As interests increase in oligonucleotide therapeutics, there has been a greater need for analytical techniques to properly analyze and quantitate these biomolecules. This article looks into some of the existing chromatographic approaches for oligonucleotide analysis, including anion exchange, hydrophilic interaction liquid chromatography, and ion pair chromatography. Some of the key advantages and challenges of these chromatographic techniques are discussed. Colloid formation in mobile phases of alkylamines and fluorinated alcohols, a recently discovered analytical challenge, is discussed. Mass spectrometry is the method of choice to directly obtain structural information about oligonucleotide therapeutics. Mass spectrometry sensitivity challenges are reviewed, including comparison to other oligonucleotide techniques, salt adduction, and the multiple charge state envelope. Ionization of oligonucleotides through the charge residue model, ion evaporation model, and chain ejection model are analyzed. Therapeutic oligonucleotides have to undergo approval from major regulatory agencies, and the impurities and degradation products must be well-characterized to be approved. Current accepted thresholds for oligonucleotide impurities are reported. Aspects of the impurities and degradation products from these types of molecules are discussed as well as optimal analytical strategies to determine oligonucleotide related substances. Finally, ideas are proposed on how the field of oligonucleotide therapeutics may improve to aid in future analysis.
A new method for reversed phase HPLC determination of thiamine and its major in vivo phosphorylation products, thiamine monophosphate (TMP) and thiamine pyrophosphate (TPP), was developed using tetrabutylammonium hydroxide as the ion-pairing agent. The separation was performed on a Phenomenex Kinetex EVO C18 column with a gradient of a phosphate-buffered aqueous solution of the ion-pair reagent and methanol. The duty cycle for the assay was 13 min and pyrithiamine was successfully used as the internal standard for the first time in a thiamine HPLC measurement protocol. Detection of the fluorescence derivatives of the analytes as well as the IS allowed for lower detection limits in order to support biological applications in cell culture models. The linearity, sensitivity, specificity, accuracy and precision of the method were evaluated and met the requirements specified by the US Food and Drug Administration. The calibration curves proved to be linear and the method was validated over the range from 1.0-4000 nM for both cells and the media where complete recovery of the analytes was also achieved.
Rationale
Cationic adduction causes poor sensitivity and increases spectral complexity during mass spectral analysis of oligonucleotides and alkylamines are used to reduce this adduction. It is unclear the effect of the physiochemical properties of the alkylamines on the reduction of the cationic adduction.
Methods
All samples were directly infused into a Synapt G2 HDMS quadrupole time‐of‐flight (TOF) hybrid mass spectrometer in negative ion electrospray ionization mode through the native built‐in fluidics system. The infusion flow rate was set to 50 μL/min. The TOFMS tuning parameters were as follows: capillary voltage −2.0 kV, cone voltage 25 V, extraction cone voltage 2 V, source temperature 125°C, desolvation temperature 450°C, cone gas flow rate 0 L/h, and desolvation gas (nitrogen) flow rate 1000 L/h.
Results
A quantitative model was created to predict the optimized alkylamine for MS analysis, while a qualitative model was generated to explain the most important physiochemical properties: proton affinity (13.83%), gas‐phase basicity (11.79%), pKa (11.47%), boiling point (10.73%), MW (10.3%), Henry's Law Constant (9.56%), and partition coefficient (logP) (9.44%). The quantitative model was applied to RNA (microRNA) and a phosphorothioate and predicts the trend of cationic adduction.
Conclusions
Two models are described to understand the physiochemical properties that contribute to the adduction and to provide users a quick mathematical tool to predict the best choice of alkylamine to lower cationic adduction and decrease spectral complexity while enhancing sensitivity.
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