ABSTRACT
For qualitative and quantitative analysis of related substances in clotrimazole cream, HPLC-Q-TOF spectrometer was used to analyze the fragmentation pathways and identify structures of the related substances. Five related substances named by BP (2018) were identified as impurity A ((2-chlorophenyl)-diphenylmethanol), impurity B (para-clotrimazole isomer), impurity E (2-chlorobenzophenone), impurity F (1-tritylimidazole) and impurity 4 (9-(2-chlorophenyl)-fluorene), respectively, by using impurity references matching and comparison with the literature data. Four related substances were detected in clotrimazole cream except impurity E, and 9-(2-chlorophenyl)-fluorene is the first identified impurity in this preparation. To establish an HPLC method for determination of the related substances in Clotrimazole Cream, the Agilent Poroshell Bonuns RP column was used (100 mm×4.6 mm, 2.7 μm) with UV detection at 215 nm. The mobile phase was acetonitrile-10 mmol·L-1 dipotassium phosphate buffer (adjusted with phosphoric acid to pH of 5.80) with a flow rate of 1.0 mL·min-1. Gradient elution was used. The column temperature was maintained at 40℃. A good linear behavior was achieved between component's concentrations and peak area for impurity A, B, E, F within the range of 0.20-10.02 μg·mL-1, 0.20-10.00 μg·mL-1, 0.20-10.10 μg·mL-1, 0.10-5.01 μg·mL-1 with the correlation coefficients were 0.999 7, 1.000 0, 1.000 0, 0.999 9, respectively. The average recoveries were 94.3%, 95.0%, 100.0%, 99.6% with RSDs were 2.8%, 2.2%, 1.1%, 2.7%, respectively (n=9). LOQ were 200.4, 200.0, 202.0, 100.2 ng·mL-1, respectively. LOD were 57.25, 57.14, 57.71, 28.63 ng·mL-1, respectively. The developed method was simple, rapid, accurate and effective for testing related substances in clotrimazole cream to control its quality, ensuring the safety of clinical medication.
ABSTRACT
<p><b>AIM</b>To study the four diastereomers of leucogen and structure of the related substances.</p><p><b>METHODS</b>LC-DAD, LC-MS/MS and LC- 1H NMR were used. LC was carried out with a Phenomnex Luna C18 (250 mm x 4.60 mm ID, 5 microm) column and a mobile phase of water-acetonitrile-glacial acetic acid (58:42: 0.3).</p><p><b>RESULTS</b>The structures of leucogen and its related substances were identified. Leucogen and the related substances were found to have four diastereomers in solution state separately. The stability and transformation of the four diastereomers were analyzed and 3R4S5R was found to be more stable than the others according to quantum calculations.</p><p><b>CONCLUSION</b>Leucogen have four diastereomers in solution state and it can transform from one diastereomer to the others, and the 3R4S5R is more stable than the others.</p>