Study design
The study took place in Bukavu city, the capital of South-Kivu province in DRC, from May to September 2019. It assessed pharmaceutical technology requirements concerning labeling, physical examination, disintegration time, friability, and mass uniformity. Thin-layer chromatography (TLC), non-aqueous protometry, and UV-spectrophotometry served to identify and assay active ingredients using referral pharmacopeial guidelines and regulations. Critical attributes were labeling quality, general tests on pharmaceutical dosage forms, content and dissolution, identification, and the presence of unidentifiable impurities in formulations.
Equipment, chemicals, and reagents
The equipment included UV spectrophotometer (Pharmacia LKB Ultrrospec Plus, Germany), chromatographic plates (pre-coated TLC sheets Alugram® Xtra Sil G/UV 254 layer: 0.20 mm silica gel 60 with fluorescent indicator UV 254), UV lamp 254 nm (Technology Transfer, Germany), analytical balance (balance digital jewelry scale, capacity 20 g, readability 0.001 g, China), friability device (manufactured by lab-line instruments. Inc. Model n°1641 China), aggregation device (Erweka-apparate bau, Germany), durometer (Schleuniger-2E, Switzerland), hot plate (Protherm Winn Leek, Holland), water bath (Gerhardt Bonn, Germany), rotavapor (Pleuger, Switzerland), centrifuge (IEC Centra-2 centrifuge, USA), and vortex shaker (Vortex-2 genie Bohemia, USA).
The solvents were distilled water; toluene R (Merck KGaA, 64271 Darmstadt, Germany), ethyl acetate R (Merck KGaA, 64271 Darmstadt, Germany), methanol R (Maprochim-Labo, 59, lot 0112, 90% 0.791, DRC), hydrochloric acid R (PANREAC Quimica SAU, E-08211, Spain), glacial acetic acid R (BN: 04H240011, Spain), acetic anhydride R (E. Merck Darmstadt, Germany), acetone R (Merck KGaA 64271 Darmstadt, Germany), perchloric acid R (70%, density 1.76, E. Merck, D-6100 Darmstadt, Germany), chloroform R (Prolabo, Belgium), potassium iodide R (Merck, D-6100 Darmstadt, Germany), and mercury chloride II R (B Zedelgem, Germany).
Method development
Uniformity of mass
It represents the average mass of 20 tablets. The deviation of the individual tablet weight from the mean weight should not exceed a minimum of ± 5.0% and a maximum of ± 10.0% for 18 and 20 tablets, respectively [18].
Friability of tablets
A device manufactured by Lab-Line Instruments Inc. Model n°1641 operating by rotation at 100 revolutions per 4 min served to measure friability, with a total of tablets corresponding, as near as possible, to 6.5 g introduced in the machine. According to the requirement, the maximum acceptable loss of mass (obtained from a single test or the mean of 3 tests) is not greater than 1.0% for the sample [19].
Hardness-resistance of tablets to crushing
A durometer (Schleuniger-2E, Switzerland) served to measure the minimum and maximum force (newton) to crush each of 10 tablets [20].
Disintegration of tablets
We used a 6-tube basket disintegrating device Erweka-apparate, containing HCl 1 N as immersion fluid maintained at 37 ± 2 °C, in triplicate. The basket filled with six tablets was rotated for 30 min and then raised to count the units not completely disintegrated. At least 16 of the 18 dosage units tested should disintegrate in time [21, 22].
Identification of quinine sulfate by Mayer reagent
A tiny amount of crushed tablets powder was mixed with distilled water, filtered, and the filtrate mixed with the Mayer reagent (5 g of potassium iodide (KI), 1.36 g of mercury chloride (HgCl2), and 100 ml of distilled water) in a test tube. The reaction was positive if a yellowish-white precipitate is formed [23].
Identification of quinine sulfate and Artemether-Lumefantrine by TLC
The protocol followed the existing procedures with slight adaptation. For QS samples [24,25,26], the stationary phase was silica gel R6; the mobile phase was methanol/ammonia (20: 0.5 v/v). The final concentration was 1.428 mg/ml. For AL samples [27, 28], the stationary phase was silica gel R6; the mobile phase was toluene/ethyl acetate/anhydrous acetic acid (18:4:2 v/v/v). The final concentration was 1.30 mg/ml of Artemether and 7.80 mg/ml of Lumefantrine. Approximately 2 μl of samples and 2 μl of standards were spotted at 1.5 cm on silica gel R6 plates. After migration, each chromatographic plate was removed, dried, and then examined with ultraviolet light (254 nm) for QS samples. The identification of AL samples required spraying the plate with sulfuric acid R/methanol R mixture (10:190 v/v) and then heating the chromatographic plate to dryness on the heating plate at 50 °C for approximately 10 min. We compared the correspondence in position, appearance, and intensity between the test samples and the standards. The retardation factor error (%Rf error) of Rf-sample and Rf-standard was calculated as follows:
$$ \% RFerror=\frac{100\times \left( Rfstandard- Rfsample\right)}{Rfstandard} $$
Rule: If Rf error ≤ 5%, the sample is considered valid; if Rferror is between 5 and 10%, the analyte is deemed doubtful; if Rf error ≥ 10%, it is invalid [2].
Quantitative analysis of quinine sulfate in tablets
Aliquots of 20 tablets crushed to a powder, equivalent to 100 mg of quinine sulfate, were gently stirred for 15 min in 40 ml of acetic anhydride R and 40 ml of anhydrous acetic acid. And we titrated with perchloric acid (0.1 mol/L) using violet crystal as the indicator (from violet to blue and apple green). Each milliliter of perchloric acid (0.1 mol/l) is equivalent to 26.10 mg of quinine sulfate [(C20H24N2O2)2, H2SO4.2H2O]. The test requires that the yield be not less than 90.0% and not more than 110.0% of the amount of quinine sulfate [29, 30].
Quantitative analysis of Artemether-Lumefantrine
The principle consists of extracting Artemether and Lumefantrine from TLC spots and measuring the absorbance at 254 nm. For the Artemether (sample and standard), the layer obtained by TLC was carefully scraped, weighed, and diluted with 2 ml of the HCl/ethanol mixture (1 mol/l) in a test tube. After homogenization on the vortex, the test tube was heated in a water bath at 55 °C for 5 h, then cooled to room temperature and centrifuged. The procedure was the same for the blank without Artemether. The resulting solution’s absorbance was measured in a 1-cm layer quartz cell at 254 nm against a solvent cell containing the blank. The dilution was 38.5 for Artemether (C16H26O5) in the sample and standard, using the absorptivity value \( {A}_{1 cm}^{1\%}=385 \) [27]. The percentage content of Artemether (C16H26O5) in the tablets was calculated as follows:
$$ Yield\%=\frac{100\times \left( mean\ Asorbance\ sample\right)}{mean\ Absorbance\ standard} $$
The test met the requirement if the yield is between 95.0 and 105.0% of the amount of Artemether (C16H26O5) and Lumefantrine (C30H32Cl3NO). For the lumefantrine analysis, the TLC layer spots were carefully scraped, weighed, and dissolved in 2 ml of methanol R in a test tube. The mixture was then homogenized on the vortex for 15 min and centrifuged. The absorbance was measured at 380 nm [28] against the blank prepared in the same way without Lumefantrine.
Data analysis
We set the score of the critical quality attributes as 1 or 0 when the test met or not the requirement—the sum of scores allowed calculating the percentages of global quality satisfaction. The quality was excellent (0.90–1.00), good (0.80–0.90), acceptable (0.70–0.80), and low (0.30–0.70), based on a modified psycho-physical Harrington’s scale of quality [31].
The risk of treatment failure, death due to untreated disease, or toxicity was high, moderate, or low concerning the absence or wrong active ingredient, identity, under-dose, over-dose, disintegration, uniformity of mass, and labeling outcomes. MS Excel 2013 was used to calculate the descriptive statistics.
