Accepted Manuscript

Title: Simultaneous determination of 3-O-acetyloleanolic acid

and oleanolic acid in rat plasma using liquid chromatography
coupled to tandem mass spectrometry
Author: Eunyoung Kim Keumhan Noh Sang Joon Lee
Beomsoo Shin Joo Tae Hwang Seung Woong Lee Mun-Chul
Rho Wonku Kang
PII:
DOI:
Reference:

S0731-7085(15)30203-X
http://dx.doi.org/doi:10.1016/j.jpba.2015.10.030
PBA 10306

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Journal of Pharmaceutical and Biomedical Analysis

Received date:
Revised date:
Accepted date:

13-8-2015
13-10-2015
19-10-2015

Please cite this article as: Eunyoung Kim, Keumhan Noh, Sang Joon Lee, Beomsoo
Shin, Joo Tae Hwang, Seung Woong Lee, Mun-Chul Rho, Wonku Kang, Simultaneous
determination of 3-O-acetyloleanolic acid and oleanolic acid in rat plasma using liquid
chromatography coupled to tandem mass spectrometry, Journal of Pharmaceutical and
Biomedical Analysis http://dx.doi.org/10.1016/j.jpba.2015.10.030
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Simultaneous determination of 3-O-acetyloleanolic acid and oleanolic acid in rat plasma

using liquid chromatography coupled to tandem mass spectrometry

Eunyoung Kima, Keumhan Noha, Sang Joon Leea, Beomsoo Shinb, Joo Tae Hwangc, Seung
Woong Leec, Mun-Chul Rhoc, Wonku Kanga,*

Center for Metareceptome Research, College of Pharmacy, Chung-Ang University, Seoul

156-756 South Korea

b

College of Pharmacy, Catholic University of Daegu, Kyoungbuk 712-702 South Korea

Bio-Material Research Institute, Korea Research Institute of Bioscience and Biotechnology,

Jeongeup 580-185, South Korea

*Corresponding author
Wonku Kang, College of Pharmacy, Chung-Ang University, Seoul 156-756 South Korea, Email address: wkang@cau.ac.kr; Mun-Chul Rho, Bio-Material Research Institute, Korea
Research Institute of Bioscience and Biotechnology, Jeongeup 580-185, South Korea, E-mail
address: rho-m@kribb.re.kr

Graphical abstract

Highlights
3-O-acetyloleanolic acid (OAA), a triterpenoid compound was determined in rat plasma
using LC/MS/MS for the first time.
A bioanalytical method validation was performed for the determination of both OAA and
its metabolite, oleanolic acid (OA).
This method was successfully applied to monitor plasma concentrations of both substances
over time following an intravenous administration of OAA in rats.

Abstract
3-O-acetyloleanolic acid (OAA) is a triterpenoid compound, and exerts an apoptosis in
cancer cell lines, an inhibition of both atopic and allergic contact dermatitis in murine model,
and a suppression of inflammatory bone loss in mice. OAA can be converted into oleanolic
acid (OA) by hydrolysis in vivo, and OA exhibits several pharmacological effects as well. A
liquid chromatographic method using tandem mass spectrometry (MS/MS) was developed for
the simultaneous determination of OAA and OA in rat plasma. After liquid-liquid extraction
with ethylacetate, both substances were chromatographed on a reversed phase column with a
mobile phase of 0.1% formic acid aqueous solution and acetonitrile (1:9, v/v). The accuracy
and precision of the assay were in accordance with FDA regulations for the validation of
bioanalytical methods. This analytical method was successfully applied to monitor plasma
concentrations of both substances over time following an intravenous administration of OAA
in rats.

Keywords: 3-O-acetyloleanolic acid, oleanolic acid, LC-MS/MS, rat plasma

1. Introduction
3-O-acetyloleanolic acid (10-acetoxy-2,2,6a,6b,9,9,12a-heptamethyl-1,3,4,5,6,6a,6b,7,8,8a,9,
10,11,12, 12a,12b,13,14b-octadecahydro-2H-picene-4a-carboxylic acid; OAA; Fig. 1) is a
triterpenoid compound isolated from the seeds of Vigna sinensis K. OAA exerts several
pharmacological effects, such as inducing apoptosis in human colon carcinoma cells, which is
mediated by upregulation of the death receptor DR5 [1], and inhibiting both atopic and
allergic contact dermatitis in a murine model [2]. The substance also inhibits
osteoclastogenesis via downregulation of PLC2-Ca2+-NFAT signaling, and suppresses
inflammatory bone loss in mice [3]. OAA at 10-50 mg/kg has been administered in mice to
investigate these activities; however, its systemic exposure has not yet been measured.
It is expected that OAA can be converted into oleanolic acid (OA), resulting from
hydrolysis of the ester at the 3-carbon position in vivo. OA has anti-inflammatory actions [4],
anti-nociceptive activities [5], and anti-tumor effects [6]. Although plasma OA concentrations
have been determined in mice [7], rats [8] and humans [9] by LC/MS/MS, there is no report
on the determination of OAA in plasma or the time course of plasma OAA concentrations.
In this study, we developed a liquid chromatographic method using tandem mass
spectrometry (MS/MS) for the simultaneous determination of OAA and OA in rat plasma.
This analytical method was successfully applied to monitor plasma concentrations of both
compounds over time following intravenous administration of OAA in rats.

2. Material and Methods

2.1 Reagents and materials

OAA was synthesized and kindly donated by Korea Research Institute of Bioscience and
Biotechnology (Jeongeup, Korea). OA and diclofenac (internal standard, IS) were purchased
from Sigma (Seoul, Korea), and acetonitrile was obtained from Burdick & Jackson
(Muskegon, MI, USA). All other chemicals and solvents were of the highest analytical grade
available.

2.2 Preparation of standards and quality controls

OAA, OA, and the internal standard (IS) were dissolved in methanol to a concentration of 1.0
mg/mL. OAA and OA solutions were then serially diluted with methanol, and 5 L of a
5

mixture of both diluted solutions was added to 90 L drug-free plasma to obtain final
concentrations of 0.01, 0.05, 0.1, 0.5, 2, and 10 g/mL for both OAA and OA. Using linear
regression, six calibration graphs were derived from the ratio between the area under the peak
of each compound and the IS. Quality control samples were prepared with 90 L blank rat
plasma by adding 5 L of serially diluted solutions of each substance to determine the lower
limit of quantification (0.01 g/mL), as well as low (0.03 g/mL), intermediate (1 g/mL),
and high concentrations (8 g/mL). These samples were used to evaluate the intra- and
interday precision and accuracy of the assay.

2.3 Characterization of product ions using MS/MS

Briefly, 10 ng/mL of OAA, OA, and the IS were individually infused into the mass
spectrometer at a flow rate of 10 l/min to characterize the product ions of each substance.
The precursor ions and fragmentation patterns of the analytes and the IS were monitored
using negative-ion mode. Major peaks in the MS/MS scan were used to quantify the three
compounds.

2.4 Analytical system

Plasma concentrations of OAA and OA were quantified using an API 4000 LC/MS/MS
system (AB SCIEX, Framingham, MA, USA) equipped with an electrospray ionization
interface. The compounds were separated on a reversed-phase column (Atlantis T3,
50 2.1 mm internal diameter, 3-m particle size; Waters, Dublin, Ireland) in the mobile
phase (0.1% formic acid aqueous solution, and acetonitrile, 1:9, v/v). The column was heated
to 30C, and the mobile phase was eluted at 0.2 mL/min using an HP 1260 series pump
(Agilent, Wilmington, DE, USA). The turbo ion spray interface was operated at 4500 V and
500C. Both the precursor ions and the product ion of OAA and OA were mainly identified
as deprotonated ions [M-H]- at m/z 497.4 and 455.6, respectively. The IS was also mainly
identified as the deprotonated ion, and its ion transition was at m/z 296.1251.0.
Quantification was performed by selective reaction monitoring of the deprotonated precursor
ions and the related product ions using the ratio of the area under the peak for each solution.
The data were processed using Analyst software (ver. 1.5.2, Applied Biosystems, Foster City,
CA, USA).

2.5 Sample preparation

The IS (50 L; 1 g/mL in methanol) was added to the spiked plasma and the mixture was
extracted with 1 mL of ethylacetate in 2.5 mL polyethylene tubes by vortex-mixing for 5 min.
After centrifugation (13,200 rpm, 10 min), the upper organic layer was transferred to a new
tube and evaporated to dryness under vacuum at 40oC. The residues were dissolved in 50 L
of the mobile phase, and 5 l was injected into the column [10,11].

2.6 Validation procedure

The validation parameters were selectivity, extraction recovery, precision, and accuracy.
Blank plasma samples obtained from five rats were screened to determine specificity. The
intra- and inter-day assay precision and accuracy were estimated using a calibration curve to
predict the concentration of quality controls. Acceptable criteria were within 15% of
precision and accuracy, except the lower limit of quantification, which was within 20%.
Recovery was determined by comparing the mean peak areas of quality controls spiked
before the liquid-liquid extraction to those spiked after pretreatment. The matrix effect was
assessed based on the percentile of the mean peak areas of quality controls spiked after
pretreatment of stock solutions.

2.7 Stability
Stability of OAA and OA in stock solutions and plasma samples was examined under
different conditions. The stock solution was checked for short-term stability after 4 h of
storage at room temperature and for long-term stability after 4 weeks at 4C. For the stability
study in plasma, control samples were made at 0.05 and 1 g/mL concentrations. Short- and
long-term stabilities were assessed after 2 h of storage at room temperature and after 2 weeks
of storage at 70C, respectively. The stability in plasma samples was also tested after three
freezethaw cycles (70C to room temperature). The stability in extracts was also examined
after 24 h of storage at 4C. The requirement for a stable analyte was that the difference
between the mean concentrations of tested samples after being placed under various
conditions was approximately 15%.

2.8 Application
Five SpragueDawley rats were administered a single intravenous injection of 2 mg/kg of
OAA dissolved in a mixture of N,N-dimethylacetamide:polyethylene glycol 400:saline (2:4:1,
7

v/v/v) [12]. Blood samples (0.25 mL) were taken serially from subclavian vein at 2, 5, 15 min,
and 0.5, 1, 1.5, 2, 3, 4, and 6 h after drug administration and collected in heparinized
centrifuge tubes. After centrifugation (13,200 rpm, 10 min), plasma samples were stored at 70C. The study was approved by the Chung-Ang University Animal Care and Use
Committee. All animals used in this study were cared for in accordance with the principles
outlined in the National Institutes of Health Guide for the Care and Use of Laboratory
Animals.

3. Results and Discussion

3.1 Quantification of compounds and method validation

The mass spectra of OAA, OA and the IS are shown in Figure 1. As previously reported [9],
OA predominantly produced a deprotonated molecule at m/z 455.6, and the fragment ion was
monitored at the same mass to charge. OAA represented the same pattern as OA: the
precursor to product ion combination was m/z 497.4497.4, from which it seemed that the
triterpenoid structure was too rigid to generate fragment ions under general mass
spectrometry conditions [13].
Figure 2 presents a typical chromatogram for blank plasma (A), plasma spiked with 1
g/mL of the IS (B), together with 20 ng/mL of OAA and OA (C), and a rat plasma sample
(D). OAA and OA eluted at 2.89 and 4.44 min, respectively, and the IS eluted at 1.18 min.
Although the baseline level at the mass transition of OAA was higher than that of OA, there
was no interference at the elution times of either analyte, and addition of the IS did not affect
the determination of analytes. The calibration curves provided a reliable response for the two
compounds. The ratio of the peak area of OAA and OA relative to that of the IS correlated
with the corresponding plasma concentration, and good linearity was observed (r2 > 0.998).
The detection limits for OAA and OA were 1 and 0.5 ng/mL at a signal-to-noise (S/N) ratio
of 3, respectively.
The relative standard deviations of the intraday assay precision were less than 11.0% and
10.4% for OAA and OA, respectively. The intraday assay accuracy was 94.598.9% for
OAA and 94.3101.9% for OA. The relative standard deviations of the interday precision
were less than 10.1% for OAA and 12.0% for OA. The interday accuracy was 94.797.6%
for OAA and 93.598.6% for OA (Table 1). The mean recoveries of OAA and OA ranged
8

from 85% to 96% and 88% to 95%, respectively, and the mean matrix effect was 89% for
OAA and 84% for OA.

3.2 Stability
OAA and OA stock solutions were stable at room temperature for 4 h and at 4C for 4 weeks.
OAA and OA in plasma were stable at room temperature for 2 h, and remained intact for up
to 2 weeks at 70C. No degradation was observed after three cycles of freezing and thawing.
The stability of the compounds in extracts was confirmed after 24 h of storage at 4C

3.3 Application of the method

The validated method described herein was used to evaluate the pharmacokinetics of OAA
and its metabolite OA in rats. Figure 3 shows the mean plasma concentrations of OAA and
OA after a single intravenous injection of 2 mg/kg OAA in five rats. The mean volume of
distribution and the terminal half-life of OAA were 255 mL/kg and 1.5 h, respectively. OA
concentration peaked (0.8 g/mL) approximately 10 min following intravenous
administration of OAA, and decayed with a terminal half-life of approximately 1 h. The mean
AUCinfs of OAA and OA were 1375 and 328 ngh/mL, respectively.

4. Conclusions
A sensitive method for the simultaneous determination of OAA and OA in rat plasma was
developed using LC/MS/MS. This method is suitable for pharmacokinetic studies of OAA in
rats.

Conflict of interest
The authors declared no conflict of interest.

Acknowledgements
This work was supported by the research grant from Korea Research Institute of Bioscience
and Biotechnology and the National Research Foundation of Korea (NRF) grant funded by
the Korea government (MSIP) (No. 2015R1A5A1008958), and by the Chung-Ang University
Research Grants in 2014.

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11

Figure Legend
Fig. 1. Mass spectra of 3-O-acetyloleanolic acid (OAA), oleanolic acid (OA) and the IS
(diclofenac).
Fig. 2. Chromatograms of 3-O-acetyloleanolic acid (OAA), oleanolic acid (OA) and the IS
(diclofenac). A: double-blank plasma; B: plasma spiked with 1 g/mL the IS; C: 20 ng/mL
OAA and OA, and 1 g/mL the IS; D: a plasma sample after an intravenous injection of 2
mg/kg OAA.
Fig. 3. Time courses of plasma3-O-acetyloleanolic acid (OAA) and oleanolic acid (OA)
concentrations in rats after a single intravenous injection of 2 mg/kg OAA (n=5).

12

Fig. 1

13

Fig. 2

14

Fig. 3

15

Table 1.Precision and accuracy of the intra- and inter dayassay (n=5).
Concentration
3-O-acetyloleanolic acid

a
b

oleanolic acid

(g/ml)

intraday

interday

intraday

interday

0.01

95.310.5a (11.0)b

96.69.8 (10.1)

94.39.8(10.4)

93.511.2 (12.0)

0.03

97.54.5 (4.6)

97.66.7 (6.9)

96.88.2 (8.5)

95.89.4 (9.8)

98.93.0 (3.0)

94.77.4 (7.8)

101.95.6 (5.5)

98.66.3 (6.4)

94.56.5 (6.9)

95.56.2 (6.5)

99.15.2 (5.2)

95.27.6 (8.0)

Accuracy (mean % S.D.)

RSD, relative standard deviation (%)

16