HPLC and NMR methods for the quantitation of the (R)‐enantiomer in (−)‐(S)‐timolol maleate drug raw materials

HPLC and 1H‐NMR methods for the quantitation of the (R)‐enantiomer in (−)‐(S)‐timolol maleate were developed and validated. The HPLC method requires a 25 cm × 4.6 mm 5 μm Chiracel OD‐H (cellulose tris‐3,5‐dimethylphenylcarbamate) column, a mobile phase of 0.2% (v/v) diethylamine and 4% (v/v) isopropanol in hexane at a flow rate of 1 ml/min and UV detection at 297 nm. A system suitability test was devised to verify the separation of the (R)‐ and (S)‐enantiomers of timolol from other drug‐related impurities. The NMR method requires the use of a high‐field NMR spectrometer (>360 MHz) and a chiral solvating agent, (−)‐(R)‐2,2,2‐trifluoro‐1‐(9‐anthrylethanol) (R‐TFAE). The limits of quantitation were 0.05% and 0.2% (m/m) for HPLC and NMR, respectively. The methods were applied to the determination of the (R)‐enantiomer in eight lots of raw material. The results for the two methods were in very good agreement, with results ranging from 0.1 to 4.1% (m/m) by HPLC and none detected to 4.3% (m/m) by NMR. The USP method for specific rotation was found to be unsuitable for detecting the presence of low levels of the (R)‐enantiomer in (−)‐(S)‐timolol maleate.