Int J Pharm Pharm Sci, Vol 7, Issue 2, 498-501Original Article
ANALYTICAL METHOD DEVELOPMENT & VALIDATION OF ARTEMETHER IN BULK DRUG BY RP- HPLC METHOD AS PER ICH GUIDELINES
M. LAXMI1, SOMSUBHRA GHOSH*1, B. V. V. RAVIKUMAR2
1 Department of Pharmaceutical Analysis & Quality Assurance Nalanda College of Pharmacy, Nalgonda, Andhra Pradesh, India, 508001, 2 Department of Pharmaceutical Analysis & Quality Assurance Roland Institute of Pharmaceutical Sciences, Berhampur, Odisha, 760010
Email: som_subhra_ghosh@yahoo.co.in
Received: 21 Nov 2014 Revised and Accepted: 14 Dec 2014
ABSTRACT
Objective: An accurate, precise, rapid & economical RP-HPLC method was developed for the estimation of Artemether as per International Conference on Harmonization (ICH) guideline in pharmaceutical dosage form using ultraviolet (UV) detector.
Methods: Elution was carried out using a mobile phase consisting of Acetonitrile & Methanol (50:50 v/v) and the flow rate was set to 1.6 ml/min at 216 nm, retention time for Artemether was found to be 1.330 min.
Results: The method was found to be linear in the concentration range of 100-600 µg/ml, in the linearity study regression equation was found to be y = 0.199x – 1.133 & correlation coefficient was found to be 0.999. This method was Rugged and Robust in different testing criteria, LOD and LOQ were found to be 23.037µg/ml, 69.809µg/ml respectively. Accuracy study was done in 3 different concentration level i. e 50, 100, 150% & % recovery of the method was found to be 99.4%, 100.4%, 99.7% respectively in 3 different levels & mean recovery was 99.8%, so method was accurate.
Conclusion: Results of all validation parameters were within the limits as per ICH guidelines.
Keywords: HPLC, Validation, Method Development, Artemether, Accuracy, Ptrecission.
INTRODUCTION
Artemether is an antimalarial agent used to treat acute uncomplicated malaria. It is administered in combination with lumefantrine for improved efficacy. This combination therapy exerts its effects against the erythrocytic stages of Plasmodium spp. and may be used to treat infections caused by Plasmodium falciparum and unidentified Plasmodium species, including infections acquired in chloroquine-resistant areas. IUPAC Name: (1R,4S,5R, 8S,9R,10S,12R,13R)-10-methoxy-1,5,9-trimethyl-11,14,15,16 tetra oxatetracyclo[10.3.1.0^{4,13}.0^{8,13}]hexadecane[1]. It Involves an interaction with ferriprotoporphyrin IX (“heme”), or ferrous ions, in the acidic parasite food vacuole, which results in the generation of cytotoxic radical species. The generally accepted mechanism of action of endo peroxide antimalarials involves interaction of the endo peroxide-containing drug with heme, a hemoglobin degradation byproduct, derived from proteolysis of hemoglobin. This interaction is believed to result in the formation of a range of potentially toxic oxygen and carbon-centered radicals[2]. According to literature review [3-16] there are very few method reported for the determination of Artemether in different instrumental techniques, out of these methods only one method was reported in single Drug by using RP- HPLC. The objective or need of the proposed method is to develop simple and accurate methods for the determination of Artemether by RP-HPLC methods in pharmaceutical dosage forms.
Fig. 1: It Shows structure of Artemether
MATERIALS AND METHODS
Standard drugs
Artemether was procured from the Hetero Pharma, Hydrabad.
Chemicals and reagents
Methanol (Finer chemical Ltd.), Acetonitrile (Rankem chemicals), Purified water ((Rankem chemicals).
Instruments
HPLC (Analytical technologies), UV (Elico SL-196), Detector (UV detector, Analytical technologies), Column (Hypersil ODS C18, (150 *4.6 mm, 5µ), Software (Analchrome, Clarity), Sonicator (Analytical technologies).
Preparation of mobile phase
Accurately measured 50 ml of Acetonitrile mixed with 50 ml of Methanol HPLC grade was degassed in an ultrasonic water bath for 10 minutes and then filtered through 0.45 µ nylon filter under vacuum filtration.
Diluent
Mobile phase was used as diluents.
Standard preparation
Accurately weighed 25 mg of Artemether was transferred in to 25 ml volumetric flask and about 10 ml of the solvent mixture was added to dissolve the compound. The solution was cooled after sonication to room temperature and diluted to the mark with the solvent mixture. From this 3 ml was transferred to 10 ml volumetric flask and made up the mark and it was used as working sample to get 300 µg/ml.
Sample preparation
Sample prepared as same as standard preparation.
Method development
Wavelength selection
The test sample was scanned for its absorbance maxima using UV spectrophotometer from 400 nm to 200 nm. The λ max was found to be 216 nm. All scans to Artemether were done using this wavelength.
Fig. 2: It Shows UV spectrum of Artemether standard
Optimized chromatographic conditions
Column - Hypersil ODS C18 (150*4.6 mm), 5 µ
Flow rate - 1.6 ml/min
Wavelength - 216 nm
Column temperature - 35̊c
Injection volume - 10 µl
Run time - 5 min
Method validation
The following parameters were considered for the analytical method validation of Artemether in bulk form.
System Suitability
Chromatograph standard preparations (6 replicate injections) and peak area responses for the analyte peak was measured and the system suitability parameters were evaluated.
Accuracy
For accuracy determination, three different concentrations were prepared separately i. e. 50%, 100% and 150% for the analyte and chromatograms were recorded for the same.
Precision
The standard solution was injected for six times and the area was measured for all six injections in HPLC. The % RSD for the area of six replicate injections was found to be within the specified limits.
Robustness
As part of the robustness, deliberate change in the temperature and flow rate variation was made to evaluate the impact on the method.
Linearity and range
Linearity of the analytical method for assay by injecting the linearity solutions prepared in the range of 100µg to 600 µg (33.3% to 200%) of test concentration, into the chromatograph, covering minimum 6 different concentrations.
Ruggedness
Establish the ruggedness of the analytical method by using the assay of 6 different sample preparations of the same batch by a different analyst using a different HPLC system.
RESULTS AND DISCUSSION
Standard preparation
The dissolved standard sample was run in 10 µl aliquots using RP HPLC and the Chromatogram of Artemether was shown in Figs 3 & 4.
And the Artemether’s retention time as a peak was found at 1.33 min during its elution in solvent mixture medium of CAN and Me (50:50v/v). The procedure was used for samples also.
Fig. 3: It Shows Chromatogram of Artemether standard
Fig. 4: It Shows Chromatogram of Artemether sample
Validation
Accuracy
Average recoveries of Artemether are 99.4%, 100.4%, 99.7%, at 50%, 100% & 150% concentrations level respectively, with mean percentage recoveries of the drug is 99.8 % which is within the limits 98-102%. So the method is accurate, accuracy data for Artemether are presented in the table 1.
Table 1: Shows Accuracy results of Artemether
| Concentration level | Amount added (mg) | Amount found (mg) |
%recovery | Average % recovery |
| 50% | 12.5 mg 12.5 mg 12.5 mg |
12.5 mg 12.4 mg 12.3 mg |
100% 99.2% 98.4% |
99.4% |
| 100% | 25 mg 25 mg 25 mg |
25.0 mg 24.9mg 25.6 mg |
100% 99.6% 102.4% |
100.4% |
| 150% | 37.5 mg 37.5 mg 37.5 mg |
37.5 mg 37.6 mg 37.3 mg |
100% 100.2% 99.4% |
99.7% |
Table 2: Shows % Recovery of Artemether
| Amount added (mg) | Amount found (mg) | Average % recovery |
| 25 mg | 24.95 mg | 99.8% |
Precision
Precision are summarized in Table 1 , respectively. The %RSD values for precession was less than 2.0%, which indicates that the proposed method is precise.
Table 3: Shows precision results of Artemether
| Sample No. | Peak area of Artemether |
| Injection 1 | 74.913 |
| Injection 2 | 72.197 |
| Injection 3 | 72.943 |
| Injection 4 | 76.015 |
| Injection 5 | 74.198 |
| Injection 6 | 73.602 |
| Mean | 73.978 |
| Standard deviation | 1.375249505 |
| %RSD | 1.8 |
Fig. 5: It Shows Calibration graph of Artemether
Linearity
The response was found linear over a concentration range of 100-600 μg/ml of Artemether. The correlation co-efficient were found to be 0.999 for Artemether. So the method is linear, data is presented in Table: 4. Linearity curve of Artemether is given in Fig.5:
Table 4: Shows linearity results of Artemether
| % level | Concentration (µg/ml) |
Peak area |
| 33 | 100 | 21.159 |
| 66 | 200 | 41.433 |
| 100 | 300 | 60.778 |
| 133 | 400 | 80.455 |
| 166 | 500 | 99.673 |
| 200 | 600 | 118.823 |
| Y Intercept | 1.133 | |
| Correlation co-efficient (r2) | 0.999 | |
| Slope | 0.197 | |
| Linearity range | 100-600 |
Robustness
Minor deliberate changes in different experimental parameters such as flow rate (±0.2 ml) and temperature (±5̊ c) did not significantly affect the retention time & peak area of Artemether indicating that the proposed method is robust which is mentioned in table – 5 & 6.
Ruggedness
The method is rugged by different time intervals and the method did not significantly affect the recoveries, peak area and retention time of all the above drugs indicating that the proposed method is rugged which is mentioned in table 7.
Table 5: Shows robustness results of Artemether
S. No. |
Flow rate |
Peak area of Artemether |
Average |
SD |
%RSD |
|
1 |
1.4 ml/min |
207.377 |
206.535 |
206.956 |
0.5953 |
0.28 |
2 |
1.6 ml/min |
103.778 |
104.889 |
104.333 |
0.7855 |
0.75 |
3 |
1.8 ml/min |
50.593 |
50.315 |
50.454 |
0.1965 |
0.38 |
Table 6: Shows robustness results of Artemether
S. No. |
Temperature |
Peak area of Artemether |
Average |
SD |
%RSD |
|
1 |
30 ̊c |
66.015 |
65.432 |
65.723 |
0.4122 |
0.63 |
2 |
35 ̊c |
103.778 |
104.889 |
104.333 |
0.7855 |
0.75 |
3 |
40 ̊c |
60.123 |
61.521 |
60.822 |
0.9885 |
1.62 |
Table 7: Shows Ruggedness results of Artemether
| Name | Peak area of Artemether |
| Ruggedness-(Day-1) | 84.758 |
| Ruggedness-(Day-2) | 84.642 |
| Ruggedness-(Day-3) | 85.466 |
| Ruggedness-(Day-4) | 85.937 |
| Ruggedness-(Day-5) | 84.591 |
| Ruggedness-(Day-6) | 85.159 |
| Average | 85.0921 |
| SD | 0.533562899 |
| % RSD | 0.62 |
LOD (limit of detection)
The limit of detection is determined by the analysis of samples with known concentration of analyte and by establishing that minimum level at which the analyte can reliably detected, The LOD are calculated by formula LOD = 3.3 x SD/ b where, SD- standard deviation of the peak area of the drugs, b -is slope of the corresponding calibration curve. LOD for Artemether was 23.037µg/ml.
LOQ
The limit of quantification is generally determined by the analysis of sample with known concentrations of analyte and by establishing the minimum level at which the analyte can be quantified with acceptable accuracy and precision, The LOQ are calculated by formula LOQ = 10 x SD/ b where, SD- standard deviation of the peak area of the drugs, b -is slope of the corresponding calibration curve. LOQ for Artemether was 69.809 µg/ml.
Table 8: Shows LOD & LOQ results of Artemether
| Parameters | Artemether |
| LOD | 23.037µg/ml |
| LOQ | 69.809µg/ ml |
From the results of all validation parameter it is shown that all the values are within the limits according to ICH guideline. Summary of the validation parameter is given below in table 9.
Table 9: Shows summary of validation parameter Results
| S. No. | Parameter | Acceptance criteria | HPLC |
| 1 | % recovery | 98-102% | 99.8% |
| 2 | Linearity range(µg/ml) | - | 100-600(µg/ml) |
| 3 | Correlation coefficient | NLT 0.999 | 0.999 |
| 4 | No. of Theoretical plates | NLT 2500 | 3457 |
| 5 | Precision | % RSD NMT 2% | 1.80 |
| 6 | Intermediate precision | % RSD NMT 2% | 0.62 |
| 7 | LOD | - | 23.037(µg/ml) |
| 8 | LOQ | - | 69.809(µg/ml) |
CONCLUSION
Method development & validation of Artemether was done by RP-HPLC method. The estimation was done by using Hypersil C18 (4.6 x 150 mm, 5µm, Make: Analytical technologies). Mobile phase was used as Acetonitrile and Methanol in (50:50) ratio at a flow rate 1.6 ml/min, retention time was 1.33 min. at λ max 216 nm. The linearity range of Artemether was found to be within 100-600 µg/ml. Mean recovery was 99.8 %, which is within 98-102%. Correlation coefficient value was 0.999, % RSD was 1.02 % which is within the limit. These results show the method is accurate, precise, sensitive, economic & rugged. The HPLC method is more rapid. The proposed method can be successfully applied to estimate bulk drug & Tablet dosage form. The method was found to be having suitable application in routine laboratory analysis with high degree of accuracy and precision.
ACKNOWLEDGEMENT
We are very thankful to authorities of Nalanda College of Pharmacy for providing the facilities to complete this research work.
CONFLICT OF INTERESTS
Declared None
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