SPDS SET 2: ASH-01

4.7) Range of quantification:

4.7.1) Definition: The range of the method is the interval between the lower and upper concentration level that have been demonstrated to be linear.

4.7.2) Assessment: It was calculated from the graph. A graph is plotted using the concentration on the X-axis and response factor on Y-axis. Response factor is the ratio between the

area and the concentration in mcg/ml.

4.7.3) Acceptance Criteria: Response factors of concentrations fall within the horizontal zone ± 7.5% .

4.7.4) Experiment:

a) Procedure: 20 µ l of each individual standard solution (refer section 4.2.4) were injected in

replicates and the chromatograms were recorded. Mean area was determined. Response

factors were calculated for all the Withanoside IV, Withanoside V, Withaferin A, 12-Deoxy

withastramonolide, Withanolide A and Withanolide B by dividing the mean area with the concentration.

The mean response factor for Withanoside IV between the concentration 20.64 to

330.3 mcg/ml is 7838.43

Table-68:

Withanoside IV (mcg/ml)

Different Concentrations

Dilution -6

Dilution -5

Dilution -4

Dilution -3

Dilution -2

Dilution -1

S. solution

(20.64)

(41.29)

(82.58)

(165.15)

(330.30)

(660.60)

(1321.20)

Mean

166024

329761

651132

1276177

2493821

4816148

9587357

7290.57

7256.55

Response factor Area / concentration Mean (Dilution 2 to 6)

8042.32

7986.95

7885.34

7727.38

7550.17

7838.43

± 5.0% of 7838 is 7446 to 8229

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