Validated analytical RP HPLC method for quantitation of trigonelline by alicejenny


									    Validated analytical RP-HPLC method for quantitation of trigonelline from
                     marketed herbal formulations containing
                       Trigonella foenum - graecum (Linn.).

         Sunita Shailajan1, Sasikumar Menon2, Ashish singh3* and Neelam Sayed3
Herbal Research Laboratory, Ramnarain Ruia College, Matunga (East), Mumbai-
          Therapeutic Drug Monitoring Laboratory, Sion (East), Mumbai- 400 022
                Ramnarain Ruia College, Matunga (East), Mumbai-400019
      Corresponding author:,
Trigonella foenum - graecum (Linn.) (Fam. – Papilionaceae) has been used as an
important ingredient of Ayurvedic and other marketed herbal formulations. The seeds
are used in many traditional systems as antibacterial agent, antidiabetic agent, gastric
stimulant, against anorexia and as galactogogue. In recent decades, several therapeutic
benefits of fenugreek seeds have been observed in animal studies as well as in human
trials. These include antidiabetic effect, hypocholesterolemic influence, antioxidant
potency, digestive stimulant action, and hepatoprotective effect. Trigonelline is one of
the major phytoconstituents found in Trigonella foenum - graecum and can be used as a
bioactive marker to establish the quality of the crude drug and its formulations.In the
present study, trigonelline-based standardization of Trigonella foenum - graecum and its
quantitation from marketed herbal formulations has been documented using RP-HPLC.
This method was validated as per ICH guidelines and was used to quantitate the content
of trigonelline in polyherbal formulations like Dibet powder and Amyron syrup. The LOD
was found to be 5.00ng/mL and the LOQ was 50.00ng/mL. The linearity range of
response was from 100.00ng/mL to 8000.00ng/mL. The validated method is found to be
sensitive, accurate, simple, reproducible and can be recommended for quality assurance
and marker-based standardization, of polyherbal formulations containing Trigonella
foenum - graecum.
Keywords:- Trigonella foenum - graecum (Linn.), Trigonellin, RP-HPLC, Dibet powder,
Amyron syrup.
1. Introduction:
       Trigonella foenum - graecum (Linn.) belonging to the family Papilionaceae
commonly known as Fenugreek is a aromatic, 30-60 cm tall, annual herb, cultivated
throughout the country. The seeds are hot, with a sharp bitter taste; tonic, antipyretic,
anthelmentic, increase the apetite, astringent to the bowels, cure leprosy, vomiting,
bronchitis, piles; remove bad taste from the mouth, useful in heart disease. The plant
and seeds are hot and dry, suppurative, aperient, diuretic, emmenagogue, useful in
dropsy, chronic cough, enlargement of the liver and the spleen. The leaves are useful in
external and internal swellings and burns; prevent the hair falling off. Fenugreek seeds
are considered carminative, tonic and aphrodisiac. Several confections made with this
the article are recommended for use in dyspepsia with loss of appetite, in the diarrhea of
puerperal women, and in rheumatism (Mehrafarin et al 2011, Toppo et al 2009). The
main chemical constituents of Trigonella foenum - graecum are fibers, flavonoids,
polysaccharides, saponins, flavonoids and polysaccharides fixed oils and some
identified alkaloids viz., trigonelline and choline (Toppo et al 2009).
       The current work is aimed at optimizing and validating a simple and reliable
HPLC method on a reversed-phase C-18 column with photodiode array detection for
monitoring the quality of Trigonella foenum – graecum seed and marketed formulation
(Dibet Powder and Amyron Syrup) (WHO 1991, EMEA 2001, USFDA 2004). In the
present paper a suitable analytical method for identification and quantification of
trigonelline from seeds of Trigonella foenum – graecum as well as from herbal
formulations (Dibet Powder and Amyron Syrup) has been developed.

2. Experimental
2.1 Materials and Reagents
       Reference standard of trigonelline were purchased from Natural remedies
(Bangalore, India). HPLC-grade methanol was purchased from Merck (Darmstadt,
Germany), and, ultra-pure water was prepared using a Milli-Q purification system
(Millipore S.A. Molsheim, France). The plant material i.e. seeds of Trigonella foenum -
graecum was procured from local market and get authenticated by one of the authors
Dr. Sunita Shailajan according to their morphological characteristics. The formulations
Dibet Powder and Amyron Syrup of Sheetal Medicare Products Pvt. Ltd (Batch No. 906)
and Aimil Pharmaceuticals (India) Ltd (Batch No. ARS-1443) respectively procured from

2.2 HPLC Analytical Conditions
       HPLC analysis was performed on a JASCO's HPLC system containing an PU-
980 liquid chromatography pump unit, a reversed-phase Cosmosil (Nacalai Tesque,
INC. Japan), Cosmosil CN-MS (250 mm x 4.6 mm, i.d.) with an extended guard column
of the same material, a autosampler (AS-1555-1 0) and a Photo diode array detector
(MD-910). The mobile phase consist of Methanol: Distilled water (95:5), pH adjusted to
3.5 using HCl. at a flow rate of 1.0 ml/min. Detection was carried out at 267 nm at room
temperature (270C ± 10C). The injection volume was 20µI for all HPLC runs. Data
acquisition and analysis were carried out using Borwin Integrator Software, version 1.21
chromatography analysis software.

2.3 Method Validation
       The developed RP-HPLC-DAD method was validated in terms of its selectivity,
peak purity, linearity, limits of detection (LOD), limits of quantification (LOQ), recovery
and precision, as per ICH guideline.

3. Results and Discussion
       Currently chemical markers or pharmacologically active components in
Polyherbal formulation are employed for evaluating the quality and authenticity of
Polyherbal formulation (Calixto et al 2000, Lianga et al 2004). Based on the experiments
done during the course of validation, it can be concluded that the intended method is
validated for the estimation of trigonelline from fruit of Trigonella foenum - graecum over
the concentration range of 100.00 ng/mL - 8000.00 ng/mL. The precision and accuracy
are within the acceptance limits. Consistent recoveries were observed for LQC, MQC
and HQC(Table 1). This method can be used for quantification of trigonelline from fruit of
Trigonella foenum - graecum and also from marketed formulations (Dibet Powder and
Amyron Syrup). This method was proved to be rugged for different Column, different
Analyst, different days, change in instrument, change in flow rate, change in injection
volume and mobile phase composition variation under specified conditions. The
developed method is rugged for quantification of trigonelline from fruit of Trigonella
foenum - graecum as well as from herbal formulations under specified limitations(Table
2). The content of trigonelline in seeds of Trigonella foenum - graecum was 98.36
mg/gm while formulations contains 0.025 mg/ml and 0.015 mg/gm in amyron syrup and
Dibet powder respectively(Table 3).

      We acknowledge the part financial assistance for the current work from NMPB,
AYUSH, Ministry of Health, Govt. of India.

1) USFDA, Guidance for Industry Botanical Drug Product, U.S. Food and Drug
Administration, Rockville, 2004.
2) EMEA, Note for Guidance on Quality of Herbal Medicinal Products, The European
Agency for the Evaluation of Medicinal Products, London, 2001.
3) WHO, Guidelines for the Assessment of Herbal Medicine World Health Organization,
Geneva, 1991.
4) Review-Quality control of herbal medicines, Yi-Zeng Lianga,., Peishan Xieb, Kelvin
Chanc ; Journal of Chromatography B, (2004); 812; 53–70.
5) Efficacy, safety, quality control, marketing and regulatory guidelines for herbal
medicines (phytotherapeutic agents) ; J.B. Calixto; Brazilian Journal of Medical and
Biological Research (2000) ; 33: 179-189.
6) Pharmacological actions and potential uses of Trigonella foenum-graecum: a review ;
Fedelic Ashish Toppo, Rachna Akhand, A.K. Pathak ; Asian Journal of Pharmaceutical
and Clinical Research (2009); 2 ; 4.
7) A Review on Biology, Cultivation and Biotechnology of Fenugreek (Trigonella foenum-
graecum L.) as a Valuable Medicinal Plant and Multipurpose ; Mehrafarin A , Rezazadeh
Sh , Naghdi Badi H, Noormohammadi Gh , Zand E, Qaderi A; Journal of Medicinal
Plants (2011); 10; 37.
Table 1. Summary of validation data

                Method characteristic             Trigonelline
      LOD                                5.0 ng/mL
      LOQ                                50 ng/mL
      Linear range (ng/mL)               100 ng/mL- 8000 ng/mL
      Mean Correlation coefficient (r)   0.9967
      Mean Slope                         22.412
      System suitability
         Retention Time(n=5) %CV         0.13
         Area(n=5) %CV                   1.05
      Precision %CV
         Within-Batch (n = 3)            1.14-1.73
         Between-Batch (n = 3)           1.69-1.96
      Recovery (%)
          LQC (n = 7)                    101.80
          MQC (n = 7)                    99.24
          HQC (n = 7)                    98.67
Table 2. Robustness/ Ruggedness

                                             %CV           %Difference
Change in Column
      Column 1 (n=3)                    0.05-1.62
                                                           -1.40 – 1.02
      Column 2 (n=3)                    0.56-1.53
Change in Analyst
       Analyst 1 (n=3)                  0.46-0.90
                                                           -0.94 - 1.09
       Analyst 2 (n=3)                  0.18-1.06
Change in Day
       Day1 (n=3)                       0.58 – 1.85
                                                           -1.44 – 0.84
      Day2 (n=3)                        0.44 – 1.30
Change in Instrument
      Instrument 1 (n=3)                0.77-1.36
                                                           -0.07- 0.39
      Instrument 2 (n=3)                0.39-1.04
Change in Flow rate
        0.95 ml/min (n=3)               0.92-1.82          -0.07- 1.96
        1.00 ml/min (n=3)               0.40-1.70
        1.05 ml/min (n=3)               0.61-1.48          -0.04- 2.05
Change in Injection volume
         15 µL (n=3)                    0.93-1.90          16.07-31.07
         20 µL (n=3)                    0.51-1.07
         25 µL (n=3)                    0.26-1.86         -21.72—29.88
Change in Composition of mobile phase
           94.5:5.5 (n=3)               0.69-1.29          -2.01-3.27
           95:5 (n=3)                   0.48-1.07
           95.5:4.5 (n=3)               0.28-1.42          -2.76-2.49

Table 3. Method Application

           Sample Name         Mean Conc.       S.D.
            Amyron Syrup       0.025 mg/ml     0.0011
            Dibet Powder       0.015 mg/g      0.0026

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