Optimized Griess Reaction for UV-Vis and Naked-eye Determination of Anti-malarial Primaquine.

Primaquine (PMQ), an important anti-malarial drug, has been recommended by the World Health Organization (WHO) for the treatment of life-threatening infections caused by P. vivax and ovale. However, PMQ has unwanted adverse effects that lead to acute hemolysis in patients with glucose-6-phosphate dehydrogenase (G6PD) deficiency. There is a need to develop simple and reliable methods for PMQ determination with the purpose of dosage monitoring. In early 2019, we have reported an UV-Vis and naked-eye based approach for PMQ colorimetric quantification. The detection was based on a Griess-like reaction between PMQ and anilines, which can generate colored azo products. The detection limit for direct measurement of PMQ in synthetic urine is in the nanomolar range. Moreover, this method has shown great potential for PMQ quantification from human serum samples at clinically relevant concentrations. In this protocol, we will describe the technical details regarding the syntheses and characterization of colored azo products, the reagent preparation, and the procedures for PMQ determination.

An unexpected Griess reaction on the important anti-malarial drug primaquine and its application for drug determination.

Primaquine (PMQ), a well-known anti-malarial drug, is of increasing importance as people moving toward global malaria eradication. PMQ has serious side effects that it often causes acute hemolytic toxicity in people with glucose-6-phosphate dehydrogenase (G6PD) deficiency. The development of simple and reliable approaches for quantitative dose monitoring is thus becoming important during malarial treatment with PMQ. Herein, an unexpected Griess reaction on PMQ was systematically studied. The reaction happened between substituted aniline and a primaquine molecule in the presence of nitrite. Both experimental measurements and theoretic calculation showed that UV-vis absorption of the azo products varied because of different electron contributing effects of substituents. Based on the optimized conditions, a novel colorimetric method has been developed for PMQ determination with excellent sensitivity and selectivity. The detection limits for PMQ in water and synthetic urine samples were down to nanomolar range. More importantly, this method has been successfully used to quantify PMQ from human serum samples within clinically relevant concentration ranges.

Metabolism of primaquine in normal human volunteers: investigation of phase I and phase II metabolites from plasma and urine using ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry.

BACKGROUND: Primaquine (PQ), an 8-aminoquinoline, is the only drug approved by the United States Food and Drug Administration for radical cure and prevention of relapse in Plasmodium vivax infections. Knowledge of the metabolism of PQ is critical for understanding the therapeutic efficacy and hemolytic toxicity of this drug. Recent in vitro studies with primary human hepatocytes have been useful for developing the ultra high-performance liquid chromatography coupled with high-resolution mass spectrometric (UHPLC-QToF-MS) methods for simultaneous determination of PQ and its metabolites generated through phase I and phase II pathways for drug metabolism. METHODS: These methods were further optimized and applied for phenotyping PQ metabolites from plasma and urine from healthy human volunteers treated with single 45 mg dose of PQ. Identity of the metabolites was predicted by MetaboLynx using LC-MS/MS fragmentation patterns. Selected metabolites were confirmed with appropriate standards. RESULTS: Besides PQ and carboxy PQ (cPQ), the major plasma metabolite, thirty-four additional metabolites were identified in human plasma and urine. Based on these metabolites, PQ is viewed as metabolized in humans via three pathways. Pathway 1 involves direct glucuronide/glucose/carbamate/acetate conjugation of PQ. Pathway 2 involves hydroxylation (likely cytochrome P450-mediated) at different positions on the quinoline ring, with mono-, di-, or even tri-hydroxylations possible, and subsequent glucuronide conjugation of the hydroxylated metabolites. Pathway 3 involves the monoamine oxidase catalyzed oxidative deamination of PQ resulting in formation of PQ-aldehyde, PQ alcohol and cPQ, which are further metabolized through additional phase I hydroxylations and/or phase II glucuronide conjugations. CONCLUSION: This approach and these findings augment our understanding and provide comprehensive view of pathways for PQ metabolism in humans. These will advance the clinical studies of PQ metabolism in different populations for different therapeutic regimens and an understanding of the role these play in PQ efficacy and safety outcomes, and their possible relation to metabolizing enzyme polymorphisms.

Fabrication of highly sensitive gold nanourchins based electrochemical sensor for nanomolar determination of primaquine.

A gold nanourchins modified glassy carbon electrode (AuNu/GCE) was developed for the determination of antimalarial drug, primaquine (PQ). The surface of AuNu/GCE was characterized by electrochemical impedance spectroscopy (EIS), scanning electron microscopy (SEM), transmission electron microscopy (TEM) and cyclic voltammetry (CV). EIS results indicated that the electron transfer process at AuNu/GCE was faster as compared to the bare electrode. The SEM and TEM image confirmed the presence and uniform dispersion of gold nanourchins on the GCE surface. Upon investigating the electrochemical behavior of PQ at AuNu/GCE, the developed sensor was found to exhibit high electrocatalytic activity towards the oxidation of PQ. Under optimal experimental conditions, the sensor showed fast and sensitive current response to PQ over a linear concentration range of 0.01-1µM and 0.001-1µM with a detection limit of 3.5nM and 0.9nM using differential pulse voltammetry (DPV) and square wave voltammetry (SWV), respectively. The AuNu/GCE showed good selectivity, reproducibility and stability. Further, the developed sensor was successfully applied to determine the drug in human urine samples and pharmaceutical formulations demonstrating its analytical applicability in clinical analysis as well as quality control. The proposed method thus provides a promising alternative in routine sensing of PQ as well as promotes the application of gold nanourchins in electrochemical sensors.

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.

Raising antibodies in chickens against primaquine, pyrimethamine, dapsone, tetracycline, and doxycycline.

Antibodies against primaquine, pyrimethamine, dapsone, tetracycline, and doxycycline were raised in chickens inoculated with each drug conjugated to a rabbit albumin carrier. Antibody titres against drug and carrier were highest during week 6 postinoculation. Affinity purified anti-primaquine antibodies did not recognise other drugs, but affinity purified anti-doxycycline and anti-tetracycline antibodies recognised both tetracycline and doxycycline in addition to primaquine. Primaquine was detected in urine from 6 to 12 hours after ingestion of therapeutic doses of the drug by anti-primaquine antibodies in a competitive ELISA. Affinity purified anti-primaquine antibodies detected primaquine in the cytoplasm and localised in organelles in monocytes that had been incubated with therapeutic concentrations of the drug.

Column liquid chromatographic analysis of quinine in human plasma, saliva and urine.

A new simple, selective and reproducible high-performance liquid chromatographic method for the determination of quinine in plasma, saliva and urine is described. The ion-pair method was carried out on a reversed-phase C18 column, using perchlorate ion as the counter ion and ultraviolet detection at 254 nm. Quinine was well resolved from its major metabolite, 3-hydroxyquinine, and the internal standard, primaquine. The limit of detection was 10 ng/ml and the recovery was greater than 90% from the three biological fluids.

Development of a magnetisable solid-phase fluoroimmunoassay for primaquine and carboxyprimaquine.

Primaquine coupled to keyhole limpet hemocyanin was used as an immunogen to produce antiprimaquine antibodies in three sheep. The antisera obtained were characterised by the increase in fluorescence polarisation found upon binding to fluorescein-labelled primaquine prepared via same route. All sheep showed a good antibody response and one antiserum was coupled to magnetisable solid-phase particles to facilitate the separation of the antibody bound from free labelled antigen and the removal of interfering components which may be present in the sample. The fluoroimmunoassay requires addition of 100 microliters of standard or sample (urine or serum) to 100 microliters tracer (150 nmol/l) followed by 100 microliters of magnetisable solid-phase particles (12.5 g/l). After one hour incubation followed by the usual washing and eluting procedures, using a magnetic rack, the fluorescence of the supernatant was measured directly in a fluorimeter. Sodium salicylate was incorporated in the tracer solution to block the non-specific binding of tracer to the protein in serum samples. Cross-reactivity studies showed that the antibodies have high specificity for the 8-aminoquinoline nucleus but not to the 8-N-aminobutyl side chain. Thus carboxyprimaquine cross-reacted equally with primaquine and the assay can be used to measure their combined level. After extraction of primaquine from a basified sample with methylene chloride, the assay may be applied for the quantitation of either primaquine (in the organic phase) or its acidic metabolites including carboxyprimaquine (in the aqueous phase) separately. This approach was applied for the determination of total primaquine (primaquine and its metabolites) and extracted primaquine in urine samples following a single oral dose of 45 mg primaquine.(ABSTRACT TRUNCATED AT 250 WORDS)

A simple colorimetric method for the determination of primaquine metabolites in urine.

A simple method of screening large numbers of urine samples for the presence of primaquine metabolites has been developed using a commercially available diazonium salt reagent and an extraction cartridge. The extraction requires only 1.0 ml of urine and is selective for acidic or neutral primaquine metabolites. With primaquine-dosed rats, primaquine metabolites could be detected 36 hours after administration of the drug. The sensitivity of the method was found to be approximately 400 mug/l.

High-performance liquid chromatographic-ultraviolet determination of primaquine and its metabolites in human plasma and urine.

A high-performance liquid chromatographic method was developed for the simultaneous determination of primaquine and its metabolites from plasma and urine samples obtained after oral administration of primaquine diphosphate. Following partial deproteinization with acetonitrile, samples were chromatographed by direct injection onto a cyano column with UV detection at 254 nm. Levels as low as 100 ng/mL per 20-microL injection were quantitated. Preliminary pharmacokinetic analysis is reported for two human volunteers after oral doses of 60 mg and 90 mg. Two apparent plasma metabolites and two possible urinary metabolites of primaquine are also reported.

Plasma kinetics and urinary excretion of primaquine in man.

1 The kinetics of primaquine have been studied in twenty volunteers after single and multiple dose regimes. 2 The kinetic parameters were similar in glucose-6-phosphate dehydrogenase (G6PD) normal Thais, G6PD deficient Thais and in Caucasians. 3 The Caucasian subjects showed about 1% of the dose was excreted in the urine. 4 The kinetic parameters obtained from multiple dose studies in Thais were very similar to those obtained from single dose studies in Thais.

Urinary primaquine excretion and red cell methaemoglobin levels in man following a primaquine:chloroquine regimen.

1 Red cell methaemoglobin levels were found to be significantly raised in healthy subjects given a 14-day course of primaquine with chloroquine on the first 3 days. 2 The methaemoglobin levels were not related to the quantity of primaquine excreted. 3 No primaquine could be detected in the plasma at 24 h following the last three daily doses.

A selected ion monitoring assay for primaquine in plasma and urine.

The antimalarial drug primaquine was analysed in plasma and urine by gas chromatography mass spectrometry, using a deuterated internal standard. After freeze-drying and extraction with trichloroethylene the sample plus internal standard was reacted with Tri Sil TBT (a 3:3:2 by volume mixture of trimethylsilylimidazole, N,O,-bis-(trimethylsilylacetamide and trimethylchlorosilane) and an aliquot injected into the gas chromatograph mass spectrometer. The gas chromatographic effluent was monitored at m/z 403 and m/z 406, the molecular ions of the bis-TMS ethers of primaquine and 6-trideuteromethoxy primaquine. Calibration curves were prepared from standards made up in plasma and urine. Data from the analysis of plasma and urine samples from a volunteer who ingested the equivalent of 45 mg primaquine are presented.