Development and Validation of First Order Derivative Spectrophotometric

Method for Simultaneous Estimation of Rutin Trihydrate and Trigonelline

Hydrochloride in Antidiabetic Herbal Formulations

PALAK CHAUDHARY*1,, DR. HARSHA U. PATEL2

1
Research Scholar, Hemchandracharya North Gujarat University, Patan, Gujarat, India.
2
Shri Satsangi Saketdham “Ram Ashram”, Group of Institutes, Faculty of Pharmacy,
Vadasma, Mehsana, Gujarat, India.
Email: palakc24@gmail.com
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 synthetic mixture and in herbal formulation. Rutin and
Trigonelline from herbal plants are used as antidiabetic drug to prevent and manage Diabetes.
The Zero crossing point (ZCP) of markers were 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.1-5
Rutin and Trigonelline from herbal plants are used as antidiabetic drug 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
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
10
Fagopyrum tataricum Seeds by HPLC and Capillary Electrophoresis , 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.
 Fig. 1: Structure of Rutin trihydrate (RUT)*

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

METERIALS AND METHODS

RUT and TRG standard markers were procured from Sigma Aldrich and herbal formulation
(Gaia Herbs from Glycimic Health) was procured from a local pharmacy. AR grade methanol
(Ranchem) was used for development purpose.
Spectroscopic Analysis was 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).
Selection of wavelength
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.

METHOD VALIDATION
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 zero order
derivative spectrum measured the absorbance at 348nm for Rutin Trihydrate and at 258 nm
for Trigonelline Hydrochloride 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.
Precision
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.
Accuracy
Take Synthetic powder equivalent to 10mg of RUT and TRG in 100ml each of four
volumetric flasks. Amount of standard drug equivalent to 80%, 100%, 120% spiked into flask
2, 3, 4 and flask 1 remain as a control. 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. Data from the
determination over three concentration levels covering the specified range was determined
and % Recovery was calculated as shown in Table 5.
Limit of Detection and Limit of Quantitation
The Limit of detection and quantitation of the developed Method was assessed by analyzing
10 replicates of standard solutions containing concentrations 10 μg/ml for RUT and 10 μg/ml
TRG. The LOD and LOQ were calculated as LOD = 3.3*σ/S, and LOQ = 10*σ/S, where σ is
the standard deviation of the lowest standard concentration and S is the slope of the standard
curve. % RSD was calculated.
Analysis of Formulation
For Synthetic Mixture:
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 10mg of RUT and TRG in 100ml 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 Gaia Herbs (capsules):
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
Abs.

-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

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)

Trigonelline
Parameters Rutin Trihydrate
Hydrochloride
Wavelength (nm) 348 nm 258 nm
Linearity 5-25 µg/ml 5-25 µg/ml
a
Linear regression equation
Intercept (c) 0.0003 0.0005
Slope (m) 0.0004 0.002
Correlation coefficient
0.999 0.999
(R2)
LOD 0.50 0.18
LOQ 1.50 0.55
a
y=mx+c
 Table 3: Intraday precision data for estimation of RUT and TRG*(n=3)]
Sr. Conc. (µg/ml) Absorbance Absorbance at
%RSD %RSD
No. at 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)]

Sr. No. Conc. (µg/ml) Absorbance Absorbance at

%RSD %RSD
at 258nm 348nm
1 10 0.020 0.028 0.008 0.001
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 Conc. Amount of Amount % Recovery ± SD

No. of Taken standard found (µg/ml)
Spiking (µg/ml) added
(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 forund (mg/100gm)
Rutin Trihydrate 10.5
Gaia Herbs
Trigonelline Hydrochloride 18.5

RESULT AND DISCUSSION

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

REFERENCES
1. Chandramohan G, Ignacimuthu S, Pugalendi KV, A novel compound from casearia esculenta
(roxb.) root and its effect on carbohydrate metabolism in streptozotocin-daibetic rats.
European Journal of Pharmacology, 2008; 590(1-3): 437-443.
2. S. Ramachandran, K. Asokkumar, M. Uma Maheswari, et al., Investigation of Antidiabetic,
Antihyperlipidemic, and In Vivo Antioxidant Properties of Sphaeranthus indicus Linn. in
Type 1 Diabetic Rats: An Identification of Possible Biomarkers. Evidence-Based
Complementary and Alternative Medicine, 2011; 1-8.
3. Velingkar VS, Dandekar VD, Murugananthan K, Synthesis and pharmacological evaluation
of some novel potent type 2 antidiabetic agents. International Journal of Pharmacy and
Pharmaceutical Science, 2009; 1(1): 149-158.
4. Bhat M, Zinjarde SS, et al, Antidiabetic Indian Plants: A Good Source of Potent Amylase
Inhibitors. Evidence-Based Complementary and Alternative Medicine, 2011; 6.
5. Dorota Zozulinska, Bogna Wierusz-Wysocka, Type 2 diabetes mellitus as inflammatory
disease. Diabetes Research and Clinical Practice, 2006; 74(2): S12-S16.
6. Niture NT, Ansari AA, Naik SR, Antihyperglycemic activity of Rutin in streptozocin induced
diabetic rats: An effect mediated through cytokines, antioxidants and lipid biomarkers. Indian
Journal of Experimental Biology, 2014; 52: 720-727.
7. Sharma S L, Ali A, Ali J, Sahani J K, Baboota S, Rutin: therapeutic potential and recent
advances in drug delivery. Expert Opinon On Investigational Drugs, 2013; 22(8):1063-79.
8. Vinayagam R and Xu Baojun, Antidiabetic properties of dietary flavonoids: a cellular
mechanism review. Nutr metab (lond), 2015; 12: 60.
9. Sorimuthu Pillai Subramanian, Gopalan Sriram Prasath, Antidiabetic and antidyslipidemic
nature of trigonelline, a major alkaloid of fenugreek seeds studied in high-fat-fed and low-
dose streptozotocin-induced experimental diabetic rats. Biomedicine and Preventive
Nutrition, 2014; 4: 475-480.
10. Vachirapatama N, Chamnankid B, Kachonpadungkitti Y, Determination of Rutin in
Buckwheat Tea and Fagopyrum tataricum Seeds by High Performance Liquid
Chromatography and Capillary Electrophoresis. Journal of Food and Drug Analysis, 2011;
19(4): 463-469.
11. Kumar A, Lakshman K, Jayaveera K, Satish K, Tripathi S, Estimation of rutin and quercetin
in Terminalia chebula by HPLC. The Internet Journal of Aesthetic and Antiaging Medicine,
2008; 2: 1.
12. Leonardo P. Landim et al, Development and validation of a HPLC method for the
quantification of three flavonoids in a crude extract of Dimorphandra gardneriana. Brazilian
Journal of Pharmacognosy, 2013; 23(1): 58-64.
13. R Shanmugam, K Gowthamarajan, DL Priyanka, et al., Development and Validation of a RP-
UFLC Method for Simultaneous Estimation of Quercetin and Rutin. Hygeia: Journal for
Drugs and Medicines, 2013; 5(1): 113-120.
14. Pawar NP, Salunkhe VR, Development and Validation of UV Spectrophotometric Method
for Simultaneous Estimation of Rutin and Gallic Acid In Hydroalcoholic Extract of Triphala
Churna. International Journal of Pharmtech Research, 2013; 5(2): 724-729.
15. G Subramanian, Meyyanathan SN, et al., Development and validation of HPLC method for
the simultaneous estimation of quercetin and rutin in Aganosma dichotoma [Roth] K. Schum.
International Journal of Pharmacy and Pharmaceutical Sciences, 2014; 6(2): 606-608.
16. Sathe P, Dighe V, HPLC method development and validation for quantitation of Trigonelline
from Mirabilis Jalapa Linn. leaves and enhancement in extraction yield from ultra fine
powder. International journal of current pharmaceutical research. 2017; 9(1): 0975-7066.
17. Shailajan S, Menon S, Singh A, et al., A validated RP-HPLC method for quantitation of
trigonelline from herbal formulations containing Trigonella foenum-graecum (L.) Seeds.
Pharmaceutical methods, 2011; 2(3): 157-60.
18. Zhuo R, Wang L, Wang L, Xiao H, Cai S, Determination of trigonelline in Trigonella
foenum-graecum L. by hydrophilic interaction chromatography. Chinese journal of
chromatography, 2010; 28(4): 379-82.
19. Kalia et al., High Performance Thin Layer Chromatography method for simultaneous
estimation of vicine, trigonelline and withaferin-a in a polyherbal antidiabetic formulation.
International journal of pharmacy and pharmaceutical sciences, 2013; 5(2): 0975-1491.
20. Nandanwadkar S, Mastiholimath V, Surlaker, HPTLC Method Development and Validation
of Antidiabetic Marker Compound from Polyherbal Formulation. Indian Journal of
Pharmaceutical Education and Research, 2016; 50(4): 657-664.
21. ICH, Q2 (R1) Validation of Analytical Procedures: Text and Methodology, International
Conference on Harmonization of Technical Requirements for the Registration of
Pharmaceutical for Human Use, Geneva, Switzerland, 2005.