Potential Misidentification of Natural Isomers and Mass-Analogs of Modified Nucleosides by Liquid Chromatography–Triple Quadrupole Mass Spectrometry

Abstract

Triple quadrupole mass spectrometry coupled to liquid chromatography (LC-TQ-MS) can detect and quantify modified nucleosides present in various types of RNA, and is being used increasingly in epitranscriptomics. However, due to the low resolution of TQ-MS and the structural complexity of the many naturally modified nucleosides identified to date (>160), the discrimination of isomers and mass-analogs can be problematic and is often overlooked. This study analyzes 17 nucleoside standards by LC-TQ-MS with separation on three different analytical columns and discusses, with examples, three major causes of analyte misidentification: structural isomers, mass-analogs, and isotopic crosstalk. It is hoped that this overview and practical examples will help to strengthen the accuracy of the identification of modified nucleosides by LC-TQ-MS.

1. Introduction

Research into post-transcriptional RNA modification is increasingly focusing on its critical impacts on RNA decay, translational efficiency, subcellular localization, quality control, RNA–protein interactions, and disease development [1,2,3]. Intensely mined information from the epitranscriptome reveals that dynamic modification of RNA is a response to physiological and environmental changes, although the biological consequences of such modifications are still being elucidated [3,4,5]. Methylation of key transcripts, for example, N⁶-methyladenosine (m⁶A), has been observed in budding yeast and was crucial to the initiation of meiosis during nitrogen starvation [6]. YTHDF1, an m⁶A reader protein that enhances translational efficiency by recruiting eukaryotic initiation factor 3, was found to be concentrated on stress granules and triggered stalled translation when arsenite was added to induce oxidative stress in HeLa cells [7]. YTHDF2 (an m⁶A reader) and FTO (an m⁶A eraser) competitively bind to the 5′-UTR of messenger (m)RNA and regulate m⁶A methylation in a mouse embryonic fibroblast (MEF) cell line, which facilitates cap-independent translation of specific transcripts under stress conditions [8]. On transfer RNAs (tRNAs), modifications at positions 34 (the wobble position), 37, and 58 can be dynamic and related to environmental factors. When taurine supply was limited, τm⁵U34 (5-taurinomethyluridine) and τm⁵s²U34 (5-taurinomethyl-2-thiouridine) on five mitochondrial transfer (mt)RNAs switched to cmnm⁵U34 (5-carboxymethylaminomethyluridine) and cmnm⁵s²U (5-carboxymethylaminomethyl-2-thiouridine) with unknown consequences [9]. Bicarbonate-free air culturing of HEK293T cells triggered the downregulation of t⁶A37 (N⁶-threonylcarbamoyladenosine) on mt-tRNAs decoding ANN codons and probably impaired the translation of mitochondrial respiratory complex I [10]. m¹A58 (1-methyladenosine) in human cytoplasmic tRNAs was influenced by the glucose concentration of the culture medium via an FTO (m¹A eraser)-dependent pathway that adjusted the decoding preference [11].

To determine the identity, quantity, and location of RNA modifications, RNA sequencing (NGS or NNGS approaches) [12,13,14], oligonucleotide mass spectrometry [15,16], and nucleoside mass spectrometry [17] are frequently used. Nuclear magnetic resonance spectroscopy has also recently been used to observe the dynamic incorporation of RNA modifications into nascent tRNA [18,19]. A generic protocol to qualitatively and quantitatively analyze modified nucleosides has been developed using high-performance liquid chromatography coupled to triple quadrupole mass spectrometry (LC-TQ-MS) [20]. Total RNA or a purified fraction is hydrolyzed and dephosphorylated to the free nucleosides using alkaline phosphatase, phosphodiesterase I, and nuclease P1. Two-step digestion is recommended as basic pH is suitable for alkaline phosphatase, while phosphodiesterase I and nuclease P1 are more effective in acidic conditions. However, some studies recommend an acidic environment for the complete digestion because certain RNA modifications are sensitive to pH. For instance, ct⁶A (cyclic N⁶-threonylcarbamoyladenosine) quickly epimerizes to t⁶A in mild alkaline buffer, causing an 18 Da mass increase [21]. The enzymes are then removed from the digestion mixture using a 10 kDa cut-off centrifugal filter unit, and the resulting nucleosides are vacuum-dried and dissolved in an appropriate solvent, usually 90% (v/v) acetonitrile or water, depending on the downstream liquid chromatography. Hydrophilic interaction liquid chromatography (HILIC) [22] and reversed-phase chromatography (RPC) [23] with semi-micro flow rates (0.1–1 mL/min) have been used to separate nucleosides, while micro-flow HPLC (5–50 μL/min) has recently been recommended for higher sensitivity without loss of reproducibility [24].

Multiple reaction monitoring (MRM) mode is used with TQ-MS when determining modified nucleosides. Collision-induced dissociation of the N-β-glycosidic bond under the appropriate collision energy (CE) yields the nucleobase production, BH₂⁺. Ideally, synthetic standards of the modified nucleosides should be used to confirm optimum CE, ion mass–to–charge ratios (m/z), specific product ions and retention times, but their preparation is expensive, time consuming and labor intensive. The m/z values for precursor (MH⁺) and product (BH₂⁺) ions can be readily calculated from their chemical structures (Figure 1). CEs for detecting modified nucleosides can be estimated using native (unmodified) adenosine (A), uridine (U), cytidine (C), and guanosine (G) standards. This may not yield exact values for the modified nucleosides but is a practical compromise. Some published LC-TQ-MS applications for RNA modifications use fewer than 20 modified nucleoside standards and determine other nucleosides using calculated MRM transitions and estimated CE values [20,25].

Over 160 natural RNA modifications have been identified to date [26]. However, this complexity and the low resolution of TQ-MS (approximately 0.5 Da) can give rise to three types of misidentification when using MH⁺ and BH₂⁺ MRM transitions to determine nucleosides. Type I mistakes result from regioisomers. For instance, five natural monomethylated adenosines have been identified. These are m¹A, m²A (2-methyladenosine), m⁶A, m⁸A (8-methyladenosine), and Am (2′-O-methyladenosine) (Figure 2), and have been found variously in mRNA, tRNA, and ribosomal (r)RNA [26]. Am can be discriminated by monitoring the transition m/z 282.1→136 because of its unmodified nucleobase, while m¹A, m²A, m⁶A, and m⁸A all give rise to the transition m/z 282.1→150 (Figure 2). High-resolution mass spectrometry can discriminate between these four monomethylated derivatives via in-source, collision-induced dissociation (CID) and negative mode ionization that produces unique patterns of fragments [27]. However, this approach requires a library of MS² and MS³ spectra for each isomer and sensitivity can be reduced for quantitative analysis.

Although synthetic standards can strengthen the identification of nucleoside isomers, some cannot be separated (or are closely eluted) by liquid chromatography because of their structural similarity. Type II mistakes with TQ-MS are nucleoside misidentifications due to similar masses (<0.5 Da mass differences). m^6,6A (N⁶,N⁶-dimethyladenosine) and f⁶A (N⁶-formyladenosine) exhibit MH⁺ masses of 296.1359 and 296.0995, respectively. Both are modified on the nucleobase (Figure 3). Low-resolution TQ-MS of m/z 296.1→164 cannot distinguish between these two compounds. Furthermore, m^6,6A has an isomer—m^2,8A (2,8-dimethyladenosine)—which can lead to the complicated situation of f⁶A/m^6,6A being present in a eukaryotic total RNA sample, or m^6,6A/m^2,8A being present in a eubacterial rRNA sample [26].

Type III misidentification arises from isotopic crosstalk, which is often not considered. For instance, the low-resolution isotopic distributions of positively ionized adenosine and inosine are shown in Figure 4. The transition 269.1→137 used to monitor inosine is subject to interference from the same transition arising from an isotopologue of adenosine. When adenosine and inosine are eluted simultaneously by a short HPLC method, the signal for inosine would be amplified significantly by isotopic mass of adenosine. Given the abundance of isotopic masses of small molecules composed of the elements C, H, O, N, and S, nucleosides differing in mass by one or two units are likely to interfere with each other. Based on the natural abundance of isotopes of these elements (13C 1.11%, 2H 0.0115%, 18O 0.205%, 15N 0.364%, and 34S 4.21%), such mass differences mainly arise from 13C and 34S. Such mass-analogs are not rare among naturally modified nucleosides: crosstalk between mcm⁵U (5-methoxycarbonylmethyluridine, 317.1→185) [28], nchm⁵U (5-carbamoylmethyl-2-thiouridine, 318.1→186) [29], and cm⁵s²U (5-carboxymethyl-2-thiouridine, 319.1→187) [30] is a good example and is illustrated in Figure 5.

These three types of misidentifications can occur in the same RNA sample; thus, correctly identifying a nucleoside by TQ-MS is not straightforward. This study illustrates this complexity by describing the analysis of 17 nucleosides and modified nucleosides using three commercially available liquid chromatography columns. Ongoing discussion of the separation of commonly modified nucleosides using reversed-phase and HILIC chromatography, and of the signals derived from TQ-MS, will help to minimize the misidentifications described above.

2. Materials and Methods

LC-MS grade formic acid and acetonitrile were purchased from Thermo Fisher (Shanghai, China) and LC-MS grade water from Sigma (Shanghai, China) for the preparation of mobile phases and standard solutions. Nucleoside standards were purchased from respected commercial manufacturers and agencies as follows: uridine (U), cytidine (C), 4-thiouridine (s⁴U), and N⁶-methyladenosine (m⁶A) from Aladdin (Shanghai, China); 1-methyladenosine (m¹A), 1-methylinosine (m¹I), N⁶,N⁶-dimethyladenosine (m^6,6A), N⁶-formyladenosine (f⁶A), pseudouridine (Y), 5-hydroxyuridine (ho⁵U), 2-thiouridine (s²U), 5-methyldihydrouridine (m⁵D), 2-thiocytidine (s²C), and N¹-methylguanosine (m¹G) from TRC (Toronto, Canada); 2-methyladenosine (m²A) from Howei Pharm (Guangzhou, China); N²-methylguanosine (m²G) from TopScience (Rizhao, China); and 7-methylguanosine (m⁷G) from Sigma (Shanghai, China).

Stock solutions of nucleosides (10–100 mM) were prepared in dimethyl sulfoxide and stored at −20 °C. Mixed standard solutions were prepared by diluting stock solutions with 0.1% formic acid in acetonitrile/water (90/10, v/v) for HILIC chromatography, or water containing 0.1% formic acid for reversed-phase chromatography.

Mass spectrometry was conducted on a QTRAP 6500 LC-MS system (AB Sciex, Redwood, CA, USA). Mobile phase A consisted of 0.1% (v/v) formic acid in water, and mobile phase B was acetonitrile containing 0.1 % (v/v) formic acid. Reversed-phase chromatographic separations were run on a Waters Atlantis T3 column (2.1 mm² × 150 mm², 3 µm) and a Supelco Discovery HS F5 column (2.1 mm² × 150 mm², 3 µm). The gradient program (0.1 mL/min flow rate) was as follows: 0–6 min, 0% B; 6–35 min, 0–90% B; 35–40 min, 90% B; 40–40.1 min, 90–0% B; and 40.1–50 min, 0% B. HILIC separations were conducted on a Waters Acquity UPLC BEH amide column (2.1 mm² × 150 mm², 1.7 µm) at 0.1 mL/min flow rate using the elution gradient: 0–5 min, 90% B; 5–35 min, 90–40% B; 35–40 min, 40% B; 40–40.1 min, 40–90% B; and 40.1–50 min, 90% B. Column temperatures were maintained at 36 ºC, the autosampler at room temperature, and the injection volume was 1 μL. MS and MS/MS detection used an electrospray ionization (ESI) source in positive ion mode and the following optimized parameters: ion-spray voltage 5.5 kV, source temperature 350 °C, curtain gas 30 psi, collision gas 8 psi, ion source gas 1 at 30 psi, ion source gas 2 at 1 psi, entrance potential 10 V, collision cell exit potential 13 V, and dwell time 10 ms. MultiQuant 3.0.3 software (AB Sciex, Redwood, CA, USA) was used for data analysis.

3. Results and Discussion

3.1. Analytical Chromatography Columns

Three analytical columns were evaluated for their ability to resolve modified nucleosides over a 50 min chromatographic runtime: Acquity BEH amide (1.7 μm, 2.1 mm × 150 mm; Waters), Discovery HS F5 (3 μm, 2.1 mm × 150 mm; Supelco), and Atlantis T3 (3 μm, 2.1 mm × 150 mm; Waters) (Figure 6). The Acquity BEH amide (HILIC) column was packed with ethylene-bridged hybrid particles covalently attached by trifunctionally-bonded amide groups, while the linker structure of BEH amide is not published by the Waters Corporation. The Discovery HS F5 column was filled with spherical silica gel and a propyl spacer-linked pentafluorophenyl (PFP) stationary phase. The Atlantis T3 column was an octadecyl silica-based (ODS), reversed-phase C18 column with optimized pore diameter, C18-ligand density, and end-capping. Excellent performance in the separation of highly polar chemicals, including carbohydrates and nucleoside triphosphates, has been reported using these columns [31,32,33].

3.2. Adenosine Derivatives

One pmol each of m¹A, m²A, m⁶A, m¹I (1-methylinosine), m^6,6A, and f⁶A were mixed and resolved on the three columns (Figure 7). Positive mode mass pair 282.1→150 was monitored to detect m¹A, m²A, and m⁶A, 283.1→151 for m¹I, and 296.1→164 for m^6,6A and f⁶A. The methylated adenosines were eluted in the order m⁶A/m²A/m¹A under HILIC conditions, but m¹A/m²A/m⁶A under PFP and ODS conditions (Figure 7A). With the PFP column, m²A and m⁶A did not achieve baseline separation, which may lead to type I misidentification.

When m/z 283.1→150 was used to detect m¹I, type III misidentification (isotopic crosstalk) could occur. Interfering signals from isotopes of m¹A, m²A, and m⁶A were evident, adjacent to the primary m¹I peak (Figure 7B). In the case of HILIC separation, a small m⁶A peak eluted close to m¹I, while a m²A peak co-eluted with m¹I under PFP conditions. Isotopic crosstalk should be carefully ruled out, especially when the target analyte is in low abundance and may be masked by an isotopologue of another nucleoside.

m^6,6A and f⁶A exhibited similar retention times in the HILIC method (Figure 7C), although baseline separation was achieved. TQ-MS is not sufficiently sensitive to distinguish the mass difference between these two nucleosides (0.0364 Da); thus, the transition 296.1→164 acquired signals from both compounds. It is noteworthy that, although one pmol of each standard was injected, the signal for f⁶A was much lower than for m^6,6A. This can be explained by the N⁶-formyl group of the f⁶A nucleobase tending to be deprotonated rather than protonated. This detection weakness under positive mode ionization increases the chance of f⁶A being misidentified as m^6,6A in the absence of synthetic standards. Therefore, it is recommended to use a PFP or ODS column to detect m^6,6A and f⁶A because of the large difference in retention times under these conditions (Figure 7C).

3.3. Uridine and Cytidine Derivatives

The m/z differences between protonated uridine and cytidine (m/z 245.1 and 244.1), and between their nucleobases (m/z 113 and 112) are approximately one. Thus, monitoring of uridine derivatives can be subject to inferring signals from isotopes of cytidine derivatives. For example, the transition m/z 259.1→127 can detect m⁵U (5-methyluridine), m³U (3-methyluridine), and isotopes of m⁵C (5-methylcytidine), m⁴C (N⁴-methylcytidine), and m³C (3-methylcytidine). Type I, II, and III misidentifications are possible, in some cases, in a single chromatogram monitoring uridine derivatives. Two standard mixtures were prepared to illustrate this complexity: firstly, 1 pmol each of U (uridine), C (cytidine), and Y (pseudouridine); and secondly, 1 pmol each of s²U (2-thiouridine), s⁴U (4-thiouridine), ho⁵U (5-hydroxyuridine), m⁵D (5-methyldihydrouridine), and s²C (2-thiocytidine). Figure 8A illustrates the monitoring of uridine (m/z 245.1→113) with isotopic crosstalk from cytidine. However, although the mass of pseudouridine is identical to uridine, the protonated nucleobase of pseudouridine is not seen. Instead, pseudouridine is uniquely identified by the transitions m/z 245.1→209/179/155. The CID fragmentation of pseudouridine is shown in Figure 8B [34]. The product ions of pseudouridine derivatives, such as m¹Y (1-methylpseudouridine) and Ym (2’-O-methylpseudouridine), can be predicted using this fragmentation pattern to avoid interference with uridine derivatives.

Type I, II, and III potential misidentifications can be observed simultaneously in the mixture of s²U, s⁴U, ho⁵U, m⁵D, and s²C standards. Figure 8C shows how peaks for all five modified nucleosides appear in one MRM channel (m/z 261.1→129): s²U and s⁴U are structural isomers (type I); s²U, s⁴U, ho⁵U, and m⁵D have similar precursor and product ion masses (<0.5 Da difference) (type II); and a cross-talking isotopologue of s²C can be observed in the primary s²U/s⁴U/ho⁵U/m⁵D channel (type III).

3.4. Guanosine Derivatives

Obstacles to the identification of guanosine derivatives center on the possible isomers, particularly among the monomethylated (m¹G, m²G, m⁷G), dimethylated (m^2,2G, m^2,7G), and trimethylated (m^2,2Gm, m^2,7Gm) guanosines. Figure 9 shows the elution sequence of m¹G, m²G, and m⁷G on various columns, from which the elution sequence of the dimethylated and trimethylated derivatives can be extrapolated. Figure 9 also illustrates the importance of analytical column choice, with only HILIC being capable of resolving the monomethylated guanosines.

4. Conclusions

Monitoring the fragmentation of protonated nucleosides into their respective nucleobases using the MRM mode of TQ-MS is useful for detecting RNA modifications. However, it is not practical to synthesize standards for many of these modified nucleosides and, consequently, three types of misidentification can result. Firstly, structural isomers can appear in the same MRM channel and may be closely eluted under HPLC (type I misidentification). Secondly, mass-analogs differing by less than 0.5 Da might also appear in the same channel and may be confused with other analytes (type II). Finally, analytes with mass differences of one or two Da can cause isotopic crosstalk (type III). This study applied a long HPLC runtime of 50 min but some nucleosides could still not be clearly resolved on-column. Therefore, the authors suggest that reports of rapid LC-MS methods for the detection of nucleosides (typically with 5–10 min runtimes) should be examined to rule out the possibility of analyte misidentifications arising from overlapping peaks. Stable isotope labeling of nucleosides can help to prevent type II and III misidentifications, as the mass shifts due to 13C or 15N provide additional molecular composition information [35,36,37]. Potential misidentifications of frequently modified nucleosides are listed in

Table 1. Nucleosides with potential type I misidentification (natural isomers).

Name	Abbreviation	Chemical Formula	Precursor Ion	Product Ion
Adenosine derivatives
1-Methyladenosine	m1A	C11H15N5O4	282.1	150
2-Methyladenosine	m2A	C11H15N5O4	282.1	150
N6-Methyladenosine	m6A	C11H15N5O4	282.1	150
8-Methyladenosine	m8A	C11H15N5O4	282.1	150
2,8-Dimethyladenosine	m2,8A	C12H17N5O4	296.1	164
N6,N6-Dimethyladenosine	m6,6A	C12H17N5O4	296.1	164
Cytidine and Uridine derivatives
3-Methylcytidine	m3C	C10H15N3O5	258.1	126
N4-Methylcytidine	m4C	C10H15N3O5	258.1	126
5-Methylcytidine	m5C	C10H15N3O5	258.1	126
3-Methyluridine	m3U	C10H14N2O6	259.1	127
5-Methyluridine	m5U	C10H14N2O6	259.1	127
1-Methylpseudouridine	m1Y	C10H14N2O6	259.1	179
3-Methylpseudouridine	m3Y	C10H14N2O6	259.1	179
2-Thiouridine	s2U	C9H12N2O5S	261.1	129
4-Thiouridine	s4U	C9H12N2O5S	261.1	129
5,2′-O-Dimethylcytidine	m5Cm	C11H17N3O5	272.1	126
N4,2′-O-Dimethylcytidine	m4Cm	C11H17N3O5	272.1	126
3,2′-O-Dimethyluridine	m3Um	C11H16N2O6	273.1	127
5,2′-O-Dimethyluridine	m5Um	C11H16N2O6	273.1	127
5-Carboxyhydroxymethyluridine	chm5U	C11H14N2O9	319.1	187
Uridine 5-oxyacetic acid	cmo5U	C11H14N2O9	319.1	187
5-(Carboxyhydroxymethyl)uridine methyl ester	mchm5U	C12H16N2O9	333.1	297
Uridine 5-oxyacetic acid methyl ester	mcmo5U	C12H16N2O9	333.1	297
3-(3-amino-3-carboxypropyl)pseudouridine	acp3Y	C13H19N3O8	346.1	214
3-(3-amino-3-carboxypropyl)uridine	acp3U	C13H19N3O8	346.1	214
Guanosine derivatives
1-Methylguanosine	m1G	C11H15N5O5	298.1	166
N2-Methylguanosine	m2G	C11H15N5O5	298.1	166
7-methylguanosine	m7G	C11H15N5O5	298.1	166
1,2′-O-Dimethylguanosine	m1Gm	C12H17N5O5	312.1	166
N2,2′-O-Dimethylguanosine	m2Gm	C12H17N5O5	312.1	166
N2,7-Dimethylguanosine	m2,7G	C12H17N5O5	312.1	180
N2,N2-Dimethylguanosine	m2,2G	C12H17N5O5	312.1	180
N2,N2,2′-O-Trimethylguanosine	m2,2Gm	C13H19N5O5	326.1	180
N2,7,2′-O-Trimethylguanosine	m2,7Gm	C13H19N5O5	326.1	180
Isowyosine	imG2	C14H17N5O5	336.1	204
Wyosine	imG	C14H17N5O5	336.1	204

,

Table 2. Nucleosides with potential type II misidentification (mass-analogs).

Name	Abbreviation	Chemical Formula	Precursor Ion	Product Ion
Adenosine derivatives
N6-Formyladenosine	f6A	C11H13N5O5	269.1	164
2,8-Dimethyladenosine	m2,8A	C12H17N5O4	269.1	164
N6,N6-Dimethyladenosine	m6,6A	C12H17N5O4	269.1	164
N6-Methyl-N6-threonylcarbamoyladenosine	m6t6A	C15H20N6O8	427.2	295
N6-Hydroxynorvalylcarbamoyladenosine	hn6A	C16H22N6O8	427.2	295
Cytidine and Uridine derivatives
2-Thiocytidine	s2C	C9H13N3O4S	260.1	128
5-Hydroxycytidine	ho5C	C9H13N3O6	260.1	128
2-Thiouridine	s2U	C9H12N2O5S	261.1	129
4-Thiouridine	s4U	C9H12N2O5S	261.1	129
5-Hydroxyuridine	ho5U	C9H12N2O7	261.1	129
5-Methyldihydrouridine	m5D	C10H16N2O6	261.1	129
5-Formylcytidine	f5C	C10H13N3O6	272.1	140
N4,N4-Dimethylcytidine	m4,4C	C11H17N3O5	272.1	140
5-Methyl-2-thiouridine	m5s2U	C10H14N2O5S	275.1	143
5-Methoxyuridine	mo5U	C10H14N2O7	275.1	143
5-Formyl-2′-O-methylcytidine	f5Cm	C11H15N3O6	286.1	140
N4,N4,2′-O-Trimethylcytidine	m4,4Cm	C12H19N3O5	286.1	140
5-Carbamoylmethyl-2-thiouridine	ncm5s2U	C11H15N3O6S	318.1	186
5-Carbamoylhydroxymethyluridine	nchm5U	C11H15N3O8	318.1	186
5-Carboxymethyl-2-thiouridine	cm5s2U	C11H14N2O7S	319.1	187
5-Carboxyhydroxymethyluridine	chm5U	C11H14N2O9	319.1	187
Uridine 5-oxyacetic acid	cmo5U	C11H14N2O9	319.1	187
5-Methoxycarbonylmethyl-2-thiouridine	mcm5s2U	C12H16N2O7S	333.1	201
5-(carboxyhydroxymethyl)uridine methyl ester	mchm5U	C12H16N2O9	333.1	201
Uridine 5-oxyacetic acid methyl ester	mcmo5U	C12H16N2O9	333.1	201
5-Carboxymethylaminomethyl-2-thiouridine	cmnm5s2U	C12H17N3O7S	348.1	216
3-(3-amino-3-carboxypropyl)-5,6-dihydrouridine	acp3D	C13H21N3O8	348.1	216

and

Table 3. Nucleosides with potential type III misidentification (isotopic crosstalk).

Name	Abbreviation	Chemical Formula	Precursor Ion	Product Ion
Adenosine derivatives
1-Methyladenosine	m1A	C11H15N5O4	282.1	150
2-Methyladenosine	m2A	C11H15N5O4	282.1	150
N6-Methyladenosine	m6A	C11H15N5O4	282.1	150
1-Methylinosine	m1I	C11H14N4O5	283.1	151
2′-O-Methyladenosine	Am	C11H15N5O4	282.1	136
2′-O-Methylinosine	Im	C11H14N4O5	283.1	137
1,2′-O-Dimethyladenosine	m1Am	C12H17N5O4	296.1	150
1,2′-O-Dimethylinosine	m1Im	C12H16N4O5	297.1	151
Cytidine and Uridine derivatives
3-Methylcytidine	m3C	C10H15N3O5	258.1	126
5-Methylcytidine	m5C	C10H15N3O5	258.1	126
N4-Methylcytidine	m4C	C10H15N3O5	258.1	126
3-Methyluridine	m3U	C10H14N2O6	259.1	127
5-Methyluridine	m5U	C10H14N2O6	259.1	127
2-Thiocytidine	s2C	C9H13N3O4S	260.1	128
5-Hydroxycytidine	ho5C	C9H13N3O6	260.1	128
2-Thiouridine	s2U	C9H12N2O5S	261.1	129
4-Thiouridine	s4U	C9H12N2O5S	261.1	129
5-Hydroxyuridine	ho5U	C9H12N2O7	261.1	129
5-Methyldihydrouridine	m5D	C10H16N2O6	261.1	129
5,2′-O-Dimethylcytidine	m5Cm	C11H17N3O5	272.1	126
N4,2′-O-Dimethylcytidine	m4Cm	C11H17N3O5	272.1	126
3,2′-O-Dimethyluridine	m3Um	C11H16N2O6	273.1	127
5,2′-O-Dimethyluridine	m5Um	C11H16N2O6	273.1	127
5-Hydroxymethylcytidine	hm5C	C10H15N3O6	274.1	142
5-Aminomethyluridine	nm5U	C10H15N3O6	274.1	142
5-Methyl-2-thiouridine	m5s2U	C10H14N2O5S	275.1	143
5-Methoxyuridine	mo5U	C10H14N2O7	275.1	143
5-Cyanomethyluridine	cnm5U	C11H13N3O6	284.1	152
N4-Acetylcytidine	ac4C	C11H15N3O6	286.1	154
5-Formyl-2′-O-methylcytidine	f5Cm	C11H15N3O6	286.1	140
N4,N4,2′-O-Trimethylcytidine	m4,4Cm	C12H19N3O5	286.1	140
2′-O-Methyl-5-hydroxymethylcytidine	hm5Cm	C11H17N3O6	288.1	142
5-Methylaminomethyluridine	mnm5U	C11H17N3O6	288.1	156
5-Aminomethyl-2-thiouridine	nm5s2U	C10H15N3O5S	290.1	158
5-Carbamoylmethyluridine	ncm5U	C11H15N3O7	302.1	170
5-Carboxymethyluridine	cm5U	C11H14N2O8	303.1	171
5-Methylaminomethyl-2-thiouridine	mnm5s2U	C11H17N3O5S	304.1	172
5-Methoxycarbonylmethyluridine	mcm5U	C12H16N2O8	317.1	185
5-Carbamoylmethyl-2-thiouridine	ncm5s2U	C11H15N3O6S	318.1	186
5-Carbamoylhydroxymethyluridine	nchm5U	C11H15N3O8	318.1	186
5-Carboxymethyl-2-thiouridine	cm5s2U	C11H14N2O7S	319.1	187
5-Carboxyhydroxymethyluridine	chm5U	C11H14N2O9	319.1	187
Uridine 5-oxyacetic acid	cmo5U	C11H14N2O9	319.1	187
5-Carboxymethylaminomethyluridine	cmnm5U	C12H17N3O8	332.1	200
5-Methoxycarbonylmethyl-2-thiouridine	mcm5s2U	C12H16N2O7S	333.1	201
5-(carboxyhydroxymethyl)uridine methyl ester	mchm5U	C12H16N2O9	333.1	201
Uridine 5-oxyacetic acid methyl ester	mcmo5U	C12H16N2O9	333.1	201
5-Carboxymethylaminomethyl-2′-O-methylu-ridine	cmnm5Um	C13H19N3O8	346.1	200
5-(carboxyhydroxymethyl)-2′-O-methyluridi-ne methyl ester	mchm5Um	C13H18N2O9	347.1	201
2′-O-Methyluridine 5-oxyacetic acid methyl ester	mcmo5Um	C13H18N2O9	347.1	201
5-(isopentenylaminomethyl)-2-thiouridine	inm5s2U	C15H23N3O5S	358.1	226
1-Methyl-3-(3-amino-3-carboxypropyl)pseud-ouridine	m1acp3Y	C14H21N3O8	360.1	228
Guanosine derivatives
7-Aminocarboxypropylwyosine methyl ester	yW-58	C19H26N6O7	451.2	319
Undermodified hydroxywybutosine	OHyWx	C18H24N6O8	453.2	321

.

Modified nucleosides can contain hydrophobic nucleobases (e.g., those with isopentenyl moieties) and hydrophilic nucleobases (e.g., those with hydroxy moieties), making reversed-phase and HILIC methods suitable for the separation of different groups of nucleosides. The PFP column (Discovery HS F5) separated uridine and cytidine derivatives effectively, and the HILIC column (Acquity BEH amide) was helpful for adenosine and guanosine derivatives. The chromatographic column should be carefully chosen and the HPLC method optimized depending on the target analyte(s). The analytical sensitivity of the nucleosides under positive mode LC-TQ-MS conditions is usually A>G>C>U (uridine being difficult to protonate under positive mode ionization due to its low pKa value). Negative mode ionization, derivatization [38], and the careful selection of productions should be considered to improve sensitivity. The degradation, oxidation [39], and spontaneous chemical derivatization [40] of nucleosides during pre-treatment procedures should also be taken into account. We recommend the establishment of strict standards for nucleoside analysis.