RP-HPLC Method Development and Validation for Simultaneous Estimation of Rutin and Quercetin in Morus alba L. leaf extract

 

Sarita Garg¹, Rubal Chahal¹, Deepak Kaushik¹, Rakesh Kumar², Vineet Mittal¹*

¹Department of Pharmaceutical Sciences, Maharshi Dayanand University, Rohtak, Haryana, India.

²Department of Pharmacy, Vaish Institute of Pharmaceutical Education and Research, Rohtak, India.

 

ABSTRACT:

Mulberry, which pertains to the Moraceae family and the genus Morus,has been used in medicine for ages because of its chemical composition and pharmacological action. The plant is recognized in traditional Chinese medicine for its antiphlogistic, diuretic, expectorant, and antidiabetic characteristics. The mulberry leaf is high in flavonoids, which have a variety of biological functions, including antioxidant potential. Quercetin, isoquercetin, rutin, isoquercitrin, quercitrin, luteolin, chlorogenic acid, and other flavonoids can be found in their leaves. The major constituents of total flavones in mulberry leaf extract are rutin and quercetin. The goal of the RP-HPLC approach was to design and validate a method for identifying these elements. HPLC (Shimadzu Technologies LC series) with UV-visible detector at 259nm and C18 column (250mm 4.6mm, 5µm particle size) was used to achieve chromatographic separation (Phenomenex Luna). Solvents (acetonitrile and 0.1 percent v/v solution of glacial acetic acid) were used in the isocratic elution mode. The run time was 10 minutes. The column temperature was 37 degrees Celsius with sample insertion volume 20µl and the flow velocity was 1ml/min. For quercetin and rutin, the devised method produced strong linearity, high accuracy, high precision, high repeatability, high robustness, and high ruggedness. For quercetin and rutin, the LOD and LOQs values were determined to be within acceptable levels.

 

 

 

INTRODUCTION: 

The importance of herbal drugs has increased in exponential manner during this Pandemic era. Quality control and standardization of herbal formulations is one of the main challenge in the popularization of traditional crude drugs. Flavonoids are plant-originated secondary substance with antioxidant and free radical-cleaning aptitudes¹. Plants rely on flavonoids for appropriate proliferation, maturation, defense against infection, and injury recovery².

 

Mulberry, a member of the Moraceae family and the genus Morus, is a traditional herb that has been used in medicines for ages due to its chemical makeup and pharmacological action^3-5. Mulberry (Morus alba L.) is endamic to many Near Eastern and Asian nations, including Turkey,

 

Azerbaijan, Iran, Pakistan, India, China, Korea, and Japan, and is widely used in traditional medicine in such countries⁶. The majority of fostered varieties in India are from the M.alba or M.indica species⁷. In traditional Chinese medicine, the plant is used for its antiphlogistic, diuretic, expectorant, anti-ulcer, and antidiabetic effects⁸. The fruit, bark, leaf, and root of the mulberry plant have all been studied for their ability to cure hyperglycemia, with the bark and root in particular being used to reduce blood sugar levels^9-10.

 

Mulberry leaf's flavonoid content confers distinct antioxidant characteristics and may assist animals to cope with reactive oxygen species stress,at stressful times like new-born, ablactation, and the periparturient phase (Figure 1A)^11-13. Moreover, it is usedto treat tearing eyes, dizziness, fever, sore throat, cough, skin problems, thirst, neurosis, ulcers, leukaemia, colon cancer, breast cancer and, as well as reduce hypertension and blood glucose levels pertaining to amino sugars, polysaccharides, alkaloids, flavonoids, and related chemicals^14-23. A few reports of the M.alba species having anti-ulcer effects have been discovered⁷.

These flavonoids have a high predilection for assimilation in the body via the intestine, with little to no negative side effects²⁴. The leaves are also fed to livestock as an adjunct to boost milk production and quality²⁵. Mulberry leaves contained significant levels of phenolic chemicals such as 1-deoxynojirimycin and melatonin^26-28. Mulberry leaves, as a result, could be used as a beneficial additive in the manufacturing of nutritious dishes²⁹.

 

The majority of earlier research focused solely on cultivar effects on phenolic chemicals in mulberry leaves. However, the interactive effects of cultivars and leaf ages have been shown to impact melatonin content in mulberry leaves²⁸. Furthermore, phenolics in spinach and berries were altered by cultivars and leaf ages. Quercetin, isoquercetin, rutin, isoquercitrin, quercitrin, luteolin, chlorogenic acid, and other flavonoids can be found in their leaves as shown in Figure 1B. Rutin and quercetin are essential constituents of mulberry leaf extract's overall flavones³⁰.

 

Figure 1: Multipurpose Roles of Mulberry plant (A);Chemical structure of Rutin and Quercetin present in M. alba (B)³⁰

 

Flavonoids are polyphenolic chemicals with a C6-C3-C6 structure that are present in a wide variety of plants, fruits, and vegetables. Anthocyanidins, flavanones, flavanols, and flavonols are all flavonoids. Glycosides and aglycones like quercetin make up the flavonols in mulberry leaves. The antioxidant potential of mulberry leaf extract varies depending on the flavonol proportions, which varies depending on the extraction process³¹.

Several more therapeutic effects of quercetin and rutin have been described, including anti-inflammatory, antihypertensive, vasodilator effects, anti-obesity, anti-hypercholesterolemic, anti-ulcer, and anti-atherosclerotic activities³². However, research on physiologically active quercetin and rutin precursors in mulberry leaves is limited, and it is unclear whether these variants might help boost biological impacts in humans in-vivo.

 

Various analytical methods, such as ultraviolet-visible spectroscopy, high-performance liquid chromatography (HPLC), mass-spectrometry (MS) and fluorescence spectroscopy have been used for the determination of flavonoids from herbal materials. But there has always been the requirement for the development of simple, fast and reliable analytical method for the qualitative and quantitative analysis of pharmacologically important flavonoids. Moreover, the concentration of plant actives in the herbal raw material largely dependent upon various exogenous and endogenous factors²⁰. Therefore, the development of novel analytical method is need of hour to establish the quality of raw material for herbal formulations. Hence, in the present research we aimed to develop and validate a novel RP-HPLC method for the simultaneous estimation of flavonoids like rutin and quercetin from the extract of selected medicinal plant.

 

MATERIALS AND METHODS:

Chemical and Reagents:

All the reagents and chemicals used for the present study were of HPLC grade. HPLC-grade acetonitrile, methanol, double distilled water and ethanol were obtained from Fisher Scientific (Fair Lawn, NJ, USA). A standard sample of quercetin and rutin were supplied from LobaChemie Pvt. Ltd. (Mumbai, Maharashtra, India). HPLC grade Glacial acetic acid was procured from Merck (Kenilworth, New Jersey).

 

Collection of Plant material:

Mulberry (Morus alba L.) leaves were taken from the Vaish Institute of Pharmaceutical Education and Research's Herbal Garden in Rohtak. The plant material was certified by the Maharshi Dayanand University's Department of Botany in Rohtak.

 

Extraction of polyphenols from Mulberry leaf:

Polyphenols were isolated from the leaves of the Mulberry (M.alba) using method given by Yang Zo et al (2013) with minor adjustments. The leaves were disinfected by washing them three times with water and then drying them in the shade at room temperature. The dried leaves were ground to a fine powder in a grinder, then sieved (40 mesh) and stored in an airtight glass container. Using the soxhlet equipment, 20 gramme of dried leaves powder was extracted for 1 hour with 2 litres of 99.9% ethanol. After that, the ethanol was evaporated, and the extract was dried at room temperature until all of the ethanol had evaporated. The acquired extract was used to design and validate a technique utilizing HPLC³³.

 

Selection of Mobile Phase and Optimization of Chromatographic condition:

Various solvent systems, such as Methanol, Water, Glacial Acetic acid, Acetonitrile, Acetate buffer pH 3, O-phosphoric acid, were attempted rather than selecting a suitable mobile phase for the estimation of Rutin and Quercetin and optimization of HPLC chromatogram. According to the literature, the offered mobile phases have constraints such as peak symmetry, retention time, method run time, and quantification^33-36. As a result, the chromatographic conditions had to be optimized.

 

Identification of polyphenolic compounds by HPLC and its quantification:

A Shimadzu Technologies LC series (Kyoto, Japan) HPLC system with a quaternary pump, a thermostatted column compartment, a vacuum degasser, and an automated injector with a UV detector was used to chromatographically estimate polyphenolic chemicals. The chromatographic settings were C18column (250 mm 4.6mm, 5µm particle size, Phenomenex Luna), Isocratic elution mode, and 50:50 mobile phase A and B solvents (acetonitrile and 0.1 percent v/v solution of glacial acetic acid). The estimations were taken at 259 nm after a 10-minute running period. The sample injection volume was 20µl, the flow rate was 1ml/min and the column temperature was 37°C. The retention period and UV spectrum of reference standards were used to identify phenolic acids. The concentrations of the substances were determined from peak area according to the calibration curves after injecting reference standards at concentrations of 10µg/ml into the HPLC apparatus. Each sample received three injections. A comparison of the retention period and ultraviolet spectra of each peak in mulberry leaf samples with those of reference compounds established their identities³⁷.

 

Method Validation for Rutin and Quercetin analysis in Mulberry leaf extract:

Following ICH guidelines, the method was evaluated for linearity, detection and quantification limits (LOD and LOQ), precision (intraday and Interday variation and repeatability), and accuracy (recovery)³⁸.

 

Linearity:

The potential of an analytical technique to produce test findings that are directly proportional to the quantity of analyte in a sample within a given range is referred to as linearity. The analytical method's range is the distance between the top and lower analyte values that have been shown to be determined with sufficient precision, accuracy, and linearity. The linearity range for rutin and Quercetin was chosen to be 1-6µg/ml.

 

Rutin was produced as a stock solution (10µg/ml) in ethanol and diluted to various quantities (1-6µg/ml). The same medium was used to generate the quercetin stock solution, which was then diluted to form serial dilutions in the same concentration range as rutin, i.e., 1-6µg/ml. For the calibration curves, the samples were filtered via a 0.2µm membrane filter and then injected. By graphing concentration versus area, the linearity was calculated. The intercept, slope, and correlation coefficient of each calibration line were calculated using linear regression analysis. The standard deviation of 'Y' intercepts of regression lines and the slope were used to calculate the limit of detection (LODs) and quantification (LOQs), using the equations:

                      3.3 𝜎                              10 𝜎

LOD = ------------------  LOQ= ----------------------

                       S                                   S

 

Where 𝜎 is the standard deviation of the response (Y-intercept) and S is the slope of the calibration curve.

 

Precision:

The ISO International Vocabulary of Basic and General Terms in Metrology (ISO-VIM) and the International Committee on Harmonization (ICH) define precision as the degree of agreement among quantity values obtained by repeated measurements of a quantity under prescribed conditions. Assessing precision entails using the standard deviation, variance, or coefficient of variation to represent quantitatively the random error or degree of dispersion of a set of individual measurements. By assessing specimen with six concentrations of rutin and quercetin, intra- and inter-day fluctuations were used to determine the precision of the devised assay. Precision was calculated as a percentage of the variance coefficient³⁵. The intra-day and inter-day variation for Rutin (0.108mg per 25mg of extract) and Quercetin (0.158mg per 25mg of extract) assessment was carried out six times on the same day and six days in a row, with the concentration of Rutin and Quercetin being used to compute the % RSD.

 

Accuracy:

The accuracy of the method was assessed by calculating the sample's percent mean recovery at three distinct levels (80-120percent). Three determinations were made at each level. The method's great accuracy is demonstrated by the low percentage relative SD value (% RSD<1).

 

Repeatability:

It is the agreement between a sequence of measurements of the same amount taken in fast succession under the same conditions (analyst, apparatus, instrument, and day). A standard Rutin solution (0.108mg per 25mg extract) was made for this experiment and examined six times using the described approach. The same methodology was used to generate and evaluate a standard solution of Quercetin (0.158mg per 25mg of extract).

 

Robustness:

The capacity to give accurate and exact outcomes under a range of settings is referred to as robustness. To determine the degree of technique resilience, the most essential parameters were swapped out while the other parameters remained the same, and the chromatographic profile was monitored and recorded in parallel. Changes in wavelength and flow rates were the parameters that were investigated. The capacity to give accurate and exact outcomes under a range of settings is referred to as robustness. To determine the degree of technique resilience, the most essential parameters were swapped out while the other parameters remained the same, and the chromatographic profile was monitored and recorded in parallel. Changes in wavelength and flow rates were the parameters that were investigated.

 

Ruggedness:

Ruggedness was tested by having two independent analyzers analyse the same samples of Rutin and Quercetin in concentration (0.108mg/25mg extract; 0.158mg/25mg extract) and recording the percentage recovery for each³⁹.

 

RESULTS:

Development and Optimization of RP-HPLC Method for estimation of Rutin and Quercetin:

Selection of Mobile Phases and Optimization of Chromatographic Conditions

Various permutations of solvent systems, such as Methanol, Water, Glacial Acetic acid, Acetonitrile, Acetate buffer pH 3, O-phosphoric acid at varying concentrations, were tried to optimize the HPLC chromatogram, and the conjunction of Acetonitrile (Solvent A) and 0.1 percent v/v solution of Glacial Acetic acid was chosen for the assessment of Rutin and Quercetin in Mulberry extract.

 

Identification of Polyphenolic compounds by HPLC:

As rutin and quercetin were thought to be important ingredients in M.alba leaves, their quantification was done using HPLC analysis. Figure 2 depicted the HPLC chromatogram of standard Rutin and Quercetin, which showed peaks at retention times of 3.239 and 5.959, respectively, and 3.304 and 5.863 (Figure 3) in M.alba extract, which were both very close to the standard values, confirming the potency of the adopted method for Rutin and Quercetin quantification.

 

Figure 2: Chromatogram of Rutin and Quercetin Standards (10 ug/ml)

 

 

Figure 3: Chromatogram of Mulberry leaf extract showing Rutin and Quercetin peaks

 

Validation of RP-HPLC method forRutin and Quercetin in Mulberry leaf extract:

System Suitability:

System suitability testing is commonly used to verify the HPLC system's reproducibility. The results obtained in the various parameters assessed are satisfactory (Table 1), indicating that the HPLC system's functionality was enough for the detection of rutin and quercetin in Mulberry.

 

Table 1. System suitability and Validation parameters for Rutin and Quercetin

Parameters	Results
	Rutin	Quercetin
Retention time	3.304	5.863
Peak Area	206749	419923
Linearity range (µg/mL)	1-6 μg/ml	1-6 μg/ml
Slope	38434	73603
Intercept	40353	48074
Correlation coefficient (R2)	0.999	0.998
Limit of detection (µg/mL)	0.059438	0.104
Limit of quantification (µg/mL)	0.180114	0.315

 

Linearity and Range:

By examining different concentrations of rutin and quercetin and determining the peak area for every concentration, a six-point calibration curve was created. The linearity curve was created by plotting the observed chromatogram peak regions against concentrations (1-6 µg/mL). Table 1 and Figure 4 summarizes the correlations (R² values).

 

 

Figure 4: Standard curve of Rutin (A) and Quercetin (B) by RP-HPLC

 

 

Limit of Detection (LOD) and Limit of Quantification (LOQ):

The specificity of the proposed technique was assessed by calculating the limit of detection (LOD) and the limit of quantification (LOQ) according to the International Council on Harmonization (ICH) criteria (LOQ). The sensitive nature of the devised approach was demonstrated by the low LOD and LOQ values given in Table 1.

 

Specificity:

When the pure chromatograms of Rutin and Quercetin were compared to those of leaf extract (Figure 2 and 3), it was clear that the contents of the extract did not interfere with the peak of pure chemicals. Furthermore, the polyphenols from the fraction, namely Rutin and Quercetin, were eluted at the same retention period as pure substances, demonstrating the method's specificity.

 

Accuracy and Precision:

Precision:

The intra-day and inter-day variation for Rutin and Quercetin detection was carried out 6 times on the same day and six days apart using concentrations of Rutin and Quercetin in the extract of 0.108mg/25mg and 0.158 mg/25 mg, respectively. The percent RSD was determinedand results weredepicted in Table 2.

 

 

Table 2: Results of Precision data of Rutin and Quercetin

Intraday Precision			Interday Precision
Rutin
Concentration(mg/25mg)	% Recovery	Mean=98.07537629 SD=1.866079558 %RSD=1.902699361	% Recovery	Mean=98.31659067 SD=1.595552294 % RSD=1.62287187
0.108	96.00693541		96.04066326
0.108	97.81498924		99.7856595
0.108	96.40444225		97.29100295
0.108	98.33415726		99.4363353
0.108	98.67926547		97.48313126
0.108	101.2124681		99.86275173
Quercetin
Concentration(mg/25mg)	% Recovery	Mean=101.0101 SD=0.855623 % RSD=0.847066	% Recovery	Mean=100.931 SD=0.695061 % RSD=0.68865
0.158	100.8884		102.0393
0.158	100.9469		101.1596
0.158	101.6544		100.8227
0.158	99.41112		100.1013
0.158	101.6811		100.3101
0.158	101.4786		101.1529

 

 

For Rutin and Quercetin, the percent RSD value determined from intraday precision was 1.902 percent and 0.847 percent, respectively. The percent RSD for both constituents was roughly 1.622 and 0.688, respectively, as a result of Interday precision. The percent RSD value obtained with both intraday and interday precision was quite low, indicating that the devised approaches were accurate.

 

Accuracy:

To assess the accuracy of the devised approach, the percent (%) mean recovery of the sample was collected at three distinct levels (80-120 %).  Percent mean recovery was calculated as shown in Table 3. The established recovery levels were between 80 and 120 percent.

According to the findings of the Accuracy analysis, the percent (%) recovery of both constituents, Rutin and Quercetin, was within the specified range of 80-120 percent, indicating that the established method was accurate. The obtained results indicated that the suggested approach was accurate and precise within the analytical range.

 

Robustness:

Changes in wavelength and flow rates were used to confirm the system's robustness and results were depicted in Table 4 for Rutin and the results with Quercetin were presented in Table 5.

 

Table 3: Accuracy data of Rutin and Quercetin

 

Table 4: Robustness data of Rutin (Change in wavelength and Flow rate)

Robustness	Change in wavelength				Change in flow rate
	257 nm		261 nm		0.80 ml/min		1.2 ml/min.
Concentration (mg/25mg)	Area	Statistical analysis	Area	Statistical analysis	Area	Statistical analysis	Area	Statistical analysis
0.108	153948	Mean = 154848 SD = 1322.7 %RSD = 0.854	136128	Mean =135549 SD =608.41 %RSD = 0.448	181517	Mean =181647 SD =2429.61 % RSD =1.33	111387 111694 110360	Mean =111147 SD =698.63 % RSD =0.628
0.108	160367		135605		179285
0.108	154230		134915		184139

 

Table 5: Robustness data of Quercetin (Change in wavelength and Flow rate)

Robustness	Change in wavelength				Change in flow rate
	257 nm		261 nm		0.80 ml/min		1.2 ml/min.
Concentration (mg/25mg)	Area	Statistical analysis	Area	Statistical analysis	Area	Statistical analysis	Area	Statistical analysis
0.158	356408	Mean = 356949 SD = 937.62 % RSD = 0.262	330424	Mean =330815 SD =791.25 % RSD = 0.239	421247	Mean =420710 SD =606.72 % RSD =0.1442	294927	Mean =295636 SD =1228 % RSD =0.415
0.158	358032		330296		420832		294927
0.158	356408		331726		420052		297054

The statistical analysis of Rutin and Quercetin was not significantly affected by a modest change in the settings, according to the robustness research. The HPLC variables did not change significantly much, showing that the HPLC approach used is trustworthy.

Ruggedness:

By altering the experimental settings, the robustness of the approaches was investigated. In this study, two analysts used the identical set of parameters to execute the procedure, and mean, standard deviation, and relative standard deviation were computed. The % RSD as shown in Table 6.

 

Table 6: Results showing Ruggedness

Rutin data
	Analyst 1		Analyst 2
Concentration (mg/25mg)	Area	Statistical analysis	Area	Statistical analysis
0.108	206917	Mean = 204969.7 SD = 2105.156 % RSD =1.027	205452	Mean =202064 SD = 2944.82 % RSD = 1.45
0.108	202736		200116
0.108	205256		200625
Quercetin data
	Analyst 1		Analyst 2
Concentration (mg/25 mg)	Area	Statistical analysis	Area	Statistical analysis
0.158	420314	Mean = 420399 SD = 1204.2 % RSD =0.286	427369	Mean =424483 SD = 2549.9 % RSD = 0.6007
0.158	421643		422535
0.158	419239		423545

 

It was observed that there were no marked changes in the HPLC parameters demonstrating that the HPLC methods developed was rugged. There were no significant changes in the HPLC parameters, indicating that the HPLC procedures established were robust.

 

DISCUSSION:

Herbal medicine standardization is riddled with difficulties. We devised a simple, optimized, and verified HPLC method for the standardization of M.alba in this study. Flavonoids, which include rutin, quercetin, and isoquercetin, are the active antioxidant components in the flora of M.salba and are responsible for pharmacological activity. The flavonoids rutin and quercetin were chosen for quantification since they are responsible for the plant's physiological effect. Calibration of analytical processes is the method of verifying those analytical techniquesthat are appropriate for their intended use and support the recognition, concentration, quality, purity, and effectiveness of pharmaceutical preparations. A new HPLC approach was devised and validated for the quantification of two flavonoids from leaves: rutin and quercetin. The isocratic elution method was used in the HPLC method to optimize the mobile and stationary phases such that rutin and quercetin peaks can be detected concurrently. Different elements such as the column, detector, mobile phase, and others influence the isolation and clarification of the HPLC process. To optimize the HPLC settings, multiple mobile phases (water, acetonitrile, buffer, glacial acetic acid) with varied ratios were tested at a detection wavelength of 259 nm. The C18 stationary phase column was used because it provided good retention, theoretical plates, and resolution between extract and pure rutin and quercetin. The mobile phase ratio and concentration were changed to provide an excellent chromatogram and a short retention period without sacrificing the method's selectivity. The acetonitrile and 0.1 percent v/v solution of glacial acetic acid (50:50) was determined to be suitable among the several mobile phases used. By isocratic elution of the selected mobile phase utilizing UV absorbance at max 259 nm, the HPLC chromatogram of Rutin and Quercetin in the produced fraction exhibited good segregation with higher peak resolution. Indeed, the produced fraction's HPLC chromatogram of rutin and quercetin was analogous to the pure drug's standard solution. The devised analytical method for detecting rutin and quercetin from M.alba leaf extracts yielded retention times of 3.304 and 5.863 minutes, respectively, at a flow rate of 1 mL/min in the current investigation. According to Ju Wan-Taek and colleagues, the retention duration for these phytoconstituents measured from Korean mulberry leaves is 13.5 minutes⁴⁰. The findings of the aforementioned study indicated that the detection of rutin requires a lengthy run time, resulting in a waste of time and solvent. In other studies, the retention times of rutin and quercetin were shown to be 28.74 and 30.90 minutes, respectively, which is significantly higher than our reported values. As a result, the proposed method might be described as cost-effective and hence new when compared to existing methods⁴¹. The rutin and quercetin calibration curves showed excellent linearity and a high regression coefficient. Furthermore, the linearity limit detected in this study indicates that both phytoconstituents may be analyzed across a broad range, which is another benefit of the suggested approach. The proposed method's accuracy and precision describe how near the experimental and actual values are⁴². The empirical inter-day and intraday accuracy and precision data represent the proposed method's accuracy and precision, with precision values of 2% for the repeated analysis of a set of quality control samples that fulfil the standard criteria of the ICH harmonized tripartite guideline⁴³. The high recovery of additional rutin and quercetin with a low percent RSD indicated that matrix interference on the extraction method was minimal. The positive stability data obtained with the new approach also suggests that rutin and quercetin in the extract are stable at room temperature for more than a day. Overall, the devised HPLC technique was shown to be adequate for rutin and quercetin extraction and quantification from leaf extract without interference from other phytoconstituents, with the desired accuracy and precision.

 

CONCLUSION:

For the simultaneous quantification of rutin and quercetin from M.Alba leaves, a simple, fast, and consistent reversed-phase-HPLC approach was designed and implemented. It is recommended for routine quality control analysis of rutin and quercetin from M.Alba leaves. Because of the high tolerance limit, the devised method is advantageous. So far, no report on the estimation of these components in M.alba has been published. As a result, determining the content of these components is critical. For Rutin and Quercetin, the new approach produced repeatability with percent RSDs of 1.41 and 0.873, respectively. After intraday precision, the percent RSD values for Rutin and Quercetin were 1.90 and 0.847, respectively, and after interday precision, they were 1.62 and 0.688, respectively. Both constituents' accuracy data were satisfactory, as the obtained values were within the specified limitations. The recovery study, which was close to 100 percent, was used to determine accuracy and reproducibility. For Rutin and Quercetin, the LOD and LOQ values were found to be 0.059 µg/ml and 0.104 µg/ml, and 0.1801 µg/ml and 0.315µg/ml, respectively. The linearity, robustness, and ruggedness values were all within acceptable limits. The developed method may be useful for routine analysis of rutin, quercetin and other phytoconstituents present in M.alba  leaves that could transformed into viable formulations for efficacious delivery of herbal drugs at targeted sites.

 

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

 

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Accepted on 14.09.2022           © RJPT All right reserved

Research J. Pharm. and Tech 2023; 16(5):2327-2335.