A Validated Chiral HPLC Method for the Enantiomeric separation of Mefloquine

 

S. Jenifer Ashwini1, S. T. Narenderan1, S. N. Meyyanathan1*, B. Babu1, B. Gowramma2

1Department of Pharmaceutical Analysis, JSS College of Pharmacy (JSS Academy of Higher Education and Research, Mysuru) Udhagamandalam, Tamil Nadu, India.

2Department of Pharmaceutical Chemistry, JSS College of Pharmacy (JSS Academy of Higher Education and Research, Mysuru) Udhagamandalam, Tamil Nadu, India.

*Corresponding Author E-mail: snmeyyanathan@jssuni.edu.in

 

ABSTRACT:

A validated direct chiral HPLC method was developed for the separation of enantiomers of Mefloquine. The separation was achieved using chiral PAK IG-3 (250 x 4.6 mm) 3μm column. The mobile phase was composed of 10 mM ammonium acetate and methanol in the ratio of 30:70, v/v. The flow rate of the mobile phase was set at 0.7 ml/min. The detection wavelength was set at 284 nm with column temperature maintained at 25°C. The retention time of both (+) and (-) enantiomers was found to be 4.59 min and 6.47 min, respectively under a runtime of 10 min.  The method was validated as per ICH guidelines. The method was found to be linear over a concentration range of 20 – 120 µg/ml and 15 – 105 μg/ml for (+) and (-) Mefloquine enantiomers, respectively. The chiral assay of Mefloquine in a pharmaceutical formulation was performed and the recoveries ranged from 99.3 to 99.9 %. The detection limit for the (+) and (-) enantiomers was found to be 5.5 μg/ml and 5μg/ml, respectively.

 

KEYWORDS: Mefloquine, Enantiomers, HPLC, Validation, ICH.

 

 


INTRODUCTION:

After the discovery of optical isomerism by Pasteur, the importance of chirality with respect to biological activity has been clearly recognized. The physiological environment within a living organism is chiral, and the biological activities of enantiomeric forms of molecules can differ dramatically. The regulatory requirements are becoming tougher and enantiomeric purity of 99.9% is deemed necessary by the regulatory authorities like Food and Drug Administration (FDA) and International Conference on Harmonization (ICH)1.Chirality now plays a major role in the development of new pharmaceuticals which is driven by both regulatory and therapeutic rationales, one-third of all marketed drugs are now sold in single isomeric form2.

 

 

In the pharmaceutical industry, much emphasis is put on chiral analysis due to their potentially different behavior of the enantiomers of a chiral drug molecule after administration. To cause a therapeutic effect, an administered molecule has to interact with a target receptor. For chiral drug molecules, only one enantiomer (the eutomer) will fit properly into this receptor, resulting in the desired therapeutic effect. The other enantiomer (the distomer) can either not interact or can interact less intense with the receptor, which generally causes a lower effect. Occasionally the distomer interacts with other receptors, causing side- or even toxic effects. As a consequence, the enantiomers of drug candidates must be subjected to supplementary investigations during development processes: the eutomer has to be distinguished from the distomer during identification and impurity determinations of the drug substance3. Racemates separation remains a challenge due to their identical properties in a chiral environment, and research on specialized separation techniques continues to be developed to resolve individual enantiomers4. Study on literatures revealed few methods on HPLC determination of Mefloquine in combination with other drugs5-6.

In this study, the Chiralpak IG-3 was used as a stationary phase to separate the Mefloquine enantiomers. The advantages of this stationary phase are rapidity, good rigidity, large column capacity and reproducibility. Mefloquine chemically is a 2-piperidinyl-2,8-bis(trifluoromethyl)-4-quinolinemethanol hydrochloride is a recently developed antimalarial agent (Fig.1). It is effective as a single oral dose against multidrug-resistant malaria in man. It also provides suppressive prophylaxis against Plasmodium vivax and P. falciparum infections in human volunteers7. Mefloquine is orally administered as a racemic mixture of (+)-(11S,12R)- and ()-(11R,12S) -MQ. It is metabolized by cytochrome P450 (CYP) 3A subfamily to 4-carboxymefloquine (CMQ), which shows no antimalarial activity in vitro8. The developed HPLC method for the enantioseparation of Mefloquinewas found to be relatively simple, rapid and highly sensitive which utilized a short run time (10 min) analysis.

 

 

Fig. 1: Structure of Mefloquine

 

MATERIALS AND METHODS

Chemicals and Reagents:

The racemic Mefloquine was obtained as a gift sample from Indian Pharmacopoeia Commission, New Delhi. Commercially available Mefloquine tablets were procured from Intas Pharmaceuticals, Ahmedabad, Gujarat, India. HPLC grade methanol and AR grade ammonium acetate were procured from Rankem Fine Chemical Ltd. HPLC grade water was procured from Milli-Q RO system (Millipore, Bedford, USA).

 

Equipment:

Enantiomeric separation was done using High-Performance Liquid Chromatography (Shimadzu gradient HPLC system) equipped with a solvent delivery system (Model-LC-10 AT-VP), Rheodyne injector (Model-7725i with 20μl loop), UV detector (Model-SPD M-10A VP). The data were recorded using class-VP data station.

 

Standard Preparation:

1 mg/ml concentration of Mefloquine working standard was prepared by dissolving 10 mg of the drug in a 10 ml volumetric flask with the mobile phase. The volume was made up with the mobile phase. The prepared standard solution was injected and the chromatogram was recorded.

 

Selection of Wavelength:

A solution of 10 µg/ml of Mefloquine in water was prepared. The UV spectrum was recorded by scanning the solution in the range of 200 to 400 nm. From the UV spectrum wavelength of 284 nm was selected at which Mefloquine showed maximum absorbance.

 

Preparation of Ammonium acetate (10 mM):

0.77 g of ammonium acetate was accurately weighed and transferred into a 1000ml volumetric flask. About 900ml of Millipore water was added and degassed by subjecting to sonication for 10 min and final volume was made up with water. The resulting buffer solution was filtered through 0.22 μm membrane filter before use.

 

Method Development:

The chromatographic separation was achieved using a Chiral PAK IG-3 (250 x 4.6 mm) 3µm column. The mobile phase was 10 mM ammonium acetate and methanol in the ratio of 30:70, v/v. The flow rate of the mobile phase was 0.7 ml/min. The column temperature was maintained at 25°C and the eluent was monitored at a wavelength of 284 nm. The injection volume was 10 µL. The typical retention time of (+) and (-) enantiomers of Mefloquinewas about 4.59 min and 6.47 min, respectively (Fig.2).

 

 

Fig. 2: Enantioselective separation of racemic Mefloquine in bulk

 

Validation of the Method:

The Method validation comprising of the parameters such as specificity, system suitability, linearity, precision, accuracy, limits of detection and quantification (LOD and LOQ), robustness and solution stability was done in accordance with the guidelines of ICH9.

 

Specificity:

Specificity is the ability to assess unequivocally the analyte in the presence of components which may be expected to be present. Typically these may include impurities, degradant, etc.

 

System Suitability:

The working solution was injected for six replicates and the chromatograms were recorded. The resolution factor, number of theoretical plates and asymmetric factor were calculated for the standard solutions.

 

Linearity:

The linearity of measurement was evaluated by analyzing the standard solutions of Mefloquine in the range 20 – 120 μg/mlfor (+) Mefloquine and 15 – 105 μg/ml for (-) Mefloquine enantiomers, respectively. The linearity of the proposed method was then evaluated by linear regression analysis. The correlation coefficient, slope and intercept were calculated from the graph

 

Assay of Mefloquine Formulation:

Twenty tablets were weighed and crushed into a fine powder and mixed with a mortar and pestle. A quantity of the powder equivalent to 250 mg of Mefloquine was weighed accurately and transferred to 10 ml volumetric flask. To this 5 ml of mobile phase was added and sonicated for 10 minutes to completely dissolve the Mefloquine tablets. The resulting solution was made upto 10 ml and mixed well. A small portion of the above solution was withdrawn and filtered through a 0.2 µm filter. The filtrate was appropriately diluted with the mobile phase before injection into the column. The blank, standard and sample solutions were injected in triplicate. Chromatograms were recorded and peak areas were then measured (Fig.3).

 

Fig. 3: Enantioselective separation of racemic Mefloquine in a pharmaceutical formulation

 

Limit of Detection (LOD):

Limit of detection is the lowest concentration of the analyte that can be detected by injecting decreasing amount, not necessarily quantity by the method, under the stated experimental conditions. The minimum concentration at which the analyte can be detected is determined from the linearity curve by applying the formula.

 

LOD = 3.3 SD/s

SD is the standard deviation of intercept

s is the slope of the calibration curve

Limit of Quantification (LOQ):

Limit of quantification is the lowest concentration of the analyte in a sample that can be estimated quantitatively by injecting a decreasing amount of drug with acceptable precision and accuracy under the stated experimental conditions of the method. Limit of quantitation can be obtained from the linearity curve by applying the following formula.

 

LOQ = 10 SD/ s

SD is the standard deviation of intercept

s is the slope of the calibration curve

 

Accuracy:

The accuracy of the proposed HPLC method was evaluated from the assay results of the components. Accuracy was done by performing the assay of samples and calculated the peak area responses of different samples by recovery method. The accuracy of the method was calculated by recovery studies at three levels low, middle and high concentration from the linearity range. The mean, standard deviation and % RSD were calculated.

 

RESULTS AND DISCUSSION:

In the present study, a reverse phase direct chiral HPLC method has been developed and validated for the enantiomeric separation of Mefloquine enantiomers. In order to develop a simple and efficient RP-HPLC method for Mefloquine enantiomers, preliminary tests were conducted to select suitable and optimum conditions. The following parameters, such as solubility, detection of wavelength, mobile phase buffers, mobile phase proportions, stationary phase, flow rate, column oven temperature were carefully evaluated. The optimized conditions were finally selected based on the criteria of peak properties like resolution factor, peak asymmetry factor, column theoretical plate number, capacity factor, peak height and peak area respectively.

The optimized chromatographic conditions are Chiralpak IG-3 (250 x 4.6) mm 3µm as the stationary phase on an isocratic elution mode. The mobile phase consisting of 10 mM ammonium acetate and methanol in the ratio of 30:70, v/v and a flow rate of 0.7 ml/min and UV detection was carried out at 284 nm. The mobile phases were filtered through 0.22 μm membrane filter before injecting into the HPLC system. The retention times of both (+) and (-) Mefloquine enantiomers were found to be 4.59 and 6.47 min, respectively.

 

Method Validation:

Linearity:

The linearity was determined by plotting peak area versus concentration in milligram per millilitre for Mefloquine enantiomers at the concentration range of 20 – 120 μg/ml for (+) Mefloquine and coefficient of regression was found to be 0.996. The coefficient of regression for(-) Mefloquine enantiomer was 0.998 at the linearity ranging from 15 – 105 μg/ml. The linearity range and graph of (-) Mefloquine and (+) Mefloquine enantiomer represented in (Fig. 4).


 

Fig.4: Calibration curve (a) (+) Mefloquine enantiomer and (b) (-) Mefloquine enantiomer

 


Accuracy:

The accuracy of the method was determined by recovery studies and the percentage recovery was calculated. The accuracy of the optimized methods was determined by absolute recovery experiments. An analysis of the results showed that the percentage recovery values were found to be for both (+) and (-) Mefloquine, respectively. The developed method was applied for the determination of Mefloquine in tablet dosage forms. The obtained results for Mefloquine were comparable with a corresponding label claim Table 1.


 

Table 1: Recovery study for (+) and (-) enantiomer of mefloquine

Label claim (mg)

Amount present (mg/tablet) ± % RSD

(n = 3)

% Recovery ± % RSD (n = 3)

(+) & (-)

(+)

(-)

(+) & (-)

(+)

(-)

 

250

248.3 ± 0.23

136 ± 0.31

111.7  ± 0.31

99.3 ± 0.23

55.0  ± 0.11

45.0  ± 0.33

248.1 ± 0.57

133.2 ± 0.15

114.8  ± 0.11

99.2 ± 0.57

53.7  ± 0.41

46.3  ± 0.13

249.7 ± 0.35

139.5 ± 0.38

110.1  ± 0.21

99.9 ± 0.35

55.9  ± 0.52

44.1  ± 0.27

 


Limit of detection and quantification:

The Detection limit for this method was found to be the lowest limit detected for the both (+) and (-) enantiomers of Mefloquine at 5.5μg/ml and 5μg/ml, respectively based on the signal-to-noise ratio 3:1. Due to the increase in the sensitivity of the method, quantification was done at 20 μg/ml and 15 μg/ml for both (+) and (-) Mefloquine enantiomer. This method found to have a high percentage of recovery at low concentration at the acceptable limit.

System Suitability:

System suitability tests were performed to chromatograms obtained from standard and test solutions to check parameters such as column efficiency, resolution factor and an asymmetric factor of Mefloquine peak. Results obtained from six replicate injections of the standard solution as per the proposed method are summarized in Table 2.

 


 

Table 2: System suitability study for (+) and (-) enantiomer of mefloquine

S. No

Parameters

(+) Mefloquine

(-) Mefloquine

1

Linearity range

20 – 120 µg/ml

15 - 105 µg/ml

2

Regression equation

y = 17221x - 1113.6

y = 2297.6x - 7666.7

3

Correlation coefficient

0.996

0.998

4

Theoretical plate/meter

7186.71

5264.74

5

Resolution factor

6.5

6

Asymmetric factor

1.1

1.1

7

LOD(µg/ml)

5.5

5

8

LOQ(µg/ml)

20

15

 

 

CONCLUSION:

A simple direct chiral reverse phase HPLC method was developed and validated as per the ICH guidelines as it provides good sensitivity and excellent precision and reproducibility. Chiralpak IG-3 (150 x 4.6) mm 3µm chiral column was found to be selective for the enantiomeric separation of Mefloquine. The developed direct chiral reverse phase HPLC method was found to be simple, rapid, accurate, precise and linear.

 

ACKNOWLEDGEMENT:

The authors are thankful to Mr. Ch. Thrupathi and Mr. K. Nagesh Kumar for their kind support in the completion of the study.

 

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Received on 23.12.2018          Modified on 10.02.2019

Accepted on 16.03.2019        © RJPT All right reserved

Research J. Pharm. and Tech. 2019; 12(5):2304-2308.

DOI: 10.5958/0974-360X.2019.00384.6