Abstract
The current technologies for substandard and counterfeit drug detection are either too expensive for low-resource settings or only provide qualitative or semi-quantitative results. GPHF minilab™ is one of them based on thin layer chromatography(TLC) principles with a semi-quantitative capability by visual observation of the spot area and intensity for medicine quality analysis. Thus, its use as a quality control tool for pharmaceutical products has limitations as spot area and intensity visual observation by the naked eye highly varies from analyst to analyst. As such, in this study, the semi-quantitative technique has been transferred to a quantitative approach by capturing the developed TLC plate image using an Android-based mobile phone inside a simple carton box. Then, the spot area was quantified using justTLC software. The quantitative results were compared with the-high performance liquid chromatography (HPLC) method as the golden standard. Accordingly, linearity was observed in the assayed range (80–120% label claim), and the correlation coefficients found were (R2 = 0.958, 0.997, 0.941, and 0.956 for Albendazole, Mebendazole, Artemether, and Lumefantrine, respectively.). The values are satisfactory. The %RSDs found were less than 2% for all drugs [intraday (n = 6) (RSD = 1.17, 1.61, 1.87, and 1.64), and interday (n = 18) (RSD = 1.16, 0.72, 1.12, and 1.18) for Artemether, Lumefantrine, Mebendazole, and Albendazole, respectively]. Moreover, comparisons of results obtained from the sophisticated CAMAG UV cabinet (R2 =0.991, 0.971, 0.946, and 0.967) and the developed simple carton box (R2 = 0.958, 0.997, 0.941, and 0.956) for Albendazole, Mebendazole, Artemether, and Lumefantrine, respectively. The values are comparable and reveal the accuracy of the method. Robustness testings' that were performed under different altered conditions revealed the robustness of the method (RSD less than 2% for all factors). Additionally the deviations from the golden HPLC results were on average −8.62% for albendazole, −3.79% for artemether, and −4.52% for lumefantrine samples. The developed method shows a satisfactory performance capability to utilize the GPHF minilab™ as a quantitative technique for medicine quality control purposes. It will be a very useful tool in a resource-limited setting. The target method profile, which encompasses a simple, low-cost, linear, precise, robust, accurate, and quantitative GPHF minilab™ system, was obtained for Albendazole, Mebendazole, and Arthemeter lumefantine combinations (Co-artem). The proposed method was successfully applied to analyze the content of the marketed medicines in the above mentioned tablets and offered acceptable deviations from the golden HPLC method. Automation of quantitative GPHF minilab™ was highly recommended to enhance the appropriateness and use of this system.
Background
According to the World Health Organization (WHO), up to 25% of medicines consumed in developing countries are either counterfeit or substandard [1]. These drugs have remained a major public health concern in many countries across the world, particularly in Africa and Southeast Asia. Poor quality medicines might also be potential major contributors to the development and spread of drug-resistant infections, such as tuberculosis (TB), malaria, and others, which are highly mortal in resource-limited settings [2]. Poor quality medicines are a cause for serious health consequences, including the risk of treatment failure, the development of antimicrobial resistance, and the occurrence of serious adverse drug events, all of which increase healthcare costs and erode public trust in the healthcare system [3].
One of the biggest challenges in which developing countries face in their fight against counterfeit and substandard medicines is building their technical capacity to regulate medicines. Suitable techniques for pharmaceutical quality control analysis duties in resource-constrained settings are essential for monitoring the legal and illegal drug markets. This greatly assists in the early detection of falsified medicines. It will potentially discourage criminal counterfeiters from bringing their products to market. The quality of medications in these settings cannot be guaranteed without efficient, cost-effective, and validated analytical technologies and methods. In response to this need, the World Health Organization (WHO) and the United States Pharmacopeia (USP) have supported the use of a portable medicine screening kit developed by the Global Pharma Health Fund (GPHF), a non-profit subsidiary company of Merck, Darmstadt, Germany, called GPHF minilab™ in several developing countries [4, 5].
GPHF minilab™ is a portable screening technique that uses the principle of thin-layer chromatography (TLC). This technology provides a relatively inexpensive, versatile, and robust means of identifying substandard drugs at a fraction of the resources required for modern laboratory medicine quality testing [6]. Within two heavy-duty flight cases, the GPHF minilab™ contains glassware, TLC chromatographic plates, developing and detection chambers, an electronic pocket balance, UV lamps with different wavelengths, a hot plate, and calliper rulers. Developing solvents, reference materials, and other necessary reagents are included in the bags. The minilabs are designed to be operable in cases where a sophisticated laboratory setup is absent. Laboratory analysts, as well as inspectors who have been particularly trained to conduct screening tests in locations where laboratory resources are insufficient for monograph testing and in situations where very highly skilled laboratory analysts are not available, the minilabs have been effectively deployed for qualitative and semi-quantitative detection of substandard and falsified (SF) medicines [7].
However, since GPHF minilab™ is a semi-quantitative technique, its applicability for quality control of pharmaceuticals is limited. To address this limitation, recent advances in densitometry quantification [8–11] and digital image technologies have been created to allow for a more complete quantitative study of the TLC plate image [12]. As a result, previously published papers demonstrate a model process for transferring qualitative/semi-quantitative thin-layer chromatography (TLC) methods used to identify pharmaceutical products with quality defects to quantitative high-performance TLC (HPTLC)–densitometry methods that can be used to support regulatory actions.
However, TLC-densitometry equipment is still quite expensive, which limits its availability in resource-limited countries. Thus, for low-income countries, it is a formidable challenge to afford HPTLC and other gold-standard analytical methods. As a result, most of the samples reach the market without proper quality control screening and confirmatory tests by regulatory authorities. In most cases, such countries lack the capacity to conduct frequent medicine quality surveillance at various levels of the drug supply chain system, which enables substandard and falsified medicines to enter the market [13].
Therefore, in resource-limited settings, it is indispensable to seek out low-cost analysis methods for TLC plate quantifications. Some of the researchers like, Hiss Amber et al., demonstrated digitally enhanced TLC as a quantitative analysis for TLC plates. This can be used like a TLC scanner in a resource-limited setting [14]. Furthermore, researchers have demonstrated quantitative analysis of pharmaceutical products using a mobile phone TLC analyzer. The demonstration was carried out by integrating a UV lamp and a TLC plate in a 3D-printed plastic cradle that can be readily interfaced with a mobile phone. The system acts as a portable scanner for performing TLC plate analysis to identify counterfeit and substandard pharmaceutical products [15, 16]. However, their work lacks a quality target and method profile that should be validated by the International Conference on Harmonization approach, and to our knowledge, no method has been developed so far for selected medicines quantitative analysis by minilab system and mobile phone TLC plate image analysis combinations. Given the pervasiveness of the problem and the substantial impact of poor-quality medicines on the health systems of low-income settings, this study reports a validated quantitative minilab system for Albendazole, Arthemether –lumefantrine combination (Co-artem), and Mebendazole. This will help to perform regular surveillance for SF medicines in low-income countries.
Furthermore, medicine regulatory authorities can use the quantitative GPHF minilab™ system proposed in this study to implement an effective regulatory system for medicine quality assurance and control for the selected medicines and others, whether imported or produced locally. It will help them screen many samples for potential counterfeit and/or grossly substandard products, as well as support regulatory measures. Pharmaceutical manufacturers can also use it to ensure the quality of their products.
Materials and methods
Materials and reagents
Albendazole, Mebendazole, and arthemether-lumefantrine (Co-artem) reference tablets supplied with the GPHF-minilab™ kit were used. Analytical solutions were prepared using analytical-grade reagents (methanol (CARLO EBRA Reagents S.A.S., France); ethyl acetate (CARLO EBRA Reagents S.A.S., France); acetic acid (Sigma Aldrich Laborchemikalien GmbH, Germany); and toluene (BDH Chemicals Ltd., England). Albendazole and Mebendazole samples were purchased from private medicine outlets in Addis Ababa, Ethiopia. Furthermore, many arthemether-lumefantrine combination (Co-artem®) and albendazole samples were collected from all over Ethiopia for validation studies.
GPHF minilab™ system
The study was carried out using a GPHF minilab™ kit, which was intended to enable the detection of mislabeled, substandard, and counterfeit products in resource-limited settings. The kit includes all of the materials and equipment required to conduct pharmaceutical product analyses. It is also equipped with complete instructions on how to utilize it. Accordingly, standards were prepared following the published outline for this GPHF minilab™ kit, with slight changes to meet our objectives, mainly in concentration levels. The solvent front line was pre-marked at 1.5 cm from the bottom edge of the chromatoplate. Two microliters of the test solutions were manually placed on the plate along the origin line as a spot using a capillary tube with the capacity of delivering exactly 2 µl. The homogeneity of all spots was then evaluated using UV light at 254 nm, and if there was a discrepancy, spotting was repeated. Following spotting, the plates were air-dried before being placed in a jar that served as a developing chamber and contained the same mobile phase as the semi-quantitative system. The developing jar was lined with filter paper and left to rest for 15 min to ensure that the chamber was saturated with solvent vapor.
The TLC plate was then carefully inserted into the jar and closed immediately, and the chromatoplate was developed until the solvent front moved for about three-quarters of the plate's length, which took about 20 min. The plate was then removed from the chamber, the solvent front was marked, and the solvent was allowed to evaporate using a hot plate. After drying, the plate image was captured in a sophisticated CAMAG UV cabinet and also in e our newly developed and constructed simple carton box. The spot area was quantified with justTLC software, and the data was entered into Minitab software for additional analysis.
Mobile phases
For TLC development, mobile phases are prepared per the minilab protocol as follows:
For arthemether, it is prepared by mixing 18 mL of toluene, 4 mL of ethyl acetate, and 2 mL of glacial acetic acid; for lumefantrine, it is prepared by combining 18 mL of ethyl acetate, 4 mL of methanol, 2 mL of glacial acetic acid; for mebendazole, 14 mL of toluene, 4 mL of ethyl acetate, and 4 mL of glacial acetic acid, and finally, for Albendazole, 10 mL of toluene, 4 mL of ethyl acetate and 8 mL of glacial acetic acid.
Preparation of standard, sample stock solutions, and working solutions for albendazole, artemether, lumefantrine, and mebendazole
Authentic reference tablets supplied with the GPHF minilab™ kit for each product was wrapped with aluminum foil and crushed to a fine powder with a pestle. Aluminum foil was carefully emptied into the laboratory glass bottle of the appropriate capacity supplied with the kit. All solid residues were washed down with 40 mL of glacial acetic acid for Albendazole, 9 mL of methanol followed by 1 mL of glacial acetic acid for Arthemeter, 45 mL of methanol followed by 5 mL of glacial acetic acid for Lumefantrine, and 20 mL of glacial acetic acid for Mebendazole by using pipettes. The bottle was closed and shaken well for about three minutes until the majority of the solids had dissolved. Undissolved residues were allowed to settle below the supernatant liquid within an additional five minutes. The solutions obtained were labeled as 'drug stock standard solutions' for each medication. They contained the required amount of total drug in mg per mL. These stock solutions were then used to prepare different concentrations of working standard solutions (120%, 110%, 100%, 90%, and 80%) that were applied to TLC plates, and a similar procedure was used to prepare sample stock and working solutions of Albendazole, Mebendazole, and arthemether-lumefantrine (Co-artem) samples.
Method development: quantification of GPHF minilab™ system as a low-cost quantitative TLC method
To develop a quantitative minilab system, the number of spot points on the TLC plate used was optimized. Four spot points on the TLC plate were already used for the semi-quantitative minilab system. We have optimized 5 spot points and 6 spot points on the TLC plate (Fig. 1). After 5 spot points on the TLC plate were optimized, 1 mL of albendazole in the stock solution and 2 mL of mebendazole in the stock solution were diluted with 7 mL and 2 mL of glacial acetic acid to prepare 100% label claim (lc) with concentrations of 1.25 mg mL−1 and 1.25 mg mL−1 of albendazole and mebendazole, respectively. Similarly, 1 mL of lumefantrine stock solution was diluted with 2 mL of methanol to prepare 100% lc of lumefantrine with a concentration of 0.8 mg mL−1. The stock standard solution of Artemether was taken without any dilution, a 100% medicine content standard solution of Artemether with a concentration of 2 mg mL−1. Two microliters (2 μL) of each 100% lc standard solution of the drug were spotted on the five-point TLC plate of the minilab. Then, the plates were run for each drug, and images of the developed plates were captured inside a simple carton box using an Android mobile phone (HUAWEI SCL-U31) (Fig. 2). The spot area on the plates was quantified for each drug using justTLC software, and the data were imported to Minitab software (Minitab® 16.2) for further analysis of the developed method. Moreover, factors that affect the spot area of the plate and associated factors affecting the simple carton box operation were optimized by a one-factor approach. These factors encompass the angle between the UV lamp and plate (30, 40, and 50°), the resolution of the camera (3264 × 2448 (8 m), 3264 × 1836 (6 m), and 2448 × 2448 (6 m), the distance between the UV lamp and plate (4 cm, 5 cm, and 6 cm), the timer (at off and 2 s), and background noise (light and dark). The largest possible calculated area of spot obtained from the software for the stated level of the factor was taken as the optimum level of response for that factor.
Typical demonstration for the design of a simple carton box used to capture the image of the TLC plate by using AutoCAD (it is simply constructed from a medicine secondary package)
Citation: Acta Chromatographica 2025; 10.1556/1326.2024.01197
Optimization of number of spot point on TLC plate. Six (6) points Five (5) points
Citation: Acta Chromatographica 2025; 10.1556/1326.2024.01197
Method validation
Validation of the method was performed based on the International Conference on Harmonization guidelines (ICH) Q2 (R1).
Linearity of calibration curve
For each drug product, serial dilutions of 80%, 90%, 100%, 110%, and 120% were prepared separately from their respective stock solutions. Calibration curves were generated for concentration vs. spot area of the plate (captured inside a simple carton box), and the resulting data were subjected to linear regression analysis.
Precision
For intraday precision, six sample solutions (n = 6) were prepared at 100% lc for each drug and analyzed by a developed quantitative minilab system. Similarly, the interday precision was evaluated over 3 consecutive days (n = 18). Concentrations for each drug (albendazole, mebendazole, and arthemether-lumefantrine combinations (Co-artem) were determined, and relative standard deviations (RSDs) were calculated.
Accuracy
The accuracy of the quantitative minilab system was demonstrated by comparing the statistical results (R2, typically) obtained from data that was subjected to linear regression analysis, in which calibration curves for concentration versus spot area of the plate (captured inside the CAMAG UV lamp cabinet) were plotted.
Robustness
A one-factor-at-time (OFAT) approach was applied to evaluate the robustness of the quantification of the GPHF minilab™ system. Five samples were prepared at 100% level content and analyzed by varying different factors that affect the quantification process, such as the angle between the UV lamp and the plate (30, 40, and 50°), camera resolution [in pixels 3264 × 2448 (8 M), 3264 × 1836 (6 M), 2448 × 2448 (6 M)], distance between the UV lamp and the plate (4 cm, 5 cm, and 6 cm), timer (off and 2 s), and background noise (light and dark). Responses to analysis (content, spot area, and relative standard deviation (%)) were evaluated under each condition.
Comparison between quantitative minilab and HPLC assay
The quantitative minilab was carried out in accordance with the spot analysis method developed in our study with JustTLC software, using the method developed for the semi-quantitative evaluation routine method following the instructions in the manual supplied with the GPHF minilab™ kit. For the comparison between quantitative Minilab and HPLC assays, the HPLC assay was performed using the USP 2015 monographic methods for the selected products.
Results and discussions
Method development
The analytical target was to develop a quantitative minilab system that can be used in resource-constrained settings and demonstrate its utility with the analysis of selected drugs (Albendazole, Mebendazole, and arthemether –lumefantrine combinations (Co-artem). Thus, transferring the pre-existing semi-quantitative minilab technique to a quantitative tool is by far the most important way to ensure compliance with regulatory requirements. The quality target method profile includes validation parameters for the method that should comply as per the International Conference on Harmonization (ICH) Q2 (R1) approach. Since the current GPHF minilab™ is a semi-quantitative technique, it has been inefficient as a quality control tool for pharmaceutical products. Thus, as a result, due to the inherent limitations in the technology related to the operator's visual acuity and/or proficiency in executing spot size comparisons on the chromatographic plates, this procedure cannot be used to support a regulatory compliance action [7]. But it is widely used as a screening technology. Furthermore, from a regulatory perspective, quantitative methods are one of the most basic requirements for any developed analytical method that is used to analyze pharmaceutical products of questioned quality circulating in resource-limited settings. Thus, we modified this semi-quantitative technique to a quantitative approach by photographing the developed image on the TLC plate with an android phone (HUAWEI SCL-U31) inside a simple carton box, using the same solvents to prepare the sample and standard solutions, using the same weights as in the semi-quantitative minilab method, and using the same mobile phase and detection mode as routinely used in the current state. This simple carton box holds the mobile phone and rear-facing camera in a fixed position relative to a UV lamp and a TLC plate (Fig. 1). Five (5) spot points on the TLC plate clearly developed, while six spot points were attached to each other (Fig. 2), thus challenging the quantification of spot area. The 100% level content standard solutions with concentrations of 1.25 mg mL−1, 1.25 mg mL−1, and 0.8 mg mL−1 of albendazole, mebendazole, arthemeter, and lumefantrine, respectively, have produced an area of spot (n = 5) for Albendazole (mean = 11,584.2, SD = 98.37, %RSD = 0.8, and SE of mean = 44.00), mebendazole (mean = 11,595, SD = 131.5, %RSD = 1.1, and SE of mean = 58.8), arthemeter (mean = 22,542.2, SD = 450.13, %RSD = 1.9, and SE of mean = 200.1), and Lumefantrine (mean = 17,827.6, SD = 177.5, %RSD = 0.99, and SE of mean = 79.4). Outputs of justTLC software for arthemether-lumefantrine combination (Co-artem®) containing arthemeter/lumefantrine, albendazole, and mebendazole finished pharmaceutical products (FPPs) that show a quantifiable spot of area (n = 5) of 100% level content standard solutions are depicted in Figs 3a–e, and other details of the information are presented in Table 1. The largest possible calculated area of spot for the stated level of factor was taken as the optimum level of response, and the details are depicted in Table 2. No other spot was observed on the developed plate than their corresponding active pharmaceutical ingredients, Albendazole, Mebendazole Arthemeter, and Lumefantrine; thus, the method is a stability-indicating method (Fig. 3e).
Quantification spot of area (n = 5) of 100% lc standard solutions of Arthemeter
Citation: Acta Chromatographica 2025; 10.1556/1326.2024.01197
Quantification spot of area (n = 5) of 100% lc standard solutions of Lumefantrine
Citation: Acta Chromatographica 2025; 10.1556/1326.2024.01197
Quantification spot of area (n = 5) of 100% label content standard solutions of albendazole
Citation: Acta Chromatographica 2025; 10.1556/1326.2024.01197
Quantification spot of area (n = 5) of 100% lc standard solutions of Mebendazole
Citation: Acta Chromatographica 2025; 10.1556/1326.2024.01197
Typical stability-indicating developed TLC plate images for all drugs
Citation: Acta Chromatographica 2025; 10.1556/1326.2024.01197
Quantification spot of area (n = 5) of 100% lc standard solutions of all selected drugs: Albendazole, Mebendazole, Lumefantrine, and Artemether
| s/n | Conc. (%) | Albendazole | Mebendazole | Lumefantrine | Artemether |
| 1 | 100 | 11,621 | 11,696 | 17,923 | 22,995 |
| 2 | 100 | 11,595 | 11,656 | 17,932 | 22,923 |
| 3 | 100 | 11,413 | 11,366 | 17,711 | 21,893 |
| 4 | 100 | 11,658 | 11,612 | 17,575 | 22,315 |
| 5 | 100 | 11,634 | 11,645 | 17,995 | 22,585 |
| Mean(n = 5) | 11,584.2 | 11,595 | 17,827.6 | 22,542.2 | |
| SD | 98.4 | 131.5 | 177.5 | 450.1 | |
| %RSD | 0.8 | 1.1 | 1.0 | 1.9 | |
| SE of mean | 44.0 | 58.8 | 79.4 | 200.1 | |
Optimization of factors that affect the spot area of the plate and associated factor affecting simple carton box
| S.N | Variables | Level | Response (mean area of five-point spot at 100% conc.) | Level optimized |
| 1 | Angle between UV lamp and plate | 30° | 11,695.5 | 40° |
| 40° | 11,762.4 | |||
| 50° | 11,728.6 | |||
| 2 | Resolution | 3264 × 2448 (8 M) | 11,769.6 | 3264 × 2448 |
| 3264 × 1836 (6 M) | 11,689.4 | |||
| 2448 × 2448 (6 M) | 11,754.6 | |||
| 3 | Distance between UV lamp and plate | 4 cm | 11,776.8 | 4 cm |
| 5 cm | 11,636.4 | |||
| 6 cm | 11,676.2 | |||
| 4 | Timer | Off | 11,719.4 | 2 s |
| 2 s | 11,761.9 | |||
| 5 | Background noise | Dark | 11,698.8 | Dark |
| Light | 11,681.2 |
Method validation
Linearity
A linear correlation was obtained between the spot area of the plate and the concentration of selected drugs in the assayed range (80–120% label content). The regression analyses are presented in Table 3 for all drugs, revealing the linearity of the calibration curve, which is by far greater than the visual determination of the result using a qualitative GPHF minilab™ kit.
Calibration curve of all drugs analyzed, in which images are captured inside carton box
| Sr.no | Concentration (%) | Albendazole | Mebendazole | Artemether | Lumefantrine |
| Area of spot | Area of spot | Area of spot | Area of spot | ||
| 1 | 80 | 11,534 | 9,212 | 20,311 | 16,720 |
| 2 | 90 | 11,592 | 10,468 | 21,793 | 17,396 |
| 3 | 100 | 11,794 | 11,835 | 22,036 | 18,215 |
| 4 | 110 | 11,824 | 13,017 | 25,069 | 20,466 |
| 5 | 120 | 11,963 | 14,649 | 26,316 | 21,413 |
| R2 | 0.958 | 0.997 | 0.941 | 0.956 | |
| Equation | y = 10.9x+10651 | y = 134x−1574 | y = 153x+7819 | y = 125x+6386 | |
| Slope ± standard error | 10.9 ± 1.32 | 134 ± 3.982 | 153 ± 22.15 | 125 ± 15.5 | |
| Intercept ± standard error | 10,651 ± 133.2 | −1,574 ± 402.2 | 7,819 ± 2,237 | 6,386 ± 1,564 | |
| F value | 68.3 | 1,134 | 47.6 | 64.7 | |
| P value | 0.004 | 0.000 | 0.006 | 0.004 | |
| N | 5 | 5 | 5 | 5 | |
Precision
The values of %RSD for intraday (n = 6) and inter-day (n = 18) variation are given in Table 4. In both cases, %RSD values were found to be well within the 2% limit, indicating that the current method is repeatable.
Intraday precision of quantitative GPHF minilab™ kit
| S/N | Conc. | Intraday (n = 6) | |||
| Artemether | Lumefantrine | Mebendazole | Albendazole | ||
| 1 | 100 | 21,999 | 17,874 | 11,271 | 11,680 |
| 2 | 100 | 22,005 | 17,361 | 11,716 | 11,678 |
| 3 | 100 | 22,330 | 17,902 | 11,743 | 11,567 |
| 4 | 100 | 22,282 | 18,188 | 11,690 | 11,529 |
| 5 | 100 | 22,578 | 17,939 | 11,357 | 11,512 |
| 6 | 100 | 22,586 | 17,611 | 11,355 | 11,163 |
| Mean | 2,2296.7 | 1,7812.5 | 11,522 | 11,521.5 | |
| SD | 259.9 | 287.4 | 215.78 | 189.92 | |
| %RSD | 1.17 | 1.61 | 1.87 | 1.64 | |
Interday precision of quantitative GPHF minilab™ kit
| S/N | Con | Interday (n = 8) | |||||||||||
| Day1 | Day2 | Day3 | |||||||||||
| ART | LUM | MBZ | ABZ | ART | LUM | MBZ | ABZ | ART | LUM | MBZ | ABZ | ||
| 1 | 100 | 21,370 | 17,926 | 11,684 | 11,575 | 22,038 | 17,613 | 11,734 | 11,338 | 22,133 | 18,169 | 11,483 | 11,855 |
| 2 | 100 | 21,351 | 18,190 | 11,356 | 11,810 | 22,055 | 18,277 | 11,760 | 11,709 | 21,934 | 18,086 | 11,406 | 11,770 |
| 3 | 100 | 22,065 | 17,553 | 11,756 | 11,576 | 22,185 | 18,098 | 11,396 | 11,152 | 22,057 | 18,165 | 11,795 | 11,412 |
| 4 | 100 | 21,638 | 18,187 | 11,385 | 11,125 | 21,805 | 17,910 | 11,624 | 11,548 | 22,334 | 18,154 | 11,876 | 11,650 |
| 5 | 100 | 21,096 | 18,156 | 11,574 | 11,691 | 22,684 | 17,915 | 11,600 | 11,353 | 22,274 | 17,913 | 11,876 | 11,874 |
| 6 | 100 | 21,068 | 18,232 | 11,720 | 11,810 | 22,690 | 17,821 | 11,631 | 11,336 | 22,418 | 17,650 | 11,712 | 11,602 |
| Mean | 21,431 | 18,041 | 11,579 | 11,597 | 22,242 | 17,939 | 11,624 | 11,406 | 22,192 | 18,022 | 11,691 | 11,693 | |
| SD | 373.8 | 262.5 | 173.0 | 254 | 365.2 | 228.3 | 129 | 194.3 | 182.3 | 208.6 | 202.1 | 175.5 | |
| %RSD | 1.70 | 1.46 | 1.50 | 1.80 | 1.64 | 1.30 | 1.11 | 1.70 | 0.81 | 1.15 | 1.72 | 1.50 | |
ART = Arthemether, LUM = Lumefantrine, MBZ = Mebendazole ABZ = Albendazole.
Accuracy
Statistical results obtained from experiments using the analysis of a plate captured inside a simple carton box and CAMAG UV light were compared for demonstration of the accuracy of the method (Table 5). The result demonstrated the accuracy of the developed method; both the simple carton box and CAMAG outputs showed comparable R2.
Accuracy of quantification of the GPHF minilab™ GPHF minilab™ process
| Sr.no | Con | Albendazole | Mebendazole | Artemether | Lumefantrine | ||||
| Cabinet | Carton B | Cabinet | Carton B | Cabinet | Carton B | Cabinet | Carton B | ||
| 1 | 80 | 13,285 | 11,534 | 5,912 | 9,212 | 9,603 | 20,311 | 12,248 | 16,720 |
| 2 | 90 | 13,725 | 11,592 | 6,114 | 10,468 | 9,662 | 21,793 | 12,404 | 17,396 |
| 3 | 100 | 14,021 | 11,794 | 6,366 | 11,835 | 10,264 | 22,036 | 12,522 | 18,215 |
| 4 | 110 | 14,530 | 11,824 | 6,708 | 13,017 | 10,821 | 25,069 | 12,829 | 20,466 |
| 5 | 120 | 14,786 | 11,963 | 6,777 | 14,649 | 10,994 | 26,316 | 13,103 | 21,413 |
| R2 | 0.991 | 0.958 | 0.971 | 0.997 | 0.946 | 0.941 | 0.967 | 0.956 | |
| Equation | y = 38.1x+10,262 | y = 10.9x+10,651 | y = 23.2x+4,051 | y = 134x−1587 | y = 39.4x+6,328 | y = 153x+7,819 | y = 21.4x+10,486 | y = 125x+6,386 | |
| N | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | |
Carton B: the simple constructed box cabinet: the Camag manufactured commercial UV cabinet.
Robustness evaluation of the developed quantitative GPHF minilab™ process
A one-factor-at-a-time approach was applied to evaluate the robustness of the quantification process of the GPHF minilab™ technique. Five factors—the angle between the UV lamp and plate, resolution of the camera on an Android mobile phone, distance between the UV lamp and plate, timer, and background noise—affect the illumination of the UV lamp, and thus the yield of quantification. The maximum quantified spot area of the plate was the target of the optimization process. The percentage of RSD of robustness testing under different altered conditions is given in Table 6, revealing that the current method is robust, and the assay for the reference drug was within the stated label claim (90–110%). This shows that the method was appropriate for the determination of the assay (Table 6). It demonstrates important implications for the deployment of the newly constructed quantitative GPHF minilab™ system.
Robustness evaluation via one factor at a time principle
| S.N | Variables | Level | Response (mean area of five point spot at 100% conc.) | Amount recovered (100%) | %RSD | Level optimized |
| 1 | Angle between UV lamp and plate | 30° | 11695.5 | 95.8% | 0.28 | 40° |
| 40° | 11762.4 | 101.9% | ||||
| 50° | 11728.6 | 98.86% | ||||
| 2 | Resolution | 3264 × 2448 (8 M) | 11769.6 | 102.6% | 0.36 | 3264 × 2448 |
| 3264 × 1836 (6 M) | 11689.4 | 95.2% | ||||
| 2448 × 2448 (6 M) | 11754.6 | 101.2% | ||||
| 3 | Distance between UV lamp and plate | 4 cm | 11776.8 | 103.2% | 0.66 | 4 cm |
| 5 cm | 11636.4 | 90.4% | ||||
| 6 cm | 11676.2 | 94.1% | ||||
| 4 | Timer | Off | 11719.4 | 98.1% | 0.26 | 2 s |
| 2 s | 11761.9 | 101.9% | ||||
| 5 | Background noise | Dark | 11698.8 | 96.1% | 0.11 | Dark |
| Light | 11681.2 | 94.5 |
Sample analysis using quantitative GPHF minilab™ kit
The developed method was successfully applied to analyze the contents of the tested-finished pharmaceutical products. The assay results of marketed samples of albendazole, mebendazole, and arthemether-lumefantrine (Co-artem) obtained in Addis Ababa are presented in Table 7. The drug content varied from 93.2% to 100.1% label claim, within the 90–180% label claim specifications recommended in the monograph. Thus, transferring the semi-quantitative minilab technique to a quantitative tool is attempted, which is by far most helpful tool in ensuring compliance with regulatory requirements.
Content of active pharmaceutical ingredient determination of albendazole, mebendazole and artemether-lumefantrine samples (n = 5 for each)
| Drug | Brands/type | Drug content (%) |
| Albendazole tablets | Generic 1 | 99.4% |
| Generic 2 | 95.6% | |
| Mebendazole tablets | Generic 1 | 99.6% |
| Generic 2 | 100.1% | |
| Co-artem tablets | Lumefantrine | 93.7% |
| Artemether | 93.2% |
Comparison between HPLC and quantitative Mini-lab
The comparison between the developed method and the gold standard HPLC method was conducted for 29 albendazole tablets and 6 artemether-lumefantrine combination tablets. As shown in Figs 4–6, the newly developed method results were compared with those of the gold standard HPLC assay method. The percent variation between the two methods is calculated by the difference between the mean percentage of the new method values and the mean percentage of the HPLC results obtained divided by the mean percentage of the HPLC results. The values that were found are on average −8.62% for albendazole, −3.79% for artemether, and −4.52% for lumefantrine samples. This indicates that the newly developed quantitative minilab method can work with less than a 10 percent deviation on average from the gold standard HPLC monograph method.
Assay result comparison of newly developed quantitative minilab method with HPLC method for albendazole tablet samples
Citation: Acta Chromatographica 2025; 10.1556/1326.2024.01197
Assay result comparison of newly developed quantitative minilab method with HPLC method for artemether in Co-artem tablet samples
Citation: Acta Chromatographica 2025; 10.1556/1326.2024.01197
Assay result comparison of newly developed quantitative minilab method with HPLC method for lumefantrine in co-artem tablet samples
Citation: Acta Chromatographica 2025; 10.1556/1326.2024.01197
Conclusions
A quantitative minilab system was developed and validated for finished pharmaceutical products of Albendazole, Mebendazole, as well as Artemether and Lumefantrine containing a fixed-dose combination of products. This finding revealed that a simple and low-cost quantitative minilab technique was successfully developed that is intended to be used in resource-limited settings. The method was validated for an analytical target profile encompassing linearity, precision, accuracy, and robustness. Moreover, the developed method was successfully applied to analyze the contents of sample-finished pharmaceutical products. The quality target method profile was validated by the International Conference on Harmonization (ICH) Q2 (R1) approach. Thus, in contrast to a semi-quantitative minilab system, this procedure can be used to support a regulatory compliance action. Therefore, it can be used by medicine regulatory agencies to support regular surveillance and rapid screening of drug quality at various levels of the drug supply chain in low-income countries.
Ethics approval and consent to participate
Jimma University's, School of Pharmacy Ethical Review Board approved this study. A letter of permission and ethical clearance was received from the School of Pharmacy, Addis Ababa University also under protocol School of pharmacy, AAU, Ethical review Board (ERB) Numbered ERB/SOP/116/07/2018 dated July 9/2018 to commence the study.
Consent for publication
Not applicable.
Availability of data and materials
The supporting documents for this study can be available from the corresponding author upon reasonable request.
Competing interests
The authors declare that they have no competing interests.
Funding
Not applicable.
Authors' contributions
GH, SS, WB and AA designed, performed experiments, extracted, analysed and interpreted the data. GH prepared the first draft manuscript. WB and AA wrote the second draft and reviewed it. All authors read and approved the final manuscript.
Acknowledgements
This study was supported by Ministry of innovation and technology, Government of Ethiopia grant to AA and Jimma University MSc studies support to GH.
Abbreviations
| FPP | Finished Pharmaceutical Product |
| GPHF | Global Pharma Health Fund |
| HPTLC | High Performance Thin Layer Chromatography |
| LMIC | Low- and middle-income countries |
| TLC | Thin layer chromatography |
| SF | Substandard and falsified |
| USP | United State Pharmacopeia |
| UV | Ultra-Violet |
| WHO | World Health Organization |
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