Cross-Reactivity of Chloroquine and Hydroxychloroquine With DRI Amphetamine Immunoassay.

BACKGROUND: Chloroquine and hydroxychloroquine are medical drugs used to treat the chemoprophylaxis of malaria and a second-line anti-inflammatory drug. METHODS: We performed a study of cross-reactivity of chloroquine and hydroxychloroquine in the DRI Amphetamine Assay inspired by a case report of a self-ingestion of chloroquine after a family dispute, that involved the following: (1) an in vitro study with control samples of healthy subjects, (2) an in vivo study with samples of patients with rheumatoid arthritis, and (3) an evaluation of the cross-reactivity of chloroquine and hydroxychloroquine in 3 additional immunoassays. RESULTS: In the case report, the Amphetamine DRI assay resulted positive both at 1000 ng/mL cutoff (1507 and 1137 ng/mL) and at 500 ng/mL cutoff (1178 and 642 ng/mL). Chloroquine urine levels were 103,900 and 100,900 ng/mL at 5 and 9 hours after ingestion. The results with control samples showed a positive cross-reactivity of chloroquine in the DRI Amphetamine Assay (approximately 0.74% and 0.89% at cutoff of 1000 and 500 ng/mL, respectively). Hydroxychloroquine did not cross-react with the DRI Amphetamine Assay up to 1,000,000 ng/mL. In patients treated with chloroquine or hydroxychloroquine, DRI Amphetamine did not produce false-positive results. The comparative assay study showed a positive cross-reactivity of chloroquine in the Emit II Plus Amphetamines Assay with control samples. CONCLUSIONS: Chloroquine can cause false-positive results in the DRI Amphetamine Assay when it is present at high concentrations. Hydroxychloroquine did not produce false-positive results neither in the DRI Amphetamine Assay nor in the others immunoassays evaluated.

A simple qualitive procedure for the detection of chloroquine in urine for use in clinical analytical toxicology in resource poor settings.

Objective: To develop and validate a simple procedure for the qualitative determination of chloroquine in urine with potential for use in developing countries lacking sophisticated analytical equipment and expensive reagents. Design: This was a laboratory based study making use of which combines a colorimetric test, Dill-Glazko's test, and UV/Visible absorbance spectrometry to confirm the presence of chloroquine. The spectrophotometric method was cross validated with the standard Baselt's method for quantification of chloroquine in biological fluids. Setting: Pharmacology laboratory at the Department of Clinical Pharmacology, College of Health Sciences, University of Zimbabwe. Main Outcome Measures: Recovery of the methods was assessed by comparing the peak absorbances and the resolution of the peaks at 329nm and 343nm. Sensitivity and specificity was determined by analysing in a blinded manner. The limits of detection of both the Dill-Glazko's test and the confirmatory test was determined. Results: In the prevalidation procedures increasing the volume of the ethylacetate and the volume of the lower aqueous layer extracted was found to increase the recovery of the confirmatory test. There was a significant difference between both the peak absorbances and the peak resolution for the two methods (p<0.0001). The confirmatory test had a sensitivity of 90% and a specificity of 100%, whereas the Baselt's method had a sensitivity of 83.3% and a specificity of 96.7%. The limit of detection of the Dill-Glazko's test was 15mg/Land that of the confirmatory test was 5mg/L. Conclusions: The confirmatory test had better recovery and was more sensitivity compared with the Baselt's method. The limit of detection of the combination method (Dill-Glazko's plus confirmatory test) was 15mg/L. The combination test showed appreciable sensitivity to be suitable for application to clinical toxicology.

Clinical Toxicological Follow-Up Analysis of a Suicide Attempt Using Chloroquine.

Chloroquine, a drug approved for the treatment of malaria, is frequently used to commit suicide. We report about a suicide attempt by ingesting a high dose of chloroquine in combination with other drugs. Findings of the emergency toxicology screening of blood and urine and those of the follow-up analyses in blood are discussed. Systematic toxicological analysis approaches revealed the presence of chloroquine, butylscopolamine, cafedrine, diazepam, lorazepam, metoclopramide, nordazepam, norephedrine and 11-nor-9-carboxy-∆9-tetrahydroxycannabinol in blood and/or urine of the subject. Suicide due to a combination of chloroquine and benzodiazepines is known as the so-called "Kusch method" in German-speaking countries. The initial chloroquine plasma concentration was determined to be 9.6 mg/L after precipitation and analysis by liquid chromatography-high-resolution tandem mass spectrometry. The analytical procedure was developed ad hoc and validated in accordance with international recommendations. Clinical toxicological follow-up analyses in blood were performed over a period of 3 weeks. The chloroquine concentration remained above the therapeutic range (up to 0.5 mg/L) for 2 weeks and dropped to 0.3 mg/L after 3 weeks. Furthermore, monodesethylchloroquine (MDCQ) and bisdesethylchloroquine (BDCQ) were determined to be the most abundant metabolites in plasma. Within 3 weeks, the area ratios of MDCQ and chloroquine increased 4-fold (from 0.090 to 0.350), and those of BDCQ and chloroquine increased 100-fold (from 0.002 to 0.218). This information may help to estimate the chloroquine excretion progress in the future.

Chronic use of chloroquine disrupts the urine concentration mechanism by lowering cAMP levels in the inner medulla.

Chloroquine, a widely used anti-malaria drug, has gained popularity for the treatment of rheumatoid arthritis, systemic lupus erythematosus (SLE), and human immunodeficiency virus (HIV). Unfortunately, chloroquine may also negatively impact renal function for patients whose fluid and electrolyte homeostasis is already compromised by diseases. Chronic administration of chloroquine also results in polyuria, which may be explained by suppression of the antidiuretic response of vasopressin. Several of the transporters responsible for concentrating urine are vasopressin-sensitive including the urea transporters UT-A1 and UT-A3, the water channel aquaporin-2 (AQP2), and the Na(+)-K(+)-2Cl(-) cotransporter (NKCC2). To examine the effect of chloroquine on these transporters, Sprague-Dawley rats received daily subcutaneous injections of 80 mg·kg(-1)·day(-1) of chloroquine for 4 days. Twenty-four hour urine output was twofold higher, and urine osmolality was decreased by twofold in chloroquine-treated rats compared with controls. Urine analysis of treated rats detected the presence chloroquine as well as decreased urine urea and cAMP levels compared with control rats. Western blot analysis showed a downregulation of AQP2 and NKCC2 transporters; however, UT-A1 and UT-A3 abundances were unaffected by chloroquine treatment. Immunohistochemistry showed a marked reduction of UT-A1 and AQP2 in the apical membrane in inner medullary collecting ducts of chloroquine-treated rats. In conclusion, chloroquine-induced polyuria likely occurs as a result of lowered cAMP production. These findings suggest that chronic chloroquine treatment would exacerbate the already compromised fluid homeostasis observed in diseases like chronic kidney disease.

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.

Evidence of Self-Medication with Chloroquine before Consultation for Malaria in the Southern Pacific Coast Region of Colombia.

Self-medication with antimalarial drugs is a major factor in the development of drug resistance, exerting subtherapeutic drug pressure on circulating parasite populations. Data on self-medication with antimalarials from the Southern Pacific coast region of Colombia, where 4-aminoquinolines resistance and political instability prevail, are vital to elimination strategies. We present results of an exploratory study of 254 individuals having malaria symptoms who sought malaria diagnosis in two hospitals in Tumaco, Department of Nariño, Colombia. Thirty-two percent (82/254) of participants had positive Saker-Solomons urine tests, indicating self-medication with chloroquine (CQ) before consultation for diagnosis. Notably, among 30 pregnant women participating in the study, 43% were Saker--Solomons positive. Molecular analysis of the K76T position encoded by the pfcrt gene revealed the mutant allele in all four samples that were both positive for Plasmodium falciparum and positive for the Saker-Solomons test, suggesting persistent CQ pressure. The high frequency of self-medication, particularly among pregnant women merits attention by public health authorities and comprehensive investigation.

Is chloroquine chemoprophylaxis still effective to prevent low birth weight? Results of a study in Benin.

BACKGROUND: In areas of stable transmission, malaria during pregnancy is associated with severe maternal and foetal outcomes, especially low birth weight (LBW). To prevent these complications, weekly chloroquine (CQ) chemoprophylaxis is now being replaced by intermittent preventive treatment with sulfadoxine-pyrimethamine in West Africa. The prevalence of placental malaria and its burden on LBW were assessed in Benin to evaluate the efficacy of weekly CQ chemoprophylaxis, prior to its replacement by intermittent preventive treatment. METHODS: In two maternity clinics in Ouidah, an observational study was conducted between April 2004 and April 2005. At each delivery, placental blood smears were examined for malaria infection and women were interviewed on their pregnancy history including CQ intake and dosage. CQ was measured in the urine of a sub-sample (n = 166). Multiple logistic and linear regression were used to assess factors associated with LBW and placental malaria. RESULTS: Among 1090 singleton live births, prevalence of placental malaria and LBW were 16% and 17% respectively. After adjustment, there was a non-significant association between placental malaria and LBW (adjusted OR = 1.43; P = 0.10). Multiple linear regression showed a positive association between placental malaria and decreased birth weight in primigravidae. More than 98% of the women reported regular chemoprophylaxis and CQ was detectable in 99% of urine samples. Protection from LBW was high in women reporting regular CQ prophylaxis, with a strong duration-effect relationship (test for linear trend: P < 0,001). CONCLUSION: Despite high parasite resistance and limited effect on placental malaria, a CQ chemoprophylaxis taken at adequate doses showed to be still effective in reducing LBW in Benin.

[Different assays for hingamine in the study of biological fluids].

The authors present a method of hingamine identification in non-biological substances (tablets, powder, syringes) and biological fluids (blood, urine). Isolation was made with chloroform in pH 10. Identification was conducted with thin-layer chromatography, gas chromatography/mass-spectrometry, high-performance liquid chromatography, spectrophotometry in UV region. The quantity was estimated with spectrophotometry in UV region, high-performance liquid chromatography and high-performance thin-layer chromatography. The results of the three methods are comparable.

A simple technique for the detection of anti-malarial drug formulations and their presence in human urine.

A simple, sensitive, specific assay technique for the detection and semi-quantification of chloroquine, amodiaquine, quinine, primaquine, sulfadoxine and pyrimethamine in formulations and in human urine by using thin layer chromatography (TLC) was developed and tested in the laboratory. The method involved developing test samples spotted on TLC chromatogram by diethylamine-toluene-isopropanol (1:4:5 v/v/v) as the eluting solvent. The solvent system diethylamine-toluene-isopropanol (1:4:5 v/v/v) enabled the elution and detection of all the tested antimalarial drugs in solution and those spiked in human urine. Detection limits for chloroquine, amodiaquine, quinine and primaquine were the lowest at 0.00025 mg/ml. Sulfadoxine exhibited a detection limit of 0.0005 mg/ml whereas that of pyrimethamine was 0.001 mg/ml. The results indicate the suitability of this technique in antimalarial drug quality and bioavailability studies. It is envisaged that this technique will adequately address the role of drug absorption and excretion in the chemotherapy of malaria as well as to detect types of antimalarial drugs commonly used in the community.

Simultaneous determination of quinine and chloroquine anti-malarial agents in pharmaceuticals and biological fluids by HPLC and fluorescence detection.

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.

Determination of the stereoisomers of hydroxychloroquine and its major metabolites in plasma and urine following a single oral administration of racemic hydroxychloroquine.

Plaquenil (hydroxychloroquine, HCQ; Sanofi Winthrop Pharmaceuticals, New York, NY) is a stereoisomeric drug administered as a racemic (50: 50) mixture of two isomeric forms--(+) and (-) HCQ. To correlate clinical efficiency accurately with dose, it is necessary to determine the fate of both isomers. This will allow for the optimization of clinical dosing. A method has been developed for the quantitation of (+) and (-) HCQ and its major metabolites, desethylhydroxychloroquine (DHCQ), desethylchloroquine (DCQ), and bisdesethylchloroquine (BDCQ), in plasma and in urine. Application of this method in a preliminary study in four human volunteers is reported. After a single oral dose of 200 mg of Plaquenil, the average enantiomeric ratio of (+) to (-) HCQ was approximately 1 over an 8-hour period. However, the average cumulative 48-hour excretion of HCQ, DHCQ, and DCQ showed stereoselective excretion.

Protective cardiovascular effects of diazepam in experimental acute chloroquine poisoning.

To assess the effects of diazepam in chloroquine poisoning, we studied pentobarbital anesthetized and mechanically ventilated pigs. All the pigs received 50 mg.kg-1 chloroquine given intravenously for 25 min. Eight pigs acted as control (group C). Another 7 were treated with diazepam given intravenously 5 min after the end of chloroquine infusion: 2 mg.kg-1 of diazepam for 2 min, then 1 mg.kg.h-1 for 25 min (group D). Thereafter, all pigs were sacrificed. In both groups the chloroquine infusion induced a large fall in arterial pressure, a decrease in heart rate, and an increase in QRS duration. No difference was observed between the 2 groups for weight, systolic and diastolic arterial pressures, heart rate, QRS and QT durations before diazepam. After diazepam, systolic and diastolic arterial pressures, heart rate, urine volume, urinary excretion of chloroquine, plasma and blood cell chloroquine levels were higher, whereas QRS duration was lower, in group D compared to group C. No difference was observed between the 2 groups for urinary concentration of chloroquine, the ratio between plasma and blood cell chloroquine levels, hepatic, cardiac, and skeletal muscle chloroquine levels, and QT duration. After diazepam, the slope of the regression curve between QRS duration and plasma chloroquine levels was reversed in group D compared to group C. We conclude that diazepam counteracts some haemodynamic and electrocardiographic changes, and increases urinary excretion of chloroquine, in acute experimental chloroquine poisoning.

Standard and reduced doses of mefloquine for treatment of Plasmodium falciparum in Tanzania: whole blood concentrations in relation to adverse reactions, in vivo response, and in vitro susceptibility.

Fifty-three asymptomatic Tanzanian school children with 400-31,000 asexual Plasmodium falciparum parasites/microliter of blood were given standard, one-half, one-quarter, or one-eighth of the recommended mefloquine treatment dose of 25 mg base/kg body weight. Mefloquine and main metabolite concentrations were determined in 100 microliters of capillary blood using a high performance liquid chromatographic method. In the standard, one-half, and one-quarter dose groups, all children cleared the parasites within three days after treatment. Reappearance was noted in one of the children in the one-quarter dose group during 49-56 days of followup. Among the children given one-eighth of a dose, two had an RII response and four had an RI response with early recrudescence. All 24-hour in vitro micro-tests (n = 30) showed full susceptibility for mefloquine. Adverse gastrointestinal reactions were reported by eight children on the first day after treatment, four of whom had been given a standard dose. These children had higher mefloquine concentrations one day after treatment than the other children in this group (P less than 0.05). In the standard dose group (n = 13), the area under the curve of capillary whole blood concentrations of mefloquine versus time was 52.4-112.1 mumol/liter x days. The highest concentration on day 1 was 2.75-7.20 mumol/liter and the median terminal half-life was 17.4 days. The highest concentrations of the main metabolite were observed 1-2 weeks after treatment and the median half-life was 18.9 days. The concentrations in the other groups were approximately proportional to those in the standard dose group both for mefloquine and the metabolite.(ABSTRACT TRUNCATED AT 250 WORDS)

Chloroquine elimination in humans: effect of low-dose cimetidine.

A controlled study was carried out in ten healthy, male volunteers (randomly distributed into control and test groups of five subjects each) to determine the effect of low-dose cimetidine on chloroquine elimination. The control group subjects received two tablets of chloroquine sulfate (300-mg base) only, while the test group subjects took 400-mg cimetidine at bedtime for four days prior to chloroquine (two tablets of chloroquine sulfate) administration and throughout the duration of the study. Blood samples, 5 mL, were collected periodically after chloroquine administration. The samples were assayed for chloroquine and monodesethylchloroquine using a combination of thin-layer chromatography and spectrophotometry. Wilcoxon's test for unpaired data at P less than .05 was used to determine if there was any significant difference in the elimination of chloroquine in the test group when compared with the control group. The apparent oral clearance rate of chloroquine was reduced from 0.49 +/- 0.04 L/d/kg in the control group to 0.23 +/- 0.02 L/d/kg in the test group, and the elimination half-life was prolonged from 3.11 days in the control group to 4.62 days in the test group. There was a 47.04% reduction in the AUC0-7d of monodesethylchloroquine, the major metabolite of chloroquine, in the test group when compared with the control group. The apparent volume of distribution at steady state was increased from 0.46 +/- 0.07 L/kg in the control group to 0.72 +/- 0.10 L/kg in the test group. All these changes were statistically significant. The conclusion is that cimetidine impairs the elimination of chloroquine in healthy subjects.

An ELISA test for detecting chloroquine in urine.

Two ELISA tests for detecting chloroquine in urine have been developed using polyclonal and monoclonal antibodies which react with the 7-chloro-4-amino-quinoline part of the chloroquine molecule and thus recognize chloroquine, its metabolites, and amodiaquine. The ELISAs were sensitive and specific and did not cross-react with other commonly used antimalarials. In a field trial the chloroquine ELISA performed better than the Dill Glazko or Haskins colorimetric tests. A small proportion of urines gave an apparently false positive reaction when tested at a dilution of 1:10, but not when tested at higher dilution.

Field evaluation of the use of an ELISA to detect chloroquine and its metabolites in blood, urine and breast-milk.

The evaluation of an enzyme-linked immunosorbent assay (ELISA) for chloroquine and its metabolites in blood, urine and breast-milk is reported. ELISA blood levels, following standard treatment with chloroquine of pregnant and non-pregnant women, showed mean values comparable to other analytical methods. Blood chloroquine concentrations were estimated at day 0, 350-400 ng/ml; day 2, 1000-1500 ng/ml; day 14, 350-400 ng/ml; day 28, 180-350 ng/ml. In a separate sample a significant association was observed between history of chloroquine use in the previous 2 weeks and blood ELISA values (P less than 0.01). Mean ELISA values in breast-milk were higher than in corresponding whole blood samples. High concentrations of chloroquine in urine were observed. There was a weak association of the ELISA of urine and blood samples collected at the same time (P = 0.076). This study confirms the low sensitivity (less than 55%) of the Dill-Glazko test in urine, which is 100-1000 times less sensitive than the ELISA; the latter can detect 10-20 ng chloroquine per ml. A cut-off value for blood positivity 2 weeks following therapeutic drug ingestion was determined (40% ELISA inhibition), which can be used to make decisions about subject selection in drug sensitivity tests or population surveys. There are several advantages with the ELISA under field conditions. These include direct measurement of whole blood concentration; avoidance of problems of urine collection; suitability of finger-prick samples, especially with young children; application to population surveys to monitor drug use; and selection of subjects for drug sensitivity tests and monitoring of blood levels during in vivo tests.

Quantitative determination of chloroquine and desethylchloroquine in biological fluids by high performance thin layer chromatography.

A high performance thin layer chromatographic (HPTLC) method for chloroquine (CQ) and desethylchloroquine (DCQ) determinations in plasma, erythrocytes and urine is described. Samples extracted by heptane from an alkaline water phase are separated on HPTLC silica gel plates, using toluene/diethylamine (9:1). The compounds are quantified by scanning fluorescent signals. The detection limit is 0.01 mumol/1 for CQ and DCQ, with an extraction efficiency of 76 +/- 7%. Further simplification could make the method suitable for use in field surveys.

Compliance with malaria chemoprophylaxis programmes in Zimbabwe.

Compliance with malaria chemoprophylaxis programmes was studied in the National Railways and two large commercial farm labour forces in Zimbabwe. Prophylaxis was primarily conducted using pyrimethamine/dapsone and the study measured compliance rates through interview and the detection of drug in the urine. Compliance rates as indicated from registers or questioning were not always reliable and were found from urine examination to be in the range of 50-60% for the farm labour force. Annual drug utilization figures also indicated that complete coverage was not being achieved. - The results are discussed in relation to the difficulties involved in implementing malaria prophylaxis programmes. The limited use of large scale chemoprophylaxis is stressed, particularly in the light of increasing drug resistance.

Testing strategies to increase use of chloroquine chemoprophylaxis during pregnancy in Malawi.

The widespread problem of low and non-compliance to antimalarial chemoprophylaxis during pregnancy demands that attention be focused on alternative approaches to programming, product acceptability and demand for preventive services. This study describes the testing of three interventions to determine their effect on use of chloroquine (currently the most widely used drug for chemoprophylaxis) during pregnancy. The strategies evolved from community-based formative research undertaken to learn about the local concept of malaria and issues surrounding malaria prevention and treatment during pregnancy. The resulting interventions were tested in four clinics, and included a change in the health education message given during antenatal sessions, distribution of a sugar-coated chloroquine tablet, and an intervention combining the two strategies. The results showed a 45% increase in chloroquine use when the health education message was changed, and a 64% increase when the product was changed. High use levels were maintained with the combined intervention; an additive effect was seen. The study shows that improving the product was the most important factor in increasing the use of the program, and that changing the health education message can also make an impact on use.

Simultaneous determination of chloroquine and metronidazole in human biological fluid by high pressure liquid chromatography.

Chloroquine (CQ) and metronidazole (MZ) were measured in human urine and plasma by HPLC with UV detection. This method was used to analyse plasma levels in 4 African volunteers after an oral dose of 1000 mg CQ and 750 mg MZ, in a European on weekly prophylaxis of 500 mg CQ, and on 50 hospital urine samples. In the Africans peak plasma levels were over 1 microgram/ml and peak time was 1 1/2-2 hr. In the European plasma levels ranged from 0.58 to 0.36 microgram/ml. Over 80% of the urine samples contained CQ, MZ or both. The assay system was found flexible and economical for the therapeutic monitoring of these two important tropical drugs.