Chloroquine Phosphate HPLC Assay and Impurity Profiling Methods Following the USP Monograph

Introduction

A rapid, accurate, and simple method was implemented for the total chromatographic purity analysis of Chloroquine Phosphate by High Performance Liquid Chromatography with a Diode Array Detector. The experimental conditions follow guidelines, with minor modifications, from the USP43-NF38 monograph methods for Chloroquine Phosphate Assay and Impurity Profiling. Chloroquine Phosphate, Chloroquine Related Compound A, Chloroquine Related Compound D, Chloroquine Related Compound E, Chloroquine Related Compound G, Hydroxychloroquine, and Phenol can be resolved with baseline separation within 16 minutes using an Ascentis Express C18 column (250 x 4.6 mm, 5 µm). A 1.4 g/L Dibasic sodium phosphate solution (pH 3.0) in water: 0.4% triethylamine in methanol (30:70 v/v) were employed as the mobile phase for the isocratic elution. Under applied conditions, system suitability criteria are met, and the method demonstrates good resolution/selectivity, reproducibility, and sensitivity.

Chloroquine Phosphate
Chloroquine Phosphate

 

Hydroxychloroquine sulfate
Hydroxychloroquine sulfate

 

Phenol
Phenol

 

Chloroquine Related Compound A
Chloroquine Related
Compound A

 

Chloroquine Related Compound D
Chloroquine Related
Compound D

 

Chloroquine Related Compound E
Chloroquine Related
Compound E

 

Chloroquine Related Compound G
Chloroquine Related
Compound G

 

Experimental Conditions      
Column Ascentis Express C18 column (25cm x 4.6mm, 5µm) Injection volume 20 µL
Detection UV @ 260 nm (analytical flow cell; 10 µL) Flow rate 1 mL/min
Buffer Dissolve 1.4 g of K2HPO4 in 1000 mL Milli-Q water and adjust to pH 3.0 using H3PO4. Temperature Column: 30°C Autosampler: 16°C
Mobile phase Buffer and 0.4% triethylamine in Methanol (30:70) v/v. Pressure 237 bar
Diluent Mobile Phase
Test solution Dissolve 50 mg of Chloroquine Phosphate in 25 mL mobile phase (2.0 mg/mL).
System suitability solution (SST) Dissolve 5.0 mg of each Chloroquine Phosphate, Phenol, Hydroxychloroquine, Chloroquine Phosphate Related Compound A, Chloroquine Phosphate Related Compound D, Chloroquine Phosphate Related Compound E, and Chloroquine Phosphate Related Compound G into 25 mL mobile phase. Then, further take 1.0 mL of this solution and dilute to 100.0 mL using diluent (5.0 µg/mL).
 
 

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System Suitability Solution (SST) (Standard)

Antibody characterization of the 16HBE14o- cell line

Chloroquine phosphate (Test solution)

Antibody characterization of the 16HBE14o- cell line
 

Specificity (System Suitability Solution)

Peaks Compound Retention Time (min) RRT RRT Reference per USP43-NF38 Resolution Requirement per USP43-NF38 Theoretical plates Tailing factor
1 Phenol 2.60 0.26 0.2 -   11484 1.1
2 Chloroquine related compound G (RCG) 3.18 0.32 0.27 5.4   11444 1.2
3 Chloroquine related compound D (RCD) 4.77 0.48 0.42 10.5   10437 1.6
4 Hydroxychloroquine sulfate 5.14 0.52 0.49 2.2   16090 1.1
5 Chloroquine related compound A (RCA) 8.49 0.86 0.73 18.8   28913 1.1
6 Chloroquine Phosphate 9.91 1.00 1.0 6.0 > 2.0 (to RCA) 20259 1.1
7 Chloroquine related compound E (RCE) 15.18 1.53 1.5 15.9   23609 1.2
 

Repeatability (System Suitability Solution)

Peaks Compound Area Response (n=3) Standard Deviation RSD (%)
1 Phenol 42.42 0.13 0.3
2 Chloroquine related compound G (RCG) 178.92 1.12 0.6
3 Chloroquine related compound D (RCD) 283.39 0.90 0.3
4 Hydroxychloroquine sulfate 195.04 0.64 0.3
5 Chloroquine related compound A (RCA) 75.60 0.22 0.3
6 Chloroquine Phosphate 152.47 0.70 0.5
7 Chloroquine related compound E (RCE) 37.97 1.57 4.1
 
     
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