Prepare an Acquisition method
Click here to see this page in full context

Prepare an Acquisition method

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.

Preparations

  • 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.

  1. Launch the instrument.

  2. Prepare two acquisition methods, one with standard conditions, one with harsh conditions. Below are the recommended LC and MSD method parameters.

Acquisition method recommendations

LC Parameters

HPLC

1260/1290 Infinity II or Infinity III

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 or G6170A

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/III 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.

×