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Indo American Journal of Pharmaceutical Research, 2017 ISSN NO: 2231-6876

DEVELOPMENT AND VALIDATION OF RP-HPLC METHOD FOR SIMULTANEOUS

ESTIMATION OF IVERMECTIN AND CLORSULON IN IVERCAM INJECTION
Vegad Kunjal L*, Paranjape Dipty B., Shah Dhwani A., Patel Ekta D., Patel Yogesh K., Patel Kaushik R.
Sharda School of Pharmacy, Gandhinagar, Pethapur.
ARTICLE INFO ABSTRACT
Article history A precise, simple, accurate and selective method was developed and validate for estimation of
Received 01/08/2017 Ivermectin and Clorsulon in Ivercam injection, Reversed phase high performance liquid
Available online chromatographic (RP-HPLC) method was developed for routine quantification of Ivermectin
30/08/2017 and Clorsulon in laboratory prepared mixtures as well as in combined dosage form.
Chromatographic separation was achieved on a BDS hypersil C18 (5μ, 250 x 4.6 mm)
Keywords utilizing mobile phase of filtered and degassed mixture of 60 phosphate buffer (pH 5.5
RP-HPLC, adjusted with 1% O-phosphoric acid) and Methanol (60:40 v/v) at a flow rate of 1 mL/min
Ivermectin, with UV detection at 234 nm. The method has been validated for linearity, accuracy and
Clorsulon, precision. In RP-HPLC method, the calibration graphs were linear in the concentration range
Ivercam Injection. of 2.5-7.5 μg/ml for Ivermectin and 25-75 μg/ml for Clorsulon with percentage recoveries of
100.34 % and 99.76% for Ivermectin and Clorsulon respectively. Conclusion: The results
obtained by RP-HPLC methods are rapid, accurate and precise. Therefore proposed method
can be used for routine analysis of Clorsulon and Ivermectin in injection.

Corresponding author
Vegad Kunjal L.
Assistant Professor,
Department of Pharmcognosy
Sharda school of Pharmacy, Gandhinagar, Pethapur
kunjalvegad11@gmail.com
9825867380

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Please cite this article in press as Vegad Kunjal L. et al. Development and Validation of RP-HPLC Method for Simultaneous
Estimation of Ivermectin and Clorsulon in Ivercam Injection. Indo American Journal of Pharmaceutical Research.2017:7(08).

Copy right © 2017 This is an Open Access article distributed under the terms of the Indo American journal of Pharmaceutical
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Research, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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Vol 7, Issue 08, 2017. Vegad Kunjal L. et al. ISSN NO: 2231-6876

INTRODUCTION
Ivermectin (IVR) is chemically 22, 23-dihydroavermectin B1a+ 22, 23-dihydroavermectin B1b (Figure: 1); is an extremely
potent semisynthetic derivative of an antinematodal principle obtained from Streptomyces avermitilis. It binds to glutamate-gated
chloride ion channels which occur in invertebrate nerve and muscle cells. This leads to an increase in the permeability of the cell
membrane to chloride ions with hyper polarization of the nerve or muscle cell, resulting in paralysis and death of the parasite [1-5].

Figure: 1 Structure of Ivermectin.

Clorsulon (CLOR) is 4-amino-6-(1,2,2-trichloroethenyl)-benzene-1,3-disulfonamide (Figure: 2); this drug has good efficacy
against mature rather than immature flukes when is given orally or via subcutaneous injection [3, 4]. Clorsulon is specific against
Fasciola spp. used for cattle at dose of 7 mg/kg to effect at great extent against fluke aged 8 weeks or older but the suggested (not
registered) dose rate against immature flukes for sheep is 15 mg/kg [6-7].

Figure: 2 Structure of Clorsulon.

Uses of dosage form

Used in effective treatment and control of internal parasites, including adult liver flukes, and external parasites in cattle.

Objective:
Development and validation of High performance liquid chromatography method for estimation of Ivermectin and Clorsulon in
injection .

Introduction to dosage form

IVERCAM injection was manufactured by Kamdhenu Stermed (Ahmedabad). Each injction claimed to contain 10mg/ml of
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Ivermectin and 100mg/ml of Clorsulon.

MATERIAL AND METHODS

Reagent and Chemicals:
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Pure Samples
Clorsulon and Ivermectin working standard was kindly gifted by NGL Fine Chem ,Mumbai.

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Chemicals used in RP-HPLC method

Acetonitrile (HPLC), Potassium dihydrogen phosphate, Water (HPLC), - Orthophosphoric acid (AR), Triethylamine (AR)
and Methanol (HPLC) were procured from Merck, Rankem.

System suitability parameters

System suitability parameters like average peak area of standards, resolution, tailing factors etc. has been measured and data
were shown in Table 1.

Table 1. System suitability parameters.

System Suitability Parameters Clorsulon Ivermectin

No. of theoretical plates 5785 7222
Retention Time (min) 3.770 5.657
Tailing factor 1.53 ± 0.016 1.38 ± 0.012
Rsolution 8.123

Wavelength determination
The detection was carried out in the UV region and wavelength selected for detection was 234 nm in mobile phase. 10µg/ml
of Clorsulon and Ivermectin working standard solutions were separately prepared in methanol. The zero order overlain spectrums of
the prepared solutions were recorded from 200 to 400 nm in double beam UV-visible spectrophotometer (Shimadzu, model 1800).
The isoabsorptive point was found at 234 nm and it was selected as wavelength of determination for both the drugs. The overlain
spectrum of Clorsulon and Ivermectin was shown in Figure: 3.

Figure: 3 Overlain UV spectra of CLOR (10μg/ml) and IVER (10μg/ml).

Chromatographic conditions
The HPLC system consisted of Shimadzu LC-20A system equipped with model LC-20AT pump, SPD- 20AT Shimadzu UV-
Vis (Diode array) detector, Hamilton syringe and DGU-20A5 online degasser, and a Rheodyne injection valve. Peak areas were
integrated using a Spinchrom Software program. Experimental conditions were optimized on a BDS Hypersil C 18 column (5μ, 250 x
4.6 mm), Thermo scientific at room temperature using 0.1 % KH2PO4: Methanol (60:40V/V) as mobile phase. Mobile phase was
flowed at 1 mL/min and all chromatographic experiments were performed at room temperature (25 ˚C ± 2˚C).

Preparation of mobile phase

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Preparation of KH2PO4:
2.72 gm of phosphate buffer was accurately weighed and dissolved in 1000ml of water, and then pH 5.5 was adjusted using
1% O-phosphoric acid.
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Mobile Phase: Prepare a filtered and degassed mixture of 60 volumes of buffer preparation (pH 5.5) and 40 volumes of Methanol.

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Preparation Standard of stock solution

a) Stock solution of Ivermectin
Standard stock solution of Ivermectin was prepared by dissolving 10 mg of drug with mobile phase in 10 ml of volumetric
flask and made up to volume to get concentration of 1000 μg/ml

b) Stock solution of Clorsulon

Standard stock solution of Clorsulon was prepared by dissolving 10 mg of drug with mobile phase in 10 ml of volumetric
flask and made up to volume to get concentration of 1000 μg/ml.

Preparation of Sample solution

1 ml solution containing 10mg Ivermectin and 100mg Clorsulon from formulation was taken and dissolved in mobile phase
and made upto 10ml. So, the concentration of Ivermectin was 1000μg/ml and Clorsulon was 10,000 μg/ml. It was further diluted to
100μg/ml and 1000μg/ml by taking 1ml from above solution and diluting it upto
10ml.Further dilutions were made according to the requirement.

Standard Solutions for linearity

Linearity was studied by preparing standard solutions at 5 different concentrations. The linearity range for Clorsulon and
Ivermectin were found to be 25-75μg/ml and 2.5-7.5μg/ml, respectively. Calibration curve were obtained by plotting respective peak
area against concentration in μg/mL and the regression equation was computed.

Validation of the developed method:

Validation of the developed method done using following parameters as per International Conference on Harmonization (ICH
Guidelines) [8-10].
1.Specificity
2. Linearity
3.Range
4.Accuracy
5.Precision
6.Detection limit
7.Quantitation limit
8.Robustness
9.System suitability testing

Method validation [11]:

Linearity and range:
The linearity response was determined by analyzing 5 independent levels of calibration curve in the range of 25-75μg/ml and
2.5-7.5μg/ml for CLOR and IVER respectively. Plot the calibration curve of Area versus respective concentration and find out
correlation co-efficient and regression line equation for CLOR and IVER.

Precision
a) Intra-day precision
For Intraday precision, it was carried out by preparing 3 replicates of 3 different concentrations, within the linearity range and
measuring the peak area of each solution on the same day. % RSD (% relative standard deviation) was calculated.

b) Inter-day precision
For Interday precision, it was carried out by preparing 3 replicates of 3 different concentrations, within the linearity range and
measuring the peak area of each solution on 3 different days. % RSD (% relative standard deviation) was calculated.

Accuracy
To a fixed amount of pre-analyzed sample of CLOR 25μg/ml and IVER 2.5μg/ml, increasing amount working standard
solution of CLOR (20 ,25 and 30 ug/ml) and IVER (2, 2.5, and 3μg/ml) were added in 10 ml volumetric flask and made up to mark
with mobile phase Samples were injected into system and analyzed as described. Calculate the mean % recovery from peak areas
obtained.

Limit of Detection (L.O.D.)

The L.O.D. was estimated from the set of 5 calibration curves used to determine method linearity. The L.O.D. may be calculated as
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LOD = 3.3 × (σ/S)

Where,
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σ = Standard deviation of the Y- intercepts of the 5 calibration curves.

S = Mean slope of the 5 calibration curves.

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Limit of Quantification (L.O.Q.)

The L.O.Q. was estimated from the set of 5 calibration curves used to determine method linearity. The L.O.Q. may be calculated as

LOQ = 10 × (σ/S)
Where,
σ = Standard deviation of the Y-intercepts of the 5 calibration curves.
S = the mean slope of the 5 calibration curves.

Robustness
For robustness evaluation of HPLC method a few parameters like flow rate, mobile phase composition and pH were
deliberately changed. Robustness of the method was done at the concentration level 50 μg/ml and 5 μg/ml for CLOR and IVER
respectively.

RESULT AND DISCUSSION

Linearity and Range
The linearity range for CLOR was found to be in the range of 25-75μg/ml and for IVER it was 2.5-7.5 μg/ml. Calibration
data for CLOR and IVER is presented in Table 2 and Regression analysis obtained are presented in Table 3. Calibration curve of
Clorsulon shown in Figure: 4 and Calibration curve of Ivermectin Shown in figure:5.

Table 2: Calibration data for Clorsulon and Ivermectin.

CLOR (μg/ml) IVER (μg/ml) Peak area Avg. area (n=5)

CLOR IVER
25 2.5 930.27 607.125
32.5 3.25 1394.17 909.35
50 5 1882.14 1229.92
62.5 6.25 2340.86 1529.93
75 7.5 2816.65 1840.54

Table 3: Regression analysis for Clorsulon and Ivermectin.

Regression analysis CLOR IVER

Correlation coefficient 0.9999 0.9999
Slope 37.75 246.99
Intercept 14.95 11.59

Figure 3: Calibration curve of Clorsulon.

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Figure 4: Calibration curve of Ivermectin.

Precision

Table: 4. Repeatability for Clorsulon and Ivermectin.

Drug Average SD RSD

Clorsulon 1879.46 1.99 0.10
Ivermectin 1237.28 20.52 1.65

Intra-day precision
The data for intraday precision of CLOR and IVER are presented in Table 5 and Table 6 respectively. The % R.S.D. for
Intra-day precision was found to be 0.11-0.15 % for CLOR and 0.201-0.203% for IVER.

Table: 5. Intraday precision for Clorsulon:

Conc.μg/ml Avg. area Standard deviation %RSD

50% 25 923.16 1.38 0.15
100% 50 1869.64 2.22 0.11
150 75 2796.60 3.36 0.12

Table: 6. Intraday precision for Ivermectin.

Conc. μg/ml Avg. area Standard deviation %RSD

50% 2.5 603.46 1.22 0.20
100% 5.0 1222.54 2.46 0.20
150 7.5 1829.48 3.71 0.20

Inter-day precision
The data for inter-day precision of CLOR and IVER are summarized in Table 7 and Table 8 respectively. The % R.S.D. for
inter-day precision was found to be 0.100-0.106 % CLOR and 0.19-0.20 % for IVER.

Table 7. Interday precision for Clorsulon.

Conc. μg/ml Avg. area Standard deviation %RSD

50% 25 924.52 0.98 0.10
100% 50 1870.63 1.91 0.10
150 75 2799.88 2.81 0.10

Table 8. Interday precision for Ivermectin.

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Conc. μg/ml Avg. area Standard deviation %RSD

50% 2.5 604.08 1.19 0.19
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100% 5.0 1223.78 2.45 0.20

150 7.5 1831.33 3.62 0.19

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Accuracy
Accuracy of the method was confirmed by recovery study from marketed formulation at three level of standard
addition. Percentage recovery for CLOR was 98.9-101.3%, while for IVER, it was found to be in range of 99.3-101.5%, The
results are shown in Table. 9 and Table. 10. Recovery greater than 98 % justifies the accuracy of the method.

Table 9 Recovery data for Clorsulon.

Amt of sample (µg/ml) Amt of std. spiked(µg/ml) Avg.Amt. recovery %recovery ± SD %RSD
80% 25 20 19.96 99.82 ± 1.3 1.32
100% 25 25 24.93 99.73 ± 0.8 0.82
120% 25 30 29.91 99.72 ± 0.6 0.64

Table 10 Recovery data for Ivermectin.

Amt of sample (µg/ml) Amt of std. spiked(µg/ml) Avg.Amt.recovery %recovery ± SD %RSD

80% 2.5 2 2.009 100.59 ± 1.12 1.12
100% 2.5 2.5 2.50 100.24 ± 0.65 0.65
120% 2.5 3 3.005 100.19 ± 0.49 0.49

Limit of Detection & Limit of Quantification

The LOD and LOQ value for CLOR and IVER are shown in Table 11.

Table 11. LOD and LOQ for Clorsulon and Ivermectin.

Clorsulon Ivermectin
LOD 0.61 0.06
LOQ 1.86 0.20

Robustness
The data for robustness are presented in Table 12 and 13.

Table 12. Robustness for Clorsulon.

Standard Variation Area %RSD

pH +0.2 1795.14 0.34
-0.2 1932.94 0.39
Mobile phase +2 1836.52 0.34
-2 1931.88 0.33
Flow rate +0.2 1836.68 0.45
-0.2 1951.61 0.37

Table 13. Robustness for Ivermectin.

Standard Variation Area %RSD

pH +0.2 1174.27 0.62
-0.2 1264.58 0.58
Mobile phase +2 1201.20 0.61
-2 1264.58 0.58
Flow rate +0.2 1200.00 0.61
-0.2 1276.90 0.58

Applicability of the Method

Applicability of the proposed method was tested by analyzing the commercially available injection formulation. The results
are shown in Table 14.
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Table 14. Analysis of Marketed Formulation.

Sr.no Label claim (mg) Result (w/w) %Assay Avg %Assay SD %RSD
1 100 99.91 99.91
Clorsulon
2 100 100.11 100.11 99.97 0.12 0.12
50µg/ml
3 100 99.88 99.88
Ivermectin 1 10 9.99 99.91
5µg/ml 2 10 10.01 100.11 100.11 0.19 0.19
3 10 10.03 100.31

Summary of Parameters
Table 15. Summary of parameters.

Result
Parameters
CLOR IVER
Linearity 0.999 0.999
Range 25-75 µg/ml 2.5-7.5 µg/ml
80% 99.82 ± 1.3 100.59 ± 1.12
Accuracy 100% 99.73 ± 0.8 100.24 ± 0.65
120% 99.72 ± 0.6 100.19 ± 0.49
Inter day %RSD=0.100-0.106 %RSD=0.19-0.20
Precision Intra day %RSD=0.11-0.15 %RSD=0.201-0.203
Inter day %RSD=0.1059 %RSD=1.6589
LOD 0.61 µg/ml 0.06 µg/ml
LOQ 1.86 µg/ml 0.20 µg/ml

CONCLUSION
The proposed RP-HPLC method was accurate and precise and applicable for the determination of Ivermectin and
Clorsulon without interference and with good sensitivity. Therefore proposed method can be used for routine analysis of
Clorsulon and Ivermectin in injection.

Recommended future work: Stability indicating study

Abbreviations
HPLC : High Performance Liquid Chromatography
UV : Ultra violet
Mm : Millimetre
μm : Micrometre
LOD : Limit of Detection
LOQ : Limit of Quantitation
ICH : International Conference on Harmonisation
mL : Millilitre
μg/mL : Microgram/Millilitre
r2 : Correlation coefficient
mg : Milligram
kg : Kilogram
% : Percentage
% RSD : Percent relative standard deviation
CLOR : Clorsulon
IVER : Ivermectin

ACKNOWLEDGEMENT
It is very great pleasure and proud sense of reverence that I express my gratitude and thanks to Dr. Yogesh K. Patel,
Principal of the Sharda School of pharmacy.
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Conflict of interest:
There is a no conflict of interest
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