6 mm i d , 5 ��m particle size) were selected to achieve good res

6 mm i.d., 5 ��m particle size) were selected to achieve good resolution and acceptable peak symmetry. Flow rates between 0.5 and 1.2 ml/min were tried. Flow rate of 1.0 ml/min was observed to be enough to get both the drugs eluted within less than 10 min. The column temperature was set at 40��C. System suitability The retention times for LAMI, TDF, and EFV using optimum conditions were 2.76, 3.96, and 10.5 min, respectively. For three of them, the peak symmetries were <1.5 and the theoretical plates�� numbers were >2000. These values are within the acceptable range of United state pharmacopoeia definition and the chromatograms obtained under optimized chromatographic conditions. Figure 4 clearly shows the ability of the method to assess the analyte in the presence of other excipients. The results obtained are shown in Table 1. Figure 4 Chromatograms of LAMI (3 ��g/ml), TDF (3 ��g/ml), and EFV (6 ��g/ml) reference substances Table 1 System suitability parameters Method validation[36] The developed method for simultaneous estimation of LAMI, TDF, and EFV has been validated in accordance with the ICH guidelines.[11] Linearity Linearity was checked by preparing standard solutions at six different concentration levels of each of LAMI, TDF, and EFV, ranging from 1 to 6 ��g/ml for each of LAMI and TDF and from 2 to 12 ��g/ml for EFV. Triplicates of 20-��l injections were made for each concentration and were chromatographed under the chromatographic conditions mentioned above. Peak areas were plotted against the corresponding concentrations to obtain the calibration graph for each compound. The regression analysis data are given in Table 2. Table 2 Linearity data Sensitivity The sensitivity of the analytical method was evaluated by determining the limits of detection (LOD) and quantitation (LOQ). The values of LOD and LOQ for LAMI, TDF, and EFV are given in Table 2. Accuracy The accuracy of the method for assay determination was checked at three concentration levels of 80%, 100%, and 120% for LAMI, TDF, and EFV. The percentage recoveries are tabulated in Table 3. The recovery was calculated from the slope and intercept of the calibration curve of each drug. As per the ICH guideline, the % recovery must be between 98% and 102%. Table 3 Repeatability precision Precision Repeatability: System repeatability was determined by replicate applications and measurements of peak area for LAMI, TDF, and EFV. One dilution in six replicates was analyzed on the same day for repeatability and results were found within acceptable limits (RSD <2) as shown in Table 4. Table 4 Result of recovery studies with static evaluation Intermediate precision: Intermediate precision was assessed by the assay of sample sets on three different days (inter-day precision). Three dilutions in three replicates were analyzed and results were found within acceptable limits (RSD <2) as shown in Table 4.

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