The precision study was also carried out at the LOQ level by injecting six individual preparations of ��-isomers and guaiacol impurities and calculating the % RSD of the area [Table 2]. Linearity Linearity test solutions were prepared by diluting the stock solutions to the required concentrations. The solutions were Tubacin molecular weight prepared at six concentration levels ranging from 0.227 to 72.046 ��g/mL for ��-isomers and 0.163�C4.8121 ��g/mL for guaiacol impurites. Calibration curves were plotted between the responses of peak vs. analyte concentrations. The correlation coefficient obtained was greater than 0.999 and % bias at 100% response was within 5% [Table 2]. The above results show that an excellent correlation existed between peak area and concentration of ��-isomer and guaiacol.

Accuracy Accuracy of the method for ��-isomer was evaluated in triplicate using six concentration levels at 0.235, 11.843, 17.765, 23.686, 44.412, and 54.281��g/mL and guaiacol at 0.163, 0.542, 0.854, 1.164, 2.871, and 3.492 ��g/mL (i.e. LOQ, 50%, 75%, 100%, 200%, and 300% level of specification, respectively). The percentage recovery of ��-isomer and guaiacol in guaifenesin samples varied from 88.3% to 108.7%. The LC chromatogram of the spiked sample at the specification level of both impurities in the guaifenesin tablet sample is shown in Figure 4. The recovery values for ��-isomers and guaiacol are presented in Table 3.

Figure 4 Typical chromatogram of sample spiked with impurities Table 3 Recovery study of the analytical method Robustness To determine the robustness of the developed method, experimental conditions were deliberately altered and the relative retention time (RRT) of ��-isomer and guaiacol with respect to guaifenesin; and system suitability parameters for guaifenesin standard was recorded. The variables evaluated in the study were pH of the mobile phase buffer (+0.2), column temperature (�� 5��C), flow rate (�� 0.2 mL/min), and % organic in the mobile phase (�� 10%). In all the deliberate varied chromatographic conditions, all analytes were adequately resolved and the elution order remained unchanged. The area ratio for the guaifenesin peak from standard solution was between 0.9 and 1.1 and the tailing factor was less than 1.1 [Table 4].

Table 4 Robustness results of the HPLC method Stability of solution and the mobile phase The solution stability of guaifenesin and its impurities AV-951 were determined by leaving test solution and standard solutions in tightly capped volumetric flasks at room temperature for 48 h and measured the amount of both impurities at every 24 h against freshly prepared standard solution. The stability of the mobile phase was also determined by freshly prepared solutions of guaifenesin and its impurities at 24 h interval for 48 h. The mobile phase was not changed during the study. The variability in the estimation of ��-isomers and guaiacol was within �� 10% during solution stability and mobile phase stability.