Common Mass Impurities Observed in Oligonucleotide Analysis
This article lists common mass impurities and adducts observed during LC-MS analysis of oligonucleotides. These signals are frequently misinterpreted as synthesis failures but are often expected chemical or solvent-related artifacts.
Many unexpected peaks detected by LC-MS are expected outcomes of synthesis chemistry, downstream processing, storage, or the mass spectrometer itself and do not necessarily indicate a synthesis failure.
Use this guide to:
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Identify common mass shifts and their origins
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Distinguish true synthesis issues from MS artifacts
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Determine when corrective action is required
Key Definitions (Important)
Impurities vs Contaminants
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Impurities
Chemical variants inherently related to synthesis or processing (e.g., truncations, incomplete deprotection, backbone variants). -
Contaminants
Foreign species introduced from solvents, salts, handling, purification, or the analytical method itself (e.g., Na⁺, K⁺, solvent adducts).
Not all detected signals represent synthesis defects, even if they are reported by analytical software.
How to Interpret Mass Shifts (Before Troubleshooting)
Always determine:
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Direction
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Positive (+) → retained groups, adducts, oxidation
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Negative (−) → deprotection, truncation, fragmentation
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Scaling
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Per base
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Per linkage
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Once per molecule
-
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Presence of Full-Length Mass
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If the expected full-length oligo mass is present, synthesis is often acceptable even if minor variants are observed.
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Protecting Group–Related Mass Shifts
5′-DMT (Dimethoxytrityl)
| Observation | Mass Shift |
|---|---|
| Successful detritylation | −298 Da |
| DMT retained | +302 Da |
Interpretation
-
−298 Da is expected after detritylation or DMT-OFF Synthesis
-
+302 Da indicates DMT-ON Synthesis or failure to remove final DMT group
Base Protecting Groups (Incomplete Deprotection)
| Protecting Group | Mass Shift | Applies To |
|---|---|---|
| Acetyl (Ac) | +42 Da per base | C |
| Benzoyl (Bz) | +104 Da per base | A, C |
| N,N-Dimethylformamidine (Dmf) | +55 Da per base | G |
| Isobutyryl (iBu) | +114 Da per base | G |
| Phenoxyacetyl (Pac) | +136 Da per base | A |
Typical Appearance
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Multiple partially deprotected species
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Laddered peaks
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Broadened charge envelopes
Action
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Extend deprotection
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Verify reagent freshness
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Confirm temperature and mixing
Backbone & Structural Modifications
Cyanoethyl (β-CE) Retention
| Shift | Meaning |
|---|---|
| +53 Da per site | Incomplete cyanoethyl removal |
| +106 Da | Two retained cyanoethyl groups |
One of the most common and benign findings if full-length mass is present.
Backbone Variants and Oxygen Substitution
| Modification | Mass Shift | Notes |
|---|---|---|
| Phosphorothioate (PS) | +/- 16 Da per site | Oxygen replaced with sulfur or sulfur replaced with oxygen |
| Methylphosphonate (MP) | +/- 14 Da per site | MP or oxygen substitution |
Unexpected +/- 16 or +/- 14 Da shifts relative to the designed sequence can indicate incomplete sulfurization or incomplete methylphosphonate incorporation, leading to oxygen-containing phosphodiester linkages instead of the intended modification.
Small-Molecule & Solvent-Related Adducts (Analytical Artifacts)
| Adduct | Mass Shift | Source |
|---|---|---|
| Sodium (Na⁺) | +22 Da | Glassware, buffers |
| Potassium (K⁺) | +38 Da | Salts |
| HFIPA adduct | +168 DA | LC-MS mobile phase buffer |
Guidance
If adduct peaks:
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Coexist with full-length mass
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Shift cleanly with charge state
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Disappear after desalting
→ No corrective action required
Side Reactions vs MS Artifacts
True Chemical Damage (Less Common)
| Reaction | Approx. Mass Shift (Da) | Notes |
|---|---|---|
| Depurination (A) | −135 | Loss of adenine base |
| Depurination (G) | −151 | Loss of guanine base |
| Depyrimidination (C) | −111 | Loss of cytosine base |
| Depyrimidination (T) | −126 | Loss of thymine base |
| Depyrimidination (U) | −112 | Loss of uracil base (RNA) |
| Transamination (Usually C) | +14 per affected base | Side reaction of strong AMA base deprotection on certain protecting groups (usually Bz) |
Risk Factors
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Very acidic detritylation conditions or extended times
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Elevated temperature
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Incorrect deprotection times or conditions
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RNA and modified-base chemistries
In-Source Fragmentation (Common, Benign)
| Fragment | Mass |
|---|---|
| Free Thymine (T) | ~125 Da |
| Free Cytosine (C) | ~110 Da |
| Free Adenine (A) | ~134 Da |
| Free Guanine (G) | ~150 Da |
| Base loss (−A, −G, −C, −T) | −90 to −150 Da |
If these signals increase with higher source energy and decrease under softer conditions, they are mass spectrometry artifacts, not synthesis defects.
Truncation & Support-Related Signals
| Issue | Mass Shift | Interpretation |
|---|---|---|
| CPG retention | ~260–300 Da | Incomplete cleavage or incorrect conditions |
| Modified CPG | 600–2000+ Da | Support-specific |
These findings often require investigation, especially if the full-length mass is weak or absent.
Common Nucleotide-Residue Losses (DNA)
| Description | Approx. Mass Shift (Da) | Notes |
|---|---|---|
| −Phosphate (−PO₃H) | −79.98 | Terminal phosphate or backbone fragmentation |
| −Adenine residue (−A) | −313.209 | dA; Loss of one nucleotide residue |
| −Cytosine residue (−C) | −289.184 | dC; Loss of one nucleotide residue |
| −Guanine residue (−G) | −329.208 | dG; Loss of one nucleotide residue |
| −Thymine residue (−T) | −304.196 | dT; Loss of one nucleotide residue |
Common Nucleotide-Residue Losses (RNA)
| Description | Approx. Mass Shift (Da) | Notes |
|---|---|---|
| −Adenine residue (−A) | ~329 | rA; Loss of one nucleotide residue |
| −Cytosine residue (−C) | ~305 | rC; Loss of one nucleotide residue |
| −Guanine residue (−G) | ~345 | rG; Loss of one nucleotide residue |
| −Uracil residue (−U) | ~306 | rU; Loss of one nucleotide residue |
Fluorophore & Solid-Support Modifications
| Modification | Mass Shift |
|---|---|
| FAM label | -537 Da |
| Biotin / specialty CPG | Varies (600–2000+) |
Mixed labeled/unlabeled populations may indicate incomplete coupling or labeling efficiency issues.
Quick Decision Guide
| Observation | Likely Cause | Action |
|---|---|---|
| Full-length mass present + minor adducts | MS artifact | None |
| +53 x | Cyanoethyl | Extend deprotection |
| +302 dominant | DMT retained | Fix final detritylation if |
| −330 ladder | Truncation | Check coupling |
| Free bases only | In-source | Lower source energy |
| No full-length mass | Real synthesis issue | Investigate process |
Storage, Handling, and Environmental Effects
Low-level mass variants may arise from:
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Oxidation (+16 Da increments)
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Moisture uptake
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Extended storage or elevated temperature
These effects often appear as minor, low-intensity peaks rather than defined truncation ladders.
Key Takeaway
Most unexpected LC-MS peaks are explainable, expected, and non-fatal.
Always prioritize:
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Presence of full-length mass
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Consistency with chemistry design
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Sensitivity to MS conditions
Only then escalate to synthesis troubleshooting.