Phytochemical Analysis

332 A. BORLOZ ET AL.

Phytochem. Anal. 17: 332–336 (2006)
Phytochemical
Published online 18 July 2006 in Wiley InterScience (www.interscience.wiley.com) DOI: 10.1002/pca.922
Analysis

The Determination of Huperzine A in European

Lycopodiaceae Species by HPLC-UV-MS
A. BORLOZ, A. MARSTON and K. HOSTETTMANN*
Laboratoire de Pharmacognosie et Phytochimie, Section des Sciences Pharmaceutiques, Université de Genève, Université de Lausanne, CH-1211
Genève 4, Switzerland

Received 20 February 2006; Revised 11 May 2006; Accepted 16 May 2006

Abstract: A rapid qualitative HPLC-UV-MS method was developed for the detection of huperzine A in Lycopodiaceae species. HPLC
coupled with mass spectrometry experiments allowed the identification of the alkaloid and enabled targeted analysis of complex
matrices such as herbal extracts. Huperzine A was detected by single ion monitoring of the pseudomolecular ion [M + H]+ at m/z
243.2. The alkaloid was also detected on TLC in a bioautographic enzyme assay with acetylcholinesterase to show the activity of
the compound. Four Lycopodiaceae species collected in Switzerland were tested by these methods and Huperzia selago (L.) Bernh.
ex Schrank et Martius was the only species found to contain huperzine A. Copyright © 2006 John Wiley & Sons, Ltd.

Key words: HPLC-UV-MS; TLC; Huperzine A; Lycopodiaceae; Huperzia selago.

INTRODUCTION advantage against other commercially available inhibitors,

In the early 1980s, huperzine A (1) was isolated
from Huperzia serrata (Thunb. ex Murray) Trevis
(Lycopodiaceae), a traditional Chinese medicine used
for the treatment of fever, inflammation states and
for memory improvement (Patocka, 1998; Shu, 1998).
Huperzine A also shows a strong reversible and
highly selective inhibitory activity against the enzyme
acetylcholinesterase (AChE) (Tang and Han, 1999).
This enzyme inactivates acetylcholine, the most
important cell-to-cell communication molecule found
in the brain. The neurotransmitter acetylcholine is
known to be essential for the memory process
(Drachman and Leavitt, 1974) and the abnormality of
cholinergic neurotransmission has been shown to
contribute to the cognitive impairment and the
behavioural disturbances of Alzheimer’s disease (AD)
patients (Cummings, 2000). Along with the inhibition
of AChE, huperzine A seems also to show other benefi-
cial effects such as a decrease in neuronal cell death
and a long duration of action (Patocka, 1998; Xiao
et al., 2002). Several clinical trials have been reported
in China and the efficacy of huperzine A has been
demonstrated, with cholinergic side effects (dizziness,
nausea and diarrhoea), but without liver or kidney
toxicity (Shu, 1998; Tang and Han, 1999). Given this
* Correspondence to: K. Hostettmann, Laboratoire de Pharmacognosie
et Phytochimie, Section des Sciences Pharmaceutiques, Université de
Genève, Université de Lausanne, CH-1211 Genève 4, Switzerland.
E-mail: kurt.hostettmann@pharm.unige.ch
Contract/grant sponsor: Swiss National Science Foundation; contract/
grant number: 2153-0066874.01/1.
the interest shown in this compound is very high.
Huperzia serrata is widely used by the local com-
munities for medicinal purposes and as a source of
huperzine A. Consequently, natural stocks are declin-
ing dramatically in China and a change in collection
practice and general attitude towards this plant is
required for its conservation (Ma et al., 2006). More-
over, total synthesis is only able to provide the racemic
mixture (±)-huperzine A, three times less potent
than the natural compound, (−)-huperzine A (Tang
et al., 1994). Therefore, it would be useful to find
other sources for this compound; the study of some
Lycopodiaceae species could provide new sources of
huperzine A.
For this purpose, an HPLC-UV-MS method was
developed and some European Lycopodiaceae species
[Diphasiastrum alpinum (L.) Holub, Lycopodium anno-
tinum L., Lycopodium clavatum L. and Huperzia selago
(L.) Bernh. ex Schrank et Martius (Mingard and Moret,
2003)] were tested in order to find huperzine A. The
results were compared with those obtained from a
bioautographic assay by TLC (Marston et al., 2002).
EXPERIMENTAL
Chemicals and solvents. Acetylcholinesterase from
electric eel was obtained from Sigma (St. Louis, MO,
USA). Tris, bovine serum albumin, α-naphthyl acetate
and ammonium acetate were purchased from Merck
(Darmstadt, Germany); Fast Blue B salt was from
Fluka (Buchs, Switzerland). The reference compound
(±)-huperzine A was also obtained from Sigma.

Copyright © 2006 John Wiley & Sons, Ltd. Phytochem. Anal. 17: 332–336 (2006)
DOI: 10.1002.pca

Ammonium hydroxide was purchased from Carlo Erba
Reagents (Rodano, Italy); acetic acid was also from
Fluka (Buchs, Switzerland); ethyl acetate, chloroform,
dichloromethane and methanol were purchased from
Schweizerhall (Avenches, Switzerland); water (HPLC
gradient grade) was from Fluka and methanol (HPLC-
MS) was from Riedel-de Haën (Seelze, Germany).
Samples of plant materials. Whole plants of Diphasia-
strum alpinum (L.) Holub. (10 g), and Huperzia selago
(L.) Bernh. ex Schrank et Martius (10 g) were collected
in La Breya (2004), Lycopodium annotinum L. (10 g) in
Champex d’en Bas (2004), and L. clavatum L. (10 g) in
La Bovine (2005), all in Wallis, Switzerland. Plant
material was dried and powdered, moistened with
concentrated ammonium hydroxide and extracted
three times with chloroform (300 mL) over 24 h, giving
0.3, 0.4, 0.7 and 0.6 g of chloroform extract for each
plant, respectively.
HPLC parameters. The HPLC system consisted of a
Hewlett Packard (HP; Palo Alto, CA, USA) 1100 unit
equipped with a binary pump, a photodiode array
high-speed spectrophotometric detector and an auto-
sampler. The various components of this system were
controlled by Agilent ChemStation 8.01 software.
Separation was performed on a Waters (Milford,
MA, USA) Symmetry® C18 column (250 × 4 mm i.d.;
Figure 1 HPLC-UV analysis of a chloroform extract of H. selago and of the standard compound huperzine A. Detection: UV at
308 nm. (For chromatographic protocol see the Experimental section.)
Copyright © 2006 John Wiley & Sons, Ltd.
DETERMINATION OF HUPERZINE A 333
5 µm). The mobile phase consisted of a gradient of
methanol:10 mM ammonium acetate (pH 3.5) from
20:80 (vv) to 50:50 in 20 min, then from 50:50 to 100:0
in 10 min and finally with 100% methanol for 10 min.
The flow rate was set to 1.0 mL/min and detection to
308 nm.
HPLC-APCI/MS conditions. Analyses were performed in
the positive mode on a Finnigan MAT (San Jose, CA,
USA) model LCQ ion-trap mass spectrometer equipped
with a Finnigan atmospheric pressure chemical ioniza-
tion (APCI) source. The APCI parameters were: capillary
temperature, 200°C; vaporiser temperature, 400°C;
corona needle current, 5.00 µA; and sheath gas pres-
sure (nitrogen), 80 arbitrary units. The scan rate
was set at 3 mscan and the detection was performed
between 100 and 500 m/z. The [M + H]+ ion of
huperzine A was detected by single ion monitoring
(SIM) at m/z 243.2.
HPLC-ESI/MS conditions. Analyses were performed with
a Finnigan MAT (San Jose, CA, USA) model LCQ
ion-trap mass spectrometer equipped with a Finnigan
electrospray ionisation (ESI) source. Prior to the
source, the flow was split: 0.1 mL/min went to the MS
and 0.9 mL/min to the waste. The transfer capillary
temperature was set to 250°C and the capillary voltage
to 20 V; the pressure of the sheath gas (nitrogen) was
Phytochem. Anal. 17: 332–336 (2006)
DOI: 10.1002.pca
334 A. BORLOZ ET AL.
Figure 2 HPLC-ESI/MS analysis of a chloroform extract of H. selago and of the standard compound huperzine A. Detection:
single ion monitoring of the specific mass m/z 243.2 ± 0.5 Da. (For chromatographic protocol see Experimental section.)
80 arbitrary units. The ESI/MS system operated in the
positive mode at 4.8 kV. The scan rate was 3 mscan
and the detection spectra were acquired from 100 to
500 m/z. Huperzine A was selectively detected by SIM
of its [M + H]+ ion (m/z 243.2).
TLC bioautographic assay. The dried extracts obtained
as above were dissolved in chloroform to prepare a
1 mg/mL solution; an aliquot (10 µL) was spotted on
pre-coated silica gel 60 F254 aluminium sheets (Merck,
Darmstadt, Germany). After migration of the sample in
ethyl acetate:methanol:water (100:13.5:10), the TLC
plate was dried. A solution of 1000 units of AChE
dissolved in 150 mL of 0.05 M Tris–hydrochloric acid
buffer (pH 7.8) with 1% bovine serum albumin was
sprayed and the plates were incubated in a humid
atmosphere at 37°C for 20 min after drying. A mixture
of 1 part α-naphthyl acetate (2.5 mg/mL in ethanol) and
four parts Fast Blue B salt (2.5 mg/mL in water) was
then applied. A purple colouration appeared after 1–2 min
and huperzine A, as well as other inhibitory com-
pounds, appeared as white spots (Marston et al., 2002).
Copyright © 2006 John Wiley & Sons, Ltd.
RESULTS AND DISCUSSION
Method development
In order to analyse various plant extracts, a fast and
robust HPLC-UV-MS method was developed involving
detection of huperzine A (1) by two different ionisation
techniques. The chromatographic separation of alkaloid
extracts was restricted by the required compatibility
with MS ionisation. The basic properties of alkaloids
lead to protonated molecules under acidic or neutral
conditions, increasing their polarity and decreasing
their retention on lipophilic stationary phases. More-
over, charged molecules are responsible for hydrogen
bonding and ionic interactions with silanol groups,
leading to additional retention and peak tailing. An
answer to this problem is either to work with a basic
solvent system or to use acid buffers with ion pair
reagents that have high affinities for protonated
alkaloids.
A gradient elution method on an octadecylsilyl column
was developed in order to optimise the separation
Phytochem. Anal. 17: 332–336 (2006)
DOI: 10.1002.pca
 DETERMINATION OF HUPERZINE A 335

between huperzine A and the other compounds in

the samples. Three mobile phases were tested by
changing the pH values (pH 3.5, 6 and 7.8); for each
one, the chromatogram showed a symmetrical peak
shape for huperzine A and a good separation in the
zone of interest. The UV spectrum of huperzine A
shows two maxima, one at 232 nm and the other at
308 nm (Fig. 1), the latter being used for detection in
order to reduce possible interactions with other plant
constituents. Typical chromatograms from huperzine A
standard and a tested sample (H. selago) are shown in
Fig. 1. The limit of detection (LOD) for huperzine A was
50 ng for a signal-to-noise ratio of 3:1. Initial analyses
with the methanol extract of L. clavatum showed the
absence of huperzine A. Therefore, in order to deter-
mine if the other plant components interfered with the
detection, the LOD was also tested for this extract co-
injected with different concentrations of huperzine A
and the same value as above was obtained.
The HPLC-MS ionisation of 1 was compared using
two different interfaces, APCI and ESI. First the pH of Figure 3 TLC bioautograph showing the inhibition of
the mobile phase was optimised to obtain the best acetylcholinesterase activity. Lane 1, chloroform extract (10 µg
ionisation. For this, a 0.01 mg/mL huperzine A solu- applied) of Diphasiastrum alpinum; lane 2, chloroform extract
(10 µg applied) of Lycopodium annotinum; lane 3, chloroform
tion was injected with the three mobile phases (pH 3.5,
extract (10 µg applied) of Lycopodium clavatum; lane 4, chloro-
6 and 7.8). The optimum was obtained at pH 3.5; at
form extract (10 µg applied) of Huperzia selago and lane 5
this pH, huperzine A was fully protonated (pKa, 8.66). huperzine A (0.01 µg applied). The assay was carried out
The different parameters for APCI/MS were established using a silica gel G60 F254 plate which had been eluted
with a flow injection analysis of a solution of huperzine with ethyl acetate:methanol:water (100:13.5:10). For assay
A at 10 µg/mL. The standard solution injection was protocol, see the Experimental section.
10 µL/min, diluted before the APCI interface by addi-
tion of methanol:10 mM ammonium acetate (pH 3.5;
(25:75) at a flow rate of 1.0 mL/min from the HPLC The different extracts tested by HPLC-UV-MS were
system by means of a T junction. The [M + H]+ ion was also tested on TLC plates for their activity against
detected by single ion monitoring (SIM) of the specific AChE, together with the reference compound huper-
mass, m/z 243.2, for a better sensitivity. The LOD for zine A. The sensitivity in this test for huperzine A
APCI/MS was 5 ng (signal:noise 3:1). is 0.2 ng. The results obtained confirm those of the
ESI/MS conditions were based on those developed HPLC-UV-MS analysis. Activity was only found in H.
by Yang et al. (2003). The LOD for huperzine A with selago. The reference compound huperzine A showed
SIM detection of the ion [M + H]+ was 5 ng in ESI/MS a spot with Rf of 0.25; a spot at the same Rf was
(signal:noise, 3:1), with the same value for the extract also found in H. selago (Fig. 3), which corresponds
co-injected with huperzine A. The SIM chromatograms with the data obtained by HPLC-UV-MS. Moreover, the
at m/z 243.2 obtained for huperzine A and for one of extract of H. selago showed several other white spots,
the tested samples are shown in Fig. 2. The sensitivity which indicate the presence of various other AChE
obtained for the TIC was the same as that for UV inhibitors.
detection. The LOD values for both ionisation sources These data suggest that even if the TLC auto-
were comparable, but the peak of huperzine A was biographic assay gives a more sensitive detection, the
better resolved with ESI. confirmation of huperzine A by HPLC-UV-MS is required
for the formal identification of the compound. While
H. selago contains huperzine A, the other Lycopodiaceae
Screening of species of Lycopodiaceae tested were devoid of this alkaloid. Other species of
Lycopodiaceae are presently being investigated.
The chloroform extracts of D. alpinum, L. annotinum,
L. clavatum and H. selago were tested under the condi-
tions described above. The SIM showed that only Acknowledgements
H. selago contains huperzine A (Fig. 2). Ma and Financial support for this work was provided by the
Gang (2004) also reported the presence of huperzine Swiss National Science Foundation (grant no. 2153-
A in this plant. 066874.01/1 to K. Hostettmann).

Copyright © 2006 John Wiley & Sons, Ltd. Phytochem. Anal. 17: 332–336 (2006)
DOI: 10.1002.pca
336 A. BORLOZ ET AL.
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