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Licensed Unlicensed Requires Authentication Published by De Gruyter September 13, 2020

Validation of a liquid chromatography tandem mass spectrometry method for the simultaneous determination of hydroxychloroquine and metabolites in human whole blood

  • Donna Austin , Catharine John , Beverley J Hunt and Rachel S. Carling ORCID logo EMAIL logo



Hydroxychloroquine (HCQ) is an anti-malarial and immunomodulatory drug reported to inhibit the Corona virus, SARS-CoV-2, in vitro. At present there is insufficient evidence from clinical trials to determine the safety and efficacy of HCQ as a treatment for COVID-19. However, since the World Health Organisation declared COVID-19 a pandemic in March 2020, the US Food and Drug Administration issued an Emergency Use Authorisation to allow HCQ and Chloroquine (CQ) to be distributed and used for certain hospitalised patients with COVID-19 and numerous clinical trials are underway around the world, including the UK based RECOVERY trial, with over 1000 volunteers. The validation of a liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the simultaneous determination of HCQ and two of its major metabolites, desethylchloroquine (DCQ) and di-desethylchloroquine (DDCQ), in whole blood is described.


Blood samples were deproteinised using acetonitrile. HCQ, DCQ and DDCQ were chromatographically separated on a biphenyl column with gradient elution, at a flow rate of 500 μL/min. The analysis time was 8 min.


For each analyte linear calibration curves were obtained over the concentration range 50-2000 μg/L, the lower limit of quantification (LLOQ) was 13 μg/L, the inter-assay relative standard deviation (RSD) was <10% at 25, 800 and 1750 μg/L and mean recoveries were 80, 81, 78 and 62% for HCQ, d4-HCQ, DCQ and DDCQ, respectively.


This method has acceptable analytical performance and is applicable to the therapeutic monitoring of HCQ, evaluating the pharmacokinetics of HCQ in COVID-19 patients and supporting clinical trials.

Corresponding author: Rachel Carling, Biochemical Sciences, Viapath, Guys & St Thomas’ NHSFT, 4th Floor, North Wing, St Thomas’ Hospital, Westminster Bridge Road, London, SE1 7EH, UK; and GKT Medical School, Kings College London, London, UK, E-mail:

  1. Research funding: None declared.

  2. Author contributions: BJH and RSC conceived the study. DA performed the laboratory work and acquired the data. DA, KJ and RSC analysed and interpreted the data. RSC was the lead author of the manuscript. All authors participated in drafting the article and revising it critically for important intellectual content. All authors have accepted responsibility for the entire content of this manuscript and approved its submission.

  3. Competing interests: Authors state no conflict of interest.

  4. Informed consent: Informed consent was obtained from all individuals included in this study.

  5. Ethical approval: Research involving human subjects complied with all relevant national regulations, institutional policies and is in accordance with the tenets of the Helsinki Declaration (as revised in 2013), and has been approved by the authors’ Institutional Review Board.


1. Kalia, S, Dutz, J. New concepts in antimalarial use and mode of action in dermatology. Dermatol Ther 2007;20:160–74. in Google Scholar

2. Williams, H, Egger, M, Singer, J, Willkens, R, Kalunian, K, Clegg, D, et al. Comparison of hydroxychloroquine and placebo in the treatment of the arthropathy of mild systemic lupus erythematosus. J Rheumatol 1994;21:1457–62.Search in Google Scholar

3. Alarcon, G, McGwin, G, Bertoli, A, Fessler, B, Calvo-Alen, J, Bastian, HM, et al. Effect of hydroxychloroquine on the survival of patients with systemic lupus erythematosus: data from LUMINA, a multiethnic US cohort (LUMINA L). Ann Rheum Dis 2007;66:1168–72. in Google Scholar

4. Ruiz-Irastorza, G, Ramos-Casals, M, Brito-Zeron, P, Khamashta, M. Clinical efficacy and side effects of antimalarials in systemic lupus erythematosus: a systematic review. Ann Rheum Dis 2010;69:20–8. in Google Scholar

5. Kuhn, A, Ruland, V, Bonsmann, G. Cutaneous lupus erythematosus: update of therapeutic options. Part I. J Am Acad Dermatol 2011;65:179–93. in Google Scholar

6. Rolain, J, Colson, P, Raoult, D. Recycling of chloroquine and its hydroxyl analogue to face bacterial, fungal and viral infections in the 21st century. Int J Antimicrob Agents 2007;30:297–308. in Google Scholar

7. Ben-Zvi, I, Kivity, S, Langevitz, P, Shoenfeld, Y. Hydroxychloroquine: from malaria to autoimmunity. Clin Rev Allergy Immunol 2012;42:145–53. in Google Scholar

8. Keyaerts, E, Vijgen, L, Maes, P, Neyts, J, Van Ranst, M. In vitro inhibition of severe acute respiratory syndrome coronavirus by chloroquine. Biochem Biophys Res Commun 2004;323:264–8. in Google Scholar

9. Vincent, M, Bergeron, E, Benjannet, S, Erickson, B, Rollin, P, Ksiazek, T, et al. Chloroquine is a potent inhibitor of SARS coronavirus infection and spread. Virol J 2 2005;2. in Google Scholar

10. Keyaerts, E, Li, S, Vijgen, L, Rysman, E, Verbeeck, J, Van Ranst, M, et al. Antiviral activity of chloroquine against human coronavirus OC43 infection in newborn mice. Antimicrob Agents Chemother 2009;53:3416–21. in Google Scholar

11. Savarino, A, Boelaert, J, Cassone, A, Majori, G, Cauda, R. Effects of chloroquine on viral infections: an old drug against today’s diseases?. Lancet Infect Dis 2003;3:722–7. in Google Scholar

12. Dyall, J, Coleman, C, Hart, B, Venkataraman, T, Holbrook, M, Kindrechuk, J, et al. Repurposing of clinically developed drugs for treatment of Middle East respiratory syndrome coronavirus infection. Antimicrob Agents Chemother 2014;58:4885–93. in Google Scholar

13. Dyall, J, Gross, R, Kindrachuk, J, Johnson, R, Olinger, G, Hensley, L, et al. Middle East respiratory syndrome and severe acute respiratory syndrome: current therapeutic options and potential targets for novel therapies. Drugs 2017;77:1935–66. in Google Scholar

14. Principi, N, Esposito, S. Chloroquine or hydroxychloroquine for prophylaxis of COVID-19. Lancet Infect Dis 2020;20:S1473–3099. 30296-9. in Google Scholar

15. Alhazzani, W, Møller, M, Arabi, YM, Loeb, M, Gong, MN, et al. Surviving Sepsis Campaign: guidelines on the management of critically ill adults with Coronavirus Disease 2019 (COVID-19). Intensive Care Med 2020;46:854–87. in Google Scholar

16. Cucinotta, D, Vanelli, M. WHO declares COVID-19 a pandemic. Acta Biomed 2020;91:157–60. in Google Scholar

17. World Health Organization. Coronavirus disease 2019 (COVID-19) situation report – 91. 2020. Available from: [Accessed 21 Apr 2020].Search in Google Scholar

18. Rosa, S, Santos, W. Clinical trials on drug repositioning for COVID-19 treatment. Rev Panam Salud Públic 2020;44:1–13. in Google Scholar

19. Lythgoe, M, Middleton, P. Ongoing clinical trials for the management of the COVID-19 pandemic. Trends Pharmacol Sci 2020;41:363–382. in Google Scholar

20. Luo, P, Liu, Y, Qiu, L, Liu, X, Liu, D, Li, J. Tocilizumab treatment in COVID‐19: a single center experience. J Med Virol 2020:1–5. in Google Scholar

21. Gautret, P, Lagier, JC, Parola, P, Hoang, V, Meddeb, L, Mailhe, M, et al. Hydroxychloroquine and azithromycin as a treatment of COVID-19: results of an open label non-randomized clinical trial. Int J Antimicrob Agents 2020;56:105949. in Google Scholar

22. Villar, J, Ferrando, C, Martínez, D, Ambrós, A, Muñoz, T, Soler, J, et al. Dexamethasone treatment for the acute respiratory distress syndrome: a multicentre, randomised controlled trial. Lancet Respir Med 2020;8:267–76. in Google Scholar

23. Liu, J, Cao, R, Xu, M, Wang, X, Zhang, H, Hu, H, et al. Hydroxychloroquine, a less toxic derivative of chloroquine, is effective in inhibiting SARS-CoV-2 infection in vitro. Cell Discovery 2020;6:1–4. in Google Scholar

24. Chen, Z, Hu, J, Zhang, Z, Jiang, S, Han, S, Yan, D. Efficacy of hydroxychloroquine in patients with COVID-19: results of a randomized clinical trial. Int J Infect Dis 2020;97:396–403. in Google Scholar

25. Yao, X, Ye, F, Zhang, M, Cui, C, Huang, B, Niu, P, et al. Vitro antiviral activity and projection of optimized dosing design of hydroxychloroquine for the treatment of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Clin Infect Dis 2020;71:732–9. in Google Scholar

26. Taccone, F, Gorham, J, Vincent, J. Hydroxychloroquine in the management of critically ill patients with COVID-19: the need for an evidence base. Lancet Respir Med 2020;8:539–41. in Google Scholar

27. Ferner, R, Aronson, J. Chloroquine and hydroxychloroquine in covid-19: use of these drugs is premature and potentially harmful. BMJ 2020;369. in Google Scholar PubMed

28. Gbinigie, K, Frie, K. Should chloroquine and hydroxychloroquine be used to treat COVID-19? A rapid review. BJGP Open 2020. in Google Scholar PubMed PubMed Central

29. US Food & Drug Administration. Emergency use authorisation. Available from: [Accessed 27 Apr 2020].Search in Google Scholar

30. Lenzer, J. Covid-19: US gives emergency approval to hydroxychloroquine despite lack of evidence. BMJ 2020;369. m1335 2020;369. in Google Scholar PubMed

31. O’Laughlin, J, Mehta, P, Wong, B. Case Rep Cardiol 2016. in Google Scholar PubMed PubMed Central

32. Chen, C, Wang, F, Lin, C. Chronic hydroxychloroquine use associated with QT prolongation and refractory ventricular arrhythmia. Clin Toxicol 2006;44:173–5. in Google Scholar PubMed

33. Marmor, M, Kellner, U, Lai, T, Lyons, J, Mieler, W. Revised recommendations on screening for chloroquine and hydroxychloroquine retinopthy. Opthalmology 2011;118:415–22. in Google Scholar PubMed

34. Marmor, M, Kellner, U, Lai, T, Melles, R, Mieler, W. Recommendations on screening for chloroquine and hydroxychloroquine retinopthy (2016 revision). Opthalmology 2016;123:1386–94. in Google Scholar

35. Tétu, P, Hamelin, A, Lebrun-Vignes, B, Soria, A, Barbaud, A, Francès, C, et al. Prevalence of hydroxychloroquine-induced side-effects in dermatology patients: a retrospective survey of 102 patients. Ann Dermatol Venereol 2018;145:395–404. in Google Scholar

36. Garcia-Cremades, M, Solans, B, Hughes, E, Ernest, J, Wallender, E, Aweeker, F, et al. Optimizing hydroxychloroquine dosing for patients with COVID-19: an integrative modelling approach for effective drug repurposing. Clin Pharmacol Therapeut 2020;108:253–63. in Google Scholar

37. Balevic, S, Green, T, Clowse, M, Eudy, A, Schanberg, L, Cohen-Wolkowiez, M. Pharmacokinetics of hydroxychloroquine in pregnancies with rheumatic diseases. Clin Pharmacokinet 2019;58:525–33. in Google Scholar

38. Munster, T, Gibbs, J, Shen, D, Baethge, BA, Botstein, GR, Caldwell, J, et al. Hydroxychloroquine concentration–response relationships in patients with rheumatoid arthritis. Arthritis & Rheumatology 2002;46:1460–6. in Google Scholar

39. Collins, K, Jackson, M, Gustafson, D. Hydroxychloroquine: a physiologically-based pharmacokinetic model in the context of cancer-related autophagy modulation. J Pharmacol Exp Therapeut 2018;365:447–59. 2018; 365(3): 447-459. in Google Scholar

40. Petri, M, Elkhalifa, M, Li, J, Magder, L, Goldman, D. Hydroxychloroquine blood levels predict hydroxychloroquine retinopathy. Arthritis & Rheumatology 2020;72:131–7. in Google Scholar

41. Williams, S, Patchen, L, Churchill, F. Analysis of blood and urine samples for hydroxychloroquine and three major metabolites by high-performance liquid chromatography with fluoresence detection. J Chromatogr B Biomed Sci Appl 1988;433:197–206. in Google Scholar

42. Tett, S, Cutler, D, Brown, K. High performance liquid chromatography assay for hydroxychloroquine and metabolites in blood and plasma using a stationary phase of polystyrene divinylbenzene and a mobile phase at pH11 with fluorometric detection. J Chromatogr B Biomed Sci Appl 1985;344:241–8. in Google Scholar

43. Wang, L, Ong, R, Chin, T, Thuya, W, Wan, S, Wong, A, et al. Method development and validation for rapid quantification of hydroxychloroquine in human blood using liquid chromatography–tandem mass spectrometry. J Pharmaceut Biomed Anal 2012;61:86–92. in Google Scholar PubMed

44. Chambliss, A, Füzéry, A, Clarke, W. Quantification of hydroxychloroquine in blood using turbulent flow liquid chromatography-tandem mass spectrometry (TFLC-MS/MS). Methods Mol Biol 2016;1383:177–84. in Google Scholar PubMed

45. Soichot, M, Mégarban, B, Houzé, P, Chevillard, L, Fonsart, J, Baud, F, et al. Development, validation and clinical application of a LC-MS/MS method for the simultaneous quantification of hydroxychloroquine and its active metabolites in human whole blood. J Pharmaceut Biomedical 2014;100:131–7. in Google Scholar PubMed

46. Food and Drug Administration. Guidance forindustry: bioanalytical method validation. Rockville, MD: US Department of Health and Human Services, FDA, Center for Drug Evaluation and Research and Center for Veterinary Medicine; 2018. in Google Scholar

47. Matuszewski, B, Constanzer, M, Chavez-Eng, C. Strategies for the assessment of matrix effect in quantitative bioanalytical methods based on HPLC-MS/MS. Anal Chem 2003;75:3019–30. in Google Scholar PubMed

Received: 2020-04-28
Accepted: 2020-08-29
Published Online: 2020-09-13

© 2020 Walter de Gruyter GmbH, Berlin/Boston

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