Development and validation of an HPLC method for the simultaneous determination of artesunate and mefloquine hydrochloride in fixed-dose combination tablets

The present study developed and validated an HPLC method for the simultaneous determination of artesunate (AS) and mefloquine hydrochloride (MQ) in fixed-dose combination tablets, according to ICH guidelines. The chromatographic separation was carried out on an XBridge C18 (250 x 4.6 mm i.d., 5 μm particle size, Waters) analytical column. The mobile phase included a 0.05 M monobasic potassium phosphate buffer (pH adjusted to 3.0 with phosphoric acid) and acetonitrile (50 + 50, v/v). The flow rate was 1.0 mL/min, and the run time was 13 minutes. A dual-wavelength approach was employed: AS detection was performed at 210 nm and MQ was detected at 283 nm, using a diode array detector. Stability of sample solutions was evaluated for 8 hours after preparation, during which time the solutions remained stable. Youden’s test was employed to evaluate robustness. The method proved to be linear (r2>0.99), precise (RSD<2.0%), accurate, selective, and robust, proving to be appropriate for routine drug quality control analysis.


INTRODUCTION
New antimalarial regimens have been deemed necessary ever since the worldwide emergence of Plasmodium falciparum's resistance to drugs used in monotherapy (Kremsner, Krishna, 2004).The main strategy to reduce and/or prevent antimalarial drug resistance is the use of drug combinations (White, 1998).The rationale is twofold: clinical efficacy is increased, and the evolution of resistance is slowed (Hastings, 2011).Artemisinin-based combination therapies (ACTs), such as artemether-lumefantrine, artesunate-amodiaquine, artesunate-sulfadoxine-pyrimethamine, and artesunatemefloquine are recommended by the World Health Organization (WHO) for the treatment of uncomplicated P. falciparum malaria (WHO, 2011a).ACT is based on the use of two drugs with different modes of action: an artemisinin-derivative that rapidly clears asexual blood stage parasites and gametocytes, as well as a partner drug that has a longer half-life, thus eliminating residual parasites (Varotti et al., 2008).
Artesunate(AS)-Mefloquine hydrochloride(MQ)--ASMQ-fixed-dose combination tablets are Brazil's ACT choice for the treatment of uncomplicated falciparum malaria.Two tablet strengths are currently available: 25 mg of AS and 55 mg of MQ (corresponding to 50 mg of mefloquine base) for children and 100 mg of AS and 220 mg of MQ (corresponding to 200 mg of mefloquine base) for adults.The use of AS and MQ in a fixed-dose tablet formulation considerably reduces the possible appearance of a resistant parasite, given that the parasites are never exposed to the artemisinin derivative alone and that mefloquine contains a small residual biomass to eradicate at a time of maximum blood concentration (White, Olliaro, 1996).Chemical structures of AS and MQ are shown on Figure 1.
To date, neither pharmacopeial monographs nor methods have been reported in scientific literature to determine AS and MQ in fixed-dose combination tablets.Analytical methods to determine AS and MQ in fixeddose combination tablets are needed to evaluate their quality in endemic areas, as well as to detect counterfeit drug products (Martino, 2010).One method in which to determine AS in tablets is described in the International Pharmacopoeia (World, 2011b), whereas a method to determine MQ in tablets is described in the Brazilian Pharmacopeia (Brasil, 2010).Methods for the single determination of AS or MQ in tablets were also found in prior scientific literature (Nogueira et al., 2011;Gaudiano et al., 2006;Ranher et al., 2010).In addition, the simultaneous assay of AS and MQ in plasma (Hodel et al., 2009) and a method presenting a single extraction procedure and two HPLC systems to determine AS and MQ in human plasma have been published in the literature (Lai et al., 2007).
Hence, the present study aimed to develop and validate an HPLC method, using dual-wavelength UV detection, to simultaneously quantify AS and MQ in fixeddose combination tablets.As AS and MQ present different maximum wavelengths in their UV spectra, detection was performed at 210 nm (for AS quantification) and 283 nm (for MQ quantification).The dual wavelength approach eliminated the standard addition procedure performed in a previous study of artemether and lumefantrine in a fixeddose combination tablet (César et al., 2008).Stability of standard and sample solutions was also investigated due the instability of AS in aqueous solutions (Gaudin et al., 2007).The validated method was applied to the analysis of tablets containing the AS and MQ associations (25 + 55 mg).

EXPERIMENTAL Material and reagents
AS and MQ reference standards were purchased from U.S. Pharmacopeial Convention (Rockville, MD, USA).ASMQ tablets containing 25 mg of AS and 55 mg of MQ were produced by Farmanguinhos/FIOCRUZ (Rio de Janeiro, Brazil).Concerning croscarmellose sodium, microcrystalline cellulose, magnesium stearate, opadry white and blue lake FDC 2, all excipients of the formulation, were donated by Farmanguinhos/ FIOCRUZ.Acetonitrile was obtained from Tedia (Fairfield, OH, USA) and from Honeywell (Seelze, Germany), the latter used only in the robustness study.Ultrapure water was obtained from a Millipore Direct-Q system (Billerica, MA, USA).Monobasic potassium phosphate and phosphoric acid were purchased from Merck (Darmstadt, Germany).

Instrumental and Analytical Conditions
The HPLC analyses were carried out using an Agilent 1100 system (Santa Clara, CA, USA), composed of quaternary pump, autosampler, diode array detector (DAD), and HP ChemStation Software.Chromatographic separation was carried out on an XBridge C18 (250 x 4.6 mm i.d., 5 µm particle size) analytical column from Waters (Milford, MA, USA), maintained at 30 o C. UV detection was performed at 210 nm for AS quantification and at 283 nm for MQ quantification.UV spectra from 200 to 400 nm were online recorded for peak identification.The injection volume for all solutions was 20 μL.The optimized mobile phase was a mixture of 0.05 M monobasic potassium phosphate (pH adjusted to 3.0 with phosphoric acid) and acetonitrile (50 + 50, v/v).To determine k, t 0 was estimated by injecting a 0.01% (w/v) NaNO 3 solution onto the chromatograph.
An Agilent 1200 system and an ACE column (250 x 4.6 mm i.d., 5 µm particle size) from Advanced Chromatography Technologies -ACT (Aberdeen, Scotland, UK) were also employed in the robustness study.

Preparation of standard solutions
Approximately 12.5 mg of AS and 27.5 mg of MQ reference standards were accurately weighed and transferred to a 25 mL volumetric flask, followed by the addition of 20 mL of mobile phase.The flask was sonicated for 10 minutes and then filled to the mark with mobile phase to obtain a solution at 0.5 mg/mL of AS and 1.1 mg/mL of MQ.

Analysis of fixed-dose combination tablets
Twenty tablets, containing 25 mg of AS and 55 mg of MQ each, were crushed, and an accurately weighed portion of the powder, equivalent to about 25 mg of AS, was transferred to a 50 mL volumetric flask followed by the addition of 40 mL of mobile phase.The flask was sonicated for 10 minutes and filled to the mark with mobile phase, with theoretical sample concentrations of AS and MQ registered at 0.5 mg/mL and 1.1 mg/mL, respectively.The amounts of AS and MQ in the samples were calculated using a one-point calibration.Standard solutions were injected five times and sample solutions once.AS and MQ peak areas were measured, and their concentration was calculated using the average standard response factor (peak area divided by standard concentration).

Selectivity
Spectral purities of AS and MQ chromatographic peaks were evaluated using the UV spectra recorded by a diode array detector.A solution containing a mixture of tablet excipients was also prepared according to the sample preparation procedure and injected onto the chromatograph to evaluate possible interfering peaks.

Linearity
Standard solutions containing 2.5 mg/mL of AS and 5.5 mg/mL of MQ were prepared in triplicate.Aliquots of these solutions were diluted in the mobile phase to five different concentrations: 0.250, 0.375, 0.500, 0.625, and 0.750 mg/mL of AS and 0.550, 0.825, 1.100, 1.375, and 1.650 mg/mL of MQ.Calibration curves of concentration versus area were plotted, and the obtained data were subjected to regression analysis using the least-squares method.

Precision
The intra-day precision was evaluated by analyzing six samples (n = 6) at 100% of the test concentration (0.5 mg/mL of AS and 1.1 mg/mL of MQ).Samples were prepared as set forth in Analysis of fixed-dose combination tablets.Similarly, the inter-day precision was evaluated on two consecutive days (n = 12).The AS and MQ contents (% of labeled amount) were determined, and the RSD was calculated.A Student's t test was employed to compare the average content of AS and MQ obtained in the two consecutive days.

Accuracy
Accuracy was investigated by means of a standard addition experiment.Amounts of AS (11.25,17.50,and 23.75 mg) and MQ (24.75,38.50,and 68.75 mg) were added to tablet powder, corresponding to 7.5 mg of AS and 16.5 mg of MQ (0.3 average tablet weight).The mixture was transferred to a 50 mL volumetric flask, and the volume was completed with mobile phase.The accuracy was investigated at three concentration levels: 75, 100, and 125 percent of the theoretical concentrations of AS and MQ.At each level, the solutions were prepared in triplicate, and the recovery percentage was calculated.

Robustness
Method robustness was assessed by determining the AS and MQ contents in tablets through a series of eight experiments following a factorial design (Youden, Steiner, 1975).The seven analytical parameters employed, as well as the introduced variations, are shown in Table I.A standard and a sample solution were prepared for each condition, and were injected three times onto the chromatograph.Peak area, retention time, tailing factor, theoretical plate number, and the contents of AS and MQ in the tablets were evaluated for each condition.

Stability of solutions
A standard and a sample solution were prepared as described above, and were injected onto the chromatograph, at each hour, for 8 consecutive hours.The presence of additional peaks that could indicate AS or MQ degradation and/or the decrease of AS or MQ peak areas were evaluated.

Optimization of the chromatographic conditions
Initial experiments were carried out with the chromatographic conditions to determine the mefloquine hydrochloride content in tablets (Nogueira et al., 2011).The mobile phase was a mixture of 0.05 M phosphate buffer pH 3.5 and methanol (40 + 60, v/v).MQ and AS retention factors (k) were 3.04 and 6.18, respectively.The resolution between the two peaks was greater than 10.0, and the run time was 40 minutes.Methanol presents a UV cutoff at 205 nm, which could therefore not be employed in the mobile phase to determine the AS content, given that AS only presents UV absorption in the initial wavelengths of the UV spectrum (200-220 nm) due to the absence of chromophores in its structure.Thus, methanol was replaced by acetonitrile in the mobile phase.The acetonitrile proportion was adjusted to 50% to maintain the same solvent strength of the previous mobile phase (methanol + buffer).The acetonitrile proportion was calculated using a nomograph.
A simultaneous quantification of AS and MQ, using the wavelength of 210 nm, proved to be impossible due to the excessive broadening of the MQ peak at this wavelength.Thus, simultaneous quantification proved to be possible, using a wavelength of 210 nm to quantify AS and a wavelength of 283 nm to quantify MQ.The run time was 13 minutes, and the retention factors (k) for MQ and AS were approximately 0.66 and 3.26, respectively.The resolution between MQ and AS peaks was 6.9.

Selectivity
Peak purity of higher than 99.0% was obtained for AS in the chromatograms of sample solutions, demonstrating that other compounds did not co-elute.MQ peak purity could not be assessed due to the large analyte concentration used.The chromatograms obtained with the mixture of tablet excipients showed no interfering peaks in the same retention time of AS and MQ.The overlaid chromatograms of the sample solution and the mixture of excipients, at 283 nm and at 210 nm, are shown in Figure 2 and Figure 3, respectively.

Linearity
A linear correlation was found between the peak areas and the concentrations of AS and MQ in the assayed range.The regression analysis data are shown in Table II.The regression coefficient (r 2 ) obtained in this phase was higher than 0.99 for both compounds, which attests to the linearity of the method.

Accuracy
Accuracy was investigated by means of a standard addition experiment.Table III shows both the expected concentrations of AS and MQ and the recovery percentages.All values were within the specified limits, between 98.0 and 102.0% of the theoretical concentration.

Robustness
Effects of the variation of analytical parameters in content, retention time, tailing factor, and theoretical plate number are shown in Table IV.In general, the variation of the flow rate and the column supplier were responsible for the greatest differences in the plate number for AS and MQ.Other factors remained practically unchanged.

Stability of solutions
No changes in the retention time could be observed for AS and MQ peaks after 8 hours.Likewise, no additional peaks were observed.A negligible reduction (about 1.2%) of the AS peak area was observed when the initial peak area (zero time) and the area of AS peak after 8 hours of preparation were compared.Despite this small reduction in the AS peak area, the recommendation was to inject the solutions as soon as they had been prepared.MQ peak areas remained practically unchanged after 8 hours of analysis.

Analysis of fixed-dose combination tablets
Samples of ASMQ fixed-dose combination tablets containing 25 mg of AS and 55 mg of MQ were analyzed using the validated method.The samples presented AS and MQ contents that proved to be very close to the labeled amount.The AS content in samples varied from 94.12% to 97.99%, while the MQ content varied from 97.80% to 102.25%.

CONCLUSION
This study is the first report which simultaneously quantifies the artesunate and mefloquine hydrochloride content in fixed-dose combination tablets.The method proved to be selective, linear, precise, accurate, and robust.
In addition, the two drugs were separated by an adequate run time of 13 minutes.The developed method proved to be a simple and suitable technique to quantify these antimalarials and can therefore be employed for routine quality control analysis.

FIGURE 2 -
FIGURE 2 -(A) -Overlay of the chromatogram of the sample solution (full line, λ = 210 nm) and the chromatogram of the mixture of excipients (dotted line), optimized mobile phase, showing both mefloquine hydrochloride (MQ) and artesunate (AS) peaks.(B) -overlay of the chromatogram of the sample solution (full line, λ = 283 nm) and the chromatogram of the mixture of excipients (dotted line), using the optimized mobile phase, showing the mefloquine hydrochloride (MQ) peak.

TABLE I -
Analytical parameters and variations for the robustness evaluation of the chromatographic method for simultaneous AS and MQ quantification

TABLE II -
Overview of the linearity data for AS and MQ

TABLE IV -
Effects of the analytical parameters in content, retention time (Rt), tailing factor (T), and theoretical plate number (N) of the chromatographic method for AS and MQ quantification Average of the values obtained at nominal conditions -average of the values obtained at altered conditions. *