456 LCGC NORTH AMERICA VOLUME 22 NUMBER 5 MAY 2004 www.chromatographyonline.

com

Rapid Determination of Terpene

Lactones in Ginkgo Biloba Commercial
Products by HPLC with Evaporative
Light-Scattering Detection
A rapid, sensitive, and reproducible high performance liquid chromatography
gradient method has been developed for the measurement of ginkgolides A, B, C,
and J, along with bilobalide, in a Ginkgo biloba commercial product. The separation
was achieved in less than 14 min, employing a water–methanol–trifluoroacetic acid
mobile phase and an evaporative light-scattering detector. No sample clean-up
procedures were used with the methanol extraction of the Ginkgo biloba dietary
supplement. The detection limit (signal-to-noise ratio  5) is less than 125 ng on-
column for each terpene lactone on a reversed-phase C18 column. Both intra- and
interday reproducibility were evaluated. Four brands of standardized Ginkgo biloba
herbal supplements were assessed for their terpene lactone content. This method is
applicable for analyzing a Ginkgo biloba dietary supplement in capsule, tablet, or
liquid form.

Tim Herring
Alltech Associates, Inc., 2701
Carolean Industrial Drive, State
College, Pennsylvania 16801,
e-mail therring@alltechemail.com
T
he world’s most ancient surviving
tree, Ginkgo biloba, originated
more than 200 million years ago.
Ginkgo, sometimes known as the
maidenhair tree, has an average life span of
1000 years and can grow to 20–40 m in
height. A monotypic tree with broad fan-
shaped leaves, Ginkgo biloba has been valued
for its medicinal properties for many cen-
turies in Asia, where it is used to treat dis-
eases such as asthma, tuberculosis, and arte-
riosclerosis. In western medicine, its leaves
and extractions are used primarily to treat
demential disorders such as concentration
difficulties and memory impairment (1).
Ginkgo biloba leaf extract has been shown to
possess antioxidant, anti-ischemic, and neu-
roprotective properties (2), and has been
shown to improve mental capacities in
Alzheimer’s patients (3).
The pharmacologically active terpene lac-
tones selectively inhibit the platelet-activat-
ing factor, preventing thrombus formation
and bronchoconstriction. Bilobalide is
reported to possess neuroprotective proper-
ties (4). Figure 1 shows the structures of the
terpene lactones in Ginkgo biloba.
In the U.S. market, Ginkgo biloba prepa-
rations were the top-selling nutraceutical in
1998 and 1999, comprising more than 20%
of the total sales in the herbal–nutraceutical
industry (5). Today, it is still the best-selling
herbal supplement worldwide. Therefore, a
rapid and reproducible method for extract-
ing and determining the active constituents
in Ginkgo biloba is very desirable in industry.
Experimental
Reagents and chemicals: Methanol (high
performance liquid chromatography
[HPLC] grade) was purchased from Fisher
Scientific Co. (Fair Lawn, New Jersey).
Deionized water was obtained with an in-
house U.S. Filter Service Deionization water
system (Pittsburgh, Pennsylvania). Trifluo-
roacetic acid (99%) was acquired from
Aldrich Chemicals Co. (St. Louis, Missouri).
Bilobalide, ginkgolide J, ginkgolide C,
ginkgolide A, and ginkgolide B standards
were obtained from Alltech Associates, Inc.
(Deerfield, Illinois). Several brands of
Ginkgo biloba supplements were bought
from a local department store. Supplies for
the preparation of standards and samples
included 5-mL Luer tip plastic syringes;
nylon 13-mm diameter, 0.45-
m porosity
polypropylene-encased syringe filters; 0.45-

m porosity, 47-mm nylon filter mem-
branes; and 4-mL HPLC vials with caps
(Alltech Associates).
Chromatographic Conditions: The
HPLC system was assembled from an on-
line degassing system (Alltech Associates), a
model L-7100 quaternary gradient HPLC
www.chromatographyonline.com MAY 2004 LCGC NORTH AMERICA VOLUME 22 NUMBER 5 457

pump (Hitachi Instruments, San Jose, Cali-

fornia), a model 7125 manual sample injec-
tor (Rheodyne, Cotati, California) with a 5-

L PEEK flex-connect sample loop (Alltech
Associates), a model ELSD 2000 evaporative
light-scattering detector (Alltech Associates),
and a PeakSimple chromatography data sys-
tem (SRI, Torrance, California). The detec-
tor was operated in the impactor off mode
(no aerosol splitting). The drift tube tem-
perature was 110 °C, and the nitrogen gas
flow was 3.1 L/min.
A 100 mm  4.6 mm, 3-
m dp Alltima
C18 column (Alltech Associates) was used
for all separations, and they were done at
ambient temperature. A binary mobile phase
was used with mobile phase A being 0.1%
trifluoroacetic acid in deionized water and
mobile phase B consisting of 0.1% trifluo-
roacetic acid in methanol (6). The gradient
was 10–55% B over 15 min. All runs were
baseline-equilibrated before the initiation of
the next run. The flow rate was 1.3 mL/min.
All HPLC injections of standards and sam-
ples had an injection volume of 5
L. All Figure 1: Structures of the terpene lactones in Ginkgo biloba. (Courtesy of the Institute for Nat-
ural Products Research.)
mobile phases were mixed, degassed, and fil-
tered before use.
Standard Solution Preparation: 1.017 ginkgolide A, and 1.004 mg of ginkgolide B 10-mL volumetric flask. Methanol was
mg of bilobalide, 1.011 mg of ginkgolide J, were weighed accurately on a microscale and added to volume, and the compounds were
1.025 mg of ginkgolide C, 1.031 mg of quantitatively transferred into a clean, dry dissolved. From this, a subsample was
458 LCGC NORTH AMERICA VOLUME 22 NUMBER 5 MAY 2004
Figure 2: Separation of terpene lactones in (a) Brand A capsule, (b) Brand B tablet, (c) Brand C
capsule, and (d) Brand D liquid extract. See the text for conditions. Peaks: 1  bilobalide, 2 
ginkgolide J, 3  ginkgolide C, 4  ginkgolide A, 5  ginkgolide B.
syringe-filtered through a 13-mm, 0.45-
m
nylon-syringe filter into a 4-mL HPLC vial
that was capped tightly and stored at 4 °C.
Sample Solutions Preparation: Four
Ginkgo biloba herbal supplements were ana-
lyzed. Samples of the supplements in cap-
sule form were prepared by emptying the
content of single capsules into clean, dry,
and tared 4-mL vials. Each vial was weighed
accurately to determine the mass of the
sample. Samples of the supplements in
tablet form were prepared by thoroughly
pulverizing single tablets with a mortar and
pestle. The powders were transferred care-
fully into individual clean, dry, and tared 4-
mL vials. Each vial was accurately weighed
to determine the mass of the sample.
All solid samples were extracted as fol-
lows: 3 mL of methanol was added to a vial
containing a solid sample. The vial was
tightly capped and sonicated for 10 min.
Table I: Terpene lactones in standard solution (n = 7)
Bilobalide Ginkgolide J
Area count 55.843 55.933
Concentration 101.7 101.1
(
g/mL)
RSD 4.22% 5.08%
The content of the vial was allowed to settle
for 10 min, and the supernatant was trans-
ferred by pipette into a 5-mL syringe fitted
with a 13-mm diameter, 0.45-
m porosity
nylon syringe filter. The liquid extract was
filtered into a clean, dry, 10-mL volumetric
flask. For each solid sample, the extraction
was repeated two more times, and the com-
bined extracts were brought to volume with
methanol (7). The samples were capped
tightly and stored at 4 °C.
The commercially prepared liquid
Ginkgo biloba extract with 2 g of leaf
extracted into 1 mL of deionized water and
ethanol (1:1) was analyzed directly by
HPLC with no sample preparation.
Quantitation: Determination of the
ginkgolides (A, B, C, and J) and bilobalide
(BB) present in the standardized Ginkgo
biloba supplement was as follows: Average
area counts (Ā) for these constituents were
Ginkgolide C Ginkgolide A Ginkgolide B
55.722 97.761 78.198
102.5 103.1 100.4
6.66% 2.86% 6.73%
www.chromatographyonline.com
obtained from the Brand A (capsule) over
three days (n  18, each injected in tripli-
cate) for the reproducibility study. The ter-
pene lactone content was also evaluated in
brands B (tablet) and C (capsule) (n  6,
each injected in triplicate). The standard-
ized liquid extract (Brand D) was injected
six times to obtain Ā. A solution of stan-
dards was injected seven times to obtain Ā
for the terpene lactones.
1. The average concentration (micrograms
per capsule or tablet) of each terpene lac-
tone (for example, BB) is:
Note: The last conversion factor (10
mL/capsule or tablet) is not used for the liq-
uid extract because it is unaltered.
2. Converting into milligrams per gram:
Reproducibility and Sensitivity: The
precision of the method was assessed by
examining sample-to-sample and day-to-
day reproducibility. Run-to-run repro-
ducibility also was evaluated for the stan-
dards. The limit of detection for the
evaporative light-scattering detector was
measured for this HPLC method by dilut-
ing the standard sample until an S/N greater
than 5 was reached.
Results and Discussion
Evaporative light-scattering detection
(ELSD) was chosen over other methods of
detection for this analysis for several rea-
sons. Independent of functional group
properties, it produces a response that gives
a closer estimate of the true sample mass
when compared with UV detection, which
depends upon the optical characteristics
(extinction coefficients) of the analytes. The
terpene lactones in Ginkgo biloba are poor
chromophores with weak absorption in the
200–220 nm range. Refractive-index detec-
tion was considered and rejected because a
stable baseline is more easily attained with
ELSD, resulting in faster equilibration. In
addition, ELSD is gradient compatible,
whereas the refractive-index detection is
not. Gas chromatography with flame ion-
ization detection is a reliable and sensitive
way to perform this analysis, but the process
requires time-consuming sample prepara-
www.chromatographyonline.com MAY 2004 LCGC NORTH AMERICA VOLUME 22 NUMBER 5 459

dependent upon the gas flow rate used in

the analysis. The optimum gas flow rate will
produce the highest S/N. In this study, the
gas flow rate of 3.1 L/min gave the highest
S/N. Then the mobile phase evaporates in a
heated stainless steel drift tube, leaving a
fine mist of dried sample particles in the sol-
vent vapor. The optimum tube temperature
will produce the most stable baseline with-
out compromising sensitivity (S/N), which

The HPLC method

Figure 3: Separation of terpene lactone
standards. See the text for conditions. Peaks: 1
 bilobalide, 2  ginkgolide J, 3  ginkgolide
C, 4 ginkgolide A, 5  ginkgolide B.
tion and derivatization, which potentially
introduces more errors into the method (8).
The unique detection principle of ELSD
involves nebulization, evaporation, and
detection of the remaining nonvolatile
solute particles. Inside the nebulizer, the
column effluent passes through a needle
and mixes with nitrogen gas to form an
aerosol consisting of a uniform distribution
of droplets. The size of each droplet is
was applied to
Ginkgo biloba
standardized
supplements in
capsule, tablet, and
liquid form.
in this application was 110.0 °C. Detection
occurs inside an optical cell, where non-
volatile sample particles interrupt a laser
light beam. The scattered light is detected
by a silicon photodiode, producing a signal
that is sent to the analog output for data
Figure 4: Limit of detection of terpene lac-
tones. See the text for conditions. Peaks: 1 
bilobalide, 2  ginkgolide J, 3  ginkgolide C,
4  ginkgolide A, 5  ginkgolide B.
collection (9).
The HPLC method was applied to
Ginkgo biloba standardized supplements in
capsule, tablet, and liquid form. The ter-
pene lactone peaks were identified by direct
comparison of the retention times of the
peaks in the standard chromatogram with
the peaks resolved in the sample chro-
matogram. The order of elution is as fol-
lows: bilobalide, ginkgolide J, ginkgolide C,
460 LCGC NORTH AMERICA VOLUME 22 NUMBER 5 MAY 2004
Figure 5: Comparison of terpene lactones in commercial Ginkgo biloba standardized
supplements.
ginkgolide A, and ginkgolide B. Figure 2
shows the chromatograms of each of the
samples. Figure 3 illustrates the separation
of the terpene lactone standards. The
methanol extraction was exhaustive, which
was verified by analyzing the third extrac-
tion in which no measurable amounts of the
terpene lactones were found.
Table I lists the average area counts with
the corresponding concentrations and the
run-to-run relative standard deviations (n 
7) for each of the terpene lactones in the
standard solution. For the reproducibility
study, Table II reports the Brand A sample
area counts along with the standard devia-
tions (SD) and relative standard devia-
tions. The relative standard deviations for
intraday samples (n  6, each injected in
triplicate) ranged from 2.31% (bilobalide,
day 3) to 9.22% (ginkgolide J, day 1). Day-
to-day relative standard deviations ranged
from 1.31% (ginkgolide A) to 11.09%
(ginkgolide J). As depicted in Figure 4, the
limit of detection (S/N  5) was less than
Table II: Terpene lactones in standard solution of brand A
Bilobalide
Day 1 (n = 6) 27.31  1.32
Intraday 4.84%
Day 2 (n = 6) 30.32  1.25
Intraday 4.12%
Day 3 (n = 6) 29.79  0.69
Intraday 2.31%
Overall (n = 3) 29.14  1.61
Interday 5.52%
125 ng on-column for each of the five ana-
lytes. Quantitation of the terpene lactones
was straightforward using equation 1, and
the results reported in Table III. Table IV
summarizes the amount of the total terpene
lactones per serving size for the Ginkgo
biloba herbal supplements.
A direct comparison illustrating the
absolute and relative amounts of terpene
lactones present in each of the four Ginkgo
biloba supplements is shown in Figure 5.
Both the amounts of the Ginkgo biloba
standardized extract and leaf content vary
widely from brand to brand, as evidenced in
Figure 4. The extract portion is standard-
ized to  6% total terpene lactones. How-
ever, the amounts of the terpene lactones are
unregulated in the leaf material. Moreover,
the terpene lactone content in the leaf can
vary considerably depending upon the sea-
son and the geographic location of cultiva-
tion (10). The total terpene lactones, the
ginkgolides A, B, C, and J, along with
bilobalide, in the leaf are not taken into
Ginkgolide J Ginkgolide C Ginkgolide A
3.47  0.32 21.92  1.39 124.88  7.22
9.22% 6.35%
3.02  0.20 21.78  0.61 126.80  5.25
6.55% 2.81%
2.79  0.15 19.67  0.55 123.55  4.00
5.53% 2.77%
3.10  0.34 21.12  1.26 125.08  1.63
11.09% 5.95%
www.chromatographyonline.com
account in the quantitation listed on the
product label, often leading to an underesti-
mation of the amount of the total terpene
lactones in the Ginkgo biloba supplement
when the leaf is present, as Brands A and C
indicate. Brand B, which contains no leaf,
contains approximately 69.1% of the total
terpene lactones it is purported to have
according to the label, while Brand D, the
liquid extract, contains 103.6% compared
to its label claim, which is within experi-
mental error. The low amount of total ter-
pene lactones in Brand B relative to the
label claim could be attributed to label inac-
Without
hydrolyzation,
determination of the
flavonol glycosides is
impossible due to
the different sizes of
sugars that are
otherwise bound to
each of the flavonol
glycosides.
curacies by the manufacturer. Perhaps less
relative surface area is present after manual
pulverization in Brand B, which is in a
tablet form along with fillers and binders.
The whole product of Brand D liquid
extract is standardized, leading to a more
accurate quantitation, as opposed to the
capsulated brands A and C, where the leaf
portion is neglected for quantitation. As a
result, the total terpene lactone content dif-
fers extensively from brand to brand, as
Table IV summarizes.
Ginkgolide B
77.17  4.67
5.78% 6.05%
75.34  2.34
4.14% 3.10%
65.80  1.73
3.24% 2.63%
72.77  6.10
1.31% 8.39%
www.chromatographyonline.com MAY 2004 LCGC NORTH AMERICA VOLUME 22 NUMBER 5 461

This HPLC method is 40% faster than Table III Determination of Terpene Lactones (TLs) in Ginkgo Biloba Supplements
other methods reviewed. Separation of the
five terpene lactones occurs in less than 14 Brand A Bilobalide Ginkgo. J Ginkgo.C Ginkgo. A Ginkgo. B Total TLs
min. Both Polymer Laboratories (Amherst,
g/capsule 541.6 55.9 388.5 1319.1 934.3 3239.4
Massachusetts) (11) and Ganzera and col- mg/g 1.40 0.145 1.06 3.41 2.42 8.43
leagues (7) managed to resolve the lactones %(w/w) 0.14% 0.014% 0.11% 0.34% 0.24% 0.84%
within 25 min. In addition, only three ter-
Brand B Bilobalide Ginkgo. J Ginkgo.C Ginkgo. A Ginkgo. B Total TLs
pene lactones were assessed in the Polymer
Laboratories study (11). The binary mobile
g/tablet 44.4 15.9 149.1 1480.5 798.1 2488.0
phase, comprising methanol, water, and tri- mg/g 0.14 0.050 0.47 4.67 2.52 7.85
fluoroacetic acid, is much simpler in this %(w/w) 0.01% 0.005% 0.05% 0.47% 0.25% 0.79%
method. A ternary gradient is used in the
Brand C Bilobalide Ginkgo. J Ginkgo.C Ginkgo. A Ginkgo. B Total TLs
Polymer application, while Ganzera and his
colleagues (7) used a buffer, an acid, two
g/capsule 790.1 91.7 639.4 1923.2 889.3 4333.7
alcohols, and water in their mobile phase. mg/g 2.88 0.335 2.33 7.02 3.25 15.82
%(w/w) 0.29% 0.034% 0.23% 0.70% 0.32% 1.57%
Sample preparation is simpler when com-
pared to other published methods. Li and Brand D Bilobalide Ginkgo. J Ginkgo.C Ginkgo. A Ginkgo. B Total TLs
Fitzloff (6) attempted to determine both the
terpene lactones and flavonol glycosides in
g/mL 4993.1 511.6 2320.8 1644.5 471.6 9941.6
mg/g 5.24 0.54 2.44 1.73 0.50 10.5
Ginkgo biloba simultaneously. Their sample %(w/w) 0.52% 0.05% 0.24% 0.17% 0.05% 1.03%
preparation involved a liquid–liquid extrac-
tion, subsequent evaporation, and resuspen-
sion. In addition, the flavonol glycosides flavonol glycoside (12). Conclusion
were not acid hydrolyzed into their respec- The limit of detection for the terpene lac- The total terpene lactones present in the
tive aglycones. Without hydrolyzation, tones using ELSD is 125 ng on-column for Ginkgo biloba supplements analyzed ranged
determination of the flavonol glycosides is the terpene lactones. This is much lower from 0.79% (w/w) for Brand B to 1.57%
impossible due to the different sizes of sug- than both Polymer Laboratories’ (11) and (w/w) for Brand C. The concentrations of
ars that are otherwise bound to each of the Ganzera and colleagues’ (7) limits of detec- the individual terpene lactones varied from
flavonol glycosides, resulting in numerous tion, which were 250 and 203 ng on-col- 0.005% (w/w) for ginkgolide J in Brand B
peaks with varying retention times for each umn, respectively. to 0.70% (w/w) for ginkgolide A in
462 LCGC NORTH AMERICA VOLUME 22 NUMBER 5 MAY 2004
Table IV: Comparison of total terpene lactones (TTLs)
Brand Serving Size (SS) ActualTTLs (µg/SS)
A 2 capsules
B 1 tablet
C 2 capsules
D 1 mL
Brand C.
The HPLC method described here allows
analysis of Ginkgo biloba much more
rapidly than has been reported previously
without compromising sensitivity and
reproducibility. As a result, routine quality
control in high-throughput laboratories
within the nutraceutical industry can be
performed much more efficiently.
Acknowledgements
The author would like to thank the follow-
ing employees of Alltech Associates:
Michael Li, Stephen Liu, Dennis McCreary,
Sharon McKinley, Gary Nixon, Cary
Thrall, Mark Waksmonski, Bob Wiedemer,
and Bob Ziegler. He would also like to
thank all of Alltech Associates for their
invaluable contributions to this project.
www.chromatographyonline.com
(9) Alltech Associates, Inc., Alltech ELSD 2000
Evaporative Light Scattering Detector Operating
Label Claim TTLs (µg/SS) Manual (2001).
6478.8 2400.0 (10) T.A. Van Beek, H.A. Scheeren, T. Rantio,
2488.0 3600.0 W.C. Melger, and G.P. Lelyveld. J. Chro-
8667.4 7200.0 matogr. 543, 375–387 (1991).
9941.6 9600.0 (11) Polymer Laboratories, Inc., The Application
Notebook, LCGC, 44–45 (February 2003).
(12) D. McCreary, personal communication (2002).
References
(1) A.Y. Leung and S. Foster, Encyclopedia of Com-
mon Natural Ingredients Used in Food, Drugs,
and Cosmetics, 2nd ed. (Wiley & Sons, Hobo- Tim Herring is with Alltech Associates, Inc.,
ken, New Jersey, 1996). 2701 Carolean Industrial Drive, State College,
(2) C. Mar and S. Bent, Western J. Med. 171, Pennsylvania 16801, e-mail therring@all-
168–171 (1999). techemail.com.
(3) P.L. Le Bars, M.M. Katz, N. Berman, T.M. Itil,
A.M. Freedman, and A.F. Schatzberg, J. Am.
Med. Assoc. 278, 1327–1332 (1997).
(4) P.F. Smith, C.L. MacLennan, and J. Darling-
ton, J. Ethnopharmacol. 50, 131–139 (1996).
(5) M. Blumenthal, Herbalgram 47, 64–65 (1999).
(6) W. Li and J. Fitzloff, J. Pharm. Biomed. Anal.
30, 67–75 (2002).
(7) M. Ganzera, J. Zhao, and I. Khan, Chem.
Pharm. Bull. 49, 1170–1173 (2001).
(8) E. Lolla, A. Paletti, and F. Peterlongo, Fitoter-
apia 69, 513–519 (1998).