Relative quantitation of oligonucleotides and impurities is commonly measured by LC-UV trace peak integration. Oftentimes multiple oligonucleotides elute within the same integrated UV peak, complicating UV quantitation. An example is shown below, with multiple coeluting oligonucleotide impurities.
The Byos UV-MS Report Tab uses the Deconvoluted MS Intensity ratio to approximately quantitate multiple oligonucleotides observed in the same UV peak. This can be observed in the UV-MS column in the Report tab. An example UV-MS Report Tab is shown below, calculating relative abundance of a 40-mer full length product and several observed impurities for two replicate samples.
The UV-MS Report Tab rows are divided into three levels. First by datafile (Sample name column 1), second by UV integrated peak for each sample (Peak # column 2), and third by deconvoluted assigned masses in each UV Peak (Oligonucleotide name, Oligo Candidate, and Delta name columns 8, 9 and 10). Only assigned deconvoluted masses are included in the UV-MS Report Tab and MS Ratio% calculation (discussed in detail below).
Four columns are included for quantitation: Normed area %, Intensity, MS Ratio%, and UV-MS.
- Normed area % is the percent Area of a UV peak relative to the total area of all other peaks measured in the sample’s UV trace.
- Intensity is the height of a deconvoluted mass peak.
- MS Ratio% is an assigned mass peak Intensity divided by the sum of all assigned masses' Intensity in the same UV peak.
- Finally, UV-MS is the product of Normed area % of a UV peak and the MS Ratio% of an assigned mass observed in the UV peak.
UV Peak # 1 in the 40-mer Rep 1 sample is used as an example to explain how UV-MS is calculated.
First, MS Ratio% is calculated for each assigned mass in UV Peak # 1. The formula below shows how MS Ratio% is calculated for the Agilent 40-mer [24-40] impurity mass assignment:
Second, UV-MS is calculated for each mass assigned to UV Peak # 1. The formula below shows how UV-MS is calculated for the Agilent 40-mer [24-40] impurity mass assignment:
Adding the UV-MS Report Tab:
As of Byos v5.4, the UV-MS Report Tab is not yet included as a preset. Please reach out to firstname.lastname@example.org for access. Direct access from Byos and further enhancements to the report template will be available in future releases.