Hydroxychloroquine (2-[[4-[(7-Chloroquinolin-4-yl) amino]pentyl](ethyl) amino]-ethanol, HCQ), an effective anti-malarial drug, has been applied in the clinics for potential treatment of severe coronavirus disease 2019 (COVID-19). Although the clinical benefits of HCQ require extensive clinical data to confirm, the existence of a chiral center in the molecule to possess two optical isomers suggests that there might be an enantiomeric difference on the treatment of COVID-19. Due to poor resolution and the inability of quantification by previously reported methods for the analysis of HCQ enantiomers, it is necessary to develop an analytical method to achieve baseline separation for quantitative and accurate determination of the enantiomeric purity in order to compare the efficacy and toxicity profiles of different enantiomer. In this study, we developed and validated an accurate and reproducible normal phase chiral HPLC method for the analysis of two enantiomers of HCQ, and the method was further evaluated with biological samples. With this newly developed method, the relative standard deviation of all analytes was lower than 5%, and the limit of quantification was 0.27 μg/ml, 0.34 μg/ml and 0.20 μg/ml for racemate, R- and S-enantiomer, respectively. The present method provides an essential analytical tool for preclinical and clinical evaluation of HCQ enantiomers for potential treatment of COVID-19.
Research Article
Development of a chiral HPLC method for the separation and quantification of hydroxychloroquine enantiomers
https://doi.org/10.21203/rs.3.rs-56796/v2
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Hydroxychloroquine
chiral HPLC
COVID-19
enantiomeric purity
rat plasma
As severe coronavirus disease 2019 (COVID-19) pandemic progresses, effective therapeutics are urgently needed to ease the outbreak and save more lives1-5. In addition to the anti-malarial use, hydroxychloroquine (HCQ) has been utilized for the treatment of systemic lupus erythematosus6,7, rheumatoid arthritis8, primary Sjogren's syndrome and other diseases. Based on in vitro evidence of inhibiting the replication of HIV-19,10 and COVID-19 virus11-14, HCQ has been thought to be effective in clinical settings to combat COVID-19 pandemic. However, the results of various clinical trials of using HCQ for COVID-19 have been contradictory15-19, due mainly to the differences on infection stage and dosing. Meanwhile, HCQ exhibits serious side effects, especially cardiac and retinal toxicities20-25. On the other hand, chloroquine, a structural analogue of HCQ, has recently been reported to be beneficial for reducing the length of hospital stay and postponing the deterioration of COVID-19 infected patients26. Given the structural similarity, same mode of action and less toxic, HCQ has drawn significant attention for its clinical usefulness for the treatment of COVID-19, and several large-scale multicenter clinical trials are in progress27.
Although HCQ was approved for clinical use as a racemate, the existence of a chiral center in HCQ may result in enantiomeric difference on the efficacy and toxicity. Indeed, previous metabolic studies have shown that the concentration of (R)-(-)-HCQ in blood was 1.6 to 2.9 times that of (S)-(+)-HCQ (Fig. 1A), and renal clearance rate of (S)-HCQ was approximately twice that of (R)-enantiomer20, strongly indicating that two enantiomers may exhibit different profiles of efficacy, toxicity and metabolic preference. Therefore, the comparison of HCQ enantiomers on the efficacy and toxicity for treating COVID-19 would clarify the differences and provide more effective and safer therapeutic option for this global health crisis.
To compare biological activities and therapeutic values of HCQ enantiomers, the separation of the enantiomers and the analysis of their optical purity are a prerequisite. Previously, two groups of researchers reported a similar analytical method for the analyses of HCQ enantiomers and their metabolites to investigate the enantiomeric preference on drug metabolism28,29. Unfortunately, in addition to the reproducibility issue, the reported resolution of HCQ enantiomers was very poor, which was unable to accurately and quantitatively analyze the actual values of respective enantiomer of HCQ. To achieve baseline separation and reproducibly analyze HCQ enantiomers, a reliable and accurate analytical method is obviously required for potential chiral switch. In the present study, we developed a Chiralpak AD-H based normal phase HPLC method after systematic optimization of the chromatographic conditions. After validation of the analytical method, we further applied this method for the determination of plasma concentration of HCQ enantiomers. This study provided a reliable, reproducible and quantitative method for the analysis of individual enantiomers of HCQ, which could be used for preclinical and clinical studies on the enantiomeric differences, developing single enantiomer as a potential new therapy for COVID-19 pandemic.
Irreproducibility and poor resolution by reported method
Previously, two studies have reported the separation of two enantiomers of HCQ with Chiralpak AD-RH column using n-hexane and isopropanol in the ratio of 92:8 (v/v) containing 0.1% diethylamine (DEA) as mobile phase. Firstly, Chiralpak AD-RH is a reversed phase column, which is not compatible to organic solvent-based mobile phase. Therefore, we recognized that the column used in the literatures could actually be Chiralpak AD-H. Next, when reported chiral HPLC conditions were used with Chiralpak AD-H column, the resolution of racemic HCQ was extremely poor as shown in Fig. 1B.
Unsuitability of chiral RP-HPLC columns
Typically, reversed phase chromatography exhibits several advantages, including better resolution, shaper peaks and aqueous based mobile phase. Thus, to develop a suitable HPLC method for baseline separation of the enantiomers of HCQ, different mobile phases and stationary phases for reversed phase HPLC (RP-HPLC) were employed and compared. Various reversed phase chiral columns, such as Chiralcel OJ-RH, Chiralcel OX-RH, Chiralcel OZ-RH, Chiralpak AD-RH, Chiralpak AS-RH, Chiralpak AY-RH, Chiralpak AZ-RH, were screened for possible resolution of HCQ enantiomers. Unfortunately, none of the columns at the experimental conditions gave meaningful resolution (data not shown). As a result, reversed phase chiral HPLC was unlikely useful for the separation and quantification of HCQ enantiomers, which might be due to the fact that the chiral center of HCQ is located in the middle of the molecule with a large steric hindrance to prevent distinct recognition and binding between different enantiomers and the stationary phase.
Optimization of chromatographic conditions with chiralpak AD-H column
Despite the poor resolution of HCQ enantiomers by reported method (Fig. 1B), little separation of the enantiomers was observed under normal phase conditions. Combined this observation and the failure of using reversed phase chiral columns, we therefore decided to optimize chromatographic conditions based on Chiralpak AD-H column.
Firstly, the influence of the ratio of n-hexane and isopropanol on the resolution was examined at 90:10, 90.5:9.5, 91:9, 91.5:8.5, 92:8, 92.5:7.5, 93:7, respectively. With the decrease of isopropanol, the retention of the enantiomers on the column was prolonged, and the separation was also better (data not shown), suggesting that the decrease of the content of polarity regulator (isopropanol) in the mobile phase increased the molecular interactions between the analytes and the stationary phase. To balance the resolution and analysis time, we chose the ratio of 93:7 (hexane-isopropanol) as the mobile phase for further optimization.
Since HCQ is a basic molecule containing multiple nitrogen atoms for ionization, the addition of a basic additive in the mobile phase could improve the resolution of the enantiomers. Thus, we compared two commonly used basic additives, diethylamine (DEA) and triethylamine (TEA). We found that the effects on the resolution by DEA were more obvious (data not shown), which is consistent with previous reports28,29. Consequently, we examined the influence on the resolution by different amounts of DEA. As shown in Supplementary Figure S1, there was no linear relationship between DEA concentration and resolution. However, a definitive trend was observed, in which the increase of DEA correlated with better resolution and 0.35% DEA in the mobile phase showed the best resolution under the experimental conditions. To precisely control DEA concentration close to the optimal value, we decided to add 0.5% DEA in n-hexane.
Given the importance of flow rate on the resolution, we next evaluated the effects of flow rate on the separation of HCQ enantiomers with established analytical method. Although slower flow rates generated broad peaks and longer analysis time, the decrease of flow rate from 1.0 ml/min to 0.8 ml/min resulted in a satisfactory resolution and reasonable analysis time, in which complete baseline separation of the enantiomers was achieved. Therefore, the optimized chromatographic conditions on Chiralpak AD-H column were the use of mobile phase system consisting n-hexane containing 0.5% DEA and isopropanol at the ratio of 93:7 (v/v) with the flow rate of 0.8 ml/min at 20 ℃. Under the optimal conditions, typical retention times of (R)-HCQ and (S)-HCQ were found to be 26 and 29 min with a resolution value of 2.08 (Fig. 2), which could accurately quantify the enantiomeric purity of two enantiomers based on their peak areas.
Validation of chiralpak AD-H based method
The linear regression curves between peak area and sample concentration were obtained for Rac-HCQ samples ranging from 1 to 25 μg/mL. Three standard curves of Rac-, S- and R-HCQ were plotted respectively, and the linear equations and the square values of the correlation coefficient (R2) were obtained to show excellent correlation (R2 >0.995, Supplementary Equation).
Using formula (1) and (2) in the section of Materials and Methods, we calculated the values of LOQ and LOD for racemate, S- and R-HCQ. The LOQ and LOD for racemate were 0.27 μg/ml and 0.09 μg/ml; the LOQ and LOD for S-HCQ were 0.20 μg/ml and 0.07 μg/ml; and the LOQ and LOD for R-HCQ were 0.34 μg/ml and 0.11 μg/ml. In addition, the specificity of this analytical method was verified by comparing plasma samples between blank and different optical isomers of HCQ (Fig. 3).
The accuracy and precision of the method were examined using linear equations. Table 1 summarizes the results of the samples tested on the same day, one day apart and four days apart. In all cases, intra-day (0 day) and inter-day (1 day) variability were lower than 5%. After 4 days, the variability compared to day 1 was above 5%. These data indicated that the present method is highly reliable and reproducible. On the other hand, the data also suggested that the samples are not suitable to store at room temperature for longer than 24 h at which the concentrations were remarkably higher than the beginning, due mainly to the volatility of the sampling solvent.
Table 1. Precision and accuracy of the method for the analysis of HCQ and its enantiomers.
|
Time/d |
Theoretical concentration |
Obtained concentration±SD* |
R.S.Da |
|
|
(μg/ml) |
(μg/ml) |
% |
||
|
0 |
Racb |
10 |
9.67±0.10 |
1.01 |
|
Rc |
9.58±0.12 |
1.25 |
||
|
Sd |
9.76±0.10 |
1.05 |
||
|
1 |
Rac |
10 |
10.48±0.41 |
3.90 |
|
R |
10.69±0.40 |
3.78 |
||
|
S |
10.69±0.19 |
1.76 |
||
|
Rac |
15 |
15.98±0.36 |
2.24 |
|
|
R |
15.70±0.86 |
2.15 |
||
|
S |
15.93±0.37 |
2.35 |
||
|
Rac |
20 |
21.93±0.43 |
1.98 |
|
|
R |
21.72±0.26 |
1.18 |
||
|
S |
22.15±0.61 |
2.77 |
||
|
4 |
Rac |
10 |
10.77±0.36 |
3.30 |
|
R |
10.88±0.33 |
3.01 |
||
|
S |
10.93±0.08 |
0.75 |
||
|
Rac |
15 |
16.18±0.82 |
5.07 |
|
|
R |
16.17±0.71 |
4.39 |
||
|
S |
16.18±0.93 |
5.74 |
||
*n=3; aRelative standard deviation; b Racemic HCQ; cR-enantiomer; dS-enantiomer.
In this study, a simple and accurate normal phase chiral HPLC method was developed for the enantiomeric separation and quantification of S- and R-HCQ. The baseline separation of two enantiomers allowed to quantitatively determine the concentrations of HCQ enantiomers, which was further validated for its accuracy, precision, sensitivity, specificity and reproducibility. The present method can be applied to quantitate HCQ enantiomers for pharmacokinetic and toxicokinetic studies, which could be useful to thoroughly assess and compare the efficacy and toxicity profiles of HCQ enantiomers in animals and patients. More importantly, the present method will be a valuable tool to assist the development of potential therapeutics for the treatment of COVID-19.
Chemicals
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.
Sample preparations
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).
Method validation
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).
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Acknowledgements
This work was supported by the grants of the MOST of China (2018YFA0902000, 2019ZX09721-001-04-01), the National Natural Science Foundation of China (No. 21778076), the “111 Project” from the Ministry of Education of China and the State Administration of Foreign Expert Affairs of China (No. 111-2-07) and the “Double First-Class” University project (CPU2018GY36).
Author contributions
YC conceived, designed and supervised the study and wrote the manuscript; XX designed and performed the experiments, analyzed the data and wrote the manuscript; KW analyzed data and interpreted the results; TT and JF performed experiments.
Additional information
Supplementary information to this article can be found online.
Competing Interests: The authors declare that they have no known competing interests.
- SupplementaryInformation.docx
Supplementary Information for
Development of a chiral HPLC method for the separation and quantification of hydroxychloroquine enantiomers
- FigureS1.jpg
Effects of DEA concentration on the enantiomeric resolution of Rac-HCQ. Chromatographic conditions: Chiralpak AD-H (4.6 mm×150 mm, 5 μm particle size); n-hexane-isopropanol (93:7, v/v) with various concentrations of DEA as mobile phase at a flow rate of 1.0 ml/min with UV detection at 343 nm at 25℃.
