Table 6 a Method robustness showing effect of column temperature. b Method robustness showing effect of change of mobile phase
Parameter | Column temperature | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|
Decrease | Normal | Increase | ||||||||
33 °C | 35 °C | 37 °C | ||||||||
RT | RRT | RES | RT | RRT | RES | RT | RRT | RES | ||
a | ||||||||||
HC | 2.91 | 0.34 | NA | 2.91 | 0.34 | NA | 2.91 | 0.35 | NA | |
HCB | 8.55 | 1.00 | 29.7 | 8.48 | 1.00 | 29.7 | 8.41 | 1.00 | 29.5 | |
HCB-21 | 9.27 | 1.08 | 3.3 | 9.20 | 1.08 | 3.3 | 9.11 | 1.08 | 3.2 | |
HC-3-methyl enol ether butyrate | 13.81 | 1.62 | 19.0 | 13.75 | 1.62 | 19.6 | 13.67 | 1.63 | 20.3 | |
HC-17,21- butyrate ortho ester | 16.51 | 1.93 | 9.2 | 16.32 | 1.92 | 9.1 | 16.12 | 1.92 | 9.0 | |
The %RSD for the HCB Area from five replicate injections of standard | 0.2 | 0.1 | 0.2 | |||||||
Tailing for HCB peak in all standard injections (max) | 1.0 | 1.0 | 1.0 | |||||||
The number of theoretical plates in all working standard injections for HCB (min) | 22,298 | 22,593 | 22,788 | |||||||
Total %RSD for the HCB Area | 0.2 | 0.1 | 0.2 | |||||||
% Assay % RC | HCB (%Assay) | 98.6 | 99.9 | 98.8 | ||||||
HC | 0.09 | 0.08 | 0.09 | |||||||
HCB-21 | 0.43 | 0.45 | 0.39 | |||||||
% Unknown max impurity | 0.05 | 0.05 | 0.05 | |||||||
% Total Impurities | 0.78 | 0.78 | 0.71 | |||||||
Parameter | Robustness [change in mobile phase-A composition] | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|
Decrease in organic | Normal organic | Increase in organic | ||||||||
ACN:Water (27:73 v/v) | ACN:Water (30:70 v/v) | ACN:Water (33:67 v/v) | ||||||||
RT | RRT | RES | RT | RRT | RES | RT | RRT | RES | ||
b | ||||||||||
HC | 3.24 | 0.35 | NA | 2.91 | 0.34 | NA | 2.64 | 0.34 | NA | |
HCB | 9.28 | 1.00 | 31.2 | 8.48 | 1.00 | 29.7 | 7.66 | 1.00 | 27.4 | |
HCB-21 | 10.01 | 1.08 | 3.4 | 9.20 | 1.08 | 3.3 | 8.33 | 1.09 | 3.1 | |
HC-3-methyl enol ether butyrate | 14.56 | 1.57 | 19.9 | 13.75 | 1.62 | 19.6 | 12.83 | 1.67 | 19.2 | |
HC-17,21- butyrate ortho ester | 17.31 | 1.87 | 9.3 | 16.32 | 1.92 | 9.1 | 15.28 | 1.99 | 8.9 | |
The %RSD for the HCB Area from five replicate injections of standard | 0.1 | 0.1 | 0.1 | |||||||
Tailing for HCB peak in all standard injections (max) | 1.0 | 1.0 | 1.1 | |||||||
The number of theoretical plates in all working standard injections for HCB (min) | 27,299 | 22,593 | 18,693 | |||||||
Total %RSD for the HCB Area | 0.1 | 0.1 | 0.1 | |||||||
% Assay % RC | HCB (%Assay) | 99.1 | 99.9 | 98.8 | ||||||
HC | 0.08 | 0.08 | 0.08 | |||||||
HCB-21 | 0.44 | 0.45 | 0.43 | |||||||
% Unknown max impurity | 0.05 | 0.05 | 0.07 | |||||||
% Total impurities | 0.77 | 0.78 | 0.81 | |||||||