Racemic hydroxychloroquine (Rac-HCQ) sulfate, (R)-(-)-HCQ sulfate and (S)-(+)-HCQ sulfate were from Desite Biopharmaceutical Co., Ltd (Chengdu, China). HPLC grade n-hexane was purchased from Aladdin (Shanghai, China). Isopropanol was from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). Methanol and acetonitrile were from Tedia (Fairfield, USA). Diethylamine (DEA) was obtained from Ling Feng Chemical Reagent Co., Ltd (Shanghai, China).
Animal welfare and ethics
All animal experiments were performed in accordance with the relevant laws and regulations on the use and management of experimental animals and the relevant provisions of the institutional animal care and use committee (IACUC) of Center of New Drug Safety Evaluation and Research of China Pharmaceutical University (CPU). The number of animals, the design of tests and the disposal of animals were approved by the IACUC and were strictly carried out. The approval number for animal ethics was B20200515-1.
Optimized chiral HPLC conditions
HPLC analyses were performed on a LC-2010A HT (Shimadzu, Kyoto Japan) consisting of an UV-Vis detector, automatic sampler and thermostatic column oven compartment. The resolution of HCQ enantiomers was achieved on a Chiralpak AD-H column (4.6 mm×150 mm, particle size 5 μm) with column oven maintained at 20 ℃. The mobile phase consisted: (A) n-hexane in the presence of 0.5% DEA, and (B) isopropanol. Two portions of mobile phases were mixed online (93:7, v/v) with an isocratic elution at a flow rate of 0.8 ml/min. The UV wavelength at 343 nm was set for detection with an injection volume of 10 μl.
Screening of different chiral columns with reversed phase chromatography
Reversed-phase chiral columns, including Chiralcel OJ-RH, OX-RH, OZ-RH and Chiralpak AD-RH, AS-RH, AY-RH, AZ-RH, were used to examine the separation of HCQ enantiomers with gradient elution at a flow rate of 0.5 ml/min. The mobile phase consisted: (A) water in the presence of 0.1% TFA, and (B) acetonitrile in the presence of 0.1% TFA. The elution was conducted as follows: 10% B in 0~5 min, 15% B in 5~20 min, 25% B in 20~30 min, and 50% B in 30~40 min.
The influence of diethylamine (DEA) on the resolution
The influence of DEA added in the mobile phase on the resolution of HCQ enantiomers was investigated under the optimal chromatographic conditions with Chiralpak AD-H column. The content of DEA was 0.0%, 0.10%, 0.15%, 0.20%, 0.25%, 0.30%, 0.35%, 0.40%, 0.45%, 0.50%, respectively.
The stock solutions of Rac-HCQ sulfate, (R)-HCQ sulfate and (S)-HCQ sulfate were prepared at 1 mg/ml in water. Working solutions of Rac-HCQ sulfate at concentrations of 1, 5, 10, 15 and 20 μg/ml were prepared by dilution with water. Working solutions of (R)-HCQ sulfate and (S)-HCQ sulfate were prepared at 10 μg/ml by dilution with water respectively. All solutions were temporarily stored at 4 ℃ before the analysis.
Preparation of plasma samples
Drug-free blood (~12 ml) from the abdominal aorta of healthy SD rats was taken in a vacuum sampling vessel containing EDTA-K2, and then centrifuged at 3000 rpm for 10 min at 4℃ to obtain the plasma (~5 ml). In 10 ml centrifuge tube, 500 μl plasma was mixed with 500 μl working solution of racemate and enantiomers of HCQ supplemented with 250 μl potassium phosphate (pH 7.4, 50 mM) and 250 μl 2% sodium bicarbonate. The above solution was mixed and incubated at room temperature for 1 h before the addition of 1.5 ml cold acetonitrile and 2 μl of 5 N sodium hydroxide (pH 10.0). After incubation for 1 h, 3 ml chloroform was added for extraction. The extracted sample was centrifuged at 3000 rpm for 5 min and the organic layer separated and evaporated by nitrogen gas. The residue was dissolved in 1 ml of hexane-isopropanol (93:7, v/v, containing 0.5% DEA).
The optimized method for Rac-HCQ and two enantiomers was validated according to ICH guidelines30. The factors for the examination included accuracy, precision (repeatability and intermediate precision), sensitivity (limit of quantification and limit of detection) and specificity.
Calibration standards of Rac-HCQ at 1, 5, 10, 15, 20, 25 μg/ml, and the concentrations of each enantiomer at half of these were freshly prepared by dilution of stock solution into the mobile phase, and the mobile phase without HCQ was used as blank. The theoretical concentrations of Rac-HCQ (free base) were 0.77, 3.87, 7.74, 11.61 and 15.48 μg/ml. Plots of the concentrations of Rac-HCQ and two enantiomers versus peak area were generated, and the equations of linear regression were applied to the determination of the concentrations of racemate and enantiomers respectively.
The sensitivity of the method was assessed by identifying the limit of quantification (LOQ). Residual standard deviation (σ) method was implemented to predict the values of LOQ and limit of detection (LOD) by following formula (1) and (2) and the precision was established at these predicted levels30.The specificity of the method was verified by comparison of blank and sample solutions.
where σ = Residual standard deviation of response; S = Slope of the calibration curve.
The accuracy and precision of this method were evaluated by same day (n=3) and different days (n=3) using Rac-HCQ (free base) at the concentrations of 10, 15 and 20 μg/ml, and the concentrations corresponding to each enantiomer were half of these concentrations. The results are shown by relative standard deviation (R.S.D).