Ibn Al-Haitham Jour. for Pure & Appl. Sci. 53 (1)2022 50 This work is licensed under a Creative Commons Attribution 4.0 International License. A Subject Review on a Some Analytical Methods for the Determination of Chloroquine and Hydroxychloroquine Drugs Department of Chemistry, College of Education for Pure Sciences/ Ibn Al-Haitham, University of Baghdad, Iraq. Abstract Chloroquine and Hydroxychloroquine drugs are widely prescribed for malaria disease. Since the end of 2019, humans have been under threat due to a disease called (COVID-19), which was first reported in China. Many methodical approaches have been reported to quantify chloroquine and hydroxychloroquine in blood, urine, plasma, serum, and pharmaceutical dosage form. Some of these techniques are spectrophotometry, liquid chromatography with a mass detector, gas chromatography, and ultra-performance, high- performance liquid chromatography (HPLC), in addition to electrochemical methods. This literature review discusses various analytical methods for the determining hydroxychloroquine and chloroquine. Keywords: Review, Hydroxychloroquine, Chloroquine, Analytical Methods. 1.Introduction Almost all types of human malaria have been commonly treated with Chloroquine (CQ) and Hydroxychloroquine (HCQ) (Figure1) [1]. Nevertheless, these medicines have been used to treat many diseases like hepatic amoebiasis, rheumatoid arthritis, and lupus erythematosus [2]. Since 1934 a chloroquine was first prescribed for the treatment of malaria; later, in 1955, hydroxychloroquine was introduced and became favored because of its safety profile [3]. Hydroxychloroquine has been considered as prospective as an active Ibn Al Haitham Journal for Pure and Applied Science Journal homepage: http://jih.uobaghdad.edu.iq/index.php/j/index Doi: 10.30526./35.1.2799 Article history: Received 24 , October, 2021, Accepted 5,December, 2021, Published in January 2022. Ali Khalil Mahmood ali.khalil.mahmood@gmail.com Khalid Waleed S. Al-Janabi Khalid.Janabi@gmail.com https://creativecommons.org/licenses/by/4.0/ mailto:ali.khalil.mahmood@gmail.com mailto:Khalid.Janabi@gmail.com Ibn Al-Haitham Jour. for Pure & Appl. Sci. 35(1)2022 51 remedy toward COVID 19 [4]. CQ is manufactured as tablets (administered orally) as its phosphate 500 mg for each tablet, while HCQ is manufactured as its sulfate with an oral dose of 200 mg [5]. In this literature review, various analytical methods are demonstrated for the quantitative determination of chloroquine and hydroxychloroquine; some of these techniques are reported, and it is given in Table 1. Figure 1: Chemical structure of A) Chloroquine, B) Hydroxychloroquine Table 1: Some analytical methods for the determination of chloroquine and hydroxychloroquine. Drug Methods sample Results Ref. No. Chloroquine high-performance liquid chromatography plasma and urine λ max = 254 and 340nm. linearity equle to (10 nmol.L-1) for uv detection), (0.5 nmol.L-1 for fluorescence detection), RSD = 12%, r2= 0.974-0.996. [6] Chloroquine Photometric, ion-pair extraction urine λ max = 410 nm. linearity up to 120 mg.l-1 [7] Chloroquine High performance thin layer chromatographic biological fluids LOD = 0.01 m.mol.L-1, extraction efficiency = 76 ±7% , silica gel plates, using toluene/diethylamine (9:1) [8] Chloroquine Column liquid chromatography human plasma, red blood cells, Urine and Blood mobile phase of phosphate buffer (pH 3.0)-acetonitrile (88:120) λ max = 340 nm, %Recovery =75% [9] Chloroquine High performance thin layer chromatographic urine Silica-gel of 60 plates (10X10)cm had impregnated fluorophore LOD = 0.25 micrograms.mL-1, Rf = 0.27 [10] Chloroquine Colorimetric and thin- layer chromatographic methods urine concentrations up to (32 mug.ml-1) LOD for modification I 0.3 mug.ml- 1, while that for modification II is 1 mug.ml-1 [11] Chloroquine colorimetric methods urine linear range up to 8 micrograms/ml. LOD =1µ.ml-1. LOD for its metabolites = 2 µ/ml [12] Chloroquine High-performance liquid chromatography biological fluids Excit. wavelength 325nm, emi. wavelength 375 nm, mobile phase contains a mixture of acetonitrile with methanol-25% and ammonia (92.7:7.5, v/v) LOD = 5ng.mL-1 [13] Chloroquine “reversed-phase ion- pair high-performance liquid chromatographic” biological fluids λ max = 254, mobile phase water - acetonitrile - methanol (78:28:4) plus 0.5M ammonium formate with 0.075M perchloric acid. nm, LOQ = 6ng.mL-1 [14] Chloroquine and Hydroxychloroquine High-performance liquid chromatography Serum and Blood Excit. wavelength, 215 nm. LOQ for CQ =(0.005-0.01)mg.L-1. LOQ for HOQ= 0.05 mg.L-1, [15] Ibn Al-Haitham Jour. for Pure & Appl. Sci. 35(1)2022 52 sulfophenylpropyl -modified silica column, volumes (50-200 micro) Chloroquine liquid chromatographic method biological fluids Linearity (0-200) ng.mL-1 (for plasma), (0- 3) ng.m-1 (for urine), r2=0.990, CV= 4.8% in plasma and 4.4% in urine. [16] Chloroquine Flow injection fluorimetric method plasma Laser λ at 355 nm, “linearity = (25- 600) µg.L-1”, r2 = 0.999, RSD = 4.3% [17] Chloroquine High-performance liquid chromatography Blood For children Concentrations between 17 and 100 nmol.L-1 (25%), 100 to 499 nmol.L1- (14%) and 500 nmol.L-1 (13%) in Young age> [18] Chloroquine laser-induced photochemical reaction and fluorescence Plasma λ max =355nm using pulsed Nd:YAG laser Linearity = (25-600) µg.L-1, r2= 0.997, LOD=8µg.L-1, RSD =4.3%, intrinsic fluorescence = 7 times. [17] Chloroquine High-performance liquid chromatography biological samples λ max = 333nm, used, C(18) column, mobile phase (methanol phosphate) buffer pH 3 and perchloric acid (v/v) (250: 747.5 : 2.5) [19] Chloroquine High-performance liquid chromatography with fluorescence detection pharmaceuticals and biological fluids Excitation wavelength 230 nm, emission wavelength 375 nm, linearity up to 0.5 ng/microL,Kromasil, C18, 5 microm column, mobile phase methanol -acetonitrile-ammonium acetate, (45:15:40). Re.+ (90.7- 105.4)% [20] Hydroxychloroquine differential pulse voltammetry and spectrophotometric pharmaceutical formulations Electrochemical, LOD = 11.2µg/mL, RSD = 0.46%, spectrophotometric, λ max = 343nm, LOD = 0.1µg.mL-1, RSD = 0.36% [21] Chloroquine High-performance liquid chromatography plasma Linearity (0-1000) ng/ml, r2=0.9987, extracted with n-hexane, 10 microl aqueous layer [22] Chloroquine capillary-LC with native laser serum (He-Cd 325 nm) detector, micro HPLC-LIF injection volumes were 200. separation time 3 min, LOD = 1.9, Re. more than 95% accuracy <10% [23] Chloroquine reverse-phase liquid chromatography dried blood spots λ max = 254nm, linearity (150 – 2500), solid-phase extraction is C18 Bond Elut cartridge) LOQ=50 ng.mL-1, Cv =10.3% [24] Chloroquine refractometry and colorimetry pharmaceutical formulations λ max = 528nm linearity (10 – 30)µL, r2=0.946, Accuracy 92- 103%, Precision (7-20)% [25] Chloroquine charge–transfer formation pharmaceutical formulations λ max = 520nm, Linearity=0.8-5 mg.100mL-1 r2=0.998. [26] Chloroquine ion pair extraction pharmaceutical formulations and urine λ max = 420nm, Linearity=1.25- 8.75 µg.mL-1 €= 4.09x104.mol-1.cm-1, r2=0.9989, RSD = 0.27 [27] Chloroquine High-performance liquid chromatography “whole blood and finger-prick λ max =256nm, mobile phase (v:v:v) “diethylamine, acetonitrile and [28] Ibn Al-Haitham Jour. for Pure & Appl. Sci. 35(1)2022 53 capillary blood” methanol” (20:55:25,), LOQ =(25- 50) ng.mL-1, r = 0.997. Re. (74- 87)% Chloroquine Ion association complex pharmaceutical formulations 1- λ max =950nm, Linearity =(50- 250) µg.mL-1 €= 1.79 × 104L.mol- 1.cm-1, r2 = 0.9992. LOD and LOQ equals (0.27 and 0.82) µg.mL-1 respectively. λ max = 420nm, Linearity = (50- 250) µg.mL-1, € = 3.09 × 104 L.mol- 1.cm-1, r2 = 0.9996, LOD and LOQ = 0.15 and 0.460 µg.mL-1 respectively. [29] Chloroquine High-performance liquid chromatography plasma λ max =331nm, Linearity = (20- 2000) nM, BDS-Hypersil (C18) 5m, 250×4.6)mm column, Mean Re. = 83.7%, r2 =0.993 [30] Chloroquine oxidation of the drug with Fe(III)-1, 10 phenanthroline pharmaceutical formulations λ max = 510nm, Linearity=20-320 µg.mL-1, €= 666.66L.mol-1.cm-1, r2 = 0.999, LOD equle 0.1915 µg.mL-1 while LOQ equle 0.5801 µg.mL-1 [31] Chloroquine pharmaceutical formulations λ max = 285 and 345nm, Linearity= 50-250 μg/ml. r2=0.999, [32] Hydroxychloroquine Uv. spectrophotometric Raw and pharmaceuticals λ max = 343nm, Linearity=1-20 µg.mL-1 €=0.2269x103L.mol-1.cm-1, r2=0.9992\RSD= 0.169%. [33] Chloroquine Uv spectral Method using 0.1N HCL pharmaceutical formulations λ max = 342nm, Linearity=2.5-25 µg.mL-1, €= 8.88x103L.mol-1.cm-1, LOD and LOQ equles 0.39 and 1.18 µg.mL-1 respectivly. [34] Chloroquine HPLC method with diode array detector whole blood and plasma λ max =343nm, Linearity= 10-5000 ng/mL (150 × 4.6) mm and 5μm (SB - CN) column, “mobile phase contain phosphate buffer” (pH 2.6) - acetonitrile 88:12, (v/v), LOD, LOQ = (10 and 4) ng.mL-1 Respectively. [35] Chloroquine Ion pair reactions pharmaceutical formulations λ max =420nm, Linearity=1-20 µg.mL-1 €= 1.79 × 104L.mol-1.cm-1, r2=0.9992. LOD =, LOQ = 0.27 and 0.82 µg.mL-1 respectively, λ max= 420nm, Linearity = 0.5 -12 µg.mL-1, €=3.09 ×104L.mol-1.cm-1, r2=0.9996, LOQ = 0.46 µg.mL-1 and LOD = 0.15 µg.mL-1. [36] Chloroquine Uv spectral Method using H2O pharmaceutical formulations λ max = 343nm, Linearity=10.88- 30.56µg.mL-1 , r2=0.99972. [37] Chloroquine liquid chromatography Blood UPLC- HSS T3, 2.5 µmand (75 mm x 2.1 mm) column, LOQ = 20 ng.ml-1, precision (-12.1 to +11.1%), sensitivity. (1.4 to 15.0%), r =0.97, [38] Hydroxychloroquine liquid chromatography with tandem mass spectrometry mouse blood and tissues λ max =420nm, average Linearity = (1–2000) ng/mL, Thermo Aquasil C18 (50 × 4.6 mm, 3μ) column, mobile phase 0.20 % formic acid in methanol, r2= 0.998. LOQ = 1.0 [39] Ibn Al-Haitham Jour. for Pure & Appl. Sci. 35(1)2022 54 ng.mL-1. Chloroquine LC-MS/MS whole blood, plasma and dried blood spots λ max =420nm, average Linearity= (1.41–1552 )ng.ml-1, Sb-Cn 3.5 μm (50× 4.6) mm, mobile phase contain “acetonitrile + ammonium formate” 20 mM and formic acid (1%) pH = 2.6 (15–85), (v/v). [40] Hydroxychloroquine sulfate ultra-HPLC (U-HPLC) method whole blood Excitation wavelength 335 nm , emission wavelength 390 nm, Linearity = (125 to 4000) ng.mL-1, U-HPLC RP18 column, mobile phase piperazine buffer, (at pH equle to 9.8) plus acetonitrile (68:32), LOQ = 10 ng/mL , accuracies = (7.90-7.85)%, imprecisions (1.14 -8.78)% [41] Hydroxychloroquine High-performance liquid chromatography Blood Excit. Wavelength = 337 nm , emi. Wavelengths = 405 nm, Linearity = (3-3000) ng/mL, column (C18), “mobile phase sodium phosphate (20 mM) buffer solution with 0.25% triethylamine and acetonitrile (60:40, v/v)”, precisions=(1.3 to 7.3.). [42] Hydroxychloroquine High-performance liquid chromatography pharmaceutical formulations Linearity = (0.200–6.004) µg mL−1, LOD and LOQ = 0.066 µg mL−1and 0.200 µg mL−1 respectively, RSD%= (98.25±1.05), r2= 0.9999. [43] Hydroxychloroquine High-performance liquid chromatography pharmaceutical formulations λ max =254nm Linearity =(25-300) μg ml-1, “Zorbax C8, 250 mm × 4.6 mm i.d., column”, RSD <1.5%, R 2 > 0.999 [44] Hydroxychloroquine LC-MS/MS method plasma Linearity = (2-1000) ng/mL, “Column (2.0 × 50 ) mm and 3 μm. For detection ESI, MRM are used”. Ion pairs m/z equle to (336.1→247.1), Re. = (88.9- 94.4)%. [45] Chloroquine and Hydroxychloroquine two-dimensional isotope-dilution liquid chromatography- tandem mass spectrometry serum Injection volume of 5 μL, accuracy ≤ 9.59 %, imprecision ≤ 11.1 % for all quality controls. [46] Hydroxychloroquine multisensing probe pharmaceutical formulations λ max = 521, 600, 620 and 670 nm, LOD = 2.61nM for optical, 0.15nM fluorescence, and 0.85 nM electrochemical method. [47] Hydroxychloroquine liquid phase microextraction-gas chromatography-mass spectrometry urine, serum and saliva LOD = 0.74 μg/kg, LOQ = 2.4 μg.kg-1 Re.= 93.9%-101.7% for serum, 95.2%-105.0%, for urine, 93.1%-102.3% for saliva. [48] 2.Conclusions The literature of different quantitative analytical methods for estimatig Chloroquine CQ and Hydroxychloroquine HCQ in their pharmaceutical preparations was carefully reviewed. Common instrumental methods for the determination of these drugs were Spectrophotometry, high-performance liquid chromatography HPLC with mass spectrometry Ibn Al-Haitham Jour. for Pure & Appl. Sci. 35(1)2022 55 or diode array detector DAD, gas- chromatography, as well as electrochemical approaches. Chromatographic methods, especially HPLC, were the most routine practice. HPLC proved to be sensitive and accurate since it usually overcomes the errors that arise from interferences in the bulk of the pharmaceutical formulation. References 1.Al-Bari, M.A.A. 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