Vol.2, No.7, 713-717 (2010) Natural Science
http://dx.doi.org/10.4236/ns.2010.27088
A sensitive, rapid and validated liquid chromatography −
tandem mass spectrometry (LC-MS-MS) method for
determination of Mimosine in Mimosa pudica Linn
Parikshit A. Champanerkar1*, Vikas V. Vaidya1, Sunita Shailajan1, Sasikumar N. Menon2
1Department of Chemistry, Ramnarain Ruia College, Mumbai University, Mumbai, India;
*Corresponding Author:
2Therapeutic Drug Monitoring Laboratory, Sion Koliwada, Mumbai, India
Received 28 March 2010; revised 24 May 2010; accepted 2 June 2010.
ABSTRACT
A rapid, sensitive and accurate liquid chroma-
tographic tandem mass spectrometric method is
described for the determination of Mimosine in
Mimosa pudica Linn. whole plant powder. Mi-
mosine was extracted from the plant using 1.0%
HCl in water. The chromatographic separation
was achieved using a Thermo Hypurity C18 (50 x
4.6 mm) 5.0 μ column interfaced with a triple
quadrapole mass spectrometer. The mobile
phase consisted of a mixture of Methanol: 10
mM ammonium formate buffer whose pH was
adjusted to 3.00 ± 0.05 with formic acid (80:20,
v/v) and was delivered at a flow rate of 1.0 mL
min-1. Electrospray ionization (ESI) source oper-
ated in the negative ion mode was used for the
quantitation. Detection was performed using an
Applied Biosystems Sciex API 3200 Mass spec-
trometer. The method was found to be simple,
precise, accurate, fast, specific and sensitive
and can be used for routine quality control
analysis of Mimosine in Mimosa pudica Linn.
Keywords: LC-MS-MS; Mimosine; Mimosa pudica
Linn
1. INTRODUCTION
Mimosa pudica Linn. (Fam. -Leguminosae) is common-
ly known as Sensitive plant in English and Lajvanti or
Chuimui in Hindi language. The plant is distributed
through out India especially in moist places. Mimosa
pudica Linn. is also said to have larvicidal property [1].
It is used to treat menorrhagia and leucorrhoea [2-4]. In
Ayurvedic system of medicine, Mimosa pudica Linn. has
been described as an indispensable drug for blood pres-
sure [5]. Phytochemical screening has revealed that the
plant contains Mimosine (alkaloid), stigmasterol, leu-
coanthocyanidin, D-xylose and D-glucuronic acid, nore-
pinephrine, D-pinitol, linoleic acid, oleic acid, palmitic
acid, stearic acid, -sitosterol and crocetin dimethyl ester.
Of all these, the major compound present in Mimosa
pudica Linn. is Mimosine [4]. Mimosine is used for trea-
ting the cutaneous effects of psoriasis and related skin
disorders [6]. It is less soluble in methanol and ethanol,
insoluble in other organic solvents, but sparingly soluble
in water. It is soluble in dilute acid and base. Structure of
Mimosine is shown in Figure 1 [7].
The quality of herbal medicine that is the profile of
the constituents in the final product has implication in
efficacy and safety. Due to the complex nature and in-
herent variability of the chemical constituents of the
plant based drugs, it is difficult to establish quality con-
trol parameters and modern analytical techniques are
accepted to help in circumventing this problem [8]. Re-
cently, the concept of marker-based standardization of
herbal drugs is gaining momentum. Identification of ma-
jor and unique compounds in herbs as markers and de-
velopment of analytical methodologies for monitoring
them are the key steps involved in marker-based stan-
dardization [9].
Quantitation of Mimosine from Mimosa pudica L.
using RP-HPTLC has been reported [10]. A method us-
ing HPLC and spectrophotometric determination of Mi-
mosine has also been reported [11,12]. Literature survey,
hence, revealed that there is no method available in the
N
O
OH
COOH
NH2
Figure 1. Structure of Mimosine.

P. A. Champanerkar et al. / Natural Science 2 (2010) 713-717
714
public domain for quantitation of Mimosine from Mi-
mosa pudica Linn. using an LC-MS-MS system. So, the
aim of the present work was to develop a simple, fast,
sensitive, precise, and accurate LC-MS-MS method for
determination of Mimosine from Mimosa pudica L. The
developed method was further validated as per ICH
guidelines to indicate its suitability [13,14].
2. Experimental
2.1. Chemicals and Preparation of Standard
Solutions
HPLC grade Methanol and acetonitrile were purchased
from J.T. Baker, Mumbai, India. Extra pure Formic acid
(99.9%) and ammonium formate was purchased from
Fluka, Steinheim, Germany. High purity deionised water
was prepared in-house using a Milli-Q water purification
system obtained from Millipore, Bangalore, India. Mi-
mosine standard (Purity 98%) was procured from Sig-
ma-Aldrich (Aldrich Division; Steinheim, Federal Re-
public of Germany).
The stock solution A of Mimosine (1,000 µg mL-1)
was prepared by dissolving 25 mg of accurately weighed
Mimosine in minimum quantity of 1.0% HCl in water
and diluting with same solution up to the mark in a 25
mL standard volumetric flask. Further solution B of
Mimosine (10 µg mL-1) was prepared by transferring
0.25 mL of stock solution A and diluting with mobile
phase up to the mark in a 25 mL volumetric flask. Dif-
ferent volumes in the range of 0.4-1.0 mL of stock solu-
tion B were transferred to 10 mL standard volumetric
flasks and diluted up to the mark with the mobile phase,
to provide a concentration range of 400-1000 ng mL-1.
2.2. Plant Material and Preparation of
Sample Solution
The plant Mimosa pudica L. was collected from Mumbai,
Maharashtra, India and was authenticated by National
Institute for Science Communication and Information
Resources (NISCAIR), New Delhi, India. The collected
plant material was dried at room temperature, under
shade and then ground in a mixer to a fine powder. This
was then passed through an ASTM BSS mesh (size 85)
and stored in an airtight container at room temperature.
25 mg of the dried powder was accurately weighed,
placed in a stoppered tube and 10 mL of Methanol was
added. The sample was vortexed for 1-2 minutes and
then mixed on a shaker for 60 minutes. The contents of
the tube were then centrifuged at 4600 rpm and filtered
through Whatmann No. 41 filter paper (E. Merck, Mum-
bai, India) and residue was collected in a 10 mL standard
volumetric flask and 1.0% HCl in water was added up to
the mark, the sample was vortexed for 1-2 minutes and
left to stand overnight at room temperature. The content
was filtered through Whatmann No. 41 filter paper and
the clear supernatant was collected in a dry tube (solu-
tion C). Further solution D was prepared by transferring
1.0 mL of solution C and diluting with mobile phase up
to the mark in a 10 mL volumetric flask. Solution D was
used for further experiments.
2.3. Instrumentation and Chromatographic
Conditions
A Hypurity C18, (50 × 4.6 mm), 5 µ obtained from
Thermo Electron, Mumbai, India was used for the com-
pound retention. The mobile phase consisted of mixture
of Methanol: 10mM ammonium formate buffer pH ad-
justed to 3.00 ± 0.05 with formic acid (80:20, v/v) and
was delivered at a flow rate of 1.0 mL min-1 by employ-
ing a Shimadzu Prominence series (Kyoto, Japan) binary
pump, at ambient temperature. Detection was achieved
using an Applied Biosystems API 3200 MS-MS appara-
tus (Applied Biosystems, Ontario, Canada) fitted with a
Turbo Ion Spray source. The instrument was interfaced
with a computer running Applied Biosystems Analyst
version 1.4.2 software. Electrospray ionization (ESI)
was performed in the negative ion mode. The spray vol-
tage and source temperature were −4500 V and 550°C
respectively. Nitrogen was used as the collision gas. The
Declustering Potential (DP), Collision Energy (CE), En-
trance potential (EP), Cell Exit Potential (CXP) were
optimized during tuning as −20, −24, −10, −4 eV for
Mimosine. The collision activated dissociation (CAD)
gas was set at 3 psi, while the curtain gas was set at 12
psi. The Applied Biosystems API 3200 LC-MS-MS ap-
paratus was operated at unit resolution in the multiple
reaction monitoring (MRM) mode, monitoring the tran-
sition of the molecular ion m/z 197.7 to the product ion
m/z 162.8 for Mimosine. The instrument response was
optimized for Mimosine by infusing a constant flow of a
standard solution (1000 ng mL-1) via a T-piece into the
stream of mobile phase eluting from the column. Figure
2 shows the product ion mass spectra obtained from col-
lision-induced dissociation of the deprotonated molecu-
lar ions of Mimosine.
3. METHOD VALIDATION
3.1. System Suitability
System suitability tests are used to ensure reproducibility
of the equipment. The test was carried out by injecting
10 µL of standard solution of Mimosine (600 ng mL-1)
six times. The % RSD was found to be 1.27% for Mi-
mosine, which was acceptable as it is less than 2%.
3.2. Linearity
In order to establish linearity, standard solutions of Mi-

P. A. Champanerkar et al. / Natural Science 2 (2010) 713-717
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715
Figure 2. Representative Spectra of product ion of Mimosine.
mosine at six different concentrations (400.0, 5‰00.0,
600.0, 700.0, 800.0 and 1000.0 ng mL-1) were prepared
in mobile phase. Each of these solutions (10 µL) was
injected and the detector response for the different con-
centrations was measured. A graph was plotted of drug
peak area against concentration. The plot was linear in
the range 400.0 ng mL-1 to 1000.0 ng mL-1 for Mimosine.
The experiment was performed five times and the mean
was used for the calculations. The equation of linear re-
gression curve obtained was y = 94.4x − 1923.3, where y
= (peak area), x = (concentration of Mimosine in ng
mL-1) with a correlation coefficient 0.9951. A typical
chromatogram of standard and plant is shown in Figure
3 and Figure 4 respectively.
3.3. Limit of Detection and Limits of Quan-
titation
The signal-to-noise ratio of 3:1 and 10:1 was used to
establish LOD and LOQ, respectively. The LOD and
LOQ of Mimosine were 100 ng mL-1 and 400.0 ng mL-1,
respectively.
3.4. Assay
The developed LC-MS-MS method was used for deter-
mination of Mimosine from whole plant powder of Mi-
mosa pudica L. The sample working solution D (10 µL)
was injected and the area of Mimosine peak was meas-
ured. From the calibration curve, the amount of Mi-
mosine in dry powder of Mimosa pudica L. was calcu-
lated. The retention time of Mimosine in sample solution
and in the standard solution was found to be 0.67 min.
The mean assay value of Mimosine was found to be
1.938 mg/g of plant powder with % RSD as 1.55%.
3.5. Precision and Accuracy
The intra-day and inter-day precision was used to study
the variability of the method. The % RSD for intra-day
and inter-day precision for Mimosine were 0.66 and
1.06%, respectively. Accuracy of the method was studied
using the method of standard addition. Standard Mi-
mosine solution were added to the extract of the whole
plant powder of Mimosa pudica L. and the percent re-
covery was determined at two different levels 50% and
100%. Mimosine content was determined and the per-
cent recovery was calculated. The results of recovery
analysis are shown in Table 1.
4. RESULTS AND DISCUSSION
The high selectivity of MS-MS detection allowed the
development of a very specific and rapid method for the
determination of Mimosine in Mimosa pudica L. whole

P. A. Champanerkar et al. / Natural Science 2 (2010) 713-717
716
Table 1. Results of recovery experiment.
Standard Level
Pre analysed
sample in
(ng mL-1)
Amount of std added
to pre analysed
sample in (ng mL-1)
Total amount of
std found in
(ng mL-1)
SD RSD (%)
(n = 7)
Recovery
(% )
0 484.59 0 479.13 6.72 1.40 98.87
50% 484.59 250 730.79 7.40 1.01 99.48
Mimosine
100% 484.59 500 979.39 10.961.12 99.47
Mean 99.28
Figure 3. Representative chromatogram of standard Mimosine at LLOQ level (400 ng mL-1).

Figure 4. Representative chromatogram of plant Mimosa pudica L.
plant powder. During method development different
options were evaluated to optimize, detection parameters
and chromatography. Electrospray ionization (ESI) was
evaluated to get better response of analytes as compared
to atmospheric pressure chemical ionization (APCI)
mode. It was found that the best signal was achieved

P. A. Champanerkar et al. / Natural Science 2 (2010) 713-717
717
717
with the ESI negative ion mode. A mobile phase con-
taining formic acid solution and Methanol in varying
combinations was tried during the initial development
stages. But the best signal for Mimosine was achieved
using a mobile phase containing 10mM ammonium for-
mate buffer pH adjusted to 3.00 ± 0.05 with formic acid
in combination with Methanol (20:80 v/v). Use of a
short Hypurity C18, (50 mm × 4.6 mm), 5 µ column re-
sulted in reduced run time of 1.5 min. Regression analy-
sis of calibration data showed that the linearity of Mi-
mosine was observer over a concentration range of 400
ng mL-1 to 1000 ng mL-1 with regression coefficient of
0.9951. The concentration of Mimosine in 1.0 g of
whole plant powder of Mimosa pudica L. was found to
be 1.938 mg.
When the method was validated in terms of instru-
mental precision, intra-assay precision and intermediate
precision, the percent RSD values were found to be less
then 2, indicating that the proposed method is precise
and reproducible. The accuracy of the method was es-
tablished by means of recovery experiments. The mean
recovery was close to 100%, which indicates that me-
thod is accurate. The low values of %COV for replicate
analyses are indicative of precision of the method. The
method is specific because it resolved the standard Mi-
mosine (Retention time = 0.67) well in presence of other
phytochemicals of whole plant powder of Mimosa pu-
dica L.
5. CONCLUSIONS
A new LC-MS-MS method has been developed for
quantification of Mimosine from whole plant powder of
Mimosa pudica L. The method developed with careful
validation was found to be fast, simple, precise, sensitive
and accurate. The linearity, precision, accuracy of the
method prove that the method is easily reproducible in
any quality control set-up provided all the parameters are
followed accurately.
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