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The derived half-normal plot graphically distinguishes significant from insignificant effect for each parameter, in that significant effects all tend to fall on a straight line through zero, whereas significant effects deviated from the straight line and are above the calculated margin of error (ME). The candidate method was found to be robust for the method parameters evaluated, and the results obtained were normally distributed, with variances conforming to that expected by chance (Figure 4).

Table 12: Method factors assessed in ruggedness trial Factor High Low water added A = 5.2 mL a = 4.8 mL Carrez solution B = 0.12 mL b = 0.08 mL stand time C = 25 min c = 15 min centrifuge speed D = 2200 rcf d = 1800 rcf extract volume E = 0.11 mL e = 0.09 mL acetonitrile volume F = 0.91 mL f = 0.89 mL dummy factor G = left-hand g = right-hand

Figure 4: Half-normal plot of robustness results

As with similar methods exploiting stable-isotope-labelled internal standard quantitation, critical method parameters include accurate measurement of sample weight, the amount of