Antisense oligonucleotides, siRNAs, miRNAs, aptamers, and other small nucleic acids are gaining prominence in the biopharmaceutical pipeline. The need for robust, accurate, and efficient ways of detecting such oligonucleotides and their metabolites in complex bodily fluids is crucially important for pharmacokinetic, pharmacodynamic, and exposure-response studies, as well as for the future of patient care.
There are several ways in which these measurements are accomplished, including by LC-MS/MS, HPLC-UV, capillary gel electrophoresis, and immunologically based approaches. Each of these methods offers certain advantages and limitations. The LC-MS/MS approach is sensitive and specific, accurate and reproducible, and is amenable to fast, high-throughput analysis. The Table summarizes some of these benefits.
An LC-MS/MS assay offers three levels of discrimination. First, samples are separated based on their mobility through the chromatography column—generally based on some combination of size, polarity, charge, and, perhaps, affinity. A typical chromatograph will indicate the amount of substance (detected by UV absorption, MS signal intensity, etc.) versus the time (or fraction number) at which it was eluted.
When a triple quadrupole mass spectrometer is used, the eluate is ionized as it enters the mass spectrometer, where it is scanned for mass/charge ratio (m/z) in Q1 based on its molecular weight and charge states. Selected ions with specific m/z from Q1 are then allowed to collide in Q2 with gas molecules, fragmenting them into pieces, which are then measured for m/z by a second detector (Q3).
On the basis of multiple specific MRM (multiple reaction monitoring) “transitions” from a compound to unique fragments, the instrument is able to discriminate among starting compounds that could not otherwise be differentiated. This is especially true for poly-ionic oligonucleotide compounds that usually have multiple unique ionic charge states in Q1 scan (Figure 1).
Scientists at Tandem Labs have developed and validated dozens of sensitive, high-throughput LC-MS/MS assays allowing us to quantify oligonucleotide molecules in bodily fluids such as urine and plasma. This tutorial presents an outline of the development of a LC-MS/MS method and an example of its use: the detection and quantification over a broad dynamic range of a model compound from human plasma.