Development and validation of high-performance thin-layer chromatographic method for the determination of Rutin from the aerial parts of Alhagi pseudalhagi (M. Bieb) Desv Mishra S. B.a, Singh S.a* and Mukerjee A.a (Received 06 January 2017) (Accepted 16 March 2017) Abstract A simple, accurate and precise HPTLC method has been developed for the estimation of rutin from the aerial parts of Alhagi pseudalhagi (M. Bieb) Desv. The method employed silica gel 60 F254 precoated plates as stationary phase and a mixture of ethyl acetate: acetic acid: formic acid: water (10:1:1:1 v/v/ v/v) as mobile phase. Densitometric scanning was performed at 366 nm after derivatizing the resolved peaks with anisaldehyde-sulphuric acid reagent. Beer’s law was obeyed in the concentration range of 400-1200ng/spot with regression equation for standard rutin as Y= 7.658x - 53.86 and r2=0.9885. The Retention factor for rutin is 0.39 ± 0.008. The % RSD (n=9) for interday, intraday precision and the recovery values were found to be 1.31% and 1.46% and 99.55-101.12%, respectively. The method was validated as per ICH Guidelines, proving its utility in estimation of rutin from the aerial parts of Alhagi pseudalhagi (M. Bieb) Desv.
Keywords: Alhagi pseudalhagi (M. Bieb) Desv, Rutin, HPTLC, Validation, Densitometric
Introduction Alhagi pseudalhagi (M. Bieb) Desv. (Alhagi maurorum Medic), is a perennial plant from fabaceae family, used traditionally as laxative, antibilious, diuretic, diaphoretic, expectorant and antiemetic. The leaves are used for fever, headache and rheumatism. The flowers are used for piles. The whole plant is used in gout and hemorrhagic disorders. The aerial parts of the plant contain flavonoids, tannins, sterols, triterpenes, saponins and anthraquinones1,2. The plant is also used as antiinflammatory, analgesic, antiproliferative, antioxidant and antiulcerogenic3-10. Rutin is classified as polyphenolic flavonoid and is isolated from the aerial parts of the plant, A. pseudalhagi (M. Bieb) Desv11,12. Apart from flavanoids, other constituents including essential oils, ketones, acid derivatives, terpenoids and hydrocarbons have also been isolated from stems and leaves of A. pseudalhagi (M.Bieb) Desv13-16. Rutin possess antioxidant, antirheumatic, antidiabetic, antiadipogenic and neuroprotective activity which have pharmacological relevance to the society hence this study has been undertakens17-21. Literature survey revealed that Reverse phase High Performance Liquid Chromatography (RP-HPLC)22-24 and HPTLC25,26
methods heve been developed for the estimation of rutin in various plant samples and polyherbal formulations, but no HPTLC method has been developed for rutin from the aerial parts of the plant A. pseudalhagi (M.Bieb) Desv. This paper describes a HPTLC method for the determination of rutin from the aerial parts of the plant.
Materials and methods Plant Material The dried aerial parts of A. pseudalhagi (M. Bieb) Desv., were collected from the local market of Allahabad and authenticated by the taxonomist at Botanical Survey of India, Allahabad. The voucher specimen (95446) has been deposited in the departmental herbarium of the BSI, Allahabad for future reference.
Solvents, chemicals and Instruments All the solvents were purchased from CDH Fine Chemicals and were of AR grade. A CAMAG HPTLC system (Muttenz, Switzerland) comprising of Hamilton 100 µl syringe, Linomat IV applicator, twin trough developing chamber (20 X 20 cm), Camag TLC Scanner 3 with wincats 3 Software, UV Cabinet with UV lamp, Silica Gel G60 F254 and 5cm X 10cm TLC Plate as stationary plate.
a
Department of Pharmacy, United Institute of Pharmacy, A-31/1, UPSIDC Industrial Area, Naini - 211 010, Allahabad, India * For correspondence: E-mail-
[email protected]
INDIAN DRUGS 55 (05) may 2018
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Preparation of Standard Solution of Rutin Standard stock solution of rutin (1000µg/ml) was prepared by dissolving 25mg rutin in 25ml of methanol. 2.0 mL aliquot of the above solution was made upto 10mL with methanol (200ng/µl). From the above solution, 2µl, 3µl, 4µl, 5µl and 6µl were spotted on the HPTLC plate, which corresponds to 400ng/spot, 600ng/spot, 800ng/spot, 1000ng/spot and 1200ng/spot, respectively.
parameters checked were linearity & range, accuracy, precision, limit of detection, limit of quantitation and robustness. The results are shown in Table II. Table II: Method Validation Parameters Parameters Linearity range (ng/spot)a Correlation coefficient (r2) Slope Intercept Intraday Precision (%RSD)n Interday Precision (%RSD)n LOD LOQ % Recovery
Extraction and Preparation of Test Samples The dried aerial parts were powdered and 200 grams of powder were extracted with 95% V/V ethanol in Soxhlet apparatus at 500C. The extract was evaporated to dryness in rotavapour to get the residue. 2 grams of the residue was dissolved in 25 mL methanol.
Chromatographic Conditions Analysis was performed on 5cm X 10cm HPTLC silica gel G60 F254 Plates. Different compositions of the mobile phase were tried using binary and ternary mixtures of solvents like toluene, methanol, chloroform, ethyl acetate, acetic acid and formic acid, with chamber saturation to achieve optimum resolution. After several trials, mixture of ethyl acetate: acetic acid: formic acid: water (10:1:1:1v/v/v/v) was chosen as the mobile phase for analysis. The details are given in Table I.
a n
Rutin 400-1200 0.9885 7.658 53.86 1.37 1.46 5.24 15.90 99.5-101.12
five concentrations nine determinations
Linearity Linearity of the method was analyzed by using five different concentrations in the range of 400-1200ng/spot for rutin. Results are expressed in terms of correlation coefficient. The calibration curve was plotted as shown in the Fig.1.
Table I: Selection of solvent System Solvent System Ratio Chloroform: methanol: water 9:1:0.1(v/v/v) Toluene : ethyl acetate : formic 7 :2 :1(v/v/v) acid Ethyl acetate :acetic acid : formic 10:1:1:2.5(v/v/ acid : water v/v) Ethyl acetate :acetic acid : formic 10:1:1:1(v/v/v/v) acid : water The plant extract and standard samples were applied on the plate as bands with the band size of 6mm and at position of 15mm. The plate was developed to a distance of 80mm in the glass chamber, previously saturated with ethyl acetate: acetic acid: formic acid: water (10:1:1:1v/v/V/V) mobile phase for 30 min. The plates were completely dried in air at room temperature, derivatized with anisaldehyde-sulphuric acid solution and scanned. The areas for sample and standard were recorded at 366nm by densitometry.
Method Validation As per the ICH guidelines27, the method validation 38
Fig. 1: Calibration graph of standard drug Rutin
Precision The precision of the method was demonstrated by intraday and interday variation studies. Three different concentrations, 400, 600 and 800 ng/spots of standard plant extract, were analyzed in a day for intraday variation and three consecutive days for inter day variation studies. The results were determined by calculating %RSD. INDIAN DRUGS 55 (05) may 2018
Limit of Quantitation and limit of detection From the calibration curve, the standard deviation (SD) of the intercept (slope) was determined. Then, LOD and LOQ were determined using the expression LOD=3.3ρ/S and LOQ=10ρ/S Where, S is the slope of the calibration curve and ρ is the standard deviation of Y-intercept of regression line.
Accuracy The accuracy of the method was determined by recovery experiments. The recovery studies were carried out at three levels of 80, 100 and 120% and the percentage recovery was calculated and presented in Table III. Table III: Recovery studies %Amount estimated 80 100 120
Amount present (ng/spot) 400 400 400
Amount recovered (ng/spot) 395.53 398.25 400.10
%Recovery ± SD
Fig. 3: Densitometric chromatogram of 95% V/V ethanolic extract of Alhagi pseudalhagi (M.Bieb) Desv. at 366nm
101.12 ± 1.40 99.55 ± 0.92 100.02 ± 0.21
Fig. 2: Densitometric chromatogram of Standard Rutin at 366nm
Fig. 4: HPTLC plate diagram (1-5) Standard Rutin spots and (6) 95% V/V ethanolic extract Alhagi pseudalhagi (M. Bieb) Desv. spot
Robustness
Assay
The robustness was determined by performing small changes in mobile phase ratio and chamber size. The effect on the RF values and peak areas were noted.
A 0.025 mL aliquot of the test solution was made upto 10-mL with methanol. 3µL was spotted on the plate for HPTLC assay. The assay was performed in triplicates and mean ± SD were calculated.
Specificity Specificity is the ability to access unequivocally the analyte in the presence of impurities and degradation products. It is done by comparing RF value of samples with that of standard rutin. The method was found to be specific. INDIAN DRUGS 55 (05) may 2018
Results and Discussion Plates were developed by linear ascending development method. The densitometric chromatogram of 39
standard rutin is shown in Fig. 2. There is no interference in analysis by presence of other components in the extract. The densitometric chromatogram of 95% V/V ethanolic extract of A. pseudalhagi (M.Bieb) Desv. is shown in Fig. 3. Spectra of standard drug also match with the sample spectra and hence it is confirmed that the method is specific. The calibration curve for rutin was obtained by plotting peak area of rutin versus concentration. The HPTLC plate is shown in Fig.4. The results of interday and intraday precision studies indicate that the developed HPTLC method was precise. The recovery studies were within the range of 100.23 ± 1.10%. For robustness parameters in case of the mobile phase ratio and the chamber change the % change in RF value was not more than 0.08% and 0.22%, respectively, and the % change in area was not more than 0.189 % & 0.117%, respectively. The method was found to be robust since the monitored parameters were not significantly affected. The content of rutin in the aerial part of the plant was found to be 0.241± 0.01ng/spot.
Conclusion The proposed HPTLC method for the estimation of Rutin in aerial parts of Alhagi pseudalhagi (M.Bieb) Desv., was found to be sensitive, accurate, precise, specific, simple and rapid. This method can also be used to estimate presence of rutin in formulations. Summary of all validation parameters confirms the consistency of the method.
Acknowledgements Authors are thankful to Mr. G. P. Sinha, Scientist, Botanical Survey of India, Allahabad for authenticating the plant. Authors are also grateful to NBRI, Lucknow for providing access to instrumentation facilities.
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