Alfuzosin Related Substances Method (HPLC) following United States Pharmacopeia Monograph on Ascentis Express C18 with UV Detection


Alfuzosin is an antagonist of the α1 adrenergic receptor and it relaxes the muscles in the prostate and bladder neck, making it easier to urinate. It is used to treat benign prostatic hyperplasia (BPH), and it was approved by the United States FDA in June 2003. Alfuzosin is branded and marketed by Sanofi Aventis as Uroxatral.

Ascentis® Express C18 columns can be used for the HPLC methods within both the Alfuzosin USP and EP Monographs.
Here, it is illustrated with analytical data for testing the organic impurities (related substances) per the USP41-NF36 guidelines. The suitability requirement for organic impurities is defined through the chromatographic resolution and the peak-to-valley ratio. The ratio of the height of the furamide analog peak to the height of the valley between the furamide analog peak and alfuzosin is not less than five (5). The relative retention time should furthermore be about 0.5, 1.0 and 1.2 for deacylated alfuzosin, alfuzosin and the furamide analogue, respectively. Same experimental conditions are used in the corresponding European pharmacopeia monograph but with slightly different system suitability requirements.




Experimental Conditions
Column Ascentis® Express C18, 150x4.6 mm, 5 Micron HPLC column Injection volume 10 µL
Detection UV @ 254 nm (analytical flow cell; 10 µL) Flow rate 1.0 mL/min
Dilute 5 mL of perchloric acid in 900 mL of water.
Adjust with 2 M sodium hydroxide to a pH of 3.5, and dilute with water to 1 L with water.
Temperature 25 °C
Mobile phase Acetonitrile, tetrahydrofuran, and buffer 20:1:80 (v/v/v)
Diluent Mobile phase Pressure drop 93 bar (1350 psi)
SST solution 0.4 mg/mL of USP Alfuzosin System Suitability Mixture RS in Mobile phase
Test solution Dissolve 40.0 mg of the sample in 100 mL of mobile phase.
Reference solution 0.40 μg/mL of Alfuzosin Hydrochloride in Mobile phase from the Sample solution

Chromatographic Data (Standard Solution)


Retention Time (min) Tailing factor Theoretical plate
Alfuzosin 6.5 1.1 18,340


Chromatographic Data (System Suitability Solution)

  Compound Retention Time (min) RRT Peak-to-valley ratio
1 Deacetylated alfuzosin 3.3 0.5 -
2 Alfuzosin 6.5 1.0 -
3 Furamide analogue 7.9 1.2 7.3

Validation and verification parameters

1. Specificity: Inject solution and determine the retention time of desired analyte in presence of other component like impurities and excipient.
    Retention Time (min) Resolution
1 Deacetylated alfuzosin 3.3 -
2 Alfuzosin 6.5 12.2
3 Furamide analogue 7.9 4.1


3. Linearity, LOD & LOQ
Concentration (μg/mL) Mean Area
0.4 18340
1 46684
5 224219
10 467163
20 916188
32 1461609
40 1837803
48 2208987
60 2719299
LOQ 0.2 ppm (S/N 10.0)
LOD 0.06 ppm (S/N 3.0 ppm)
2. Standard Repeatability (0.4 ppm)
STD 1 18340
STD 2 18185
STD 3 18436
STD 4 18392
STD 5 18231
Mean 18317
Standard Deviation 106.2
RSD (%) 0.6


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