Validation Parameters MP PP LRD
Specificity Specific Specific Specific
System Suitability      
Retention time (min.) 2.4 3.6 8.7
Resolution factor (Rs) --- 7.665 34.13
No. of theoretical plates (N) 5075 7067 37695
Tailing factor (Asymmetry) 1.0693 0.9955 0.9298
Precision (Repeatability) %RSD      
System (n=6) 0.479 0.488 0.490
Method (n=6) 0.763 0.718 0.756
Reproducibility      
Intra-day (n=6) 1.304 1.204 1.258
Inter-day (n=6) 1.221 1.324 1.098
Accuracy (% Recovery mean ± SD)      
Spike level I (n=3) 101.87 ± 0.097 101.64 ± 0.19 101.36 ± 0.309
Spike level II (n=3) 101.57 ± 0.217 101.44 ± 0.269 101.70 ± 0.256
Spike level III (n=3) 101.93 ± 0.049 101.60 ± 0.310 101.31 ± 0.471
Linearity and Range (µg/mL)      
Response range 120-180 12-18 80-120
r2 0.9998 0.9999 0.9999
Y-Intercept -257647 -8780.7 -25617
Slope 102422 91165 49078
Sum of residual squares 3.6E+009 9004199 1.5E+008
Limit of detection/quantitation      
LOD (µg/mL) 7.15 0.06 0.46
LOQ (µg/mL) 21.68 0.19 1.40
Robustness (% assay mean ± SD)      
Change in flow rate      
0.8 mL/min (n=3) 100.72 ± 1.147 100.50 ± 1.003 100.43 ± 0.806
1.2 mL/min (n=3) 100.98 ± 0.911 100.61 ± 0.857 100.29 ± 0.734
Change in detection wavelength      
261 nm (n=3) 100.69 ± 1.195 100.44 ± 1.061 100.04 ± 0.994
265 nm (n=3) 100.81 ± 1.047 100.55 ± 0.935 100.35 ± 0.677
Change of column      
Merck (n=3) 100.83 ± 1.032 100.58 ± 0.900 100.59 ± 0.694
Teknokroma (n=3) 100.72 ± 1.177 100.43 ± 1.085 100.66 ± 0.625
Change in column temperature      
23°C (n=3) 100.87 ± 0.972 100.63 ± 0.846 100.24 ± 0.776
27°C (n=3) 100.85 ± 0.989 100.51 ± 0.991 100.20 ± 0.814
Table 2: Summary of validation parameters.
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