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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.

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., Competing Interests: Declaration of Competing Interest The authors report no declarations of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

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.

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 (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., (Copyright © 2013 Elsevier B.V. All rights reserved.)

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).

Capillary electrophoresis (CE) methods for chiral resolution of five antimalarial drugs (primaquine, tafenoquine, mefloquine, chloroquine and quinacrine) were developed by using a wide selection of neutral and anionic cyclodextrin (CD) derivatives. The use of sulfobutyl-β-CD and carboxymethyl-β-CD (CMBCD) resulted in good resolution of quinacrine and tafenoquine, respectively. New results are presented for resolutions of chloroquine and mefloquine. Application of carboxyalkyl- and sulfobutyl-CD derivatives provided improved resolution for primaquine. The impurity in primaquine sample detected by CE was identified as quinocide by MS and NMR. CMBCD provided not only the best separation of primaquine from quinocide but also the simultaneous complete resolution of both compounds., (Copyright © 2010 Elsevier B.V. All rights reserved.)

The focus of this study is identification and characterization of major unknown impurities in chloroquine (CQ) and hydroxychloroquine (HCQ) bulk drug samples using liquid chromatography/ion trap mass spectrometry (LC/IT/MS) and liquid chromatography/time of flight mass spectrometry (LC/TOF/MS). The newly developed LC/MS method was employed for the analysis of both the drugs. The analysis revealed the presence of two impurities in each of the drugs. The impurities are designated as CQ-I, CQ-II (for chloroquine); HCQ-I and HCQ-II (for hydroxychloroquine). Three of the impurities, CQ-II, HCQ-I and HCQ-II were unknown have not been reported previously. Accurate masses of the impurities were determined by using Q-TOF mass spectrometer and fragmentation behavior was studied by an ion trap mass spectrometer. Based on the spectrometric data and synthetic specifics the structures of CQ-II, HCQ-I and HCQ-II were proposed as 1,4 pentanediamine, N(4)(7-chloro-4-quinolinyl), N(4)-chloromethyl, N(4)-ethylamine; 2-(4-(7-chloroquinolin-4-ylamino) pentylamino) ethanol and [[4-[(7-chloro-4-quinolyl) amino] N-pentyl] N-chloromethyl-N-ethylamino] ethanol respectively. The impurities were isolated by semi-preparative HPLC and structures were confirmed by NMR spectroscopy. The formation and through characterization of known CQ-I impurity is also discussed.

A simple, sensitive, and inexpensive single drop liquid-liquid-liquid microextraction combined with isocratic RP-HPLC and UV detection was developed for the determination of anti-malaria drug, chloroquine. The target compound was extracted from alkaline aqueous sample solution (adjusted to 0.5 mol/L sodium hydroxide) through a thin layer of organic solvent membrane and back-extracted to an acidic acceptor drop (adjusted to 0.02 mol/L phosphoric acid) suspended on the tip of a 25 microL HPLC syringe in the organic layer. This syringe was also used for direct injection after extraction. The linear range was 1-200 microg/L. The LOD and LOQ were 0.3 and 1.0 microg/L, respectively. Intra-and inter-day precisions were less than 2.0 and 2.3%, respectively. The real samples were successfully analyzed using the proposed method. The recoveries of spiked samples were more than 94.6%.

Background & Objectives: The aim of the present study is to investigate the physicochemical equivalence of seven brands of tablets containing chloroquine phosphate, an antimalarial purchased from different retail pharmacy outlets., Methods: The quality and physicochemical equivalence of seven different brands of chloroquine phosphate tablets were assessed. The assessment included the evaluation of uniformity of weight, friability, crushing strength, disintegration and dissolution tests as well as chemical assay of the tablets., Results: All the seven brands of the tablets passed the British Pharmacopoeia (BP) standards for uniformity of weight, disintegration and crushing strength. One of seven brands failed the friability test. One of the brands did not comply with the standard assay of content of active ingredients. Dissolution test passes the pharmacopoeial standards for chloroquine phosphate tablets. There were no significant differences in the amounts of chloroquine phosphate released from the different brands., Interpretation & Conclusion: Out of the seven brands of anti-malarial chloroquine phosphate tablets only one brand fails to meet BP quality specifications which shows constant market monitoring of new products to ascertain their equivalency to pharmacopoeial standards.

Even nowadays millions of people suffer and even die each year from malaria and hundreds of millions of people especially in tropical countries. Quinine (Q) a natural occurring alkaloid and chloroquine (CQ) a synthetic drug are widely used as anti-malarial agents. Herein an isocratic reversed-phase high performance liquid chromatographic (RP-HPLC) method is described for the simultaneous determination of quinine and chloroquine, at low concentrations, in pharmaceuticals and biological fluids. The present method is characterized by higher sensitivity and analytes are separated in less time than the already published methods. The analytical column, an MZ Kromasil, C18, 5 microm, 250 x 4mm, was operated at ambient temperature with backpressure values of 230 kg/cm(2). Mobile phase consisted of methanol-acetonitrile-0.1 mol/L ammonium acetate, (45:15:40 v/v) at a flow rate of 1.0 mL/min. Fluorescence detection was performed at excitation 325 nm and emission 375 nm, respectively. Salicylic acid was used as internal standard at a concentration of 0.5 ng/microL, resulting in a detection limit of 0.3 ng, while upper limit of linear range was 0.7 ng/microL for quinine and 0.5 ng/microL for chloroquine. Separation was completed within 5 min. The statistical evaluation of the method was examined performing intra-day (n=8) and inter-day calibration (n=8) and was found to be satisfactory, with high accuracy and precision results. Solid phase extraction provided high relative extraction recoveries from biological matrices: 92.1% for quinine and 105.4% for chloroquine from blood serum and 101.8% for quinine and 90.7% for chloroquine from urine.

Poly (vinyl chloride) membrane electrodes that responded selectively towards the antimalarial drug chloroquine are described. The electrodes were based on the use of the lipophilic potassium tetrakis(4-chlorophenyl)borate as ion-exchanger and bis(2-ethylhexyl)adipate (BEHA), or trioctylphosphate (TOP) or dioctylphenylphosphonate (DOPP) as plasticizing solvent mediator. All electrodes produced good quality characteristics such as Nernstian- and rapid responses, and are minimally interfered with by the alkali and alkaline earth metal ions tested. The membranes were next applied to a flow-through device, enabling it to function as flow-injection analysis (FIA) detector. The performance of the sensor after undergoing the FIA optimization was further evaluated for its selectivity characteristics and lifetime. Results for the determination of chloroquine in synthetic samples that contained common tablet excipients such as glucose, starch, and cellulose, and other foreign species such as cations, citric acid or lactic acid were generally satisfactory. The sensor was also successfully used for the determination of the active ingredients in mock tablets, synthetic fluids and biological fluids. The sensor was applied for the determination of active ingredients and the dissolution profile of commercial tablets was also established.

A combination Kit for antirelapse treatment of P. vivax malaria, consisting of Chloroquine phosphate tablets and Bulaquine capsules has been recently developed, and marketed under the trade name Aablaquine. Bulaquine is prepared from Primaquine. Several methods of analysis are reported for each drug separately as well two drugs in combination but no method for simultaneous estimation of these three drugs is known. Therefore, the present study was undertaken to develop a sensitive and reproducible high performance liquid chromatographic as well as high performance thin layer chromatographic assay method for the simultaneous estimation of Chloroquine, Primaquine and Bulaquine.

A 19-year-old woman suspect of a suicidal drug intoxication was exhumed after a 10-month earth-grave, because the police was accused of manslaughter and neglected help by the relatives of the deceased. Toxicologic analysis revealed as the cause of death an acute chloroquine intoxication. An expert opinion had to deal with the question if the woman would have been saved if the police had appeared earlier. Therefore the duration of agonal period after suicidal chloroquine ingestion was important. An estimation of the time since death was possible on the one hand ex-post from the development of cadaveric changes and supravital reactions and on the other hand, based on premortal changes detectable on the body together with the findings of the authorities. Taking into account all evidence the woman was probably already dead at or prior to the arrival of the police (110 min after ingestion), at least this could not be excluded. Chloroquine has to be considered to be useful for fatal poisoning, which is also recommended in some publications on methods to commit suicide.

In order to assay the antipaludic capsule of the Service de Santé des Armées (SSA), that contains two antimalarial drugs, i.e. chloroquine sulfate (CQS, cp1) and proguanil hydrochloride (PGH, cp5), a HPLC procedure was developed. A reversed-phase ion-pair high-performance liquid chromatography (HPLC) method with an ultraviolet detection at 254 nm was set up and validated. Elution system includes programming of both organic concentration and flow-rate known as 'dual-mode gradient'. This method allows the simultaneous determination of both active compounds and separation of four process related substances. The method is simple, rapid, selective and accurate, and the precision is good with an inter- and intra-assay of <2%. The sensitivity is particularly suitable for pharmaceutical quality control.

In a number of developing countries there is reportedly a high incidence of the availability of substandard drugs. The majority of these reports do not contain quantitative data to support these claims, nor do they describe the methodology employed for the quality assessment. Many assume counterfeiting as the reason for the poor quality and in some cases this is not justified. We collected 96 samples of chloroquine and selected antibacterials from Nigeria and Thailand in a controlled and methodical manner and analysed them using appropriately validated methods based on high-performance liquid chromatography capable of detecting drug-related impurities and quantifying active drug(s). The results indicate that 36.5% of the samples were substandard with respect to pharmacopoeial limits. Decomposition was the cause of poor quality in a number of the samples but overall, poor manufacturing appeared to be prevalent. The analyses generated little evidence to indicate fraudulent manufacturing. Treatment failure and drug-resistance are possible consequences of the use of substandard drugs.

We have developed a rapid and cost-effective method for the analysis of chloroquine (CQ) and desethylchloroquine in biological samples. The extraction procedure is a simple two-step process that has eliminated the need for costly extraction and evaporation equipment. The mobile phase consists largely of a buffer that was mostly water, making the method cheap to run. The calibration curves were linear from 0 to 200 ng/ml for plasma and from 0 to 3,000 ng/ml for urine. The regression coefficients were of the order of 0.99 and above in all instances. The interassay coefficient of variation (CV) for the assay of CQ was 4.8% in plasma and 4.4% in urine. The intraassay CV for CQ was 3.8% in plasma and 4.2% in urine. There were no interfering peaks from presently used antimalarial drugs. Baseline resolution and separation were very good. The method was found to be applicable for pharmacokinetic studies.

The antimalarial agent chloroquine is known for high affinity for melanin. This 4-aminoquinoline derivative was examined for anti-melanoma activity and uptake into melanoma cells. Chloroquine inhibited growth of cultured melanoma cells; the effect was much greater to a moderately pigmented cell line HMV-II than to a nonpigmented HMV-I. Treatment with chloroquine at a dose of 62 mg/kg i.p. for 12 days prolonged by 71% the life span of mice bearing B16 melanoma, while 24-day treatment at 31 mg/kg resulted in a 81% increase in life span. HMV-II cells showed a two-fold increase in uptake of chloroquine as compared with HMV-I cells. Chloroquine, 24 hr after administration to mice implanted s.c. with B16 melanoma, was selectively accumulated in the pigmented tissues, melanoma and eyes. Other nonpigmented tissues such as the liver, lung, and kidney showed rapid uptake (within 1 hr) and release. These results suggest that chloroquine is toxic to pigmented melanoma cells, the process being partly mediated by binding to melanin.