Analytica Chimica Acta 471 (2002) 61–75

High-performance liquid chromatographic analysis of Black

Cohosh (Cimicifuga racemosa) constituents with in-line
evaporative light scattering and photodiode array detection
Wenkui Li a,b,c , Shaonong Chen a,d , Daniel Fabricant a,b,d , Cindy K. Angerhofer e ,
Harry H.S. Fong a,b,c,d , Norman R. Farnsworth a,b,d , John F. Fitzloff a,b,c,∗
a Program for Collaborative Research in the Pharmaceutical Sciences (PCRPS, m/c 877), College of Pharmacy,
University of Illinois at Chicago, 833 South Wood Street, Chicago, IL 60612-7231, USA
b Department of Medicinal Chemistry and Pharmacognosy (m/c 781), College of Pharmacy, University of Illinois at Chicago,
833 South Wood Street, Chicago, IL 60612-7231, USA
c Functional Food for Health (FFH) Core Analytical Laboratory, College of Pharmacy, University of Illinois at Chicago,

833 South Wood Street, Chicago, IL 60612-7231, USA

d NIH/UIC Center for Botanical Dietary Supplements Research, College of Pharmacy, University of Illinois at Chicago,

833 South Wood Street, Chicago, IL 60612-7231, USA

e Tom’s of Maine Corporation, Lafayette Center, P.O. Box 710, Kennebunk, ME 04043-0710, USA

Received 3 April 2002; received in revised form 7 August 2002; accepted 7 August 2002

Abstract
A validated and reproducible high-performance liquid chromatography (HPLC) method with in-line evaporative light scat-
tering and photodiode array detection was developed for the analysis of major constituents in Black Cohosh (Cimicifuga
racemosa L.). The method is based on the baseline chromatographic separation of 18 compounds reported to be present in
Black Cohosh on a C-18 column (5 ␮m, 4.6 mm × 250 mm) with water (0.05% trifluoroacetic acid, TFA)–acetonitrile–water
as the mobile phase, and their in-line detection using photodiode array detector (PDA) and evaporative light scattering
detector (ELSD). Sixteen of these (caffeic acid, ferulic acid, isoferulic acid, cimicifugoside H-1, cimiracemoside A,
cimicifugoside H-2, (26R)-actein, 26-deoxycimicifugoside, (26S)-actein, 23-epi-26-deoxyactein, 23-OAc-shengmanol-3-O-
␤-d-xyloside, 26-deoxyactein, 25-OAc-cimigenol-3-O-␣-l-arabinoside, 25-OAc-cimigenol-3-O-␤-d-xyloside, cimigenol-
3-O-␣-l-arabinoside, cimigenol-3-O-␤-d-xyloside) were detected to be present and quantifiable. The reputed two flavonoid
constituents (kaempferol and formononetin) were not detectable.
The validation of the method included tests for sensitivity, linearity, reproducibility, recovery and stability. The detection
limits were found to be in the range of 26–55 ng (10 ␮l per injection) in ELSD for the 16 constituents of Black Cohosh. The
exponential linear calibration curves were observed for all the constituents tested with r2 being >0.99. The reproducibility
of the method was evaluated by analyzing three sets of controls on three consecutive days (n = 3) with R.S.D. (%) and
relative error (%) <8.14 and 11.27%, respectively. The observed recovery rates were better than 91.75%. The stability of
Black Cohosh methanolic sample solution was assessed by consecutively analyzing the methanolic solutions prepared from
liquid extract (Sample A), powered extract (Sample B), milled plant material (Sample C) and one of the commercial product
(Sample D1) for the content of 16 major constituents with the relative standard deviations (R.S.D., %) <6.39%, indicating
the sample matrix of Black Cohosh is stable.

∗ Corresponding author. Tel: +1-312-355-4660; fax: 312-996-7107.

E-mail address: fitzloff@uic.edu (J.F. Fitzloff).

0003-2670/02/$ – see front matter © 2002 Elsevier Science B.V. All rights reserved.
PII: S 0 0 0 3 - 2 6 7 0 ( 0 2 ) 0 0 7 7 6 - 6
62 W. Li et al. / Analytica Chimica Acta 471 (2002) 61–75
Contrary to literature reports, kaempferol and formononetin were not detected in either our field collected plant materials
or commercial products of Black Cohosh in the current assay.
© 2002 Elsevier Science B.V. All rights reserved.
Keywords: Black Cohosh; Cimicifuga racemosa; Triterpene glycosides; Phenolic acids; Flavonoids; HPLC; PDA; ELSD
1. Introduction
Cimicifuga racemosa (L.) Nutt., or Actaea race-
mosa (Ranunculaceae) [1,2], commonly known as
Black Cohosh, is a perennial herb whose subterranean
portion consists of a thick knotted rhizome system and
aerial stem attains a height of 1–2.5 m. Black Cohosh
is native to North America, with its distribution pri-
marily in the mountain regions of the Eastern United
States (Blue Ridge and Great Smoky Mountains) and
Southeastern Canada [3]. This plant has been used
traditionally by Native Americans for gynecological
conditions long before the arrival of European set-
tlers in the New World [4]. Various preparations of
this plant have also been used for the treatment of
menopausal disorders in Germany for over 50 years
[5], and are presently available in the USA as dietary
supplements. A number of clinical studies have been
published in support of the use of Black Cohosh as al-
ternative treatment of menopausal symptoms [6–10].
Chemically, Black Cohosh extracts contain at
least two major natural product groups: cycloartane
triterpene glycosides, such as actein, 26-deoxyactein
[11–14], and phenolic compounds, such as ferulic
acid and isoferulic acid [15]. The flavonoids, such as
formononetin, have also been reported as present in
Black Cohosh [16].
In spite of extensive chemical, biological and clin-
ical studies, neither the active constituents nor the
mode of action of Black Cohosh have been deter-
mined. Currently, the standardization of Black Cohosh
preparations is based on the content of triterpene gly-
cosides, calculated as 26-deoxyactein [9]. Since the
most common commercial preparation is a powdered
root extract and since the biological active constituents
have not been identified, product standardization
should be considered from an aggregate of its major
constituents, rather than on a single compound. The
availability of a robust method allowing the analysis
of such a mixture of compounds in an extract is highly
desirable.
In general, direct reverse-phase high-performance
liquid chromatography (RP-HPLC) with UV detec-
tion could be, and is used for the analysis of Black
Cohosh samples. However, the detection of triterpene
glycosides using UV is well known for its insensitiv-
ity because of the weak chromophoric functionality of
triterpene glycosides in the 200–210 nm region. In ad-
dition, the complex matrix of Black Cohosh extracts
also interferes and challenges the sensitivity and se-
lectivity of the HPLC-UV methodology [12]. LC–MS
or LC–MS–MS methods may work well for the iden-
tification and quantification of the wide range of triter-
pene glycoside and phenolic compounds in the matrix
[12]. However, such equipment is expensive and may
not be readily available. Thus, the development of an
alternative HPLC method with a reliable and repro-
ducible detection is desirable. The inexpensive evapo-
rative light scattering detector (ELSD) may be suitable
for the routine analysis of Black Cohosh preparations.
As a mass detection method, the ELSD is based
on the nebulization of the LC column effluent into
droplets by the nebulizing gas and the entrance of the
resulting vapor into a temperature-controlled evapora-
tor tube, where the evaporation of mobile phase takes
place. The resulting “clouds” of solid microparticles
are then directed towards a narrow light beam. As a re-
sult, light is scattered by microparticles and measured
using a photomultiplier or photodiode. A plot of detec-
tor response versus analyte concentration is sigmoidal,
and the peak area (I) is related to the sample size and
shape, but not the chemical identity of the residual
particles passing through the light beam, by the fol-
lowing relationship: I = amb , where b is the slope of
the response line, m is the mass of the compound in-
jected, and a is the response factor. Plots of the peak
area versus the analyte concentration with logarithmic
co-ordinates are linear [17]. ELSD has been applied
to a wide range of UV-transparent analytes includ-
ing lipids [18,19], peptides [20], carbohydrates [21],
and botanical bioactive compounds [17,22]. Recently,
Ganzera et al. reported an HPLC–ELSD method for
 W. Li et al. / Analytica Chimica Acta 471 (2002) 61–75
the determination of actein, 26-deoxyactein, and
cimiracemoside A in Black Cohosh extact with LOD
of 105, 156 and 316 ng of them on the column, re-
spectively [23]. However, taking into consideration
of the fact that >18 triterpene glycosides and a num-
ber of phenolic acids have been reported from Black
Cohosh [11–14], it is necessary to expand on the ex-
isting procedure and develop a more comprehensive
method for routine analysis of the constituents in
Black Cohosh and its commercial products.
In the development of a broad based analytical pro-
cedure, all chemical constituents in plant materials or
commercial products must be considered. Flavonoids,
such as formononetin, have been reported to be present
in Black Cohosh [16], but their presence have been
questioned because isoflavonoids (formononetin) are
known to be rich in the Legume family (Leguminosae
or Fabaceae), but are relatively rare in other plant fam-
ilies [24–27]. Recent attempts to validate the presence
of formononetin and kaempferol in isopropyl alcohol
and ethanol extracts of Black Cohosh failed to either
identify their presence or to show their being present in
any significant level [28]. Thus, further investigations
to determine the presence or absence of flavonoids in
Black Cohosh are warranted.
In the present study, an HPLC–PDA–ELSD method
was developed for the quantitative and qualitative
analysis of 16 major constituents of Black Cohosh
along with two potential constituents (kaempferol,
formononetin) in one single HPLC run. This paper
describes the details of the methodology.
2. Experimental
2.1. Reagents
Methanol, acetonitrile, trifluoroacetic acid (TFA)
were HPLC grade from Fisher Scientific Co. (Fair
Lawn, NJ, USA). Deionized water was obtained with
an in-house Nano-pure® water system (Barnstead,
Newton, MA, USA).
2.2. Preparation of standard solutions and samples
Reference cimiracemoside A was purchased from
ChromaDex Inc., Laguna Hills, CA 92653, USA;
Reference caffeic acid was obtained from Sigma
63
Company, St. Louis, MO 63178, USA; Reference
kaempferol and formononetin were obtained from
Indofine Chemical Company, Somerville, NJ 08876,
USA; Standard (26S)-actein, (26R)-actein, ferulic acid
[15], isoferulic acid [15], cimicifugoside H-1 [29],
cimicifugoside H-2 [29], 26-deoxycimicifugoside
[30], 23-epi-26-deoxyactein [31], 23-OAc-sheng-
manol-3-O-␤-d-xyloside [32], 26-deoxyactein [31],
25-OAc-cimigenol-3-O-␣-l-arabinoside [12], 25-
OAc-cimigenol-3-O-␤-d-xyloside [32], cimigenol-3-
O-␣-l-arabinoside [12], cimigenol-3-O-␤-d-xyloside
[33] were isolated and purified from Cimicifuga race-
mosa roots. The identities and of purity of the iso-
lates were characterized by means of spectral (NMR
and MS) analyses and/or X-ray crystallography. The
structures of these reference standards are shown in
Fig. 1.
All reference standards were dissolved in methanol
at a range of concentrations as described under
Section 3.2. For reproducibility determination, three
sets of control solutions containing (26S)-actein,
cimiracemoside A, ferulic acid, isoferulic acid, cimi-
cifugoside H-1, cimicifugoside H-2, 26-deoxyci-
micifugoside, 23-epi-26-deoxyactein, 26-deoxyactein,
25-OAc-cimigenol-3-O-␣-l-arabinoside and 25-OAc-
cimigenol-3-O-␤-d-xyloside were prepared and stored
at −20 ◦ C and brought to room temperature before
use.
Black Cohosh samples: (A) liquid extracts dissolved
in 15% EtOH and 40% glycerin was provided by
Tom’s of Maine Corporation (Kennebunk, ME 04043);
(B) dried powdered extract was provided by Tom’s
of Maine Corporation (Kennebunk, ME 04043); (C)
plant materials was collected by one of the authors and
milled after dried; and (D) Black Cohosh commercial
dietary supplement products were purchased from lo-
cal pharmacies in Chicago, IL, USA. To protect the
manufacturers’ identity, the sample sources are labeled
D1–7. Products (D1–7) were claimed to contain from
9 to 40 mg of Black Cohosh extract (per serving), stan-
dardized to 2.5–8% triterpene glycosides, and contain
other herbal ingredients, including soy isoflavonoids,
red clover extract, licorice extract, kudzu extract, and
others.
Sample preparation was carried out according to
the nature of samples. (A) Liquid extract: 1.0 ml of
liquid formula of Black Cohosh was filtered through a
0.22 ␮m of membrane filter into a 1 ml HPLC sample
64 W. Li et al. / Analytica Chimica Acta 471 (2002) 61–75

Fig. 1. Structures of reference standards.

 W. Li et al. / Analytica Chimica Acta 471 (2002) 61–75
vial; (B) dried powdered extract: 30 mg of dried
powdered extract was dissolved in 1 ml of methanol
using sonicator and the resulting solution was fil-
tered through a 0.22 ␮m of membrane filter into a
1 ml HPLC sample vial; (C) milled plant materials:
0.5–1.0 g of samples was accurately weighed into a
20 ml PTFE-capped sample vial, 15 ml of methanol
was added and intimately mixed followed by sonica-
tion at 25–30 ◦ C for 30 min. After cooling to room
temperature, the resulting mixture was filtered through
a Whatman #1 filter paper into a 250 ml round-bottom
flask. The residue was returned to the vial, a second
15 ml of methanol was added, sonicated and filtered
as described, This procedure was repeated a third time
as described. The combined methanol extracts were
evaporated to dryness, under vacuum, at 45–50 ◦ C.
The residue was re-dissolved in HPLC grade methanol
(4 ml × 2 ml), transferred to a 10 ml volumetric flask,
and made up to the volume with methanol. The
sample solution was filtered through a 0.22 ␮m mem-
brane filter just prior to HPLC analysis; (D) Black
Cohosh commercial dietary supplement products: one
dosage of the product (the content of one capsule,
caplet or tablet) was accurately weighed into a 20 ml
PTFE capped sample vial and extracted with vary-
ing concentration of methanol (100% methanol for
capsule, 80% methanol for caplet, 50% methanol for
tablet formulations) under sonication at 25–30 ◦ C for
60 min. After cooling, the resulting mixture was fil-
tered through a Whatman #1 filter paper into a 250 ml
round-bottom flask, and the residue was returned
to the vial and treated as described for milled plant
materials with varying concentration of methanol,
dependent on the formulation as stipulated above.
The resulting sample solution was filtered through a
0.22 ␮m membrane filter just prior to HPLC analysis.
To evaluate the recovery rate, Black Cohosh root
powder (Sample C, 3.0 g) was extracted in a 50 ml
tared flask with 25 ml of methanol by sonication for
30 min. After filtration, the residue was returned to the
same flask and sonicated in 25 ml of methanol. The
process was repeated until the last filtrate was shown
to be free of detectable substances by HPLC–ELSD
as described above. The residue was thoroughly dried
and a portion (0.5 g) was accurately weighed into
a 20 ml PTFE capped sample vial. To this residue
in the vial, 1 ml of standard working solution con-
taining 0.749, 1.362, 0.54, 0.157 and 0.532 mg,
65
respectively, of reference isoferulic acid, cimicifugo-
side H-1, 26-deoxyactein, 25-OAc-cimigenol-3-O-␣-
l-arabinoside and cimigenol-3-O-␤-d-xyloside and
thoroughly mixed. This material was then extracted
with methanol and sonicated as previously described
for the Sample C. Three replicate recovery samples
were prepared for analysis. A blank recovery sam-
ple was prepared and analyzed for the chemically
exhausted root powder for comparative analysis.
To assess the stability of the major constituents of
Black Cohosh in sample matrix. The sample solutions
prepared from liquid extract (Sample A), powered
extract (Sample B), milled plant material (Sample C)
and one of the commercial product sample (D1)
were stored at room temperature (20 ◦ C) in dark
and analyzed on consecutive days (24, 48 and 72 h)
as described above. The relative standard deviation
(R.S.D., %) for the content of each constituent was
obtained by comparing the content measured on 24 h
(day 1), 48 h (day 2) and 72 h (day 3), respectively.
To facilitate the clean-up and identification of the
flavonoids in the analyte, 1 ml of Sample A solution
was absorbed on polyamide CC-6 powder (1g, 0.05–
0.16 mm, Macherey Nagel, Doren, Germany), dried
and loaded onto a column (1 cm × 45 cm) prepared
with 10 g of polyamide CC-6 in water and developed
with water and 20% methanol, consecutively (200 ml
each). Finally, the column was eluted with 200 ml
of methanol (100%). Methanol fractions were evapo-
rated under reduced pressure at 40 ◦ C and the resulting
residue was dissolved in 1 ml of methanol. Triplicate
samples were prepared and 10 ␮l of sample solution
was subjected to HPLC–PDA–ELSD analysis.
2.3. Chromatography
A Waters 2690 Alliance HPLC system (Waters
Corporation, Milford, MA, USA), equipped with a
996 photodiode array detector, an on-line degasser
and an autosampler, and a Waters YMC ODS-AQTM
RP-18 column (5 ␮m, 120 Å, 4.6 mm × 250 mm, se-
rial # 0425503853) was used as in the current study,
along with a Waters Delta-Pak RP-18 guard column
(4.6 mm × 5 mm). UV detection was achieved in the
range of 200–400 nm on a photodiode array detector
(PDA). After the PDA, the chromatographic column
effluent was subjected to the detection by an evap-
orative light scattering detector (ELSD) (Sedex 75,
66 W. Li et al. / Analytica Chimica Acta 471 (2002) 61–75
Alfortville, France). Nebulization of the effluent in
the ELSD was provided by a stream of pressurized
air (3.4 bar) at room temperature, and the nebulized
effluent was evaporated at 43 ◦ C. The detector was set
at gain 11, with output interfaced, via a SATIN box
to the Waters Millennium 2000® chromatographic
manager system (Waters) equipped with on a Compaq
6400X/10000/CDS computer (Houston, TX, USA)
for data handling and chromatogram generation.
The chromatographic elution was accomplished by
a gradient solvent system consisting of water contain-
ing 0.05% TFA (A), acetonitrile (B) and water (C). The
gradient conditions were: 0–8 min, 80% A, 20% B;
8–15 min, 32% B, 68% C; 15–55 min, 64% B, 36% C;
55–65 min, 95% B, 5% C, kept to 70 min; 70–85 min,
back to 80% A and 20% B. The flow rate was kept at
1.6 ml/min, and the injection volume was 10 ␮l (injec-
tion loop 50 ␮l). The eluted constituents were identi-
fied by comparison of the retention time in both ELSD
and PDA chromatograms and by comparison of the
UV spectra of the peaks in the samples with those of
reference standards. To simplify the computation of
the chromatograms, peaks detected by the PDA were
integrated at the wavelength of 254 nm, which is nor-
mally used for the detection of aromatic compounds.
Prior to each run, the HPLC–PDA–ELSD system was
allowed to warm up and the baseline was monitored
until stable before sample analysis.
3. Results and discussion
3.1. Detection
Chromatograms of all 18 reference standards gen-
erated by ELSD and PDA detection are presented in
Fig. 2. Baseline separation of all these reference stan-
dards was obtained by ELSD detection as shown in
Fig. 2A. UV detection at 203 and 254 nm in the PDA
detector are presented in Fig. 2B and C.
Due to their being poor chromophores, most of
the Black Cohosh triterpene glycosides gave very
weak UV absorption at 203 nm (Fig. 2B) and no
absorption at 254 nm (Fig. 2C), when compared to
same concentration of reference standards detected
by the ELSD (Fig. 2A), which confirmed the useful-
ness of the latter method for the detection of poor
absorbing or non-absorbing compounds [17]. The
lowest limits of detection (S/N > 3) of the 18 refer-
ence standards by ELSD detection were: (26S)-actein
(43 ng), (26R)-actein (24 ng), caffeic acid (50 ng),
kaempferol (50 ng), formononetin (53 ng), ferulic
acid (50 ng), isoferulic acid (52 ng), cimicifugoside
H-1 (49 ng), cimiracemoside A (55 ng), cimicifugo-
side H-2 (49 ng), 26-deoxycimicifugoside (26 ng),
23-epi-26-deoxyactein (45 ng), 23-OAc-shengmanol-
3-O-␤-d-xyloside (46 ng), 26-deoxyactein (50 ng),
25-OAc-cimigenol-3-O-␣-l-arabinoside (46 ng), 25-
OAc-cimigenol-3-O-␤-d-xyloside (52 ng), cimigenol-
3-O-␣-l-arabinoside (32 ng), and cimigenol-3-O-␤-d-
xyloside (31 ng) [34]. On the other hand, it is not
surprising that the UV detection at 254 nm showed
a very good sensitivity for the detection of pheno-
lic compounds with lowest measurable quantities
of 1 ng of caffeic acid, ferulic acid, isoferulic acid,
kaempferol and formononetin, each, on column. This
sensitivity is acceptable for the detection of potential
kaempferol and formononetin in the samples tested.
3.2. Linearity
Data obtained for the analysis of each reference
standards in 6–11 concentrations individually or in
a mixture were employed from at least nine experi-
ments. In the ELSD mode, a second-order polynomial
calibration (peak area against amount) was observed.
After log-transformation, the data provided a linear
function for all reference standards following the
equation: Y = a + bX with Y being the log value of
the peak area, X the log value of sample amount, a
the intercept and b the slope (Table 1) [17].
3.3. Reproducibility
The precision and accuracy of the method were as-
sessed by within and between run validations. The
reproducibility was evaluated by injecting three sets
of controls on three separate days. By substituting
the peak-area into the calibration curve equation from
the same run, the measured concentrations were ob-
tained. By comparing observed and theoretical con-
centrations, the relative errors (RE, %) were obtained.
The relative standard deviation (R.S.D., %) was cal-
culated by comparing the observed concentrations. As
shown in the Table 2, the R.S.D. and the RE were
found to be <8.14 and 11.27%, respectively.
W. Li et al. / Analytica Chimica Acta 471 (2002) 61–75 67
68 W. Li et al. / Analytica Chimica Acta 471 (2002) 61–75

Fig. 2. (Continued ).
 W. Li et al. / Analytica Chimica Acta 471 (2002) 61–75 69

Table 1
Linearity of assays for 16 constituents of Black Cohosh and two flavonoids in HPLC–ELSD by regression analysis
# Name Slope Intercept r2 Rangea

1 Caffeic acid 0.6439 1.9709 0.9975 10–100

2 Ferulic acid 1.2014 1.7943 0.9988 10–100
3 Isoferulic acid 0.8347 1.8361 0.9980 10–100
4 Cimicifugoside H-2 1.1703 1.6881 0.9949 10–340
5 Kaempferol 1.1567 1.7082 0.9976 10–100
6 Cimiracemoside A 1.066 1.7323 0.9988 11–110
7 Formononetin 1.3553 1.7089 0.9984 10.5–105
8 Cimicifugoside H-1 1.0327 1.7005 0.9972 10–340
9 (26R)-Actein 1.3651 1.7708 0.9966 3.5–12
10 26-Deoxycimicifugoside 1.2818 1.7733 0.9974 5.3–53
11 (26S)-Actein 0.9752 1.6845 0.9972 8–80
12 23-epi-26-Deoxyactein 1.4261 1.6507 0.9953 10–320
13 23-OAc-shengmanol-3-O-␤-d-xyloside 1.1486 1.6465 0.9952 9–90
14 26-Dexoyactein 1.007 1.6080 0.9964 10–100
15 25-OAc-cimigenol-3-O-␣-l-arabinoside (24S) 1.1772 1.6154 0.9970 9–90
16 25-OAc-cimigenol-3-O-␤-d-xyloside (24S) 1.1850 1.6246 0.9971 10–100
17 Cimigenol-3-O-␣-l-arabinoside (24S) 0.7324 1.8148 0.9960 6.5–125
18 Cimigenol-3-O-␤-d-xyloside (24S) 0.9898 1.7995 0.9982 6.5–65
Y: log value of peak area; X: log value of concentration (␮g/ml).
a Lowest and highest concentration in ␮g/ml.

3.4. Recovery side H-2 (3.39%), 26-deoxycimicifugoside (4.72%),

The three sets of recovery samples were analyzed as
described in Section 2. The average recovery (%) and
R.S.D. (%) for the reference standards were: isoferulic
acid (97.37, 1.26), cimicifugoside H-1 (98.50, 1.50),
26-deoxyactein (99.96, 1.34), 25-OAc-cimigenol-O-
␣-l-arabinoside (95.38, 4.16) and cimigenol-3-O-␤-d-
xyloside (91.75, 5.54). Similar recoveries for closely
related compounds would be expected.
3.5. Stability
In the current study, the consecutive analyses of the
major constituents in the sample solutions prepared
from liquid extract (Sample A), powered extract (Sam-
ple B), milled plant material (Sample C) and one of
the commercial product (Sample D1) revealed that the
methanolic solutions of different Black Cohosh sam-
ple (Samples A, B, C and D1) are stable at room tem-
perature with the highest relative standard deviations
(R.S.D., %) observed as follows: (26S)-actein (4.23%),
(26R)-actein (4.27%), caffeic acid (3.58%), ferulic
acid (3.26%), isoferulic acid (3.91%), cimicifugoside
H-1 (3.07%), cimiracemoside A (4.41%), cimicifugo-
23-epi-26-deoxyactein (3.85%), 23-OAc-shengmanol-
3-O-␤-d-xyloside (3.77%), 26-deoxyactein (4.61%),
25-OAc-cimigenol-3-O-␣-l-arabinoside (6.39%), 25-
OAc-cimigenol-3-O-␤-d-xyloside (4.64%), cimige-
nol-3-O-␣-l-arabinoside (2.51%), and cimigenol-3-
O-␤-d-xyloside (4.93%). However, the presence of
phenolic acids such as caffeic acid, ferulic acid,
isoferulic acid in the sample solutions might affect
the stability of triterpene glycosides when the sam-
ple solutions were put at room temperature for long
time. Thus, the long-term stability of Black Cohosh
preparation needs further investigation.
3.6. Application
A commercial liquid extract (A) and a dried pow-
dered extract (B) as well as a milled field collected
plant material (C) of Black Cohosh were analyzed in
triplicate. Most of the constituents in these samples
were detectable and quantifiable. The content of the
major constituents in these materials is presented in
Table 3. The typical HPLC chromatograms of liquid
extract (A) are shown in Fig. 3 ((A) HPLC–ELSD;
(B) HPLC-UV at 203 nm; (C) HPLC-UV at 254 nm).
70 W. Li et al. / Analytica Chimica Acta 471 (2002) 61–75
 W. Li et al. / Analytica Chimica Acta 471 (2002) 61–75 71

Fig. 3. Typical HPLC chromatograms of Black Cohosh liquid extract (Sample A) ((A) HPLC–ELSD; (B) HPLC-UV at 203 nm; (C)
HPLC-UV at 254 nm).
72 W. Li et al. / Analytica Chimica Acta 471 (2002) 61–75

Fig. 3. (Continued ).

Table 3
To determine the feasibility of the present method
Content (%) of major constituents in liquid extract (Sample A),
dried powdered root extract (Sample B) and milled field collected for post marketing analysis of Black Cohosh products,
samples (Sample C) of Black Cohosha seven commercial dietary supplement preparations
(Sample D) were analyzed. The results of the anal-
Constituent Liquid extract Powdered Field collected
(mg/ml) root extract crude material ysis (Table 4) showed triterpene glycosides content
(%, w/w) (%, w/w) in the range of 0.13–5.97 mg per serving. As previ-
1 0.07 0.05 0.01 ously indicated, Black Cohosh extract is generally
2 0.75 0.93 0.01 standardized to 2.5% triterpene glycosides, i.e. if the
3 0.36 4.94 0.07 product contains 40 mg of standardized extract per
4 0.11 1.31 0.01 serving (capsule, caplet or tablet), the assumed con-
6 0.40 0.09 0.05
8 0.73 5.01 0.01
tent of triterpene glycosides will be 1 mg per serving.
9 0.34 ND ND However, the current assay revealed that some Black
10 0.25 0.13 0.02 Cohosh preparations did not contain as much of the
11 2.80 1.08 0.17 triterpene glycosides as the manufacturers claimed.
12 1.25 1.24 0.09 On the other hand, the production of Black Cohosh
13 1.04 1.18 0.22
14 0.20 0.55 0.04
product, like any other botanical dietary supplement,
15 0.13 0.20 ND involves the acquisition of source plant materials by
16 0.10 0.63 ND cultivation or field collection, processing of the plant
17 ND ND 0.0001 materials into a standardized extract, and manufactur-
18 ND ND 0.0009 ing the standardized extract into suitable dosage forms
a ND: not detected. such as capsule, caplet, tablet, etc. Under the Dietary
W. Li et al. / Analytica Chimica Acta 471 (2002) 61–75 73
74 W. Li et al. / Analytica Chimica Acta 471 (2002) 61–75

Table 4
Total triterpene glycosides content (per serving) in commercial Black Cohosh products
Source Amount/serving Label claim Total triterpene Triterpene glycosides detecteda
glycosides found
(mg) per serving
D1 500 mg of root powder NA 5.97 4, 6, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18
D2 9 mg of extract Triterpene glycosides 1.18 4, 6, 8, 11, 14, 15, 17, 18
in extract (8%)
D3 40 mg of extract Teriterpene glycoside 1.12 4, 6, 8, 11, 12, 13, 14, 15, 16, 17, 18
in extract (2.5%)
D4 40 mg of extract NA 1.33 4, 6, 8, 11, 12, 13, 14, 15, 16, 18
D5 40 mg of extract NA 0.87 4, 6, 8, 11, 14, 17, 18
D6 40 mg of extract Triterpene glycosides 3.23 4, 6, 8, 10, 11, 12, 13, 15, 16, 17, 18
in extract (2.5%)
D7 20 mg of extract NA 0.13 6, 11, 12
a For ID # of each constituent, please refer to Table 1.

Supplement Health and Education Act (DSHEA) of 4. Conclusion

1994, Black Cohosh products are marketed as dietary
supplements in the USA. Since DSHEA only places
the responsibility of safety, but not the burden of
proof for the product on the manufacturers, Black Co-
hosh products have not been subjected to mandated
quality assurance (QA) standards. As a consequence,
it is not uncommon to see that the product differ
from brand to brand, even from lot to lot. In view of
the variability of the major constituents detected in
the commercial samples (D1–7), a better regulation
and effective control (GAP, GLP and GMP) is highly
desirable.
In contrast to their reputed presence in the lit-
erature [16], the flavonoids, kaempeferol and for-
mononetin were not detected in the current study,
based on comparison of the retention times and UV
spectral library (200–400 nm) with the reference stan-
dards. In order to eliminate interfering substances in
the materials tested, the samples were subjected to
a “clean-up” procedure as described in Section 2.
Kaempferol and formononetin were still not detected
in the purified fraction. Although the HPLC–PDA
chromatogram of a purified fraction of Black Cohosh
sample (A) showed several potential peaks with re-
tention times close to those of kaempferol (22.96 min)
and formononetin (26.39 min) reference standards
(Fig. 4), their UV spectra (200–400 nm), however,
are very different. Confirmation of the presence of
these flavonoids in Black Cohosh requires further
investigation.
The present study describes a validated analytical
method for the quantitation of major triterpene glyco-
sides and phenolic compounds in the Black Cohosh
samples. The limits of detection of those compounds
have been determined to be between 26 and 55 ng.
Kaempferol and formononetin were not detected in the
Black Cohosh extracts and in the commercial prod-
ucts, contrary to literature reports in the current assay.
In the current study, some Black Cohosh commercial
products did not meet the label claim for triterpene
glycosides.
Acknowledgements
This study was partially supported by funds from
NIH/NCCAM Grants no. 5950AT00155 and the
University of Illinois Functional Food for Health
Program.
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