ejpmr, 2016,3(1), 324-335 SJIF Impact Factor 2.

026
Research Article
Salunkhe et al. EUROPEAN JOURNAL OF PHARMACEUTICAL
European Journal of Pharmaceutical and Medical Research
AND MEDICAL RESEARCH ISSN 3294-3211
www.ejpmr.com EJPMR

UV AND HPLC METHOD DEVELOPMENT OF AZADIRACHTIN AND GYMNEMIC

ACID IN POLYHERBAL CHURNA AND ITS VALIDATION
Dr. Vijay R. Salunkhe1* and Dr. C. S. Magdum2
1*,2
Kasegaon Education Societys, Rajarambapu College of Pharmacy, Kasegaon Dist. Sangli (MS), Affiliated to Shivaji
University Kolhapur. Maharastra.

*Correspondence for Author: Dr. Vijay R. Salunkhe

Kasegaon Education Societys, Rajarambapu College of Pharmacy, Kasegaon Dist. Sangli (MS), Affiliated to Shivaji University Kolhapur.
Maharastra.

Article Received on 19/11/2015 Article Revised on 10/12/2015 Article Accepted on 01/01/2016

ABSTRACT
The present research work is associated with UV and HPLC method development of Azadirachtin and Gymnemic
acid in polyherbal churna and its validation .UV method for simultaneous estimation of Azadirachtin and
Gymnemic acid was developed using 95% methanol as a solvent. By scanning, the each solution was in the range of
200-400 nm. 210 nm was selected as a wavelength for Azadirachtin while 217 nm for Gymnemic acid. Method was
validated by linearity ,range ,accuracy, precision (intraday and interday),LOD LOQ. HPLC method for simultaneous
estimation of Azadirachtin and Gymnemic acid was developed using HPLC system of JASCO UV -2075 with
C18Intresil, 4.6(i.d.) x 263 nm columns. Chromatogram for marker was developed using mobile phase methanol
:acetonitrile in the ratio of 60:40 v/v. Separation was achieved with good resolution as 6.3 , Retention time as 1.9417
,3.2083,asymmetry 1.15,0.87 and theoretical plates 1180,7955 for Azadirachtin and Gymnemic acid respectively.
Method was validated by parameters as linearity, range, accuracy, precision (intraday and interday), LOD LOQ
,robustnees . Churna was analyzed in comparision with standard Azadirachtin and Gymnemic acid. The
quantification of Azadirachtin and Gymnemic acid in churna chromatogram was done by comparing peak areas
from chromatogram of standard Azadirachtin and Gymnemic acid.

KEYWORDS: UV, HPLC, method development, Azadirachtin, Gymnemic acid, polyherbal churna, validation.

INTRODUCTION been reported for Azadirachtin and Gymnemic acid by

Quality control[1] of herbal preparation or proprietary UV and HPLC, Therefore it wasour intention to develop
products however is much more difficult than synthetic the suitable method for the same, which gives high
drugs because of the chemical complexity of the degree of assurance with better strength, identity and
ingredients. As herbal preparation comprise hundreds of purity of both the compounds. UV and HPLC method
mostly unique, or species-specific, compounds, it is development and validation is important tool in
difficult to completely characterize all of these analytical area .Hence simultaneous estimation of
compounds. It is also equally difficult to know precisely Azadirachtin and Gymnemic acid in MMC is challenging
which one is responsible for the herbs or herbal investigation in era of herbal drug analysis.
preparation’s therapeutic action because these
compounds often work synergistically in delivering EXPERIMENTAL
therapeutic effects. Thus maintaining consistent quality UV Analysis[6,8]
in herbal preparation, both from batch to batch and over It is must to observe pattern of UV absorbance with prior
time is as problematical as it is necessary and has drawn to HPLC method development for target materials.
serious attention recently as challenging analytical[2]
task. Small scale and large scale producers of herbal Instrument Used
products are proceed large numbers of Ayurvedic SHIMADZU 1800 UV/Visible double beam
proprietary medicine. As our nation is except of high spectrophotometer, with pair of matched quartz cells
market potential in future standardization of such corresponding to 1 cm path length was used for
medicine by advanced analytical techniques is the most measurement of absobance. Elder digital balance used
essential tool for quality assurance[3-5] of the same. Its for weighing, ultra sonicator of Prama instrument was
quantitative and qualitative determination by UV and used for sonicating the drug and sample solution.
HPLC will be a choice of method development of two
active marker compound. Such proposed work with its UV Method Development for Markers (Azadirachtin
assurance of quality of such products in herbal industry and Gymnemic acid)
is currently having great significance. There is no any Selection of Common Solvents: Solubility studies were
precise and economic simulations estimation method has carried out with a view to find a suitable solvent in which

www.ejpmr.com 324
Salunkhe et al. European Journal of Pharmaceutical and Medical Research

the markers are completely soluble and stable. Solvents minutes. Filtered the solution; to get concentration of
like water, 95% ethanol, methanol, acetonitrile were tried 1000 μg/mL. Further dilution was made to get final
for checking solubility of AZA and GYM. After concentration of 10 μg/mL. Absorbance was determined
assessing the solubility of drug in different solvent 95% at 210nm wavelength for Azadirachtin and 217nm
methanol has been selected and finalized as common wavelength for Gymnemic acid Concentration of both
solvent to observe spectral characteristics. markers was estimated by using following formula,
C=A/ba
Selection of Sampling Wavelength for Simultaneous Whereas,
Analysis: The aliquot portions of stock standard C = concentration of AZA/GYM μg/mL , b=path length
solutions of AZA and GYM were diluted appropriately A = absobance of mixture at 210nm/217nm.
with solvent 95% methanol to obtain concentration 10
μg/mL of AZA and 10 μg/mL GYM solution, both the a = absorptivity of AZA at 210nm/absorptivity of GYM
solutions were scanned in the range of 200-400 nm in 10 at 217 nm
mm cell against solvent blank. The absorption maxima
was determined, a representative spectrum of AZA and Method validation[7,8]
GYM in 95% methanol is shown in figure 29, 30 Following parameters were used to validate the method.
respectively. The study of spectrum revealed that
Azadirachtin showed a well defined λmax at 210nm Linearity Study: Linearity was studied by preparing
whereas Gymnemic acid showed at 217nm. These two serial dilutions using standard stock solution in 10 mL
wavelengths were selected for development of volumetric flask. I.C.H. Recommends that for the
simultaneous equation. establishment of linearity, a minimum of 5 concentration
normally used . The various dilutions used for linearity
Preparation of Standard Stock Solution and Study of study are as follows. And the further study was carried
Beer-Lamberts Law out.
Standard Azadirachtin Stock Solution: An accurately
weighed quantity of Azadirachtin (AZA) 10 mg was LOD and LOQ: Limit of detection (LOD) is the lowest
dissolved in 95% methanol in 100 mL volumetric flask amount of an analyte that can be detected but not
and volume was made up to the mark with the same necessarily as an exact value.
solvent to get final concentration of 100 μg/ mL.
Limit of quantification (LOQ) is the lowest amount of an
Standard Gymnemic acid Stock Solution analyte in a sample that can be quantitatively determined
An accurately weighed quantity of Gymnemic acid with suitable precision and accuracy. The LOD and LOQ
(GYM) equivalent to 10 mg was dissolved in 95% were separately determined which is based on calibration
methanol in 100 mL volumetric flask and volume was curve. The S.D. of y intercept of regression line may be
made up to the mark with the same solvent to get final used as S.D. The given concentration was used for the
concentration of 100 μg/ mL. this and the absorbance was taken and then calculate the
standard deviation and slope.
Study of Beer-Lambert's Law
Aliquots of working stock solution of AZA and GYM LOD = 3.3 X σ/s
were prepared with 95% methanol to get concentration LOQ= 10 X σ/s
range of 2-12μg/ mL for AZA and 2-12μg/ mL for GYM. Where, σ =Standard deviation of y intercept of
The absorbance of resulting solutions was measured at regression lines, S =Slope of calibration curve.
their respective wavelength. A calibration curve was
constructed to study the Beer-Lambert's Law and Accuracy: Accuracy of an analytical method is the
regression equation. Dilution used for calibration curve closeness of test results obtained by the method to the
as below. true value. It was ascertained on the basis of recovery
studies performed by standard addition method at 50,
Determination of Absorptivity Values of Markers At 100 and 150 % of test concentration. Known amount of
Selected Wavelengths: Aliquot portions of AZA and standard drugs were added to analyzed sample and
GYM stock standard solutions were diluted with 95% subjected to the developed UV method. Then the
methanol to obtain different concentrations of each drug. absorbance was taken and further calculation was carried
The absorbance of each solution was measured at 210 out. The recovery study was performed three times at
nm and 217 nm. A (1%, 1cm) values were calculated each level.
using following formula, Absorbance
A (1%, 1cm) = Precision: Precision of an analytical method is the
Conc.(g/100ml) degree of agreement among individual results when the
Estimation of Concentration of Azadirachtin and method is applied repeatedly to multiple readings of a
Gymnemic acid In Polyherbal Formulation (MMC) homogeneous sample. It is expressed as %R.S.D. of
10 mg extract of MMC was dissolved in 10 mL 95% series of measurements. The interday's and intra
methanol in 10 mL volumetric flask, sonicated for 15

www.ejpmr.com 325
Salunkhe et al. European Journal of Pharmaceutical and Medical Research

precision was determined using 10 μg/mL concentration ay2 = Absorptivity value of GYM at 217nm
of each marker. A1 = Absorbance of sample at 210nm
A2 = Absorbance of sample at 217nm
Intraday's: It was carried out by estimating the
corresponding responses (absorbance) three times on the HPLC Analysis[10,11,12]
same day between three hrs interval, measurement of Instrument used: HPLC system (Jasco UV-2075
responses (absorbance) was expressed in terms of % model) consisting manual injector having capacity of 20
Relative Standard Deviation (%RSD).The given dilution μL with detector LC-UV-2075 UV/VIS detector. The
was used and the absorbance was taken by different software used was chromenav .Column Intersil ODS-3V
intervals in the same day like 11pm ,1pm ,3pm and (250 x 4.6mm) C18Column packed with 5μm diameter
further calculation was done. particles size from GL science Inc. (made in Japan) was
used for experiments. Elder digital balance used for
Interday (Different days): It was carried out by weighing, pH meter of Hanna instrument, Ultrasonicator
estimating the corresponding responses (absorbance) of of Prama instrument was used for sonicating the drug
same sample was recorded on three different days, and sample solution.
measurement of responses (absorbance) was expressed in
terms of % Relative Standard Deviation (%RSD). The HPLC Method Development For Markers
given dilution was used and the absorbance was taken by (Azadirachtin and Gymnemic acid)
different intervals in a different day like first day, second Selection of Analytical Wavelength
day, third day and further calculation was done. The wavelength for detection was selected by preparing
the individual solution of l0μg/mL of Azadirachtin and
Robustness: The robustness of analytical method is a Gymnemic acid. Each solution was scanned in the range
measure of its capacity to remain unaffected by small but of 200-400 nm and they were overlaid. The wavelength
deliberate variations in method parameters and provides selected for the analysis 263 nm at which both drug
an indication of its reliability during normal usage. By showed significant absorbance.
using this dilution the absorbance was taken by changing
the concentration then the further calculation was carried Selection of Stationary Phase
out. On the-basis of reversed phase HPLC mode, stationary
phase column with C18 bonded phase i.e. - Intersil ODS-
Ruggedness: The ruggedness of an analytical method is 3V (250 x 4.6mm) with particle size 5 μm from GL
the degree of reproducibility of test results obtained by science Inc (made in japan) was used for separation.
the analysis of the same samples under a variety of
conditions, such as different laboratories, different Optimization of mobile phase
analysts, different instruments, different lots of reagents, Different mobile phases were tried in order to find best
different elapsed assay times, different assay condition for separation of AZA and GYM. Following
temperature, different days, etc. By using this dilution composition mobile phases were tried,
the absorbance was taken and the further calculation was 1. Water: Methanol (60:40 v/v).
carried out. 2. Water: Acetonitrile (30:70 v/v)
3. Water: Acetonitrile: Methanol (40:30:30 v/v/v)
Analysis polyherbal formulation (MMC) 4. Methanol: Acetonitrile (75:25 v/v)
simultaneous equation method: An accurately weighed 5. Methanol: Acetonitrile (80:20 v/v)
quantity of polyherbal formulation (MMC) extract l00mg 6. Methanol: Acetonitrile (95:5v/v)
was taken in 100 ml volumetric flask and dissolved in 7. Methanol: Acetonitrile (90:10v/v)
95% methanol by vigorous shaking. The volume was 8. Methanol: Acetonitrile (60:40v/v)
made up to the mark with same solvent and further
dilutions were made to get final concentration of about Buffer and mobile phase preparation
10 μg/mL of extract.The absorbances of the resulting • Buffer preparation
solutions were measured at 210nm and 217nm gainst 10 mM phosphate buffer was prepared by adding 1.360
blank. From this extract amount of each drug was gm potassium dihydrogen phosphate in 1000 ml double
determined using simultaneous equation as mentioned as, distilled water. The solution was filtered through 0.45μm
filter and sonicated for 15 min.
A2ay1-A1ay2
C x = Ax2ay1-ax1ay2, A1ax 2-A2ax1 • Preparation of mobile phase: Mobile phase was
C y = Ax2ay1-ax1ay2 prepared by mixing methanol: Acetonitrile in the ratio of
Where, 60:40v/v. pH of mobile phase was adjusted to 6.1 with
C x = Concentration of AZA in μg/ml othophosphoric acid.
C y = Concentration of GYM in μg/ml
ax1 = Absorptivity value of AZA at 210nm • Degassing of the mobile phase
ax2 = Absorptivity value of AZA at 210nm The prepared mobile phase was degassed by
ay1 = Absorptivity value of GYM at 217nm ultrasonication for 20 min, so as to avoid the
disturbances caused by dissolved gases.

www.ejpmr.com 326
Salunkhe et al. European Journal of Pharmaceutical and Medical Research

• Filtration of mobile phase Calibration curve

The degassed mobile phase was filtered through 0.45μ Calibration curves were prepared by taking appropriate
filter to avoid the column clogging due to smaller aliquots of standard AZA and GYM stock solutions in
particles. different 10 mL volumetric flask and diluted up to the
mark with mobile phase to obtain final concentrations in
Final chromatographic condition the range of 10-60 μg/mL of AZA and 10-60 μg/mL of
System used: JASCO UV -2075 GYM The mobile phase was allowed to equilibrate with
stationary phase until steady baseline was obtained. Then
Software: Chromnav each dilution of both the markers was injected and peak
area recorded. Calibration curve was constructed by
Column used: C18 intersil, 4. 6(i.d.)x 250mm plotting the peak area vs. the drug concentration and
regression equation was computed, dilutions used for
Mobile phase used: Methanol: Acetonitrile calibration curve are as below, (Mobile phase used -
Methanol : Acetonitrile 60:40).
Flow rate: 1ml/min
Method Validation[14,15]
UV detection: 263nm The proposed method was validated as per ICH
guidelines. The solutions of the drugs were prepared as
Condition: Gradient condition per the earlier adopted procedure given in the
experiment.
Sample preparation
Standard stock solution containing Azadirachtin (AZA) Specificity
and Gymnemic acid (GYM) was prepared by dissolving Specificity was measured as ability of the proposed
10 mg of (AZA) and (GYM )separately in 100 mL of method to obtain well separated peak for AZA and GYM
mobile phase and to get stock solution containing 100 without any interference from other constituents of plant.
μg/mL of AZA, 100 μg/ml of GYM in different 100 mL Retention time for AZA -1.9417min. and GYM -3.2083
volumetric flasks. Suitable dilutions were made and the min. The each dilution of both the markers was injected
sample was filtered through 0.2 μ nylon membrane filter. and the peak area was recorded .The values obtained
Aliquots of 20 μL of the clear filtrate were injected into were very close to that in standard laboratory mixture
the HPLC column. indicates no interference from the other constituents of
plant.(Mobile phase used - Methanol : Acetonitrile
Priming of the system 60:40).
Air in the conducting tubes was removed by manual
method to obtain the continuous flow and to avoid the Linearity and Range: According to USP extract
backpressure on the pump, avoiding the damage to the equivalent to 80, 90, 100, 110, 120 % of test
column. concentration was taken and dissolved in mobile phase,
diluted appropriately to obtain a concentration in the
Conditioning of the column range of 80%-120% of the test concentration. Then each
Before a new run on system, warm HPLC grade water dilution of both the markers was injected and
was run at flow rate of 1 mL/ min-1 for 1 hr, so as to chromatogram was recorded. The chromatograms of the
remove water soluble impurities from the column. Then resulting solutions were recorded and by observing the
methanol and water in 50:50 ratio was run at the same chromatogram if this appears to be linear relationship,
flow for 30 min. Conditioning of the column was done then the test result was appropriate . (Mobile phase used
by passing methanol at 1 mL/min-1 flow rate for 30 min. - Methanol: Acetonitrile 60:40).
So as to remove the remains of the previous run.
LOD and LOQ: The LOD and LOQ were separately
Loading of mobile phase determined which is based on calibration curve. The S.D.
Filtered and degassed mobile phase was filled in the of y intercept of regression line may be used as S.D.
reservoir. Priming was done for each freshly prepared LOD = 3.3 x σ/s
mobile phase. LOQ = 10xσ/s
Where, σ= Standard deviation of y intercept of
Baseline stabilization regression lines
The detector was turned on for an hour before the actual S = Slope of calibration curve.
run so as to obtain the stable UV light. The mobile phase
run was started at required flow rate and the run was Then each dilution of both the markers was injected and
continued so as to obtain stable baseline. chromatogram was recorded. The chromatograms of the
resulting solutions was recorded and further calculation
Loading of samples was done. (Mobile phase used - Methanol: Acetonitrile
Properly prepared, filtered and sonicated samples were 60:40).
loaded into the manual injector port with the help of
syringe and the sample was injected.

www.ejpmr.com 327
Salunkhe et al. European Journal of Pharmaceutical and Medical Research

Precision: It is expressed as % R.S.D. of series of Effect of mobile phase composition variation

measurements. The interday and intra precision was Robustness of method was checked by using mobile
determined using 10 μg/mL concentration of each phase composition 65:35and 70:30 instead of 60:40 by
marker. (Mobile phase used - Methanol: Acetonitrile injecting the three replicate injection of standard
60:40) (l0μg/ml Azadirachtin and l0μg/ml Gymnemic acid) at
65:35 and 70:30.
Intraday
It was carried out by estimating the corresponding Ruggedness
responses (peak area) three times on the same day The ruggedness of an analytical method is the degree of
between three hrs intervals. Measurement of responses reproducibility of test results obtained by the analysis of
(peak area) was expressed in terms of % Relative the same samples under a variety of conditions, such as
Standard Deviation (%RSD). Then each dilution of both different laboratories, different analysts, different
the markers was injected and chromatogram was instruments, different lots of reagents, different elapsed
recorded at different intervals in the same day like 11 assay times, different assay temperature, different days,
pm, 1pm, 3pm and further calculation was done. etc. Then each dilution of both the markers was injected
and The chromatograms of the resulting solutions was
Interday (Different days) recorded and further calculation was carried out. (Mobile
It was carried out by estimating the corresponding phase used - Methanol: Acetonitrile 60:40)
responses (peak area) of same sample were recorded on
three different days. Measurement of responses (peak System suitability test
area) was expressed in terms of % Relative Standard System suitability test is a pharmacopoeial requirement
Deviation (%RSD). Then each dilution of both the and is used to verify, whether the resolution and
markers was injected and chromatogram was recorded at reproducibility of the chromatographic system are
different intervals in a different day like first day, second adequate for analysis to be done. The tests were
day, third day and further calculation was done. performed by collecting data from five replicate
injections of standard drug solution. Then each dilution
Accuracy: This involved the addition of known of both the markers was injected and The chromatograms
quantities of markers into analyzed sample .Three of the resulting solutions was recorded and then calculate
concentration levels were tested (50%, 100%, 150%). At the peak area, retention time, therotical plate,
each level, samples were prepared in triplicates and Asymmetry, Resolution along with their standard
analyzed according to previously described procedure. deviation .(Mobile phase used - Methanol : Acetonitrile
Accuracy was expressed as % recovery. Then each 60:40).
dilution of both the markers was injected and The
chromatograms of the resulting solutions was recorded RESULT AND DISCUSSUION
and further calculation was carried out. The recovery UV Analysis
study was performed three times at each level. (Mobile Selection of analytical wavelength: Azadirachtin
phase used - Methanol: Acetonitrile 60:40). showed maximum absorbance at 210nm and 212nm and
Gymnemic acid acid showed maximum absorbance at
Robustness 217nm, 220nm but 210nm and 217nm was selected for
It is measure of its capacity to remain unaffected by Azadirachtin and Gymnemic acid respectively.
small but deliberate change in method parameters and
provides an indication of its reliability in normal usage. Absorptivity values of Markers at selected
The parameters for HPLC method include the variation wavelengths
in flow rate, wavelength and mobile phase composition. Aliquot portions of AZA and GYM stock standard
The Retention time and asymmetry were considered for solutions were diluted with solvent 95%methanol to
robustness. obtain different concentrations of each drug . the
absorbance of each solution was measured at 210nm and
Effect of flow rate variation 217nm. A(1%1cm) values were calculated using
Robustness of method was checked by changing flow following formula
rate from 0.9 mL/min-1 to 1.1 mL/min-1 instead of 1 Absorbance: A(1%1cm)= Conc.(g/100ml
mL/min-1 by injecting the three replicate injection of
standard (l0μg/mL Azadirachtin and l0μg/mL Gymnemic Estimation Of Concentration Of Azadirachtin And
acid ) at 0.9 mL/min-1 and 1.1 mL/min-1. Gymnemic Acid In Polyherbal Formulation By UV
Spectroscopy
Effect of wavelength variation Concentration of both markers estimated by using
Robustness of method was checked by using wavelength formula
258nm and 268nm instead of 263nm by injecting the C=A/ba
three replicate injections of standard (l0μg/mL
Azadirachtin and l0μg/mL Gymnemic acid ) at 258nm Whereas,
and 268nm. C= Concentration of AZA/GYM μg/ml

www.ejpmr.com 328
Salunkhe et al. European Journal of Pharmaceutical and Medical Research

A=Absorbance of mixture at 210nm/217nm corresponding responses three times on the same day and
a= Absorptivity of AZA at 210nm/ Absorptivity of GYM on three different days and expressed as % RSD. The
at 217nm results for intraday and interday's precision of extract is
b=path length shown in table as below. The low values of % R.S.D. (<
2%) for intra and inter day's variation, which suggested
Method validation an excellent precision of method.
Linearity study
Linearity was studied by preparing serial dilution using Robustness: The robustness of analytical method is a
standard stock solution as shown in dilution schem. The measure of its capacity to remain unaffected by small but
linearity range for Azadirachtin and Gymnemic acid deliberate variations in method parameters and provides
were found to be 2 μg/ml-20 μg/ml and 4μg/ml-36μg/ml an indication of its reliability during normal usage. By
,respectively. Graph no 2 and 3. using this dilution the absorbance was taken by changing
the concentration. Low values of SD and % RSD
Linear regression data for calibration curve of obtained after introducing small deliberate changes in the
Azadirachtin and Gymnemic acid developed HPLC method indicated the robustness of
The calibration plot Azadirachtin and Gymnemic acid method.
follow Beer-Lambert's law at all selected wavelengths
indicates response is linear function of concentration in Ruggedness: The ruggedness of an analytical method is
the range of 2-20ug/mL for Azadirachtin and 4-36ug/mL the degree of reproducibility of test results obtained by
for Gymnemic acid in UV spectrophotometric method . the analysis of the same samples under a variety of
Graph no 1. conditions, such as different laboratories, different
analysts, different instruments, different lots of reagents,
LOD and LOQ different elapsed assay times, different assay
Limit of detection is the lowest amount of an analyte that temperature, different days, etc. By using this dilution
can be detected but not necessarily as an exact value. the absorbance was taken and the further calculation was
Limit of quantification is the lowest amount of an analyte carried out.
in a sample that can be quantitatively determined with
suitable precision and accuracy. The LOD and LOQ HPLC Analysis
were separately determined which is based on calibration Selection of analytical wavelength: After overlapping
curve. The S.D. of y intercept of regression line may be the individual spectra of AZA and GYM, 263 nm was
used as S.D. selected for simultaneous analysis, at which both the
LOD = 3.3 xσ/s marker shows significant absorbance. Graph no 4.
LOQ-10xσ/s
Where, σ = Standard deviation of y intercept of Optimization of HPLC method
regression lines, Different mobile phases were tried in order to find best
S = Slope of calibration curve. condition for separation of AZA and GYM.
Following mobile phases were tried,
The LOD was found to be 0.09 μg /mL and LOQ was 1. Water:Methanol (60:40 v/v).
found to be 0.27 μg /mL for Azadirachtin and LOD was 2. Water;Acetonitrile (30:70 v/v)
found to be 0.105ug /mL and LOQ was found to be 3. Water:Acetonitrile:Methanol (40:30:30 v/v/v)
0.318pg /mL for Gymnemic acid . The limit of detection 4. Methanol:Acetonitrile (75:25 v/v).
(LOD) and quntification (LOQ) were near about 5. Methanol:Acetonitrile(80:20 v/v)
0.09μg/mL, 0.27 μg/mL for Azadirachtin and 0.105 6. Methanol:Acetonitrile:(95:5v/v)
μg/mL, 0.318 μg/mL for Gymnemic acid, which 7. Methanol:Acetonitrile(60:40 v/v)
indicates adequate sensitivity of method.
Final mobile phase was developed as shown in Graph
Accuracy no 5.
Accuracy of an analytical method is the closeness of test
results obtained by the method to the true value. It was Method validation[12]
ascertained on the basis of recovery studies performed by Specificity: Extract was analyzed to the specificity of the
standard addition method at 50, 100 and 150 % level, optimized method in the presence of impurities and other
known amount of standard drugs were added to analyzed ingredients. The representative chromatograms did not
sample and subjected them to the proposed UV method . show any other peaks , which confirmed the specificity
Results from recovery studies were within acceptable of method . In extract AZA and GYM eluted at the same
limits 98.50-99.39% and 99.01-99.37% for Azadirachtin retention time even in presence of the other constituents
Gymnemic acid respectively indicating accuracy of of plant.
method was good.
Constituents of plant
Precision: The intraday and inter day's precision study Azadirachtin - 1.7414
of AZA and GYM was carried out by estimating the Gymnemic acid -3.3734

www.ejpmr.com 329
Salunkhe et al. European Journal of Pharmaceutical and Medical Research

Linearity and Range responses three times on the same day and on three
According to USP extract equivalent to 80, different days and expressed as % RSD. The results for
90,100,110,120 % of test concentration was dissolved in intraday and interday's precision of extract is shown in
mobile phase,diluted appropriately to obtain a table as below. The low values of % R.S.D. (< 2%) for
concentration in the range of 80%-120% of the intra and inter day's variation, which suggested an
concentration. The calibration plot Azadirachtin and excellent precision of method. It is shown in table no 10.
Gymnemic acid follow Beer-Lambert's law, The
correlation coefficient, intercept and slope were 0.9992, Accuracy
0.125, 83644 and 0.994, 0.142, 63501 for Azadirachtin Accuracy of an analytical method is the closeness of test
and Gymnemic acid respectively. The good correlation results obtained by the method to the true value. It was
coefficient indicates the method is linear over the ascertained on the basis of recovery studies performed by
concentration range. It is shown in table no 1 to 5. standard addition method at 50, 100 and 150 % level,
known amount of standard drugs were added to analyzed
LOD and LOQ: Limit of detection is the lowest amount sample and subjected them to the proposed UV method.
of an analyte that can be detected but not necessarily as Results from recovery studies were within acceptable
an exact value. Limit of quantification is the lowest limits 98.26-99.35% and 98.32-99.27% for Azadirachtin
amount of an analyte in a sample that can be and Gymnemic acid respectively indicating accuracy of
quantitatively determined with suitable precision and method was good. It is shown in table no 11.
accuracy. The LOD and LOQ were separately
determined which is based on calibration curve. The S.D. Robustness: Each factor selected was changed at three
of y intercept of regression line may be used as S.D. different levels. One factor at one time was changed to
LOD = 3.3 xσ/s estimate the effect. The parameter included variation in
LOQ-10xσ/s flow rate, wavelength of detection and composition of
Where, σ = Standard deviation of y intercept of mobile phase. Results are presented in table as below.
regression lines, Low values of SD and % RSD obtained after introducing
S = Slope of calibration curve. small deliberate changes in the developed HPLC method
indicated the robustness of method. It is shown in table
The LOD was found to be 0.26 μg /mL and LOQ was no 12.
found to be 0.804μg /mL for Azadirachtin and LOD was
found to be 0.25ug /mL and LOQ was found to be System suitability Test: This mobile phase gives
0.78μg /mL for Gymnemic acid, which indicates resolved peaks with symmetry within limits and
adequate sensitivity of method. It is shown in table no 5. significant retention time. System suitability parameters
were studied in order to determine the suitability of
Precision chromatographic system for analysis to be done. It is
The intraday and inter day's precision study of AZA and shown in table no 13.
GYM was carried out by estimating the corresponding

Graph no 1

www.ejpmr.com 330
Salunkhe et al. European Journal of Pharmaceutical and Medical Research

Graph no 2

Graph no 3

Graph no 4

Graph no 5

www.ejpmr.com 331
Salunkhe et al. European Journal of Pharmaceutical and Medical Research

Table 1: Absorbance values for calibration curve of Azadirachtin at λmax 210nm

Mean absorbance
Sr no Concentration (μg/ml)
*+SD
1 2 0.112
2 4 0.225
3 6 0.335
4 8 0.445
5 10 0.559
6 12 0.585

Table 2: Absorbance values for calibration curve of Gymnemic acid at λmax 217nm
Sr no Concentration (μg/ml) Mean absorbance *+SD
1 2 0.075
2 4 0.145
3 6 0.254
4 8 0.328
5 10 0.417
6 12 0.478

Table 3
Sr no Conc.gm/100ml Absorbance A(1%1cm)
210nm 217nm 210nm 217nm
1 0.0010 0.575 0.456 544.71 456.7
2 0.0020 1.089 0.918 546 459.3
3 0.0030 1.657 1.378 546.3 459.2
4 0.0040 2.156 1.827 544.7 456.7
5 0.0050 2.748 2.295 549.0 459.0
6 0.0060 3.294 2.745 548.1 456.3
Mean 546.6 458.3
SD 1.9572 1.2721

Table 4
Sr no Conc.gm/100ml Absorbance A(1%1cm)
254nm 273nm 254nm 273nm
1 0.0010 0.280 0.290 280.0 290.0
2 0.0020 0.558 0.578 279.0 289.0
3 0.0030 0.832 0.860 277.3 286.6
4 0.0040 1.110 1.418 277.5 287.6
5 0.0050 1.388 1.435 277.6 287.0
6 0.0060 1.683 1.741 280.6 290.1
Mean 278.6 288.2
SD 1.4130 1.6055

Table 5
Sr no Parameter Azadirachtin Gymnemic acid
1 Slope 0.014 0.015
2 Intercept 0.046 0.032
3 R2 0.999 0.994
4 Range 2 μg/ml-20 μg/ml 4μg/ml-36μg/ml

Table 6: Linear regression data for calibration curve of Azadirachtin and Gymnemic acid
Recovery of Azadirachtin
Spiked Level (%) Mean % Recovery* SD % RSD
50 99.39 0.1705 0.171
100 98.50 0.5701 0.578
150 99.50 0.0812 0.08

www.ejpmr.com 332
Salunkhe et al. European Journal of Pharmaceutical and Medical Research

Recovery of Gymnemic acid

Spiked Level (%) Mean % Recovery SD % RSD
50 99.39 0.1705 0.171
100 98.50 0.5701 0.578
150 99.50 0.0812 0.080

Table 7: Intra and interday´s precision for Azadirachtin

Formulation Intra-day precision (n= 3) Inter-day precision (n=3)
SD of response % RSD SD of response % RSD
Extract 0.002 0.76 0.0041 0.56

Intra and interday´s precision for Gymnemic acid

Formulation Intra-day precision (n= 3) Inter-day precision (n=3)
SD of response % RSD SD of response % RSD
Extract 0.001 0.132 0.0014 0.51

Table 8: Result of Ruggedness studies

Drug Lable claim(μg/ml) Amount Found (μg/ml) % lable claim ±SD*
Analyst 1 Azadirachtin 0.007 μg 0.00699 99.95±0.0975
Gymnemic acid 10 μg 10.02 100.6±0.647
Analyst 2 Azadirachtin 0.007 μg 0.0070 100.08±0.427
Gymnemic acid 10 μg 10.03 100.57±0.457

Table 9: Analysis of formulation by simultaneous equation method

Mean*%
Sr no Drug SD %RSD
estimation
1 Azadirachtin 99.32% 0.9550 0.962
2 Gymnemic acid 99.65% 0.1573 0.151

Chromatographic parameter for Mixture

Name R.T.(min) Therotical plate Asymmetry Resolution
Azadirachtin 1.9417 2145 1.52445 0.0000
Gymnemic acid 3.2083 4332 1.15953 6.3084

Linear regression data

Sr no Parameter Azadirachtin Gymnemic acid
1 Slope 0.014 0.015
2 Intercept 0.046 0.032
3 R2 0.999 0.994

Table 10: Intra and interday´s precision for Azadirachtin

Formulation Intra-day precision (n= 3 Inter-day precision (n=3
SD of response % RSD SD of response % RSD
Extract 46370.2 1.78 46499.5 1.43

Intra and interday´s precision for Gymnemic acid

Formulation Intra-day precision (n= 3 Inter-day precision (n=3
SD of response % RSD SD of response % RSD
Extract 29745.9 1.13 40137.4 1.22

Table 11: Recovery of Azadirachtin

Spiked Level (%) Mean % Recovery* SD % RSD
50 98.26 0.31 0.31
100 99.35 0.314 0.314
150 98.45 0.298 0.298

www.ejpmr.com 333
Salunkhe et al. European Journal of Pharmaceutical and Medical Research

Recovery of Gymnemic acid

Spiked Level (%) Mean % Recovery SD % RSD
50 98.63 0.291 0.291
100 98.35 0.210 0.210
150 99.27 0.221 0.221

Table 12: Robustness evaluation of method of AZA and GYM

Parameter Azadirachtin Gymnemic acid
Retention time Asymmetry Retention time Asymmetry
A: Flow rate
0.9ml/min-1 1.8424 1.23 3.1478 0.86
1 ml/min-1 1.9417 1.19 3.2083 0.87
1.1 ml/min-1 1.8447 1.19 2.9549 0.88
MeanSD(n=3) 1.8762±023 1.20±0.023 3.1036±0.12 0.87±0.01
B: Wavelength
256nm 1.8742 1.39 3.4756 0.95
263nm 1.9427 1.19 3.2083 0.87
258nm 1.7434 0.95 2.8948 0.97
MeanSD(n=3) 1.8534±0.15 1.29±0.10 3.1929±0.18 0.93±0.052
C: mobile phase composition
70:30 1.8546 1.20 3.2541 0.88
60:40 1.9427 1.19 3.2083 0.87
65:35 1.8475 1.23 3.4212 0.85
Mean SD(n=3) 1.8816±0.24 1.20±0.02 3.2945±0.11 0.86±0.015

Table 13: Result of System suitability Test

Sr no Peak area R.T.( Min) Therotical plate Asymmetry Resolution
AZA GYM AZA GYM AZA GYM AZA GYM
1 8364389 6350090 1.94 3.208 2042 4332 1.159 0.88 6.307
2 8364396 6350140 1.92 3.24 2040 4330 1.189 0.876 6.318
3 8364399 6350099 1.943 3.20 2046 4336 1.177 0.867 6.329
4 8364439 63500153 1.924 3.21 2048 4334 1.189 0.878 6.337
Mean 8364427 6350127 1.92 3.26 2044 4332 1.157 0.875 6.319
SD 25.2037 30.7457 0.0074 0.0027 1.3631 2.0945 0.010 0.049 0.0131
% RSD 0.0003 0.0004 0.318 0.0604 0.523 0.0284 0.9238 0.657 0.274

SUMMARY HPLC method for simultaneous estimation of

In spite of the great advances observed in modern Azadirachtin and Gymnemic acid was developed by
medicine in recent as medicines for the treatment of using HPLC system of JASCO UV-2075 with C18
range of diseases. Madhu-Mehantak churna (MMC) is Intersil, 4.6 (i.d.) x 250 mm column. During optimization
one of the polyherbal formulation as an antidiabetic and of mobile phase number of trials were carried out as
consist of nine ingredients including Curcuma longa, Water : Methanol (60:40 v/v), water : Acetonitrile (30:70
Azardichata indica, Emblica officinalis, Syzygium v/v), Water : Acetonitrile : Methanol (40:30:30
jambulanum,, Gymnema sylvestre, Momordica v/v/v),Methanol:Acetonitrile(75:25 v/v),Methanol:
charantia. Acetonitrile (80:20 v/v), Methanol: Acetonitrile :
(95:5v/v), Methanol: Acetonitrile (90:10v/v),
UV method for simultaneous estimation of Azadirachtin Methanol:Acetonitrile(85:15v/v). Finally this method
and Gymnemic acid was developed by using the includes use of C18 Intersil, 4.6 (i.d.) x 250 mm column,
spectrum mode of analysis of SHIMADZU 1800 mobile phase consist of mixture of methanol:acetonitrile
UV/Visible double beam spectrophotometer. 95 % (pH 6.1) in the ratio of 60:40 v/v at flow rate 1ml min".
methanol was selected as a suitable solvent The pH of mobile phase was adjusted with OPA. Retention
absorbance range of Azadirachtin and Gymnemic acid time was found to be 1.9417,3.2083 for Azadirachtin and
was found to be 0.1 to 1.0 and 0.07 to 1. The method, Gymnemic acid respectively. Theoretical plates was
obeys Beer's and Lambert law. Method was validated found to be 8210, 7955 for Azadirachtin and Gymnemic
with the help of parameter as linearity, range, accuracy, acid respectively. Method was validated with the help of
precision (intraday and interday's), LOD, LOQ. This parameter as linearity, range, accuracy, precision
method is useful as primary approach for HPLC method (intraday and interday's), LOD, LOQ, robustness. The
development. developed HPLC method for Azadirachtin ,Gymnemic

www.ejpmr.com 334
Salunkhe et al. European Journal of Pharmaceutical and Medical Research

acid and simultaneous estimation of Azadirachtin and Pharmaceutical Sciences and Drug Research 2013;
Gymnemic acid is simple, accurate, precise, robust. 5(3): 113-119.
6. Sethi P. D., Quantitative Analysis of Drugs in
CONCLUSION Pharmaceutical Formulation, 1997, 3rd EDn, CBS
It is concluded that, Azadirachtin and Gymnemic acid Publication and Distributors, New Delhi, 1-28.
identity and its estimation might be sufficient for 7. Skoog DA, Holler FJ, Timothy A, Nieman.Principle
standardization of churna. Any herbal dosage form may of Instrumental Analysis 1998, 5th EDn., Stanford
contain several constituent may have impossible and University, Saunders College Publication ,London,
tedious work to a certain the quality of such product. 1-4; 300- 325.
It will be possible to analyze one or each component in 8. Beckett AH, Stenlake JB ,Practical Pharmaceutical
each and every crude drug by analytical technique and it chemistry , part –II, 4th EDn, CBS Publication and
will be better to get a satisfactory protocol for correct Distributors , New Delhi, 2002; 1-8: 157-166.
identification and standardization of the same. The 9. Ewing GW., Instrumenal method of chemical
present work done is partial fulfillment of research on analysis, 1985, 5th EDn., Megraw Hill International
analysis of crude material in polyherbal formulation. Edition., chemistry series, 340-347.
10. Kar A. Pharmaceutical Drug Analysis, 2nd EDn,
FUTURE PROSPECTS New age international publication, 2005; 452-467.
It is necessary to screen phytochemical investigation of 11. Snyder LR. Practical HPLCmethod development,
marketed antidiabetic polyherbal formulation Madhu- 2nd EDn., John Wiley and Sons , 1-97; 161-168:
Mehantak churna by various method like detailed 653-660.
macroscopy, microscopy, quantitative microscopy, 12. ICH Topic Q 2 (R1) Validation of Analytical
physical determination, proximate chemical analysis, Procedures , Text and Methodology June 1995.
HPTLC fingerprint, estimation of volatile oil principles 13. Murugan S, Pravallika N, Sirisha P and Chandrakala
by gas chromatography, preclinical trial and clinical trial. K,A review on bioanalytical method development,
It is also must to subject same for short term, accelerated, Journal of Chemical and Pharmaceutical Sciences,
and long term stability study of such herbal dosage form 2013; 6(1): 41-45.
as per ICH guidelines or any other regulatory 14. Siladitya Behera , Subhajit Ghanty, Fahad Ahmad
recommendation. Stability studies may include detailed and Saayak Santra , UV-Visible Spectrophotometric
standardization by all applicable and critical parameters Method Development and Validation ,J Anal
at different conditions. Real time stability study of such Bioanal Techniques, 2013; 2(3): 541-549.
herbal dosage form may be carried out by keeping the 15. Kapil Kalra, Method Development and Validation of
packs of formulation at 30 ± 2 °c and 65 % relative Analytical Procedures,,International Journal of
humidity at an interval of 0, 3, 6, 9, 12 ,18, 24 months for Pharma Research and Development, 2010; 1(1):
long period. Stability data may give indication of 257-269.
presence of original phytoconstituents, any chemical
degradation, microbial contamination, presence of active
chemical entity throughout the period.

REFERANCES
1. Srivastava Shruti and Maurya Umashanker,
Traditional indian herbal medicine used
asantipyretic, antiulcer, anti-diabetic and anticancer:
a review, International journal of research in
pharmacy and chemistry, 2011; 1(4): 521-524. 3.
2. Kamboj V.P., Herbal medicine, current science,
2000; 78(1): 725-729. 4. Kuldip Raj Kohli , current
scenario of ayurvedic industry and the way
forward,current science, 2006; 2(1): 625-629.
3. Sanjoy Kumar Pal and Yogeshwer Shukla,Herbal
Medicine: Current Status and the Future, Asian
Pacific Journal of Cancer Prevention, 2003; 4(2):
281-287.
4. Swapnil G. Patil ,Anita S.Wagh, Ramesh C.Pawara
and Sandeep M.Ambore ,Standard Tools for
Evaluation of Herbal Drugs: AnOverview , The
Pharma innovation journal, 2013; 2(9): 528-534.
5. J Sojitra,P Dave, K Pandya, V Parikh, P Patel and G
Patel , Standardization Study of Poly Herbal
Formulation, International Journal of

www.ejpmr.com 335