As serious but neglected public health problems, poor quality medicines, i.e. for antimalarial medicines, urged to be fought. One of the approaches is to consider the analytical chemistry and separative techniques. In this study, a generic liquid chromatographic method was firstly developed for the purpose of screening 8 antimalarial active ingredients, namely amodiaquine (AQ), piperaquine (PPQ), sulfalene (SL), pyrimethamine (PM), lumefantrine (LF), artesunate (AS), artemether (AM) and dihydroartemisinine (DHA) by applying DoE/DS optimization strategy. Since the method was not totally satisfying in terms of peak separation, further experiments were undergone applying the same development strategy while splitting the 8 ingredients into five groups. Excellent prediction was observed prior to correlation between retention times of predicted and observed separation conditions. Then, a successful geometric transfer was realized to reduce the analysis time focusing on the simultaneous quantification of two WHO’s recommended ACTs in anti-malarial fixed-dose combination (AM-LF and AS-AQ) in tablets. The optimal separation was achieved using an isocratic elution of methanol-ammonium formate buffer (pH 2.8; 10 mM) (82.5:17.5, v/v) at 0.6 ml/min through a C18 column (100 mm × 3.5 mm, 3.5 μm) thermostated at 25 ℃. After a successful validation stage based on the total error approach, the method was applied to determine the content of AM/LF or AS/AQ in seven brands of antimalarial tablets currently marketed in West, Central and East Africa. Satisfying results were obtained compared to the claimed contents.
Poor quality medicines are serious but neglected public health problems. Anti-infective medicines are particularly afflicted [
Fast acting artemisinin-based compounds are combined with a drug from a different class. Companion drugs include lumefantrine, mefloquine, amodiaquine, sulfadoxine/pyrimethamine, piperaquine and chlorproguanil/ dapsone. The artemisinin derivatives usually used including dihydroartemisinin, artesunate and artemether. Implementation of the recommendation to use ACTs is limited by the small number of available and affordable co- formulated anti-malarial drugs, but most countries are now starting to implement this regimen. A co-formulated drug is one in which two different drugs are combined in one tablet; this is important to ensure both drugs are used.
Artemether/lumefantrine was the first fixed-dose artemisinin-based combination therapy recommended and pre-qualified by WHO for the treatment of uncomplicated malaria caused by P. falciparum. It has been shown to be effective both in sub-Saharan Africa and in areas with multi-drug resistant P. falciparum in Southeast Asia. It is currently recommended as first-line treatment for uncomplicated malaria in several countries. However, its complex treatment regimen of two doses daily for three days could affect patient adherence to treatment. A fixed- dose combination of amodiaquine-artesunate was launched in February 2007 [
According to WHO 200,000 deaths over one million that occur from malaria annually would be avoidable if the available medicines were effective, of good quality and used correctly [
In this context, analytical chemistry and especially separative screening methods such as liquid chromatography (LC) methods are suitable to help fighting against such medicines and therefore can be used [
Recently, Debrus et al. published interesting work on an innovative HPLC method development for the screen- ing of 19 antimalarial drugs based on a generic approach, using design of experiments, independent component analysis and design space. That method was found somewhat time consuming due to the gradient mode [
In the present study, several HPLC separations considering isocratic mode (short run time) were optimized for targeted subsets of 8 antimalarial active ingredients (AAI) used alone or in combination.
The first objective was the optimization of the separation conditions (screening method) for these 8 AAI among which were 4 companion drugs (amodiaquine (AQ), piperaquine (PPQ), sulfalene (SL), pyrimethamine (PM) lumefantrine (LF)) and Artemisinin derivatives include dihydroartemisinin (DHA), artesunate (AS) and artemether (AM). Their chemical structures are presented in
The second objective was the simultaneous determination of artemether, lumefantrine, artesunate, amodiaquine in fixed dose combination tablets as recommended by WHO. As suggested in ICH Q8 (R2) and previously successfully tested by Debrus et al. [
The third objective was to validate the transferred method using the accuracy profile as decision tool for the simultaneous quantitation of artemether and lumefantrine; artesunate and amodiaquine in fixed dose combina- tion (FDC) tablets.
Finally, the validated method was used to analyze several antimalarial drugs marketed in Benin (West Africa), DRC (Central Africa) and Rwanda (East Africa).
Methanol (HPLC gradient grade), formic acid (98% - 100%) and orthophosphoric acid Eur Ph. grade (85%) were purchased from Merck (Darmstadt, Germany). Ammonium formate (99%) was provided by BDH Prolabo (Almere, Netherlands). Ultrapure water was obtained from a Milli-Q Plus 185 water purification system from Millipore (Billerica, MA, USA). Artesunate (99.8%) and dihydroartemisinin alpha and beta (100.0%) were purchased from Apoteket AB (Stockholm, Sweden). Lumefantrine (99.4%) and artemether (99.5%) were kindly donated by Fourrts laboratories (Chennai, India) and Meridian Pharmacare Pvt Ltd. (Bangalore, Inde). Amodiaquine hydrochloride (99.0%), Piperaquine tetraphosphate (99.2%) and Pyrimethamine (99.0%) were purchased from Sigma Aldrich (St. Louis, MO, USA). Sulfalene (100.0%) was purchased from Fagron NV/SA (Waregem, Belgium). For the preparation of validation standards, a matrix formulation of tablets containing 20 mg of AM and 120 mg of LF was provided by Fourrts laboratories (Kanchipuram, Inde). Mefanther® 20/120 mg tablet were kindly donated by the same laboratories. Antimalarial drugs containing AS and AQ 50/150 were purchased in drugstore located in DRC (Kinshasa). Antimalarial drugs containing AM (20, 40 or 80 mg) and LF (120, 240 or 480 mg) were purchased in drugstore located in Benin (Cotonou), DRC (Kinshasa) and Rwanda.
Individual stock solutions of AM, AS and DHA at 5 mg/ml and of AQ, PPQ, PM 1mg/ml were prepared in methanol. A stock solution of LF at 100 µg/ml was prepared in methanol acidified by phosphoric acid (0.1% acid phosphoric in methanol (w/v)). Mixture solutions were prepared by diluting stock solutions in methanol-water (50:50, v/v) to achieve the following concentrations: 2.5 mg/ml for AM, AS, DHA; 50 µg/ml for LF, SL and 25 µg/ml for PPQ, PM and AQ.
A stock solution of calibration standards (CS) of AM (240 µg/ml) and LF (1440 µg/ml) was prepared in methanol acidified by acid orthophosphoric. A stock solution of AS (240 µg/mL) and AQ (720 µg/mL) was prepared in methanol. Dilutions were performed in methanol-water (50:50) in order to obtain solutions at 3 different concentration levels:
Level 1(40%): 80 µg/ml (AM) - 480 µg/ml (LF) and 80 µg/ml (AS) - 240 µg/ml (AQ);
Level 3 (80%): 160 µg/ml (AM) - 960 µg/ml (LF) and 160 µg/ml (AS) - 480 µg/ml (AQ);
Level 5 (120%): 240 µg/ml (AM) - 1440 µg/ml (LF) and 240 µg/ml (AS) - 720 µg/ml (AQ).
The levels of the concentration were chosen in order to allow construction of different regression models that will determine back-calculated concentrations of validation standards. For each concentration level three replications were run for three days corresponding to three series (p = 3).
The validation standards were prepared in matrices, here tablets, obtained by the manufacturers of the corresponding medicines in order to better simulate the sample preparation in routine analysis. Stock solutions were obtained as in the case of calibration standards to which is added a corresponding amount of the matrix. Dilutions were performed in methanol-water (50:50) in the same way as described for the CS in order to obtain solutions at 5 different concentration levels.
Level 1 (40%): 80 µg/ml (AM) - 480 µg/ml (LF) and 80 µg/ml (AS) - 240 µg/ml (AQ);
Level 2 (60%): 120 µg/ml (AM) - 720 µg/ml (LF) and 120 µg/ml (AS) - 360 µg/ml (AQ);
Level 3 (80%): 160 µg/ml (AM) - 960 µg/ml (LF) and 160 µg/ml (AS) - 480 µg/ml (AQ);
Level 4 (100%): 200 µg/ml (AM) - 1200 µg/ml (LF) and 200 µg/ml (AS) - 600 µg/ml (AQ);
Level 5 (120%): 240 µg/ml (AM) - 1440 µg/ml (LF) and 240 µg/ml (AS) - 720 µg/ml (AQ).
Three independent preparations (n = 3) were carried out per each of the five concentration levels (m = 5). All these preparations were repeated for three days corresponding also to three series (p = 3).
For routines analyses, the concentrations of reference standards were 200 µg/ml of AM and 1200 μg/mL of LF in a mixture, 200 µg/ml of AS and 600 μg/mL of AQ in another mixture. For the sample tablets, powdered portions were taken and treated in the same way as reference solutions to give final expected concentrations of 200 µg/ml (AM) - 1200 μg/mL (LF) for AM-LF combination and 200 µg/mL (AS) - 600 μg/mL (AQ) for AS- AQ combination. The solutions were freshly prepared and protected from light. They were filtered through 0.45 µm PTFE syringe filtration disks prior to their analysis onto the liquid chromatographic system.
The experiments for optimization of the LC conditions, for the validation work and for the routine analysis were carried out on a LC system from Waters 2695 (Waters, Milford, USA) composed of a Waters selector 7678, autosampler, photodiode array detector (PDA) Waters 2996 and Empower 2.0 software. The analytical column for optimization was an XBridge C18 (250 × 4.6 mm i.d.; 5 µm particle size) preceded by a guard column XBridge guard C18 (20 × 4.6 mm i.d.; 5 µm particle size) both from Waters. The optimized conditions were transferred to an XBridge C18 (100 × 4.6 mm i.d.; 3.5 µm particle size), 4 µl for injection volume. Peak analytes were monitored at 230 nm during optimization and at 210 nm during validation and routine application. However, the UV spectra were recorded online from 210 nm to 400 nm to allow the peak identification at all the experiments. The injection volume was 10 µl for all tested experimental conditions. The buffer solution of the isocratic mobile phase consisted of 10 mM ammonium formate (pKa = 3.8) adjusted to pH of 2.8 with formic acid.
Design of experiments (DoE) was used to define the Design of Space (DS). Flow of mobile phase (F), column temperature (T˚C) and proportion of methanol in the mobile phase (%OM) were selected as the factors to investigate (see
Because of the temperature control problem that might be encountered in that kind of environment we de- cided to include that factor in the study and to extend the range for test. A total of 29 experimental conditions were defined as shown in
Empower 2.0 for Windows was used to control the HPLC and to record the signals from the detector and interpret the chromatograms. An algorithm was set up to develop a Bayesian model and to compute the DS.
The algorithm was written in R2.13, which is available as free-ware from: http://www.rproject.com.
HPLC calculator V3.0 (University of Geneva, Switzerland) was used to carry out the necessary computations for the geometric transfer methodology.
The accuracy profiles as well as the statistical calculations including the validation results and uncertainty esti- mates were obtained using e-noval® V3.0 software (Arlenda, Belgium).
Due to acidic and alkaline comportments of the AAI to test, and considering literature data we choose to perform the experiments in acidic media. Preliminary tests allowed setting the pH to 2.8 as well as setting up the range and the levels of each factor (see
The influence of the critical factors on the separation of the chromatographic peaks was then assessed by means of full factorial design. As can be noticed in
For better reliable prediction of the chromatographic conditions of each AAI, modeling was performed using the
Factors | Levels | ||
---|---|---|---|
Organic modifier (%) | 80 | 85 | 90 |
Flow rate (ml/min) | 0.3 | 0.5 | 0.7 |
Temperature of the column oven (˚C) | 25.0 | 30.0 | 35.0 |
Trial | Experimental design | Experimental set up | ||||
---|---|---|---|---|---|---|
X1 | X2 | X3 | Organic modifier (%) | Flow rate (mL/min) | Temperature (˚C) | |
1 | −1 | −1 | 0 | 80 | 0.3 | 30 |
2 | 1 | 0 | 1 | 90 | 0.5 | 35 |
3 | 0 | 0 | 0 | 85 | 0.5 | 30 |
4 | 1 | −1 | 1 | 90 | 0.3 | 35 |
5 | 0 | −1 | 1 | 85 | 0.3 | 35 |
6 | −1 | 1 | 1 | 80 | 0.7 | 35 |
7 | −1 | 1 | −1 | 80 | 0.7 | 25 |
8 | 1 | −1 | 0 | 90 | 0.3 | 30 |
9 | 0 | 0 | 0 | 85 | 0.5 | 30 |
10 | 0 | 0 | 0 | 85 | 0.5 | 30 |
11 | −1 | −1 | −1 | 80 | 0.3 | 25 |
12 | 1 | 1 | −1 | 90 | 0.7 | 25 |
13 | −1 | 1 | 0 | 80 | 0.7 | 30 |
14 | −1 | 0 | 1 | 80 | 0.5 | 35 |
15 | 1 | 0 | −1 | 90 | 0.5 | 25 |
16 | 0 | 0 | −1 | 85 | 0.5 | 25 |
17 | 0 | 0 | 1 | 85 | 0.5 | 35 |
18 | 0 | −1 | 1 | 85 | 0.3 | 35 |
19 | 0 | −1 | −1 | 85 | 0.3 | 25 |
20 | 0 | 1 | 1 | 85 | 0.7 | 35 |
21 | −1 | 0 | 0 | 80 | 0.5 | 30 |
22 | 1 | 0 | 0 | 90 | 0.5 | 30 |
23 | 1 | 1 | 1 | 90 | 0.7 | 35 |
24 | 0 | −1 | 0 | 85 | 0.3 | 30 |
25 | 1 | −1 | −1 | 90 | 0.3 | 25 |
26 | 0 | 1 | 0 | 80 | 0.7 | 30 |
27 | −1 | −1 | 1 | 85 | 0.3 | 35 |
28 | 1 | 1 | 0 | 90 | 0.7 | 30 |
29 | −1 | 0 | −1 | 80 | 0.5 | 25 |
retention time of each strategic part of the chromatographic peak, i.e. the beginning, the apex and the end [
The good relationship between the predicted retention times and those obtained allowed validating the linear re- gression model and optimizing selected criteria. The separation between the peaks of the critical pair has been chosen as a critical quality attribute (CQA) for the evaluation of quality chromatogram [
Over the experimental domain, as shown on
This low probability of peak separation led us to split molecules with similar chromatographic behavior in 4 separated groups (
These five groups were experimented with the same design tested before applying the same corresponding factors levels as mentioned in
Group | Subgroups | Molecules |
---|---|---|
Group 1 | - | AM, LF, DHA-1, DHA-2 and PPQ |
Group 2 | - | AM, LF, AS and AQ |
Group 3 | - | AM, LF, DHA-1, DHA-2 and PM |
Group 4 | - | AM, LF, AS and SL |
Group 5 | 1 | AM and LF |
2 | AS and AQ |
Legend: AM = Artemether, LF = Lumefantrine, DHA = Dihydroartemisinin, PPQ = Piperaquine, AS = Artesunate, AQ = Amodiaquine, PM = Pyrimethamine, SL = Sulfalene.
Optimal condition by group | Final optimal conditions | ||||||
---|---|---|---|---|---|---|---|
Optimal conditions | Optimal P (S > 0) | Flow rate (F in mL/min) | Organic modifier (OM in %) | Temperature (T in ˚C) | |||
Groups | Subgroups | ||||||
1 | - | 68.0% | 0.45 (0.45 - 0.61) | 81.3 (80.0 - 82.1) | 25.0 (25.0 - 35.0) | OM: 80.0% F: 0.5 mL/min T: 25˚C | |
2 | - | 99.5% | 0.70 (0.55 - 0.70) | 80.0 (80.0 - 81.8) | 32.5 (25.0 - 34.5) | ||
3 | - | 73.0% | 0.45 (0.41 - 6.50) | 81.3 (81.0 - 82.1) | 25.0 (25.0 - 27.0) | ||
4 | - | 92.9% | 0.65 (0.48 - 7.00) | 80.0 (80.0 - 81.5) | 32.5 (25.0 - 35.0) | ||
5 | 1 | 98.5% | 0.70 (0.61 - 0.70) | 81.3 (80.5 - 82.0) | 26.3 (25.0 - 35.0) | OM: 82.5% F: 0.6 mL/min T: 25˚C | |
2 | 99.9% | 0.40 (0.55 - 0.70) | 88.8 (87.5 - 90.0) | 25.0 (25.0 - 35.0) | |||
One can say that a large temperature robust range (25˚C to 35˚C, except for Group 3 (25˚C to 27˚C)) is important for applying easily the methods in the laboratories without an efficient temperature control system that is often met in resource-restraint environments.
In order to facilitate the screening of AAI in Groups 1 to 4, a single method was generated by computing DS obtained only for these groups. One single method was also generated for Groups 5.1 and 5.2. The optimal con- ditions are given in
To support the ability of DS to predict analytical conditions that permit chromatographic separation for the AAI in the 5 groups, we tested the mixture of these AAI in each optimal condition using an XBridge C18 (250 × 4.6 mm i.d.; 5 µm particle size), preceded by a guard column XBridge guard C18 (20 × 4.6 mm i.d.; 5 µm particle size). The experimental and the predicted chromatograms are given in Figures 6-11 where it can be noticed a close agreement between the different predicted chromatograms and the corresponded experimental ones.
The correlation between the predicted retention times and observed for the chromatograms recorded at the optimal condition was very good. Indeed, in all cases, the linear correlation coefficient was very close to the unit, validating the accuracy of the prediction. Concerning the two WHO's recommended ACTs, the liquid chromatography method developed for the simultaneous quantification offered the advantage of being used in isocratic mode, unlike the methods of the American pharmacopoeia and international pharmacopoeia offering the gradient mode and are time consuming [
In order to reduce the analysis time and thus the solvent consumption, the geometric transfer was performed for each developed method following geometric transfer methodology while checking their robustness [
The chromatograms in Figures 6-11 and the results in
It was found important to highlight that the same optimal condition can be used to analyze dihydroartemisinin-piperaquine because of the very good separation observed (data not shown). By cons, the optimized method cannot be used to analyze the associations such as sulfalene-pyrimethamine-dihydroartemisinin and artesunate- sulfalene-pyrimethamine, due to the co-elution of the chromatographic peaks corresponding to sulfalene and pyrimethamine. These associations of antimalarial drugs are also marketed in certain African countries.
In current practice, after the optimization step, it becomes increasingly obvious and essential to demonstrate
Compounds | HPLC Optimal | HPLC Transfer | Relative observed retention times error | ||||
---|---|---|---|---|---|---|---|
Predicted retention times | Observed retention times | Relative predicted retention times | Relative observed retention times | Observed retention times | Relative observed retention times | ||
AM | 21.529 | 20.335 | 0.791 | 0.746 | 8.572 | 0.572 | 0.184 |
AQ | 5.505 | 5.270 | 0.202 | 0.193 | 2.297 | 0.153 | 0.040 |
AS | 10.142 | 9.731 | 0.373 | 0.357 | 4.006 | 0.268 | 0.089 |
DHA-1 | 11.016 | 10.032 | 0.405 | 0.368 | 4.100 | 0.274 | 0.094 |
DHA-2 | 13.946 | 12.662 | 0.513 | 0.464 | 5.226 | 0.349 | 0.115 |
LF | 27.195 | 27.274 | 1.000 | 1.000 | 14.976 | 1.000 | 0.000 |
PM | 6.724 | 6.087 | 0.247 | 0.223 | 2.621 | 0.175 | 0.048 |
PPQ | 6.486 | 5.680 | 0.238 | 0.208 | 2.461 | 0.164 | 0.044 |
SL | 5.966 | 5.686 | 0.219 | 0.208 | 2.159 | 0.144 | 0.064 |
through a method validation that optimized method provides reliable results. In this work, the transferred method was also validated using the accuracy profile as decision tool and for the simultaneous quantitation of the couples artemether/lumefantrine and artesunate/amodiaquine in fixed dose combination (FDC) tablets [
An analytical method is specific if it guarantees that the measured signal is only related to the substance intended to be analyzed (targeted compound) and if it allows quantitation of a physicochemical parameter or a chemical group from a single or several substance(s) in the sample [
Secondly, we investigated the response function of the method. It is the existing relationship between the response (signal) and the concentration (quantity) of the analyte sample within the range of concentrations tested. The calibration curve was the most appropriate response function.
The selected calibration model is linear regression due to his high level of accuracy index. The concentrations results were back-calculated using the calibration curves. These concentrations were used to determine the relative bias, the precision (repeatability and intermediate precision), the β-expectation tolerance intervals at 95% probability level, and the linearity. The accuracy profiles for the four compounds are given in
The acceptance limits have been set at ±10% according to the International Pharmacopeia and the intended use of the analytical procedure [
Trueness refers to the closeness of agreement between a conventionally accepted value or reference value and a mean experimental one. It gives information on systematic error. As shown in
Precision is the closeness of agreement among measurements from multiple sampling of a homogeneous sample under the recommended conditions. It gives some information on random errors and it can be evaluated at two levels: repeatability and intermediate precision. As can be seen in
The linearity of an analytical method is the ability within a definitive range to obtain results directly proportional to the concentration (quantity) of the analyte in the sample. A linear regression model is fitted on the
Active ingredient | Model | Indexes for: | |||
---|---|---|---|---|---|
Precision | Trueness | Dosing range | Accuracy | ||
Artemether | Linear regression through 0 fitted using the highest level only | 0.830 | 0.746 | 0.414 | 0.635 |
Linear regression through 0 fitted using the level 1.0 only | 0.662 | 0.866 | 0.569 | 0.688 | |
Weighted (1/X) linear regression | 0.461 | 0.970 | 1.000 | 0.765 | |
Linear regression | 0.485 | 0.962 | 0.982 | 0.771 | |
Lumefantrine | Linear regression through 0 fitted using the highest level only | 0.763 | 0.864 | 0.498 | 0.690 |
Linear regression through 0 fitted using the level 1.0 only | 0.657 | 0.845 | 1.000 | 0.828 | |
Weighted (1/X) linear regression | 0.674 | 0.868 | 0.645 | 0.723 | |
Linear regression | 0.648 | 0.864 | 1.000 | 0.824 | |
Artesunate | Linear regression through 0 fitted using the highest level only | 0.725 | 0.957 | 0.944 | 0.869 |
Linear regression through 0 fitted using the level 1.0 only | 0.713 | 0.952 | 0.999 | 0.878 | |
Weighted (1/X) linear regression | 0.689 | 0.974 | 0.997 | 0.874 | |
Linear regression | 0.713 | 0.952 | 1.000 | 0.877 | |
Amodiaquine | Linear regression through 0 fitted using the highest level only | 0.884 | 0.001 | 0.521 | 0.001 |
Linear regression through 0 fitted using the level 1.0 only | 0.931 | 0.001 | 0.157 | 0.001 | |
Weighted (1/X) linear regression | 0.662 | 0.996 | 1.000 | 0.870 | |
Linear regression | 0.660 | 0.996 | 1.000 | 0.870 |
Validation Criteria | Level | Artemether-Lumefantrine | Artesunate-Amodiaquine | ||
---|---|---|---|---|---|
Artemether | Lumefantrine | Artesunate | Amodiaquine | ||
Trueness: Absolute bias (µg/mL) (Relative bias (%)) | 1 | 1.76 (2.20) | −9.83 (−2.05) | 1.40 (1.74) | 0.79 (0.33) |
2 | 0.83 (0.69) | −42.54 (−5.91) | −0.97 (−0.81) | 0.33 (0.08) | |
3 | −4.34 (−2.71) | 36.58 (3.81) | −0.04 (−0.03) | 3.78 (0.79) | |
4 | −4.34 (−2.17) | 25.12 (2.09) | −4.76 (−2.38) | −2.71 (−0.45) | |
5 | 3.22 (1.34) | −44.56 (−3.18) | 1.77 (0.74) | 8.21 (−0.33) | |
Precision: Repeatability (RSD in %)/ Intermediate precision (RSD in %) | 1 | 3.28/3.28 | 0.56/0.77 | 3.29/3.29 | 3.24/3.24 |
2 | 2.04/2.37 | 0.44/0.67 | 0.80/0.80 | 1.28/1.28 | |
3 | 2.18/2.18 | 2.11/2.11 | 0.53/0.53 | 1.01/1.01 | |
4 | 1.24/1.24 | 1.81/1.81 | 1.19/1.24 | 1.08/1.08 | |
5 | 1.63/1.89 | 0.97/0.99 | 1.03/1.41 | 1.13/1.33 | |
Accuracy: β-expectation tolerance interval (in µg/mL) (Relative β-expectation tolerance interval (in %)) | 1 | 75.33 - 88.19 (−5.83/10.24) | 459.30 - 481.10 (−4.32/0.22) | 74.96 - 87.85 (−6.31/9.81) | 221.80 - 259.80 (−7.60/8.26) |
2 | 113.30 - 128.40 (−5.60/6.97) | 662.20 - 692.70 (−8.02/−3.80) | 116.70 - 121.40 (−2.76/1.14) | 349.10 - 371.50 (−3.04/3.21) | |
3 | 147.10 - 164.2 (−8.05/2.63) | 947.00 - 1046.00 (−1.35/8.97) | 157.90 - 162.00 (−1.32/1.27) | 471.90 - 495.60 (−1.68/3.26) | |
4 | 189.60 - 201.70 (−5.20/0.86) | 1172.00 - 1278.00 (−2.32/6.51) | 189.00 - 201.40 (−5.48/0.72) | 581.40 - 613.10 (−3.09/2.19) | |
5 | 231.2 - 255.2 (−3.67/6.35) | 1321.00 - 1390.00 (−5.64/−0.73) | 231.00 - 251.30 (−3.42/4.90) | 708.30 - 748.20 (−1.63/3.91) | |
Uncertainty: Relative expanded uncertainty (%) | 1 | 6.92 | 1.69 | 6.94 | 6.83 |
2 | 5.12 | 1.49 | 1.68 | 2.69 | |
3 | 4.60 | 4.45 | 1.12 | 2.13 | |
4 | 2.61 | 3.81 | 2.64 | 2.28 | |
5 | 4.09 | 2.10 | 3.10 | 2.39 | |
Linearity: | Slope | 0.994 | 1.002 | 0.992 | 1.010 |
Intercept | 0.329 | −8.938 | 0.703 | −2.659 | |
R2 | 0.994 | 0.988 | 0.997 | 0.998 |
back-calculated concentrations as a function of the introduced concentrations. The good linearity of the results was illustrated (
Accuracy refers to the closeness of agreement between the test result and the accepted reference value, namely the conventionally true value. The accuracy takes into account the total error, i.e. systematic and random errors, related to the test result. It is assessed from the accuracy profile illustrated in
The limit of detection (LOD) is the smallest quantity of the targeted substance that can be detected, but not accurately quantified in the sample. Reported values were: 24.05, 75.17, 3.285 and 11.47 µg/ml for AM, LF, AS and AQ, respectively.
The lower limit of quantification (LOQ) is the smallest quantity of the targeted substance in the sample that can be assayed under experimental conditions with well defined accuracy. The definition can also be applicable to the upper limit of quantitation which is the highest quantity of the targeted substance in the sample that can be assayed under experimental conditions with well defined accuracy. The limits of quantitation were obtained by calculating the smallest and highest concentrations beyond which the accuracy limits or β-expectation limits go outside the acceptance limits. The dosing range is the interval between the lower and the upper limits where the procedure achieves adequate accuracy. Dosing ranges were 82.90 to 240 µg/mL for AM, 480 to 1440 µg/mL for LF, 80 to 240 µg/mL for AS and 240 to 720 µg/mL for AQ, respectively.
The uncertainty is a parameter associated with the result of a measurement that characterizes the dispersion of the values that could reasonably be attributed to measurand. As shown in
The validated method was then used to determine the content of the four targeted compounds found in two sets of tablets samples with fixed dosage combinations. The first set consisted to five different brands coded A1, A2, A3, A4, A5, respectively, and claimed to contain artemether and lumefantrine while the second set coded B1, B2 was claimed to contain artesunate and amodiaquine. The results obtained for the analyses are presented in
In the perspective of fighting against poor quality antimalarials, we undertake the development and validation of one generic procedure of dosage (HPLC-UV/Isocratic mode) for the simultaneous quantification of two WHO’s recommended ACTs in anti-malarial fixed-dose combination (artemether-lumefantrine and artesunate-amodia- quine) tablets by using the DoE/DS optimization strategy. Three Analytical factors were selected for the experimental design namely: Flow rate of mobile phase (F), column temperature (T˚C) and proportion of methanol in the mobile phase (%OM). The experiments showed that only the Flow rate of mobile phase (F) and proportion of methanol in the mobile phase (%OM) had significant effects on peak separations within the explored experi-
Drug | Artemether (AM)―Lumefantrine (LF) | Artesunate(AS)―Amodiaquine (AM) | Country of Sampling | ||
---|---|---|---|---|---|
Artemether | Lumefantrine | Artesunate | Amodiaquine | ||
A1 | 20 mg 100.6% ± 1.8% | 120 mg 99.9% ± 0.6% | - | - | Benin |
A2 | 20 mg 100.1% ± 0.9% | 120 mg 98.0% ± 0.4% | - | - | Benin |
A3 | 20 mg 100.2% ± 1.2% | 120 mg 98.2% ± 0.8% | - | - | Benin |
A4 | 20 mg 99.1% ± 1.5% | 120 mg 94.8% ± 0.9% | - | - | Rwanda |
A5 | 80 mg 100.5% ± 0.5% | 480 mg 94.6% ± 0.3% | - | - | DRC |
B2 | - | - | 50 mg 100.7% ± 0.7% | 153 mg 93.4% ± 0.2% | DRC |
B3 | - | - | 100 mg 99.3% ± 0.3% | 270 mg 104.4% ± 0.5% | DRC |
mental domain. Design space strategy led to the development of one fast HPLC method able to screen 9 AAI and one for the simultaneous quantitation of two WHO’s recommended ACTs in anti-malarial FDC (AM-LF and AS-AQ) tablets. The LC method developed for the simultaneous quantitation offers the advantage of being used in isocratic mode, unlike the methods of the American and international pharmacopoeias offering the gradient mode and are time consuming. This method was then successfully validated prior to selectivity, linearity, accuracy, trueness and precision, for simultaneous quantitation of AM, LF, AS and AQ using the approach based on total error and accuracy profile as decision tool. This method can be applied in the routine regulatory quality control of β-artemether and lumefantrine, artesunate and amodiaquine containing FDC drug products. Application to 7 commercial antimalarial formulations marketed in Benin (West Africa), DRC (Central Africa) Rwanda (East Africa) and containing AM/LF or AS/AQ per tablet gave a content in good agreement with the declared content. This study was the first report of simultaneous determination of artemether lumefantrine arte- sunate and amodiaquine in fixed dose combination tablets.
Many thanks are due to the “ARES-CCD (Académie de Recherche et d’Enseignement supérieur―Commission de la Coopération au Développement) or previous Commission Universitaire pour le Développement” of Belgium for fellowship grant of Jérémie Mbinze Kindenge. The authors would like to thank Fourrts laboratories (Chennai, India) and Meridian Pharmacare Pvt Ltd. (Bangalore; Inde) for the provision of the reference sub- stances and the excipients.