INTRODUCTION:
This species grows worldwide and has been cultivated since long ago, in ancient Egypt, Mesopotamia, China and India (2). Rosemary extracts, enriched in phenolic compounds are effective antioxidants due to their phenolic hydroxyl groups but they also possess plenty of other beneficial effects like antimicrobial, antiviral, anti-inflammatory, anticarcinogenic activities and is also known to be an effective chemopreventive agent (3) Rosmary is rich source of phenolic compound such as Rosmarinic acid carnosnic acid carnosol, diterpenes as well as vitamins, mineral sand essential oils.
Fig. 1: Structure of Rosmarinic acid
Rosmarinic acid is a naturally occurring polyphenolic compound. Rosmarinic acid is an ester of caffeic acid and 3, 4-dihydroxyphenyllactic acid.
Rosmarinic acid have antioxidant, anti-inflammatory, as well as help in boost immunity, improve digestion, reduce stress and prevent Alzheimer. Literature survey revealed that the estimation of Rosmarinic acid in marketed herbal formations using validated simple rapid, precise and accurate HPTLC method for estimation of Rosmarinic acid in herbal formulation (.4-5-6)
MATERIAL AND METHODS:
Chemicals and reagents:
Standard Rosmarinic acid was obtained from Global Pharma Ltd., Mumbai. All chemical and reagents used were of analytical grade. Rosemary capsules were purchased from local market.
Instrumentation:
Chromatographic separation was performed on A Camag HPTLC system comprising of Camag linomat –v semiautomatic sample applicator, twin trough development chamber, Camag 100µl syringe, Camag TLC Scanner -3, WinCAT software version 1.4.3.6336 were used during the study.
Preparation of standard solution:
An accurately weighed quantity of Rosmarinic acid 10 mg was transferred to 10ml volumetric flask and dissolves in methanol and made the volume up to 10ml (1000ug/ml).
Preparation of sample solution:
For analysis of herbal Rosmary capsule formulation, 20 capsules were weighed and emptied. Take a powder sample equivalent to 10mg of Rosmarinic acid accurately weighed and transferred into 10ml of volumetric flask. Volume was make up with methanol up to 10ml. The solution was sonicated for 30 min and filtered through Whatman filter paper.
Optimization of mobile phase:
As mobile phase plays important role in the chromatographic method, the first step for development of successful method is to optimize the solvent system for good extraction efficiency. Method that gives compact spot with significant value for determination of Rosmarinic acid.in formulation. To optimize mobile phase different combination and ratio of mobile phase was studied. Use of Toluene: ethyl acetate: formic acid (4:5:1) resulted in sharp well defined Rosmarinic acid peak.
Selection of wavelength:
Rosmarinic acid solution was scan in UV visible spectroscopy at wavelength 400-200nm; absorbance maxima obtained at 330nm was used for estimation of drug.
Chromatographic conditions:
The sample were spotted in form of band of 5mm with a camag microlite syringe on pre-coated silica gel aluminum plate 60 F254 mobile phase was composed of toluene: ethyl acetate: formic acid (4:5:1v/v). The optimized chamber saturation time for mobile phase was 10 min at room temperature. The developed TLC plate were dried with the help of an air dryer. The source of radiation utilized was deuterium lamp emitting continuous UV-spectrum between 400 to 200nm.
Method validation
Linearity:
To determine the linearity, calibration curves were plotted. A concentration of standard stock solution ranging from 200-1200ng/band. The calibration curve was obtained using peak areas against each band concentration. (7-8)
Precision:
Six replicates of same concentration of Rosmarinic acid (600ng/band) was used for determination of instrumental precision and the repeatability of methanol was estimated by carrying out intra-day and inter-day precision and % RSD values were calculated (9-10).
Accuracy:
The pre-analyzed samples were spiked with 80, 100, and 120% of standard. Rosmarinic acid and the resulting solution were analyzed by the developed method using three replicates of each level and %RSD values were calculated.
LOD and LOQ:
Detection limit (LOD and limit qualification (LOQ) was calculated using the equation
LOD:
and
LOQ:
Where,
= Standard deviation of y intercepts of regression
line.
S = Slope of calibration curve.
Robustness:
Robustness was evaluated using 600ng/band by making small deliberate change in saturation time (±5%) of mobile phase, the amount of phase (±1). The effect of various deliberate changes was studied on retention factor and peak area, the % RSD was calculated. (7-8)
RESULT AND DISCUSSION:
A wavelength of 330nm was chosen for quantification. The Rf value of Rosmarinic acid after development with mobile phase toluene: ethyl acetate: Formic acid (4:5:1) was 0.55 ± 5%.
Fig. 2: Chromatogram of Standard Rosmarinic acid
Fig. 3: Chromatogram of rosmary capsule containing Rosmarinic acid.
Linearity:
Linearity of an analytical method is its ability within a given range to obtain test results that are directly or through a mathematical transformation proportional to concentration of analyte (9).
A good linear relationship between response (peak area) and amount was obtained over the range of 200-1200ng/band. Linear regression data for the calibration plot as correction coefficients (r) was found to be 0.994. (Fig. no.4) The regression equation for Rosmarinic acid was found to be Y= 4028.127 + 9.113X.
The regression was used to estimate the amounts of Rosmarinic acid in Rosmary capsule.
Fig. 4: 3D overlay of HPTLC chromatogram of Rosmarinic acid
Precision:
In order to control scanner parameters, that is, repeatability of measurement of peak area, instrumental precision was check by repeated scanning (n=6) of the same band of Rosmarinic acid (600ng/band), and was expressed as %RSD and was found to be less than 3% as shown table no(2). In the inter day intra day precision studies six replicate injection of standard samples solutions were made and the response factor of standard compounds and %RSD were calculated. The result are shown in table no (1).
Table no.1: Statistical evaluation for system precision and method precision
|
Sr. No |
System precision |
Method precision |
||
|
Peak area |
% Rosmarinic acid |
Peak area |
% Rosmarinic acid |
|
|
Mean |
11757.035 |
3.33 |
11250 |
3.15 |
|
S.D |
73.66 |
0.02 |
160.35 |
0.045 |
|
RSD. |
1.59 |
1.6 |
0.75 |
0.78 |
Accuracy:
Accuracy of an analytical method is closeness of test results to true value analyte (10). It was determined by application of analytical procedure studies. The marketed formulation was spiked with 80, 100, and 120% of Rosmarinic acid standards. The mixtures were analyzed in triplicate. Results is shown in Table no (2).
Table no: 2 Statistical validation for recovery study
|
Level of recovery |
% Recovery |
S. D (±) |
R. S. D |
|
80% |
99.47 |
0.46 |
0.4 |
|
100% |
100.18 |
0.55 |
0.5 |
|
120% |
99.60 |
0.58 |
0.5 |
Limits of detection and quantification:
Limit of detection (LOD) and limit of quantification (LOQ) of the developed method were determined by injecting progressively low concentrations of the standard solutions using the developed HPTLC method. The LOD and LOQ of Rosmarinic acid calculated 0.64(ng/Band) and 1.94 (ng/Band) respectively.
Robustness:
The robustness of the method was determined by making slight changes in the chromatographic conditions (mobile phase volume, saturation time). It was observed that there was no marked change in the chromatograms (Table no.3).
Assay of marketed formulation:
For the analysis of herbal formulation, 20 capsule (Rosmary capsule) were weighed and finely powdered. The capsule powder equivalent to 10mg of Rosmarinic acid was accurately weighed and transferred into a 10ml volumetric flask and methanol was added and the solution was sonicated for 30 min and filtered through Whatman filter paper 41 and then the volume was made up to the mark with methanol (1000µg/ml). From the above solution, 6µl was applied (n=6) on the TLC plate and the percentage content of Rosmarinic acid was calculated from the calibration curve.
Table 3: Result of robustness
|
Factor |
Level |
Area |
Rf |
|
Saturation time |
|
|
|
|
5 min |
-5 |
11755.71 |
0.52 |
|
10 min |
0 |
11748.73 |
0.55 |
|
15 min |
+5 |
11750.65 |
0.50 |
|
|
S. D± R. S. D |
|
1.9 ± 0.34 |
|
Total mobile phase |
level |
Area |
Rf |
|
9ml |
- 1 |
11680.75 |
0.55 |
|
10 ml |
0 |
11690.13 |
0.55 |
|
11 ml |
+1 |
11685.04 |
0.54 |
|
|
S. D± R. S. D |
|
1.5 ± 0.26 |
Table no 4: Results of assay of rosmary capsule formulation by HPLC method
|
S. No |
Wt. of Rosmarinic acid equivalent to 10 (mg) |
Peak area |
Amount of Rosmarinic acid Found (mg) |
% of Label Claim |
|
1. |
350 |
11118.2 |
11.08 |
3.1 |
|
2. |
350 |
11397.2 |
11.39 |
3.3 |
|
3. |
350 |
11080.7 |
11 |
3.1 |
|
4. |
350 |
11160.6 |
11.06 |
3.1 |
|
5. |
350 |
11497.1 |
11.04 |
3.4 |
|
6. |
350 |
11123.5 |
11.06 |
3.1 |
|
Mean content (%) |
3.2 |
|||
|
% R.S.D. |
0.2 |
|||
|
% S.D. |
0.13 |
|||