Page 22 - Oligonucleotide Therapeutics Solution Guide

Characteristic analysis
Quality Control

Molecular Weight Confirmation & Quantitation LCMS-8060NX

Features
Quantitation and Confirmation of Molecular
LCMS-8060 NX is the culmination of Shimadzu’s expertise in triple quadrupole mass spectrometry which boasts increased robustness while Modification
Weight of Oligonucleotide Therapeutics maintaining the high sensitivity and ultra-fast acquisition speeds that are the hallmark of Shimadzu’s technology. Target selection
In response to work-flow changes and the need for cost reductions in recent years, the market for LC-MS systems has diversified, and there is a
click here demand for highly efficient systems that are robust and easy to operate in addition to being highly sensitive. Therefore, LCMS -8060 NX adopts
a unique new ion source, the Ion Focus unit (Patent No. 6593548, US 10546740), that efficiently introduces ions into the mass spectrometer and
removes unnecessary neutrals. In addition, technologies such as UF-Qarray II and UF-Lens II, which have been reengineered to be even more resistant
• High sensitivity quantitation and molecular weight confirmation are possible to contamination, are incorporated to minimize equipment downtime and improve laboratory operational efficiency.
with one system.
benefits • Ease of maintenance helps minimize analytical downtime.
UFsweeper III collision cell
UFsweeper is a unique Shimadzu technology which Excision
Methods and Results Ultra-fast power supply effectively sweeps ions from the collision cell without Ultra-fast detector Unprotected
deceleration. Crosstalk is virtually eliminated and
The unique high-voltage power supply enables high-speed polarity high-sensitivity analysis is maintained, even at high A detector boasting ultra-fast response
switching in 5 ms for high-throughput simultaneous analysis of acquisition speeds. The high-speed transport enables sensitive and stable signal detection Oligomer synthesis
Sample 5'-mG-mC*-mC*-mU*-mC*-dA-dG-dT-dC*-dT-dG-dC*-dT- multiple analytes. technology of UFsweeper III minimizes ion loss even even with a short pause time, dwell time and
dT-dC*-mG-mC*-mA-mC*-mC*-3‘ at the highest data acquisition rate of 0.8 msec. polarity switching time.
m: 2'-O-(2-methoxyethyl) nucleoside (2'-MOE) Increase laboratory throughput with an MRM speed
* : Indicates 5-C or 5-U methylation IonFocus unit of 555 channels per second.
D : 2'-deoxynucleoside (newly-developed ESI probe)
Average mass : 6436.39
Conc., Volume 1 ~ 300 ng/mL, 2 µL
Preparation Dilution in ultrapure water to the concentrations above.
Analytical Table 1 shows the HPLC and MS method conditions. An ionpair
Conditions reagent is generally used in reversed phase separation of
oligonucleotides.
Results Fig. 1 shows a representative chromatogram acquired in Quadrupole rods Purification
MRM mode and the calibration curve. The calibration A high-performance hyperbolic mass
curve was prepared from 1 to 300 ng/mL. Fig. 2 shows the filter with a proven track record
mass chromatogram measured in scan mode. In the mass Figure 2 Mass Chromatogram of Oligonucleotide Therapeutics maintains high ion transmittance
chromatogram, hexavalent, heptavalent, and octavalent ions and high sensitivity, even at a
were selected. Figure 3 shows the mass spectrum. Pentavalent to scanning rate of 30,000 u/s.
octavalent ions were detected. As shown in the deconvolution
results, the estimated molecular weight was determined to be
6436.37 (theoretical value: 6436.39).
Table 1 Analysis Conditions
[HPLC conditions] (Nexera)
Column: C18 Column (50 mm × 2.1 mm I.D., 2.5 µm)
Mobil phases: A) 50 mmol/L HFIP and 10 mmol/L DIPEA B) Acetonitrile
Gradient Program: B Conc. 5%(0-0.5 min) – 15%(0.5-3 min)
Flow rate: 0.2 mL/min
Column Temp.: 60 °C
Injection volume: 2 µL UF-Qarray II, UF-Lens II Quality Control
Re-engineered ion guide improves Characteristic analysis
[MS conditions] (LCMS-8060) robustness without compromising UF-Qarray II UF-Lens II
Ionization: ESI(Negative mode) on sensitivity. Maintenance of
Probe Voltage: -4 kV UF-Qarray II and UF-Lens II can be
Mode: Full-scan (m/z 500 - 2000) MRM(m/z 803.5 > 95.0) performed easily without tools.
CID gas: 330 kPa
Nebulizing gas flow: 3.0 L/min Drying gas flow: 8.0 L/min
Heating gas flow: 12.0 L/min DL Temp.: 300 °C Figure 3 Mass Spectra of Oligonucleotide Therapeutics
Heat Blok Temp.: 450 °C Interface Temp.: 250 °C
Ion Focus
The ESI (Electrospray ionization) method, which is widely used as an DDS
ionization method for LC/MS, applies a high voltage to a nebulized
sample spray, creating charged droplets. Desolvation results in gas Pharmacokinetics
phase ions which are introduced into the mass spectrometer. However,
neutrals and matrix can enter the mass spectrometer, causing
contamination and a decrease in sensitivity. If the spray position is
moved further from the MS inlet in order to mitigate contamination,
both neutral contaminants and ions of interest are effected, resulting in
reduced sensitivity.
The new Ion Focus unit has been designed to solve this problem. The
Figure 1 MRM Chromatograms of Oligonucleotide Therapeutics and focus electrode for ion transport allows only ions of interest to be
Calibration Curve Figure 4 Results of Multivalent Ion Analysis drawn into the mass spectrometer. As a result, unnecessary solvents
Conclusions and matrix can be removed, making it possible to achieve both high Other
sensitivity analysis and an extremely robust system.
High-speed, high-sensitivity triple-quadrupole mass spectrometry enables molecular weight estimation and quantitative analysis of oligonucleotide
therapeutics

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