Corresponding author: Nguyen Duc Hanh (
Tongkat Ali (
Epimedin C (
Standardization is one of the difficult tasks for quality control of herbal medicine due to the complex nature of constituents of plant-based drug. In recent years, several analytical technologies have been developed to overcome these problems. Currently, high performance liquid chromatography equipped with a diode-array detector (
Chemical structures of (a) eurycoumanone, (b) epimedin C and (c) icarriin.
The
High performance liquid chromatography (
Mobile phase composition and gradient elution conditions.
|
|
|
|
Condition I | 0–15 | 89.5 | 10.5 |
15–17 | 89.5 → 69 | 10.5 → 31 | |
17–20 | 69 → 72 | 31 → 28 | |
20–35 | 71 | 29 | |
35.01–45 | 30 | 70 | |
45.01–55 | 89.5 | 10.5 | |
Condition II | 0–16 | 90.5 | 9.5 |
16–18 | 90.5 à 71 | 9.5 → 29 | |
18–21 | 71 → 73 | 29 → 27 | |
21–35 | 72 | 28 | |
35.01–45 | 30 | 70 | |
45.01–55 | 90.5 | 9.5 | |
Condition III | 0–16 | 91.5 | 8.5 |
16–18 | 91.5 → 71 | 8.5 → 29 | |
18–21 | 71 → 73 | 29 → 27 | |
21–35 | 73 | 27 | |
35.01–45 | 30 | 70 | |
45.01–55 | 91.5 | 8.5 |
Test solutions: 20
Standard solutions:
The thin layer chromatography (
Condition A: 10 µl of each solution (
Condition B: 40 µl of each solution (
The best solvent was selected based on its capability of simultaneous extraction of
Two different ratios were screening as follows:
The ratio A: 750 mg of EE tablet powder was extracted by 25 ml of the selected solvent.
The ratio B: 750 mg of EE tablet powder was extracted by 50 ml of the selected solvent.
The
Contents of three markers in the
Contents of three markers in the |
|||
---|---|---|---|
Ratio |
|
|
|
A | 0.3686 | 6.0408 | 0.5758 |
0.3656 | 5.8947 | 0.5702 | |
0.3574 | 5.9577 | 0.5650 | |
B | 0.3657 | 5.9657 | 0.5729 |
0.3641 | 6.0417 | 0.5717 | |
0.3658 | 5.9880 | 0.5720 |
The analysis method was validated according to International Conference on Harmonisation (
Specificity was evaluated using the optimized
System suitability was carried out by injecting six replicates of the test solution. The theoretical plate number (N), resolution (
The mixed standard stock solution was prepared by exactly weighing and simultaneously dissolving
The sensitivity of the analysis method was determined by lower limit of quantification (
The intra-day precision was evaluated by analyzing six replicates of standard solutions within one day. The inter-day precision was determined on three consecutive days. The intra- and inter-day precision were evaluated by the relative standard deviation (
The accuracy of the method was determined by adding standard markers into the test solution. The samples were spiked at three different levels (80%, 100%, and 120%) of the three targeted markers. The contents of each marker were calculated using the calibration curve and the recovery percentages were determined.
The maximum absorption wavelength of
UV spectra (200–400 nm) of
The
In order to optimize the extraction solvent for preparation of test solution, the extraction efficiency of various solvents (
The purities of (a)
The developed method resulted in the elution of
The results of system suitability.
tR (min) | S (mAU.s) |
|
|
|
N | ||
---|---|---|---|---|---|---|---|
|
Mean | 15.92 | 106338 | 1.62 | 1.52 | 0.99 | 8251 |
% |
0.61 | 1.37 | |||||
|
Mean | 32.59 | 2278305 | 1.52 | 1.52 | 1.06 | 44367 |
% |
0.10 | 0.83 | |||||
|
Mean | 35.68 | 183768 | 1.64 | 1.54 | 0.91 | 44568 |
% |
0.11 | 1.18 |
There was no peak in the blank sample at the retention times of the
Table
Linearity and sensitivity of three investigated markers in
Components | Linear range (µg/ml) | Regression equation | Correlation coefficient (r2) | ||
---|---|---|---|---|---|
|
7.3–58 | y = 7673.9x | r2 = 0.9987 | 2.2 | 6.5 |
|
72.3–578.4 | y = 9300.1x | r2 = 0.9997 | 10.8 | 32.5 |
|
6.8–54.4 | y = 7951.7x | r2=0.9987 | 2.0 | 6.1 |
y: peak areas (mAU), x: concentrations of markers (µg/ml)
The inter- and intra-day precision data at working concentrations are summarized in Table
Analytical results of inter- and intra-day test.
Precision | Tablet content (mg/tablet) | |||
---|---|---|---|---|
|
|
|
||
Intra-day (n = 6) | Mean | 0.3287 | 5.9641 | 0.5677 |
0.48 | 1.60 | 1.59 | ||
Inter-day (n = 6) | Mean | 0.3345 | 5.9897 | 0.5620 |
1.88 | 1.41 | 1.67 |
The
The developed method had good accuracy with the recovery ranged from 98.51 to 104.58% for
Analytical results of accuracy test.
Compound | Spiked amount (mg) | Measured amount (mg) | Recovery (%) | % |
---|---|---|---|---|
|
0.2610 | 0.2671 | 102.32 | 2.00 |
0.2593 | 99.33 | |||
0.2571 | 98.51 | |||
0.3197 | 0.3331 | 104.19 | 2.35 | |
0.3241 | 101.37 | |||
0.3180 | 99.45 | |||
0.4000 | 0.4139 | 103.47 | 1.84 | |
0.4035 | 100.89 | |||
0.4183 | 104.58 | |||
|
4.85 | 4.6668 | 96.22 | 0.62 |
4.7232 | 97.39 | |||
4.7085 | 97.08 | |||
6.05 | 6.3107 | 104.31 | 2.60 | |
6.3407 | 104.80 | |||
6.0460 | 99.93 | |||
7.21 | 7.1990 | 99.85 | 2.42 | |
7.3151 | 101.46 | |||
6.9747 | 96.74 | |||
|
0.45 | 0.4667 | 103.71 | 2.62 |
0.4556 | 101.25 | |||
0.4429 | 98.42 | |||
0.54 | 0.5265 | 97.50 | 1.55 | |
0.5365 | 99.35 | |||
0.5430 | 100.56 | |||
0.68 | 0.6878 | 101.14 | 2.16 | |
0.6475 | 101.11 | |||
0.7137 | 104.96 |
The analytical method for simultaneous determination of
The developed
The contents of three markers in three batches of
Compound | Contents (mg/tablet) | |||
---|---|---|---|---|
Batch No. 01NMDDH | Batch No. 02NMDDH | Batch No. 03NMDDH | Mean ± SD | |
|
0.338 | 0.304 | 0.296 | 0.313 ± 0.022 |
|
5.966 | 5.800 | 5.834 | 5.867 ± 0.088 |
|
0.568 | 0.525 | 0.546 | 0.546 ± 0.022 |
The contents of three markers in 3 batches of
The
This study was supported by the grant from Ho Chi Minh City Department of Science and Technology, Vietnam.