The phenyl vinyl sulfone reference standard (claim 99.32%) was procured from Fortune Pharma, Hyderabad, as a gift sample. All HPLC grade solvents were obtained from the Merck India Limited, Mumbai, India. All the solvents and solutions used were filtered through millipore (0.25 μm) filters.
Optimized LC-MS method conditions
The Waters LC-MS with auto sampling system was used to perform the method. Separation of the phenyl vinyl sulfone was successfully achieved by the Symmetry C18 (50 × 4.6 mm, 3.5 μm) column and a mobile phase composition of 0.1%v/v ammonia buffer to methanol (5:95 v/v), using 0.45 ml/min flow rate and 20 μl of injection volume, with methanol used as diluent. The temperature maintained in the auto sampler and column was 5 °C and ambient respectively. MS parameters were mentioned in the Table 1.
Preparation of phenyl vinyl sulfone standard stock solution (1000 ppm)
Two hundred fifty milligrams of phenyl vinyl sulfone standard substance was weighed accurately and transferred into 10 ml volumetric flask and diluted with diluent. The 0.4 ml of above solution was further diluted to 10 ml to get a concentration of 1000 ppm.
Preparation of standard solution (18.0 ppm)
0.36 ml of the standard stock solution was diluted to 20 ml to obtain a standard concentration of 18 ppm.
Preparation of test solution
The test solution accurately weighed about 100.00 mg of the eletriptan test sample and was diluted to 10 ml with diluent.
Method validation
System suitability test
The system suitability of the method was performed by injecting blank solution once and 100% level standard solution of phenyl vinyl sulfone for six times into LC-MS system. The system suitability was confirmed by assessing the % RSD.
Linearity
The linearity of a method represents the direct proportional relationship between concentration and test result. It was conducted for the phenyl vinyl sulfone in the range of LOQ level (4.767 ppm) to about 150% of limit (27.00 ppm) by injecting each level of concentration two times and plotted a curve between average peak area versus concentration in ppm to find out the regression coefficient value.
Accuracy
The accuracy of the method has been done by performing the recovery studies, at LOQ level, 50%, 100%, and 150% level. A known amount of phenyl vinyl sulfone spiked separately to pre-analyzed samples of the mentioned levels. Each spiked level was injected for three times into the LC-MS system and calculated the percent recovery of each level.
Method precision
The method precision was performed by spiking the sample with phenyl vinyl sulfone at 100% of the specified limit with respect to the sample concentration. Six homogenous replicates were injected and calculated the content of phenyl vinyl sulfone to determine the % RSD.
Intermediate precision
The intermediate precision was performed by spiking the sample with phenyl vinyl sulfone at 100% of the specified limit with respect to the sample concentration in six preparations. The intermediate precision study was carried out on different days with different analysts. We calculated the content of phenyl vinyl sulfone in spiked preparations and determined the % RSD.
Sensitivity (LOD and LOQ)
Limit of detection (LOD) and limit of quantification (LOQ) of the analytical method were determined by using signal to noise ratio. The LOD solution was prepared in such a way to obtain the S/N ratio is about 3:1 to 5:1. Based on the concentration of LOD, the LOQ solution was prepared (3 times to LOD concentration) to obtain the signal to noise ratio of about 10:1 to 15:1.