DEVELOPMENT AND VALIDATION OF A RP HPLC METHOD FOR THE SIMULTANEOUS DETERMINATION OF VITEXIN 2″ ORHAMNOSIDE AND HYPEROSIDE IN LEAVES AND FLOWERS OF HAWTHORN

DEVELOPMENT AND VALIDATION OF A RP-HPLC METHOD FOR

THE SIMULTANEOUS DETERMINATION OF VITEXIN-2

″

-O-RHAMNOSIDE AND HYPEROSIDE IN LEAVES AND

FLOWERS OF HAWTHORN

Andriy Gudzenko*

State Laboratory for Quality Control of Medicines, State Institution “Institute of

Pharmacology and Toxicology of National Medical Academy of Science of Ukraine”, Kiyv,

Ukraine. 14, Eugene Potie str., 03680, Kyiv, Ukraine.

ABSTRACT

A method for separation and quantification of two flavonoids by

reverse-phase high performance liquid chromatography (HPLC) was

developed and validated. Flavonoids present in extract of the leaves

with flowers of hawthorn (Crataegus pentagyna Waldst. et Kit) were

analyzed. Vitexin-2″-O-rhamnoside and hyperoside were used as

calibration standards.In this study, an excellent linearity was obtained

with r higher than 0.99. Besides, the chromatographic peaks showed

good resolution. With other validation data, including precision,

specificity, accuracy and robustness, this method demonstrated good

reliability and sensitivity, and can be conveniently used for the

quantification of vitexin-2″-O-rhamnoside and hyperoside of the

extract of the leaves with flowers of hawthorn. In summary, the

method above can be considered specific, exact, precise, linear, robust

and easy to perform. Further this method can be applied to a

standardization of multicomponent herbal remedies, that incorporate

leaves with flowers of hawthorn.

KEYWORDS: leaves with flowers of hawthorn (Crataegus pentagyna Waldst. et Kit), HPLC, hyperoside, vitexin-2″-O-rhamnoside, validation.

Volume 2, Issue 5, 1270-1282. Research Article ISSN 2277 – 7105

Article Received on 17 June 2013,

Revised on 22 July 2013, Accepted on 25 August 2013

*Correspondence for

Author:

Dr. Andriy Gudzenko

State Laboratory for Quality

Control of Medicines, State

Institution “Institute of

Pharmacology and Toxicology

of National Medical Academy

of Science of Ukraine.

1. INTRODUCTION

Hawthorn is the common name for Crataegus species in Rosaceae family. There are over

1000 species of Crataegus distributed primarily in Asia, Europe and North America. It has

been used as a medicinal material and food for hundreds of years both in Europe and in

China[1]. Clinical investigations and other research suggest that extracts of hawthorn have

multiple health effects including anti-atherosclerotic, hypotensive, cardioprotective,

antidiabetic, antioxidant and radical scavenging activities[2-5].

The diversity and complexity of the phytochemical composition of Crataegus species may

explain their polyvalent pharmacological activity. The raw material of hawthorn contains

triterpene acids, procyanidins, lignans, phenolic acids, sugars, sugar alcohols and

flavonoids[1, 6-10].

Flavonoids constitute one of the most important groups of pharmacologically active

principles in hawthorn. It is suggested that anti-inflammatory, free-radical-scavenging, and

antiviral activities of hawthorn are mainly attributed to the flavonoid complex[11-13].

The main components of the flavonoid fraction of leaves and flowers of Crataegus pentagyna

Waldst. et Kit - hawthorn species, which is widespread in Ukraine[14], are hyperoside and

vitexin-2″-O-rhamnoside[15, 16]. Structure of these flavonoids shown in the Fig.1. These

flavonoids have a wide spectrum of biological activity.

Hyperoside has a variety of pharmacological effects including viral, oxidative,

anti-apoptotic and anti-inflammatory activities[17-18]. Vitexine-2″-O-rhamnoside has

antithrombotic, anti-inflammatory and antioxidant activities[11-13, 19].The choice of these two

flavonoids was based on other studies, which reported the presence of these compounds in

other species of flowers and leaves of Crataegus[1, 16, 20-22]. Thus, the objective of this study

was to develop and validate a method for the separation and simultaneous quantitative

analysis of hyperoside and vitexin-2″-O-rhamnoside by HPLC, obtained from an extract of

leaves and flowers of Crataegus pentagyna Waldst. et Kit. The method was validated

according to ICH Harmonised Tripartite Guidelines “Validation of analytical procedures:

Text and Methodology Q2(R1)”[23].

The following validation characteristics were assessed: specificity, linearity, limit of

Hyperoside

Vitexin-2″-O-rhamnoside

Figure 1: Chemical structure of hyperoside and vitexin-2″-O-rhamnoside.

2. MATERIALS AND METHODS 2.1 Plant material

The leaves and flowers of Crataegus pentagyna Waldst. et Kit was collected in the region of

Borispol (Kyiv region, Ukraine) in may 2011. Identification of the species was confirmed in

State Laboratory for Quality Control of Medicines, State Institution "Institute of

pharmacology and toxicology National Academy of Medical Sciences of Ukraine" (Ukraine).

A voucher specimen (CF011-15) was deposited at the herbarium in this laboratory.

2.2 Chemicals and reagents

All reagents and solvents were analytical and HPLC grades (Fluka, USA). Ultra-pure water

was used in all experiments. Hyperoside and vitexin-2″-O-rhamnoside (Roth, Germany) of

the highest grade (purity>98.0%) were used as the external standards.

2.3 Instrumentation and chromatographic conditions

The analyses were carried out using an HPLC system (Shimadzu, Japan) consisting of a

solvent delivery pump (Model LC-20 AD), a diode array detector (Model SPD-20A), an

auto-injector (Model SIL-20A) and system controller (Model CBM-20A). Data collection

and analyses were performed using LCsolution (ver. 1.22SP1). A gradient elution was

performed on a Phenomenex Luna C18(2) (250 x 4.6 mm i.d., 5 µm particle size)

(Phenomenex, USA). The mobile phase consisted of two different solutions, solution A and

solution B. Solution A: 0.1% trifluoroacetic acid solution in acetonitrile. Solution B: 0.1%

trifluoroacetic acid in water. All solutions were degassed and filtered through a 0.45 µm pore

size filter (Millipore, USA). Separations were effected by a gradient elution program as

follows: from 0 to 5 min, B was isocratic at 95%; from 5 to 35 min, solution B followed a

linear change from 95% to 75%; from 35 to 40 min, B was isocratic at 75%; from 40 to 60

min, B linearly changed from 75% to 50%, from 60 to 65 min, B linearly changed from 50%

to 20%, from 65 to 70 min, B was isocratic at 20%; and from 70 to 85 min, B was isocratic at

95%. The mobile phase flow rate was 1 mL/min and the injection volume was 5 µL. UV

detection was performed at 338 nm.

Using these chromatographic conditions, it was possible to confirm the retention time of

hyperoside and vitexin-2″-O-rhamnoside by injection of each standard separately.

2.4 Sample preparation

Plant samples (1.0 g) were extracted two times, each time for 45 min at 100°C, with a 50 %

ethanol, by use of a hot reflux equipment, and the extracts were combined in a 100 ml flask

with 50 % ethanol.

Samples were purified by solid phase extraction as described in[24].

2.5 Preparation of standard solution

Accurately weighed appropriate amounts of the reference compounds (hyperoside HYP;

vitexin-2″-O-rhamnoside VIT) were mixed and dissolved in methanol in a 100-mL

volumetric flask, to obtain a stock solution. The concentration of the two compounds in this

solution was 308.0 µg/mL (HYP), and 199.6 µg/mL (VIT). Besides, external standards were

according to the level estimated in the plant sample. Working solutions were prepared by

stepwise dilution of the stock solution with methanol.

2.6 Method validation

In the validation of the analytical method used for the quantification of hyperoside and

vitexin-2″-O-rhamnoside in leaves and flowers of hawthorn, the following parameters were

determined: specificity, linearity, sensitivity, accuracy, precision and robustness.

2.7 Specificity

Specificity is the ability of a method to discriminate between the study analyte(s) and other

components in the sample. The specificity was demonstrated by running a procedural blank.

In addition, the resolution between the peaks of the main flavonoids that could be found in

extracts of leaves and flowers of hawthorn was determined by analysis of chromatograms of

the standard solution and the sample solution.

2.8 Linearity

The linearity between peak area and concentration was analyzed using two calibration curves

obtained with standard solutions at seven different concentrations of each standard HYP, and

VIT. The concentrations of the two compounds in the solution that was considered 100% was

154.0 µg/mL (HYP), and 99.8 µg/mL (VIT). The other concentration levels used to construct

calibration curves were 10%, 50%, 75%, 100%, 125%, 150 % and 200% of the concentration

mentioned above. The data for peak area versus drug concentration were treated by linear

regression analysis.

2.9 Sensitivity

The limit of detection (LOD) and the limit of quantification (LOQ) were determined from the

calibration curves of the HYP and VIT standards. LOD was calculated according to the

expression DPx3/IC, where DP is the standard deviation of the response and IC is the slope

of the calibration curve. LOQ was established by using the expression DP x10/IC[23].

2.10 Accuracy

The accuracy was evaluated by means of recovery assays carried out by adding known

amounts of the HYP and VIT standard to the sample, at three different levels (5%, 10% and

Average recoveries were calibrated by the formula recovery (%) = {(amount found - original

amount)/amount spiked} x 100.

2.11 Precision

The precision of the method was investigated with respect to repeatability, intermediate

precision (inter-day variation) and reproducibility by determination of standard solution at

100% of the test concentration. To assess the intra-day precision (repeatability) of the

method, the sample was injected six times within a day. The inter-day precision was

determined with the sample assayed on different days and by another analyst. Precision was

expressed as the relative standard deviations (% RSD) of the concentrations of each

compound, HYP and VIT.

2.12 Robustness

Three sample solutions were prepared and analyzed under the conditions established and by

changing the wavelength parameter from 336 nm to 370 nm, by using columns from different

suppliers and by changing the mobile phase composition (±5% change organic solvent) and

(±5% change trifluoroacetic acid concentration)[23].

2.13 Statistical analysis

The data were submitted to statistical analysis using Excel® software.

3. RESULTS AND DISCUSSION

The HPLC method carried out in this study was aimed at developing a chromatographic

system, capable of eluting and resolving flavonoid compounds in leaves and flowers of

hawthorn. In the development of the HPLC method for determination of hyperoside and

vitexin-2″-O-rhamnoside in leaves and flowers of hawthorn, several solvent systems

(methanol-water-trifluoroacetic acid, acetonitrile-water-trifluoroacetic acid,

tetrahydrofuran-water-trifluoroacetic acid) and separation columns Phenomenex Luna C18(2) (250 x 4.6 mm

i.d., 5 µm particle size), WatersX-Terra C18 column (250 x 4.6 mm i.d., 5 µm particle size),

Macherey-Nagel Nucleosil 100-5 C18 (250 x 4.6 mm i.d., 5 µm particle size) were evaluated

and compared. The Phenomenex Luna C18 column provided better separation of the plant

extract than with other specifications or brands of columns.

The choice of detection wavelength was determined by performing a screening with 10 ppm

were recorded from 220 to 380 nm and exhibited maximum wavelengths at 268 nm and 338

nm. It was carried out an analysis on HPLC with the two wavelengths and which provided

better response even for the others compounds (vitexin-2″-O-rhamnoside) was at a

wavelength of 338 nm.

The results for quantification of the flavonoids in the sample were 153,37 µg/mL of

hyperoside and 96,91 µg/mL of vitexin-2″-O-rhamnoside, which means, 0.839% and 0.526%

of each compound contained in the leaves and flowers of Crataegus pentagyna Waldst. et

Kit, based on the dried raw, respectively.

System suitability test showed that critical parameters such as retention time, area, number of

theoretical plates and resolution between HYP and VIT met the acceptance criteria on all the

experimental days (Table 1).

Table 1: System suitability test.

Compound Parameter Acceptance Average %RSD Status

Hyperoside

Retention time % RSD < 2 31.90 0.87 Passed

Peak area % RSD < 2 444269 1.54 Passed

No. of Plates > 50000 265376 3.41 Passed

Asymmetry factor < 2 1.243 2.34 Passed

Resolution

VIT/HYP

> 2 4.33 2.41 Passed

Vitexin-2″

-O-rhamnoside

Retention time % RSD < 2 30.42 0.52 Passed

Peak area % RSD < 2 767457 1.21 Passed

No. of Plates > 50000 107673 5.12 Passed

Asymmetry factor < 2 1.287 2.88 Passed

The specificity of the method was evaluated by analysis of blank, standard and sample

solution chromatograms (Figure 2). Good separation between the peaks of VIT and HYP was

achieved, with the retention times, 30,42 min for vitexin-2″-O-rhamnoside and 31.90 min for

hyperoside. Furthermore, the chromatographic peaks showed good resolution (around 4.33

between VIT and HYP). In relation to asymetry, the peaks showed values 1.287 for VIT and

Blank

Standard

Sample

Linearity was evaluated by the correlation coefficient r, and all values for the two compounds

were greater than 0.999, showing that responses for the standard in the concentration ranges

examined (from 10 to 200%) were linear. Besides, according to[23], the minimum acceptable

correlation coefficient is 0.990.

As shown in Table 2, the LOD values were 0.02 µg/mL for the compounds VIT and HYP,

while the LOQ values were 0.07 µg/mL.

Table 2: Calibration curve parameters, limit of detection (LOD), limit of quantification (LOQ) for hyperoside and vitexin-2”-O-rhamnoside.

Compound Calibration curve equation

Correlation coefficient (r)

Linear range (µg/mL)

LOD(µg/mL) LOQ(µg/mL)

Hyperoside y = 2838782x

+ 383

0.99943 15.4-308.0 0.02 0.08

Vitexin-2″

-

O-rhamnoside

y = 3748733x

- 4380

0.99925

9.98-199.60

0.02 0.08

The recovery of the compounds HYP and VIT was determined by spiking the extracts of

leaves and flowers of hawthorn with known amounts of HYP and VIT standards. Recovery of

each substance was obtained from the calculated amount found and original amount. The

results are presented in Table 3 and conform with the recommendations of[23].

The data of the precision are shown in Tables 4, 5. The results display a coefficient of

variation less than that recommended by [23] whose limit is 5%.

Also, there were no significant differences between assay results, indicating that the precision

of the proposed method was satisfactory.

Robustness was evaluated to ensure that the HPLC method is insensitive to small changes in

the experimental conditions. In this study, the wavelength, column supplier and pH of the

mobile phase were changed. None of the modifications caused any significant change in the

resolution or response of the HYP and VIT peaks.

All results were displayed according to the ICH Harmonised Tripartite Guidelines

Table 3: Results of accuracy determination by analyzing of the hyperoside and vitexin-2”-O-rhamnoside of known concentrations.

Compound/Initial concentration

Theoretical

concentration after dilution added in the extract (µg/mL) Amount recovered (µg/mL) Recovery (%)

Mean (%) RSD (%)

Hyperoside

(Concentration

measured in the

sample = 153,37

µg/mL)

7.70

159.18 98.83

98.88 0.81 158.03 98.11

160.60 99.71

15.40

167.69 99.36

98.60 0.75 166.34 98.56

165.21 97.89

23.10

176.68 100.12

98.90 1.07 173.35 98.23

173.54 98.34

Vitexin-2”-O-rhamnoside

(Concentration

measured in the

sample = 96,91

µg/mL)

5.01 99.52 97.65 98.79 1.11

100.79 98.89

101.75 99.83

10.02 107.31 100.36

99.10 1.16 104.91 98.11

105.68 98.83

15.03 111.88 99.95

98,46 1,36 109.82 98.11

108.96 97.34

Table 4: Results of the repeatability.

Compound Mean (µg/mL)±standard deviation (n=6) RSD (%)

Hyperoside 153,37±3.87 1.12

Vitexin-2”-O-rhamnoside

Table 5: Results of the intermediate precision.

Compound Mean (µg/mL)±standard deviation (n=18) RSD (%)

Hyperoside 152,87±4.15 1.36

Vitexin-2”-O-rhamnoside

96,55±2.98 1.65

4. CONCLUSION

The quantitative method developed here was successfully applied in the simultaneous

analysis of two different compounds in extracts of f leaves and flowers of hawthorn. Taking

into account the results obtained in this study, the proposed method can be conveniently used

for the analysis of hyperoside and vitexin-2”-O-rhamnoside in extracts of leaves and flowers

of hawthorn.

The proposed method demonstrated high specificity at 338 nm detection for the extracts of

leaves and flowers of hawthorn showing reliability in the quantification of HYP and VIT.

Further, this method can be applied to a standardization of multicomponent herbal remedies,

that incorporate leaves and flowers of hawthorn. In summary, the method above can be

considered specific, exact, precise, linear, robust and easy to perform.

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