Chloroquine is an antimalarial drug used in the treatment of Plasmodium vivax malaria. Three methods to quantify chloroquine and its metabolite in blood matrices were developed and validated.
Methodology & results:
Different high-throughput extraction techniques were used to recover the drugs from whole blood (50 μl), plasma (100 μl) and dried blood spots (15 μl as punched discs) followed by quantification with LC-MS/MS. The intra- and inter-batch precisions were below 15% and thus meet regulatory acceptance criteria.
The developed methods demonstrated satisfactory validation performance with high sensitivity and selectivity. The assays used simple and easy to automate extraction techniques. All methods were reliable with robust performance and demonstrated to be suitable to implement into a high-throughput routine analysis of clinical pharmacokinetic samples.
Malaria is still a major public health problem worldwide, resulting in an estimated 445,000 annual deaths in 2016. Chloroquine was once the most extensively used antimalarial drug, due to its low cost, high efficacy and relative safety. It was later discontinued for the treatment of Plasmodium falciparum infections in most countries worldwide due to increasing drug resistance. However, it has been reported that a prolonged absence of chloroquine in endemic areas can lead to a reversal of resistance in the parasite population, providing a renewed potential to treat P. falciparum infections. Chloroquine is still the main first-line therapy recommended for the treatment of Plasmodium vivax infections. However, chloroquine resistance has been reported in P. vivax in Brazil, Ethiopia, Indonesia, Malaysia (Borneo), Myanmar, Thailand, Papua New Guinea and Peru. Chloroquine belongs to the 4-aminoquinoline group of antimalarial drugs. The major active metabolite of chloroquine, generated by CYP450 CYP2C8 and CYP3A4/5 enzymes, is desethylchloroquine. Both chloroquine and desethylchloroquine are slowly eliminated, with a terminal elimination half-life of approximately 30–60 days. They are mainly bound to platelets, erythrocytes, thrombocytes and granulocytes, similar to other quinoline antimalarial drugs, resulting in increased concentrations in infected or uninfected blood cells that are about two- to five-times higher than what can be found in plasma. Figure 1 shows the molecular structure of chloroquine and desethylchloroquine.
Several quantification methods of chloroquine in biological matrices have been described previously. Early publications of bioanalytical methods commonly used extraction procedures, such as protein precipitation and liquid–liquid extraction, which often leave protein residues in the extracted sample. Liquid–liquid extraction is also labor intensive and time consuming. A separation and detection method consisting of LC coupled with UV or fluorescence detection are easy to operate but provide only low sensitivity and selectivity, and often require large sample volumes to achieve adequate sensitivity for quantification of clinical pharmacokinetic samples. A recent publication with diode array detector used a more powerful sample extraction technique (e.g., SPE). However, this method still proved less sensitive (LLOQ of 10 ng/ml) using a large injection volume (50 μl) for improved sensitivity. The introduction of mass spectrometric detection has become popular for its high sensitivity and selectivity that is useful for pharmacokinetic studies. Several mass spectrometric methods have been published for chloroquine determination, but those were simultaneous analysis with other antimalarial drugs. Simultaneous analysis often leads to compromises, for example, a general extraction method needs to be used that can lead to severe matrix effects. A large injection volume to improve sensitivity and prolonged analysis time, for example, 19–21 min/sample due to added chromatography column washout period, to reduce memory effects or sample carryover, were some of the compromises met.
Here we present an optimized protocol for the quantification of chloroquine and its metabolite, desethylchloroquine, in plasma, whole blood and dried blood spots (DBS) using LC–MS/MS. Three different extraction techniques were used to ensure high-throughput and optimal recoveries of the drugs from the different biological matrices. The use of MS for the detection of the drug molecules provides higher sensitivity, selectivity, and requires smaller sample volumes. The methods described here were developed and optimized for implementation in high-throughput routine settings and were validated in accordance to the Guidance for Industry, Bioanalytical Method Validation (US FDA, 2001) and the Guidance on Bioanalytical Method Validation (European Medicines Agency, London, UK, 2012).
Materials & methods
Chemicals & reagents
Chloroquine and desethylchloroquine were obtained from AlsaChim (Illkirch, France). The stable isotope-labeled internal standards, chloroquine-D4-diphosphate salt and desethylchloroquine-D4, were obtained from Santa Cruz Biotechnology (TX, USA). All solvents and chemicals were of MS grade, except ethyl acetate, which was HPLC grade, and ammonia solution (25%), which was analytical grade. Water, acetonitrile, methanol and ethyl acetate were obtained from JT Baker (NJ, USA). Formic acid (98–100%) and ammonium formate were obtained from Fluka (Sigma-Aldrich, MO, USA). Ammonia solution (25%) was used to prepare ammonium hydroxide 0.5 M (Merck, Darmstadt, Germany). Blank whole blood and plasma were obtained from Thai Red Cross, Bangkok, Thailand with citrate phosphate dextrose as anticoagulant. For other anticoagulants, EDTA, fluoride-oxalate, fluoride-heparin, Na-heparin and Li-heparin were collected from healthy volunteers at the Faculty of Tropical Medicine, Mahidol University, Thailand. Ethical approval for the method development and validation was sought from the Ethics Committee of the Faculty of Tropical Medicine, Mahidol University, Thailand (certificate no. MUTM 2017-014-01 and approval no. TMEC 16–095).
The following SPE columns were used for sample extraction: 100 mg, 1 ml, carboxylic acid bonded sorbent (CBA) fixed 96-wellplate (Biotage, Uppsala, Sweden) for whole blood, Phree Phospholipids Removal 96-wellplate, 8E-S133-TGB (Phenomenex, CA, USA) for DBS and ISOLUTE® SLE+ 96-well plate, 820-0200-P01, IST (Biotage, Uppsala, Sweden) for plasma. A Freedom Evo 200 platform liquid handler (TECAN, Mannedorf, Switzerland) was used to automate the sample preparation and extraction. A Robotic Punch Instrument (BSD600-Duet Semi-Automated, Queensland, Australia) was used to obtain samples from the DBS. A TurboVap®96 (Biotage) was used to evaporate the eluted sample.
Preparation of standards, working solutions, calibration standards & quality control samples
Stock solutions (1 mg/ml) of chloroquine, desethylchloroquine and their stable isotope-labeled internal standards were prepared in acetonitrile–water (50–50, v/v) containing 0.5% formic acid and stored at -80°C. Working solutions were prepared from the stock solution using acetonitrile–water (50–50, v/v) as dilution solution and then used for the spiking of whole blood, plasma and whole blood for DBS.
Unless otherwise stated, blank blood from healthy volunteers with EDTA as anticoagulant was used. Plasma was obtained by centrifugation of whole blood at 1500–2000 × g for 10 min. Whole blood applied on chromatography filter paper Whatman (31 ET Chr, DMPK-C, 903 Protein saver and 3 MM Chr; Whatman, Buckinghamshire, UK) and an alternative brand, Ahlstrom 226 (PerkinElmer, MA, USA) was used for DBS technique. The calibration curves of chloroquine/desethylchloroquine were 2.56–1220/3.36–1220 ng/ml, 1.41–610/1.41–610 ng/ml and 1.82–1552/2.95–1552 ng/ml in whole blood, plasma and DBS, respectively. The final volume of working solution in blank blood was kept below 5% in all samples.
Whole blood, plasma or punched discs of DBS were aliquoted into 96-well plates and processed using an automated liquid handler platform (Freedom Evo 200) as described below.
Whole blood (50 μl) was aliquoted into a 96-wellplate and 100 μl of water containing stable isotope-labeled internal standard (desethylchloroquine-D4 25.8 ng/ml and chloroquine-D4 72.5 ng/ml) was added, followed by 450 μl of ammonium carbonate 20 mM. The plate was mixed on Mixmate (Eppendorf, Hamburg, Germany) at 1000 r.p.m. for 2 min and centrifuged at 1100 × g for 2 min (i.e., extraction-ready samples). CBA-fixed SPE 96-wellplate cartridges were conditioned with methanol (1 ml) followed by ammonium carbonate 20 mM (1 ml). Each buffer-diluted whole blood sample (200 μl) was loaded onto the conditioned CBA SPE 96-wellplate and subsequently washed with ammonium carbonate 20 mM (1 ml), ammonium carbonate 20 mM–methanol (20–80, v/v; 1 ml) and methanol–water (50–50, v/v; 1 ml). Full vacuum (10-inch Hg) was applied for 40 min to dry the wells and any liquid left on the SPE cartridge tips was removed. The bound fraction was eluted by adding 900 μl of elution solvent (2% formic acid in methanol), followed by evaporation of the eluent at 70°C under nitrogen gas. The dried samples were reconstituted in 800 μl of mobile phase; acetonitrile-ammonium formate 20 mM with 1% formic acid (15/85, v/v).
Plasma (100 μl) was aliquoted into a 96-wellplate and diluted with 350 μl ammonium hydroxide (0.5 M) containing stable isotope-labeled internal standards (48.1 ng/ml of desethylchloroquine-D4 and 22.7 ng/ml of chloroquine-D4). The plate was mixed on a Mixmate at 1000 r.p.m. for 2 min and centrifuged at 1100 × g for 2 min (i.e., extraction-ready samples). The extraction samples (200 μl) were transferred to a supported liquid extraction, SLE+, 96-well plate. Vacuum of 3–4 inch Hg was applied for 30 s to allow the sample to absorb to the cartridge. The bound fraction was eluted with ethyl acetate (800 μl) followed by evaporation of the eluent at 70°C under nitrogen gas. The dried samples were reconstituted in 800 μl of mobile phase; acetonitrile-ammonium formate 20 mM with 1% formic acid (15–85, v/v).
From one DBS of approximately 50 μl, five discs of 3.2 mm in diameter were punched out (equivalent to 15 μl of whole blood) into a 96-wellplate. Acetonitrile-water with 0.5% formic acid (50-50, v/v; 200 μl) containing stable isotope-labeled internal standards (3.4 ng/ml of desethylchloroquine-D4 and 9.6 ng/ml of chloroquine-D4) was added to each sample, and the plate was mixed on a Mixmate at 1000 r.p.m. for 10 min and centrifuged at 1100 × g for 2 min. Acetonitrile (200 μl) was added to each sample and the plate was mixed on a Mixmate at 1000 r.p.m. for 2 min and centrifuged at 1100 × g for 2 min (i.e., extraction-ready samples). The extraction samples (250 μl) were loaded on a Phree Phospholipids Removal 96-wellplate. Vacuum was applied until the entire sample volume passed through the column, and the collected eluate was diluted with 170 μl of water.
Instrumentation & chromatographic conditions
The LC system was an Agilent 1260 infinity system consisting of a binary LC pump, a vacuum degasser, a temperature-controlled microwell plate autosampler set at 4°C and a temperature-controlled column compartment set at 40°C (Agilent technologies, CA, USA). Data acquisition and processing were performed using Analyst 1.6.2 (Sciex, MA, USA). The analytes were separated on a Zorbax SB-CN 50 mm × 4.6 mm, I.D. 3.5 μm (Agilent Technologies), with a precolumn CN AJO-4305 4 mm × 3 mm, I.D. 3.5 μm (Phenomenex), at a flow rate of 700 μl/min. The mobile phase consisted of (A) acetonitrile-ammonium formate 20 mM with 1% formic acid pH approximately 2.6 (15–85, v/v) and (B) methanol–acetonitrile (75–25, v/v). The mobile phase gradient was A: 0–2 min, B: 2.2–3.7 min and A: 3.9–6.5 min (with 0.2 min linear gradient switch), resulting in a total runtime of 6.5 min per sample. The injection volume was 2 μl.
An API 5000 triple quadrupole mass spectrometer (Sciex) with a TurboV ionization source interface, operating in positive ion mode, was used for the MS/MS analysis. Ion spray voltage was set to 5500 V, with a drying temperature at 650°C. The curtain gas was 25 psi and the nebulizer (GS1) and auxiliary (GS2) gases were 60 psi.
The assays were validated according to the FDA, 2001 and European Medicines Agency, 2012 on bioanalytical method validation.
Accuracy and precision of the methods were determined by analyzing five replicates of samples at the LLOQ and ULOQ, as well as quality control (QC) samples at three concentrations. Four (whole blood and plasma) to six (DBS) independent runs were performed and evaluated. Accuracy was calculated as mean relative error (%) by comparing the measured average concentration at each QC level with the nominal concentration. Precision of the method (within-run, between-run and total-assay variability) was calculated using a single factor analysis of variance (ANOVA), and expressed as the coefficient of variation (%). The ability to dilute samples above the ULOQ (i.e., dilution integrity of over the curve samples) was investigated by analyzing five replicates at 2–3 × ULOQ for chloroquine and desethylchloroquine by 1:5 dilutions for whole blood and DBS methods, and 1:10 dilutions for plasma method.
The calibration curve was assessed by analyzing four to six separate runs (the same as accuracy and precision determination). The best performing linear regression model (nonweighted, 1/x-weighted and 1/x2-weighted) was chosen based on the accuracy and precision of back-calculated concentrations of calibration standards and QC samples. Calibration standards and QC samples contributed equally to the selection of regression model by a ranking approach as previously described.
Selectivity was evaluated by analyzing six blank samples from six different donors for each matrix and the chromatograms were evaluated for any signal that potentially could interfere with the drug identification and measurement. Potentially interfering co-administered antimalarial drugs were investigated in a similar way by injecting 2 μl of individual piperaquine, pyronaridine, artesunate, primaquine and carboxyprimaquine at 30 ng/ml. The same experiment was then repeated while performing postcolumn infusion of chloroquine, desethylchloroquine and their stable isotope-labeled internal standards mix solution (20 ng/ml) for any signs of signal enhancement or suppression.
Absolute extraction recovery was determined by comparing the average response of extracted QC samples (five replicates at each level) with that of postextraction spiked blank blood samples at the same nominal concentration as the QC samples.
Matrix effects were investigated for different donors and anticoagulants using postcolumn infusion experiments. Blood from six different donors were collected using EDTA and from one of the donors, different anticoagulants (Na-heparin, Li-heparin, fluoride-heparin, citrate phosphate dextrose and fluoride oxalate) were also collected. All blank blood extracted samples from six different donors and different anticoagulants were investigated for ion suppression or enhancement caused by the matrix.