Quality assessment of common anti-malarial medicines marketed in Gambella, National Regional State, South Western-Ethiopia.

BACKGROUND: Over the past years, there has been a growing concern that a considerable amount of anti-malarial supply in the underdeveloped world particularly in the private sector, is of poor quality. The World Health Organization (WHO) has received about 1500 reports that mentions instances of substandard and falsified products since 2013. The majority of the reports concerned antibiotics and anti-malarials. The majority of reports (42%) originate from the WHO African region. OBJECTIVE: This study intends to assess the quality of the most widely used anti-malarial medications [artemether-lumefantrine tablets, chloroquine phosphate tablets, primaquine phosphate tablets, artesunate, and artemether injections] in Gambella, South-West, Ethiopia. METHODS: A total of 52 samples were collected on June 2022 from Gambella National Regional State, Ethiopia. Half of the districts (six) located in the four zones of the region were chosen using simple random sampling technique. All drug retail outlets available in the selected districts (locally known as woredas) were included. The samples were subjected to visual inspection with a tool adopted from the joint WHO/FIP/ USP checklist. The pharmacopeial tests for identification, uniformity of dosage forms, assay, thickness, diameter, hardness, friability, disintegration test, dissolution, and sterility tests were carried out according to the USP 44-NF 39 and International Pharmacopoeia 11th edition, 2022 monographs. RESULTS AND DISCUSSION: Only 25% of the samples were registered on the Ethiopian Food and Drug Authority (EFDA's) electronic regulatory/ registration system (ERIS). Besides, 88.8% of artemether injection products were presented in clear glass ampoules. This might expose the products to photochemical degradation that leads to in loss of anti-plasmodial activity. In addition, 50% of the artemether products assessed were not bioequivalent with the comparator product in the in vitro dissolution comparison tests. Overall, the study findings reveal a high prevalence (58.3%) of substandard anti-malarial drugs in the region. The stated percent of the samples had failed in one or more of the quality test parameters assessed in this study. CONCLUSION: The study findings reveal a high prevalence (58.3%) of substandard anti-malarial drugs in the region. Only a quarter were registered and 38% of the unregistered products failed the quality tests. Hence, the national, regional medicine regulatory bodies and other stake holders should perform the required roles to circumvent presence of Substandard and Falsified (SF) anti-malarial drugs in the study sites.

Black tea kombucha: Physicochemical, microbiological and comprehensive phenolic profile changes during fermentation, and antimalarial activity.

This study shows the changes in physicochemical and microbiological composition, and in the phenolic profile of black tea kombucha during fermentation. In addition, the antimalarial potential of the kombucha was evaluated. Ultra-performance liquid chromatography-mass spectrometry multiplex analysis (UPLC-MSE) results revealed a 1.7 log2 fold-change increase in phenolics with the fermentation time, with emphasis on the increase of phenolic acids (0.3 log2 fold-change). Over time there was degradation of flavonoids such as nepetin, hesperidin and catechin 5-O-gallate, to the detriment of the increase in phenolic acids such as gallic acid and cinnamic acid. In addition, black tea kombucha presented antiplasmodic activity against the 3D7 (sensitive chloroquine) and W2 (resistant to chloroquine) strains. Therefore, important changes in the black tea kombucha phenolic profile take place during fermentation, which may help in the development of kombuchas with higher bioactive potential and contribute to a better understanding of the kombucha fermentation process.

Quadruple isotope dilution gas chromatography-mass spectrometry after simultaneous derivatization and spraying based fine droplet formation liquid phase microextraction method for the accurate and sensitive quantification of chloroquine phosphate in human serum, urine and saliva samples at trace levels.

This study presents an accurate and precise analytical strategy for the determination of chloroquine phosphate at trace levels in human body fluids (urine, serum, and saliva). Simultaneous derivatization-spraying based fine droplet formation-liquid phase microextraction (SD-SFDF-LPME) method was used to derivatize and preconcentrate the analyte prior to gas chromatography-mass spectrometry (GC-MS) measurements. Acetic anhydride was employed as derivatizing agent in this study. After optimizing the SD-SFDF-LPME method, the limit of detection (LOD) and limit of quantitation (LOQ) were found to be 0.16 and 0.53 mg/kg, respectively. Quadruple isotope dilution (ID4) was coupled to the SD-SFDF-LPME method in order to alleviate matrix effects and promote accuracy/precision of the method. Chloroquine acetamide-d3 was firstly synthesized in our research laboratory and used as the isotopic analogue of the analyte in the ID4 experiments. Superior percent recovery results (99.4% - 101.0%) with low standard deviation values were obtained for the spiked samples. This validated the developed SD-SFDF-LPME-ID4-GC-MS method as highly accurate and precise for the determination of chloroquine phosphate at trace levels. In addition, the isotopic analogue of the analyte was obtained via the acetamide derivative of the analyte, which is an alternative to obtain isotopic analogues of organic compounds that are not accessible or commercially available.

HPLC methods for choloroquine determination in biological samples and pharmaceutical products.

OBJECTIVE: Review and assess pharmaceutical and clinical characteristics of chloroquine including high-performance liquid chromatography (HPLC)-based methods used to quantify the drug in pharmaceutical products and biological samples. EVIDENCE ACQUISITION: A literature review was undertaken on the PubMed, Science Direct, and Scielo databases using the following keywords related to the investigated subject: 'chloroquine', 'analytical methods', and 'HPLC'. RESULTS: For more than seven decades, chloroquine has been used to treat malaria and some autoimmune diseases, such as lupus erythematosus and rheumatoid arthritis. There is growing interest in chloroquine as a therapeutic alternative in the treatment of HIV, Q fever, Whipple's disease, fungal, Zika, Chikungunya infections, Sjogren's syndrome, porphyria, chronic ulcerative stomatitis, polymorphic light eruption, and different types of cancer. HPLC coupled to UV detectors is the most employed method to quantify chloroquine in pharmaceutical products and biological samples. The main chromatographic conditions used to identify and quantify chloroquine from tablets and injections, degradation products, and metabolites are presented and discussed. CONCLUSION: Research findings reported in this article may facilitate the repositioning, quality control, and biological monitoring of chloroquine in modern pharmaceutical dosage forms and treatments.

Composition analysis of falsified chloroquine phosphate samples seized during the COVID-19 pandemic.

The proliferation of falsified medicines can cause serious public health issues, particularly in the context of a global pandemic such as the actual COVID-19 pandemic. Our study involved eight chloroquine phosphate medicines seized in Cameroon, Democratic Republic of Congo and Niger during March and May 2020. These suspect samples were first analyzed in a screening phase using field tools such as handheld Raman spectroscopy (TruScan) and then in a confirmation phase using laboratory tools such as hyperspectral Raman imaging and High Performance Liquid Chromatography (HPLC). The results confirmed the falsified nature of the samples, highlighting the presence of metronidazole at low dose in four samples (16.6, 15.2, 15.2 and 14.5 mg/tab), too low levels of chloroquine in two samples (2.4 and 20.2 mg/tab), and substitution of chloroquine phosphate by paracetamol in one sample (255.7 mg/tab). The results also confirmed that four samples had been adulterated with paracetamol in trace amounts and two of them presented traces of chloramphenicol.

Liquid Chromatographic Methods for COVID-19 Drugs, Hydroxychloroquine and Chloroquine.

COVID-19 has been a threat throughout the world since December 2019. In attempts to discover an urgent treatment regime for COVID-19, hydroxychloroquine (HCQ) and chloroquine (CQ) have been on solidarity clinical trial. However, many countries have pulled HCQ and CQ from their COVID-19 treatment regimens recently, some countries still continue using them for patients who have previously started HCQ and CQ and they may complete their course under the supervision of a doctor. HCQ and CQ are 4-aminoquinoline drugs and it is safe to use them for autoimmune diseases, rheumatoid arthritis, systemic lupus erythematosus and malaria as well. Determination of CQ, HCQ and their metabolites in biologic fluids and in pharmaceuticals has great importance, especially for pharmacokinetics, pharmacodynamics and epidemiological studies. In this review, liquid chromatographic methods developed in the last 10 years were summarized focusing on sample preparation and detection methods for HCQ and CQ determination in biological fluids and pharmaceutical preparations. It is hoped that this article could be helpful to facilitate the use of these drugs in clinical trials or drug research studies as it provides comprehensive information on the reported analytical methods.

Identification of Falsified Chloroquine Tablets in Africa at the Time of the COVID-19 Pandemic.

Reports that chloroquine and hydroxychloroquine may be effective against COVID-19 have received worldwide attention, increasing the risk of the introduction of falsified versions of these medicines. Five different types of falsified chloroquine tablets were discovered between March 31, 2020 and April 4, 2020, in Cameroon and the Democratic Republic of Congo by locally conducted thin layer chromatographic analysis. Subsequent investigation by liquid chromatography and mass spectrometry in Germany proved the absence of detectable amounts of chloroquine and the presence of undeclared active pharmaceutical ingredients, that is, paracetamol and metronidazole, in four of the samples. The fifth sample contained chloroquine, but only 22% of the declared amount. Such products represent a serious risk to patients. Their occurrence exemplifies that once medicines or vaccines against COVID-19 may be developed, falsified products will enter the market immediately, especially in low- and middle-income countries (LMICs). Timely preparations for the detection of such products are required, including the establishment of appropriate screening technologies in LMICs.

A metal organic framework-functionalized monolithic column for enantioseparation of six basic chiral drugs by capillary electrochromatography.

Poly(glycidyl methacrylate)-co-(ethylene dimethacrylate) [poly(GMA-co-EDMA)] monoliths were used as a support to grow a zeolitic imidazolate framework-8 (ZIF-8) via layer-by-layer self-assembly. Pepsin, acting as as chiral selector, was covalently linked to the surface of the amino-modified ZIF-8 through the Schiff base method. The material was characterized by scanning electron microscopy, thermogravimetric analysis, X-ray diffraction, Fourier transform infrared spectroscopy and elemental analysis. The pepsin-ZIF-8-poly(GMA-co-EDMA) column was utilized to the enantioseparation of the racemic forms of hydroxychloroquine (HCQ), chloroquine (CHQ), hydroxyzine (HXY), nefopam (NEF), clenbuterol (CLE) and amlodipine (AML). In comparison with a pepsin-poly(GMA-co-EDMA) monolithic column (without self-assembled ZIF-8 nanoparticles), the resolution is strongly enhanced (HCQ: 0.34 → 2.50; CHQ: 0.45 → 1.97; HXY: 0.39 → 1.43; NEF: 0.27 → 0.81; CLE: 0 → 0.81; AML: 0.16 → 0.72). Effects of self-assembly layers of ZIF-8, pepsin concentration, buffer pH values and applied voltage were investigated with hydroxychloroquine as the model analyte. The reproducibility of run-to-run, day-to-day and column-to-column were explored, and found to be satisfactory. Graphical abstractSchematic representation of capillary electrochromatography (CEC) systems with a pepsin-zeolitic imidazolate framework-8 (ZIF-8) modified poly(glycidyl methacrylate)-co-(ethylene dimethacrylate) [poly(GMA-co-EDMA)] monolithic column as stationary phases for separation of basic racemic drugs. ZIF-8 modified column was prepared via layer-by-layer self-assembly.

In vivo efficacy of top five surveyed Ghanaian herbal anti-malarial products.

BACKGROUND: Anti-malarial herbal preparations (HPs) continue to enjoy high patronage in Ghana despite reports that the artemisinin-based combination therapy (ACT), the recommended first choice for treatment of uncomplicated malaria in the country, remains efficacious. A major issue with the use of these preparations is inadequate or unreliable data on their efficacy and quality. An assessment of the potency and quality of the most popular commercial anti-malarial HPs in Ghana was, therefore, carried out. The outcome of this investigation is herein discussed preceded by a short literature review of herbal medicines in Ghana. METHODS: Using a questionnaire survey of 344 individuals in parts of Ghana, five of the most frequently used HPs were identified and selected for test of their efficacy and quality. The effect of the selected compounds on Plasmodium berghei in vivo was assessed using standard methods. RESULTS: All five tested HPs (HP-A, HP-B, HP-C, HP-D and HP-E) showed chemo-suppressive activity against P. berghei in vivo. However the degree of parasites inhibition is significantly lower compared to the WHO-recommended artemether-lumefantrine combination (p < 0.05, 99.9% chemosuppression/activity, 28 days survival). Using the Solomon Saker's Test, two of the preparations were found to contain chloroquine or compounds with chemical properties like that of chloroquine. CONCLUSION: Popular anti-malarial HPs used in southern Ghana were found to have chemo-suppressive properties. Intentional addition of chloroquine or SCs to these preparations in order to enhance their effectiveness has serious public health concerns as it may induce cross resistance to amodiaquine, one of the partner drugs in the recommended ACT for use in Ghana.

Feasibility of amlodipine besylate, chloroquine phosphate, dapsone, phenytoin, pyridoxine hydrochloride, sulfadiazine, sulfasalazine, tetracycline hydrochloride, trimethoprim and zonisamide in SyrSpend(®) SF PH4 oral suspensions.

The objective of this study was to evaluate the feasibility of 10 commonly used active pharmaceutical ingredients (APIs) compounded in oral suspensions using an internationally used suspending vehicle (SyrSpend(®) SF PH4 liquid): (i) amlodipine, (as besylate) 1.0mg/mL; (ii) chloroquine phosphate,15.0 mg/mL; (iii) dapsone, 2.0 mg/mL; (iv) phenytoin, 15.0 mg/mL; (v) pyridoxine hydrochloride, 50.0 mg/mL; (vi) sulfadiazine, 100.0 mg/mL; (vii) sulfasalazine, 100.0 mg/mL; (viii) tetracycline hydrochloride, 25.0 mg/mL; (ix) trimethoprim, 10.0 mg/mL; and (x) zonisamide, 10.0 mg/mL. All suspensions were stored both at controlled refrigeration (2-8 °C) and controlled room temperature (20-25 °C). Feasibility was assessed by measuring the percent recovery at varying time points throughout a 90-day period. API quantification was performed by high-performance liquid chromatography (HPLC-UV), via a stability-indicating method. Given the percentage of recovery of the APIs within the suspensions, the expiration date of the final products (API+vehicle) was at least 90 days for all suspensions with regard to both the controlled temperatures. This suggests that the vehicle is stable for compounding APIs from different pharmacological classes.

Simultaneous quantitation of chloroquine and primaquine by UPLC-DAD and comparison with a HPLC-DAD method.

BACKGROUND: Chloroquine and primaquine are the first-line treatment recommended by World Health Organization for malaria caused by Plasmodium vivax. Since the problem of counterfeit or substandard anti-malarials is well established all over the world, the development of rapid and reliable methods for quality control analysis of these drugs is essential. Thus, the aim of this study was to develop and validate a novel UPLC-DAD method for simultaneously quantifying chloroquine and primaquine in tablet formulations. METHODS: The UPLC separation was carried out using a Hypersil C18 column (50 × 2.1 mm id; 1.9 µm particle size) and a mobile phase composed of acetonitrile (A) and 0.1% aqueous triethylamine, pH 3.0 adjusted with phosphoric acid (B), at a flow rate 0.6 mL/min. Gradient elution was employed. UV detection was performed at 260 nm. UPLC method was fully validated and the results were compared to a conventional HPLC-DAD method for the analysis of chloroquine and primaquine in tablet formulations. RESULTS: UPLC method was shown to be linear (r2 > 0.99), precise (CV < 2.0%), accurate (recovery rates from 98.11 to 99.83%), specific, and robust. No significant differences were observed between the chloroquine and primaquine contents obtained by UPLC and HPLC methods. However, UPLC method promoted faster analyses, better chromatographic performance and lower solvent consumption. CONCLUSIONS: The developed UPLC method was shown to be a rapid and suitable technique to quantify chloroquine and primaquine in pharmaceutical preparations and may be successfully employed for quality control analysis.

Quality of antimalarial drugs and antibiotics in Papua New Guinea: a survey of the health facility supply chain.

BACKGROUND: Poor-quality life-saving medicines are a major public health threat, particularly in settings with a weak regulatory environment. Insufficient amounts of active pharmaceutical ingredients (API) endanger patient safety and may contribute to the development of drug resistance. In the case of malaria, concerns relate to implications for the efficacy of artemisinin-based combination therapies (ACT). In Papua New Guinea (PNG), Plasmodium falciparum and P. vivax are both endemic and health facilities are the main source of treatment. ACT has been introduced as first-line treatment but other drugs, such as primaquine for the treatment of P. vivax hypnozoites, are widely available. This study investigated the quality of antimalarial drugs and selected antibiotics at all levels of the health facility supply chain in PNG. METHODS AND FINDINGS: Medicines were obtained from randomly sampled health facilities and selected warehouses and hospitals across PNG and analysed for API content using validated high performance liquid chromatography (HPLC). Of 360 tablet/capsule samples from 60 providers, 9.7% (95% CI 6.9, 13.3) contained less, and 0.6% more, API than pharmacopoeial reference ranges, including 29/37 (78.4%) primaquine, 3/70 (4.3%) amodiaquine, and one sample each of quinine, artemether, sulphadoxine-pyrimethamine and amoxicillin. According to the package label, 86.5% of poor-quality samples originated from India. Poor-quality medicines were found in 48.3% of providers at all levels of the supply chain. Drug quality was unrelated to storage conditions. CONCLUSIONS: This study documents the presence of poor-quality medicines, particularly primaquine, throughout PNG. Primaquine is the only available transmission-blocking antimalarial, likely to become important to prevent the spread of artemisinin-resistant P. falciparum and eliminating P. vivax hypnozoites. The availability of poor-quality medicines reflects the lack of adequate quality control and regulatory mechanisms. Measures to stop the availability of poor-quality medicines should include limiting procurement to WHO prequalified products and implementing routine quality testing.

Anti-malarial activity and HS-SPME-GC-MS chemical profiling of Plinia cerrocampanensis leaf essential oil.

BACKGROUND: Plinia cerrocampanensis is an endemic plant of Panama. The leaf essential oil of this plant has shown antibacterial activity. However, anti-malarial activity and chemical profiling by HS-SPME-GC-MS of this essential oil have not been reported before. METHODS: Anti-malarial activity of the essential oil (EO) was evaluated in vitro against chloroquine-sensitive HB3 and chloroquine-resistant W2 strains of Plasmodium falciparum. Synergistic effect of chloroquine and the EO on parasite growth was evaluated by calculating the combination index. A methodology involving headspace solid phase microextraction and gas chromatography-mass spectrometry (HS-SPME-GC-MS) was developed to investigate the composition of Plinia cerrocampanensis EO. RESULTS: Plinia cerrocampanensis EO showed a high anti-malarial activity and a synergistic interaction with chloroquine. The Plinia cerrocampanensis EO inhibited P. falciparum growth in vitro at an IC50 of 7.3 µg/mL. Chloroquine together with the EO decreased the IC50 of chloroquine from 0.1 µg/mL to 0.05 µg/mL, and of the EO from 7.3 µg/mL to 1.1 µg/mL. The measured combination index was 0.58, which clearly indicates that the EO acts synergistically with chloroquine. Since the EO maintained its inhibitory activity on the chloroquine-sensitive strain of the parasite, it could be acting by a different mechanism of action than chloroquine. The best HS-SPME-GC-MS analytical conditions were obtained when the temperature of extraction was 49°C, incubation time 14 min, and the time of extraction 10 min. This method allowed for the identification of 53 volatile constituents in the EO, including new compounds not reported earlier. CONCLUSIONS: The anti-malarial activity exhibited by the Plinia cerrocampanensis EO may lend support for its possible use as an alternative for anti-malarial therapy.

Chloroquine-N-oxide, a major oxidative degradation product of chloroquine: identification, synthesis and characterization.

Chloroquine (CQ) (1) which has endured as one of the most powerful antimalarial drugs was subjected to oxidative stress conditions and the degradation profile was studied. The oxidative stress condition of CQ furnished one major degradation product along with other minor degradation products. The unknown major degradation product was identified in HPLC and pure impurity was isolated using column chromatography. The structure of this major product was elucidated using UV, FT-IR, (1)H NMR, (13)C NMR, 2D NMR (HSQC) and mass spectral data. Based on the results obtained from the different spectroscopic studies, it was confirmed that the N-oxide was formed at the tertiary amine nitrogen instead of the pyridine nitrogen. Subsequently, an efficient and simple synthetic approach was developed for the synthesis of chloroquine-N-oxide using a work-up procedure that does not require chromatography techniques for further purification. It was observed that the spectral data of the isolated degradation product coincided appropriately with the synthesized product spectral data.

Analysis of chloroquine and metabolites directly from whole-body animal tissue sections by liquid extraction surface analysis (LESA) and tandem mass spectrometry.

The rapid and direct analysis of the amount and spatial distribution of exogenous chloroquine (CHQ) and CHQ metabolites from tissue sections by liquid extraction surface sampling analysis coupled with tandem mass spectrometry (LESA-MS/MS) was demonstrated. LESA-MS/MS results compared well with previously published CHQ quantification data collected by organ excision, extraction and fluorescent detection. The ability to directly sample and analyze spatially resolved exogenous molecules from tissue sections with minimal sample preparation and analytical method development has the potential to facilitate the assessment of target tissue penetration of pharmaceutical compounds, to establish pharmacokinetic/pharmacodynamic relationships, and to complement established pharmacokinetic methods used in the drug discovery process during tissue distribution assessment.

Development and validation of a capillary electrophoresis assay for the determination of the stereoisomeric purity of chloroquine enantiomers.

A stereoselective CE assay for the determination of the enantiomeric purity of (R)-(-)-chloroquine and (S)-(+)-chloroquine was developed and validated. The separations were performed in a 50.2/40 cm uncoated fused silica capillary at 20°C using a 100 mM sodium phosphate buffer, pH 2.5, containing 30 mg/mL sulfobutylether(VII)-ß-cyclodextrin as background electrolyte operated at an applied voltage of -25 kV and 20°C. The detection wavelength was 225 nm. Carbamazepine was used as internal standard. The assay was validated in the range of 0.05-1.0% for the respective minor chloroquine enantiomer based on a concentration of 3 mg/mL of the major enantiomer, either (R)-(-)-chloroquine or (S)-(+)-chloroquine. The method was applied to analyze the stereoisomeric purity of synthetic samples of the chloroquine enantiomers.

Development of ELISA-based methods to measure the anti-malarial drug chloroquine in plasma and in pharmaceutical formulations.

BACKGROUND: In Central and South America and Eastern and Southern Africa, Plasmodium vivax infections accounts for 71-81% and 5% of malaria cases, respectively. In these areas, chloroquine (CQ) remains the treatment of choice for P. vivax malaria. In addition, CQ has recently proven to be an effective HIV-1 therapeutic agent. There is a dire need to continue monitoring quality of CQ as there is a major influx of substandard and fake formulations into malaria-endemic countries. The use of fake/substandard drugs will result in sub-therapeutic levels endangering the patient and possibly select for parasite resistance. The aim of this study was to develop an inexpensive, simple antibody-based ELISA to measure CQ concentrations in tablets and in plasma. METHODS: A monoclonal antibody (MAb) that reacts with the N-side chain of the CQ molecule was prepared by use of a CQ analogue. A specific and reliable ELISA for detection of CQ was developed. The developed assay was validated by measuring CQ in tablets sold in Denmark, India and Sudan. Furthermore, kinetics of CQ concentrations in plasma of four volunteers, who ingested two tablets of Malarex® containing, 250 mg CQ base, were measured before drug intake, three hours later and thereafter at days 1, 3, 7, 14, 21 and 28. The same plasma samples were simultaneously measured by high performance liquid chromatography (HPLC). RESULTS: The ELISA proved an easy-to-handle and very sensitive tool for the detection of CQ with a lower limit of detection at 3.9 ng/ml. ELISA levels of CQ in plasma showed high agreement with the levels obtained by HPLC (r = 0.98). The specificity in the negative control group was 100%. CONCLUSION: The developed ELISA can be used for quality screening of CQ in pharmaceutical formulations and for drug monitoring in malaria and in other infectious diseases, such as HIV, where CQ proved to be an effective therapeutic agent. The methodology has been exploited to develop monoclonal antibodies for the drugs used in artemisinin-based combination therapy (ACT).

Distribution of chloroquine in ocular tissue of pigmented rat using matrix-assisted laser desorption/ionization imaging quadrupole time-of-flight tandem mass spectrometry.

In pharmacology and toxicology, localization of the distribution of a drug molecule in its target tissue provides very important in vivo biological information. Traditionally, this has been examined using autoradiography (ARG). However, there are significant limitations in this application. One is the synthesis and use of radiolabeled compounds, the other is that the image generated expresses an undifferentiated mixture of the parent drug and/or its metabolites. The objective of the study was to define the specific distribution of the parent drug in rat ocular tissue containing melanin (e.g. the retina) using non-labeled chloroquine by MALDI Imaging tandem mass spectrometry (MS/MS). After single oral administration (at 20 mg/kg) of chloroquine, sections (10 µm) of rat eye tissue were prepared at 24 h. The MS system used was a quadrupole time-of flight (Q-TOF) tandem mass spectrometer (MALDI Synapt™, Waters, Milford, MA, USA). Tissue sections were sprayed with CHCA (α-cyano-4-hydroxycinnamic acid, 5 mg/mL) in 80% acetonitrile (ACN) containing 5% formic acid (FA) using either a manual sprayer (airbrush) or an automated sprayer (TM-Sprayer™, HTX Technologies, Carrboro, NC, USA). Chloroquine was readily detected in the MS/MS mode by monitoring one of its major fragment ions (m/z 247.10) and imaged through the rat eye tissue. The image of the specific distribution within the retina in the rat eye tissue was confirmed, and found to be similar to autoradiograms after oral administration of (14)C-chloroquine reported previously.

HPLC with ultraviolet detection for the determination of chloroquine and desethylchloroquine in whole blood and finger-prick capillary blood dried on filter paper.

A simple, sensitive, selective and reproducible method based on liquid chromatography was developed for the determination of chloroquine (CQ) and its active plasma metabolite desethylchloroquine (DECQ) in finger-pricked capillary blood spot onto filter paper (DBS) and whole blood samples. Both were separated from the internal standard quinine on a reversed phase C18 column, with the mobile phase consisting of a mixture of 1% diethylamine, acetonitrile and methanol (20:55:25, v:v:v) running at a flow rate of 1.0ml/min. Retention times of QN, DECQ and CQ were 4.5, 5.7 and 6.4min, respectively. Ultraviolet detection was at the wavelength 256nm. Sample preparation was done by extraction with hexane and tert-butyl methyl ether (1:1, v:v). Good precision and accuracy were obtained for both within-day repeatability and day-to-day reproducibility. Limit of quantification (LOQ) for both CQ and DECQ was accepted as 50ng/ml using 80µl DBS sample and 25ng/ml using 150µl whole blood sample. The mean recoveries for CQ, DECQ and internal standard for both whole blood and DBS were between 74 and 87%. The method was successfully applied for a pharmacokinetic study of CQ and DECQ in patients with Plasmodium vivax. Excellence correlation (r=0.997) was observed between the analysis of both CQ and DECQ in paired whole blood and DBS samples.