Objectives: By using high performance liquid chromatography (HPLC), develop and validate an analytical method for astilbin. Methods: Development of chromatographic conditions for astilbin quantification in Rhizoma Smilacis glabra. The method was validated according to ICH guidelines.
Trang 1QUANTITATIVE STUDY FOR DETERMINATION OF ASTILBIN
IN SMILAX GLABRA BY HIGH PERFORMANCE LIQUID
CHROMATOGRAPHY
Le Viet Duc*; Vu Binh Duong**; Trinh Nam Trung**; Pham Thi Thanh Huong**
SUMMARY
Objectives: By using high performance liquid chromatography (HPLC), we develop and
validate an analytical method for astilbin Methods: Development of chromatographic conditions
for astilbin quantification in Rhizoma Smilacis glabra The method was validated according to
ICH guidelines Results: We investigated HPLC chromatographic conditions, including
Phenomenex Gemini RP C18 column (4.6 mm x 250 mm, 5 µm) at a flow rate of 1.0 mL/min,
detection wavelength of 291 nm, and an isocratic elution of 0.1% aqueous acetic acid - acetonitrile
(70:30, v/v) The method displayed an acceptable precision (RSD ≤ 1.37%) and accuracy
The detection and quantification limits were 0.5 μg/ml and 1.5 μg/ml respectively Conclusion:
The HPLC analytical method of astilbin was validated to meet requirements of ICH guidelines
* Key words: Smilax glabra; High performance liquid chromatography; Astilbin
INTRODUCTION
Smilax glabra is a wild herb grown in the
northern mountainous provinces of Vietnam
According to Vietnamese traditional medicine,
Smilax glabra is often used in treatment of
tendon diseases, worms, ulcers, diabetes,
and detoxification [1] Concerning to
compositions of Smilax glabra, astilbin, which
is the main active constituent (figure 1) with
high content, has been revealed to have
hypouricemic, anti-inflammatory, analgesic
anti-oxidation effects [2, 3] Thus, quantitation
for studies on extraction as well as quality
control is very important
Figure 1: The chemical structure
of astilbin
Vietnam Military Medical University has formulated several pharmaceutical products that contains Smilax glabra, such as: “Thong Phong Khang”, “Kien khop tieu thong”
* Minishy of Defense
** Vietnam Military Medical University
Corresponding author: Vu Binh Duong (vbduong2978@gmail.com)
Trang 2to serve public health care In order to
standardize Smilax glabra as well as its
products, development and validation of
astilbin quantitative method is required
In this study, we developed and validated
a quantitative analysis of astilbin in
Smilax glabra using high-performance
liquid chromatography (HPLC)
MATERIALS AND METHODS
1 Materials and equipment.
Smilax glabra was obtained from Son
Lam Pharmaceutical Company complying
the forth Vietnamese Pharmacopoeia
Astilbin standard was purchased from
Sigma Aldrich, 99.9% HPLC grade methanol
(MeOH), acetonitrile (ACN), distilled water
were purchased from Merck All reagents
used were of analytical grade (PA) High
performance liquid chromatography system
(Waters 1525 Binary HPLC pump; USA)
was applied in this study
2 Methods
* Sample preparation:
- Standard preparation: Powder of
standard astilbin is dissolved in MeOH to
make the stock solution of 1.000 µg/mL
Dilute the stock solution to get working
standard solutions
- Sample preparation: Accurately weighed
about 1.0 g of powdered Smilax glabra,
performed ultrasonic extraction using MeOH
solvent (8 ml x 3 times) The extracts were
centrifuged and poured into 200 mL
volumetric flask and diluted up to the mark
with methanol Then diluted ten-folded using
10 mL volumetric flask, filtered through 0.22 µm membrane before HPLC analysis
* Chromatographic conditions:
There were several reports on HPLC analysis of astilbin [5, 6] In this study, we selected chromatographic conditions to develop an appropriate analytical method
* Validation of analysis method:
Validation of analytical method was carried out in accordance with the guideline of ICH (International Conference
on Harmonisation) [4] for raw materials and finished products with the following criteria: system suitability, selectivity - specificity; linearity; accuracy; precision; limit of detection (LOD) and limit of quantification (LOQ)
RESULTS
1 Chromatographic conditions
Based on published researches [5, 6],
we developed chromatographic conditions for astilbin analysis which consisted of C18 column (4.6 x 250 mm, 5 μm) using a mobile phase of 0.1 aqueous acetic acid - ACN (70:30, v/v) at a flow rate of 1 mL/min
UV - VIS detector was employed at 291 nm Injection volume of 10 µL was subjected
to HPLC system For the proposed conditions, the chromatograms on the figure 2 showed well-resolved and symmetrical peaks of astilbin with retention time of approximately 19.7 minutes, which is suitable for analyzing
astilbin in Smilax glabra So, we choose
these conditions of chromatographic method for validation
Trang 32 Method validation
* System suitability, specificity:
Figure 2: Chromatogram of blank solution (a),
standard solution (b), test solution (c)
Six replicate injections of a working
standard solution of 5 µg/mL were
injected Retention time and peak area
were evaluated The results indicated that
relative standard derivations of retention
time (19.43 ± 1.77, RSD = 1.77) and peak area (11,0192 ± 797, RSD = 1.77) were not more than 2% Therefore, the method
is comply to system suitability criteria.
Analyze the standard, blank and test solutions of astilbin simultaneously The spectrum of the peak at retention time of 19.7 of standard and test solutions proved
that it was surely of astilbin (figure 3)
Figure 3: UV-Vis spectrum of peaks at
19.7 minute of standard solution (a)
and test solution (b)
In figure 2, peak of astilbin was completely separated from peaks of impurities, symmetric and narrow width
The retention time of astilbin in the chromatogram of the test solution and standard solution were the same There weren’t any corresponding peak in the chromatogram of blank solution Therefore, the method showed high selectivity and specificity
a
a
b
c
b
Trang 4* Linearity:
The working standard solutions from 5 to 100 µg/mL was injected to HPLC system Results showed that in the test range, there was a linear regression between peak area and the concentration of astilbin The linear equation was y = 22172 - 15877, with correlation coefficient R2 of 0.999 (figure 4).
Concentration g/ml
Concentration g/ml
Figure 4: Behavior of peak area vs.concentration of astilbin
* LOD and LOQ:
LOD was determined to be 0.5 µg/mL and LOQ was 1.5 mg/mL
* Precision:
The precision of the method was carried out at 3 concentrations: LQC, MQC and HQC The results showed that the relative standard deviations at the concentrations during
intra and interday were less than 2% (table 1) Thus, the accuracy of the method was satisfactory to analyze astilbin
Table 1: Intraday and interday precision of standard solutions.
Intermediate
precision
Concentration (µg/mL)
Trang 5* Accuracy:
The accuracy of the method is performed
by spiking of 16 mg, 20 mg and 24 mg to
1.0 g of powdered Smilax glabra Extraction
and chromatography analysis are conducted
using the proposed procedure The recovered
percentages of astilbin were compared
with the original and determined as accuracy
The results indicated that the percentages
of recovery were found in the acceptance
criteria of 80 to 120% (table 2) Therefore,
the quantitative methods completely meet
the requirements of ICH guidelines
Table 2: Accuracy of astilbin in herbal
samples
4.6
100.0 ± 2.30
102.5 ± 1.00
CONCLUSION
The HPLC method has been developed
to determine astilbin in Smilax glabra using
Phenomenex Genimi RP - C18 column,
mobile phase containing 0.1% acetic acid -
ACN (70:30, v/v), flow rate of 1 mL/min,
UV - VIS detector at a wavelength of 291 nm,
and the injection volume of 10 µl The
validated quantitative method has high
precision (98.3 ± 4.6% to 102.5 ± 1.00%)
and accuracy (RSD ≤ 1.37%), the correlation
coefficient (R2) of 0,999 Thus, the proposed
method is suitable to quantify astilbin in
Smilax glabra as well as pharmaceutical products containing this compound
REFERENCES
1 Loi Do Tat Vietnamese Medicinal Plants
and Herbs Medical Publishing House 2004, pp.498-499
2 Qing-Feng Zhang, Zhong-Rong Zhang,
of Rhizoma Smilacis Glabrae extracts and its key constituent-astilbin Food Chemistry
3 Wen-Ai Xu et al Study on the correlation
between constituents detected in serum from Rhizoma Smilacis Glabrae and the reduction
of uric acid levels in hyperuricemia Journal of Ethnopharmacology 2013, Vol 150, Issue 2, pp.747-754
4 ICH Validation of analytical procedures,
Text and Methodology Q2R1 2005
5 Liang Chen, Ye Yin, Hongwei Yi, Qiang Xu, Ting chen Simultaneous quantification of five
major bioactive flavonoids in Rhizoma Smilacis Glabrae by high-performance liquid chromatography Journal of Pharmaceutical and Biomedical Analysis 2007, 43, pp.1715-1720
6 Qing-Feng Zhang, Yu-Xian Guo, Xinchen Shangguan, Guodong Zheng and Wen-Jun Wang Identification and quantification of
polyphenols in Rhizoma Smilacis chinae by HPLC/DAD/ESI-MS/MS Journal of Liquid Chromatogrhaphy & Related Technologies
2013, 36, pp.2251-2260