PHYTOCHEMICAL ANALYSIS

302 Phytochem. Anal. 16, 302–306 (2005) M. E. GARÍN-AGUILAR ET AL.

Published online 14 July 2005 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002.pca.821

High-performance Liquid Chromatography–Mass

Spectrometric Analysis of Alkaloids Extracted
from Seeds of Erythrina herbacea

M. E. Garín-Aguilar,1 G. Valencia del Toro,1,3 M. Soto-Hernández2* and G. Kite4

1
UNAM, Facultad de Estudios Superiores Iztacala. Av. de los Barrios s/n, Los Reyes Iztacala, Tlalnepantla Edo 54690, Mexico
2
Colegio de Postgraduados, Montecillo, Edo. de México, 56230, Mexico
3
UPIBI-IPN, Av. Acueducto s/n, Barrio La Laguna, Ticoman 07340, Mexico
4
Royal Botanic Gardens Kew, Richmond, Surrey, UK

A sensitive, reverse-phase HPLC-MS method for the analysis of the alkaloids of Erythrina has been developed.
The method is based on the use small amounts of crude extracts (20 mg) and is sufficiently sensitive to detect the
presence of the typical alkaloids, such as erysodine, erysovine, erythraline, erysopine and the hexoside of
erysopine, that are representative of the title species. Copyright © 2005 John Wiley & Sons, Ltd.
Keywords: HPLC-MS; Erythrina alkaloids; Erythrina herbacea.

consuming and the accuracy of identification at low con-

INTRODUCTION
Since the 1940s the genus Erythrina has received
much research attention mainly owing to the widespread
presence of alkaloids that show curariform activity. The
systematic study of 50 Erythrina species showed that
all of them contain alkaloids with paralysing activity,
and the more pharmacologically potent components,
i.e. β-erythrodine and dihydro-β-erythroidine, have been
studied in some detail (Garín Aguilar et al., 1999; García-
Mateos et al., 2000). The interest in these alkaloids is
related to their structures as tertiary amines that show
high activity and few side effects (Craig, 1955).
The flowers and seeds of Erythrina herbacea
(Pemoche) are used by the Tenek ethnia in San Luis
Potosi, México, as a tranquilliser agent (Garín-Aguilar
et al., 2000) and the effect is attributed to the alkaloids
present. Although Folkers and Koniuszy (1940) were
able to isolate erysodine and erysopine from the seeds
of this species, little could be established about their
structures at that time. As these constituents are present
only in minor quantities in the seeds, accurate, fast and
reproducible analytical methods with high sensitivity are
required for their analysis. Although TLC is still em-
ployed as a major tool in alkaloid analysis, alternative
HPLC methods have recently been developed; however,
HPLC with detection based solely upon UV absorption
is not sufficiently selective. GC methods are also used
for the analysis of alkaloids, but the low volatility of
these compounds and their instability at high tempera-
tures has limited the application of this technique. The
Erythrina alkaloids have been analysed by GC and
by GC-MS (García-Mateos et al., 1999) but, although
these techniques provide interesting data, they are time-
* Correspondence to: M. Soto-Hernández, Colegio de Postgraduados,
Montecillo, Edo. de México, 56230, Mexico.
Email: msoto@colpos.mx
centrations of alkaloids is poor.
The development of a rapid and sensitive method for
the analysis of Erythrina alkaloids would enable further
elucidation of the alkaloids present in Erythrina seeds,
especially in those species previously reported to contain
only trace amounts. For these reasons the coupling of
HPLC with mass spectrometry (MS) is of considerable
interest for the analysis of alkaloids. The present paper
describes a fast, accurate sensitive and selective proce-
dure for the HPLC-MS analysis of the alkaloids charac-
teristic of E. herbacea.
EXPERIMENTAL
Plant material. Seeds of Erythrina herbacea L.
(Fabaceae) were collected in San Pedro de las Anonas,
San Luis Potosí, México (500 miles north of Mexico City)
in September 2002. The identification of the plant was
carried out by Dr. Mario Souza Sánchez of the MEXU
herbarium (National University of Mexico) at which in-
stitute a voucher specimen has been deposited (classifi-
cation number 599010).
Isolation and extraction of alkaloids. Alkaloid fractions
were obtained from the seeds of E. herbacea according
to the method described by Games et al. (1974). Three
main crude fractions were obtained: free alkaloids in
hexane, free alkaloids in methanol, and liberated alka-
loids. Seeds were air-dried, milled separately and ex-
tracted with hexane by Soxhlet extraction for 48 h.
Alkaloids in the hexane fraction were washed with 1 m
sulphuric acid (3 × 50 mL), and the aqueous acidic phase
adjusted to pH 8 with solid sodium bicarbonate. The al-
kaline solution was then extracted with dichloromethane
(3 × 100 mL) to give the free alkaloids in hexane frac-
tion (0.22%). The defatted flour of each fraction was next

Copyright © 2005 John Wiley & Sons, Ltd. Received

Phytochem. 20 302–306
Anal. 16: December 2003
(2005)
Copyright © 2005 John Wiley & Sons, Ltd. Revised 10 September 2004
Accepted 22 September 2004
 HPLC ANALYSIS OF THE ALKALOIDS OF ERYTHRINA HERBACEA
extracted in a Soxhlet for 48 h with methanol, the extract
evaporated under vacuum, and the residue taken up
in 2% sulphuric acid. The acidic solution was extracted
with dichloromethane to remove traces of fat, the
aqueous phase was basified with sodium bicarbonate
to pH 8 and then extracted with dichloromethane
(3 × 100 mL) to give the free alkaloids in methanol
(1.13%). The solvent was evaporated and the residue
was dried in a desiccator and weighed. The remain-
ing aqueous phase was re-acidified to pH 2 with hydro-
chloric acid and heated in steam bath for 3 h to
hydrolyse the esterified alkaloids. The process of
neutralisation, basification to pH 8 and extraction
with dichloromethane was repeated to yield the liberated
alkaloid fraction.
These three main fractions were screened firstly by
TLC on silica gel layers (Merck, Darmstadt, Germany;
15–40 µm) with dichloromethane:ethanol (9:1) as eluent.
The main alkaloids of the free alkaloid samples and those
of the liberated alkaloid fraction were separated by col-
umn chromatography over silica gel (0.063–0.200 mm
mesh) eluted with mixtures of dichloromethane and
methanol of increasing polarity.
GC analysis. Prior to analysis the crude alkaloid mixture
(1–2 mg) was derivatised to produce the trimethylsilyl
(TMS) derivatives by treatment for 30 min with N,O-
bis(trimethylsilyl)acetamide (25 µL) in acetonitrile
(25 µL) in a Teflon-lined screw-cap vial to prevent
evaporation. The mixed alkaloid derivatives were then
analysed by GC on a Varian (Walnut Creek, CA, USA)
model 2700 chromatograph equipped with a flame ioni-
sation detector, a glass column (4 m × 2 mm i.d.) packed
with 3% OV-17 on Gas Chrom Q-100-200 mesh (Pierce
Chemical Co., Rockford, IL, USA). The injector and
detector temperatures were maintained at 250 and
280 °C, respectively, while the oven temperature was
programmed from 225 to 285 °C at a rate of 2 °C/min;
the flow-rate of the carrier gas (nitrogen) was maintained
at 30 mL/min.
HPLC-MS/MS analysis. The analytical system con-
sisted of a Waters (Milfard, Massachusetts, USA)
chromatograph (model 600 E pumps and controller)
interfaced to a Thermo Finnigan (Winsford, Cheshire,
UK) LCQ classic quadrupole ion trap mass spectrometer
via an atmospheric chemical ionisation (APCI) source.
The alkaloids were separated on a Supelco (Bellefonte,
PA, USA) Discovery C18 column (250 × 4.6 mm i.d.;
5 µm) eluted at 1 mL/min with a linear gradient mobile
phase programmed from three solvent reservoirs, A
(0.1% ammonium acetate, pH 7.4), B (methanol) and
C (acetonitrile), as follows: 0 min, 75:20:5 (A:B:C);
10 min, 50:45:5 (A:B:C); and 15 min, 50:45:5 (A:B:C).
The vaporiser of the APCI source was set to 450°C with
sheath and auxiliary nitrogen gas pressures of 80 and 20
psi, respectively, and the needle current was 5 µA; the
heated capillary temperature was 150°C. The MS was
programmed to survey ions with m/z in the range 150–
500 and to subject the most intense ion in the survey scan
to MS/MS analysis, followed by MS/MS/MS analysis of
the most intense product ion in the MS/MS scan. The
collision energy was 40% using an ion isolation width of
3 amu. The alkaloids were identified by comparison of
the various CID spectra and retention times with those
of isolated standards or were tentatively identified from
Copyright © 2005 John Wiley & Sons, Ltd.
303
their molecular weights by reference to published data
for previously reported alkaloids. Further confirmation
of the identity of a compound was through 1H-NMR
recorded in CD3OD on a Bruker (Coventry, UK) model
300 spectrometer operating at 360 MHz.
RESULTS AND DISCUSSION
The conventional techniques of alkaloid isolation, involv-
ing large-scale extraction, chromatography and crystalli-
sation, are time-consuming. In order to avoid this
process, HPLC-MS techniques have been developed that
allow high speed, accuracy and the ability to work with
mg quantities of crude extracts. In order to prepare such
extracts from the plant material, the procedure devel-
oped by Games et al. (1974) was mainly followed. In this
method, the ground seeds are extracted first with hexane
to remove fats and then with methanol to extract the
alkaloids. It was observed that the hexane fraction
contained small amounts of alkaloids and it is therefore
important not to discard this fraction without first deter-
mining the alkaloids present. The methanolic extract
yields the free alkaloid fraction, i.e. those alkaloids that
are not esterified with sugars or organic acids. After
acidic hydrolysis of the esters, the remaining liberated
alkaloid fraction is obtained.
For extracts obtained from the seeds of E. herbacea,
screening of the three main fractions by TLC provided
clear evidence of the presence of alkaloids, mainly in the
free alkaloid and in the liberated alkaloid fractions,
showing at least three distinct alkaloid components.
More specific analyses involving GC, HPLC-MS and 1H-
NMR were conducted on the basis of this information in
order to determine the structure of the alkaloids present
in the samples. In the GC analysis, standards of erysovine
(1), erysodine (2) and erysopine (6) were run followed
by samples of the alkaloid extracts; the sensitivity range
of the GC allowed the presence of 1 and 2 to be detected
mainly in the free and liberated alkaloid fractions. In
the free alkaloid fraction in hexane, compounds 1 and 2
were detected in smaller amounts, and only a trace of
erysotrine (3) was observed.
The analysis of the alkaloids in crude samples by
HPLC-MS/MS showed interesting tendencies in their
profile and a better sensitivity compared with GC. In
the free alkaloid fraction in hexane, four compounds
(Fig. 1) were detected being, in order of increasing
retention time, erysovine (1), erysodine (2), erysotrine
Phytochem. Anal. 16: 302–306 (2005)
304 M. E. GARÍN-AGUILAR ET AL.

Figure 1. HPLC-MS TIC chromatogram of the free alkaloids in hexane fraction from the seeds of Erythrina herbacea showing
the main components: peak 1, erysovine (Rt = 14.73 min; MS base peak = 300.19); peak 2, erysodine (Rt = 15.01 min; MS base
peak = 300.17); peak 3, erysotrine (Rt = 17.81 min; MS base peak = 314.13); and peak 4, erythraline (Rt = 20.55 min; MS base peak
= 298.23). (For extraction and analytical protocols see Experimental section.)

(3) and erythaline (4); the latter was the main alkaloid in
this fraction. The free alkaloid fraction in methanol
showed (Fig. 2) the presence of 1–4 and glucoerysopine
(5), with 1 and 2 the most abundant alkaloids in
this fraction. In contrast, the liberated alkaloid frac-
tion (Fig. 3), obtained after acidic hydrolysis of the
conjugated alkaloids, showed the presence of three
alkaloids, namely, 1, 2 and erysopine (6); the latter was
the main alkaloid in this fraction.
Since 1, 2 and 6 were the most abundant alkaloids
detected mainly in the free alkaloid and liberated
alkaloid fractions, they were analysed by 1H-NMR
following purification by column chromatography over
silica gel. Their 1H-NMR spectra were in full agreement
with those reported in the literature (Soto-Hernández,
1989).
The HPLC-MS method developed showed high
sensitivity and allowed the detection of the presence
of erysopine, erysodine, erysovine, erysotrine and
erythraline in seeds of E. herbacea. Glucoerysopine has
not been described before in this species, and this finding
was not observed with GC. Clearly, there is potential
for optimising the resolution of particular mixtures of
Erythrina alkaloids by modifying the HPLC elution
gradient, but the technique has proven to be a powerful
analytical tool for analysing extracts obtained from very
small amounts of plant tissue.
Acknowledgements
Work supported by PACPA, PPPit 216803 and 247504 D64PA-UNAM.

Copyright © 2005 John Wiley & Sons, Ltd. Phytochem. Anal. 16: 302–306 (2005)
 HPLC ANALYSIS OF THE ALKALOIDS OF ERYTHRINA HERBACEA 305

Figure 2. HPLC-MS TIC chromatogram of the free alkaloids in methanol fraction from the seeds of Erythrina herbacea show-
ing the main components: peak 1, erysovine (Rt = 15.20 min; MS base peak = 300.15); peak 2, erysodine (Rt = 15.68 min; MS
base peak = 300.15); peak 3, erysotrine (Rt = 18.13 min; MS base peak = 314.12); peak 4, erythraline (Rt = 20.71 min; MS base
peak = 298.14); and peak 5, glucoerysopine (Rt = 12.22 min; MS base peak = 462.20). (For extraction and analytical protocols
see Experimental section.)

Copyright © 2005 John Wiley & Sons, Ltd. Phytochem. Anal. 16: 302–306 (2005)
306 M. E. GARÍN-AGUILAR ET AL.

Figure 3. HPLC-MS TIC chromatogram of the liberated alkaloids from the seeds of Erythrina herbacea showing the main com-
ponents: peak 1, erysovine (Rt = 11.00 min; MS base peak = 300.14); peak 2, erysodine (Rt = 11.40 min; MS base peak = 300.12);
and peak 6, erysopine (Rt = 7.07 min; MS base peak = 286.13). (For extraction and analytical protocols see Experimental section.)

REFERENCES

Craig L. 1955. Erythrina alkaloids. In The Alkaloids, Vol. V,
Manske RH, Holmes HL (eds). Academic Press: London;
281–287.
Folkers K, Koniuszy F. 1940. Isolation and characterization of
erysodine, erysopine, erysocine and erysovine. J Am
Chem Soc 62: 1677–1682.
Games DE, Jackson AH, Kahn NA, Millington DJ. 1974. Alka-
loids of African, Asian, Polynesian and Australian species
of Erythrina. Lloydia 37: 581–588.
García-Mateos R, Soto-Hernández M, Martínez-Vazquez M,
Villegas Monter A. 1999 Isolation of alkaloids of Ery-
thrina from tissue culture. Phytochem Anal 10: 12–
16.
García-Mateos R, Garín-Aguilar ME, Soto-Hernández M,
Martínez-Vázquez M. 2000. Effect of β-dihydroerythroidine
from Erythrina americana on rats aggressive behaviour.
Pharm Pharmacol Lett 10: 34–37.
Garín-Aguilar ME, Valencia del Toro G, Soto-Hernández M,
García-Avila J, Servín-Morales E. 1999. Determinación de
la dosis letal media (LD50) y caracterización química
preliminar de fracciones de alcaloides de semillas de
Erythrina herbacea. In Productos Naturales, Vol. IV.
Perspectivas Biotecnológicas, Cruz-Sosa F, Lechuga JM
(eds). UAM Iztapalapa: México; 2–5.
Garín-Aguilar ME, Ramírez Luna LJE, Soto-Hernández M,
Valencia del Toro G, Martínez Vázquez M. 2000. Effect of
crude extracts of Erythrina americana Mill. on aggressive
behaviour in rats. J Ethnopharmac 69: 189–196.
Soto-Hernández M. 1989. Chemical and structural studies of
Erythrina alkaloids PhD Thesis, University of Cardiff.

Copyright © 2005 John Wiley & Sons, Ltd. Phytochem. Anal. 16: 302–306 (2005)