Inter. J. of Phytotherapy / Vol 4 / Issue 2 / 2014 / 73-83.

e - ISSN - 2249-7722
Print ISSN - 2249-7730

International Journal of Phytotherapy

www.phytotherapyjournal.com

PROPOSAL OF A HPLC-PAD METHOD FOR THE

DETERMINATION OF GRANDIFLORENIC ACID AND KAURENIC
ACID FROM COESPELETIA TIMOTENSIS
Alida Perez1, José F Ovalles*2, Ana Medina3, Rosa Aparicio1, Alfredo Usubillaga1
1
Instituto de Investigaciones, 2Departamento de Análisis y Control, 3Departamento de Ciencias de los Alimentos, Facultad
de Farmacia y Bioanálisis, Universidad de Los Andes, Mérida, Venezuela.

ABSTRACT
A simple, rapid, and selective high-performance liquid chromatography (HPLC) method with photo diode
array detection (PAD) was developed for the determination of grandiflorenic acid (G-ac), kaurenic acid (k-ac) and
iso-kaurenic acid (iK-ac) in Coespeletia timotensis. Effective chromatographic separation was achieved using a
Waters-XBridge C18 (3 mm x 50 mm; 3.5 μm particle size) column, maintained at 50 ºC in an oven, with a mobile
phase composed of 0.1% phosphoric acid solution, acetonitrile, and methanol in the ratio of 30:49:21 (v/v). The
mobile phase was pumped isocratically at a flow rate of 0.6 mL min -1, and quantification of each ent-kaurenoic acid
was based on measuring its peak area at 220 nm. The retention time for G-ac was about 3.0 min with acceptable
resolution from K-ac (4.0 min) and iK-ac (4.4 min) in a total run of just 10 min. The reliability and analytical
performance of the proposed HPLC procedure were statistically validated. Peak identity was confirmed using pure
standards of ent-kaurenoic acids. Calibration curves were linear in the range of 10 – 50 ng μL-1 with an acceptable
correlation coefficient. The validated HPLC method was successfully applied to the determination of G-ac in resin´s
acidic fraction of aerial parts of C. timotensis.

Key words: Reversed phase liquid chromatography, HPLC-PAD, Grandiflorenic acid, Kaurenic acid, kaurenoic
acid, Andean giant rosette, Frailejón, Coespeletia timotensis.

INTRODUCTION
The subtribe Espeletiinae (Asteraceae) are
resinous plants that grow from an altitude of 2,500 m,
some even higher than 4,000 m above sea level, on the
Andes of Colombia, Ecuador and Venezuela. Celestino
Mutis, a spanish priest and botanist who lived in Popayán
(Colombia) described these plants and created the genus
Espeletia to pay homage to José Ezequiel de Espeleta,
viceroy of New Granada (1789-1796) [1]. Humboldt, who
met Mutis when he visited Northern South America,
collected near Bogota in 1801 three species of Espeletia
and published their descriptions in Plantae Aequinociales
[2]. In 1976 José Cuatrecasas, eminent spanish botanist,
grouped all the frailejones or Andean giant rosette
(common names) species in the subtribe Espeletiinae, and
divided it in eight genera [3, 4]. So far, more than 180
species have been described, 74 from Venezuela.
About 20 Espeletiinae species have been thus far
inspected phytochemically and it has been found that their
resin composition is rather homogeneous containing the
same type of diterpenes but in different proportions.
Moreover, all of them have been found to contain
mixtures of ent-kaur-9(11)16-diene-19-oic acid, known as
grandiflorenic acid (G-ac), and ent-kaur-16-en-19-oic
acid, known either as kaurenic acid or kaurenoic acid (K-

Corresponding Author:- José Fernando Ovalles Email: ovallesd@ula.ve

~ 73 ~

ac) as main constituents [5].
Kaurane diterpenes are compounds of great
interest because some of them can be used to obtain
therapeutic agents. The therapeutic significance of these
species is attributed to K-ac [6, 7], whereas G-ac has
uterotonic properties that could be considered either an
undesired property or alternatively a property of
pharmaceutical interest [8]. Nevertheless, lastly G-ac has
found new therapeutic applications [9-11].
It is possible to separate these two compounds by
fractional crystallization but to obtain them 100% pure it
is necessary to perform AgNO3 chromatography on the
methyl esters. It is interesting to point out that the subtribe
Espeletiinae also contain other valuable ent-kauranes like
ent-15α-hydroxy-kaur-16-en-19-oic acid (15-OH-K-ac),
as it has been determined by analysis of the methyl esters
by GC-MS [12].
Since kauranes are a valuable natural resource it
was considered convenient to develop a HPLC method to
perform a rapid screening of the extracts of species
belonging to the subtribe Espeletiinae, without having to
resort to the time consuming task of methylation. On the
other hand, it is interesting to point out that there are
numerous species pertaining to the Asteraceae as well as
to other families that contain kauranes that could make a
profitable use of such method.
A few reversed phase (RP) HPLC methods have
been developed for the quantitative determination of
kaurane diterpenes in plants. Consequently, not many RP-
HPLC methods have been reported for the quantitative
determination of both G-ac and K-ac in extracts of herbs
[13, 14]. In spite that G-ac and K-ac have been
chromatographically separated, one from each other, the
reported procedures suffer from low resolution in regards
to other eluted compounds, peak tailing, and long
retention times [15-17]. Therefore, some of these
shortcomings need improvement. Peak purity in RP-
HPLC is particularly important in analysis of herbs due to
the expected complex matrix composition. To overcome
this difficulty pH-dependent liquid-liquid extraction has
been proposed in previous works [18]. We also used to
separate acid and neutral fractions until GC-MS sample
analyses in order to purify the target analytes from
pigments, kaurane glycosides and other possible
interfering matrix compounds [19].
This paper describes a rapid analytical method
based on the advantages of combining RP-HPLC and
photodiode array (PDA) detection to analyze acid
fractions of resinous samples of the Espeletiinae species.
Coespeletia timotensis was selected in this preliminary
work in order to develop the proposed RP-HPLC-PAD
method. Most of the qualitative data were undertaken by
GC-MS previous to the HPLC analysis. The proposed
method was optimized and validated in order to ensure
data validity. Four kaurane diterpenes were identified in
C. timotensis and two of them were quantified under the
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Inter. J. of Phytotherapy / Vol 4 / Issue 2 / 2014 / 73-83.
developed chromatographic conditions. Figure 1 shows
the chemical structure of three of the mentioned ent-
kaurenoic acids.
MATERIAL AND METHODS
Instrumentation
Liquid chromatography. The HPLC system
consisted of a modular design of Finnigan Surveyor Plus
conformed by autosampler, quaternary pump with
vacuum degasser, and photo diode array (PDA) detector
connected to a computer loaded with ChromQuest
Software. The Finnigan Surveyor PDA Plus detector has
up to five times the sensitivity of conventional diode array
detectors (DAD) by using a 5 cm optical path length at a
cell volume of only 10 µL. Thermo’s Autosampler
technology features temperature-controlled sample trays,
injection valve (5-25 µL), and integrated column oven to
maintain sample integrity before and during analysis. The
column used was the Waters-XBridge C18, 3 mm x 50
mm, 3.5 μm particle size (Water Corporation, Milford,
MA, USA). Preliminary studies were carried out using a
HPLC model Waters Delta Prep 3000 System, Waters
486 Tunable Absorbance Detector, Waters Prep Pump,
Waters Prep LC Controller.
General instrumentation for qualitative analysis.
Melting points were determined on a Fisher-Johns
melting point apparatus and were uncorrected. IR spectra
were measured on a Perkin Elmer FT-1710 instrument, as
KBr disks. NMR spectra were recorded with a Bruker
Avance 400 MHz instrument for solutions in CDCl 3. The
Gas chromatographic mass spectrometry analysis was
performed on a Hewlett-Packard MSD 5973 instrument
fitted with a 5 % phenylmethyl polysiloxane fused-silica
column (HP-5MS, 30 m, 0.25 mm, film thickness 0.25
μm). The initial analysis temperature was 250 ºC, which
was increased at 5 ºC min-1 to a final temperature of 300
ºC. Analytical thin-layer chromatography was performed
on E. Merck aluminum-backed silica gel foils (F254).
Flash chromatography was performed on silica gel E.
Merck grade 60, 63-200 mesh, by gradient elution with n-
hexane and n-hexane-EtOAc mixtures.
Materials
Reference substances. Grandiflorenic acid (G-
ac), kaurenic acid (K-ac), iso-kaurenic acid (iK-ac), and
15-OH-kaurenic acid (15-OH-K-ac) were isolated,
purified and identified in our laboratory. The identity and
purity of these ent-kaurane diterpenes were confirmed by
FTIR, 1H and 13C NMR, and GC-MS analysis.
Standard solutions. G-ac stock solution (1000 μg
mL-1), K-ac stock solution (1000 μg mL-1), iK-ac stock
solution (500 μg mL-1), and 15-OH-K-ac stock solution
(500 μg mL-1) were prepared in organic mixture
constituted by CH3CN and CH3OH (70:30, v/v).
Complete dissolution was obtained with the aid of
sonication. After 15 min in an ultrasonic bath, they were

cooled at room temperature. The intermediate solution
was obtained by diluting each stock solution in 10 mL
dissolving mixture using a volumetric flask. The
dissolving mixture was prepared by mixing CH 3CN,
CH3OH, and 0.1% H3PO4 solution (28:12:60, v/v). The
working solutions were prepared by dilution of the
intermediate stock solution with HPLC-mobile phase
consisting of CH3CN, CH3OH, and 0.1% H3PO4 solution
(49:21:30, v/v) to reach the concentration range of 10 μg
mL-1 - 250 μg mL-1, for each ent-kaurenoic acid. Each
standard solution was filtered through a filter of 0.45 µm
porous size previous to the injection in the
chromatograph.
Reagents. HPLC-grade acetonitrile (Fischer
Scientific, New Jersey, USA). HPLC-grade methanol
(J.T. Baker, Xalostoc, México), Analytical grade of
orthophosphoric acid ACS reagent, ≥ 85 wt. % in water
(Sigma-Aldrich, St. Louis, MO, USA), hydrochloric acid
(puriss p.a. Riedel-de Haên, Hamburg, Germany), sodium
hydroxide Riedel-de Haên, Hamburg, Germany), sodium
azide (Sigma-Aldrich, St. Louis, MO, USA), and high-
purity distilled water (Milli-Q, Millipore Corp. USA)
were used.
Plant material. Samples of aerial parts of C.
timotensis were collected when the plants were blooming
at Páramo Pico del Águila located two hours’ drive from
Mérida in the Venezuelan Andes (8° 50' 26'' North and
70° 49' 40'' West). Voucher specimens of each plant were
deposited at the Herbarium of the Faculty of Pharmacy
and Bioanalysis, University of Los Andes, Mérida,
Venezuela. The species were identified by direct
comparison with Espeletiinae specimens at the above
mentioned herbarium. The leaves were dried, ground, and
extracted with an n-hexane-diethyl ether mixture (3:1,
v/v). The acid fraction was obtained by shaking each
extract with 0.5 N NaOH solution.
Sample solution. A portion of the dried extract
acid resin sample was accurately weighed in order to
obtain a final concentration of 1030 µg mL-1 in organic
mobile phase composed of CH3CN and CH3OH (70:30,
v/v). Complete dissolution was obtained with the aid of
sonication. After 15 min in an ultrasonic bath, it was
cooled at room temperature. A working sample solution
was prepared by adding 250 µL of a dissolvent mixture
composed of CH3CN, CH3OH, and 0.1% H3PO4 solution
(28:12:60, v/v) to 500 µL of stock sample solution, and
finally diluted up to 1000 µL with mobile phase
consisting of CH3CN, CH3OH, and 0.1% H3PO4 solution
(49:21:30, v/v). Each working sample solution was
filtered through an Acrodisc syringe filter for HPLC
samples (Ann Arbor, MI, USA) of 0.45 µm pore size and
13 mm effective filtration area before injecting sample
into the liquid chromatograph. For the standard addition
assay, sample solutions (500 µL of stock sample solution)
were spiked with aliquots of standard solutions, G-ac and
K-ac, to obtain total concentrations within the previously
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Inter. J. of Phytotherapy / Vol 4 / Issue 2 / 2014 / 73-83.
specified ranges by dilution up to 1000 µL with a final
equivalent dissolution to the HPLC mobile phase.
General chromatographic conditions
The mobile phase was prepared by mixing
orthophosphoric acid 0.1% (by volume), CH3CN, CH3OH
in the ratio of 30:49:21 (v/v). Sodium azide (0.02%, w/v)
was used as preservative of the aqueous phase. The
mobile phase was filtered by passing it through a 0.45 μm
pore size membrane filter and degassed by sonication
prior to use. The flow rate was 0.6 mL min-1. The eluent
was monitored by a PDA detector from 200 to 350 nm,
and chromatograms were recorded at 210, 220 and 225
nm, respectively. Separation of all the ent-kaurenoic acids
was performed in a column maintained at 50 °C.
Triplicate 25 µL injections of each solution were made,
and chromatographed under the previously described LC
conditions. The peak areas were plotted against the
corresponding concentrations to plot the calibration
graphs. Recovered concentrations were calculated from
the corresponding calibration graphs obtained by
comparing the ent-kaurenoic acid response with the
increment response reached after the addition of the
standard.
RESULTS AND DISCUSSION
Optimization of chromatographic conditions
Mobile phase selection. The usual way to
separate kaurane diterpenes in plants by RP-HPLC is
using CH3CN in water as mobile phase in isocratic mode
[13, 15, 16]. The observed disadvantages through this
approach are the resolution and run time. These
previously published studies did not demonstrate the
selectivity of the methods to allow the resolution of G-ac
from their related substances, mainly K-ac. Our present
work resembles those previously published in that we
used aqueous CH3CN in isocratic mode, but we explored
the inclusion of CH3OH (proton-donor ability) and other
parameters that used to improve selectivity to overcome
most of the shortages observed in previous works.
Mobile phase pH. As described above
quantitative determination by RP-HPLC of G-ac and K-ac
in extracts of plants has been conducted isocratically by
using 60% - 80% CH3CN in just water [13, 15, 16].
Additionally, other authors have proposed elution with
acetonitrile in aqueous solutions containing acids, such as
acetic, trifluoroacetic (TFA), phosphoric, and
hexanesulfonic [14, 20]. In this particular case, the
inclusion of an acid in the mobile phase is important in
order to maintain not ionized the ent-kaurenoic acids.
When control at a lower pH (2-3 units) is desired in
HPLC, phosphoric acid is perhaps the most common used
option. Therefore, we selected this last mentioned acid at
0.1% in order to guarantee a pH value close to 3 units.
Isocratic optimization. Firstly, it is important to
point out that the content of water in order to control the

retention of ent-kaurenoic acids was a limiting factor
because solubility of the analytes should be checked
before changing the mobile phase composition. As a
compromise among retention capacity, resolution and ent-
kaurenoic acids solubility we found that 30% aqueous
composition could guarantee the needed chromatographic
conditions. In this regard, mobile phase (MP) strength (%
CH3CN) was changed from 80 % to 60 % until an
acceptable retention range was achieved (within 1 – 10, k
values), i.e. until early eluting peaks may run after the
unretained peak or matrix components and their
quantitation may be possible and reproducible. Further
work on improving selectivity (α) was achieved by
changing mobile phase organic modifier. In this sense,
methanol was incorporated in the organic MP by using
mixtures of CH3OH and CH3CN; a ratio of 21:49 (v/v)
improved selectivity without affecting negatively the
solubility of the ent-kaurenoic acids. Summarizing, the
selected MP was orthophosphoric acid 0.1% (v/v),
CH3CN, and CH3OH in the ratio of 30:49:21 (v/v) instead
of the pioneered MP composed of H2O - CH3CN in the
ratio of 20:80 (v/v) [15].
Stationary phase selection. One of the most
important aspects in the RP-HPLC method development
is the achievement of sufficient resolution in a reasonable
analysis time. To achieve this goal, several experiments
were carried out in order to select the appropriate column.
At the beginning, various RP-HPLC C18 columns were
checked using a preparative chromatographic system
suited for analytical HPLC. Keeping in mind that the goal
was to get the best resolution in a shorter analysis time, a
decreasing of the column length was considered in this
study. A decrease in column length may require a
decrease in packing particle size because a column of
shorter length will reduce the plate number and
resolution. Figure 1(a) displays a representative
chromatogram obtained using a longer column than the
most popular column size, 150 mm x 4.6 mm i.d. (5.0 μm
particle size), while Fig. 1(b) shows one of the
chromatograms obtained using a C18 column of both
shorter length and smaller particle size. This last column
was a modern trifunctionally bonded C18 ligand, called
XBridge from Waters Corporation. It was observed that
both short and long C18 column lengths did not lead to
discontinuous changes of retention and separation
selectivity. Since the column of shorter length gave a
reasonable analysis time with acceptable resolution, it was
selected and used in further studies.
Column temperature. Effect of column
temperature on the separation of kaurenic diterpenoids
from an acid fraction of C. timotensis resin was
conducted. Figure 2 shows the chromatograms where
separation of the ent-kaurenoic acids on the column, with
temperature changing from 30 ºC to 55 ºC, can be
observed. Good baseline separations of the ent-kaurenoic
acids were achieved in the lower temperature limit. As
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Inter. J. of Phytotherapy / Vol 4 / Issue 2 / 2014 / 73-83.
expected, increasing the column temperature will shorten
the retention time of all the peaks but K-ac and its isomer
iK-ac were affected differently than G-ac. An increase on
column temperature up to 45 ºC caused loss of resolution
between K-ac peak and iK-ac peak, resulting in a change
in selectivity. Unexpected, higher increase on column
temperature up to 55 ºC, but decreasing slightly the flow
rate, improves the resolution between K-ac peak and iK-
ac peak. Finally, a temperature of 50 ºC was chosen as a
compromise between speed, resolution, and peak purity.
Table 1 lists the calculated peak purity match factors for
G-ac peak, K-ac peak, and iK-ac peak, demonstrating the
good separation of the analytes for the analysis of a real
sample at a column temperature of 50 °C.
Flow rate. The option of using a short column
allowed decreasing the flow rate without affecting
negatively the factors involved in the Van Deemter
equation. Decreasing the flow rate (from 1.0 mL min-1 to
0.6 mL min-1) allowed also decreasing slightly the column
temperature in order to obtain better separation between
K-ac and iK-ac.
Wavelength selection. Taking into account both
the analytes and the sample matrix, and considering also
the mobile phase composition, a scanning diode array
detector was used in full UV-scan mode to scan all
relevant wavelengths of the components of the sample
(i.e. 200 nm to 350 nm). Prominent peak wavelengths
with the maximum signal to noise (S/N) ratio was
determined by reviewing the spectra data (Fig. 3). All of
them exhibited absorption over the range of 200 nm - 230
nm with a maximum near 220 nm. The ent-kaurenoic acid
with the highest absorption was G-ac followed by iK-ac
and K-ac, respectively. Consequently, only one
wavelength channel, i.e. 220 nm, was selected for
quantification of all the analytes.
System suitability test (SST)
This test is commonly used to verify resolution,
column efficiency, and repeatability of a chromatographic
system to ensure its adequacy for a particular analysis.
Figure 3(a) shows a typical chromatogram for the
separation of compounds found in an acid fraction from a
C. timotensis sample extract. The identification of the ent-
kaurenoic acid peaks in the chromatogram of the sample
was ensured registering a chromatogram belonging to a
mixture of pure standards (Fig. 3(b)) and by enrichment
of a representative sample of C. timotensis. The identified
peaks showed in Fig. 3(b), G-ac, k-ac and iK-ac eluted at
retention times (n = 6) 3.003 (± 0.005) min, 3.948 (±
0.006) min, and 4,388 (± 0.007) min, respectively.
The suitability was evaluated as the relative
standard deviations of peak areas and retention times.
Assuming the usual value of 1.5 for resolution, separation
between two consecutive peaks was obtained for the three
ent-kaurenoic acids. An improved resolution could be
obtained but at the expense of run time. Resolution and
 Inter. J. of Phytotherapy / Vol 4 / Issue 2 / 2014 / 73-83.

selectivity for the mixture under analysis are given in
Table 1. The measure of peak symmetry was found close
to the unity (reference value for perfectly symmetrical
peak), which was also reflected in integration, and hence
in high precision. Replicate injections of a mixture of
standards resulted in a relative standard deviation lesser
than 1.5% (both peak area and retention time) for all the
ent-kaurenoic acids. Finally, column performance,
expressed by the number of theoretical plates (N), and
capacity factor were also registered (Table 1). Since all
the requirements in the SST were acceptable,
accompanied also by the robustness evaluation, analyses
of samples were carried out under the below described
chromatographic conditions (Table 1).

Fig. 1. Chromatographic behavior of the ent-kaurenoic acids in a Waters-Spherisorb C18 (4 mm x 250 mm, 5.0 μm
particle size) column using an alternative chromatograph with conventional UV detector; MP: phosphoric acid 0.1%
and CH3CN (30:70, v/v) at a flow rate of 1.5 mL min -1 (at room temperature); detection at 200 nm (a).
Chromatographic behavior of the ent-kaurenoic acids in a Waters-XBridge C18 (3 mm x 50 mm, 3.5 μm particle size);
chromatographic conditions are those selected in the present study (b)

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Table 1. System suitability test and robustness

Obtained results
Parameter Specification
G-ac K-ac iK-ac
Resolution (USP)a of one analyte from others in the mixture > 1.5 1.79 4.50 1.70
Relative retention > 1 1.36 1.36 1.12
Repeatability (RSD, n = 10)b <2% 0.67 0.82 1.19
Peak asymmetry (Tailing at 10 % height)  2.00 1.11 1.09 0.96
Theoretical plates Column performance 3877 4661 4467
Capacity factor Column performance 11.13 14.99 16.69
Mobile phase pH alteration by 0.1 units RSD  5 % 2.30 1.98 -
Mobile phase flow rate alteration (5 %) RSD  5 % 3.72 4.25 -
Robustness: Column oven temperature alteration (5 %) RSD  5 % 3.98 4.60 -
Percent organic content (5 % CH3CN) RSD  5 % 4.25 3.85 -
Percent organic content (5 % CH3OH) RSD  5 % 3.99 3.28 -
a
United States Pharmacopoeia. b Relative standard deviation.

Fig. 2. Representative stack chromatograms on the effect of column temperature on the separation of ent-kaurenoic
acid diterpenoids from an acid fraction of a C. timotensis sample extract. Chromatographic conditions: SP, Waters-
XBridge C18 (3 mm x 50 mm, 3.5 μm particle size); MP, H3PO4 0.1%, CH3CN, CH3OH (30:49:21, v/v); PDA detection
at 220 nm.

Validation of the proposed method
The method was validated according to the
guidelines for the validation of analytical methods for
active constituents, agricultural and veterinary chemical
products of the Australian pesticides & veterinary
medicines authority [21], and the guidelines on the
validation of analytical methods from Lab-Compliance
Tutorials [22].
Linearity and concentration ranges
The linearity of the proposed HPLC procedure
was evaluated by analyzing a series of different
concentrations for G-ac and K-ac in the range of 10 ng
µL-1 - 250 ng µL-1 (Fig. 4). Availability of a pure standard
quantity of iK-ac for preparing solutions in order to plot a
calibration curve was a limiting factor is this study.
Therefore, it was not possible to quantify with accuracy
the referred ent-kaurenoic acid. All determinations were
done in triplicate injections. The linear regression
equations were generated by least squares treatment of the
calibration data. Under the optimized conditions
described above, the measured peak areas at 220 nm were
found to be proportional to the concentrations of the ent-
kaurenoic acids in the range of 10 ng µL-1 – 100 ng µL-1.

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Table 2 shows both obtained data and selected data in this supported by the statistical analysis taken from the
study. Regression analysis showed acceptable linearity as analysis of variance (ANOVA) test.
indicated from the correlation coefficient values and

Fig. 3. Representative RP-HPLC-PAD chromatogram obtained for a real sample solution of an acid resin fraction of
C. timotensis; insert: spectra of the analytes (a). Representative RP-HPLC-PAD chromatogram obtained for a mixture
of standards: 50 ng µL-1 G-ac and 50 ng µL-1 K-ac; insert: spectra of the analytes (b). The PDA collects and displays
three simultaneous channels concurrent with 3-D spectral data for sample identification and automated purity
analysis

Precision and accuracy concentration within one day. Similarly, the between-day
Precision and accuracy for the proposed method (inter-day) precision and accuracy were tested by
were studied at three concentration levels for each analyzing the same three concentrations for each
compound using three replicate determinations for each compound using three replicate determinations repeated

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on three days. In all cases, results were better than the other components which absorb at wavelengths far away
acceptable maximum limit (Table 2). Recoveries were from or near the peak wavelengths.
calculated using the corresponding regression equations
and they were satisfactory only for G-ac. In this last Fig. 5. Representative RP-HPLC-PAD chromatogram
particular case, the relative standard deviation did not obtained for the analysis of the acid fraction of an C.
exceed 1 %, proving the high repeatability and acceptable timotensis sample extract (250 ng µL-1) against stack
accuracy of the HPLC developed method for the chromatograms of standards: 50 ng µL-1 K-ac, 50 ng
estimation of G-ac in the analyzed acid fraction of a C. µL-1 iK-ac, and 25 ng µL-1 G-ac
timotensis sample extract (Table 2). On the contrary,
although a reliable result was found for K-ac standard, its
determination in a real sample required the technique of
standard addition since matrix effect was detected. This
last approach was suggested because it was not possible
to prepare an equivalent blank sample matrix without the
presence of the referred ent-kaurenoic acid.

Fig. 4. Calibration graphs in the studied range in

order to select the appropriate linear range for the two
selected ent-kaurenoic acids of the acid fraction of a C.
timotensis sample extract

Selectivity and specificity

Initially, both unresolved peaks and hidden peaks
were explored changing mobile phase composition,
stationary phase, flow rate, and column temperature.
Hidden peaks were not detected during this evaluation.
Increasing column temperature from 30 ºC to 55 ºC and
simultaneously decreasing the flow rate allowed us to
obtain acceptable resolution between K-ac and its isomer
iK-ac in a reasonable run time; this last test without
affecting negatively the chromatographic behavior of G-
ac (Fig. 2).
Method specificity was explored somehow by
monitoring the recorded spectra during chromatographic
peak elution using a PDA detector. The PDA collects and
displays three simultaneous channels concurrent with 3-D
spectral data for sample identification and automated
purity analysis. The full scanning mode of the detector
was used throughout the analysis in order to detect any

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Table 2. HPLC method validation
Parametera Strategy
Instrumental variability (10
injections of one sample
solution)
Intra-assay precision (3
replicate samples with 3
Precision
injections)
Precision of the accuracy (3
concentrations with 3
injections in the addition
standard calibration)
Linear calibration
Linear range (10 - 100) ng µL-1
Correlation coefficient
A test for an intercept
significantly different from
zero by applying the Student t-
test. H0: µa = 0
Linearity A test for a slope significantly
different from zero by applying
the Student t-test. H0: µb = 0
(lack of correlation)
Goodness of fit by comparing
the variance of the lack of fit
against the pure-error variance
Correlation coefficient
(technique of standard
additions)
Technique of standard
additions
Accuracy
[21]
Testing for equal lines by
using z-tests. H0: b1 = b2
√ freedom = 18
a
Validation of Analytical Methods and Procedures: Lab-Compliance Tutorials [22].
The above referred concomitant compounds
could add or subtract signal from the main peak making it
appear to be more or less concentrated (or more or less
pure) than it actually could be found. This study was
complemented by peak purity determination to show that
the ent-kaurenoic acid chromatographic peak was not
attributable to more than one component. Table 1 lists the
calculated peak purity match factors for the three
identified compounds found in the sample extract,
demonstrating in some way the selectivity under the
Inter. J. of Phytotherapy / Vol 4 / Issue 2 / 2014 / 73-83.
Obtained results
Specification
G-ac K-ac
< 1 % (RSD) 0.67 % 0.82 %
5.3 % – 7.3 % (RSD) 4,17 % 5,84%
Low = 2.41 %
Low = 0.44 %
Intermediate = 2.46
< 5 % (RSD) Intermediate = 0.45 %
%
High = 0.23 %
High = 0.58 %
Y = a + b [X] Y = 64336 + 38270 [X] Y = 2232 + 17254 [X]
Dynamic range 10 - 50 10 - 50
R > 0.99 0,9967 0,9986
t-test: if calculated t-
Calculated t-value = Calculated t-value =
value > tabulated t-
1.76 0.63
value, the null
t(10, 0.975) = 2.23 t(13, 0.975) = 2.16
hypothesis is rejected
t-test: if calculated t-
Calculated t-value = Calculated t-value =
value > tabulated t-
38.60 41.53
value, the null
t(10, 0.975) = 2.23 t(13, 0.975) = 2.16
hypothesis is rejected
F-test: if calculated F-
Calculated F-value = Calculated F-value =
value >> critical F-
1490 1725
value, the calibration
Critical F-value (P = Critical F-value (P =
model is considered
0.05) = 3.2x10-12 0.05) = 3.3x10-15
suitable
R > 0.99 0.9987 Matrix effect
Low (50 %) = 119.3
Acceptable mean
Intermediate (100 %) =
recovery: -
111.3
80 % - 120 %
High (150 %) = 106.3
Calculated acceptable Experimental “d” value
zone: = 1.49 (α = 0.05)
-
(-2.10)  z  (+2.10) Calculated degrees of
described optimized chromatography conditions.
Additionally, any other compound that could be present in
the sample, and stronger retained than the target analytes,
was checked during a run of 60 minutes; no other peaks
were observed after 10 min run that could appear in the
next sample injection as a ghost peak.
The compound 15-OH-K-ac showed in Fig. 1(a)
was discarded for analytical purposes because it was
retained very close to the unretained material and also it
was not well separated from other related compounds
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 Inter. J. of Phytotherapy / Vol 4 / Issue 2 / 2014 / 73-83.

appearing in the matrix composition following the
proposed experimental conditions.
Robustness
This criterion was examined by evaluating the
influence of small variations in different conditions such
as those presented in Table 1. Relative standard deviation
for the measured peak areas using these deliberated
variations did not exceed 5% demonstrating that these
disparities did not have any significant effect on either the
measured response or the chromatographic resolution.
Samples and standards in their original solid-states were
kept at room temperature. The stock solutions prepared in
a dissolvent mixture composed of CH3CN and CH3OH
(70:30, v/v) were stored refrigerated at 4 ºC. Under these
conditions retention times and peak areas of the ent-
kaurenoic acids remained relatively unchanged and no
significant degradation was observed during at least one
week.
Analysis of real samples
The optimized RP-HPLC-PAD method was
applied for the assay of G-ac in an acid fraction of a C.
timotensis sample extract; percentages (by weight, n = 3)
of 13.4 ± 0.1, 14.6 ± 0.1, and 14.0 ± 0.5 were found for
three independently prepared samples. The proposed
method was also used in the determination of K-ac by
extrapolation in the standard addition calibration;
percentage (by weight, n= 3) of 9.9 ± 0.4 were found for
the analyzed sample of C. timotensis. Figure 5 illustrates
chromatographically the ability of the method to
accurately measure the analyte response in the presence of
all potential sample components.
CONCLUSIONS
The present work describes the development of a
RP-HPLC-PAD method for the qualitative and
quantitative determination of G-ac in samples of C.
timotensis. A significant advantage in this study was the
separation of G-ac from the other most abundant kaurane-
type diterpenes from the ent-series, identified as K-ac and
iK-ac, in a reasonably short run time. The HPLC method
described in the present work allows for the direct
detection and quantification of G-ac in the acid fraction of
the resin obtained from the aerial parts of C. timotensis
without the prerequisite of derivatization required by the
GC methodologies commonly applied in the analysis of
the species of the subtribe of Espelettinae.
The described method resulted also superior to
the previously reported analytical methods for
determining ent-kaurenoic acids in other herbs. This last
statement was based on comparing visually the resolution
of the target analytes. A cleaner sample matrix was also
employed since the proposal involved the analysis of just
one fraction of the total extract of the sample, improving
somehow the peak purity, as it was checked by PDA
detection.
The PDA detector used in the developed RP-
HPLC method allowed confirming the presence of iK-ac
in an apparent relatively big amount for C. timotensis.
This finding could be considered of importance since iK-
ac is of very restricted occurrence in the plant kingdom as
it has been previously recognized [23].
Finally, the results provided the basis for
developing in a near future a more versatile method for
the simultaneous quantitative analysis of G-ac, K-ac, and
iK-ac for subsequent use in ent-kaurenic acids profile
determination of species belonging to the genus
Coespeletia.
ACKNOWLEDGMENTS
The authors are grateful to the Council for
Scientific, Humanistic, Technological, and Artistic
Development of the University of Los Andes, Mérida,
Venezuela for providing financial support through the
CDCHTA-FA-509-11-08-A Project. The authors are also
thankful to the National Fund for Science, Technology
and Innovation of Venezuelan Ministry of Science and
Technology for providing financial support through the
Science Mission ULA-2007000881 Project.

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