Page 20 - Oligonucleotide Therapeutics Solution Guide

Characteristic analysis
Quality Control

Molecular Weight Confirmation & Quantitation LCMS-9030

Features
Molecular Weight Determination and Quantitation of Oligonucleotide
LCMS-9030 is a quadrupole time-of-flight (Q-TOF) mass spectrometer with two types of ion mass separation mechanisms: quadrupole and time-of- Modification
Therapeutics Using Quadrupole Time-of- Flight Mass Spectrometer LCMS-9030 flight. Unique technologies abound, such as technology to improve ion utilization efficiency, processing technology to produce high-strength fine Target selection
grid electrodes, high-precision temperature control, and optimization of potential distribution, enabling data acquisition with both high sensitivity
click here and high resolution while maintaining stable mass accuracy.
Since oligonucleotide therapeutics have a molecular weight of 6000 or more at 20 bases, high-precision mass spectrometers such as Q-TOF mass
spectrometers are used to confirm the molecular weight.
• High-resolution mass spectra provide more accurate molecular weight
confirmation.
benefits • Achieves stable mass accuracy without frequent mass calibration. Excision

Methods and Results Unprotected Oligomer synthesis

Sample 5'-mG-mC*-mC*-mU*-mC*-dA-dG-dT-dC*-dT-dG-dC*-dT-
dT-dC*-mG-mC*-mA-mC*-mC*-3‘
m: 2‘-O-(2-Methoxyethyl) nucleoside (2-MOE)
* : 5-methylated derivatives of C and U
D : 2’-deoxyribonucleoside
Monoisotopic mass: 6431.7239
Conc., Volume 1 ~ 1000 ng/mL, 5 µL
Preparation Dilution in ultrapure water to the concentrations above.
Analytical The HPLC and MS analysis conditions are shown in
Conditions Table 1. In this analysis, HFIP (1,1,1,3,3,3-Hexafluoro-2-propanol) Purification
and DIPEA (N, N-diisopropylethylamine) were used in the mobile
phase to allow highly sensitive measurement to be performed.
Results Figure 1 shows the mass spectrum extracted from the scan mode
data. In the mass spectrum, multi-charged ions such as those at Figure 1 Mass Spectra of Oligonucleotide Therapeutics
m/z 1071.6, 918.4 and 803.5 were detected.
The m/z values of these multi-charged ions were deconvoluted
and Figure 2 shows the results of molecular mass calculation. As
shown in the deconvolution spectrum, the monoisotopic mass
was 6431.72 with a mass error of 3 mDa (0.05 ppm).
The calibration curve is shown in Figure 3 insert, The calibration
curve was prepared from 1 - 1000 ng/mL. The correlation
2
coefficient (r ) was 0.996. Figure 3 shows representative
chromatograms obtained using multiple reaction monitoring
(MRM) mode. The octavalent ion at m/z
803.4626 was selected as a precursor ion. The product
ion at m/z 94.9358 (PSO2-) was used for quantitation. Simple Ionization Unit Quality Control
Table 1 Analysis Conditions In addition to the standard ESI ionization unit, Characteristic analysis
[HPLC conditions] (Nexera) optional APCI and Dual Ion Source (DUIS) probes
Column: Shim-pack Scepter C18 (75 mm × 2.0 mm I.D.,1.9 µm) are available for the LCMS-9030 to meet various
Mobile phases: A)50 mmol/L HFIP and 10 mmol/L DIPEA Figure 2 Deconvolution spectra analytical needs. Shimadzu's DUIS offers an
B)Acetonitrile
Gradient Program B5％ (0-0.5 min) – 15%(0.5-6 min) efficient combination of ESI and APCI ionization
Flow rate : 0.2 mL/min capabilities.
Column Temp. : 50 °C
Injection volume : 5 µL
[MS conditions] (LCMS-9030) DDS
Ionization: ESI(Negative mode)
Probe Voltage: -3 kV Calibrant Delivery Pharmacokinetics
Mode: Full-scan （m/z 500-3000）
MRM(803.4626>94.9358) System (CDS)
Nebulizing gas flow: 3.0 L/min
Drying gas flow: 10.0 L/min The CDS allows calibration standards to be introduced via a separate ionization probe that
Heating gas flow: 10.0 L/min functions independently from the main probe. This optional sub-ionization unit is installed in the
DL Temp.: 250 °C main probe housing and is available for all probe types (ESI/APCI/DUIS). By having two probes
Heat Block Temp.: 400 °C
Interface Temp.: 350 °C Figure 3 MRM Chromatograms of Oligonucleotide Therapeutics and Calibration Curve in one system, high-concentration calibration standards can be introduced only when needed
without switching flow lines, and are kept in isolation from the sample stream, eliminating
contamination.
Conclusions Other

Accurate mass spectrometry using the Q-TOF mass spectrometer LCMS-9030 determined the molecular mass with an error of 0.05 ppm.
Additionally, linearity was observed within a range of 1 - 1000 ng/mL.

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