Table 4 Summary of validation parameters for simultaneous estimation of THH and PIO

Linearity study (n = 5)

Linearity range (µg/mL)

40–200 µg/mL

30–150 µg/mL

Regression line equation

1754 x – 17,654

4232 x -134.56

Correlation coefficient

0.9994

0.9996

Precision study (% RSD)

Repeatability sample injection (n = 7)

0.75

0.83

Repeatability of sample measurement (n = 7)

0.45

0.38

Intra-day variation

0.87–0.98

0.59–0.92

Inter-day variation

0.92–1.12

0.77–1.04

Accuracy study (%) (n = 3)

% recovery

99.32–99.45

98.12–99.32

LOD & LOQ (n = 5)

Limit of detection (LOD)

5 µg/mL

3 µg/mL

Limit of quantitation (LOQ)

15 µg/mL

9 µg/mL

Robustness study (%RSD) (n = 3)

Mobile phase variation

0.73–0.92

0.89–1.03

Wavelength variation

0.65–1.12

0.77–1.05

Flow rate variation

0.62–1.05

0.83–1.03

Column temperature variation

0.81–1.14

0.94–1.21

Specificity study by PDA detector (correlation-coefficient)

Peak start (s)

0.99923

0.99934

Peak apex (a)

0.99928

0.99944

Peak end (m)

0.99956

0.99933