Pharmacognosy Magazine

: 2020  |  Volume : 16  |  Issue : 5  |  Page : 486--491

Method development on reverse-phase high-performance liquid chromatography for the improvement of routine analysis of flavonoids on agricultural and food products

Paranthaman Ramakrishnan, Chakkaravarthi Kamalanathan, Vidyalakshmi Rajagopal 
 Indian Institute of Food Processing Technology, Ministry of Food Processing Industries, Government of India, Thanjavur, Tamil Nadu, India

Correspondence Address:
Vidyalakshmi Rajagopal
Associate Professor & Head, Department of Biotechnology Indian Institute of Food Processing Technology, Ministry of Food Processing Industries, Government of India, Pudukkottai Road, Melavasthachavadi, Thanjavur - 613 005, Tamil Nadu


Objectives: The objective of this work was to investigate the effect of reversed-phase high-performance liquid chromatography conditions for the determination of flavonoids in the plant extract. Methods: The analytical conditions (total flow rate, mobile phase, colum temperature, and detection wavelength) optimized for gallic acid, rutin, and quercetin. The optimized method was compared with the control method and evaluated with the use of reference compounds. Results: The result shows that the maximum flavonoids recovery was in observed on modified chromatographic conditions, Colum: Octadecyl-silica C18column (250 mm × 4 mm, 5 μm) Mobile Phase solution water-acetic acid (25:1) and methanol. Mobile Phase solution B was a Mobile phase – 4th min - 20%, 10th min - 80%, and a flow rate of 0.5 mL/min. Colum temperature 35°C and detection wavelength was 260 nm. Conclusion: In this work, the proposed method will increase the analysis time but minimize the mobile solvents and have the potential of being useful for the routine analysis of flavonoids.

How to cite this article:
Ramakrishnan P, Kamalanathan C, Rajagopal V. Method development on reverse-phase high-performance liquid chromatography for the improvement of routine analysis of flavonoids on agricultural and food products.Phcog Mag 2020;16:486-491

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Ramakrishnan P, Kamalanathan C, Rajagopal V. Method development on reverse-phase high-performance liquid chromatography for the improvement of routine analysis of flavonoids on agricultural and food products. Phcog Mag [serial online] 2020 [cited 2022 May 17 ];16:486-491
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The current work has been performed to study the effect of chromatographic conditions for the estimation of flavonoids in the plant extract. The ultra-high-performance liquid chromatography conditions such as flow rate, mobile phase, and colum temperature exposure wavelength was optimized for the easy identification of flavonoids (gallic acid, rutin, and quercetin). The modified conditions were compared with the reference method and evaluated. The highest recovery of flavonoids observed in optimized chromatographic conditions and this method will minimize the utilization of high-performance liquid chromatography grade solvents and have the possibility of being useful for the regular study of phenolic compounds.


Abbreviations used: RP-HPLC: Reverse-Phase High-performance liquid chromatography; ODS: Octadecyl-silica; GA: Gallic acid; RU: Rutin, QU: Quercetin; C: Control method; M: Modified method.


High-performance liquid chromatography (HPLC) has been the most widely used chromatographic technique in the flavonoid analysis. It has added a new aspect to the examination of flavonoids in food and plant extracts and gives high resolution and sensitivity.[1],[2],[3],[4],[5] Gallic acid, rutin, and quercetin are phenolic compounds, and they have been proved to have potential preventive and beneficial effects in many diseases, where the oxidative stress has been concerned, as well as cardiovascular diseases, cancer, and neurodegenerative disorders.[6],[7],[8],[9],[10] The reverse phase HPLC with UV detector is a significant investigative method with well-built chromophores that attract light in the wavelength area from 200 to 800 nm.[11] The aim of the present work was to investigate the effect of reversed-phase (RP) HPLC conditions for the determination of flavonoids and improve the HPLC conditions for increasing the recovery. In this article described to develop and validate a liquid chromatographic method that can be used for the usual analysis of gallic acid, rutin, and quercetin on agricultural and food products.

 Materials and Methods


HPLC system consisted of a Shimadzu Corporation (Make), Japan, LC-10ATvp (Model) connected to SPD-10A vp UV detector with dual wavelength and column oven was used in this study. The LC solution classvp software was used for the data analysis and extracts the chromatograms. A Phenomenex Luna C18 column (250 mm·4.6 mm id, 5 lm,) was used to perform the separation, with a C18 Phenomenex, 4 3.0 mm Id, security guard column. The mobile phase was filtered through the millipore filter assembly attached with a vacuum pump. The mobile phase was sonicated with ultrasonic cleaner. The gradient elution of mobile phase a (water-acetic acid [25:1 v/v]) and solvent B (methanol) with gradient proportions, solvent B was improved to 50% in 4 min, and then increased to 80% in 10 min at a flow rate of 1.0 mL/min. The detection wavelength was 280 nm.[12],[13]

Modification of reverse phase high-performance liquid chromatography conditions

In this research, the packed HPLC C18 column (octadecyl silica [ODS]) was used for flavonoids purification. In arrange to avoid pressure increasing of column, acetonitrile was used as inorganic reagent because of its low viscosity. The flow rate, column temperature, mobile phase variations, and wavelength take part in an important responsibility in the reverse-phase liquid chromatographic method given in [Table 1]. In this investigation, these experiments were standardizing for the determination of flavonoids.{Table 1}

Method validation

The chromatographic condition was validated using the ICH guidelines[14] Q2(R1) with linearity, precision, accuracy, detection limit, and quantification limit.

 Results and Discussion

In this study, different HPLC conditions were investigated, and the modified analytical technique was developed to RP HPLC with UV detector, including detection wavelength, mobile phase, total flow rate, and colum temperature. The chromatographic conditions were optimized on the Phenomenex ODS C18 column (250 mm × 4 mm,) 5 μm column. The optimized method is summarized on [Table 2], and validation and system suitability studies results are given in [Table 3].{Table 2}{Table 3}

Effect the Flow rate on retention time

The total flow rate of the mobile phase has an important effect on retention time and peak area and little effect on separation for gallic acid, rutin, and quercetin. The result shows the gradient scaling of flow rates from 0.5 to 1.50 mL/min using RP-HPLC and well-resolved peaks were observed for GA, RU, and QU. The elution order and the retention times for control method gallic acid, rutin, and quercetin were 35.742, 10.525, and 12.41, and modified method, gallic acid, rutin, and quercetin were 11.500, 17.300, and 19.650. The 0.5 ml/min flow rate produced high peak area percent gallic acid (76.06%) compares to control flow rate 1.0 ml/min [Figure 1]a and [Figure 2].{Figure 1}{Figure 2}

The flow rate may influence the detection sensitivity of flavonoids. The increasing of flow rates and volumes of solvent also increased by Beer's law, adsorption decreases. Low flow rates lead to the usage of small volumes of solvent and adsorption and sensitivity increase. The major reason that narrow-bore HPLC columns enhance detection sensitivity is because they are run at low flow rates and flavonoids are eluted in small volumes of solvent.

Effect the colum temperature

The result shows gradient scaling of colum temperature from 25°C to 45°C with reverse phase C18 colum (250–4.6 mm id, 5 lm) and well-resolved peaks were observed for GA, RU, and QU. The separation of flavonoids on the reverse-phase chromatographic conditions and linearity of column temperature versus peak area were obtained in the temperature range of 25°C–45°C. The colum oven temperature 35°C was produced high-peak area percent gallic acid (74.45%), rutin (25.54%), and quercetin (70.41%) compare to control colum temperature 40°C, gallic acid (53.60%), rutin (17.23%), and quercetin (29.16%). Previous literature reported that the optimum temperature is 35°C for the extraction of flavonoids.[15] The chromatogram efficiency reached a maximum at the temperatures in 35°C [Figure 1]b. This maximum of peak ability shows that the decrease in peak area was smaller than the decline in peak width at the short temperature range.

Variations in the mobile phase

One important factor which influences on selectivity is mobile phase compositions. The exact mobile phase compositions will give suitable polarity with respected compounds separated. The result shows that the mobile phase concentrations of control and modified methods no variations and quercetin shows 29.16 percent of high peak area. Increasing the methanol composition can improve the resolution for the determination of flavonoids.[16] Polarity and strength elution of mobile phase were more appropriate at the methanol level <80% [Figure 1]c.

Variations in detection wavelength mobile phase concentration at 0.5 ml flow rate and 35°C

The wavelength of the HPLC UV detector has an important effect on the detection of peak area for gallic acid, rutin, and quercetin. The result shows the level of detection wavelength from 260 nm to 300 nm using the UV detector RP-HPLC-and highest peaks area were observed for quercetin (45.08%) and rutin (27.52%) compare to control detection wavelength [Figure 1]d.

Calibration details of the improved method

The calibration curve was prepared by plotting the peak area using a modified method [Table 2] and [Table 3] and was linear in the range of 5–50 mg/Kg. The observed data were focused to linear regression analysis to calculate the calibration equation and correlation coefficients. The regression equation was found as gallic acid (r2 = 0.9915, n = 5), rutin (r2 = 0.9847, n = 5), and quercetin (r2 = 0.9971, n = 5). The goodness-of-fit (r2) was found to be 0.99, representing a linear connection between the concentration and peak area of the flavonoids. The HPLC standard chromatogram and validation data of gallic acid, rutin, and quercetin are presented in Table 4.{Figure 3}{Figure 4}{Table 4}

Standard accuracy of the method

The different concentration (2–100 mg/kg) mixed flavonoids of certified reference materials was analyzed using optimized HPLC conditions. The standard accuracy percentage calculated by compared with injected standard value and obtained instrument obtained values. The results show that the injected standard concentration, instrument reading value, and recovery percentage given in [Figure 4].

The accuracy of an optimized HPLC conditions was expressed the nearness of obtained outcome by modified RP-HPLC conditions to the accurate value. The repeatability results produced good RDS values in the range of 0.06 %–0.70% as given in [Table 6]. The standard accuracy results showed in the range of 95.71%–114.16% and percentage of RDS values were in the range of 1.39%–1.46%, as given in [Table 6]. The results exhibits that the recovery percentage and RSD% were within the accepted limits, and it indicates the applicability of the method for phytochemical evaluation in plants.{Table 5}{Table 6}


The importance of the work is the improvement the HPLC conditions designed for the simultaneous analysis of flavonoids in plant extracts, and it will be applied for short-type HPLC column. The mobile solvent economy uniqueness of HPLC method is very advantageous, compared to the most widely used control HPLC technique. The modified reverse phase HPLC method was competent in quantifying phytochemicals in commercial agriculture and food products.


The authors would like to show their thanks Dr. C. Anandharamakrishnan, Director, Indian Institute of Food Processing Technology, Ministry of Food Processing Industries, Government of India, for his constant encouragement and granting me permission to pursue this study.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.


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