Rapid and simple method to determine chloroquine and its desethylated metabolites in human microsomes by high-performance liquid chromatography with fluorescence detection

doi:10.1016/S0378-4347(97)00273-9

Journal of Chromatography B: Biomedical Sciences and Applications

Volume 698, Issues 1–2, 26 September 1997, Pages 243-250

https://doi.org/10.1016/S0378-4347(97)00273-9 Get rights and content

Abstract

A sensitive and selective method was developed for the simultaneous determination of chloroquine (CQ) and its desethylated metabolites monodesethylchloroquine (DCQ) and bisdesethylchloroquine (BDCQ) in human liver microsomes. Analytes were separated on a C₁ column using methanol-water (70:30, v/v) and triethylamine (0.1% v/v) as the mobile phase. The fluorescence detector was set at 250 (excitation) and 380 nm (emission). Following protein precipitation with ice-cold acetonitrile, microsomal incubation supernatants were directly injected into the HPLC system. Typically, 200 μl of incubate were diluted with 200 μl of acetonitrile and 15 μl were injected. The limit of quantitation was 78 nM of CQ or metabolite. Intra-day variability averaged 2.9% for CQ, 1.5% for DCQ and 2.5% for BDCQ. Inter-day variability was 3.1% for CQ, 3.5% for DCQ and 3.7% for BDCQ. Mean accuracies were 100% for CQ and BDCQ and 102% for DCQ.

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Validation of a method for the simultaneous quantification of chloroquine, desethylchloroquine and primaquine in plasma by HPLC-DAD
2014, Journal of Pharmaceutical and Biomedical Analysis
Citation Excerpt :
Desethylchloroquine (DSCQ) is the major metabolite of CQ, which has antimalarial activity [8–10]; and the major plasma metabolite of primaquine is the inactive carboxyprimaquine [11,12]. A large number of methods have been described to quantify CQ and DSCQ in different biological matrices [13–21]. Regarding CQ, various quantification studies have been reported for P. falciparum infections and some for P. vivax [22–24].
One of the most important aspects regarding the therapeutic efficacy of antimalarials is its quantification in biologic fluids. The detection and measurement of antimalarial drug levels is important for demonstrating (1) adequate absorption of the drug being given, (2) compliance in taking the full regimen required for treatment and (3) the level of drug in the blood at any time during the test period that parasites reappear. There is a lack of validated methods that simultaneously quantify different antimalarials administered at the same time, such as the use of chloroquine (CQ) and primaquine (PQ) in infections caused by Plasmodium vivax. In this study, a bioanalytical method was validated for the simultaneous quantification of primaquine (PQ), chloroquine (CQ) and desethylchloroquine (DSCQ) in human plasma using liquid–liquid extraction and high performance liquid chromatography with a diode array detector (HPLC-DAD). The PQ was evaluated over a concentration range of 100–3000 nM and the CQ and DSCQ was evaluated over a concentration range of 20–2000 nM. The selectivity of the method was verified by checking for interference by commonly used antimalarials and plasma samples. The accuracy and precision of the method was assessed for drugs spiked into human plasma and recoveries of 83.7%, 92.3%, and 76.5% were obtained for CQ, DSCQ, and PQ, respectively. The applicability of this method was also demonstrated with blood samples from patients with vivax malaria that received combination CQ plus PQ treatment. The simultaneous detection and accurate measurement of CQ, DSCQ, and PQ levels in human plasma provides an important and economical method for validating and monitoring sensitivity/resistance of P. vivax to more common treatment regimen.
Chloroquine-N-oxide, a major oxidative degradation product of chloroquine: Identification, synthesis and characterization
2013, Journal of Pharmaceutical and Biomedical Analysis
Citation Excerpt :
For the analysis and quantification of degradation products, related compounds and impurities in CQ, several methods have been developed and reported in the literature such as electron-impact and chemical ionization (ionization gas ammonia) mass spectrometry [9,10] 1H NMR [11,12], LC–MS [13] and CE with end-column electrochemiluminesence (ECL) detection [14]. Different LC methods are reported for the quantification of CQ in human whole blood, plasma and urine [15–19], in liver cells [20] and tissues [21] with combination of other antimalarial drugs [22] and to identify products of photodegradation [23]. Current drug stability test guideline Q1A(R2) and photostability testing Q1B issued by the ICH [24,25] suggested that stress studies should be carried out on a drug to establish its inherent stability, which can lead to the identification of degradation products.
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, ¹H NMR, ¹³C 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.
HPLC with ultraviolet detection for the determination of chloroquine and desethylchloroquine in whole blood and finger-prick capillary blood dried on filter paper
2011, Journal of Pharmaceutical and Biomedical Analysis
Citation Excerpt :
The earlier methods with ultraviolet detection lack sensitivity and specificity as they were unable to differentiate the parent CQ from DECQ [2–5]. HPLC methods with fluorescence detection achieve the highest sensitivity and selectivity [6–11]. Several methods (HPLC with UV or fluorescence detection, UV spectrometer, TLC densitometer) have also been developed for simultaneous determination of CQ and its metabolites with other antimalarials.
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 1.0 ml/min. Retention times of QN, DECQ and CQ were 4.5, 5.7 and 6.4 min, respectively. Ultraviolet detection was at the wavelength 256 nm. 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 50 ng/ml using 80 μl DBS sample and 25 ng/ml using 150 μl 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.
Voltammetric determination of some anti-malarial drugs using a carbon paste electrode modified with Cu(OH)<inf>2</inf> nano-wire
2009, Talanta
A sensitive electroanalytical methodology for the determination of chloroquine and primaquine using differential pulse voltammetry (DPV) at a Cu(OH)₂ nano-wire-modified carbon paste electrode is presented. The cyclic voltammetric and DPV pulse voltammetric techniques are compared. The effects of scan rate and pH on current were investigated and an optimal scan rate of 50 mV s⁻¹ and a pH 5.5, 0.1 mol L⁻¹ phosphate buffer solution (PBS), were used. Additions of chloroquine and primaquine using DPV show linear ranges from 0.068 to 6.88 μg mL⁻¹ with a detection limit of 0.01 μg mL⁻¹ for chloroquine and 0.58–5.89 μg mL⁻¹ with a detection limit of 0.25 μg mL⁻¹ for primaquine. The method was then successfully utilized for the determination of chloroquine and primaquine in a real sample of their tablets and a recovery of 95% was obtained without interference from tablet matrix.
Enantioselective analysis of oxybutynin and N-desethyloxybutynin with application to an in vitro biotransformation study
2008, Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences
An enantioselective method using liquid-phase microextraction (LPME) followed by HPLC analysis was developed for the determination of oxybutynin (OXY) and its major metabolite N-desethyloxybutynin (DEO) in rat liver microsomal fraction. The LPME procedure was optimized using multifactorial experiments. Under the optimal extraction conditions, the mean recoveries were 61 and 55% for (R)-OXY and (S)-OXY, respectively, and 70 and 76% for (R)-DEO and (S)-DEO, respectively. The validated method was employed to an in vitro biotransformation study using rat liver microsomal fraction. The results demonstrated the enantioselective biotransformation of OXY.
Capillary electrophoresis with end-column electrochemiluminescence for the analysis of chloroquine phosphate and the study on its interaction with human serum albumin
2007, Journal of Chromatography A
This paper describes a novel and sensitive method for the estimation of chloroquine phosphate (CQ) using capillary electrophoresis with end-column electrochemiluminescence (ECL) detection. Under the optimized condition, linear calibration curve was obtained for the system over two orders of magnitude with a detection limit of 3 × 10⁻⁷ M (S/N = 3). The relative standard deviations of the ECL intensity and the migration time were 1.4% and 0.05%, respectively (n = 6, 5 × 10⁻⁵ M CQ). Successful separation of chloroquine phosphate, difenidol hydrochloride and clomifene citrate was obtained at pH 7.0. Using the proposed electrochemiluminescence system, a simple method was proposed to study the interaction between chloroquine phosphate and human serum albumin (HSA), and the number of binding sites and binding constant were estimated to be 32.6 and 7.7 × 10³ M⁻¹, respectively.

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¹: Present address: Astra Research Centre Montreal, Montreal, Quebec, Canada.

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Regular paperRapid and simple method to determine chloroquine and its desethylated metabolites in human microsomes by high-performance liquid chromatography with fluorescence detection

Abstract

J. Chromatogr.

Lancet

Am. J. Med.

J. Chromatogr.

J. Chromatogr.

J. Chromatogr.

J. Chromatogr.

J. Chromatogr.

J. Chromatogr.

J. Chromatogr.

J. Chromatogr.

N. Eng. J. Med.

Eur. J. Clin. Pharmacol.

J. Pharm. Exp. Ther.

Br. J. Clin. Pharmacol.

J. Clin. Pharmacol.

Eur. J. Clin. Pharm.

Eur. J. Clin. Pharmacol.

Regular paper
Rapid and simple method to determine chloroquine and its desethylated metabolites in human microsomes by high-performance liquid chromatography with fluorescence detection