INTRODUCTION:

Artemether (ARM) is a semisynthetic polyoxygenated amorphene (Fig 1A) containing a peroxide bridge that confers potent antimalarial activity. A disadvantage however with the endoperoxides is that, they have short half-lives ^1,2 and effective levels in plasma are sustained for only relatively brief periods ³. Therefore, the WHO recommends their use in combination with long acting antimalarial drugs such as lumefantrine (LUM) or mefloquine to manage drug resistance, recrudescence, and non compliance ⁴. ARM is practically insoluble in water; very soluble in dichloromethane, chloroform and acetone and freely soluble in ethyl acetate, dehydrated ethanol and methanol. ARM is official in Indian Pharmacopoeia, British Pharmacopoeia and United State Pharmacopoeia. LUM (Fig 1B) is used in the treatment of uncomplicated falciparum malaria. LUM is not official in any Pharmacopoeia except USP SALMOUS.

It is practically insoluble in water; freely soluble in ethyl acetate; soluble in dichloromethane; slightly soluble in ethanol and methanol.

Figure 1

Both ARM and LUM act as blood schizontocides. ARM is concentrated in the food vacuole. It

then splits its endoperoxide bridge as it interacts with haem, blocking conversion to haemozoin, destroying existing haemozoin and releasing haem and cluster of free radicals into the parasite ⁵.

LUM is thought to interfere with the haem poly-merization process, a critical detoxifying pathway for malaria parasite. The combination is available as injection (Larither injection), capsules (Larither Capsule), tablets (Lumearax, Arte Plus CD), syrup (Artevil plus).

Literature survey indicates that ARM and LUM were estimated by UV ^6,7, HPLC ^4,8-10, HPTLC ^11,12, GC-MS ¹³, Chemiluminescence and electrochemical detection and capillary electrophoresis techniques¹⁴. Spectrophotometrically, both drugs are estimated separately. ARM, as such lacks strongly absorbing chromophores and so its acid decomposition product is made use of in its assay by UV Spectrophotometry. LUM having chromophoric group can be analysed as such spectrophotometrically ¹⁵. However, No method is available for the simultaneous estimation of these drugs in combination using Derivative Spectrophotometry.

Derivative spectroscopy is one of the powerful tools used to tackle many problems related to quantitative analysis of many drug combinations in Pharmaceutical formulations ¹⁶. In recent years, derivative spectrophotometry has been found to be a useful method in the determination of mixture with two or more components having overlapping spectra and in eliminating interference from formulation matrix by using zero crossing technique ¹⁷. Derivative spectroscopy is simple, rapid, accurate and rugged which has been adopted for the routine analysis of pharmaceutical formulations as an alternative to complicated and time consuming HPLC methods where ever possible.

The aim of the present work is to develop and validate an accurate, specific, economical and reproducible Derivative UV Spectrophotometric method for determination of ARM and LUM as bulk drug and in solid dosage forms. So, a successful attempt has been made for the first time to estimate these drugs simultaneously by first order derivative spectroscopy method. The method developed was validated for its simplicity, rapidity, accuracy, reproducibility and ruggedness. Other validation parameters like Linearity, Precision, accuracy, LOD, LOQ were also checked.

MATERIALS AND METHODS:

Materials and Instruments:

ARM and LUM were procured as a gift sample from IPCA Laboratories Ltd. Mumbai, India. Methanol and HCL was purchased from Ranbaxy Ltd, India. Whatman filter no. 42 was used to filter solutions. Commercially available formulation Arte plus CD, was procured from local drug store (Marketed Formulation containing 80mg ARM and 480mg LUM, Themis Medicare Limited, Haridwar, Uttarakhand). Spectrophotometric measurements were made on a Shimadzu, model UV- 1700 Pharma Spec double beam UV Visible Spectrophotometer with a fixed slit of 1nm Coupled with computer loaded with UV Probe PC software version 2.31, A Mettler Toledo AG, (AB265- S/ FACT) electronic analytical balance was used for weighing the sample. Ultrasonic Cleaner, Steryl Medi-Equip Systems (40050). The derivative spectrophotometric determinations were performed at slow scan speed in the wave length range 400-200 nm using First Derivative order, Delta Lamba; 2, Scaling Factor: 10.

Preparation of stock Solutions:

10mg of drug (ART or LUM) was accurately weighed and were dissolved in 5ml of 0.1 M methanolic HCl in separate 10ml volumetric flasks. The solution was heated on water bath for 3 hours at temperature 60±2^o C and then was allowed to cool at room temperature and volume was then made up to the mark with methanolic HCl. These formed standard stock solutions containing 1 mg/ml of each drug.

Preparation of standard solutions:

Four series of standard solutions of ARM and LUM alone and in combinations were prepared using the prepared stock solutions by dilution using 0.1 M methanolic HCl. The series A contained a varying concentration of ARM (30- 80µg/mL), series B contained a varying concentration of LUM (4-12µg/mL), series C contained a varying concentration of ARM (30-80µg/mL) and a constant concentration of LUM (12µg/mL), series D contained a constant concentration of ARM (80µg/mL) and a varying concentration of LUM (4-12µg/mL). Blank was prepared by the same method as explained above without adding the drugs.

Procedure for Analysis:

The zero-order absorption spectra of ARM and LUM solutions in methanolic HCl are shown in Fig. 2. The spectra display overlapping in the region of 200-400 nm. This makes the determination of ARM in the presence of LUM by conventional UV spectrophotometry difficult, but the determination of LUM in the region of 200 to 400 nm is possible without the interference from ARM.

Figure 2

Figure 2: Overlay Zero-order spectra of ARM , LUM and ARM+LUM taken in the range between 200 to 400 nm wavelength.

First-order Derivative:

The derivative spectrophotometry technique was, hence, chosen for the determination of both the drugs since it could remove broadband contributions from excipients and may also overcome the interference from peak overlapping. The experiments showed that the first-derivative spectra of ARM and LUM were simple and gave encouraging results with suitable precision (with delta lamda 2 and scaling factor of 10). Overlay first derivative spectra of ARM and LUM showing zero crossing of ARM at 286.2nm and LUM at 260 nm are shown in fig 3. This clearly indicates that using first derivative spectrum; ARM can be estimated at 260 nm without the interference of LUM and LUM can be estimated at 286.2 nm without the interference of ARM. Accordingly, first derivative spectra of different concentrations of ARM and of LUM were taken for the generation of calibration curve. Regression analysis was carried out for the data obtained with all the series (A to D) to determine slope, intercept and correlation coefficient.

Figure 3

Figure 3: Overlay spectra of ARM and LUM showing Zero crossing of ARM at 286.2nm and LUM at 260 nm

Combined estimation of ART and LUM in tablets:

The developed and validated method of combined estimation of ART and LUM was applied for their estimation in commercial tablet. Arte Plus CD (Zydus Cadila) which has label claim to contain 80 mgs of ART and 480 mgs of LUM per tablet was selected for the estimation.

Preparation of Test Solution from Tablet Powder:

Six tablets were accurately weighed and finely powdered. A quantity equivalent to 40 mg of ARM and 240 mg of LUM was transferred to 100ml volumetric flask and mixed with 50 ml of methanolic HCL and the solution was sonicated for 15 minutes and then the volume was made up with the same solvent. The solution was filtered through Whatmann filter paper No. 42. Then 0.5ml filtrate was taken and transferred to another 100ml of volumetric flask. 50 ml of methanolic HCl was added and was subjected to heat for 3 hours at 60 ± 2^o C. The solution was allowed to cool at room temperature and then volume was made up to the mark with methanolic HCl. Absorption readings were taken for suitably diluted samples and ART and LUM content were determined using the calibration plots given in Fig 4.

Validation:

After optimizing the conditions of the first derivative spectroscopic method for the analysis of ART with LUM, the parameters of linearity, accuracy, inter-day precision, intra-day precision, limit of detection (LOD) and limit of quantification (LOQ) were evaluated to validate the process. Linearity was established by regressing the analytical date to obtain regression equation and correlation co-efficient (r²). Accuracy of the method was determined by the recovery studies conducted with the tablet formulation containing ART and LUM by the addition of known quantities of standard drug solution to pre-analyzed samples. Experiments were repeated three times in a day to determine intra-day precision and on three different days to establish inter-day precision. The relative standard deviation (RSD) was calculated in each analysis. LOD and LOQ were calculated by repeating the blank measurements six times at first order lmax determined previously for ART(260 nm) and LUM(286.2).

RESULTS AND DISCUSSION:

ART-LUM combination has become very important in the treatment of malaria and WHO recommends the use of this combination to manage drug resistance, recrudescence, and non compliance. Accordingly, ART-LUM formulations are commercially available in regular strength tablets with 20mg/120 mg dose and 80mg/480 mgs as high strength tablets. The combination is also available as injection, capsules and syrups. Assay of ART and LUM in fixed dose combination tablets by HPLC method was reported by Donald et.al. ¹⁸ but the method needed different column conditions, mobile phase and detector wave length for estimation. The assay of LUM employed a C18 reverse phase column with an isocratic mixture of methanol and 1% trifluro acetic acid in water (90:10) as mobile phase while, were estimated by combined method by HPLC but Spectrophotometrically, both drugs are estimated separately. However, No method is yet established for the simultaneous estimation of these drugs in combination using Derivative Spectrophotometry.

The ART lack strongly absorbing chromophore and so absorb weakly in the low wavelength region and hence makes its quantification difficult. The method adopted here makes use of its acid decomposition product, α b unsaturated decalone which absorbs strongly at a wavelength of 254 nm ¹⁹. However, the conditions of the acid decomposition reported vary. For example, IP method for the estimation of pure sample of ART alone and in its formulation reports the use of 1M ethanolic HCl and heating at 55^oC for 5 hours. Method developed by Srivastava et.al. ⁷ recommends heating at 60^oC for three hours while, method developed by Green and Mount ²⁰ involves acid decomposition in presence of a dye to yield a colored derivative which absorbs at 420 nm. However, all these methods are applied for the estimation of ART alone and not in combination with LUM. In spite of these methods ART could not be estimated in presence of LUM due to the interference of LUM on the analysis of ART by spectrophotometry. Simultaneous analysis of ART and LUM in fixed dose combination formulation is described in USP SALMOUS edition by HPLC involving gradient elution using acetonitrile as ion pairing reagent.

Results of our study showed the wavelength of maximum absorbance (lmax) of ART at 249.0 and that of LUM at 235.0nm (Fig. 2). Because of overlapping of both spectra, simultaneous estimation of ARM and LUM was not possible using zero order spectrum method. However, their first derivative spectra (Fig 3) showed a zero crossing point at 286.2nm and lmax at 260.0 nm for ARM while that of LUM showed a zero crossing point at 260 nm and lmax at 286.2 nm Therefore 260.0 nm was selected as a wavelength for estimation of ARM and 286.2 for LUM as at these wavelengths mutual interference does not exist. Hence, first derivative spectra of ARM in the concentration range between 30 to 80 mg were taken (fig 4a). Similarly, first derivative spectra of LUM in the concentration range between 4 to 12 mg were taken (fig 4b).

Figure 4

(a)

(b)

Figure 4: Overlay First Derivative Spectra of (a) ARM at concentrations (mg) of 30 (A), 40(B), 50(C), 60(D), 70(E) and 80(F) at 260nm; (b) LUM at concentrations (mg) of 4(A), 6(B), 8(C), 10(D) and 12(E) at 286.2nm

These spectra indicated that the signals at 260 nm (zero-crossing point of LUM) were proportional to the ARM concentration (Fig 4a) and the signals at 286.2 nm (ARM reads zero) were proportional to the LUM concentration (Fig 4b). Absorbance vs. concentration data at these wave lengths (260 and 286.2 nm) were plotted for the estimation of ARM (Fig 5A) and LUM (Fig 5B). Regression analysis was carried out for the data obtained with all the series (A to D) to determine slope, intercept and correlation coefficient.

Based on the first derivative spectra of both the drugs the calibration curves (Fig. 5) were generated for estimation of ART and LUM simultaneously.

Figure 5

Figure 5: Standard curve for the estimation of ARM (A) and LUM (B) using First Derivative Spectrophotometric method.

Validation:

The method developed was validated for all validation parameters like linearity, accuracy, intra-day and inter-day precision with relative standard deviation, limit of detection and limit of quantization and are tabulated in table 1.

Table1. Summary of validation Parameters

Parameters		First derivative Values
		Artemether	Lumefantrine
Wavelength(nm)		260.0	286.2
Linearity range (μg/ml)		30-80	4-12
Regression equation Slope		y=0.0028x-0.0009 0.0028	y= 0.0051x-0.0002 0.0051
Intercept		-0.0009	-0.0002
Regression Coefficient (r²)		0.9993	0.9999
Accuracy(% recovery)		99.215± 2.2695	99.563±1.6379
LOD(μg/ml)		0.3606	0.1145
LOQ(μg/ml)		1.0928	0.3470
Precision (%RSD)	Intraday	0.944	0.890
	Interday	1.124	1.009
% purity(w/w)		95.23%	94.48%

Linearity:

ARM showed good linearity with regression coefficient (r2) of 0.9993 (Fig 5A) over a range of 30-80μg/ml. LUM showed linearity with regression coefficient of 0.9999 (Fig 5B) over the range of 4-12μg/ml. Linear regression analysis for series A to D is shown in Table 2.

Precision:

The intraday precision of the method was evaluated by means of six determinations at 100% of their respective test concentration given in table 1. In the analysis of the ART, the RSD values for intra-day (n=6) and inter-day (n=6) precision were 0.994 and 1.124 respectively. Similarly, the values for LUM were 0.890 and 1.009 respectively. Thus the precision was demonstrated for both the drugs, since all the obtained RSD values were less than 2.0%.

Table2.Regression Analysis Data of the Calibration curves prepared using Series A, B, C, D solutions.

Series Code	Concentration of Solution(μg/ml)		Linear regression Equation (y=mx+c)	Regression coefficicent ( r²)
Series Code	ARM	LUM	Linear regression Equation (y=mx+c)	Regression coefficicent ( r²)
A	30-80	0	y= 0.0028x-0.0009	0.9993
B	0	4-12	y= 0.0051x-0.0002	0.9999
C	30-80	12	y= 0.0028x-0.0011	0.9999
D	80	4-12	y= 0.0049x-0.0018	0.9991

Accuracy:

Accuracy was evaluated by the standard addition method for both ART and LUM. The mean percentage recovery obtained were 99.215± 2.2695 and 99.563±1.6379 for ART and LUM respectively. These recovery values indicate the accuracy of the method developed.

Selectivity

The selectivity of the method was assessed by comparing the regression equations obtained with series containing one drug (Example: ARM) alone with the series containing the drug with variable concentration of the other (Example ARM with LUM). Linear regression analysis for series A to D is shown in Table 2. Regression equation for series A (ARM) and C (ARM+LUM) showed no difference in the slope values (0.0028 for both) and almost same intercept values (0.0009 and 0.0011 for series A and C respectively). This result indicates that there is no interference of LUM in determination of ARM. Similarly, Regression equations for series B and D showed no difference in the equations indicated that there was no interference of ARM on measurement of LUM.

Limit of detection (LOD) and limit of quantization (LOQ):

The determination of these limits was carried out using an equation that considers the parameters of the analytical curve. The resulting values of LOD were 0.3606 μg/ml for ARM and 0.1145 μg/ml for LUM. These results indicate higher sensitivity of the method for LUM in comparison to ARM obviously due to low absorptivity of ARM. However these limits are quite sufficient for analysis in the commercial samples of the drug combination. The values of LOQ were 1.0928 μg/ml and 0.3470 μg/ml for ARM and LUM respectively.

CONCLUSIONS:

A simple, rapid and accurate method of analysis for the simultaneous estimation of ARM and LUM by first order derivative spectroscopy was developed and validated. The analytical method showed linearity in the range of 30 to 80mg and 4 to 12mg for ART and LUM respectively. The method was validated using parameters like intra-day and Inter-day precision, accuracy, selectivity and LOD and LOQ levels. The method was also applied effectively for the simultaneous estimation of both the drugs in commercial fixed dose combination tablet.

ACKNOWLEDGEMENTS:

Authors acknowledge the courtesy of M/s IPCA Laboratories Ltd. Mumbai, India for providing the gift samples of artemether and lumefantrine for the study. Authors also acknowledge Mr. Praveen Garg, Chairman, I.S.F. College of Pharmacy for providing facilities to carry out the work.

CONFLICT OF INTEREST STATEMENT:

No conflict of interest exists.

REFERENCES:

1. Muhia DK, Mberu EK, Wat kins WM. Differential extraction of artemether and its metabolite dihydroartemisinin from plasma and determination by high-performance liquid chromatography .J. Chromatogr. B 660 (1994) 196–199.

2. Karbwang J, Na-Bangchang K, Molunto P, Banmairuroi V, Congpuong K. Pharmacokinetics of intramuscular artemether in patients with severe falciparum malaria with or without acute renal failure. J. Chromatogr. B 690 (1997) 259–265.

3. Meshnick SR, Taylor TE, Kamchonwongpaisan S, Artimisinin and the antimalarial endoperoxides: From herbal remedy to targeted chemotherapy, Microbiological review, 1996, 60(2), 301-15.

4. Chimanuka, B., Gabriels, M., Detaevernier, M.R., Plaizier-Vercammen, J.A., 2002 Preparation of beta-artemether liposomes, their HPLC-UV evaluation and relevance for clearing recrudescent parasitaemia in Plasmodium chabaudi malaria-infected mice. J. Pharm. Biomed. Anal. 28, 13–22

5. Aditya NP, Patankar S, Madhusudhan B, Murthy RSR, Souto EB. Artemether loaded lipid nanoparticles produced by modified-thin film hydration: Pharmacokinetics, toxicological and in vivo antimalarial activity. European Journal of Pharmaceutical Sciences.2010; 40:448-455.

6. Arun R. and Anton S A. Development of analytical method for Lumefantrine by UV-Spectrophtometry. Int. J. Res. Pharm. Sci. 2010;1(3):321-4

7. Shrivastava A, Nagori BP, Saini P, Issarani R, Gaur S. New Simple and Economical Spectrophotometric Method for Estimation of Artemether in Pharmaceutical Dosage Forms. Asian J. Research Chem. (2008)1, 1-19.

8. Cesar C, Noqueira FH, Antonio P G. Simultaneous determination of Artemether and Lumefantrine in fixed dose combination tablets by HPLC with UV detection. . J. Pharm. Biomed. Anal. 48(1)10 2008, Pages 223–226

9. Thomas CG, Ward SA, Edwards G. Selective determination, in plasma, of artemether and its major metabolite, dihydroartemisinin, by high-performance liquid chromatography with ultraviolet detection. J. Chromatogr. B 583 (1992) 131–136.

10. Khalil IF, Abildrup U, Alifrangis LH, Magia D, Alifrangis M, Hoegberg L, Vestergaarde LS, Persson OP, Nyagonde N, Lemnge MM, Theander TG, Bygbierg IC. developed the measurement of Lumefantrine and its metabolite in plasma by HPLC with UV detection. J Pharm Biomed Anal, 2011 Jan 5;54(1) : 168-72.

11. Gabriёls M, Plaizier-Vercammen JA,. Densitometric Thin-Layer Chromatographic Determination of Artemisinin and its Lipophilic Derivatives, Artemether and Arteether. J Chromatogr Sci. (2003), 41(7):359-66.

12. Gabriёls M, Plaizier-Vercammen JA,. Development of a Reversed-Phase Thin-Layer Chromatographic Method for Artemisinin and Its Derivatives, .J. Chromatogr. Sci. 42 (2004) 341–347.

13. Verbeken M, Sultan S, Bram B, Vangheluwe E , Sylvia VD, Christian Burvenich, Luc-Duchateau, Frans H J and Bart D S. Stability-indicating HPLC-DAD/UV- EST/MS impurity profiling of the anti-malarial drug Lumefantrine. Malarial J.2011;10: 51.

14. Hulst AD, Augustijns P, Arens S, Van Parijs L, Colson S, Verbeke N, Kinget R. Determination of Artesunate by Capillary Electrophoresis with Low UV Detection and possible application to analogues. J. Chromatogr. Sci. 41 (1996)34.

15. Varma J K, Syed H. A. Spectrophotometric method for determination of Lumefantrine in pharmaceutical formulation. J. Pharm. Res. 2009;2(9):1550-1.

16. Beckett AH, Stenlake JB, Practical Pharmaceutical Chemistry. 4th ed. Delhi: CBS Publisher and Distributors, 1997.

17. Patel PM, Suhagia BN, Shah SA, Marolia BP, Bodiwala KB, Prajapati PB, Simultaneous estimation of etoricoxib and paracetamol in the synthetic mixture and Pharmaceutical dosage forms by derivative spectophotometry, Journal of Pharmacy Research, 2010, 3(8), 1967-70.

18. Donald W, A Practical handbook of preparative HPLC, Elsevier publisher, New York, 2006, p. 37-45.

19. Thomas CG, Ward SA, Edwards G, J. Selective determination, in plasma, of artemether and its major metabolite, dihydroartemisinin, by high-performance liquid chromatography with ultraviolet detection, Chromatogr. 1992, 583, 131-6. Green MD, Mount DL, Wirtz RA., Authentication of artemether, artesunate and dihydroartemisinin antimalarial tablets using a simple colorimetric method., Trop. Med. Int. Health, 2001 Dec;6(12):980-82.

Received on 05.02.2013 Modified on 08.02.2013

Asian J. Research Chem. 6(3): March 2013; Page 226-231