ISSN 0975-2366

Research Article https://doi.org/10.31838/ijpr/2019.11.02.092

Development and Validation of First Order

Derivative Spectrophotometric Method for
Simultaneous Estimation of Rutin Trihydrate and
Trigonelline Hydrochloride in Antidiabetic Herbal
Formulation
PALAK CHAUDHARY* 1 , DR. HARSHA U. PATEL 2
1
Research Scholar, Hemchandracharya North Gujarat University, Patan, Gujarat, India.
2
Shri Satsangi Saketdham “Ram Ashram”, Group of Institutions, Faculty of Pharmacy, Vadasma, Mehsana,
Gujarat, India.
Email id : palakc24@gmail.com
Received: 26.04.19, Revised: 26.05.19, Accepted: 26.06.19

ABSTRACT
A simple, fast, accurate, reliable and economical first order derivative spectrophotometric method was developed for
the simultaneous determination of Rutin trihydrate (RUT) and Trigonelline hydrochloride (TRG) in herbal
formulation. Rutin and Trigonelline are present as markers in antidiabetic herbal formulations to prevent and manage
diabetes. The Zero crossing point (ZCP) of markers was determined at which no interference from the other
marker. The developed method gives best results in terms of linearity, accuracy, precision, limit of detection (LOD)
and limit of quantification (LOQ). The method was validated as per ICH guidelines. The linearity range for Rutin
trihydrate and Trigonelline hydrochloride were found to be 5 to 25 μg/ mL and 5 to 25 μg/mL respectively. The
values of LOD were 0.50 μg/ mL and 0.18 μg/mL and the values of LOQ were found to be 1.50 μg /mL and 0.55 μg/
mL for Rutin trihydrate and Trigonelline hydrochloride respectively.

Keywords: Rutin trihydrate (RUT), Trigonelline hydrochloride (TRG), First order derivative spectroscopy, Validation

INTRODUCTION
Diabetes mellitus (DM) is a group of metabolic
disorder characterized by hyperglycemia resulting
from the defects in insulin secretion, insulin action or
both. A chronic hyperglycemic condition in diabetes
is associated with long term damage, dysfunction,
and failure of various organs, such as eyes, kidneys,
nerves, heart and blood vessels. It is the most
common serious metabolic disorder and is
considered to be one of the five leading causes of
death in the world. DM is classified into two major
categories: type 1 and type 2 diabetes. Type 2 DM is
a chronic and progressive syndrome characterized by
metabolic abnormalities such as insulin resistance
and decreased pancreatic β-cell function that
modifies fuel-sensing processes in the body.1-5Rutin
and Trigonelline are markers present in many
antidiabetic herbal formulations used to prevent and
manage DM. Rutin is a polyphenolic bioflavonoid
and has wide range of pharmacological applications
due to its significant antioxidant properties. Rutin has
multispectrum pharmacological benefits for the
treatment of various chronic diseases such as cancer,
diabetes, hypertension and hypercholesterolemia.6-8
Trigonelline, a major alkaloid component of
fenugreek, is reported to be responsible for most of
its pharmacological activities. Fenugreek (Trigonella
foenum graecum) is one of the most widely used
medicinal plants in folk medicine. It is known to have
diuretic, cardio tonic, hypotensive, hypoglycemic and
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hypolipidemic effect.9 From the literature it is
revealed that various analytical methods for the
determination of RUT and TRG have been reported,
which include Determination of Rutin in Buckwheat
Tea and Fagopyrum tataricum Seeds by HPLC and
Capillary Electrophoresis 10, Estimation of rutin and
quercetin in Terminalia chebula by HPLC11, HPLC
method for the quantification of three flavonoids in a
crude extract of Dimorphandra gardneriana12, RP-
UFLC Method for Simultaneous Estimation of
Quercetin and Rutin13, Development and Validation
of UV Spectrophotometric Method for Simultaneous
Estimation of Rutin and gallic acid in Hydroalcoholic
Extract of Triphala Churna14, development and
validation of HPLC method for the simultaneous
estimation of quercetin and rutin in aganosma
dichotoma [roth] k. Schum15, HPLC method for
quantification of trigonelline16-18, HPTLC
method. 19,20
A comprehensive literature survey
revealed that there is no suitable method reported for
simultaneous estimation of Rutin trihydrate and
Trigonelline hydrochloride in synthetic mixture and
polyherbal formulation.
Materials and methods
RUT and TRG standard markers were procured from
Sigma Aldrich and herbal formulation (Gaia Herbs
from Glycimic Health) was purchased from a local
pharmacy. AR grade methanol (Ranchem) was used
 Palak et al / Development and Validation of First Order Derivative Spectrophotometric Method

for development purpose.Spectroscopic Analysis was Selection of wavelength

carried out on a UV/VISIBLE 2450 (Shimadzu) double
beam UV-Visible spectrophotometer with software of
UV Probe version 2.34. The zero order absorption
spectra were recorded over the wavelength range of
200-400 nm, against solvent blank, in quartz
Cuvettes with 1 cm diameter. A Semi micro analytical
balance (Sartorius CD2250, Germany) was used for
weighing purpose. Overlay in zero order spectra of
10 µg/ml RUT and 10 µg/ml TRG using methanol as
solvent (1:1 Ratio) were taken (Fig. 3).
Preparation of standard solutions
Accurately weighed RUT (10 mg) and TRG (10 mg)
were individually transferred to a 100 mL
volumetric flask, dissolved in and diluted to the mark
with methanol to obtain a standard stock solution
(100 µg/ml).
The zero order spectra of RUT (10µg/ml), TRG
(10µg/ml) and their standard mixture (10µg/ml) were
taken in the range of 200-400 nm (Fig.3). The
spectra for first order derivative method were
scanned between 200-400 nm. For determination of
TRG, wavelength of 258 nm was selected where no
interference of RUT (zero absorbance of RUT). For
determination of RUT, wavelength of 348 nm was
selected where no interference of TRG (zero
absorbance of TRG). Figure 3 & 4 shows the
spectroscopic graphs for 10µg/ml RUT, 10µg/ml
TRG and 10µg/ml of their standard mixture using
methanol as solvent.

Fig. 1: Structure of Rutin trihydrate (RUT)*

Fig. 2: Structure of Trigonelline hydrochloride (TRG)*

Method Validation Precision

Linearity
The linearity response was determined by analyzing
5 independent levels of calibration curve in the range
of 5-25μg/ml and 5-25μg/ml for RUT and TRG
respectively (n=6). A first order derivative spectrum
measured the absorbance at 348nm for RUT and at
258 nm for TRG against a reagent blank solution
(Methanol). The calibration curve of absorbance vs
concentration was plotted and regression coefficients
and equations were derived for the same.
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Intraday precision
The mixed solution containing concentrations 10, 15,
20 μg/ml for RUT and 10, 15, 20 μg/ml for TRG was
analysed with three replicate (n=3) each on same
day and %RSD was calculated. Intraday precision
data presented in Table 3.
Interday Precision
The mixed solution containing concentrations 10, 15,
20 μg/ml for RUT and 10, 15, 20 μg/ml for TRG was
analysed with three replicate (n=3) per day for
consecutive 3 days and %RSD was calculated.
Interday precision data presented in Table 4.
 Palak et al / Development and Validation of First Order Derivative Spectrophotometric Method

Accuracy determined and % Recovery was calculated as shown

Take Synthetic powder equivalent to 10mg of RUT in Table 5.
and TRG in 100ml each of four volumetric flasks. Limit of Detection and Limit of Quantitation
Amount of standard drug equivalent to 80%, 100%, The Limit of detection and quantitation of the
120% spiked into flask 2, 3, 4 and flask 1 remain as developed Method was assessed by analyzing five
a control. Dissolve it in 25ml methanol. Sonicate for replicates of standard solutions containing
15 min and make upto the mark with methanol. concentrations 10 μg/ml for RUT and 10 μg/ml TRG.
Shake vigorously and filter the solution. Finally the The LOD and LOQ were calculated as LOD =
solution had the concentration 100μg/ml and 3.3*σ/S, and LOQ = 10*σ/S, where σ is the
100μg/ml respectively for RUT and TRG. After that standard deviation of the lowest standard
from this solution 1ml was pipette out and diluted up concentration and S is the slope of the standard
to 10 ml with methanol. So the concentration was curve. % RSD was calculated.
10μg/ml and 10μg/ml for RUT and TRG respectively. Analysis of Formulation
Data from the determination over three For Synthetic Mixture
concentration levels covering the specified range was The synthetic Mixture is prepared as
Table 1: Composition of Synthetic Mixture
Sr. no. Ingredients Amount(mg)
1 Rutin Trihydrate 10
2 Trigonelline Hydrochloride 10
3 Talc q.s.
Total 50

Take Synthetic powder equivalent to 10 mg of RUT
and TRG in 100 ml volumetric flask. Dissolve it in
25ml methanol, sonicate for 15 min and make upto
the mark with methanol. Shake vigorously and filter
the solution. Finally the solution had the
concentration 100μg/ml and 100μg/ml respectively
for RUT and TRG. After that from this solution 1ml
was pipette out and diluted up to 10 ml with
methanol. So the concentration was 10μg/ml and
10μg/ml for RUT and TRG respectively. RUT and TRG
were measured and assay was calculated (Table 6).
For Glycemic Health capsules (Gaia Herbs)
Take the capsules and remove content from it. From
that take 5 gm of powder formulation and dissolve in
50 ml of methanol. Macerate for 1 hour. Sonicate for
60 min at room temperature. Filter the extract and
estimate the content of Trigonelline and Rutin present
in formulation (Table 7).

Fig. 3: Overlain zero order spectra of RUT (10µg/ml), TRG (10µg/ml) and their standard mixture
(10µg/ml) using Methanol as solvent
0 . 0 3 6

R U T + T R I
0 . 0 2 0

0 . 0 0 0
T R I
R U T
A bs.

-0 . 0 2 0

-0 . 0 4 0

-0 . 0 5 3
2 0 0 . 5 6 2 5 0 . 0 0 3 0 0 . 0 0 3 5 0 . 0 0 4 0 0 . 0 0
n m .

Fig. 4: First Order spectra of RUT (10µg/ml), TRG (10µg/ml) and their standard mixture (10µg/ml) using
Methanol as solvent

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 Palak et al / Development and Validation of First Order Derivative Spectrophotometric Method

Fig. 5: Overlain First Order spectra of RUT and TRG in Ratio (1:1)

Fig. 6: Calibration curve of Trigonelline Hydrochloride

Fig. 7: Calibration curve of Rutin Trihydrate

Table 2: Linear Regression Data (n=6)

Parameters Rutin Trihydrate Trigonelline Hydrochloride
Wavelength (nm) 348 nm 258 nm
Linearity 5-25 µg/ml 5-25 µg/ml
Linear regression equation a
Intercept (c) 0.0003 0.0005
Slope (m) 0.0004 0.002
2
Correlation coefficient (R ) 0.999 0.999
LOD 0.50 0.18
LOQ 1.50 0.55
a
y=mx+c

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 Palak et al / Development and Validation of First Order Derivative Spectrophotometric Method

Table 3: Intraday precision data for estimation of RUT and TRG*(n=3)]

Conc. (µg/ml) Absorbance at Absorbance at

Sr. No. %RSD %RSD
258nm 348nm

1 10 0.020 0.050 0.008 0.075

2 15 0.030 0.019 0.011 0.051

3 20 0.040 0.025 0.016 0.037

Table 4: Interday precision data for estimation of RUT and TRG*(n=3)]

Absorbance at Absorbance at
Sr. No. Conc. (µg/ml) %RSD %RSD
258nm 348nm

1 10 0.020 0.028 0.008 0.081

2 15 0.029 0.020 0.011 0.051

3 20 0.041 0.014 0.016 0.037

Table 5: Recovery Data for Accuracy (n=3)

Sr. Level of Conc. Amount of Amount found % Recovery ± SD
No. Spiking Taken standard added (µg/ml)
(µg/ml) (µg/ml)
RUT TRG RUT TRG RUT TRG

1 Control 10 - - 10.07 10.02 100.67±0.401 100.20±0.360

2 80% 10 8 8 18.01 18.02 100.07±0.170 100.11±0.222

3 100% 10 10 10 20.01 20.01 100.03±0.126 100.05±0.132

4 120% 10 12 12 22.02 22.02 100.09±0.164 100.09±0.182

Table 6: Assay Recovered of Synthetic Mixture

Sr. No. Drugs % Assay

1 Rutin Trihydrate 99.92±0.761

2 Trigonelline Hydrochloride 100.50±0.995

Table 7: Result of analysis of formulation

Formulation Drug Amount found (mg/100gm)

Rutin Trihydrate 18.5

Glycemic Health
(Gaia Herbs)
Trigonelline Hydrochloride 10.5

Result And Discussion

RUT and TRG obeyed the Beer’s law between the with no other interference (Fig. 4-5). Finally, the
range of 5-25 µg/ml and 5-25 µg/ml respectively. developed first derivative method was validated for
The first order derivative graph for the same gives different validation parameters. The results for the
good resolution with easy sample preparation and same are presented in above figures and table 2-7.

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 Palak et al / Development and Validation of First Order Derivative Spectrophotometric Method
The lower value of RSD shows the method is accurate
and precise.
Conclusion
In summary, the method developed is rapid,
sensitive, specific, accurate and repeatable. It
possesses significant linearity, precision within
acceptable range of RSD and no interference from
the excipients. As no method is available for
combined estimation, this method can be used for
the routine QC analysis of Antidiabetic herbal
formulation having RUT and TRG. The method is
simple, inexpensive, requires an easy sample
preparation and gives reliable results for estimation.
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