Table 7 Comparison between published (Ref.5) and newly developed methods
Parameters | Published method (Ref.5) | Developed methods | ||||
|---|---|---|---|---|---|---|
HPLC | TLC- densitometric | |||||
SER.DMP | DMP | SER.DMP | DMP | SER.DMP | DMP | |
Chromatographic conditions | ||||||
Mobile phase | Trifluoroacetic acid: acetonitrile (70:30 v/v) | 5 mM phosphate buffer (pH 5.5): acetonitrile (40:60 v/v) | methanol: chloroform (70:30 v/v) | |||
Detection wavelength | 265 nm | 265 nm | 220 nm | 220 nm | 220 nm | 220 nm |
Run time | 7.33 min | 7.33 min | 7.75 min | 7.75 min | –- | –- |
Retention time Rt or | 2.71 min | 7.33 min | 7.75 min | 4.82 min | –- | –- |
Flow rate (Rf) | – | –- | – | –- | 0.24 min | 0.62 min |
Forced degradation (% degradation) | ||||||
HCl (0.1N) | – | – | – | – | ||
Normal condition | 3.1% | 3.3% | ||||
HCl (0.1N) | ||||||
Reflux for 2 h at 60 °C | – | – | 100% verified by TLC, three degradants | 100% verified by TLC, one degradant | 100% verified by TLC, three degradants | 100% verified by TLC, one degradant |
HCl (1N) | ||||||
Normal condition | 22% | 21.5% | N/A | |||
NaOH (0.1N) | ||||||
Normal condition | 2.7% % | 2.5% | – | – | – | – |
NaOH (0.1N) | ||||||
Reflux for 2 h at 60 °C | – | – | 100% verified by TLC, two degradants | 100% verified by TLC, two degradants | 100% verified by TLC, two degradants | 100% verified by TLC, two degradants |
NaOH (1N) | ||||||
Normal condition | 24.9% | 20.3% | N/A | |||
Degradation products | ||||||
Separation | ||||||
Identification of acid degradants | – | – | + | + | + | + |
Separation | ||||||
Identification of alkaline degradants | – | – | + | – | + | – |
Confirmation of degradation pathway | – | – | confirmed using TLC, IR, 1H-NMR and mass spectroscopy | |||
Method validation | ||||||
Linearity range/sensitivity (μg/mL) | 4.2–63 | 0.9to3.5 | 2.5–25 | 5–25 | 5–25 | 5–25 |
LOD (μg/mL) | 0.042 | 0.009 | 0.051 | 0.098 | 0.184 | 0.115 |
LOQ (μg/mL) | 0.42 | 0.09 | 0.165 | 0.186 | 0.202 | 0.237 |
Selectivity (application to lab prepared mixtures in presence of degradants) | Not tested | Not tested | No interference from degradation products,up to 80% acidic & 70% alkaline | No interference from degradation products, up to 80% ( acidic) | No interference from degradation products, up to 80% acidic & 70% alkaline | No interference from degradation products, up to 80% acidic |
Application of standard addition techniquea | – | – | + | + | – | – |
Verification of practical outcomes by MDSTb | – | – | + | + | – | – |