Analysis of Thiopurines - AppNote
November 3, 2014
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Separation using LCMS Gradient Method
The Cogent Diamond Hydride Column with gradient elution was used for analysis of Thiopurines. Figure A shows the Peak of 6-TG, which also can be analyzed using Isocratic conditions. Good retention and symmetrical peak shape were obtained under the analysis conditions.
Figure B represents two separated Thiopurines (Mono and Di- Phosphate forms). Figure C shows two Inosine compounds, one with an additional Methyl group, being separated.
Column: Cogent Diamond Hydride™, 4μm, 100Å
Catalog No.: 70000-15P-2
Dimensions: 2.1 x 150mm
Mobile Phase:
--A: DI Water / 50% Methanol / 0.1% Formic Acid (v/v)
--B: Acetonitrile / 0.1% Formic Acid (v/v)
Gradient:
Post Time: 2 minutes
Flow rate: 0.4 mL/minute
Detection: ESI – POS - Agilent 6210 MSD TOF Mass Spectrometer
Injection vol.: 1μL
Sample Preparation: 0.4 mg/mL solutions in DI Water. For MS analysis, samples were diluted 1:100 into 50% Acetonitrile / 50% DI Water mixture. Before injection, samples were filtered through a 0.45µm Nylon Syringe Filter (MicroSolv Tech Corp.).
Attachment
No 322 Analysis of Thiopurines pdf 0.3 Mb Download File
The Cogent Diamond Hydride Column with gradient elution was used for analysis of Thiopurines. Figure A shows the Peak of 6-TG, which also can be analyzed using Isocratic conditions. Good retention and symmetrical peak shape were obtained under the analysis conditions.
Figure B represents two separated Thiopurines (Mono and Di- Phosphate forms). Figure C shows two Inosine compounds, one with an additional Methyl group, being separated.
Peaks:
A: Thioguanine (6-TG) at m/z = 168.0338 [M+H]+
B: 6-Thioguanosine -5´-Phosphate (TGMP) at m/z =380.3, 6-Thioguanosine -5´-Diphosphate (TGDP) at m/z= 460.3
C: 6-Methyl-Thioinosine-5´-Monophosphate (meTIMP) at m/z = 379.3 and 6-Thioinosine-5´-Triphosphate (TITP) at m/z = 525
Column: Cogent Diamond Hydride™, 4μm, 100Å
Catalog No.: 70000-15P-2
Dimensions: 2.1 x 150mm
Mobile Phase:
--A: DI Water / 50% Methanol / 0.1% Formic Acid (v/v)
--B: Acetonitrile / 0.1% Formic Acid (v/v)
Gradient:
| Time (minutes) | %B |
| 0 | 100 |
| 12 | 30 |
| 14 | 30 |
| 15 | 0 |
| 19 | 0 |
| 20 | 100 |
Flow rate: 0.4 mL/minute
Detection: ESI – POS - Agilent 6210 MSD TOF Mass Spectrometer
Injection vol.: 1μL
Sample Preparation: 0.4 mg/mL solutions in DI Water. For MS analysis, samples were diluted 1:100 into 50% Acetonitrile / 50% DI Water mixture. Before injection, samples were filtered through a 0.45µm Nylon Syringe Filter (MicroSolv Tech Corp.).
Attachment
No 322 Analysis of Thiopurines pdf 0.3 Mb Download File