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Development and Validation of an HPTLC-Densitometric


Method for the Estimation of Trigonelline in the Leaves of
Abrus precatorius L. and Its Formulation

Article  in  Acta Chromatographica · September 2012


DOI: 10.1556/AChrom.24.2012.3.13

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Acta Chromatographica 24(2012)2, 263–270
DOI: 10.1556/AChrom.24.2012.2.9

Development and Validation of an HPTLC–


Densitometric Method for the Estimation of
Trigonelline in the Leaves of Abrus precatorius L.
and Its Formulation
B.B. KUMBHALKAR, A.A. RAJOPADHYE, AND A.S. UPADHYE*
Botany Group, Plant Science Division, Agharkar Research Institute,
G.G. Agarkar Road, Pune 411 004, India
E-mail: upadhye.anuradha@gmail.com

Summary. A new, simple, and reproducible HPTLC–densitometric method has been es-
tablished and validated for estimation of trigonelline in the leaves of Abrus precatorius L.
and its herbal formulation Chatak® using ICH guidelines. Accelerated solvent extraction
(ASE) as an alternative to conventional techniques was also explored for rapid extrac-
tion. The methanol extracts of leaves, its formulation, and standard solution of trigonel-
line were applied on silica gel F254 HPTLC plates and developed in a twin chamber using
the mobile phase toluene–ethyl acetate–formic acid–methanol (2:3:1.8:3.8, v/v/v/v). The
plates were scanned at 268 nm (λmax of trigonelline) using a Camag TLC scanner 3 with
the CATS 4 software. A linear relationship was obtained between the response (peak
area) and the amount of trigonelline in the range 40–200 ng per spot; the correlation coef-
ficient was 0.9957. The ASE method gives higher extraction efficiency in less time com-
pared to Soxhlet extraction. The HPTLC–densitometric method showed good linearity,
recovery, and high precision. It is useful to analyze trigonelline in A. precatorius leaves
and routine quality control of its marketed formulation.

Key Words: trigonelline, Abrus precatorius L., accelerated solvent extraction, HPTLC–
densitometry, ICH guidelines

Introduction
Abrus precatorius L. (Fabaceae) is an important medicinal plant commonly
known as “Indian liquorice” and found throughout India. In Indian tradi-
tional systems of medicine, leaves of A. precatorius are used for the treat-
ment of gonorrhea, jaundice, biliousness, hemoglobinuric bile, leucoderma,
itching, skin diseases, etc. [1–3]. Pharmacological activities such as anti-
inflammatory [4], antimicrobial [5], neuromuscular [6], and toxicity studies
[7] have been reported for its leaves. It is also a major ingredient of many
marketed polyhedral digestive mixtures such as Chatak®.
Leaves are the foremost source of an alkaloid trigonelline [8] having
diverse pharmacological activities such as hypoglycemic, hypocholes-
terolemic, antiseptic, antimigraine, antitumor, mutagenic, and osmoregula-
tory [9]. Hence, the active principal trigonelline has been used as a marker
0231–2522 © 2012 Akadémiai Kiadó, Budapest
264 B.B. Kumbhalkar et al.

compound for quality control and standardization of the A. precatorius


leaves and its formulations.
A perusal of the literature showed no reports on any analytical meth-
ods for the analysis of trigonelline in the leaves of A. precatorius, its extracts,
or its commercial formulations. The present study is focused on the devel-
opment and validation of a simple and precise high-performance thin-layer
chromatography (HPTLC)–densitometry method for the analysis of trigo-
nelline in a methanol extract of A. precatorius leaves as well as its commer-
cial product Chatak® using the ICH guidelines [10]. In addition to this, the
applicability of accelerated solvent extraction (ASE) as an alternative
to Soxhlet extraction was also explored for the rapid and exhaustive
extraction.

Experimental
Plant Material
The leaves of A. precatorius were collected during the winter of 2009 from
Pune, India. The plant sample was identified, authenticated, and deposited
in the crude drug repository of Agharkar Research Institute, Pune (Voucher
specimen number L-049). The sample was powdered, passed through an 80-
mesh sieve, and stored in an airtight container at 25°C for further studies.

Chemicals
Standard trigonelline (CAS 535-83-1; purity ≥98.0%) was purchased from
Fluka-Aldrich Chemical (Steinheim, Germany). Silica gel F254 HPTLC plates
were purchased from Merck (Darmstadt, Germany). Chatak®, a digestive
mixture (Aishwarya Products, Pune, India), was procured from the local
market. Other analytical-grade solvents and reagents were obtained from
SD Fine Chemicals (Mumbai, India).

Preparation of Sample Solutions

Soxhlet Extraction
Accurately weighted (2.5 g) powdered leaves and the Chatak® sample were
extracted exhaustively with methanol (50 mL) using a Soxhlet apparatus for
6–10 h. The optimum yield was obtained in methanol with an extraction
time of 8 h. The extracts were concentrated under reduced temperature and
Development of an HPTLC–Densitometric Method 265

pressure using a rotary evaporator. The yields of the methanol extract of the
leaves and Chatak® samples were 0.9475 and 0.67 g, respectively.

Accelerated Solvent Extraction

Powdered leaves and the Chatak® sample (2.5 g) were extracted with
methanol and placed in the stainless steel cell of a Dionex ASE™ 100 system
(Sunnyvale, CA, USA). The extraction was performed at 100 bar and at
three temperatures, 60, 80, and 100°C, for 15 min (1 cycle). The optimum
yield was obtained at 80°C. In the experiment at 80°C, five replicate cycles
were performed. The extracts were concentrated under reduced tempera-
ture and pressure using a rotary evaporator. The yields of the methanol ex-
tract of the leaves and the Chatak® sample were 0.969 and 0.703 g, respec-
tively.

Chromatography

HPTLC was performed on aluminum-backed HPTLC plates 10 × 10 cm


coated with a 0.2-mm layer of silica gel 60 F254 (E. Merck, Germany). Sam-
ples were applied to the plate with a band width 6 mm using a Linomat IV
sample applicator (Camag, Switzerland) fitted with a microliter syringe.
Linear ascending development of the plates to a distance of 80 mm was per-
formed with the mobile phase toluene–ethyl acetate–formic acid–methanol
(2:3:1.8:3.8, v/v/v/v) in a twin-trough glass chamber previously saturated
with mobile phase vapor for 10 min at 25°C. The dried plate was scanned at
a wavelength of 268 nm (λmax of trigonelline) using a Camag TLC scanner 3
with the CATS 4 software.
A variety of mobile phases were tried for the analysis of trigonelline in
the methanolic extracts of leaves and the commercial formulation. This in-
cluded toluene–ethyl acetate–formic acid (5:3:2, v/v/v), toluene–ethyl ace-
tate–formic acid–methanol (4:3:1.5:1.5, v/v/v/v), toluene–ethyl acetate–
methanol (4:3:1:2, v/v/v), and toluene–ethyl acetate–formic acid–methanol
(2:3:1.8:3.8, v/v/v/v).
For the calibration curve, a standard stock solution (1 mg mL–1) was
prepared by dissolving 10 mg accurately weighted trigonelline in methanol
and diluting it to 10 mL in a volumetric flask. Working standard trigonel-
line solutions of 2, 4, 6, 8, and 10 μg mL–1 of different concentrations of 40,
80, 120, 160, and 200 ng, respectively, were prepared by diluting the stock
solution. Each solution (20 μL) was applied on the plate, which was devel-
oped under predetermined conditions as described above. The procedure
266 B.B. Kumbhalkar et al.

was repeated thrice to plot a graph of the response (peak area) versus the
amount of trigonelline.

Quantification of Trigonelline

Suitably diluted solutions of test samples (10 μL) were applied in triplicate
on an HPTLC plate along with the standard. The plate was developed un-
der predetermined conditions as described above and scanned at 268 nm
(λmax of trigonelline). The peak areas were recorded, and the trigonelline
content in the samples was calculated using the calibration plot.

Validation of the Method


The method was validated according to the ICH guidelines [10] by deter-
mining the peak purity, limit of detection (LOD), limit of quantitation
(LOQ), precision, and recovery of trigonelline from the samples. LOD and
LOQ were determined by diluting known concentrations of standard stock
solution until the average responses were approximately 3 or 10 times the
response of blank.
Instrument precision was checked by repeated scanning of the trigonel-
line band (160 ng) six times and expressed as relative standard deviation
(%RSD). Precision was studied by analyzing six bands of sample solutions
per plate on three plates (intraday precision) and six bands of sample solu-
tion per plate on three consecutive days (interday precision) at three differ-
ent amounts (80, 120, and 160 ng) and calculating the %RSD. The accuracy
of the method was tested by determining the recovery at three levels. Pre-
analyzed samples were spiked with extra trigonelline (50%, 100%, and
150%) and the mixtures were reanalyzed. The robustness of the method was
studied at three different concentrations, namely, 80, 120, and 160 ng per
band trigonelline, by introducing small, deliberate changes in the
mobile phase composition toluene–ethyl acetate–formic acid–methanol
(2.2:2.8:1.6:3.6, 1.8:3.1:2:3.6, 2.1:2.9:1.9:3.5, v/v/v/v). The repeatability of the
method was assessed by the analysis of 120 ng per band of standard solu-
tion of trigonelline (n = 6) and expressed as %RSD of the peak areas. Per-
centage recovery and standard deviation (SD) were calculated for each con-
centration level. LOD and LOQ were determined by the SD method from
the slope (S) of the calibration plot and the SD of a blank sample (blank
methanol spotted three times) by use of the equations: LOD = 3.3 × SD/S
and LOQ = 10 × SD/S.
Development of an HPTLC–Densitometric Method 267

Results and Discussion


For the development of a rapid sample preparation method, along with
Soxhlet, ASE was studied. ASE has been applied for the first time for the ex-
traction of trigonelline in A. precatorius leaves. ASE at 80°C gave almost the
same results in 15 min as Soxhlet in 8 h. Of the various solvent systems
tried, the one containing toluene–ethyl acetate–formic acid–methanol
(2:3:1.8:3.8, v/v/v/v) gave optimal result with the sharp, symmetrical, and
well-resolved peak of trigonelline at RF 0.19 from other components of the
sample extract (Fig. 1). A linear relationship was obtained between the re-
sponse (peak area) and amount of trigonelline in the range 40–200 ng
per spot; the correlation coefficient was 0.9957 (y = 309.78x + 801.85).
The amount of trigonelline was found in the range 4.21–4.46 and 0.083–
0.088 mg g–1 using ASE-extracted leaf samples and the herbal product
Chatak® at 80ºC, whereas Soxhlet-extracted yield of trigonelline was 4.5–
4.67 and 0.086–0.091 mg g–1. The compound yield of Soxhlet extraction was
found to have almost similar efficiency. However, taking into consideration
the time of extraction and solvent consumption, ASE proved to be a promis-
ing alternative to Soxhlet.

Fig. 1. HPTLC chromatograms of leaves of A. precatorius and the herbal product


methanolic extract along with standard trigonelline: (A, B) standard trigonelline
solution; (C) methanolic Soxhlet extract of the leaves; (D) methanolic Soxhlet extract of
Chatak®; (E) methanolic ASE 80°C extract of the leaves; (F) methanolic ASE extract
at 80°C of Chatak®
268 B.B. Kumbhalkar et al.

The method was validated in terms of peak purity, precision, LOD,


LOQ, and accuracy (Tables I–III). The method was specific for the analysis of
the active principal trigonelline because it resolved the compound at RF
0.19. The purity of the trigonelline peak was checked from the samples by
recording the UV spectra. The identified trigonelline spot was confirmed
from the sample extract by overlaying the UV absorption spectrum of the
samples with that of the standard at 268 nm (Fig. 2). The instrument preci-
sion was studied by scanning the same spot of trigonelline six times
(%RSD = 0.51). Intraday and interday precision were studied by triplicate
assay of three different concentrations of trigonelline (80, 120, 160 ng per
band) on the same day and on different days. The low RSD values (Table II)
confirmed that the method was precise. Small changes in the mobile phase
composition had no significant effect on the chromatographic results. The
low RSD values of the peak areas calculated indicate the robustness of the
method (Table I). The accuracy of the method was determined at three levels
(50%, 100%, and 150%) by adding known amounts of trigonelline to the
sample extract. Recovery of trigonelline at the three levels is presented in
Table III. The high recovery indicates that the proposed method is reliable
and reproducible. The LOD and LOQ were 13.33 and 40 ng per spot, respec-
tively.

Fig. 2. Overlay of the UV absorption spectra of standard trigonelline solution


Development of an HPTLC–Densitometric Method 269

Table I. Validation data of HPTLC method for the estimation of trigonelline

Instrumental precision (%RSD, n = 6) 0.51


Calibration range (ng per spot) 40–200
Regression equation y = 309.78x + 801.85
Correlation coefficient 0.9957
Repeatability of standards (%RSD, n = 6) 0.67
Repeatability of samples (%RSD, n = 6) 0.93
Limit of detection (LOD) (ng per spot) 13.33
Limit of quantitation (LOQ) (ng per spot) 40
Robustness (%RSD, n = 3) 0.81

Table II. Results from study of intraday and interday precision for trigonelline

Concentration (ng per band) Intraday (%RSD, n = 6) Interday (%RSD, n = 6)

80 1.09 0.94
120 0.89 0.83
160 1.18 1.23

Table III. Recovery study of the method for trigonelline


Amount of Amount of
Amount of Average
trigonelline trigonelline Recovery
Compound trigonelline recovery
in sample found in (%)a
added (ng) (%)
(ng)a mixture (ng)a
Leaves methanol extract (SX)
256 ± 8.33 128 378.65 ± 4.33 98.67 ± 2.22
Trigonelline 256 ± 8.33 256 483.58 ± 5.87 94.45 ± 3.45 96.45
256 ± 8.33 384 615.87 ± 9.03 96.23 ± 7.85
Chatak® methanol extract (SX)
89 ± 2.12 44.5 125.46 ± 3.84 94.34 ± 0.25
Trigonelline 89 ± 2.12 89 169.06 ± 4.23 94.98 ± 2.45 95.29
89 ± 2.12 133.5 214.34 ± 3.08 96.55 ± 1.56
Leaves methanol extract (ASE 80°C)
256 ± 8.33 128 367.37 ± 2.34 95.67 ± 2.35
Trigonelline 256 ± 8.33 256 485.88 ± 3.98 94.90 ± 1.56 95.23
256 ± 8.33 384 608.76 ± 2.45 95.12 ± 2.56
Chatak® methanol extract (ASE 80°C)
89 ± 2.12 44.5 122.98 ± 2.34 92.67 ± 5.67
Trigonelline 89 ± 2.12 89 171.28 ± 4.14 96.23 ± 2.53 94.91
89 ± 2.12 133.5 213.35 ± 3.67 95.89 ± 2.5

aMean ± SD (n = 3)
270 B.B. Kumbhalkar et al.

Conclusion
A new, simple, accurate, precise, reproducible, and sensitive HPTLC–
densitometric method has been established for the determination of
trigonelline in leaves of A. precatorius and its commercial product Chatak®.
Comparison of the extraction yields of the trigonelline reveals that ASE is a
simple and efficient method which is helpful for standardization and rou-
tine quality control of raw materials and herbal products containing A. pre-
catorius leaves as ingredients. It provides significant advantages in terms of
greater specificity and rapid analysis.

Acknowledgments
The authors are grateful to the Director and the In-charge, Botany Group,
Agharkar Research Institute, Pune, India, for providing the facilities and en-
couragement throughout the work.

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