Analytical Development and HPTLC Method Validation of Mefloquine Hydrochloride in Bulk and Dosage Form

Mefloquine is a drug used to prevent or cure malaria. It is marketed under several trade names, including Lariam¹.Mefloquine is an orally administered blood schizontocide.² Mefloquine hydrochloride, cu-[2,8_bis(trifluoromethyl)-4-quinolyll--(2-piperidyl) methanol hydrochloride³. is administered orally in the prophylaxis and treatment of chloroquine-resistant falciparum malaria⁴.mefloquine hydrochloride is an odourless, white powder⁵. Its melting point is 259– 260 degree Celsius⁶. Mefloquine hydrochloride is a first-line medication that is taken orally for the prevention, treatment, and emergency management of malarial infections⁷. In addition to its longer half-life as compared to others antimalarial drugs, in the late 1980’s the first report of mefloquine hydrochloride use in pregnant women were published. MQ is safe in the second half of pregnancy⁸. Mefloquine targets the Plasmodium falciparum 80S ribosome and functions as an antimalarial in the cytoplasm of protozoa. (+)-erythro-mefloquine has been shown to interact with PfuL13 residues like Leu59 and Glu55. Cytochrome P450 3A4 metabolises mefloquine slowly in the liver (half-life: 3 weeks) ⁹. The chemical formula of mefloquine hydrochloride C₁₇H₁₆F₆N₂O and molecular weight is 414.8. Under the brand name Lariam, mefloquine is a synthetic analogue of Quinine and quinidine-is sold as racemic form. Mefloquine is essentially quinine and quinidine in a more structurally simplified form. Mefloquine differs from chichona alkaloids in that it has a piperidine ring rather than the bicyclo quinuclidine ring, has a different substitution on the quinoline ring and does not have a vinyl group¹⁰. Rare but serious neuropsychiatric problems have been associated with its use¹¹.

Fig.No.1. structure of Mefloquine hydrochloride

MATERIALS AND METHODOLOGY

Chemical And Reagent:

Working standards for analytically pure mefloquine hydrochloride were obtained from the central drug testing laboratory with defined potency.  Lariam tablet (Mefloquine 250 mg) was procured from Central Drug Testing Laboratory, Mumbai. HPLC-grade acetonitrile, Merck Life Science's methanol, Finar Chemical’s glacial acetic acid, Rankem's dichloromethane, and Toluene.

Instrument:

Bands were applied to pre coated silica gel 60 F254 HPTLC plates (20´10cm) with a thickness of 250µm using a sample applicator CAMAG Linomat 5 (CAMG, Muttenz, Switzerland) with a nitrogen aspirator and a CAMAG Hamilton Bonaduz schwetz microsyringe (100µl) from Sigma-Aldrich. With a closed cover, a CAMAG twin trough glass chamber utilised for development and saturation.  The CAMAG TLC Plate Heater was used for the activation of HPTLC plates. An air dryer was used in a wooden room with sufficient ventilation to dry the plates. Spectrodensiometric study was performed using the CAMAG TLC Scanner 4 using a deuterium lamp. CAMAG TLC Visualiser 2 was utilised for picture documentation, while CAMAG visionCATS software version 3.0 was employed for chromatographic evaluation. For all weighing requirements, a Sartorius Analytical Balance was used.

Preparation Of Mobile Phase:

To achieve clear, well-defined peaks and optimise the mobile phase composition, several tests were carried out. The mobile phase was chosen to comprise acetonitrile, toluene, and glacial acetic acid in a 6:3:1 ratio.

Preparation Of Standard Stock:

Standard stock (A)

Accurately weigh 10 mg of the reference standard mefloquine hydrochloride, then pour it into volumetric flask of 100ml. After adding roughly 50ml of diluent, sonicate for 15 minutes. With diluent, adjust the volume to 100 ml.

Standard solution (B)

Pipette 5ml of the standard stock A into 10ml capacity volumetric flask. Then, add diluent to bring the volume up to 10ml.

Preparation Of Sample Solution:

A single 250 mg tablet of mefloquine hydrochloride was put it into 100ml capacity volumetric flask. Sonicate for 15 to 20 minutes after adding 50 millilitres of diluent. With diluent, adjust the volume to 100 ml. Remove 1 millilitre of the solution using a pipette, then pour it into 50-millilitre capacity of volumetric flask and top it off with the same diluent.

Determination Of Wavelength:

A 100 ml volumetric flask was filled with around 10.0 mg of active mefloquine hydrochloride, and the volume was adjusted using 100% methanol (diluent). To achieve 10ug/ml of the working standard, required portions of standard stock solution of Mefloquine hydrochloride have been suitably diluted with diluent. The 200-400 nm wavelength range was used to scan the solutions. The maximum absorption for Mefloquine hydrochloride was observed at 222nm. Consequently, the ultimate wavelength for method development and validation was determined to be 222nm.

Figure 2: Detection wavelength of Mefloquine hydrochloride

The CAMAG TLC Plate heater was used to activate the pre-coated silica gel F254 plates at 110 degrees Celsius for five to ten minutes. The CAMAG Linomat 5 sample applicator was used to spot the sample, which appeared as narrow bands. The distance between two bands was kept 20mm. Each band spotted was 5 µl. The chromatogram was developed up to 80 mm in a linear increasing fashion. A CAMAG Twin trough chamber was used for chamber saturation for 10 minutes. The plate was developed with a closed lid. The plate was scanned at 222nm for spectrodensitometric analysis of Mefloquine Hydrochloride using CAMAG TLC Scanner 4. Various proportion of mobile phase were tried by using trial and error method. A mobile phase comprising glacial acetic acid, toluene, and acetonitrile in a 6:3:1 ratio was employed. Mefloquine hydrochloride was found to have an Rf value of 0.524.

Validation Parameters for Developed Method:

In accordance with ICH Q2 (R1) recommendations, developed technique was validated for specificity, accuracy, robustness, linearity, limit of quantification, limit of detection and precision.

 System Suitability:

Six replicate bands of standard solutions were applied, developed, and analyzed in an HPTLC system, and acceptance criteria for all parameters were studied.

 Specificity:

Mefloquine hydrochloride standard and sample solutions were analysed to demonstrate the method's specificity. The band for mefloquine hydrochloride was verified by comparing the standard's band spectrum and Rf value.

Linearity:

By observing varying concentrations of the standard solution of mefloquine hydrochloride, the linearity of measurement was assessed.

LOD and LOQ

LOD is the lowest or minimum concertation of the substance that can be detected in a sample.

Limit of quantification is lowest analyte concentration that can be quantified with stated accuracy and precision.

Accuracy:

Mefloquine hydrochloride is estimated using the suggested method from pharmaceutical dosage forms after it was being spiked with a working standard solution. The procedure was accurate, as evidenced by the good recovery of the added drug at each concentration. The pre-analyzed formulation solution was mixed with a known quantity of standard powdered mefloquine hydrochloride (100, 110, and 120 percent). Data were obtained by repeating the experiment over two injections of each concentration.

Precision

The degree of closeness of multiple set of measurements to each other. It describes how reproducible a measurement is when repeated under same conditions Intermediate precision, reproducibility and repeatability are the three levels of precision that can be distinguished.

Robustness

An analytical procedure's robustness indicates its dependability within the normal range and is a measure of its ability to withstand minor but intentional changes in technique parameters. The specifications changed were chamber saturation period by ± 2 minutes, distance travelled by solvent by ±5mm, volume of mobile phase by ± 5ml, and mobile phase ratio by ± 2ml.

Analysis Of Pharmaceutucal Formulation

A formulation of mefloquine hydrochloride tablets was analysed using the established analytical method. The test of tablets revealed a resolved peak at an Rf value of 0.524 in the optimised HPTLC chromatogram of mefloquine hydrochloride. The % assay of tablets was found to be 98.54% of the label claim of the Lariam 250mg tablets of Mefloquine hydrochloride.

RESULT AND DISCUSSION

System Sutability Test

In order to assess the system's appropriateness and performance, six replicate bands of a reference standard solution containing 50ng/bands were developed and scanned. Standard solutions in the HPTLC system are made using a single band of blank preparation. Using chromatograms, the Rf value of mefloquine hydrochloride was determined to be 0.524, and the percentage RSD of the SST parameters fell within the permissible range.Table.No. 1 provides a summary of the system suitability statistics.

Table.No.1. System suitability study for Mefloquine hydrochloride

Fig.No.3. 3D Chromatogram of Mefloquine Hydrochloride

For the specificity blank, a standard solution of 50ng/band and a sample solution of 50ng/band were developed and scanned into the HPTLC system and evaluated for interference from the main peak; no interference was found. The band for mefloquine hydrochloride was verified by comparing its Rf value and spectra to those of the standard. The peak purity of mefloquine hydrochloride was ascertained by comparing the spectra in three distinct band regions: peak start (S), peak apex (M) and peak end (E). The chromatograms of blank, standard, and sample are shown below.

Fig.No.4. The HPTLC Densitogram for Blank

Fig.No.5. The HPTLC Densitogram for Standard

Fig.No.6. The HPTLC Densitogram for sample

By observing varying concentrations of the standard solution of mefloquine hydrochloride, the linearity of measurement was assessed. Results were found linear at six different concentrations ranging from 10-100ng/band with correlation coefficient of 0.9992.

Table.No.2. The Linearity of Mefloquine HCL

Figure.No.7. Graph of Mefloquine Hydrochloride

Mefloquine hydrochloride was found to have a LOD of 0.94µg/ml and a LOQ of 2.84µg/ml.                 

Table.No.3. LOD and LOQ data of Mefloquine HCL

Accuracy:

Following a spike with a working standard solution, the suggested approach is used to estimate the amount of mefloquine hydrochloride in pharmaceutical dosage forms. At every concentration, a good recovery of the added drug was achieved, proving the accuracy of the procedure. To the formulation's pre-analyzed solution, a known quantity of standard powdered mefloquine Hcl (100, 110, and 120%) was added. Data were obtained by repeating the experiment over two injections of each concentration. Table 4 shows the recovery result for mefloquine hydrochloride from the commercial formulation.

The method's precision was assessed using intermediate precision and repeatability. Six applications of a standard solution of mefloquine hydrochloride at a concentration of 50 ng/band were used to calculate the percentage RSD for repeatability. The precision study's overall percentage RSD was found to be within acceptable limits. Table No. 5 displays the findings of the mefloquine hydrochloride precision repeatability studies.

By adjusting the experimental parameters in minor but intentional ways, robustness is verified. The changes were made in chamber saturation time (10min+/-5 Min), mobile phase composition, change in mobile phase volume (20ml+/-5ml) and change in distance travelled by solvent (80mm+/-5mm). Effect on peak area was checked. The method's % RSD was less than 2, indicating its robustness.