Author: admin_dev

Equipment

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.

Extraction procedure

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.

Validation procedure

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.