Analysis of primaquine and its metabolite carboxyprimaquine in biological samples: enantiomeric separation, method validation and quantification

The clinical formulation of primaquine (PQ) is a mixture of (−)‐(R)‐ and (+)‐(S)‐ primaquine enantiomers which may show different pharmacokinetic and pharmacodynamic properties. To assess the efficacy and toxicity of primaquine enantiomers, a method using LC‐MSD‐TOF has been developed. The enantiomers were well separated using a Chiralcel OD column (250 × 4.6 mm, 10 µm) with a linear gradient of mobile phase consisting of acetonitrile (0.1% formic acid) and aqueous ammonium formate (20 mm ; 0.1% formic acid) adjusted to pH 5.9 at a flow rate of 0.7 mL/min. The method was validated for linearity, precision, accuracy and limits of detection and quantification. The calibration curves were linear with all correlation coefficients being >0.999. The average recoveries of (−)‐(R)‐ and (+)‐(S)‐primaquine and (−)‐(R)‐ and (+)‐(S)‐carboxyprimaquine were 88 and 92%, respectively, in spiked human plasma and 89 and 93% respectively in spiked mouse plasma samples. The RSD of (−)‐(R)‐ and (+)‐(S)‐primaquine and (−)‐(R)‐ and (+)‐(S)‐carboxyprimaquine were 2.15, 1.74, 1.73 and 2.31, respectively, in spiked human plasma and 2.21, 1.09, 1.95 and 1.17% in spiked mouse plasma, respectively. The intra‐day and inter‐day precisions expressed as RSD were lower than 10% in all analyzed quality control levels. The method as reported is suitable for study of the pharmacokinetic and pharmacodynamic properties of the enantiomers of primaquine. The method was successfully applied to study plasma pharmacokinetic profile of enantiomers of primaquine and carboxyprimaquine in mice administered with primaquine in racemic form. The analytical method was found to be linear, accurate, precise and specific. Copyright © 2010 John Wiley & Sons, Ltd.     

Development of a capillary electrophoresis method for the enantioselective estimation of primaquine in pharmaceutical formulations

A capillary electrophoresis (CE) method has been developed that allows the separation and estimation of primaquine enantiomers using hydroxypropyl-gamma-cyclodextrin (HP-gamma -CD) as a chiral selector. The influence of chemical and instrumental parameters on the separation, such as type and concentration of CD, buffer concentration, buffer pH, applied voltage, capillary temperature, and injection time, were investigated. Good separation of the racemic mixture of primaquine was achieved using a fused-silica capillary (52.5 cm effective length x 50 microm id) and a background electrolyte composed of tris-phosphate buffer solution (50 mM, pH 2.5) containing 15 mM HP-gamma-CD as a chiral selector. The recommended applied voltage, capillary temperature, and injection time were 15 kV, 25 degrees C, and 6 s, respectively. Within-day and interday reproducibility of peak area and migration time gave relative standard deviation values ranging from 1.05-3.30%. Good recoveries (range of 96.8-104.9%) were obtained from the determination of placebos that were spiked with 0.25-1.00 mg/L primaquine. The proposed CE method was successfully applied to the assay of primaquine diphosphate in pharmaceutical formulations (tablets).     

Enantioselective analysis of primaquine and its impurity quinocide by capillary electrophoresis

A capillary electrophoretic (CE) method for the baseline separation of the enantiomers of primaquine diphosphate (PQ) and quinocide (QC) (a major contaminant) in pharmaceutical formulations is proposed. Both components were separated under the following conditions: 50 mm tris phosphate buffer (pH 3.0) containing 15 mm hydroxypropyl-gamma-cyclodextrin (HP-gamma-CD) as background electrolyte; applied voltage, 16 kV; capillary temperature, 25 degrees C; detection wavelength, 254 nm; hydrostatic injection, 10 s. The separations were conducted using a 35 cm length and 50 microm i.d. uncoated fused silica capillary column. Under the optimized conditions, the components were successfully separated in about 5 min. Intraday precision of migration time and corrected peak areas when expressed as relative standard deviation ranged from 0.17 to 0.45 and 2.60 to 3.94%, respectively, while the interday precision ranged from 2.59 to 4.20 and 3.15 to 4.21%, respectively. After the validation exercise, the proposed method was applied for the determination of QC impurity in PQ formulations.     

Dissociation and electrooxidation of primaquine diphosphate as an approach to the study of anti-chagas prodrugs mechanism of action

This paper describes the voltammetric behavior of primaquine as a previous support to the further understanding of the delivery and action mechanisms of its respective synthesized prodrugs. There are few papers describing the drug behavior and most of the time no correlation between oxidation process and pH is done. Our results showed that primaquine oxidation is a one-step reaction involving two electrons with the charge transfer process being strongly pH-dependent in acid medium and pH-independent in a weak basic medium, with the neutral form being easily oxidized. This leads to the conclusion that quinoline nitrogen ring neutralization is a determinant step to the formation of the oxidized primaquine form. The existence of a relationship between the primaquine dissociation equilibrium and its electrooxidation process is shown. This work points the importance of voltammetric methodology as a tool for further studies on quantitative relationship studies between chemical structure and biological activity (QSAR) for electroactive drugs.     

Enantioselective analysis of primaquine and its metabolite carboxyprimaquine by capillary electrophoresis

An enantioselective capillary electrophoresis method for the simultaneous determination of primaquine (PQ) and carboxyprimaquine (CPQ) in rat liver mitochondrial fraction, suitable for in vitro metabolism studies is presented. The drug and metabolite were extracted by liquid-liquid extraction using ethyl ether. The enantiomers were resolved in a fused-silica capillary, 50 microm inside diameter (ID) and 24 cm of effective length, using an electrolyte solution consisting of a 20 mmol/L sodium phosphate solution, pH 3.0, and 10% w/v maltodextrin. Hydrodynamic sample injection was used with a 10 s injection time at 50 mbar pressure. The applied voltage was 22 kV and the capillary temperature was controlled at 20 degrees C. Detection was carried out at 264 nm. Under these conditions, the enantiomeric fractions of the drug and of its metabolite were analyzed within 6 min. The extraction procedure was efficient in removing endogenous interferents and low values (<10%) for the coefficients of variation and deviation from theoretical values were demonstrated for both within-day and between-day assays. The method described allows the determination of PQ and CPQ enantiomers as low as 100 and 40 ng/mL, respectively. After validation, the method was used for an in vitro metabolism study of PQ. The results showed that the enantiomer (-)-PQ was preferentially metabolized to (-)-CPQ.     

Determination of quinocide as impurity in primaquine tablets by capillary zone electrophoresis

A capillary zone electrophoretic method has been developed and validated for the determination of the impurity quinocide (QC) in the antimalarial drug primaquine (PQ). Different buffer additives such as native cyclodextrins and crown ethers were evaluated. Promising results were obtained when either β‐cyclodextrin (β‐CD) or 18‐crown‐6 ether (18C6) were used. Their separation conditions such as type of buffer and its pH, buffer additive concentration, applied voltage capillary temperature and injection time were optimized. The use of 18C6 offers slight advantages over β‐CD such as faster elution times and improved resolution. Nevertheless, migration times of less than 5 min and resolution factors (R_s) in the range of 2–4 were obtained when both additives were used. The method was validated with respect to selectivity, linearity, limits of detection and quantitation, analytical precision (intra‐ and inter‐day variability) and repeatability. Concentrations of 2.12 and 2.71% (w/w) of QC were found in pharmaceutical preparations of PQ from two different manufacturers. A possible mechanism for the successful separation of the isomers is also discussed. Copyright © 2008 John Wiley & Sons, Ltd.     

Photophysical properties and photobiological behavior of amodiaquine, primaquine and chloroquine

This article describes the results of a coupled photophysical and photobiological study aimed at understanding the phototoxicity mechanism of the antimalarial drugs amodiaquine (AQ), primaquine (PQ) and chloroquine (CQ). Photophysical experiments were carried out in aqueous solutions by steady-state and time-resolved spectrometric techniques to obtain information on the different decay pathways of the excited states of the drugs and on the transient species formed upon laser irradiation. The results showed that all three drugs possess very low fluorescence quantum yields (10(-2)-10(-4)). Laser flash photolysis experiments proved the occurrence of photoionization processes leading to the formation of a radical cation in all three systems. In the case of AQ the lowest triplet state was also detected. Together with the photophysical properties the photobiological properties of the antimalarial drugs were investigated under UV irradiation, on various biological targets through a series of in vitro assays. Phototoxicity on mouse 3T3 fibroblast and human keratinocyte cell lines NCTC-2544 was detected for PQ and CQ but not for AQ. In particular, PQ- and CQ-induced apoptosis was revealed by the externalization of phosphatidylserine. Furthermore, upon UV irradiation, the drugs caused significant variations of the mitochondrial potential (Deltapsi(mt)) measured by flow cytometry. The photodamages produced by the drugs were also evaluated on proteins, lipids and DNA. The combined approaches were useful in understanding the mechanism of phototoxicity induced by these antimalarial drugs.     

Assignments of dissociation constants of primaquine by 13C-NMR spectroscopy

The first and second dissociation constants of primaquine have been determined by titration in acetonitrile-water mixtures and estimated in pure water by using linear regression analysis. The assignments of dissociation constants were unambiguously achieved by studying the ¹³C nmr spectral data obtained on the mono-, di-, and trihydrochloride salts.     

Unanticipated stereoselectivity in the reaction of primaquine alpha-aminoamides with substituted benzaldehydes: a computational and experimental study

Imidazolidin-4-ones are commonly employed as skeletal modifications in bioactive oligopeptides, either as proline surrogates or for protection of the N-terminal amino acid against aminopeptidase- and endopeptidase-catalyzed hydrolysis. Imidazolidin-4-one synthesis usually involves the reaction of an alpha-aminoamide moiety with a ketone or an aldehyde to yield an imine, followed by intramolecular cyclization. We have unexpectedly found that imidazolidin-4-one formation is stereoselective when benzaldehydes containing o-carboxyl or o-methoxycarbonyl substituents are reacted with alpha-aminoamide derivatives of the antimalarial drug primaquine. A systematic computational and experimental study on the stereoselectivity of imidazolidin-4-one formation from primaquine alpha-aminoamides and various substituted benzaldehydes has been carried out, and they have allowed us to conclude that intramolecular hydrogen-bonds involving the C=O oxygen of the o-substituent play a crucial role.     

Synthesis of imidazolidin-4-one and 1H-imidazo[2,1-a]isoindole-2,5(3H,9bH)-dione derivatives of primaquine: scope and limitations

The synthesis of imidazolidin-4-one derivatives of primaquine as potential antimalarial agents is described. The target compounds were synthesized in three steps: (i) condensation of (±)-primaquine with N^α-protected amino acids, (ii) removal of the N^α-protecting group, and (iii) reaction of the N-acylprimaquine with a carbonyl compound: acetone, three cyclic ketones and veratraldehyde. Using 2-formylbenzoic acid in the third step afforded 1H-imidazo[2,1-a]isoindole-2,5(3H,9bH)-diones. All products were isolated in good to excellent yields. Whereas imidazolidin-4-ones were formed as mixtures of all possible diastereomers in equal amounts, 1H-imidazo[2,1-a]isoindole-2,5(3H,9bH)-diones were produced in a stereoselective fashion. The compounds hydrolyse very slowly (t_1/2 5-30 d) in pH 7.4 buffer to release primaquine. These primaquine derivatives are being submitted to biological assays, and preliminary results of their antimalarial activity are quite encouraging.     

Synthesis of N4-(5-methoxy-7-benzofuranyl)-1,4-pentanediamine,an oxygen isostere of primaquine

N⁴-(5-Methoxy-7-benzofuranyl)-1,4-pentanediamine, an oxygen isostere of primaquine was synthesized and found devoid of antimalarial activity.     

Determination of the Antimalarial Primaquine in Whole Blood and Urine by Normal-Phase High-Performance Liquid Chromatography

Abstract

A method has been developed to estimate primaquine in whole blood and urine by sensitive and selective high-performance liquid chromatography. Using the linear chain analogue of primaquine as the internal standard, a single-step extraction, normal-phase silica column with a basic mobile phase, levels down to 1 ng/ml of primaquine could be measured with good precision. Other anti-malarials like amodiaquine and pyrimethamine did not interfere in the assay. The major carboxylic acid metabolite of primaquine did not elute under the normal-phase chromatographic conditions. The method is suitable for use in clinical pharmacokinetic studies with primaquine.     

Simplified and Rapid Determination of Primaquine and 5,6-Orthoquinone Primaquine by UHPLC-MS/MS: Its Application to a Pharmacokinetic Study

The method for the determination of primaquine (PQ) and 5,6-orthoquinone primaquine (5,6-PQ), the representative marker for PQ active metabolites, via CYP2D6 in human plasma and urine has been validated. All samples were extracted using acetonitrile for protein precipitation and analyzed using the ultra-high-performance liquid chromatography–tandem mass spectrometry (UHPLC-MS/MS) system. Chromatography separation was carried out using a Hypersil GOLD^TM aQ C₁₈ column (100 × 2.1 mm, particle size 1.9 μm) with a C₁₈ guard column (4 × 3 mm) flowed with an isocratic mode of methanol, water, and acetonitrile in an optimal ratio at 0.4 mL/min. The retention times of 5,6-PQ and PQ in plasma and urine were 0.8 and 1.6 min, respectively. The method was validated according to the guideline. The linearity of the analytes was in the range of 25–1500 ng/mL. The matrix effect of PQ and 5,6-PQ ranged from 100% to 116% and from 87% to 104% for plasma, and from 87% to 89% and from 86% to 87% for urine, respectively. The recovery of PQ and 5,6-PQ ranged from 78% to 95% and form 80% to 98% for plasma, and from 102% to from 112% to 97% to 109% for urine, respectively. The accuracy and precision of PQ and 5,6-PQ in plasma and urine were within the acceptance criteria. The samples should be kept in the freezer (−80 °C) and analyzed within 7 days due to the metabolite stability. This validated UHPLC-MS/MS method was beneficial for a pharmacokinetic study in subjects receiving PQ.     

Profiling primaquine metabolites in primary human hepatocytes using UHPLC‐QTOF‐MS with 13C stable isotope labeling

Therapeutic efficiency and hemolytic toxicity of primaquine (PQ), the only drug available for radical cure of relapsing vivax malaria are believed to be mediated by its metabolites. However, identification of these metabolites has remained a major challenge apparently due to low quantities and their reactive nature. Drug candidates labeled with stable isotopes afford convenient tools for tracking drug‐derived metabolites in complex matrices by liquid chromatography‐tandem mass spectrometry (LC‐MS‐MS) and filtering for masses with twin peaks attributable to the label. This study was undertaken to identify metabolites of PQ from an in vitro incubation of a 1:1 w/w mixture of ¹³C₆‐PQ/PQ with primary human hepatocytes. Acquity ultra‐performance LC (UHPLC) was integrated with QTOF‐MS to combine the efficiency of separation with high sensitivity, selectivity of detection and accurate mass determination. UHPLC retention time, twin mass peaks with difference of 6 (originating from ¹³C₆‐PQ/PQ), and MS‐MS fragmentation pattern were used for phenotyping. Besides carboxy‐PQ (cPQ), formed by oxidative deamination of PQ to an aldehyde and subsequent oxidation, several other metabolites were identified: including PQ alcohol, predictably generated by oxidative deamination of PQ to an aldehyde and subsequent reduction, its acetate and the alcohol's glucuronide conjugate. Trace amounts of quinone‐imine metabolites of PQ and cPQ were also detected which may be generated by hydroxylation of the PQ/cPQ quinoline ring at the 5‐position and subsequent oxidation. These findings shed additional light on the human hepatic metabolism of PQ, and the method can be applied for identification of reactive PQ metabolites generated in vivo in preclinical and clinical studies. Copyright © 2013 John Wiley & Sons, Ltd.     

Determination of three metabolites of a new angiotensin-converting enzyme inhibitor, imidapril, in plasma and urine by gas chromatography-mass spectrometry using multiple ion detection.

A specific and sensitive gas chromatographic-mass spectrometric method for the determination of three metabolites of the angiotensin-converting enzyme inhibitor, imidapril, in plasma and urine was developed. The metabolites were isolated from plasma and urine using a Bond Elut C18 solid-phase extraction cartridge. The isolated metabolites were converted to sensitive derivatives by pentafluorobenzyl bromide and heptafluoro-n-butyric acid anhydride. Following derivatization, the sample solutions were analysed by wide-bore column gas chromatography-mass spectrometry with multiple ion detection. The detection limits of the three metabolites were each 1 ng/ml in plasma and 5 ng/ml in urine. Analysis of the spiked plasma and urine samples demonstrated the good accuracy and precision of the method. This method was very useful for use in pharmacokinetic and bioavailability studies of the three metabolites of imidapril in humans.     

Determination of amodiaquine in pharmaceuticals by reaction with periodate and spectrophotometry or by high-performance liquid chromatography

Two methods are described for the determination of amodiaquine in pharmaceuticals. In the first method, amodiaquine was treated with periodate at pH 7.5-8.5 to yield a chromogen, which is believed to be a 1,4-benzoquinone imine derivative, and extracted into chloroform for spectrophotometric assay. The maximum molar absorptivity was found to be 2.17 x 10(3) l mol-1 cm-1 at 442 nm. Primaquine and chloroquine, if present also, do not interfere. The second method, which was used for the simultaneous determination of amodiaquine, primaquine and chloroquine, is based on high-performance liquid chromatography on an octadecylsilane column using a mobile phase of methanol-water (80 + 20) containing 0.5% anhydrous acetic acid and an over-all concentration of sodium dodecyl sulphate of 0.5 mM, with detection at 340 nm.     

Analysis of baclofen by capillary electrophoresis with laser-induced fluorescence detection

A new analytical method for baclofen (4-amino-3-p-chlorophenylbutyric acid) based on capillary electrophoretic separation and laser-induced fluorescence detection has been developed. Naphthalene-2,3-dicarboxaldehyde was used for precolumn derivatization of the non-fluorescent drug. Optimal separation and detection were obtained with an electrophoretic buffer of 50 mM sodium borate (pH 9.5) and a He-Cd laser (excitation at 442 nm, emission at 500 nm). Linearity (r > or = 0.99) over three orders of magnitude was generally obtained and the concentration limit of detection was in the nanomolar level. Coupled with a simple cleanup procedure, the method was successfully applied to the analysis of baclofen in human plasma. Recovery of spiked baclofen in plasma was 98%. The relative standard deviation values on peak size and migration time were 7.9% and 0.4%, respectively. The limit of detection of baclofen in plasma was 10 ng/ml.     

Analysis of quinocide in unprocessed primaquine diphosphate and primaquine diphosphate tablets using gas chromatography–mass spectrometry with supersonic molecular beams

Malaria is one of the most widespread and deadly diseases on the planet. Every year, about 500 million new cases are diagnosed, and the annual death toll is about 3 million. Primaquine has strong antiparasitic effects against gametocytes and can therefore prevent the spread of the parasite from treated patients to mosquitoes. It is also used in radical cures and prevents relapse. Consequently, primaquine is an often-used drug. In this study the separation of unprocessed primaquine from the contaminant quinocide based on gas chromatography–mass spectrometry with supersonic molecular beam (SMB) is presented and 7.5 mg primaquine diphosphate tablets were analyzed. We present a novel method for fast determination of quinocide which is an isomer of primaquine as the main contaminant in unprocessed primaquine and in its medical form as tablets by gas chromatography–mass spectrometry with SMB (also named supersonic GC–MS). Supersonic GC–MS provides enhanced molecular ion without any ion source related peak tailing plus extended range of compounds amenable for GC–MS analysis. In addition, major isomer mass spectral effects were revealed in the mass spectra of primaquine and quinocide which facilitated the unambiguous identification of quinocide in primaquine tablets. Fast GC–MS analysis is demonstrated with less then 2 min elution time of the drug and its main contaminants.     

Determination of midazolam in rabbit plasma by GC and LC following nasal and ocular administration

GC with nitrogen phosphorus detection and HPLC with UV detection were used to determine midazolam (MDZ) levels in rabbit plasma following ocular and nasal administration. For GC with nitrogen phosphorus detection, the analyte was extracted from the plasma using a three‐step liquid–liquid extraction including extraction with an isopropanol/butyl chloride mixture in an alkaline solution, followed by extractions with 1 M HCl, and finally with an alkaline solution of butyl chloride. The recovery of MDZ was dependent on the sample alkalization time prior to the final extraction. The procedure increased the recovery of MDZ up to 99.6%. Improved sample preparation led to a significant increase in the sensitivity of the determination by GC with nitrogen phosphorus detection. The achieved detection limit was 0.34 ng/mL, which is ten times lower than that obtained using HPLC with UV detection. The small plasma volume was another advantage of the GC with nitrogen phosphorus detection method (200 μL per assay). Both administration routes of the anesthetic (nasal and ocular) resulted in comparable plasma MDZ levels. Kinetic simulation of the MDZ plasma was performed for both administration routes.     

Capillary electrophoresis analysis of fosfomycin in biological fluids for clinical pharmacokinetic studies

A feasible capillary zone electrophoresis (CZE) method with indirect UV and contactless conductivity detection was developed for the determination of fosfomycin, an antibiotic, in human plasma and microdialysis samples. Samples were collected from test persons during a clinical trial. The background electrolytes used consisted of 25 mM benzoic acid and 0.5 mM hexadecyltrimethylammonium bromide, adjusted with tris(hydroxymethyl)aminomethane solution to pH 6.95 for plasma, and to pH 8.05 for microdialysis samples. CZE separations of the anionic analyte were carried out with reversed electroosmotic flow directed towards the anode. The limit of detection was between 0.6 and 2 microg/mL, depending on the matrix and the detection method. No sample preparation was needed for microdialysis samples; for plasma samples, proteins were precipitated with methanol (1+2, v+v), and the supernatant was analyzed. The yield determined with spiked samples was about 100%, the reproducibility of the entire method, expressed by the RSD% of three independent determinations of fosfomycin in triplicate after spiking Ringer's solutions and plasma samples, respectively, was better than 8%. The method is thus well-suited for clinical studies for the determination of the antibiotic in biological fluids.