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38 Pharmaceutical Technology ® The Real Message Behind Commercial mRNA Products April eBook 2023 PharmTech.com AnAly tics acid-based therapeutics, including today's complex, multi-faceted structures. Issues of st abi l it y, del iver y, a nd bi nd i ng have resulted in the development of RNA therapeutics t hat of ten i nvolve sign i f ica nt mod i f icat ion(s) to the basic oligoribonucleic acid structure. Figure 1 provides several examples of these modified RNA structures. Changes to the phosphodiester linkage have included replacement of the free oxygen with sulfur (producing the more stable phosphorothioate linkage) and substitution with amide and borono- phosphate linkages (12). Similarly, multiple, diverse structural alterations to both sugar and base moi- eties have been described, including methylation, methoxyethylation, f luorination, and cyclization of the 2' sugar hydroxyl, resulting in so called "locked nucleic acid" (LNA) (10). Often, several of these mod- i f icat ions may be i ncor porated w it h i n t he sa me therapeutic. In addition, linking of these RNA-based therapeutics with a wide range of conjugates has been exploited as a means to significantly enhance and direct their delivery and greatly improve their effectiveness. A notable example of such modifica- tion is the conjugation of siRNAs with N-acet ylga- lactosamine providing specific binding to asialoly- coprotien receptors in hepatocytes. This structural concept has led to the successful development of the drug, givosiran, which treats hepatic porphyria by si lencing t he expression of aminolev ulinate syn- t hase 1 m RNA in t he liver (13). Many ot her conju- gates have been described such as lipids, peptides, antibodies (complete or fragments), vitamins, and a range of other molecular classes (14). Currently, the most widely used hyphenated an- aly tical technique is LC–MS. Multiple separation methodologies have been reported including ion-pair reversed-phase chromatography, hydrophilic inter- action liquid chromatography, ion-exchange chro- matography, and size exclusion chromatography. A report by Santos and Brodbelt (15) provides a compre- hensive review of these and other related techniques as applied to nucleic acids. Two-dimensional LC has also been described as a means to overcome incom- patibility between mobile-phase composition and MS detection (16), while 3D-LC–MS—incorporating in-line digestion and hydrophilic interaction liquid chromatography (HILIC) separations—has been used for the sequencing of CRISPR-guide RNAs (17). The majority of mass spectrometric applications involving nucleic acids have utilized either laser de- sorption (matrix or surface assisted) or electrospray ion izat ion (ESI) tech n iques. Bot h a re considered "soft," thereby reducing in-source fragmentation of these large biomolecules and providing a relatively high ion current for the intact molecular species. Al- most all analyzer types have been employed, includ- ing magnetic sector, time of f light, quadrupole, ion trap, and combinations of each. Today, a technology that has gained considerable acceptance both for quantitative and qualitative investigations is the so- called Orbitrap that was introduced commercially in 2005 and is shown in Figure 2. Combining ion trap and/or quadrupole mass filters, orbitrap-based FIGURE 1. Examples of modified RNA structures. FIGURE 2. SGS uses liquid chromatography tandem mass spectrometry (LCMSMS) Orbitrap technology to solve complex analytical problems. FIGURES COURTESY OF THE AUTHOR.