Detection Methods of Urapidil hydrochloride and innner impurities

Introduction

Urapidil Hydrochloride (Figure 1) is a highly selective a-receptor blocker. Active pharmaceutical ingredient of Urapidil Hydrochloride Injection is phenylpiperazine substituted ureacil derivative,which is mainly used for the treatment of Hypertension crisis and perioperative hypertension. Urapidil Hydrochloride has broad market application prospect.

Determination methods of Urapidil Hydrochloride

Report 1: (1) The HPLC method for the determination of related substances in Urapidil Hydrochloride Injection is established. The column is Zorbax Bonus-RP (4.6mm x 150mm, 3.5um). 0.02 mol/L sodium perchlorate solution (pH 2.0) as mobile phase A, methanol as mobile phase B are used in gradient elution. The detection wavelengths are 236 nm and 270 nm. The flow rate is 1.0 mL/min.The column temperature is 40°C. The injection volume is 10 uL. The resolution between Urapidil Hydrochloride and all other impurities were good. Urapidil hydrochloride and impurities are within LOQ ~ 200%, All known impurities had good linearity in appropriate concentration ranges and the correlation coefficients were greater than 0,9996. The limits of quantitation of impurities C is 0.3 g/mL and the other impurities and Urapidil Hydrochloride were 0.2 g/mL. The average recoveries of all known impurities are in the range of 92.16%-102.11%, with RSD of less 5.0%. The determination results show that the specificity, system applicability and other verification items of the determination method of relevant substances meet the requirements. So the specific and accurate method can effectively and quickly detect related substances in Urapidil Hydrochloride Injection.

(2) The HPLC method for the determination of assay in Urapidil Hydrochloride Injection is established. The column is Zorbax Bonus-RP (4.6mmx 150mm, 3.5 um). The mobile phase is 0.02 mol/L sodium perchlorate solution (pH 2.0) - methanol (70: 30). The column temperature is 40°C and the detection wavelength is 270 nm. The injection volume is 10 uL. The resolution between Urapidil Hydrochloride and all other impurities is greater than 5.314. The limits of quantitation of Urapidil Hydrochloride is 0.2266 g/mL. Urapidil Hydrochloride Injection has good linearity in appropriate concentration ranges between 0.0002266~0.1680g/mL and the correlation coefficients are greater than 0.9998. The average recoveries of all known impurities were in the range of 100.13%~101.81%, with RSD of less 0.57%.The validation results show the high specificity and excellent accuracy of the method.[1]

Determination of genotoxic impurities in urapidil hydrochloride by HPLC-MS/MS method

To establish an HPLC-MS/MS method for the determination of genotoxic impurities bis (2-chloroethyl) amine hydrochloride and O-anisidine hydrochloride in urapidil hydrochloride. Chromatographic separation was performed on a Horizon Amide 18 column (100 mm×2.1 mm, 3 μm) using amobile phase consisting of 0.1% formic acid aqueous solution (A) and acetonitrile (B) with gradient elution. The flow rate was 0.3 mL/min and the column temperature was 30°C. Detection was carried out on a Vanquish-TS Quantis Plus triple quadrupole mass spectrometer equipped with an electrospray ionization source (ESI),operating in positive ion mode, using multiple reaction monitoring. Results: The linear range of bis (2-chloroethyl)amine hydrochloride was 0.47-120 ng/mL (r=0.999 7). The limit of detection was 0.24 ng/mL, the limit of quantitation was 0.47 ng/mL. The recovery rates at three concentration levels ranged from 75.7% to 110.2% with RSDs of 2.9%-7.8% (n=3). The calibration curve for O-anisidine hydrochloride was linear over the range of 2.34-120 ng/mL (r=0.999 9). The limit of detection was 0.59 ng/mL, and the limit of quantitation was 2.34 ng/mL. The recovery rates at three concentration levels ranged from 79.6% to 110.9% with RSDs of 1.8% to 7.9% (n=3). In three batches of urapidil hydrochloride samples, the contents of bis (2-chloroethyl) amine hydrochloride were 0.19,0.17, and 0.27 μg/g, while the contents of O-anisidine hydrochloride were 1.54, 1.57, and 2.35 μg/g all of which complied with the specification limit (＜5.48μg/g). The method is rapid, specific, sensitive and accurate, and can be effectively applied to the determination of bis (2-chloroethyl) amine hydrochloride and O-anisidine hydrochloride in urapidil hydrochloride.[2]

Determination of Bacterial Endotoxin Content in Urapidil Hydrochloride by micro-kinetic chromogenic method

To investigate the feasibility for quantitative determination of bacterial endotoxin in Urapidil Hydrochloride Injection by micro-kinetic chromogenic method. Micro-kinetic chromogenic method of limulus amebocyte lysate reagent was used to assess the reliability of the bacterial endotoxin standard curve. The interference test was conducted to determine the non-interference concentration. The bacterial endotoxins of six batches of Urapidil Hydrochloride Injection were detected，and the results were compared with those obtained by the gel method. Results The reliability detection range of standard curve was 0. 05-5. 0 EU/mL，the regression equation of micro-kinetic chromogenic method of limulus amebocyte lysatere agents from the two manufacturers were lgT₁ =2. 9230-0. 2832 lgC1；lgT₂=2. 9866-0. 2229 lgC2，respectively，with the correlation coefficients of - 0.9996 and - 0.9999，respectively. Micro-kinetic chromogenic method of limulus amebocyte lysatere agent was used to detect the bacterial endotoxin in Urapidil Hydrochloride Injection. When Urapidil Hydrochloride Injection was diluted to the concentration of 0. 15625 mg/mL or lower，there were non - interference with the agglutination reaction between micro - kinetic chromogenic of limulus amebocyte lysate reagent and bacterial endotoxin，and the recovery rate was 50% - 200%.The endotoxin test results in six batches of test samples were consistent with the gel method，which all met the specified requirements. Micro - kinetic chromogenic method can be used for the quantitative determination of bacterial endotoxin in Urapidil Hydrochloride Injection，and the results are accurate，and the sensitivity is high.[3]

References

[1]Wang CJ. Quality Research of Urapidil Hydrochloride Injection[D].Nanjing University of Chinese Medicine,2022.DOI:10.27253/d.cnki.gnjzu.2022.000358.

[2]Zhong A,et al.Determination of genotoxic impurities in urapidil hydrochloride byHPLC-MS/MS method[J].Chin J Pharm Anal,2025,45(10):1826-1832.DOI:10.16155/j.0254-1793.2024-1146.

[3]Li FC,et al.Determination of Bacterial Endotoxin Content in Urapidil Hydrochloride Injection by Micro -Kinetic Chromogenic Method[J].China Pharmaceuticals,2026,35(04):101-105.

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