Oligo Analysis Accelerator was developed for analysis of data acquired using Agilent Single Quadrupole LC/MSD detectors. This ensures that the LC/MS can be run at full scan, profile mode with acceptable sensitivity to quantify low-level impurities with limits of quantitation greater than or equal to 0.2 % by peak areas.
Prerequisites
To be able to carry out the procedure as described, you need the privileges Project Management > Edit content of project and Acquisition Method > Create and modify acquisition method. Privileges are configured in the Control Panel.
Launch the instrument.
Prepare two acquisition methods, one with standard conditions, one with harsh conditions. Below are the recommended LC and MSD method parameters.
LC Parameters
HPLC |
1260/1290 Infinity II |
Column |
Agilent AdvanceBio Oligonucleotide, 2.1 x 150 mm, 2.7 µm |
Mobile Phase |
A:10 % ACN, 5 mM Tributylammonium Acetate, 1 µm EDTA B: 80 % ACN, 5 mM Tributylammonium Acetate, 1 µm EDTA |
Flow-rate |
0.25 mL/min |
Column Temp |
50 °C |
Gradient |
45-80 % B in 22 minutes |
UV | 260 nm | Bandwidth 4 nm Ref: 400 nm| Bandwidth 80 nm |
MSD Parameters
MSD |
G6135C |
Data Storage |
Full, Profile Mode |
Source |
ESI |
Drying Gas Flow |
12.0 L/min (standard) 13.0 L/min (harsh) |
Gas Temp |
260 °C (standard) 350 °C (harsh) |
Nebulizer pressure |
25 psig |
Capillary Voltage |
4000 V |
Mode |
Negative |
Scan |
FLP, -350 to +150 m/z e.g., for 1728.1, 1820-2320 m/z, Profile |
Scan time |
1149 ms (standard) 975 ms (harsh) |
Fragmentor |
100 V |
Gain Factor |
2 |
Although some variation may be acceptable, the following are imperative to ensure robust and reproducible results:
Although many oligo methods use the Agilent Jet Stream source, using standard ESI ensures in-source fragmentation is kept to acceptable levels.
Optimize in-source fragmentation to minimize abasic or depurinated impurities. This includes the source temperature (typically 250-260 °C for Standard MS conditions) and fragmentor voltage (100-150 V).
Using a bio-inert system such as the Agilent 1290 Infinity II BioLC is recommended to reduce metal adducts. This is critical for adducts which may spectrally overlap with other known impurities, such as iron (Fe+) and CNET (N3-(2-cyanoethyl)thymine).
Tributylammonium acetate is the recommended ion pairing reagent, as this ensures the 4-charge state is predominant.
Ensure the UV detector does not induce photooxidation. This may occur with diode array detectors.