1. INTRODUCTION:

Artemetheris used as Antimalarial drug. It is chemically named as: (1R,4S,5R,8S,9R,10S,12R, 13R) -10-methoxy-1,5,9-trimethyl-11, 14, 15, 16 tetraoxatetracyclo [10.3.1.0⁴^,¹³.0⁸^,¹³] hexadecane. It is official in Indian Pharmacopoeia 2018.¹Artemether interact with ferriprotoporphyrin IX (“heme”), or ferrous ions, in the acidic parasite food vacuole, which results in the generation of cytotoxic radical species.²

Fig. 1: Chemical Structure of Artemether

Lumefantrine is used as Antimalarial drug. Lumefantrine is chemically named as: 2-(dibutyl amino)-1-[(9Z)-2,7-dichloro-9-[(4-chlorophenyl) methylidene]-9H-fluoren-4-yl] ethan-1-ol. It is official in USP 2018.³The exact mechanism by which lumefantrine exerts its antimalarial effect is unknown. However, available data suggest that lumefantrine inhibits the formation of β-hematin by forming a complex with hemin and inhibits nucleic acid and protein synthesis.

Fig. 2: Chemical Structure of Lumefantrine

Artemether and lumefantrine combination therapy is indicated for the treatment of acute uncomplicated malaria caused by plasmodium falciparum, including malaria acquired in Chloroquine-resistant areas. It may be used to treat uncomplicated malaria when the plasmodium has not been identified. Artemether has a rapid onset of action and is rapidly cleared from the body and it provides rapid symptomatic relief by reducing the number of malarial parasites.Lumefantrine has a much longer half-life and is believed to clear residual parasites.⁴ Analytical quality by design (AQbD) and Central composite design (CCD) help in regulatory compliancefor RP-HPLC method development, stress testing, or stability indicating methods.^5-8 UV spectrophotometric methods (simultaneous equation method/Vierodt') and LC-MS are widely acceptable for simultaneous estimation of pharmaceutical combinations.^9-11The presence of impurities critically affectsthe stability and pharmacological action of pharmaceutical API and drug products.^12-16The purpose of stability and related substance study is to provide evidence on how the quality of a drug substance or drug product varies with time under the influence of a variety of environmental factors.^17-18

Literature survey reveals that this combination is not official in any pharmacopoeia but both individual drug as well as in the combination of this drug have been determined by various methods including UV spectrophotometry, RP-HPLC (assay), RP-HPLC (stability indicating), HPTLC, LC-MS-MS and GC.^19-28However, not a single UPLC method has been reported.UPLC method provides benefit for estimation of large number of samples within a short period of time, which saves more time with very less amount of solvent consumption. UPLC method is superior as compared to other methods as it improves the selectivity, sensitivity, resolution, reduces cost and speed for their detection.²⁹In vivo dissolution.³¹The proposed method is validated as per ICH guideline Q2(R1).³⁰Thus, it is worthwhile to develop the RP-UPLC-PDA method for the simultaneous estimation of Artemether and lumefantrine in API and tablet dosage form.

2. MATERIALS AND METHODS:

2.1 Materials:

Working Standard of Artemether and Lumefantrine was gifted by Vital LaboratoriesPrivate Ltd., Mumbai, India. Artemether and Lumefantrine tablets containing 20mg and 120mg and the inactive ingredient used in drug matrix were procured from (Trade name: ARH-L, Manufacturedby Lincoln Pharma, India) Market.

2.2 Instrumentation:

The Waters Acquity UPLC H-class system (Waters, Milford, USA) consisting of a quaternary gradient pump, an inbuilt auto sampler, Photo diode array detector (PDA) and column oven connected to a multi-instrument data acquisition and processing system with Empower 3 version, analytical balance (Mettler Toledo, XP 504) and pH meter (Mettler Toledo).

2.3 Chromatographic Conditions:

Chromatographic analysis was performed on Acquity UPLC BEH C18 column (50mm, 2.1mm, and 1.7µm). The mobile phase consisted of ACN: 20mM Hexane sulfonic acid sodium salt adjust pH 2.5±0.05 with OPA (70:30 %V/V). The ﬂow rate of the mobile phase was adjusted to 0.3ml/min, Temperature 40℃ and the injection volume was 5µl. Detection was performed at 218nm.

Diluent: Acetonitrile is used as diluent.

2.4 Preparation of standard solution and sample solution:

Standard Solution: Approximately weigh and prepare 160μg/ml of Artemether and 960μg/ml of Lumefantrine in diluent.

Sample Solution: Five tablets of Artemether and Lumefantrine were weighed and finely powdered. A quantity equivalent to 4mg of Artemether and 24mg of Lumefantrine was transferred into 25ml volumetric flask and make up with diluent to obtain solution of 160μg/ml of Artemether and 960μg/ml of Lumefantrine.The contents were sonicated to dissolve it and filtered through 0.45µm membrane filter.

3. Method Validation:

3.1 Specificity:

The specificity of the method was ascertained by analysing diluent, placebo, standard and tablet formulation to examine the % interference of excipients and their impurities with analytes peak.

3.2 Linearity:

Five-point calibration solutions were prepared in the concentration range 80-240μg/mL for Artemether and 480-1440μg/mL for Lumefantrine.

3.3 Accuracy:

An Accuracy study was performed by adding known amounts of Artemether and Lumefantrine to the placebo preparation. Recovery of the method was evaluated at three different concentration levels (corresponding to 50%, 100% and 150% of test preparation concentration). For each concentration level, three sets were prepared and % recovery was calculated.

3.5 Precision:

The precision of the method was evaluated in terms of repeatability by carrying out six independent of test sample preparation and RSD was calculated. The intra-day and inter-day precision of the method was determined by analyzing the sample solution in 3 sets each of 50%, 100% and 150% in the same day and 3 different days, at 80, 160 and 240μg/mL and 480, 960 and 1440μg/mL for Artemether and Lumefantrine respectively.

3.6 Robustness:

The robustness of the proposed method was determined by carrying out the analysis, during which change in Flow rate ±0.1 unit (0.3ml/min ±0.1), Temperature ± 1^oC (i.e. 39^oC and 41^oC), Mobile phase buffer pH ±0.1-unit (i.e. 2.4 and 2.6) andOrganic Phase ratio of mobile phase ±2% absolute. (i.e. Buffer solution: Acetonitrile (32:68) for -2% and Buffer solution: Acetonitrile (28:72) for +2%) were altered, andfor each parameter, three sets were prepared and the peak areas, retention times and number of theoretical plates were noted.

4. RESULTS AND DISCUSSIONS:

4.1 Optimized UPLC conditions:

The chromatographic conditions of UPLC method were optimized with respect to suitability parameters. Acceptable separation of both drug with short run time was achieved on Acquity UPLC BEH C₁₈ column (50 mm X 2.1mm, 1.7µm). Various proportions of Acetonitrile, Trifluroaceticacid, Hexane sulfonic acid sodium salt and Ortho phosphoric acid in mobile phase at different composition, pH and different flow rate were investigated. After trials, adequate result was obtained using the mobile phase ACN: 20 mM Hexane sulfonic acid sodium salt adjust pH 2.5±0.05 with OPA(70:30% V/V) at a flow rate of 0.3ml/min and temperature 40℃. Typical chromatogram recorded at 218nm is shown in Fig. 3. Theretention time of Artemether and Lumefantrine were found to be 0.908 min and 1.916 min respectively. The analyte peaks were well resolved and were free from tailing (< 2 for both the analytes).

4.2 System Suitability:

Five replicate injections of standard preparation were injected and asymmetry, retention time, resolution, theoretical plate and RSD of peak area were determined for same. Results of system suitability is shown in table 1.

4.3 Method Validation:

The proposed method was validated with respect to various parameter including Linearity, Accuracy, Precision, Specificity and Robustness according to ICHQ2(R1) guideline.

4.3.1 Linearity:

The method was found linear in the range of 80-240 µg/ml for Artemether and 480-1140 µg/ml for lumefantrine. The linear regression equation was y = 5004.3x + 7052.4 with correlation coefﬁcient 0.999 for Artemether and y = 2330.1x + 11940 with correlation coefficient 0.999 for Lumefantrine.

4.2.2 Specificity:

The developed method is specific as there was no interference of excipients at Artemether and Lumefantrine peak.

4.2.3 Accuracy:

The percentage recovery for Artemether was in range of 98.80-100.56and for Lumefantrine was in range of 98.15-101.51. Thus, the recovery study of both drugs is acceptable, as shown in Table 2.

4.2.4 Precision:

The developed method was found to be precise as the 002RSD values for the repeatability and intermediate precision studies were less than 2. The results of precision as shown in table 2.

4.2.5 Robustness:

The robustness of the suggested method was confirmed by performing the analysis with modifications to the Flow rate, Temperature, Mobile phase buffer pH and Organic Phase ratio of mobile phase. RSD of peak area were found to be less than 2%, indicating that the method is robust. Results remained unaffected by small variations of these parameters.

4.3 Assay of Tablet Dosage form:

Analysis of marketed tablets ARH-L(n=3) (Artemether 20 mg and Lumefantrine 120 mg) was carried out using the developed and validated method. The % drug content of Artemether and lumefantrine in tablet dosage form were found to be 98.75 and 99.56 respectively. The results of assay are acceptable as shown in Table 3.

Fig. 3: Optimized Chromatogram of Artemether and Lumefantrine

Table 2: Validation summary report

Sr. No.	Validation Parameters	Artemether		Lumefantrine
1.	Accuracy (n=5)
	Level	Conc. µg/ml	% Recovery	Conc. µg/ml	% Recovery
	50 %	80	98.80	480	99.62
	100 %	160	99.46	960	98.15
	150 %	240	100.56	1440	101.51
2.	Precision (n=3)	RSD		RSD
	System precision	0.65		0.41
	Intra-day precision	0.54		0.55
	Inter-day precision	0.59		0.49
3.	Robustness	RSD		RSD
	Flow rate	0.77		0.39
	Temperature	0.69		0.45
	Mobile phase pH	0.56		0.52
	Mobile phase ratio	0.62		0.76

Fig 4: Calibration curve of Artemether and lumefantrine

Table 3: Analysis of marketed formulation

Brand name	Drug	Label Claim (mg)	RSD (n=3)	% Assay
ARH-L	Artemether	20	0.92	98.75
ARH-L	Lumefantrine	120	0.66	99.56

5. CONCLUSION:

The proposed RP-UPLC-PDA method is rapid, specific, accurate, precise and robust for the simultaneous estimation of Artemether and Lumefantrine in tablet dosage form. The method has been found to be better, because of its wide range of linearity, use of readily available mobile phase, lack of extraction procedures, less solvent consumption,and less time consuming.By injecting the standard six times, system suitability parameters were studied, and the results were considerably below the acceptance criteria. Linearity study of artemether and lumefantrine were found to of 80-240μg/ml and 480-1440µg/mlwith correlation coefficient of 0.999. By RP-UPLC-PDA method, the accuracy of artemether and lumefantrine were found to be in the range of 98-101% and 99-102% respectively.It was discovered that precision was less than 2% in term of RST. The method can be successfully used for routine analysis of Artemether and Lumefantrine in API and tablet dosage forms.

6. ACKNOWLEDGEMENTS:

The authors are gratefully acknowledging Vital Laboratories, Mumbai, for providing Artemether and Lumefantrine as gift samples for this work. The authors thank Piramal enterprise Ltd., Ahmedabad for providing equipment and guidance for the study. The authors thank the Quality Assurance department of B.K. Mody Government Pharmacy College, Rajkot to provide guidance for the study.

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Received on 27.06.2022 Modified on 16.10.2022

Research J. Pharm. and Tech 2023; 16(8):3936-3940.

DOI: 10.52711/0974-360X.2023.00647

Parameters (n=5)	Artemether	Lumefantrine
SST	0.4	0.3
Resolution	0.0	5.7
Retention time	0.908	1.916
Tailing factor	1.1	1.1
Theoretical Plates	5158	7234