Prepared a mixture of Dimethyl acetamide and water in the ratio of 1:1 (%volume/volume) mixed well and sonicated to degas.

Transferred 35.0 µL of Bromoethane standard into a 100 mL volumetric flask containing about 50 mL of diluent and make up to volume with diluent.

Further transferred 1.0 mL of the above solution into a 100 mL volumetric flask containing about 50 mL of diluent and make up to volume with diluent.

Accurately weighed 500 mg of the test sample into a 20 mL head space vial added 5.0 mL of diluent and seal the vial immediately.

Accurately weighed 500 mg of the test sample into a 20 mL head space vial added 5.0 mL of standard solution and seal the vial immediately.

The sample diluent is important for a static Headspace GC method as it affects the sensitivity and accuracy by influencing the equilibrium between the analyte in the liquid phase and the analyte in the headspace. Several sample diluents were evaluated including dimethyl sulfoxide, dimethyl acetamide, dimethyl formamide and water alone and in combination. The use of water alone as the sample diluent led to irreproducible results, water solubility of drug (55.0 mg/mL). Overall solubility of the drug Vigabatrin, was also determined in DMA-water and DMSO-water system and the values were found to be 56.23 mg/mL and 50.03 mg/mL respectively. A combination of water with the organic diluents 1:1 ratio was studied. Under different head space oven temperature at 80 °C, 90 °C and 100 °C. Under these GC headspace conditions, the reproducibility of Bromoethane spiked Vigabatrin was evaluated, and the results demonstrated that the DMA: water 1:1 v/v sample diluent at a headspace temperature of 90 °C showed the best sensitivity and selectivity.

One of the key objectives of the method development was to achieve adequate sensitivity for low level Bromoethane analysis. The Bromoethane method sensitivity was further optimized by the evaluating the effect of split ratio on the noise level and S/N value of a 50 ppm Bromoethane standard solution. Several GC injection split ratios including 5:1, 10:1 and 30:1 was studied. The optimal noise level and adequate signal was observed using the 5:1 spilt ratio injection parameter.

For head space GC, vial equilibration temperature 90 °C, Loop temperature 95 °C, transfer-line temperature 100 °C, equilibration time 10 min, pressurization time 0.2 min and injection time of sample 1.0 min. GC cycle time 40 minutes. The flame ionization detector (FID) detector was used split ratio 5:1, injection port temperature 200 °C; detector temperature 230 °C; temperature program was set to 40 °C for hold time 8 minutes, then raised to 150 °C with rate 10 °C per minute hold time zero minutes and then maintained at 230 °C for with rate 25 °C per minute hold time 11 minutes and carrier gas was used nitrogen.

The method specificity was validated for potential interference from blank, standard, sample, and spiked sample solution. There are no detectable peaks in the chromatograms of blank, standard, sample, and spiked sample. The Bromoethane peak in the chromatogram of 50 ppm Bromoethane spiked sample solution is sufficiently resolved from all other peaks before and after Bromoethane peak. The retention time of Bromoethane in the chromatogram of 50 ppm Bromoethane spiked sample solution matches well with that from 50 ppm Bromoethane standard solution (Figs 3, 4, 5, 6).

Figure 3. 

Typical chromatogram of blank.

Figure 4. 

Typical chromatogram of standard.

Figure 5. 

Typical chromatogram of sample.

Figure 6. 

Typical chromatogram of spiked sample.

System precision was demonstrated by preparing standard solution as per method and chromatographed the same into GC system in six replicated injections of standard solution. The peak areas of analyte were recorded for these standard injections. The system precision was evaluated by computing the % Relative standard deviation for the peak area of these standard injections (see Table 1).

A solution containing 10.80 ppm of Bromoethane standard was injected six times. The RSD of areas and S/N ratios for each standard were calculated. A solution containing 3.57 ppm of Bromoethane standard was injected three times. The worst found signal to noise ratio for each peak was greater than 3 in each injection. All the peaks were detected in all the three injections (Figs 7, 8).

Figure 7. 

Typical chromatogram of LOQ.

Figure 8. 

Typical chromatogram of LOD.

Therefore, the quantitation limit (QL) and the detection limit (DL) was thus set at 10.80 ppm and 3.57 ppm, respectively (see Table 1). These S/N ratios are much greater than ICH recommended S/N value for DL (S/N~3) and QL (S/N~10).

Precision (repeatability) was evaluated from the recovery data. Recovery data was determined by injecting six sample solutions spiked Bromoethane 50 ppm at specification level (see Table 1). The samples were prepared as per the analytical method.

The linearity of Bromoethane was evaluated from 10.80 ppm to 77.18 ppm (six levels with duplicate preparations at each level). The peak areas were plotted against the corresponding concentrations and the linear regression was performed. The range 10.80–77.18 ppm was established by meeting the acceptable criteria of linearity, accuracy, and precision for the entire concentration interval study (see Table 1).

Accuracy was determined by analyzing the triplicate preparation of Bromoethane standard at low 10.80 ppm, 25.0 ppm, 50.0 ppm and 75.0 ppm levels in the presence of Vigabatrin drug substance, as per the analytical method. The accuracy as % recovery was calculated from the experimental concentrations of Bromoethane standards by the theoretical concentrations. The recovery of ranged from 95.3% to 106.8% were obtained for the three concentrations levels (see Table 1).

The stability of standard, sample and spiked sample solutions were prepared in duplicate and stored at ambient laboratory conditions (25±5 °C), refrigeration (2–8 °C), respectively. Therefore, the standard solution, sample solution and spiked sample solution were stable for 24 hrs at both room and refrigerated temperature conditions.