Table 7. Summary of the Main Validation Results Using the Improved Method Reported in Table 5.

Precision of the System
RSD of the prednisolone peak area (n = 6) at 0.25 μg/mL (LOQ)			3.6%	≤10%
RSD of the prednisolone peak area (n = 6) at 2.50 μg/mL			0.6%	≤5%
Precision of the Method for the Active Ingredient (Real Sample)
RSD for impurity F (tR = 5.357 min)			2.3%	≤10%
RSD for impurity B (tR = 7.387 min)			7.8%	≤10%
RSD for impurity A (tR = 8.747 min)			2.5%	≤5%
RSD for unknown impurity (tR = 11.317 min)			7.4%	≤10%
RSD for total impurities			2.1%	≤5%
LOD and LOQ
LOD	0.025% of the working concentration (0.125 μg/mL)
LOQ	0.05% of the working concentration (0.25 μg/mL)
Linearity of the Method
parameter		prednisolone	impurity A	impurity B	impurity C
range (μg/mL)		0.26–6.33	0.27–6.34	0.28–6.54	0.28–6.58
slope (mL/μg)		28.310	28.150	26.029	24.236
intercept		0.1495	0.0972	0.1420	0.6132
R		0.9998	0.9999	0.9999	0.9999
correction factora		1.00	1.01	1.09	1.17
Accuracy of the Method
% of the working concentration		prednisolone	impurity A	impurity B	impurity C
0.05 (maximal LOQ)	average recovery (%)b	101.3 ± 12.8	102.9 ± 12.7	97.3 ± 12.6	82.0 ± 17.1
	RSD (%)	5.1	5.0	5.2	8.4
0.1	average recovery (%)b	101.3 ± 4.9	97.5 ± 4.5	88.7 ± 4.7	82.3 ± 6.1
	RSD (%)	2.0	1.9	2.1	3.0
0.3	average recovery (%)b	97.1 ± 0.5	98.4 ± 0.5	89.0 ± 3.8	82.2 ± 12.1
	RSD (%)	0.2	0.2	1.7	5.9
0.5	average recovery (%)b	98.2 ± 3.0	96.1 ± 1.9
	RSD (%)	1.2	0.8
1.0	average recovery (%)b	100.3 ± 5.9	95.5 ± 0.8
	RSD (%)	2.4	0.3
1.3	average recovery (%)b	96.2 ± 1.3
	RSD (%)	0.5
Selectivity
substance	retention time (min)		relative retention time
prednisolone	8.462		1.00
impurity A	8.883		1.05
impurity B	7.507		0.89
impurity C	15.534		1.84
impurity F	5.514		0.65
impurity J	10.970		1.30

^aCorrection factors for impurities were not included in the calculations as they were within the acceptable 0.8–1.2 range.

^bAverage recovery is reported along with a 95% confidence interval.