Table 2.
Chromatographic parameters used in the LC/MS and LC-MS/MS analysis.
| Parameter | Aqueous Fractions Analysed Using ZORBAX SB-AQ Column | Aqueous Fractions Analysed Using ZORBAC HILIC Column | ||
|---|---|---|---|---|
| Ionization mode | Positive MS and positive AutoMSMS | Negative MS and Negative AutoMSMS | Positive MS and positive AutoMSMS | Negative MS and Negative AutoMSMS |
| Mobile phase | Mobile phase A: Water with 0.2% acetic acid Mobile phase B: Methanol with 0.2% acetic acid |
Mobile phase A: Water with 1 mM ammonium fluoride acetic acid Mobile phase B: 100% Acetonitrile |
Mobile phase A: 90:10 of Acetonitrile: 50 mM ammonium acetate Mobile phase B: 50:40:10 of acetonitrile:water:50 mM ammonium acetate |
Mobile phase A: 90:10 of Acetonitrile:50 mM ammonium acetate Mobile phase B: 50:40:10 of acetonitrile:water:50 mM ammonium acetate |
| LC gradient | Time (min) | % mobile phase B | Time (min) | % mobile phase B |
| 1.00 | 5.0 | 3.00 | 0.0 | |
| 10.0 | 35.0 | 10.0 | 100.0 | |
| 11.0 | 95.0 | 13.0 | 100.0 | |
| 13.0 | 95.0 | 13.10 | 0 | |
| 13.1 | 5.0 | 17.00 | 0 | |
| 15.0 | 5.0 | |||
| Parameter | Organic fractions analysed using ZORBAX EP-C18 | Organic fractions analysed using ZORBAX EP Phenyl Hexyl | ||
| Ionization mode | Positive MS and positive utoMSMS | Negative MS and Negative AutoMSMS | Positive MS and positive AutoMSMS | Negative MS and Negative AutoMSMS |
| Mobile phase | 95:5 of water: Methanol with 0.1% formic acid and 5 mM ammonium formate | 65:30:5 of Isopropanol:methanol: water with 0.1% formic acid and 5 mM ammonium formate | 95:5 of water: Methanol with 0.1% formic acid and 5 mM ammonium formate | 65:30:5 of Isopropanol:methanol: water with 0.1% formic acid and 5 mM ammonium formate |
| LC gradient | Time (min) | % of mobile phase B | Time (min) | % of mobile phase B |
| 1.00 | 60.0 | 1.00 | 60.0 | |
| 8.0 | 100.0 | 8.0 | 100.0 | |
| 11.0 | 100.0 | 11.0 | 100.0 | |
| 11.10 | 60.0 | 11.10 | 60.0 | |
| 14.00 | 60.0 | 14.00 | 60.0 | |