7
/ 95 |
C18 |
18 |
MS
(negative) |
[25] |
1 |
Grown
from seeds |
8
/ 150 |
C18 |
36 |
UV:
280 |
[43] |
7 |
Commercial
products off shelf |
3g / 15h |
C18 |
32 |
UV:
270 |
[45] |
8 |
Research
Center for Medicinal Plant Resources, Tsukuba & commercial sources |
15
/ 180 |
C18 |
24 |
UV:
277 |
[46,47] |
1 |
Grown
from seeds |
ASEg |
C18 |
33 |
UV:
270 |
[48] |
1 |
Tissue
culture from seeds |
10
/ 185 |
C18 |
60 |
MS
(positive) |
[49] |
2 |
AHP
& commercial raw material |
7
/ 170 |
C18 |
30 |
UV:
280 |
aNumber of S. lateriflora samples analyzed
bNumber of sample preparation steps involved (see Table 6 in Appendix 1)
cEstimated based on description provided in the reference (see Table 6 in Appendix 1)
dNot including the time used to return to initial conditions and equilibrate
eMethod has been used with over 20 authenticated reference samples and commercial products as part of QC
fExtraction with ethanol-water (7:3,
v/v)
gASE = accelerated solvent extraction
hIndications refer to tinctures only
Table 5. Comments on the published
HPLC methods for S. lateriflora
Reference |
Comments |
Upton, [6]
Bergeron [15] |
The
method was validated by 2 independent laboratories, and has good separation
and peak shapes. The sample extraction was optimized for flavone-glucuronides
(unpublished data) but is quite lengthy and therefore addition of
citric/ascorbic acid is needed to prevent degradation. The Zorbax column is
superior to the Phenomenex column, but some peaks (e.g., 6) are not resolved. There is no indication about detection
levels of Teucrium species. System
suitability parameters have not been published. |
Li [16] |
This
is a reliable fingerprinting method, and the MS fractionation gives excellent
data on the peak identity for phenolics. There are no data on Teucrium species detection. The sample
preparation time of over 2 hours with a number of handling steps is not ideal
for a routine lab method. The run time is lengthy without noticeable
improvement in peak separation over other methods. The method calls for the injection
of a fairly concentrated solution, so samples high in 1 could lead to column overload. There are no data on system
suitability parameters and the method has not been validated. |
Lin [17] |
The
chromatogram includes both flavonoids and diterpenes. The method provides good
separation but the high injection volume is likely the reason for the tailing
observed with 1. The sample
extraction is easy and quick, optimized for flavone-glucuronides. Some
diterpenes may not be very soluble in 60% aqueous methanol. Teucrium species at levels as low as
1% in S. lateriflora can be
detected. The run time is lengthy. The sample size is small (100 mg) and
sonication (1 hr) longer than in other methods. There are no data on system
suitability parameters and the method has not been validated. |
Zhang [18] |
This
is a validated method and the conditions give good peak shapes. The sample
preparation is specific to research project, but is not applicable in routine
QC. There are no data on Teucrium species
detection, but 8 can be quantified
down to 0.5 mg/g (500 ppm) of plant material. The method is lengthy (no peaks
of interest are eluting in the first 30 min) and the separation between
chrysin and oroxylin A is insufficient. There are no data on system
suitability parameters. |
Sun [24] |
This method has a short run time due to use of
UPLC-MS. The sample preparation method is easy and quick, although the sampling
size (10 mg) is low for a quantitative method and therefore some of the precision
may be lost. There is no indication on detection levels of Teucrium species. The chromatogram contains some
unresolved peaks. There are no data on system suitability parameters and the method has not been validated. |
Islam [25] |
The
HPLC-UV method is reasonably short (the HPLC-MS method is very short) and
shows a good separation. Some broadening of later eluting peaks (e.g.,
chrysin) is observed. The internal standard (digoxin) for HPLC-MS is
chemically very different from the target analytes. MS parameters are not
fully detailed and there is no indication about detection levels of Teucrium species; however, 8 can be quantified down to 0.51 mg/g
(510 ppm, UV/vis) and 0.38 mg/g (380 ppm, MS) dry plant material. There are
no data on system suitability parameters and the method has not been
validated. |
Gao [43] |
The
method is validated, reasonably short, and has good peak shapes. It has been
tested only on tinctures. Only very small volumes are used for sample
preparation (as low as 50 µL), possibly leading to lower precision. The internal
standard (IS) helps to correct for imprecisions in the injection system, but
the additional dilution due to the IS addition using small volumes might
actually introduce a larger error than any contribution from injection
imprecisions. There are no data on Teucrium species detection. Not all peaks are well separated. Data on system
suitability parameters are lacking. |
Makino [45] |
This
is an official method (JP XV for S.
baicalensis). It is short and gives good peak shapes. The extraction
procedure using MeCN-phosphoric acid is labor intensive. There are no data on Teucrium species detection. Not all
peaks are well separated. Data on system suitability parameters are lacking. |
Parajuli, [46]
Tascan [47] |
The
method is reasonably short and has good peak shapes. The sample preparation
is specific to the research project, and not applicable in routine QC. Addition
of HCl during the sample preparation may hamper stability of phytochemicals; the
HCl concentration is unclear. There are no data on Teucrium species detection. The method works only for
medium-to-low polar compounds. Flavone-glucuronides are not sufficiently well
separated. There is no information on column temperature. Data on system
suitability parameters are lacking and the method has not been validated. |
Cole [48] |
The
high injection volume (50 µL) into the 100% aqueous initial mobile phase may
lead to precipitation of compounds on column. Materials analyzed were from
tissue cultures, which will differ from wild-crafted or cultivated plant
material. The HPLC parameters are unclear: the text describes a 45 min run
time but states that the elution of peaks was monitored up to 60 min, which
would mean eluting for 30 min with 100% MeCN. The HPLC-MS trace also shows a
run time of 60 min, with no peak of interest eluting after 25 min. MS
detection parameters and validation data for skullcap flavonoids are not
available. No Teucrium samples were
analyzed. There are no data on system suitability parameters. |
Brock [49] |
The
chromatography is reasonably short. The sample preparation time is short; and
the sonication should be 30 min rather than twice for 15 min (personal
communication to S. Gafner, September 19, 2013). There are no data on the
method’s ability to detect Teucrium species.
The separation is insufficient and a reprint of the chromatogram shows prominent
peak tailing (indicative of contamination, bad column, or some precipitation
of flavonoids in the mobile phase during the injection process). There are no
data on system suitability parameters and the method has not been validated. |
Note: The use
of the term “validated” indicates a method has been validated for quantitative
analysis, not for qualitative identification according to LaBudde and Harnly. [52]
Table 6. Various sample process steps
and time requirements. If no specific duration is given in a
paper, the time is estimated according to the table (e.g., if the authors
sonicate a sample for 15 min, then 15 min is used for calculation rather than
30 min as indicated in the table). Plant collection, drying, and grinding are
not included in order to better compare the actual processing time among the
various methods.
Processing step |
Time
[min] |
Cooling
down
Combining
fractions
Decanting
and centrifugation
Dissolving
Diluting
Evaporation
(organic solvents only)
Evaporation
(solvent mixtures containing water)
Filling
to volume
Filtration
(paper, organic solvents only)
Filtration
(paper, solvent mixtures containing water)
Filtration
(0.2 or 0.45 µm
HPLC filters)
Initial
addition of solvent
Lyophilization
(including time to freeze the sample)
Mixing
(by inversion)
Partitioning
Sonication
Sonication
(including solvent addition, e.g., in repeated extractions)
Washing
(flasks, beakers)
Weighing |
30
5
10
5
5
60
120
0
30
60
5
5
720
0
60
30
30
5
5 |