• ALANKAR SHRIVASTAVA Department of Pharmaceutical Quality Assurance, KIET School of Pharmacy, KIET Group of Institutions, Ghaziabad



Hydroxychloroquine, Spectrophotometry, Chromatography methods, Electroanalytical methods, Methods for determination of Hydroxychloroquine, Determination of hydroxychloroquine


Hydroxychloroquine (HCQ) is classified under the class of drugs called antimalarials. This is used for preventions and treatment of malaria. HCQ is also used in the treatment of DLE (Discoid Lupus Erythematosus) or SLE (Systemic Lupus Erythematosus) and RA (Rheumatoid Arthritis). Recently, this drug attracts its attention by scientists of all of the worlds for its potential activity in the improvement of conditions of covid patients. There are many clinical trials are under process to prove its activity against this dangerous virus. The presented review describes different analytical procedures for the analysis of HCQ in various components available in the currently available literature. The paper will be certainly helpful for the scientists and researchers engaged in research, especially in the development of formulation or quality assurance of HCQ. The results of any clinical trial also includes the determination of drug in body fluids for interpretation of data. The analytical methods described here are explained in three parts; spectrophotometry, chromatography and other (including capillary electrophoresis and electroanalytical methods)


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Author Biography

ALANKAR SHRIVASTAVA, Department of Pharmaceutical Quality Assurance, KIET School of Pharmacy, KIET Group of Institutions, Ghaziabad

Associate Professor,

Department of Quality Assurance


Cannella AC, O’Dell JR. Traditional DMARDs. Kelley and Firestein’s Textbook of Rheumatology; 2017. p. 958–82. DOI:10.1016/b978-0-323-31696-5.00061-9.

British Pharmacopoeia. Available from: https://www. [Last assessed on 18 Apr 2020].

Jordan P, Brookes JG, Nikolic G, Le Couteur DG. Hydroxychloroquine overdose: toxicokinetics and management. J Toxicol Clin Toxicol 1999;37:861-4.

National Centre for Biotechnology Information. PubChem Database. Hydroxychloroquine, CID=3652. Available from: [Last accessed on 18 Apr 2020].

Collins KP, Jackson KM, Gustafson DL. Hydroxychloroquine: a physiologically-based pharmacokinetic model in the context of cancer-related autophagy modulation. J Pharmacol Exp Ther 2018;365:447-59.

Fox RI. Mechanism of action of hydroxychloroquine as an antirheumatic drug. Semin Arthritis Rheum 1993;23(2 Suppl 1):82-91.

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;6:16.

WHO Director-General’s opening remarks at the media briefing on Covid-19; 2020. Available from: dg/speeches/detail/who-directorgeneral-s-opening-remarks-atthe-media-briefing-on-covid-19 [last accessed on 11 Mar 2020].

Pedersen SF, Ho YC. SARS-CoV-2: a storm is raging. J Clin Invest 2020;13pii:137647. DOI:10.1172/JCI137647.

Yavuz S, Unal S. Antiviral treatment of Covid-19. Turk J Med Sci 2020;50:611-9.

LiverTox: Clinical and research information on drug-induced liver injury. Bethesda (MD): National Institute of Diabetes and Digestive and Kidney Diseases; 2012.

Sinha N, Balayla G. Hydroxychloroquine and covid-19. Postgrad Med J 2020. DOI:10.1136/postgradmedj-2020-137785.

Saqrane S, El Mhammedi MA. Review on the global epidemiological situation and the efficacy of chloroquine and hydroxychloroquine for the treatment of Covid-19. New Microbes New Infect 2020;35:100680.

Taghizadeh Hesary F, Akbari H. The powerful immune system against powerful Covid-19: A hypothesis. Med Hypotheses 2020;140:109762.

Krejner Bienias A, Grzela K, Grzela T. Do novel drugs for diabetes will help in COVID‐19? Another brick in the wall? 2020. DOI:10.1111/1753-0407.13050.

Inamo J. How should we overcome the threat by the pandemic of 2019-nCoV? Epidemic simulation using the SIRS model. Clin Rheumatol 2020;9:1-3.

Singh AK, Singh A, Shaikh A, Singh R, Misra A. Chloroquine and hydroxychloroquine in the treatment of Covid-19 with or without diabetes: a systematic search and a narrative review with a special reference to India and other developing countries. Diabetes Metab Syndr 2020;14:241-6.

Rathi S, Ish P, Kalantri A, Kalantri S. Hydroxychloroquine prophylaxis for COVID-19 contacts in India. Lancet Infect Dis 2020. DOI:10.1016/S1473-309930313-3.

Choudhary R, Sharma AK, Choudhary R. Potential use of hydroxychloroquine, ivermectin and azithromycin drugs in fighting Covid-19: trends, scope and relevance. New Microbes New Infect 2020:100684. DOI:10.1016/j.nmni.2020.100684.

Sinha N, Balayla G. Hydroxychloroquine and covid-19. Postgraduate Med J 2020. DOI:10.1136/postgradmedj-2020-137785.

McKee DL, Sternberg A, Stange U, Laufer S, Naujokat C. Candidate drugs against SARS-CoV-2 and Covid-19. Pharmacol Res 2020;157:104859.

Patil VM, Singhal S, Masand N. A systematic review on use of aminoquinolines for the therapeutic management of Covid-19: Efficacy, safety and clinical trials. Life Sci 2020. DOI:

Germer TA, Zwinkels JC, Tsai BK. Spectrophotometry accurate measurement of optical properties of materials. In: Experimental methods in the physical sciences. Elsevier Inc 2014;46:2-533.

Ferraz LRM, Santos FLA, Ferreira PA, Maia-Junior RTL, Rosa TA, Costa SPM, et al. Quality by design in the development and validation of analytical method by ultraviolet-visible spectrophotometry for quantification of hydroxychloroquine sulfate. Int J Pharm Sci Res 2014;5:4666-76.

Singh A, Kishora R, Sharma PK, Gupta R, Mondal N, Kumar S, et al. Development and validation of UV-spectrophotometric method for the estimation of hydroxychloroquine sulphate. Indian J Chem Technol 2016;23:237-9.

USP29–NF24 Page 1086.

Mehta SS, Patel MB. Development and validation of UV spectrophotometric method for the determination of hydroxychloroquine in bulk and tablet formulation. Pharma Sci Monitor 2012;3: Suppl 2:2959-67.

Poole CF. The essence of chromatography. First Edition. Elsevier Science; 2003. p. 2, 4.

Tett SE, Cutler DJ, Brown KF. High-performance liquid chromatographic assay for hydroxychloroquine and metabolites in blood and plasma, using a stationary phase of poly(styrene divinylbenzene) and a mobile phase at pH 11, with fluorimetric detection. J Chromatogr 1985;344:241-8.

Brown RR, Stroshane RM, Benziger DP. High-performance liquid chromatographic assay for hydroxychloroquine and three of its major metabolites, desethylhydroxychloroquine, desethylchloroquine and bidesethylchloroquine, in human plasma. J Chromatogr 1986;377:454-9.

Williams SB, Patchen LC, Churchill FC. Analysis of blood and urine samples for hydroxychloroquine and three major metabolites by high-performance liquid chromatography with fluorescence detection. J Chromatogr 1988;433:197-206. DOI:10.1016/s0378-434780598-8.

Iredale J, Wainer IW. Determination of hydroxychloroquine and its major metabolites in plasma using sequential achiral-chiral high-performance liquid chromatography. J Chromatogr 1992;573:253-8.

Fieger H, Iredale J, Wainer IW. Enantioselective determination of hydroxychloroquine and its major metabolites in urine and the observation of a reversal in the (+)/(-)-hydroxychloroquine ratio. Chirality 1993;5:65-70.

Yimin Wei, Gloria A Nygard, Shoukry KW Khalil. A HPLC method for the separation and quantification of the enantiomers of hydroxychloroquine and its three major metabolites. J Liq Chromatr 1994;17:3479-90.

Croes K, McCarthy PT, Flanagan RJ. Simple and rapid HPLC of quinine, hydroxychloroquine, chloroquine, and desethylchloroquine in serum, whole blood, and filter paper-adsorbed dry blood. J Anal Toxicol 1994;18:255-60.

Volin P. Simple and specific reversed-phase liquid chromatographic method with diode-array detection for simultaneous determination of serum hydroxychloroquine, chloroquine and some corticosteroids. J Chromatogr B Biomed Appl 1995;666:347-53.

Cardoso CD, Bonato PS. Enantioselective analysis of the metabolites of hydroxychloroquine and application to an in vitro metabolic study. J Pharm Biomed Anal 2005;37:703-8.

Wang LZ, Ong RY, Chin TM, Thuya WL, Wan SC, Wong AL, et al. Method development and validation for rapid quantification of hydroxychloroquine in human blood using liquid chromatography-tandem mass spectrometry. J Pharm Biomed Anal 2012;61:86-92.

Soichot M, Megarbane B, Houze P, Chevillard L, Fonsart J, Baud FJ, 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 Pharm Biomed Anal 2014;100:131-7.

Singh A, Roopkishora, Singh CL, Gupta R, Kumar S, Kumar M. Development and validation of reversed-phase high performance liquid chromatographic method for hydroxychloroquine sulphate. Indian J Pharm Sci 2015;77:586-91.

Qu Y, Noe G, Breaud AR, Vidal M, Clarke WA, Zahr N, et al. Future sci OA. 2015;1:FSO26.

Armstrong N, Richez M, Raoult D, Chabriere E. Simultaneous UHPLC-UV analysis of hydroxychloroquine, minocycline and doxycycline from serum samples for the therapeutic drug monitoring of Q fever and Whipple’s diseases. J Chromatogr B: Anal Technol Biomed Life Sci 2017;1060:166-72.

Qu Y, Brady K, Apilado R, O'Malley T, Reddy S, Chitkara P, et al. Capillary blood collected on volumetric absorptive microsampling (VAMS) device for monitoring hydroxychloroquine in rheumatoid arthritis patients. J Pharm Biomed Anal 2017;140:334-41.

Charlier B, Pingeon M, Dal Piaz F, Conti V, Valentini G. Development of a novel ion-pairing HPLC-FL method for the separation and quantification of hydroxychloroquine and its metabolites in whole blood. Biomed Chromatogr 2018;32:e4258.

Chhonker YS, Sleightholm RL, Li J, Oupický D, Murry DJ. Simultaneous quantitation of hydroxychloroquine and its metabolites in mouse blood and tissues using LC-ESI-MS/MS: An application for pharmacokinetic studies. J Chromatogr B: Anal Technol Biomed Life Sci 2018;1072:320-7.

El-Koussi WM, Atia NN, Saleh GA, Hammam N. Innovative HPTLC method for simultaneous determination of ternary mixture of certain DMARDs in real samples of rheumatoid arthritis patients: an application of quality by design approach. J Chromatogr B: Anal Technol Biomed Life Sci 2019;1124:135-45.

Shrivastava A. Analytical methods for venlaflaxine hydrochloride and metabolites determinations in different matrices. Syst Rev Pharm 2012;3:42-50.

Arguelho ML, Andrade JF, Stradiotto NR. Electrochemical study of hydroxychloroquine and its determination in plaquenil by differential pulse voltammetry. J Pharm Biomed Anal 2003;32:269-75.

Dickow Cardoso C, Polisel Jabor VA, Sueli Bonato P. Capillary electrophoretic chiral separation of hydroxychloroquine and its metabolites in the microsomal fraction of liver homogenates. Electrophoresis 2006;27:1248-54.

de Oliveira AR, Cardoso CD, Bonato PS. Stereoselective determination of hydroxychloroquine and its metabolites in human urine by liquid-phase microextraction and CE. Electrophoresis 2007;28:1081-91.

Khoobi A, Ghoreishi SM, Behpour M, Shaterian M, Salavati Niasari M. Design and evaluation of a highly sensitive nanostructure-based surface modification of glassy carbon electrode for electrochemical studies of hydroxychloroquine in the presence of acetaminophen. Colloids Surf B 2014;123:648-56.

Deroco PB, Vicentini FC, Oliveira GG, Rocha Filho RC, Fatibello Filho O. Square-wave voltammetric determination of hydroxychloroquine in pharmaceutical and synthetic urine samples using a cathodically pretreated boron-doped diamond electrode. J Electroanal Chem 2014;719:19-23.

Khalil MM, Issab YM, El Sayed GA. Modified carbon paste and polymeric membrane electrodes for the determination of hydroxychloroquine sulfate in pharmaceutical preparations and human urine. RSC Adv 2015;5:83657-67.

Khalil MM, Issab YM, El Sayed GA. Development of a new coated graphite electrode for hydroxychloroquine sulfate determination in pharmaceutical preparations and human urine. Inter J Adv Res 2015;3:592-603.

Rolain JM, 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.

Dousa KM, Malavade SS, Furin J, Gripshover B, Hatszegi M, Hojat L, et al. SARS-CoV-2 infection in a patient on chronic hydroxychloroquine therapy: implications for prophylaxis. ID Cases 2020:e00778. DOI:10.1016/j. idcr.2020.e00778.

Dabic D, Babic S, Skoric I. The role of photodegradation in the environmental fate of hydroxychloroquine. Chemosphere 2019;230:268-77.

Wei Y, Nygard GA, Ellertson SL, Khalil SK. Stereoselective disposition of hydroxychloroquine and its metabolite in rats. Chirality 1995;7:598-604.

Ducharme J, Fieger H, Ducharme MP, Khalil SK, Wainer IW. Enantioselective disposition of hydroxychloroquine after a single oral dose of the racemate to healthy subjects. Br J Clin Pharmacol 1995;40:127-33.

Crowley TE. High-performance liquid chromatography. Purification and Characterization of Secondary Metabolites; 2020. p. 49–58.

Sadiq NW, Beauchemin D. Liquid chromatography. Sample Introduction Systems in ICPMS and ICPOES; 2020. p. 213–54.

de Koster CG, Schoenmakers PJ. History of liquid chromatography-mass spectrometry couplings. Hyphenations of Capillary Chromatography with Mass Spectrometry; 2020. p. 279-95.

Tonnesen H, Grislingaas AL, Woo SO, Karlsen J. Photochemical stability of antimalarials. I. Hydroxychloroquine. Inter J Pharm 1988;43:215-9.

Saini B, Bansal G. Characterization of four new photodegradation products of hydroxychloroquine through LC-PDA, ESI-MSn and LC-MS-TOF studies. J Pharm Biomed Anal 2013;84:224-31.

Dabic D, Babic S, Skoric I. The role of photodegradation in the environmental fate of hydroxychloroquine. Chemosphere 2019;230:268-77.



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