Impressive Sensitivity and Linearity of Oligonucleotides on a Xevo QTof MS

The sensitivity demonstrated in this application note illustrates that Waters has achieved approximately three to six times higher sensitivity when compared to previous-generation MS instrumentation. This will potentially prove useful for those laboratories that wish to push the levels of sensitivity further. The ability to obtain a linear calibration curve over multiple orders of magnitude at this low level is useful for those who need to quantify oligonucleotides at low levels.

This work highlights the improved performance characteristics of Waters biopharmaceutical system solutions for oligonucleotides analysis. The ability to detect at better sensitivity and to provide a linear range for quantitation is demonstrated using UPLC Technology with the Xevo QTof MS. Organizations that are able to simultaneously quantify and identify therapeutic oligonucleotides will realize business advantages because they will have a better characterization of their product, and will be able to get products to customers more rapidly.

Oligonucleotides are generally quantified by UV methods because the absorbance measured at 260 nM is not dependent on sequence or structure. Although quantitation by UV may be preferable, Waters’ new generation of benchtop mass spectrometer, the Xevo QTof MS, makes comparisons of quantitative and qualitative data more achievable.

In this technical note, we demonstrate that Waters can detect LC-MS oligonucleotides peaks to the low-femtomolar level. The example illustrated shows linear detection in the range of 5 picomolar to 25 femtomolar on-column of a multi-T oligomer.

A standard mix made by Waters that contains a range of oligomers of 15-mer, 20-mer, 25-mer, 30-mer and 35-mer length (186004135) was used for the analysis. This mixture is used to demonstrate the separation capability of the ACQUITY UPLC System with a UPLC Oligonucleotide Separation Technology (OST) Column and to demonstrate the ability of the Xevo QTof MS to detect a mixture of oligomers in 15 minutes. This separation and MS detection is challenging to achieve in HPLC mode.

Figure 1 shows the calibration curve obtained for the 20-mer oligomer in the range 25 to 500 femtomolar using 12 acquisitions (scans) across the chromatographic peak apex. The signals of the other oligomers in the standard mixture are normalized to that of the 20-mer oligomer and therefore quantities can be extrapolated for the other oligomers.

At the lowest loading level, shown in Figure 3, the most abundant charge state (3-) of the 20-mer is shown to indicate the good signalto-noise ratio of approximately 10:1 (Raw data with no smoothing in continuum mode).

The raw spectrum of the 3- charge state can be compared to the theoretical data at 10,000 RP. This is shown in Figure 3 where 12 scans at the chromatographic peak apex were averaged to obtain the spectrum. The agreement with the theoretical spectrum for that charge state is 0.016 Da and the deconvoluted data shows agreement with the theoretical isotopic masses. Figure 4 shows the raw and smoothed data compared to the MaxEnt1 deconvoluted data.

It is important to note that being able to achieve these separations relies on the chromatographic system. UPLC OST Column technology has been specifically designed for the ACQUITY UPLC System and for these types of sample. The lack of carryover is shown in Figure 5, where a blank run and a 25 femtomolar sample are shown.

The sensitivity demonstrated in this application note illustrates that Waters has achieved approximately three to six times higher sensitivity when compared to previous-generation MS instrumentation. This will potentially prove useful for those laboratories that wish to push the levels of sensitivity further. The ability to obtain a linear calibration curve over multiple orders of magnitude at this low level is useful for those who need to quantify oligonucleotides at low levels.