Chemicals and reagents
Artemether (98.0%) standard reference (RS), Artesunate (RS at 98.0%), Lumefantrine (RS and 98.0% of purity) and Sulfadoxine (RS at 98.0%) were all obtained from Tokyo Chemical Industry (TCI) in Japan. dihydrated quinine sulfate (99.0%) was afforded from VWR Prolabo Chemicals and Pyrimethamine (RS at 98.0%) was obtained from Sigma-Aldrich. Methanol and Toluene were purchased from Merck (Plv) Ltd in South Africa, Ethyl Acetate and Acetic acid were obtained from Merck KGaA in Germany. Ammonia 99.98% (RS) was obtained from Sigma Aldrich, in France and Iodine (RS) was purchased from Altichem in France. Samples made of Quinine sulphate 500 mg tablets, Quinine drops and syrup were purchased from Pharmakina (Kinshasa, D.R. Congo) ; Artemether + Lumefantrine 20mg/120mg (Branded Lumiter®) was afforded from a local distributor AMT (Kinshasa, D.R. Congo) and Artesunate Injectable 60mg branded Armadoc 60®, and purchased from Doctor Pharma (Kinshasa, D.R. Congo).
Instrumentation
Camag® Linomat 5 automat spotter, Camag® twin trough chamber, Camag® chromatogram immersion device 3, TLC plate (Silica gel F254), Khöler® TLC visualizer (two UV tubes for illumination at 254 nm and 360 nm) and Capillary tube.
Preparation of standards and sample solutions
1. Artemether standard preparation
A 100 mg quantity of the standard was weighed using an electronic balance and dissolved in 10ml of Methanol for analysis, to produce a solution at the concentration of 10 mg/ml.
2. Artesunate standard preparation
A 50 mg quantity of the standard was weighed using an electronic balance and dissolved in 10ml of Methanol for Analysis, to afford a 5 mg/ml solution.
3. Lumefantrine standard preparation
A 10 mg quantity of the standard was weighed using an electronic balance and dissolved in 10ml of Methanol for Analysis, to produce a 1 mg/ml solution.
4. Dihydrated quinine sulphate solution
A 50 mg quantity of the standard was weighed using an electronic balance and dissolved in 10ml of Methanol for Analysis, to obtain a solution at the concentration of 25 mg/ml.
5. Sulfadoxin standard solution preparation
A 100 mg quantity of the standard was weighed using an electronic balance and dissolved in 10ml of Methanol for Analysis, to produce a solution at the concentration of 10 mg/ml.
6. Pyrimethamin standard solution preparation
A 50 mg quantity of the standard was weighed using an electronic balance and dissolved in 10ml of Methanol for Analysis, to obtain a solution at 5 mg/ml.
Common standards solution (contrôle solution)
The control solution was prepared by dissolving 250mg of Arthemeter powder, 125mg of Artesunate, 25mg of Lumefantrine, 250mg of Sulfadoxine, 125mg of Pyrimethamine and 125mg of Quinine in a 25ml volume of Methanol for analysis obtained from LAM, to give the concentration of 10mg/ml, 5mg/ml, 1mg/ml, 10mg/ml, 5mg/ml and 5mg/ml respectively for each SCR present in solution.
Matrix preparations
1. Tablets matrix
This was made of following components :
10 mg of maize starch, 10 mg of gelatin, 10 mg of lactose, 10 mg of Magnesium stearate, 10 mg of CMC, 10 mg areosil, 10 mg of talc, 10 mg of sodium saccharinate and 10 mg of citric acid.
Operating protocol : simply mix the powders; take a 10mg sample to be dissolved in 20ml Methanol; filter and proceed with the analysis.
2. Syrup matrix
Made of :
Nipagine 0.01%, Nipasone 0.01%, Sodium benzoate 0.01%, Simple syrup 20 ml and distilled water (suffiscient quantity to make 100ml).
Operating protocol : Mix the powders; dissolve the powder mixture in 20ml of simple syrup; then bring the solution to 100ml with distilled water; take a 10ml test sample in 10ml of Methanol then proceed with the analysis.
3. Injectable solution matrix
This was made of NaCl 0.9% and NaHCO3
Operating protocol : Mix the powders; dissolve the powder mixture in 20ml distilled water; then bring the volume to 100ml with distilled water; take a 6ml test sample in 10ml Methanol then proceed with the analysis.
Calibration and Validation standards
This was established only for the Quinine following the protocol below :
Calibration standard
Stock solution (Quinine at 5 mg/ml) :
The stock solution for calibration was obtained by weighing 100mg of Quinine sulfate dihydrate, the reference substance, dissolved in 20ml of Methanol for analysis, to give a concentration of 5mg/ml.
Dilution series were prepared in different levels as follow
Level 1 (50%) : Obtained by withdrawing exactly 1ml of the calibration stock solution (quinine 5mg/ml) into a 10ml volumetric flask, then making up to the mark with methanol to obtain a final concentration of 0.5mg/ml.
Level 2 (100%) : Obtained by withdrawing exactly 2ml of the calibration stock solution (quinine 5mg/ml) into a 10ml volumetric flask, then making up to the mark with methanol to obtain a final concentration of 1mg/ml.
Level 3 (150%) : Obtained by withdrawing exactly 3ml of the calibration stock solution (quinine 5mg/ml) into a 10ml volumetric flask, then making up to the mark with methanol to obtain a final concentration of 1.5mg/ml.
Validation standard
Stock solution (Quinine at 5 mg/ml) :
The stock solution for validation was obtained by mixing 100mg of Quinine sulfate dihydate, the reference substance with 10mg of powder constituting the tablet matrix, then dissolving the powder mixture in 20ml of Methanol for Analysis, to obtain a concentration of 5mg/ml and filtering.
Dilution series : prepared in same conditions as the calibration standards as follow,
Level 1 (50%) : Obtained by withdrawing exactly 1ml of the calibration stock solution (quinine 5mg/ml) into a 10ml volumetric flask, then making up to the mark with methanol to obtain a final concentration of 0.5mg/ml.
Level 2 (100%) : Obtained by withdrawing exactly 2ml of the calibration stock solution (quinine 5mg/ml) into a 10ml volumetric flask, then making up to the mark with methanol to obtain a final concentration of 1mg/ml.
Level 3 (150%) : Obtained by withdrawing exactly 3ml of the calibration stock solution (quinine 5mg/ml) into a 10ml volumetric flask, then making up to the mark with methanol to obtain a final concentration of 1.5mg/ml.
Chromatographic conditions :
The chromatographic conditions applied to develop our method are presented in table I below:
Table I. Chromatographic conditions of the method
Procedure
1. TLC Tank preparation
Place the previously prepared mobile phase in the bottom of the vessel and cover it for at least 15 minutes to saturate the atmosphere with solvent vapour.
2. Analytical samples preparation
a. TLC plate preparation :
Using a pencil, draw the start line (generally 1cm from the bottom of the plate) and an end line for migration (generally 1cm from the top of the plate), as well as the points where the sample spots will be applied to the migration start line.
b. Samples spotting on plate :
Using a micropipette set at 10microliters, apply the various substances to be analyzed, then dry the spots using an electronic dryer or hot plate.
3. Elution :
Place the plate in the cuvette, close and allow the eluent to diffuse (approx. 15 minutes). Stop the TLC when the eluent front has reached the migration end line (1cm from the top of the plate), then dry the plate to remove the elution solvent.
4. Visualizing : Depending on the type of compound, revelation may be carried out using UV light at 254 nm and/or iodine.
5. Calculation and interpretation : Using the mathematical formula, determine the frontal ratio for each eluted compound.
Data acquisition and processing
The following tools and software were used to acquire and process analytical data :
- FIJI Image J 1.54 C Densitometric Analysis software
- ANOVA 2 software
- JMP experimental design software
- Microsoft Excel software
- E-Noval software for validation