20
May/June 2013
Compound 1 2 3 4
Retention time (min) 1.37 2.65 3.13 3.57
tR %RSD 0.19%
0.11% 0.13% 0.10% Table 2: Retention time, precision data, and resolution obtained using a Hypercarb 5 µm, 100 x 2.1 mm column (method 1.) Chromatographic method 1:
Instrument: Thermo Scientific™ Accela™ 600 and Open Autosampler
Column: Thermo Scientific Hypercarb™ 5µm, 100× 2.1mm
Mobile Phase:
A: 100 mM KH2P04 in LC-MS grade water B: 100 mM KH2P04 : acetonitrile (LC-MS grade)( 50:50, v/v)
Gradient conditions:
T (min) % A %B 0.0 0.6 5.0 5.5 9.0
95 95 0
95 95
5 5
100 5 5
Flow rate: 400µL / min UV: 195nm Wash solvent: Water: Acetonitrile (95:5, v/v) Injection volume: 1µL Temperature: Ambient Pressure (t=0): 252 bar
Method 2: Test mix preparation:
Approximately 3.0mg of compounds 1, 2, 3, 5 and 6 were dissolved in 100mL of sample diluents diluents [water / acetonitrile (95:5 v/v)] to give a concentration of 0.03mg/mL impurity solution. Then, 1.5mg of compound 4 was dissolved in 1mL impurity solution to give the final ratio of 2.0% impurity to parent compound (4).
Chromatographic method 2: Instrument: Conventional HPLC System Column: Hypercarb™ 5µm, 100 × 4.6mm
Mobile Phase: A: 100 mM KH2P04 in LC-MS grade Water
B: 100 mM KH2P04 : Acetonitrile (LC-MS grade) (50:50 v/v)
Gradient conditions:
T(min) % A %B 0.0
15.0 17.0 17.1 27.0
95 0 0
95 95
5
100 100 5 5
Flow rate: 1.50mL/min UV: 195nm Wash solvent: water: acetonitrile (95:5 v/v) Injection volume: 5µL Temperature: 60˚C Pressure (t=0): 74 Bar
Results and discussion Method 1
With the smaller ID column and fewer compounds, the analysis was carried out in less than 9 min (Figure 4). The peaks were eluted in less than 4 min and very high resolution could be achieved. To check the reproducibility of the method, over 40 runs were carried out. Some of the chromatograms are overlaid in Figure 5. The method is very robust and consistent. The RSD values on retention time from the 39 runs (excluding the first run) were calculated to be less than 0.2% for all four compounds. The results are recorded in Table 2.
Method 2
The need for a method suitable for use on a conventional HPLC system with high dwell volume and high dead volume required the use of a 4.6 mm ID column. Two additional compounds (analytes 5
0
and 6) were also added to the method requirements. The result of this was that the analysis had to be extended to 27 minutes. With this method, all of the peaks were separated with excellent resolution (Figure 6).
The method reproducibility was also established with 40 replicate injections and overlaid chromatograms of seven injections from through the run are shown in Figure 7. After the column equilibrated sufficiently, consistent results could be obtained.
Further method robustness and reproducibility was assessed by investigating column-to-column variability. Three columns with different batches of stationary phase were obtained and the application SST (containing analytes 1-6) was analysed on each. After each column had been suitably equilibrated each column produced equivalent chromatograms.
Norm. 120
Resolution
14.05 6.58 5.45
100
80
60
40
20
0
5
10
Figure 6: The separation of 6 extremely polar compounds on Hypercarb 5 µm, 100 x 4.6 mm column (method 2).
min
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