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Gazi University Journal of Science GU J Sci 25(2):377-383 (2012) ORIGINAL ARTICLE
A Validated HPLC Method for Separation and
Determination of Mefloquine Enantiomers
in Pharmaceutical Formulations
Ola A. SALEH,1 Aida A. EL-AZZOUNI1♠, Amr M. BADAWEY2, Hassan Y. ABOUL-ENEIN2,♠ 1Medicinal and Pharmaceutical Chemistry Department, Pharmaceutical and Drug Industries Research Division, National Research Centre, Dokki, Cairo, Egypt, 2Analytical Chemistry Department, Faculty of Pharmacy, Cairo University, Cairo, Egypt. Received 27,09,2011 Accepted:14,11,2011 ABSTRACT

A simple, rapid and validated method for separation and determination of mefloquine enantiomers was
developed. Mefolquine was separated and quantitated on cyclodextrin chiral column Quest-CM carboxymethyl-
BCD (250x4mm i.d., 5µm particle size) using a mixture of acetonitrile: 1% triethylammonium acetate buffer
(pH = 4.5) (20:80 v∕ v) as a mobile phase at 20 oC and a flow rate of 1 mL /min. The UV-detector was set at 240
nm. The applied HPLC method allowed the separation and quantification of mefloquine enantiomers with good
linearity (r > 0. 999) in the studied range. The relative standard deviations (RSD) were 0.865 and 0.907 for the
mefloquine enantiomers with accuracy of 100.00 and 100.68. The limit of detection and limit of quantification
of mefloquine enantionmers were found to be 5 and 15 µg/mL, respectively. The method was validated through
the parameters of linearity, accuracy, precision and robustness. The HPLC method was applied for the
quantitative determination of mefloquine in pharmaceutical formulations.
Keywords Chiral separation, antimalarials, mefloquine, pharmaceutical analysis.

1. INTRODUCTION

quinolone methanol, Figure 1, with two asymmetric carbon atoms has been used as a racemic mixture in Although many chiral drugs are clinically used as prophylaxis and treatment of the resistant strains of racemates, it has been demonstrated that in most cases Plasmodium faciparum. Various studies demonstrated the two enantiomers of a chiral drugs have different that MFQ pharmacokinetics are highly stereoselective pharmacological activities [1]. Mefloquine (MFQ), rac- erythro- α -(2- piperidyl)-2,8- bistrifluoromethyl)-4- ♠Corresponding author, e-mail: : haboulenein@yahoo.com and elazzounyaida@yahoo.com GU J Sci, 25(2):377-383 (2012)/ Ola A. SALEH, Aida EL-AZZOUNI, Amr BADAWEY, Hassan ABOUL-ENEIN (+)-Mefloquine 11R,2 S (-)-Mefloquine 11S,2 R Figure 1. Stereochemical structure of mefloquine enantiomers The peak concentration and the area under the curve Instrumentation and Analytical Conditions
(AUC) of the (-)-MFQ have been significantly higher The HPLC unit was a Agilent 1100 series apparatus than those of the (+)-MFQ in blood and plasma after equipped with a quaternary pump, a vacuum degasser, a oral administration [3-5]. Higher concentration of the column oven, a diode array UV-detector, and a HP (+)-MFQ in rat plasma after oral administration has also Chemstation. The column used was cyclodextrin chiral been reported [6]. column Quest-CM carboxymethyl-BCD (250x4mm i.d., There are conflicting reports about the antimalarial 5µm particle size) as a gift obtained from Cyclolab Ltd., activity of MFQ enantiomers. No significant difference Budapest, Hungary. is observed between antimalarial activities of The mobile phase consisted of acetonitrile, 1% enantiomers against Plasmodium bergheior and triethylammonium acetate buffer pH= 4.5 (20:80% v∕v). Plasmodium yoelli in rodents [4,7]. In vitro activity of The flow rate was 1mL/min. All the samples were MFQ enantiomers on two chloroquine resistant and measured at wavelength 240 nm at 20 0C. susceptible strains of Plasmodium falciparum showed similar activities for both enantiomers [8]. In another Preparation of the standard solutions
report, the (+) -enantiomer of MFQ has shown 1.69-1.81 times more active than the (-)-MFQ against Mefloquine reference standard (50 mg) was accurately weighed, transferred to 50 mL volumetric flask, and chloroquine resistant (Indochina w-2) strains of dissolved in 20 mL methanol, and then completed to Plasmodium falciparum in vitro [9]. Because volume with methanol (final concentration 1mg /mL). The resulting solution was sonicated for 10min and pharmacological activities, the objective of this work is diluted to volume. All solutions were freshly prepared. to develop a validated method of separation and Determination of Mefloquine Enantiomers
enantioselective analysis of mefloquine enantiomers in the pharmaceutical tablet formulation. All the validation For construction of the calibration graph, take aliquot parameters are performed including accuracy, precision portions (0.5-5 mL) of 1mg/mL mefloquine standard and robustness besides linearity, limit of quantification solution into a series of 25 mL measuring flasks, and (LOQ) and limit of detention (LOD) using HPLC on complete to volume with methanol. Inject 20 µL of the cyclodextrin chiral column Quest-CM carboxymethyl- solution from each flask and record the chromatograms, BCD (250x4mm i.d., 5µm particle size). The mobile maintaining the flow rate at 1mL/min and wavelength phase used was acetonitrile: 1% triethylammonium 240nm. Measure the ratio of peak area corresponding to acetate buffer (pH = 4.5) in the ratio 20: 80 (v∕ v). concentration of each. Construct a calibration graph representing the relation between concentration and 2. EXPERIMENTAL
peak area. Concentration of unknown samples could be Chemicals
derived from the calibration graph or calculated from the following regression equation. Mefolquine hydrochloride was obtained from Aldrich (Schnelldorf, Germany, Cat. No. M2319). Methanol Enantiomer 1:
Y = 0.0018X + 0.0981 r = 0.9990 (HPLC-grade) was obtained from Merck (Darmstadt Enantiomer 2:
Y = 0.0018X + 0.0841 r = 0.9997 Germany). Acetonitrile and triethylamine of analytical grade were delivered from Sigma chemicals (St. Louis, Where: Y= Peak area of sample. X= concentration of mefloquine in µg/mL. Pharmaceutical preparation, Lariam® Tablets, was r = Correlation coefficient. labeled to contain 250 mg mefloquine per tablet, manufactured by Roche Company. B.N. 1163BO1. During the chromatographic analysis, the following parameters were measured. GU J Sci, 25(2):377-383 (2012)/ Ola A. SALEH, Aida EL-AZZOUNI, Amr BADAWEY, Hassan ABOUL-ENEIN k1 and k2: Capacity factors of the first and second eluted Linearity
enantiomers and were 0.49 and 1.76, respectively. The calibration curve was obtained with six α: selectivity factor, α= k2 / k1 = 3.61 concentrations of the standard solution 50-500 µg/mL. The solutions were prepared in triplicate. The linearity Rs: resolution factor was found to be 2.61, calculated was evaluated by linear regression analysis, which was according to the following equation, Rs = 2(t2 – t1) / w1 calculated by the least square regression method. Precision
Where w is the baseline band width obtained by drawing tangents to the inflexion points of the The precision of the assay was determined by chromatographic peak. repeatability (intra-day) and intermediate precision (inter-day). Intra-day precision was evaluated by Detetermination of Mefloquine Enantiomers in
assaying the sample, at the same concentration and Lariam ® Tablets
during the same day-six sample solutions (100µg/mL) Weight 10 tablets individually , grind in a mortar and were prepared and assayed. The intermediate precision weigh powdered tablets equivalent to 50 mg of (inter-day) was studied by comparing the assays on mefloquine. Extract with 30mL methanol using a different days (3-days). magnetic stirrer and complete to volume with methanol Accuracy
then filter. Determine mefloquine concentration by taking 0.5-5 mL into 25mL measuring flasks. Complete The accuracy of an analytical method is determined by to volume with methanol and proceed as previously how close the test results obtained by that method come described before. to the true value. It can be determined by application of the analytical procedure to an analyte of known purity 3. METHOD VALIDATION
(for the drug substance) or by recovery studies, where a The methods were validated according to the known amount of standard is spiked in the placebo (for International Conference on Harmonization guidelines drug product). In this study, a number of different for validation of analytical procedures (ICH, 1996). solutions were prepared with a known added amount of ANOVA was used to verify the validity of the methods. drug substance and injected in triplicate. Percent recoveries of response factor (area and concentration) were calculated as shown in Table 1, which indicates Table 1. Intra- Day and inter- Day Accuracy and Precision Data of HPLC Method for Mefloquine Theoretical
Intra-Day
Inter- Day
Accuracy %
Precision a
Accuracy
Precision a
Enantiomer 1
Enantiomer 2
a Mean of five determinations for each concentration. Robustness
in the flow rate (0.8-1.2 mL/ min), in the temperature of the column (18-25○C), and in the wavelength (240-260 The robustness of the HPLC method was determined by analysis of samples under a variety of conditions by making small changes in the mobile phase composition, GU J Sci, 25(2):377-383 (2012)/ Ola A. SALEH, Aida EL-AZZOUNI, Amr BADAWEY, Hassan ABOUL-ENEIN Limit of Detection and Limit of Quantification
This research deals with the enantiomeric separation and quantitation of mefloquine enantiomers in bulk and LOD is defined as the lowest concentration of an in pharmaceutical tablets using a new cyclodextrin analyte in a sample that can be detected, but not based weak cation-exchange chiral column Quest-CM necessarily quantified and LOQ was defined as the carboxymethyl-BCD (250x4mm i.d., 5µm particle size) lowest concentration of analyte in a sample that can be stationary phase as a chiral selector. determined with acceptable precision and accuracy. The chromatographic conditions were optimized in 4. RESULTS AND DISCUSSION
order to provide a reliable assay performance. Mobile Currently, there is a great interest within the phase selection was based on peak parameters, runtime, pharmaceutical laboratories to develop single isomer ease of preparation and cost. A typical chromatogram is formulations and also analytical methods to determine shown in Figure 2 for the analysis and separation of a the enantiomeric purity of drugs. Figure 2 Chromatogram of Mefloquine 300µg/ml on a cyclodextrin chiral column Quest-CM carboxymethyl-BCD (250x4mm i.d., 5µm particle size) using a mixture of acetonitrile, 1% triethylammonium acetate buffer pH= 4.5 (20: 80% v∕v) as a mobile phase and flow rate 1ml/min at 240 nm.) The retention time was observed at 5.38min for
enantiomer 1 and 6.76min for enantiomer 2. The LOD
and LOQ were obtained using the slope and standard
deviations of the intercept from three curves and
determined by the linear regression line and were 5 and
15µg/mL, respectively. These values were also used in
an experimental assay confirming the calculation.
The calibration curves for mefloquine enantiomers were constructed by plotting concentration versus the ratio of peak area and showed good linearity in the 50 -500 µg / mL range as shown in Figures 3 and 4. Fıgure 3. Linearity of concentration of mefloquine
enantiomer 1 to peak area of mefloquine enantiomer 1
(500µg/ml) as an external Standard.
GU J Sci, 25(2):377-383 (2012)/ Ola A. SALEH, Aida EL-AZZOUNI, Amr BADAWEY, Hassan ABOUL-ENEIN Accuracy and precision of the proposed method were assessed by performing triplicate analyses of the standard solutions. Three different concentrations, diluted with the mobile phase, were prepared in the linear range of the calibration curve and analyzed to determine intra-day variability and accuracy. The inter- and intra-day precisions were calculated as the RSD%. The results and the mean values were shown in Table 1 demonstrating good precision and accuracy. When chromatographic conditions were intentionally altered, no significant effect was observed in the chromatogram, confirming the robustness of the method. The intraday-precision obtained by the proposed method showed a RSD of 0.45 and 0.62 % for both
enantiomer 1 and enantiomer 2, respectively.
Fıgure 4 Linearity of concentration of mefloquine
enantiomer 2 to peak area of mefloquine enantiomer 2
Inter–day variability was calculated and showed a RSD (500µg/ml) as an external standard of 0.71 and 0.84% for both enantiomers, respectively, as shown in Table 1. The representative linear equations were: Results of the determination of mefloquine in Lariam®, Y = 0.0018 X + 0.0981 for enantiomer 1 and Y=
the pharmaceutical tablets formulation, are shown in 0.0018X + 0.084 for enantiomer 2 with high correlation
coefficients r = 0.9990 and r= 0.9997, respectively. Table 2 Results of Determination of Mefloquine in Lariam® Tablets by HPLC Sample (µg)
Amount a (µg)
Enantiomer 1
Enantiomer 2
a Mean of five determination for each concentration. Table 3 Results of Standard Addition of Authentic Mefloquine to Lariam® Tablets. Rcovery %
Found Autheutic µg/mL
Recovery % (X) for
Found Authent µg/mL
Authentic
for Enantiomer 1
Enantiomer 1
for Enantiomer 2
Enantiomer 2
The tablets excipients did not interfere with the analysis was 100.00±0.865 and 100.68±0.907%, respectively. of mefloquine entantiomers and it was found that the The results are expressed in Table 4. accuracy of the HPLC method for enantiomers 1 and 2
GU J Sci, 25(2):377-383 (2012)/ Ola A. SALEH, Aida EL-AZZOUNI, Amr BADAWEY, Hassan ABOUL-ENEIN Table 4 Determination of authentic mefloquine via the suggested HPLC method. Found Authentic µg/ml
Recovery % of
Found Authentic
Recovery % of
Authentic
of Enantiomer 1
Enantiomer 1
µg/ml of Enantiomer 2
Enantiomer 2
a Average of at least three separate determination. significance level, respectively Table 5. The calculated The proposed analytical method was compared with F-value for both enantiomers (F cal =2.99) and (F reference method using statistical analysis [10]. The cal=2.68) were found to be less than the tabulated F- calculated t-value for both enantiomers (t cal =0.26) and value (F tab = 9.28 ) and (F tab=9.28) at a 1% (t cal=0.62) were found to be less than the tabulated t- significance level, respectively Table 5. value (t tab = 2.45) and (t tab= 2.45) at a 1% Table 5 Statistical Comparison of the Results obtained by Adopting the Proposed Method as Compared with the Reference Method a for Analysis is of Mefloquine Technique
Mean ± RSD
Variance
Student (t) test
Reference method HPLC for Enantiomer 1
HPLC for Enantiomer 2
a Quautitative UV spectrophotometry in ethanol using A (1%,1cm) at 282.5 nm for the determination of mefloquine. b The figures in parenthesis are the theoretical t and F values at (P = 0.05). The methanolic solution of mefloquine was stable all Hungary for providing us with the chiral column used in through the period required for analysis and did not show sign of degradation products. REFERENCES
The proposed method described discusses a fully Aboul-Enein,H.Y.;Abou Basha, L.I. Chirality and drug hazards, chapter 1 in "The impact of enantiomers in pharmaceutical tablets formulations. stereochemistry in drug development and use", 5. CONCLUSION
Aboul-Enein, H.Y. and Wainer, I.W. (eds.), John
Wiley &Sons,
New York, 1-19 (1997).
The proposed HPLC method described a quantitative determination 2- Palmer, K.J.; Holiday, S.M.; Brogden, R.N. enantiomers in bulk drug and in pharmaceutical tablets "Mefloquine, a review of its antimalarial activity, formulations. The proposed HPLC method is fast, precise, accurate, and efficient and can be applied for efficacy", Drugs., 45:430- 475(1993).
routine analysis in quality control laboratories. 3- Gimenez, F.; Farinotti, R.; Thuilier, A.; 6. ACKNOWLEDGMENTS
Hazerbroucq, G.; Wainer, I.W. "Determination of the enantiomers of mefloquine in plasma and The authors are sincerely indebted and profoundly whole blood using a coupled a chiral-chiral high grateful to Professor Dr. Mohamed Nabil Aboul-Enein, performance liquid chromatographic system", J.
Professor of Pharmaceutical Chemistry, Department of chromatogr. 529, 339-346 (1990).
Medicinal and Pharmaceutical Chemistry, National Research Centre, for his endless support, guidance, and [4] 4- Martin, C.; Gimenez, F.; Bangchang, K.N.; unlimited valuable advice throughout this work. Also, Karbwang, J.; Wainer, I.W. "Whole blood the authors would like to thank Mrs Gyrgyi Herczkn and Ms. Csilla Fada of Cyclolab Ltd., Budapest inhealthy Thai volunteers", Eur. J. Clin.
Pharmacol.
47: 85-87 (1994).
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[6] Souri, E.; Farsam, H.; Jamali, F. "A preliminary stereo-selectivity [9] Basco, L.K.; Gillotin, C.; Gimenez, F.; Farinotti, enantiomers in rat." Acta Med Iran, 36: 133-137
R.; Le Bras, J. "In vitro activity the enantiomers of mefloquine, halofantrine and eniproline against
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[7] Corroll, F.L.; Blackwell, J.T., "Optical isomers of 33, 517-520 (1992). aryl-2 piperidyl methanol antimalarial agents. Preparation, stereochemistry" J. Med. Chem. 17, 210-219
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