Analysis of Tris(Hydroxymethyl)Aminomethane in Oligonucleotide Active Pharmaceutical Ingredient (API) Using Ion Chromatography

Significance of the topic

This application note addresses reliable quantification of Tris(hydroxymethyl)aminomethane (Tris) in oligonucleotide active pharmaceutical ingredient (API) matrices using non-suppressor ion chromatography (IC). Tris is commonly used in buffer systems during oligonucleotide synthesis but is considered an undesired residual in final API material; sensitive and selective measurement is therefore important for quality control and regulatory oversight, particularly when coexisting inorganic cations (e.g., Na+, K+, Mg2+, Ca2+) are present.

Objectives and overview of the study

The primary goals were:

• Develop an IC method capable of simultaneous separation and quantification of Tris and six common cations (Li+, Na+, NH4+, K+, Mg2+, Ca2+) in oligonucleotide API matrices.
• Maintain good chromatographic throughput while preserving separation, especially resolving Tris from sodium.
• Demonstrate method linearity, repeatability, and recovery in a real oligonucleotide API sample.

Methodology

The approach used a cation-exchange column operated in a non-suppressor conductivity detection mode with specially modified eluents. Key methodological principles included leveraging crown ether additives to tune cation retention and using a minor fraction of organic solvent to accelerate elution without losing resolution.

Used instrumentation

• System: HIC-NS non-suppressor ion chromatograph.
• Column: Shim-pack IC-C4, 150 mm × 4.6 mm I.D., 7 µm; Guard: Shim-pack IC-C4(G), 10 mm × 4.6 mm I.D., 7 µm.
• Detection: Conductivity (non-suppressed).
• Chromatographic conditions (representative): flow 1.0 mL/min, column temperature 40 °C, injection volume 50 µL.
• Eluent composition (Analytical conditions 1): 2.5 mmol/L methanesulfonic acid with 5 mmol/L 18-crown-6 and 5 mmol/L 15-crown-5 (crown ethers added to 1 L as specified in the application note preparation instructions).
• Eluent modification (Analytical conditions 2): same composition with 5% (v/v) acetonitrile (eluent:acetonitrile = 95:5) to shorten retention times.

Main results and discussion

Separation strategy and mechanism:

• Crown ethers (18-crown-6 and 15-crown-5) form inclusion complexes with specific cations. Complexation increases hydrophobic character of the included species, allowing selective modulation of retention in reversed-phase-like interactions on the IC stationary phase.
• 18-crown-6 is commonly used for cation manipulation; however, increasing 18-crown-6 alone to resolve Tris from Na+ prolongs elution of other cations. Addition of 15-crown-5 (5 mmol/L) alongside 18-crown-6 (5 mmol/L) selectively delays sodium elution, permitting Tris/Na separation without unduly extending elution of Mg2+, K+, and Ca2+.

Analytical performance:

• Calibration: Mixed-standard calibration covered a practical concentration range for all analytes (example standards from 0.02–0.4 mg/L for Li to 0.4–4 mg/L for other cations; Tris from 0.5–10 mg/L across STD1–STD5).
• Linearity: All analytes exhibited excellent linearity with r2 ≥ 0.9994 (most ≥ 0.9998), indicating reliable quantitative response across the tested ranges.
• Repeatability: Peak area %RSDs from six consecutive analyses (STD3 level) ranged from 0.35% (Li) to 2.12% (Mg), demonstrating robust precision suitable for QC use.
• Sample application: An oligonucleotide API was analyzed (600 mg/L solution); native Tris was not detected and sodium was present at ≈30 mg/L. A spike-and-recovery test with 3 mg/L Tris gave 94.3% recovery, supporting method accuracy in the API matrix.
• Throughput improvement: Adding 5% acetonitrile to the eluent accelerated elution; calcium was eluted within 20 minutes under the acetonitrile-containing conditions versus longer runtimes without organic modifier, while maintaining separation quality.

Benefits and practical applications of the method

• Simultaneous quantification: Enables concurrent determination of Tris and common inorganic cations in a single injection, simplifying sample workflows and minimizing sample consumption.
• Selective control of retention: Use of combined crown ethers allows selective adjustment of sodium retention to resolve it from Tris without broadly increasing runtimes for other cations.
• Improved throughput: Small amounts of acetonitrile shorten analysis time while preserving chromatographic performance, increasing laboratory productivity.
• Applicability to oligonucleotide APIs: Demonstrated spike recovery and matrix tolerance indicate suitability for QC testing of therapeutic oligonucleotide materials where Tris residues may be of concern.

Future trends and potential applications

• Broader adoption of crown-ether modified eluents in pharmaceutical ionic impurity testing for complex biologics and oligonucleotide products where amine-containing buffers are common.
• Optimization of organic modifier percentage or use of alternative solvents to further reduce run times while retaining resolution, enabling higher throughput QC environments.
• Extension of the approach to automated high-throughput platforms and coupling with mass spectrometric detection for orthogonal confirmation when needed.
• Method adaptation to regulated validation workflows (limits of detection/quantification, robustness, stability) to support formal release testing as regulatory expectations for medium-molecular-weight therapeutics evolve.

Conclusion

The described non-suppressor IC method using combined 18-crown-6 and 15-crown-5 additives provides an effective solution for simultaneous separation of Tris and six common cations in oligonucleotide API matrices. The method delivers excellent linearity and repeatability, accurate recovery in spiked API samples, and can achieve shorter run times by inclusion of a small fraction of acetonitrile. This approach offers a practical analytical route for monitoring residual Tris and inorganic cations in development and QC of oligonucleotide therapeutics.

References

• Shimadzu Application News: Analysis of Tris(hydroxymethyl)aminomethane in Oligonucleotide API Using Ion Chromatography, First Edition Mar. 2026 (Application note 01-01065-EN).
• Related Shimadzu Application News on non-suppressor cation analysis: Application News No. 01-00274-JP.
• Acknowledgment: Juzen Chemical Co., Ltd. for sample provision (as noted in the original application note).